Elucidating the Mechanisms of Lipid Droplet Protein Degradation

阐明脂滴蛋白质降解的机制

• 批准号: 9891849
• 负责人: Melissa Roberts
• 金额: $ 4.1万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9891849
• 关键词: Address; Affect; Bacteria; Biochemistry; Biogenesis; Biological Assay; CRISPR screen; Candidate Disease Gene; Cardiovascular Diseases; Cell Line; Cells; Cellular biology; Cholesterol Esters; Complement; Defect; Degradation Pathway; Development; Disease; Endoplasmic Reticulum; Enzymes; Epidemic; Exhibits; Fatty Acids; Fatty Liver; Fibrinogen; Flow Cytometry; Fluorescence; Fluorescence Microscopy; Follow-Up Studies; Genes; Genetic; Genus Hippocampus; Goals; Green Fluorescent Proteins; Guide RNA; Health; Heart; Homeostasis; Human; Hydrophobicity; Impairment; Individual; Insulin Resistance; Knock-out; Knowledge; Lipids; Lipolysis; Liver; Malignant Epithelial Cell; Measures; Mediating; Metabolic; Metabolic Diseases; Metabolism; Molecular; Obesity; Organelles; Pathogenesis; Pathology; Pathway interactions; Phospholipids; Plants; Play; Prevalence; Primary carcinoma of the liver cells; Process; Proteins; Proteome; Public Health; Regulation; Reporter; Reporting; Research; Role; System; Tissues; Triglycerides; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitination; Western Blotting; base; candidate validation; energy balance; enzyme pathway; functional genomics; genetic regulatory protein; genome wide screen; genome-wide; insight; lipid metabolism; monolayer; multicatalytic endopeptidase complex; non-alcoholic fatty liver; novel; novel therapeutics; overexpression; oxidation; perilipin; protein degradation; response; screening; ubiquitin-protein ligase

PROJECT SUMMARY The growing global epidemic of metabolic disease is a pressing public health issue. As the prevalence of disorders such as obesity, insulin resistance, and nonalcoholic fatty liver continue to climb, the need for a thorough understanding of cellular lipid storage mechanisms has become increasingly imperative. Most metabolic disorders involve the aberrant accumulation of lipid in tissues such as the liver and heart, leading to devastating systemic health effects. Within cells, lipids are stored in cytosolic organelles called lipid droplets (LDs), which consist of a neutral lipid core of triacylglycerols and cholesterol esters bounded by a phospholipid monolayer. Associated with the monolayer are multiple regulatory proteins and enzymes that control the dynamic sequestration and release of the lipid reserves, allowing LD proteins to influence the metabolism of the entire cell. Although LD proteins have essential roles in maintaining cellular lipid homeostasis, little is known regarding the regulation of LD proteins themselves – in particular, the pathways that control LD protein abundance. Although several studies report a role for the ubiquitin-proteasome system (UPS) in modulating LD protein levels, the identities of the required ubiquitination machinery (e.g. E3 ubiquitin ligases and E2 ubiquitin conjugating enzymes) and the pathways by which they exert control remain unclear. To address these fundamental questions, I propose 1) a genome-wide, fluorescence-based CRISPR/Cas9 screen to identify the degradation pathway of the LD protein perilipin-2 (PLIN2), and 2) follow-up studies to interrogate how impaired PLIN2 degradation impacts global cellular metabolism. I have characterized a human hepatoma Cas9-expressing fluorescent reporter cell line in which endogenous PLIN2 is tagged with GFP. Flow cytometry, Western blotting, and fluorescence microscopy analyses confirmed that PLIN2-GFP is expressed at endogenous levels, localizes to LDs, and is degraded by the proteasome. The validated PLIN2-GFP cell line was employed in a pilot screen of a 10-guide-per-gene lentiviral sublibrary of single guide RNAs (sgRNAs) enriched in UPS genes. This screen identified several candidate UPS factors that I hypothesize are involved in PLIN2 degradation. My proposed studies include the completion of a comprehensive, genome-wide screen, validation of candidate genes, characterization of PLIN2 degradation pathways, and examination of the functional consequences of impaired PLIN2 clearance. These studies will elucidate the mechanisms of LD protein regulation by the UPS, providing novel insights into how cells maintain lipid homeostasis. Knowledge of LD protein degradation pathways will allow for an understanding of how alterations in LD protein regulation contribute to the pathogenesis of metabolic disease.

项目摘要 代谢疾病的全球流行病日益严重，这是一个紧迫的公共卫生问题。作为流行 肥胖，胰岛素抵抗和非酒精性脂肪肝等疾病继续攀升，需要 对细胞脂质储存机制的透彻了解变得越来越急切。最多 代谢性疾病涉及脂肪在肝脏和心脏等组织中的异常积累，导致 毁灭性的系统性健康影响。在细胞内，脂质存储在称为脂质液滴的胞质细胞器中 （LDS），由三酰甘油的中性脂质核和由磷脂界定的胆固醇酯和胆固醇酯组成 单层。与单层相关的是多种调节蛋白和控制动态的酶 脂质储量的固相和释放，从而使LD蛋白能够影响整个的代谢 尽管LD蛋白在维持细胞脂质体内稳态方面具有重要作用，但关于 LD蛋白本身的调节 - 特别是控制LD蛋白丰度的途径。 尽管几项研究报告了泛素 - 蛋白酶体系统（UPS）在调节LD蛋白水平中的作用，但 所需的泛素化机制的身份（例如E3泛素连接酶和E2泛素结合 酶）及其施加控制的途径尚不清楚。解决这些基本 问题，我建议1）全基因组，基于荧光的CRISPR/CAS9屏幕以识别降解 LD蛋白Peripin-2（PLIN2）的途径和2）随访研究，以询问PLIN2如何受损 降解会影响全球细胞代谢。我已经表征了人类肝癌的表达 内源性PLIN2用GFP标记的荧光报告细胞系。流式细胞仪，蛋白质印迹， 荧光显微镜分析证实，PLIN2-GFP在内源水平上表达，本地化 到LDS，并被蛋白酶体降解。经过验证的PLIN2-GFP细胞系在飞行员屏幕上进行 富含UPS基因的单个指南RNA（SGRNA）的每条慢病毒s蓝曲线的10指导性sublibrary。这个屏幕 确定了我假设的几个候选UPS因素与PLIN2降解有关。我提出的 研究包括全面，全基因组筛查的完整性，验证候选基因的验证， PLIN2降解途径的表征以及检查受损的功能后果 PLIN2间隙。这些研究将通过UPS阐明LD蛋白调节的机制，提供 关于细胞如何保持脂质稳态的新颖见解。 LD蛋白降解途径的知识将 允许了解LD蛋白调节中的改变如何有助于代谢的发病机理 疾病。