Regulation of mitochondrial glycerol-3-P acyltranferase

说明(由申请人提供)本项目将在阿根廷实施，作为父母赠款DK56598的扩展(4/15/99-3/31/03)。甘油磷酸酰基转移酶(GPAT)催化甘油脂合成的第一步，也就是3-磷酸甘油的酰化为溶血磷脂酸。我们先前证明GPAT的线粒体异构体(MitoGPAT)在两个不同的细胞系中指导三酰甘油(Tag)的细胞合成，而不是磷脂的合成，这表明mitoGPAT可能是一个很好的候选药物来控制肥胖和糖尿病的病理条件下Tag的合成。MitoGPAT是一种具有两个跨膜区的线粒体外膜蛋白。该蛋白含有胞质N-端区的活性部位，面对线粒体膜间间隙的84个氨基酸的环，以及一个238个氨基酸的C-端区，其功能未知。我们以前的结果表明，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-末端结构域的调节。拟议的实验将分析CHO细胞中表达的不同截短和突变的丝裂原GPATs的活性。我们将确定mitoGPAT的哪些片段是其正常功能所必需的。我们还将确定可能的蛋白质-蛋白质或蛋白质内相互作用，通过在CHO细胞中共表达标记了不同表位的mitoGPATs结构来调节酶的活性。共表达丝裂原GPATs的细胞膜部分(通常是全长的活性蛋白和修饰的非活性蛋白，或者两个标记了不同表位的全长活性蛋白)将被交联并探测每种蛋白表达的表位。因此，我们将能够阐明是否在修饰的蛋白质中阻止了膜上mitoGPAT与其他特定蛋白质或功能寡聚体的物理接触。这些结果将阐明三酰甘油合成途径的调节，并可能提出不同于针对mitoGPAT活性部位的酶抑制的新策略。

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.