Department of Chemistry, Boston College, Chestnut Hill, MA 02467, USA.
Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA; Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Cell Chem Biol. 2016 Jun 23;23(6):727-37. doi: 10.1016/j.chembiol.2016.05.008. Epub 2016 Jun 9.
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.
S-亚硝基谷胱甘肽（GSNO）是一种内源性的转亚硝基供体，参与多种细胞蛋白的 S-亚硝基化，从而调节多种蛋白质功能。定量蛋白质组学方法是建立哪些半胱氨酸残基对 GSNO 介导的转亚硝基化最敏感所必需的。在这里，实施了一种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 介导的转亚硝基化的敏感性。该平台确定了一组半胱氨酸残基，它们对半胱氨酸残基具有很高的 GSNO 介导的转亚硝基化倾向。对以前未注释的 S-亚硝基化位点的功能特征分析表明，HADH2 活性位点远端半胱氨酸残基的 S-亚硝基化会损害催化活性。同样，溶酶体天冬氨酸蛋白酶组织蛋白酶 D（CTSD）中非催化半胱氨酸残基的 S-亚硝基化抑制了蛋白水解激活。总之，这些研究揭示了两个以前未表征的半胱氨酸残基，它们调节蛋白质功能，并建立了一个化学蛋白质组学平台，具有确定其他细胞转亚硝基化剂底物特异性的能力。
