Regulation of NADH:ubiquinone oxidoreductase (complexI)

描述(由申请人提供)：线粒体NADH：泛醌氧化还原酶(复合体I)是所有好氧细胞中生物能量机制的关键基本组成部分。哺乳动物复合体I是线粒体呼吸链的最大组成部分，由至少46个亚基和多达10个不同的氧化还原中心组成。该酶结构的复杂性与其不同的催化和调节特性是一致的。在特定的代谢条件下，该酶存在两种缓慢平衡的形式，A(活性)和D(失活)，并且每种形式对抑制和/或激活配体的敏感性不同。本项目是我们在分子和生理水平上对复杂的I/A/D转变的合作研究的扩展。将研究游离脂肪酸对络合物I和其他生理相关的膜扰动化合物的抑制作用的机制。脂肪酸及其衍生物的抑制和/或激活作用将与它们对A/D转换和络合物I产生超氧化物的调节作用有关。这些研究将在完整的线粒体和由内向外紧密耦合的亚线粒体颗粒中进行。计划进行实验，以确定其他能够产生超氧化物和过氧化氢的基质定位氧化还原酶(S)与复合体I的活性有关。这项研究将主要在俄罗斯实验室进行。所获得的结果将被用作进一步阐述生理水平的A/D转换的指南，使用的是朗恩多夫灌流的大鼠心脏缺血/再灌注模型，这是一种在美国实验室很好地开发的技术。美国实验室将协助俄罗斯科学家建立朗宁多夫灌流心脏模型，并识别参与调节A/D转换的复合体I的特定亚基。俄罗斯研究小组提出的两核苷酸结合位点(S)模型将在美国实验室的帮助下，使用美国实验室最近发现的一种新的紧密核苷酸结合位点定向抑制物进行测试。这个项目的生物医学意义在于确定参与细胞呼吸调节和线粒体相关病理发展的关键因素，如缺氧、2型糖尿病和神经退行性疾病。复合体I的缺陷已被证明与帕金森氏症有关，这些研究可能有助于阐明这种重要的代谢酶是如何调节的。

DESCRIPTION (provided by applicant): The mitochondrial NADH:ubiquinone oxidoreductase (Complex I) is a key essential component of the bioenergetic machinery in all aerobic cells. The mammalian Complex I is the largest component of mitochondrial respiratory chain; composed of at least 46 individual subunits and up to 10 distinct redox centers. The structural complexity of the enzyme is consistent with its varied catalytic and regulatory properties. Two slowly equilibrating forms of the enzyme, A (active) and D (deactive) exist under given metabolic conditions and each form has different sensitivity to inhibitory and/or activating ligands. This project is to extend our previous collaborative studies on the Complex I A/D transition at the molecular and physiological level. The mechanisms responsible for the inhibitory effects of free fatty acids on Complex I and other physiologically relevant membrane perturbing compounds will be studied. The inhibitory and/or activating effects of fatty acids and their derivatives will be correlated with their modulating effects on the A/D transition and superoxide production by Complex I. These studies will be done in intact mitochondria and inside-out tightly coupled submitochondrial particles. Experiments designed to identify other matrix-located redox enzyme(s) capable of superoxide and hydrogen peroxide production as related to Complex I activity are planned. This research will be primarily done in the Russian laboratory. The results obtained will be used as a guideline for further elaboration at the physiological level of the A/D transition using a model of ischemia/reperfusion in Langendorf-perfused rat hearts, a technique well developed in the U.S. laboratory. The U.S. laboratory will assist the Russian scientists with the Langendorf-perfused heart model and in identifying particular subunits of Complex I that are involved in the regulatory A/D transition. A two-nucleotide binding site(s) model proposed by the Russian group will be tested with the help of the US laboratory by use of a new tight nucleotide-binding site-directed inhibitor recently discovered in the U.S. laboratory. The biomedical significance of this project is to identify the key players involved in regulation of cellular respiration and in development of mitochondria-related pathologies such as occur during oxygen supply deficiency, type 2 diabetes, and neurodegenerative diseases. Defects in Complex I have been shown to contribute to Parkinson's disease and these studies may shed light on how this important metabolic enzyme is regulated.