Lipid droplet regulation and proteome dynamics

脂滴调控和蛋白质组动力学

• 批准号: 10365414
• 负责人: JAMES A OLZMANN
• 金额: $ 32.29万
• 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/10365414
• 关键词: Anabolism; Antibacterial Response; 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; Homeostasis; Human; Hydrophobicity; Immune response; Knowledge; Label; Libraries; Life; Lipids; Lipolysis; Lipopolysaccharides; Malignant Neoplasms; Mediating; Metabolic; Metabolic Diseases; Metabolism; Nerve Degeneration; Obesity; Organelles; Pathologic; Pathway interactions; Peripheral; Phenotype; Phospholipids; Play; Proteins; Proteome; Regulation; Reporter; Research; Resources; Role; Series; Surface; Testing; Therapeutic; Triglycerides; Ubiquitin; Ubiquitination; Validation; Viral; base; cell type; experimental study; genome-wide; genomic locus; induced pluripotent stem cell; innovation; insight; interest; lipid metabolism; monolayer; non-alcoholic fatty liver disease; novel; pathogen; pathogenic bacteria; perilipin; prevent; protein degradation; repository; response; sterol ester; 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.

项目摘要 /摘要 脂质液滴（LDS）是中性脂质储存细胞器，充当脂质稳态的细胞中心。 LD功能的失调与肥胖症，糖尿病，诸如肥胖症，糖尿病等流行的代谢疾病有关 心血管疾病和非酒精性脂肪肝病（NAFLD）。确实，是病理性的标志 NAFLD是大型肝LDS的积累。除代谢性疾病外，LDS也是 与癌症的增殖和生存，宿主 - 病原体相互作用和神经退行性有关。因此， 了解如何调节LD的潜力有可能广泛影响我们对人类健康的理解 和疾病。 LDS是具有独特超微结构的ER衍生的细胞器，由中性的核心组成 脂质被磷脂单层包围，并装饰有整体和外周蛋白。最近 发现已经提出了我们对LD生物发生的理解，如何在不同的代谢下调节LD 条件以及LD蛋白质组的组成如何保持不足。克服这些关键 知识的差距并定义了调节中性脂质存储的机制，我们进行了一系列 在人类细胞中使用基于荧光的中性脂质报告剂在不同代谢下的CRISPR-Cas9屏幕 状况。我们还采用了遗传筛选来检查调节PLIN2的机制，即接近 无处不在的II类LD蛋白在调节LD稳定性中起重要作用。我们的发现建立了一个 中性脂质存储调节剂的汇编，揭示了特定于条件的有趣的新型调节剂。 此外，我们确定了几个影响中性脂质储存和稳定性的泛素化因素 PLIN2。当前的建议旨在基于我们广泛的初步数据提供的基础 表征LD调节的新机制。在AIM 1中，我们将完成验证实验以建立 对于社区而言，一种广泛的，表型丰富的资源是假设产生的。我们还将检查 代谢状态依赖性LDS的概念是细胞脂质的重要原因 稳态。最后，我们将表征IPSC衍生的肝细胞中的高优先级候选者，并检查 假设调节剂的子集控制LD稳定性，这是宿主对病原体反应的一部分。在AIM 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，从而研究假设 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 将提供新的全球和机械见解，以在不同 代谢条件。