The role of acyl-CoA binding proteins in selective autophagy

酰基辅酶A结合蛋白在选择性自噬中的作用

• 批准号: 8704394
• 负责人: Taras Y. Nazarko
• 金额: $ 11.6万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8704394
• 关键词: Acyl Coenzyme A; Address; Adipose tissue; Adult; American; Amino Acid Motifs; Arabidopsis; Autophagocytosis; Autophagosome; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Biology; Biometry; Cell Line; Cells; Co-Immunoprecipitations; Collaborations; Complex; Consultations; Coronary heart disease; Cytoplasm; Cytosol; Development; Diazepam Binding Inhibitor; Eating; Electron Microscopy; Faculty; Family; Fatty acid glycerol esters; Fluorescence Microscopy; Genes; Goals; Grant; Health; Hela Cells; Hepatocyte; Homeostasis; Homologous Gene; Human; Immunofluorescence Microscopy; In Vitro; International; Journals; Knock-out; Knowledge; Lecithin; Lipid Binding; Lipids; Liposomes; Lysosomes; Malignant neoplasm of cervix uteri; Mammalian Cell; Mammals; Manuscripts; Mediating; Membrane; Mentored Research Scientist Development Award; Mentors; Modeling; Molecular; Monitor; Mutate; Names; National Institute of Diabetes and Digestive and Kidney Diseases; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Organelles; Orthologous Gene; Pathway interactions; Phosphatidylethanolamine; Phospholipids; Physiological; Pichia; Pilot Projects; Positioning Attribute; Prevention; Process; Proteins; Public Health; Public Speaking; Publications; Rattus; Reporter; Research; Research Personnel; Risk Factors; Role; Stroke; Structure; Study models; Techniques; Teratocarcinoma; Testing; Training; Vacuole; Vesicle; Western Blotting; Work; Writing; Yarrowia lipolytica; Yeast Model System; Yeasts; career development; design; lipid metabolism; meetings; mutant; novel; novel strategies; novel therapeutic intervention; novel therapeutics; obesity treatment; peroxisome; programs; protein complex; receptor; research and development; yeast two hybrid system

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.

描述（由申请人提供）：肥胖是21世纪的主要健康问题。发现预防和治疗肥胖症的新方法对公共卫生具有重要意义。肥胖具体指的是过量的脂肪组织，其中脂质储存在称为脂滴（LD）的专门细胞器中。自噬领域的最新研究表明，LD可以选择性地递送到溶酶体并被溶酶体降解。这一发现开启了一个令人兴奋的新机会，通过调节脂噬作用，即LD的选择性自噬（或细胞的“自食”）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，并确定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（ACBPs），Acb 1和另一种蛋白质，我们确定为Atg 36，是必不可少的选择性自噬过氧化物酶体（pexophagy）和Ape 1复合物（Cvt途径）在毕赤酵母。因此，本课题的第一个目标是阐明ACBPs在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。我们将描述ACBD 5和DBI，Atg 36和Acb 1的哺乳动物直系同源物，分别在pexophagy和lipophagy中的作用。第三个目标是开发肥胖模型酵母，解脂耶氏酵母，作为一个简单的脂肪吞噬模型，并研究ACBPs在脂肪吞噬中的分子作用。我的长期目标是发展一个独立的研究计划，对脂肪吞噬，揭示其分子机制和ACBPs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愈人类肥胖。为了完成我从（1）酵母到哺乳动物细胞的转变，（2）从pexophagy到lipophagy，（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pexophagy和lipophagy的培训。我将通过与Till博士的合作以及与Cuervo博士的咨询来完成，Cuervo博士分别是哺乳动物的食粪和食脂专家。我将通过咨询ACBP专家卢米斯博士来扩大我对ACBP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接受脂质生物学方面的广泛培训，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突变体的脂质分析和研究ACBP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LD的知识，我将继续参加自噬和脂质生物学会议。在公开演讲的训练中，我将在国际演讲会上练习演讲。2012年，我将在Gordon自噬研究研讨会（Ventura，CA）上发表关于Atg 36的演讲。为了成为一名成功的独立调查员，我还将接受生物统计学，科学管理，写作和赠款写作方面的培训，通过上课和与UCSD教师的个人会议。我将撰写关于ACBPs在选择性自噬中的作用的手稿，并在K 01奖的第二年年底将其提交给排名最高的期刊之一。此外，我将写我的第一个R 01基金在脂肪吞噬的分子机制，并提交给NIDDK的第三年年底。我将在第四年开始寻找终身教职，并在4年K 01奖结束时接受录取。在发展了一个关于脂肪吞噬的独立研究方向后，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的作用。我的导师Subramani博士将在K 01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。目标1将解决ACBPs在巴斯德毕赤酵母中选择性自噬中的两种替代但不相互排斥的作用：（1）通过ACBP与吞噬细胞蛋白的相互作用，在桥接货物和吞噬细胞（自噬隔离膜）中的作用，Atg8（酵母双杂交和免疫共沉淀研究）和（2）通过ACBP与磷脂的相互作用和吞噬细胞膜的重塑在产生吞噬细胞膜弯曲中的作用（体外过滤和脂质体结合试验，ACBP突变体的脂质分析）。目的2探讨ACBPs在哺乳动物细胞选择性自噬中的生理作用。我们将使用人宫颈癌、肝癌和畸胎癌细胞以及一种新的噬菌体报告基因mRFP-GFP-SKL来研究ACBD 5和DBI在噬菌体中的作用。我们的初步研究表明，人类ACBD 5是必不可少的pexophagy。我还将使用已建立的大鼠肝细胞系和脂肪吞噬测定来解决ACBD 5和DBI在脂肪吞噬中的作用。最后，目标3将使用Y.解脂我们会消灭Y Lipolytica ATG 36和ACB 1基因，并使用荧光显微镜和新的Tgl 3-GFP加工测定阐明它们在脂肪吞噬中的作用。我们期望酵母和哺乳动物细胞中ACBPs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。