Structure-function analysis of RanBP2 in the neuroretina

RanBP2在神经视网膜中的结构-功能分析

• 批准号: 6888021
• 负责人: PAULO A FERREIRA
• 金额: $ 2.12万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6888021
• 关键词: affinity chromatography; binding proteins; cell cycle; gender difference; genetic translation; genetically modified animals; intracellular transport; laboratory mouse; mass spectrometry; neurogenesis; neurophysiology; protein biosynthesis; protein protein interaction; protein structure function; protein transport; retina; retina degeneration; southern blotting; visual photosensitivity

DESCRIPTION (provided by applicant): Protein trafficking is a fundamental biological process across all eukaryotic cells. A central question in cell physiology is how the cell manages to accommodate the trafficking of a plethora of cargoes with a very limited number of motor proteins. A wide variety of modulators mediate the intracellular transport of a plethora of endogenous cargoes between subcellular compartments, and pathogens often hijack components of this transport machinery. Emerging evidence also supports this process is vital for triggering defined cellular events such as the onset of mitosis and cell death. Protein kinesis assumes special relevance in neuronal systems in light of the exquisite compartmentalization and polarization of subcellular compartments and organelles across neurons, and significant burden imposed on neurons for the long distances cargoes often need to travel to reach their final destinations. A large and growing number of genetic lesions in trafficking components and interacting cargoes, lead to a host of neuropathies and allied maladies in the human. In this proposal, the neuroretina is the experimental system of choice to study protein kinesis because it is a diverse but extremely well defined biological system highly amenable to manipulations in vitro and in vivo. In addition, there are a large number of neurodegenerative retinal dystrophies, which affect directly and indirectly numerous facets of processes mediating protein trafficking. The focus of this proposal is to understand the role of an unique and large scaffold vertebrate protein, Ran-binding protein 2 (RanBP2/Nup358), in mediating and integrating key steps of intracellular trafficking processes and protein biogenesis, and implications of these processes in retinal cell physiology and etiology of pathological states. Past work has implicated RanBP2 in mediating in vivo the functional production of light-receptors and nuclear protein import. We have mapped and characterized domains of RanBP2, identified associated molecular partners and determined the spatial localization of these among and within retinal neurons. Moreover, a large number of molecular, biochemical, immunological and genetic tools were developed. These, in combination with other interdisciplinary approaches, will now be used to probe in vitro and in vivo specific and emerging trafficking pathways mediated by RanBP2 within retinal neurons, to identify novel partners and cargoes, regulatory mechanisms modulating the assembly of RanBP2 complexes, and to dissect further the molecular basis of neuronal-restricted transport pathways mediated by RanBP2. Finally, we will investigate the physiological effects of these components and processes in the pathogenesis of retinal dystrophies.

描述（由申请人提供）： 蛋白质运输是所有真核细胞的基本生物过程。细胞生理学的一个核心问题是细胞如何设法用数量非常有限的运动蛋白来适应大量货物的运输。多种调节剂介导亚细胞区室之间大量内源性货物的细胞内运输，而病原体经常劫持这种运输机制的组件。新出现的证据也支持这一过程对于触发特定的细胞事件（例如有丝分裂和细胞死亡）至关重要。鉴于神经元之间亚细胞区室和细胞器的精致划分和极化，以及货物通常需要长距离运输才能到达最终目的地给神经元带来的巨大负担，蛋白质运动在神经元系统中具有特殊的相关性。贩运成分和相互作用的货物中存在大量且不断增加的遗传损伤，导致人类出现一系列神经病和相关疾病。在该提案中，神经视网膜是研究蛋白质运动的实验系统，因为它是一个多样化但定义极其明确的生物系统，非常适合体外和体内的操作。此外，还有大量的神经退行性视网膜营养不良，它们直接和间接影响介导蛋白质运输过程的许多方面。该提案的重点是了解独特的大型脊椎动物支架蛋白 Ran 结合蛋白 2 (RanBP2/Nup358) 在介导和整合细胞内运输过程和蛋白质生物发生的关键步骤中的作用，以及这些过程在视网膜细胞生理学和病理状态病因学中的影响。过去的研究表明 RanBP2 介导体内光受体的功能性产生和核蛋白输入。我们绘制并表征了 RanBP2 的结构域，识别了相关的分子伙伴，并确定了这些分子在视网膜神经元之间和内部的空间定位。此外，还开发了大量分子、生物化学、免疫学和遗传工具。这些方法与其他跨学科方法相结合，现在将用于探测视网膜神经元内 RanBP2 介导的体外和体内特异性和新兴运输途径，以确定新的伙伴和货物、调节 RanBP2 复合物组装的调节机制，并进一步剖析 RanBP2 介导的神经元限制运输途径的分子基础。最后，我们将研究这些成分和过程在视网膜营养不良发病机制中的生理作用。