Exploration of zinc-binding groups for the design of inhibitors for the oxytocinase subfamily of M1 aminopeptidases

The oxytocinase subfamily of M1 aminopeptidases consists of three members, ERAP1, ERAP2 and IRAP that play several important biological roles, including key functions in the generation of antigenic peptides that drive human immune responses. They represent emerging targets for pharmacological manipulation of the immune system, albeit lack of selective inhibitors is hampering these efforts. Most of the previously explored small-molecule binders target the active site of the enzymes via strong interactions with the catalytic zinc(II) atom and, while achieving increased potency, they suffer in selectivity. Continuing our earlier efforts on weaker zinc(II) binding groups (ZBG), like the 3,4-diaminobenzoic acid derivatives (DABA), we herein synthesized and biochemically evaluated analogues of nine potentially weak ZBGs, based on differential substitutions of functionalized pyridinone- and pyridinethione-scaffolds, nicotinic-, isonicotinic-, aminobenzoic- and hydrazinobenzoic-acids. Crystallographic analysis of two analogues in complex with a metalloprotease (MMP-12) revealed unexpected binding topologies, consistent with the observed affinities. Our results suggest that the potency of the compounds as inhibitors of ERAP1, ERAP2 and IRAP is primarily driven by the occupation of active-site specificity pockets and their proper orientation within the enzymes.     

Novel selective inhibitors of aminopeptidases that generate antigenic peptides

Endoplasmic reticulum aminopeptidases, ERAP1 and ERAP2, as well as Insulin regulated aminopeptidase (IRAP) play key roles in antigen processing, and have recently emerged as biologically important targets for manipulation of antigen presentation. Taking advantage of the available structural and substrate-selectivity data for these enzymes, we have rationally designed a new series of inhibitors that display low micromolar activity. The selectivity profile for these three highly homologous aminopeptidases provides a promising avenue for modulating intracellular antigen processing.     

Structural Basis for Antigenic Peptide Recognition and Processing by Endoplasmic Reticulum (ER) Aminopeptidase 2

Endoplasmic reticulum (ER) aminopeptidases process antigenic peptide precursors to generate epitopes for presentation by MHC class I molecules and help shape the antigenic peptide repertoire and cytotoxic T-cell responses. To perform this function, ER aminopeptidases have to recognize and process a vast variety of peptide sequences. To understand how these enzymes recognize substrates, we determined crystal structures of ER aminopeptidase 2 (ERAP2) in complex with a substrate analogue and a peptidic product to 2.5 and 2.7 Å, respectively, and compared them to the apo-form structure determined to 3.0 Å. The peptides were found within the internal cavity of the enzyme with no direct access to the outside solvent. The substrate analogue extends away from the catalytic center toward the distal end of the internal cavity, making interactions with several shallow pockets along the path. A similar configuration was evident for the peptidic product, although decreasing electron density toward its C terminus indicated progressive disorder. Enzymatic analysis confirmed that visualized interactions can either positively or negatively impact in vitro trimming rates. Opportunistic side-chain interactions and lack of deep specificity pockets support a limited-selectivity model for antigenic peptide processing by ERAP2. In contrast to proposed models for the homologous ERAP1, no specific recognition of the peptide C terminus by ERAP2 was evident, consistent with functional differences in length selection and self-activation between these two enzymes. Our results suggest that ERAP2 selects substrates by sequestering them in its internal cavity and allowing opportunistic interactions to determine trimming rates, thus combining substrate permissiveness with sequence bias.     

The internal sequence of the peptide-substrate determines its N-terminus trimming by ERAP1

Background

Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims N-terminally extended antigenic peptide precursors down to mature antigenic peptides for presentation by major histocompatibility complex (MHC) class I molecules. ERAP1 has unique properties for an aminopeptidase being able to trim peptides in vitro based on their length and the nature of their C-termini.

Methodology/Principal Findings

In an effort to better understand the molecular mechanism that ERAP1 uses to trim peptides, we systematically analyzed the enzyme''s substrate preferences using collections of peptide substrates. We discovered strong internal sequence preferences of peptide N-terminus trimming by ERAP1. Preferences were only found for positively charged or hydrophobic residues resulting to trimming rate changes by up to 100 fold for single residue substitutions and more than 40,000 fold for multiple residue substitutions for peptides with identical N-termini. Molecular modelling of ERAP1 revealed a large internal cavity that carries a strong negative electrostatic potential and is large enough to accommodate peptides adjacent to the enzyme''s active site. This model can readily account for the strong preference for positively charged side chains.

Conclusions/Significance

To our knowledge no other aminopeptidase has been described to have such strong preferences for internal residues so distal to the N-terminus. Overall, our findings indicate that the internal sequence of the peptide can affect its trimming by ERAP1 as much as the peptide''s length and C-terminus. We therefore propose that ERAP1 recognizes the full length of its peptide-substrate and not just the N- and C- termini. It is possible that ERAP1 trimming preferences influence the rate of generation and the composition of antigenic peptides in vivo.     

Concerted In Vitro Trimming of Viral HLA-B27-Restricted Ligands by Human ERAP1 and ERAP2 Aminopeptidases

In the classical human leukocyte antigen (HLA) class I antigen processing and presentation pathway, the antigenic peptides are generated from viral proteins by multiple proteolytic cleavages of the proteasome (and in some cases other cytosolic proteases) and transported to the endoplasmic reticulum (ER) lumen where they are exposed to aminopeptidase activity. In human cells, two different ER-resident enzymes, ERAP1 and ERAP2, can trim the N-terminally extended residues of peptide precursors. In this study, the possible cooperative effect of generating five naturally processed HLA-B27 ligands by both proteases was analyzed. We identified differences in the products obtained with increased detection of natural HLA-B27 ligands by comparing double versus single enzyme digestions by mass spectrometry analysis. These in vitro data suggest that each enzyme can use the degradation products of the other as a substrate for new N-terminal trimming, indicating concerted aminoproteolytic activity of ERAP 1 and ERAP2.     

Allotypic variation in antigen processing controls antigenic peptide generation from SARS-CoV-2 S1 spike glycoprotein

Population genetic variability in immune system genes can often underlie variability in immune responses to pathogens. Cytotoxic T-lymphocytes are emerging as critical determinants of both severe acute respiratory syndrome coronavirus 2 infection severity and long-term immunity, after either recovery or vaccination. A hallmark of coronavirus disease 2019 is its highly variable severity and breadth of immune responses between individuals. To address the underlying mechanisms behind this phenomenon, we analyzed the proteolytic processing of S1 spike glycoprotein precursor antigenic peptides across ten common allotypes of endoplasmic reticulum aminopeptidase 1 (ERAP1), a polymorphic intracellular enzyme that can regulate cytotoxic T-lymphocyte responses by generating or destroying antigenic peptides. We utilized a systematic proteomic approach that allows the concurrent analysis of hundreds of trimming reactions in parallel, thus better emulating antigen processing in the cell. While all ERAP1 allotypes were capable of producing optimal ligands for major histocompatibility complex class I molecules, including known severe acute respiratory syndrome coronavirus 2 epitopes, they presented significant differences in peptide sequences produced, suggesting allotype-dependent sequence biases. Allotype 10, previously suggested to be enzymatically deficient, was rather found to be functionally distinct from other allotypes. Our findings suggest that common ERAP1 allotypes can be a major source of heterogeneity in antigen processing and through this mechanism contribute to variable immune responses in coronavirus disease 2019.     

A common single nucleotide polymorphism in endoplasmic reticulum aminopeptidase 2 induces a specificity switch that leads to altered antigen processing

Endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2) cooperate to trim antigenic peptide precursors for loading onto MHC class I molecules and help regulate the adaptive immune response. Common coding single nucleotide polymorphisms in ERAP1 and ERAP2 have been linked with predisposition to human diseases ranging from viral and bacterial infections to autoimmunity and cancer. It has been hypothesized that altered Ag processing by these enzymes is a causal link to disease etiology, but the molecular mechanisms are obscure. We report in this article that the common ERAP2 single nucleotide polymorphism rs2549782 that codes for amino acid variation N392K leads to alterations in both the activity and the specificity of the enzyme. Specifically, the 392N allele excises hydrophobic N-terminal residues from epitope precursors up to 165-fold faster compared with the 392K allele, although both alleles are very similar in excising positively charged N-terminal amino acids. These effects are primarily due to changes in the catalytic turnover rate (k(cat)) and not in the affinity for the substrate. X-ray crystallographic analysis of the ERAP2 392K allele suggests that the polymorphism interferes with the stabilization of the N terminus of the peptide both directly and indirectly through interactions with key residues participating in catalysis. This specificity switch allows the 392N allele of ERAP2 to supplement ERAP1 activity for the removal of hydrophobic N-terminal residues. Our results provide mechanistic insight to the association of this ERAP2 polymorphism with disease and support the idea that polymorphic variation in Ag processing enzymes constitutes a component of immune response variability in humans.     

Secretion of endoplasmic reticulum aminopeptidase 1 is involved in the activation of macrophages induced by lipopolysaccharide and interferon-gamma

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme with an important role in processing antigenic peptides presented to class I major histocompatibility complex in the endoplasmic reticulum. In this study, we found that endoplasmic reticulum-retained ERAP1 was secreted from macrophages in response to activation by treatment with lipopolysaccharide (LPS) and interferon (IFN)-γ and enhanced their phagocytic activity. Enhancement of the phagocytic activity of murine macrophage RAW264.7 cells induced by LPS/IFN-γ was inhibited by a potent aminopeptidase inhibitor, amastatin. The addition of recombinant wild-type but not inactive mutant ERAP1 to culture medium enhanced phagocytosis. These results suggest that enhancement of phagocytic activity is at least in part mediated by secreted ERAP1 through the generation of active peptides processed by the enzyme. Our data reveal ERAP1-mediated activation of macrophages for the first time and will provide new insights into the role of this enzyme in innate immunity.     

A recyclable assay to analyze the NH(2)-terminal trimming of antigenic peptide precursors

The proteasome plays an essential role in the production of MHC class I-restricted antigenic peptides. Recent results have indicated that several peptidases, including tripeptidyl peptidase II and puromycin-sensitive aminopeptidase, could act downstream of the proteasome by trimming NH(2)-terminal extensions of antigenic peptide precursors liberated by the proteasome. In this study, we have developed a solid-phase peptidase assay that allowed us to efficiently purify and immobilize proteasome, tripeptidyl peptidase II, and puromycin-sensitive aminopeptidase. Whereas the first peptidase was active against small fluorogenic peptides, the latter two could also digest antigenic peptide precursors and could be used repeatedly with different precursors. Using three distinct antigenic peptide precursors, we found that tripeptidyl peptidase II never cleaved within the antigenic peptide sequence, suggesting that, aside from its proteolytic activities, it may also play a role in protecting antigenic peptides from complete hydrolysis in the cytosol. This method should be valuable for high throughput screenings of substrate specificity and potential inhibitors.     

Cutting Edge: Coding single nucleotide polymorphisms of endoplasmic reticulum aminopeptidase 1 can affect antigenic peptide generation in vitro by influencing basic enzymatic properties of the enzyme

ER aminopeptidase 1 (ERAP1) customizes antigenic peptide precursors for MHC class I presentation and edits the antigenic peptide repertoire. Coding single nucleotide polymorphisms (SNPs) in ERAP1 were recently linked with predisposition to autoimmune disease, suggesting a link between pathogenesis of autoimmunity and ERAP1-mediated Ag processing. To investigate this possibility, we analyzed the effect that disease-linked SNPs have on Ag processing by ERAP1 in vitro. Michaelis-Menten analysis revealed that the presence of SNPs affects the Michaelis constant and turnover number of the enzyme. Strikingly, specific ERAP1 allele-substrate combinations deviate from standard Michaelis-Menten behavior, demonstrating substrate-inhibition kinetics; to our knowledge, this phenomenon has not been described for this enzyme. Cell-based Ag-presentation analysis was consistent with changes in the substrate inhibition constant K(i), further supporting that ERAP1 allelic composition may affect Ag processing in vivo. We propose that these phenomena should be taken into account when evaluating the possible link between Ag processing and autoimmunity.     

A continuous fluorigenic assay for the measurement of the activity of endoplasmic reticulum aminopeptidase 1: Competition kinetics as a tool for enzyme specificity investigation

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a recently discovered enzyme that plays critical roles in antigen presentation and the immune response. Unlike other aminopeptidases, ERAP1 displays strong sequence preferences for residues distal to the peptide-substrate’s N terminus. This unusual substrate specificity necessitates the development of new assays that are appropriate for the study of such aminopeptidases. Here we describe a continuous fluorigenic assay suitable for the analysis of the enzymatic properties of ERAP1. In this assay, signal is generated by the excision of an internally quenched N-terminal tryptophan residue from a 10mer peptide by the aminopeptidase, resulting in the enhancement of tryptophan fluorescence in the solution. This method overcomes the limitations of previously used fluorigenic and high-performance liquid chromatography (HPLC)-based assays and is appropriate for small molecule inhibitor screening as well as for rapid substrate specificity analysis by kinetic competition experiments. Such efficient peptidic fluorigenic substrates like the ones described here should greatly simplify specificity analysis and inhibitor discovery for ERAP1 and similar aminopeptidases.     

The crystal structure of human endoplasmic reticulum aminopeptidase 2 reveals the atomic basis for distinct roles in antigen processing

Endoplasmic reticulum aminopeptidases ERAP1 and ERAP2 cooperate to trim a vast variety of antigenic peptide precursors to generate mature epitopes for binding to major histocompatibility class I molecules. We report here the first structure of ERAP2 determined at 3.08 ? by X-ray crystallography. On the basis of residual electron density, a lysine residue has been modeled in the active site of the enzyme; thus, the structure corresponds to an enzyme-product complex. The overall domain organization is highly similar to that of the recently determined structure of ERAP1 in its closed conformation. A large internal cavity adjacent to the catalytic site can accommodate large peptide substrates. The ERAP2 structure provides a structural explanation for the different peptide N-terminal specificities between ERAP1 and ERAP2 and suggests that such differences extend throughout the whole peptide sequence. A noncrystallographic dimer observed may constitute a model for a proposed ERAP1-ERAP2 heterodimer. Overall, the structure helps explain how two homologous aminopeptidases cooperate to process a large variety of sequences, a key property of their biological role.     

Functional Interaction of the Ankylosing Spondylitis-associated Endoplasmic Reticulum Aminopeptidase 1 Polymorphism and HLA-B27 in Vivo

The association of ERAP1 with ankylosing spondylitis (AS)¹ among HLA-B27-positive individuals suggests that ERAP1 polymorphism may affect pathogenesis by altering peptide-dependent features of the HLA-B27 molecule. Comparisons of HLA-B*27:04-bound peptidomes from cells expressing different natural variants of ERAP1 revealed significant differences in the size, length, and amount of many ligands, as well as in HLA-B27 stability. Peptide analyses suggested that the mechanism of ERAP1/HLA-B27 interaction is a variant-dependent alteration in the balance between epitope generation and destruction determined by the susceptibility of N-terminal flanking and P1 residues to trimming. ERAP1 polymorphism associated with AS susceptibility ensured efficient peptide trimming and high HLA-B27 stability. Protective polymorphism resulted in diminished ERAP1 activity, less efficient trimming, suboptimal HLA-B27 peptidomes, and decreased molecular stability. This study demonstrates that natural ERAP1 polymorphism affects HLA-B27 antigen presentation and stability in vivo and proposes a mechanism for the interaction between these molecules in AS.The mechanism underlying the strong association of HLA-B27 with ankylosing spondylitis (AS) remains unknown. Three main possibilities, each one based on a different molecular feature of HLA-B27, are currently being investigated. The arthritogenic peptide hypothesis (1), based on the canonic antigen-presenting properties of Major Histocompatibility Complex class I (MHC-I) molecules, assumes that a peptide epitope of external origin would activate HLA-B27-restricted T-cells, whose cross-reactivity with a self-derived HLA-B27 ligand would result in autoimmune damage. The misfolding hypothesis (2) is based on the slow folding and tendency to misfold of HLA-B27 (3, 4). An accumulation of misfolded heavy chains (HCs) in the endoplasmic reticulum (ER) would elicit an unfolded protein response and activate pro-inflammatory pathways. The surface homodimer hypothesis (5, 6) is based on the expression of HLA-B27 HC homodimers at the cell surface and their recognition by leukocyte receptors (7), which leads to immunomodulation of inflammatory responses. Because the constitutive binding of endogenous peptides by MHC-I molecules determines not only their antigen-presenting specificity, but also their folding and stability, it was proposed that the HLA-B27 peptidome, through its global influence on the biological behavior of the molecule, is critical to its pathogenetic role (8). This idea found strong support with the discovery of the association of ER aminopeptidase (ERAP) 1 with AS (9) in HLA-B27-positive, but not B27-negative, disease (10). With an estimated population attributable risk of 26%, ERAP1 is the non-MHC gene most strongly associated with AS. Given that ERAP1 is involved in the N-terminal trimming of peptides to their optimal size for MHC-I binding (11–13), its association with AS suggests a pathogenetic mechanism of functional interaction with HLA-B27 that influences peptide binding and antigen presentation. ERAP1 trimming is limited by peptide size, becoming highly inefficient for 8-mers and shorter peptides (13, 14). This is a seemingly unique feature of ERAP1 that is not even shared by its analog ERAP2 (14, 15). The only putative exception, which has not been entirely ruled out, might be insulin-regulated amino peptidase (IRAP), an endosomal analog of ERAP1 involved in cross-presentation, but probably not in processing of constitutive MHC-I ligands (16, 17). IRAP degrades peptides to smaller products than ERAP1 in vitro (18). The three-dimensional structure of ERAP1 reveals a substrate binding cavity close to the catalytic site, as well as four domains; the conformational rearrangement between an open and a closed conformation, presumably induced upon substrate binding, regulates its enzymatic activity (19, 20). The polymorphic residues found among natural ERAP1 variants (21), and often co-occurring in complex allotypes, are located in various topological regions, including some in close proximity to the catalytic site, the substrate binding cavity, or domain junctions. Therefore, they might alter ERAP1 activity by directly affecting catalysis, altering substrate binding, or modulating domain rearrangements. The association of ERAP1 with AS does not by itself reveal the specific feature(s) determining the pathogenetic role of HLA-B27. Indeed, ERAP1 might influence the generation of specific pathogenetic epitopes; have a general effect on the HLA-B27 peptidome, altering the stability or other features of the molecule; or both. This study investigated general effects of ERAP1 polymorphism on the HLA-B27 peptidome by comparing the size distribution, molecular features, and N-terminal flanking sequences of peptides from human cells expressing the AS-associated B*27:04 subtype and different natural variants of ERAP1.     

Enhanced recombinant expression and purification of human IRAP for biochemical and crystallography studies

Insulin-regulated aminopeptidase (IRAP) in humans is a membrane bound enzyme that has multiple functions. It was first described as a companion protein of the insulin-responsive glucose transporter, Glut4, in specialized vesicles. The protein has subsequently been shown to be identical to the oxytocinase/aminopeptidase or the angiotensin IV (Ang IV) receptor (AT₄ receptor). Some AT₄ ligand peptides, such as Ang IV and LVV-hemorphin-7, have been shown to act as IRAP inhibitors that exert memory-enhancing properties. As such IRAP has been a target for developing cognitive enhancers. To facilitate detailed mechanistic studies of IRAP catalysis and inhibition, and to pave the way for biophysical and structural studies of IRAP in complex with peptide inhibitors, we report here an optimized expression and purification system using High Five insect cells. We also report biochemical characterizations of the purified recombinant IRAP with a standard aminopeptidase substrate and an optimized IRAP peptide inhibitor with a Ki of 98 nM.     

Binding of longer peptides to the H-2Kb heterodimer is restricted to peptides extended at their C terminus: refinement of the inherent MHC class I peptide binding criteria.

MHC class I molecules usually bind short peptides of 8-10 amino acids, and binding is dependent on allele-specific anchor residues. However, in a number of cellular systems, class I molecules have been found containing peptides longer than the canonical size. To understand the structural requirements for MHC binding of longer peptides, we used an in vitro class I MHC folding assay to examine peptide variants of the antigenic VSV 8 mer core peptide containing length extensions at either their N or C terminus. This approach allowed us to determine the ability of each peptide to productively form Kb/beta2-microglobulin/peptide complexes. We found that H-2Kb molecules can accommodate extended peptides, but only if the extension occurs at the C-terminal peptide end, and that hydrophobic flanking regions are preferred. Peptides extended at their N terminus did not promote productive formation of the trimolecular complex. A structural basis for such findings comes from molecular modeling of a H-2Kb/12 mer complex and comparative analysis of MHC class I structures. These analyses revealed that structural constraints in the A pocket of the class I peptide binding groove hinder the binding of N-terminal-extended peptides, whereas structural features at the C-terminal peptide residue pocket allow C-terminal peptide extensions to reach out of the cleft. These findings broaden our understanding of the inherent peptide binding and epitope selection criteria of the MHC class I molecule. Core peptides extended at their N terminus cannot bind, but peptide extensions at the C terminus are tolerated.     

Balancing selection maintains a form of ERAP2 that undergoes nonsense-mediated decay and affects antigen presentation

A remarkable characteristic of the human major histocompatibility complex (MHC) is its extreme genetic diversity, which is maintained by balancing selection. In fact, the MHC complex remains one of the best-known examples of natural selection in humans, with well-established genetic signatures and biological mechanisms for the action of selection. Here, we present genetic and functional evidence that another gene with a fundamental role in MHC class I presentation, endoplasmic reticulum aminopeptidase 2 (ERAP2), has also evolved under balancing selection and contains a variant that affects antigen presentation. Specifically, genetic analyses of six human populations revealed strong and consistent signatures of balancing selection affecting ERAP2. This selection maintains two highly differentiated haplotypes (Haplotype A and Haplotype B), with frequencies 0.44 and 0.56, respectively. We found that ERAP2 expressed from Haplotype B undergoes differential splicing and encodes a truncated protein, leading to nonsense-mediated decay of the mRNA. To investigate the consequences of ERAP2 deficiency on MHC presentation, we correlated surface MHC class I expression with ERAP2 genotypes in primary lymphocytes. Haplotype B homozygotes had lower levels of MHC class I expressed on the surface of B cells, suggesting that naturally occurring ERAP2 deficiency affects MHC presentation and immune response. Interestingly, an ERAP2 paralog, endoplasmic reticulum aminopeptidase 1 (ERAP1), also shows genetic signatures of balancing selection. Together, our findings link the genetic signatures of selection with an effect on splicing and a cellular phenotype. Although the precise selective pressure that maintains polymorphism is unknown, the demonstrated differences between the ERAP2 splice forms provide important insights into the potential mechanism for the action of selection.     

Identification of modulating residues defining the catalytic cleft of insulin-regulated aminopeptidase.

Inhibition of insulin-regulated aminopeptidase (IRAP) has been demonstrated to facilitate memory in rodents, making IRAP a potential target for the development of cognitive enhancing therapies. In this study, we generated a 3-D model of the catalytic domain of IRAP based on the crystal structure of leukotriene A4 hydrolase (LTA4H). This model identified two key residues at the 'entrance' of the catalytic cleft of IRAP, Ala427 and Leu483, which present a more open arrangement of the S1 subsite compared with LTA4H. These residues may define the size and 3-D structure of the catalytic pocket, thereby conferring substrate and inhibitor specificity. Alteration of the S1 subsite by the mutation A427Y in IRAP markedly increased the rate of substrate cleavage V of the enzyme for a synthetic substrate, although a corresponding increase in the rate of cleavage of peptide substrates Leu-enkephalin and vasopressin was was not apparent. In contrast, [L483F]IRAP demonstrated a 30-fold decrease in activity due to changes in both substrate affinity and rate of substrate cleavage. [L483F]IRAP, although capable of efficiently cleaving the N-terminal cysteine from vasopressin, was unable to cleave the tyrosine residue from either Leu-enkephalin or Cyt6-desCys1-vasopressin (2-9), both substrates of IRAP. An 11-fold reduction in the affinity of the peptide inhibitor norleucine1-angiotensin IV was observed, whereas the affinity of angiotensin IV remained unaltered. In additionm we predict that the peptide inhibitors bind to the catalytic site, with the NH2-terminal P1 residue occupying the catalytic cleft (S1 subsite) in a manner similar to that proposed for peptide substrates.     

Conformational and structural characteristics of peptides binding to HLA-DR molecules.

A fundamental characteristic of MHC class I and class II proteins is their unusual capacity to form stable complexes with a wide spectrum of peptide ligands. In this study, sets of peptide analogues containing long chain-biotinylated lysine individually substituted for each amino acid in the sequence have been used to explore the structural requirements for the formation of peptide-MHC class II protein complexes. Based on the ability of the analogs to bind both the MHC protein and fluorescent streptavidin, receptor contact residues were identified and from their spacing the conformation of the bound peptides could be inferred. Six separate peptides were studied; three defined by HLA-DR1Dw1-restricted T cells, and three identified by T cells restricted through alleles other than HLA-DR1Dw1. The similar patterns of fluorescent signals observed when the former three peptides were studied indicated that they shared conformational features when bound to HLA-DR1Dw1. In contrast when the latter three peptides were examined, the data indicated that they shared some but not all of the conformational features characteristic of the peptides known to elicit HLA-DR1Dw1-restricted T cells. When the peptide sequences were aligned based on the critical contact residues, two positions of structural homology were apparent. In each sequence, an amino acid with a bulky hydrophobic side chain could be identified separated by four residues from a small amino acid. These minimal structural requirements were consistent with recent experiments demonstrating that only a small number of side chains in the peptide were necessary for binding to the MHC protein.     

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).