Elucidating the Mechanisms of Lipid Droplet Protein Degradation

项目摘要 日益增长的全球流行的代谢性疾病是一个紧迫的公共卫生问题。普遍存在 肥胖、胰岛素抵抗和非酒精性脂肪肝等疾病的发病率继续攀升， 对细胞脂质储存机制的透彻理解已变得越来越迫切。最 代谢紊乱涉及脂质在组织如肝脏和心脏中的异常积累，导致 破坏性的全身健康影响。在细胞内，脂质储存在称为脂滴的胞质细胞器中 (LDs)，其由被磷脂结合的三酰基甘油和胆固醇酯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，它们控制着细胞的动力学。 隔离和释放的脂质储备，使LD蛋白质影响整个代谢 cell.虽然LD蛋白在维持细胞脂质稳态中具有重要作用，但对LD蛋白的功能和作用机制知之甚少。 LD蛋白本身的调节-特别是控制LD蛋白丰度的途径。 虽然一些研究报道了泛素-蛋白酶体系统（UPS）在调节LD蛋白水平中的作用， 所需的泛素化机制（例如E3泛素连接酶和E2泛素缀合酶）的特性 酶）和它们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）全基因组，基于荧光的CRISPR/Cas9筛选，以确定降解 LD蛋白周脂蛋白-2（PLIN 2）通路，以及2）后续研究，以询问受损的PLIN 2 降解会影响整体细胞代谢。我已经表征了表达Cas9的人肝癌 荧光报告细胞系，其中内源PLIN 2用GFP标记。流式细胞术，蛋白质印迹， 和荧光显微镜分析证实PLIN 2-GFP在内源水平表达，定位于 转化为LD，并被蛋白酶体降解。验证的PLIN 2-GFP细胞系用于中试筛选 在UPS基因中富集的单向导RNA（sgRNA）的每基因10个向导的慢病毒亚文库中。此屏幕 确定了几个候选UPS因素，我假设参与PLIN 2降解。我建议的 研究包括完成全面的全基因组筛选，验证候选基因， PLIN 2降解途径的表征，以及对受损的PLIN 2降解途径的功能后果的检查。 PLIN 2清除。这些研究将阐明UPS对LD蛋白的调控机制，为进一步研究LD蛋白的功能提供理论依据。 对细胞如何维持脂质稳态的新见解。LD蛋白降解途径的知识将 允许了解LD蛋白调节的改变如何有助于代谢性疾病的发病机制。 疾病

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.