Angiotensin-II, GTPCH1 and 26S Protesomes

描述(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重排为二氢蝶呤三磷酸，该物种随后通过6-丙酮酰四氢生物蝶呤合成酶和海风蝶呤还原酶的顺序作用转化为四氢生物蝶呤(BH4)。与后两种酶相比，GTPCH1是大多数组织中的限速酶，使其成为细胞内BH4含量的主要决定因素。GTPCH1在血管细胞中有结构性表达；然而，GTPCH1是否对维持这些细胞中的BH4水平至关重要尚不清楚。最近的一些研究表明，BH4缺乏是高血压时内皮型一氧化氮合酶(ENOS)解偶联的原因，这一发现表明，联合使用BH4的前体sepiapterin可以有效地预防高血压及其相关的eNOS解偶联。补充BH4或GTPCH1增加BH4合成可恢复脱氧皮质酮醋酸酯(DOCA)-盐性高血压小鼠的BH4水平并使eNOS功能正常化。在这些小鼠中，GTPCH1的活性以及BH4水平都降低了。然而，eNOS解偶联和GTPCH1之间的体内因果关系仍有待研究，而GTPCH1是eNOS介导的内皮功能保护的关键。特别是，目前尚不清楚高血压等病理刺激如何降低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。我们令人兴奋的新的初步数据使我们假设Ang II通过ONOO-等氧化剂增加26S蛋白酶体介导的GTPCH1的降解，导致BH4缺乏、eNOS解偶联和血压升高。这一中心假设将通过组合实验方法在三个相互关联的特定目标上进行验证，包括纯化蛋白、培养细胞和几个体内高血压模型。这些研究具有重要意义，因为它们将加深我们对BH4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生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：目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提供关于GTPCH上游调控的新知识，GTPCH是BH4水平的限速酶，以及氧化应激和泛素-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。一旦获得了这样的知识，我们对GTPCH1调节血管生物学在健康和疾病中的基本理解将有望取得重大进展。此外，有希望开发新的方法来测试GTPCH1/BH4通路作为与高血压相关的血管疾病的治疗靶点的真正潜力，如果蛋白酶体抑制剂MG132，一种最近FDA批准的癌症治疗方案，可以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.