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

项目总结/摘要 脂肪组织和肝脏中三酰甘油（TAG）储存和动员之间的平衡对代谢健康至关重要，因为肥胖期间的失调可导致肌肉和肝脏中脂质的异位积累，导致糖尿病和脂肪肝疾病（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，以发现新的治疗方法。Patatin-Like Phospholipase Domain Containing 3（PNPLA 3）是一种在脂肪生成组织（如脂肪和肝脏）中高度表达的蛋白质，并且在促进脂肪储存的条件下高度上调。重要的是，PNPLA 3的常见遗传变异体I148 M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水解酶结构域蛋白5（ABHD 5），一种酶共激活剂，与PNPLA 3强烈相互作用，致病I148 M变体是一种功能获得性变体。此外，PNPLA 3 I148 M和ABHD 5之间的相互作用在促进细胞TAG保留方面特别有效，这可能在疾病进展中起核心作用。重要的是，ABHD 5/PNPLA 3相互作用可以通过内源性脂肪酸和合成ABHD 5配体动态调节。这项工作将研究ABHD 5如何调节脂肪细胞和肝细胞中PNPLA 3和I148 M变体的功能的基本机制，使用高分辨率成像技术，邻近蛋白质组学和代谢示踪剂/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综合面板。我们的具体目标是：1）使用高分辨率荧光和透射电子显微镜确定ABHD 5与WT PNPLA 3和I148 M相互作用的分子基础以及ABHD 5复合物在脂肪细胞和肝细胞中的亚细胞定位和动态运输。2)使用无偏邻近蛋白质组学和定向免疫沉淀法确定ABHD 5/PNPLA 3和ABHD 5/PNPLA 3 I148 M代谢子的位置和配体依赖性蛋白质组成。3)使用功能获得和丧失遗传学以及选择性内源性和合成ABHD 5配体结合同位素示踪剂和脂质组学，剖析脂肪细胞和肝细胞中ABHD 5/PNPLA 3和ABHD 5/PNPLA 3 I148 M代谢子的代谢功能。这些目标与NIH的使命一致，将在一个发现平台内实现，该平台最大限度地整合了分析水平（分子、细胞器、细胞和组织），以提供ABHD 5/PNPLA 3功能的稳健分析，从而提高我们对脂质水平如何调节的理解，并确定肥胖相关疾病治疗干预的新点。

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.