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（由申请人提供）：提出了新的，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，用于合成丙二酰-COA的脂肪酸。序列和功能分析表明，核编码的，线粒体靶向的脂肪酸合酶（MITFAS）成分是单个蛋白质类似于原核生物的FASS，因此与真核细胞质的FASS有所不同，其中所有酶都存在于单二肽链上。 MITFAS ACP与呼吸链的复合物1有关。最近在真菌中描述了类似的系统。这种MITFAS系统的破坏会导致呼吸不足的表型，这表明及时确定这种“新”代谢途径是否也可能在人类线粒体功能中起关键作用。该提案具有三个目标：识别，隔离和表征人类Mitfas的各个组成部分，以识别其产生的产品并评估线粒体功能的途径的重要性。 （1）通过对人类序列数据库的爆炸搜索确定的候选MITFA组件将被克隆，表达，纯化，并确定其底物特异性。确认它们是真实的线粒体蛋白，才能证明仅当存在假定的N末端靶向序列时，在人类细胞中表达的mitfas-green荧光蛋白嵌合体才能运输到线粒体。 （2）MITFAS途径形成的产物将以将透明性线粒体暴露于各种放射性标记的底物并在色谱上识别产物来表征。特别关注该途径产生八坦酸盐的可能性，lipoic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，通过沉默的MITFA成分表达来评估线粒体功能途径的重要性，并确定对细胞形态，线粒体呼吸能力和线粒体磷脂成分的影响。线粒体功能的失败与晚期发展神经退行性疾病的发病机理有关，例如帕金森氏症，阿尔茨海默氏症和亨廷顿的疾病，但许多线粒体蛋白质的作用仍然未知。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。