Lipid droplets as protein sequestration sites

描述(申请人提供)：脂滴是普遍存在的脂肪储存细胞器，在健康和疾病的脂质代谢中发挥核心作用。这些复杂的、动态的细胞器被认为与细胞功能有关，远远超出了脂质稳态。特别是，它们被认为是有毒或不稳定蛋白质的临时储存场所，大分子复合体的组装平台，或者作为细胞内运送蛋白质的载体。尽管脂滴显然在几种病毒的生命周期中扮演着重要的角色，并可能促进病毒的组装，但它们是否作为内源蛋白的瞬时蛋白隔离位置仍未解决。一长串来自其他细胞隔室的蛋白质被证明以一种受调控的方式重新定位到脂滴上，但这些蛋白质是否在液滴表面发挥新的功能，或者确实受到液滴结合的调节尚不清楚。对封存模型的关键测试对我们理解基本的细胞和发育过程以及管理由脂肪异常储存引起的人类疾病具有重要意义，包括脂肪营养不良和肥胖。这个项目的目标是解决这个问题，特别是 提出在果蝇早期胚胎中蛋白质隔离、组蛋白储存在脂滴上的挑衅性案例。在这个模型系统中，可以结合遗传学、生物化学和活体成像来解决这一普遍问题。在这些胚胎中，大量的某些组蛋白与脂滴短暂地联系在一起；这种联系是由一种新的蛋白质Jabba介导的，它可能是组蛋白在液滴上的锚。在没有Jabba的情况下，胚胎中的组蛋白水平会显著降低，当新的组蛋白合成受到轻微影响时，胚胎死亡，其表型表明缺乏足够的组蛋白供应。这些观察结果支持这样一种模型，即组蛋白存储在脂滴中，供以后在发育中使用。这个项目的目标是测试这个模型的中心预测。使用结构功能方法，将确定JABA介导液滴和组蛋白结合的结构域，并将确定液滴上组蛋白的物理状态。这些信息将被用来产生缺乏特定相互作用或错误定位到其他细胞室的Jabba突变体；这些突变体将被测试其拯救高组蛋白水平和正常发育的能力。活体成像和液滴移植将研究组蛋白从脂滴转移到细胞核的速度，以及Jabba是否保持稳定。最后，组蛋白的过度表达将被用来解决液滴是否正常缓冲组蛋白供应和防止组蛋白过量的问题。如果成功，拟议的研究将为组蛋白新陈代谢和脂滴功能提供新的见解。最重要的是，这些研究将为来自其他隔室的蛋白质如何以及为什么短暂地隔离在脂滴上提供一个范例。

DESCRIPTION (provided by applicant): Lipid droplets are ubiquitous fat storage organelles and play central roles in lipid metabolism in both health and disease. These complex, dynamic organelles have been implicated in cellular functions well beyond lipid homeostasis. In particular, they have been proposed to serve as temporary storage sites for toxic or unstable proteins, as assembly platforms for macromolecular complexes, or as vehicles to deliver proteins intracellularly. Although lipid droplets clearly play an important role in the life cycle f several viruses and likely promote viral assembly, whether they act as transient protein sequestration site for endogenous proteins remains unresolved. A long list of proteins from other cellular compartments has been shown to relocate to lipid droplets in a regulated manner, but whether these proteins perform novel functions at the droplet surface or are indeed regulated by droplet association is unclear. A critical test of the sequestration model has important implications for our understanding of basic cellular and developmental processes as well as for the management of human diseases resulting from aberrant fat storage, including lipodystrophies and obesity. The goal of this project is to resolve this issue for one particularly provocative case of proposed protein sequestration, histone storage on lipid droplets in early Drosophila embryos. In this model system, it is possible to combine genetics, biochemistry and live imaging to address this general question. In these embryos, massive amounts of certain histones associate transiently with lipid droplets; this association is mediated by the novel protein Jabba, the putative histone anchor on droplets. In the absence of Jabba, histone levels in embryos are dramatically reduced and - when new histone synthesis is mildly compromised - embryos die with phenotypes indicating lack of sufficient histone supplies. These observations support a model that histones are stored on lipid droplets for later use in development. The goal of this project is to test central predictions of this model. Using a structure-function approach, the domains of Jabba that mediate droplet and histone binding will be identified and the physical state of histones on the droplets will be determined. This information will be employed to generate Jabba mutants lacking specific interactions or mislocalized to other cellular compartments; these mutants will be tested for their ability to rescue high histone levels and normal development. Live imaging and droplet transplantation will address how quickly histones are transferred from lipid droplets to nuclei and whether Jabba remains stably behind. Finally, histone overexpression will be employed to address if droplets normally buffer the histone supply and protect against histone overabundance. If successful, the proposed studies will provide novel insights into both histone metabolism and lipid-droplet function. Most importantly, these studies will provide a paradigm for how and why proteins from other compartments are transiently sequestered on lipid droplets.