Lipid droplet regulation and proteome dynamics

项目总结/摘要 脂滴是一种中性的脂质储存细胞器，是细胞内脂质平衡的枢纽。 LD功能的失调与普遍的代谢疾病如肥胖、糖尿病， 心血管疾病和非酒精性脂肪肝（NAFLD）。事实上， NAFLD是大的肝脏LD的积聚。除了代谢性疾病，LD也被 与癌症增殖和存活、宿主-病原体相互作用和神经变性有关。因此，在本发明中， 了解LD是如何调节的，有可能广泛影响我们对人类健康的理解。 和疾病LD是ER衍生的细胞器，具有独特的超微结构，由中性的 由磷脂单层包围的脂质，磷脂单层由整体和外周蛋白质装饰。虽然最近 这些发现推进了我们对LD生物发生的理解，即LD在不同代谢条件下如何调节， 条件以及LD蛋白质组的组成仍然知之甚少。为了克服这些关键 知识的空白，并确定调节中性脂质储存的机制，我们进行了一系列的， 在不同代谢条件下使用基于荧光的中性脂质报告基因在人类细胞中进行CRISPR-Cas9筛选 条件我们还采用了遗传筛选来检查调节PLIN 2的机制，PLIN 2是一种近 普遍存在的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 中性脂质储存调节剂的纲要，揭示了有趣的新的调节剂，是条件特异性。 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子， PLIN 2.目前的建议旨在根据我们广泛的初步数据提供的基础， 描述LD调节的新机制。在aim 1中，我们将完成验证实验， 一个广泛的，表型丰富的资源，为社区的假设产生。我们亦会研究 LD的代谢状态依赖性调节是细胞脂质的重要贡献者， 体内平衡最后，我们将在iPSC衍生的肝细胞中表征高优先级候选物，并检查 这是一个假设，即一个子集的监管机构管理LD稳定性的一部分，主机响应病原体。在目标2中， 将定义新的泛素化途径在调节脂质体内平衡中的作用，检验以下假设： 所鉴定的因子通过在脂解过程中控制PLIN 2的降解来调节LD稳定性。这些发现 将提供新的全球和机制的见解，以LD蛋白质组重塑和调节下，不同的 代谢条件。

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.