Complex I: Role of L Subunit in Proton Translocation

描述(申请人提供)：复合体I(NADH：苯醌氧化还原酶)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，用于将泛苯二酚还原为泛喹酚，然后泛喹酚作为下游酶的电子载体。这种还原是通过一种未知的机制与跨膜的质子转移相耦合的。这就产生了用于合成三磷酸腺苷的电化学质子梯度。在人类中，复合体I在线粒体中扮演着额外的角色，与氧化应激、细胞信号和细胞死亡有关。例如，阻断电子传递可以增加络合物I产生的超氧化物，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有关。复合I亚基是触发细胞死亡过程的蛋白酶的靶标。复合体I蛋白亚基的突变是一系列疾病和障碍的危险因素，如多发性硬化症、2型糖尿病和躁郁症。已知一些这样的等位基因是致命性疾病的病因，如Leber遗传性视神经病变(LHON)、Leigh病、线粒体脑肌病和乳酸中毒伴卒中样发作(MELAS)。很难理解线粒体突变对复合体I的影响，因为人们对其膜亚单位的组装和功能知之甚少。最近对细菌复合体的结构研究I揭示了关于膜亚单位的新细节。这个项目的目标是检验这样一种假设，即沿着膜横向定位的异常长的阿尔法螺旋是质子转移的关键组成部分。细菌模型系统将被用于进行这些研究，因为它能够在进行质子转移的疏水蛋白质中构建突变。这项提议的具体目的是首先确定功能复合体I是否可以与截短的L亚基组装--一个缺少部分或全部羧基末端延伸的亚基，该末端延伸被认为在构象偶联中充当活塞。由于L亚基的这一区域的丢失可能会阻止集会，因此计划增加两个目标。其次，将通过构建L亚基中的半胱氨酸残基作为与其他亚基的光亲和交联点来检验活塞式运动的重要性。第三，为了测试侧螺旋是否需要完整地发挥功能，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，可以了解到质子转运是否需要L亚基的活塞式运动，这将使我们深入了解与复合体I的膜亚单位有关的许多疾病。 与公共健康相关：最近的研究表明，线粒体不仅是细胞的能量产生中心，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，其中几个基因编码蛋白质，这些蛋白质是一种名为复合体I的极其重要的酶的组成部分。这种酶既是细胞死亡的靶点，也是细胞死亡的起始者，它的多态是许多退行性疾病的风险因素或致病因素。本提案中描述的工作将使人们能够更好地了解复合体I的运作的关键方面。

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.