Mechanistic Issues Related to Aging on Human 2-Oxoglutarate Dehydrogenase Complex

 描述(申请人提供)：本R15提案的主要目标是研究依赖硫胺素二磷酸的人2-氧戊二酸脱氢酶复合体(OGDHc，也称为α-酮戊二酸脱氢酶；它由三种组成的酶组成，分别表示为E1o-h、E2O-h和E3-h)，这是柠檬酸循环(也称为三羧酸或Krebs循环)中的限速酶，是正常大脑葡萄糖代谢的关键酶。阿尔茨海默病患者的大脑中OGDHc活性降低，其特征是葡萄糖代谢降低和氧化应激标志物水平升高。OGDHc活性降低也与其他神经退行性疾病有关，包括帕金森氏病、亨廷顿病、Wernike-Korsakoff病和进行性亚核瘫痪。众所周知，OGDHc是氧化应激过程中产生的各种氧化剂的敏感靶标，包括过氧亚硝酸盐和一氧化氮，以及活性氧物种(ROS)、超氧化物和过氧化氢等试剂对OGDHc的抑制。OGDHc不仅是ROS的敏感靶点，还能在脑线粒体中产生超氧化物歧化酶和过氧化氢。虽然早期的研究将OGDHc产生的ROS归因于E3-h组分，但PI和他的合作者现在已经明确地表明：(A)依赖ThDP的E1o-h组分实际上也产生超氧阴离子和过氧化氢，(B)伴随而来的是电子顺磁共振谱(EPR)表征的ThDP结合的自由基。这些产物是与ThDP结合的中间体与氧气反应的结果。我们的贡献和这一建议之所以成为可能，是因为PI的团队最近成功地以纯形式和大量生产了这一重要复合体的人E1o-h和E2O-h组件的全长、活性重组版本，而其他小组主要解决了这个问题，主要是关于完整的脑线粒体，而不是人类OGDHc的单个蛋白质成分。OGDHc-h位于线粒体的基质中，与线粒体内膜结合，催化2-氧戊二酸转化为琥珀酰辅酶A，式为：2-氧戊二酸+NAD++CoA→琥珀酰辅酶A+NADH+H++CoA(1) 我们研究的首要目标是从原子水平解释：哪种成分(E10-h和E3-h)是ROS产生的主要来源，这与医学上发现的大脑中OGDHc活动随着年龄的增长而减少是一致的。我们的长期目标是通过确定导致ROS形成的特定残基以及人类OGDHc所有三个成分上的ROS修饰残基，为大脑中的ROS发现提供原子解释。我们在R15计划范围内的近期目标是启动一系列合理的项目，以实现我们的长期目标，包括：(A)使用我们团队开发的一系列动力学和光谱最先进的方法以及EPR和ROS检测新方法，检测和动力学表征E1o-h上的硫胺相关中间体和E2O-h上的硫胺结合中间体。我们将确定OGDHc-h组装的影响，以及OGDHc-h的三个单独成分对ROS生成的贡献。目前还没有对人类OGDHc进行这样的研究。这些研究的机理意义在于了解氧与烯胺反应生成的THDP-烯胺自由基在E1o-h上如何以及在哪里受到特殊的稳定；以及E1o-h的哪些残基是OGDHc-h产生ROS的主要贡献者。(B)开发一门名为“现代生化研究导论”的高级实验室课程，对本科生和研究生进行最先进的方法学培训，实验严格与为研究计划建议的实验相平行，并与我们计划中研究的酶相平行；将教授的工具包括动力学、光谱学(圆二色谱、MS、核磁共振、EPR)以及分子生物学和生物化学的基本工具。纽瓦克的任何罗格斯大学校区都没有这样的课程，因此它将服务于纽瓦克的文理学院和医学院的需求。这门课程还将使化学系最终能够提供生物化学课程；这样的实验课程也是AM强烈推荐的化学。社会。 意义和创新。了解OGDHc产生ROS的机制以及ROS如何改变OGDHc的活性是PI的一个重要目标，因为它可以为神经退行性疾病提供洞察力。这就是我们所提出的研究将有助于填补的知识空白。针对特定目标的所有实验的方法都在PI的实验室中到位，并在出版的材料中，因此拟议的实验风险很小。阿尔伯特·爱因斯坦医学院的加里·格芬已经同意合作进行EPR研究。被提议的课程和被提议的研究之间的密切关系肯定是创新的，并且符合R-15计划的精神。

 DESCRIPTION (provided by applicant): The PI"s goals with this R15 proposal are studies on the thiamin diphosphate (ThDP)-dependent human 2-oxoglutarate dehydrogenase complex (OGDHc, also known as α-ketoglutarate dehydrogenase; it comprises three component enzymes denoted with E1o-h, E2o-h and E3-h), the rate-limiting enzyme in the citric acid cycle (also known as the tricarboxylic or Krebs cycle), an enzyme critical for glucose metabolism in normal brain. OGDHc activity is diminished in the brain of Alzheimer's disease patients, which is characterized by reduced glucose metabolism and increased level of oxidative stress markers. A diminished OGDHc activity had also been correlated with other neurodegenerative diseases, including Parkinson's disease, Huntington disease, Wernike-Korsakoff disease and progressive subnuclear palsy. It is known that OGDHc is a sensitive target for a variety of oxidants generated during oxidative stress, including inhibition of OGDHc by peroxynitrite and nitric oxide, and by the reactive oxygen species (ROS), superoxide and H2O2, among other agents. OGDHc is not only a sensitive target of ROS, it can also produce superoxide and H2O2 in brain mitochondria. While earlier research attributed ROS generation by OGDHc to the E3-h component, the PI and collaborators have now shown definitively that (a) The ThDP-dependent E1o-h component in fact also produces superoxide anion and hydrogen peroxide, (b) Concomitantly, there is produced a ThDP-bound radical characterized by electron paramagnetic resonance spectroscopy (EPR). These products result from reaction of a ThDP-bound intermediate with dioxygen O2. Our contributions, and this proposal were made possible by the PI's group's recent success in producing full length, active recombinant versions of the human E1o-h and E2o-h components of this important complex in pure form and in significant amounts for the proposed research, while other groups address this issue mostly on intact brain mitochondrion, rather than with individual protein components of the human OGDHc. The OGDHc-h is located in the matrix of the mitochondrion where it is associated with the inner mitochondrial membrane, catalyzing the conversion of 2-oxoglutarate to succinyl-CoA according to: 2-oxoglutarate+NAD++CoA → succinyl-CoA+NADH+H++CO2 (1) The overarching goal of our studies is an atomic-level explanation of: Which component (E1o-h and E3-h) is the major source of ROS production, consistent with the medical finding of reduced OGDHc activity in the brain with aging. Our long term goal is to provide an atomic explanation for the ROS findings in brain by identifying specific residues responsible for ROS formation, and those modified by ROS on all three components of the human OGDHc. Our immediate goals within the boundaries of the R15 program, are to initiate a logical sequence of projects, which will lead to achieving of our long-term goals, include: (a) Detection and kinetic characterization of thiamin-related intermediates on E1o-h and lipoamide-bound intermediates on E2o-h using a battery of kinetic and spectroscopic state-of-the-art methods developed in our group, as well as new methods EPR and ROS detection. We will determine the effect of OGDHc-h assembly, and of the contribution of the three individual components of OGDHc-h to ROS generation. No such studies are yet done on the human OGDHc. The mechanistic significance of these studies is to provide an understanding of how and where the ThDP-enamine radical formed by reaction of dioxygen with the enamine, receives special stabilization on the E1o-h; and which residues of E1o-h are the major contributors to ROS generation by OGDHc-h. (b) Development of an advanced laboratory course titled Introduction to Modern Biochemical Research to train both undergraduate and graduate students in state-of-the-art methodologies, with experiments strictly paralleling those proposed for the research program, and with the enzymes studied in our program; the tools to be taught will include, kinetics, spectroscopy (circular dichroism, MS, NMR, EPR), and basic tools of molecular biology and biochemistry. No such course exists at any Rutgers campus in Newark, hence it will serve the needs of both arts and sciences and the medical school in Newark. This course will also enable the Chemistry Department to at last offer a Biochemistry track; such a lab course is also highly recommended by the Am. Chem. Society. Significance and Innovation. An understanding of the mechanism by which OGDHc produces ROS, and how OGDHc activity could be altered by ROS is an important goal of the PI since it could provide insight to neurodegenerative diseases. This is the gap in our knowledge that our proposed research will help to fill. Methods for all experiments in the Specific Aim are in place i the PI's laboratories and are in published material, hence the proposed experiments carry little risk. Gary Gerfen at Albert Einstein College of Medicine has agreed to collaborate on the EPR studies. The close relationship between the course being proposed and the research being proposed is surely innovative and is in the spirit of the R-15 program.