Characterizing tafazzin and Barth syndrome mutant tafazzins

为了开始剖析心磷脂（CL）重塑的过程，本建议侧重于 表征假定的CL酰基转移酶，tafazzin（Tazlp），与 Barth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，哪些步骤受到调节，以及两者正常的后果 以及线粒体的异常CL重塑！功能这将为理解 Taz 1 p在BTHS中的作用此外，这项工作将阐明其他心肌病，包括 糖尿病性心肌病，最近已被证明涉及CL合成的缺陷和增加 CL猫是一个男孩。第一个具体目标的目标是识别直接相互作用的脂质和蛋白质， 或与Taz 1 p结合形成复合物。初步结果表明，Taz 1 p非共价结合至 磷脂和/或脂肪酰基链，并组装成几个大的大分子复合物。到 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Tazlp、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。双亲和标记的Taz 1 p构建体将另外用于揭示相互作用 通过标准生物化学程序测定蛋白质。这一目标的结果预计将提供深入了解 Tazlp参与的CL重塑途径，包括潜在地鉴定底物， Taz 1 p的靶向特异性、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，其存在于相同或保守的残基处 人和酵母直系同源物之间的关系，将研究它们定位于和在细胞内的能力。 正确地，与目的1中鉴定的蛋白质和/或脂质结合，并发挥功能。通过 与野生型Taz 1 p的系统比较，Taz 1 p突变体和BTHS的分子基础将是 提供了对于那些发生在人Taz 1 p独特残基处的BTHS突变， 系统将被开发，这将允许BTHS突变体的这一子集的分子解剖;的目标， 具体目标3.这项研究的结果对公共卫生很重要，因为心脏病会影响 一般人群，这项研究将提供深入了解导致心脏病的基本机制。

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.