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，作为母基金DK 56598（4/15/99-3/31/03）的一项扩展。甘油磷酸酰基转移酶（GPAT）催化甘油脂质合成中的第一个关键步骤，即sn-甘油-3-磷酸酰化为溶血磷脂酸。我们以前证明，线粒体亚型的GPAT（mitoGPAT）指导细胞合成的三酰甘油（TAG），但不是磷脂，在两个不同的细胞系，这表明mitoGPAT可能是一个很好的候选人的药物开发控制TAG合成的病理条件下的肥胖症和糖尿病。MitoGPAT是具有两个跨膜结构域的线粒体外膜蛋白。该蛋白质在胞质N-末端结构域中含有活性位点，面向线粒体膜间隙的84个氨基酸的环，以及没有已知功能的238个氨基酸的C-末端结构域。我们以前的研究结果表明，通过在环中插入表位标签或在环区域的末端截短来灭活酶。我们建议，以确定如何mitoGPAT活性调节的环和C-末端结构域，不形成催化位点。所提出的实验将分析在CHO细胞中表达的不同截短和突变的mitoGPAT的活性。我们将确定哪些片段的mitoGPAT是必不可少的适当功能。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细胞中共表达标记有不同表位的mitoGPAT构建体来调节酶活性。将共表达mitoGPAT的细胞的膜部分（通常是全长活性蛋白和修饰的无活性蛋白，或用不同表位标记的两种全长活性蛋白）交联并探测与每种蛋白一起表达的表位。因此，我们将能够阐明是否在膜中的mitoGPAT的物理接触，无论是与其他特定的蛋白质或功能性寡聚体，被阻止在修饰的蛋白质。这些结果将阐明三酰甘油合成途径的调节，并可能提出新的酶抑制策略，不同于那些针对mitoGPAT的活性位点。

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.