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.

新兴证据支持线粒体呼吸链（MRC）通过有组织的多重复合结构称为超复合物。但是，动态和调节超复合组件尚未得到充分研究。特别是，几乎没有任何调节蛋白质因子已经确定了参与超复合组装。我们的长期目标是了解线粒体呼吸机械及其底底调节机制。这个特定的目的应用是确定线粒体伴侣是否75 kDa葡萄糖调节蛋白（GRP75）起作用在调节超复合组装并进一步确定这一重要的涉及的其他蛋白质因素过程。自普通酵母以来系统可以用作强大的遗传学系统来识别推定的监管因素，缺乏复杂I是哺乳动物超复合物的重要组成部分。我们以前已经建立了有效的方法隔离携带线粒体DNA（mtDNA）突变的细胞并进一步产生了几个具有调节/更改超复合组件的细胞模型，可能是由于增强/稳定的超复合物与调节因子之间的相互作用。这些细胞系的特征分子和蛋白质组学方法都暗示了分子伴侣GRP75超复合物集会。有趣的GRP 75以前涉及帕金森氏病（PD），这是由于1）。 GRP 75 PD患者已经确定了突变。 2）在PD患者的大脑中发现低GRP 75表达； 3）。我们的初步研究表明，杂合的GRP75小鼠表现出与有缺陷的超复合组件。该应用程序的中心假设是GRP75是必不可少的部分调节超级复合物和超复合组件缺陷的机械与缺乏GRP 75相关的将导致神经脱生。为了检验这一假设，我们建议追求以下三个特定目的：1）表征GRP75在超复合组装中的作用。在特别是，我们将遵循单个复合物和超复合物的逐步组装和降解在实验室中新开发的方法的存在和不存在GRP75的情况下； 2）确定调节通过识别与调节超复合物有关的新蛋白质因子，GRP75在超复合物上的机制集会。蛋白质分析与GRP75和复合物I相互作用的蛋白质组学分析具有调节/改变超级复杂组件的细胞模型中的超复合物，我们旨在隔离新型涉及调节超复合组装的蛋白质因子。 3）用更改的鼠标模型表征 GRP75的表达。有缺陷的超复合动力学在神经元变性中的含义将是进一步在杂合和神经元特异性GRP75敲除小鼠模型中进行了探索。我们将调查从GRP75缺陷到超复合缺陷到神经元的基本分子途径退化。该方法具有创新性，因为它结合了我们独特的细胞模型上调具有新开发的分析方法的超复合动力学，以了解呼吸超复合组件。建立具有有缺陷超复合的新型鼠标模型动力学应为研究神经元系统和神经脱生的生物能学开辟新的可能性。我们认为，我们处于表征呼吸超超复合组件的强烈位置。研究很重要，因为阐明这种机制可以为调节氧化的调节提供新的见解。磷酸化机制。此外，我们预计我们的工作还将有助于识别新型风险基因参与与线粒体功能障碍相关的神经退行性疾病。