Structures and mechanisms of nuclear import and export

描述（由申请人提供）：在人类细胞中，大分子的核胞质转运是由19个核蛋白家族成员（Kap2s/Importins/Exportins）介导的。Kap2s通过不同的核定位或输出信号（NLSs或NESs）结合其转运底物，并通过核孔复合物（NPC）进行转运。已知的NLS和NES的唯一类别是短的基本经典-NLS， PY-NLS和富含亮氨酸的NES (LR-NES)，由Kap?/Kap？1, Kap ?2（或Transportin）和CRM1。大多数其他Kap？S识别不同的序列，这些序列掩盖了它们信号的同一性。核输运比大多数Kap？S可以通过多个底物结合位点识别多个信号。本文描述了export-Kap？CRM1和进口因子Kap?2。CRM1是最通用和通用的出口kap ？对crm1 -底物复合物的结构研究将揭示识别多种NES类的一般原理，并从机制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。Kap吗?2向细胞核输入大量mRNA结合蛋白。我们最近在这些蛋白质中定义了一种新的NLS，称为PY-NLS，并发现了81个新的潜在Kap？生物信息学试点项目中的2种基质。在这里，我们提出了Kap？2 .与不同的PY-NLS相互作用，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：该资助旨在发现蛋白质货物如何被核输出因子CRM1识别并从细胞核输出。CRM1是迄今为止已知的最普遍、最通用的核输出因子，我们的工作将为这一基本细胞过程提供信息，并解释一个被破坏时可能导致心脏病和癌症的过程。我们还将研究核输入因子kap - β 2/转运蛋白如何识别蛋白质货物的逆向过程。

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.