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）：拟议的研究的目的是测试名为Mub的未表征的泛素样蛋白家族的能力，该家族是膜锚定的泛素折叠的，以将激活的泛素融合到质膜上的关键调节反应中。在质膜上，在泛素，单，多泛素化中确定泛素链长度和定位是在泛素化领域的一个重要问题，我们将在拟议的研究中解决。具体而言，我们计划定义MUBS与泛素/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。许多关键的信号蛋白受到泛素（76氨基酸蛋白）的共价附着的调节。在许多情况下，泛素化信号信号蛋白质蛋白质降解受泛素/26S蛋白酶体系统的降解。泛素/26S蛋白酶体系统的核心是广泛的表征，包括三个酶级联-E1，E2，E3和大型多蛋白水解26S蛋白酶体本身。鲜为人知的是如何协调泛素/26S蛋白酶体系统，以允许在负责蛋白质降解的系统以及包括内吞作用，自噬，过氧化物酶体生物发生和DNA修复在内的其他过程中的时间和空间分辨率。 MUB在结构上与泛素相似，但以羧基末端信号序列（称为CAAX盒）区分，该信号序列被称为CAAX盒，该信号盒通过蛋白质前化系统募集疏水膜锚定。来自几种模型生物和人类的高度保守的MUB在体外被先进。我们建议测试MUB蛋白有助于协调UPS在质膜上的活性的假设。特别是R，蛋白质相互作用研究在内，包括下拉和NMR以表征体外相互作用，以及将使用荧光互补和共免疫沉淀测定法，以确认体内的这些相互作用。将执行各种泛素/26S蛋白酶系统蛋白的酶活性测定，以确定如何通过MUB蛋白调节它们。最后，将进行共免疫沉淀测定法，质谱和遗传分析，以表征与MUB突变表型相关的途径。这些目标的执行将揭示泛素/26S蛋白酶体系统中的新组织和特异性。这项研究解决了研究的关键领域，因为质膜中的许多信号传导蛋白受到泛素化的调节，但是支持该过程的机制尚不清楚。描述的研究将出于技术原因在植物拟南芥中进行。但是，我们预计我们的结果将广泛适用于迄今为止研究的多细胞真核生物中发现的高度保守的MUB蛋白家族。公共卫生相关性：这项研究的结果将广泛适用于迄今为止研究的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表征蛋白质降解途径的细胞组织的长期目标，这会影响许多人类健康问题，包括众所周知的疾病，例如亨廷顿，帕金森氏症，阿尔茨海默氏症和各种癌症。