Table_1_Elucidation of the Signatures of Proteasome-Catalyzed Peptide Splicing.xlsx

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.

蛋白酶体催化内源性蛋白降解为寡肽，但同时可通过连接先前非连续的肽片段来生成剪接寡肽。近期研究揭示了蛋白酶体催化肽剪接（PCPS）在生成非基因组模板化的人类白细胞抗原I类（HLA-I）结合的顺式剪接肽中的重要作用，这些肽可被CD8+ T细胞在癌症和感染中靶向。然而，定义PCPS反应的机制尚不明确。在本研究中，我们通过使用定制的大规模质谱法从头测序工作流程，考察了一系列病毒和自源多肽底物的体外蛋白酶体消化物，从而实验性地定义了蛋白酶体催化顺式剪接反应的生化约束。我们发现正向和反向PCPS反应展现出独特的剪接特征，这些特征由特定氨基酸残基的优选融合和严格的肽长度分布所定义，这表明剪接反应物对蛋白酶体底物结合口袋的序列和尺寸依赖性可及性。我们的数据为更深入理解PCPS的机制提供了基础，这将有助于未来从蛋白质序列预测剪接肽。