Lipid droplet regulation and proteome dynamics

脂滴调控和蛋白质组动力学

• 批准号: 10662522
• 负责人: JAMES A OLZMANN
• 金额: $ 31.16万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10662522
• 关键词: Anabolism; Antibacterial Response; Bacterial Infections; Biogenesis; Biology; CRISPR screen; Cardiovascular Diseases; Cell Line; Cell physiology; Cells; Cellular biology; Communities; Custom; Data; Degradation Pathway; Diabetes Mellitus; Disease; Endoplasmic Reticulum; Etiology; Exhibits; Family member; Fatty Acids; Fatty acid glycerol esters; Fluorescence; Foundations; Genes; Genetic Screening; Goals; Health; Heart Diseases; Hepatic; Hepatocyte; Histones; Homeostasis; Human; Hydrophobicity; Immune response; Knowledge; Label; Libraries; Life Cycle Stages; Lipids; Lipolysis; Lipopolysaccharides; Malignant Neoplasms; Mediating; Metabolic; Metabolic Diseases; Metabolism; Nerve Degeneration; Neurons; Nonesterified Fatty Acids; Obesity; Organelles; Pathologic; Pathway interactions; Peripheral; Pharmaceutical Preparations; Phenotype; Phospholipids; Play; Proliferating; Proteins; Proteome; Proteomics; Regulation; Reporter; Research; Resources; Role; Series; Surface; Testing; Therapeutic; Triglycerides; Ubiquitin; Ubiquitination; Validation; Viral; cell type; experimental study; genome-wide; genomic locus; induced pluripotent stem cell; innovative technologies; insight; interest; lipid metabolism; monolayer; multicatalytic endopeptidase complex; non-alcoholic fatty liver disease; novel; pathogen; pathogenic bacteria; perilipin; prevent; programs; protein degradation; repository; response; sterol ester; technology platform; transcription factor; ubiquitin-protein ligase

PROJECT SUMMARY / ABSTRACT Lipid droplets (LDs) are neutral lipid storage organelles that act as cellular hubs of lipid homeostasis. Dysregulation in LD function has been implicated in prevalent metabolic diseases such as obesity, diabetes, cardiovascular disease, and non-alcoholic fatty liver disease (NAFLD). Indeed, the pathological hallmark of NAFLD is the accumulation of large hepatic LDs. In addition to metabolic diseases, LDs have also been implicated in cancer proliferation and survival, host-pathogen interactions, and neurodegeneration. Thus, understanding how LDs are regulated has the potential to broadly impact our understanding of human health and disease. LDs are ER-derived organelles that have a unique ultrastructure, consisting of a core of neutral lipid surrounded by a phospholipid monolayer decorated with integral and peripheral proteins. While recent findings have advanced our understanding of LD biogenesis, how LDs are regulated under different metabolic conditions and how the composition of the LD proteome remain poorly understood. To overcome these critical gaps in knowledge and define the mechanisms that regulate neutral lipid storage, we performed a series of CRISPR-Cas9 screen in human cells using a fluorescence-based neutral lipid reporter under different metabolic conditions. We also employed genetic screens to examine the mechanisms that regulate PLIN2, a near ubiquitous Class II LD protein that plays important roles in regulating LD stability. Our findings establish a compendium of neutral lipid storage regulators, revealing interesting novel regulators that are condition specific. Furthermore, we identify several ubiquitination factors that influence neutral lipid storage and the stability of PLIN2. The current proposal aims to build on the foundation provided by our extensive preliminary data to characterize new mechanisms of LD regulation. In aim 1, we will complete our validation experiments to establish an extensive, phenotypic-rich resource for the community that is hypothesis generating. We will also examine the concept that metabolic state-dependent regulation of LDs is a significant contributor to cellular lipid homeostasis. Finally, we will characterize high priority candidates in iPSC-derived hepatocytes and examine the hypothesis that a subset of regulators governs LD stability as part of a host response to pathogens. In Aim 2, we will define the role of new ubiquitination pathways in regulating lipid homeostasis, examining the hypothesis that the identified factors regulate LD stability by controlling the degradation of PLIN2 during lipolysis. These findings will provide new global and mechanistic insights in to LD proteome remodeling and regulation under different metabolic conditions.

项目概要/摘要 脂滴（LD）是中性脂质储存细胞器，充当脂质稳态的细胞中心。 LD 功能失调与肥胖、糖尿病、 心血管疾病和非酒精性脂肪肝疾病（NAFLD）。事实上，病理学特征 NAFLD 是大量肝脏 LD 的积累。除了代谢性疾病外，LD 还被 与癌症增殖和存活、宿主-病原体相互作用和神经变性有关。因此， 了解 LD 的监管方式有可能广泛影响我们对人类健康的理解 和疾病。 LD 是 ER 衍生的细胞器，具有独特的超微结构，由中性核心组成 脂质被磷脂单层包围，磷脂单层装饰有整体蛋白和外周蛋白。虽然最近 研究结果加深了我们对 LD 生物发生、LD 在不同代谢条件下的调控的理解。 LD 蛋白质组的条件和组成仍然知之甚少。为了克服这些关键 由于知识差距和定义调节中性脂质储存的机制，我们进行了一系列 使用基于荧光的中性脂质报告基因在不同代谢下对人体细胞进行 CRISPR-Cas9 筛选 状况。我们还采用遗传筛选来检查调节 PLIN2 的机制，PLIN2 是一种近 普遍存在的 II 类 LD 蛋白，在调节 LD 稳定性中发挥重要作用。我们的研究结果建立了 中性脂质储存调节剂纲要，揭示了针对特定条件的有趣的新型调节剂。 此外，我们还确定了几个影响中性脂质储存和稳定性的泛素化因子。 PLIN2。当前的提案旨在建立在我们广泛的初步数据提供的基础上 描述 LD 调节的新机制。在目标 1 中，我们将完成验证实验以确定 为假设生成的社区提供广泛、表型丰富的资源。我们还将检查 LD 的代谢状态依赖性调节是细胞脂质的重要贡献者的概念 体内平衡。最后，我们将表征 iPSC 衍生肝细胞中的高优先级候选者，并检查 假设一部分调节因子控制 LD 稳定性，作为宿主对病原体反应的一部分。在目标 2 中，我们 将定义新的泛素化途径在调节脂质稳态中的作用，检验以下假设： 确定的因素通过控制脂解过程中 PLIN2 的降解来调节 LD 稳定性。这些发现 将为不同条件下的 LD 蛋白质组重塑和调控提供新的全局和机制见解 代谢条件。