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Complex I: Role of L Subunit in Proton Translocation

复合物 I：L 亚基在质子易位中的作用

基本信息

批准号：
8180161
负责人：
STEVEN B VIK
金额：
$ 31.64万
依托单位：
SOUTHERN METHODIST UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-01 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8180161
关键词：
Abbreviations Acridines Affinity Alanine Alleles Amino Acids Bacterial Model Benzophenones Biological Assay Biological Models Bipolar Disorder Cations Cell Death Cell Death Process Cells Citric Acid Cycle Cleaved cell Code Complex Coupled Coupling Crosslinker Cysteine Degenerative Disorder Disease Electron Transport Electrons Engineering Enzymes Escherichia coli Gel Genes Genetic Genetic Polymorphism Glycine Human Human Genome Lactic Acidosis Lateral Learning Leber&apos s Hereditary Optic Neuropathy Leigh Disease Link Lipids Location Membrane Mitochondria Mitochondrial Encephalomyopathies Motion Multiple Sclerosis Mutate Mutation NADH NADH dehydrogenase (quinone)NADH oxidase Non-Insulin-Dependent Diabetes Mellitus Operon Oxidation-Reduction Oxidative Stress Parkinson Disease Peptide Hydrolases Peripheral Play Positioning Attribute Production Protein Complex Subunit Proteins Protons Reactive Oxygen Species Research Resistance Risk Factors Role Running Series Side Site Stroke Structure Superoxides Surface TEV protease Testing Ubiquinone Western Blotting Work alpha helix antiporter arm carbon compound crosslink enzyme activity expression vector insight intercellular communication oxidative DNA damage prevent ubiquinol

项目摘要

DESCRIPTION (provided by applicant): Complex I (NADH:quinone oxidoreductase) is the entry point of NADH into the electron transport chain of mitochondria. It oxidizes NADH to NAD+ which allows the citric acid cycle to oxidize carbon compounds. Its extraction of two electrons from NADH is used to reduce ubiquinone to ubiquinol, which then functions as an electron carrier to downstream enzymes. This reduction is coupled to the translocation of protons across the membrane, by an unknown mechanism. This generates the electrochemical proton gradient that is used to synthesize ATP. In humans Complex I plays additional roles in the mitochondrion related to oxidative stress, cell signaling, and cell death. For example, blockage of electron transport can increase the production of superoxide by Complex I, leading to oxidative damage of DNA, lipids, and protein. Loss of Complex I function is associated with the onset of idiopathic Parkinson's disease. Complex I subunits are targeted by proteases that trigger cell death processes. Mutations in the protein subunits of Complex I are risk factors for a wide range of diseases and disorders, such as multiple sclerosis, Type 2 diabetes, and bipolar disorder. Some such alleles are known to be causative for fatal disorders such as Leber's hereditary optic neuropathy (LHON), Leigh disease, and mitochondrial encephalomyopathy and lactic acidosis with stroke-like episodes (MELAS). It has been difficult to understand the effect of mitochondrial mutations in Complex I because so little is known about the assembly and function of its membrane subunits. Recent structural studies of bacterial Complex I have revealed new details about the membrane subunits. The objectives of this project are to test the hypothesis that an unusually long alpha-helix, positioned laterally along the membrane, is a key component of proton translocation. A bacterial model system will be used to carry out these studies because of the ability to construct mutations in the hydrophobic proteins that carry out proton translocation. The specific aims of this proposal are first to determine if a functional Complex I can be assembled with a truncated L subunit- one which lacks some or all of its carboxy terminal extension that has been proposed to act as a piston in conformational coupling. Since the los of this region of subunit L might prevent assembly, so two additional aims are planned. Second, the importance of piston-like motion will be tested by constructing cysteine residues in subunit L as sites for photo-affinity cross-linking to other subunits. And third, to test whether the lateral helix needs to be intact for function, protease sites will be constructed at several locations, allowing cleavage after assembly of the enzyme. From the results of this research it will be possible to learn if piston-like motion by subunit L is necessary for proton translocation, which will give insight into many of the diseases associated with the membrane subunits of Complex I. PUBLIC HEALTH RELEVANCE: Recent studies have revealed that mitochondria not only are the energy production centers of cells, but also are key players in the control of cell death. Mitochondria contain a small subset of the human genome, and several of these genes code for proteins that are components of an extremely important enzyme called Complex I. This enzyme is both a target and an initiator of cell death, and its polymorphisms are risk factors or causative agents for many degenerative diseases. The work in described in this proposal will allow greater understanding of key aspects of the functioning of Complex I.

描述（由申请人提供）：复合物I（NADH：醌氧化还原酶）是NADH进入线粒体电子传递链的入口点。它将NADH氧化成NAD+，从而允许柠檬酸循环氧化碳化合物。它从NADH中提取两个电子用于将泛醌还原为泛醇，然后泛醇作为下游酶的电子载体。这种减少与质子跨膜的易位有关，其机制未知。这产生用于合成ATP的电化学质子梯度。在人类中，复合物I在与氧化应激、细胞信号传导和细胞死亡相关的线粒体中发挥额外的作用。例如，电子传递的阻断可以增加复合物I产生超氧化物，导致DNA、脂质和蛋白质的氧化损伤。复合体I功能的丧失与特发性帕金森病的发病相关。复合物I亚基被触发细胞死亡过程的蛋白酶靶向。复合体I的蛋白质亚基的突变是多种疾病和病症的危险因素，例如多发性硬化症、2型糖尿病和双相情感障碍。已知一些这样的等位基因是致死性疾病如Leber遗传性视神经病变（LHON）、Leigh病和线粒体脑肌病和乳酸酸中毒伴中风样发作（MELAS）的病因。很难理解线粒体突变对复合体I的影响，因为对其膜亚基的组装和功能知之甚少。最近对细菌复合物I的结构研究揭示了关于膜亚基的新细节。这个项目的目标是测试一个异常长的α-螺旋，沿膜横向沿着定位，是质子易位的关键组成部分的假设。细菌模型系统将用于进行这些研究，因为能够在进行质子易位的疏水蛋白中构建突变。该提议的具体目的是首先确定功能性复合物I是否可以与截短的L亚基组装，所述截短的L亚基缺乏其羧基末端延伸的部分或全部，所述羧基末端延伸已被提出在构象偶联中充当活塞。由于亚基L的这一区域的缺失可能会阻止组装，因此计划了两个额外的目标。第二，活塞样运动的重要性将通过构建半胱氨酸残基在亚基L作为网站的光亲和交联到其他亚基进行测试。第三，为了测试侧螺旋是否需要完整的功能，将在几个位置构建蛋白酶位点，允许在酶组装后进行切割。从这项研究的结果中，将有可能了解亚基L的活塞样运动是否是质子转运所必需的，这将使我们深入了解与复合物I的膜亚基相关的许多疾病。公共卫生相关性：最近的研究表明，线粒体不仅是细胞的能量生产中心，而且是控制细胞死亡的关键角色。线粒体包含人类基因组的一小部分，其中几个基因编码的蛋白质是一种极其重要的酶（称为复合体I）的组成部分。这种酶既是细胞死亡的靶点，也是细胞死亡的引发剂，其多态性是许多退行性疾病的危险因素或致病因子。本提案中所述的工作将使人们更好地了解综合体I运作的关键方面。