Molecular and cellular analysis of the ABHD5/PNPLA3 metabolon in lipid homeostasis

项目摘要/摘要 脂肪组织和肝脏中三酰甘油(Tag)的储存和动员之间的平衡对代谢健康至关重要，因为肥胖过程中的失调会导致肌肉和肝脏中脂肪的异位聚集，从而导致糖尿病和脂肪肝(FLD)的进展。因此，一个重要的长期科学目标是了解控制TAG代谢的组织特异性分子机制，以便发现新的治疗方法。Patatin样磷脂酶结构域包含3(PNPLA3)是一种在脂肪和肝脏等成脂组织中高表达的蛋白质，在促进脂肪储存的条件下高度上调。重要的是，PNPLA3的一种常见的基因变异I148M是发生FLD及其病理后遗症的最大已知风险因素。在最近发表的初步数据中，我们证明了α/β水解酶结构域包含蛋白5(ABHD5)是一种酶共激活因子，它与PNPLA3强烈相互作用，导致疾病的I148M变体是一种功能获得。此外，PNPLA3 I148M和ABHD5之间的相互作用在促进细胞标签保留方面特别有效，这可能在疾病进展中发挥核心作用。重要的是，ABHD5/PNPLA3的相互作用可以由内源脂肪酸和合成的ABHD5配体动态调节。这项工作将利用高分辨率成像技术、邻近蛋白质组学和代谢示踪物/脂质组学，结合强大的遗传模型和内源性脂肪酸配体和化学探针的整合小组，研究ABHD5如何调节脂肪细胞和肝细胞中PNPLA3和I148M变体的功能的基本机制。我们的具体目标是：1)用高分辨荧光和透射电子显微镜研究ABHD5与WT、PNPLA3和I148M相互作用的分子基础，以及ABHD5复合体在脂肪细胞和肝细胞中的亚细胞定位和动态转运。2)利用无偏邻近蛋白质组学和直接免疫沉淀技术确定ABHD5/PNPLA3和ABHD5/PNPLA3 I148M代谢物的位置和配体依赖的蛋白质组成。3)利用获得和丧失功能遗传学、选择性内源性和合成的ABHD5配体，结合同位素示踪剂和脂质组学，剖析ABHD5/PNPLA3和ABHD5/PNPLA3 I148M代谢物在脂肪细胞和肝细胞中的代谢功能。这些目标与NIH的使命非常一致，将在一个发现平台内实施，该平台将最大限度地整合不同水平的分析(分子、细胞器、细胞和组织)，以提供ABHD5/PNPLA3功能的可靠分析，从而提高我们对血脂水平调节的理解，并为肥胖相关疾病的治疗干预确定新的切入点。

Project Summary/Abstract The balance between triacylglycerol (TAG) storage and mobilization in adipose tissue and liver is critical to metabolic health as the dysregulation during obesity can produce ectopic accumulation of lipids in muscle and liver, resulting in the progression to diabetes and fatty liver disease (FLD). Thus, an important long-term scientific goal is to understand the tissue-specific molecular mechanisms that control TAG metabolism in order to discover novel therapies. Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) is a protein that is highly expressed in lipogenic tissues, like fat and liver, and is highly upregulated under conditions that promote fat storage. Importantly, a common genetic variant of PNPLA3, I148M, is the greatest known risk factor for developing FLD and its pathological sequelae. In recently published and preliminary data, we demonstrate that α/β hydrolase domain containing protein 5 (ABHD5), an enzyme co-activator, strongly interacts with PNPLA3 and the disease-causing I148M variant is a gain-of-function. Furthermore, the interaction between PNPLA3 I148M and ABHD5 is particularly effective in promoting cellular TAG retention, which likely plays a central role in disease progression. Importantly, the ABHD5/PNPLA3 interaction can be dynamically regulated by endogenous fatty acids and synthetic ABHD5 ligands. This work will examine the basic mechanism of how ABHD5 regulates the function of PNPLA3 and the I148M variant in adipocytes and hepatocytes using high resolution imaging techniques, proximity proteomics and metabolic tracers/lipidomics in conjunction with robust genetic models and an integrative panel of endogenous fatty acid ligands and chemical probes. Our Specific Aims are: 1) To determine the molecular basis for the interaction of ABHD5 with WT PNPLA3 and I148M and the subcellular location and dynamic trafficking of ABHD5 complexes in adipocytes and hepatocytes using high resolution fluorescence and transmission electron microscopy. 2) To determine the location and ligand-dependent protein composition of the ABHD5/PNPLA3 and ABHD5/ PNPLA3 I148M metabolons using nonbiased proximity proteomics and directed immunoprecipitation. 3) To dissect the metabolic function of the ABHD5/PNPLA3 and ABHD5/PNPLA3 I148M metabolons in adipocytes and hepatocytes using gain- and loss- of-function genetics and selective endogenous and synthetic ABHD5 ligands in conjunction with isotope tracers and lipidomics. These goals, which are well aligned with the mission of the NIH will be implemented within a discovery platform that maximizes integration across level of analysis (molecular, organelle, cells and tissues) to provide a robust analysis of ABHD5/PNPLA3 function, thereby improving our understanding of how lipids levels are regulated and identify novel points for therapeutic intervention in obesity related disorders.