State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, China.
School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
Nucleic Acids Res. 2020 Jul 9;48(12):6799-6810. doi: 10.1093/nar/gkaa471.
Structure and/or function of proteins are frequently affected by oxidative/nitrosative stress via posttranslational modifications. Aminoacyl-tRNA synthetases (aaRSs) constitute a class of ubiquitously expressed enzymes that control cellular protein homeostasis. Here, we found the activity of human mitochondrial (mt) threonyl-tRNA synthetase (hmtThrRS) is resistant to oxidative stress (H2O2) but profoundly sensitive to nitrosative stress (S-nitrosoglutathione, GSNO). Further study showed four Cys residues in hmtThrRS were modified by S-nitrosation upon GSNO treatment, and one residue was one of synthetic active sites. We analyzed the effect of modification at individual Cys residue on aminoacylation and editing activities of hmtThrRS in vitro and found that both activities were decreased. We further confirmed that S-nitrosation of mtThrRS could be readily detected in vivo in both human cells and various mouse tissues, and we systematically identified dozens of S-nitrosation-modified sites in most aaRSs, thus establishing both mitochondrial and cytoplasmic aaRS species with S-nitrosation ex vivo and in vivo, respectively. Interestingly, a decrease in the S-nitrosation modification level of mtThrRS was observed in a Huntington disease mouse model. Overall, our results establish, for the first time, a comprehensive S-nitrosation-modified aaRS network and a previously unknown mechanism on the basis of the inhibitory effect of S-nitrosation on hmtThrRS.
蛋白质的结构和/或功能经常通过翻译后修饰受到氧化/硝化应激的影响。氨酰-tRNA 合成酶（aaRSs）是一类广泛表达的酶，控制细胞的蛋白质稳态。在这里，我们发现人线粒体（mt）苏氨酰-tRNA 合成酶（hmtThrRS）的活性对氧化应激（H2O2）有抗性，但对硝化应激（S-亚硝基谷胱甘肽，GSNO）非常敏感。进一步的研究表明，GSNO 处理后，hmtThrRS 中的四个半胱氨酸残基被 S-亚硝基化修饰，其中一个残基是合成活性位点之一。我们分析了单个半胱氨酸残基修饰对 hmtThrRS 体外氨酰化和编辑活性的影响，发现这两种活性都降低了。我们进一步证实，mtThrRS 的 S-亚硝基化可以在体内人细胞和各种小鼠组织中很容易地检测到，并且我们系统地鉴定了大多数 aaRSs 中的数十个 S-亚硝基化修饰位点，从而分别建立了体外和体内的线粒体和细胞质 aaRS 物种。有趣的是，在亨廷顿病小鼠模型中观察到 mtThrRS 的 S-亚硝基化修饰水平降低。总的来说，我们的结果首次建立了一个全面的 S-亚硝基化修饰 aaRS 网络和一个以前未知的机制，即在 S-亚硝基化对 hmtThrRS 的抑制作用的基础上。
