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隔，为细胞内信号转导创造了一个关键控制点。每当特定基因的表达被调节以响应细胞内或细胞外信号时，信息就会跨越核膜传递。在许多情况下，这种信息以特定蛋白质的形式传递，进入细胞核以引起基因表达的变化。尽管这一过程具有重要的生物学意义，但这种依赖信号的核靶向和转位的机制在分子水平上尚不清楚。为了使这些事件成为治疗靶点，必须描述详细的分子机制。 这一提议的广泛的长期目标是了解可溶性运输因子如何与核孔复合体合作来调节双向核运输。这项研究结合了对萌芽酵母中高度保守的运输因子的体内分析，以及蛋白质-蛋白质相互作用的定量分析和非洲爪哇卵母细胞的显微注射，以了解货物是如何被定位并运送到细胞核中的。这项建议的具体目的是：1)分析NTF2与核孔之间的分子相互作用，并利用这一分析来区分当前通过核孔复合体运输NTF2的模型；2)研究NLS货物进入细胞核的机制；以及3)研究NLS序列中的磷酸化如何调节蛋白质的输入。这些实验的结果将为核质运输的机制提供新的见解。 这项提议与健康有关有两个方面。首先，激活的信号转导通路将信号发送到细胞核，以响应刺激并激活转录。这一运输步骤可能代表了一个未被利用的目标，用于阻断特定的细胞信号以及在转化细胞中出现的未受调控的信号。其次，感染人类细胞的病毒利用内源性核运输机制进入细胞核。更详细地了解调节核运输的机制可能会为抗病毒疗法提供新的靶点。

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.