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Characterization of the Cohen syndrome protein COH1 as a membrane-associated scaffold protein for intracellular transport processes in development and maintenance of neuronal cells

科恩综合征蛋白 COH1 作为膜相关支架蛋白的表征，用于神经元细胞发育和维持的细胞内运输过程

基本信息

批准号：
223309248
负责人：
Dr. Wenke Seifert
金额：
--
依托单位：
Institut für Zell- und Neurobiologie (CCM)
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2012
资助国家：
德国
起止时间：
2011-12-31 至 2016-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/223309248?language=en
关键词：
Characterization Cohen syndrome protein COH1

项目摘要

Intracellular transport controls a wide spectrum of biological processes such as brain development and maintenance. Core determinants of regular brain function are neuronal differentiation and integration into an efficient network. Studying inherited neurological disorders may help to define molecular mechanisms underlying these processes. The autosomal recessive Cohen syndrome is mainly characterized by non-progressive mental retardation in combination with postnatal microcephaly and is caused by mutations in the COH1 (VPS13B) gene. COH1 encodes a protein of 3997 amino acids (450 kDa) length with partial sequence homology to yeast Vps13p. Recently, we established COH1 as Golgi-associated matrix protein regulating Golgi morphology and Golgi-associated membrane tubulation activity. Our preliminary data show that Coh1 is a positive regulator of neuritogenesis, suggesting that Cohen syndrome is caused by defective terminal neuronal differentiation and integration. To further investigate the molecular mechanisms controlled by COH1, we performed initial co-immunoprecipitations showing that COH1 similar to yeast Vps13p interacts with the Golgi-associated small GTPase RAB6 and the guanine nucleotide exchange factor subunit RIC1. This result implicates a functional network composed of COH1, RAB6 and the heteromeric complex RIC1/RGP1 in mammalian cells and suggests that disturbed Golgi-associated antero- and retrograde intracellular transport processes underlie defective terminal neuronal differentiation and integration in Cohen syndrome.The proposed project aims to identify further functional targets of COH1 to complete our understanding of the hitherto assumed COH1-RIC1/RGP1-RAB6 protein complex using different biochemical approaches, such as yeast-two-hybrid screens, GST-pull down and co-immunoprecipitations. Functionally, we will complement these biochemical analyses by assessment of Golgi-associated transport events and its impact on neurite extension and final synaptic integration, using different genetic and cell biological manipulations to perturb the function of COH1, RAB6, RIC1, and RGP1 and advanced imaging techniques. Finally, we aim to establish a conditional knockout mouse model to corroborate our in vitro studies with in vivo analyses of Coh1. Research within the proposed project is expected to provide profound insights into the molecular machinery that is controlled by COH1 to facilitate Golgi function and intracellular transport. Combined biochemical, genetic and cell biological approaches ensure the integration of our research to formulate a pathomechanistic model of Cohen syndrome. At the same time, our results will significantly contribute to the general comprehension of Golgi-associated transport processes for terminal neuronal differentiation and integration.

细胞内转运控制着广泛的生物过程，如大脑发育和维持。正常脑功能的核心决定因素是神经元分化和整合到一个有效的网络中。研究遗传性神经系统疾病可能有助于确定这些过程背后的分子机制。常染色体隐性科恩综合征主要表现为非进行性智力迟钝合并出生后小头畸形，由COH1 （VPS13B）基因突变引起。COH1编码一个长度为3997个氨基酸（450 kDa）的蛋白，部分序列与酵母Vps13p同源。最近，我们确定了COH1作为高尔基相关基质蛋白调节高尔基形态和高尔基相关膜管活性。我们的初步数据显示，Coh1是神经生成的正调节因子，提示Cohen综合征是由末梢神经元分化和整合缺陷引起的。为了进一步研究COH1控制的分子机制，我们进行了初步的共免疫沉淀，结果显示COH1与酵母Vps13p相似，与高尔基相关的小GTPase RAB6和鸟嘌呤核苷酸交换因子亚基RIC1相互作用。这一结果表明，哺乳动物细胞中存在一个由COH1、RAB6和异质复合体RIC1/RGP1组成的功能网络，并表明高尔基蛋白相关的细胞内前、逆行运输过程受到干扰，是Cohen综合征终末神经元分化和整合缺陷的基础。该项目旨在通过不同的生化方法，如酵母-双杂交筛选、GST-pull - down和共免疫沉淀，进一步确定COH1的功能靶点，以完成我们对迄今为止假设的COH1- ric1 /RGP1-RAB6蛋白复合物的理解。在功能上，我们将通过评估高尔基蛋白相关转运事件及其对神经突延伸和最终突触整合的影响来补充这些生化分析，使用不同的遗传和细胞生物学操作来干扰COH1、RAB6、RIC1和RGP1的功能以及先进的成像技术。最后，我们的目标是建立一个条件敲除小鼠模型，通过体内分析Coh1来证实我们的体外研究。该项目的研究有望对COH1控制的分子机制提供深刻的见解，以促进高尔基体功能和细胞内运输。结合生物化学，遗传和细胞生物学方法，确保我们的研究整合，制定科恩综合征的病理机制模型。同时，我们的研究结果将显著有助于理解高尔基蛋白相关的转运过程对终端神经元分化和整合的影响。