Peptidomics for Studying Limited Proteolysis
Peptidomics for Studying Limited Proteolysis
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用于研究有限蛋白水解的肽组学
DOI：
10.1021/acs.jproteome.5b00820
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发表时间：
2015
影响因子：
4.4
通讯作者：
Kazuki Sasaki
中科院分区：
文献类型：
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作者：
Takashi Tsuchiya;Tsukasa Osaki;Naoto Minamino;Kazuki Sasaki
有限的蛋白水解是调节蛋白质功能的关键机制。识别生理或病理生理学相关的切割位点有助于开发可用于诊断或治疗的分子工具。在分泌蛋白和膜蛋白的蛋白水解过程中，部分裂解蛋白被释放并注定要经历降解，但应保留由蛋白水解酶产生的原始裂解位点。我们分析了在原代培养的大鼠心脏成纤维细胞条件培养基中积累 4 小时的内源肽。来自 94 个分泌蛋白和膜蛋白的总共 3916 个冗余肽序列用于识别有限的切割位点（带注释和未注释），用于信号肽或前肽去除、肽激素加工、胞外域脱落和调节的膜内蛋白水解。在细胞外基质蛋白和肽激素前体肾上腺髓质素 ADM 等典型蛋白质中发现了错误预测的信号切割位点。通过鉴定侧翼肾上腺髓质素原 N 端肽的主要分子形式，对所揭示的 ADM 信号肽裂解位点进行了实验验证。我们认为，内源肽的分析（如转录组序列读取）在成纤维细胞等常规细胞中有意义，并且肽组学可以深入了解蛋白水解调节的蛋白质功能。
Limited proteolysis is a pivotal mechanism regulating protein functions. Identifying physiologically or pathophysiologically relevant cleavage sites helps to develop molecular tools that can be used for diagnostics or therapeutics. During proteolysis of secretory and membrane proteins, part of the cleaved protein is liberated and destined to undergo degradation but should retain original cleavage sites created by proteolytic enzymes. We profiled endogenous peptides accumulated for 4 h in media conditioned by primary cultured rat cardiac fibroblasts. A total of 3916 redundant peptide sequences from 94 secretory proteins and membrane proteins served to identify limited cleavage sites, both annotated and unannotated, for signal peptide or propeptide removal, peptide hormone processing, ectodomain shedding, and regulated intramembrane proteolysis. Incorrectly predicted signal cleavage sites are found in typical proteins such as extracellular matrix proteins and the peptide hormone precursor adrenomedullin ADM. The revealed signal peptide cleavage site for ADM was experimentally verified by identifying the major molecular form of flanking proadrenomedullin N-terminal peptide. We suggest that profiling of endogenous peptides, like transcriptome sequence reads, makes sense in regular cells such as fibroblasts and that peptidomics provides insight into proteolysis-regulated protein functions.