Characterizing tafazzin and Barth syndrome mutant tafazzins

描述(由申请人提供)： 为了开始剖析心磷脂(CL)重塑的过程，这项建议集中在推测的CL酰基转移酶Tafazzin(Tazlp)的特征上，Tafazzin(Tazlp)是Barth综合征(BTHS)相关的突变基因产物。这项研究的长期目标是确定CL是如何重塑的，参与这一过程的分子角色，调控哪些步骤，以及正常和异常CL重塑对线粒体功能的影响。这将为理解Taz1p在BTHS中的作用提供基础。此外，这项工作将阐明其他心肌病，包括糖尿病心肌病，最近被证明与CL合成缺陷和CL分解增加有关。第一个特定目标的目标是识别与复合体中的Taz1p直接相互作用或关联的脂类和蛋白质。初步结果表明，Taz1p以非共价方式结合到磷脂和/或脂肪酰链上，并组装成几个大的大分子络合物。为了确定潜在的脂类配体，将采用一系列的生化和遗传学方法，包括使用重组Taz1p、定义的磷脂、ELISA、脂肪印迹、BN-PAGE和双亲和标记的Taz1p构建。双亲和标记的Taz1p结构还将被用于通过标准的生化程序揭示相互作用的蛋白质。这一目标的结果有望为深入了解Taz1p参与的CL重塑途径提供线索，包括潜在地确定Taz1p的底物和靶标特异性，参与这一过程的其他成分，以及Taz1p潜在的新功能。对于第二个特定目标，将研究一组BTHS突变体，它们发生在人和酵母同源基因之间相同或保守的残基上，研究它们正确定位到线粒体和线粒体内的能力，与目标1中确定的蛋白质和/或脂结合，以及功能。通过与野生型Taz1p的系统比较，为Taz1p突变体和BTHS提供分子基础。对于那些发生在人类Taz1p特有残基上的BTHS突变，将开发一种细胞培养模型系统，将允许对BTHS突变的这一子集进行分子解剖；特定目标3的目标。这项研究的结果对公共卫生很重要，因为心脏病影响普通人群，这项研究将为深入了解导致心脏病的基本机制提供帮助。

DESCRIPTION (provided by applicant): To begin dissecting the processes of cardiolipin (CL) remodeling, this proposal focuses on the characterization of the putative CL acyltransferase, tafazzin (Tazlp), the mutant gene product associated with Barth syndrome (BTHS). The long-term goal of this research is to ascertain how CL is remodeled, the molecular players involved in this process, what steps are regulated, and the consequences of both normal and abnormal CL remodeling with respect to mitochondrial function. This will provide a basis to understand the role of Taz1 p in BTHS. Moreover, this work will shed light on additional cardiomyopathies, including diabetic cardiomyopathy, which has recently been shown to involve deficits in CL synthesis and increased CL cataboIism. The goal of the first specific aim is to identify lipids and proteins that either directly interact or associate with Taz1 p in a complex. Preliminary results indicate that Taz1 p binds noncovalently to phospholipids and/or fatty acyl chains and assembles into several large macromolecular complexes. To identify potential lipid ligands, a battery of biochemical and genetic approaches will be employed including use of recombinant Taz1p, defined phospholipids, ELISA, Fat Blots, BN-PAGE, and a dual affinity tagged Taz1 p construct. The dual affinity tagged Taz1 p construct will additionally be employed to reveal interacting proteins by standard biochemical procedures. Results from this aim are expected to provide insight into the CL remodeling pathway in which Taz1p participates, including potentially identifying the substrate and target specificity of Taz1 p, other components involved in this process, and potentially novel functions of Taz1 p. For the second specific aim, a panel of BTHS mutants, occurring at identical or conserved residues between the human and yeast orthologs, will be investigated for their ability to localize to and within the mitochondrion correctly, associate with proteins and/or lipids as identified in aim 1, and function. Through the systematic comparison with wild type Taz1 p, the molecular basis for a Taz1 p mutant and BTHS will be provided. For those BTHS mutations that occur at residues unique to human Taz1 p, a cell culture model system will be developed that will allow the molecular dissection of this subset of BTHS mutants; the goal of specific aim 3. Results from this study are important for public health because cardiac disease affects the general population and this study will provide insight into basic mechanisms leading to cardiac disease.