Lipid droplets as protein sequestration sites

脂滴作为蛋白质隔离位点

基本信息

批准号：
10653248
负责人：
MICHAEL Andreas WELTE
金额：
$ 38.27万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-04-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10653248
关键词：
Affect Affinity Binding Biological CDC2 gene Cell Cycle Cell Nucleus Cell physiology Cells Chromatin Chromatin Modeling Cytoplasm Cytosol Data Defect Development Disease Drosophila genus Embryo Embryonic Development Fatty acid glycerol esters Fertilization Future Gene Expression Regulation Generations Genetic Transcription Goals Grant Health Health Promotion Histone H2A Histones Homeostasis Importins In Vitro Laboratories Life Cycle Stages Lipids Measures Mediating Membrane Metabolism Modeling Molecular Chaperones Nuclear Nuclear Import Nuclear Proteins Nurses Oocytes Oogenesis Organelles Ovary Phosphorylation Physiological Play Process Protein Dephosphorylation Protein Dynamics Proteins Quantitative Reverse Transcriptase PCR Regulation Role Signal Transduction Site Testing Time Transcript Viral Viral Proteins Work blastocyst dosage human disease in vivo insight lipid metabolism mutant premature prevent recruit transcription factor transcriptome transcriptome sequencing

项目摘要

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.

项目摘要脂质液滴是脂肪储存的细胞内部位，具有脂质代谢和能量的重要功能贮存。他们在控制特定蛋白质的命运方面也有第二个主要作用，即介导其成熟，运输，重新折叠，存储和营业额。虽然在解散如何取得了进步液滴控制脂质代谢，对其蛋白质处理作用的研究仍然有限。这个项目的目标是通过服用该蛋白质处理功能的机制，调节和生理相关性这种现象的最佳特征示例的优势，组蛋白H2AV固结至脂质果蝇卵巢和胚胎中的液滴。 H2AV是动态存储的，在脂质之间不断交换通过细胞质的液滴。脂质液滴固换可防止卵母细胞中的H2AV降解及其过早进口到胚胎中的核。它也受到发展的调节，在中间的胚胎中关闭囊泡过渡。在最后一个授予期内的进展将Importins Impa2和IPO9确定为关键介导H2AV交换的因素，发现了细胞周期激酶CDK1的活性之间的相关性， Impa2的磷酸化状态和H2AV交换的速率，发现过多的核H2AV 极大地改变了转录组。这些见解导致有关H2AV机制的新模型交换，调节及其生物学作用，当前应用建议测试。这是假设Impa2通过与Jabba进行物理相互作用并减少了H2AV交换与H2AV的亲和力和IPO9充当细胞质伴侣，并伴随着H2AV的旅程 LDS。该模型将通过确定两个进口蛋白的细胞内分布和动力学来测试卵巢和胚胎以及通过分析关键功能域中的突变体如何影响H2AV交换。到了解发育法规，将产生Impa2中的点突变体以预防或模仿磷酸化和CDK1活性将受到抑制。这些操纵对H2AV的后果 Expertins，H2AV锚Jabba和H2AV之间的交换和物理相互作用将是决定。在缺乏jabba的护士细胞中，缺乏隔离会导致核H2AV水平升高。使用RNA SEQ分析，H2AV剂量的操纵和无法结合H2AV的JABBA突变体，它将是确定改变的H2AV水平是否导致转录组发生变化。如果成功，这些研究将测试有关脂质液滴对H2AV隔离的机制，调节和功能的关键预测并开发一般的范式，用于如何控制脂质液滴的蛋白质处理能够控制其他地方的过程细胞。因此，这些研究将为确定是否以及如何通过脂质液滴在健康和疾病中起着重要作用。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos.

DOI：
10.3791/63291
发表时间：
2021-12-13
期刊：
Journal of visualized experiments : JoVE
影响因子：
0
作者：
Kilwein MD;Welte MA
通讯作者：
Welte MA

Visualizing Lipid Droplets in Drosophila Oogenesis.

果蝇卵子发生过程中脂滴的可视化。

DOI：
10.1007/978-1-0716-2970-3_12
发表时间：
2023
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
0
作者：
White,RogerP;Welte,MichaelA
通讯作者：
Welte,MichaelA

Sequestration to lipid droplets promotes histone availability by preventing turnover of excess histones.

脂滴的隔离通过防止过量组蛋白的周转来促进组蛋白的可用性。

DOI：
10.1242/dev.199381
发表时间：
2021
期刊：
Development (Cambridge, England)
影响因子：
0
作者：
Stephenson,RoxanA;Thomalla,JonathonM;Chen,Lili;Kolkhof,Petra;White,RogerP;Beller,Mathias;Welte,MichaelA
通讯作者：
Welte,MichaelA

Lipid droplet motility and organelle contacts.