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

描述（由申请人提供）：本研究的重点是人丙酮酸脱氢酶复合物（PDHc-h），由六种蛋白质组成，包括硫胺素二磷酸（ThDP）依赖的丙酮酸脱氢酶（E1， α2ß2异四聚体），二氢脂酰胺转乙酰化酶（E2，具有两个脂酰基结构域），二氢脂酰胺脱氢酶（E3），独特的E3结合蛋白（E3BP）和两种调节酶丙酮酸脱氢酶激酶（PDK1-4）和磷酸酶（PDP1-2）。具体来说，PDK1通过E1磷酸化调控PDHc，因为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，可以为抗癌药物的设计提供新的靶点。由于PDKs位于整个复合物的E2•E3BP核心（与E2•E3BP结合更强），特异性抑制PDK1-E2•E3BP相互作用为治疗癌症提供了一种可能的新方法。最近，Jordan小组在2015年1月的《生物化学》特刊《激酶研究前沿》上发表了第一篇论文，通过两种互补的方法，即hd -交换质谱（HDX-MS）和多核核磁共振（NMR）光谱，定义了PDK1（和PDK2）和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在复杂的多步骤途径中PDK1对E1磷酸化的影响在何处表达；在这个目标中，沿着复杂途径检测ThDP中间体将通过既定的方法进行，即CD光谱，Tittmann-Hubner (TH) NMR分配和停止流动CD。该提案的第二个主要目标将集中在后续实验上，以验证最近Jordan小组出版物中确定的E2•E3BP上与PDK1相互作用的位点，这将通过两个连续的步骤完成：(i)对E2•E3BP相互作用图中确定的氨基酸进行定点诱变研究，并在最近的论文中作为“热点”，随后（ii）利用HDX-MS、等温滴定量热法（ITC）和荧光滴定对取代的E2•E3BP与PDK1和PDK2的蛋白质-蛋白质相互作用进行研究。这些测量结果将确认所询问的“热点”是否确实对E2•E3BP与PDK1的相互作用很重要，以及通过将这些测量结果与PDK2进行比较，它是否仅针对PDK1。这些实验将确定“真正的”热点，并据此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.