The role of acyl-CoA binding proteins in selective autophagy

描述(申请人提供)：肥胖是21世纪的一个主要健康问题。确定预防和治疗肥胖症的新方法对公众健康非常重要。肥胖特指过多的脂肪组织，脂肪储存在被称为脂滴(LDs)的专门细胞器中。最近在自噬领域的研究表明，LDS可以选择性地递送到溶酶体并被其降解。这一发现打开了一个令人兴奋的新机会，通过调节脂噬作用，即选择性的自噬(或细胞的“自噬”)来纠正人类的脂类代谢。在过去的12年里，我一直在研究选择性自噬，并鉴定了几种蛋白质，这些蛋白质是选择性降解另一种细胞器--过氧化物酶体--所需的。最近，我们发现酰基辅酶A结合蛋白(ACBPS)，Acb1和另一种我们鉴定为Atg36的蛋白，在毕赤酵母中对过氧化物体(Pexphagy)和APE1复合体(CVT途径)的选择性自噬是必不可少的。因此，我的项目的第一个目标是阐明ACBPS在巴斯德毕赤酵母选择性自噬中的分子作用。我们预测ACBPS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ACBPS在哺乳动物细胞中的生理作用。我们将表征ACBD5和DBI，分别是Atg36和Acb1的哺乳动物同源基因，在吞噬和脂噬中的作用。第三个目标是发展肥胖模型酵母，解脂雅罗威，作为一个简单的脂噬模型，并研究ACBPS在脂噬中的分子作用。我的长期目标是开发一个关于吞脂性的独立研究计划，揭示其分子机制和ACBPS在这一过程中的作用。了解LDS自噬降解过程中具体涉及的蛋白质可能有助于设计新的治疗方法来治愈人类肥胖。为了完成我从(1)酵母到哺乳动物细胞的过渡，(2)从吞噬到脂噬的过渡，以及(3)指导我进行一项独立研究，我将继续接受与哺乳动物细胞系合作以及监测哺乳动物吞噬和脂噬的培训。我将通过与蒂尔博士的合作和与库尔沃博士的咨询来完成这项工作，库埃沃博士分别是哺乳动物食性吞噬和吞脂性方面的专家。我将通过咨询ACBP专家卢米斯博士来扩大我对ACBPS结构和功能的了解。我还将在我的共同导师菲尔德博士的实验室里接受广泛的脂质生物学培训，菲尔德博士是细胞脂质和脂质结合蛋白的专家。这种训练对于完成ACBP突变体的脂质分配和研究ACBPS与磷脂的相互作用是必不可少的。为了增加我对选择性自噬和LDS的了解，我将继续参加自噬和脂类生物学会议。为了进行演讲训练，我将在国际演讲会上练习演讲。2012年，我将在戈登自噬研究研讨会(加利福尼亚州文图拉)上发表关于Atg36的演讲。为了成为一名成功的独立调查员，我还将通过上课和与加州大学圣地亚哥分校教职员工的私人会议，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ACBPS在选择性自噬中的作用的手稿，并在K01奖第二年结束时将其提交给排名靠前的期刊之一。此外，我将写下我的第一份关于吞噬脂肪的分子机制的R01拨款，并在第三年结束前提交给NIDDK。我将在第四年开始寻找终身教职，并在四年K01奖结束时接受邀请。在发展了吞脂性的独立研究方向后，我将继续揭示其分子机制以及ACBPS在酵母和哺乳动物细胞中这一过程中的作用。我的导师Subramani博士将在K01颁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将阐述ACBPS在巴斯德毕赤酵母选择性自噬中的两个不同但不是相互排斥的作用：(1)ACBPS与吞噬蛋白Atg8相互作用在货物和吞噬隔膜(自噬隔离膜)中的作用(酵母双杂交和免疫共沉淀研究)和(2)ACBPS与磷脂相互作用和吞噬体膜重塑(体外过滤和脂体结合分析，ACBP突变体的脂谱分析)在产生吞噬体膜曲率中的作用。目的2探讨ACBPS在哺乳动物细胞选择性自噬中的生理作用。我们将研究ACBD5和DBI在人宫颈癌、肝癌和畸胎癌细胞吞噬中的作用，以及一种新的吞噬报告MRFP-GFP-SKL。我们的初步研究表明，人类ACBD5对于食欲症是必不可少的。我还将利用已建立的大鼠肝细胞系和脂噬实验来研究ACBD5和DBI在脂噬中的作用。最后，目标3将开发第一个利用解脂耶尔森菌进行吞脂作用的简单模型。我们将敲除解脂耶尔森菌ATG36和ACB1基因，并利用荧光显微镜和一种新的Tgl3-GFP加工方法来阐明它们在脂噬中的作用。我们期望在酵母和哺乳动物细胞中对ACBPS的研究将扩大我们对细胞器动态平衡的理解，并为人类肥胖的预防和治疗提供新的靶点。

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.