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Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims antigenic peptide precursors to generate mature antigenic peptides for presentation by major histocompatibility complex class I (MHCI) molecules and regulates adaptive immune responses. ERAP1 has been proposed to trim peptide precursors both in solution and in preformed MHCI-peptide complexes, but which mode is more relevant to its biological function remains controversial. Here, we compared ERAP1-mediated trimming of antigenic peptide precursors in solution or when bound to three MHCI alleles, HLA-B*58, HLA-B*08, and HLA-A*02. For all MHCI-peptide combinations, peptide binding onto MHCI protected against ERAP1-mediated trimming. In only a single MHCI-peptide combination, trimming of an HLA-B*08-bound 12-mer progressed at a considerable rate, albeit still slower than in solution. Results from thermodynamic, kinetic, and computational analyses suggested that this 12-mer is highly labile and that apparent on-MHC trimming rates are always slower than that of MHCI-peptide dissociation. Both ERAP2 and leucine aminopeptidase, an enzyme unrelated to antigen processing, could trim this labile peptide from preformed MHCI complexes as efficiently as ERAP1. A pseudopeptide analogue with high affinity for both HLA-B*08 and the ERAP1 active site could not promote the formation of a ternary ERAP1/MHCI/peptide complex. Similarly, no interactions between ERAP1 and purified peptide-loading complex were detected in the absence or presence of a pseudopeptide trap. We conclude that MHCI binding protects peptides from ERAP1 degradation and that trimming in solution along with the dynamic nature of peptide binding to MHCI are sufficient to explain ERAP1 processing of antigenic peptide precursors.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that optimizes the peptide cargo of major histocompatibility class I (MHC-I) molecules and regulates adaptive immunity. It has unusual substrate selectivity for length and sequence, resulting in poorly understood effects on the cellular immunopeptidome. To understand substrate selection by ERAP1, we solved 2 crystal structures of the enzyme with bound transition-state pseudopeptide analogs at 1.68 Å and 1.72 Å. Both peptides have their N terminus bound at the active site and extend away along a large internal cavity, interacting with shallow pockets that can influence selectivity. The longer peptide is disordered through the central region of the cavity and has its C terminus bound in an allosteric pocket of domain IV that features a carboxypeptidase-like structural motif. These structures, along with enzymatic and computational analyses, explain how ERAP1 can select peptides based on length while retaining the broad sequence-specificity necessary for its biological function.

Processing of N-terminally elongated antigenic peptide precursors by Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) is a key step in antigen presentation and the adaptive immune response. Although ERAP1 can efficiently process long peptides in solution, it has been proposed that it can also process peptides bound onto Major Histocompatibility Complex I molecules (MHCI). In a previous study, we suggested that the occasionally observed “ontο MHCI” trimming by ERAP1 is likely due to fast peptide dissociation followed by solution trimming, rather than direct action of ERAP1 onto the MHCI complex. However, other groups have proposed that ERAP1 can trim peptides covalently bound onto MHCI, which would preclude peptide dissociation. To explore this interaction, we constructed disulfide-linked MHCI-peptide complexes using HLA-B*08 and a 12mer kinetically labile peptide, or a 16mer carrying a phosphinic transition-state analogue N-terminus with high-affinity for ERAP1. Kinetic and biochemical analyses suggested that while both peptides could access the ERAP1 active site when free in solution, they were unable to do so when tethered in the MHCI binding groove. Our results suggest that MHCI binding protects, rather than promotes, antigenic peptide precursor trimming by ERAP1 and thus solution trimming is the more likely model of antigenic peptide processing.

Endoplasmic Reticulum aminopeptidase 1 and 2 are two homologous enzymes that help generate peptide ligands for presentation by Major Histocompatibility Class I molecules. Their enzymatic activity influences the antigenic peptide repertoire and indirectly controls adaptive immune responses. Accumulating evidence suggests that these two enzymes are tractable targets for the regulation of immune responses with possible applications ranging from cancer immunotherapy to treating inflammatory autoimmune diseases. Here, we review the state-of-the-art in the development of inhibitors of ERAP1 and ERAP2 as well as their potential and limitations for clinical applications.

Presentation of antigenic peptides by MHCI is central to cellular immune responses against viral pathogens. While adaptive immune responses versus SARS-CoV-2 can be of critical importance to both recovery and vaccine efficacy, how protein antigens from this pathogen are processed to generate antigenic peptides is largely unknown. Here, we analyzed the proteolytic processing of overlapping precursor peptides spanning the entire sequence of the S1 spike glycoprotein of SARS-CoV-2, by three key enzymes that generate antigenic peptides, aminopeptidases ERAP1, ERAP2 and IRAP. All enzymes generated shorter peptides with sequences suitable for binding onto HLA alleles, but with distinct specificity fingerprints. ERAP1 was the most efficient in generating peptides 8-11 residues long, the optimal length for HLA binding, while IRAP was the least efficient. The combination of ERAP1 with ERAP2 greatly limited the variability of peptide sequences produced. Less than 7% of computationally predicted epitopes were found to be produced experimentally, suggesting that aminopeptidase processing may constitute a significant filter to epitope presentation. These experimentally generated putative epitopes could be prioritized for SARS-CoV-2 immunogenicity studies and vaccine design. We furthermore propose that this in vitro trimming approach could constitute a general filtering method to enhance the prediction robustness for viral antigenic epitopes.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that is important for the generation of antigenic epitopes and major histocompatibility class I-restricted adaptive immune responses. ERAP1 processes a vast variety of different peptides but still shows length and sequence selectivity, although the mechanism behind these properties is poorly understood. X-ray crystallographic analysis has revealed that ERAP1 can assume at least two distinct conformations in which C-terminal domain IV is either proximal or distal to active site domain II. To improve our understanding of the role of this conformational change in the catalytic mechanism of ERAP1, we used site-directed mutagenesis to perturb key salt bridges between domains II and IV. Enzymatic analysis revealed that these mutations, although located away from the catalytic site, greatly reduce the catalytic efficiency and change the allosteric kinetic behavior. The variants were more efficiently activated by small peptides and bound a competitive inhibitor with weaker affinity and faster dissociation kinetics. Molecular dynamics analysis suggested that the mutations affect the conformational distribution of ERAP1, reducing the population of closed states. Small-angle X-ray scattering indicated that both the wild type and the ERAP1 variants are predominantly in an open conformational state in solution. Overall, our findings suggest that electrostatic interactions between domains II and IV in ERAP1 are crucial for driving a conformational change that regulates the structural integrity of the catalytic site. The extent of domain opening in ERAP1 probably underlies its specialization for antigenic peptide precursors and should be taken into account in inhibitor development efforts.

Endoplasmic reticulum aminopeptidase 2 assists with the generation of antigenic peptides for presentation onto Major Histocompatibility Class I molecules in humans. Recent evidence has suggested that the activity of ERAP2 may contribute to the generation of autoimmunity, thus making ERAP2 a possible pharmacological target for the regulation of adaptive immune responses. To better understand the structural elements of inhibitors that govern their binding affinity to the ERAP2 active site, we cocrystallized ERAP2 with a medium activity 3,4-diaminobenzoic acid inhibitor and a poorly active hydroxamic acid derivative. Comparison of these two crystal structures with a previously solved structure of ERAP2 in complex with a potent phosphinic pseudopeptide inhibitor suggests that engaging the substrate N-terminus recognition properties of the active site is crucial for inhibitor binding even in the absence of a potent zinc-binding group. Proper utilization of all five major pharmacophores is necessary, however, to optimize inhibitor potency.

The efficacy of cancer immunotherapy, including treatment with immune-checkpoint inhibitors, often is limited by ineffective presentation of antigenic peptides that elicit T-cell-mediated anti-tumor cytotoxic responses. Manipulation of antigen presentation pathways is an emerging approach for enhancing the immunogenicity of tumors in immunotherapy settings. ER aminopeptidase 1 (ERAP1) is an intracellular enzyme that trims peptides as part of the system that generates peptides for binding to MHC class I molecules (MHC-I). We hypothesized that pharmacological inhibition of ERAP1 in cells could regulate the cellular immunopeptidome. To test this hypothesis, we treated A375 melanoma cells with a recently developed potent ERAP1 inhibitor and analyzed the presented MHC-I peptide repertoire by isolating MHC-I, eluting bound peptides, and identifying them using capillary chromatography and tandem mass spectrometry (LC-MS/MS). Although the inhibitor did not reduce cell-surface MHC-I expression, it induced qualitative and quantitative changes in the presented peptidomes. Specifically, inhibitor treatment altered presentation of about half of the total 3204 identified peptides, including about one third of the peptides predicted to bind tightly to MHC-I. Inhibitor treatment altered the length distribution of eluted peptides without change in the basic binding motifs. Surprisingly, inhibitor treatment enhanced the average predicted MHC-I binding affinity, by reducing presentation of sub-optimal long peptides and increasing presentation of many high-affinity 9–12mers, suggesting that baseline ERAP1 activity in this cell line is destructive for many potential epitopes. Our results suggest that chemical inhibition of ERAP1 may be a viable approach for manipulating the immunopeptidome of cancer., NCSRD—INRASTES research activities in the framework of the national RIS3.” (MIS 5002559) which is implemented under the “Action for the Strategic Development on the Research and Technological Sector”, funded by the Operational Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014-2020) and co-fnanced by Greece and the European Union (European Regional Development Fund)

Simple Summary A new class of drugs, termed Immune Checkpoint Inhibitors, has revolutionized cancer therapy during the last few years. Unfortunately, these drugs are only effective for a subset of patients and cancer types. Recent work has suggested that how well cancer cells present some of their molecules to the immune system is critical for patient responses to immunotherapy with immune checkpoint inhibitors. Here, we review the role of the biochemical pathway of antigen presentation in cancer and discuss how it can be modulated to enhance the efficacy of cancer immunotherapy. Abstract Recent clinical successes of cancer immunotherapy using immune checkpoint inhibitors (ICIs) are rapidly changing the landscape of cancer treatment. Regardless of initial impressive clinical results though, the therapeutic benefit of ICIs appears to be limited to a subset of patients and tumor types. Recent analyses have revealed that the potency of ICI therapies depends on the efficient presentation of tumor-specific antigens by cancer cells and professional antigen presenting cells. Here, we review current knowledge on the role of antigen presentation in cancer. We focus on intracellular antigen processing and presentation by Major Histocompatibility class I (MHCI) molecules and how it can affect cancer immune evasion. Finally, we discuss the pharmacological tractability of manipulating intracellular antigen processing as a complementary approach to enhance tumor immunogenicity and the effectiveness of ICI immunotherapy.

Insulin-regulated aminopeptidase (IRAP) is an enzyme with several important biological functions that is known to process a large variety of different peptidic substrates, although the mechanism behind this wide specificity is not clearly understood. We describe a crystal structure of IRAP in complex with a recently developed bioactive and selective inhibitor at 2.53 Å resolution. In the presence of this inhibitor, the enzyme adopts a novel conformation in which domains II and IV are juxtaposed, forming a hollow structure that excludes external solvent access to the catalytic center. A loop adjacent to the enzyme's GAMEN motif undergoes structural reconfiguration, allowing the accommodation of bulky inhibitor side chains. Atomic interactions between the inhibitor and IRAP that are unique to this conformation can explain the strong selectivity compared to homologous aminopeptidases ERAP1 and ERAP2. This conformation provides insight on IRAP's catalytic cycle and reveals significant active-site plasticity that may underlie its substrate permissiveness.

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 A, respectively, and compared them to the apo-form structure determined to 3.0 A. 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.

Aminopeptidases that generate antigenic peptides influence immunodominance and adaptive cytotoxic immune responses. The mechanisms that allow these enzymes to efficiently process a vast number of different long peptide substrates are poorly understood. In this work, we report the structure of insulin-regulated aminopeptidase, an enzyme that prepares antigenic epitopes for cross-presentation in dendritic cells, in complex with an antigenic peptide precursor analog. Insulin-regulated aminopeptidase is found in a semiclosed conformation with an extended internal cavity with limited access to the solvent. The N-terminal moiety of the peptide is located at the active site, positioned optimally for catalysis, whereas the C-terminal moiety of the peptide is stabilized along the extended internal cavity lodged between domains II and IV. Hydrophobic interactions and shape complementarity enhance peptide affinity beyond the catalytic site and support a limited selectivity model for antigenic peptide selection that may underlie the generation of complex immunopeptidomes.

The 5-methyl cytosine (5meC) genomic methylation patterns play crucial roles in mammalian development and are altered in cancer. The enzymes that create, maintain and modify the DNA methylation patterns are the DNA methyltransferases (Dnmts) which are all encoded by essential genes. The de novo Dnmts -Dnmt3a and Dnmt3b- establish the DNA methylation patterns early in mammalian development by introducing DNA methylation marks where no previous methylation exists. These enzymes do not exhibit affinity for specific DNA sequences, thus their recruitment to specific DNA loci and their activities must be tightly regulated. In particular, Dnmt3a2 -one of the two protein isoforms produced by the Dnmt3a locus- is the most abundant DNA methyltransferase in mouse Embryonic Stem Cells. To identify Dnmt3a (and DNA methylation) regulators we have searched for Dnmt3a2 interacting proteins in mESCs by pull down and Mass Spectrometry. The DEAD box p68/Ddx5 RNA helicase was identified to directly interact with Dnmt3a1 and Dnmt3a2 in vitro and in vivo. We have created a mutant Ddx5 (Ddx5 MUT ) protein exhibiting an altered nuclear localization pattern compared to the wt protein. Both wt and mutant Ddx5 interact directly in vitro and co localize in vivo with Dnmt3a proteins. Our data suggest that the Dnmt3a/Ddx5 interaction might be significant for modulating the DNA methylation/demethylation dynamics in vivo.

Dnmt3a1 and Dnmt3a2 are two de novo DNA methyltransferases expressed in mouse embryonic stem cells (mESCs). They differ in that a 219-amino-acid (aa) amino (N)-terminal noncatalytic domain is present only in Dnmt3a1. Here, we examined the unique functions of Dnmt3a1 in mESCs by targeting the coding sequence of the Dnmt3a1 N-terminal domain tagged with enhanced green fluorescent protein (GFP) for insertion into the mouse Rosa26 locus. Using these targeted cells (GFP-3a1Nter), we showed that Dnmt3a1 was efficiently recruited to the silenced Oct3/4 and activated Vtn (vitronectin) gene promoters via its unique N-terminal domain. This recruitment affected the two genes in contrasting ways, compromising Oct3/4 gene promoter DNA methylation to prevent consolidation of the silent state while significantly reducing Vtn transcription. We used this negative effect of the Dnmt3a1 N-terminal domain to investigate the extent of transcriptional regulation by Dnmt3a1 in mESCs by using microarrays. A small group of all-trans retinoic acid (tRA)-inducible genes had lower transcript levels in GFP-3a1Nter cells than in wild-type mESCs. Intriguingly, this group included genes that are important for fetal nutrition, placenta development, and metabolic functions and is enriched for a distinct set of imprinted genes. We also identified a larger group of genes that showed higher transcript levels in the GFP-3a1Nter-expressing cells than in wild-type mESCs, including pluripotency factors and key regulators of primordial germ cell differentiation. Thus, Dnmt3a1 in mESCs functions primarily as a negative and to a lesser extent as a positive regulator of transcription. Our findings suggest that Dnmt3a1 positively affects transcription of specific genes at the promoter level and targets chromosomal domains to epigenetically silence gene clusters in mESCs.