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<jats:title>Abstract</jats:title><jats:p>Proteasomes catalyse 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-catalysed peptide splicing (PCPS) in the generation of non-genomically templated human leukocyte antigen class I (HLA-I)-bound <jats:italic>cis-</jats:italic>spliced peptides that can be targeted by CD8<jats:sup>+</jats:sup> T cells in cancer and infection. However, the mechanisms defining PCPS reactions are poorly understood. Here, we experimentally define the biochemical constraints of proteasome-catalysed <jats:italic>cis</jats:italic>-splicing reactions by examination of <jats:italic>in vitro</jats:italic> proteasomal digests of a panel of viral- and self-derived polypeptide substrates using a tailored mass-spectrometry-based <jats:italic>de novo</jats:italic> 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.</jats:p>

Original publication




Journal article


Frontiers in Immunology


Frontiers Media

Publication Date