Functional Characterization of Membrane-Anchored Ubiquitin-Fold Protein Complexes

膜锚定泛素折叠蛋白复合物的功能表征

基本信息

批准号：
8037421
负责人：
BRIAN Patrick DOWNES
金额：
$ 28.5万
依托单位：
SAINT LOUIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-01 至 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8037421
关键词：
26S proteasome Active Sites Address Affect Alzheimer&apos s Disease Amino Acids Animal Model Area Autophagocytosis Binding Biogenesis Biological Assay Boxing Cell membrane Co-Immunoprecipitations Complex DNA Repair Data Degradation Pathway Disease Endocytosis Enzymes Eukaryota Family Fluorescence Goals Health Human Huntington Disease In Vitro Length Malignant Neoplasms Mass Spectrum Analysis Membrane Modeling Mono-S Mouse-ear Cress Names Parkinson Disease Pathway interactions Peptide Signal Sequences Phenotype Physiological Plants Positioning Attribute Process Protein Binding Protein Family Protein Isoprenylation Proteins Reaction Recruitment Activity Regulation Research Resolution Signal Transduction Signaling Protein Specificity System Testing Transcriptional Regulation Ubiquitin Ubiquitin Like Proteins Ubiquitin-Conjugating Enzymes Ubiquitination enzyme activity genetic analysis in vivo mutant peroxisome protein complex protein degradation protein folding

项目摘要

DESCRIPTION (provided by applicant): The goal of the proposed research is to test the ability of an uncharacterized ubiquitin-like protein family named MUB, for Membrane-anchored Ubiquitin-fold, to funnel activated ubiquitin into critical regulatory reactions at the plasma membrane. How ubiquitin chain length and positioning is determined for poly-, mono-, and multi- ubiquitination at the plasma membrane is an important question in the ubiquitination field, which we will address in the proposed research. Specifically, we plan to define plasma membrane localized interactions between MUBs and the Ubiquitin/26S Proteasome System, ultimately, to better understand the regulation of eukaryotic signal transduction. Many key signaling proteins are regulated by covalent attachment to ubiquitin, a 76 amino acid protein. In many cases, ubiquitination signals protein degradation by the Ubiquitin/26S Proteasome System. The core of the Ubiquitin/26S Proteasome System is extensively characterized and includes a three enzyme cascade - E1, E2, E3, and the large multiproteolytic 26S proteasome itself. It is less well known how the Ubiquitin/26S Proteasome System is coordinated to allow the temporal and spatial resolution incumbent on a system responsible for protein degradation, and other processes including endocytosis, autophagy, peroxisome biogenesis, and DNA repair. MUBs are structurally similar to ubiquitin, but distinguished by a carboxyl terminal signal sequence, called a CaaX box, which recruits a hydrophobic membrane-anchor via the protein prenylation system. Highly conserved MUBs from several model organisms and humans are prenylated in vitro. We propose to test the hypothesis that MUB proteins help to coordinate the activity of the UPS at the plasma membrane. In particular r, protein interaction studies including pull-downs and NMR to characterize interactions in vitro, and fluorescence complementation and co-immunoprecipitation assays to confirm these interactions in vivo will be used. Enzyme activity assays of various Ubiquitin/26S Proteasome System proteins will be performed to determine how they are regulated by MUB proteins. Finally, co-immunoprecipitation assays, mass spectrometry, and genetic analysis will be performed to characterize pathways related to MUB mutant phenotypes. Execution of these aims will reveal new organization and specificity within the Ubiquitin/26S Proteasome System. This research addresses a key area of research because many signaling proteins in the plasma membrane are regulated by ubiquitination, but the mechanisms supporting this process are unknown. The described studies will be conducted in the plant Arabidopsis thaliana for technical reasons. Yet, we expect that our results will be broadly applicable to the highly conserved MUB protein family found across multi- cellular eukaryotes examined to date. PUBLIC HEALTH RELEVANCE: The results of this study will be broadly applicable to the highly conserved MUB protein family found across multi-cellular eukaryotes examined to date. These studies will ultimately contribute to the long term goal of characterizing the cellular organization of the protein degradation pathways, which impact many human health issues including well known diseases such as Huntington's, Parkinson's, Alzheimer's and various cancers.

描述（由申请人提供）：拟议研究的目标是测试名为 MUB 的未表征的类泛素蛋白家族（用于膜锚定泛素折叠）将活化的泛素汇集到质膜上关键调节反应的能力。如何确定质膜上多泛素、单泛素和多泛素化的泛素链长度和定位是泛素化领域的一个重要问题，我们将在拟议的研究中解决这个问题。具体来说，我们计划最终定义 MUB 和泛素/26S 蛋白酶体系统之间的质膜局部相互作用，以更好地了解真核信号转导的调节。许多关键信号蛋白通过与泛素（一种 76 个氨基酸的蛋白质）共价连接来调节。在许多情况下，泛素化标志着泛素/26S 蛋白酶体系统的蛋白质降解。泛素/26S 蛋白酶体系统的核心经过广泛表征，包括三酶级联 - E1、E2、E3 和大型多蛋白水解 26S 蛋白酶体本身。鲜为人知的是，泛素/26S 蛋白酶体系统如何协调，以实现负责蛋白质降解以及其他过程（包括内吞作用、自噬、过氧化物酶体生物合成和 DNA 修复）的系统所承担的时间和空间分辨率。 MUB 在结构上与泛素相似，但其区别在于羧基末端信号序列（称为 CaaX 盒），它通过蛋白质异戊二烯化系统招募疏水性膜锚。来自几种模式生物和人类的高度保守的 MUB 在体外被异戊二烯化。我们建议检验 MUB 蛋白有助于协调质膜 UPS 活性的假设。特别是，将使用蛋白质相互作用研究，包括下拉和核磁共振来表征体外相互作用，以及荧光互补和免疫共沉淀测定来确认这些体内相互作用。对各种泛素/26S 蛋白酶体系统蛋白进行酶活性测定，以确定它们如何受到 MUB 蛋白的调节。最后，将进行免疫共沉淀分析、质谱分析和遗传分析来表征与 MUB 突变表型相关的途径。这些目标的实现将揭示泛素/26S 蛋白酶体系统内的新组织和特异性。这项研究涉及一个关键的研究领域，因为质膜中的许多信号蛋白受到泛素化的调节，但支持这一过程的机制尚不清楚。出于技术原因，所描述的研究将在拟南芥植物中进行。然而，我们预计我们的结果将广泛适用于迄今为止在多细胞真核生物中发现的高度保守的 MUB 蛋白家族。公共健康相关性：这项研究的结果将广泛适用于迄今为止在多细胞真核生物中发现的高度保守的 MUB 蛋白家族。这些研究最终将有助于实现表征蛋白质降解途径的细胞组织的长期目标，蛋白质降解途径影响许多人类健康问题，包括众所周知的疾病，如亨廷顿舞蹈症、帕金森病、阿尔茨海默病和各种癌症。

项目成果

期刊论文数量（1）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（1）

A MUB E2 structure reveals E1 selectivity between cognate ubiquitin E2s in eukaryotes.

MUB E2 结构揭示了真核生物中同源泛素 E2 之间的 E1 选择性。

DOI：
发表时间：
2016-08-23
期刊：
影响因子：
16.6
作者：
Lu, Xiaolong;Malley, Konstantin R;Brenner, Caitlin C;Koroleva, Olga;Korolev, Sergey;Downes, Brian P
通讯作者：
Downes, Brian P