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用数量非常有限的马达蛋白来容纳过多的货物的运输。多种调节剂介导过多的内源性货物在亚细胞区室之间的细胞内转运，并且病原体经常劫持这种转运机制的组分。新出现的证据也支持这一过程对于触发定义的细胞事件至关重要，例如有丝分裂和细胞死亡的开始。蛋白质动力学在神经系统中具有特殊的意义，因为亚细胞区室和细胞器在神经元中具有精致的区室化和极化，并且由于货物通常需要长途运输才能到达其最终目的地，因此对神经元施加了重大负担。在运输组分和相互作用的货物中，大量且越来越多的遗传病变导致人类的许多神经病和相关疾病。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，因为它是一个多样的，但非常明确的生物系统，非常适合在体外和体内的操作。此外，还有大量的神经退行性视网膜营养不良，其直接和间接地影响介导蛋白质运输的过程的许多方面。本提案的重点是了解一个独特的和大的支架脊椎动物蛋白，RAN结合蛋白2（RanBP 2/NUP 358），在介导和整合细胞内运输过程和蛋白质生物合成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的病因学中的意义。过去的研究表明RanBP 2在体内介导光受体和核蛋白输入的功能性产生。我们已经绘制和表征了RanBP 2的结构域，鉴定了相关的分子伴侣，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，还开发了大量的分子、生物化学、免疫学和遗传学工具。这些，与其他跨学科的方法相结合，现在将被用来探测在体外和体内特定的和新兴的贩运途径视网膜神经元内的RanBP 2介导的，以确定新的合作伙伴和货物，调节机制的组装RanBP 2复合物，并进一步剖析神经元限制的转运途径的分子基础RanBP 2介导的。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。

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.