Defining the role of ubiquitination in the regulation of lipid droplets

定义泛素化在脂滴调节中的作用

• 批准号: 8911916
• 负责人: JAMES A OLZMANN
• 金额: $ 6.27万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8911916
• 关键词: Address; Adipocytes; Affinity; Atherosclerosis; Award; Biochemical; Biochemistry; Biological; Biological Assay; Biology; Cardiovascular Diseases; Cell physiology; Cells; Cellular biology; Collaborations; Complex; Coupled; Cytoplasmic Organelle; Data; Development; Diabetes Mellitus; Disease; Endoplasmic Reticulum; Energy Metabolism; Environment; Enzymes; Epidemic; Essential Fatty Acids; Eukaryotic Cell; Fatty Acids; Fatty Liver; Fatty acid glycerol esters; Foundations; Goals; Grant; Growth; Health; Hydrolysis; Immunoblot Analysis; Institutes; Lead; Link; Lipids; Lipodystrophy; Lipolysis; Maps; Measurement; Measures; Mediating; Membrane; Mentors; Mentorship; Metabolic; Metabolic Control; Metabolic Diseases; Methodology; Molecular; Multiprotein Complexes; Obesity; Organelles; Pathway interactions; Peptides; Phase; Phospholipids; Physiologic pulse; Positioning Attribute; Post-Translational Protein Processing; Process; Proteins; Proteome; Proteomics; RNA Interference; Regulation; Research; Research Personnel; Research Training; Role; Signal Transduction; Sucrose; System; Techniques; Testing; Training; Triglycerides; Ubiquitin; Ubiquitination; Universities; Work; abstracting; base; career; career development; combat; design; experience; human disease; innovation; lipid metabolism; monolayer; novel therapeutics; overexpression; p97 ATPase; programs; protein complex; protein degradation; response; sensor

Project Summary / Abstract In order to develop novel therapeutic strategies to combat the increasing epidemic of metabolic diseases (eg. obesity, diabetes, and hepatic steatosis) an in-depth understanding of the molecular mechanisms underlying the storage and mobilization of fat is critical. In cells, fat is stored as triacylglycerol in lipid droplets (LDs), an endoplasmic reticulum-derived organelle composed of a neutral lipid core encircled by a phospholipid monolayer decorated with embedded proteins. Despite the clear importance of the LD proteome in controlling LD function, the mechanisms involved in regulating their activities and levels are poorly understood. Posttranslational conjugation with ubiquitin, which occurs via an enzymatic cascade involving an E1, E2, and E3 enzyme, regulates virtually every cellular process. Our preliminary findings identify a metabolically regulated LD ubiquitination pathway that involves UBXD8-mediated recruitment of p97/VCP and regulation LD turnover catalyzed by ATGL, the rate-limiting enzyme in lipolysis. These preliminary data establish the functional importance of this pathway in LD biology and provide a foundation for further mechanistic studies. The studies described in this application are designed to test the hypothesis that UBXD8 functions as a sensor for fatty acids that stabilizes LDs in response to metabolic signals by targeting the enzymes required for lipolysis for Ub-dependent silencing. Specifically, the studies proposed in this grant will elucidate the mechanism of UBXD8-mediated inhibition of ATGL function (Aim 1), will construct a comprehensive map of the LD-associated ubiquitination networks (Aim 2), and will identify LD ubiquitination targets and their regulation by the metabolic state of the cell (Aim 3). I have extensive experience in the application of cell biology and biochemistry approaches to understand the role of the ubiquitin system in various aspects of cellular protein quality and quantity control. As a postdoctoral scholar I have expanded my research training to include systems-level strategies, quantitative proteomics methodologies, and LD functional assays. During the mentored K99 phase of this award I will work closely with my mentors Dr. Ron Kopito and Dr. Robert Farese Jr., recognized experts in the fields of ubiquitin biology and lipid metabolism, to perform the proposed research and to implement my research and career plan. Under their mentorship I will benefit from the combined environments of Stanford University and the Gladstone Institute of Cardiovascular Disease, which together provide an environment rich with opportunities for experimental training, intellectual growth, collaborations, networking, career development, and innovative research. My short-term goal is to obtain a position as an independent investigator, and during the R00 period of the award I will develop a robust and original research program focused on the contribution of ubiquitin to LD function in health and disease. My long-term goal is to function as a scientific leader and mentor to promote pioneering research that advances the fundamental understanding of the molecular mechanisms regulating lipid metabolism and that ultimately leads to the successful development and implementation of efficacious strategies to treat metabolic diseases.

项目摘要 /摘要 为了制定新型的治疗策略，以打击代谢疾病的日益流行（例如。 肥胖，糖尿病和肝脂肪变性）对分子机制的深入了解 脂肪的存储和动员至关重要。在细胞中，脂肪被储存为三酰基甘油中的脂肪液滴（LDS） 内质网源性细胞器，由磷脂包围的中性脂质芯组成 单层装饰有嵌入式蛋白质。尽管LD蛋白质组在控制中显然很重要 LD功能，调节其活动和水平的机制知之甚少。 翻译后与泛素结合，这是通过涉及E1，E2和 E3酶，几乎调节每个细胞过程。我们的初步发现在代谢上确定 涉及UBXD8介导的P97/VCP募集和调节LD的调节LD泛素化途径 营业额由ATGL催化，ATGL是脂解的限速酶。这些初步数据建立了 该途径在LD生物学中的功能重要性，并为进一步的机械研究提供了基础。 本应用中描述的研究旨在检验UBXD8作为传感器的假设 对于脂肪酸，可以通过靶向对代谢信号稳定LDS的脂肪酸。 脂解，用于UB依赖性沉默。具体而言，本赠款中提出的研究将阐明 UBXD8介导的ATGL功能的抑制机制（AIM 1）将构建一张综合图 与LD相关的泛素化网络（AIM 2），并将通过 细胞的代谢状态（AIM 3）。 我在细胞生物学和生物化学方法的应用方面有丰富的经验 了解泛素系统在细胞蛋白质质量和数量控制的各个方面的作用。 作为博士后学者，我将研究培训扩展到包括系统级策略， 定量蛋白质组学方法和LD功能测定。在指导的K99阶段 奖项我将与我的导师Ron Kopito博士和小罗伯特·法雷斯博士紧密合作，这是公认的专家 泛素生物学和脂质代谢领域，进行拟议的研究并实施我 研究和职业计划。在他们的指导下，我将从斯坦福的联合环境中受益 大学和格拉德斯通心血管疾病研究所，共同提供富裕的环境 有实验培训，智力增长，合作，网络，职业的机会 开发和创新研究。我的短期目标是获得独立的职位 调查员，在奖励的R00期间，我将制定一个强大而原始的研究计划 专注于泛素对LD功能在健康和疾病中的贡献。我的长期目标是运作 作为促进开拓性研究的科学领导者和导师 了解调节脂质代谢的分子机制，并最终导致 成功开发和实施有效治疗代谢疾病的策略。