Angiotensin-II, GTPCH1 and 26S Protesomes

描述（由申请人提供）：鸟苷5 ′-三磷酸环化水解酶I（GTPCH 1）是由25-kDa亚基组成的同源十聚体蛋白，其酶催化GTP重排为二氢新蝶呤三磷酸，二氢新蝶呤三磷酸是随后通过6-异戊酰四氢生物蝶呤合酶和sepiapterin还原酶的顺序作用转化为四氢生物蝶呤（BH 4）的种类。与后两种酶相反，GTPCH 1是大多数组织中的限速酶，使其成为细胞内BH 4含量的主要决定因素。GTPCH 1在血管细胞中组成型表达;然而，GTPCH 1是否对维持这些细胞中的BH 4水平至关重要尚不清楚。最近的几项研究表明，BH 4缺乏是高血压期间内皮型一氧化氮合酶（eNOS）解偶联的原因，如通过共同施用BH 4的前体sepiapterin有效预防高血压和相关eNOS解偶联的发现所见。补充BH 4或通过GTPCH 1增加BH 4合成恢复BH 4水平，并使醋酸脱氧皮质酮（DOCA）盐高血压小鼠的eNOS功能正常化。在这些小鼠中，GTPCH 1活性以及BH 4水平降低。然而，eNOS解偶联和GTPCH 1之间的体内因果关系，这是eNOS介导的内皮功能保护的关键，还有待研究。特别是，目前尚不清楚病理刺激，如高血压降低GTPCH 1水平，以及如何刺激，如血管紧张素II（Ang II）和高血压调节蛋白酶体功能。我们令人兴奋的新的初步数据使我们假设，血管紧张素II，通过氧化剂，如ONOO-，增加26 S蛋白酶体介导的降解GTPCH 1，导致BH 4缺乏，eNOS解偶联，血压升高。这一中心假设将在三个相互关联的具体目标进行测试，通过使用实验方法的组合，包括纯化的蛋白质，培养的细胞，在体内高血压的几个模型。拟议的研究是重要的，因为它们将加深我们对BH 4水平的上游调节，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。特别是，这些研究将为MG 132的治疗效果提供线索，MG 132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：本申请的目的是剖析高血压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提供新的知识，上游调控GTPCH，BH 4水平的限速酶，以及在血管损伤的发展中的氧化应激和泛素-蛋白酶体系统的贡献和氧化应激在血压调节中的贡献。一旦获得这些知识，我们对健康和疾病中GTPCH 1调节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GTPCH 1/BH 4通路作为高血压相关血管疾病治疗靶点的真正潜力，以及蛋白酶体抑制剂MG 132（最近FDA批准的癌症治疗方案）是否可用于治疗高血压中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.