Digestion of protein substrates by 20S proteasomes
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion, Q Exactive HF
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Michele Mishto Dr. Juliane Liepe
PROVIDER: MSV000092631 | MassIVE | Wed Aug 09 01:39:00 BST 2023
SECONDARY ACCESSION(S): PXD044463
REPOSITORIES: MassIVE
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Project description: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
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