Production of spliced peptides by the proteasome

Abstract

CD8 cytolytic T lymphocytes are essential players of anti-tumor immune responses. On tumors, they recognize peptides of about 8-to-10 amino acids that generally result from the degradation of cellular proteins by the proteasome. Until a decade ago, these peptides were thought to solely correspond to linear fragments of proteins that were liberated after the hydrolysis of the peptide bonds located at their extremities. However, several examples of peptides containing two fragments originally distant in the protein sequence challenged this concept and demonstrated that proteasome could also splice peptides together by creating a new peptide bond between two distant fragments. Unexpectedly, peptide splicing emerges as an essential way to increase the peptide repertoire diversity as these spliced peptides were shown to represent up to 25% of the peptides presented on a cell by MHC class I. Here, we review the different steps that led to the discovery of peptide splicing by the proteasome as well as the lightening offered by the recent progresses of mass spectrometry and bioinformatics in the analysis of the spliced peptide repertoire.

Noncontiguous T cell epitopes in autoimmune diabetes: From mice to men and back again

“…Although evidence suggests that noncontiguous CD8 + T cell epitopes are generated primarily by proteasome-mediated peptide splicing involving transpeptidation ( 75 ), the generation of HIPs remains more of a biochemical mystery to date. The sites of formation of HIPs are also under investigation; this knowledge could shed light on how they are produced.…”

In vitro proteasome processing of neo-splicetopes does not predict their presentation in vivo

“…Nevertheless, what applied to non-spliced epitopes also seemed to be valid for spliced HLA class I epitopes generated in vitro by PCPS. Thus, FGF-5, SP110, and several gp100-derived spliced epitopes that are recognized by CD8 + T cells on the cell surface were demonstrated to be produced also in in vitro PCPS assays ( Ebstein et al, 2016 ; Vigneron et al, 2019 ). Although in these cases the generation efficiency of spliced epitopes in in vitro PCPS assays seemed to be in line with the in vivo situation, it should be noted, however, that the abundance of spliced epitopes presented at the cell surface is a matter of substantial controversy ( Liepe et al, 2016 ; Liepe et al, 2019 ; Mylonas et al, 2018 ; Paes et al, 2019 ; Rolfs et al, 2019 ).…”

“…Therefore, in vitro generation of epitopes using purified 20S proteasomes and synthetic polypeptide substrates encompassing the epitope(s) of interest, in combination with mass spectrometric analyses and both in vitro and in vivo CD8 + T cell assays still represent the most frequently used tool to validate the generation of immune relevant non-spliced peptides, assuming that it closely simulates the in vivo (in cellulo) situation with respect to both quality and relative amounts of the epitope (Kessler et al, 2001;Kessler & Melief, 2007;Sijts & Kloetzel, 2011). Thus, FGF-5, SP110 and several gp100 derived spliced epitopes that are recognized by CD8 + T cells on the cell surface were demonstrated to be produced also in in vitro PCPS assays (Ebstein et al, 2016;Vigneron et al, 2019). Although in these cases the generation efficiency of spliced epitopes in in vitro PCPS assays seemed to be in line with the in vivo situation, it should be noted, however, that the abundance of spliced epitopes presented at the cell surface is a matter of substantial controversy (Liepe et al, 2016;Mylonas et al, 2018;Paes et al, 2019;Rolfs et al, 2019).…”

Main Strategies for the Identification of Neoantigens

“…Unlike the genomics-based approach, which only provides for neoantigen prediction, mass-spectrometry allows us to take a real snapshot of the total MHC-bound protein interactome. Additionally, it could reveal not only neoantigens that originate from somatic mutation variants but also those which arise due to proteasome-mediated peptide splicing [ 157 , 158 ]. Using mass-spectrometry, it was shown that the proportion of spliced peptides relative to peptides displayed by HLA class I varies from 2-6% reported in [ 159 ] to 30% reported in [ 60 ].…”