The role of acyl-CoA binding proteins in selective autophagy

描述（由申请人提供）：肥胖是21世纪的主要健康问题。确定预防和治疗肥胖的新方法对公共卫生具有重要意义。肥胖特指脂肪组织过多，脂质储存在称为脂滴（ld）的专门细胞器中。最近在自噬领域的研究表明，ld可以选择性地递送到溶酶体并被溶酶体降解。这一发现打开了一个令人兴奋的新机会，通过调节脂噬，即脂噬（或细胞的“自噬”）来纠正人类的脂质代谢。在过去的12年里，我一直在研究选择性自噬，并发现了另一种细胞器（过氧化物酶体）选择性降解所需的几种蛋白质。最近，我们发现酰基辅酶a结合蛋白（acbp）、Acb1和另一种我们鉴定为Atg36的蛋白在毕赤酵母过氧化物酶体（pexophagy）和Ape1复合物（Cvt途径）的选择性自噬中是必不可少的。因此，我的项目的第一个目标是阐明acbp在P. pastoris选择性自噬中的分子作用。我们预测acbp在选择性自噬中的作用从酵母到哺乳动物都是保守的。因此，第二个目标是阐明acbp在哺乳动物细胞中的生理作用。我们将分别描述Atg36和Acb1的哺乳动物同源物ACBD5和DBI在食腐和脂噬中的作用。第三个目标是将肥胖模型酵母脂溶耶氏菌作为简单的脂噬模型，研究acbp在脂噬中的分子作用。我的长期目标是建立一个独立的研究项目，研究脂肪吞噬，揭示其分子机制和acbp在这一过程中的作用。了解特异性参与ld自噬降解的蛋白质可能有助于设计新的治疗方法来治疗人类肥胖。为了完成我从(1)酵母到哺乳动物细胞，(2)食腐到食脂，(3)指导独立研究，我将继续接受哺乳动物细胞系工作和监测哺乳动物食腐和食脂的培训。我将与Till博士合作，并咨询Cuervo博士，分别是哺乳动物食腐和食脂方面的专家。我将通过咨询ACBP专家Loomis博士，扩大我对ACBP结构和功能的了解。我还将在我的共同导师Field博士的实验室接受关于脂质生物学的广泛培训，他是细胞脂质和脂质结合蛋白方面的专家。这种训练对于完成ACBP突变体的脂质分析和研究ACBP与磷脂的相互作用是必不可少的。为了增加我在选择性自噬和ld方面的知识，我将继续参加自噬和脂质生物学的会议。为了训练公开演讲，我将在国际演讲会练习演讲。2012年，我将在戈登自噬研究研讨会（Ventura， CA）上发表关于Atg36的演讲。为了成为一名成功的独立研究者，我还将通过上课和与加州大学圣地亚哥分校教师的个人会议，接受生物统计学、科学管理、写作和拨款写作方面的培训。我将撰写关于acbp在选择性自噬中的作用的稿件，并在K01奖第二年年底前提交给排名靠前的期刊之一。另外，我将在第3年年底前撰写我的第一篇关于脂噬分子机制的R01 grant，并提交给NIDDK。我将在第四年开始寻找一个终身教职的职位，并在4年K01奖结束前接受offer。在脂噬方面已经形成了一个独立的研究方向，我将继续在酵母和哺乳动物细胞中揭示其分子机制和acbp在这一过程中的作用。我的导师Subramani博士将在K01奖期间密切关注我的研究和职业发展。我的建议包括三个具体目标。目的1将讨论两种可选的，但不是相互排斥的，acbp在巴斯德酵母选择性自噬中的作用：(1) ACBP与噬菌体蛋白Atg8（酵母双杂交和共免疫沉淀研究）相互作用，在架起cargo和吞噬体（一种自噬隔离膜）之间的桥梁作用；(2)ACBP与磷脂的相互作用和吞噬体膜的重塑，在产生吞噬体膜曲率方面的作用（体外过滤器和脂质体结合试验，ACBP突变体的脂质谱分析）。目的2将探讨acbp在哺乳动物细胞选择性自噬中的生理作用。我们将利用人宫颈癌、肝癌、畸胎癌细胞和新的噬瘤报告细胞mRFP-GFP-SKL，研究ACBD5和DBI在噬瘤中的作用。我们的初步研究表明，人类ACBD5对食腐至关重要。我还将利用已建立的大鼠肝细胞细胞系和脂噬实验来解决ACBD5和DBI在脂噬中的作用。最后，Aim 3将开发第一个简单的脂噬模型。我们将敲除聚脂Y. ATG36和ACB1基因，并利用荧光显微镜和一种新的Tgl3-GFP处理实验来阐明它们在脂噬中的作用。我们期望对酵母和哺乳动物细胞中acbp的研究将扩展我们对细胞器稳态的理解，并为预防和治疗人类肥胖提供新的靶点。

DESCRIPTION (provided by applicant): Obesity is a major health problem of the 21st century. Identification of novel approaches for prevention and treatment of obesity is of great importance for public health. Obesity specifically refers to an excessive amount of adipose tissue, where lipids are stored in specialized organelles called lipid droplets (LDs). Recent studies in the autophagy field suggest that LDs can be selectively delivered to and degraded by lysosomes. This discovery has opened an exciting, new opportunity to correct lipid metabolism in humans by modulating lipophagy, the selective autophagy (or the cell's "self-eating") of LDs. I have studied selective autophagy for the past 12 years and identified several proteins required for the selective degradation of another organelle, the peroxisome. Recently, we found that acyl-CoA binding proteins (ACBPs), Acb1 and another protein we identified as Atg36, are essential for selective autophagy of peroxisomes (pexophagy) and Ape1 complexes (Cvt pathway) in the Pichia pastoris yeast. Therefore, the first goal of my project is to elucidate the molecular role o ACBPs in selective autophagy in P. pastoris. We predict that the role of ACBPs in selective autophagy is conserved from yeast to mammals. Thus, the second goal is to elucidate the physiological role of ACBPs in mammalian cells. We will characterize the role of ACBD5 and DBI, the mammalian orthologs of Atg36 and Acb1, respectively, in both pexophagy and lipophagy. The third goal is to develop the obesity model yeast, Yarrowia lipolytica, as a simple lipophagy model and study the molecular role of ACBPs in lipophagy. My long- term goals are to develop an independent research program on lipophagy, uncover its molecular mechanism and the role of ACBPs in this process. Knowledge of the proteins specifically involved in the autophagic degradation of LDs might help to design new therapeutic approaches to cure obesity in humans. To complete my transition from (1) yeast to mammalian cells, (2) pexophagy to lipophagy and (3) mentored to an independent research, I will continue to receive training in working with mammalian cell lines and in monitoring mammalian pexophagy and lipophagy. I will do it through collaboration with Dr. Till and consultation with Dr. Cuervo, experts on mammalian pexophagy and lipophagy, respectively. I will expand my knowledge on the structure and function of ACBPs through consultation with an ACBP expert, Dr. Loomis. I will also receive extensive training on lipid biology in the lab of my co-mentor, Dr. Field, an expert on cellular lipids and lipid-binding proteins. This training is essential to accomplish lipid profiing of ACBP mutants and to study interactions of ACBPs with phospholipids. To increase my knowledge on selective autophagy and LDs, I will continue to attend both autophagy and lipid biology meetings. For the training in public speaking, I will practice giving talks at the Toastmasters International. In 2012, I will give a talk on Atg36 at the Gordon Research Seminar on Autophagy (Ventura, CA). To become a successful independent investigator, I will also receive training in biostatistics, scientific management, writing and grant writing by taking classes and through personal meetings with UCSD faculties. I will write the manuscript on the role of ACBPs in selective autophagy and submit it to one of the top-ranked journals by the end of the 2nd year of the K01 award. Additionally, I will write my first R01 grant on the molecular mechanism of lipophagy and submit it to NIDDK by the end of the 3rd year. I will start searching for a tenure-track faculty position during the 4th year and accept an offer by the end of the 4-year K01 award. Having developed an independent research direction on lipophagy, I will continue to uncover its molecular mechanism and the role of ACBPs in this process in both yeast and mammalian cells. My mentor, Dr. Subramani, will closely monitor my research and career development during the K01 award. My proposal consists of the three Specific Aims. Aim 1 will address two alternative, but not mutually exclusive, roles of ACBPs in selective autophagy in P. pastoris: (1) the role in bridging cargo and the phagophore, an autophagic isolation membrane, via interaction of ACBPs with the phagophore protein, Atg8 (yeast two-hybrid and co-immunoprecipitation studies) and (2) the role in generating phagophore membrane curvature via the interaction of ACBPs with phospholipids and remodeling of the phagophore membrane (in vitro filter and liposome binding assays, lipid profiling of ACBP mutants). Aim 2 will explore the physiological role of ACBPs in selective autophagy in mammalian cells. We will study the role of ACBD5 and DBI in pexophagy using human cervical cancer, hepatocarcinoma and teratocarcinoma cells and a new pexophagy reporter, mRFP-GFP-SKL. Our pilot studies suggest that human ACBD5 is essential for pexophagy. I will also address the role of ACBD5 and DBI in lipophagy using an established rat hepatocyte cell line and lipophagy assays. Finally, Aim 3 will develop the first simple model for lipophagy using Y. lipolytica. We will knock out the Y. lipolytica ATG36 and ACB1 genes, and elucidate their roles in lipophagy using fluorescence microscopy and a novel Tgl3-GFP processing assay. We expect studies on ACBPs in yeast and mammalian cells will extend our understanding of organelle homeostasis and provide new targets for the prevention and treatment of obesity in humans.