Defining the role of ubiquitination in the regulation of lipid droplet function

描述（由申请人提供）：为了开发新的治疗策略来对抗日益流行的代谢性疾病（如肥胖、糖尿病和肝脂肪变性），深入了解脂肪储存和动员的分子机制至关重要。在细胞中，脂肪以三酰基甘油的形式储存在脂滴（ld）中，脂滴是一种内质网衍生的细胞器，由中性脂核组成，脂核被包裹着嵌入蛋白质的磷脂单层所包围。尽管LD蛋白组在控制LD功能中具有明确的重要性，但调节其活性和水平的机制尚不清楚。泛素与泛素的翻译后偶联是通过涉及E1、E2和E3酶的酶级联发生的，它调节着几乎所有的细胞过程。我们的初步研究结果确定了代谢调节的LD泛素化途径，该途径涉及ubxd8介导的p97/VCP募集和脂肪分解限速酶ATGL催化的LD转换调节。这些初步数据证实了该通路在LD生物学中的重要功能，并为进一步的机制研究奠定了基础。本应用程序中描述的研究旨在验证UBXD8作为脂肪酸传感器的假设，通过靶向脂解所需的酶来稳定ld，以响应代谢信号，从而实现ub依赖性沉默。具体而言，本基金提出的研究将阐明ubxd8介导的ATGL功能抑制机制（Aim 1），将构建LD相关泛素化网络的综合图谱（Aim 2），并将确定LD泛素化靶点及其代谢调控

DESCRIPTION (provided by applicant): In order to develop novel therapeutic strategies to combat the increasing epidemic of metabolic diseases (e.g. 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.! PUBLIC HEALTH RELEVANCE: Relevance to public health: How cells store and mobilize fatty acids from lipid droplets is a fundamental question with broad relevance for the treatment of metabolic diseases such as obesity and diabetes. Ubiquitination is an important posttranslational modification that regulates virtually every known process in the cell, but the role of ubiquitination in lipid droplet biology is poorly understood. The aims described in this proposal will utilize recent technological advances to define the components and downstream targets of a new ubiquitination pathway that regulates lipid droplet function, and will lay the foundation for the development of novel therapeutic strategies.!