Data_Sheet_1_Elucidation of the Signatures of Proteasome-Catalyzed Peptide Splicing.PDF

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质降解为寡肽，但可同时通过连接先前非连续的肽片段生成剪接寡肽。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的人白细胞抗原I类（HLA-I）结合的顺式剪接肽中的重要作用，这些肽可以被CD8+ T细胞在癌症和感染中靶向。然而，定义PCPS反应的机制尚不清楚。在本研究中，我们通过使用定制的基于质谱的从头测序工作流程，对一系列病毒和自源性多肽底物的体外蛋白酶体消化物进行考察，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条件。我们发现，正向和反向PCPS反应表现出独特的剪接特征，这由特定氨基酸残基的优先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地理解PCPS的机制提供了基础，并将有助于未来从蛋白质序列预测剪接肽。