调控线虫脂滴融合的一个脂肪酸水化酶DROP-1的表达与功能分析

We have isolated a series of mutants with enlarged lipid droplets from a genetic screen of chemically mutagenized C. elegans. We have cloned one of the genes, drop-1. drop-1 encodes a putative fatty acid hydratase conserved in species from yeast to human. In humans, the DROP-1 enzyme was reported to catalyze fatty acid hydration, a necessary step for fatty acid oxidation and catabolism. However, there is no report for its fatty acid substrate specificity or a role in lipid droplet regulation. Our research data demonstrates that C. elegans DROP-1 negatively regulates lipid droplet fusion and loss-of-function mutation of drop-1 leads to enhanced lipid droplet fusion and large lipid droplet formation in intestinal cells. Our working hypothesis is that DROP-1 regulates lipid droplet fusion through controlling the oxidation of specific fatty acids and hence changing the phospholipid composition of lipid droplet monolayer membrane. This project will focus on characterizing the tissue-specific expression and subcellular localization, the function in fatty acid metabolism, and the role in regulating lipid droplet membrane fusion of DROP-1. This project will employ a combination of molecular genetics, cell biology, biochemistry, lipid analytical chemistry, and other approaches when necessary.

我们通过化学诱导基因突变，筛选出一系列的具有脂滴变大表型的秀丽隐杆线虫突变体。其中的一个基因突变drop-1已经被我们克隆，drop-1编码一个从酵母到人体内都保守的脂肪酸水化酶，参与脂肪酸氧化分解。在人体内，这个酶已被报道具有水化脂肪酸的活性，但其脂肪酸底物的特异性并不清楚，也没有报道表明它能调控脂滴。我们的前期结果表明，线虫DROP-1蛋白负调控脂滴融合，drop-1基因缺失导致肠道细胞脂滴融合过程加强，脂滴变大。我们的科学猜想是：DROP-1蛋白调控某些脂肪酸的氧化分解，决定脂滴单层膜上的磷脂成分，从而调控脂滴与脂滴之间的膜融合过程。本课题将运用分子遗传学、细胞生物学、生物化学、脂质分析化学等方法手段，集中研究DROP-1的组织表达和亚细胞定位、脂肪酸代谢功能、以及调控脂滴膜融合过程的分子机理。

在亚细胞水平上，脂肪储存在脂滴中，脂滴的大小与脂代谢疾病紧密相关。在本项目研究过程中，我们筛选并克隆了3个调控线虫脂滴大小的基因突变：drop-1、drop-8、drop-31，这三个基因分别编码CYP-37A1（一个细胞色素P450蛋白）、EMB-8（细胞色素P450氧化还原酶）、DAF-12（一个核激素受体）。CYP-37A1和EMB-8蛋白合成一种脂溶性激素，该激素抑制DAF-12的促脂滴融合功能。当cyp-37A1、emb-8基因失去功能后，DAF-12介导的脂滴融合增强，该融和过程在30 oC高温条件下最强，导致超大脂滴形成。总而言之，本项目研究发现了一个新颖的脂滴融合过程，即热敏型脂滴融合，并且发现了调控热敏型脂滴融合的一条新颖的核激素受体信号通路。