Duke University Medical Center, Durham, USA.
Redox Biol. 2015 Aug;5:414. doi: 10.1016/j.redox.2015.09.015. Epub 2015 Dec 30.
The Ras family of small GTPases cycle between an inactive, GDP-bound state and an active, GTP-bound state. When bound to GTP, Ras engages and activates a number of effectors that mediate proliferative and survival signals. Ras is mutated in over 30% of human cancers, usually at codons 12, 13, or 61, to remain in this active, GTP-bound state, which promotes tumorigenesis. One of these oncogenic mutations that commonly occurs in lung cancer is G12C. Recently, it was shown that alkylating agents that react with the thiol functional group of this mutant amino acid can inactivate oncogenic Ras.
Given that Cys of Ras is accessible to thiol alkylating agents and forms interactions within the electrostatic phosphoryl-binding loop of Ras, we postulated that Cys may possess an altered pKa, potentially allowing this residue to be modified by NO and other cellular oxidants.
We conducted several biochemical analyses to determine whether nitrosylation of Ras alters its activity and structure in vitro. We also determined the biological effects of increasing NO production on the tumorigenic growth of cells transformed by Ras.
We found that Cys has a depressed pKa of 7.4, which increases the susceptibility of the thiol to modification by oxidation or nitrosylation at physiological pH. We also found that coexpressing active eNOS and Ras accelerated tumorigenic growth of human and murine cell line xenografts.
Modification of Cys in mutant, oncogenic Ras may promote its tumorigenic activity.
Ras 家族的小分子 GTP 酶在非活性、GDP 结合状态和活性、GTP 结合状态之间循环。当与 GTP 结合时，Ras 与许多效应物结合并激活，这些效应物介导增殖和存活信号。Ras 在超过 30%的人类癌症中发生突变，通常在密码子 12、13 或 61 处，以保持这种活性、GTP 结合状态，从而促进肿瘤发生。在肺癌中常见的一种致癌突变是 G12C。最近，研究表明与该突变氨基酸的巯基官能团反应的烷化剂可以使致癌 Ras 失活。
鉴于 Ras 的半胱氨酸可被巯基烷化剂接近，并在 Ras 的静电磷酸结合环内形成相互作用，我们推测半胱氨酸可能具有改变的 pKa，可能允许该残基被 NO 和其他细胞氧化剂修饰。
我们进行了几项生化分析，以确定 Ras 的硝化是否会改变其体外活性和结构。我们还确定了增加 NO 产生对 Ras 转化的细胞致瘤生长的生物学影响。
我们发现半胱氨酸的 pKa 为 7.4，降低了其在生理 pH 下被氧化或硝化修饰的敏感性。我们还发现，共表达活性 eNOS 和 Ras 加速了人源和鼠源细胞系异种移植物的致瘤生长。
突变型致癌 Ras 中半胱氨酸的修饰可能促进其致瘤活性。
