Human mitochondrial fatty acid synthase

描述(由申请人提供)：新的，令人信服的证据表明，人类线粒体包含一个依赖于酰基载体蛋白(ACP)的系统，用于从丙二酰辅酶A合成脂肪酸。序列和功能分析表明，核编码的线粒体靶向脂肪酸合成酶(MitFas)组分是与原核生物的Fass相似的单个蛋白质，因此不同于真核细胞胞质的Fass，在真核细胞中，所有的酶都存在于单一的多肽链上。MitFas ACP与呼吸链的复合体1相关。最近在真菌中也描述了类似的系统；这个mitFAS系统的破坏会导致呼吸缺陷的表型，这表明现在是时候确定这一新的代谢途径是否也在人类线粒体功能中发挥关键作用。该提案有三个目标：识别、分离和鉴定人类mitFas的单个成分，识别它们产生的产物，并评估该途径对线粒体功能的重要性。 (1)通过对人类序列数据库的BLAST搜索确定的候选mitFas组分将被克隆、表达、纯化并确定其底物特异性。为了证实它们是真正的线粒体蛋白，将通过证明在人类细胞中表达的mitFas-绿色荧光蛋白嵌合体只有在假定的N-末端靶向序列存在时才被运输到线粒体。(2)mitFas途径形成的产物的特征是将被渗透的线粒体暴露在各种放射性标记的底物上，并对产物进行层析鉴定。将特别注意该途径产生辛酸、硫辛酸前体和/或可用于线粒体磷脂生物合成的长链脂肪酸的可能性。(3)通过RNA介导的干扰沉默mitFas组分的表达，并测定其对细胞形态、线粒体呼吸能力和线粒体磷脂组成的影响，以评估该途径对线粒体功能的意义。线粒体功能障碍与帕金森氏病、阿尔茨海默病和亨廷顿病等晚期神经退行性疾病的发病机制有关，但许多线粒体蛋白的作用尚不清楚。阐明mitFas系统在线粒体功能中的作用可能有助于理解这些疾病的病因。

DESCRIPTION (provided by applicant): New, compelling evidence is presented indicating that human mitochondria contain an acyl carrier protein (ACP)-dependent system for synthesis of fatty acids from malonyl-CoA. Sequence and functional analyses indicate that nuclear-encoded, mitochondrially-targeted fatty acid synthase (mitFAS) components are individual proteins resembling the FASs of prokaryotes and thus differ from the eukaryotic cytosolic FASs, in which all of the enzymes are present on a single polypeptide chain. The mitFAS ACP is associated with complex 1 of the respiratory chain. A similar system has recently been described in fungi; disruption of this mitFAS system results in respiratory deficient phenotypes, indicating that it is timely to determine whether this 'new' metabolic pathway may also play a critical role in human mitochondrial function. The proposal has three objectives: to identify, isolate and characterize individual components of the human mitFAS, to identify the products they generate and to assess the significance of the pathway to mitochondrial function. (1) Candidate mitFAS components, identified by BLAST searches of the human sequence database, will be cloned, expressed, purified and their substrate specificities determined. Confirmation that they are authentic mitochondrial proteins will be sought by demonstrating that mitFAS-green fluorescent-protein chimeras expressed in human cells are transported to the mitochondria only when putative N-terminal targeting sequences are present. (2) The products formed by the mitFAS pathway will be characterized by exposing permeabolized mitochondria to various radiolabeled substrates and identifying the products chromatographically. Particular attention will be paid to the possibility that the pathway generates octanoate, the precursor of lipoic acid, and/or long chain fatty acids that could be used in the biosynthesis of mitochondrial phospholipids. (3) The significance of the pathway to mitochondrial function will be assessed by silencing expression of mitFAS components, through RNA-mediated interference, and determining the effect on cellular morphology, mitochondrial respiratory capacity and mitochondrial phospholipid composition. Failure in mitochondrial function has been implicated in the pathogenesis of late developing neurodegenerative disorders such as Parkinson's, Alzheimer's, and Huntington's diseases, yet the role of many mitochondrial proteins is still unknown. Elucidation of the role of the mitFAS system in mitochondrial function may aid in understanding the etiology of these disorders.