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ERAP1 allotypes 2 and 10 differentially regulate the immunopeptidome of melanocytes

ABSTRACT: Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a polymorphic enzyme that shapes the peptide repertoire presented by MHC class I molecules and can regulate adaptive immune responses in cancer and autoimmunity. Common missense polymorphisms in ERAP1 modulate its activity and are found in specific allotypes in humans. ERAP1 allotypes are linked to predisposition to HLA-associated inflammatory diseases. ERAP1 allotypic variation has been correlated with the development of psoriasis and Bechet’s disease, through the generation of specific CD8+ T cell populations targeting disease-specific HLAs. Given the established broad effects of ERAP1 activity on the cellular immunopeptidome, we hypothesized that ERAP1 allotypic variation may lead to broad immunopeptidome shifts that underlie antigenic responses. To test this hypothesis, we generated two A375 melanoma cell lines, each one expressing one of the most common, disease-associated, ERAP1 allotypes, namely allotypes 2 or 10. Comparison of the immunopeptidome of these two cell lines showed only minor differences in peptide sequences presented but extensive changes in abundance that included alterations in length distribution, binding affinity and sequence motifs. Our results suggest that enzymatic differences between ERAP1 allotypes are reflected primarily on the quantitative composition of the cellular immunopeptidome. These quantitative changes may constitute a mechanism that underlies ERAP1-allotypic associations with HLA-associated autoimmunity and variable anti-tumor responses.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Martina Samiotaki

LAB HEAD: Efstratios Stratikos

PROVIDER: PXD066752 | Pride | 2025-11-04

REPOSITORIES: Pride

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Distinct modulation of cellular immunopeptidome by the allosteric regulatory site of ER aminopeptidase 1

Project description:ER aminopeptidase 1 (ERAP1) is an ER-resident aminopeptidase that excises N-terminal residues off peptides that then bind onto Major Histocompatibility Complex I molecules (MHC-I) and indirectly modulates adaptive immune responses. ERAP1 contains an allosteric regulatory site that accommodates the C-terminus of only some peptide substrates, raising questions about its exact influence on antigen presentation and the potential of allosteric inhibition for cancer immunotherapy. We used an inhibitor that targets this regulatory site to study its effect on the immunopeptidome of a human cancer cell line. The immunopeptidomes of allosterically inhibited and ERAP1 knockout cells contain high-affinity peptides with sequence motifs consistent with the cellular HLA class I haplotypes, but were strikingly different in peptide composition. Compared to knockout cells, allosteric inhibition did not affect the length distribution of peptides and skewed the peptide repertoire both in terms of sequence motifs and HLA allele utilization, indicating significant mechanistic differences between the two ways of disrupting ERAP1 function. These findings suggest that the regulatory site of ERAP1 plays distinct roles in antigenic peptide selection, which should be taken into consideration when designing therapeutic interventions targeting the cancer immunopeptidome.

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Project description:Recent studies have linked the activity of ER aminopeptidase 2 (ERAP2) to increased efficacy of immune-checkpoint inhibitor cancer immunotherapy, suggesting that pharmacological inhibition of ERAP2 could have important therapeutic implications. To explore the effects of ERAP2 inhibition on the immunopeptidome of cancer cells we treated MOLT4 T lymphoblast leukaemia cells with a recently developed selective ERAP2 inhibitor, isolated Major Histocompatibility class I molecules (MHCI) and sequenced bound peptides by tandem liquid chromatography mass spectrometry. Inhibitor treatment induced significant shifts on the immunopeptidome so as more than 20% of detected peptides were either novel or significantly upregulated. Most of the inhibitor-induced peptides were 9mers and had sequence motifs and predicted affinity consistent with being optimal ligands for at least one of the MHCI alleles carried by MOLT4 cells. Such inhibitor-induced peptides could serve as triggers for novel cytotoxic responses against cancer cells and synergize with the therapeutic effect of immune-checkpoint inhibitors.

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ERAP1 as a regulator of the immunopeptidome, proteome and cellular homeostasis of cancer cells. Immunopeptdome_PartII

Project description:Endoplasmic reticulum aminopeptidase 1 (ERAP1) metabolizes peptides inside the ER and shapes the peptide repertoire available for binding to Major Histocompatibility Class I molecules (MHC-I). Moreover, it may have additional effects on cellular homeostasis, which have not been explored. To address these questions, we utilized genetic silencing of ERAP1 expression as well as a selective allosteric ERAP1 inhibitor to probe changes in the immunopeptidome and proteome of the A375 melanoma cancer cell line. We observed significant immunopeptidome shifts upon both methods of ERAP1 functional disruption, which were distinct for each method. Both methods of inhibition led to mild enhancement of cancer cell killing by stimulated human PBMCs from a healthy donor. ERAP1 functional disruption also resulted in significant proteomic alterations that focused on pathways related to metabolism and cellular stress. Similar proteomic changes were also observed in the leukemia cell line THP-1. Biochemical analyses suggested that ERAP1 inhibition affected ER stress, reactive oxygen species production and mitochondrial metabolism. Although the proteomics shifts were significant, their potential in shaping immunopeptidome shifts was limited since only 15.8% of differentially presented peptides belonged to proteins with altered expression and 4.8% of proteins with altered expression were represented in the immunopeptidome shifts. Taken together, our findings suggest that inhibition of ERAP1 can generate unique immunopeptidomes, primarily due to altered peptide processing in the ER, but also induce compensatory changes in the cellular proteome and metabolic state which may have additional effects on cancer cells.

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ERAP1 as a regulator of the immunopeptidome, proteome and cellular homeostasis of cancer cells. A375 Immunopeptidome, Part I.

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ERAP1 activity modulates the immunopeptidome but also affects the proteome, metabolism and stress responses in cancer cells

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ERAP1 activity modulates the immunopeptidome but also affects the proteome, metabolism and stress responses in cancer cells

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