Molecular Basis of Ran-Mediated Nuclear Transport

描述（由申请人提供）：真核细胞核内遗传物质的绝对区室化为细胞内信号传导创造了关键控制点。当特定基因的表达受到细胞内或细胞外信号的调控时，信息就通过核膜传递。在许多情况下，这种信息作为特定的蛋白质进入细胞核，引起基因表达的变化。尽管这一过程的生物学重要性，但这种信号依赖性核靶向和易位的机制在分子水平上尚不清楚。为了使这些事件作为治疗靶点，必须描述详细的分子机制。 这个提议的广泛的长期目标是了解可溶性转运因子如何与核孔复合物合作来介导双向核转运。本研究结合了芽殖酵母S.酿酒酵母，定量分析蛋白质-蛋白质相互作用和显微注射到非洲爪蟾卵母细胞，以了解货物是如何针对和交付到细胞核。该提案的具体目的是：1）分析NTF 2与核孔之间的分子相互作用，所述核孔是通过孔转运NTF 2所需的，并利用该分析来区分当前通过核孔复合物转运的模型; 2）检查NLS货物递送到核中的机制;以及3）研究NLS序列内的磷酸化如何调节蛋白质输入。这些实验的结果将提供新的见解核质转运的机制。 这项建议的健康相关性是双重的。首先，激活的信号转导通路向细胞核发送信号，以响应刺激并激活转录。该转运步骤可能代表用于阻断特定细胞信号以及在转化细胞中出现的未调节信号的未利用的靶标。其次，感染人类细胞的病毒利用内源性核转运机制进入细胞核。更详细地了解介导核转运的机制可能会为抗病毒治疗提供新的靶点。

DESCRIPTION (provided by applicant): The absolute compartmentalization of the genetic material within the nucleus of the eukaryotic cell creates a critical control point for intracellular signaling. Whenever expression of specific genes is regulated in response to an intra- or extraceullar signals, information is transferred across the nuclear envelope. In many cases this information is transmitted as specific proteins that enter the nucleus to elicit changes in gene expression. Despite the biological importance of this process, the mechanism of this signal dependent nuclear targeting and translocation is not understood at the molecular level. In order for these events to serve as therapeutic targets the detailed molecular mechanism must be delineated. The broad long-term objective of this proposal is to understand how soluble transport factors cooperate with the nuclear pore complex to mediate bidirectional nuclear transport. This study combines in vivo analyses of the highly conserved transport factors in the budding yeast, S. cerevisiae, with quantitative analysis of protein-protein interactions and microinjection into Xenopus oocytes to learn how cargoes are targeted to and delivered into the nucleus. The specific aims of this proposal are to: 1) Analyze molecular interactions between NTF2 and the nuclear pores that are required to translocate NTF2 through pores and exploit this analysis to distinguish between current models for transport through the nuclear pore complex; 2) Examine the mechanism of NLS cargo delivery into the nucleus; and 3) Investigate how phosphorylation within NLS sequences modulates protein import. Results from these experiments will provide novel insights into the mechanism of nucleocytoplasmic transport. The health-relatedness of this proposal is two-fold. First, activated signal transduction pathways send signals to the nucleus in order to respond to stimuli and activate transcription. This transport step may represent an unexploited target for blocking specific cellular signals as well as the unregulated signals that arise in transformed cells. Second, viruses that infect human cells exploit the endogenous nuclear transport machinery to gain entry to the nucleus. A more detailed understanding of the machinery that mediates nuclear transport may provide novel targets for anti-viral therapies.