Regulation of mitochondrial glycerol-3-P acyltranferase

描述（由申请人提供）本项目将在阿根廷进行，作为父母补助金DK56598的扩展（1999年4月15日- 2003年3月31日）。甘油磷酸酰基转移酶（GPAT）催化甘油脂合成的第一步，即sn- 3-磷酸甘油酰化为溶血磷脂酸。我们之前已经证明，在两种不同的细胞系中，GPAT的线粒体同型体（mitoGPAT）指导三酰甘油（TAG）的细胞合成，而不是磷脂，这表明mitoGPAT可能是开发药物来控制肥胖和糖尿病病理条件下TAG合成的良好候选物。MitoGPAT是线粒体外膜蛋白，具有两个跨膜结构域。该蛋白在胞质n端结构域中含有一个活性位点，一个面向线粒体膜间空间的84个氨基酸环，以及一个238个氨基酸的功能未知的c端结构域。我们之前的结果表明，通过在环中插入表位标签或在环区末端截断该酶来灭活该酶。我们建议确定mitoGPAT活性如何受到不形成催化位点的环和c端结构域的调节。本实验将分析CHO细胞中表达的不同截断和突变的mitoGPATs的活性。我们将确定mitoGPAT的哪些片段对其正常功能至关重要。我们还将确定可能的蛋白-蛋白或蛋白内相互作用，通过在CHO细胞中共表达带有不同表位标记的mitoGPATs构建物来调节酶活性。共表达mitoGPATs的细胞的膜组分（通常是全长活性蛋白和一个修饰的无活性蛋白，或两个全长活性蛋白标记不同的表位）将被交联并探测每个蛋白表达的表位。因此，我们将能够阐明是否在修饰蛋白中阻止了mitoGPAT在膜中的物理接触，无论是与其他特定蛋白还是与功能性低聚物。这些结果将阐明三酰甘油合成途径的调控，并可能提出新的酶抑制策略，不同于针对mitoGPAT活性位点的酶抑制策略。

DESCRIPTION (provided by applicant) This project will be carried out in Argentina as an expension of the parent grant DK56598 (4/15/99-3/31/03). Glycerol phosphate acyltransferase (GPAT) catalyzes the first and committed step in glycerolipid synthesis, the acylation of the sn-glycerol-3-phosphate to lysophosphatidic acid. We previously demonstrated that the mitochondrial isoform of GPAT (mitoGPAT) directs the cellular synthesis of triacylglycerol (TAG), but not phospholipids, in two different cell lines, suggesting that mitoGPAT may be a good candidate for the development of drugs to control TAG synthesis in the pathological conditions of obesity and diabetes. MitoGPAT is an outer mitochondrial membrane protein with two transmembrane domains. The protein contains an active site in the cytosolic N-terminal domain, a 84 amino acid loop that faces the mitochondrial intermembrane space, and a 238 amino acid C-terminal domain that has no known function. Our previous results showed that the enzyme is inactivated by inserting an epitope tag in the loop or by truncating at the end of the loop region. We propose to determine how mitoGPAT activity is regulated by the loop and C-terminal domains which do not form the catalytic site. The proposed experiments will analyze the activity of different truncated and mutated mitoGPATs expressed in CHO cells. We will determine which fragments of the mitoGPAT are essential for its proper function. We will also determine possible protein-protein or intraprotein interactions that regulate the enzyme activity by co-expressing in CHO cells mitoGPATs constructs tagged with different epitopes. The membrane fractions of the cells co-expressing mitoGPATs (typically the full-length active one and a modified-inactive one, or two full-length active proteins tagged with different epitopes) will be cross-linked and probed for the epitopes expressed with each protein. Thus, we will be able to elucidate whether the physical contact of mitoGPAT in the membrane, either with other specific proteins or with functional oligomers, is prevented in the modified proteins. These results will elucidate the regulation of the pathway of triacylglycerol synthesis, and may suggest novel strategies for enzyme inhibition, different from those targeted to mitoGPAT's active site.