Structures and mechanisms of nuclear import and export

描述(申请人提供)：在人类细胞中，大分子的核质运输是由Kap2s/Importins/Exportins蛋白家族的19个成员介导的。KAP2通过不同的核定位或输出信号(NLSS或NESS)与其运输底物结合，并通过核孔复合体(NPC)运输它们。已知的NLS和NES类仅有Kap？/Kap？1、Kap？2(或Transportin)和CRM1分别识别的短碱性经典NLS、PY-NLS和富亮氨酸NES(LR-NES)。大多数其他的卡普-S识别出不同的序列，这些序列遮挡了他们信号的身份。核运输更加复杂，因为大多数KAP-S可以通过多个底物结合位点识别多个信号。本提案描述了EXPORT-KAP？底物识别的结构和生化分析。CRM1和进口因子Kap？2.CRM1是最通用、用途最广的出口因子Kap？CRM1-底物复合体的结构研究将揭示识别多种NES类物质的一般原理，并从力学上解释核输出的不同步骤，如核内的输出复合体组装和细胞质中的底物解离。KAP？2将大量的信使核糖核酸结合蛋白导入细胞核。我们最近在这些蛋白质中定义了一种新的NLS，称为PY-NLS，并在一项生物信息学试点计划中发现了81种新的潜在Kap？2底物。在这里，我们建议对Kap？2与不同的PY-NLS相互作用进行结构和热力学研究，以加深对PY-NLS识别的生物物理理解，并为未来准确和全面地预测基因组中Kap？2底物的计算工作生成结构和热力学训练数据集。公共卫生相关性：这笔赠款旨在发现蛋白质货物是如何被核出口因子CRM1识别并从细胞核输出的。CRM1是迄今已知的最通用和最多功能的核输出因子，我们的工作将介绍这一基本的细胞过程，并解释当干扰时可能导致心脏病和癌症的过程。我们还将研究核进口因子Kap-beta2/Transportin识别蛋白质货物的反向过程。

DESCRIPTION (provided by applicant): In human cells, nucleocytoplasmic transport of macromolecules is mediated by 19 members of the Karyopherin2 (Kap2s/Importins/Exportins) protein family. Kap2s bind their transport substrates through distinct nuclear localization or export signals (NLSs or NESs), and transport them through the nuclear pore complex (NPC). The only known classes of NLS and NES are the short basic classical-NLS, the PY-NLS and the leucine- rich NES (LR-NES), recognized by Kap?/Kap?1, Kap?2 (or Transportin) and CRM1, respectively. Most other Kap?s recognize diverse sequences that occlude the identity of their signals. Nuclear transport is further complicated as most Kap?s can recognize multiple signals through multiple substrate binding sites. This proposal describes structural and biochemical analyses of substrate recognition by export-Kap? CRM1 and import factor Kap?2. CRM1 is the most general and versatile export-Kap?. Structural studies of CRM1-substrate complexes will reveal general principles for recognition of multiple NES classes and mechanistically explain different steps of nuclear export such as export complex assembly in the nucleus and substrate dissociation in the cytoplasm. Kap?2 imports numerous mRNA binding proteins into the nucleus. We have recently defined a new NLS in these proteins termed PY-NLS and uncovered 81 new potential Kap?2 substrates in a pilot bioinformatics initiative. Here, we propose structural and thermodynamics studies of Kap?2 interactions with diverse PY-NLSs to deepen biophysical understanding of PY-NLS recognition and to generate structural and thermodynamics training datasets for future computational work to accurately and comprehensively predict Kap?2 substrates in genomes. Public Health Relevance: This grant aims to discover how protein cargoes are recognized by nuclear export factor CRM1 to be exported from the cell nucleus. CRM1 is the most general and versatile nuclear export factor known to date and our work will inform on this basic cellular process as well as explain a process that when disrupted can result in heart disease and cancer. We will also study the reverse process of how protein cargoes are recognized by nuclear import factor Kap-beta2/Transportin.