Lipid droplets as protein sequestration sites

项目总结 脂滴是细胞内脂肪储存的场所，在脂类代谢和能量方面具有重要功能 储藏室。它们在控制特定蛋白质的命运方面也有第二个主要作用，即调节它们的 成熟、运输、重折叠、储存和周转。虽然在解开如何 液滴控制脂肪代谢，但对其处理蛋白质的作用的研究仍然有限。这个项目的目标是 是剖析这种蛋白质处理功能的机制、调节和生理相关性，通过采取 这一现象最典型的例子的优点是组蛋白H_2Av与脂质的隔离 果蝇卵巢和胚胎中的液滴。H_2Av是动态存储的，在脂质之间不断交换 液滴通过细胞质。脂滴隔离预防卵母细胞中H_2Av的降解及其早熟 输入到胚胎的细胞核中。它也受发育调节，在胚胎发育中期被关闭。 胚泡过渡。上一次批准期的进展表明Impa2和Ipo9是至关重要的 介导H_2Av交换的因素，揭示了细胞周期激酶CDK1的活性与 Impa2的磷酸化状态，以及H_2Av交换的速率，发现过量的核H_2Av 极大地改变了转录组。这些洞察导致了关于H_2Av机制的新模型 交易所、它的监管和它的生物学作用，是目前的应用程序建议测试的模型。它是 假设Impa2通过与Jabba的物理相互作用促进H_2Av交换 与H_2Av的亲和力和Ipo9作为细胞质伴侣，伴随H_2Av在 LDS。这一模型将通过确定这两个重要蛋白在细胞内的分布和动态来检验 通过分析关键功能区域的突变如何影响H_2Av交换。至 了解发育规律，Impa2中会产生点突变来阻止或模仿 磷酸化和CDK1活性将受到抑制。这些操纵对H_2Av的后果 重要蛋白、H2 Av锚定Jabba和H2 Av之间的交换和物理相互作用将是 下定决心。在缺乏JABBA的哺育细胞中，缺乏隔离会导致核H_2Av水平增加。 利用RNA序列分析，操纵H_2Av剂量，以及Jabba突变体无法结合H_2Av，就会 确定改变的H_2Av水平是否导致转录组的变化。如果成功，这些研究 将测试关于脂滴隔离H_2Av的机制、调节和功能的关键预测 并开发了通过脂滴处理蛋白质如何控制体内其他过程的通用范例 牢房。因此，这些研究将为确定蛋白质是否以及如何通过 脂滴有助于它们在健康和疾病中的突出作用。

Project summary Lipid droplets are the intracellular sites for fat storage, with essential functions in lipid metabolism and energy storage. They also have a second major role in controlling the fate of specific proteins, namely mediating their maturation, transport, refolding, storage, and turnover. While there has been great progress in unraveling how droplets control lipid metabolism, studies of their protein-handling roles remain limited. The goal of this project is to dissect the mechanism, regulation, and physiological relevance of this protein handling function, by taking advantage of the best characterized example of this phenomenon, the sequestration of histone H2Av to lipid droplets in Drosophila ovaries and embryos. H2Av is dynamically stored, exchanging constantly between lipid droplets via the cytoplasm. Lipid droplet sequestration prevents H2Av degradation in oocytes and its premature import into nuclei in embryos. It is also developmentally regulated, being switched off in embryos at the mid- blastula transition. Progress in the last granting period identified the importins Impa2 and Ipo9 as critical factors mediating H2Av exchange, uncovered a correlation between the activity of the cell cycle kinase Cdk1, the phosphorylation state of Impa2, and the rate of H2Av exchange, and discovered that excess nuclear H2Av dramatically alters the transcriptome. These insights led to new models about the mechanism of H2Av exchange, its regulation, and its biological role, models that the current application proposes to test. It is hypothesized that Impa2 promotes H2Av exchange by physically interacting with Jabba and reducing its affinity to H2Av and that Ipo9 acts as cytoplasmic chaperone, accompanying H2Av on its journey between LDs. This model will be tested by determining intracellular distribution and dynamics of the two importins in ovaries and embryos and by analyzing how mutants in key functional domains affect H2Av exchange. To understand the developmental regulation, point mutants in Impa2 will be generated to prevent or mimic phosphorylation and Cdk1 activity will be inhibited. The consequences of these manipulations for H2Av exchange and the physical interactions between importins, the H2Av anchor Jabba, and H2Av will be determined. In nurse cells lacking Jabba, absence of sequestration results in increased levels of nuclear H2Av. Using RNA seq analysis, manipulation of H2Av dosage, and Jabba mutants unable to bind H2Av, it will be determined whether the altered H2Av levels result in changes to the transcriptome. If successful, these studies will test key predictions about the mechanism, regulation, and function of H2Av sequestration by lipid droplets and develop general paradigms for how protein handling by lipid droplets can control processes elsewhere in the cell. These studies will thus provide the groundwork for determining if and how protein sequestration by lipid droplets contributes to their prominent roles in health and disease.