Hemopressin: a novel bioactive peptide derived from the alpha1-chain of hemoglobin

Hemopressin (PVNFKFLSH), a novel bioactive peptide derived from the alpha1-chain of hemoglobin, was originally isolated from rat brain homogenates. Hemopressin causes hypotension in anesthetized rats and is metabolized in vivo and in vitro by endopeptidase 24.15 (EP24.15), neurolysin (EP24.16), and angiotensin-converting enzyme (ACE). Hemopressin also exerts an antinociceptive action in experimental inflammatory hyperalgesia induced by carrageenin or bradykinin via a mechanism that is independent of opioids. These findings suggest that this peptide may have important regulatory physiological actions in vivo.     

Confocal microscopy reveals thimet oligopeptidase (EC 3.4.24.15) and neurolysin (EC 3.4.24.16) in the classical secretory pathway

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) and neurolysin (EC 3.4.24.16; EP24.16) are closely related enzymes involved in the metabolic inactivation of bioactive peptides. Both of these enzymes were previously shown to be secreted from a variety of cell types, although their primary sequence lacks a signal peptide. To investigate the mechanisms responsible for this secretion, we examined by confocal microscopy the subcellular localization of these two enzymes in the neuroendocrine cell line AtT20. Both EP24.15 and EP24.16 were found by immunohistochemistry to be abundantly expressed in AtT20 cells. Western blotting experiments confirmed that the immunoreactivity detected in the soma of these cells corresponded to previously cloned isoforms of the enzymes. At the subcellular level, both enzymes colocalized extensively with the integral trans-Golgi network protein, syntaxin-6, in the juxtanuclear region. In addition, both EP24.15 and EP24.16 were found within small vesicular organelles distributed throughout the cell body. Some, but not all, of these organelles also stained positively for ACTH. These results demonstrate that both EP24.15 and EP24.16 are present within the classical secretory pathway. Their colocalization with ACTH further suggests that they may be targeted to the regulated secretory pathway, even in the absence of a signal peptide.     

Endopeptidases 3.4.24.15 and 24.16 in endothelial cells: potential role in vasoactive peptide metabolism

The closely related metalloendopeptidases EC (EP24.15; thimet oligopeptidase) and 24.16 (EP24.16; neurolysin) cleave a number of vasoactive peptides such as bradykinin and neurotensin in vitro. We have previously shown that hypotensive responses to bradykinin are potentiated by an inhibitor of EP24.15 and EP24.16 (26), suggesting a role for one or both enzymes in bradykinin metabolism in vivo. In this study, we have used selective inhibitors that can distinguish between EP24.15 and EP24.16 to determine their activity in cultured endothelial cells (the transformed human umbilical vein endothelial hybrid cell line EA.hy926 or ovine aortic endothelial cells). Endopeptidase activity was assessed using a specific quenched fluorescent substrate [7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-d-Lys(2,4-dinitrophenyl)], as well as the peptide substrates bradykinin and neurotensin (assessed by high-performance liquid chromatography with mass spectroscopic detection). Our results indicate that both peptidases are present in endothelial cells; however, EP24.16 contributes significantly more to substrate cleavage by both cytosolic and membrane preparations, as well as intact cells, than EP24.15. These findings, when coupled with previous observations in vivo, suggest that EP24.16 activity in vascular endothelial cells may play an important role in the degradation of bradykinin and/or other peptides in the circulation.     

The intracellular distribution and secretion of endopeptidases 24.15 (EC 3.4.24.15) and 24.16 (EC 3.4.24.16)

Endopeptidase 24.15 (EC 3.4.24.15; EP24.15) and endopeptidase 24.16 (EC 3.4.24.16; EP24.16) are enzymes involved in general peptide metabolism in mammalian cells and tissues. This review will focus on morphological and biochemical aspects related to the subcellular distribution and secretion of these homologous enzymes in the central nervous system. These are important issues for a better understanding of the functions of EP24.15 and EP24.16 within neuroendocrine systems.     

Metalloendopeptidases EC 3.4.24.15 and EC 3.4.24.16: potential roles in vascular physiology

Summary The zinc metalloendopeptidases EC 3.4.24.15 (EP 24.15) and EC 3.4.24.16 (EP 24.16) are closely related ubiquitous enzymes, which have well-defined in vitro activities in generation and degradation of a range of specific peptide targets. Despite this, little is known regarding their roles in whole animal physiology. One of the peptides degraded by these enzymes in vitro is bradykinin, a mediator with potent effects on the vasculature at both systemic and local levels. This review summarises the work that has examined the role of EP 24.15/24.16 in regulation of the vascular effects of bradykinin in vivo. This work was made possible by the development of a specific stable inhibitor of these enzymes, JA-2. Use of this inhibitor has shown that EP 24.15/24.16 are capable of regulating responses induced by exogenous bradykinin. This effect was observed at a systemic level with an increase in the hypotensive effect of intravenous bradykinin. Further work is required to determine whether these enzymes also regulate bradykinin produced endogenously.     

Metalloendopeptidases EC 3.4.24.15 and EC 3.4.24.16: potential roles in vascular physiology

The zinc metalloendopeptidases EC 3.4.24.15 (EP24.15) and EC 3.4.24.16 (EP 24.16) are closely relatedubiquitous enzymes, which have well-defined in vitroactivities in generation and degradation of a range ofspecific peptide targets. Despite this, little is knownregarding their roles in whole animal physiology. One of thepeptides degraded by these enzymes in vitro isbradykinin, a mediator with potent effects on the vasculatureat both systemic and local levels. This review summarises thework that has examined the role of EP 24.15/24.16 inregulation of the vascular effects of bradykinin invivo. This work was made possible by the development of aspecific stable inhibitor of these enzymes, JA-2. Use of thisinhibitor has shown that EP 24.15/24.16 are capable ofregulating responses induced by exogenous bradykinin. Thiseffect was observed at a systemic level with an increase inthe hypotensive effect of intravenous bradykinin. Further workis required to determine whether these enzymes also regulatebradykinin produced endogenously.     

Bradykinin analogues with beta-amino acid substitutions reveal subtle differences in substrate specificity between the endopeptidases EC 3.4.24.15 and EC 3.4.24.16.

The closely related zinc metalloendopeptidases EC 3.4.24.15 (EP24.15) and EC 3.4.24.16 (EP24.16) cleave many common substrates, including bradykinin (BK). As such, there are few substrate-based inhibitors which are sufficiently selective to distinguish their activities. We have used BK analogues with either alanine or beta-amino acid (containing an additional carbon within the peptide backbone) substitutions to elucidate subtle differences in substrate specificity between the enzymes. The cleavage of the analogues by recombinant EP24.15 and EP24.16 was assessed, as well as their ability to inhibit the two enzymes. Alanine-substituted analogues were generally better substrates than BK itself, although differences between the peptidases were observed. Similarly, substitution of the four N-terminal residues with beta-glycine enhanced cleavage in some cases, but not others. beta-Glycine substitution at or near the scissile bond (Phe5-Ser6) completely prevented cleavage by either enzyme: interestingly, these analogues still acted as inhibitors, although with very different affinities for the two enzymes. Also of interest, beta-Gly8-BK was neither a substrate nor an inhibitor of EP24.15, yet could still interact with EP24.16. Finally, while both enzymes could be similarly inhibited by the D-stereoisomer of beta-C3-Phe5-BK (IC50 approximately 20 microM, compared to 8 microM for BK), EP24.16 was relatively insensitive to the L-isomer (IC50 12 approximately microM for EP24.15, >40 microM for EP24.16). These studies indicate subtle differences in substrate specificity between EP24.15 and EP24.16, and suggest that beta-amino acid analogues may be useful as templates for the design of selective inhibitors.     

Metalloendopeptidases EC 3.4.24.15/16 regulate bradykinin activity in the cerebral microvasculature

Bradykinin is a vasoactive peptide that has been shown to increase the permeability of the cerebral microvasculature to blood-borne macromolecules. The two zinc metalloendopeptidases EC (EP 24.15) and EC (EP 24.16) degrade bradykinin in vitro and are highly expressed in the brain. However, the role that these enzymes play in bradykinin metabolism in vivo remains unclear. In the present study, we investigated the role of EP 24.15 and EP 24.16 in the regulation of bradykinin-induced alterations in microvascular permeability. Permeability of the cerebral microvasculature was assessed in anesthetized Sprague-Dawley rats by measuring the clearance of 70-kDa FITC dextran from the brain. Inhibition of EP 24.15 and EP 24.16 by the specific inhibitor N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Aib-Tyr-p-aminobenzoate (JA-2) resulted in the potentiation of bradykinin-induced increases in cerebral microvessel permeability. The level of potentiation was comparable to that achieved by the inhibition of angiotensin-converting enzyme. These findings provide the first evidence of an in vivo role for EP 24.15/EP 24.16 in brain function, specifically in regulating alterations in microvessel permeability induced by exogenous bradykinin.     

Substrate phosphorylation affects degradation and interaction to endopeptidase 24.15, neurolysin, and angiotensin-converting enzyme

Recent findings from our laboratory suggest that intracellular peptides containing putative post-translational modification sites (i.e., phosphorylation) could regulate specific protein interactions. Here, we extend our previous observations showing that peptide phosphorylation changes the kinetic parameters of structurally related endopeptidase EP24.15 (EC 3.4.24.15), neurolysin (EC 3.4.24.16), and angiotensin-converting enzyme (EC 3.4.15.1). Phosphorylation of peptides that are degraded by these enzymes leads to reduced degradation, whereas phosphorylation of peptides that interacted as competitive inhibitors of these enzymes alters only the K(i)'s. These data suggest that substrate phosphorylation could be one of the mechanisms whereby some intracellular peptides would escape degradation and could be regulating protein interactions within cells.     

Role of calcium in the release of EC 3.4.24.15 and EC 3.4.24.16 from endothelial cells

Summary The two closely related soluble zinc metalloendopeptidases EC 3.4.24.15 (EP24.15) and EC 3.4.24.16 (EP24.16) readily hydrolyze the vasocative peptide bradykinin in vitro, and therefore may play a role in cardiovascular regulation. Although primarily soluble cytosolic enzymes, both secreted and membrane-associated forms of both peptidases have been reported. However, these enzymes have neither a transmembrane domain nor a signal sequence; thus, the mechanisms of membrane anchoring and secretion are unknown. In the present study, secreted/released EP24.15 and EP24.16 activity from aortic endothelial cells in culture was assessed by the cleavage of a specific quenched fluorescent substrate. An increase in enzyme activity released from endothelial cells, which express both peptidases, was seen following incubation with calcium-free media. In the AtT-20 endocrine cell (mouse pituitary corticotrope), which predominantly expresses EP24.15, the release of activity into media was unaffected by calcium removal. The release of enzyme activity from endothelial cells was inversely proportional to calcium concentrations ranging between 0.01 mM (activity equivalent to calcium-free media) and 0.5 mM (activity equivalent to normal media). Cleavage of the EP24.16-specific substrate AcNT_8–13 indicated that the increase in enzyme activity released upon incubation with calcium-free medium was due at least in part to the release of EP24.16. These results suggest that EP24.15 and EP24.16 are secreted from endothelial cells, and that removal of calcium selectively enhances the release of EP24.16 by an as yet unknown mechanism.     

Role of calcium in the release of EC 3.4.24.15 and EC 3.4.24.16 from endothelial cells

The two closely related soluble zinc metalloendopeptidases EC 3.4.24.15 (EP24.15) and EC 3.4.24.16 (EP24.16) readily hydrolyze the vasoactive peptide bradykinin in vitro, and therefore may play a role in cardiovascular regulation. Although primarily soluble cytosolic enzymes, both secreted and membrane-associated forms of both peptidases have been reported. However, these enzymes have neither a transmembrane domain nor a signal sequence; thus, the mechanisms of membrane anchoring and secretion are unknown. In the present study, secreted/released EP24.15 and EP24.16 activity from aortic endothelial cells in culture was assessed by the cleavage of a specific quenched fluorescent substrate. An increase in enzyme activity released from endothelial cells, which express both peptidases, was seen following incubation with calcium-free media. In the AtT-20 endocrine cell (mouse pituitary corticotrope), which predominantly expresses EP24.15, the release of activity into media was unaffected by calcium removal. The release of enzyme activity from endothelial cells was inversely proportional to calcium concentrations ranging between 0.01 mM (activity equivalent to calcium-free media) and 0.5 mM (activity equivalent to normal media). Cleavage of the EP24.16-specific substrate AcNT_8-13 indicated that the increase in enzyme activity released upon incubation with calcium-free medium was due at least in part to the release of EP24.16. These results suggest that EP24.15 and EP24.16 are secreted from endothelial cells, and that removal of calcium selectively enhances the release of EP24.16 by an as yet unknown mechanism.     

14-3-3 epsilon modulates the stimulated secretion of endopeptidase 24.15

Endopeptidase 24.15 (ep24.15: EC3.4.24.15), a secreted protein involved in peptide metabolism, is unusual in that it does not contain a signal peptide sequence. In this work, we describe the physical interaction between ep24.15 and 14-3-3 epsilon, one isoform of a family of ubiquitous phosphoserine/threonine-scaffold proteins that organizes cell signaling and is involved in exocytosis. The interaction between ep24.15 and 14-3-3 epsilon increased following phosphorylation of ep24.15 at Ser(644) by protein kinase A (PKA). The co-localization of ep24.15 and 14-3-3 epsilon was increased by exposure of HEK293 cells (human embryonic kidney cells) to forskolin (10 microm). Overexpression of 14-3-3 epsilon in HEK293 cells almost doubled the secretion of ep24.15 stimulated by A23187 (7.5 microm) from 10%[1.4 +/- 0.24 AFU/(min 10(6) cells)] to 19%[2.54 +/- 0.24 AFU/(min 10(6) cells)] (p < 0.001) of the total intracellular enzyme activity. Treatment with forskolin had a synergistic effect on the A23187-stimulated secretion of ep24.15 that was totally blocked by the PKA inhibitor KT5720. The ep24.15 point mutation S644A reduced the co-localization of ep24.15 and 14-3-3 in stably transfected HEK293 cells. Indeed, secretion of the ep24.15 S644A mutant from these cells was only slightly stimulated by A23187 and insensitive to forskolin, in contrast to that of the wild type enzyme. Together, these data suggest that prior interaction with 14-3-3 is an important step in the unconventional stimulated secretion of ep24.15.     

Inhibitors of metalloendopeptidase EC 3.4.24.15 and EC 3.4.24.16 stabilized against proteolysis by the incorporation of beta-amino acids

The enzyme EC 3.4.24.15 (EP 24.15) is a zinc metalloendopeptidase whose precise function in vivo remains unknown but is thought to participate in the regulated metabolism of a number of specific neuropeptides. The lack of stable and selective inhibitors has hindered the determination of the exact function of EP 24.15. Of the limited number of EP 24.15 inhibitors that have been developed, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (CFP) is the most widely studied. CFP is a potent and specific inhibitor, but it is unstable in vivo due to cleavage between the alanine and tyrosine residues by the enzyme neprilysin (EP 24.11). This cleavage by EP 24.11 generates a potent inhibitor of angiotensin converting enzyme, thereby limiting the use of CFP for in vivo studies. To develop specific inhibitors of EP 24.15 that are resistant to in vitro and potentially in vivo proteolysis by EP 24.11, this study incorporated beta-amino acids replacing the Ala-Tyr scissile alpha-amino acids of CFP. Both C2 and C3 substituted beta-amino acids were synthesized and substituted at the EP 24.11 scissile Ala-Tyr bond. Significant EP 24.15 inhibitory activity was observed with some of the beta-amino acid containing analogues. Moreover, binding to EP 24.11 was eliminated, thus rendering all analogues containing beta-amino acids resistant to degradation by EP 24.11. Selective inhibition of either EP 24.15 or EP 24.16 was also observed with some analogues. The results demonstrated the use of beta-amino acids in the design of inhibitors of EP 24.15 and EP 24.16 with K(i)'s in the low micromolar range. At the same time, these analogues were resistant to cleavage by the related metalloendopeptidase EP 24.11, in contrast to the alpha-amino acid based parent peptide. This study has therefore clearly shown the potential of beta-amino acids in the design of stable enzyme inhibitors and their use in generating molecules with selectivity between closely related enzymes.     

Zinc coordination and substrate catalysis within the neuropeptide processing enzyme endopeptidase EC 3.4.24.15. Identification of active site histidine and glutamate residues.

Endopeptidase EC 3.4.24.15 (EP24.15) is a zinc metalloendopeptidase that is broadly distributed within the brain, pituitary, and gonads. Its substrate specificity includes a number of physiologically important neuropeptides such as neurotensin, bradykinin, and gonadotropin-releasing hormone, the principal regulatory peptide for reproduction. In studying the structure and function of EP24.15, we have employed in vitro mutagenesis and subsequent protein expression to genetically dissect the enzyme and allow us to glean insight into the mechanism of substrate binding and catalysis. Comparison of the sequence of EP24.15 with bacterial homologues previously solved by x-ray crystallography and used as models for mammalian metalloendopeptidases, indicates conserved residues. The active site of EP24.15 exhibits an HEXXH motif, a common feature of zinc metalloenzymes. Mutations have confirmed the importance, for binding and catalysis, of the residues (His473, Glu474, and His477) within this motif. A third putative metal ligand, presumed to coordinate directly to the active site zinc ion in concert with His473 and His477, has been identified as Glu502. Conservative alterations to these residues drastically reduces enzymatic activity against both a putative physiological substrate and a synthetic quenched fluorescent substrate as well as binding of the specific active site-directed inhibitor, N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate, the binding of which we have shown to be dependent upon the presence, and possibly coordination, of the active site zinc ion. These studies contribute to a more complete understanding of the catalytic mechanism of EP24.15 and will aid in rational design of inhibitors and pharmacological agents for this class of enzymes.     

Intracellullar peptides as putative natural regulators of protein interactions

Extralysosomal proteolysis by multicatalytic complexes such as the 26S proteasome produces large amounts of peptides in the cytosol, mitochondria and nuclei of eukaryotic cells, and there is increasing evidence that the resulting free intracellular peptides can modulate specific protein interactions. The demonstration that free peptides added to the intracellular milieu can regulate cellular functions mediated by protein interactions suggests new putative roles for these molecules in gene regulation, metabolism, cell signaling and protein targeting. Such interactions frequently involve specific consensus amino acid sequences that can be predicted based on similarities in domain composition. We have recently developed a new strategy for identifying novel natural peptides, the sequences of which correspond to fragments of intracellular proteins and contain putative post-translational modification sites. In this review, we examine the evidence that intracellular peptides released by proteasomes may be involved in regulating protein interactions. In particular, the role of endopeptidase 24.15 (thimet oligopeptidase; EC 3.4.24.15) is discussed in detail as this enzyme has been implicated in intracellular peptide metabolism in vivo in concert with the 26S proteasome.     

A structure-based site-directed mutagenesis study on the neurolysin (EC 3.4.24.16) and thimet oligopeptidase (EC 3.4.24.15) catalysis

Neurolysin (EP24.16) and thimet oligopeptidase (EP24.15) are closely related metalloendopeptidases. Site-directed mutagenesis of Tyr(613) (EP24.16) or Tyr(612) (EP24.15) to either Phe or Ala promoted a strong reduction of k(cat)/K(M) for both enzymes. These data suggest the importance of both hydroxyl group and aromatic ring at this specific position during substrate hydrolysis by these peptidases. Furthermore, the EP24.15 A607G mutant showed a k(cat)/K(M) of 2x10(5) M(-1) s(-1) for the Abz-GFSIFRQ-EDDnp substrate, similar to that of EP24.16 (k(cat)/K(M)=3x10(5) M(-1) s(-1)) which contains Gly at the corresponding position; the wild type EP24.15 has a k(cat)/K(M) of 2.5x10(4) M(-1) s(-1) for this substrate.     

Analysis of Intracellular Substrates and Products of Thimet Oligopeptidase in Human Embryonic Kidney 293 Cells

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is an intracellular enzyme that has been proposed to metabolize peptides within cells, thereby affecting antigen presentation and G protein-coupled receptor signal transduction. However, only a small number of intracellular substrates of EP24.15 have been reported previously. Here we have identified over 100 peptides in human embryonic kidney 293 (HEK293) cells that are derived from intracellular proteins; many but not all of these peptides are substrates or products of EP24.15. First, cellular peptides were extracted from HEK293 cells and incubated in vitro with purified EP24.15. Then the peptides were labeled with isotopic tags and analyzed by mass spectrometry to obtain quantitative data on the extent of cleavage. A related series of experiments tested the effect of overexpression of EP24.15 on the cellular levels of peptides in HEK293 cells. Finally, synthetic peptides that corresponded to 10 of the cellular peptides were incubated with purified EP24.15 in vitro, and the cleavage was monitored by high pressure liquid chromatography and mass spectrometry. Many of the EP24.15 substrates identified by these approaches are 9–11 amino acids in length, supporting the proposal that EP24.15 can function in the degradation of peptides that could be used for antigen presentation. However, EP24.15 also converts some peptides into products that are 8–10 amino acids, thus contributing to the formation of peptides for antigen presentation. In addition, the intracellular peptides described here are potential candidates to regulate protein interactions within cells.Intracellular protein turnover is a crucial step for cell functioning, and if this process is impaired, the elevated levels of aged proteins usually lead to the formation of intracellular insoluble aggregates that can cause severe pathologies (1). In mammalian cells, most proteins destined for degradation are initially tagged with a polyubiquitin chain in an energy-dependent process and then digested to small peptides by the 26 S proteasome, a large proteolytic complex involved in the regulation of cell division, gene expression, and other key processes (2, 3). In eukaryotes, 30–90% of newly synthesized proteins may be degraded by proteasomes within minutes of synthesis (3, 4). In addition to proteasomes, other extralysosomal proteolytic systems have been reported (5, 6). The proteasome cleaves proteins into peptides that are typically 2–20 amino acids in length (7). In most cases, these peptides are thought to be rapidly hydrolyzed into amino acids by aminopeptidases (8–10). However, some intracellular peptides escape complete degradation and are imported into the endoplasmic reticulum where they associate with major histocompatibility complex class I (MHC-I)³ molecules and traffic to the cell surface for presentation to the immune system (10–12). Additionally, based on the fact that free peptides added to the intracellular milieu can regulate cellular functions mediated by protein interactions such as gene regulation, metabolism, cell signaling, and protein targeting (13, 14), intracellular peptides generated by proteasomes that escape degradation have been suggested to play a role in regulating protein interactions (15). Indeed, oligopeptides isolated from rat brain tissue using the catalytically inactive EP24.15 (EC 3.4.24.15) were introduced into Chinese hamster ovarian-S and HEK293 cells and were found capable of altering G protein-coupled receptor signal transduction (16). Moreover, EP24.15 overexpression itself changed both angiotensin II and isoproterenol signal transduction, suggesting a physiological function for its intracellular substrates/products (16).EP24.15 is a zinc-dependent peptidase of the metallopeptidase M3 family that contains the HEXXH motif (17). This enzyme was first described as a neuropeptide-degrading enzyme present in the soluble fraction of brain homogenates (18). Whereas EP24.15 can be secreted (19, 20), its predominant location in the cytosol and nucleus suggests that the primary function of this enzyme is not the extracellular degradation of neuropeptides and hormones (21, 22). EP24.15 was shown in vivo to participate in antigen presentation through MHC-I (23–25) and in vitro to bind (26) or degrade (27) some MHC-I associated peptides. EP24.15 has also been shown in vitro to degrade peptides containing 5–17 amino acids produced after proteasome digestion of β-casein (28). EP24.15 shows substrate size restriction to peptides containing from 5 to 17 amino acids because of its catalytic center that is located in a deep channel (29). Despite the size restriction, EP24.15 has a broad substrate specificity (30), probably because a significant portion of the enzyme-binding site is lined with potentially flexible loops that allow reorganization of the active site following substrate binding (29). Recently, it has also been suggested that certain substrates may be cleaved by an open form of EP24.15 (31). This characteristic is supported by the ability of EP24.15 to accommodate different amino acid residues at subsites S4 to S3′, which even includes the uncommon post-proline cleavage (30). Such biochemical and structural features make EP24.15 a versatile enzyme to degrade structurally unrelated oligopeptides.Previously, brain peptides that bound to catalytically inactive EP24.15 were isolated and identified using mass spectrometry (22). The majority of peptides captured by the inactive enzyme were intracellular protein fragments that efficiently interacted with EP24.15; the smallest peptide isolated in these assays contained 5 and the largest 17 amino acids (15, 16, 22, 32), which is within the size range previously reported for natural and synthetic substrates of EP24.15 (18, 30, 33, 34). Interestingly, the peptides released by the proteasome are in the same size range of EP24.15 competitive inhibitors/substrates (7, 35, 36). Taken altogether, these data suggest that in the intracellular environment EP24.15 could further cleave proteasome-generated peptides unrelated to MHC-I antigen presentation (15).Although the mutated inactive enzyme “capture” assay was successful in identifying several cellular protein fragments that were substrates for EP24.15, it also found some interacting peptides that were not substrates. In this study, we used several approaches to directly screen for cellular peptides that were cleaved by EP24.15. The first approach involved the extraction of cellular peptides from the HEK293 cell line, incubation in vitro with purified EP24.15, labeling with isotopic tags, and analysis by mass spectrometry to obtain quantitative data on the extent of cleavage. The second approach examined the effect of EP24.15 overexpression on the cellular levels of peptides in the HEK293 cell line. The third set of experiments tested synthetic peptides with purified EP24.15 in vitro, and examined cleavage by high pressure liquid chromatography and mass spectrometry. Collectively, these studies have identified a large number of intracellular peptides, including those that likely represent the endogenous substrates and products of EP24.15, and this original information contributes to a better understanding of the function of this enzyme in vivo.     

Evidence that enzymatic conversion of N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate, a specific inhibitor of endopeptidase 24.15, to N-[1 (R,S)-carboxy-3-phenylpropyl]-Ala-Ala is necessary for inhibition of angiotensin converting enzyme

N-[1 (R,S)-Carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (cFP-AAF-pAB) is a potent, substrate-related, specific inhibitor of endopeptidase 24.15, an enzyme involved in the metabolism of bioactive peptides including bradykinin, neurotensin, and proenkephalin, and prodynorphin-derived enkephalin precursors. The observation that this inhibitor causes a pronounced decrease in blood pressure after intravenous infusion into normotensive rats posed the question of the mechanism of this hypotensive response. It was suggested previously that cFP-AAF-pAB is an inhibitor of angiotensin converting enzyme (ACE) and that this function can account for the hypotensive response to the inhibitor. We present here evidence that cFP-AAF-pAB has no intrinsic ACE-inhibitory activity. The previously observed inhibition is shown to be dependent on cleavage of the Ala-Phe bond in the inhibitor by endopeptidase 24.11 (enkephalinase, EC 3.4.24.11), a contaminant of some ACE preparations.     

Inhibition of phospholipases by Met-Leu-Phe-Ile-Leu-Ile-Lys-Arg-Ser-Arg-His-Phe, C terminus of middle-sized tumor antigen

The N and C terminals and tyrosine-phosphorylating site of the middle-sized tumor antigen of polyoma virus were chemically synthesized. The sequences of these peptides were Met-Asp-Arg-Val-Leu-Ser-Arg-Ala-Asp-Lys (N-MT), Met-Leu-Phe-Ile-Leu-Ile-Lys-Arg-Ser-Arg-His-Phe (C-MT), and Glu-Glu-Glu-Glu-Tyr-Met-Pro-Met-Glu (MT-Tyr), respectively. Among these peptides, the C-MT peptide inhibited phospholipase A2 (EC 3.1.1.4), phospholipase C (EC 3.1.4.3), and phospholipase D (EC 3.1.4.4). In addition, phosphatidylinositol-specific phospholipase C (EC 3.1.4.10) was also inhibited by this peptide. To study the mechanism of the inhibition, kinetic analysis was performed using phospholipase A2 from porcine pancreas. The degree of inhibition of phospholipase was dose dependent, and maximal inhibition was observed at pH 8.8. This peptide inhibited phospholipase A2 in a competitive manner for low-affinity sites of Ca2+, and in a noncompetitive manner for phospholipid substrates. When a fatty acid in the 2 position of the glycerol moiety of phosphatidylcholine was replaced by palmitic acid (C16:0), oleic acid (C18:1), linoleic acid (C18:2), eicosatrienoic acid (C20:3), or arachidonic acid (C20:4), the degree of inhibition of phosphatidylcholine hydrolysis by the C-MT peptide decreased. Inhibition of phospholipase A2 by the C-MT peptide was reversed by low concentrations of sodium deoxycholate but not by Triton X-100 or Nonidet P40, nonionic detergents. These detergents and the modification of acyl groups altered the micellar state of phospholipids. These results, taken together, suggest that the binding of the C-MT peptide near the low-affinity Ca2+ binding sites modifies the interaction of phospholipid substrates with the active center of phospholipase A2.