Proteolysis and physiological regulation

Molec. Aspects Med. Vol. 9, pp. 173-287, 1987 Printed in Great Britain. All rights reserved.

0098-2997,'87 $0.00 + .50 Copyright (~ 1987 Pergamon Journals Ltd.

PROTEOLYSIS AND PHYSIOLOGICAL REGULATION Judith S. Bond

Department of Biochemistryand MolecularBiophysics, Virginia CommonwealthUniversity, Richmond VA 23298, U.S.A

Robert J. Beynon

Department of Biochemistry, Universityof Uverpoo/, PO Box 147, Uverpool L69 3BX, U.K.

Contents

Preface

175

CHAPTER

1

INTRODUCTION AND OVERVIEW i.I Classification and nomenclature 1.2 Diversity of proteases 1.3 Regulation of proteases 1.4 Proteases in disease processes

176 176 179 182 183

CHAPTER

2

STRUCTURE AND ENZYMOLOGY OF PROTEASES 2.1 Structural features 2.2 Mechanisms of peptide bond hydrolysis 2.3 Kinetic features 2.4 Evolutionary relationships

184 184 186 189 191

CHAPTER

3

SPECIFICITY OF PROTEASES 3.1 The meaning of specificity 3.2 Primary specificity 3.3 Subsite specificity 3.4 Higher order specificity

193 193 194 196 199

173

174 CHAPTER

J. S. Bond and R. Jo Beynon 4

INHIBITORS OF PROTEASES 4.1 Introduction 4.2 Mammalian protease inhibitors 4.3 Microbial protease inhibitors 4.4 Synthetic protease inhibitors 4.5 Protease inhibitors as therapeutic

agents

CHAPTER

5

PROTEASES: MEASUREMENT AND MEDICAL USES 5.1 Principles of protease determination 5.2 Measurement of proteases as antigens 5.3 Protease determinations in diagnosis 5.4 Proteases as therapeutic agents

CHAPTER

6

PROTEASES AS MEDIATORS OF PHYSIOLOGICAL PROCESSES 6.1 An overview of proteolytic regulation 6.2 Proteolytic generation of biologically active molecules 6.3 Proteolytic inactivation of biologically active molecules 6.4 Integration of proteolysis with metabolic regulation

201 201 201 206 209 211 213 213 216 217 219 222 222 225 227 228

CHAPTER

7

INTRACELLULAR PROTEOLYSIS 7.1 Processing of nascent proteins 7.2 Intracellular protein degradation 7.3 Regulation and dysfunction of intracellular proteolysis

230 230 231 238

CHAPTER

8

PROTEASES IN CELL-CELL INTERACTIONS 8.1 Introduction 8.2 Proteases and the nervous system 8.3 Proteases and cell proliferation/ tumour formation 8.4 Proteases and growth factors 8.5 Protease nexins 8.6 Proteases in the reproductive system 8.7 Other processes involving proteases

242 242 243 245

CHAPTER

EXTRACELLULAR PROTEOLYTIC SYSTEMS 9.1 Coagulation/fibrinolysis 9.2 Complement 9.3 The kallikrein-kinin system 9.4 Cellular defence processes 9.5 Inflammation 9.6 The renin/angiotensin system 9.7 Proteases and inhibitors in pulmonary emphysema

9

Concluding

remarks

246 247 247 248 250 250 253 254 255 25'6 259 261 263

Acknowledgments

264

References

264

Preface

We are all familiar with the enzymic hydrolysis of peptide bonds, at least in the context of gastrointestinal digestion of ingested proteins. Our view of the proteolytic enzymes and their mode of action is often dominated by our understanding of the enzymes of the digestive tract - a view that obscures the true nature of proteolysis as a pervasive and exquisitely controlled process, capable of fine regulation of a variety of biological events. Proteases, enzymes that attack other polypeptides, have a special position in the history of biochemistry, physiology and medicine. Pepsin was the first enzyme to be named, by Schwann, in 1825. Proteases, derived from diverse sources such as plants, pancreatic and gastric juices, and the mouth parts of maggots have been used in medicine as aids to digestion, wound debridement, treatment of burns and ulcers, in cosmetic products and for deproteinisation of contact lenses. This review attempts to bring together many examples of proteolysis and to draw from them general principles of their mode of action, control and dysfunction. The review is broadly divided into two parts. The first six chapters provide the background of the enzymology, protein chemistry and measurement of proteases. Additionally, inhibition of proteases and the general principles of protease-mediated regulation are discussed. The second half of the review takes as the main themes various physiological events and the role of proteases in their mediation. Here we have attempted to include discussion of pathology of proteases and the consequences of their dysfunction. The literature in this area is extensive and the diversity of biological events that involve proteolysis is such that treatment in detail is impossible. Thus, we have emphasised the underlying principles rather than the details, using the latter in an illustrative fashion. Inevitably, we have treated some aspects superficially and have neglected some. The review concentrates on proteolysis in m ~ a l ian systems, human where possible. Hopefully, the base of the coverage is broad enough and the literature cited extensive enough that readers can further pursue areas of specific interest.

175

Chapter 1

Overview

1.1 Classification and nomenclature Proteases are hydrolases; i.e. they cleave peptide bonds with the addition of water and are therefore placed within category 3.4 ('Acting on peptide bonds') of the International Union of Biochemistry formal enzyme classification (EC) system (Webb, 1984). Proteases are further classified as exo- or endopeptidases and each protease has been given a number according to Fig. I.I. The exopeptidases fall under the subcategories 3.4.11 - 3.4.19 and they are grouped according to their substrate specificity; they include amino- and carboxypeptidases (peptidases whose action is directed by the amino- or carboxy-terminus of the peptide, respectively) and dipeptidases. The carboxypeptidases (EC 3.4.16-18) have been further subdivided according to their mechanisms of action as indicated by the catalytic residue at the active site (serine, cysteine or a metal ion). Most of the exopeptidases act on peptides that are not blocked by substitutions of the termini. The newest group of exopeptidases (omega peptidases, 3.4.19.) are able to remove amino acids from the termini of a peptide that lack a free alpha-amino group or alpha-carboxyl group; for example, 3.4.19.1 can remove an N-acylamino acid from the N terminus of a peptide. Tripeptidylpeptidases have not yet been assigned an EC number but future classification schemes will include these enzymes (McDonald, 1985). The endopeptidases (that cleave peptide bonds internally in peptides and usually will not accommodate the charged amino or carboxyl termini amino acids at the active site) are classified according to mechanism, rather than substrate specificity. There are four basic catalytic mechanisms for endopeptidases that can be distinguished most readily by sensitivity to protease inhibitors: these are the serine, cysteine, aspartic and metallo-proteinases. According to the latest classification scheme from the International Union of Biochemistry (Webb, 1984), the serine proteinases (EC 3.4.21) are most abundant in eukaryotic organisms (approximately 50 different enzymes have been identified); the other proteinase groups each have about 15 entries. There has been some confusion over the trivial terms used to describe proteolytic enzymes. Most of this confusion has arisen from the lack

176

Proteolysis and Physiological Regulation Exopeptidases 3.4.11.

-amino acylpeptide hydrolases (aminopeptidases) e.g. leucine aminopeptidase (3.4.1 1.1)

3.4.13.

dipsptide hydrolases e.g. cysteinyl glycine dipeptidase (3.4.13.6)

3.4.14.

dipeptidylpeptide hydrolases (amin odipeptidases) e.g. dipeptidyl peptidase 1 (3.4.1 4.1)

3.4.15.

peptidyldipeptide hydrolases(carboxydipeptidases) e.g. dipeptidyl carboxypeptidase I (3.4.15,1)

3.4,16.

serine carboxypeptidases e.g. tyrosine carboxypeptidase (3. 4.16.3)

3.4.17.

metailo-carboxypeptidase(carboxypeptidases) e.g. carboxypeptidase A (3.4.17.1)

3.4.18.

cysteine carboxypeptidases (carboxypeptidases) e.g. lysosomai carboxypeptidase B (3.4.18.1)

3.4.19.

omega psptidases (exopeptidases th at remove sub6tituted N- or C- terminal amiro acid residues) e.g. peptidyl glycinamidase (3.4.1 9.2)

3.4.--.

tripeptidylpeptidases e.g. tripeptidyl peptidase (3.4.--.--)

Endopeptidases

O- ............. ~

o=~l,,,q,,oll~ ,lllIBDH=Hll~ ,=l=lHOHllll~ C-O~,,,,,,,,,,,,,..O

............. --O-O

3.4.21.

serine proteinases contain an active site serine residue e.g. trypsin (3.4.21.4)

3.4.22.

cysteine proteinases (formerly thiol proteases) e.g. ~thepsin B (3.4.22.1)

3.4.23.

asparti¢ proteinases (formerly acid or ¢arboxyl proteases) e.g. pepsin A (3.4.23.1)

3.4.24.

metailo-proteinases (formerly metal proteases) e.g. vertebrate collagenase (3.4.2 4.7)

3.4.99.

proteinases of unknown mechanism e.g. angiotensinase (3.4.99.3)

Fig. 1.1. Classification of proteases and their relationship within the enzyme nomenclature scheme

177

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J.S. Bond and R.J. Beynon

of a sharply defined specificity o f t h e s e enzymes, s u c h t h a t e x o p e p t i d a s e s can d i g e s t p r o t e i n s and e n d o p e p t i d a s e s can e x h i b i t exopeptidase activity. For the purposes of this review and in keeping with the recommendations of other authorities in this field (Barrett, 1986), we shall adopt the scheme shown in Fig. 1.2. Thus, all p r o t e o l y t i c enzymes fall within the scope of the general term 'protease' or 'peptide hydrolase'. They are further subdivided into e n d o ~ p t l d a s e s : those that hydrolyse internal peptide bonds within a peptide chain, or e x ~ p t l d a a e s : those that are restricted to the hydrolysis of peptides at or near the N- or C- terminal of the peptide or protein.

Peptide hydrolases/proteases

I

Endopeptidases (proteinases)

I

I

Exopeptidases

Fig. 1.2. Recommended usage of common terms for peptide hydrolases.

The action of exopeptidases is directed by the amino terminus (aminopeptidases) or the carboxyl terminus (carboxypeptidases) of the substrate. Enzymes that have both endo- and exo-peptidase activity are generally classified as endopeptidases and the term proteinase is reserved for, and can be used interchangeably with the term 'endopeptidase'. The 20 amino acids found in proteins can give rise to 400 different dipeptides and hence, 'flavours' of peptide bonds (alphabetically, from Ala-Ala, Ala-Asp .... Val-Val) and it is probable that all of these bonds exist in vivo. As will be d i s c u s s e d in Chapter 3, proteases are rarely absolutely specific in their action and any protease has the potential to hydrolyse more than one substrate. This ability to attack more than one 'type' of peptide bond has one consequence for the naming of proteases. Many enzymes are named according to the substrates that they act upon and the reaction that is catalysed - lactate dehydrogenase, DNA ligase etc. The earliest proteases to be studied were rather unspecific and were given names that did not imply a preference for one p a r t i c u l a r protein; terms such as trypsin, thrombin, papain, and cathepsin were used. As it has become more apparent that proteases are involved in a wide range of p h y s i o l o g i c a l processes the tendency to name enzymes on the basis of substrate has become more con~mon. Trivial names such as 'insulindegrading proteinase' or 'enkephalinase' are sometimes chosen on the basis of the interests of the research group and can imply a specificity that may not be realised in vivo (Turner et al, 1985; Kenny, 1986). We caution those with a peripheral interest in proteases that trivial names, whilst useful as a reference to enzymes that have yet to be formally classified, can be m i s l e a d i n g and should not be taken too literally.

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179

1.2 Diversity of proteases The truism that 'proteases hydrolyse peptide bonds' conceals the range of events that involve proteolysis. At one end of the continuum, the goal is to bring about complete hydrolysis of a mixture of proteins and generate amino acids. The digestive system springs to mind most readily but it is worth noting that the process of intracellular protein catabolism is highly active and that more protein is broken down inside cells than in the lumen of the gastrointestinal tract. Another site of extensive protein hydrolysis is the proximal tubule of the kidney where much of the protein that enters the glomerular filtrate is hydrolysed and recovered as small peptides or amino acids.

Table 1.1. Diversity of processes involving proteolysis Process

Tissue

Examples of proteases involved.

Coagulation

plasma

thrombin, Factor X, Factor VIII

Fibrinolysis

plasma

plasmin, plasminogen activators

Immune response

plasma

complement C3

Plasma pressure regulation

plasma

renin, angiotensinconverting enzyme

Defence against foreign matter

haemopoieticceils

cathepsin G, elastase, chymase

Cell-cell communication

nervous system, many membrane proteases

Digestion

gastrointestinaltract

trypsin,chymotrypsin, carboxypeptidases

Nitrogen economy

kidney, lysssomes

proximal tubule peptidases, oathepsins

Fertillsation

reproductive organs

acrosin

Uterine involution

uterus

collagenases, cathepsins

Intrasellular protein catabolism

all cells

cathepsins, others

Intracellular protein processing

all calls

signal peptidase, others

To contrast these processes of extensive hydrolysis, there are many examples whereby the cleavage of a single peptide bond elicits profound physiological consequences. Formation of kinins by hydrolysis of kininogens, activation of zymogens, the generation, processing and inactivation of angiotensins all involve controlled and restricted action of proteases. Table i.i lists some of the processes that involve proteolysis. The list is not intended to be exhaustive but serves to emphasise the multiple roles that are played by proteases in the body. Detailed treatment of any of the processes listed in ~he table is beyond the scope of this review. Proteinases catalyse

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J.S. Bond and R.J. Beynon

reactions by formation of covalent intermediates of enzyme and substrate (e.g. serine and cysteine proteases) and the formation of non-covalent tetrahedral intermediates (e.g. metallo-proteases). The mechanisms of most of the mammalian proteases have not yet been studied in detail and are often assumed to be analogous to the few abundant and well-defined proteases that have been crystallised and analysed by multiple techniques. Many of the recently discovered mammalian proteases are larger and more complex enzymes (in terms of subunit structure) than their prokaryotic counterparts and may have more complicated mechanisms and superimposed controls. Our perception of proteases can be attributed to the detailed study of a subset that are small and monomeric. However, proteases are globular proteins ranging in molecular mass from approximately 20,000 to >600,000 Daltons and the more recently discovered, larger members are not yet understood to the same degree. The minimal requirements of an extended substrate binding site and the correct environment for functional groups seem to be met within a mass of approximately 25,000 Daltons. Even in the larger proteases that have been studied, the catalytic domain, recognised by homology with other smaller proteases, is roughly the same size as in the small proteases. It seems likely that the non-catalytic sequences regulate catalytic activity and macromolecular recognition. A good example is provided by the intracellular calcium-activated protease, calpain, that comprises four domains; a catalytic domain homologous to other smaller cysteine proteases, a regulatory calcium-binding domain that is homologous to ca!modulin and two central domains of unknown function. Intrinsic membrane proteases will retain anchor sequences that insert into the phospholipid bilayer. In some instances the high molecular mass of some proteases can be attributed to an oligomeric structure but the consequences of quaternary structure in proteases are unknown. Finally, as our knowledge of proteases increases it is apparent that they share most of the complex structural features of other proteins, including disulphide bridges and carbohydrate residues. There is considerable heterogeneity in both the type and amount of proteases associated with tissues, cells and subce!lular organelles. For example, many of the plasma proteases are specifically synthesised in the liver and are secreted into plasma. Other cells, such as mast cells and leukocytes express specific proteases that are associated with their special roles. By contrast, most cells synthesise the full complement of lys0somal proteases but vary greatly in the relative amounts of the enzymes. This heterogeneity extends to the different cells that constitute the tissue, a complication that has been the source of considerable confusion when a protease derived from a minor cell type is ascribed to the major cell type. There are different types of proteases associated with subcellular structures (Fig. 1.3). The !ysosome, a subcellular organelle that contains hydrolytic enzymes capable of degrading most of the macromolecules of cells, contains very high concentrations of cathepsins. The endoplasmic reticulum contains specialised peptidases that are involved in the correct processing of nascent proteins. Thus, the diversity of type, amount and compartmentalisation of proteases provides yet another way of extending the potential of this class of enzymes to mediate biological events.

Proteolysis and Physiological Regulation

181

j 'Plasmamembrane:inactivationand] Lysosome:completedegradationof hydrolysisofpeptidesandproteinsI endocytosedproteins(heterophagy)and I ..... ,,,,,,,,

intracellularproteins(autopha~y)

~anCytosolCytoskeletaJ : remodelling I d degradationof cellularproteins I

0

I

Nucleus:regulationof gene expressionby proteolysisof chromatinproteins.

©

O

ot

MJtochondria:processingand 1 degradationof some mitochon. ddal proteins.

JTransportvesicles:zymogensand active J to extracellular

PernOteoaSnnemS;r~.route '

'

I

! Endoplasmicreticulum:processing I of nascentproteins.

Fig. 1.3 Subcellular locations and roles of proteases

182

J.S. Bond and R.J. Beynon

1.3 Regulation of proteases Proteolysis is to all intents and purposes an irreversible reaction which, together with the lack of absolute specificity, introduces the potential for adventitious and irrecoverable damage. It follows that the activity of this class of enzymes must be stringently controlled. It is well established that the action of proteases is restricted through several devices and these are d i s c u s s e d in detail in later chapters. Here we present a brief introduction to control of proteases with the objective of emphasising the diversity of the mechanisms that have evolved. Many biological events require that proteolysis be restricted spatially. Examples are the localised activation of plasma coagulation and the release of proteases at a site of inflammation. In the first example, localisation is attained by restricted access of a pervasive system to the site of action and is controlled by localised release and activation of zymogens, restricted substrate specificity and inhibitors that define the outer limit of the sphere of effect. In the second, cells migrate to the site of action and release proteases at that site. Intracellularly, proteases are denied free access to many potential substrates by compartmentalisation, by the action of inhibitors or by the loss of a degree of freedom through membrane association. Some evidence for the potentially damaging consequences of adventitious proteolysis can be derived from the recognition that protease inhibitors are almost as common and w i d e s p r e a d as proteases. Naturally occuring protease inhibitors are typically large peptides or proteins and are usually capable of supressing the activity of more than one protease. Current thinking supports the view that the inhibitors function primarily to restrict the activity of proteases and thus act in a protective fashion. There is little evidence for modulation of biological events by alteration of the concentration or activity of an inhibitor under conditions where the protease level remains constant (although some disease states are elicited by a deficiency of inhibitor under conditions where protease levels are normal). Inhibitors are often found in close proximity to their target enzymes - plasma is a rich source of inhibitors that can react with all of the plasma proteinases in addition to those that a c c i d e n t a l l y leak out into the interstitial fluid from other cells. Similarly, the lysosomes are surrounded by cytoplasm that contains inhibitors to the cysteine proteases. Protease activity can also be restricted by the simple expedient of physicochemical inactivation of the enzyme in a foreign environment. The best known example is the denaturation of pepsin in the neutralalkaline environment of the duodenum. Escape of cathepsins from the acidic environment of the lysosome is a c c o m p a n i e d by rapid inactivation of some of the proteases at cytoplasmic pH values. Many proteases are initially synthesised as inactive precursors (zymogens) that are subsequently a c t i v a t e d at the functional site. Reasons for the evolution of zymogens are not hard to find. First, they protect the secretory cell from the p o t e n t i a l l y harmful effects of a protease that must be stored in a secretory vesicle. Secondly, m a i n t e n a n c e of a high concentration of an inactive zymogen allows for rapid activation and a correspondingly prompt response to an external

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183

stimulus (such as the response of the coagulation system to tissue damage). Zymogen activation systems are a feature of extracellular processes - it is likely that intracellular proteolytic events are continuous and are controlled in a less episodic fashion than is possible by activation of zymogens. However, a number of intracellular proteases (such as the cathepsins) are synthesised as precursors and thus, may be considered as "zymogens" that prevent action of the protease before it has been installed in the correct cellular location. U n r e s t r i c t e d action of a protease has the oportunity to deplete the pool of substrate to a great extent. This has some potential as a control process and shifts the primary determinant of regulation to availability of substrate. Thus, the rate of synthesis, secretion or uptake of substrate may control the effect of a protease that has the role of rapid depletion of a substrate pool. This type of control may be of relevance in cell-cell communication where the duration of the response to a signalling peptide is determined by alteration of the rate of secretion in the context of an u n c h a n g i n g potential for degradation.

1.4 Proteases in disease processes Proteases are fundamental to, or involved in the pathogenesis of a great number of diseases. That this class of enzymes should participate in so many diseases is not surprising in view of the their multiple roles in normal cellular function. In several instances the aetiology of the disease can be ascribed to a specific deficiency of a p r o t e a s e - f o r example, Christmas Disease. In other diseases such as congenital disposition to emphysema, inflammatory diseases and metastasis the involvement of proteases is indicated strongly but may not be the primary defect. Many diseases (insulin-dependent diabetes, cancer, nephrotic syndrome, septicaemia and post-surgical trauma) elicit a generalised wasting of tissues (cachexia); this must involve the processes of intracellular protein catabolism. Additionally, proteases may be involved in the expression of tissue specific diseases such as muscular dystrophies. The w i d e s p r e a d involvement of proteases in disease processes has led to extensive research on the therapeutic value of protease inhibitorso Indeed, there are several successful therapies based upon administration of protease inhibitors, including anticoagulant therapy, the treatment of hypertension and septic shock. Design of inhibitors is greatly facilitated by knowledge of the mechanisms and structure of target enzymes; this is discussed in more detail in Chapter 4. At the same time, the absence of stringent substrate specificity of proteinases can create problems in the design of an inhibitor that is targeted to a single protease. However, protease inhibitors have considerable potential as drugs (Barrett, 1980a) and we anticipate that as understanding of the role of proteases in pathogenesis accumulates, intervention in their activity will become more and more feasible.

Chap~r2 Structure and enzymology of proteases

2.1 Structural features Before the 1980's proteases were thought of, in general, as small (molecular weights of 15,000 to 35,000), stable enzymes. Indeed there are many examples that fit in this category: such as the mammalian proteases trypsin, chymotrypsin, carboxypeptidase A, pepsin, and the bacterial proteases subtilisin and thermolysin. These enzymes are among the best-understood of any enzymes and the fine structures of a few have been determined by x-ray diffraction to a resolution of about 0.2 rim. They are similar in that they are monomeric and relatively stable enzymes; properties that contributed in important ways to the ease of their characterisation. They are all secreted from cells and act extracellularly. For example, the gastric and pancreatic enzymes are active in the gastrointestinal lumen and the bacterial enzymes are secreted into the pericellu!ar environment. The extracellular enzymes often contain disulfide bridges which contribute to their stability. The lysosomal proteases (cathepsins) are among the best-characterised cellular proteases and are relatively small enzymes; molecular weights in the range of 21,000 - 42,000. By contrast, a surprising number of the recently discovered mammalian cellular proteases are large, we define a large polypeptide as >80,000 Daltons, derived from the findings of Kiehn and Holland (1970) and Srere (1984) who have shown that the majority of polypeptides in cells (from Escherichia co!i to HeLa cells) have subunit molecular weights of 30,000 to 50,000 and that only 3 to 5% of the proteins have subunit molecular weights greater than 80,000. The small proteins (single polypeptide chains of <20,000 Daltons) were generally found to be secreted proteins, hormones or extracellular proteases. The large proteins were either structural proteins or enzymes which catalyze complex reactions (multifunctional enzymes). Proteases span the size range of known enzymes and the larger ones may well have evolved regulatory properties, sites for interaction with macromolecular complexes, increased substrate specificity and surface areas large enough for multiple interactions (Srere, 1984; Cornish-Bowden, 1986).

184

Proteolysis and Physiological Regulation

185

There are many examples of mammalian proteases that do not fit the generalisation of small, monomeric and stable enzymes. Many of the exopeptidases have molecular weights of the order of 300,000 and typically are relatively unstable glycoproteins with complex subunit structures (McDonald, 1985). The endopeptidase calpain (E.C. 3.4.22.17), present in the cytosol of many cells, has a molecular weight of 110,000 and is a heterodimer with subunits of 80,000 and 30,000 (Murachi, 1983); the ATP-dependent proteases have molecular weights of >500,000 and are composed of several polypeptide chains (Hershko and Ciechanover, 1982; Waxman et al., 1985); several other high-molecular weight cytosolic proteases have been described (Dahlmann, 1985; Dahlmann et al., 1985; Rivett, 1985; Rosin et al., 1984). Several plasma membrane-associated peptidases are also large proteins. For example, enteropeptidase (E.C. 3.4.21.9), the enzyme that acts on trypsinogen to form active trypsin, has a molecular weight of 145,000; endopeptidase 24.11 (formally E.C. 3.4.24.11), which acts on insulin B chain and small biologically active peptides, has a subunit molecular weight of 90,000 and probably exists as a dimer (Kerr and Kenny, 1974); meprin, which acts on insulin B chain and large proteins, also has a subunit molecular weight of approximately 90,000 and is active as a dimer or tetramer (Beynon et al., 1981). The large proteases are not restricted to cellular enzymes, there are several examples of large complex extracellular proteases as well. For example, the human blood coagulation Factor XI (Plasma thromboplastin antecedent) a precursor of protease Factor XIa has a molecular weight of 160,000 (Jackson and Nemerson, 1980). Kallikrein, the protease that converts kininogen to bradykinin, is active as a 90,000 molecular weight protein. In addition, the large proteases are not restricted to mammalian tissues. Several prokaryotic proteases are large proteins: e.g., Escherichia coli ATP-dependent protease La is a tetramer of four identical 94,000 Daltons subunits (Waxman and Goldberg, 1985), and Rhodococcus erythropolis contains a protease of 90,000 Daltons (Shannon et al., 1982). Thus proteases from many different sources are quite diverse in terms of size. The stability of proteases also spans a wide range. Most of our information on proteases comes from studies of those that are stable to heat and harsh environmental conditions; unsurprisingly the stable proteases are easier to purify and work with. Thus, the lysosomal proteases are often purified from mammalian tissues after an autolysis step in which many other proteins are destroyed or denatured. The purification of some proteases associated with renal brush borders includes a process that involves treatment with toluene and trypsin, a procedure that the proteases survive whilst other enzymes are inactivated and destroyed. Yet there are a large number of proteases that are quite unstable and difficult to purify. Many of the large cytosolic proteases seem to be in this latter catagory. One common feature of cytosolic endopeptidases that may relate to their instability is the presence of cysteine residues at the active site as well as in other areas of the protein. The potential for cysteine residues to be oxidised or to react with metal ions may create problems. In addition, the fact that these proteins are large indicates that there may be a variety of domains that interact with other molecules in addition to a number of sites that may be vulnerable to proteolysis (either by other proteases or through autolysis).

186

J.S. Bond and R.J, Beynon

Evidence is accumulating that indicates that the large size of some proteases is a consequence of gene fusion and that this has resulted in the evolution of proteases from simple digestive enzymes to regulatory enzymes (Neurath, 1984). The proteases of the blood coagulation cascade are good examples of this phenomenon. The domain organisation of human blood coagulation Factor IX (Christmas Factor) and protein C, for instance, are strikingly similar. They both contain similar pre-pro-leader sequences, activation peptide domains, catalytic domains (both are serine proteases), growth f~ctor domains (homologous to epidermal growth factor, EGF) and CaZ+-binding domains (Katayama et al., 1979). The catalytic domains are clearly homologous to each other and to the pancreatic serine proteases trypsin and chymotrypsin, as well as to the catalytic domains of several other blood coagulation enzymes. While the active sites of all these proteases are similar, the substrate specificity of the enzymes is different and the multiple domains confer different regulatory properties on the enzymes. Some of the plasma proteases (e.g., prothrombin and plasminogen) have 'kringle' domains (Patthy, 1985). Kringles are triple-looped, disulphide-crosslinked domains that may appear once or in multiple copies in one protein and are thought to be important for binding of mediators, such as membranes, other proteins, or phospholipids. The multiple domains of the coagulation proteases convert a simple digestive enzyme into a complex one, endow these enzymes with the ability to interact with, and to be regulated by, large (e.g., other proteins) and small ligands (e.g., CaZ+).

2.2 Mechanisms of peptide bond hydrolysis Endopeptidases can be conveniently subdivided into four mechanistic classes depending on the functional groups at their active sites: serine, cysteine, aspartic and metallo-peptidases (Barrett and McDonald, 1980; Webb, 1984). The serine proteases are the most abundant proteinases (numerically) of those discovered thus far. This group includes trypsin, chymotrypsin, elastase, the coagulation factors that are proteases (at least 5), plasmin, thrombin, all the complement protease components (at least 7 different enzymes), plasminogen activator, kallikrein, tryptase, and chymase. These enzymes contain essential serine and histidine residues at their active site. The mechanisms proposed for their action on substrates involve the covalent binding of substrates to the serine residue and the participation of the histidine in the reaction as a general base catalyst (Kraut, 1977). Chymotrypsin is probably the most thoroughly characterised member of this group and some classic studies have come from investigations of this enzyme (e.g., Spencer and Sturtevant, 1959). Studies with protease inhibitors definitively showed that a serine residue (Set-195 in chymotrypsin) was at the active site and essential for activity. The use of the active site-directed inhibitor diisopropyl fluorophosphate (DFP, DIFP or diisopropyl phosphofluoridate, DipF) was used to establish the presence of an active site serine residue in chymotrypsin and subsequently for many serine proteases. DFP forms a stable inhibitorenzyme complex with serine enzymes in a ratio of 1 DFP to 1 molecule of enzyme. There are 28 serine residues in chy~notrypsin, but DFP will only react with the active site residue. DFP will not react with chymotrypsinogen, the inactive precursor of chymotrypsin, denatured chymotrypsin, serine alone or serine in small peptides. Thus, there is

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something unique about the serine residue at the active site that makes it particularly reactive and facilitates the covalent binding to DFP. The formation of a one to one complex of a serine protease with DFP completely prevents the binding of substrates to the enzyme and there is a good experimental evidence to justify the designation of DFP as an "active site" reagent. There are other enzymes in addition to proteases that contain active site serine residues, e.g. esterases, phosphatases, that are also totally inactivated by DFP, so this reagent is not specific for proteases. Indeed, the potent inhibition by DFP of acetylcholinesterase means that it is highly toxic, a factor that probably contributes to the wider use of the less effective reagent phenylmethanesulphonylfluoride (PMSF). Several lines of evidence implicate a histidine residue as an active participant in chymotrypsin action. Some of the evidence derives from work with another active site reagent, tosyl-L-phenylalanyl chloromethane (TPCK) (Schoellmann and Shaw, 1963). TPCK is structurally similar to substrates for chymotrypsin and reacts stochiometrically with the enzyme; one histidine reacts per chymotrypsin molecule. Again TPCK will not react with inactive forms of chymotrypsin (chymotrypsinogen, denatured chymotrypsin) or histidine alone in solution. The TPCK-enzyme complex is stable and analyses have identified the amino acid residue that reacts to be His-57. TPCK will not react with the enzyme if it has been inhibited beforehand with DFP and from many studies, including crystallograpic studies, the close proximity of Ser195 and His-57 at the active center has been established. The mechanism of serine proteases involves the reaction of a substrate (e.g., the model substrate tyrosine-glycinamide) with the hydroxyl group of the active site serine residue. For chymotrypsin, the nonprotonated imidazole group of His-57 increases the reactivity of Ser195, by attracting the H atom of the serine-OH group and making the oxygen more nucleophilic (i.e., more negatively charged). The serine oxygen attacks the carbonyl carbon of the substrate (the carbonyl carbon of tyrosine in the tyrosine-glycinamide substrate for chymotrypsin), and forms an acyl-enzyme complex between the substrate binding subsite on the enzyme (Ser-195) and the P1 site (tyrosyl residue) on the substrate. The C-terminal portion of the substrate (glycinamide) is the leaving group and the acyl-enzyme intermediate is then hydrolyzed. For the breakdown of the acyl-enzyme complex it has been suggested that the positively charged imidazole group of His-57 attracts an hydroxyl group of water and this hydroxyl group acts as the nucleophile to split the ester bond linkage of the enzymesubstrate intermediate complex. This hydrolysis results in the formation and release of the substrate (as tyrosine in the above example) and the return of the enzyme to its original form. The cysteine proteases are also abundant in nature and occur in plant, bacterial and animal cells. This group includes papain and chymopapain (from papaya), ficin (from figs), bromelain (from pineapple), actinidin (from kiwi fruit), the lysosomal cathepsins B, H, and L, the calpains, the ATP-dependent proteases of bacteria, mitochondria and reticulocytes, as well as high molecular cytosolic proteases of mammalian cells. Papain is the best characterised of the cysteine proteases. Papain is a single polypeptide chain with a molecular weight of 23,400. As with all cysteine proteases, the enzyme is completely inactivated by thiol blocking reagents such as iodoacetic acid and

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p-hydroxymercuribenzoic acid. The catalytic residue at the active site (Cys-25) forms an acyl-enzyme intermediate with substrates in a manner similar to that described for active site serine residues. Similarly, a histidine residue, His-159, is important for increasing the reactivity of the active site thiol and the acylation of the enzyme involves the attack of the negatively charged sulphydryl of Cys-25 on the substrate. The rate-determining step of the reaction appears to be the deacylation reaction and the mechanism of this reaction is similar to that for the serine proteases. The active site of papain is formed from two domains of the protein with a cleft between them with Cys-25 and His-159 in close proximity but on opposite sides of the cleft. The substrate binding site on the enzyme interacts with approximately seven amino acid residues on the substrate and while Cys-25 is the binding site (S I) for the intermediate complex, the substrate specificity is determined primarily by other subsites. Some of the cysteine peptidases show evidence of both endopeptidase and exopeptidase activity. Thus cathepsin H hydrolyzes internal bonds in polypeptide chains and can also act as an aminopeptidase; it is referred to as an endoaminopeptidase (Lorand, 1981). Cathepsin B has endopeptidase activity as well as peptidyldipeptidase activity depending on the substrate (McKay et al., 1983). The aspartic proteases, formerly called acid proteases because of their activity at low pH values, are less abundant than the serine and cysteine proteases and are synthesised only by eukaryotic organisms. Examples of this group include pepsins, chymosin (rennin), renin and cathepsins D and E. These enzymes contain two aspartic acid residues at their active sites and are all inhibited by pepstatin (a small peptide isolated from Actinomycetes spp.) and diazo compounds, such as diazoacetyl-L-phe-methyl ester, that react with carboxyl residues. The best characterised member of this group is pepsin but the reaction mechanism of aspartic proteases is the least understood of the four classes. Pepsin is a single polypeptide chain of molecular weight 34,644. The substrate binding site interacts with four or five amino acid residues on substrates and the enzyme has a preference for hydrophobic amino acid residues on both sides of the scissile bond (this seems to be true of all aspartic proteases). For pepsin, Asp-32 and Asp-215 are the two catalytically active residues and there is evidence that they are hydrogen-bonded. The details of the mechanism of the catalysis by pepsin is as yet unknown but it does not appear to involve the formation of covalent enzyme-substrate intermediates. General acid-base catalysis is probably involved. The metallo-proteases are widespread in nature, occur in prokaryotes and eukaryotes but relatively few have been purified and wellcharacterised (Bond and Beynon, 1985). This class includes thermolysin, collagenases, gelatinases, and plasma membrane peptidases. Most of the metallo-proteinases contain zinc as the metal ion that is essential for activity. The metallo-peptidases should be distinguished from the metal-activated peptidases; metallo-peptidases contain metals as an integral part of their structure (this means that the binding constant between enzyme and metal is large and the metal does not easily dissociate), by contrast, metal-activated peptidases bind metals loosely and thus metal ions readily dissociate and must be

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Proteolysis and Physiological Regulation added to solutions to activate the enzyme. Metal-activated are cysteine or serine proteases, not metallo-proteases.

proteases

Thermolysin is a bacterial metallo-proteinase with a molecular weight of 34,600. The three dimensional structure of the enzyme has been analyzed (Matthews et al., 1972). There is one zinc atom per enzyme molecule at the active site that is important in catalysis. Thermolysin also contains four calcium ions per molecule and these ions play a role in enhancing the stability of the enzyme. The enzyme contains an extended cleft that binds the substrate polypeptide and the binding at the active site involves a complex between zinc, histidine and glutamate residues. The role of zinc is to enhance the reactivity of water or hydroxyl ions and polarize the peptide bond prior to nucleophilic attack. The metallo-proteinases that have been described range in molecular weights from 17,000 to 800,000 and are also very diverse in terms of substrate specificity. The collagenases, for example, show a high degree of specificity for native collagen and have very little proteolytic activity against most other substrates; by contrast, thermolysin and meprin are general peptidases that hydrolyse a broad range of polypeptide substrates. Most of the metallo-proteinases described are secreted from cells or are bound to the plasma membrane of cells (this includes thermolysin, collagenases, the kidney brush border peptidases). By virtue of their location, they are not exposed to the reducing environment of the cell and they contain disulphide bridges. Intrasubunit disulphide bridging is also found in other classes of proteases (e.g., in the serine peptidase chymotrypsin and the cysteine peptidase cathepsin B). At least two metallo-peptidases (meprin and enteropeptidase) contain intersubunit disulphide bridges resulting in the formation of covalently linked subunits (Bond and Beynon, 1986; Liepnieks and Light, 1979). Proteinases of unknown mechanisms, that do not fit into one of the four catagories above, have been recognised as a new group (EC 3.4.99; Webb, 1984). Proteases are usually classified mechanistically by their ability to be inhibited by group specific inhibitors (metal chelators, sulphydryl reagents, serine active site reagents) and for the great majority of proteases this has worked well and yielded simple, definitive answers. There are some proteinases, however, that are difficult to classify because they react with several different classes of inhibitors or with none of the inhibitors usually used. These unclassified proteases may not have been characterised well enough to fit into the known groups, and might, for instance, represent mixtures of proteases, or they may truly represent classes of proteinases that are different from the four known classes. There appear to be a few examples of metallo-cysteine proteases (e.g., Rosin et al., 1984) and signal peptidases that are not sensitive to the standard protease inhibitors (e.g., Wolfe et al., 1982).

2.3 Kinetic features Two factors define the activity of proteases: the affinity for the substrate (described in terms of Km, the Michaelis-Menten constant) and the speed at which catalysis can be brought about (described in terms of kcat, the "turnover number" of an enzyme or maximum number of molecules of product formed per molecule of enzyme per min under defined conditions). These kinetic constants are usually determined

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J.S. Bond and R.J, Beynon

using purified enzyme preparations in vitro and give us some idea of the preference of enzymes for substrates and the efficiency of catalysis under specific conditions of pH, buffers, temperature. This type of information has allowed the determination of the specificity of enzymes for substrates. Both the Km and kcat values are important because they determine the ultimate rate of the enzymatic reaction a% a variety of substrate concentrations. If an enzyme has a very low Km (high affinity) but also a small kcat, the rate of hydrolysis of the substrate may be very low. Likewise if the enzyme has a very high Km (low affinity) but a high kcat, the substrate may be considered a poor substrate at low concentrations, but a good substrate at high concentrations. The specificity of an enzyme for a substrate therefore depends on several factors. A good way to express specificity is in terms of the parameter kcat/Km which considers both rate and binding terms. As already mentioned and detailed in Chapter 3, the proteases span a wide range of specificity. Several examples have been mentioned above (e.g., collagenase is highly specific for substrate, thermolysin has a broad specificity) and examples of highly specific and non-specific enzymes can be found in all the classes of proteases. It should be remembered, however, that the kinetics and specificity of proteases in vivo may be quite different from that in vitro. For instance, cathepsin activity against substrates is usually measured using catalytic amounts of enzyme (micrograms, < I~M), whereas in the lysosome where these enzymes act, the concentrations of the cathepsins are estimated to be in the range of ImM (25 mg/ml). These large differences in enzyme concentration may substantially alter the ability of the cathepins to hydrolyze substrates and our interpretation of information obtained from studies in v i t r o must be mindful of this fact. Also when enzymes are extracted from membranes, often using harsh treatments, the activity of the enzymes against specific substrates may be altered. It is clear that pH affects enzymatic activity, and many of the proteases operate in cellular and extracellular compartments where their activity will be optimal. The most familiar examples are pepsin which works well in the strongly acidic environment of the stomach, trypsin and chymotrypsin that have pH optima of approximately 8 for most substrates and therefore work well in the slightly alkaline environment of the small intestine, and the cathepsins that hydrolyse polypeptides best in the acid range (pH 3-6) and therefore are suited for the acid environment of the lysosome. The activity of the serine peptidases is usually neutral to slightly alkaline and this is appropriate for the many peptidases of this group in the plasma. The cytosolic cysteine proteases, such as calpain and the ATP-dependent proteases, also work optimally at neutral to slightly alkaline pH values in cells. The pH of a compartment may also limit the activity of certain proteases; thus the neutral pH of the cytosol will cause the lysosomal cathepsins to be inactive and unstable. The concentration of specific substrates for proteases is another factor that is important for the physiological action of proteases. For example, many proteases may be capable of hydrolysing neuropeptides that are present in the plasma and in the external environment of cells, but at the low concentrations that these peptides are present, only specific proteases may degrade the neuropeptides and interactions with receptor proteins may be favoured over those with proteases. Receptor proteins may also effectively

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191

the concentration of peptides in the membrane and thereby the availability of the substrate to proteases.

The presence of inhibitors of proteases and other ligands that may modulate activity (e.g., calcium ions) may also affect the activity of proteases in vivo. There are a great variety of inhibitors present in and outside of cells that may totally inhibit proteolysis at inappropriate times or places and may act in a counterpoised system to regulate the amount of hydrolysis normally (See Chapters 4 and 6).

2.4 Evolutionary relationships Within each class of proteinase there seem to be evolutionarilyrelated families of enzymes. Many of the serine proteinases have been sequenced and the picture that emerges is that two superfamilies exist within this classs: the chymotrypsin family and the subtilisin family (Barrett and Salvesen, 1986). The subtilisin family is only found in prokaryotes and shows no homology with trypsin and chymotrypsin. The chymotrypsin family is the one that is most commonly found and enzymes of this family exist in prokaryotic and eucaryotic cells; examples of this family include trypsin, elastase, thrombin, kallikrein, the blood coagulation factors and complement proteases. Comparative structural studies of chymotrypsin, trypsin and elastase have shown that the polypeptide backbones of these enzymes are almost superimposable and small differences in the substrate binding site can explain their different specificities (Chapter 3). The three enzymes have approximately 50% amino acid homology, which is surprisingly low since their three dimensional structures are so similar. Further analyses indicate that the internal amino acids are considerably more conserved (60%) than the exterior amino acids (10% homology). This is an example of divergent evolution from a con~non ancestor (Fersht, 1985). The catalytic domains of the larger, more complex serine proteinases of higher organisms, again show significant homology with the simple digestive serine proteinases. Thus the catalytic domains of thrombin and Factor Xa are 38% and 50% homologous with trypsin. The fibrinolytic and blood coagulation systems also have large noncatalytic domains and the function of these domains are for activation and interaction with small and large molecules which regulate the proteolytic activity and determine biological specificity (Jackson and Nemerson, 1980). The noncatalytic domains (calcium-binding, kringle, growth factor, and finger domains) correspond to amino acid sequences that occur in several proteins in addition to proteases, and it is possible that the genes coding for such domains have fused with those coding for proteases in different ratios at different times during evolution (Patthy, 1985). The cysteine proteinases can also be divided into families but in this instance four or more families exist (Barrett and Salvesen, 1986). The most common family in plants and eukaryotic cells is the papain family. Lysosomal cathepsins B, H, and L have a very high degree of homology with papain (90% around the active site cysteine) making it likely they have evolved from a common ancester. The amino acid sequence near the catalytic cysteine residue of calpain, the calciumdependent protease of many cell types, has approximately 33% homology with papain; while this is a very significant degree of homology, it is much lower than that found for papain and the cathepsins. Thus it has been suggested that calpain may be more closely related to one of

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the other families of cysteine proteinases than papain (Metrione, 1986). The other families of cysteine proteinases are represented streptococcal proteinase, clostripain and a viral proteinase.

by a

Calpain is a good example of a protease that has probably evolved by gene fusion (Ohno et al., 1984). The 80,000 Dalton subunit of calpain contains four domains: domain II is the catalytic domain which is homologous to other cysteine proteases, domain IV is a regulatory domain that binds calcium ions and which is homologous to calmodulin and the function of the other two domains is not yet known. By virtue of the fusion of these domains, a simple digestive protease has been converted to an enzyme that is regulated by ligands such as calcium ions and has a much more restricted substrate specificity than its less complex digestive counterpart. The aspartic proteinases appear to belong to one family. Amino acid sequencing and structural data on pepsin, cathepsin D and other aspartic proteinases indicate that some of the domains of these proteinases resulted from gene duplication and that there is considerable homology among these enzymes (Huang, et al., 1980). The evolutionary relationships of the metallo-proteases are not yet known because very few have been sequenced. Data from the endoproteinase thermolysin and the exopeptidase carboxypeptidase A indicate that these two enzymes have no sequence or structural homologies and yet their active sites and catalytic mechanisms are very similar (Fersht, 1985). They might represent examples of convergent evolution.

Chap~r3

Specificity of proteases

3.1 The meaning of specificity Mammalian proteases hydrolyse trans-peptide bonds linking L-amino acids. Thus, they are specific in the broadest sense of the word, hydrolysing neither cis-peptide bonds nor those involving D-~mino acids. Whilst some proteases exhibit a high degree of primary or substrate specificity, most exhibit preferences rather than absolute requirements. Thus, chymotrypsin preferentially cleaves peptide bonds in which the carbonyl group is provided by bulky hydrophobic ~mino acids. Specificity is an overused term that has several meanings (Fersht, 1977). The statement 'protease A is specific for substrate B' is often encountered and indeed, it is not uncommon to see, in this context, A referred to as 'B-ase'. Unfortunately, this type of statement often tells us little or nothing about the behaviour of the system in vivo. Under appropriate conditions in vitro, proteases can act as esterases, peptide synthetases and peptidyl transferases; yet it is never claimed that they exert all of these functions in vivo. Similarly, the observation that a protease can attack one substrate in vitro, in an optimised system, may have little relevance to the functional role of the protease. Competing substrates, their relative rates of hydrolysis and the location of the enzyme and substrate may conspire to diminish the rate of a reaction in vivo that appeared to be very powerful in vitro. We warn therefore that many claims of substrate specificity must be seen in a wider context and that this is a concept that can be misconstrued in discussions of protease function. Other aspects of protease specificity can be discussed with confidence. First, we can consider the substrate as a linear sequence of amino acids and establish preferences for the type of amino acids surrounding the scissile bond. Related to this is the preference of some proteases (the exopeptidases) for a free and ionised amino or carboxyl terminal. These aspects we define as primary specificity. Secondly, many of the critical regulatory processes involve the interaction of proteases with substrate molecules that have a strictly

193

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J.S. Bond and R. J. Beynon

controlled three dimensional structure. Under these circumstances i~ is inappropriate to consider such molecules purely as a linear sequence of amino acids; higher order structure must also be considered and its effect on specificity of proteases is a subject that merits detailed investigation.

3.2 Primary specificity As has been discussed in Chapter 2, a common feature of most proteases whose active sites have been examined by X-ray crystallography is a cleft (more or less clearly defined) running across the surface of the enzyme and into which the substrate fits. The cleft can accomodate many amino acids of the substrate (usually 6-8) but only one of the bonds in this extended sequence is hydrolysed. Thus, a distinction must be made between the active site, where the peptide bond is cleaved, and the substrate binding site that may extend a considerable distance beyond the active site. The classification of proteases was discussed in Chapter I. A major division of proteases separates the endopeptidases and the exopeptidases. This subdivision is based upon the ability of the protease to bind substrates at such a position that the free amino or carboxyl terminus interacts with the substrate site. Fig. i.i illustrates the differences between endopeptidases and exopep tidases. Tripeptidyl peptidases represent the limit of terminal removal that characterises the exopeptidases, based on the reasonable assumption that the terminal group is still interacting with the substrate binding site. For endopeptidases, the amino or carboxy! termini may be far removed from the site of hydrolysis, but this does not exclude the possibility that endopeptidases may exhibit some exopeptidase activity. In these instances, strong binding of some residues near the scissile bond may overcome weak or unfavourab!e interactions with other residues. The evolution of the two classes of proteases can be readily reasoned. First, because exopeptidases work on the 'ends' of a polypeptide chain, it follows that complete digestion of a large protein would be sequential, inefficient and would only be able to form small products (amino acids, dipeptides and tripeptides). Endopeptidase action, on the other hand can effect the rapid fragmentation of a polypeptide chain, generating a range of smaller peptides. Further, as the endopeptidase action continues, more and more new termini are formed. Endopeptidases are in general poor exopeptidases and thus, the additional activity of exopeptidases upon these termini completes the hydrolysis of the protein and releases metabolically valuable amino acids. Concerted action of endopeptidases and exopeptidases not only allows complete digestion of proteins but also permits generation or destruction of high or low molecular weight biologically active molecules by the removal of small or large peptides. Proteolytic attack upon a substrate may either endow it with a new biological activity or destroy or modify that activity. Either process may involve the action of endopeptidases or exopeptidases. In general, inactivating processes involve, or are followed by extensive digestion whereas activation of a substrate might be expected to require only limited modification of the substrate molecule. Thus, the specificity of the interactions must be regulated in accordance with the types of reaction that are elicited. Examples of these types of behaviour are provided in Table 3.1.

Proteolysis and Physiological Regulation

Table 3.1 Physiological regulation by limited or extensive proteolysis These examples illustrate the varying extents of proteolysis that are involved in biological processes in vivo. Note that pto~sses in which a biologically active molecule is generated are limited in extent of attack, imp4yinga greater specific'ty of action than those processes in which biologically active molecules are de6troyed. Enzyme

Substrate

Products

Biological effect

enteropeptidase

trypsinogen

trypsin, activation peptide

activation of zymogen

pancreatic proteinases, cathepsins

proteins

small peptides (variable)

digestion

'converting enzymes'

proenkephalin

met-enkephalin, other peptides

formation of neuropeptides

signal peptidases

nascent proteins

native protein, signal peptide (ca 20 amino acids)

biogenesis of proteins

enkephalin degrading enzymes

enkephalins

tripsptides, dipeptides

inactivation of neuropeptides

renin

angiotensinogen

angiotensin I

activation

angiotensin converting enzyme

angiotansin I

angiotensin II

activation

angiotensin converting enzyme

kinins

small fragments

inactivation

angiotensinases

angiotensin II

small peptides

inactivation

brush border exopeptidases of kidney and intestine

small psptides

amino acids

inactivation, digestion

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J. S. Bond and R.J. Beynon

196

3.3 Subsite specificity All proteases exhibit some preference for the type of amino acids than flank the scissile bond. Put another way, there is no protease that will hydrolyse all peptide bonds with equal efficiency. A totally nonspecific protease would have to possess a substrate binding site that was capable of interacting with all twenty amino acid residues large, hydrophobic groups such as phenylalanine, acidic functional groups such as aspartate and basic functional groups such as arginine. Such a binding site, if it existed, would be very poor; the variety of side chains in amino acids dictate that a binding site designed for one group would discourage the binding of others. Thus, a recurrent objective in the study of proteases is the definition of preference for particular amino acid side chains at proximal or distal positions relative to the scissile bond.

(Phe)

(Lys)

dl

m m

Chyrnom/psin

/

(Ala)

Ik

)",i c.,m

Trypsin

Elastase

Fig. 3.1 Relationship between pdmary specificity and substrate binding sites of the pancreatic serine proteases.

By way of example, the three pancreatic endopeptidases- trypsin, chymotrypsin and elastase are collectively able to attack many of the peptide bonds of ingested protein (Fig. 3.1). They achieve this by sharing the task; each of them is only able to hydrolyse a subset of all possible peptide bonds. These three enzymes are very similar in size, show sequence homology (see Chapter 2) and undoubtedly have arisen by divergent evolution from a common ancestor. Despite remarkable similarities in their three-dimensional shape and in their mechanism of hydrolysis of peptide bonds, they differ dramatically in their primary specificity, directed towards the amino acid that binds to S t (Fig. 3.2). In chymotrypsin, a deep pocket, lined with hydrophoblc residues, accomodates the side chain of this amino acid. Strong binding of the substrate is attained by hydrophobic interactions (the exclusion of solvent water molecules from the highly unfavourable hydrophobic environment around the substrate residue and within the pocket). Thus, chymotrypsin demonstrates highest affinity for hydrophobic residues. In trypsin, the residue in the base of the pocket is no longer neutral and hydrophobic but is an aspartyl residue. Thus, a strong negatively charged group has been introduced

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197

R

I

. N-C.C-N_C.C-N-C.CTN.C.C-N-C.C-N.C-C-N-C-C-N-CC H I II H j ~ H j II i H I il H J Jl H J II H j ff H J U HO HO HO i HO HO HO HO HO

P3

P2

|

PI!

PI'

P2'

P3'

P4'

I

Scissile bond Fig. 3.2 The subsite nomenclature of Schecter & Berger (1967)

at the base of the pocket and positively charged, basic residues bind electrostatically in the pocket. The pocket is still largely hydrophobic and excludes water molecules; this has advantages because electrostatic 'salt bridges' between two oppositely charged residues are strengthened in a solvent-free environment. Finally, elastase retains the hydrophobic pocket but access to the bottom of the pocket is prevented by replacement of small residues at the mouth of the pocket by larger residues, such that the pocket is occluded. E!astase can therefore only hydrolyse bonds adjacent to small hydrophobic residues. In the example above, the amino residue at the active site has 5he strongest effect on affinity between the enzyme and substrate. However, as mentioned previously, most proteases are sufficiently large to accomodate additional residues of the substrate and the binding of these residues may also contribute to the interaction with the enzyme. In order to describe the effects of these distal interactions Schechter and Berger (1967) introduced a system of subsite nomenclature. The substrate binding site is discussed in terms of subsites, each of which interacts with one amino acid residue of the substrate. The amino acid residues on the amino terminal side of the scissile bond are numbered PI, P2, P3 .... counting outwards; on the carboxy terminal side they are numbered PI', P2', P3' ..... again counting outwards. By analogy, the subsites on the surface of the enzyme are numbered similarly; ...$3-$2-SI-SI'-$2'-$3'... (Fig. 3.2) Using this nomenclature, it is possible to describe the preferences in the extended substrate binding sites of proteases. In some proteases, such as the pancreatic enzymes, the residue interacting with S i is the primary determinant of binding. However, even in the presence of such

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J.S. Bond and R.J. Beynon

a strong binding site there are preferences for certain amino acid residues in S 2 and S 3. Such interactions can be probed by examination of the bonds that are preferentially hydrolysed in proteins, by the activity of the enzyme towards model peptide substrates or by the use of inhibitors that map the active site. Other proteases, such as cathepsin B, have the highest affinity for the residue binding to S 2. Most serine and cysteine proteases are not demanding of the residues occupying SI',$2' , etc, this allows non-peptidic moieties to be linkei to peptides such that they are valuable chromogenic or fluorogenic reporters of hydrolytic activity (Chapter 5). By contrast, neither aspartic nor metallo-proteases tolerate non-peptidic groups on either side of the scissile bond; hence there are few good chromogenic substrates for these classes of enzymes. In some of these enzymes the residue binding to S I' or S 2' becomes paramount. Finally, in exopeptidases the substrate binding site must be formed in such a way as to discourage occupancy by all but the terminal sequences of the substrate. Extension of the specificity of the substrate binding site has the potential for increased complementarity between the substrate and the protease - a key observation in physiological regulation. There are 400 possible dipeptide sequences, 8000 possible tripeptide sequences, 160000 possible tetrapeptide sequences and 3200000 pentapeptide sequences. Thus, a protease restricted in action to a pentapeptide sequence has a low probability of encountering the same sequence by chance in another protein (it has been suggested that on stochastic grounds, pentapeptide sequences may be considered as virtually unique). This does not exclude the possibility that the sequence may have evolved in more than one substrate. In reality, it is rare for a protease to be that specific (exceptions may be intestinal enteropeptidase, cleaving Val-(AsP) 4-Lys from trypsinogen and renin, cleaving angiotensin I from angiotensinogen) and thus most proteases are active towards more than one peptide sequence. In part this reflects the ability of the substrate binding subsites to accomodate more than one amino acid residue - it is probable that all of the aliphatic amino acids can bind to a hydrophobic subsite, albeit with differing affinities. The proteases of the blood clotting system, although highly controlled and acting in an amplification cascade, have the potential to hydrolyse a number of physiological substrates. Thrombin, for example, can hydrolyse peptide bonds in a variety of substrates (Elodi eC al., 1984) including, amongst others, Arg-Gly in fibrinogen and Factor XIII, Arg-Thr in prothrombin, Arg-Val in fibrinogen, Arg-Ser in prothrombin and antithrombin III, Arg-Ile in Factor VII and Lys-A!a in actin. Three important points can be inferred from such data. First, the P1 residue which must be basic and preferably arginine) is more important than the PI' residue (which can vary from Gly to His; Elodi et al., 1984). Secondly, the limited attack by thrombin upon each of these substrates implies that most of the bonds are inaccessible to the enzyme; each of the substrates might be expected on stochaszic grounds to possess additional bonds that would be susceptible to the action of the enzyme (see below). Finally, although thrombin has a relatively broad primary specificity, uncontrolled hydrolysis of other proteins must somehow be prevented; this aspect is addressed in other chapters.

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a) Substrate held in rigid conformation complementary to binding sites.

b)

Flexible substrate must first adopt the correct conformation to bind to enzyme.

Fig 3.3 The role of substrate conformation in proteolysis If the substrate is held in a conformation that is complementary to the binding site on the protease, productive interactions will be favoured more fTerquently than if a flexible substrate is required to fold into the correct conformation. Such folding may however be directed by the active site, following binding of an amino acid residue to a subsite on the enzyme surface.

3.4 Higher order specificity Primary specificity in not enough to define all aspects of the interaction of proteases and their substrates. The conformation of the substrate is an important determinant of the rate and extent of proteolysis (Rupley, 1963). Trypsin, for example exerts a strong preference for peptide bonds of the Lys-X or Arg-X type, with much less preference for the surrounding amino acids. On average, a protein might be expected to possess about 10% basic (Arg or Lys) residues and thus, digestion of any protein with trypsin should be anticipated to produce extensive fragmentation. However, examples abound whereby the digestion in vitro of a native protein by trypsin causes the hydrolysis of very few of the potentially vulnerable peptide bonds. An example in vivo is provided by the tryptic activation of the zl~r,ogens chymotrypsinogen and proelastase. Implicit in this model is the generation of derivatives that exhibit similar or greater resiszance to further proteolytic attack; in this example the derivatives are

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J.S. Bond and R.J. Beynon

chymotrypsin and elastase. Limited proteolysis is a well-documented phenomenon that has important roles, both as a biochemical tool (Mihalyi, 1978, Beynon, 1980, Beynon et al., 1985) and as a regulatory phenomenon (Neurath and Walsh, 1976). In other instances, rapid and complete digestion will occur, presumably because the initial attack destabilises the protein and increases the availability of other vulnerable bonds. To illustrate, intracellular protein breakdown or intestinal digestion must bring about the complete hydrolysis of proteins that are destined for catabolism (Chapter 7). The significance and consequences of these different types of behaviour are discussed in detail in Chapter 6; for now it is sufficient to emphasise that the three dimensional structure of the protease and its substrate can be critical factors in eliciting specificity of response. It is important to appreciate that the 'specificity' of a protease-substrate action may be most strongly linked to the availability of the substrate or to the exposure of a vulnerable region of the substrate molecule (Alexandrov, 1969). It may rarely be necessary to demand that a proteinase has evolved in such a way as to restrict its action to one peptide sequence. Indeed, in the examples where this might be the case, the protease and substrate have evolved together into a complementary pair where both make important contributions to their interaction. A fine example is provided by the notable specificity of action of renin upon the e-globulin angiotensinogen (Ondetti and Cushman, 1982). The minimal substrate that is hydrolysed appreciably by renin corresponds to an octapeptide occupying S 5 .... S 3' - it is interesting to calculate the probability of an octapeptide being unique in nature. However, it is also likely that the interaction between renin and angiotensinogen is facilitate/ by the structure of angiotensinogen that may enhance binding. Possibly, the higher molecular weight substrate serves to 'lock' the hydrolysed region in a conformation that enhances binding to the enzyme. Such an interaction would be more energetically favourable than one in which a flexible peptide had to sacrifice flexibility in order to b i n d to the active site (Fig. 3.3).

Chapter4 Inhibitors ofproteases

4.1 Introduction Most mammalian proteases can be inhibited in vivo by at least one, and often by several endogenous inhibitors. Inhibition of proteases is physiologically important, both in the regulation of their action and in the defence of tissues against unwanted proteolytic action. Further, successful therapeutic strategies have been based upon drugs that are protease inhibitors and many more have been the subject of speculation and preliminary testing. As might be expected from the diversity of proteases, the inhibitors span a broad range of molecular weight, mechanism of action and source. Many are derived from microorganisms and still more have been produced by chemical synthesis. The importance of the inhibitors is as great as the proteases and a review on the physiological roles of proteases cannot be discussed without a balanced treatment of their down-regulation by inhibition. This chapter presents a basic review of the diverse group of protease inhibitors. The aim here is to provide background information on inhibitor diversity, mechanisms of inhibition and roles. Further information can be obtained from several reviews that have appeared recently (Katunuma et al., 1983; Travis and Salvesen, 1983; Barrett and Salvesen, 1986).

4.2 Mammalian protease inhibitors Endogenous mammalian protease inhibitors are exclusively proteins with molecular weights of at least 5000 and of those described to date are all directed towards endopeptidases. Further, although there are several examples of inhibitors of soluble serine, cysteine and metalloproteases there are no well documented examples of specific inhibitors of aspartic proteases or of membrane-bound endopeptidases. The aspartic proteases, renin, cathepsin D and pepsin are all restricted in their action, either by virtue of their intolerance of the high pH of the surrounding milieu or by virtue of an extremely restricted substrate specificity. Thus, they may not require inhibition, either for regulation or protection against uncontrolled proteolytic activity. Similarly, membrane-bound proteases are inherently restricted

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in their action; the loss of a degree of freedom attendant upon binding to the membrane means that they no longer enjoy unlimited access to substrate. Under these conditions, the substrate must be delivered to the protease and thus, regulation is primarily controlled by substrate availability. There are at least nine protease inhibitor$ in plasma, collectively constituting over 10% of plasma protein (Table 4.1) They are directed towards serine, cysteine and metallo-proteases and those that are inhibitory towards the first class are by far the best represented. Intraceilular inhibitors are much less well defined and those that have been well characterised are active towards cysteine proteases. As our understanding of the diversity of protease inhibitors develops, it becomes increasingly apparent that there are probably less inhibitors than are currently thought to exist. Thus, ~-i anticollagenase is probably identical to the tissue inhibitor of metallo-proteinases (TIMP). An unusual and unexpected identity relates ~-i cysteine proteinase inhibitors and kininogens, all of which are part of the cystatin superfamily (MOller-Esterl et al., 1986; Barrett, 1986). Additionally, other inhibitors may be secreted by cells into interstitial spaces and act in close proximity to the site of release. The secretory pancreatic trypsin inhibitor, protease nexins (Knauer and Cunningham, 1984), tissue inhibitor of metallo-proteinases (Cawston et el., 1983; Bunning et el., 1984) and bronchial inhibitor fall into this category. However, some of these inhibitors can also be observed in plasma although it is not clear whether they are released directly into this fluid or whether they 'leak' in the same way as many other intracellular constituents enter the plasma pool. Intracellular protease inhibitors are all endopeptidase inhibitors, although the cystatins can inhibit dipeptidyl peptidase IV (Barrett, 1986). Here, a major question arises in the identification of the true location of the inhibitor. All tissues are vascularised, and are thus perfused by all of the plasma proteinase inhibitors. Thus, intracellular inhibitors have to be determined within the background of plasma contamination. Nonetheless, there is good evidence for the existence of several intracellular protease inhibitors. The Type 1 cystatins, also known as 'Stefins' are at least partially intracellular and probably serve to prevent adventitious extralysosomal proteolysis by the lysosomal cysteine proteases. The cystatins (with the exception of one domain of extracellular T?~e 3 oystatins, otherwise kininogens) do not inhibit calpains, which are also intracellular cysteine proteases. However, calpains are specifically inhibited by calpastatin, an intracellular inhibitor that binds calpains strongly. Calpastatin is normally in excess over calpains and the failure to detect calpain activity in tissue homogenates is largely attributable to combination with calpastatin (Murachi, 1983; 1985). Other, less well characterised inhibitors have been described (Carney et el., 1980, Afting, 1983). Endogenous inhibitors generally do not display an absolute specificity for a single protease. These inhibitors are capable of inhibiting many proteases in vitro. For example, cystatins and =-I cysteine pro%ease inhibitors can suppress the activity of plant cysteine proteases. Other factors that determine inhibitor specificity come into effect in vivo. Kinetic factors may diminish the chance of an interaction, either because the inhibitor reacts more rapidly with another protease or alternatively, because another inhibitor reacts more rapidly with

Proteolysis and Physiological Regulation Table 4.1 M a m m a l i a n protease inhibitors Inhibitors marked with an asterix probably act intracellularly. Inhibitor

Target protease(s)

Probable role

c¢-1 proteinase inhibitor

elastase>>other serine proteinases

control of elastolytic processes

antithrombin III

thrombin>>Factor IXa, Factor Xa, Factor Xla

control of coagulation

=ol antichymotrypsin

cathepsinG,chymase

control of inflammatory response

~z-1 antiplasmin

plasmin

control of fibrinolysis

C1-inhibitor

activated complement C1 -r, C1 -s, kaHikrein ?

control of complement, bradykinin release ?

inter-cotrypsin inhibitor

pancreatic serine proteinases

unknown

~-1 anticollagenase

coUagenases

see TIMP

~-cysteine inhibitor, high Mr kininogen

¢athepsin H,L > cathepsin B

protection from cysteine proteinases

c~-2macroglobulin

mostproteinases

inactivation and removal of endogenous or exogenous proteinases

secretory pancreatic trypsin inhibitor

trypsin

protection from premature activation of trypsin

tissue inhibitor of metalloproteinases (TIMP)

collagenases, other metallopmteinases

control of connective tissue turnover

protease nexin-1

arginine-specifi¢ proteinases

cell-cell interactions, growth regulation

pmtease nexin-2

epidermal growth factor binding protein

protease nexin-3

nerve growth factor gamma subunit

°cystatins A

cathepsins ...

control of intracellul~ action of cathepsins

cystatins B

cathepsins..,

control of extracellular action of cysteine proteases

°calpsstatin

calpains I and II

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the protease (Bieth, 1984). One measure of the efficiency of interaction of proteases and inhibitors is the half-time needed for formation of the enzyme-inhibitor complex. A half-time of no more than i00 ms has been proposed as a rough guide to discriminate between physiological and artifactual interactions. Many protease/inhibitor pairs for which a physiological significance is most likely have halftimes one or two orders of magnitude below this figure. By contrast, other interactions for which there is little evidence of physiological significance yield half-times of many seconds (Travis and Salvesen, 1983). The relative concentrations of the different inhibitors are also critical in this calculation, especially if a pathological process serves to diminish the levels of one of them. When such kinetic factors are taken into consideration, it becomes clear that there are a number of protease/inhibitor pairs that have evolved a high degree of complementarity and thus, can be considered as specific. One mammalian plasma protease inhibitor, e-2 macroglobulin, differs from all of the others in that it binds a number of proteases with roughly equal avidity. The mechanism whereby this deliberately broad specificity is achieved is discussed in the next section. A few generalisations may be drawn from the interactions of proteases and their endogenous protein inhibitors. With the exception of ~-2 macroglobulin inhibition is competitive, meaning that the inhibitor binds to the substrate binding site of the enzyme and, as a consequence, inhibits the enzyme by exclusion of substrate. Many of the inhibitors are substrates themselves and are hydrolysed by the protease as a part of their mechanism of action (Laskowski and Kato, 1980). It follows that the inhibitors must exhibit structural features that predispose a productive binding of a specific region of the molecule to the active site of the enzyme. The conversion of an unproteolysed 'virgin' inhibitor to 'modified' inhibitor is often a slow process, indicative that the proteinase is prevented from completing the hydrolysis efficiently. X-ray crystallographic data indicate that the proteolytic reaction has 'stalled' at an intermediate stage and that the protease-inhibitor complex resen%bles a transition intermediate in the proteolytic reaction. Clearly, the binding of the inhibitor differs from the binding of the normal substrates in such a way as to prevent completion of catalysis. However, inhibition of the target protease can also be achieved with the modified inhibitors, and virgin inhibitors can bind tightly to proteases in which catalysis is prevented by chemical modification of active site residues (Travis and Salvesen, 1983). These data imply a high degree of complementarity between the three dimensional structure of the inhibitor and the shape of the substrate binding site in the protease. The mechanism of protease inhibition by one sub-group; the ~-i proteinase inhibitor family (~-I proteinase inhibitor, ~-I antichymotrypsin and antithrombin III) differs from that described above in two respects. First, the inhibitor-protease complex becomes covalently linked through the formation of an acyl enzyme intermediate (Chapter 2). Secondly, the modified inhibitor, having undergone this reaction, is unable to inhibit further protease molecules (Travis and Salvesen, 1983). The members of the cystatin superfamily (cystatins,

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Stefins and kininogens) are not cleaved by the target proteinase. They bind tightly to the proteinase in a mechanism that does not appear to require formation of a covalent linkage between protease and inhibitor. Finally, e-2 macroglobulin is a major component of plasma (250mg/100 ml) and is remarkable in its lack of specificity for proteinases. Most proteinases can be inhibited by ~-2 macroglobulin and a curious feature of the inhibition is that the enzyme:inhibitor complex retains activity against low molecular weight substrates but loses all activity against macromolecular substrates. A popular description of the mode of action of this inhibitor is described by the 'trap hypothesis' (Starkey and Barrett, 1977; Travis and Salvesen, 1983). In this model, the protease recognises a 'bait' region of the molecule that is labile to hydrolysis. When this region is hydrolysed, the inhibitor molecule (which is very large, Mr=725 000) physically entraps the protease, hindering access to macromolecular substrates. The bait region is contained within a 27-amino acid region of the molecule and all proteinases that are inhibitable by ~-2 macrogiobulin attack the inhibitor at this region. The only proteinases that are not inhibited are either very large (and may not be 'entrappable') or have such a restricted substrate specificity that they fail to hydro!yse the bait region. Because the bait region requires endoproteolytic attack, ~-2 macroglobulin is not an inhibitor of exopeptidases. Descriptions of endogenous inhibitors and their target proteases can be found in other chapters. Here, it is sufficient to make generalisations about the role of these inhibitors. In the least specific terms they can be seen as having two roles. First, they act to regulate the activity of target proteases in a controlled fashion. Many biological phenomena require that proteases operate in a continous fashion rather than in an 'all or none' fashion. One facet of the control of proteolysis in this manner involves restriction or moderation of the balance of protease action and inhibition and it is possible, for example, that this type of regulation operates in intracellular protein catabolism. Similar roles may be postulated for the protease nexins (Chapter 8). Without such controls, a minor initiation of the coagulation cascade should result in clotting of the whole bloodstream! A second role of many of the inhibitors is protection against excessive action of proteases. Restriction of the zone of influence of a protease is in part protective but can also be regulatory. However, the role of ~-2 macroglobulin is undoubtedly the prevention of undesired proteolysis. Its lack of specificity is critical as it can react not only with endogenous proteinases but also those introduced by, for example, venoms or as a consequence of tissue injury. The cystatins are probably present in the circulation to inhibit cysteine proteases that have escaped from the intracellular environment. Similarly, the intracellular cystatins may operate to supress the activity of extralysosomal but intracellular cathepsins. Further evidence for the roles of the mammalian inhibitors derives from the study of individuals who express a faulty inhibitor molecule or who are totally deficient in such proteins. Best understood are perhaps the variants of ~-i proteinase inhibitor (Chapter 9) but

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deficiencies of a number of plasma protease inhibitors are known, including m-2 antiplasmin (Kluft et al., 1984) and antithrombin III (Sambrano et al., 1986). At least three types of antithrombin III deficiency have been recognised, characterised by either decreased production of functionally normal inhibitor levels (Type i) or production of high levels of functionally abnormal inhibitor molecules (Types 2 and 3; Nagy and Losonczy, 1984).

4.3 Microbial protease inhibitors Many microbial species secrete secondary metabolites that are potent enzyme inhibitors, a property that has been capitalised upon in the development of antibiotics and other pharmacologically active drugs. During the 1970's Umezawa and colleagues embarked upon a systematic search of bacterial products that acted as protease inhibitors. Their search was highly successful and led to the identification of many peptides and peptide analogues that were potent inhibitors of all of the classes of endopeptidases and also, some of the classes of exopeptidases (Umezawa and Aoyagi, 1977; Umezawa, 1981; Umezawa and Aoyagi, 1983). Table 4.2 lists some of the inhibitors that have been used widely as research tools and also have undergone preliminary investigation of their potential as therapeutic agents. Limitations of space prevent detailed discussion of each of the inhibitors. However, there are relatively few mechanisms whereby inhibition is achieved and they form the basis of a useful generalisation. Peptide aldehydes (leupeptin, antipain, chymostatin, ~-MAPI, elastatinal, strepin P1 and thiolstatins) are characterised by reduction of the carboxyl group on the C-terminal amino acid to an aldehyde. This aldehyde group forms a hemiacetal or hemithioacetal with active site serine or cysteine residues, thus mimicking a tightly bound transition state analogue. The specificity of the inhibitor is largely dictated by the carboxy terminal amino acid that binds to S 1 or S 2 in serine and cysteine proteinases. Thus, leupeptin, antipain and strepin P1 (all containing carboxy terminal arginine aldehyde) inhibit trypsin and trypsin-like serine proteinases (Umezawa and Aoyagi, 1977; Umezawa, 1982; Ogura et al., 1985). However, the C-terminal amino acid cannot be the only determinant of inhibitory activity as thiolstatin D (N-acetyl-phenylalanyl-arginine aldehyde, Murao et al., 1985)) is not inhibitory to trypsin. Chymostatin and ~-MAPI, possessing a phenylalanine aldehyde residue, inhibit chymotrypsin-like proteinases. Finally, elastatinal, as might be expected, exhibits an alanine aldehyde residue in the carboxy terminal position. Granulocyte elastase, but not pancreatic elastase, is also inhibited by a nonpeptidic inhibitor, elasnin. Cysteine proteases are also vulnerable to inhibition by peptide epoxides. The naturally Occuring inhibitor E-64 contains such an epoxide residue that reacts with the active site cysteine residue to form a thioether. E-64 has no activity towards serine proteases and will not react with active cysteine residues in other classes of enzymes. Cathepsins B and L and calpains are all strongly inhibited by E-64 (Hanada et al., 1983). Inhibitors of metallo-proteases contain functional groups that are directed to the most obvious site of inhibition; the active site divalent metal ion. In general terms, the inhibitors consist of a

Proteolysis and Physiological Regulation Table 4.2 Microbial protease inhibitors Endopeptidase inhibitors are identified by the mechanistic class (S: serine, C: cysteine, M: metaJlo-and A: aspartic) and representatives of the enzymes of that class that are inhibited. Few of the inhibitors are truly diagnostic of a particular class; there are well documented examples of proteases that are not susceptible to an inhibitor of the appropriate class. Inhibitor

Structural type

Class:proteases inhibited

Leupeptins

peptide aldehyde

S.'trypsin,plasmin,others C:cathepsins

Antipain

peptide aldehyde

S.'trypsin, C:cathepsins

Chymostatin

peptide aldehyde

S:chymotrypsin

8-MAPI

peptide aldehyde

S:chymotrypsin

Elastatinal

peptide aldehyde

S:elastases

Strepin-P1

peptide aldehyde

S.'trypsin C:cathepsins, caJpain

Thiolstatin D

peptide aldehyde

C:

Elasnin

non-peptide

S:granulocyte elastase

Pepstatins

transition state analogue

A:cathepsin D,renin, pepsin

Phosphoramidon

sugar

M:endopeptidase 24.11

Talopeptin

sugar N-phosphoramidate

M:

FMPt

N-phosphoramidate

M:

Ep-64

peptide epoxide

C:cathepsins B,H,L, caipaJns

Best=in

modified peptide

arninoexopeptidases

Amastatin

modified peptide

aminosxopeptidasas

Arphamenines

modif~KI peptides

aminopeptidase B

Diprotins A,B

modified peptide

dipeptidyl peptidase IV

(S)c¢Benzylmalic acid

non-peptide

carboxypeptidase A

Muraosin

sugar peptide

angiotensin converting enzyme

Aspergillomarasmine

non.peptide

angotensin converting enzyme

A5&365 A,B

non-peptide

angiotensin converting enzyme

N-phosphoramidate

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J.S. Bond and R.J. Beynon

peptidic region that presumably allows binding of the inhibitor and a powerfully electronegative group that counters the strongly electropositive metal ion (Bond and Beynon, 1985). The phosphoramidates, of which phosphoramidon is the best-known, are dipeptides, N-linked to a phosphate group that in turn is esterified to a sugar residue (rhamnose or talose). It is clear that the sugar residue is totally unnecessary and indeed, removal of rhanu~ose from phosphoramidon potentiates its inhibitory activity. Other metallo-proteases, notably exopeptidases, are inhibited by other modified peptides or nonpeptides by mechanisms that are obscure. Many of them appear to couple a potentially tight binding substrate with a non-hydrolysable bond in place of the scissile peptide bond. In particular, a common theme is the replacement of the potentially vulnerable CO-NH bond by the undegradable CO-CH 2 . Aspartic proteases are inhibited by the pepstatins, a group of peptide derivatives that contain, in particular, the statine residue that is responsible for the strong inhibition of the aspartic proteases. When bound to the enzyme, the statyl residue is thought to resemble the structure of the transition state for normal hydrolysis and thus, binds tightly, cannot be hydrolysed and effectively locks the enzyme into a transition state that is thus inactive. Microbial species, and particularly Streptomyces species, have ~he ability to produce inhibitors of almost all of the classes of endopeptidases and exopeptidases. Little is known of the value to the organism in the production of such inhibitors. The genes encoding enzymes for leupeptin production are encoded on a plasmid and thus, are readily transferred from one Streptomyces species to another. No differences have been observed in cellular proteolysis of leupeptinproducing or non-producing cells. In general, the inhibitors are not grossly toxic and have no antimicrobial activity (Umezawa, 1982). However, the possibility remains that the inhibitors are protective, defending the organism from aggressive or nutritive proteases produced by other microorganisms or by the host cell. The discovery of these inhibitors was a major advance in the development of research on mammalian proteases. The availability of inhibitors that were non-toxic, usually water soluble and to a qreater or lesser extent enzyme specific, permitted experiments that had not previously been possible. Many of the inhibitors listed in Table 4.2 have been administered to primary or stable cell lines and their consequences on intracellular protein catabolism assessed (see for example Khairallah et al., 1985). They have also been of tremendous value in establishing the class and type of proteinase in characterisation and comparative studies. Many researchers use them to prevent unwanted adventitious proteolysis of a protein of interest (Beynon, 1987). However, they have been relatively ineffective in the control of proteolysis in intact animals, due in part to their relatively broad specificity. Commercially available, they form part of the armamentarium of the scientist who is interested in activity and function of proteases or even those who consider that proteolysis is an unacceptable and irritating nuisance.

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4.4 Synthetic pro~aseinhibito~ Because of the widespread involvement of proteases in biological processes it is unsurprising that many protease inhibitors have been synthesised in the hope of finding specific and effective inhibitors that can be used as therapeutic agents. Many hundreds of putative protease inhibitors have been prepared and their inhibitory profiles assessed in vitro. Few have been developed to the stage where they are tested extensively in vivo and still fewer have found application as drugs. It is inappropriate to detail here the many protease inhibitors that have been created by the organic chemists. Of greater significance is an outline of the principles that underlie the design of a protease inhibitor. Many factors must be taken into account in the design of a novel and effective protease inhibitor (Knight, 1977). The lack of absolute specificity of proteases introduces the possibility that an inhibitor directed towards one protease may also be effective towards others. Certainly, this is true in general of the microbial protease inhibitors (Umezawa, 1982; Umezawa and Aoyagi, 1977). The degree of specificity that must be introduced into the inhibitor structure is dictated by the number of proteases, other than the target, that are susceptible to it. The requirement for absolute specificity may also be diminished if the consequences of inhibition of other enzymes are relatively minor. Irreversible inhibitors form a covalent linkage with the target enzyme. By contrast, reversible inhibitors remain bound to the enzyme by virtue of the strength of the non-covalent association between enzyme and inhibitor. The synthesis of an irreversible inhibitor must incorporate two structural features. Firstly, some structural moieties cause the initial tight association between enzyme and the inhibitor and secondly, a powerfully reactive moiety will react covalently with the enzyme and form the inactivating linkage. By contrast, a reversible inhibitor can only be effective by virtue of tight association with the enzyme. Powerful inhibition is thus attained by introduction of many groups that are complementary to many of the substrate-binding subsites on the enzyme surface. A very tightbinding but reversible inhibitor can appear to be irreversible if sufficient interactions can be introduced. However, a highly complementary peptide would be a substrate, not an inhibitor and therefore, additional modifications must be introduced in order to circumvent destruction of the inhibitor by the enzyme. The design of a successful protease inhibitor must ensure a) tight binding to the substrate binding site of the enzyme and b) covalent or non-covalent interactions with the active site residues. It follows that the strategy of inhibitor design will depend upon the mechanistic class to which the protease belongs. Suicide inhibitors are mechanism based inhibitors that require activation by the target enzyme before they become inhibitory, thus conferring specificity of action. Lactone-based inhibitors of serine proteases have been described; these rely upon enzymic hydrolysis of 6chloro-2-pyrones to generate a reactive species, a haloenol lactone that subsequently inactivates the enzyme covalently (Chakravarty et al., 1982; Westkaemper and Abeles, 1983).

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Serine proteases offer two main potential targets to inhibitors. First, the nucleophilic active site serine residue is vulnerable to attack by a variety of reactive groups, including non-peptide molecules such as DipF (Chapter 2). However, such reagents are lacking in specificity (e.g., DipF also inhibits serine esterases such as acetlycholinesterase). Peptide aldehydes were recognised to be potential inhibitors of serine proteases before the microbial products were discovered (Thompson, 1973). New peptide aldehydes, modelled on the naturally-occurring inhibitors have proven to be almost as effective as the natural compounds (Galpin et al., 1983; Mulligan et al., 1985). Secondly, the histidine residue that participates in the charge relay system is well located for attack by alkylating or other reagents, such as the halomethylketones (Kettner and Shaw, 1981). Inhibition of specific serine proteases can be achieved by compounds that model the residue that binds to S I. Thus, benzamidine and related derivatives mimic an arginine residue and are inhibitors of trypsinlike proteases. Careful selection of the substituents bound to zhe benz~midine nucleus can, for example, generate inhibitors that exhibit differing affinities for thrombin and trypsin (Sturzebecher, 1984). Similarly to serine proteases, the primary targets for inhibition of cysteine proteases are the active site cysteine residue and the proximal histidine residue. Peptide aldehydes are able to inhibit cysteine proteases by formation of a tetrahedral adduct. A number of synthetic inhibitors, based upon the epoxide inhibitor E-64 have been prepared. A second class of cysteine protease inhibitors that have been used in studies with cultured cells are the peptide diazomethyl ketones such as benzyloxycarbonyl-phenylalanyl-phenylalanyl-CHN 2 and benzyloxycarbonyl-phenylalanyl-alanyl-CHN 2 (Shaw and Green, 198!; Shaw and Dean, 1980; Kettner and Shaw, 1981; Grinde, 1983). The obvious site of inhibition of metallo-proteases is the active site metal ion. In general, the inhibitors of this class of enzymes consist of a powerfully electronegative group that is located in close disposition to the active site zinc ion. The precise disposition is achieved by incorporation of the reactive group into a peptide-like structure that favours productive binding to the subsites on the enzyme surface. The metal-reactive moiety is electronegative and amongst those that have been successfully employed are carboxylate, thiol, phosphate and hydroxamate groups. Many metallo-protease inhibitors have been described in a recent review (Bond and Beynon, 1985). A number of these inhibitors are analogues of the tetrahedral transition state, the intermediate structural form of the substrate during hydrolysis (Hangauer et al., 1984; Chu and Orlowski, 1984). One group of metallo-protease inhibitors that are in use as therapeutic agents are those directed towards angiotensin converting enzyme; captopril and elanaprilic acid (normally administered as the esterified inhibitor, alanapril; Ondetti and Cushman, 1985; Cleary and Taylor, 1986). Thiorphan (modelled on captopril) has proven to be effective as an inhibitor of the enkephalin-degrading endopeptidase 24.11; this compound exerts antinociceptive effects in vivo. Inhibitors of aspartio proteinases have not been developed to the same extent as those of the other classes. Many of them are based on the natural inhibitor, pepstatin and possess additional desirable properties such as enhanced solubility (Kay, 1985a). As might be anticipated, there has been considerable investment in the development of renin inhibitors (Haber, 1984). These inhibitors are largely based upon the sequence of angiotensinogen (the natural substrate) but often

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incorporate an analogue such as the statine residue originally observed in pepstatin to mimics the transition state complex or which replace the normally hydrolysed peptide bond with a non-hydrolysable bond (Kay, 1985b; Leckie et al., 1985).

4.5 Protease inhibitors as therapeutic agents Few protease inhibitors have been developed to the point where they are used therapeutically (Barrett, 1980a). The lack of specificity of the inhibitors or target proteases makes effective control of a protease-mediated process rather difficult. Many of the therapies based upon protease inhibitors are administered in response to acute episodes in which the plasma proteolytic systems have become uncontrolled; under such unstable and often life-threatening conditions, the use of relatively unspecific inhibitors is warranted. However, control of homeostasis under chronic disease conditions demands greater subtlety of inhibitor design to elicit the required selectivity and pharmacological behaviour. Antihypertensive therapy is largely based upon administration of inhibitors of one of the enzymes responsible for generation of angiotensin II, angiotensin converting enzyme (ACE). Early studies assessed peptides that would compete with the substrate (Ondetti and Cushman, 1985) but a breakthrough was achieved with the development of new peptide analogues that introduced a powerful electronegative sulphur atom near the active site zinc atom of ACE. One member of this family of inhibitors, captopril, is orally active and has since become a widely used antihypertensive drug (Salvetti et al., 1985). It is also of value in the treatment of congestive heart failure (Ondetti and Cushman, 1985). Many other putative ACE inhibitors have been prepared and tested and from these has developed elanapril, the ester precursor of elanaprilic acid. In elanapril, the metal-reactive group is a carboxylate group that may eliminate some of the side effects of captopril that were ascribed to the sulphydryl group (Cleary and Taylor, 1986). New ACE and also renin inhibitors are continually under development (e.g. Thorsett et al., 1986). However, the very high specificity of the aspartic protease (and hence, the efficacy of reninspecific inhibitors) may be moderated by the additional difficulties of designing effective inhibitors of this class of proteases (Haber, 1984; Leckie et al., 1985). Another blood protease system, the coagulation cascade, is a target for antiprotease intervention. Excess activation of the cascade can lead to disseminated intravascular coagulation; insufficient activation of coagulation or overactivity of fibrinolysis can lead to excessive blood loss. Thus, protease inhibitors can be used to restore imbalances in either direction. Inhibitors of fibrinolysis are indicated in a wide range of circumstances (Verstraete, 1985) and one of the most common is the basic proteinase inhibitor from bovine tissues, aprotinin. Aprotinin (Trasylol TM) is a polypeptide of 58 amino acids and is an effective inhibitor of plasmin but because it is a protein and therefore vulnerable to the digestive processes, it cannot be administered orally. Aprotinin is also of value in supression of uncontrolled proteolysis in acute episodes such as septicaemia (Jochum et al., 1986) and pancreatitis (Lankish, 1984); Eglin C, a protease inhibitor from the leech, has also been investigated as a potential antiproteolytic drug (Snider et al., 1984; Schnebli et al., 1985).

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Other fibrinolysis inhibitors that can be administered orally are tranexamic acid and c-amino caproic acid (Verstraete, 1985). Neither of these compounds are particularly effective as protease inhibitors p e r se but interfere specifically with plasmin action. These compounds complex with plasminogen at the lysine-binding sites on the zymogeno The tight association of zymogen and substrate is therefore reduced, the stimulation of plasminogen activation by fibrin disappears and the dissociated plasminogen, even if activated, is rapidly inhibited by ~-2 antiplasmin. Finally, it has been suggested that protease inhibitors may be valuable as agents that, when administered simultaneously, prevent the proteolytic inactivation of other peptidic drugs. A number of elastase inhibitors have been developed is the search for effective for therapy of pulmonary emphysema (Powers and Bengali, 1986).

Chapter 5 Proteases: measurement and medical uses

5.1 Principles of protease determination The principles behind the measurement of proteolytic activity are the same as those for enzymes in general. The object is to choose an assay that is sensitive and selective for the protease of interest. Usually this involves estimating the rate of hydrolysis of a specific substrate, by measuring the rate of disappearance of substrate or rate of appearance of a product (Wagner, 1986). Catalytic amounts of the protease (tissue sample) are used under specific conditions of substrate concentration, buffer, pH and temperature. Although proteases do not contain coenzymes (organic cofactors), other compounds are often necessary for optimal proteolytic activity. NaCI or KC! are sometimes added to proteinase assays (usually to physiological concentrations) since ionic strength markedly affects proteolytic activity (see for example Beynon et al., 1981). For cysteine proteases it is often necessary to include a reducing agent in the assay, such as dithiothreitol, to activate the enzyme or to prevent the oxidation of the active site sulphydryl groups. The activity of some proteases can only be measured when activating ligands such as calcium ions or ATP are included in the assay. Enzyme activity is ideally expressed in terms of 'units'. The units of enzyme activity must be defined for each assay. Frequently one unit of an enzyme is defined as the ~r.ount which will catalyze the hydrolysis of 1 ~mol of substrate per minute under defined conditions (this is the definition of the International Enzyme Unit, although the katal (i mol/s) is the more correct term using S.I. units). The specific activity of a protease is often expressed in terms of micromoles of substrate hydrolyzed per minute per mg of protein. The temperature should be stated and it has been suggested by the International Union of Biochemistry that the temperature of reaction mixtures be 30°C when practical. It is also recommended that initial reaction rates be measured (initial velocity, before reaction products accumulate to any marked extent compared to substrate). This is particularly important for proteases where the products of the reaction may compete with the substrate for the active site and inhibit activity in the forward direction. The assay of proteases in crude tissue samples may also be problematic because of the presence of inhibitors or other peptides/proteins that compete with

213

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J.S, Bond and R.J. Beynon

substrate for the protease. However, if the tissue extract can be used in iow enough concentrations and the assay is a sensitive one, extraneous proteins added with the protease become less of a problem. Colorimetric, spectrophotometric, fluorimetric, radiochemical and bioassays have been used to measure protease activity. A brief discussion of some of the most common methods used will be given below. The assays measure the hydrolysis of protein or synthetic substrates, as loss of substrate or appearance of product. Readers are advised to consult methodological references for details of specific assays (Lorand, 1981; Barrett, 1977; Wagner, 1986; Ruddy, 1986). Measurements of proteolytic activity against a protein substrate include determination of: (i) an increase in ninhydrin-positive or fluorescamine-positive material due to the liberation of primary amines as peptide bonds are cleaved (Udenfriend et al., 1972), (2) formation of acid-soluble peptides from proteins; often the substrate protein is coupled with dyes (e.g., azo-casein), fluorogens (methylumbelliferyl-casein), or radiolabeled compounds ([14C]-labeled haemoglobin; [125I]-labeled insulin B chain) (Beynon, 1987; Khalfan et al., 1983; Roth et al., 1971) (3) inactivation of enzymes incubated with the protease, (4) loss of the substrate and appearance of products by high pressure liquid chromatography or sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Because of the ready availability of proteins such as haemoglobin and casein, they are often used as proteinase substrates either unmodified or coupled to agents that are easily detectable (mentioned above). Denatured haemoglobin is a good substrate for proteases that are active in the acidic pH range and casein is better as a substrate in the neutral and basic pH range. For proteins that exhibit a high degree of substrate specificity, haemoglobin and casein are not generally good substrates and other proteins/peptides must be used. For example, renin specifically cleaves a leu-leu bond in angiotensinogen to form angiotensin I and it has little or no general proteolytic activity; thus angiotensinogen or angiotensinogen dodecapeptide is the usual substrate for assay of renin. Sometimes it is desirable to measure a protease activity by a specific direct assay. For instance, when the protease of interest is present in a tissue extract that contains several different proteases, it is useful to have a labeled polypeptide where the substrate and products can be measured. The conversion of [125I]plasminogen to plasmin by plasminogen activator, for example, can be followed by measuring the decrease in radioactivity associated with the substrate band and an increase in the radioactive product bands on SDS-PAGE (Christman et al., 1977). With substrates such as collagen (the most appropriate substrate for collagenase), the detection of an unusual amino acid may be used to advantage; collagenase activity can be assayed by measuring hydroxyproline-containing peptides solubilised during incubation. Synthetic substrates have provided sensitive assays where the reaction product has a characteristic absorption spectrum different from its substrate. Peptide derivatives of 4-nitroanilides or methylcoumarins fit this catagory; the release of 4-nitroaniline from substrates can be monitored colorimetrically at 405 nm while the release of aminomethylcoumarin can be measured fluorimetrically (Pozsgay et al., 1978; Barrett, 1980b; Huseby and Smith, 1980). Automated enzyme assays are available with spectrophotometric and fluorometric detection systems for clinical analyses of many samples (Pearson et al., 1981). Most of

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the substrates that are available are designed for the measurement of specific types of proteases (e.g., leu-4-nitroanilide for aminopeptidases; N-blocked substrates such as Z-Phe-Arg-7-amido-4-methyl coumarin for endopeptidases that are capable of cleaving peptide bonds containing a basic amino acid residue in the P! position). Other sensitive assays take advantage of the fact that the reaction product can be coupled to a compound resulting in a complex that has a characteristic absorption spectrum. For example, many synthetic peptides containing a naphthylamide group linked to the C-terminal amino acid are available commercially, and when the bond between the naphthylamide and the amino acid is cleaved by a protease, 2-naphthylamine is released. Naphthylamine can be detected fluorimetrically or, after a coupling reaction with a diazonium salt, such as Fast Garnet, colorimetrically (Barrett and Kirschke, 1980). The diazocoupling results in a very sensitive assay and there are many naphthylamide substrates available for both exopeptidases and endopeptidases. Unfortunately, the naphthylamides are carcinogenic and for this reason are not substrates of choice. Many other protease substrates with easily detectable leaving groups are available (Wagner, 1986; Hemker, 1983). Many serine and cysteine proteases have esterolytic activity in addition to proteolytic activity (trypsin, chymotrypsin, plasminogen activator). For example, the hydrolysis of methyl ester substrates has been detected by measuring the disappearance of the substrate spectrophotometrically, the appearance of methanol, or the formation of acid during hydrolysis (Christman et al., 1977). The complement proteases (e.g., C2a, B) are quantitatively measured by haemolytic assays (Ruddy, 1986; Whaley, 1985). Erythrocytes coated with antibodies are haemolysed by the complement cascade of reactions and the degree of haemolysis can be quantitatively related to the amount of antigen or complement components. To assay individual complement proteases, plasma from patients deficient in the proteases to be measured (or all the necessary purified components of the complement cascade) are added in excess to the erythrocytes except the protease to be measured; the test sample then supplies the missing component in the haemolytic system. For assays of C2, for instance, erythrocytes sensitised by antibody and an excess of components C1 and C4 are incubated with either serial dilutions of component C2 (to establish a standard curve) or serial dilutions of test serum. The degree of lysis of the cells is measured by haemoglobin release and the amount of C2 in the sample can be quantitatively determined. Blood coagulation proteases or, more correctly, zymogens of the proteases in the blood can be measured by coagulant assays (Lorand, 1981). Again these assays are based on addition of the test sample to a coagulation system that contains all necessary ingredients execept the compound to be determined; plasma deficient in the individual coagulation proteases is available. For example, for plasma prekallikrein assays, the test sample is incubated with buffer and prekallikrein-deficient plasma. Other factors, such as calcium ions are added and the clotting time determined. The activity is calculated from a calibration curve where the log of prekallikrein concentration is plotted against the log of the clotting time. Radiometric assays are now also available for blood coagulation factors. The principle of the radiometric assays is that zymogens of the blood coagulation

216

J.S. Bond and R.J. Beynon

proteases are labelled by tritiation of their sialic acid residues, the rate of activation can be measured as solubilised tritium-labeled activation peptides. The zymogen substrates are labeled by reductive tritiation and then incubated with the appropriate enzymes and cofactors, samples are transferred to a solution of calcium-chelator to stop the reaction, trichloroacetic acid is added and the soluble tritiated activation peptide is measured in the supernatant fraction after centrifugation.

5.2 Measurement of proteases as antigens It is sometimes desirable to measure the amount of immunoreactive proteinase present in a tissue rather than the amount of proteinase activity. Proteases may be complexed to inhibitors and, thus, can not always be measured in tissue extracts by monitoring their enzymatic activities. The inhibitors may not be in contact with the protease of interest in vivo, however, upon homogenisation or other preparations of tissues for assay, the disruption of cellular structure may bring the enzymes and inhibitors into contact. Methods have therefore been devised to allow the determination of the amount of immunoreactive protein that is present. The measurement of trypsin in serum to diagnose pancreatic diseases provides a good example of an assay based on immunoreactive material. The diagnosis of pancreatitis is often difficult and because trypsin is found exclusively in the pancreas, i.e. it is organ specific, it is ideal to detect pancreatic injury or abnormalities. Trypsin activity is not, however, measurable in the serum because of the abundance of protease inhibitors present in the serum such as ~-2-macroglobulin and e-l-proteinase inhibitor. Therefore immunoassays have been developed that recognize trypsin, trypsinogen and trypsin bound to protease inhibitors such as e-2-macroglobulin (Koop, 1984). Several groups have developed assays for immunoreactive trypsin (IRT) and while they have obtained different quantitative results, they are all successful in detecting increases in trypsin in the serum in acute pancreatitis. Increases in immunoreactive trypsin are also found in renal insufficiency and decreases are found in chronic pancreatitis. The latter observation is due to exocrine insufficiency in the chronic form of the disease. For the diagnosis of acute pancreatitis, amylase activity in the serum is usually measured because of the high sensitivity of the assay for this enzyme, but if there is any doubt, immunoreactive trypsin is measured to confirm the diagnosis. Note that irreversibly inhibited proteases must be used to immunise the animal and generate antiserum; otherwise the potent antiproteolytic systems in the body will ensure very rapid removal of the protease. To develop a radio-immunoassay for a protein or peptide, it necessary to have (i) a sample of the pure protein (antigen), (2) a method to iodinate the protein so that it is not appreciably altered with regard to immunogenicity, (3) a specific antibody to the protein and (4) a method of separating the antigen-antibody complex from the antigen itself. The methodology for each of these parts has been extensively described for an number of antigens (e.g., Clausen, 1981). In general the procedure involves mixing the antibody and radioactive antigen, allowing time for the antibody-antigen complex to form, separating the antibody-bound (B) and free (F) antigen, and measuring the ratio of

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217

B/F. Various known amounts of unlabeled antigen are added to compete with and displace the radiolabeled antigen in order to calibrate the system, and this calibration curve is used to determine the amount of antigen present in 'unknown' samples. Radial immunodiffusion assays have also been developed for the complement proteins. Monspecific antibodies for a complement protein (these are available for most of the complement proteins) are incorporated in agarose gels, and the test sample is added to holes punched in the gel. A ring of immune precipitate forms around the hole as the antigenic complement protein diffuses into the gel. The size of the precipitin ring is proportional to the concentration of the complement protein in the sample.

5.3 Protease determinations in diagnosis Several proteases are currently measured in clinical laboratories to diagnose diseases or follow the course of treatments or physiologic conditions of patients (see Table 5.1) Several heritable deficiencies of human proteases have been documented by use of these assays, abnormalities in the tissue distribution or activity of proteases has been found to be associated with several diseased states, and protease activity may be used to monitor the course of pregnancy (see Table 5.1). Serum immunoreactive trypsin (IRT) is used to ascertain the occurence of acute pancreatitis (Koop, 1984) and chymotrypsin activity against Nbenzoyl-L-tyrosyl-4-aminobenzoic acid (PABA-peptide) is used for the diagnosis of exocrine pancreatic insufficiency (Arvanatakis and Greenberger, 1976). Serum leucine aminopeptidase is assayed in the diagnosis of various liver diseases (Fleischer et al., 1964). Elevated serum levels of dipeptidyl peptidase A are diagnostic of certain lung diseases (Lieberman et al, 1979) The assay of isoenzyme (different gene products with the same enzymatic activity) patterns of soluble alanyl aminopeptidase (EC 3.4.11.14) in blood has been suggested as a potentially useful method to determine diseased tissues/organs (McDonald and Barrett, 1986). The serum concentrations of different forms of aminopeptidases are elevated in particular diseases (adenocarcinoma of the colon and pancreas, nephrotic syndrome). The enzymes from different tissues often differ in their isoelectric point and can be separated by electrophoretic techniques that permit diagnostically useful discrimination between them. In two human genetic diseases, I-cell disease and pseudo-Hurler polydystrophy (mucolipidosis III), there is an intracellular deficiency of lysosomal enzymes, including cathepsin D, accompanied by the secretion of these enzymes from cells (Hasilick and Neufeld, 1980). These diseases can be ascertained by measuring cathepsin D associated with and secreted from human fibroblasts in culture. Chediak-Higashi syndrome, a rare human disorder characterised by giant granules in many cells, is associated with very low or undetectable amounts of leukocyte elastase (Vassalli et al., 1978). A congenital deficiency of enteropeptidase is associated with failure to thrive, diarrhoea, anaemia and hyperproteinaemic oedema (Hadorn et al., 1969).

218

J.S. Bond and R. J, Beynon

Table 5,1 Peptidase measurements which are of diagnostic value

Catheplin D. Normally in lysosomes is deficient in fibroblastsof I-call disease or pseudo-Hurlerpolydystrophy (mucolipidosieIII) patients;extracellularexoessof cathepsinD and other lysosomalhydroiesesare found in fibroblasts of culturesfrom patients, ChymotrylP~n. N-benzoyI-L-tyro6yl-4-aminobenozoicacid (PABA-peptide)hydrolaseactivityin the gut is used for the diagnosis of exocdne pancreaticinsufficiency. Coagulatkm Factor IXa. This is a serine proteinase,also caJledactivatedChristmasfactor, antihaemophilicfactor B. and plasma thrombop4astincomponent.Deficiencyin this pmtease causes severe bleedingdisorders. Coagulation Factor Xa. This is a serine protalnase,also called a,utoprothmmblnC, thrombokinase,and prothrombinase.Deficiencyof factor X causes Stuart disease, anotherbleedingdisorder. Complementcomponent C2. An enzyme in the dassicaJpathway of the activation of complementin response to an antigen. C2 is a zymogenthat is convertedto C2a, an active sedne proteinase.Deficiancyof C2 is the most common of the human complementdeficienciesand is inheritedas an autosomal recessivetrait. Cy~nyl amlnopeptJdase (CAP). Serum values of this enzyme can monitorthe course of gestation;the enzyme is syntheskaKIprimarily by the placenta and released into the matsmal drculatJondudng the course of pregnancy.The ease of assay makes this a good way to follow feoto-placentalfunction. Leukocyte etaldase. Low or undectablein Chediak-Higashisyndrome;patients are susceptibleto infections. E n t e r o l ~ l d a s e (enterokinue). This enzyme cleaves a peptide bond in trypsinogenand thereby forms active trypsin. Measurementof its activity is useful in patients with gastrointestinaldiseases.Congenitaldeficienciesof the enzyme are aseocia~:lwith diarrhoea, anaemia and hypoproteinasmicoedema; secondarydeficienciesare also found with ¢oeliacdisease. Increasedactivity of the enzyme has been observed in childrenwith cystic fibrosis ar¢l adults with chronic alcoholicpancreatitlsor cancer of the pancreas. Leuclrm 8minopepUdau. The serum activity of this exopeptidaseis measuredto diagnose liver disease.The enzyme is greatly elevated in the serum in patientswith obstructivejaundice. Moderateelevationsare also found in acute hepatitis, hepatic cirrhosis, choiecystitis,and in the carcinomatousmetastasesof the liver, Peptldyl dipeptidaseA (anglotensin converting enzyme). Serum levels increase in patientswith active pulmonarysarcoidosis but not in other lung diseases and thereforeit is useful in distinguishinglung abnormalities. ~ocollagen N-pep'lkl~N. Deficiencyof this enzyme in patientsis aascciatedwith Type VII Ehiers-Danlos syndrome.Procollagan is not proc___=Jss~__correctlyto the m=tureform of collagen resultingin connectivetissue problemswith symptoms such as stretchable,velvety skin and multipledislocationsof joints. The enzyme may be assayed in skin fibroblasts. Prollne d l p e ~ d u e . Deficiencyof this enzyme is associatedwith skin disorders (espedally skin ulcerations), mental retardation,inabilityto break down dipeptidescontainingprollne, and impropercollagen metabolism. Trypsin (serum Immunoreactlvetrypsin; IRT). This enzyme is elevated in =cute pancreatitisand depressedin pancreatici ~ n c y .

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219

Several inherited human diseases result in abnormal collagen metabolism, some of which are a result of protease deficiencies. For example, the heritable deficiency of procollagen N-peptidase, which cleaves N-terminal extension peptides from procollagen, results in Type VII Ehlers-Danlos syndrome (Prockop and Kivirikko, 1984). Several different molecular defects in the metabolism of collagen result in similar clinical syndromes; these inborn errors of metabolism have contributed greatly to our understanding of collagen metabolism. Human deficiencies in the enzyme proline dipeptidase are known (Butterworth and Priestman, 1985). In this inborn error of metabolism large amounts of Gly-Pro and other imidodipeptides are excreted in the urine and skin collagen metabolism is abnormal. Some of the symptoms of this abnormality are chronic skin ulceration, premature graying and recurring infections. The disease can be diagnosed by measuring the activity of proline dipeptidase in red and white blood cells or skin fibroblasts. Immune disorders and blood clotting disorders may also be associated with plasma protease deficiencies (See Chapter 9). There are heritable deficiencies of most of the complement protein components and these deficiencies are inherited in an autosomally dominant manner. Complement deficiencies are often associated with rheumatic diseases such as glomerulonephritis, lupus-like syndrome and systemic lupus (Ruddy, 1985).

5.4 Proteases as therapeutic agents There are a number of reasons why proteases are not widely used as therapeutic agents. First, their lack of specificity introduces considerable problems of uncontrolled and damaging proteolysis. Secondly, the armamentarium of protease inhibitors, and especially ~-2 macroglobulin, ensure that the action of administered proteases is quickly limited. Thirdly, being proteins, proteases are potential immunogens and unless proteases of human origin are used, repeated systemic administration may activate an anaphylactic response. Restricted groups of proteases have been used as therapeutic agents for many years. Some are used topically and as such do not raise such serious problems of immunogenicity or toxicity. Those that are administered systemically are either human enzymes or tend to be used in acute life-threatening episodes such that repeated administrations are less likely. There are a number of instances in which proteases are used to bring about extensive digestion. Proteases have been formulated in aids to digestion for many years, and pancreatic extracts, given orally, are effective in the treatment of pancreatic exocrine insufficiency. A major problem in such therapies is the inactivation of the enzymes in the extract by the acid environment of the stomach; such problems may be corrected partially by simultaneous administration of antacids or gastric acid secretion inhibitors. Wound healing is facilitated by removal of surrounding necrotic tissue. ;un important aspect of topical treatment is the removal of such debris, including fibrin, pus and blood crusts; proteases are used with considerable success for this purpose. As with the previous

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J.S. Bond and R,J. Beynon

example, the protease preparations are used in concentrations that bring about extensive and rapid digestion of material. Factors that mitigate against effective protease action are the presence of protease inhibitors in tissue exudate and the failure to maintain optimal conditions for enzyme action, such as moisture or activating/ stabilising ligands, e.g. calcium ions. Commercially available preparations of debriding agents vary widely in composition. Some contain proteases, including trypsin or collagenase; others comprise protease/nuclease mixtures such as fibrinolysin/deoxyribonuclease or streptokinase/streptodornase. In addition to enhancing the healing process, proteases appear to enhance the delivery of antibiotics to the wound site. Other areas in which localised but extensive proteolysis is used include freeing of an opaque lens in the treatment of cataracts, and in the removal of effusions in oto-rhino-laryngology. The importance of fine control of blood coagulation is evidenced by the many diseases in which under- or over-coagulation episodes are harmful. In both instances, there is the potential to use proteases therapeutically; in the first to enhance the activity of the proteolyric coagulation cascade, in the second to stimulate the fibrinolytic system. The most common circumstances in which bleeding disorders result from low levels of coagulation factors are the haemophilias and other deficiencies (Chapter 9). Excessive or specificially localised activation of the coagulation cascade leads to thromboses. Prophylactic therapy relies upon contro! at the level of platelet function or the coagulation cascade (Cazenave et al., 1984) but thrombolytic therapy is based upon the removal of a preformed thrombus. The logical treatment is to activate the fibrinolytic pathway (Mullertz, 1984; Tilsner, 1986); in turn this requires activation of plasminogen to fibrin-dissolving plasmin. However, generation of circulating plasmin in the plasma results in rapid inhibition by ~-2-antiplasmin that may deplete the body reserves of plasminogen and inhibitor to an unacceptable level. It is well established that fibrin has a high affinity for plasminogen and ideally, fibrinolytic therapy would specifically activate thrombus plasminogen but leave the circ-ulating zymogen intact. Additionally, in its brief existence the circulating plasmin can degrade other plasma proteins, the con-sequences of which may be far reaching. For example, plasmin is able to degrade fibrinogen, Factor V, Factor VIII and some complement components. Classically, treatment of thrombotic disease has employed two proteases; streptokinase and urokinase, both of which are plasminogen activators. Streptokinase is a protease produced by cultures of Group C ~-haemolytic Streptococci. It does not cleave plasminogen directly but forms a i:i complex that is able to activate other plasminogen molecules (Nissen, 1984; Sherry and Gustafson, 1985). Streptokinase shows no preference for thrombus plasminogen and treatment results in a transient hyperplasminaemia. Further, streptokinase is a foreign protein and is therefore immunogenic. High levels of streptokinase may be needed to overcome the titre of antibodies in patients who have incurred streptococcal infections or experienced previous streptokinase therapy. Urokinase is a trypsin-like serine protease isolated from human urine or kidney cell cultures. Urokinase has the added advantages of being less immunogenic and is more specific for thrombus plasminogen.

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More recently, there has been growing interest in tissue p!asminogen activator (t-PA) in treatment of thrombosis (Collen and Lijnen, 1984; Collen, 1986). t-PA has a high affinity for fibrin; attributed to the fibrinonectin-like domain in the molecule. In turn, fibrin bound t-PA is potently active in the conversion of plasminogen to plasmin. Thus, therapy with t-PA gives thrombus-specific activation of the fibrinolytic mechanism and has no dramatic haemostatic effect, t-PA has been prepared by recombinant DNA techniques and three clinical trials have demonstrated that t-PA is more effective than streptokinase and induces less fibrinogen breakdown (Collen, 1986). One protease that has been used for a very specific application is chymopapain in the treatment of back pain caused by intervertebral disk diseases. Chymopapain is a plant cysteine protease derived from Caricas papaya latex. When injected into the disk, chymopapain brings about restricted destruction of surrounding tissue in a process refered to as chemonucleolysis (Einarson et al., 1984). It is probably restricted to the site of action by tight association with glycosaminoglycan-protein complexes; the high isolelectric point of the enzyme undoubtedly contributes to this interaction (Watts e t a ! . , 1975). Destruction of the tissue removes or reduces pressure on the nerve root; often giving immediate relief of pain that can persist for up to one year. Overall, this process can offer relief for approximately 70% of patients (over 20,000 have been treated). However, a recent commentary suggests that chymopapain treatment is being replaced by more selective surgical procedures (Merz, 1986). There are a number of adverse reactions to chymopapain preparations, the most serious being anaphylaxis resulting from prior sensitisation by meat tenderiser, a preparation of proteolytic enzymes that are also derived from C. papaya (Einarson et al., 1984). Further information on chymopapain treatment can be found in a recent series of papers (Dabezies et al., 1986 et seq).

Chapter 6

Proteases as mediators of physiological processes

6.1 An overview of proteolytic regulation The previous chapters have discussed proteases primarily in terms of their enzymology and physicochemical characteristics. Some understanding of their mode of action and specificity are necessary before they can be discussed as effectors of biological events. In the ensuing chapters we shall attempt to place proteases in the different context of functionality and illustrate the ways in which they are able to act. Again, some generalisations can be made and the goal here is to discuss principles and establish the basis that underlies the succeeding chapters. More detailed treatments of physiological regulation by proteases can be found in Reich et al., (1975); Ribbons and Brew, (1976) and Evered and Whelan, (1980). One aspect not covered here is the regulation of transcriptional or translational events that lead to changes in the rate of synthesis of proteases. Surprisingly little is known about the turnover of the proteases as a group - how, for example, are the lysosomal proteases degraded? Any protein or polypeptide has been synthesised from amino acids and unless it is lost from the body will be broken down and regenerate its constituent amino acids. Proteolysis must therefore be involved at some stage in the lifetime of most proteins or peptides. The terminal proteolysis that finally regenerates amino acids often has a predominantly nutritive role, generating energy-yielding molecules or precursors for new proteins, and may be less concerned with physiological regulation. The initial proteolytic events in breakdown might however exert an important regulatory influence. The role of peptide bond hydrolysis is best appreciated if it is considered in terms of the stages of the lifetime of a protein in the cell. For the present purposes we may assume that there are three stages. The first represents all events preceeding the formation of the biologically active molecule and includes biosynthesis, posttranslational processing and activation phenomena. The second phase is the stage during which the product of stage 1 is exerting its biological effect. Finally, the terminal phase represents hydrolysis

222

Proteolysis and Physiological R e g u l a t i o n

223

and inactivation of the active molecule. As with any model, there is a danger of over-extrapolation but many of the p r o t e a s e - m e d i a t e d processes fit this crude description rather well. Fig. 6.1 illustrates the three-phase model and lists some of the events that might constitute the individual phases for representative biological systems. At the simplest level, therefore, proteolysis has one of two roles; the generation of a biologically active molecule or its removal and/or inactivation. The whole can be v i e w e d as a biphasic process, with the ratio of the rates of the first and second stages exerting a profound controlling effect on the concentration and duration of action of the active form of the protein/peptide. 'Pre-active'formof molecule

® a) zymogen

'Post-active'form

Activefotrn

0 ~

acliveenzyme

~

degradedenzyme

b) nascent protein

.~

ac~veprotein

h.~ ~

degradation products

c) pre-neuropep~de

h.~ neuropep~e

~r

degradation products

Fig 6.1 Sequential proteolysis and physiological regulation Two sequential proteolytic processes can be employed to generate an intermediate, partially proteolysed form. The transience of the intermediate is dictated by the relative rates of the two hydrolytic events; three examples might be case a) zymogen activation, case b) intracellular protein degradation and case c) formation of transient signal peptides.

If phase 1 is much more rapid than phase 2 the active form of the molecule will accumulate. Moreover, its concentration will only diminish when the supply of precursor (Fig. 6.1a) has been exhausted, when the rate of A -> B is reduced or B -> C is increased. This type of behaviour is most readily seen in zymogen activation, when a p r e c u r s o r of an enzyme is activated by limited proteolysis. A priori, one might expect that the active enzyme should be allowed time to act and it is reasonable therefore to assume that B -> C will be

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J.S. Bond and R.J. Beynon

relatively slow. Other examples are provided by processing hydrolytic events that occur in the biogenesis of many proteins or peptides, including the excision of active molecules from larger precursors. By contrast, if the second phase is dramatically faster than the first, then molecule B will have a relatively transient existence and will never attain a concentration as high as might be expected from measurement of its rate of synthesis (Fig 6.1b). Such a situation might apply to short-lived proteins and biologically active peptides which are formed by limited proteolysis and inactivated by a second protease that guarantees the transience of the signal elicited by that peptide. Intermediate between these two extremes, the rates of the two processes might be approximately balanced such that the level of B is maintained at significant levels over a long period of time (Fig. 6.1c). Note that maintenance of this level can be attained purely by approximate balancing of the rates of the two processes, irrespective of their magnitude. If the rates are low then flux through B will also be low, but as A -> B and B -> C are increased, flux also increases. At first glance, this rapid cycling of amino acids through a protein might appear to be very wasteful of energy. Although proteolysis is exergonic, the formation of a peptide bond demands the input of substantial metabolic energy. Thus, a molecule with a high turnover rate places a drain upon the energy reserves of the body and generates heat. What is gained from this is regulatory potential. A protein undergoing rapid turnover is able to change its concentration quickly in either direction. Either or both of the processes of formation and removal can be modulated to effect such a change. Proteolytically-regulated events cannot function in isolation and must integrate with other types of metabolic regulation. These links are, for many processes, still uncertain, although it is becoming increasingly apparent that a number of the well-recognised metabolic effectors are also able to influence the activity of proteases. Finally, in contrast to many other groups of enzymes, proteases are unusual in that many of them have the potential to be controlled by protein inhibitors (Chapter 4). Most proteases lack rigid substrate specificity and in many instances a good case can be made for retaining the ability to hydrolyse a large number of substrates. The case is most evident in those digestive processes that cause complete hydrolysis of proteins, but less so in the many physiological events that depend upon protease-mediated alterations in properties of the substrate. For example, three major protease systems of blood; coagulation, fibrinolysis and kinin formation have the potential for a sursprising degree of crossreactivity. Some of the interactions are summarised in Fig. 6.2. Most interestingly, appropriate stimuli can, for example, initiate both fibrin formation and fibrinolysis. At first glance these are contradictory effects but it is likely that they form part of the exquisite regulation of the highly complex blood system. Activation of both pathways would permit changes in the rate of fibrin deposition in a system that is far more controlled and responsive than if one of the processes was enhanced in isolation. Similarly, the ability of angiotensin-converting enzyme to generate a vasoconstrictor, angiotensin II is balanced by its ability to inactivate the vasodilator bradykinin providing a second mechanism for it to mediate the appropriate

Proteolysis and Physiological Regulation

plasminogen

Factor Xll

tt

v"- plasmin

11

I

I

v

Factor Xlla ......

i

kallikrein I

prekallikrein High molecular weight kininogen

225

~

~ bradykinin

(surface contact)

Fig. 6.2 Integration of proteolytic processes in blood protease systems The processes of coagulation, kinin formation and fibrinolysis are closely associated and linked at the initiaJphase of surface activation, via Factor XII& Note that the system has the potentiaJ to initiate opposing processes, in addition to increasing blood flow via kinins. Activation of plasmin may proceed via plasminogen activators, themselves zymogens that are activated by kaJlikrein and factor Xlla.

p h y s i o l o g i c a l response. At the same time, kallikrein, the bradykininforming enzyme can catalyse the formation of renin from its circulating precursor, thus enhancing the supply of angiotensin I to the converting enzyme. That such sophisticated mechanisms have evolved testifies to the need for subtle and continuous control of a complex system. Each of the protease-mediated systems has evolved the appropriate 'inter-connections' by retaining the ability to attack more than one substrate.

6.2 Proteolytic generation of biologically active molecules Many proteins or peptides are synthesised as inactive, higher m o l e c u l a r weight forms that must be proteolysed to generate the b i o l o g i c a l l y active form. Implicit in this process is the limited action of a protease to generate a molecule that is resistant to further d i g e s t i o n - ' l i m i t e d proteolysis' (Chapter 3). Limited proteolysis is an irreversible process and other mechanisms must operate to diminish the consequences of zymogen activation, such as inactivation or clearance of the protease. Zymogen activation allows a p o t e n t i a l l y harmful protease to be 'stored' in an innocuous form, such

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that it may be activated rapidly by specific cleavage of one of a fe~ peptide bonds (reviewed in Kay, 1980). The classical example is provided by the activation of pepsinogen and the pancreatic proteases. The zymogens are activated in one of two ways. They may diffuse to the activating protease, usually membrane-bound or otherwise limited in its sphere of influence. Enteropeptidase, on the intestinal brush border membrane, can only activate trypsinogen that is delivered to that site. Regulation is thus achieved by control of secretion of the zymogens; activation of the zymogens is obligatory and generally considered to be complete. The autoactivation of trypsinogen by trypsin is an additional device that ensures the maximal response of the system. Alternatively, the primary event may be exposure of a previously cryptic zymogen activation signal. Initiation of the intrinsic pathway of coagulation relies upon interaction between an active surface (immobile) and a complex of prekallikrein, Factors XI and XII and high molecular weight kininogen (circulating) (Neurath, 1984; Jackson and Nemerson, 1980). Premature activation of the zymogens is prevented in part by the spatial restriction of the activating signal and also in part by the constant surveillance of inhibitors (secretory pancreatic trypsin inhibitor, plasma proteinase inhibitors) that suppress the activity of prematurely or adventitiously activated zymogens. Similarly, secretion of plasminogen activators, that subsequently generate the powerful proteinase plasmin from its zymogen, may be an important event in processes such as cell-cell interaction (Reich, 1978; Dano et al., 1985) and ovulation (Chapter 8). Amplification cascades are a feature of several zymogen activation processes (Neurath and Walsh, 1976; Neurath, 1984). The blood coagulation cascade is a complicated series of zymogen activation processes, linked serially such that the zymogen of one step is the substrate for the enzyme generated in the previous step. Because each of the proteases is able to activate more than one zymogen molecule, the net effect is a rapid acceleration of the process, such that the ultimate response (thrombin catalysed conversion of fibrinogen to fibrin) occurs optimally and at a rate far faster than could be obtained if thrombin had to be synthesised de n o v o or released from a secretory vesicle. In addition to amplification, inappropriate activation of the cascade can be blocked (probably by inhibitors) at early stages, before the magnitude of the signal becomes overwhelming. Relatively little is known about the mechanisms whereby many of the inactive zymogens are converted into active proteases (Neurath, 1984). The best studied group are the pancreatic proteases. Pepsinogen, chymotrypsinogen and trypsinogen, for example have been demonstrated to possess a weak but definite hydrolytic activity (Kay, 1980). Further, the zymogens are capable of reacting with a series of active site inhibitors, demonstrating that the catalytic site in the zymogens is largely preformed. Further evidence has suggested that the low activity of zymogens is predominantly due to a poor ability to bind substrates (Kay, 1980). Many biologically active molecules other than proteases are secreted in an inactive form and are subsequently activated by limited proteolysis. Collagen is initially secreted as a procollagen molecule containing additional N-terminal and C-terminal extensions. The Cterminal segments ensure correct registration of the t h r e e chains that

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constitute the collagen triple helix whereas the N-terminal segment prevents the spontaneous aggregation of procollagen inside the cell. After secretion, both of the segments are removed and the collagen fibril can be correctly laid down. Procollagen N-peptidase and procollagen C-peptidase are both secreted into the extracellular space in order to bring about these changes. A number of physiologically-active peptides are contained within much larger proteins, such that activation requires excision of the peptide. Angiotensinogen must be acted upon by renin to release the decapeptide angiotensin I, the immediate precursor of the octa-peptide angiotensin II (Ondetti and Cushman, 1982). Here, the ad-vantage probably lies in maintenance of a 'store' of a peptide that is intrinsically unstable in vivo, ensuring transience of response yet avoiding the need for a high rate of turnover. Similarly, circulating kininogens provide a reserve of kinin, released by the action of kininogenases such as kallikrein (MOller-Esterl and Fritz, 1984). Limited proteolysis makes an important contribution to co- and posttranslational processing of proteins. The release of an N-terminal signal peptide of approximately 20 hydrophobic amino acids is an early event in the biosynthesis of proteins by the rough endoplasmic reticulum-Golgi route. A specific 'signal peptidase' has been postulated. Here the role of the limited proteolysis is to clip off the part of the nascent polypeptide chain that has served the purpose of directing the route of synthesis of the protein. Additionally, within the Golgi and the secretory vesicles, further proteolytic events may take place, resulting in excision of the final, biologically active product. Such behaviour has been developed to a fine degree in the processing of neuropeptide precursors (Loh et al., 1984).

6.3 Proteolytic Inactivation of biologically active molecules In a closed biological system, mechanisms for the termination of the effect of a biologically active polypeptide must exist. The critical step is that at which the biological activity is lost, either because the polypeptide is denied access to the site of action or because it has been modified in such a way that it no longer possess that biological activity. Proteolytic inactivation falls into the latter category, although it should be appreciated that digestion of otherwise sequestered or inactivated polypeptides is almost always obligatory. Many polypeptides and proteins are internalised by cells by more or less specific endocytic mechanisms, a process that brings about their 'functional inactivation' and which will be followed by complete proteolysis when the endosome fuses with a primary lysosome. Of greater relevance here is the role of proteolysis as the 'functional inactivator', that destroys the biological activity without first segregating substrates and products. Representative examples are provided by plasma protein C that inactivates the protein cofactors of the coagulation cascade; Factors V and VIII (Brandt, 1984) and the inactivation of neuropeptides (McKelvy and Blumberg, 1986), angiotensin II and kinins. The initial, inactivating proteolysis might be expected to be limited and, inasmuch as it must destroy the biological activity of the substrate, to be directed to a site that contributes to that biological activity, directly or indirectly. If the substrate is a small

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J.S. Bond and R.J. Beynon

peptide then proteolytic attack might generate two products that dissociate and the products might exhibit a lowered affinity for the receptor. On the other hand, if the target substrate is a protein the products of the reaction may remain associated, possibly even linked by disulphide bridges, and in this instance inactivation is attained in one of two ways. Either, proteolysis causes direct destruction of the part of the molecule that is biologically active or cleavage of another part of the molecule results in transmission of a conformational change to the active site, destroying its function. Limited proteolysis is irreversible and thus, the behaviour referred to above leads to the formation of a biologically-inactive, fragmented protein or peptide. If the product of such inactivating proteolysis was refractory to further digestion the net result would be an accumulation of inactive polypeptides. It might be expected therefore that in addition to destroying the activity of the protein, limited proteolysis would also accentuate the lability of the protein to further digestion, either by the same protease or by different proteases. For example, collagenases act very specifically on the collagen triple helix, cleaving it at a position approximately 75% of the way along its length and generating products that can then be acted upon by gelatinases and other proteases (Bauer et al., 1983). In general terms, endopeptidase attack increases the opportunity for exopeptidase action and this may potentiate further hydrolysis. This model does not necessarily imply that the product of limited attack is intrinsically more labile. Equally feasible is the suggestion that the limited proteolysis enhances the opportunities for further digestion, for example by releasing the products from an immobile system (the extracellular matrix or a protein-membrane complex). The products would thus be exposed to new, soluble phase or remote proteolytic systems to which they were previously inaccessible. One such system may operate in intracellular protein catabolism, in which limited cytoplasmic proteolysis could enhance lysosomal uptake and completion of digestion of the target protein within the lysosome (Khairallah et al., 1985; Beynon and Bond, 1986). In a related fashion, limited attack of a protein may result in glomerular filtration of the products, a step that commits most polypeptides to luminal hydrolysis by the brush border peptides and/or endocytic uptake (Bennet and McMartin, 1979; Chertow, 1981).

6.4 Integration of proteolysis with metabolic regulation No one aspect of metabolic regulation can be considered in isolation from all other aspects. Metabolic regulation requires the delicate and finely controlled interaction of many processes in order that the correct physiological response can be elicited in all biological systems that are involved. There is strong evidence for interactions between those processes that are mediated by proteases; for example the coagulation, kinin and complement pathways in plasma. At present however, the links between protease action and other regulated processes are rather tenuous. Regulation of metabolism relies upon a series of signalling molecules that operate under different conditions, intracellularly or extracellularly, but the mechanisms whereby these signals might mediate proteolysis are not yet established. A major site of

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regulation may turn out to be at the level of the protein digested by the proteaseo There is a comprehensive body of data supporting the contention that the vulnerability of a protein to proteolysis can be modulated by conformational changes in that substrate (see for example Mihalyi, 1978). These conformational changes can be pro-duced by interaction of the protein with substrates, inhibitors or other allosteric ligands, by the association with or dissassociation from a macromolecular complex, post-translationl modification or by any of the other conformational excursions that are undergone by the protein in the course of its action. This modulation of susceptibility may be particularly important in the regulation of intracellular protein catabolism (Beynon, 1980; Beynon and Bond, 1986). Modulation of proteolytic activity may also take place at the level of the protease. A number of proteases can bind low molecular weight ligands that subsequently modify the activity of the protease (Holzer and Heinrich, 1980). Calcium ions affect both intracellular and extracellular proteolysis. The synthesis of prothrombin, Factors VII, IX and X all involve vitamin K-dependent post-translational conversion of glutamate residues to y-carboxy-glutamate residues (Gla residues). The Gla residues form strong calcium binding sites and this cation is an essential component of all of the steps in coagulation that involve vitamin K-dependent factors (Suttie, 1980). Additional roles for calcium ions in coagulation have also been postulated (Jackson and Nemerson, 1980). Calcium ions prevent autolysis of trypsin and may bind to several other endopeptidases (Bond and Beynon, 1986). Calcium ions are essential for the activity of a family of intracellular proteases, the calpains. The role of calcium in the activity of calpains is complex and not fully understood. Calcium ions stimulate hydrolytic activity but also initiate autolytic inactivation of the enzyme; calcium is an essential cofactor for the binding of calpains to their specific inhibitor, calpastatin (Murachi, 1953; 1985; Pontremoli and Melloni, 1986). It is clear that calcium sensitivity of this protease is brought about by a calmodulin-related domain that has been identified in the cDNA sequence (Ohno et al., 1984). A similar calcium binding domain has now been identified in the small subunit of calpain (Sakihama et al., 1985). The energy dependence of intracellular proteolysis has resulted in the search for a link between nucleotides, ATP in particular, and proteases (see Chapter 7). One ATP-dependent proteolytic system first identified in reticulocytes involves the conjugation of a 9000 Dalton protein, ubiquitin, to substrate proteins. The role of ATP is to activate the ubiquitin conjugation system rather than act as a cofactor that stim-ulates the protease activity directly. However, evidence for prot-eases, directly stimulated by ATP in vitro, has been accumulating (Khairallah et al., 1985; Reider et al., 1985; W a ~ a n et al., 1985). Some proteases are stabilised by nucleotides and the distinction between activation and stabilisation needs to be examined more closely (Dahlmann, 1985). Finally, competitive inhibition between different substrates is an area that has yet to receive close attention but may well be an important factor in the regulation of proteases i n vivo (Labella et al., 1985).

Chapter 7

Intracellular proteolysis

7.1 Processing of nascent proteins Proteolytic processing, or the limited degradation of proteins leading to the synthesis of mature, active proteins, is the most general of all covalent modifications of proteins (Wold, 1981). Proteins may undergo several types of covalent modifications (e.g., phosphorylation, glycosylation, methylation) in addition to proteolysis, but the latter is the most common because most proteins are proteolytically modified after synthesis from the precursor sequence encoded by the mRNA. In addition to the removal of amino acid sequences which serve as signals to direct proteins into correct subcellular compartments and removal of pro- or zymogen sequences which may be required to activate a protein, formation of active proteins/peptides often requires disassembly or assembly of protein complexes which involves proteolysis. For example, some hormones are synthesised within a large polypeptide containing several physiologically active entities which must be released by proteolysis after synthesis. By contrast, formation of macromolecular particles and aggregates such as virus particles and collagen may require prior proteolysis. At all stages that proteolysis is involved in the maturation of a molecule, it is an irreversible process and represents a commitment to a biological function. The proteases that are involved in the processing of mammalian proteins are, in general, not well characterised (Pontremoli and Melloni, 1986; Bond and Butler, 1987). These proteases have been difficult to purify and characterise because they are present in low concentrations in cells (unlike the lysosomal proteases), some are highly specific for substrate and therefore difficult to detect with general proteolytic substrates, and they may be physico-chemically unstable enzymes when removed from their natural environment. Considerable progress has been made in elucidating the processing of some polypeptides, however, such as polypeptide-hormones, neuropeptides, lysosomal enzymes, mitochondrial enzymes, and secretory proteins (Habener, 1985; Schwartz, 1986; Thomas and Bradshaw, 1981; yon Figura and Hasilik, 1986; Prockop and Kivirikko, 1984; Zimmerman et al., 1980).

230

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231

All proteins are synthesised on ribosomes and contain information within their sequences that directs them to the correct subcellular particle or to the external environment (Blobel, 1980). For example, most proteins destined for mitochondria are synthesised on ribosomes and contain a peptide sequence (leader signal) that directs them into mitochondria (Reid, 1985). That sequence determines the transport of mitochondrial proteins through the cytosol and the mitochondrial membrane and is then removed from the protein (post-translational import) or directly from ribosomes bound to mitochondria through the outer mitochondrial membrane (co-translational import). Once within the outer mitochondria, proteins must be targeted for the correct submitochondrial space and this requires additional signal sequences and processing mechanisms. Plasma membrane or secreted proteins are also synthesised with signal peptides on ribosomes; these signal peptides have high affinities for membrane receptor proteins and direct ribosomes from the cytosol to the endoplasmic reticulum (ER). As the proteins are synthesised, they are transferred through the ER membrane into the lumen and then into the Golgi apparatus (Walter et al., 1984; Rodriguez-Boulan et al., 1985). Proteins that are destined to remain on membranes are thought to maintain an attachment to the ER membrane and transport vesicles; other proteins detach from the membranes and after folding and glycosylation are secreted from the cell. Some signal peptidases purified from bacteria are metalloendopeptidases; some are not affected by the inhibitors usually used to classify proteases and may therefore involve new mechanisms (Wolfe et al., 1982). Neuropeptides (e.g., enkephalins) are synthesised from large precursor proteins sometimes referred to as pre-pro-proteins. The amino acid sequence on the N-terminus is the signal sequence, 15 - 30 amino acids long, and contains a high percentage of hydrophobic amino acid residues. The signal sequence (pre-sequence) is cleaved off as the polypeptide is being synthesised on the ER; the pro-sequence is cleaved off in the Golgi apparatus or in secretory granules. The signal peptidases have not yet been purified. Initial studies of these enzymes indicate they are metallo-proteinases and that cleavage of the pre-protein usually occurs at sites adjacent to small amino acid residues such as alanine, glycine and serine. The enzymes responsible for the propeptide cleavage, sometimes called 'converting enzymes', often act optimally at acidic pH values; however some proenzyme and prohormone activating enzymes are serine proteinases that act at neutral pH values. The proprotein converting enzymes cleave substrates at sites containing a pair of basic residues (lys-lys, lys-arg, arglys, or arg-arg). There is some controversy about the properties of the converting enzymes and their relation to known cellular proteases (Loh et al., 1984).

7.2 Intracellular protein degradation Most mammalian cells are capable of internalising extracellular proteins, in the process known as endocytosis, and degrading them to amino acids in the process known as heterophagy. Further, all mammalian cells are capable of degrading their own resident proteins to amino acids in a highly controlled manner. The evidence that lysosomes are the major site of degradation of extracellular proteins brought into the cell by endocytosis is very good and much is known

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about the mechanisms involved in this process (Dean and Barrett, 1976; de Duve, 1983; Bond and Aronson, 1983). By contrast, our knowledge about the proteases, cellular location and mechanisms involved in the degradation of intracellular proteins is limited (Beynon and Bond, 1986; Mayer and Doherty, 1986). There is little doubt that lysosomes have the capability to degrade cellular proteins and that under certain situations (e.g., nutritional deprivation, pathological states) they are very active in the bulk hydrolysis of cell protein (Mortimore and Poso, 1984; Kominami et al., 1983; Ahlberg and Glaumann, 1985; Glaumann and Ballard, 1986). However, the role of the lysosomal and non-lysosomal proteinases under normal physiological circumstances is not yet known. Endocytosis includes the processes of phagocytosis and pinocytosis (Chapter 9; Silverstein et al., 1977; Pastan and Willingham, 1985). Pinocytosis is common to most cells and involves the uptake of fluid and contents into the cell. One form of pinocytosis is receptormediated and permits selective uptake of macromolecules into cells. The sites on the cell membrane that are involved in this process appear as invaginations (pits) coated on the cytoplasmic side with filamentous material (consisting of clathrin). The external material is internalised and the contents are either delivered to the lysosome for digestion, to other subcellular sites (such as the Golgi system), or to the external environment (exocytosis). In receptor-mediated pinocytosis the receptor proteins are often recycled back to the membrane. There is also a form of non-selective pinocytosis, or fluid phase pinocytosis, in which small vesicles take up fluid and its contents into the cell without selecting or concentrating any components by membrane binding. The portions of the plasma membrane involved in this process also appear to be recycled back to the membrane after delivering the fluid contents. There is a substantial body of literature on endocytosis and heterophagy and its role in health and disease (de Duve, 1983; Steinman et al., 1983; Goldstein e~ al., 1978). All cells regulate the content of their resident proteins through synthesis and degradation (the two processes together are termed 'protein turnover'). The concept of the 'dynamic state' of proteins was first put forth by Schoenheimer (1942). It is now realised that the turnover of proteins within mammalian cells is a continuous process that can be regulated by metabolites, hormones and drugs and that regulation of the process for individual proteins and bulk proteins is highly controlled and energy-dependent. Individual proteins in cells have characteristic half-lives that can be measured by a variety of methods. That means that the rate of disappearance of proteins in cells is a first order process that can be described in terms of half-life (the time it takes for the half the protein molecules to be replaced) or rate constant for degradation (first order rate constant). Mathematically this can be expressed as: d[P]/dt = kd [P], where [P] is the protein concentration, t is time, and kd is the rate constant for degradation. Half-lives (tl/2 = 0.693/kd) of proteins have been estimated in a variety of ways and readers are referred to several references for methodological considerations (Garlick et al., 1976; Zak et al., 1979; Waterlow et al., 1978; Duncan and Bond, 1981; Butler et al., 1985; Watkins et al., 1987). Most techniques depend on measuring loss of biological activity (of, for instance, an induced protein or after protein synthesis is arrested) or loss of immunologically identifiable protein. When a monospecific antibody is available, proteins may be

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Table 7.1 Half-lives of some enzymes in mammalian tissues References: a, Russell and Snyder, 1969;b, Bond, 1976;c, Kuehl and Surnsion, 1970; d, Watkins eta/., 1986;e, Sinenskyand Logel, 1983; f, Chandler and Ballard, 1985;g, Butler eta/., 1985.

Enzyme

tl/2

Ref.

Rat liver

Alanine aminotransferase

3

Glyceraldehyde-phosphatedehydrogenase Lactate dehydrogenase Arginase NAD-glycohydrolase

3 3.5 4-5 20

min hours hours hours da d~ rS da rs da rs da rs da rs da rs da Is da rs

Chinese hamsterfibroblasts 3-hydroxy-3-methylglutarylCoA Reductase

2.5

hours e

HE (39) diploid human fibroblasts acetyI-CoAcarboxyla.se pyruvate ca.,'boxylase methyl crotonyI-CoAcarboxylase propionyI-CoAcarboxylase

1.3 1.7 2.2 3.6

d~s d~s d~s

f f f

d~s

f

Mouse skeletaJmuscle Glycogen phosphorylase

12

days

g

Omithine decarboxylase Tyrosine aminotransferase Sefine dehydratase Oihydroorotase Glucokinase Aldolase Catalase Cytochrome P-450

11 1.5 5-20 12 1 1-2 2.5 2.5

a b b b b C b d b c c b

b

labelled by exposure of cells to radiolabeled precursors (amino acids or cofactors) during the synthetic process, followed by a chase of the same compound (unlabeled) to dilute the radiolabel, and then the loss of the radiolabel from the protein is measured by immunoprecipitation with time. There appears to be a rate-limiting step in the degradation of proteins that initiates extensive degradation so that intermediate degradation products do not accumulate; proteins are rapidly degraded to amino acids and small peptides after that initial event (Beynon and Bond, 1986). The half-lives of proteins in mammalian cells are usually quite short relative to the life of the cell (See Table 7.1 for examples). For cytosolic proteins of rat liver cells, the half-lives of proteins range from i0 min to 20 days; mitochondrial proteins have half-lives of 1 hour to 8 days; the average protein half-life in a liver cell is about 3 days. That means that half of the resident proteins of a liver cell are degraded and replaced by new protein every 3 days. In the life-time of a liver cell, which may be the same as the life of an adult rat (approx. 3 years), the proteins of the cells turn over many

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J.S. Bond and R.J.

Beynon

times. Proteins in other tissues may turn over at different rates. For example, in rats the average half-life of soluble proteins in liver is estimated to be 0.9 days, in kidney 1.7 days, in heart 4.1 days, in brain 4.6 days and in skeletal muscle 10.7 days (Waterlow et al, 1978). In all these tissues, although the rates may vary there is extensive degradation of cellular proteins, the rates for different proteins are heterogeneous, and the nutritional, hormonal and developmental status of the animal will affect turnover. The half-life of a protein has some role in determining how fast that protein concentration will fluctuate in a cell in response to an increase or decrease in protein synthesis and degradation (Berlin and Schimke, 1965). Proteins that have short half-lives in cells (< 1 day), are usually present at low concentrations and can be induced rapidly in response to an increase in synthesis; they often can be induced by hormones or other short-acting signals. Likewise these same proteins will rapidly decrease in concentration when the rate of synthesis decreases to basal levels. Thus, the short-lived proteins fluctuate rapidly and may adapt readily to environmental or metabolic conditions. This is an important concept because intercellular signals, such as those mediated by hormones, are often short-lived and must evoke a response in a cell in a short span of time. Often, quick responses to hormonal signals are controlled by activation/inactivation of enzymes rather than changes in the concentration of enzymes. However, the existence of short-lived enzymes allows the concentrations of a select group of proteins to be controlled rapidly and to play some role in short-term hormonal effects as well. Interestingly, short-lived enzymes are often at important control points in m e t a b o l i s m and thus regulatory. Long-lived proteins, much more sluggish in response to changes in protein synthesis, are often 'household' or constitutive proteins that do not play regulatory roles. While most subcellular compartments of cells have proteins with heterogeneous turnover rates, there is some bulk turnover of subcellular structures. All the components of peroxisomes, for instance, appear to be degraded as a unit and in certain conditions, such as in diseased states or when pharmacological doses of glucagon are a ~ i n istered, m i t o c h o n d r i a and other subcellular structures are degraded in bulk in lysosomes. The processes that initiate and control the degradation of individual or bulk protein of a tissue are far from understood (Grisolia and Wheatley, 1984; Mayer and Doherty, 1986; Beynon and Bond, 1986). Because heterogeneous rates of protein turnover are observed, it has been suggested that the intrinsic properties of individual proteins determines the stability of proteins in vivo. In general, large cytosolic proteins have shorter half-lives than small proteins; proteins with acidic pI values (the pI value reflects the net charge of a protein) are shorter-lived that those with basic pI values; and shortlived proteins are generally more hydrophobic than long-lived proteins (Momany et al., 1976; Duncan et al., 1980; Bohley et al., 1981). In addition to these parameters, enz!nnes that are short-lived in vivo, tend to be relatively unstable proteins in vitro. They are more readily inactivated by low pH and high temperatures than long-lived proteins, and they are more vulnerable to proteolytic attack (Bond, 1975). The correlations between the physico-chemical properties of a protein and its biological half-life have yet to be satisfactorily

Proteolysisand Physiological Regulation explained in their general suggest that short peptide 1986; Rogers (Bachmair et

235

mechanistic terms, and there is some controversy over applicability. Of great interest are recent studies that the rate of degradation of a protein is a function of sequences, analogous to signal peptides (Dice e t a ! . , et al., 1986), or even the N-terminal amino acid al., 1986).

Ligands that alter the stability of a protein can alter its half-life in vivo. For example, methyltryptophan stabilizes tryptophan oxygenase in vitro and can increase the half-life of the enzyme in vivo; mevinolin stabilizes hydroxymethylglutaryl-CoA reductase in vitro and in vivo (Schimke et al., 1965; Sinensky and Logel, 1983). On the other hand when abnormal proteins are created by incorporation of amino acid analogues such as fluorotryptophan into proteins, protein structures are altered and these abnormal proteins are degraded more rapidly than their normal counterparts. These types of observations imply that at least some of the selectivity of the degradative process in the cell is determined by protein structure or conformation of the substrates themselves. It has also been suggested by several groups of investigators that the initial event in the degradation of a protein involves a nonproteolytic covalent modification of the protein that 'marks' or 'brands' the protein for degradation. The branding events that have been suggested include ubiquitin conjugation, oxidation, deamidation, acetylation, carbamylation, methylation, glycosylation-deglycosylation and phosphorylation-dephosphorylation (Beynon, 1980; Beynon and Bond, 1986; Rivett, 1986). A role for ubiquitin in the process of intracellular proteolysis has been proposed by Hershko and colleagues (1982). Ubiquitin is a 9,000 molecular weight protein present in all cells. Ubiquitin can be covalently linked to the c-amino groups of lysine residues in proteins by an ATP-dependent catalyzed reaction. It is proposed that ubiquitinconjugation to proteins marks proteins for degradation and the conjugation reaction accounts for the ATP-dependence of proteo!ysis. The system that has been best studied in support of this hypothesis is the reticulocyte. In the reticulocyte, denatured forms of haemoglobin can be found conjugated to one or more ubiquitin molecules and the ubiquitin-tagged forms of haemoglobin are more readily hydrolyzed by reticulocyte proteases. There is evidence that ubiquitin-conjugation is a signal for degradation of abnormal proteins and short-lived proteins in several cell types in addition to reticulocytes (HeLa cells, mouse-derived cell cycle mutants, plant cells) but for other types of cells (liver, muscle) the evidence is not yet strong (Saus et al., 1982; Haas et al., 1985; Etlinger et al., 1985; Ciechanover et al., 1984; Vierstra et al., 1985; Bond and Butler, 1987). While the conjugation enzymes of the ubiquitin-dependent, ATP-dependent proteolytic system are well characterised, the protease(s) that act in accordance with the system are poorly characterised. The whole system is a conglomerate of several proteins with 6-8 polypeptides, and a molecular weight of over 600,000. Ubiquitin-conjugated proteins are degraded down to amino acids and thus the process is likely to include endo- and exo-peptidases. However, there is evidence that other ATPdependent proteases do not require substrate ubiquitinylation (Desautels and Goldberg, 1982; Watabe and Kimura, 1985). Also it is not known whether most cellular proteases can degrade ubiquitinconjugated proteins more readily than unconjugated proteins, or if this is only true of specific protease(s). Note that the general

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J . S . B o n d and R. J. B e y n o n

T a b l e 7.2 Molecular d e t e r m i n a n t s of protein d e g r a d a t i o n

Physlcochemical properties There may be correlations between physicochemicaJ properties of proteins and their degradative rates. Some of the suggested correlations are listed below with comments on the underlying mechanisms that might give rise to the correlations, The correlalJons may not apply to all classes of proteins or subcallular compartments but they remain as observations that have yet to be explained or disproved.

Subunit molecular weighL Larger proteins are degraded more rapidly. They may be more vulnera~e to denaturation, expose a higher proportion of hydrophobic amino acids on their surface or be intrinsically more susceptible to proteolysis because of the number of available peptide bonds on the surface. Isoelec~icpoint. Acidic proteins are degraded more rapidly than basic proteins. Proteins with a high proportion of acid residues may bind to 'receptors' that commit them to degradation. Cellular proteases may have a preference for acidic residues. I-/ydrophobicity. Hydrophobic proteins are more rapidly degraded than hydrophilic proteins. Exposure of hydrophobic residues on the surface of a protein may potentiate aggregation or association with membranes. ProteolylJ'c vu/nerabili~y. Long-lived proteins may be inherently resistant to attack by cellular proteases, a property reflected in their intractability to proteolysis in vitro. Thermodynamic stability. Thermodynamic stability is a reflection of mobility within the protein molecule. Sho~lived proteins may exhibit a higher flexibility that could in turn expose paptide bonds to protease attack or hydrophobic areas to a recognition site. Modification reactions, Post-translational modifications of proteins have the potential to initiate or modula',e the entry of that protein into the degradative pathway. Such 'marking' or 'branding' reactions may have multiple consequences, including alteration of degradation rate. However, there is no evidence that these modifications act as the obligatory first step in the degradative pathway.

Ubiquitiny/ation. Conjugation of ubiquitin to cetlular proteins is a well-astablished post.transletional modificstion that may mark a protein for degradation by specific pmteases. Oxidation. Oxidative species attack proteins adventitiously, modifying or fragmenting them. The oxidised protein may be more vulnerable to degradation and hence be removed. Deamidation. Asparagine and glutamine residues are deamidated in a non-enzymatic process that makes proteins more acidic (see above) and which might enhance susceptibility to the degradative system. Amides in different sequences are deamidated at different rates, introducing the opportunity for variation of degradation rate. Phosphorylab'on. Many proteins are reversibly phosphorylated. If the two forms are degraded at different rates then degradation may be influenced by the partition between the two froms. Sequence motifs. Recent research has suggested that short amino acid sequences may function to control rate and route of degradation of a protein. As yet, there are no strong 'concensus' sequences that can be widely

ar~ied. N-terminal amino acid. The N-terminal amino acid may influence the half-life of a protein. This correlation has only been demonstrated with one protein thus far and its wider applicability is unknown. 'PEST'. The 'PEST' concensus refers to sequences containing a high concentration of proline, glutamic add, serine and threonine. "Lysosomalsignalpeptide'. A pentapeptide sequence has been identified that commits proteins to lysosornal degradation under nutritional step-down conditions.

Proteolysis and PhysiologicalRegulation applicability (Saus et al.,

of the ubiquitin-dependent 1982; Kolata, 1986)

system has been questioned

Oxidation of specific residues in proteins has been suggested by several groups of investigators as a covalent modification that initiates the degradative process (Rivett, 1986; McKay and Bond, 1985). Oxidation of cellular proteins by a variety of mechanisms has been found to increase the vulnerability of those proteins to proteolytic attack. The oxidation of several enzymes by mixed-function oxidation systems (non-enzymatic systems containing Fe(II) and 02 or ascorbate, Fe(III) and 02 ) inactivates them and increases their susceptibility to proteases (Stadtman, 1986). Most of the enzymes affected by the mixed-function oxidation system require a divalent cation for activity, possess a nucleotide binding site, and have an essential histidine residue at their active site. Glutemine synthetase has been most thoroughly studied in this system and it was found that the initial oxidation of one histidine residue abolishes enzymatic activity and marks the enzyme for degradation. Other forms of oxidation inactivate and destabilize other enzymes. Oxidation of cysteine residues by biological disulphides inactivates several muscle enzymes (e.g., aldolase) and increases the vulnerability of the enzymes to proteolysis (Offermann et al., 1984). Also oxygen-derived free radicals can fragment proteins and thereby inactivate them and may initiate the degradative process (Wolff et al., 1986). Oxidative processes may be important in initiating bulk degradation of proteins in oxygen toxicity, oxidative bursts when polymorphonuclear leukocytes are exposed to bacteria, oxidative damage to heart tissue after hypoxia and reoxygenation (Guarnieri et al., 1980), and oxidative damage to lung tissue (Deneke et al., 1985). Whatever the nature of the initiating events (Table 7.2), they are probably succeeded by the processes of sequestration and autophagy (internalization and digestion of cellular components in vacuoles or autophagosomes and fusion with lysosomes) (Mortimore and Ward, 1981; Ahlberg and Glaumann, 1985; Seglen et al., 1985). Autophagy is prevalent in nutritionally-deprived cells or in the presence of pharmacological concentrations of glucagon; whole mitochondria and other subcellular organelles can be observed in vacuoles and secondary lysosomes in these situations. In well-nourished cells, autophaglc vacuoles and other evidence of autophagy are less obvious microscopically. Nonetheless, there is biochemical evidence that lysosomes take part in basal proteolysis and it is likely that some sort of autophagy or microautophagy (sequestration of small portions of ~he cell contents and fusion with lysosomes) occurs normally. Autophagic processes can account for the random, basal level of degradation observed in cells but do not readily account for the selective nature of protein degradation under normal circumstances. Therefore mechanisms have been proposed to explain selectivity within the context of autoghagy. Some possibilities are: a) proteins selectively bind to the lysosomal membrane and are therefore more likely to be engulfed, b) denatured or hydrophobic proteins associate with cell membranes and are selectively vacuolised and fused with lysosomes, or c) specific binding and sequestration proteins (receptors?) inactivate and bind cytosol proteins. The lysosome is one site of degradation of cell 'proteins but is not the only site capable of extensive degradation of cellular proteins. Microinjection studies have demonstrated that some proteins can be

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totally degraded in the cytosol (Bigelow et al., 1981). In addition, in cells such as mammalian red blood cells and bacteria, that have no lysosomes, all proteolysis must be extra-lysosomal. There are many endo- and exo-peptidases in the cytosol (e.g., ATP-dependent proteases, other high molecular proteases, calpains) and cellular membranes (meprin, aminopeptidase M) that could act on cellular proteins and whose concerted action could lead to degradation to amino acids. Currently many believe that the initial events in the degradation of proteins occur outside of the lysosome but the final stages of protein catabolism are intralysosomal. The committed step in the degradative process may be a covalent modification caused by a proteolytic cleavage or a non-proteolytic modification such as u b i q u i t i n y l a t i o n or oxidation; the final stages must be proteolytic but may also involve the action of glycohydrolases and lipases.

7.3 Regulation and dysfunction of Intracellular proteolysis In our view, there are multiple pathways for the degradative process. The route taken by any one protein or groups of proteins may vary with cell type, nutritional status, hormonal status and environmental conditions. Within one cell type different proteins may have different routes and rates of degradation but probably have common final pathways for degradation. It is known that hormones have a profound influence on the degradation of cellular proteins. For the liver, studies with perfused tissues and cells in culture have shown that insulin decreases the rate of degradation of proteins in general and glucagon enhances degradation (Mortimore and Mondon, 1970; Ward et al., 1977; Hopgood and Bal!ard, 1980). These hormones exert at least a part of their action by affecting the lysosomal system. Insulin, corticosteroids and thyroid hormones also affect muscle proteolysis; insulin retards proteolysis while corticosteroids and thyroid hormone (T 3) enhance proteolysis (Long et al., 1984; Millward, 1985). While the effects of the hormones are clear in vitro or in isolated cell systems, the effects in vivo are much more difficult to establish because of the interactions that occur between hormones and the fact that the effects may differ depending on the nutritional state of the animal. For example, increases in muscle proteolysis are observed with increasing amounts of T in w e l l - f e d animals whereas decreases in T 3 and proteolysis are observed in protein deficiency; however, there are instances where proteolysis increases with decreased amounts of T 3 (e.g., in starvation, infection). The effects of T 3 also seem to be mediated by lysosomes and, as with all hormonal repsonses, depend on many factors in addition to the concentration of the hormone (Millward, 1985). Exercise, or muscular activity, plays an important role in regulating muscle protein turnover (Waterlow, 1984; Rennie et al., 1981; Dohm et al., 1980; 1982). The effects of short bouts of strenuous physical activity and long-term training are very different, however, and the nutritional status of the animal also has a great influence on the response. Increased proteolysis is generally associated with hypertrophy and short periDds of strenuous physical activity. There has been disagreement over the effects of strenuous exercise on muscle protein breakdown, but this seems to be due to biphasic responses to exercise (Dohm et al., 1985). During exercise there is first a decrease in muscle protein breakdown and then an increase over the

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resting rate of muscle proteolysis. Exercise can also cause an increase in nitrogen excretion ~ssociated with the loss of protein from the liver and it has been suggested that increased muscle protein degradation is due to proteolfsis of damaged muscle tissue by lysosomal enzymes (Kasperek, 1985). There has been considerable interest on the effects of ageing on proteolysis in man and in model systems (McKay and Bond, 1986; Young et al., 1982; Rothstein, 1982). There is a loss of muscle mass in the elderly but it has been difficult to establish whether this observation is due to effects on protein synthesis or degradation. The balance between the rate of synthesis and the rate of degradation will determine the total amount of body protein, and the relative contributions of the two processes to the observed decrease in muscle mass with ageing is unknown. There are specific proteins that change with age due to post-translational processing: increased collagen crosslinking and accumulation of Heinz bodies in red blood cells are examples. But it is not known why they accumulate in elderly individuals and not young ones. Model systems for ageing (e.g., mammalian cells in culture, reticulocytes, nematodes) also indicate that posttranslational modifications of proteins contribute to the accumulation of altered proteins in ageing cells. In some systems, the reticulocyte and the nematode, there is an age-related loss of proteolytic activity and this could explain the inability of cells to degrade altered proteins. A strong correlation between senescence and a diminished degradative capacity remains to be proven. Nonetheless, the accumulation of altered enzymes in ageing cells indicates that either some critical proteases are diminished or other mechanisms that initiate degradation of intracellular proteins are lacking. Muscular dystrophies in man and animals are characterised by muscle atrophy and weakness, with a progressive loss of muscle mass (Kar and Pearson, 1978). The decrease in muscle mass is accompanied by an increase in necrotic cells, fat and connective tissue. Although the initial events or aetiology of the disease are unknown, there is substantial evidence that proteolysis is abnormal in dystrophic tissue and that several proteolytic activities are elevated in the tissue. Some studies have indicated that the rate of protein synthesis is not impaired or elevated in dystrophic muscle, and that the loss of muscle protein can be attributed to an accelerated rate of protein degradation (Ionasescu et al., 1971). However, other studies on humans have implied that the major cause of muscle loss in Duchenne dystrophy is a marked depression in protein synthesis (Rennie et al., 1982). A recent study on glycogen phosphorylase demonstrated accelerated degradation of this enzyme in dystrophic mouse skeletal muscle compared to controls (Butler et al., 1985). Lysosomal cathepsins are elevated 2 to 6fold and calcium-activated proteases are also significantly higher than normal in dystrophic human muscle (Kar and Pearson, 1978; Katunuma et al., 1983). By contrast, a decrease in the lysosomal enzyme dipeptidyl aminopeptidase (DAP-I) has been found in cultured skin fibroblasts of patients with Duchenne muscular dystrophy and it has been suggested that this decrease is associated with a lysosomal abnormality in non-muscle cells of dystrophic patients (Gelman et al., 1981). Increases in-proteolytic activities in muscle tissue may be associated with secondary degenerative changes, as the disease progresses, rather than primary events (Bond and Bird, 1974). In either instance, protease inhibitors may be useful as therapeutic agents to retard the destructive consequences and progression of the disease

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(Katunuma et al., 1983). Leupeptin has been reported to prevent or delay the onset of muscular dystrophy in mice with a genetically transmitted form of the disease (Sher et al., 1981) and Ep-453, an analog of the cysteine proteinase inhibitor E-64, also has beneficial effects on muscle necrosis when administered to dystrophic hamsters (Hanada et al., 1983). By contrast, epoxide inhibitors had no beneficial effect on dystrophic chickens (Sugita et al., 1980). The use of protease inhibitors as therapeutic agents in wasting diseases is currently an active and important area of research and a better understanding of the basic mechanisms involved in intracellular proteolysis may be helpful in the rational design of drugs for treatment of these diseases. The juvenile or insulin-dependent form of diabetes mellitus is also associated with tissue wasting and impaired body growth. Studies of protein turnover in diabetic patients or diabetic animals has established that there is an enhanced excretion of urea, a negative nitrogen balance and body wasting in diabetics despite an increased dietary intake (Bond, 1980). Once again the net loss of protein mass can be due to a decrease in protein synthesis, an increase in protein degradation or relative effects on both processes. The precise effects of diabetes on protein turnover are not simple to uncover because different tissues appear to be affected differently. In addition, the stage and severity of the disease affects the results; the nutritional status of animals may be markedly different (diabetic animals are hyperphagic and may consume 3 times more than normal controls if fed ad libitum) and the disease affects the levels of several hormones (insulin, glucagon, corticosteroids, thyroxine) which can affect protein turnover. Nonetheless, in general, the rates of protein synthesis are decreased (whole body and in muscle) while the rates of degradation are not changed (Sloan et al, 1980; Ashford and Pain, 1986). The rate of synthesis of liver proteins was decreased in diabetic mice and the rate of degradation of these proteins not altered or decreased (Duncan and Bond, 1981). For individual proteins the effects of diabetes on turnover may be quite different from the average; enhanced synthesis and degradation of some specific proteins such as hepatic glycogen synthetase and phosphorylase has been observed in diabetes (Bahnak and Gold, 1982). However, the loss of protein mass in diabetes seems to be due primarily to a marked decrease in the synthesis of proteins with little or no change in protein catabolism. The finding that diabetes mainly disturbs protein synthesis and not protein degradation was unexpected because there is substantial evidence that insulin deficiency enhances protein degradation in liver and muscle tissues (Mortimore and Mondon, 1970; Long et al., 1984). Also hepatic ultrastructural changes that indicate autophagy and enhanced degradation have been observed in severe forms of diabetes and in response to glucagon (which may be elevated in diabetes) (Garfield and Cardell, 1979). There is little question that urea production is great ly elevated in diabetic animals; this is associated with enhanced amino acid metabolism. If animals are hyperphagic, as is the case before the terminal stages of diabetes, but fail to incorporate amino acids into protein as rapidly as usual, the enhanced degradation of dietary proteins and amino acids could explain increased urea production and the sparing o f body protein. Also hepatic degradation of extrinsic proteins taken up by the liver is enhanced in diabetes and this may spare the degradation of intracellular protein (Hutson et al., 1982).

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Studies of proteases in diabetic tissues have also revealed discreparties. No significant increase in lysosomal proteases have been found (McElligott and Bird, 1981; Duncan and Bond, 1981) and decreases in some proteolytic activities have been reported (Stauber and Fritz, 1985). An alkaline protease activity associated with mast cells increases in diabetic muscle but the significance of this observation is unknown (McElligott and Bird, 1981). Injuries, such as surgical trauma or burns result in negative nitrogen balance and it has been assumed that this is due to an increase in protein degradation, particularly in muscle (Cuthbertson, 1976). Muscle protein breakdown may increase if the injury is severe but after moderate surgical trauma the negative nitrogen balance is mainly attributable to a fall in whole body protein synthesis, with no change in breakdown (Waterlow et al., 1978). Elevated proteolytic activities may be found in the plasma after traumatic shock and protease inhibitors, such as eglin C, prolong survival of rats presumably by inhibiting these activities (Hock and Lefer, 1985). Muscular atrophy induced by burns is caused by an increase in the rate of protein breakdown with no change in synthesis (Odessey et al., 1983). Protease activities at neutral and acidic pH values increased in rat muscle in response to local burn injury; this may have been due to invasion of the injured tissue by macrophages and mast cells (Odessey, 1985). Severe weight loss is observed in host tissues of patients with solid tumors (cancer cachexia) and in some instances of diabetic neuropathy (Lundholm et al., 1976; Ellenberg, 1974). Progressive weight loss in these patients cannot be explained by a decrease in food intake. In a study of cancer patients, hydrolytic enzymes such as cathepsin D were elevated in the host skeletal muscle tissue whereas other enzyme activities (e.g., hexokinase, lactate dehydrogenase) were decreased. In addition, the rate of incorporation of leucine into protein was decreased and the fractional degradation rate of protein increased in muscle. Other studies indicate that cathepsin D is elevated in several host tissues (heart, kidney, lung and spleen) of tumor bearing animals (Greenbaum and Sutherland, 1983). These studies indicate that cachexia is associated with elevations in protein degradation and lysosomal enzyme activities and it has been suggested that lysosomal protease inhibitors might have beneficial effects as anti-cachexia agents. Ischaemia, caused by the obstruction of arterial flow, can cause severe and irreversible damage to many tissues. In the heart, ischaemia leads to a decrease in tissue pH and a decrease in the rate of proteolysis (Chua et al., 1979). Myocytes in culture release lysosomal enzymes such as cathepsin D in response to ischaemia and it has been suggested that this release leads to tissue damage (Wildenthal and Crie, 1980). Administration of a cysteine protease inhibitor (NCO-700) to dogs before and during ischaemia inhibited some of the degradation of myofibrillar proteins and may have some therapeutic value (Sashida and Abiko, 1985). In the kidney, ischaemia leads to a rapid loss of microvilli protein (Paddock et al., 1981) and decreases in several enzyme activities are found in liver ischaemia. In general the detailed effects of ischaemia on protein metabolism are not known but some of the most damaging effects may be due to the generation of free radicals (superoxides, hydrogen peroxide, hydroxyl radicals) during the ischaemic process or during reperfusion of ischaemic tissue.

Chapter8

Proteases In cell-cell Interactions

8.1 Introduction A controlled multicellular existence demands that cells communicate with their neighbours. Diverse mechanisms have evolved to allow these cell-cell interactions and many of them are mediated by the action of proteins or peptides. In turn, this introduces the opportunity for protease involvement in the generation or removal of the peptide messengers that communicate between the different cells. Many biologically active peptides and proteins are generated by limited proteolysis that occurs intracellularly, prior to release into the intercellular milieu and thus, precedes transmission of the signal. However, in other instances, the secreted polypeptide is a precursor that must subsequently be activated by proteolytic attack. Once released and activated, the messenger molecule must ultimately have its action terminated. Suppression of the secretion/activation mechanism does not resolve the problem of the persistence of previously released material and once again proteolysis is involved. Receptor-mediated or fluid-phase uptake by endocytosis is one option for removal of biologically active peptides and proteins; these molecules are ultimately degraded by the lysosomal hydrolytic apparatus. Extracellular proteases or externally-facing membrane proteases can terminate the biological activity of the peptide whilst it remains at the functional site. Extracellular proteolysis need not elicit complete degradation of the messenger protein/peptide; it is sufficient to effect limited hydrolysis so that it is no longer capable of binding to the receptor and thus exerting its effect, it is possible to dissociate binding of the peptide from manifestation of biological effect; limited proteolysis might therefore generate a product that can bind to the receptor but no longer bring about the structural changes in the receptor that produce the required response. Such modified peptides would be competitive inhibitors of the active peptide. In such circumstances, the response would be governed by the balance of newly-secreted active effector, the rate of proteolysis that generates modified competitive molecules and the rate of removal of both forms by uptake, further proteolysis or diffusion. Such subtle

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control mechanisms are as yet largely unexplored but we suggest that they might operate over time scales and distances that are highly significant in terms of cell-cell interactions. In this chapter we examine the role of proteolysis in regulation of interactions between cells that are physically close and also, in the modulation of the action of biologically active proteins or peptides that continually bombard the surface of the cell. The latter include long-range interactions mediated by peptide hormones distributed via the systemic circulation. Finally, the cell is in close contact with underlying connective tissue; the need to control this contact is well illustrated by the role of connective tissue destruction in cell proliferation during malignancy.

8.2 Proteases and the nervous system Peptides are a major class of mediators in the nervous system. In descriptions of the action of these peptides (at least 30 have been described in vertebrate systems) there has been a discernible tendency to invoke a model based upon the cholinergic synapse, in which the transmitter, acetylcholine, is released and rapidly hydrolysed by a specific esterase. By analogy, the postulate includes specific peptidases, located at the post-synaptic site, that are responsible for the rapid inactivation of released peptides. Almost as soon as the neuropeptides were described there were reports of peptidases that were capable of inactivating them in vitro. There have since been many studies that describe neuropeptide inactivating enzymes, often dignifying such activities with names such as 'substance P degrading enzyme', 'enkephalinase' and so forth. Enkephalinase was first described as a metallo-protease, a dipeptidylcarboxydipeptidase, converting for example, leu-enkephalin (tyr-gly-gly-phe-leu) into tyr-glygly and phe-leu (Schwartz et al., 1981). Thiorphan (3-mercapto-2benzylpropanoyl glycine), an enkephalinase inhibitor, potentiates the action of exogenous and released endogenous enkephalins and exerts antinociceptive effects (Roques et al., 1980). However, the criteria used to establish that enkephalinase was a dipeptidylcarboxydipeptidase did not exclude the possibility that the enzyme was an endopeptidase, and evidence has accumulated that supports the suggestion that enkephalinase activity is due to a metalloendopeptidase (Orlowski and Wilk, 1981; Almenoff et al., 1981; Malfroy and Schwartz, 1982). Additional studies of substrate specificity and inhibitor susceptibility have established that enkephalinase is identical to a well-characterised protease that was first purified from kidney; endopeptidase 24.11 (Fulcher et al., 1982; Matsas et al., 1983). This enzyme hydrolyses a range of peptides and is widely distributed throughout mammalian tissues; thus the claims of specificity of enkephalinase, either in terms of location or preferred substrate, are untenable (Mckelvy and Blumberg, 1986; Kenny, 1986). Whilst the ultimate fate of neuropeptides is to be hydrolysed to amino acids the precise role of proteolysis in the regulation of their action remains uncertain (Orlowski, 1983). There is a lack of understanding of peptidergic synapses, compounded by the need to assess the precise relationship between peptide-releasing cells and inactivating peptidases. It is unlikely that a single protease is responsible and there is evidence for the involvement of aminopeptidases and angiotensin converting enzyme in neuropeptide degradation

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(Dua et al., 1985; Ulrich and Hersh, 1985). There is some evidence for a close association between the opiate receptor and an aminopeptidase (Hui et al., 1985); such an interaction might ensure that free peptides were rapidly removed. By contrast, the time scale of action of some of the neuropeptides is such that simple diffusion of the peptides from their site of action could elicit a sufficiently rapid decay of activity (McKelvy and Blumberg, 1986). Such a model might relegate the role of proteases to that of 'scavengers'; ensuring ultimate destruction of the peptide. Further, the intrinsic susceptibility of each peptide to the degrading enzymes may also dictate the time domain of their response. Whilst this area of research has yet to produce a definitive role for the action of membrane-bound peptidases in the nervous system, the potential for control of the enzymes by exogenous inhibitors may offer opportunities for therapeutic intervention. Relatively little is known about the role of intracellular proteolysis in neural tissue. Neural tissue has a lysosomal system (Pope and Nixon, 1984) and cytoplasmic calpains (Yanasigawa et al., 1983; Ishizaki et al., 1985)). Many, if not all of the neuropeptides are excised from larger precursor translation products; such cleavages often occur at dibasic residues (-lys-arg-, -arg-arg-, -lys-lys- or arg-lys). Serine and cysteine proteases from neural tissue are capable of eliciting the correct excision of peptides but the enzymes responsible for such hydrolyses in vivo remain to be established (Loh et al., 1984). Abnormal (increased) degradation of myelin basic protein has been reported in multiple sclerosis and the deposition of abnormal neurofilament proteins in Alzheimer's presenile dementia may reflect a failure or overwhelming of the normal processes of intracellular protein degradation (Pope and Nixon, 1984). The role of proteases in inflammatory demyelinating diseases has been reviewed recently (Beret and Whitaker, 1985). There is increasing evidence for the role of proteases in the deposition and maintenance of memory. Synaptic membrane preparations from hippocampal tissue respond to exogenous calcium by increasing the numbers of glutamate receptors, the receptors that are considered to be associated with long term potentiation of neuronal interactions during memory deposition. An additional effect of calcium administration is stimulation of the micromolar form of calpain and consequent degradation of fodrin, a cytoskeletal protein that is exquisitely sensitive to proteolytic attack (Siman et al., 1984). Further, there is good evidence that fodrin proteolysis is linked to enhancement of glutamate receptors (Siman et al., 1985), probably by disruption of the interaction between the cell membrane and the underlying cytoskeletal network. Interestingly, leupeptin, an inhibitor of calpains (but also of other cellular proteases), impairs mazelearning ability in rats (Lynch and Baudry, 1984), providing yet another piece of evidence for the role of proteolysis in memory deposition. The proponents of this fascinating idea suggest that calcium-stimulated proteolysis has two additional features that make it attractive in this role. First, it would be possible to achieve selective proteolysis and increases in glutamate receptors at specific patches on the cell surface, allowing modification of specific intercellular comm~unication channels without disturbing others. Additionally, the relative insensitivity of calpain to calcium ions might provide a threshold that prevents retention of every event that impinges on the conciousness.

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8.3 Proteases and cell proliferation/tumour formation Maligant cells have a markedly incresed ability to invade surrounding tissue. Further, if the cells are metastatic they are also able to destroy tissue at the secondary site. Tumour cells have long been recognised as capable of degrading the surrounding connective tissue substratum upon which they are cultured. The processes of intravasation and extravasation during metasasis both require attack of connective tissue by the malignant cell. Since such uncontrolled destruction of surrounding tissue is not a feature of normal cells, many studies have focussed on the role of proteases that elicit this attack. Although at present there is little direct evidence for the involvement of tumour specific proteases, a general model, outlined below, has been proposed that takes into account many of the features of tumour cell destruction of surrounding tissue. Migration of a tumour cell across vascular or lymphatic endothelial surfaces requires that it degrades the connective tissue components that form the barrier. These components are predominantly collagen, proteoglycans and glycoproteins. It is unsurprising therefore that the malignant phenotype is characterised by the production of increased levels of extracellular proteases, including collagenases and other connective tissue-degrading enzymes. A further common observation of an increase in intracellular proteases is less easy to explain and may simply reflect the higher growth rate of the cancerous cells associated with a higher level of intrinsic protein turnover. However, it has been suggested that the release of lysosomal enzymes is associated with a "sublethal autolysis" leading to cell detachment (Pauli and Kuettner, 1984). In particular, cathepsin B has been implicated in the metastatic process and for some groups of tumour cells there is a strong positive correlation between the levels of secreted cathepsin B and the malignancy of the cell (Sloane and Honn, 1984). Although cathepsin B is a normal lysosomal cysteine protease, an extracellular form of the enzyme has also been implicated in neoplastic and inflammatory diseases (Sloane and Honn, 1984; Mort et al., 1984). However, the lysosomal form of cathepsin B would be labile to the neutral pH of the extracellular matrix. High molecular weight precursor forms of cathepsin B are secreted into ascites fluid and are stable at neutral pH (Sloane and Honn, 1984; Mort and Recklies, 1986). These forms are relatively poor proteases and their proteolytic potential remains to be elucidated. Collagen degradation by tumour cells is well established. The two potential sources of collagenase activity are precursor procollagenases in the interstitial fluid (derived from other cells) and collagenases (active or precursor forms) secreted by the neoplastic cells. Degradation of basement membrane components by tumour cells grown in culture demonstrates that at least part of the enzyme activity capable of attacking Type IV collagen (basement membrane) is derived from neoplastic cells, In addition, neoplastic cells synthesise collagenases capable of attacking Type I and Type III collagens of interstitial connective tissue (Liotta, 1984; Wooley, 1984). Collagenase inhibitors have anti-invasive properties and the complex mechanisms for collagenase regulation undoubtedly contribute to the range of behaviour of neoplastic cells (Harris et al., 1984). Less invasive tumours have been shown to produce more TIMP (tissue inhibitor of metallo-proteinases) than invasive tumours (Halaka et al., 1983). The roles of other connective tissue-degrading enzymes is still uncertain

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but evidence points to the secretion of a wide range of connectivetissue degrading proteases by malignant cells (Liotta, 1984). It is likely that the spectrum of proteases will differ for different tumour cells and there is a danger of extrapolating too far from the behaviour of a single cell type (Pauli and Kuettner, 1984). Plasminogen activators, defined by virtue of the ability to activate plasminogen by limited proteolysis, have also been implicated in the neoplastic process (Reich, 1978a). Plasminogen is the circulating precursor of plasmin, the fibrinolytic but relatively non-specific trypsin-like serine protease. It is now well established (from enzymic, immunological and molecular cloning studies) that there are two types of plasminogen activators. One is referred to as urokinase-like plasminogen activator, u-PA, because it was first identified as urokinase in urine; the other was first discovered in tissue extracts and is referred to as tissue plasminogen activator, t-PA (Dano et al., 1985). Plasminogen activators are involved in several biological processes in addition to fibrinolysis, and including mammary gland involution, disruption of the follicle wall during ovulation and implantation of the blastocyst (Bachmann and Kruithof, 1984). They have been implicated in a wide range of non-neoplastic conditions in which tissue damage ensues (reviewed in Dano et al., 1985). Many neoplastic tissues, explants and cells secrete plasminogen activator, usually (but not exlusively) of the u-PA type. Additionally, transformation by oncogenic viruses can elicit an increased production of u PA. The role most commonly proposed for the released plasminogen activators is enhanced degradation of the surrounding tissue. This degradation can be attained by plasmin degradation of the substrate or by plasmin-mediated activation of latent collagenase. However, other roles for the secreted protease, or of the products of its action, should not be discounted. For example, the mitogenic effects of proteases have not yet been fully explored.

8.4 Proteases and growth factors Growth factors are polypeptide signals secreted by one cell that affect the growth and differentiation of other target cells. Proteases are intimately involved with the processing and perhaps action of these factors. For example, nerve growth factor is stored and secreted as a multisubunit complex with a molecular weight of 140,000; containing two e-subunits, one ~-subunit and two ~ s u b u n i t s (Thomas et al., 1981). The ~ s u b u n i t s are processing endopeptidases; they have molecular weights of approximately 26,000, have no general proteinase activity and are highly specific for the nerve growth-factor proprotein. EGF-binding protein is also synthesised and packaged in secretory granules with a serine proteinase that is highly specific for processing and release of this growth factor (Taylor et al., 1974). While the endopeptidases for processing NGF and EGF are very similar (they have approximately 70% sequence identity), they will not substitute for each other in processing (Thomas and Bradshaw, 1981). The processing proteases appear to be part of a family of argininespecific serine proteases that include proteases such as glandular kallikrein, and process polypeptide hormones and kinins as well as growth factors. The endopeptidases derive from a multigene family that have distinct mRNAs that encode for the different enzymes and are selectively expressed in different tissues (Ashley and MacDonald, 1985). Although the relationship between proteolysis and growth factor

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action is not yet fully understood, their interaction with protease nexins may be one method of either potentiating or restricting their action (Knauer and Cunningham, 1984; Nichols and Shooter, 1985).

8.5 Protease nexins Protease nexins (PN) are a family of protease inhibitors that are secreted by many cells, although most information has derived from studies of human fibroblasts (Knauer and Cunningham, 1984). There are at least three members of the family: PN-I is relatively non-specific and will bind many trypsin-like serine proteases; PN-II appears to be more specific and binds to the epidermal growth factor binding protein and PN-III binds the y subunit of nerve growth factor. Inhibition of the proteases results in cleavage of the nexin and formation of a stable acyl-enzyme complex. The nexin-protease complex binds to (nexin) receptors on the cell surface and is subsequently internalised and degraded. Roles for nexins are unclear, but they have the potential to control growth factor mediated events, to limit the coagulation process in the pericellular environment or to regulate the activity of plasminogen activators.

8.6 Proteases in the reproductive system Much of the evidence for the involvement of proteases in the reproductive system is circumstantial and is often based either upon the measurement of an activity at a particular site or on the effect of protease inhibitors of varying degrees of specificity. There are a number of events in mammalian reproduction in which extracellu!ar or intracellular proteolysis can play a significant role and this section will deal briefly with those aspects that are best understood. Considerable interest has been shown in the proteases of the acrosome, the m e m b r a n e - l i m i t e d organelle that covers the anterior part of the nucleus of the sperm. The acrosome can be considered as a modified lysosome that contains hydrolytic enzymes to aid fertilisation (Morton, 1977). Just prior to fertilisation, the acrosome undergoes dramatic changes in morphology, including vesiculation and loss of the external membrane and release of acrosomal contents; the 'acrosome reaction' There is some evidence for the involvement in this reaction of a t r y p s i n - l i k e protease that is distinct from acrosin (Meize!, 1984). The best studied acrosomal protease is acrosin, a trypsin-like serine protease that is present, predominantly as a zymogen, at high concentrations in this organelle. Current thinking holds acrosin responsible for dissolution of the zona pellucida, the acidic glycoprotein matrix that surrounds the ovum. Such a role has been inferred from inhibitor experiments, many of which must be interpreted with caution (Morton, 1977). To ilustrate, some synthetic inhibitors affect sperm motility or react with non-acrosomal regions of the spermatozoa. Finally, it is worth noting that several other roles that have been proposed for acrosin (Morton, 1977). However, any role that dem~ands the release of acrosin from the spermatozoa must explain how the enzyme escapes inhibition by the powerful acrosin inhibitors that are present in seminal plasma. Considerable interest has been shown in acrosin inhibitors as anti-fertility agents (Zaneveld, 1982). These

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include the family of peptide arginine aldehydes (Borin et al., 1981) and arginine analogues (Kaminski et al., 1985), unsurprising since acrosin, being trypsin-like, hydrolyses peptide bonds of the Arg-X and Lys-X type. Antifertility activity, probably associated with acrosin inhibition, is also a property of compounds that are not obvious substrate analogues, such as protected glycine esters (Hall et al., 1979; Drew et al., 1981) and sterol sulphates (Burck and Zimmerman, 1980; Burck et al., 1982). The human prostate-specific antigen, used to monitor prostatic cancer and as a post-coitial test in rape investigations, is markedly homologous with serine proteases of the kallikrein family and is indeed, an active protease (Watt et al., 1986). There are a number of events in the female reproductive process that may be mediated by proteases, or which are associated with changes in the level of expression of protease activity (Morton, 1977). There are several events characterised by controlled but extensive tissue destruction. The first is the lysis of the follicular wall that is attendant upon ovulation. This process bears some resemblance to the sequence of events associated with neoplastic tissue invasiveness. Preovulatory follicles possess a high level of tissue plasminogen activator that has the potential to activate the plasminogen that is a component of follicular fluid (Canipari and Strickland, 1986). The inhibitor of plasminogen activation, trans-aminomethylcyclohexanecarboxylic acid (tranexamic acid) suppresses ovulation in rats but aprotinin (Trasyloln~), an inhibitor of plasmin but not plasminogen activators, was unable to suppress ovulation (Akazawa et al., 1983) Additionally, collagenases are secreted under hormonal control (Curry et al., 1986). Secondly, implantation of the fertilised blastocyst in the uterine wall is accompanied by invasion of trophoblasts and again, the analogy has been drawn between this process and the growth of neoplastic tissues (Dano et al., 1985). There appears to be good evidence for the involvement of plasminogen activators in the destruction of the extracellular matrix. A third process that must involve extensive proteolysis is the massive tissue resorption that occurs during uterine involution post-partum. Since much of the increase in uterine mass is due to hypertophy as well as hyperplasia it follows that involution must require a potent intracellular proteolytic system. The details of this intracellular system, in common with all intracellular proteolysis, are unknown but there is good evidence for the involvement of the lysosomal system, and possibly a neutral protease/antiprotease system (Afting, 1983). Enhanced extracellular proteolysis is probably attained by activation of the ubiquitous plasmin/collagenase system (Harris and Cartwright, 1977; Shimada et al., 1985). Finally, cessation of lactation is accompanied by involution of the mammary gland, again by a process that probably involves urinary plasminogen activator (Dano et al., 1984; Saksela, 1985).

8.7 Other processes Involving proteases There are many other processes in which the involvement of proteases has been surmised, usually by virtue of suppression of the process by protease inhibitors (Bond and Butler, 1987). Evidence is accumulating

Proteolysis and Physiological Regulation for a link between entry of calcium ions and activation of proteinase(s) that may mediate a variety of biological events. Several roles have been advanced for intracellular calcium activated proteolysis (Ishiura, 1981). Proteolysis of membrane proteins may be an intrinsic component of cell-fusion (Ahkong et al., 1978). Endopeptidases have been implicated in fusion of rat myoblasts (Couch and Strittmatter, 1983;1984), synaptic transmission (Baxter et al., 1983) and receptormediated exocytosis in mast cells and adrenal chromaffin cells (Mundy and Strittmatter, 1985). These studies provide interesting suggestions for the role of proteases in events at the cell surface and in communication between cells. The mechanisms by which such proteases exert their effects are enigmatic, although all of the possibilities discussed in Chapter 6 should be considered.

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Chapter 9

Extracellular proteolyUcsystems

9.1 Coagulation/flbrinolysis The blood coagulation system provides an excellent example of a finely controlled cascade of proteolytic reactions (Jackson and Nemerson, 1980). The system involves 13 or 14 plasma proteins plus several other factors, such as calcium ions, platelets and membrane surfaces. At least 7 of the proteins involved in blood coagulation exist in the plasma as precursors of serine proteases: prekallikrein, Factor XII (Hageman Factor), Factor XI, Factor IX (Christmas Factor), Factor X (Stuart-Prower Factor), Factor VII and prothrombin. The system can be described in terms of an Intrinsic and an Extrinsic Pathway that both ultimately lead to the conversion of fibrinogen to fibrin, and hence the clot, but there are multiple factors that influence the reactions and several interactions between the two pathways and the actual process is extremely complicated (Seegers, 1978). Fig. 9.1 presents the serine proteinases (in boldface) and precursors that are involved in these pathways. The reactions are sequential; kallikrein catalyses the conversion of Factor XII to XIIa, Factor XIIa catalyses the conversion of Factor XI to XIa, etc., and the final reactions of the two pathways (Factor X...fibrin) are common to both. The scheme shown is greatly simplified and does not include many essential factors for the process. Also it should be noted that whilst a tremendous amount of information is available about the components and individual reactions in blood coagulation, critical questions about initiation and control of the process remain unanswered. In Fig. 9.1 two types of proteolysis are illustrated; first the conversion of an inactive precursor to an active enzyme by the removal of a peptide(s) which makes the catalytic site available to substrates, and second, the conversion of a protein (fibrinogen) to one that polymerises (fibrin). In the latter reaction, four small peptide chains (totalling 6,000 Daltons) are removed from fibrinogen (340,000 Daltons) by thrombin and this exposes aggregation sites on the fibrin molecule. A third type of proteolytic reaction that occurs in the blood coagulation scheme, that is not represented in Fig. 9.1,

250

Proteolysis and Physiological Regulation Intrinsic Pathway Extrinsic Pathway Stimulus

Prekallikrein ~

kallikrein

Factor Xll ~ (Hageman Factor)

Factor Xlla..J. i

Factor Xl

Factor Xla

251

i

Stimulus

~

Factor IX ~ (Christmas Factor)

i

Factor IXa ~ Factor X

FactorVII ~

_~--~":

Prothrombin._L__~

Factor Vlla i

FactorXa

Thrombin i

Fibrinogen

'

~

Fibrin

Fig. 9.1 Blood coagulation proteases The blood coagulation system has traditionally been divided into two pathways, the intrinsic and extrinsic pathways with common final reactions. The system includes sequential reactions in which precursors proteins are converted into active proteinases (shown in boldface).

is the conversion of a protein to one that enhances the rate of one of the proteases. Examples of the latter are the conversion of Factors V and VIII to Factors Va and VIIIa, respectively, by thrombin. Factor Va is an accessory component in the proteolysis of prothrombin to form thrombin and VIIIa enhances the proteolysis of X to Xa by IXa. Active thrombin can act on accessory components in the system to enhance the rate of activation of more prothrombin molecules to thrombin; a positive feedback system that ensures rapidity of response. All of the zymogens of the serine proteases are normal plasma components. In the process of coagulation each protease converts, by limited cleavages at specific sites, a precursor of another proteinase to its enzymatically active form. The rates of the reactions are actually very slow unless accessory proteins and other ligands are present, and it is the combination of protease specificity and control by physiological factors that regulates the cascade of the reactions that occurs. The primary structures of the blood coagulation precursor proteases have been determined. The zymogens range in molecular weight from 45,000 to 160,000 and they all contain disulphide bonds and serine proteinase domains of about 25,000. The serine protease domains are

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J.S. Bond and R.J. Beynon

homologous with the chymotrypsin family of serine proteases and these domains are always located at the C-terminus of the zymogen. Thus while the catalytic domains of the different zymogens are very similar in size, the non-catalytic domains vary considerably (Patthy, 1985). The activation of the zymogens can involve a large change in mass (e.g., prothrombin decreases from 72,500 to 37,000 Daltons during activation) or a very small change (Factor VII and XI) . The specificity of the blood coagulation proteases is conferred by multipoint subsite interactions between the enzymes and substrates. The substrate specificity of the blood coagulation proteases has been studied extensively and has led to the development of oligopeptide synthetic substrates(Huseby and Smith, 1980). Assay of the individual proteases has many potential diagnostic functions such as monitoring anti-coagulant therapies. Inhibitors play an important role in controlling the coagulation process. Antithrombin III is an important physiological inhibitor of thrombin and reduction of the concentration of this inhibitor in blood is associated with fatal thrombotic complications. Heparin inhibits coagulation by increasing the thrombin/antithrombin interaction and also by direct inhibition of thrombin. Plasminogen is the precursor of the enzyme plasmin which degrades fibrin. Plasminogen is a single polypeptide chain of 90,000 Daltons. Upon activation to plasmin by other proteinases (tissue plasminogen activators, urokinase-like plasminogen activators), two polypeptide chains are created; a light chain, of 25,000 Daltons, which is homologous to the chymotrypsin family of serine proteases and a heavy chain, molecular weight 60,000 Daltons, which is homologous to nonthrombin domains of prothrombin. Plasminogen can also be activated by plasmin kallikrein, and streptokinase. Interest in plasminogen activators has been stimulated by the finding that they are released in large amounts by neoplastic cells (See Chapter 8; Reich, 1978a,b) . Normally, the blood clotting system is in a dynamic state such that fibrin clots are constantly being formed and broken down in an orderly, regulated manner. There are, however, many situations that result in blood clotting abnormalities and only a few are mentioned here. Increases in the plasma concentration of plasminogen activators, due to release of these enzymes from tissues, occurs in several cancers and shock. Abnormally high levels of plasmin disrupt the normal formation of clots and lead to fibrinolytic disorders (Sherry, 1972). Disseminated intravascular coagulation occurs in response to infections, malignant diseases of the liver, and trauma (Rapaport, 1972). Deficiencies in many of the blood coagulation proteinases or their inhibitors are known and most, but not all result in blood clotting abnormalities. The classical haemophilia (haemophilia A) results from a heritable deficiency of Factor VIII (which is not a proteinase but rather a protein that enhances the activity of one of the blood coagulation proteinases). Haemophilia B, Christmas disease, is caused by a heritable deficiency of the proteinase Factor IX; this form of haemophilia tends to be a less severe bleeding disease than haemophilia A. Other blood clotting abnormalities occur in response to deficiencies in the proteinases Factor X (Stuart Factor deficiency), Factor XI (PTA deficiency), and Factor XII (Hageman trait). The molecular basis of these deficiencies is usually an inability to syn~hesise these protein factors; in some instances an abnormal protein is synthesised that disrupts the normal clotting process (Ratnoff, 1972).

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Reductions in the plasma concentration of antithrombin III, the major inhibitor of thrombin, results in thrombosis (e.g., Monnier et al., 1978). Decreased concentrations of antithrombin III may be inherited or can result after trauma such as major surgery or septic shock, in which uncontrolled activation of proteases consumes inhibitors faster than they can be replaced. Antithrombin III is reduced in patients with diabetes mellitus and this could be responsible for increased intravascular clotting associated with this disease.

9.2 Complement The complement system is composed of approximately 18 plasma proteins that mediate antigen-antibody reactions and leads to the destruction and removal of foreign substances from the blood. The reactions are involved in a nunlber of inflammatory responses in addition to bacteriolysis, including increased vascular permeability, chemotaxis, lysosomal enzyme secretion, and increased intracellular degradation of immune complexes (Whaley, 1985). The proteins of the complement system exist in the blood as zymogens and by a series of sequential reactions, involving limited proteolysis, active serine proteases are formed in a cascade similar to the blood coagulation system (Ruddy, 1986). Again there are at least two pathways ("classic" and "alternative") leading to a final common pathway and both are initiated by antibody aggregates (Fig. 9.2). The proteinases in the classical pathway are: Clr, Cls, and C2a; in the alternative pathway, D, and Bb are the proteinases. Complement proteases Clr, CIs and D are approximately 25,000 Daltons and Bb and C2a have molecular weights of approximately 60,000, but these proteins are only active as enzymes in association with other proteins. For example, Clr is only active in a complex of Clr2, calcium ions, Cls2, and Clq. In addition to the complement proteases and protein components that link or activate the proteases, there are protein inhibitors (e.g., C1 inhibitor) that bind strongly to some components and limit activity. Both pathways lead to the hydrolysis of C3 and formation of C3b with removal of a 8,000 Dalton C3a. C3b provides a binding site for C5 and this leads to the cleavage of C5 and formation of complexes that cause membrane damage and cell lysis. Complement components are synthesised primarily in the liver and have a half-life in the plasma of approximately 24 hours. The c o m p l ~ e n t pro-proteinases are all glycoproteins and have large molecular weights (i00,000 to 190,000) except for D which is a 23,500 Dalton protein. Structural variations due to genetic polymorphisms are commonly observed in the complement components and they are usually identified by electrophoretic techniques. Three of the complement components (C2, C4 and B) are coded for by genes in the Major Histocompatibility Complex (MHC) of man and several other species and are designated as class III MHC gene products. The significance of the association of these genes with the MHC is not known, however, because polymorphism in the complement components does not affect graft survival. Nonetheless, several diseases such as multiple sclerosis, juvenile onset diabetes, psoriasis, coeliac disease and rheumatoid arthritis are associated with altered MHC antigens and immune responses; these may be related to polymorphisms in the complement components (Whaley, 1985).

254

J. S. Bond and R. J. Beynon (antibody-antigen complex)

Clr Cls

Clr-Cls-Clq

C3bBb

v

°'

,, c3

C3bBbP

I

II

PI

I c3

I I

v

C5

I

(activation of lytic system) Fig. 9.2 Complement activation pathways The complement system is composed of two pathways, the classic and alternative pathways that lead to cleavage and activation of C3. Subeequently, a common sequence of events (not shown) assembles the 'membrane.attack complex' that causes membrane damage. The active protease components of the pathways are shown in boldface.

Inherited deficiencies have been found for most of the complement components (Ruddy, 1985). C2 deficiency is associated with a predisposition to rheumatic diseases, particularly systemic lupus erythaematosus. Deficiencies in the other complement proteases also result in increased susceptibility to systemic lupus and infectious diseases.

9.3 The kallikrein-kinin system The kallikrein/kinin system mediates the production of the powerfully vasoactive nonapeptide, bradykinin (Iwanaga et al., 1980; MOllerEsterl and Fritz, 1984). Bradykinin is a potent vasodilator, causes smooth muscle contraction and increases vascular permeability. Bradykinin is excised from two larger precursor molecules; high and low molecular weight kininogens (HM-K and I/M-K). HM-K comprises a 'light' chain of 58,000 Daltons and a 'heavy chain' of 63,000 Daltons; LM-K has a light chain of 5,000 Daltons and a heavy chain of 62,000 Daltons. In both, the kinin sequence is located at the carboxyl terminus of the heavy chain. Release of kinins from kininogens is catalysed by kallikrein, a trypsin-like serine protease. Two forms of kallikein are recognised. The first, tissue kallikrein (30,000

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Daltons), is associated with major secretory glands and other tissues and is found in secretions such as urine, saliva and pancreatic juice. The pig enzyme has recently been crystallysed and is structurally homologous to trypsin (Bode et al., 1983). Tissue kalikrein exhibits a preference for LM-K, releasing the decapeptide kallidin (lys-bradykinin) that is subsequently converted to bradykinin by amino-peptidase attack. The other kallikrein is secreted into plasma as a zymogen; prokallikrein. Activation of prokallikrein is closely linked to the contact phase of the coagulation cascade. The complex that builds up on damaged and exposed endothelial surfaces comprises Factor XIi, a HM-K:prokallikrein complex and possibly Factor XI. Anchorage of this complex to the exposed endothelial surface is mediated by the histidine-rich region of HM-K. The whole complex undergoes reciprocal activation whereby kallikrein facilitates the formation of Factor XIIa which in turn activates prokallikrein. The kallikrein thus formed releases bradykinin from the complex. Two functions are thus integrated: activation of coagulation and release of a mediator that increases vascular permeability and which therefore may stimulate tissue repair. Kallikrein is also able to effect other reactions, the significance of which is less clear. It can activate prorenin (and hence, stimulate angiotensin production), activate complement and convert plasminogen into plasmin. The kinins are inactivated by further proteolysis, particularly by the action of angiotensin converting enzyme, a dipeptidyl carboxypeptidase (Ondetti and Cushman, 1982). There is some evidence for kinin inactivation by other endopeptidases (Kouyoumdjian et al., 1984). It has now been established that LM-K and HM-K are encoded by the same gene and are produced by alternative processing of the transcribed RNA into distinct mRNAs (Nakanishi et al., 1985). As mentioned in Chapter 4, kininogens contain sequences that are highly homologous to o~her inhibitors of cysteine proteases; LM-K and ~ cysteine proteinase inhibitor are the same protein (Salvesen et al., 1986). It has been suggested that the large size of the kininogens has evolved to provide kinin precursors that are persistent in plasma, but it is not clear whether the use of cystatin-like domains has any other significance. It is tempting to speculate upon a relationship between tissue d~mage, release of cysteine proteases and activation of kininogens.

9.4 Cellular defence processes The primary function of phagocytic cells in animals is to defend the host against foreign material such as bacteria and viruses; phagocytes accomplish this by endocytosis and degradation of potentially dangerous materials. The classical studies of Metchnikoff documented the importance of phagocytes in immunity and also compared the phagocytic cells of vertebrates to unicellular organisms such as amoeba. The protozoa depend on endocytosis for survival, i.e., they engulf and degrade bacteria for food. For phagocytic cells of vertebrates, however, the main function of the process is to defend the host by eliminating bacteria and other foreign particles from the animal and thus benefit the whole organism rather than the individual cell. Leukocytes may in fact engulf large amounts of foreign material and die in the process.

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J.S. Bond and R.J. Beynon

Two of the most important phagocytic systems are the polymorphonuclear (PMN) leukocytes of the blood and mononuclear cells of the reticuloendothelial system. The latter system is comprised of a variety of cells such as the Kupffer cells of the liver, macrophages of the alveoli, neuroglia and 'wandering' macrophages of many tissues (Cohn et al., 1963). Leukocytes and many mononuclear macrophages have the ability to migrate through the walls of small blood vessels to areas of inflammation, a very important aspect of their function. These cells are well equipped for endocytosis and degradation of macromolecules. During the process of endocytosis the cell membrane surrounds particles such as bacteria and internalises the particles in endocytic vesicles, often referred to as phagosomes. Many good reviews are devoted totally to this process (e.g., Silverstein et al., 1977). The hydrolysis of the majority of endocytosed material takes place in the lysosome after phagosomes (also referred to as endosomes) merge with lysosomes. There is some presorting of endocytosed material so that not all that is ingested is destined to reach the lysosome, but the material that is delivered to the lysosome is usually hydrolysed to small compounds by the high concentration of acid hydrolases. The lysosomes contain a very high concentration of proteinases; cathepsin B and D in human liver, for example, have been estimated to be present at approximately 1 mM (25 mg/ml) in the intralysosomal space (Dean ani Barrett, 1976). There is little recognisable structure in the iysosome and because the lysosomal proteinases appear to be fully active within the lysosome, it is unknown why the proteinases do not undergo rapid autolysis or digest the lysosomai membrane. There is a slow turnover of lysosomal enzymes (estimated half-lives of some are 8 days in rat liver) and newly synthesised lysosomal enzymes are delivered to lysosomes from the Golgi apparatus by fusion of particles that pinch off from this apparatus. Thus the lysosome and its contents are in a dynamic state and constantly merging with other membrane enclosed particles. The products of lysosomal digestion, such as amino acids, diffuse out of the lysosome into the cytosol. Indigestible material accumulates in the lysosome and may remain there for the life of the cell. The alveoli macrophages will accumulate particulate material in the lung and eventually die with lysosomes filled with residual bodies; in this instance the cells are removed from the lungs with the sputum. There are many examples of inherited lysosomal enzyme deficiency diseases where specific products (such as lipids or complex sugars) build up in lysosomes because of a missing hydrolase; these diseases are lethal. Interestingly, there are no known cases of a deficiency of a specific proteolytic lysosomal enzyme. Lysosomal protease deficiences may not be noticeable either because of the many different proteases with overlapping specificities that exist in the lysosome or because a deficiency in a lysosomal protease is totally incompatible with normal foetal development.

9.5 Inflammation Proteinases are important components of inflammatory responses. The tissue destruction and degradation of the intercellular matrix and debris concomitant with inflammation are caused mainly by proteinases released from neutrophils and macrophages. Neutrophil leukocytes contain a high concentration of serine proteinases that are active at neutral pH values, namely elastase and cathepsin G; these enzymes are distinctly different from the lysosomal hydrolases that act optimally

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at low pH values (Starkey, 1977). The serine proteinases of leukocytes are stored in subcellular particles called azurophil granules and are released from the cells in the process of phagocytosis. Elastase and cathepsin G may have some function intracellularly to combat bacterial infection and digest immune complexes, but in many situations they act extracellularly and are probably the most important enzymes involved in the initiation of inflammation. These enzymes are able to hydrolyse the major connective tissue proteins, elastin and proteoglycans and to solubilise collagen. Neutrophils also contain cathepsin B and D in azurophil granules but these are present at lower concentrations than the serine proteinases (Baggiolini et al., 1980). Other subcellular particles of the neutrophil contain collagenase, plasminogen activator and acid cathepsins; all of these enzymes are released from granules during the course of phagocytosis. The process of phagocytosis is initiated by the interaction of particles with receptors of the plasma membrane. As the cell forms pseudopodia around the particle, the granules of the neutrophil begin to release their contents into the area containing the particle. In the process some of the proteinases are released into the extracellular space and may then damage structures such as basement membranes and articular cartilage. The plasma inhibitors =-2-macroglobulin and ~-l-proteinase inhibitor would be capable of inhibiting the serine proteinases of the neutrophil. However, they must be supplied and replenished by the circulatory system and thus the proteinases can act in the early stages of inflammmation. Lung tissue presents good examples of a situation in which the proteases of neutrophils and macrophages are important in the normal maintenance and cleaning of the tissue. For example macrophages actively rid the alveoli of bacteria and viruses brought in by inspired air and controlled proteolysis helps maintain the intricate connective tissue scaffold necessary for respiration (Gadek et al., 1984). The balance between proteases and protease inhibitors in the lung is crucial to healthy lung tissue; imbalances of the protease/antiprotease activity can cause massive destruction of the connective tissue matrix. The consequence of acute or chronic inflammation in the lung can be disruption of lung structure and this results when the release of proteases exceeds the capacity of the inhibitors to control the proteolysis. Patients with cystic fibrosis often suffer from chronic lung infections with Pseudomonas aeruginosa and contain high concentrations of proteolytic enzymes in their sputum (Goldstein and Doting, 1986). The proteases derive from neutrophils, rather than from the bacteria, and it has been suggested that the lung damage that occurs in these patients is due to the destructive effects of neutrophil serine proteinases on lung tissue and oxidative or proteolytic damage to protease inhibitors in the lung. Macrophages do not contain elastase and cathepsin G, but they do secrete lysosomal acid proteinases and plasminogen activator during the process of phagocytosis. The lysosomal enzymes are released into phagosomes in a manner similar to that described for neutrophil granule release; the plasminogen activator is secreted directly into the extracellular space of the activated macrophages and generates local plasmin activity (which can subsequently activate latent collagenase). Monocytes and PMN (polymorphonuclear) leukocytes, in addition to macrophages, secrete plasminogen activator under a variety of conditions and it has been proposed that this proteinase is important in initiating tissue remodelling by controlled proteolysis in a local extracellular environment (Reich, 1978a,b).

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Rheumatoid arthritis is another form of inflammation (Harris, 1984). It is initiated by a persistent immune response localised to joints. The initial phases of the disease and the antigens that trigger the immune response are not understood but the immune response begins in the synovium and monocytes and lymphocytes become activated here. Large amounts of PMN leukocytes are attracted to the joint site and phagocytosis of immune complexes can result in spillage of neutral serine proteases into the synovium and consequent destruction of normal articular structures. Lysosomal proteases and metalloproteinases are also present in cell-free rheumatoid synovial fluid and it is not clear which proteases are responsible for the massive destruction of joint structures (Dingle, 1984). There are proteinase inhibitors present in synovial fluid, the major ones are a-l-proteinase inhibitor and a-2-macroglobulin, and proteinase/proteinase inhibitor complexes are also found. The inhibitors may limit proteolysis of normal tissue but unfortunately they do not prevent the destructive effects of the proteinases in this disease. Periodontal disease provides another example of the role of proteases in a chronic inflammatory process (Sandholm, 1984). The proteolytic enzymes that destroy the gum tissue may in part derive from bacteria associated with plaque but most of the destruction is thought to derive from proteases of PMN leukocytes that are drawn to the site of inflammation. The bacteria themselves, endotoxin and products of the bacteria may induce phagacytosis and the release of PMN granules. The neutral serine proteases and collagenases that are released from these cells can destroy vascular basement membranes, collagen, elastin and proteoglycans of the gingival crevices. Severe inflammatory processes, such as septic shock, occur after a major trauma (burns, abdominal surgery) and usually affect organs that contain high concentrations of proteases (lung, liver, kidney). Clinical symptoms of generalised inflammatory processes usually include high fever, high leukocyte counts, elevated plasma proteinase/inhibitor complexes, disseminated intravascular coagulation and anaphylactic responses induced by the activation of the complement system (e.g., Fritz, 1980). These symptoms may be induced by endotoxins of bacteria or other foreign organisms and the resulting reactions indicate the proteinase/antiproteinase balance of plasma and other tissues are disturbed (Jochum et al., 1986). Measurements of the major plasma proteinase inhibitors and clotting and complement factors have indicated that these proteins are generally decreased in the plasma of patients suffering from septic shock. The decrease in the plasma proteins is thought to be due to complexation and degradation of the proteins primarily by the leukocyte serine proteinases, cathepsin G and elastase. The therapeutic use of inhibitors of these enzymes (e.g., eglln C) is suggested to prevent plasma protein and tissue destruction. There are several plasma proteins, including some proteinase inhibitors, that increase in concentration in response to traumas; these proteins are called 'acute phase proteins' (Killingsworth, 1982). They are synthesised in the liver and their rate of synthesis and secretion is inGreased in response to surgery, infections (bacterial, viral, fungal), myocardial infarction and other traumas. Examples of acute-phase proteins are: C-reactive protein, haptoglobulin, a-l-antitrypsin, a-l-antichymotrypisn, a-l-acid

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glycoprotein (orosomucoid). They are indicators of the inflammatory response and have some diagnostic value (Bienvenu et al., 1982; Fisher and Gill, 1975).

9.6 The renin-angiotensin system The octapeptide, angiotensin II is one of the most important mediators of blood pressure regulation. It also affords an excellent example of the involvement of proteases in the generation and destruction of a Angioter~inogen Renin

Angioteneln I Anglotenllln convortlng enzyme

Anoiotenlin

II

Angiotonsinme

I~ive

fl'aQrnerltl

Fig. 9.3 Proteases in the regulation of blood pressure biologically active molecule (Reid et al., 1978; Ondetti and Cushman, 1982). Indeed, the system is so specific that it has been possible to design protease inhibitors that exhibit enzymological and pharmacological properties appropriate for therapeutic control of blood pressure (Salvetti et al., 1985). Fig. 9.3 summarizes those aspects of blood pressure control that are mediated by proteases (Peach, 1977; Cleary and Taylor, 1986). The precursor of biologically active angiotensin (a high molecular weight protein, angiotensinogen) is cleaved in a single position by a circulating endopeptidase, renin, to generate a decapeptide, angiotensin I. Angiotensin I is further cleaved by a dipeptidyl carboxypeptidase, angiotensin converting enzyme (ACE), located on the endothelial surfaces of several tissues (notably lung) to generate the active angiotensin II molecule. Angiotensin II is the biologically active molecule that is a potent vasoconstrictor and which stimulates the secretion of aldosterone by the adrenal cortex. The angiotensin II molecule is subsequently inactivated by further proteolysis, catalysed by enzymes of unknown specificity; angiotensinsases (Ondetti and Cushman, 1982). Angiotensinogen is synthesised in the liver. It is a glycoprotein and the human protein is approximately 420 amino acids long. The sequence of angiotensinogen is known and it shares significant homology with s-1proteinase inhibitor and antithrombin III (Nakanishi, 1985). Thus, it is a member of the superfamily of protease inhibitors although it is not known whether it is a specific inhibitor of a serine protease.

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J.S. Bond and R.J. Beynon

Renin is an aspartic protease but differs from many aspartic proteases in that is has a neutral pH optimum and is highly specific for angiotensinogen. It is synthesised as prorenin in the kidney, accumulates in secretory vesicles (renin granules) in that tissue and is released into the circulation. Activation of prorenin is itself proteolytic and is catalysed by a number of proteases, including kallikrein, providing the first link with the kinin system (Inagami et al., 1984). As far as is known, angiotensinogen is the only physiological substrate of renin. This specificity is undoubtedly due to the extended subsite requirements of the enzyme. The smallest peptide that is hydrolysed appreciably by renin is the octapeptide 6-13; His-Pro-Phe-His-Leu-LeuVal-Tyr; there is a very high probability of a sequence of this length being unique. Although the three dimensional structure of renin is unknown, there is strong sequence homology between it and other aspartic proteases. Prospective renin models have therefore been built, based upon the known structures of other aspartic proteases. A structure that is readily accomodated makes the renin molecule bilobal, the two domains being separated by a long and extended cleft that represents the extended substrate binding site of this enzyme (Hemmings et al., 1985; Carlson et al., 1985). Such information will be valuable in the development of new renin inhibitors. Angiotensin converting enzyme (ACE) is a membrane-bound metalloprotease that has considerably less specificity than renin. A number of biologically active peptides are substrates for ACE. The major site of ACE action is thought to be the vascular bed of the lung although the enzyme can be detected in many other tissues. Indeed, both brain and kidney contain all of the components needed to express an intracerebral or intrarenal renin-angiotensin system (Phillips, 1983; Navar, 1986). In view of the specificity of the renin-angiotensinogen system it is not particularly clear why the product of renin action, angiotensin I should require further proteolytic attack before it attains full biological activity. There appears to be little physiological control over ACE and the conversion of angiotensin I to angiotensin II is very rapid. It may be that the function of this second step is not to increase the concentration of angiotensin II but to reduce the plasma concentration of angiotensin I and hence eliminate some undesirable property of the latter molecule. A decapeptide such as angiotensin I would be susceptible to many exopeptidases and it is possible that this is an example of adventitious proteolysis and that the primary role of ACE is not to effect this reaction but to bring about the inactivation of other peptides such as kinins or neuropeptides. The whole system is exquisitely balanced. ACE causes formation of angiotensin If, a vasoconstrictor and destruction of bradykinin, a potent vasodilator. Yet activation of the kinin system via kallikrein has the potential to increase the levels of active renin and thus, the opposing processes of kinin and angiotensin formation are activated simultaneously, a common occurrence in biological control. Another aspect, not fully understood is the competition that could occur between the two ACE substrates, bradykinin and angiotensin I. Possibly, the main role of angiotensin I is to act as a competitor of bradykinins? The inter-relationships between the renin-angiotensin system and the kinin system serve to emphasise the complexity of regulation that can be achieved by the irreversible control mechanism of proteolysis. However, it must be emphasised that bidirectional control can only be

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attained if the effectors are somehow removed. Angiotensin II is small, passes readily into the glomerular filtrate and is removed from the systemic circulation. Why then are angiotensinases needed? The several reasons that may be advanced underly many other processes that involve proteolysis. First, tissue-associated inactivating enzymes would destroy the biological activity of the octapeptide near the site of generation, removing the need for diffusion of the peptide into the bloodstream prior to inactivation and thus eliminating the possibility of unwanted effects. The intrarenal renin-angiotensin system may also operate in other tissues, particularly the nervous system (Navar, 1986). Short range, localised degradative processes may be particularly important in such systems. Secondly, the time scale of glomerular filtration may be too slow for effective control of angiotensin concentration. In this context, it is worth remembering that high-turnover systems give highly responsive control. Finally, biologically active peptides that are filtered may elicit unwanted responses in the urogenital tract; this may explain in part the apparently excessive proteolytic capacity of the brush border of the proximal tubule (Bennett and McMartin, 1979; Chertow, 1981). The renin-angiotensin system has proven to be a valid target for the control of hypertension. Although most interest, and indeed, the drugs that are currently available, have targeted on ACE; the lack of specificity of this exopeptidase may be the cause of the relatively minor side effects (ACE inhibitors are discussed in Chapter 4). Attention is now being directed towards the highly specific renin as the next therapeutic target. Substrate analogues containing nonhydrolysable bonds have been prepared (Haber, 1984; Leckie et al., 1985) and we may anticipate the rapid development of potent, orally active renin inhibitors.

9.7 Proteases and inhibitors in pulmonary emphysema Emphysema is defined as the dilatation of the respiratory airspaces, associated with the destruction of the connective tissue components of the alveolar matrix (Snider, 1984; Janoff, 1985). All connective tissue components are normally in a dynamic steady state, maintained by balanced synthesis and breakdown. In emphysema, an imbalance between the two processes ensues and brings about the gradual destruction of the elastic tissue. Inasmuch as proteases must catalyse this tissue destruction their role in emphysema has been the subject of extensive research. The protease that is considered to be primarily responsible for tissue damage is elastase, derived from pulmonary macrophages and neutrophils (Gadek et al., 1984). In normal tissue, the activity of elastase is constrained by the inhibitory action of ~-i proteinase inhibitor. In emphysema, the protease-antiprotease balance is disturbed, primarily through supression of inhibitor levels but exacerbated by protease release as a consequence of the inflammatory repsonse of the lung. The two conditions that are mainly responsible for this inbalance are a congenital deficiency of a-i proteinase inhibitor and heavy cigarette smoking. As mentioned above, a-I proteinase inhibitor (API) is a potent inhibitor of leukocyte elastase (the older term, alpha-I antitrypsin, is a misnomer). There are two common genetic variants of API. The two variants are referred to as the severe Z and the less severe S genotypes. Individuals of the ZZ phenotype have API levels of

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approximately 15% of normal whilst the SS genotype is characterised by AgI levels some 60% of normal. ZZ, and possible SZ individuals have a pronounced tendency to develop emphysema, a degenerative lung condition in which attack of the elastic tissue of the lung by leukocyte elastase leads to a progressive loss of pulmonary function (Gadek et al., 1984). The ZZ genotype is due to a mutation leading to replacement of Glu 342 with a lysine residue. The consequence of this substitution is incorrect folding of the protein molecule, such that it accumulates in the rough endoplasmic reticulum, probably because of a failure in intracellular transport (Bathurst et al., 1983; Bathurst et al., 1984; Riley et al., 1985). Curiously, the misfolded protein retains the inhibitory activity of the normal product. Current hypotheses are based upon the insolubility or membrane attachment of the mutant protein (Carrell et al., 1985, Carrell and Travis, 1985). The tendency to develop pulmonary emphysema in ZZ and SZ individuals is dramatically exacerbated if the individuals smoke cigarettes. Cigarette smoke stimulates invasion of lung tissue by leukocytes, increasing the proteolytic burden on an already diminished inhibitory capacity. Further, there is good evidence that the lung of cigarette smokers is subject to an additional oxidative stress, either from components of the smoke or as a result of the release of free radicals by the increased population of leukocytes. The reactive site of API contains a methionine residue that fits the active site of elastase. Oxidative attack upon the methionine residue converts it to methionine sulphoxide that no longer fits into the active site of the enzyme; thus it becomes ineffective as an elastase inhibitor (Carrell and Travis, 1985). Why should such a crucial proteinase inhibitor have evolved a structure that is susceptible to oxidative attack? Current thinking suggests that this provides a mechanism to switch off the inhibitor in close proximity to a site of tissue injury. Invading leukocytes must secrete free radicals to kill bacteria and release proteases to hydrolyse the necrotic material to allow tissue regeneration. Release of oxidative species can therefore prevent local inhibition of the released proteases; widespread inactivation of API is prevented by the short lifetime of the reactive species that cannot diffuse far beyond the site of injury.

Concluding remarks

The field of proteolysis and physiological regulation is so wideranging that even a review of this size can only scratch the surface Proteases abound in mammalian systems, they range from the nonspecific and destructive to those that generate molecules with new biological activities. It has become increasingly apparent that they are highly regulated, an appreciation that has developed from the original concept of proteases as crude and undiscriminatory. Yet, despite a detailed understanding of the mechanisms of some proteases in vitro, we lack knowledge of their action and control in vivo. Our goal in this review has been to demonstrate that although the complexity of proteolytic regulation may appear daunting, common themes recur. Enzymology is entering a new era, in which new methodologies such as molecular cloning are providing deeper insights into the molecular basis of normal and disease processes, including those that involve proteases and their inhibitors. These techniques will have profound consequences; we anticipate with excitement the new understanding of protease function that will emerge.

263

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J. S. Bond and R.J. Beynon Acknowledgments

The collaboration that led to this review was made possible by a NATO Research Grant (#0574/82). Work from our laboratories was supported by NIH (Grant DK 19691), Medical Research Council (GR 840/7575), Muscular Dystrophy Group of Great Britain and Northern Ireland (RA3/162) and the Science and Engineering Research Council (GR/D/26955).

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