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

人丙酮酸脱氢酶复合物与其激酶 1 的机制和相互作用。

• 批准号: 9099514
• 负责人: Elena Luisa Guevara Talarico
• 金额: $ 2.34万
• 依托单位: RUTGERS THE STATE UNIV OF NJ NEWARK
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9099514
• 关键词: Acetyl Coenzyme A; Acetylation; Acids; Amino Acids; Antineoplastic Agents; Binding; Binding Proteins; Biochemistry; Breast Adenocarcinoma; Calorimetry; Communication; Complex; Decarboxylation; Detection; Drug Design; Enzymes; Family; Family member; Fluorescence; Functional disorder; Genes; Glioblastoma; Gluconeogenesis; Goals; Health; Hot Spot; Human; Isoenzymes; Jordan; Knowledge; Lead; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Measurement; Measures; Methods; Microscopic; Mitochondria; Molecular; Multienzyme Complexes; Mutation; NMR Spectroscopy; Non-Insulin-Dependent Diabetes Mellitus; Nuclear Magnetic Resonance; Outcome; Oxidoreductase; PDH kinase; PDPK1 gene; Paper; Pathway interactions; Patients; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Positioning Attribute; Primary carcinoma of the liver cells; Production; Protein phosphatase; Proteins; Publications; Publishing; Pyruvate; Pyruvate Dehydrogenase Complex; Pyruvate Dehydrogenase E1; Reaction; Recruitment Activity; Regulation; Renal Cell Carcinoma; Research; Research Project Grants; Scheme; Site; Site-Directed Mutagenesis; Spectrum Analysis; Structure; Testing; Textbooks; Thiamine Pyrophosphate; Time; Titrations; Validation; Visit; Warburg Effect; aerobic glycolysis; cancer cell; cancer therapy; design; dihydrolipoamide dehydrogenase; follow-up; frontier; malignant stomach neoplasm; molecular mass; novel; novel strategies; overexpression; oxidation; protein protein interaction; pyruvate dehydrogenase; research study

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.

描述（申请人提供）：本研究的重点是人丙酮酸脱氢酶复合物（PDHc-h），它由六种蛋白质组成，包括二磷酸硫胺素（ThDP）依赖的丙酮酸脱氢酶（E1，α 2 β 2-异四聚体），二氢硫辛酰胺转乙酰酶（E2，具有两个硫辛酰基结构域），二氢硫辛酰胺脱氢酶（E3），一种独特的E3结合蛋白（E3 BP）和两种调节酶丙酮酸脱氢酶激酶（PDK 1 -4）和磷酸酶（PDP 1 -2）。具体来说，将通过PDK 1磷酸化E1来调节PDHc，因为PDK 1已被强烈暗示为多种癌细胞中的过表达罪魁祸首;因此，抑制PDK 1与E1或E2·E3 BP的相互作用可以为抗癌药物的设计提供新的靶点。由于PDK位于整个复合物的E2·E3 BP核心上（具有更强的结合），特异性抑制PDK 1-E2·E3 BP相互作用提供了一种可能的治疗癌症的新方法。最近，Jordan小组在2015年1月题为“Frontiers in Kinase Research”的Biochemistry特刊上发表了第一篇论文，通过两种互补方法，HD交换质谱（HDX-MS）和多核核磁共振（NMR）光谱，定义了PDK 1（和PDK 2）与E2·E3 BP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沿着复杂的多步途径中，表达了E1通过PDK 1磷酸化的作用;在该目标中，将通过已建立的方法，即CD光谱、Tittmann-Hubner（TH）NMR分配和停流CD，检测ThDP中间体沿着复杂途径。该提案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出版物中确定的与PDK 1相互作用的E2·E3 BP上的站点，这将通过两个连续步骤来实现：（i）对E2·E3 BP相互作用图谱中鉴定为“热点”的氨基酸进行定点诱变研究，（ii）用HDX-MS、等温滴定量热法（ITC）和荧光滴定法研究了PDK 1和PDK 2取代的E2·E3 BP的蛋白质-蛋白质相互作用。这些测量将确认所询问的“热点”对于E2·E3 BP与PDK 1的相互作用是否确实重要，以及通过将这些测量与PDK 2进行比较，它是否对PDK 1特异。这些实验将确定“真实的”热点，据此可以进行合理的药物设计。