Identification and validation of a lipophagy regulator

 描述（由申请人提供）：我们身体的细胞在称为脂滴（LD）的细胞器中积累储存脂质。LDs在脂质代谢中起重要作用。它们的丰度由经由胞质脂肪酶的脂解作用和脂肪吞噬作用（将LD递送至溶酶体/空泡脂肪酶的自噬运输途径）控制。脂肪分解或脂肪吞噬的缺乏会产生过量的LD，导致肥胖和肥胖相关疾病。最近发现的脂肪吞噬开辟了一个新的机会，治疗肥胖，但我们的知识，这一途径仍然非常有限。在K01奖励项目期间，我们分离出具有高度升高的基础噬脂性的酵母突变体。因此，本项目的第一个目标是鉴定和验证负责这种表型的基因。如果该基因在哺乳动物中是保守的，我们也将验证该基因在哺乳动物脂肪吞噬调节中的作用。我们的第二个目标是确定形态中间体和蛋白质的脂肪吞噬作用。这将为我们随后的R01资助申请和深入研究所确定的调节剂在脂肪吞噬中的作用提供必要的框架。达到这些目标也可能为预防和治疗肥胖提供新的有希望的靶点，因为对脂肪吞噬抑制剂的药物抑制将增加脂肪吞噬的速率并减少LD的体积。我们的项目有两个具体目标。目的1致力于鉴定和验证脂肪吞噬的负调节因子。这是K01目标3的逻辑延伸，在该目标3中，我们开发了巴斯德毕赤酵母作为简单的噬脂模型，并分离了受噬脂负调控影响的nrl1突变体。我们将对nrl1细胞的基因组进行测序，并确定受突变影响的基因。然后，我们将通过基因缺失分析将它们缩小到脂肪吞噬抑制基因。为了验证所鉴定的基因为脂肪吞噬抑制基因，我们将进行基因过表达研究。如果所鉴定的基因在哺乳动物中是保守的，我们还将通过使用大鼠肝细胞和已建立的脂肪吞噬测定的基因敲除和过表达研究来验证其在脂肪吞噬中的作用。目的二是确定噬脂作用的形态学中间体和分子结构。在K01的Aim 3中，我们发现巴斯德毕赤酵母中的噬脂作用依赖于自噬相关蛋白Atg 5和Atg 8以及液泡隔离膜。在这里，我们将检查是否吞噬LD的空泡隔离膜涉及形成一个桥接自噬膜，吞噬细胞。我们还将定义所有已知的Atg蛋白的脂肪吞噬要求，并确定在每个Atg突变体中阻断的途径的阶段。最后，我们将建立一个具有潜在的正调控和负调控点的脂肪吞噬分子模型，这将在R01基金申请中进一步探索。

 DESCRIPTION (provided by applicant): The cells of our bodies accumulate storage lipids in the organelles called lipid droplets (LDs). LDs play an important role in lipid metabolism. Their abundance is controlled by lipolysis via cytosolic lipases and by lipophagy, the autophagic trafficking pathway that delivers LDs to lysosomal/vacuolar lipases. The lack of lipolysis or lipophagy creates an excess of LDs causing obesity and obesity-related disorders. The recent discovery of lipophagy opened a new opportunity for treatment of obesity, but our knowledge of this pathway is still very limited. During the K01 award project, we isolated a yeast mutant with highly elevated basal lipophagy. Therefore, the first goal of this project is to identify and validte the gene responsible for this phenotype. If this gene is conserved in mammals, we will also validate the role of this gene in the regulation of mammalian lipophagy. Our second goal is to identify the morphological intermediates and proteins essential for lipophagy. This will provide us with essential framework for a subsequent R01 grant application and in-depth studies on the role of the identified regulator in lipophagy. Reaching these goals might also provide a new promising target for prevention and treatment of obesity, since pharmaceutical inhibition of the lipophagy suppressor will increase the rates of lipophagy and decrease the volume of LDs. Our project has two specific aims. Aim 1 is dedicated to identification and validation of the negative regulator of lipophagy. It is a logical extension of the K01's Aim 3 where we developed the Pichia pastoris yeast as a simple lipophagy model and isolated the nrl1 mutant affected in the Negative Regulation of Lipophagy. We will sequence the genome of nrl1 cells and identify the gene(s) affected by mutation(s). Then, we will narrow them down to the gene of the lipophagy suppressor by gene deletion analysis. To validate the identified gene as the gene of the lipophagy suppressor, we will perform the gene overexpression studies. If the identified gene is conserved in mammals, we will also validate its role in lipophagy through gene knockdown and overexpression studies using rat hepatocytes and established lipophagy assays. Aim 2 will identify the morphological intermediates and molecular architecture of lipophagy. In the K01's Aim 3, we found that lipophagy in P. pastoris depends on the autophagy-related proteins, Atg5 and Atg8, and on the vacuolar sequestering membranes. Here, we will examine if engulfment of LDs by the vacuolar sequestering membranes involves formation of a bridging autophagic membrane, the phagophore. We will also define the lipophagy requirements of all known Atg-proteins and identify the stage of the pathway blocked in each atg-mutant. Finally, we will build a molecular model of lipophagy with potential points of positive and negative regulation, which will be further explored in the R01 grant application.