TETRAHYDORBIOPTERIN BIOSYNTHESIS BY DOPAMINE NEURONS

描述（改编自申请人摘要）： 酶GTP环化水解酶I（GTPCH）催化第一和限制性步骤 在四氢生物蝶呤（BH 4）的合成中， 单胺（MA）神经递质的产生。BH 4可用性是一个控制点 MA的合成，并通过GTPCH基因表达的变化进行调节。GTPCH 基因表达是由信号转导途径控制的， GTPCH启动子。其中一些信号通路利用第二个 信使cAMP和蛋白激酶A（PKA）。为了更好地理解 cAMP和PKA控制GTPCH基因的转录，我们已经克隆，测序， 开始表征5.8kb的大鼠GTPCH启动子。我们重点抓了 GTPCH核心启动子内的顺式作用CRE和邻近的CCAAT盒元件 因为我们已经证明它们对基础和cAMP依赖性的 转录。本申请的总体目标是牢固确立 反式作用因子ATF-4和NF-Y被这些DNA应答结合 元件，并通过PKA的作用，用于介导cAMP依赖性 GTPCH转录的增强，我们已经在PC 12细胞中观察到。 具体目标我将使用重组ATF-4和NF-Y，EMSA，supershift，并在 体外和体内DNA足迹技术分析蛋白质复合物 CRE和CCAAT-box成员招募。本目标还将调查 CRE和CCAAT框之间的空间关系以及 非典型的TATA盒转录。Specific i Aim 2将使用TPCH 与野生型的强制表达组合的启动子报告基因构建体， ATF-4和NF-Y的显性阴性形式，以检验这些 蛋白质和共激活因子CBP在基础和 cAMP依赖性转录。Specifie Aim 3将使用GTPCH启动子报告基因 构建沿着与野生型和显性阴性的fored表达 PKA的肽抑制剂和PKA缺陷细胞系， 确定PKA在控制转乳中的作用。这一目标还将 研究ATF-4和NF-Y是否在体外和体内被磷酸化， PKA。 由于多巴胺（DA）的合成是由人 黑质纹状体神经元对GTPCH基因突变选择性易感 这会导致遗传性进行性肌张力障碍（HPD），一种运动障碍， 主要影响年轻女孩。了解eAMP耦合信号如何 DA能神经元内GTPCH基因转录调控的转导途径 因此对于我们理解HPD的根本原因至关重要 表型和其他可能涉及黑质纹状体DA神经元的疾病， 比如帕金森病

DESCRIPTION(Adapted from applicant's abstract): The enzyme GTP cyclohydrolase I (GTPCH) catalyzes the first and limiting step in the synthesis of tetrahydrobiopterin (BH4), the required cofactor for monoamine (MA) neurotransmitter production. BH4 availability is a control point for MA synthesis and is regulated by changes in GTPCH gene expression. GTPCH gene expression is controlled by signal transduction pathways that converge on the GTPCH promoter. Some of these signaling pathways utilize the second messenger cAMP and protein kinase A (PKA). In order to better understand how cAMP and PKA control GTPCH gene transcription we have cloned, sequenced and begun to characterize 5.8 kb of the rat GTPCH promoter. We have focused on the cis-acting CRE and adjacent CCAAT-box elements within the GTPCH core promoter because we have already shown them to be critical for basal and cAMP-dependent transcription. The overall goal of this application is to firmly establish that the trans-acting factors ATF-4 and NF-Y are bound by these DNA response elements and, through the actions of PKA, serve to mediate the cAMP-dependent enhancement of GTPCH transcription that we have observed in PC12 cells. Specific Aim I will use recombinant ATF-4 and NF-Y, EMSA, supershift, and in vitro and in vivo DNA footprinting techniques to analyze the protein complexes recruited by the CRE and CCAAT-box elements. This Aim will also investigate the spatial relationship between the CRE and CCAAT-box as well as the role of the non-canonical TATA-box in transcription. Specific i Aim 2 will use TPCH promoter reporter constructs combined with foreed expression of wild type and dominant negative forms of ATF-4 and NF-Y to test the hypothesis that these proteins and the eo-activator CBP play an essential role in basal and cAMP-dependent transcription. Specifie Aim 3 will use GTPCH promoter reporter eonstruets along with foreed expression of wild type and dominant negative subunits of PKA, a peptide inhibitor of PKA and a PKA-defieient eell line to establish the role of PKA in the control of transeription. This Aim will also investigate whether ATF-4 and NF-Y are phosphorylated in vitro and in vivo by PKA. This researeh is important because dopamine (DA) synthesis by human nigrostriatal neurons is selectively vulnerable to genetic mutations in GTPCH that produce hereditary progressive dystonia (HPD), a disorder of movement that primarily effects young girls. Knowledge of how eAMP-eoupled signal transduetion pathways eontrol GTPCH gene transeription within DA neurons is therefore critical to our understanding of the underlying causes of the HPD phenotype and possibly other illnesses that involve nigrostriatal DA neurons, such as Parkinson's Disease.