alpha-突触核蛋白通过PP2A调节TH磷酸化的结构学机理

Parkinson's disease is the second most common neurodegenerative.disorder after Alzheimer's disease, and affects 2% of people over the age of 60. The main cause of the movement disorder is the dopamine depletion, which results from greatly reduced activity of dopamine-secreting cells caused by cell death in the pars compacta region of the substantia nigra. Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, is stimulated by N-terminal phosphorylation, which determines the dopamine biosynthesis activity. We and others have previously demonstrated that tyrosine hydroxylase is almost exclusively dephosphorylated and consequently inhibited by protein serine/threonine phosphatase 2A (PP2A), and the alpha-synuclein(α-syn) may potentiate PP2A's ability of dephosphorylation, resulting more inhibition of TH and less dopamine biosynthesis. However, now the structural mechanism underlying the α-syn-and-PP2A-mediated TH phosphorylation modulation is still elusive. In this project, we plan to employ the site-directed mutagenesis, gel-filtration, pull-down, and so on, to study the structure-function relationships between the PP2A, TH and α-syn, to identify the binding sites and even finally the exact corresponding residues in the respective protein. Through this approach we may get novel insight into the precise manners by which α-syn binds to PP2A to stimulate PP2A activity, which vigorously dephosphorylate the TH and control the dopamine biosynthesis. Shortly, the primary goal of this project is to explore the structural mechanism of α-syn potentiating the ability of PP2A to reduce TH phosphorylation. Delineating these mechanisms holds promise to help deeply understand the PD and aid in the design of novel therapies to optimize dopaminergic neurotransmission for the treatment of PD.

帕金森病是仅次于阿兹海默症的第二大神经退行性疾病。黑质纹状体多巴胺递质水平低下是该病运动功能异常的主要原因。酪氨酸羟化酶(TH)是儿茶酚胺类物质合成限速酶,磷酸化水平决定其多巴胺合成的活性。本组及他人都证明蛋白磷酸酶2A(PP2A)能够使TH去磷酸化从而抑制它的活性,而且α-突触核蛋白（α-syn）能够上调PP2A的去磷酸化活性从而降低多巴胺的生物合成。但是这种调控背后的分子结构学机理并不清楚。本研究试图采用定点突变，凝胶过滤及Pull-down等方法，对PP2A，TH以及α-syn进行结构-功能分析，研究TH与PP2A的结合方式，找出在PP2A上TH的结合位点；同时揭示α-syn和PP2A结合作用方式，洞察二者起结合作用的结构域及具体的氨基酸位点。最终明确α-syn通过上调PP2A活性而使TH磷酸化水平降低的结构学机理，为深入理解帕金森病的发病机制和开发潜在药物靶点提供新思路。

帕金森病是仅次于阿兹海默症的第二大神经退行性疾病。黑质纹状体多巴胺递质水平低下是该病运动功能异常的主要原因。酪氨酸羟化酶(TH)是儿茶酚胺类物质合成限速酶,磷酸化水平决定其多巴胺合成的活性。本组及他人都证明α-突触核蛋白（α-syn）能够上调PP2A的活性从而使TH去磷酸化进而降低多巴胺的生物合成，但是这种调控背后的分子结构学机理并不清楚。本研究试图采用定点突变，凝胶过滤及Pull-down等方法，对PP2A和α-syn进行结构-功能分析，揭示α-syn和PP2A结合作用方式，洞察二者起结合作用的结构域及具体的氨基酸位点，最终明确α-syn通过上调PP2A活性而使TH磷酸化水平降低的结构学机理。首先，分别在大肠杆菌和昆虫细胞中过表达α-syn和PP2A 的催化亚基Cα，通过亲和层析和凝胶过滤等方法将其纯化，而后应用GST Pull-down法在体外检测α-syn和PP2A Cα的直接相互作用，并用活性比色法证明α-syn可以上调PP2A Cα的活性。然后，分别在α-syn及PP2A Cα上进行一系列突变，结合蛋白分子结构模拟、GST Pull-down及酶活分析法证明我们的实验假设：α-syn的NAC区可以结合到PP2A Cα的疏水中心从而调节PP2A的活性。最后，我们还确认了PP2A Cα疏水中心的I123位点对于α-syn的结合和活性调节起着重要的作用。通过上述系列实验得出结论：α-syn的NAC区和PP2A Cα的疏水中心（尤其是I123位点）存在直接结合作用，而且这些结构域/氨基酸位点对α-syn上调PP2A 活性从而降低TH 磷酸化水平起着重要作用。在此基础上，我们还发现过表达α-Syn可以使胞内产生氧化应激，从而使烟碱性乙酰胆碱受体（nAChR）α4β2失活，进而探究了其背后的机理。通过这些研究，为深入理解帕金森病的发病机制和开发潜在药物靶点提供了新思路。.在该项目执行期间，本课题组已经完成全部计划的研究内容。上述成果已发表SCI论文5篇（其中三篇IF>5），另投送出去SCI论文1篇（目前在评审中）；发表两篇中文核心期刊文章；申请相关国家发明专利1项，现处于公报阶段；参加国内外学术交流活动共2次；培养硕士生4名。