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Molecular mechanisms that regulate lysosomal protein transport

调节溶酶体蛋白转运的分子机制

基本信息

批准号：
9892564
负责人：
Anjon Audhya
金额：
$ 4.77万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-01 至 2021-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9892564
关键词：
ATP phosphohydrolase Address Animals Architecture Attenuated Binding Biochemical Biogenesis Biological Assay Biological Models Caenorhabditis elegans Carrier Proteins Cell surface Cells Complex Computer Simulation Cryoelectron Microscopy Data Defect Deposition Development Disease Down-Regulation Electron Microscopy Electron Transport Complex III Embryo Embryonic Development Endosomes Engineering Eukaryotic Cell Exhibits Fertilization Filament Freezing Frontotemporal Dementia Future Genetic Goals Grant Homeostasis Hormone Receptor Huntington Disease Image Immune System Diseases Immunoelectron Microscopy In Vitro Individual Integral Membrane Protein Intervention Investigation Lead Lipid Bilayers Location Lysosomes Malignant Neoplasms Mammalian Cell Mediating Membrane Membrane Proteins Methodology Methods Microscopy Microtomy Modeling Molecular Movement Mutagenesis Nature Neck Neurodegenerative Disorders Neurons Oocytes Organelles Organism Outcome Parkinson Disease Pathway interactions Physiological Play Polymers Process Production Proteins RNA Interference Research Resolution Role Signal Transduction Site Sorting - Cell Movement Specific qualifier value System Testing Time Transgenes Ubiquitin Vesicle Work base cryogenics electron tomography endosome lumen endosome membrane experimental study gene replacement genetic approach genetic manipulation human stem cells imaging genetics in vivo intracellular protein transport live cell imaging lysosomal proteins molecular dynamics molecular modeling mutant oocyte maturation pressure prevent protein complex protein transport reconstitution therapeutic target tool trafficking zygote

项目摘要

ABSTRACT The long-term goal of this proposal is to define molecular mechanisms that regulate the trafficking of integral membrane proteins to the lysosome for degradation. The ESCRT machinery, a set of conserved endosomal protein complexes, is proposed to bind directly to ubiquitinylated membrane proteins and govern their entry into vesicles that bud into the lumen of specialized multivesicular endosomes (MVEs). This process is particularly important for the downregulation of hormone receptors and to prevent constitutive signaling, which can lead to developmental abnormalities and disease. How the late-acting components of the ESCRT machinery coordinate the formation of intralumenal vesicles at MVEs will be addressed in this proposal. The C. elegans germline and early embryo are powerful model systems to study membrane dynamics in an intact, developing animal. Specific proteins can be efficiently depleted from oocytes using RNA interference. Additionally, oocyte maturation and fertilization reproducibly trigger the internalization and ESCRT-mediated degradation of multiple transmembrane proteins, providing an ideal, physiologically relevant system for studying lysosomal protein transport. C. elegans is highly amenable to genetic manipulation and can be engineered easily to stably express transgenes for gene replacement strategies. Additionally, we have established methods to high pressure freeze animals at specific timepoints during embryo development to enable the stepwise characterization of de novo MVE biogenesis using electron microscopy (EM)-based approaches. Given the stereotypic nature of early embryo development, we can correlate these EM data directly with our findings using live cell imaging assays, which we have pioneered in this system. Taking advantage of this unique combination of attributes, the specific aims of this first renewal application are to: 1) define regulatory mechanisms that specify the site of ILV formation on MVEs, 2) determine mechanisms that promote the nucleation of ESCRT-III filaments, and 3) define regulatory mechanisms that control ESCRT-III polymer dynamics. The genetic and biochemical studies conducted during the first period of grant support defined new methods and tools to study ESCRT-III polymer assembly, raising intriguing hypotheses regarding how this process is controlled. Using a combination of in silico molecular modeling, in vitro reconstitution experiments, and in vivo high resolution microscopy-based assays, we will define new mechanisms that regulate ESCRT-III complex assembly during MVE formation. These studies will provide a key framework for future investigation into highly related pathways in mammalian cells.

摘要这项提案的长期目标是确定调节整合素转运的分子机制。膜蛋白被运送到溶酶体进行降解。ESCRT机制，一套保守的内体蛋白复合物，被认为是直接结合到泛素化的膜蛋白，并控制他们的进入进入囊泡，芽入特化多囊内体（MVEs）的腔中。这个过程是对于激素受体的下调和防止组成性信号传导特别重要，会导致发育异常和疾病ESCRT的晚期作用成分机械协调MVE管腔内囊泡的形成将在本提案中得到解决。梭线虫种系和早期胚胎是研究完整细胞膜动力学的有力模型系统，发展中的动物利用RNA干扰可以有效地从卵母细胞中去除特定的蛋白质。此外，卵母细胞成熟和受精可重复地触发内化和ESCRT介导的降解多个跨膜蛋白，提供了一个理想的，生理相关的系统，研究溶酶体蛋白质运输C.秀丽隐杆线虫非常适合基因操作，易于工程化以稳定表达用于基因置换策略的转基因。此外，我们还有在胚胎发育期间的特定时间点高压冷冻动物的既定方法，使用基于电子显微镜（EM）的逐步表征从头MVE生物发生接近。考虑到早期胚胎发育的固有性质，我们可以将这些EM数据直接与我们的发现使用活细胞成像分析，这是我们在这个系统中开创的。以利用这种独特的属性组合，第一次更新申请的具体目标是：1）定义指定MVE上ILV形成位点的调节机制，2）确定促进ESCRT-III丝的成核，和3）定义控制ESCRT-III的调节机制聚合物动力学在第一个资助期内进行的遗传和生化研究定义了新的方法和工具来研究ESCRT-III聚合物组装，提出了有趣的假设，这个过程是如何控制的。使用计算机分子建模、体外重构实验，并在体内高分辨率显微镜为基础的测定，我们将定义新的机制，在MVE形成过程中调节ESCRT-III复合物组装。这些研究将提供一个关键框架，进一步研究哺乳动物细胞中高度相关的通路。

项目成果

期刊论文数量（15）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Hrs and STAM function synergistically to bind ubiquitin-modified cargoes in vitro.

DOI：
10.1016/j.bpj.2014.11.004
发表时间：
2015-01
期刊：
Biophysical journal
影响因子：
3.4
作者：
Hirohide Takahashi;J. Mayers;Lei Wang;J. Edwardson;A. Audhya
通讯作者：
Hirohide Takahashi;J. Mayers;Lei Wang;J. Edwardson;A. Audhya

Phosphoinositide signaling during membrane transport in Saccharomyces cerevisiae.

酿酒酵母膜转运过程中的磷酸肌醇信号传导。

DOI：
10.1007/978-94-007-3015-1_2
发表时间：
2012
期刊：
Sub-cellular biochemistry
影响因子：
0
作者：
Schuh,AmberL;Audhya,Anjon
通讯作者：
Audhya,Anjon

The midbody ring scaffolds the abscission machinery in the absence of midbody microtubules.

DOI：
10.1083/jcb.201306036
发表时间：
2013-11-11
期刊：
The Journal of cell biology
影响因子：
0
作者：
Green RA;Mayers JR;Wang S;Lewellyn L;Desai A;Audhya A;Oegema K
通讯作者：
Oegema K

Regulated lipid synthesis and LEM2/CHMP7 jointly control nuclear envelope closure

DOI：
10.1083/jcb.201908179
发表时间：
2020-04
期刊：
The Journal of Cell Biology
影响因子：
0
作者：
Lauren Penfield;Raakhee Shankar;E. Szentgyörgyi;A. Laffitte;M. Mauro;A. Audhya;T. Müller-Reichert;Shirin Bahmanyar
通讯作者：
Lauren Penfield;Raakhee Shankar;E. Szentgyörgyi;A. Laffitte;M. Mauro;A. Audhya;T. Müller-Reichert;Shirin Bahmanyar

The ESCRT machinery: from the plasma membrane to endosomes and back again.