Lipid droplet regulation and proteome dynamics

项目摘要/摘要 脂滴是中性的脂类储存细胞器，是脂类动态平衡的细胞中枢。 LD功能失调与常见的代谢性疾病，如肥胖、糖尿病、 心血管疾病和非酒精性脂肪肝(NAFLD)。事实上，该病的病理特征是 NAFLD是大的肝脏LDs的积聚。除了代谢性疾病外，LDS还 与癌症的增殖和存活、宿主与病原体的相互作用和神经退化有关。因此， 了解LDS是如何受到监管的，可能会广泛影响我们对人类健康的理解 和疾病。LDS是内质网衍生的细胞器，具有独特的超微结构，由中性的核心组成 被磷脂单分子层包围的脂类，磷脂单分子层装饰着完整的和外围的蛋白质。虽然是最近的 这些发现加深了我们对LD生物发生的理解，即LD在不同代谢下是如何调节的 条件和LD蛋白质组的组成仍然知之甚少。要克服这些关键问题 知识差距和定义调节中性脂肪储存的机制，我们进行了一系列 不同代谢条件下人细胞CRISPR-Cas9荧光中性脂报告筛选 条件。我们还利用基因筛查来检查调节PLIN2的机制，PLIN2是一种近乎 普遍存在的II类LD蛋白，在调节LD稳定性方面发挥重要作用。我们的发现建立了一个 中性脂肪储存调节剂概要，揭示了有趣的新调节器，这些调节剂是特定条件的。 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.目前的建议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建立 为正在生成假设的社区提供广泛的、表型丰富的资源。我们还将检查 LDS的代谢状态依赖性调节是细胞脂质的重要贡献者 动态平衡。最后，我们将鉴定IPSC来源的肝细胞中的高优先级候选细胞，并检查 一种假说，即作为宿主对病原体反应的一部分，调节因子的子集管理LD的稳定性。在目标2中，我们 将定义新的泛素化途径在调节脂质动态平衡中的作用，检验以下假设 识别的因素通过控制PLIN2在脂解过程中的降解来调节LD的稳定性。这些发现 将为不同条件下LD蛋白质组的重塑和调控提供新的全局和机械性见解 新陈代谢状况。

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.