Faculty of Natural Sciences and Medicine, Ilia State University, Tbilisi, Georgia.
Department of Biochemistry, I. Beritashvili Center of Experimental Biomedicine, Tbilisi, Georgia.
Cell Biochem Funct. 2019 Oct;37(7):545-552. doi: 10.1002/cbf.3431. Epub 2019 Aug 19.
Ras-GTPases regulate many central signalling pathways in the cell. Hypoxia induces nitrosative/oxidative stress and dysregulates Ras-dependent downstream processes. H-Ras possesses two cysteine residues (C181 and C184) in the C-termini, which are palmitoylated once or twice. Palmitoylation is sufficient for promoting stable plasma membrane localization. We hypothesized that high concentrations of hypoxia-formed nitric oxide could induce terminal cysteine S-nitrosylation, followed by depalmitoylation and H-Ras mislocalization. We investigated the action of a 100-μM nitric oxide-donor (sodium nitroprusside [SNP]) and a 100-μM palmitoylation inhibitor (2-bromopalmitate) on the distribution of membrane-bound S-nitrosylated and palmitoylated H-Ras under hypoxic/normoxic conditions in undifferentiated/differentiated pheochromocytoma (PC12) cells. We found that under normoxic conditions, SNP increases membrane-bound H-Ras nitrosylation only in differentiated cells, whereas under hypoxic conditions, SNP stimulates H-Ras nitrosylation in both differentiated and undifferentiated cells. SNP greatly decreases the palmitoylation of H-Ras under hypoxic conditions in both undifferentiated and differentiated cells, while under normoxic conditions, the effect of SNP is more negligible. Furthermore, Western blot analyses have shown that SNP decreases ERK phosphorylation under hypoxic conditions, in parallel with an elevation in hypoxia-induced factor activity and intracellular succinate concentration. We propose that high concentrations of hypoxia-formed nitric oxide can nitrosylate H-Ras terminal cysteines, which induce H-Ras activity dysregulation and alter the cellular response to hypoxia. SIGNIFICANCE OF THE STUDY: To our knowledge, these observations may be important for cancer prevention and therapy because cancer is one of the most prevalent disorders caused by the misregulation of Ras activity by a redox agent. Oncogenic activation of the H-Ras gene has been found in a wide variety of neoplastic transformations, and thus, investigation of the redox regulation of H-Ras activity is significant for cancer research as well.
Ras-GTPases 调节细胞内的许多中央信号通路。缺氧诱导硝化/氧化应激，并使 Ras 依赖性下游过程失调。H-Ras 在 C 末端有两个半胱氨酸残基（C181 和 C184），它们被棕榈酰化一次或两次。棕榈酰化足以促进稳定的质膜定位。我们假设，高浓度的缺氧形成的一氧化氮可以诱导末端半胱氨酸 S-亚硝化，随后去棕榈酰化和 H-Ras 定位错误。我们研究了 100μM 一氧化氮供体（硝普钠[SNP]）和 100μM 棕榈酰化抑制剂（2-溴棕榈酸）在缺氧/常氧条件下对未分化/分化嗜铬细胞瘤（PC12）细胞中膜结合 S-亚硝化和棕榈酰化 H-Ras 分布的作用。我们发现，在常氧条件下，SNP 仅在分化细胞中增加膜结合 H-Ras 的亚硝化，而在缺氧条件下，SNP 刺激未分化和分化细胞中的 H-Ras 亚硝化。SNP 在缺氧条件下大大降低了未分化和分化细胞中 H-Ras 的棕榈酰化，而在常氧条件下，SNP 的作用则更微不足道。此外，Western blot 分析表明，SNP 降低了缺氧条件下 ERK 的磷酸化，同时缺氧诱导因子活性和细胞内琥珀酸浓度升高。我们提出，高浓度的缺氧形成的一氧化氮可以亚硝化 H-Ras 末端半胱氨酸，从而诱导 H-Ras 活性失调，并改变细胞对缺氧的反应。研究的意义：据我们所知，这些观察结果可能对癌症的预防和治疗很重要，因为癌症是由氧化还原剂对 Ras 活性的失调引起的最常见疾病之一。H-Ras 基因的致癌激活已在多种肿瘤转化中发现，因此，研究 H-Ras 活性的氧化还原调节对癌症研究也具有重要意义。
