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Peptide Degradation (Neprilysin and Other Regulatory Peptidases)
Chapter 241
Peptide Degradation (Neprilysin and Other Regulatory Peptidases) Anthony J. Turner and Natalia N. Nalivaeva
ABSTRACT Turning off peptide signaling is principally mediated by a group of cell-surface peptidases whose active sites face the extracellular space (ectopeptidases). Two groups of metallopeptidases are the best characterized as peptide degrading enzymes: the neutral endopeptidase or neprilysin (NEP) family and the angiotensinconverting enzyme (ACE) family. NEP and its homologs are important in degrading both brain peptides (e.g. enkephalins, tachykinins) and cardioactive peptides (e.g. atrial natriuretic peptides). They also metabolize the Alzheimer amyloid β-peptide (Aβ). In the cardiovascular system, ACE and ACE2 act to counterbalance each other in controlling angiotensin peptide levels and may also participate in the metabolism of other peptides. Prolyldirected peptidases such as dipeptidyl peptidase IV and prolyl oligopeptidase play highly specific roles in peptide metabolism. This chapter summarizes current developments in our understanding of the roles and the regulation of these key mediators of peptide signaling, their roles in disease and as potential therapeutic targets.
INTRODUCTION Proteolysis, which regulates the production and removal of biologically active peptides, represents a key biological process underpinning events from fertilization, through development to cell death. The ubiquity and diversity of peptidases are indicated by the existence of some 28,000 specific peptidases and their homologs characterized in >6000 known organisms (http://merops.sanger.ac.uk/). In the human genome of approximately 24,000 proteincoding genes, 686 genes of putative peptidases are known alongside 423 of noncatalytic homologs. Many of these are orphan peptidases for which the biologically active peptide substrates have not yet been identified. Such a profusion of proteolytic events requires specifically controlled regulation. Hence, although genes for peptidases constitute about 2.8% of the human genome, a significantly higher number (7.4%) of genes encode protein inhibitors of peptidases. Handbook of Biologically Active Peptides. http://dx.doi.org/10.1016/B978-0-12-385095-9.00241-4 Copyright © 2013 Elsevier Inc. All rights reserved.
The term “peptidase” refers to enzymes that hydrolyze peptide bonds and, as such, all known biologically active peptides represent potential substrates for these enzymes. According to the location of the peptide bond cleaved by the peptidases, they are conventionally subdivided into “endopeptidases” that cleave the bond within the polypeptide chain, or exopeptidases that release amino acids from the Nor C-terminus of the substrate. However, some peptidases, e.g. angiotensin-converting enzyme (ACE), can fulfill both endopeptidase and exopeptidase functions. The physiological role of peptidases is very diverse. In general, peptidases do not cleave their substrates at random but participate in very well-controlled, specific processing of peptide molecules. The term “limited proteolysis” was originally introduced by Linderstrøm-Lang to distinguish the restricted specificity of certain peptidases, particularly those involved in posttranslational modification of proprotein precursors, from the random proteolysis accompanying protein digestion and degradation. All peptidases have been divided into six classes (metallo-, serine, cysteine, aspartic, threonine, and glutamate peptidases) with cysteine-, serine-, and metallopeptidases representing the major categories. A seventh class involving asparagine peptidases has recently been reported.24 Aberrant functioning of these peptidase genes is implicated in such diverse pathologies as inflammatory and cardiovascular diseases, cancer, and neurodegeneration. Understanding the normal functional roles of peptidases and their regulation and restoration in disease is hence important for the design of novel and selective therapeutics. This chapter is focused primarily on two major families of zinc peptidases participating in the specific degradation of biologically active peptides. The prototypes for these peptidases are neprilysin (neutral endopeptidase, NEP) and ACE. The roles of peptidases directed specifically toward prolyl residues are also reviewed. Table 1 summarizes the diversity of peptide substrates hydrolyzed by this cohort of enzymes. 1757
Chapter | 241 Peptide Degradation (Neprilysin and Other Regulatory Peptidases)
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TABLE 1 Typical Biologically Active Peptides Degraded by Peptidases Discussed in this Chapter
NEUTRAL ENDOPEPTIDASE (NEPRILYSIN) FAMILY
Substrate
Peptidase
Name and History
Adrenomedullin
NEP
Amyloid β-peptide
NEP, NEP II, ECE-1, IDE, ACE
Amyloid precursor protein intracellular domain (AICD)
IDE
Angiotensin I
ACE, NEP, ACE2, POP
Angiotensin II
ACE2, NEP, POP
Apelin
ACE2
Atrial natriuretic peptide
NEP, IDE
β-Endorphin
IDE, POP
Big endothelin-1
ECE-1, ECE-2, NEP, NEP II
Bradykinin
ACE, ECE-1, ECE-2, IDE, NEP, APP2, POP
Brain natriuretic peptide
IDE
Cholecystokinin-8
ACE, NEP
Corticotropin
ACE, NEP
Dynorphins
NEP, IDE, ECE-2
Endomorphin
DPP IV, NEP
Enkephalins
NEP, ACE
Endothelin-1
NEP
Gastrin
NEP
Ghrelin
ACE2
Glucagon-like peptide-1
DPP IV
Neprilysin activity was first characterized from rat kidney brush border membranes as an enzyme hydrolyzing insulin B chain at neutral pH, and hence termed “neutral endopeptidase” (NEP), although it plays no role in the physiological insulin inactivation, which is mediated by another peptidase, insulin-degrading enzyme (IDE). NEP is a zinc metallopeptidase resembling the bacterial enzyme thermolysin, including its sensitivity to the inhibitor, phosphoramidon. NEP has a diverse range of biological roles and hence has been rediscovered in different proteolytic contexts. For example, after the discovery of the enkephalins, NEP was rediscovered in brain as a membrane enzyme hydrolyzing these opioid peptides and was termed enkephalinase. It is also the principal enzyme inactivating many other neuropeptides (e.g. substance P) and cardioactive peptides (atrial natriuretic peptide (ANP) family) and hence represents the first general “neuropeptidase.” NEP plays an important role in the immune system where it is referred to as the common acute lymphoblastic leukemia antigen (CALLA or CD10), although its substrate(s) have not been identified. NEP also contributes to the metabolism of the Alzheimer amyloid β-peptide (Aβ) in the brain.12 The human genome contains at least seven NEP-like enzymes, including the biosynthetic enzyme endothelin-converting enzyme (ECE-1). The family is even more abundant in Drosophila (24 predicted members) and Caenorhabditis elegans (22 members).4 NEP is also known as MME, for membrane metalloendopeptidase, in the Human Gene Nomenclature database.
Gonadoliberin-1
ACE, POP
Insulin
IDE
Insulin-like growth factor I
DPPIV
Insulin-like growth factor II
IDE
Luliberin
NEP, ACE
Neuropeptide Y
DPPIV, NEP, APP2
Neurotensin
ACE, ACE-2, ECE-1, ECE-2, NEP, POP
Oxytocin
NEP, POP
Somatostatin
NEP, ECE-1, IDE
Substance P
NEP, ACE, DPPIV, ECE-1, ECE2, POP
VIP
NEP, ACE, IDE
Properties NEP is exclusively localized to the plasma membrane (type II integral membrane protein) with the active site facing the extracellular space (ectoenzyme) where it is poised to inactivate circulating peptides. It is a glycoprotein of approximately Mr 90,000. The enzyme is classified as a “gluzincin” peptidase with the typical His-Glu-Xaa-Xaa-His (HEXXH) zinc-binding motif. The glutamate in this motif (Glu584 in the human enzyme) is essential for catalysis and an additional glutamate (Glu646) constitutes the third zinc ligand. NEP is an archetypal peptidase rather than proteinase because it generally only hydrolyzes peptides up to about 50 amino acids long. The 3D structure of the NEP catalytic domain in complex with its inhibitor phosphoramidon has been deduced aiding the design of novel inhibitors and providing a better understanding of its substrate specificity and catalytic mechanism.20 The active site region is restricted in size preventing access of large peptides and hence explaining its oligopeptidase nature.
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In general, NEP hydrolyzes peptidases on the aminoterminal side of hydrophobic residues, e.g. the Gly3-Phe4 bond of enkephalins, giving it a very broad specificity for known biologically active peptides although its physiologically validated substrates are more restricted. In addition to opioids, tachykinins, and the ANP family, it inactivates endothelins, bradykinin, and somatostatin. Other NEP substrates include glucagon, the vasoactive intestinal peptide family and adrenomedullin. NEP may also regulate bone metabolism through its presence on the surface of human osteoblasts and its ability to metabolize osteogenic peptides such as calcitonin and calcitonin gene-related peptide. Because inhibition of NEP can, in theory, potentiate the action of any of its peptide substrates, much emphasis has been directed toward developing novel and potent NEP inhibitors, with thiorphan originally being synthesized as an inhibitor of its enkephalin-hydrolyzing activity, and this is still the most commonly used NEP inhibitor. Many others have subsequently been developed as potential therapeutics in cardiovascular disease by prolonging the actions of ANP. The combined actions of NEP and ACE inhibitors have improved benefits over ACE inhibitors alone. The prodrug of thiorphan, acetorphan (racecadotril), is an effective antidiarrheal agent through its potentiation of the antisecretory effects of enkephalins in the intestine. However, the NEP substrate that has attracted by far the most interest over the last decade has been the Alzheimer amyloid-β peptide (Aβ).
Distribution/Localization NEP and its homologs are widely distributed enzymes in the animal kingdom reflecting the general need for terminating the action of circulating regulatory peptides. Many invertebrate peptides are inactivated by NEP-like peptidases where it often plays a role in development and reproduction. In C. elegans, NEP seems to be an effector of locomotion. In mammals, the enzyme is particularly abundant in the kidney where its expression levels in brush border membranes are several orders of magnitude higher than in brain synaptic membranes. It is also highly expressed in the reproductive system (testis, ovary), although its functional role is unknown. Elsewhere NEP, although widespread, is expressed at much lower levels. In the brain, NEP inactivates peptide neurotransmitters, especially opioid and tachykinin peptides, after their release into the synapse. Given the role of these peptides in pain mechanisms, NEP inhibitors have antinociceptive actions. However, most attention has been focused on the role that NEP plays, alongside other peptidases such as ECE, insulin-degrading enzyme, and possibly ACE, in detoxifying the Aβ peptide that predisposes to Alzheimer’s disease (Fig. 1). The susceptibility of Aβ is perhaps not surprising given the
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FIGURE 1 Main cleavage sites of amyloid-β peptide by peptidases. The Aβ-(1–42) sequence is shown in black with the flanking amino acids of the amyloid precursor protein (APP) shown in gray. The principal peptidases involved in Aβ peptide metabolism are indicated in black and the other activities in gray. N, neprilysin; N2, neprilysin-2; A, angiotensin-converting enzyme; E, endothelin-converting enzyme-1; I, insulin-degrading enzyme. Letters in circles indicate peptidases that cleave Aβ at the same site.
specificity of NEP, as it is a highly hydrophobic peptide. Indeed, under normal conditions there is considerable evidence that Aβ itself may be a regulatory peptide, and it is only in pathological conditions when it accumulates excessively that it becomes toxic. This process is enhanced in aging because there is a well-documented age-related decline in NEP expression. The levels of brain Aβ are hence increased in NEP-deficient mice. NEP expression is also diminished after periods of ischemia or hypoxia. Infusion of an NEP inhibitor into rat brain leads to a significant accumulation of the more neurotoxic Aβ42 peptide, and hence, upregulation of NEP has been proposed as a potential therapeutic strategy in Alzheimer’s disease.12,18 NEP gene transfer can decrease amyloid-like pathology and cognitive decline in transgenic mice.16 Given the potential of NEP inhibitors to increase brain Aβ levels and hence to induce Alzheimer’s disease-like symptoms, interest in the development of novel NEP inhibitors as potential cardiovascular therapeutics has considerably diminished. In the brain, NEP is mainly localized on neuronal cells, especially in the striatonigral pathway consistent with its role in turning off opioid peptide and tachykinin signaling, and it is also located in the hippocampus, where it functions to inactivate somatostatin and in cortical regions where it may also act to degrade Aβ . The enzyme is also present on activated astrocytes and on Schwann cells in the peripheral nervous system where it may function in nerve development and/or regeneration. Unlike many proteases, NEP is not synthesized in an inactive proenzyme form and is not generally regulated by endogenous inhibitors although sialorphin, the peptide product of the Vcsa1 gene, has been reported to inhibit the enzyme in vivo.26 In most tissues, much of the NEP expression is constitutive but recent demonstration of epigenetic regulation of NEP has opened up the possibility of new approaches to control NEP levels in brain.3,21 The Aβ peptide is formed by proteolytic processing of the amyloid precursor protein (APP). Another metabolite of APP is its intracellular domain (AICD), a 50–59 amino acid peptide, which can be translocated to the nucleus where it can bind to the NEP promoter (and promoters of some other genes) displacing
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histone deacetylases which act as transcriptional repressors, and hence upregulating gene expression.3 This effect seems to be mediated preferentially by the neuronal isoform of APP (APP695).2 Through a related mechanism, histone deacetylase inhibitors such as trichostatin A and valproic acid, can enhance NEP expression in vitro and in vivo and may therefore have therapeutic value in Alzheimer’s disease.3 NEP is an important diagnostic marker in certain forms of childhood leukemia and has also been implicated in cancer progression, particularly of the prostate. NEP gene transcription is androgen-regulated in androgen-responsive tissues. Hence, the downregulation of NEP through hypermethylation of the promoter contributes to androgen- independent progression of prostate cancer because of a reduced ability to metabolize mitogenic peptides, particularly endothelin-1. NEP may also be important in the development of obesity and its expression levels associate with insulin resistance and the metabolic syndrome.28
Related Peptidases Seven NEP-like proteins have been identified in the human genome.4 Most of these are of unknown biological function, and their substrate specificity is unclear. The most close in similarity to NEP is a peptidase referred to as NEP II in the rat, “soluble secreted endopeptidase” (SEP) or NEP-like 1 (NL1) in the mouse, or MMEL2 (human). The best-characterized NEP homologs are the endothelinconverting enzymes (ECE-1, ECE-2). ECE-1 participates in the conversion of big endothelin to the vasoactive peptide, endothelin-1 (see the chapter on Cardiovascular Peptides and below). ECE-2 probably plays a role as a processing enzyme in the biosynthesis of regulatory peptides and can process such peptides at non-classical processing sites. Mice deficient in ECE-2 have learning and memory deficits and show abnormal responses to morphine and have altered peptide levels in the spinal cord.17 NEP II was first identified in mice deficient in ECE-1 as a potential endothelin-processing enzyme.11 Although it is the most similar peptidase to NEP, it has many distinct properties; for example, it occurs as both a soluble, secreted enzyme and an alternatively spliced form retained in the endoplasmic reticulum. NEP II is particularly localized to neuronal populations in the brain and spinal cord and is also abundant in reproductive tissue—male mice deficient in NEP II show reduced fertility. Originally, NEP II was reported to have a broad repertoire of substrates, similar to those for NEP itself,12 although its physiological role apart from male fertility, remains unresolved. However, rodent NEP II exhibits a much wider substrate specificity than the human enzyme.33 For example, only substance P and angiotensin I are hydrolyzed at comparable rates by human NEP and NEP II.33 The inhibitor thiorphan is also more potent
toward NEP than NEP II. A physiological role for NEP II has emerged alongside NEP in the degradation of Aβ peptide. NEP II has now been shown to contribute significantly to Aβ degradation both in vitro and in vivo in gene knockout and transgenic studies (Fig. 1).9 ECE-1, like NEP, is a membrane-bound metalloprotease, which is found at the cell surface and is also present in the trans-Golgi network and endosomal system. The early history of ECE-1 discovery is summarized in Ref. 30. ECE-1 and NEP are both inhibited by phosphoramidon, but ECE-1 is relatively insensitive to the action of thiorphan. ECE-1 is undoubtedly the principal enzyme generating endothelin-1 from its inactive precursor, big endothelin. However, it is capable of hydrolyzing a wide range of biologically active peptides with a specificity broadly similar to that of NEP, although peptides smaller than hexapeptides (e.g. enkephalins) are hydrolyzed poorly, if at all. Among peptides hydrolyzed by ECE-1 are angiotensin I, atrial natriuretic peptide, bradykinin, calcitonin, calcitonin gene–related peptide (CGRP), neurotensin, somatostatin peptides, and substance P. A unique role for ECE-1 in peptide metabolism is its ability to degrade neuropeptides (e.g. CGRP, tachykinins, somatostatin peptides) within endosomes to control receptor recycling.25 This property is consistent with an acidic pH optimum for the enzyme. ECE-1 can also hydrolyze the amyloid peptides Aβ1–40 and Aβ1–42 at three major cleavage sites (Fig. 1) and seems to have a physiological role in Aβ metabolism as shown in mice deficient in the peptidase.7 In addition to its cardiovascular role ECE-1 is important developmentally, especially for neural crest development, again through its ability to generate endothelin-1. ECE-1 has also been implicated in numerous cancers: thyroid, ovarian, endometrial, lung, colon, and especially prostate. Indeed, it has been shown that siRNA-mediated knockdown of ECE-1 reduces prostate cancer cell invasion in culture.14
THE ACE FAMILY (ACE AND ACE-2) As described in the angiotensin chapter in the Cardiovascular Peptides section of the book, ACE was identified >50 years ago by Leonard Skeggs and colleagues as the enzyme responsible for converting angiotensin I into the vasoconstrictor peptide, angiotensin II (termed hypertensins I and II at that time). In parallel, it inactivates the vasodilator and inflammatory peptide bradykinin, which is the most favorable peptide substrate for ACE, being hydrolyzed approximately 25 times more efficiently than is angiotensin I. Hence, the inhibition of ACE has a powerful antihypertensive effect, and the enzyme has therefore been a major cardiovascular target for many years leading to the development of numerous potent and specific inhibitors (e.g. captopril, lisinopril). The catalytic activity of ACE is predominantly as a “peptidyl dipeptidase” or carboxydipeptidase, removing
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dipeptides from the C-terminus of a susceptible peptide substrate. However, ACE can act as an endopeptidase in the hydrolysis of some peptides (e.g. both substance P and luteinizing hormone-releasing hormone), although far less efficiently. ACE is a type I integral membrane protein and zinc metallopeptidase with the typical HEXXH zinc-binding motif, which is expressed on the surface of both endothelial and epithelial cells. Mammalian ACE exists as two distinct forms, arising from the use of alternative promoters. The simplest is the testis-specific form of ACE, which is essential for male fertility, and which carries a single zincbinding and catalytic domain. Elsewhere in the body, the somatic form of ACE has arisen by gene duplication and has two homologous but not identical active sites (N-domain and C-domain sites) with an overall Mr of 150–180 kDa. Hence the two sites differ somewhat in substrate specificity and inhibitor sensitivity. For example, the hemoregulatory peptide Ac-Ser-Asp-Lys-Pro (AcSDKP) and angiotensin-(1-7) are preferred substrates of the N-domain catalytic site whereas angiotensin I is mainly metabolized by the C-domain. A soluble form of ACE exists in serum and other body fluids and is derived from the membrane form of ACE by proteolytic shedding (for a review on ACE see Ref. 13). Elucidation of the structure of ACE eluded investigators for many years because of the heavy glycosylation of the glycoprotein hindering crystallization. It was not until 2003 that the first X-ray structures of ACE were determined for the testicular form with and without inhibitor (lisinopril) bound.19 Analysis of the structures has revealed that ACE most closely resembles a neurotensin-degrading zinc endopeptidase known as neurolysin rather than NEP, or carboxypeptidase A, on whose structure the design of ACE inhibitors was originally based. Subsequent structure determination of the N-domain1 has allowed comparisons of the two active sites and aided the development of domain- selective inhibitors. An unexpected substrate identified for ACE has been the Aβ peptide with the enzyme apparently cleaving the Asp7Ser8 bond of Aβ1–40,10 although this did not seem to fit with the known specificity of ACE. Subsequently, other sites of cleavage have been identified within Aβ, including the ability of ACE to convert Aβ1–42 to Aβ1–40, which is more consistent with its carboxydipeptidase specificity35 (Fig. 1). This cleavage would be beneficial in removing the more toxic Aβ1–42 species. This study also showed that treatment of transgenic mice that develop Alzheimer-like pathology with an ACE inhibitor significantly increased the deposition of brain Aβ1–42. However, there is no epidemiological evidence that human patients on ACE inhibitor therapy are any more prone to the development of Alzheimer’s disease. The only known functional homolog ofACE in humans was discovered in 2000 through genomics-based approaches.29 ACE2, like ACE, is a transmembrane glycoprotein
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but with only a single catalytic site equivalent to the C-domain of ACE. Although ACE2 is highly similar in sequence to ACE around the active site, it displays a somewhat different substrate specificity and is not sensitive to inhibition by ACE inhibitors. ACE2 behaves as a simple carboxypeptidase removing a single C-terminal amino acid from a susceptible substrate. Hence, angiotensin I is converted to angiotensin-(1–9) by ACE2 rather than to angiotensin II (angiotensin-(1–8)). However, ACE2 acts much more efficiently on angiotensin II converting it to the vasodilatory angiotensin-(1–7) and hence serving to counterbalance the actions of ACE (see the section on Cardiovascular Peptides in this Handbook). Angiotensin-(1–7) can also be formed directly from angiotensin I through the action of NEP. Of 126 biologically active peptides tested as ACE2 substrates, only 11 were hydrolyzed by the enzyme, the most efficient being angiotensin II, apelin-13, and dynorphin A-(1-13).32 Apelin-13 may be a physiologically relevant ACE2 substrate. A number of potent and specific ACE2 inhibitors have been reported, of which the first were the non-peptide inhibitor, MLN-4760, and a peptide identified from phage library screening, DX600.
INSULIN-DEGRADING ENZYME (INSULYSIN, IDE) Insulin-degrading enzyme (IDE, insulysin) is another zinc endopeptidase involved in the degradation of biologically active peptides although it differs substantially from the ACE and NEP families in location and catalytic mechanism. IDE was originally isolated as an enzyme regulating plasma levels of insulin, hence its name, and is therefore seen as of potential significance as a therapeutic target in diabetes. In addition to insulin, IDE can hydrolyze a number of biologically active peptides including insulin-like growth factor II, tumor growth factor-α, and atrial natriuretic peptide.27 Another important peptide substrate of IDE is the amyloid precursor protein intracellular domain, AICD, which acts as a transcriptional regulator controlling the expression of NEP and some other genes as discussed above.3 IDE also degrades somatostatin, a neuropeptide that declines in aging and Alzheimer’s disease.5 Unlike the other peptidases described above, IDE is mainly a soluble cytoplasmic enzyme although it also exists as a secreted form.34 There is also a splice-variant of IDE targeted to mitochondria. The mechanisms responsible for the subcellular targeting and secretion of IDE remain unresolved. The secreted form of IDE seems to play a role in the metabolism of Aβ peptide, was the first such peptidase identified and remains as a well validated Aβ-degrading enzyme from both in vitro and in vivo studies, including the use of transgenic approaches.18 Hence, IDE is also seen as a potential target for upregulation in the treatment of Alzheimer’s disease.
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IDE differs from ACE and NEP in possessing an inverted zinc-binding motif (His-Xaa-Xaa-Glu-His) and shows unusual allosteric behavior with small peptides. The structural solution of IDE27 has shown the active site to be enclosed within a flexible clamshell-like structure and ATP is required to facilitate the opening of the catalytic chamber. Until recently, there were no potent and specific IDE inhibitors to facilitate studies of the role of the enzyme in the metabolism of biologically active peptides but rationally designed inhibitors based on the structural information have now been produced which confirm the role of IDE in regulating insulin signaling.15 Such inhibitors may well have potential for the treatment of type 2 diabetes.
PROLINE-DIRECTED PEPTIDASES Numerous biologically active peptides are partially protected from exopeptidase degradation by the presence of highly conserved prolyl residues especially at or near to the peptide N- and C-termini. Hence, very specific peptidases are required to initiate their metabolism because the conformational constraint imposed by the cyclic imino structure of proline hinders proteolysis.31 The Xaa-Pro sequence is present at the N-terminus of >100 biologically active peptides or proteins, including peptide hormones, neurotransmitters, immunopeptides, and kinins. The bestcharacterized prolyl peptidase is the cell-surface serine peptidase, dipeptidyl peptidase IV (DPP IV) which, as its name suggests, hydrolyzes a dipeptide from the N-terminus of peptides provided that the second residue is a proline (or less favorably an alanine or serine). DPP IV is therefore one of only a few enzymes that have specificity directed toward prolyl residues. DPP IV was originally described as a rat liver enzyme releasing naphthylamine from Gly-Pro2-naphthylamide and postulated to be involved in collagen metabolism. However, it was unable to cleave collagen and its physiological role remained unclear. Subsequent studies showed it to be an abundant peptidase of the renal and intestinal brush borders although it is present on most cell types. It is identical with the T cell-activating antigen (CD26) and, on T cells, it associates with the immunodeficiency-related protein adenosine deaminase via a cluster of charged amino acid residues. It is a type II membrane protein with only a short cytoplasmic tail (six residues) with the bulk of the enzyme facing the extracellular space where it can participate in the metabolism of a diverse range of biologically active peptides. DPP IV is also found as a soluble protein in serum whose levels are altered in cancer and some other diseases. The soluble form is most probably shed from the membrane-bound form although the protease responsible for this process is unknown. The enzyme is discussed in further detail, especially in relation to the immune response in this Handbook (see the chapter in Immune/Inflammatory Peptides section).
DPP IV is a typical serine peptidase, and a member of the “prolyl oligopeptidase” family, possessing the catalytic triad residues Ser/Asp/His (in rat Ser631/Asp709/His741) and the enzyme functions as a homodimer and can also form functional tetramers. The crystal structure reveals that each subunit consists of two domains, an α/β-hydrolase domain and an eight-bladed β-propeller domain23 with the catalytic site located in a cavity formed between the two domains both of which participate in substrate/inhibitor binding. The human DPP IV gene consists of 26 exons and, unusually among serine proteases, the nucleotides that encode the active site sequence (Gly-Xaa-Ser-Xaa-Gly) are split between two exons. DPP IV may play a digestive role in the digestion and uptake of prolyl-containing peptides. Most interest has focused, however, on the role of the enzyme in metabolizing biologically active peptides. In this capacity, it can either generate active peptide from a precursor, convert one active peptide to another with different receptor specificity (e.g. peptide YY or neuropeptide Y), or inactivate peptides. It is the latter role that has allowed the development of DPP IV inhibitors as the first new therapeutic class of orally active antihyperglycemic drug for type 2 diabetes mellitus for a long time through the ability of DPP IV to inactivate incretin hormones (glucagon-like peptide-1; glucosedependent insulinotropic peptide).6 Almost 50 biologically active peptides have been identified as substrates for DPP IV most of which have the N-terminal Xaa-Pro sequence, but some have Xaa-Ala or less commonly Xaa-Ser. Another cell-surface peptidase capable of hydrolyzing peptides with a penultimate N-terminal prolyl residue is aminopeptidase P (APP2 to distinguish it from a cytosolic prolyl aminopeptidase, APP1). The enzyme removes the N-terminal residue leaving a free prolyl residue at the N-terminus. Examples of good substrates include bradykinin and neuropeptide Y. The specific inhibitor, apstatin, can reduce blood pressure by potentiating the action of bradykinin. A prolyl residue in the third position of a substrate renders the peptide resistant to the action of DPP IV (e.g. bradykinin, Arg-Pro-Pro-…). APP2 is a zinc peptidase and is unusual among cell-surface peptidases in being anchored to the cell surface via a glycosyl-phosphatidylinositol membrane anchor. Unlike DPP IV and APP2, which are exopeptidases, another prolyl peptidase functions as an endopeptidase and can hydrolyze a wide range of small (<30 amino acids) biologically active peptides, at least in vivo. This is a serine peptidase originally known as postproline cleaving enzyme or proline endopeptidase, which cleaves peptides on the carboxy-terminal side of prolyl residues. It is now known as prolyl oligopeptidase (POP) and was originally implicated in oxytocin metabolism. Other potential substrates include substance P, angiotensins, bradykinin, β-endorphins, neurotensin, and thyrotropin releasing hormone (TRH). To evaluate the role of POP, numerous potent and relatively specific
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small molecule inhibitors have been developed, which exhibit a number of interesting physiological properties, for example, in enhancing memory, which is perhaps consistent with vasopressin also being a substrate for the enzyme. There are many reports that POP may have a role in psychiatric and neurodegenerative diseases although it does not seem to play a role in Aβ metabolism.22 The evidence for a physiological role for POP in peptide degradation is, in most cases, lacking (see Ref. 8 for review) and a topological problem is that POP is a cytosolic enzyme and therefore unlikely to interact with circulating peptides. Another related proline-specific peptidase (acylpeptide hydrolase), which can be secreted from neuronal cells, may however contribute to Aβ metabolism. Two specific areas where POP may also participate are in inositol phosphate signaling, and in angiogenesis and hence progression of malignancies. POP is able to enhance inositol phosphate signaling and cell sensitivity to lithium acting via the enzyme inositol polyphosphate phosphatase and hence regulate gene expression. POP is also able to cleave thymosin-β4 to generate the hemoregulatory peptide AcSDKP, itself a substrate for the N-domain of ACE (see above). Both AcSDKP and POP activity are substantially increased in certain malignancies.
CONCLUSIONS Unlike the high specificity of ligand–receptor interactions, peptidases that degrade biologically active peptides tend to have multiple substrates, and hence, relatively few are needed to control the circulating levels of the multiplicity of such peptides described in this Handbook. They mostly exist as cell-surface ectoenzymes, or in a soluble, secreted form. The properties of some of the key ones, especially of the neprilysin family, have been described in this chapter, as well as methods for manipulating their activity. Structures are now available for most of these peptidases allowing rational design of potent and specific inhibitors. The promiscuity of these peptidases might suggest that inhibiting their activity could have undesirable side effects. However, in practice, this does not seem to be the case, as best exemplified by ACE inhibitors. NEP inhibitors also have a role to play in many conditions, although their chronic use could potentially lead to a build-up of brain Aβ peptide. There still remain numerous orphan peptidases, however, for which their physiological peptide substrates remain to be identified. Comparative in vivo peptidomic approaches in animals deficient in a particular peptidase now provide a powerful tool to categorize physiological substrate profiles for individual enzymes. We can expect to have a much clearer picture of the roles of peptidases in the metabolism of biologically active peptides in the next few years.
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REFERENCES 1. Anthony CS, Corradi HR, Schwager SL, Redelinghuys P, Georgiadis D, Dive V, et al. The N domain of human angiotensin-I-converting enzyme: the role of N-glycosylation and the crystal structure in complex with an N domain-specific phosphinic inhibitor, RXP407. J Biol Chem 2010;285:35685–93. 2. Belyaev ND, Kellett KA, Beckett C, Makova NZ, Revett TJ, Nalivaeva NN, et al. The transcriptionally active amyloid precursor protein (APP) intracellular domain is preferentially produced from the 695 isoform of APP in a β-secretase-dependent pathway. J Biol Chem 2010;285:41443–54. 3. Belyaev ND, Nalivaeva NN, Makova NZ, Turner AJ. Neprilysin gene expression requires binding of the amyloid precursor protein intracellular domain to its promoter: implications for Alzheimer disease. EMBO Rep 2009;10:94–100. 4. Bland ND, Pinney JW, Thomas JE, Turner AJ, Isaac RE. Bioinformatic analysis of the neprilysin (M13) family of peptidases reveals complex evolutionary and functional relationships. BMC Evol Biol 2008;8:16. 5. Ciaccio C, Tundo GR, Grasso G, Spoto G, Marasco D, Ruvo M, et al. Somatostatin: a novel substrate and a modulator of insulin-degrading enzyme activity. J Mol Biol 2009;385:1556–67. 6. Demuth H-U, McIntosh CH, Pederson RA. Type 2 diabetes-therapy with dipeptidyl peptidase IV inhibitors. Biochim Biophys Acta 2005; 1751:33–44. 7. Eckman EA, Watson M, Marlow L, Sambamurti K, Eckman CB. Alzheimer’s disease β-amyloid peptide is increased in mice deficient in endothelin-converting enzyme. J Biol Chem 2003;278:2081–4. 8. Garcia-Horsman JA, Männistö PT, Venäläinen JI. On the role of prolyl oligopeptidase in health and disease. Neuropeptides 2007;41:1–24. 9. Hafez D, Huang JY, Huynh AM, Valtierra S, Rockenstein E, Bruno AM, et al. Neprilysin-2 is an important β-amyloid degrading enzyme. Am J Pathol 2011;178:306–12. 10. Hu J, Igarashi A, Kamata M, Nakagawa H. Angiotensin-converting enzyme degrades Alzheimer amyloid β-peptide (Aβ); retards Aβ aggregation, deposition, fibril formation; and inhibits cytotoxicity. J Biol Chem 2001;276:47863–8. 11. Ikeda K, Emoto N, Raharjo SB, Nurhantari Y, Saiki K, Yokoyama M, et al. Molecular identification and characterisation of a novel membrane-bound metalloprotease, the soluble form of which hydrolyzes a variety of vasoactive peptides. J Biol Chem 1999;274: 32469–77. 12. Iwata N, Tsubuki S, Takaki Y, Watanabe K, Sekiguchi M, Hosoki E, et al. Identification of the major Aβ1–42 degrading catabolic pathway in brain parenchyma: Suppression leads to biochemical and pathological deposition. Nature Med 2000;6:143–50. 13. Lambert DW, Clarke NE, Turner AJ. Not just angiotensinases: new roles for the angiotensin-converting enzymes. Cell Mol Life Sci 2010;67:89–98. 14. Lambert LA, Whyteside AR, Turner AJ, Usmani BA. Isoforms of endothelin-converting enzyme-1 (ECE-1) have opposing effects on prostate cancer cell invasion. Br J Cancer 2008;99:1114–20. 15. Leissring MA, Malito E, Hedouin S, Reinstatler L, Sahara T, AbdulHay SO, et al. Designed inhibitors of insulin-degrading enzyme regulate the catabolism and activity of insulin. PLoS One 2010:5:e10504. 16. Marr RA, Rockenstein E, Mukherjee A, Kindy MS, Hersh LB, Gage FH, et al. Neprilysin gene transfer reduces human amyloid pathology in transgenic mice. J Neurosci 2003;23:1992–6.
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17. Miller L, Hou X, Gagnidze K, Sweedler JV, Sweedler JV, Wetsel WC, et al. Mice deficient in endothelin-converting enzyme-2 exhibit abnormal responses to morphine and altered peptide levels in the spinal cord. J Neurochem 2011;119:1074–85. 18. Nalivaeva NN, Fisk LR, Belyaev ND, Turner AJ. Amyloid-degrading enzymes as therapeutic targets in Alzheimer’s disease. Curr Alzheimer Res 2008;5:212–24. 19. Natesh R, Schwager SL, Sturrock ED, Acharya KR. Crystal structure of the human angiotensin-converting enzyme–lisinopril complex. Nature 2003;421:551–4. 20. Oefner C, D’Arcy A, Hennig M, Winkler FK, Dale GE. Structure of human neutral endopeptidase (neprilysin) complexed with phosphoramidon. J Mol Biol 2000;296:341–9. 21. Pardossi-Piquard R, Petit A, Kawarai T, Sunyach C, Alves da Costa C, Vincent B, et al. Presenilin-dependent transcriptional control of the Aβ-degrading enzyme neprilysin by intracellular domains of βAPP and APLP. Neuron 2005;46:541–54. 22. Petit A, Barelli H, Morain P, Checler F. Novel proline endopeptidase inhibitors do not modify Aβ40/42 formation and degradation by human cells expressing wild-type and Swedish mutated β-amyloid precursor protein. Br J Pharmacol 2000;130:1613–7. 23. Rasmussen HB, Branner S, Wiberg FC, Wagtmann N. Crystal structure of human dipeptidyl peptidase IV/CD26 in complex with a substrate analog. Nat Struct Biol 2003;10:19–25. 24. Rawlings ND, Barrett AJ, Bateman A. Asparagine peptide lyases: a seventh catalytic type of proteolytic enzymes. J Biol Chem 2011; 286:38321–8. 25. Roosterman D, Cottrell GS, Padilla BE, Muller L, Eckman CB, Bunnett NW, et al. Endothelin-converting enzyme 1 degrades neuropeptides in endosomes to control receptor recycling. Proc Natl Acad Sci U S A 2007;104:11838–43.
26 Rougeot C, Messaoudi M, Hermitte V, Rigault AG, Blisnick T, Dugave C, et al. Sialorphin, a natural inhibitor of rat membrane-bound neutral endopeptidase that displays analgesic activity. Proc Natl Acad Sci U S A 2003;100:8549–54. 27. Shen Y, Joachimiak A, Rosner MR, Tang WJ. Structures of human insulin-degrading enzyme reveal a new substrate recognition mechanism. Nature 2006;443:870–4. 28. Standeven KF, Hess K, Carter AM, Rice GI, Cordell PA, Balmforth AJ, et al. Neprilysin, obesity and the metabolic syndrome. Int J Obes (Lond) 2011;8:1031–40. 29. Tipnis S R, Hooper N M, Hyde R, Karran E , Christie G, Turner A J. A human homolog of angiotensin-converting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase. J Biol Chem 2000;275:33238–43. 30. Turner AJ, Murphy LJ. Molecular pharmacology of endothelin converting enzymes. Biochem Pharmacol 1996;51:91–102. 31. Vanhoof G, Goossens F, De Meester I, Hendriks D, Scharpé S. Proline motifs in peptides and their biological processing. FASEB J 1995;9:736–44. 32. Vickers C, Hales P, Kaushik V, Dick L, Gavin J, Tang J, et al. Hydrolysis of biological peptides by human angiotensin-converting enzymerelated carboxypeptidase. J Biol Chem 2002;277:14838–43. 33. Whyteside AR, Turner AJ. Human neprilysin-2 (NEP2) and NEP display distinct subcellular localisations and substrate preferences. FEBS Lett 2008;582:2382–6. 34. Zhao J, Li L, Leissring MA. Insulin-degrading enzyme is exported via an unconventional protein secretion pathway. Mol Neurodegener 2009;4:4. 35. Zou K, Yamaguchi H, Akatsu H, Sakamoto T, Ko M, Mizoguchi K, et al. Angiotensin-converting enzyme converts amyloid β-protein 1-42 (Aβ1-42) to (Aβ1-40), and its inhibition enhances brain Aβ deposition. J Neurosci 2007;27:8628–35.
Peptide Degradation (Neprilysin and Other Regulatory Peptidases) Anthony J. Turner and Natalia N. Nalivaeva
ABSTRACT Turning off peptide signaling is principally mediated by a group of cell-surface peptidases whose active sites face the extracellular space (ectopeptidases). Two groups of metallopeptidases are the best characterized as peptide degrading enzymes: the neutral endopeptidase or neprilysin (NEP) family and the angiotensinconverting enzyme (ACE) family. NEP and its homologs are important in degrading both brain peptides (e.g. enkephalins, tachykinins) and cardioactive peptides (e.g. atrial natriuretic peptides). They also metabolize the Alzheimer amyloid β-peptide (Aβ). In the cardiovascular system, ACE and ACE2 act to counterbalance each other in controlling angiotensin peptide levels and may also participate in the metabolism of other peptides. Prolyldirected peptidases such as dipeptidyl peptidase IV and prolyl oligopeptidase play highly specific roles in peptide metabolism. This chapter summarizes current developments in our understanding of the roles and the regulation of these key mediators of peptide signaling, their roles in disease and as potential therapeutic targets.
INTRODUCTION Proteolysis, which regulates the production and removal of biologically active peptides, represents a key biological process underpinning events from fertilization, through development to cell death. The ubiquity and diversity of peptidases are indicated by the existence of some 28,000 specific peptidases and their homologs characterized in >6000 known organisms (http://merops.sanger.ac.uk/). In the human genome of approximately 24,000 proteincoding genes, 686 genes of putative peptidases are known alongside 423 of noncatalytic homologs. Many of these are orphan peptidases for which the biologically active peptide substrates have not yet been identified. Such a profusion of proteolytic events requires specifically controlled regulation. Hence, although genes for peptidases constitute about 2.8% of the human genome, a significantly higher number (7.4%) of genes encode protein inhibitors of peptidases. Handbook of Biologically Active Peptides. http://dx.doi.org/10.1016/B978-0-12-385095-9.00241-4 Copyright © 2013 Elsevier Inc. All rights reserved.
The term “peptidase” refers to enzymes that hydrolyze peptide bonds and, as such, all known biologically active peptides represent potential substrates for these enzymes. According to the location of the peptide bond cleaved by the peptidases, they are conventionally subdivided into “endopeptidases” that cleave the bond within the polypeptide chain, or exopeptidases that release amino acids from the Nor C-terminus of the substrate. However, some peptidases, e.g. angiotensin-converting enzyme (ACE), can fulfill both endopeptidase and exopeptidase functions. The physiological role of peptidases is very diverse. In general, peptidases do not cleave their substrates at random but participate in very well-controlled, specific processing of peptide molecules. The term “limited proteolysis” was originally introduced by Linderstrøm-Lang to distinguish the restricted specificity of certain peptidases, particularly those involved in posttranslational modification of proprotein precursors, from the random proteolysis accompanying protein digestion and degradation. All peptidases have been divided into six classes (metallo-, serine, cysteine, aspartic, threonine, and glutamate peptidases) with cysteine-, serine-, and metallopeptidases representing the major categories. A seventh class involving asparagine peptidases has recently been reported.24 Aberrant functioning of these peptidase genes is implicated in such diverse pathologies as inflammatory and cardiovascular diseases, cancer, and neurodegeneration. Understanding the normal functional roles of peptidases and their regulation and restoration in disease is hence important for the design of novel and selective therapeutics. This chapter is focused primarily on two major families of zinc peptidases participating in the specific degradation of biologically active peptides. The prototypes for these peptidases are neprilysin (neutral endopeptidase, NEP) and ACE. The roles of peptidases directed specifically toward prolyl residues are also reviewed. Table 1 summarizes the diversity of peptide substrates hydrolyzed by this cohort of enzymes. 1757
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TABLE 1 Typical Biologically Active Peptides Degraded by Peptidases Discussed in this Chapter
NEUTRAL ENDOPEPTIDASE (NEPRILYSIN) FAMILY
Substrate
Peptidase
Name and History
Adrenomedullin
NEP
Amyloid β-peptide
NEP, NEP II, ECE-1, IDE, ACE
Amyloid precursor protein intracellular domain (AICD)
IDE
Angiotensin I
ACE, NEP, ACE2, POP
Angiotensin II
ACE2, NEP, POP
Apelin
ACE2
Atrial natriuretic peptide
NEP, IDE
β-Endorphin
IDE, POP
Big endothelin-1
ECE-1, ECE-2, NEP, NEP II
Bradykinin
ACE, ECE-1, ECE-2, IDE, NEP, APP2, POP
Brain natriuretic peptide
IDE
Cholecystokinin-8
ACE, NEP
Corticotropin
ACE, NEP
Dynorphins
NEP, IDE, ECE-2
Endomorphin
DPP IV, NEP
Enkephalins
NEP, ACE
Endothelin-1
NEP
Gastrin
NEP
Ghrelin
ACE2
Glucagon-like peptide-1
DPP IV
Neprilysin activity was first characterized from rat kidney brush border membranes as an enzyme hydrolyzing insulin B chain at neutral pH, and hence termed “neutral endopeptidase” (NEP), although it plays no role in the physiological insulin inactivation, which is mediated by another peptidase, insulin-degrading enzyme (IDE). NEP is a zinc metallopeptidase resembling the bacterial enzyme thermolysin, including its sensitivity to the inhibitor, phosphoramidon. NEP has a diverse range of biological roles and hence has been rediscovered in different proteolytic contexts. For example, after the discovery of the enkephalins, NEP was rediscovered in brain as a membrane enzyme hydrolyzing these opioid peptides and was termed enkephalinase. It is also the principal enzyme inactivating many other neuropeptides (e.g. substance P) and cardioactive peptides (atrial natriuretic peptide (ANP) family) and hence represents the first general “neuropeptidase.” NEP plays an important role in the immune system where it is referred to as the common acute lymphoblastic leukemia antigen (CALLA or CD10), although its substrate(s) have not been identified. NEP also contributes to the metabolism of the Alzheimer amyloid β-peptide (Aβ) in the brain.12 The human genome contains at least seven NEP-like enzymes, including the biosynthetic enzyme endothelin-converting enzyme (ECE-1). The family is even more abundant in Drosophila (24 predicted members) and Caenorhabditis elegans (22 members).4 NEP is also known as MME, for membrane metalloendopeptidase, in the Human Gene Nomenclature database.
Gonadoliberin-1
ACE, POP
Insulin
IDE
Insulin-like growth factor I
DPPIV
Insulin-like growth factor II
IDE
Luliberin
NEP, ACE
Neuropeptide Y
DPPIV, NEP, APP2
Neurotensin
ACE, ACE-2, ECE-1, ECE-2, NEP, POP
Oxytocin
NEP, POP
Somatostatin
NEP, ECE-1, IDE
Substance P
NEP, ACE, DPPIV, ECE-1, ECE2, POP
VIP
NEP, ACE, IDE
Properties NEP is exclusively localized to the plasma membrane (type II integral membrane protein) with the active site facing the extracellular space (ectoenzyme) where it is poised to inactivate circulating peptides. It is a glycoprotein of approximately Mr 90,000. The enzyme is classified as a “gluzincin” peptidase with the typical His-Glu-Xaa-Xaa-His (HEXXH) zinc-binding motif. The glutamate in this motif (Glu584 in the human enzyme) is essential for catalysis and an additional glutamate (Glu646) constitutes the third zinc ligand. NEP is an archetypal peptidase rather than proteinase because it generally only hydrolyzes peptides up to about 50 amino acids long. The 3D structure of the NEP catalytic domain in complex with its inhibitor phosphoramidon has been deduced aiding the design of novel inhibitors and providing a better understanding of its substrate specificity and catalytic mechanism.20 The active site region is restricted in size preventing access of large peptides and hence explaining its oligopeptidase nature.
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In general, NEP hydrolyzes peptidases on the aminoterminal side of hydrophobic residues, e.g. the Gly3-Phe4 bond of enkephalins, giving it a very broad specificity for known biologically active peptides although its physiologically validated substrates are more restricted. In addition to opioids, tachykinins, and the ANP family, it inactivates endothelins, bradykinin, and somatostatin. Other NEP substrates include glucagon, the vasoactive intestinal peptide family and adrenomedullin. NEP may also regulate bone metabolism through its presence on the surface of human osteoblasts and its ability to metabolize osteogenic peptides such as calcitonin and calcitonin gene-related peptide. Because inhibition of NEP can, in theory, potentiate the action of any of its peptide substrates, much emphasis has been directed toward developing novel and potent NEP inhibitors, with thiorphan originally being synthesized as an inhibitor of its enkephalin-hydrolyzing activity, and this is still the most commonly used NEP inhibitor. Many others have subsequently been developed as potential therapeutics in cardiovascular disease by prolonging the actions of ANP. The combined actions of NEP and ACE inhibitors have improved benefits over ACE inhibitors alone. The prodrug of thiorphan, acetorphan (racecadotril), is an effective antidiarrheal agent through its potentiation of the antisecretory effects of enkephalins in the intestine. However, the NEP substrate that has attracted by far the most interest over the last decade has been the Alzheimer amyloid-β peptide (Aβ).
Distribution/Localization NEP and its homologs are widely distributed enzymes in the animal kingdom reflecting the general need for terminating the action of circulating regulatory peptides. Many invertebrate peptides are inactivated by NEP-like peptidases where it often plays a role in development and reproduction. In C. elegans, NEP seems to be an effector of locomotion. In mammals, the enzyme is particularly abundant in the kidney where its expression levels in brush border membranes are several orders of magnitude higher than in brain synaptic membranes. It is also highly expressed in the reproductive system (testis, ovary), although its functional role is unknown. Elsewhere NEP, although widespread, is expressed at much lower levels. In the brain, NEP inactivates peptide neurotransmitters, especially opioid and tachykinin peptides, after their release into the synapse. Given the role of these peptides in pain mechanisms, NEP inhibitors have antinociceptive actions. However, most attention has been focused on the role that NEP plays, alongside other peptidases such as ECE, insulin-degrading enzyme, and possibly ACE, in detoxifying the Aβ peptide that predisposes to Alzheimer’s disease (Fig. 1). The susceptibility of Aβ is perhaps not surprising given the
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FIGURE 1 Main cleavage sites of amyloid-β peptide by peptidases. The Aβ-(1–42) sequence is shown in black with the flanking amino acids of the amyloid precursor protein (APP) shown in gray. The principal peptidases involved in Aβ peptide metabolism are indicated in black and the other activities in gray. N, neprilysin; N2, neprilysin-2; A, angiotensin-converting enzyme; E, endothelin-converting enzyme-1; I, insulin-degrading enzyme. Letters in circles indicate peptidases that cleave Aβ at the same site.
specificity of NEP, as it is a highly hydrophobic peptide. Indeed, under normal conditions there is considerable evidence that Aβ itself may be a regulatory peptide, and it is only in pathological conditions when it accumulates excessively that it becomes toxic. This process is enhanced in aging because there is a well-documented age-related decline in NEP expression. The levels of brain Aβ are hence increased in NEP-deficient mice. NEP expression is also diminished after periods of ischemia or hypoxia. Infusion of an NEP inhibitor into rat brain leads to a significant accumulation of the more neurotoxic Aβ42 peptide, and hence, upregulation of NEP has been proposed as a potential therapeutic strategy in Alzheimer’s disease.12,18 NEP gene transfer can decrease amyloid-like pathology and cognitive decline in transgenic mice.16 Given the potential of NEP inhibitors to increase brain Aβ levels and hence to induce Alzheimer’s disease-like symptoms, interest in the development of novel NEP inhibitors as potential cardiovascular therapeutics has considerably diminished. In the brain, NEP is mainly localized on neuronal cells, especially in the striatonigral pathway consistent with its role in turning off opioid peptide and tachykinin signaling, and it is also located in the hippocampus, where it functions to inactivate somatostatin and in cortical regions where it may also act to degrade Aβ . The enzyme is also present on activated astrocytes and on Schwann cells in the peripheral nervous system where it may function in nerve development and/or regeneration. Unlike many proteases, NEP is not synthesized in an inactive proenzyme form and is not generally regulated by endogenous inhibitors although sialorphin, the peptide product of the Vcsa1 gene, has been reported to inhibit the enzyme in vivo.26 In most tissues, much of the NEP expression is constitutive but recent demonstration of epigenetic regulation of NEP has opened up the possibility of new approaches to control NEP levels in brain.3,21 The Aβ peptide is formed by proteolytic processing of the amyloid precursor protein (APP). Another metabolite of APP is its intracellular domain (AICD), a 50–59 amino acid peptide, which can be translocated to the nucleus where it can bind to the NEP promoter (and promoters of some other genes) displacing
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histone deacetylases which act as transcriptional repressors, and hence upregulating gene expression.3 This effect seems to be mediated preferentially by the neuronal isoform of APP (APP695).2 Through a related mechanism, histone deacetylase inhibitors such as trichostatin A and valproic acid, can enhance NEP expression in vitro and in vivo and may therefore have therapeutic value in Alzheimer’s disease.3 NEP is an important diagnostic marker in certain forms of childhood leukemia and has also been implicated in cancer progression, particularly of the prostate. NEP gene transcription is androgen-regulated in androgen-responsive tissues. Hence, the downregulation of NEP through hypermethylation of the promoter contributes to androgen- independent progression of prostate cancer because of a reduced ability to metabolize mitogenic peptides, particularly endothelin-1. NEP may also be important in the development of obesity and its expression levels associate with insulin resistance and the metabolic syndrome.28
Related Peptidases Seven NEP-like proteins have been identified in the human genome.4 Most of these are of unknown biological function, and their substrate specificity is unclear. The most close in similarity to NEP is a peptidase referred to as NEP II in the rat, “soluble secreted endopeptidase” (SEP) or NEP-like 1 (NL1) in the mouse, or MMEL2 (human). The best-characterized NEP homologs are the endothelinconverting enzymes (ECE-1, ECE-2). ECE-1 participates in the conversion of big endothelin to the vasoactive peptide, endothelin-1 (see the chapter on Cardiovascular Peptides and below). ECE-2 probably plays a role as a processing enzyme in the biosynthesis of regulatory peptides and can process such peptides at non-classical processing sites. Mice deficient in ECE-2 have learning and memory deficits and show abnormal responses to morphine and have altered peptide levels in the spinal cord.17 NEP II was first identified in mice deficient in ECE-1 as a potential endothelin-processing enzyme.11 Although it is the most similar peptidase to NEP, it has many distinct properties; for example, it occurs as both a soluble, secreted enzyme and an alternatively spliced form retained in the endoplasmic reticulum. NEP II is particularly localized to neuronal populations in the brain and spinal cord and is also abundant in reproductive tissue—male mice deficient in NEP II show reduced fertility. Originally, NEP II was reported to have a broad repertoire of substrates, similar to those for NEP itself,12 although its physiological role apart from male fertility, remains unresolved. However, rodent NEP II exhibits a much wider substrate specificity than the human enzyme.33 For example, only substance P and angiotensin I are hydrolyzed at comparable rates by human NEP and NEP II.33 The inhibitor thiorphan is also more potent
toward NEP than NEP II. A physiological role for NEP II has emerged alongside NEP in the degradation of Aβ peptide. NEP II has now been shown to contribute significantly to Aβ degradation both in vitro and in vivo in gene knockout and transgenic studies (Fig. 1).9 ECE-1, like NEP, is a membrane-bound metalloprotease, which is found at the cell surface and is also present in the trans-Golgi network and endosomal system. The early history of ECE-1 discovery is summarized in Ref. 30. ECE-1 and NEP are both inhibited by phosphoramidon, but ECE-1 is relatively insensitive to the action of thiorphan. ECE-1 is undoubtedly the principal enzyme generating endothelin-1 from its inactive precursor, big endothelin. However, it is capable of hydrolyzing a wide range of biologically active peptides with a specificity broadly similar to that of NEP, although peptides smaller than hexapeptides (e.g. enkephalins) are hydrolyzed poorly, if at all. Among peptides hydrolyzed by ECE-1 are angiotensin I, atrial natriuretic peptide, bradykinin, calcitonin, calcitonin gene–related peptide (CGRP), neurotensin, somatostatin peptides, and substance P. A unique role for ECE-1 in peptide metabolism is its ability to degrade neuropeptides (e.g. CGRP, tachykinins, somatostatin peptides) within endosomes to control receptor recycling.25 This property is consistent with an acidic pH optimum for the enzyme. ECE-1 can also hydrolyze the amyloid peptides Aβ1–40 and Aβ1–42 at three major cleavage sites (Fig. 1) and seems to have a physiological role in Aβ metabolism as shown in mice deficient in the peptidase.7 In addition to its cardiovascular role ECE-1 is important developmentally, especially for neural crest development, again through its ability to generate endothelin-1. ECE-1 has also been implicated in numerous cancers: thyroid, ovarian, endometrial, lung, colon, and especially prostate. Indeed, it has been shown that siRNA-mediated knockdown of ECE-1 reduces prostate cancer cell invasion in culture.14
THE ACE FAMILY (ACE AND ACE-2) As described in the angiotensin chapter in the Cardiovascular Peptides section of the book, ACE was identified >50 years ago by Leonard Skeggs and colleagues as the enzyme responsible for converting angiotensin I into the vasoconstrictor peptide, angiotensin II (termed hypertensins I and II at that time). In parallel, it inactivates the vasodilator and inflammatory peptide bradykinin, which is the most favorable peptide substrate for ACE, being hydrolyzed approximately 25 times more efficiently than is angiotensin I. Hence, the inhibition of ACE has a powerful antihypertensive effect, and the enzyme has therefore been a major cardiovascular target for many years leading to the development of numerous potent and specific inhibitors (e.g. captopril, lisinopril). The catalytic activity of ACE is predominantly as a “peptidyl dipeptidase” or carboxydipeptidase, removing
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dipeptides from the C-terminus of a susceptible peptide substrate. However, ACE can act as an endopeptidase in the hydrolysis of some peptides (e.g. both substance P and luteinizing hormone-releasing hormone), although far less efficiently. ACE is a type I integral membrane protein and zinc metallopeptidase with the typical HEXXH zinc-binding motif, which is expressed on the surface of both endothelial and epithelial cells. Mammalian ACE exists as two distinct forms, arising from the use of alternative promoters. The simplest is the testis-specific form of ACE, which is essential for male fertility, and which carries a single zincbinding and catalytic domain. Elsewhere in the body, the somatic form of ACE has arisen by gene duplication and has two homologous but not identical active sites (N-domain and C-domain sites) with an overall Mr of 150–180 kDa. Hence the two sites differ somewhat in substrate specificity and inhibitor sensitivity. For example, the hemoregulatory peptide Ac-Ser-Asp-Lys-Pro (AcSDKP) and angiotensin-(1-7) are preferred substrates of the N-domain catalytic site whereas angiotensin I is mainly metabolized by the C-domain. A soluble form of ACE exists in serum and other body fluids and is derived from the membrane form of ACE by proteolytic shedding (for a review on ACE see Ref. 13). Elucidation of the structure of ACE eluded investigators for many years because of the heavy glycosylation of the glycoprotein hindering crystallization. It was not until 2003 that the first X-ray structures of ACE were determined for the testicular form with and without inhibitor (lisinopril) bound.19 Analysis of the structures has revealed that ACE most closely resembles a neurotensin-degrading zinc endopeptidase known as neurolysin rather than NEP, or carboxypeptidase A, on whose structure the design of ACE inhibitors was originally based. Subsequent structure determination of the N-domain1 has allowed comparisons of the two active sites and aided the development of domain- selective inhibitors. An unexpected substrate identified for ACE has been the Aβ peptide with the enzyme apparently cleaving the Asp7Ser8 bond of Aβ1–40,10 although this did not seem to fit with the known specificity of ACE. Subsequently, other sites of cleavage have been identified within Aβ, including the ability of ACE to convert Aβ1–42 to Aβ1–40, which is more consistent with its carboxydipeptidase specificity35 (Fig. 1). This cleavage would be beneficial in removing the more toxic Aβ1–42 species. This study also showed that treatment of transgenic mice that develop Alzheimer-like pathology with an ACE inhibitor significantly increased the deposition of brain Aβ1–42. However, there is no epidemiological evidence that human patients on ACE inhibitor therapy are any more prone to the development of Alzheimer’s disease. The only known functional homolog ofACE in humans was discovered in 2000 through genomics-based approaches.29 ACE2, like ACE, is a transmembrane glycoprotein
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but with only a single catalytic site equivalent to the C-domain of ACE. Although ACE2 is highly similar in sequence to ACE around the active site, it displays a somewhat different substrate specificity and is not sensitive to inhibition by ACE inhibitors. ACE2 behaves as a simple carboxypeptidase removing a single C-terminal amino acid from a susceptible substrate. Hence, angiotensin I is converted to angiotensin-(1–9) by ACE2 rather than to angiotensin II (angiotensin-(1–8)). However, ACE2 acts much more efficiently on angiotensin II converting it to the vasodilatory angiotensin-(1–7) and hence serving to counterbalance the actions of ACE (see the section on Cardiovascular Peptides in this Handbook). Angiotensin-(1–7) can also be formed directly from angiotensin I through the action of NEP. Of 126 biologically active peptides tested as ACE2 substrates, only 11 were hydrolyzed by the enzyme, the most efficient being angiotensin II, apelin-13, and dynorphin A-(1-13).32 Apelin-13 may be a physiologically relevant ACE2 substrate. A number of potent and specific ACE2 inhibitors have been reported, of which the first were the non-peptide inhibitor, MLN-4760, and a peptide identified from phage library screening, DX600.
INSULIN-DEGRADING ENZYME (INSULYSIN, IDE) Insulin-degrading enzyme (IDE, insulysin) is another zinc endopeptidase involved in the degradation of biologically active peptides although it differs substantially from the ACE and NEP families in location and catalytic mechanism. IDE was originally isolated as an enzyme regulating plasma levels of insulin, hence its name, and is therefore seen as of potential significance as a therapeutic target in diabetes. In addition to insulin, IDE can hydrolyze a number of biologically active peptides including insulin-like growth factor II, tumor growth factor-α, and atrial natriuretic peptide.27 Another important peptide substrate of IDE is the amyloid precursor protein intracellular domain, AICD, which acts as a transcriptional regulator controlling the expression of NEP and some other genes as discussed above.3 IDE also degrades somatostatin, a neuropeptide that declines in aging and Alzheimer’s disease.5 Unlike the other peptidases described above, IDE is mainly a soluble cytoplasmic enzyme although it also exists as a secreted form.34 There is also a splice-variant of IDE targeted to mitochondria. The mechanisms responsible for the subcellular targeting and secretion of IDE remain unresolved. The secreted form of IDE seems to play a role in the metabolism of Aβ peptide, was the first such peptidase identified and remains as a well validated Aβ-degrading enzyme from both in vitro and in vivo studies, including the use of transgenic approaches.18 Hence, IDE is also seen as a potential target for upregulation in the treatment of Alzheimer’s disease.
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IDE differs from ACE and NEP in possessing an inverted zinc-binding motif (His-Xaa-Xaa-Glu-His) and shows unusual allosteric behavior with small peptides. The structural solution of IDE27 has shown the active site to be enclosed within a flexible clamshell-like structure and ATP is required to facilitate the opening of the catalytic chamber. Until recently, there were no potent and specific IDE inhibitors to facilitate studies of the role of the enzyme in the metabolism of biologically active peptides but rationally designed inhibitors based on the structural information have now been produced which confirm the role of IDE in regulating insulin signaling.15 Such inhibitors may well have potential for the treatment of type 2 diabetes.
PROLINE-DIRECTED PEPTIDASES Numerous biologically active peptides are partially protected from exopeptidase degradation by the presence of highly conserved prolyl residues especially at or near to the peptide N- and C-termini. Hence, very specific peptidases are required to initiate their metabolism because the conformational constraint imposed by the cyclic imino structure of proline hinders proteolysis.31 The Xaa-Pro sequence is present at the N-terminus of >100 biologically active peptides or proteins, including peptide hormones, neurotransmitters, immunopeptides, and kinins. The bestcharacterized prolyl peptidase is the cell-surface serine peptidase, dipeptidyl peptidase IV (DPP IV) which, as its name suggests, hydrolyzes a dipeptide from the N-terminus of peptides provided that the second residue is a proline (or less favorably an alanine or serine). DPP IV is therefore one of only a few enzymes that have specificity directed toward prolyl residues. DPP IV was originally described as a rat liver enzyme releasing naphthylamine from Gly-Pro2-naphthylamide and postulated to be involved in collagen metabolism. However, it was unable to cleave collagen and its physiological role remained unclear. Subsequent studies showed it to be an abundant peptidase of the renal and intestinal brush borders although it is present on most cell types. It is identical with the T cell-activating antigen (CD26) and, on T cells, it associates with the immunodeficiency-related protein adenosine deaminase via a cluster of charged amino acid residues. It is a type II membrane protein with only a short cytoplasmic tail (six residues) with the bulk of the enzyme facing the extracellular space where it can participate in the metabolism of a diverse range of biologically active peptides. DPP IV is also found as a soluble protein in serum whose levels are altered in cancer and some other diseases. The soluble form is most probably shed from the membrane-bound form although the protease responsible for this process is unknown. The enzyme is discussed in further detail, especially in relation to the immune response in this Handbook (see the chapter in Immune/Inflammatory Peptides section).
DPP IV is a typical serine peptidase, and a member of the “prolyl oligopeptidase” family, possessing the catalytic triad residues Ser/Asp/His (in rat Ser631/Asp709/His741) and the enzyme functions as a homodimer and can also form functional tetramers. The crystal structure reveals that each subunit consists of two domains, an α/β-hydrolase domain and an eight-bladed β-propeller domain23 with the catalytic site located in a cavity formed between the two domains both of which participate in substrate/inhibitor binding. The human DPP IV gene consists of 26 exons and, unusually among serine proteases, the nucleotides that encode the active site sequence (Gly-Xaa-Ser-Xaa-Gly) are split between two exons. DPP IV may play a digestive role in the digestion and uptake of prolyl-containing peptides. Most interest has focused, however, on the role of the enzyme in metabolizing biologically active peptides. In this capacity, it can either generate active peptide from a precursor, convert one active peptide to another with different receptor specificity (e.g. peptide YY or neuropeptide Y), or inactivate peptides. It is the latter role that has allowed the development of DPP IV inhibitors as the first new therapeutic class of orally active antihyperglycemic drug for type 2 diabetes mellitus for a long time through the ability of DPP IV to inactivate incretin hormones (glucagon-like peptide-1; glucosedependent insulinotropic peptide).6 Almost 50 biologically active peptides have been identified as substrates for DPP IV most of which have the N-terminal Xaa-Pro sequence, but some have Xaa-Ala or less commonly Xaa-Ser. Another cell-surface peptidase capable of hydrolyzing peptides with a penultimate N-terminal prolyl residue is aminopeptidase P (APP2 to distinguish it from a cytosolic prolyl aminopeptidase, APP1). The enzyme removes the N-terminal residue leaving a free prolyl residue at the N-terminus. Examples of good substrates include bradykinin and neuropeptide Y. The specific inhibitor, apstatin, can reduce blood pressure by potentiating the action of bradykinin. A prolyl residue in the third position of a substrate renders the peptide resistant to the action of DPP IV (e.g. bradykinin, Arg-Pro-Pro-…). APP2 is a zinc peptidase and is unusual among cell-surface peptidases in being anchored to the cell surface via a glycosyl-phosphatidylinositol membrane anchor. Unlike DPP IV and APP2, which are exopeptidases, another prolyl peptidase functions as an endopeptidase and can hydrolyze a wide range of small (<30 amino acids) biologically active peptides, at least in vivo. This is a serine peptidase originally known as postproline cleaving enzyme or proline endopeptidase, which cleaves peptides on the carboxy-terminal side of prolyl residues. It is now known as prolyl oligopeptidase (POP) and was originally implicated in oxytocin metabolism. Other potential substrates include substance P, angiotensins, bradykinin, β-endorphins, neurotensin, and thyrotropin releasing hormone (TRH). To evaluate the role of POP, numerous potent and relatively specific
SECTION | XX Handbook of Biologically Active Peptides: Peptide Biosynthesis/Processing
small molecule inhibitors have been developed, which exhibit a number of interesting physiological properties, for example, in enhancing memory, which is perhaps consistent with vasopressin also being a substrate for the enzyme. There are many reports that POP may have a role in psychiatric and neurodegenerative diseases although it does not seem to play a role in Aβ metabolism.22 The evidence for a physiological role for POP in peptide degradation is, in most cases, lacking (see Ref. 8 for review) and a topological problem is that POP is a cytosolic enzyme and therefore unlikely to interact with circulating peptides. Another related proline-specific peptidase (acylpeptide hydrolase), which can be secreted from neuronal cells, may however contribute to Aβ metabolism. Two specific areas where POP may also participate are in inositol phosphate signaling, and in angiogenesis and hence progression of malignancies. POP is able to enhance inositol phosphate signaling and cell sensitivity to lithium acting via the enzyme inositol polyphosphate phosphatase and hence regulate gene expression. POP is also able to cleave thymosin-β4 to generate the hemoregulatory peptide AcSDKP, itself a substrate for the N-domain of ACE (see above). Both AcSDKP and POP activity are substantially increased in certain malignancies.
CONCLUSIONS Unlike the high specificity of ligand–receptor interactions, peptidases that degrade biologically active peptides tend to have multiple substrates, and hence, relatively few are needed to control the circulating levels of the multiplicity of such peptides described in this Handbook. They mostly exist as cell-surface ectoenzymes, or in a soluble, secreted form. The properties of some of the key ones, especially of the neprilysin family, have been described in this chapter, as well as methods for manipulating their activity. Structures are now available for most of these peptidases allowing rational design of potent and specific inhibitors. The promiscuity of these peptidases might suggest that inhibiting their activity could have undesirable side effects. However, in practice, this does not seem to be the case, as best exemplified by ACE inhibitors. NEP inhibitors also have a role to play in many conditions, although their chronic use could potentially lead to a build-up of brain Aβ peptide. There still remain numerous orphan peptidases, however, for which their physiological peptide substrates remain to be identified. Comparative in vivo peptidomic approaches in animals deficient in a particular peptidase now provide a powerful tool to categorize physiological substrate profiles for individual enzymes. We can expect to have a much clearer picture of the roles of peptidases in the metabolism of biologically active peptides in the next few years.
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