半胱氨酸在结核分枝杆菌氧化还原稳态中的功能研究及靶标发现

Tuberculosis (TB) still remains a serious threat to human health. With the increasing incidence of multi-drug resistant (MDR-TB) and extensively drug resistant TB (XDR-TB), it’s seriously urgent to find new anti-TB drugs with new mechanism. Cysteine is widely involved in Mycobacterium tuberculosis redox signaling pathways through a number of reversible and irreversible modifications, and plays an important role in maintaining the redox homeostasis of Mycobacterium tuberculosis. Reversible modifications (e.g., S-nitrosylation, disulfide bonds, and sulfenic acid) are used to protect proteins from oxidative attack and maintain cellular homeostasis, while irreversible oxidations (e.g., sulfinic acid and sulfonic acid) serve as hallmarks of oxidative stress. Proteomic analysis of cysteine enriched peptides coupled with reduction of oxidized thiols can be used to measure the oxidation states of cysteine, which is helpful for elucidating the role that oxidative stress plays in biology and disease. This project intends to use proteomics methods to analyze the levels of S-nitrosylation and S-sulfinylation of Mycobacterium tuberculosis under different oxidant stress conditions, in order to obtain important cysteine sites and key proteins that have marked changes in stress status, to evaluate their ppossibility of becoming a drug target, and to provide new ideas for the development of anti-tuberculosis drugs with new mechanisms.

结核病（TB）仍然对人类健康有严重威胁。随着耐多药（MDR-TB）和泛耐药结核病（XDR-TB）发病率的增加，寻找具有新机制的抗结核药物日益迫切。半胱氨酸通过许多可逆和不可逆的修饰广泛参与结核分枝杆菌氧化还原信号传导途径，对维持结核分枝杆菌氧化还原稳态具有重要作用。可逆修饰（如S-亚硝基化、二硫键和次磺酸）用于保护蛋白免受氧化攻击并保持细胞稳态，而不可逆氧化（如亚磺酸和磺酸）则是结核分枝杆菌氧化应激的标志。半胱氨酸肽段富集结合硫醇氧化还原的蛋白质组学分析手段可用于检测结核分枝杆菌应激状态下半胱氨酸残基的修饰变化，这有助于阐明氧化应激在生物学和疾病中的作用。本项目拟采用蛋白组学的方法对结核分枝杆菌对不同氧化剂应激条件下的半胱氨酸残基S-亚硝基化和S-亚磺酰化的修饰水平进行分析，以期得到具有标志性应激状态变化的半胱氨酸位点和关键蛋白，并对其成靶性进行评价，为新机制抗结核药物研发提供新思路。

随着耐药结核分枝杆菌的不断发展，寻找具有新靶点新机制的抗结核药物十分迫切。近期研究显示，半胱氨酸的可逆和不可逆的修饰在维持细菌的氧化还原稳态中具有非常重要的作用。本项目借助比较蛋白组学的手段、同位素标记的技术以及抗体富集的方法对结核分枝杆菌在氧化应激状态下总体蛋白半胱氨酸的氧化修饰进行研究，发现脂肪酸代谢及胆固醇降解等相关蛋白的半胱氨酸S-亚磺酰化修饰与细菌的氧化应激密切相关，提示其成为新靶标的潜力。此外，我们对半胱氨酸残基在结核分枝杆菌CYP142A1结构和功能中的作用进行了进一步探索，发现六个非中心位的半胱氨酸残基的取代会导致酶的二级结构、热稳定性和氧化效率发生许多变化。这些结果有助于我们理解半胱氨酸残基在结核分枝杆菌细胞色素P450酶的结构和功能中的作用。