Angiotensin-II, GTPCH1 and 26S Protesomes

描述（由申请人提供）：鸟苷5'-三磷酸环水解酶I （GTPCH1）是一种由25 kda亚基组成的同十聚体蛋白，该酶催化GTP重排为三磷酸二氢蝶呤，随后通过6-丙酮酰基四氢蝶呤合成酶和七氢蝶呤还原酶的顺序作用转化为四氢生物蝶呤（BH4）。与后两种酶相反，GTPCH1在大多数组织中是限速酶，使其成为细胞内BH4含量的主要决定因素。GTPCH1在血管细胞中组成性表达；然而，GTPCH1是否对这些细胞中BH4水平的维持至关重要尚不清楚。最近的一些研究表明BH4缺乏是高血压期间内皮型一氧化氮合酶（eNOS）解偶联的原因，正如发现高血压和相关的eNOS解偶联可以通过联合给药sepapterin （BH4的前体）有效预防。补充BH4或通过GTPCH1增加BH4合成可恢复BH4水平，使醋酸脱氧皮质酮盐高血压小鼠eNOS功能正常化。GTPCH1活性以及BH4水平在这些小鼠中降低。然而，eNOS解耦与GTPCH1之间的体内因果关系尚未研究，GTPCH1对eNOS介导的内皮功能保护至关重要。特别是，目前尚不清楚高血压等病理性刺激如何降低GTPCH1水平，以及血管紧张素-II （Ang II）和高血压等刺激如何调节蛋白酶体功能。我们令人兴奋的新初步数据使我们假设Ang II通过氧化剂（如ONOO-）增加26S蛋白酶体介导的GTPCH1降解，导致BH4缺乏，eNOS解偶联和血压升高。这一中心假设将在三个相互关联的特定目标中进行测试，通过使用实验方法的组合，包括纯化蛋白、培养细胞和几种体内高血压模型。这些研究将加深我们对BH4水平的上游调控、氧化应激和泛素-蛋白酶体系统在血管损伤发展中的作用以及氧化应激在血压调节中的作用的理解，具有重要意义。特别是，这些研究将为MG132的治疗效果提供线索，MG132是一种蛋白酶体抑制剂，最近被FDA批准用于癌症治疗。公共卫生相关性：当前应用的目的是剖析高血压患者血管内皮功能障碍的分子机制。三个相互关联的目标的完成将为GTPCH的上游调控提供新的知识，GTPCH是BH4水平的限速酶，氧化应激和泛素-蛋白酶体系统在血管损伤发展中的作用以及氧化应激在血压调节中的作用。一旦获得这些知识，我们对GTPCH1在健康和疾病中调控血管生物学的基本理解将取得重大进展。此外，有希望开发新的模式来测试GTPCH1/BH4途径作为高血压相关血管疾病治疗靶点的真正潜力，以及是否蛋白酶体抑制剂MG132（最近fda批准的癌症治疗方案）可用于治疗高血压的血管内皮功能障碍。

DESCRIPTION (provided by applicant): Guanosine 5'-triphosphate cyclohydrolase I (GTPCH1) is a homodecameric protein consisting of 25-kDa subunits, which enzyme catalyzes the rearrangement of GTP to dihydroneopterin triphosphate, a species subsequently converted to tetrahydrobiopterins (BH4) through the sequential action of 6-pyruvoyltetrahydrobiopterin synthase and sepiapterin reductase. In contrast to the latter two enzymes, GTPCH1 is the rate-limiting enzyme in most tissues, making it the major determinant of intracellular BH4 content. GTPCH1 is constitutively expressed in vascular cells; however, whether GTPCH1 is critical for the maintenance of BH4 levels in these cells is unknown. Several recent studies suggest that BH4 deficiency is responsible for endothelial nitric oxide synthase (eNOS) uncoupling during hypertension, as seen by the finding that hypertension and related eNOS uncoupling are effectively prevented by co- administration of sepiapterin, a precursor for BH4. BH4 supplementation or increased BH4 synthesis by GTPCH1 restores BH4 levels and normalizes eNOS function in the deoxycorticosterone acetate (DOCA)-salt hypertensive mice. GTPCH1 activity, as well as BH4 levels, is reduced in these mice. However, the in vivo cause-effect relationship between eNOS uncoupling and GTPCH1, which is critical for eNOS-mediated protection of endothelial function, has yet to be investigated. In particular, it is unclear how pathological stimuli such as hypertension reduce GTPCH1 levels and how stimuli such as angiotensin-II (Ang II) and hypertension modulate proteasome function. Our exciting new preliminary data have led us to hypothesize that Ang II, via oxidants such as ONOO-, increases 26S proteasome-mediated degradation of GTPCH1, resulting in BH4 deficiency, eNOS uncoupling, and the elevation of blood pressure. This central hypothesis will be tested in three interrelated specific aims by using a combination of experimental approaches including purified proteins, cultured cells, and several models of hypertension in vivo. The proposed studies are significant, as they will deepen our understanding of the upstream regulation of BH4 levels, the contribution of oxidative stress and ubiquitin-proteasome systems in the development of vascular injury, and the contribution of oxidative stress in blood pressure regulation. In particular, these studies will provide clues as to the therapeutic effects of MG132, a proteasome inhibitor that was recently approved by the FDA for cancer therapy. PUBLIC HEALTH RELEVANCE: The aim of the current application is to dissect the molecular mechanisms for vascular endothelial dysfunction in hypertension. Completion of three interrelated aims will provide new knowledge regarding the upstream regulation of GTPCH, a rate-limiting enzyme for BH4 levels as well as the contributions of oxidative stress and ubiquitin- proteasome systems in the development of vascular injury and the contribution of oxidative stress in blood pressure regulation. Once such knowledge is gained, significant advances in our fundamental understanding of GTPCH1 regulation of vascular biology in health and disease can be expected. In addition, there is the promise that new modalities can be developed to test the true potential of the GTPCH1/BH4 pathway as a therapeutic target in vascular diseases associated with hypertension and if proteasome inhibitor, MG132, a recent FDA-approved therapeutic regimen for cancer, can be used in treating vascular endothelial dysfunction in hypertension.