26S proteasomes and immunoproteasomes produce mainly N-extended versions of an antigenic peptide

Protein degradation by proteasomes is the source of most antigenic peptides presented on MHC class I molecules. To determine whether proteasomes generate these peptides directly or longer precursors, we developed new methods to measure the efficiency with which 26S and 20S particles, during degradation of a protein, generate the presented epitope or potential precursors. Breakdown of ovalbumin by the 26S and 20S proteasomes yielded the immunodominant peptide SIINFEKL, but produced primarily variants containing 1-7 additional N-terminal residues. Only 6-8% of the times that ovalbumin molecules were digested was a SIINFEKL or an N-extended version produced. Surprisingly, immunoproteasomes which contain the interferon-gamma-induced beta-subunits and are more efficient in antigen presentation, produced no more SIINFEKL than proteasomes. However, the immunoproteasomes released 2-4 times more of certain N-extended versions. These observations show that the changes in cleavage specificity of immunoproteasomes influence not only the C-terminus, but also the N-terminus of potential antigenic peptides, and suggest that most MHC-presented peptides result from N-terminal trimming of larger proteasome products by aminopeptidases (e.g. the interferon-gamma-induced enzyme leucine aminopeptidase).     

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.     

Sequences that flank subdominant and cryptic epitopes influence the proteolytic generation of MHC class I-presented peptides

The proteasome has been shown to make the proper C-terminal cleavage for the generation of several immunodominant class I-presented peptides whereas aminopeptidases generate their proper N termini. In this study, we show that these two distinct proteolytic processes are also involved in generating a subdominant OVA peptide KVVRFDKL (K-L). Moreover, proteasome inhibitors did not enhance the presentation of any K-L construct, suggesting that destruction of this peptide by proteasomes, if any, does not limit its presentation. We have further examined in intact cells the influence of residues flanking this epitope on these proteolytic processes. When the N-terminal flanking residues of K-L are fused to an immunodominant OVA peptide SIINFEKL (S-L), the presentation of S-L is reduced as compared with a construct with its natural flanking sequence and was not inhibited (or enhanced) by proteasome inhibitors. Similarly, a reduction in presentation was observed when the C-terminal flanking residues of the subdominant epitope were attached to S-L. A detailed analysis revealed that the Pro at the P1' position of K-L was responsible for this reduction, and presentation of these C-terminally extended constructs was sensitive to proteasome inhibitor. The study suggests that both the N- and C-terminal flanks of the subdominant peptide are suboptimal for Ag presentation. Moreover, three of four C-terminal residues that flank other subdominant or cryptic epitopes in OVA reduced the presentation of S-L. Therefore, the residues that flank the C termini of several subdominant and cryptic epitopes are often suboptimal for cleavage and may contribute to the phenomenon of immunodominance.     

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.     

Anti-peptide antibody blocks peptide binding to MHC class I molecules in the endoplasmic reticulum

The finding that MHC class I molecules are physically associated with the TAP transporter has suggested that peptides may be directly transported into the binding groove of the class I molecules rather than into the lumen of the endoplasmic reticulum (ER) where they subsequently would encounter class I molecules by diffusion. Such a mechanism would protect peptides from peptidases in the ER and/or escaping back into the cytoplasm. However, we find that an anti-peptide Ab that is cotranslationally transported into the ER prevents TAP-transported peptides from being presented on class I molecules. The Ab only blocks the binding of its cognate peptide (SIINFEKL) but not other peptides (KVVRFKDL, ASNENMETM, and FAPGNYPAL). Therefore, most TAP-transported peptides must diffuse through the lumen of the ER before binding stably to MHC class I molecules.     

A role for a novel luminal endoplasmic reticulum aminopeptidase in final trimming of 26 S proteasome-generated major histocompatability complex class I antigenic peptides

Peptides presented to cytotoxic T lymphocytes by the class I major histocompatability complex are 8-11 residues long. Although proteasomal activity generates the precise C termini of antigenic epitopes, the mechanism(s) involved in generation of the precise N termini is largely unknown. To investigate the mechanism of N-terminal peptide processing, we used a cell-free system in which two recombinant ornithine decarboxylase (ODC) constructs, one expressing the native H2-K(b)-restricted ovalbumin (ova)-derived epitope SIINFEKL (ODC-ova) and the other expressing the extended epitope LESIINFEKL (ODC-LEova), were targeted to degradation by 26 S proteasomes followed by import into microsomes. We found that the cleavage specificity of the 26 S proteasome was influenced by the N-terminal flanking amino acids leading to significantly different yields of the final epitope SIINFEKL. Following incubation in the presence of purified 26 S proteasome, ODC-LEova generated largely ESIINFEKL that was efficiently converted to the final epitope SIINFEKL following translocation into microsomes. The conversion of ESIINFEKL to SIINFEKL was strictly dependent on the presence of H2-K(b) and was completely inhibited by the metalloaminopeptidase inhibitor 1,10-phenanthroline. Importantly, the converting activity was resistant to a stringent salt/EDTA wash of the microsomes and was only apparent when transport of TAP, the transporter associated with antigen processing, was facilitated. These results strongly suggest a crucial role for a luminal endoplasmic reticulum-resident metalloaminopeptidase in the N-terminal trimming of major histocompatability complex class I-associated peptides.     

A Novel Aminopeptidase with Highest Preference for Lysine

Neuropeptides are formed from sedentary precursors to smaller, active peptides by processing enzymes cleaving at paired basic residues. The process generates peptide intermediates with additional Lys or Arg residues at their NH(2) and COOH termini; the N-terminal basic amino acids are later removed by specific aminopeptidases. We report here a novel lysine-specific aminopeptidase (KAP) of ubiquitous distribution. The enzyme was resolved from puromycin-sensitive aminopeptidase (PSA), aminopeptidase B (APB), and neuron-specific aminopeptidase (NAP). It was purified by FPLC after (NH(4))(2)SO(4) precipitation. The purified KAP had a K(m) of 333 microM with a V(max) of 0.7 nmol Lys ssNA/min/mg protein. N-terminal basic amino acids, Lys in particular, were its favorable substrates. KAP was inhibited by chelating agents and by serine protease inhibitors. It was highly sensitive to aminopeptidase inhibitor bestatin, but insensitive to puromycin and amastatin, showing that KAP is distinct from PSA, NAP, and aminopeptidase A (APA). The 62,000-Da enzyme had a pH optimum at 7.5 and NaCl was its strongest activator. However, metals could not restore KAP's activity after it was dialyzed against EGTA. Our data indicated that rat KAP did not resemble any aminopeptidases as well as the microbial lysine aminopeptidases.     

Positioning of aminopeptidase inhibitors in next generation cancer therapy

Aminopeptidases represent a class of (zinc) metalloenzymes that catalyze the cleavage of amino acids nearby the N-terminus of polypeptides, resulting in hydrolysis of peptide bonds. Aminopeptidases operate downstream of the ubiquitin–proteasome pathway and are implicated in the final step of intracellular protein degradation either by trimming proteasome-generated peptides for antigen presentation or full hydrolysis into free amino acids for recycling in renewed protein synthesis. This review focuses on the function and subcellular location of five key aminopeptidases (aminopeptidase N, leucine aminopeptidase, puromycin-sensitive aminopeptidase, leukotriene A₄ hydrolase and endoplasmic reticulum aminopeptidase 1/2) and their association with different diseases, in particular cancer and their current position as target for therapeutic intervention by aminopeptidase inhibitors. Historically, bestatin was the first prototypical aminopeptidase inhibitor that entered the clinic 35 years ago and is still used for the treatment of lung cancer. More recently, new generation aminopeptidase inhibitors became available, including the aminopeptidase inhibitor prodrug tosedostat, which is currently tested in phase II clinical trials for acute myeloid leukemia. Beyond bestatin and tosedostat, medicinal chemistry has emerged with additional series of potential aminopeptidases inhibitors which are still in an early phase of (pre)clinical investigations. The expanded knowledge of the unique mechanism of action of aminopeptidases has revived interest in aminopeptidase inhibitors for drug combination regimens in anti-cancer treatment. In this context, this review will discuss relevant features and mechanisms of action of aminopeptidases and will also elaborate on factors contributing to aminopeptidase inhibitor efficacy and/or loss of efficacy due to drug resistance-related phenomena. Together, a growing body of data point to aminopeptidase inhibitors as attractive tools for combination chemotherapy, hence their implementation may be a step forward in a new era of personalized treatment of cancer patients.     

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.     

Processing of exogenous antigens for presentation by class I MHC molecules involves post-Golgi peptide exchange influenced by peptide-MHC complex stability and acidic pH

Vacuolar alternate class I MHC (MHC-I) Ag processing allows presentation of exogenous Ag by MHC-I molecules with binding of antigenic peptides to post-Golgi MHC-I molecules. We investigated the role of previously bound peptides and their dissociation in generating peptide-receptive MHC-I molecules. TAP1-knockout macrophages were incubated overnight with an initial exogenous peptide, producing a large cohort of peptide-K(b) complexes that could influence subsequent peptide dissociation/exchange. Initial incubation with FAPGNYPAL, KVVRFDKL, or RGYVYQGL enhanced rather than reduced subsequent binding and presentation of a readout peptide (SIINFEKL or FAPGNYPAL) to T cells. Thus, K(b) molecules may be stabilized by an initial (stabilizing) peptide, enhancing their ability to bind readout peptide and implicating peptide dissociation/exchange. In contrast, incubation with SIINFEKL as stabilizing peptide reduced presentation of readout peptide. SIINFEKL-K(b) complexes were more stable than other peptide-K(b) complexes, which may limit their contribution to peptide exchange. Stabilizing peptides (FAPGNYPAL, KVVRFDKL, or RGYVYQGL) enhanced alternate MHC-I processing of HB101.Crl-OVA (Escherichia coli expressing an OVA fusion protein), indicating that alternate MHC-I Ag processing involves peptide dissociation/exchange. Stabilizing peptide enhanced processing of HB101.Crl-OVA more than presentation of exogenous OVA peptide (SIINFEKL), suggesting that peptide dissociation/exchange may be enhanced in the acidic phagosomal processing environment. Furthermore, exposure of cells to acidic pH increased subsequent binding and presentation of readout peptide. Thus, peptide dissociation/exchange contributes to alternate MHC-I Ag processing and may be influenced by both stability of peptide-MHC-I complexes and pH.     

Trafficking of exogenous peptides into proteasome-dependent major histocompatibility complex class I pathway following enterotoxin B subunit-mediated delivery

The B-subunit component of Escherichia coli heat-labile enterotoxin (EtxB), which binds to cell surface GM1 ganglioside receptors, was recently shown to be a highly effective vehicle for delivery of conjugated peptides into the major histocompatibility complex (MHC) class I pathway. In this study we have investigated the pathway of epitope delivery. The peptides used contained the epitope either located at the C terminus or with a C-terminal extension. Pretreatment of cells with cholesterol-disrupting agents blocked transport of EtxB conjugates to the Golgi/endoplasmic reticulum, but did not affect EtxB-mediated MHC class I presentation. Under these conditions, EtxB conjugates entered EEA1-positive early endosomes where peptides were cleaved and translocated into the cytosol. Endosome acidification was required for epitope presentation. Purified 20 S immunoproteasomes were able to generate the epitope from peptides in vitro, but 26 S proteasomes were not. Only presentation from the C-terminal extended peptide was proteasome-dependent in cells, and this was found to be significantly slower than presentation from peptides with the epitope at the C terminus. These results implicate the proteasome in the generation of the correct C terminus of the epitope and are consistent with proteasome-independent N-terminal trimming. Epitope presentation was blocked in a TAP-deficient cell line, providing further evidence that conjugated peptides enter the cytosol as well as demonstrating a requirement for the peptide transporter. Our findings demonstrate the utility of EtxB-mediated peptide delivery for rapid and efficient loading of MHC class I epitopes in several different cell types. Conjugated peptides are released from early endosomes into the cytosol where they gain access to proteasomes and TAP in the "classical" pathway of class I presentation.     

The use of peptide analogs with improved stability and MHC binding capacity to inhibit antigen presentation in vitro and in vivo

The identification of a core region for OVA 323-339, which is critical in determining binding to IAd, has enabled us to generate a series of analog peptides in which this core region was extended at both the N and C termini with different amino acid residues. When assessed for binding capacity, several peptides were shown to have increased affinity for IAd compared with the parent sequence, and in addition, some peptides had acquired binding specificities for class II MHC haplotypes not present for OVA 323-339. These peptides were next examined for their ability to inhibit T cell responses in vitro and in vivo. The correlation between binding and the ability to inhibit T cell activation in vitro was good. However, when assessed in vivo, it was clear that high Ia binding was not sufficient in itself to define the inhibitory capacity of a given peptide. That this discrepancy was due to differences in degradation of the core-extended peptides was suggested by 1) results from an inhibition of Ag presentation assay, in which the pulse period with Ag and inhibitor was extended to 20 h; and 2) direct analysis of peptide stability by using reverse phase HPLC. Finally, by protecting the peptide from degradation with N- and C-terminal substitutions of D-amino acids, the inhibitory capacity of an unstable core-extended peptide in vitro could be greatly enhanced. These data indicate that the core extension approach may be one method by which antagonists for MHC class II molecules may be generated.     

Crystal structure of TET protease reveals complementary protein degradation pathways in prokaryotes

Protein degradation is an essential and strictly controlled process with proteasome and functionally related proteases representing its central part. Tricorn protease (TRI) has been shown to act downstream of the proteasome, degrading produced peptides. Recently, a novel large prokaryotic aminopeptidase oligomeric complex, named TET, has been identified. This complex degrades peptides of different length in organisms where TRI is not present. We determined the crystal structure of TET from the thermophilic archaeon Pyrococcus horikoshii at 1.6 A resolution in native form and in complex with the inhibitor amastatin. We demonstrate that, beside the novel tetrahedral oligomerisation pattern, TET possesses a unique mechanism of substrate attraction and orientation. TET sequentially degrades peptides produced by the proteasome to single amino acids. Furthermore, we reconstituted in vitro the minimal protein degradation system from initial unfolding of labelled protein substrates, up to release of free amino acids. We propose that TET and TRI act as functional analogues in different organisms, with TET being more widely distributed. Thus, TET and TRI represent two evolutionarily diverged pathways of peptide degradation in prokaryotes.     

Modification of the amino terminus of a class II epitope confers resistance to degradation by CD13 on dendritic cells and enhances presentation to T cells

Dendritic cells and human B cell lines were compared for ability to present synthetic peptides corresponding to residues 145-159 and 188-203 of human Ig kappa-chains to peptide-specific mouse T cell hybridomas restricted by HLA-DR4Dw4. B cell lines presented both peptides, but dendritic cells could only efficiently present the latter epitope. In this paper, we show that dendritic cells degrade the 145-159 peptide, removing four residues from the amino terminus. Binding of the peptide to the class II restriction element is not required for this process. The degradation product is resistant to further cleavage, accumulates in the culture supernatant, and does not bind to HLA-DR4Dw4 or stimulate T cell reactivity. Cleavage can be blocked with bestatin, but not with other protease inhibitors tested, or by a mAb directed against aminopeptidase N (CD13). Addition of an acetyl group to the amino terminus of peptide 145-159 also blocks degradation, and allows dendritic cells to present the peptide to specific T cells with greatly increased efficiency. These results demonstrate that CD13 on dendritic cells is able to selectively and efficiently degrade exogenously provided peptide Ags, in a process that can be blocked by addition of an acetyl group to the amino terminus of the peptide. Modification of the amino terminus of peptide epitopes susceptible to degradation may prove to be useful as a general strategy for enhancing their immunogenicity.     

Degradation and debittering of a tryptic digest from beta-casein by aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2.

The mode of action of purified aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2 on a complex peptide mixture of a tryptic digest from bovine beta-casein was analyzed. The oligopeptides produced in the tryptic digest before and after aminopeptidase N treatment were identified by analysis of the N- and C-terminal amino acid sequences and amino acid compositions of the isolated peptides and by on-line liquid chromatography-mass spectrometry. Incubation of purified peptides with aminopeptidase N resulted in complete hydrolysis of many peptides, while others were only partially hydrolyzed or not hydrolyzed. The tryptic digest of beta-casein exhibits a strong bitter taste, which corresponds to the strong hydrophobicity of several peptides in the tryptic digest of beta-casein. The degradation of the "bitter" tryptic digest by aminopeptidase N resulted in a decrease of hydrophobic peptides and a drastic decrease of bitterness of the reaction mixture.     

Analysis of the role of bleomycin hydrolase in antigen presentation and the generation of CD8 T cell responses

Long oligopeptides (>10 residues) are generated during the catabolism of cellular proteins in the cytosol. To be presented to T cells, such peptides must be trimmed by aminopeptidases to the proper size (typically 8-10 residues) to stably bind to MHC class I molecules. Aminopeptidases also destroy epitopes by trimming them to even shorter lengths. Bleomycin hydrolase (BH) is a cytosolic aminopeptidase that has been suggested to play a key role in generating MHC class I-presented peptides. We show that BH-deficient cells from mice are unimpaired in their ability to present epitopes from N-extended precursors or whole Ags and express normal levels of MHC class I molecules. Similarly, BH-deficient mice develop normal CD8(+) T cell responses to eight epitopes from three different viruses in vivo. Therefore, BH by itself is not essential for the generation or destruction of MHC class I peptides. In contrast, when BH(-/-) mice are crossed to mice lacking another cytosolic aminopeptidase, leucine aminopeptidase, the resulting BH(-/-)leucine aminopeptidase(-/-) progeny show a selective increase in CD8(+) T cell responses to the gp276 epitope from lymphocytic choriomeningitis virus, whereas the ability to present and respond to several other epitopes is unchanged. Therefore, BH does influence presentation of some Ags, although its role is largely redundant with other aminopeptidases.     

Tripeptidyl peptidase II is the major peptidase needed to trim long antigenic precursors, but is not required for most MHC class I antigen presentation

Recent reports concluded that tripeptidyl peptidase (TPPII) is essential for MHC class I Ag presentation and that the proteasome in vivo mainly releases peptides 16 residues or longer that require processing by TPPII. However, we find that eliminating TPPII from human cells using small interfering RNA did not decrease the overall supply of peptides to MHC class I molecules and reduced only modestly the presentation of SIINFEKL from OVA, while treatment with proteasome inhibitors reduced these processes dramatically. Purified TPPII digests peptides from 6 to 30 residues long at similar rates, but eliminating TPPII in cells reduced the processing of long antigenic precursors (14-17 residues) more than short ones (9-12 residues). Therefore, TPPII appears to be the major peptidase capable of processing proteasome products longer than 14 residues. However, proteasomes in vivo (like purified proteasomes) release relatively few such peptides, and these peptides processed by TPPII require further trimming in the endoplasmic reticulum (ER) by ER aminopeptidase 1 for presentation. Taken together, these observations demonstrate that TPPII plays a specialized role in Ag processing and one that is not essential for the generation of most presented peptides. Moreover, these findings reveal that three sequential proteolytic steps (by proteasomes, TPPII, and then ER aminopepsidase 1) are required for the generation of a subset of epitopes.     

Degradation of Dynorphin-Related Peptides by the Puromycin-Sensitive Aminopeptidase and Aminopeptidase M

Abstract: The degradation of dynorphin-related peptides by the puromycin-sensitive aminopeptidase and aminopeptidase M was examined using these peptides as alternate substrate inhibitors. K _i determinations showed that both aminopeptidases exhibit a higher affinity for longer dynorphin-related peptides, i.e., K _i for dynorphin A-17 = 23–30 n M with the K _i increasing to 25–50 µ M for the enkephalin pentapeptides. Binding appears dependent not only on peptide length, but also on its sequence. With aminopeptidase M, as the peptide size increases from five to 10 amino acids, k _cat remains relatively constant; however, as the peptide size increases beyond a decapeptide, k _cat decreases significantly. With the puromycin-sensitive aminopeptidase, similar results were obtained except that k _cat was greatest for the pentapeptide. Thus, if one considers k _cat/ K _m as the relevant kinetic constant for estimating in vivo peptide hydrolysis, these results are consistent with the involvement of aminopeptidase M and the puromycin-sensitive aminopeptidase in the degradation of extended dynorphin-related peptides.     

Characterizing and optimizing protease/peptide inhibitor interactions, a new application for spot synthesis

A new method is presented that uses parallel peptide array synthesis on cellulose membranes to characterize protease/peptide inhibitor interactions. A peptide comprising P5-P4' of the third domain of turkey ovomucoid inhibitor was investigated for both binding to and inhibition of porcine pancreatic elastase. Binding was studied directly on the cellulose membrane, while inhibition was measured by an assay in microtiter plates with punched out peptide spots. The importance of each residue for binding or inhibition was determined by substitutional analyses, exchanging every original amino acid with all other 19 coded amino acids. Seven hundred eighty individual peptides were investigated for binding behavior to porcine pancreatic elastase, and 320 individual peptides were measured in inhibition experiments. The results provide new insights into the interaction between the ovomucoid derived peptide and subsites in the active site of elastase. Combining these data with length analysis we designed new peptides in a step-wise fashion which in the end not only inhibited elastase 400 times more strongly than the original peptide, but are highly specific for the enzyme. In addition, the optimized inhibitor peptide was protected against exopeptidase attack by substituting D-amino acids at both termini.     

Proteasome Inhibitors Alter Levels of Intracellular Peptides in HEK293T and SH-SY5Y Cells

The proteasome cleaves intracellular proteins into peptides. Earlier studies found that treatment of human embryonic kidney 293T (HEK293T) cells with epoxomicin (an irreversible proteasome inhibitor) generally caused a decrease in levels of intracellular peptides. However, bortezomib (an antitumor drug and proteasome inhibitor) caused an unexpected increase in the levels of most intracellular peptides in HEK293T and SH-SY5Y cells. To address this apparent paradox, quantitative peptidomics was used to study the effect of a variety of other proteasome inhibitors on peptide levels in HEK293T and SH-SY5Y cells. Inhibitors tested included carfilzomib, MG132, MG262, MLN2238, AM114, and clasto-Lactacystin β-lactone. Only MG262 caused a substantial elevation in peptide levels that was comparable to the effect of bortezomib, although carfilzomib and MLN2238 elevated the levels of some peptides. To explore off-target effects, the proteosome inhibitors were tested with various cellular peptidases. Bortezomib did not inhibit tripeptidyl peptidase 2 and only weakly inhibited cellular aminopeptidase activity, as did some of the other proteasome inhibitors. However, potent inhibitors of tripeptidyl peptidase 2 (butabindide) and cellular aminopeptidases (bestatin) did not substantially alter the peptidome, indicating that the increase in peptide levels due to proteasome inhibitors is not a result of peptidase inhibition. Although we cannot exclude other possibilities, we presume that the paradoxical increase in peptide levels upon treatment with bortezomib and other inhibitors is the result of allosteric effects of these compounds on the proteasome. Because intracellular peptides are likely to be functional, it is possible that some of the physiologic effects of bortezomib and carfilzomib arise from the perturbation of peptide levels inside the cell.