Chemoproteomic Strategy to Quantitatively Monitor Transnitrosation Uncovers Functionally Relevant S-Nitrosation Sites on Cathepsin D and HADH2.
Chemoproteomic Strategy to Quantitatively Monitor Transnitrosation Uncovers Functionally Relevant S-Nitrosation Sites on Cathepsin D and HADH2.
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DOI：
10.1016/j.chembiol.2016.05.008
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发表时间：
2016-06-23
影响因子：
8.6
通讯作者：
Weerapana E
中科院分区：
文献类型：
--
作者：
Zhou Y;Wynia-Smith SL;Couvertier SM;Kalous KS;Marletta MA;Smith BC;Weerapana E
S-亚硝基谷胱甘肽（GSNO）是一种内源性转亚硝化供体，参与多种细胞蛋白质的S-亚硝化，从而调节多种蛋白质功能。定量蛋白质组学方法是必要的，以确定哪些半胱氨酸残基是最敏感的GSNO介导的转亚硝化。在此，实施竞争性半胱氨酸反应性分析策略以定量测量>600个半胱氨酸残基对GSNO的转亚硝化的敏感性。该平台鉴定了半胱氨酸残基的子集，其具有高的GSN 0介导的转亚硝化倾向。以前未注释的S-亚硝化位点的功能表征表明，S-亚硝化的半胱氨酸残基的远端的3-羟酰辅酶A脱氢酶2型（HADH 2）活性位点损害催化活性。同样，S-亚硝化的非催化半胱氨酸残基的溶酶体乙酰化蛋白酶组织蛋白酶D（CTSD）抑制蛋白水解活化。总之，这些研究揭示了两个以前未表征的半胱氨酸残基，其调节蛋白质功能，并建立了一个化学蛋白质组学平台，能够确定其他细胞转亚硝化剂的底物特异性。Zhou等人采用反应性半胱氨酸分析策略，通过对S-亚硝基谷胱甘肽（GSNO）的敏感性对约600个半胱氨酸残基进行排序。先前未表征的HADH 2和CTSD上的GSNO敏感性半胱氨酸被证明分别对催化活性和蛋白水解加工是重要的。
S-nitrosoglutathione (GSNO) is an endogenous transnitrosation donor involved in S-nitrosation of a variety of cellular proteins, thereby regulating diverse protein functions. Quantitative proteomic methods are necessary to establish which cysteine residues are most sensitive to GSNO-mediated transnitrosation. Here, a competitive cysteine-reactivity profiling strategy was implemented to quantitatively measure the sensitivity of >600 cysteine residues to transnitrosation by GSNO. This platform identified a subset of cysteine residues with a high propensity for GSNO-mediated transnitrosation. Functional characterization of previously unannotated S-nitrosation sites revealed that S-nitrosation of a cysteine residue distal to the 3-hydroxyacyl-CoA dehydrogenase type-2 (HADH2) active site impaired catalytic activity. Similarly, S-nitrosation of a non-catalytic cysteine residue in the lysosomal aspartyl protease cathepsin D (CTSD) inhibited proteolytic activation. Together, these studies revealed two previously uncharacterized cysteine residues that regulate protein function and established a chemical-proteomic platform with capabilities to determine substrate specificity of other cellular transnitrosation agents. Zhou et al. apply a reactive-cysteine profiling strategy to rank ~600 cysteine residues by sensitivity to S-nitrosoglutathione (GSNO). Previously uncharacterized GSNO-sensitive cysteines on HADH2 and CTSD were shown to be important for catalytic activity and proteolytic processing, respectively.