Human mitochondrial fatty acid synthase

人线粒体脂肪酸合酶

• 批准号: 7090013
• 负责人: STUART SMITH
• 金额: $ 41.6万
• 依托单位: CHILDREN'S HOSPITAL & RES CTR AT OAKLAND
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7090013
• 关键词: RNA interference; antiserum; cell component structure /function; cell morphology; cellular respiration; chromatography; confocal scanning microscopy; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; fatty acid biosynthesis; fatty acid synthase; gene induction /repression; immunologic substance development /preparation; laboratory rabbit; long chain fatty acid; malonyl coA; mitochondria; molecular biology information system; molecular cloning; northern blottings; phospholipids; short chain fatty acid; western blottings

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.

描述（由申请方提供）：提供了新的令人信服的证据，表明人线粒体含有酰基载体蛋白（ACP）依赖性系统，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，神经靶向脂肪酸合成酶（mitFAS）组件是类似于原核生物的FAS的个别蛋白质，因此不同于真核细胞胞质FAS，其中所有的酶都存在于一个单一的多肽链。mitFAS ACP与呼吸链复合体1相关。最近在真菌中描述了一个类似的系统;该mitFAS系统的破坏导致呼吸缺陷表型，这表明确定这种“新”代谢途径是否也可能在人类线粒体功能中发挥关键作用是及时的。该提案有三个目标：识别、分离和表征人类线粒体FAS的各个组成部分，识别它们产生的产物，并评估线粒体功能途径的重要性。 (1)将克隆、表达、纯化通过人序列数据库的BLAST检索鉴定的候选mitFAS组分，并测定其底物特异性。通过证明仅当存在推定的N-末端靶向序列时，在人细胞中表达的mitFAS-绿色荧光蛋白嵌合体才被转运至线粒体，来确认它们是真实的线粒体蛋白。(2)通过将透化代谢的线粒体暴露于各种放射性标记底物并通过色谱法鉴别产物，对mitFAS途径形成的产物进行表征。将特别注意的可能性，该途径产生辛酸，硫辛酸的前体，和/或长链脂肪酸，可用于线粒体磷脂的生物合成。(3)通过RNA介导的干扰沉默mitFAS组分的表达，并确定对细胞形态、线粒体呼吸能力和线粒体磷脂组成的影响，评估该途径对线粒体功能的重要性。线粒体功能的失效与晚期神经退行性疾病如帕金森病、阿尔茨海默病和亨廷顿病的发病机制有关，但许多线粒体蛋白的作用仍是未知的。阐明mitFAS系统在线粒体功能中的作用可能有助于了解这些疾病的病因。