Elucidation of the Signatures of Proteasome-Catalyzed Peptide Splicing.

Wayne Paes,Nicola Ternette,German Leonov,Persephone Borrow,Thomas Partridge,Annalisa Nicastri

doi:10.3389/fimmu.2020.563800

Abstract

Proteasomes catalyze the degradation of endogenous proteins into oligopeptides, but can concurrently create spliced oligopeptides through ligation of previously non-contiguous peptide fragments. Recent studies have uncovered a formerly unappreciated role for proteasome-catalyzed peptide splicing (PCPS) in the generation of non-genomically templated human leukocyte antigen class I (HLA-I)-bound cis-spliced peptides that can be targeted by CD8+ T cells in cancer and infection. However, the mechanisms defining PCPS reactions are poorly understood. Here, we experimentally define the biochemical constraints of proteasome-catalyzed cis-splicing reactions by examination of in vitro proteasomal digests of a panel of viral- and self-derived polypeptide substrates using a tailored mass-spectrometry-based de novo sequencing workflow. We show that forward and reverse PCPS reactions display unique splicing signatures, defined by preferential fusion of distinct amino acid residues with stringent peptide length distributions, suggesting sequence- and size-dependent accessibility of splice reactants for proteasomal substrate binding pockets. Our data provide the basis for a more informed mechanistic understanding of PCPS that will facilitate future prediction of spliced peptides from protein sequences.

Highlights

Proteasomes are multi-subunit enzyme complexes in eukaryotic cells that selectively degrade “unwanted” endogenous proteins into oligopeptides
By employing a tailored MSbased de novo sequencing approach to identify peptides generated by proteasomal splicing in vitro, our study has provided novel insight into both the steric length constraints and biochemical preferences of proteasome-catalyzed peptide splicing (PCPS) mediated by the constitutive proteasome (CP)
Systematic analysis of PCPS has been hampered by the limited number of spliced peptides that have been experimentally validated to date

Summary

Introduction

Proteasomes are multi-subunit enzyme complexes in eukaryotic cells that selectively degrade “unwanted” endogenous proteins into oligopeptides. Their activity is important for protein quality control and for regulation of many intracellular processes including cell cycle progression, signaling pathways and transcription [1]. The oligopeptides generated by proteasomes can be degraded to provide a source of amino acids (aas) for protein synthesis, but a subset of these peptides is translocated from the cytoplasm into the endoplasmic reticulum (ER) by the transporter associated with antigen presentation (TAP), where they may associate with newly-synthesized human leukocyte antigen class I (HLA-I) molecules [2]. The peptides comprising the cellular HLA-bound repertoire (the immunopeptidome) were classically thought to be contiguous sequences originating from self- or foreign proteins

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Results

Conclusion