Biochemical and cellular functions of Karyopherins - Revision - 1

授权摘要R35 GM 141461： 二十种同源的Karyopherin-β（Kap）蛋白介导大部分蛋白质转运 在细胞核和细胞质之间。鸡实验室是了解 Kaps控制大分子交通的机制。我们达成这样的共识 通过结构，生物化学，细胞生物学和生物信息学分析的组合， Kap-ligand相互作用我们对核输入受体Kapβ2的研究发现， 脯氨酸-酪氨酸核定位信号（PY-NLS），它识别。这项研究导致 一个新的概念--许多核靶向信号不仅仅是由序列定义的 而是通过一系列的物理特征--这解释了Kapβ2是如何识别数百个 不同序列的组合，并使我们能够设计第一个核输入抑制剂。虽然 最近已经确定了另外两种NLS类型，大多数进口商都能识别货物， 包括Importins-4、-7、-8、-9和-11，尽管它们占50%，但仍不被理解 核进口。他们的货物很少是已知的，他们认识的NLS类是 未知缺失的知识模糊了我们对大部分的 缺乏可识别的NLS的蛋白质组被运输到细胞核中。我们的目标是发现 新的货物，类NLS和途径特异性抑制剂的所有输入。我们将联合收割机 这些知识与生物信息学分析和实验，以确定完整的货物库 每个输入，从而映射他们各自控制的流量和蜂窝进程。 我们发现，除了介导核输入，importins也作为伴侣。 我们将了解Kap β 2分子伴侣如何与RNA结合蛋白结合，防止其聚集， 理解组蛋白中Importin-9组蛋白伴侣功能的功能意义 储存和核小体组装。在我们对核出口的研究中，我们第一个发现 CRM 1如何识别唯一已知的核输出信号（内斯）。这项工作是 抗癌药物Selinexor的开发基础，以及物理/化学 我们后来揭示的抑制机制有助于FDA批准该药物。我们 还揭示了内斯的序列和结构简并性，解释了CRM 1是如何被 能够识别数百种不同的内斯序列。CRM 1还出口了数千个 mRNAs，但对其机制知之甚少。我们将研究CRM 1-mRNA的输出 复合物来阐明基因表达的这一关键步骤。最后，我们将扩大研究 输出蛋白Msn 5，它结合固有的无序和磷酸化的片段， 多件货物。这项工作将揭示第二类新的内斯。

Abstract of grant R35GM141461: Twenty homologous Karyopherin- (Kap) proteins mediate the majority of protein transport between the nucleus and the cytoplasm. The Chook Lab is a leader in understanding the mechanisms by which Kaps control macromolecular traffic. We achieve this understanding through a combination of structural, biochemical, cell biological and bioinformatic analyses of Kap-ligand interactions. Our studies of the nuclear import receptor Kapβ2 led to discovery of the proline-tyrosine nuclear localization signal (PY-NLS), which it recognizes. This study led to a new concept—that many nuclear targeting signals are defined not merely by sequence but by a collection of physical features—which explained how Kapβ2 can recognize hundreds of different sequences, and enabled us to design the first nuclear import inhibitor. Although two additional NLS types have recently been determined, cargo recognition by most importins, including Importins-4, -7, -8, -9 and -11 is not understood even though they account for 50% of nuclear import. Few of their cargos are known and the NLS classes that they recognize are unknown. The missing knowledge obscures our understanding of how a significant portion of the proteome lacking recognizable NLSs are trafficked into the nucleus. We aim to discover new cargos, classes of NLSs and pathway-specific inhibitors for all importins. We will combine this knowledge with bioinformatic analyses and experiments to define the full cargo repertoires of each importin, and thus map both the traffic and cellular processes that they each control. We showed that in addition to mediating nuclear import, importins also act as chaperones. We will learn how Kap2 chaperones RNA-binding proteins, preventing their aggregation and understand the functional significance of the Importin-9 histone-chaperone function in histone storage and nucleosome assembly. In our studies of nuclear export, we were the first to reveal how CRM1 recognizes the only known nuclear export signal (NES). This work was foundational for development of the anti-cancer drug Selinexor, and the physical/chemical mechanisms of inhibition that we later revealed contributed to FDA-approval of the drug. We also revealed the sequence and structural degeneracy of the NES, explaining how CRM1 is able to recognize hundreds of diverse NES sequences. CRM1 also exports thousands of mRNAs, but little is known about the mechanism. We will study CRM1-mRNA export complexes to shed light on this critical step of gene expression. Finally, we will expand study to the exportin Msn5, which binds intrinsically disordered and phosphorylated segments of multiple cargos. This work will reveal a second, new class of NES.