Complex I: Role of L Subunit in Proton Translocation

描述（由申请人提供）：复合体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氧化为NAD+这使得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。这种还原是通过一种未知的机制与质子跨膜的易位相结合的。这就产生了用于合成ATP的电化学质子梯度。在人类中，复合物I在线粒体中起着与氧化应激、细胞信号传导和细胞死亡相关的额外作用。例如，电子传递受阻会增加复合体I产生超氧化物，导致DNA、脂质和蛋白质的氧化损伤。复合物I功能的丧失与特发性帕金森病的发病有关。复合物I亚基是触发细胞死亡过程的蛋白酶的靶标。复合体I蛋白亚基的突变是多种疾病的危险因素，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可导致致命性疾病，如Leber氏遗传性视神经病变（LHON）、Leigh病、线粒体脑肌病和乳酸酸中毒伴卒中样发作（MELAS）。由于对复合体I的膜亚基的组装和功能知之甚少，因此很难理解复合体I中线粒体突变的影响。最近对细菌复合体I的结构研究揭示了膜亚基的新细节。这个项目的目的是验证一个假设，即沿膜横向定位的异常长的α -螺旋是质子易位的关键组成部分。细菌模型系统将用于开展这些研究，因为能够在执行质子易位的疏水蛋白中构建突变。本建议的具体目的是首先确定功能性复合物I是否可以与截断的L亚基组装-一个缺乏部分或全部羧基末端延伸的亚基，该亚基被认为是构象耦合中的活塞。由于亚基L的这一区域的丢失可能会妨碍组装，因此计划另外两个目标。其次，将通过在亚基L中构建半胱氨酸残基作为与其他亚基光亲和交联的位点来测试活塞状运动的重要性。第三，为了测试侧螺旋是否需要完整的功能，蛋白酶位点将在几个位置构建，以便在酶组装后进行切割。从这项研究的结果中，我们将有可能了解到L亚基的活塞式运动是否对质子易位是必要的，这将使我们深入了解与复合体I膜亚基相关的许多疾病。

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.