四氢生物蝶呤合成酶GTPCH1和DHFR的泛素化降解与尼古丁诱导内皮功能紊乱的机制

内皮功能紊乱是吸烟导致血管疾病的关键病理生理环节。吸烟及尼古丁削弱内皮依赖的血管舒张功能。预实验显示：尼古丁显著降低一氧化氮合酶（eNOS）重要辅因子四氢生物蝶呤（BH4）水平。尼古丁诱导BH4生成中两种关键酶GTP环化水解酶1（GTPCH1）和二氢叶酸还原酶（DHFR）泛素化降解，E3泛素连接酶Skp2可能发挥重要作用。我们推测：尼古丁促进GTPCH1和DHFR的泛素化降解，阻断BH4的从头和补救合成通路，导致eNOS脱偶联，而诱导内皮功能紊乱。本课题将围绕这一假说开展工作：1）明确BH4缺乏在尼古丁诱导内皮功能紊乱中的作用；2）阐明尼古丁对BH4从头合成及补救生成通路的影响及其机制；3）解析尼古丁诱导GTPCH1和DHFR泛素化降解的机制；4）探讨E3连接酶Skp2在内皮功能紊乱中的作用。本课题将在基因、蛋白及整体水平阐明泛素化在内皮功能中的新机制，为吸烟导致血管疾病提供新的干预靶点。

Endothelial dysfunction is a key pathophysiological step in cigarette smoking-induced vascular diseases. The studies showed that smoking and nicotine impaired endothelium-dependent relaxation. Our preliminary results showed that nicotine decreased the level of tetrahydrobiopterin (BH4), a cofactor of endothelial NO synthase (eNOS). In addition, we found that nicotine induced the ubiquitinations and degradation of GTP cyclohydrolase 1 (GTPCH1) and dihydrofolate reductase (DHFR), two key molecules for the bioavailability of BH4. E3 ligase S-phase kinase-associated protein 2 (Skp2) might play important roles during this process. Thus, the central hypothesis is that nicotine lowers BH4 level, induces eNOS uncoupling and endothelial dysfunction by decreasing the levels of GTPCH1 and DHFR via a mechanism involving ubiquitination-proteasome pathway. We will examine this hypothesis by addressing the following specific aims: 1) To explore the role of BH4 in nicotine-induced endothelial dysfunction. 2) To examine the effects and mechanisms of nicotine on the de novo biosynthetic and salvage pathways of BH4. 3) To define the mechanisms of nicotine-induced GTPCH1 and DHFR ubiquitination. 4) To characterize the role of E3 ligase Skp2 in nicotine instigated endothelial dysfunction. These studies will reveal a novel mechanism by which protein ubiquitinations modify endothelial functions and provide a new therapeutic target for smoking-related vascular diseases.