Characterizing tafazzin and Barth syndrome mutant tafazzins

为了开始解剖心磷脂(CL)重塑的过程，这项建议侧重于 可能的CL酰基转移酶Tafazzin(Tazlp)相关突变基因产物的鉴定 患有巴特综合征(BTHS)。这项研究的长期目标是确定CL是如何重塑的， 分子参与者参与了这个过程，哪些步骤受到了调控，以及两者的后果都正常 线粒体异常CL重塑！功能。这将为理解 Taz1p在BTHS中的作用。此外，这项工作将阐明其他心肌病，包括 糖尿病心肌病，最近被证明涉及CL合成缺陷和增加 我是一只小猫。第一个特定目标的目标是识别直接相互作用的脂类和蛋白质 或与复合体中的Taz1p相关联。初步结果表明Taz1p以非共价结合到 磷脂和/或脂肪酰基链，并组装成几个大的大分子复合体。至 确定潜在的脂质配体，将采用一系列生化和遗传方法，包括 使用重组Tazlp、定义的磷脂、酶联免疫吸附试验、脂肪印迹、BN-PAGE和双亲和标记 Taz1 p构造。另外还将使用双亲和标记Taz1p结构来揭示相互作用 通过标准的生化程序提取蛋白质。这一目标的结果预计将提供对 Tazlp参与的CL重塑途径，包括潜在地识别底物和 Taz1p的靶标特异性，参与这一过程的其他成分，以及潜在的新功能 对于第二个特定目的，一组BTHS突变体，出现在相同或保守的残基上 在人类和酵母的同源基因之间，将被研究他们定位到和在 线粒体正确地与目标1中确定的蛋白质和/或脂类相联系，并发挥作用。穿过 与野生型Taz1p的系统比较，Taz1p突变体和BTHS的分子基础将是 如果是这样的话。对于那些发生在人类Taz1p特有残基上的BTHS突变，细胞培养模型 将开发一种系统，允许对BTHS突变体的这一子集进行分子解剖；目标是 具体目标3.这项研究的结果对公众健康很重要，因为心脏病会影响 这项研究将提供对导致心脏病的基本机制的洞察。

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 mitochondria! 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 cat a bo I ism. 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 Tazlp, 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 Tazlp 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.