Structures and mechanisms of nuclear import and export

描述（由申请方提供）：在人细胞中，大分子的核质转运由核转运蛋白2（Kap 2s/输入蛋白/输出蛋白）蛋白家族的19个成员介导。Kap 2通过不同的核定位或输出信号（NLS或NES）结合其转运底物，并通过核孔复合物（NPC）转运它们。唯一已知的NLS和内斯类别是短的碱性经典NLS、PY-NLS和富含亮氨酸的内斯（LR-内斯），由Kap？/凯普？1、Kap？2（或Transportin）和CRM 1。大多数其他卡普？它们识别各种各样的序列，这些序列掩盖了它们信号的同一性。核运输是进一步复杂的，因为大多数卡普？S可以通过多个底物结合位点识别多种信号。该提案描述了通过输出Kap？进行底物识别的结构和生化分析。CRM 1和进口因素Kap？2. CRM 1是最通用和通用的出口Kap？. CRM 1-底物复合物的结构研究将揭示识别多个内斯类的一般原理，并从机械上解释核输出的不同步骤，如输出复合物在细胞核中的组装和底物在细胞质中的解离。凯普？2将许多mRNA结合蛋白导入细胞核。我们最近定义了一个新的NLS在这些蛋白质称为PY-NLS和发现81个新的潜在的Kap？在生物信息学试点计划中使用2种底物。在这里，我们提出的结构和热力学研究的KAP？2与不同PY-NLS的相互作用，以加深对PY-NLS识别的生物物理理解，并为未来的计算工作生成结构和热力学训练数据集，以准确和全面地预测Kap？基因组中的2个底物。公共卫生相关性：该基金旨在发现蛋白质货物如何被核输出因子CRM 1识别并从细胞核中输出。CRM 1是迄今为止已知的最普遍和最通用的核输出因子，我们的工作将为这个基本的细胞过程提供信息，并解释一个当被破坏时可能导致心脏病和癌症的过程。我们还将研究蛋白质货物如何被核输入因子Kap-beta2/Transportin识别的反向过程。

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.