Proteomic and biochemical investigation of the effects of ERAP1 functional disruption in melanoma cells

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is located in the endoplasmic reticulum (ER) and plays a crucial role in shaping adaptive immune responses. Its main function involves the removal of N-terminal amino acids from antigenic peptide precursors, aiding in the optimal presentation of these peptides by Major Histocompatibility Complex I (MHC I) molecules. While ERAP1's activity is necessary for generating many antigenic peptides, excessive trimming of some peptides has also been observed. In the case of cancer-specific epitopes, over-trimming could potentially lead to diminished cytotoxic T-cell responses and cancer evasion. This pivotal role in immune evasion, coupled with observations that inhibiting ERAP1 alters the immunopeptidome of cells, has highlighted ERAP1 as a promising target for cancer immunotherapy. Besides its role in antigen processing and presentation, ERAP1 has also been suggested to affect ER stress and to participate in innate immunity. The potential multi-faceted role of ERAP1 necessitates an increased understanding of the cellular processes that may be affected by ERAP1 inhibition before its establishment as a pharmacological target.

To investigate the effects of ERAP1 inhibition on cellular processes, we employed a data-independent acquisition (DIA) approach, followed by library-free search, to examine alterations in expressed proteins following genetic manipulation (ERAP1 knockout) or allosteric inhibition of ERAP1 in A375 melanoma cells. Proteomic analysis revealed the identification of 5575 proteins in A375 cells. Disruption of ERAP1 led to significant shifts in the proteome, with 1375 proteins showing differential expression in inhibitor-treated and knockout cells compared to wild-type cells (FDR= 0.05, S0=0.5). Interestingly, inhibitor treatment induced less pronounced proteome changes, with most of these changes also observed in knockout cells. Investigation of the differentially expressed proteins indicated alterations in cellular processes such as antigen processing and presentation, cellular response to stress and the metabolic pathways of glycolysis and oxidative phosphorylation. Western blot analysis confirmed the upregulation of protein disulfide isomerase A3, which is part of the peptide loading complex that participates in MHC I mediated antigen presentation. Moreover, DCF assay suggested a small alteration in the levels of reactive oxygen species after ERAP1 functional inhibition, while extracellular flux analysis suggested limited effects on mitochondrial metabolism. Our results suggest that the reduction of ERAP1-trimming activity in cancer cells may affect peptide homeostasis in the ER and lead to limited compensatory changes in multiple cellular pathways.