Department of Biochemistry, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, 700019, West Bengal, India.
Department of Biochemistry, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, 700019, West Bengal, India.
Arch Biochem Biophys. 2022 Oct 15;728:109358. doi: 10.1016/j.abb.2022.109358. Epub 2022 Jul 22.
Protein tyrosine nitration (PTN), a highly selective post translational modification, occurs in both prokaryotic and eukaryotic cells under nitrosative stress. However, its physiological function is not yet clear. Like many gut pathogens, Vibrio cholerae also faces nitrosative stress, which makes its proteome more vulnerable to PTN. Here, we report for the first time in-vivo PTN in V. cholerae by immunoblotting and LC-ESI-MS/MS proteomic analysis. Our results indicated that in-vivo PTN in V. cholerae was culture media independent. Surprisingly, in-vivo PTN was reduced in V. cholerae proteome under anaerobic or hypoxic condition in a nutrient deprived state. Interestingly, intracellular nitrate content was more than the nitrite content in V. cholerae under anaerobic conditions. Additionally, biochemical measurement of GSH/GSSG ratio, activities of catalase and SOD, ROS and RNS imaging by confocal microscopy confirmed a relative intracellular oxidizing environment in V. cholerae under anaerobic conditions. This altered redox environment favors the oxidation of nitrite which may be generated from protein denitration enriching the intracellular nitrate pool. The cell survival of V. cholerae can finally be facilitated by nitrate reductase (NapA) utilizing that nitrate pool. Our cell viability study using wild type and ΔnapA strain of V. cholerae also supported the role of NapA mediated cell survival under nutrient deprived anaerobic conditions. In spite of having nitrate reductase (NapA), V. cholerae lacks any nitrite reductase (NiR). Hence, in-vivo nitration may provide an avenue for toxic nitrite storage and also may help in nitrosative stress tolerance mechanism preventing further unnecessary protein nitration in V. cholerae proteome.
蛋白质酪氨酸硝化（PTN）是一种高度选择性的翻译后修饰，在硝化应激下发生在原核和真核细胞中。然而，其生理功能尚不清楚。像许多肠道病原体一样，霍乱弧菌也面临硝化应激，这使得其蛋白质组更容易受到 PTN 的影响。在这里，我们通过免疫印迹和 LC-ESI-MS/MS 蛋白质组学分析首次报道了霍乱弧菌中的体内 PTN。我们的结果表明，霍乱弧菌中的体内 PTN 与培养基无关。令人惊讶的是，在营养剥夺状态下的厌氧或缺氧条件下，霍乱弧菌的体内 PTN 在蛋白质组中减少。有趣的是，在厌氧条件下，霍乱弧菌细胞内的硝酸盐含量高于亚硝酸盐含量。此外，通过共聚焦显微镜对 GSH/GSSG 比、过氧化氢酶和 SOD 活性、ROS 和 RNS 成像的生化测定证实了厌氧条件下霍乱弧菌细胞内相对氧化环境。这种改变的氧化还原环境有利于亚硝酸盐的氧化，这可能是由蛋白质去硝化产生的，从而丰富了细胞内的硝酸盐池。硝酸盐还原酶（NapA）最终可以利用该硝酸盐池促进霍乱弧菌的细胞存活。我们使用野生型和ΔnapA 霍乱弧菌菌株进行的细胞存活率研究也支持了 NapA 介导的细胞在营养缺乏的厌氧条件下存活的作用。尽管霍乱弧菌具有硝酸盐还原酶（NapA），但它缺乏任何亚硝酸盐还原酶（NiR）。因此，体内硝化可能为有毒亚硝酸盐的储存提供了途径，并且可能有助于硝化应激耐受机制，防止霍乱弧菌蛋白质组中进一步发生不必要的蛋白质硝化。
