Lipid droplets as sites for protein sequestration

描述（由申请人提供）：脂滴是细胞内细胞器，以其在脂质储存和肥胖中的作用而闻名。本提案的目的是确定脂滴是否作为发育调节的蛋白质隔离位点具有一般的新功能。在果蝇胚胎中，母体提供的组蛋白似乎储存在液滴上，直到发育后期才需要。文献中的各种暗示表明，在从酵母到哺乳动物的真核生物中，对脂滴的调节封存——不仅是组蛋白，还有其他蛋白质——广泛存在。这种隔离可以提供一种新的发育机制，通过时间或空间控制的方式释放或灭活蛋白质。它还可能提供一种通用的方法，使细胞保护自己免受有害蛋白质的侵害。然而，封存的普遍性和机制尚未确立，封存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是否在果蝇胚胎中被隔离，将采用两种策略：将确定候选蛋白Importin alpha2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），以及蛋白质毒性应激是否诱导特异性蛋白的隔离（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是否携带标志性的翻译后修饰，以及组蛋白与液滴上的哪些蛋白质结合。这些研究将要么确定现有的破坏封存的突变体，要么提出如何产生这种突变体的策略，从而立即或在不久的将来对封存的生物学作用进行功能测试。

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.