TETRAHYDORBIOPTERIN BIOSYNTHESIS BY DOPAMINE NEURONS

描述(改编自申请人的摘要)： 酶GTP环水解酶I(GTPCH)催化第一步也是限制性步骤 在四氢生物蝶呤(BH4)的合成中，所需的辅因子 单胺(MA)神经递质的产生。BH4的可用性是一个控制点 通过GTPCH基因表达的变化来调节MA的合成。GTPCH 基因表达受汇聚在一起的信号转导通路控制 GTPCH启动子。这些信号通路中的一些利用第二个 信使cAMP和蛋白激酶A(PKA)。为了更好地了解 CAMP和PKA调控GTPCH基因转录 开始鉴定5.8kb的大鼠GTPCH启动子。我们将重点放在 GTPCH核心启动子中顺式作用的Cre和邻近的CCAAT-box元件 因为我们已经证明它们对于基础和cAMP依赖是至关重要的 抄写。这个应用程序的总体目标是坚定地确立 反式作用因子ATF-4和NF-Y被这些DNA反应结合 元素，并通过PKA的行动，起到调解cAMP依赖的作用 我们在PC12细胞中观察到GTPCH转录增强。 具体目标I将使用重组ATF-4和NF-Y、EMSA、SuperShift和In 利用体内外DNA足迹技术分析蛋白质复合体 被CRE和CCAAT-BOX成员招募。这一目标还将调查 Cre和CCAAT-box之间的空间关系以及CCAAT-box的作用 转录中的非规范塔塔盒。SPICAL I AIM 2将使用TPCH 启动子报告结构与野生型和外源表达的结合 ATF-4和NF-Y的显性否定形式来检验以下假设 蛋白质和EO激活剂CBP在基础和 CAMP依赖的转录。Specifie Aim 3将使用GTPCH启动子报告 野生型和显性负性表达的结构 PKA亚单位，PKA的多肽抑制物和PKA定义的细胞系 确立PKA在转运控制中的作用。这一目标还将 研究ATF-4和NF-Y在体外和体内是否被磷酸化 PKA. 这项研究很重要，因为人类合成多巴胺(DA) 黑质纹状体神经元选择性地易受GTPCH基因突变的影响 导致遗传性进行性肌张力障碍(HPD)，这是一种运动障碍， 主要影响年轻女孩。了解eAMP如何耦合信号 转导途径控制GTPCH基因在DA神经元内的转导 因此对我们理解HPD的根本原因至关重要 表型和可能涉及黑质纹状体DA神经元的其他疾病， 比如帕金森氏症。

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.