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Molecular chaperones in the regulation of the intermediate filament cytoskeleton

中间丝细胞骨架调节中的分子伴侣

基本信息

批准号：
BB/R003335/1
负责人：
Paul Chapple
金额：
$ 55.93万
依托单位：
Queen Mary University of London
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2018
资助国家：
英国
起止时间：
2018 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FR003335%2F1
关键词：
Molecular chaperones regulation intermediate filament

项目摘要

The cytoskeleton is a dynamic network of filaments that pervades the cytoplasm of animal cells. It acts to regulate cellular shape and internal organisation, while providing the mechanical support that enables cells to divide and move. It consists of microtubules and actin filaments, which are polymers of single types of proteins (actin and tubulin, respectively), as well as intermediate filaments, which are composed of a family of related proteins (e.g. vimentin, keratin, desmin and neurofilament) that have cell type specific expression. For example, in mammals, alpha-keratins are expressed in epitheial cells that make horn, hooves, nails and hair.Knowledge of how intermediate filaments are organised and regulated is important for our understanding of many aspects of cell biology, such as the initiation, progression and metastatic spread of cancers. Moreover, alterations in the metabolism and/or organisation of intermediate filaments is linked to disease. This includes neurodegenerative conditions such as amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, giant axonal neuropathy and Parkinson's disease.Recently we have identified loss of function of a protein called sacsin leads to dramatic alterations in the organisation of the vimentin intermediate filament cytoskeleton, while other researchers have reported alterations in neurofilaments. The function of sacsin is unknown but it has previously been shown to have regions of homology to proteins known as molecular chaperones. These modulate the folding, degradation and complex assembly/diassembly of other proteins.This has led to the overarching hypothesis of this proposal, that sacsin is part of a molecular chaperone system required for normal intermediate filament dynamics and function. This hypothesis will be tested through four interlinked, yet independent, research objectives. We will identity cytoskeletal proteins and regulators of cytoskeletal proteins that interact with sacsin. The effects of loss of sacsin on mechanisms that are known to alter vimentin network organisation and dynamics will also be tested. Chaperones do not function in isolation, but rather as components of chaperone machines, therefore we will also determine if sacsin functions with other chaperones to influence vimentin. Finally, we will investigate a spectrum of different types of intermediate filaments to determine which ones are effected by loss of sacsin.The proposed research is multi-disciplinary and will exploit our expertise in molecular cell biology and proteomics. It will utilise techniques including live cell imaging and mass spectrometry, to define the mechanism through which loss of sacsin impacts vimentin network organisation in cell models we have previously generated. These include cells where sacsin has been knocked out by a technique called genome editing and cells derived from individuals that have the neurodegenerative disease autosomal recessive ataxia of Charlevoix Saguenay (ARSACS), where sacsin is mutated.The key predicted outcome of this work will be elucidation of a novel mechanism regulating intermediate filament organisation. This is likely to be relevant to multiple types of intermediate filament as initial studies show loss of sacsin effects both vimentin and neurofilament. The work will also help to define the molecular pathogenesis of ARSACS, a childhood onset disease where patients suffer reduced manual dexterity, speech difficulties, increasing problems with walking - such that they normally require a wheelchair - and decreased life expectancy. Moreover, the research may also give insights into neurodegenerative and other diseases where intermediate filament abnormalities are a feature.

细胞骨架是一种充满活力的细丝网络，遍布动物细胞的细胞质。它的作用是调节细胞的形状和内部组织，同时提供机械支持，使细胞能够分裂和移动。它由微管和肌动蛋白丝以及中间丝组成，微管和肌动蛋白丝是单一类型蛋白质（分别为肌动蛋白和微管蛋白）的聚合物，中间丝由具有细胞类型特异性表达的相关蛋白质家族（例如波形蛋白、角蛋白、结蛋白和神经丝）组成。例如，在哺乳动物中，α-角蛋白在制造角、蹄、指甲和毛发的上皮细胞中表达。了解中间丝是如何组织和调节的，对于我们理解细胞生物学的许多方面非常重要，例如癌症的发生、发展和转移扩散。此外，中间丝的代谢和/或组织的改变与疾病有关。这包括神经退行性疾病，如肌萎缩侧索硬化症，腓骨肌萎缩症，巨大轴突神经病和帕金森氏病。最近，我们已经确定了一种称为sacsin的蛋白质功能的丧失导致波形蛋白中间丝细胞骨架组织的显着变化，而其他研究人员已经报道了神经丝的变化。sacsin的功能尚不清楚，但先前已显示其具有与称为分子伴侣的蛋白质同源的区域。这些调节其他蛋白质的折叠，降解和复杂的组装/解组装。这导致了本提案的总体假设，即sacsin是正常中间丝动力学和功能所需的分子伴侣系统的一部分。这一假设将通过四个相互关联但又相互独立的研究目标进行检验。我们将确定与糖蛋白相互作用的细胞骨架蛋白和细胞骨架蛋白的调节因子。还将测试sacsin缺失对已知改变波形蛋白网络组织和动力学的机制的影响。伴侣蛋白不是单独起作用的，而是作为伴侣蛋白机器的组成部分，因此我们也将确定sacsin是否与其他伴侣蛋白一起影响波形蛋白。最后，我们将研究不同类型的中间丝的光谱，以确定哪些是受到sacsin的损失。拟议的研究是多学科的，将利用我们在分子细胞生物学和蛋白质组学的专业知识。它将利用包括活细胞成像和质谱分析在内的技术，来定义我们以前生成的细胞模型中sacsin损失影响波形蛋白网络组织的机制。这些细胞中的sacsin已经被敲除的技术称为基因组编辑和细胞来自个人的神经退行性疾病的常染色体隐性共济失调的Charlevoix Saguenay（ARSACS），其中sacsin突变。这项工作的关键预测结果将是阐明一种新的机制，调节中间丝组织。这可能与多种类型的中间丝有关，因为初步研究表明，sacsin对波形蛋白和神经丝的作用都丧失了。这项工作还将有助于确定ARSACS的分子发病机制，ARSACS是一种儿童期发病的疾病，患者的手部灵活性降低，言语困难，行走问题增加-因此他们通常需要轮椅-预期寿命缩短。此外，这项研究还可以深入了解神经退行性疾病和其他疾病，其中中间丝异常是一个特征。