Understanding the roles of diacylglycerol acyltransferases in triacylglycerol biosynthesis and storage

三酰甘油(TG)是大多数生物体内最重要的代谢能量储存形式。TGS由三种脂肪酸连接的甘油骨架组成。由于它们高度还原且无水，TGS能够提供比碳水化合物或蛋白质多得多的能量。真核生物将TGS储存在细胞内的脂滴中，在营养缺乏期间，当能量需求得不到满足时，这些脂滴可以被新陈代谢。植物在种子中储存了大量的TGS，这些种子被用作幼苗生长发育的能量。甘油三酯是由一种名为二酰甘油酰基转移酶(DGAT)的酶合成的，该酶以二酰甘油和脂肪酰辅酶A为底物。现在已经鉴定出两种没有序列相似性的DGAT酶：DGAT1和DGAT2。虽然这两种酶在体外催化相同的生化反应，但它们在细胞中似乎没有多余的功能。DGAT1和DGAT2是为了产生不同的TG池而提出的。这两种酶都存在于细胞的内质网中；然而，DGAT2的独特之处在于它也转移到脂滴中，脂滴是储存甘油三酯的细胞器。DGAT2在脂滴上的存在被认为允许局部的TG合成，导致脂滴的生长。人们对DGAT酶如何在细胞中促进TG的合成和储存知之甚少。因此，结合蛋白质组学和脂类组学，使用生化和分子技术的综合方法将被用来理解DGAT1和DGAT2是如何合成甘油三酯的。拟议研究计划的具体目标是：(1)鉴定DGAT2的脂滴靶向/结合结构域，(2)表征参与蛋白质转运到脂滴的脂肪酸信号，(3)确定DGAT1和DGAT2是否产生不同的TGS池，以及(4)使用酵母表达系统比较和对比DGAT1和DGAT2的酶特性。拟议的研究将解决脂代谢中的基本问题，并提供对甘油三酯合成的分子机制的新见解，这可能会引起制药和农业的兴趣。由于加拿大肥胖率的惊人增长，加拿大的制药公司对开发治疗化合物以减少甘油三酯的合成和储存产生了浓厚的兴趣。在植物中，甘油三酯大量存在于油籽中，如油菜籽，这是加拿大饮食消费和工业用途的宝贵资源。由于许多甘油三酯合成途径从哺乳动物到植物都是保守的，因此我们的研究也有可能将知识转移到农业部门，目标是修改油料种子作物的甘油三酯生产。这可能会对加拿大的植物生物技术和农业产生重大影响。

Triacylglycerol (TG) is quantitatively the most important storage form of metabolic energy for most living organisms. TGs consist of a glycerol backbone to which three fatty acids are attached. Because they are highly reduced and anhydrous, TGs are capable of providing much more energy than carbohydrate or protein. Eukaryotic organisms store TGs in cytosolic lipid droplets that can be metabolized during periods of nutrient deprivation when energy requirements are not being satisfied. Plants store large quantities of TGs in seeds that are used as energy for seedling growth and development. TG is synthesized by an enzyme called diacylglycerol acyltransferase (DGAT) which uses diacylglycerol and fatty acyl CoA as substrates. Two DGAT enzymes that share no sequence similarity have now been identified: DGAT1 and DGAT2. Although these two enzymes catalyze the same biochemical reaction in vitro, they appear to not have redundant functions in cells. DGAT1 and DGAT2 are proposed to produce distinct pools of TG. Both enzymes are present in the endoplasmic reticulum in cells; however, DGAT2 is unique in that it also translocates to lipid droplets, the organelle in which TG is stored. The presence of DGAT2 on lipid droplets is thought to allow for localized TG synthesis, leading to lipid droplet growth.Little is known about how the DGAT enzymes function in cells to promote the synthesis and storage of TG. Therefore, an integrated approach using biochemical and molecular techniques in parallel with proteomics and lipidomics will be used to understand how DGAT1 and DGAT2 work to synthesize TG. The specific objectives of the proposed research program are to: (1) Identify the lipid droplet targeting/binding domain of DGAT2, (2) Characterize the fatty acid signals involved in protein translocation to lipid droplets, (3) Determine if DGAT1 and DGAT2 produce distinct pools of TGs, and (4) Compare and contrast the enzymatic properties of DGAT1 and DGAT2 using a yeast expression system. The proposed studies will address fundamental questions in lipid metabolism and provide new insights into the molecular mechanism responsible for TG synthesis that may be of interest to both the pharmaceutical and agricultural industries. Due to the alarming increase in the prevalence of obesity in Canada, there is intense interest of pharmaceutical companies in Canada to develop therapeutic compounds to reduce TG synthesis and storage. In plants, TG is found in large quantities in oil seeds such as canola which represents a valuable resource for dietary consumption and industrial uses in Canada. Since many of the pathways for TG synthesis are conserved from mammals to plants, there is also the potential for knowledge transfer of our research to the agricultural sector with the goal of modifying TG production in oil seed crops. This could have a significant impact on plant biotechnology and the agricultural industry in Canada.