硫巯基化修饰RIPK3调控腹膜透析相关腹膜纤维化的机制研究

One of the characteristics in fibrosis related diseases is metabolic disturbance, and peritoneal fibrosis is difficult to be sloved. Theraputical effect of Hydrogen sulfide (H2S) has been proved in multiple fibrosis realted diseases, while the clinical trails with H2S has been conducted in respiratory disorders. Preliminary data indicated that the H2S could improve the pritoneal dialysis related pritoneal fibrosis, as well as the metabolic disturbance in peritoneum. Biological effects of H2S are mainly performanced via S-sulfhydration modification. Mass spectrum revealed hunreds proteins with the possbility of S-sulfhydration modification in rodent peritoneum. One of them is receptor-interacting serine/threonine-protein kinase 3 (RIPK3) , with the capacity of interaction with key enzymes of amino acid metabolism, such as glutamine synthetase. Thus the study of RIPK3 modification and site of S-sulfhydration modification should be explored firstly. Then the site mutant, Co-IP, luciferase reported gene and targeted metabolomics have been used to verify the functional change in glutamine synthetase. Finally, the possbility of metabolic regualtion remedy peritoneal fibrosis would be evaluated. The present study could provide a reference for effective and safety approach of peritoneal fibrosis and be a expand of H2S related theraputic prospect.

纤维化病变的重要特征之一是代谢异常，腹膜纤维化是纤维化病变治疗中的难点。硫化氢（H2S）对多种纤维化病变均有治疗作用，预实验发现H2S不单可以改善腹膜透析相关腹膜纤维化，同时还能调节纤维化相关的代谢失衡，而H2S主要通过修饰靶蛋白半胱氨酸上巯基，即S-硫巯基化作用来发挥生物学作用。质谱技术鉴别到腹膜组织中存在多种可能发生S-硫巯基化的蛋白质，受体相互作用蛋白激酶3（RIPK3）是其中之一，而且已知RIPK3可以和谷氨酰胺合成酶（GLUL）等蛋白发生相互作用。为此课题组展开研究，首先阐明H2S对RIPK3半胱氨酸巯基的修饰作用，以及修饰作用的位点；进一步利用点突变、Co-IP，报告基因及定量代谢组学等方法明确RIPK3 S-硫巯基化对GLUL功能的影响；由此评价通过调控谷氨酰胺代谢来干预腹膜纤维化进程的可能。有望拓展硫化氢的治疗领域，也为寻找安全高效的腹膜纤维化治疗策略提供参考。

腹膜纤维化是实质脏器纤维化病变的难点，项目组通过体内、外实验发现硫化氢（H2S）对腹膜间皮细胞纤维化的治疗性作用较为明确，进一步研究的重心在于H2S延缓腹膜纤维化的分子靶点以及H2S制剂的剂型和给药途径。.项目组聚焦于H2S通过修饰靶蛋白半胱氨酸上巯基，即S-硫巯基化作用来发挥生物学作用这一机制。利用质谱技术鉴别到腹膜组织中存在多种可能发生S-硫巯基化的蛋白质，受体相互作用蛋白激酶3（RIPK3）是其中之一，同时明确了RIPK3可以和谷氨酰胺合成酶（GLUL）发生相互作用，进而调控了谷氨酰胺代谢，从而影响腹膜纤维化。同时还发现半胱氨酸上的巯基不仅可以发生S-硫巯基化，还可以发生N-亚硝基化，且两者存在一定的平衡关系，进一步利用质谱分析发现了丝氨酸/苏氨酸蛋白激酶31（Serine/threonine-protein kinase 31, STK31）和E3泛素连接酶MIB1（E3 Ubiquitin ligase Mindbomb1, MIB1）同时可能发生S-硫巯基化和N-亚硝基化。尤其是STK31的S-硫巯基化与N-亚硝基化平衡对间皮细胞纤维化过程中的细胞周期调控产生重要影响。.本项目的开展，为H2S防治腹膜纤维化的分子机制提供了重要的理论参考，在H2S通过翻译后修饰水平调控细胞功能上提出了新的思考及研究逻辑，为后续研究奠定了扎实的基础。