Characterizing tafazzin and Barth syndrome mutant tafazzins

描述（由申请人提供）： 开始解剖心磷脂（CL）重塑的过程中，这个建议的重点是表征的假定CL酰基转移酶，tafazzin（Tazlp），突变基因产物与巴斯综合征（BTHS）。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参与者，调节哪些步骤，以及正常和异常CL重塑对线粒体功能的影响。这将为进一步了解Taz 1 p在BTHS中的作用提供基础。此外，这项工作将阐明其他心肌病，包括糖尿病性心肌病，最近已被证明涉及CL合成缺陷和CL分解增加。第一个具体目标的目标是鉴定在复合物中与Taz 1 p直接相互作用或缔合的脂质和蛋白质。初步结果表明，Taz 1 p非共价结合到磷脂和/或脂肪酰基链和组装成几个大的大分子复合物。为了鉴定潜在的脂质配体，将采用一系列生化和遗传方法，包括使用重组Taz 1 p、确定的磷脂、ELISA、脂肪印迹、BN-PAGE和双重亲和标记的Taz 1 p构建体。双重亲和标记的Taz 1 p构建体将另外用于通过标准生物化学程序揭示相互作用蛋白。来自该目的的结果预期提供对Taz 1 p参与的CL重塑途径的深入了解，包括潜在地鉴定Taz 1 p的底物和靶特异性、参与该过程的其他组分以及Taz 1 p的潜在新功能。对于第二个特定目的，一组BTHS突变体，其发生在人和酵母直向同源物之间的相同或保守残基处，将研究它们正确定位于线粒体并在线粒体内的能力、与目标1中确定的蛋白质和/或脂质结合的能力以及功能。通过与野生型Taz 1 p的系统比较，为Taz 1 p突变体和BTHS的研究提供分子基础。对于那些在人Taz 1 p特有的残基处发生的BTHS突变，将开发细胞培养模型系统，其将允许对BTHS突变体的该子集进行分子解剖;具体目标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，这项研究将深入了解导致心脏病的基本机制。

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.