Lipid droplets as sites for protein sequestration

描述（由申请人提供）：脂滴是细胞内细胞器，最为人所知的是其在脂质储存和肥胖中的作用。这项建议的目标是确定脂滴是否作为发育调节蛋白质螯合的位点提供一般的新功能。在果蝇胚胎中，母体提供的组蛋白似乎储存在液滴中，直到发育后期需要。文献中的各种暗示表明，受调节的螯合-不仅是组蛋白，而且是其他蛋白质-脂滴广泛分布在从酵母到哺乳动物的真核生物中。这种隔离可以通过以时间或空间控制的方式释放或灭活蛋白质来提供新的发育机制。它还可以提供细胞保护自己免受有害蛋白质侵害的一般手段。然而，无论是普遍性还是固碳机制都尚未确立，因此固碳的功能和重要性尚未得到解决。为了确定组蛋白以外的蛋白质是否被隔离在果蝇胚胎中，将采用两种策略：将确定候选蛋白输入蛋白α 2是否以发育调节的方式被隔离到液滴中（通过免疫定位和纯化组分的Western分析确定其细胞内分布）以及蛋白毒性应激是否诱导特异性蛋白的螯合（通过分离液滴的蛋白质组学分析）。为了揭示隔离的机制，将确定隔离的组蛋白和输入蛋白α 2是否携带签名翻译后修饰以及组蛋白结合液滴上的哪些蛋白质。这些研究将确定现有的突变体，破坏螯合或建议策略如何产生这样的突变体，从而导致螯合的生物作用的功能测试，无论是立即或在不久的将来。

DESCRIPTION (provided by applicant): Lipid droplets are intracellular organelles best known for their role in lipid storage and obesity. The goal of this proposal is to determine whether lipid droplets serve a general new function as sites of developmentally regulated protein sequestration. In Drosophila embryos, maternally provided histones appear to be stored on droplets until needed later in development. Various hints in the literature suggest that regulated sequestration - not only of histones but also of other proteins - to lipid droplets is wide spread in eukaryotes from yeast to mammals. Such sequestration could provide a novel mechanism of development, by releasing or inactivating proteins in a temporally or spatially controlled manner. It may also provide a general means by which cells protect themselves from harmful proteins. However, neither the generality nor the mechanisms of sequestration have been established, leaving the function and importance of sequestration unresolved. To determine whether proteins other than histones are sequestered in Drosophila embryos, two strategies will be pursued: it will be determined whether the candidate protein Importin alpha2 is sequestered to droplets in a developmentally regulated manner (by determining its intracellular distribution via immunolocalization and Western analysis of purified fractions) and whether proteotoxic stress induces sequestration of specific proteins (by proteomic analysis of isolated droplets). To uncover mechanisms of sequestration, it will be determined whether sequestered histones and Importin alpha2 carry signature post-translational modifications and which proteins on droplets the histones bind to. These studies will either identify existing mutants that disrupt sequestration or suggest strategies how to generate such mutants, thus leading to a functional test of the biological roles of sequestration, either immediately or in the near future.