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The role of Grp75 in supercomplex assembly and neurodegeneration

Grp75 在超复合物组装和神经退行性变中的作用

基本信息

批准号：
9762142
负责人：
Yidong Bai
金额：
$ 28.98万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9762142
关键词：
ATP Synthesis Pathway Aging Animals Attention Binding Bioenergetics Brain Brain Stem Candidate Disease Gene Cell Line Cell model Cells Complex Data Defect Degenerative Disorder Disease Electron Transport Eukaryotic Cell Exhibits Genetic Goals Individual Investigation Knock-out Knockout Mice Lead Light Malignant Neoplasms Mammalian Cell Methods Mitochondria Modeling Molecular Molecular Chaperones Molecular Structure Molecular Weight Motor Activity Mus Mutation Nerve Degeneration Neurodegenerative Disorders Neurons Organelles Oxidative Phosphorylation Oxidative Regulation Parkinson Disease Pathogenesis Pathologic Pathway interactions Patients Phenotype Play Positioning Attribute Process Protein Analysis Protein translocation Proteins Proteomics Protons Public Health Quality Control Regulation Research Respiratory Chain Role Structure System Technology Testing Work Yeasts analytical method base genetic regulatory protein glucose-regulated proteins innovation insight mitochondrial DNA mutation mitochondrial dysfunction mouse model mutant nervous system disorder novel oligomycin sensitivity-conferring protein protein complex protein folding respiratory respiratory protein risk variant screening tool yeast genetics

项目摘要

Emerging evidence supports the proposition that the mitochondrial respiratory chain (MRC) functions via organized multicomplex structures called supercomplexes. However the dynamics and regulation of supercomplex assembly have not been fully investigated. In particular, hardly any regulatory protein factors involved in supercomplex assembly have been identified. Our long term goal is to understand the dynamics of mitochondrial respiratory machinery and its underling regulatory mechanism. The objective of this particular application is to determine if the mitochondrial chaperon, 75 kDa glucose regulated protein (Grp75) plays a role in regulating supercomplex assembly and further to identify additional protein factors involved in this important process. The study of mammalian respiratory supercomplex assembly has been difficult since common yeast systems, which could be utilized as a powerful genetics system to identify putative regulatory factors, lack Complex I an essential component of mammalian supercomplexes. We have previously established an efficient method to isolate cells carrying mitochondrial DNA (mtDNA) mutations and further generated several cell models with regulated/altered supercomplex assembly, probably due to the enhanced/stabilized interactions between supercomplexes and regulative factor(s). Characterizations of these cell lines employing both molecular and proteomics approaches have implicated the molecular chaperone Grp75 supercomplex assembly. Interesting Grp 75 has previous been implicated in Parkinson's diseases (PD) due to 1). Grp 75 mutations have been identified in PD patients; 2) Low Grp 75 expression was found in brains of PD patients; 3). Our preliminary studies showed heterozygous Grp75 mice exhibited lower motor activities associated with defective supercomplex assembly. The central hypothesis for this application is that Grp75 is an essential part of machinery which regulates the assembly of supercomplexes, and defective of supercomplex assembly associated with deficient Grp 75 would lead to neuro-degeneration. To test this hypothesis, we propose to pursue the following three specific aims: 1) Characterize the role of Grp75 in supercomplex assembly. In particular, we will follow the step-wise assembly and degradation of individual complexes and supercomplexes in presence and absence of Grp75 with newly developed approaches in the lab; 2) Determine the regulatory mechanisms of Grp75 on supercomplex by Identify novel protein factors involved in regulating supercomplex assembly. With proteomic analysis of proteins interacting with Grp75 and Complex I containing supercomplexes in the cell models with regulated/altered supercomplex assembly, we aim to isolate novel protein factors involved in regulating supercomplex assembly. 3) Characterize the mouse models with altered expression of Grp75. The implications of defective supercomplex dynamics in neuronal degeneration will be further explored in heterozygous and neuronal-specific Grp75 knockout mouse models. We will investigate the underlying molecular pathways derived from Grp75 defect to supercomplex deficiency to neuronal degeneration. The approach is innovative, because it combines our unique cell models exhibiting upregulated supercomplex dynamics with newly-developed analytical methods to allow understanding of the complexity of respiratory supercomplex assembly. The establishment of novel mouse models with defective supercomplex dynamics should open new possibilities to study bioenergetics in neuronal system and neuro-degeneration. We believe that we are in a strong position to characterize respiratory supercomplex assembly. The research is significant, because elucidating this mechanism could provide new insights into the regulation of oxidative phosphorylation machinery. In addition, we anticipate our work will also help to identify novel risk genes involved in neurodegenerative diseases associated with mitochondrial dysfunction.

新出现的证据支持线粒体呼吸链（MRC）通过有组织的多复合体结构称为超复合体。然而，超复杂组装尚未得到充分研究。特别是，几乎没有任何调节蛋白因子参与了超复杂组装。我们的长期目标是了解线粒体呼吸机制及其潜在的调节机制。本特别报告的目的应用是确定线粒体伴侣，75 kDa的葡萄糖调节蛋白（Grp 75）是否发挥作用在调节超复合物组装，并进一步确定其他蛋白质因子参与这一重要的过程哺乳动物呼吸超复合体组装的研究从普通酵母开始就一直是个难点系统，这可能是一个强大的遗传学系统，以确定推定的调控因素，缺乏复合体I是哺乳动物超复合体的重要组成部分.我们以前建立了一个一种有效的方法来分离携带线粒体DNA（mtDNA）突变的细胞，并进一步产生了几个具有调节/改变的超复合物组装的细胞模型，可能是由于增强/稳定的超复合物和调节因子之间的相互作用。这些细胞系的表征采用分子和蛋白质组学方法都表明，组装件.有趣的是，Grp 75此前曾因1）而与帕金森病（PD）有关。Grp 75 突变已在PD患者中鉴定; 2）在PD患者的脑中发现低Grp 75表达; 3）。我们的初步研究表明，杂合子Grp 75小鼠表现出较低的运动活动，有缺陷的超复杂装配。该应用的中心假设是Grp 75是一个重要的部分，调节超复合体组装的机械，以及超复合体组装的缺陷与缺乏Grp 75相关的神经退行性变将导致神经退行性变。为了验证这一假设，我们建议本论文的主要目的是：1）研究Grp 75在超复合物组装中的作用。在特别是，我们将遵循个别复合物和超复合物的逐步组装和降解在存在和不存在Grp 75的情况下，在实验室中使用新开发的方法; 2）确定通过鉴定参与调节超复合物的新蛋白质因子来研究Grp 75对超复合物的作用机制组装件.通过对与Grp 75和含有Grp 75的复合物I相互作用的蛋白质的蛋白质组学分析，在具有调节/改变的超复合物组装的细胞模型中，我们的目标是分离新的参与调节超复合物组装的蛋白质因子。3)表征具有改变的 Grp 75的表达。在神经元变性中有缺陷的超复杂动力学的影响将是在杂合和神经元特异性Grp 75敲除小鼠模型中进一步探索。我们将调查从Grp 75缺陷到神经元超复合物缺陷的潜在分子途径退化这种方法是创新的，因为它结合了我们独特的细胞模型，表现出上调的细胞增殖。超复杂动力学与新开发的分析方法，使理解的复杂性，呼吸超复合体组装一种新型超复合体缺陷小鼠模型的建立动力学为研究神经系统和神经退行性变的生物能量学提供了新的可能性。我们相信，我们是在一个强有力的地位，以表征呼吸超复合体组装。研究是重要的，因为阐明这一机制可以提供新的见解调节氧化磷酸化机制此外，我们预计我们的工作也将有助于确定新的风险基因与线粒体功能障碍相关的神经退行性疾病有关。