Elucidating the Mechanisms of Lipid Droplet Protein Degradation

阐明脂滴蛋白质降解的机制

基本信息

  • 批准号:
    9760318
  • 负责人:
  • 金额:
    $ 4.05万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 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

项目摘要

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.
项目概要 全球日益流行的代谢性疾病是一个紧迫的公共卫生问题。随着流行 肥胖、胰岛素抵抗和非酒精性脂肪肝等疾病持续攀升,对 彻底了解细胞脂质储存机制变得越来越必要。最多 代谢紊乱涉及肝脏和心脏等组织中脂质的异常积累,导致 破坏性的系统健康影响。在细胞内,脂质储存在称为脂滴的胞质细胞器中 (LD),由由磷脂结合的三酰甘油和胆固醇酯组成的中性脂质核心组成 单层。与单层相关的是多种调节蛋白和酶,它们控制动态 脂质储备的封存和释放,使 LD 蛋白能够影响整个细胞的代谢 细胞。尽管 LD 蛋白在维持细胞脂质稳态方面具有重要作用,但人们对 LD 蛋白知之甚少。 LD 蛋白本身的调节——特别是控制 LD 蛋白丰度的途径。 尽管一些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, 所需泛素化机制的身份(例如 E3 泛素连接酶和 E2 泛素缀合酶) 酶)及其发挥控制作用的途径仍不清楚。为了解决这些基本问题 问题,我建议 1) 进行全基因组、基于荧光的 CRISPR/Cas9 筛选来识别降解 LD 蛋白 perilipin-2 (PLIN2) 的通路,以及 2) 后续研究,以探究 PLIN2 受损的程度 降解影响整体细胞代谢。我已经表征了表达 Cas9 的人类肝癌 荧光报告细胞系,其中内源性 PLIN2 带有 GFP 标记。流式细胞术、蛋白质印迹、 荧光显微镜分析证实 PLIN2-GFP 在内源水平表达,定位 为 LD,并被蛋白酶体降解。经过验证的 PLIN2-GFP 细胞系用于试点筛选 每个基因 10 个引导的单引导 RNA (sgRNA) 慢病毒子文库富含 UPS 基因。这个画面 确定了几个候选 UPS 因素,我假设这些因素与 PLIN2 降解有关。我的提议 研究包括完成全面的全基因组筛选、候选基因的验证、 PLIN2 降解途径的表征以及受损的功能后果的检查 PLIN2 间隙。这些研究将阐明 UPS 调节 LD 蛋白的机制,提供 关于细胞如何维持脂质稳态的新见解。了解 LD 蛋白降解途径将 有助于了解 LD 蛋白调节的改变如何促进代谢性疾病的发病机制 疾病。

项目成果

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Melissa Roberts其他文献

Melissa Roberts的其他文献

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{{ truncateString('Melissa Roberts', 18)}}的其他基金

Elucidating the Mechanisms of Lipid Droplet Protein Degradation
阐明脂滴蛋白质降解的机制
  • 批准号:
    9891849
  • 财政年份:
    2019
  • 资助金额:
    $ 4.05万
  • 项目类别:

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