Mechanism and Interactions of Human Pyruvate Dehydrogenase Complex with its Kinase 1.

描述(由申请人提供)：这项研究的重点是人丙酮酸脱氢酶复合体(PDHc-h)，由六种蛋白质组成，包括依赖于THDP的丙酮酸脱氢酶(E1，α2ü2-异构体)、二氢硫辛酰胺转乙酰酶(E2，具有两个硫氨基结构域)、二氢硫辛酰胺脱氢酶(E3)、一个独特的E3结合蛋白(E3BP)和两个调节酶丙酮酸脱氢酶(PDK1-4)和磷酸酶(PDP1-2)。具体地说，由于PDK1在多种癌细胞中被认为是过度表达的罪魁祸首，因此抑制PDK1与E1或E2·E3BP的相互作用可能为抗癌药物的设计提供新的靶点，因此将通过PDK1对E1的磷酸化来调节PDHc。由于PDK位于整个复合体的E2·E3BP核心上(与之有较强的结合)，因此特异性地抑制PDK1-E2·E3BP的相互作用为治疗癌症提供了一种可能的新途径。最近，Jordan小组在2015年1月题为《Kinase研究的前沿》的生物化学特刊上发表了第一篇论文，用两种互补的方法--HD-MS和多核核磁共振光谱--定义了PDK1(和PDK2)与E2.E3BP核心之间的相互作用位点。该提案的第一个主要目标是确定PDK1在复杂的多步途径上的什么地方表达了E1磷酸化的影响；在这个目标中，沿着复杂途径的ThDP中间体的检测将通过既定的方法来执行，即CD光谱、ITH-HUBNER(TH)核磁共振指认和停流CD。该建议的第二个主要目标将侧重于后续实验，以验证约旦小组最近发表的文章中确定的与PDK1相互作用的E2·E3BP上的位点，这将通过两个连续的步骤来完成：(1)定点突变研究最近论文中确定为“热点”的E2·E3BP相互作用图中的氨基酸，然后(2)使用HDX-MS、等温滴定热法(ITC)和荧光滴定研究被取代的E2·E3BP与PDK1和PDK2的蛋白质-蛋白质相互作用。这些测量将通过与PDK2进行比较，确认所询问的“热点”是否确实对E2.E3BP与PDK1的相互作用重要，以及它是否是PDK1所特有的。这些实验将确定“真正的”热点，可以针对这些热点进行合理的药物设计。

DESCRIPTION (provided by applicant): The focus of this study is on the human pyruvate dehydrogenase complex (PDHc-h) consisting of six proteins including thiamin diphosphate (ThDP)-dependent pyruvate dehydrogenase (E1, α2ß2-heterotetramer), the dihydrolipoamide transacetylase (E2, with two lipoyl domains), dihydrolipoamide dehydrogenase (E3), a unique E3-binding protein (E3BP) and two regulatory enzymes pyruvate dehydrogenase kinases (PDK1-4) and phosphatases (PDP1-2). Specifically, the regulation of PDHc via phosphorylation of E1 by PDK1 will be pursued, as PDK1 has been strongly implicated as an overexpressed culprit in multiple cancer cells; hence inhibition of the interaction of PDK1 with the E1 or E2•E3BP, could provide novel targets for the design of anti- cancer agents. Since the PDKs reside on (have a stronger binding to) the E2•E3BP core of the entire complex, specific inhibition of the PDK1-E2•E3BP interaction provides a possible new approach to treatment of cancer. Recently, the Jordan group published the first paper defining the interaction loci between the PDK1 (and PDK2) and the E2•E3BP core by two complementary methods, HD-exchange mass spectrometry (HDX-MS) and multinuclear nuclear magnetic resonance (NMR) spectroscopy in a special issue of Biochemistry titled `Frontiers in Kinase Research' in January 2015. The first principal goal of the proposal is to determine where along the complex multistep pathway the effect of E1 phosphorylation by PDK1 is expressed; in this goal the detection of ThDP intermediates along the complex pathway will be performed by established methods, namely, CD spectroscopy, the Tittmann-Hubner (TH) NMR assignments and stopped-flow CD. The second principal goal of the proposal will focus on follow-up experiments to validate the sites on E2•E3BP identified to interact with PDK1 in the recent Jordan group publication, which will be accomplished by two consecutive steps: (i) site- directed mutagenesis studies on the amino acids identified in the interaction maps of E2•E3BP as `hot spots' in the recent paper, followed by (ii) protein-protein interaction studies on the substituted E2•E3BP with PDK1 and PDK2 using HDX-MS, isothermal titration calorimetry (ITC) and fluorescence titrations. These measurements will confirm whether the `hot spot' interrogated indeed is important for the interaction of E2•E3BP with the PDK1, and whether it is specific to PDK1 by comparing such measurements with PDK2. These experiments will identify `real' hot spots, against which rational drug design could be undertaken.