Molecular diseases of limited proteolysis

284 Con~;ributions to Biochemical Hematology

Molecular Diseases of Limited Proteolysis M.A. COLETrI-PREVIERO, B. DESCOMPS*, A. MOLLA and A. PREVIERO U 147 INSERM, 60 rue de Navacelles, Montpellier (France) PROTEASES WERE AMONG THE FIRST ENZYMES to be recognized ''2 as p r i m a r i l y associated with polypeptide digestion. They were classified in t e r m s of s p e c i f i c i t y , based on the chemical n a t u r e of the amino acids n e i g h b o u r i n g the s u s c e p t i b l e bonds. A more sophisticated view has since emerged, t a k i n g into account the amino acid residues more d i s t a n t from the site of hydrolysis and generally the structure of the protein s u b s t r a t e in t e r m s of threed i m e n s i o n a l s t r u c t u r e and a c c e s s i b i l i t y of the cleavable bond ( " t o p o g r a p h i c a l s p e c i f i c i t y " ) ~'4. This behaviour, r e f e r r e d to as " l i m i t e d p r o t e o l y s i s~'8'', is a r e s t r i c t e d cleavage of a p e p t i d e chain, i n d u c i n g or l i m i t i n g a l a r g e v a r i e t y of biological reactions, by an i r r e v e r s i b l e process, which is simply summarized in Table 1. When an a l t e r a t i o n of the control p r o t e a s e s occurs, be it an e n h a n c e m e n t or an inhibition, it can r e s u l t e i t h e r in a s i n g l e c h a n g e or in a c a s c a d e of u n p r e d i c t a b l e events, e v e n t u a l l y c a u s i n g a d e f e c t or a disease. In this paper we wish to p r e s e n t a u n i f i e d view of the d i f f e r e n t events and t h e i r relat i o n s h i p s with a l t e r a t i o n s of l i m i t e d p r o t e o l y s i s . I) Kallikrein-kinin system K a l l i k r e i n s are s e r i n e p r o t e a s e s found in mamm a l i a n p l a s m a and in some g l a n d tissues, which by limited p r o t e o l y s i s l i b e r a t e k i n i n s from kininogen. K i n i n s (e.g. b r a d y k i n i n ) are the most p o t e n t vasodil a t o r p e p t i d e s known. A l t e r a t i o n in the level of t h e components of the s y s t e m and of t h e i r n a t u r a l in-

*Centre Hospitalo-Universitaire de Montpellier

h i b i t o r s have been closely associated with several pathologic processes like anaphylactic shock, edema 8 and p o s t - t r a u m a t i c s e p s i s 9. A p l a s m a disease, known as F l e t c h e r t r a i t , which r e s u l t s as a d e f i c i e n c y of coag u l a t i v e '°, f i b r i n o l y t i c and c h e m o t a c t i c T M a c t i v i t i e s has been d e s c r i b e d and seems to be due to an absence of k a l l i k r e i n , since n o r m a l v a l u e s of all activities were r e s t o r e d by a d d i t i o n of the enzyme TM. In a d d i t i o n , a system, which p a r a l l e l s t h e kallik r e i n - k i n i n one is found in c e r t a i n p a t h o l o g i c a l fluids, such as a s c i t i c f l u i d f r o m n e o p l a s t i c d i s e a s e 1., and is r e f e r r e d to as the l e u k o k i n i n - g e n e r a t i n g system.

2) Blood Coagulation and Fibrinolysis The c a s c a d e m e c h a n i s m of blood c o a g u l a t i o n involves the i n t e r a c t i o n of n u m e r o u s p r o t e i n s more t h a n h a l f of which are c o n v e r t e d to enzymes, a c t i n g stepwise on specific p r o t e i n s u b s t r a t e s by highly selective proteolysis. In normal blood, this is an explosive reaction, whose m o l e c u l a r m e c h a n i s m shows a high level of f u n c t i o n a l i n t e g r a t i o n and which i n t e r a c t s w i t h o t h e r c a s c a d e s y s t e m s of p h y s i o l o g i c a l i m p o r t a n c e . P a t h o l o g i c a l a c t i v a t i o n of blood coagulation r e s u l t s in d i s s e m i n a t e d i n t r a v a s c u l a r coagulation (DIC), a s o m e t i m e s c a t a s t r o p h i c c l i n i c a l syndrome 's: v a r i o u s t r i g g e r m e c h a n i s m s , i n c l u d i n g p r o t e o l y s i s , can be considered, b u t r e g a r d l e s s of the initial cause, the final common m o l e c u l a r p a t h w a y involves an a l t e r e d action of t h r o m b i n , a limitedp r o t e o l y t i c enzyme. I n t i m a t e l y l i n k e d w i t h the blood c o a g u l a t i o n process is the plasminogen-plasmin system, respon-

TABLE 1 PROTEOLYTIC ACTIVATION

PRE-PRO-PROTEIN

PRO-PROTEIN

~

PROTEIN (active)

ZYMOGEN

~

ENZYME

Ex: Trysinogen PROHORMONE

•

~0~

PROTEOLYTIC LIBERATION

Ex: Proinsulin

PRECURSOR

~ ¢" 0 m

Trypsin )

HORMONE Insulin

> NEUROPEPTIDE

Ex: Lipotropin

Endorphin

PRECURSORS cascade Ex: Blood coagulation

ACTIVE PROTEIN

PROTEOLYTIC DEGRADATION >

FRAGMENTS Inactive or less active

285 sible for the lysis of fibrin clots. This system is significantly altered during neoplastic t r a n s f o r m a t i o n of the cell" by means of an increased production of plasminogen activator, the enzyme which liberates plasmin f r o m plasminogen by limited proteolysis. Besides plasma, plasminogen activator is also present in h u m a n embryonic lung cells '~. A neonatal disease, respiratory distress syndrome (RDS), is characterized by an impaired fibrinolytic system which results in the inability of the i n f a n t to remove the fibrin-containing membrane f r o m the lungs, at birth. The molecular deficiency is a lack of plasminogen, but the primordial cause of the disease is an excess of plasminogen activator production prior to birth 18. Another system of control on p l a s m a limitedproteolysis enzymes is represented by protease inhibitors in blood. I t acts on the dynamic equilibrium between the various enzymatic systems and an alteration of these inhibitors results in a, sometime severe, hereditary pathological state 19"~. 3) Collagen Formation Collagen is synthesized as a precursor, which undergoes a n u m b e r of post-translational proteolytic conversions of its primary structure before yielding the functional protein. Defects in these excision mechanisms result in diseases identifiable by structural alterations in the collagen molecule '3. O n e of the best biochemically identified diseases is dermatosparaxis, hereditary malformation in cattle", sheep" and m a n 2e inducing fragile skin and skeletal deformity. This pathological picture is not due to intrinsic abnormality in the collagen structure itself but to a defect of one of the essential post-translational modifications, the limited proteolysis of the N-terminal part of the collagen precursor, the key pathogenic event being a deficiency of the enzyme procollagen N-protease".

4) Polypeptide Hormones and Neuropeptides Limited proteolysis plays an i m p o r t a n t role in the liberation of polypeptide hormones f r o m t h e i r precusors". The i n t r a c e l l u l a r localisation and the precise m e c h a n i s m of this highly specific proteolytic process is still an open question. One striking feat u r e has a p p e a r e d to always c h a r a c t e r i z e the cleavage site of p r o h o r m o n e s : the presence of two diaminoacids side by side which are also shared by opiate neuropeptides (endorphins). An important, still unanswered, question is whether this t r a n s f o r m a t i o n is achieved by a similar or identical limited protease or if d i f f e r e n t enzymes exist f o r each hormone or each peptide. No precise pathological examples directly related to an alteration of this proteolytic liberation have been described so far, but they will p r o b a b l y show up with the increase of our knowledge in this field. The f u n d a m e n t a l problem in e v a l u a t i n g the correlations between limited proteolysis and disease is to determine t h e i r causal relationships. I f f o r some pathological conditions the altered biochemical p a r a m e t e r s are clearly disease-related, o t h e r diseases, less well defined f r o m the molecular point of view,

seem to interact with some limited proteolysis system. T u m o r promotion 29, inflammatory 3°, demyelinating 3', degradative ~2, and hepatic "~3diseases can be recalled as examples.

DISCUSSION It is increasingly evident that proteases with restricted specificity are mediators of important biological processes and that their action needs a high level of functional integration since any disturbance of the balanced system will result in a physiologic defect. The two major features qualifying their action are specificity and timing. In fact their specificity is a function of the substrate structure, around and/or distant from the bond to be cleaved, of the availability of the susceptible area and of the physico-chemical situation of the enzyme itself. Their timing is regulated in a very fine w a y since formation and inactivation of the active polypeptide must occur at a single m o m e n t in a cascade of biological events. It is evident that modifications will not have the same impact on the involved functions. S o m e will lead to only minute functional variations, others to more important changes. The disease represents the final point of a function impaired by lack of a proteolytic action on a physiologically important polypeptide. The molecular mechanism of the disease might include either altered responses of enzyme amount to regulation or altered responses of the biochemical activity to controls. The primary cause m a y not therefore be immediately evident from the study of the disease, since the identification of the molecular defect is just the first step in the comprehension of the molecular mechanism underlying it. It is noteworthy that molecular pathology in its classical examples (e.g. hemoglobinopathies) diverges from that of limited proteolysis. In the former, disease is due to structural defects, directly linked to genetic alteration ; in the latter it is a consequence of post-translational modification of the peptide substrate, not under direct genetic control. The visible consequences are, in classical examples, impairment of the function directly linked to the defective protein, whereas in limited-proteolysis they consist of the impairment of a function which should have been generated by the protease. A knowledge of the precise mechanism of the defect is a necessary prerequisite for an approach to therapy of diseases at the molecular level, as far as altered restricted proteolysis is concerned. H o w ever w h e n a polypeptide or a protein, normally produced by this process is lacking or produced in some inactive form, an eventual substitutive therapy is restricted by considerable difficulties owing to the immunogenetic properties of the protein material to be supplied and to the possibilities of its administration, the latter being usually the parenteral route. The development of n e w techniques of administration by including the protein molecule in special vehicles, such as liposomes u, might be a first advance. Another w a y to facilitate the therapeutical applications is to use synthetic molecules specially designed to escape abnormal proteolysis

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a n d ' r e a c h t a r g e t tissues with all their activity: such molecules can be analogues, agonists or effectors able to modulate the activity of the enzyme implicated in the degradative or in the biosynthetic process altered by the disease. ACKNOWLEDGEMENTS This work has been supported by a g r a n t from the Fondation pour la Recherche M4dicale Fran~aise. M.A.C.P. belongs to CNRS. RF~ERENCES

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