Identification and validation of a lipophagy regulator

 描述(申请人提供)：我们身体的细胞在细胞器中积累储存的脂质，称为脂滴(LDS)。低密度脂蛋白在脂质代谢中起着重要作用。缺乏脂肪分解或吞脂作用会造成过多的低密度脂蛋白，从而导致肥胖和肥胖相关的疾病。最近发现的吞脂作用为肥胖症的治疗打开了新的机会，但我们对这一途径的了解仍然非常有限。在K01获奖项目中，我们分离到一株基础吞脂力高度升高的酵母突变株。因此，本项目的第一个目标是鉴定和验证与这种表型有关的基因。如果这个基因在哺乳动物中是保守的，我们也将验证这个基因在哺乳动物脂噬调节中的作用。我们的第二个目标是鉴定吞脂性所必需的形态中间体和蛋白质。这将为随后的R01赠款申请和深入研究已确定的调节子在吞脂性中的作用提供必要的框架。我们的项目有两个具体目标。AIM 1致力于鉴定和验证吞脂性的负调控因子。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(S)(S)。然后，我们将通过基因缺失分析将它们缩小到脂噬抑制基因。为了验证所鉴定的基因是否为脂噬抑制基因，我们将进行基因过表达研究。目的2鉴定噬脂体的形态中间体和分子结构。在这里，我们将检查液泡隔离膜对LDS的吞噬是否涉及形成桥式自噬膜，即吞噬体。我们还将定义所有已知ATG-蛋白的吞脂要求，并确定每个ATG-突变体所阻断的途径的阶段。最后，我们将建立一个具有潜在正负调节点的吞脂性分子模型，这将在R01拨款申请中进一步探索。

 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.