An in vivo study of novel bioactive peptides that inhibit the growth of Escherichia coli.

We have created a system in which synthetically produced novel bioactive peptides can be expressed in vivo in Escherichia coli. Twenty thousand of these peptides were screened and 21 inhibitors were found that could inhibit the growth of E. coli on minimal media. The inhibitors could be placed into one of two groups, 1-day inhibitors, which were partially inhibitory, and 2-day inhibitors, which were completely inhibitory. Sequence analysis showed that two of the most potent inhibitors were actually peptide-protein chimeras in which the peptides had become fused to the 63 amino acid Rop protein which was also contained in the expression vector used in this study. Given that Rop is known to form an incredibly stable structure, it could be serving as a stabilizing motif for these peptides. Sequence analysis of the predicted coding regions from the next 10 most inhibitory peptides showed that four of the 10 peptides contained one or more proline residues either at or very near the C-terminal end of the peptide which could act to prevent degradation by peptidases. Collectively, based on what we observed in our screen of synthetic bioactive peptides that could prevent the growth of E. coli and what has been learned from structural studies of naturally occurring bioactive peptides, the presence of a stabilizing motif seems to be important for small peptides, if they are to be biologically active.     

Thimet oligopeptidase (EC 3.4.24.15), a novel protein on the route of MHC class I antigen presentation

The initial processing of antigens leading to major histocompatibility complex (MHC) class I antigenic peptides is carried out by the proteasome. However, how the final epitopes are generated and protected from degradation by cytosolic peptidases remains unknown. Coincidentally, peptides associated with the MHC class I molecules range from 8 to 13 amino acid residues, similarly to the optimum substrate size required for the cytosolic thimet oligopeptidase. Here we have investigated the putative intracellular function of thimet oligopeptidase related to antigen presentation. Using a well-characterized antigen-presenting cell system, we were able to demonstrate either inhibition or stimulation of CD8 T cell proliferation and cytotoxicity, manipulating intracellular thimet oligopeptidase levels with its specific inhibitor cFP-Ala-Ala-Tyr-pAb or loading the enzyme itself into the antigen-presenting cells. Our results suggest that thimet oligopeptidase should take an important function in the pathway of antigen presentation via MHC class I through a mechanism yet unknown.     

Degradation of proline peptides in peptidase-deficient strains of Salmonella typhimurium.

A mutant strain of Salmonella typhimurium that lacks two proline-specific peptidases (peptidases P and Q) could not complete the degradation of proline peptides formed as intermediates in starvation-induced protein breakdown. The wild-type strain produced free proline as the product of degradation of proline-labeled proteins. The pepP pepQ mutant, however, produced a mixture of small proline peptides. In the absence of peptidase Q only, peptidase P could complete the degradation of most of the proline peptide intermediates formed. In the absence of peptidase P only, about 50% of the proline-labeled, acid-soluble products were proline peptides. These results are consistent with in vitro specificity data indicating that peptidase Q hydrolyzes X-Pro dipeptides only, whereas peptidase P attacks both X-Pro dipeptides and longer peptides with X-Pro at their N-termini. A mutant strain lacking four broad-specificity peptidases (peptidases N, A, B, and D), but containing peptidases P and Q, also produced proline peptides as products of protein breakdown. This observation suggests that broad-specificity peptidases are required to generate the X-Pro substrates of peptidases P and Q. A strain lacking six peptidases (N, A, B, D, P, and Q) was constructed and produced less free proline from protein breakdown than either the pepP pepQ strain or the pepN pepA pepB pepD strain. These observations suggest that the degradation of peptide intermediates involves the sequential removal of N-terminal amino acids and requires both broad-specificity aminopeptidases (peptidases N, A, and B) and the X-Pro-specific aminopeptidase, peptidase P.     

Pathway for degradation of peptides generated by proteasomes: a key role for thimet oligopeptidase and other metallopeptidases

The degradation of cellular proteins by proteasomes generates peptides 2-24 residues long, which are hydrolyzed rapidly to amino acids. To define the final steps in this pathway and the responsible peptidases, we fractionated by size the peptides generated by proteasomes from beta-[14C]casein and studied in HeLa cell extracts the degradation of the 9-17 residue fraction and also of synthetic deca- and dodecapeptide libraries, because peptides of this size serve as precursors to MHC class I antigenic peptides. Their hydrolysis was followed by measuring the generation of smaller peptides or of new amino groups using fluorescamine. The 14C-labeled peptides released by 20 S proteasomes could not be degraded further by proteasomes. However, their degradation in the extracts and that of the peptide libraries was completely blocked by o-phenanthroline and thus required metallopeptidases. One such endopeptidase, thimet oligopeptidase (TOP), which was recently shown to degrade many antigenic precursors in the cytosol, was found to play a major role in degrading proteasome products. Inhibition or immunodepletion of TOP decreased their degradation and that of the peptide libraries by 30-50%. Pure TOP failed to degrade proteasome products 18-24 residues long but degraded the 9-17 residue fraction to peptides of 6-9 residues. When aminopeptidases in the cell extract were inhibited with bestatin, the 9-17 residue proteasome products were also converted to peptides of 6-9 residues, instead of smaller products. Accordingly, the cytosolic aminopeptidase, leucine aminopeptidase, could not degrade the 9-17 residue fraction but hydrolyzed the peptides generated by TOP to smaller products, recapitulating the process in cell extracts. Inactivation of both TOP and aminopeptidases blocked the degradation of proteasome products and peptide libraries nearly completely. Thus, degradation of most 9-17 residue proteasome products is initiated by endoproteolytic cleavages, primarily by TOP, and the resulting 6-9 residue fragments are further digested to amino acids by aminopeptidases.     

Hydrolysis of alphas1- and beta-casein-derived peptides with a broad specificity aminopeptidase and proline specific aminopeptidases from Lactococcus lactis subsp. cremoris AM2

Aminopeptidase hydrolysis of alpha(s)1 - and beta-casein-derived synthetic peptides containing non-consecutive and consecutive proline residues was characterised. Aminopeptidase P (Pep P) (EC 3.4.11.9) or post-proline dipeptidyl aminopeptidase (PPDA) (EC 3.4.14.5) along with lysine-paranitroanilide hydrolase (KpNA-H) (EC 3.4.11.1) activities are required in the degradation of peptides containing non-consecutive proline residues. However, both Pep P and PPDA along with KpNA-H are required for hydrolysis of peptides containing consecutive proline residues. The results demonstrate the mechanism by which combinations of purified general and proline specific aminopeptidases from Lactococcus lactis subsp. cremoris AM2 hydrolyse peptides containing proline residues.     

Puromycin-sensitive aminopeptidase is the major peptidase responsible for digesting polyglutamine sequences released by proteasomes during protein degradation

Long stretches of glutamine (Q) residues are found in many cellular proteins. Expansion of these polyglutamine (polyQ) sequences is the underlying cause of several neurodegenerative diseases (e.g. Huntington's disease). Eukaryotic proteasomes have been found to digest polyQ sequences in proteins very slowly, or not at all, and to release such potentially toxic sequences for degradation by other peptidases. To identify these key peptidases, we investigated the degradation in cell extracts of model Q-rich fluorescent substrates and peptides containing 10-30 Q's. Their degradation at neutral pH was due to a single aminopeptidase, the puromycin-sensitive aminopeptidase (PSA, cytosol alanyl aminopeptidase). No other known cytosolic aminopeptidase or endopeptidase was found to digest these polyQ peptides. Although tripeptidyl peptidase II (TPPII) exhibited limited activity, studies with specific inhibitors, pure enzymes and extracts of cells treated with siRNA for TPPII or PSA showed PSA to be the rate-limiting activity against polyQ peptides up to 30 residues long. (PSA digests such Q sequences, shorter ones and typical (non-repeating) peptides at similar rates.) Thus, PSA, which is induced in neurons expressing mutant huntingtin, appears critical in preventing the accumulation of polyQ peptides in normal cells, and its activity may influence susceptibility to polyQ diseases.     

Osmoprotection of Bacillus subtilis through Import and Proteolysis of Proline-Containing Peptides

Bacillus subtilis can attain cellular protection against the detrimental effects of high osmolarity through osmotically induced de novo synthesis and uptake of the compatible solute l-proline. We have now found that B. subtilis can also exploit exogenously provided proline-containing peptides of various lengths and compositions as osmoprotectants. Osmoprotection by these types of peptides is generally dependent on their import via the peptide transport systems (Dpp, Opp, App, and DtpT) operating in B. subtilis and relies on their hydrolysis to liberate proline. The effectiveness with which proline-containing peptides confer osmoprotection varies considerably, and this can be correlated with the amount of the liberated and subsequently accumulated free proline by the osmotically stressed cell. Through gene disruption experiments, growth studies, and the quantification of the intracellular proline pool, we have identified the PapA (YqhT) and PapB (YkvY) peptidases as responsible for the hydrolysis of various types of Xaa-Pro dipeptides and Xaa-Pro-Xaa tripeptides. The PapA and PapB peptidases possess overlapping substrate specificities. In contrast, osmoprotection by peptides of various lengths and compositions with a proline residue positioned at their N terminus was not affected by defects in the PapA and PapB peptidases. Taken together, our data provide new insight into the physiology of the osmotic stress response of B. subtilis. They illustrate the flexibility of this ubiquitously distributed microorganism to effectively exploit environmental resources in its acclimatization to sustained high-osmolarity surroundings through the accumulation of compatible solutes.     

A Group of Weakly Bound to Neurons Extracellular Metallopeptidases (NEMPs)

We have found that isolated from mammalian brain (rat, bovine) axonal endings (synaptosomes) degrade peptides of different composition. With the use of low concentration of non ionic detergent Triton X-100 (0.05–0.1?%) four low specific metallopeptidases were detached from synaptosomes. These peptidases were named Neuronal EctoMetalloPeptidases (NEMPs). Using specially designed test-peptides they were characterized as: carboxypeptidase (NEMP1), aminopeptidase (NEMP2) and endopeptidases NEMP3 and NEMP4. NEMPs are true peptidases (oligopeptidases), because they are able efficiently degrade peptides containing less than 40 amino acid residues. Specific properties of some NEMPs were revealed. NEMP1 is a small protein (molecular mass of about 10 kDa), which tends to dynamic oligomerization. NEMP3 needs activation. Some amino acids activate this enzyme. As far as we know, these properties were not ascribed to the known similarly localized peptidases. A possible physiological function of low specific NEMPs is participation in control of wide range of neuropeptides secreted in the synaptic cleft. However, NEMPs also due to their low specificity can destroy introduced in brain therapeutic peptides. The data obtained in this study open new opportunities for the protection of synthetic therapeutic peptides in brain and, possibly, in other tissues.     

The utilization of prolyl peptides by Escherichia coli

Peptides that have an N-terminal proline residue are taken up by Escherichia coli and are degraded by intracellular peptidases. A mutant that is unable to transport oligopeptides with N-terminal alpha-amino acids is also unable to transport the peptides with N-terminal proline. Dipeptides and oligopeptides can prevent the uptake of the corresponding prolyl peptides and the converse competitive interactions are also observed. Although the peptide alpha-amino group is essential to the process of peptide transport, the results with the prolyl peptides indicate that the dipeptide and oligopeptide permeases can handle peptides with either an alpha-amino or alpha-imino group.     

Structural studies of a signal peptide in complex with signal peptidase I cytoplasmic domain: the stabilizing effect of membrane-mimetics on the acquired fold

A protein destined for export from the cell cytoplasm is synthesized as a preprotein with an amino-terminal signal peptide. In Escherichia coli, typically signal peptides that guide preproteins into the SecYEG protein conduction channel are subsequently removed by signal peptidase I. To understand the mechanism of this critical step, we have assessed the conformation of the signal peptide when bound to signal peptidase by solution nuclear magnetic resonance. We employed a soluble form of signal peptidase, which laks the two transmembrane domains (SPase I Δ2-75), and the E. coli alkaline phosphatase signal peptide. Using a transferred NOE approach, we found clear evidence of a weak peptide-enzyme complex formation. The peptide adopts a U-turn shape originating from the proline residues within the primary sequence that is stabilized by its interaction with the peptidase and leaves key residues of the cleavage region exposed for proteolysis. In dodecylphosphocholine (DPC) micelles the signal peptide also adopts a U-turn shape comparable with that observed in association with the enzyme. In both environments this conformation is stabilized by the signal peptide phenylalanine side chain-interaction with enzyme or lipid mimetic. Moreover, in the presence of DPC, the N-terminal core region residues of the peptide adopt a helical motif and based on PRE (paramagnetic relaxation enhancement) experiments are shown to be buried within the membrane. Taken together, this is consistent with proteolysis of the preprotein occurring while the signal peptide remains in the bilayer and the enzyme active site functioning at the membrane surface.     

The final N-terminal trimming of a subaminoterminal proline-containing HLA class I-restricted antigenic peptide in the cytosol is mediated by two peptidases

The proteasome produces MHC class I-restricted antigenic peptides carrying N-terminal extensions, which are trimmed by other peptidases in the cytosol or within the endoplasmic reticulum. In this study, we show that the N-terminal editing of an antigenic peptide with a predicted low TAP affinity can occur in the cytosol. Using proteomics, we identified two cytosolic peptidases, tripeptidyl peptidase II and puromycin-sensitive aminopeptidase, that trimmed the N-terminal extensions of the precursors produced by the proteasome, and led to a transient enrichment of the final antigenic peptide. These peptidases acted either sequentially or redundantly, depending on the extension remaining at the N terminus of the peptides released from the proteasome. Inhibition of these peptidases abolished the CTL-mediated recognition of Ag-expressing cells. Although we observed some proteolytic activity in fractions enriched in endoplasmic reticulum, it could not compensate for the loss of tripeptidyl peptidase II/puromycin-sensitive aminopeptidase activities.     

S 17092-1, a highly potent, specific and cell permeant inhibitor of human proline endopeptidase

Several lines of evidence indicate that proline endopeptidase (PE) could participate to the symptomatology and/or etiology of Alzheimer's disease. Thus, proline endopeptidase appears to contribute to the degradation of neuropeptides involved in learning and memory and could also control the production of the amyloidogenic peptide Abeta. Therefore the design of potent, selective and permeant inhibitors of human PE should lead to potential probes to assess the genuine contribution of this enzyme in Alzheimer's pathology. A novel perhydroindol carboxylic derivative, S17092-1 inhibits the hydrolysis of Z-Gly-Pro-7AMC-hydrolysing activity present in human brain nuclei with a high affinity (Ki = 1 nM) and behaves as a highly potent (Ki = 1.5 nM) inhibitor of partially purified human PE. By contrast, S17092-1 is unable to affect a series of other peptidases including aminopeptidases B and M, dipeptidylaminopeptidase IV, endopeptidases 3.4.24.11, 3.4.24.15, 3.4.24.16, calpains and angiotensin-converting enzyme. Furthermore, we show that the embryonic human kidney 293 cell line displays an intracellular PE-like activity that is blocked after preincubating cells with S17092-1, indicating that this inhibitor penetrates in HEK293 cells and could affect intracellular human PE. Altogether, we establish that S17092-1 behaves as a highly potent, specific and cell permeant inhibitor of human proline endopeptidase and can be seen as a probe to examine PE contribution in Alzheimer's disease.     

P-I class metalloproteinase from Bothrops moojeni venom is a post-proline cleaving peptidase with kininogenase activity: Insights into substrate selectivity and kinetic behavior

Snake venom metalloproteinases (SVMPs) belonging to P-I class are able to hydrolyze extracellular matrix proteins and coagulation factors triggering local and systemic reactions by multiple molecular mechanisms that are not fully understood. BmooMPα-I, a P-I class SMVP from Bothrops moojeni venom, was active upon neuro- and vaso-active peptides including angiotensin I, bradykinin, neurotensin, oxytocin and substance P. Interestingly, BmooMPα-I showed a strong bias towards hydrolysis after proline residues, which is unusual for most of characterized peptidases. Moreover, the enzyme showed kininogenase activity similar to that observed in plasma and cells by kallikrein. FRET peptide assays indicated a relative promiscuity at its S₂–S′₂ subsites, with proline determining the scissile bond. This unusual post-proline cleaving activity was confirmed by the efficient hydrolysis of the synthetic combinatorial library MCA-GXXPXXQ-EDDnp, described as resistant for canonical peptidases, only after Pro residues. Structural analysis of the tripeptide LPL complexed with BmooMPα-I, generated by molecular dynamics simulations, assisted in defining the subsites and provided the structural basis for subsite preferences such as the restriction of basic residues at the S₂ subsite due to repulsive electrostatic effects and the steric impediment for large aliphatic or aromatic side chains at the S₁ subsite. These new functional and structural findings provided a further understanding of the molecular mechanisms governing the physiological effects of this important class of enzymes in envenomation process.     

Translocating proline-rich peptides from the antimicrobial peptide bactenecin 7

The intracellular delivery of most peptides, proteins, and nucleotides to the cytoplasm and nucleus is impeded by the cell membrane. To allow simplified, noninvasive delivery of attached cargo, cell-permeant peptides that are either highly cationic or hydrophobic have been utilized. Because cell-permeable peptides share half of the structural features of antimicrobial peptides containing clusters of charge and hydrophobic residues, we have explored antimicrobial peptides as templates for designing cell-permeant peptides. We prepared synthetic fragments of Bac 7, an antimicrobial peptide with four 14-residue repeats from the bactenecin family. The dual functions of cell permeability and antimicrobial activity of Bac 7 were colocalized at the N-terminal 24 residues of Bac 7. In general, long fragments of Bac(1-24) containing both regions were bactericidal and cell-permeable, whereas short fragments with only a cationic or hydrophobic region were cell-permeant without the attendant microbicidal activity when measured in a fluorescence quantitation assay and by confocal microscopy. In addition, the highly cationic fragments were capable of traversing the cell membrane and residing within the nucleus. A common characteristic shared by the cell-permeant Bac(1-24) fragments, irrespective of their number of charged cationic amino acids, is their high proline content. A 10-residue proline-rich peptide with two arginine residues was capable of delivering a noncovalently linked protein into cells. Thus, the proline-rich peptides represent a potentially new class of cell-permeant peptides for intracellular delivery of protein cargo. Furthermore, our results suggest that antimicrobial peptides may represent a rich source of templates for designing cell-permeant peptides.     

Major histocompatibility complex class I-presented antigenic peptides are degraded in cytosolic extracts primarily by thimet oligopeptidase

Nearly all peptides generated by proteasomes during protein degradation are digested rapidly to amino acids, but a few proteasomal products escape this fate and are presented to the immune system on cell surface major histocompatibility complex class I molecules. To test whether these antigenic peptides may be inherently resistant to cytosolic peptidases, six different antigenic peptides were incubated with HeLa cell extracts. All six were degraded rapidly by a process involving o-phenanthroline-sensitive metallopeptidases. One antigenic peptide, FAPGNYPAL, was rapidly destroyed in the extracts by a bestatin-sensitive exopeptidase, apparently by the puromycin-sensitive aminopeptidase. The disappearance of the other five was reduced 30-90% by a specific inhibitor of the cytosolic endopeptidase, thimet oligopeptidase (TOP) (EC ), whose physiological function(s) have been unclear and controversial. All these peptides were sensitive to pure recombinant TOP. Furthermore, upon fractionation of the extracts, the major peptidase peak that degraded the ovalbumin-derived epitope, SIINFEKL, co-purified with TOP. In the extracts, TOP also catalyzed rapid degradation of N-extended variants of SIINFEKL and of other antigenic peptides, which in vivo can serve as precursors of these major histocompatibility complex-presented epitopes. This enzyme (unlike cell proteins that promote production of antigenic peptides) is not regulated by interferon-gamma. TOP seems to be primarily responsible for the rapid breakdown of antigenic peptides in cytosolic extracts, and our related studies (A. X. Y. Mo, K. Lemerise, W. Zeng, Y. Shen, C. R. Abraham, A. L. Goldberg, and K. L. Rock, submitted for publication) indicate that TOP by destroying such peptides limits antigen presentation in vivo.     

Recycling or regulation? The role of amino-terminal modifying enzymes

Post-translational modifications are essential for a variety of functions, such as the translocation, activation, regulation, and, ultimately, degradation of proteins. The amino-terminal (N-terminal) region is a particularly active area for such alterations. Three types of reactions predominate: limited proteolysis to remove one or more amino acids; modification of the alpha-amino group; and side-chain-specific changes. The N-terminal peptidases expose penultimate residues, providing new substrates for peptidase or transferase action. These enzymes can act sequentially or competitively to influence a protein's longevity, location or activity. N-terminal modifying enzymes (NTMEs) might target a protein for ubiquitination and degradation or protect a protein from rapid turnover. The N-terminal peptidases might also have important roles in processing the peptides that are released from the proteasome. Plant NTMEs have roles in senescence, meiosis and defense, and proposed roles in polar auxin transport.     

Inhibition of a prolyl hydroxylase domain (PHD) by substrate analog peptides

Oxygen dependent degradation of hypoxia-inducible factor (HIF)-1α is triggered with hydroxylation by proline hydroxylase domain 2 (PHD2) under normoxic conditions. Some of previously developed PHD2 inhibitors show a considerable potency against factor inhibiting HIF (FIH), the HIF asparagine hydroxylase. For specific inhibition of PHD2, we have synthesized peptides containing 556-575 residues of HIF-1α with modifications at the Pro-564 and examined their inhibitory effect against PHD2. Adopting fluorescence polarization-based assays, we evaluated inhibitory potency of the peptides and selected potent inhibitors. These PHD2 inhibitor peptides showed no significant potency against FIH, demonstrating their specific inhibitory effect on PHD2.     

Side chain-backbone hydrogen bonding contributes to helix stability in peptides derived from an alpha-helical region of carboxypeptidase A

Recently, Presta and Rose proposed that a necessary condition for helix formation is the presence of residues at the N- and C-termini (called NTBs and CTBs) whose side chains can form hydrogen bonds with the initial four amides and the last four carbonyls of the helix, which otherwise lack intrahelical hydrogen bonding partners. We have tested this hypothesis by conformational analysis by circular dichroism (CD) of a synthetic peptide corresponding to a region (171-188) of the protein carboxypeptidase A; in the protein, residues 174 to 186 are helical and are flanked by NTBs and CTBs. Since helix formation in this peptide may also be stabilized by electrostatic interactions, we have compared the helical content of the native peptide with that of several modified peptides designed to enable dissection of different contributions to helix stability. As expected, helix dipole interactions appear to contribute substantially, but we conclude that hydrogen bonding interactions as proposed by Presta and Rose also stabilize helix formation. To assist in comparison of different peptides, we have introduced two concentration-independent CD parameters which are sensitive probes of helix formation.