Structure-function analysis of RanBP2 in the neuroretina

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学过程。细胞生理学中的一个中心问题是，细胞如何设法适应数量非常有限的马达蛋白的大量货物的运输。多种调节剂调节大量内源性货物在亚细胞间的细胞内运输，病原体经常劫持这种运输机械的部件。新出现的证据也支持这一过程对于触发明确的细胞事件至关重要，如有丝分裂和细胞死亡的开始。蛋白质运动在神经元系统中具有特殊的相关性，因为神经元上的亚细胞室和细胞器的精细划分和极化，以及货物通常需要长途旅行才能到达最终目的地的重大负担。在贩运部件和相互作用的货物中，大量且数量不断增加的遗传损害导致了人类的一系列神经疾病和相关疾病。在这个提议中，神经视网膜是研究蛋白质运动的实验系统的选择，因为它是一个多样化但定义非常明确的生物系统，非常容易在体外和体内进行操作。此外，还有大量的神经退行性视网膜营养不良，直接和间接地影响到调节蛋白质运输的许多方面的过程。这项建议的重点是了解一种独特的大型脊椎动物支架蛋白RAN结合蛋白2(RanBP2/Nup358)在调节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意义。过去的工作表明，RanBP2在体内参与了光受体的功能性产生和核蛋白的输入。我们已经定位和表征了RanBP2的结构域，确定了相关的分子伙伴，并确定了这些结构域在视网膜神经元之间和内部的空间定位。此外，还开发了大量的分子、生化、免疫学和遗传工具。这些结合其他跨学科的方法，现在将被用来探索在体外和体内由RanBP2介导的视网膜神经元内特定的和新兴的运输途径，以识别新的伙伴和货物，调节RanBP2复合体组装的调控机制，并进一步剖析RanBP2介导的神经元限制性运输途径的分子基础。最后，我们将探讨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。

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.