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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.

项目摘要脂滴是细胞内储存脂肪的场所，在脂质代谢和能量方面具有重要功能存储.它们在控制特定蛋白质的命运方面也有第二个主要作用，即介导它们的成熟、运输、再折叠、储存和周转。虽然在揭示如何解决这个问题上已经取得了很大的进展，虽然微滴控制脂质代谢，但对其蛋白质处理作用的研究仍然有限。这个项目的目标是剖析这种蛋白质处理功能的机制、调节和生理相关性，这种现象的最佳表征实例的优点是，组蛋白H2Av与脂质的隔离果蝇卵巢和胚胎中的水滴。H2Av是动态储存的，在脂质之间不断交换，液滴通过细胞质。脂滴隔离防止卵母细胞中的H2Av降解及其过早成熟输入到胚胎细胞核中。它也受到发育调节，在胚胎发育中期被关闭，囊胚转变上一个授予期的进展将Impa2和Ipo9确定为关键介导H2Av交换的因子，揭示了细胞周期激酶Cdk 1活性， Impa2的磷酸化状态和H2Av交换速率，并发现过量的核H2Av 极大地改变了转录组这些见解导致了关于H2Av机制的新模型交换、其调节和其生物学作用是本申请提出测试的模型。是假设Impa2通过与Jabba发生物理相互作用并减少其 Ipo9作为细胞质伴侣，伴随H2Av在H2Av与H2Av之间的旅程，身份证该模型将通过确定两种输入蛋白在细胞内的分布和动力学来测试，卵巢和胚胎，并通过分析关键功能域的突变体如何影响H2Av交换。到了解发育调控，将产生Impa2中的点突变体，以防止或模仿磷酸化和Cdk1活性将被抑制。这些操作对H2Av的影响交换和物理之间的相互作用的importins，H2Av锚贾巴，和H2Av将是测定在缺乏贾巴的营养细胞中，缺乏隔离导致核H2Av水平增加。使用RNA测序分析，操纵H2Av剂量，以及无法结合H2Av的Jabba突变体，确定改变的H2Av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H2Av的机制、调节和功能的关键预测并为脂滴处理蛋白质如何控制其他地方的过程开发了一般范例。牢房因此，这些研究将为确定蛋白质是否以及如何被螯合提供基础。脂滴有助于它们在健康和疾病中的突出作用。

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

Lipid droplet motility and organelle contacts.

DOI：
10.1177/2515256419895688
发表时间：
2019-01-01
期刊：
Contact (Thousand Oaks (Ventura County, Calif.))
影响因子：
0
作者：
Kilwein, Marcus D;Welte, M A
通讯作者：
Welte, M A

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 functions beyond energy storage.