Biochemical and cellular functions of Karyopherins

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The Chook Laboratory aims to understand mechanisms of how Karyopherin- proteins recognize binding partners and map partner/cargo repertoires. We aim to understand nuclear-cytoplasmic transport, other karyopherin functions, and discover how they organize and regulate cellular functions, in health and in disease. 20 homologous human and 14 S. cerevisiae Kaps mediate the majority of nuclear transport. We have studied the importin Karyopherin-β2 extensively. We discovered the PY-NLS that it recognizes, characterized the physical organization of this signal and designed the first nuclear import inhibitor. Karyopherin-β2 and other well-characterized importins, Importin-/β, Importin-5 and Transportin-SR, have distinct specificities and bind entirely different NLS types. However, the remaining importins: Importin-4, Importin-7, Importin-8, Importin-9 and Importin-11, are under-studied with few known cargos. Scarcity of cargos has prevented comparative biochemical/structural definition of their NLSs. We aim to discover new cargos and classes of NLSs for understudied importins and map the traffic they control. We showed that in addition to importing cargos, importins also act as chaperones to prevent aggregation of RNA-binding proteins or act as histone chaperone to prevent histone H2A-H2B aggregation and assist in nucleosome assembly. We will address the mechanism of Kap2 chaperone functions, and how Kap114 imports and chaperones H2A-H2B in the presence of canonical histone chaperones. In the study of nuclear export, we have contributed significantly to the understanding of how CRM1 binds NESs and small molecule inhibitors, but there are many more questions given CRM1’s importance in many cellular processes and disease states. CRM1 inhibitor Selinexor causes apoptosis of cancer cells, but it is not known which cargos are targeted in different cancers. Most of the >1000 NES-containing CRM1 cargos are not known. Accurate NES prediction could help identify new cargos, but diverse NES sequences and vague consensus that describes sequences ubiquitous in most helix- containing proteins make sequence-based NES prediction inefficient. To improve prediction, we are developing a structure- and energy-based NES predictor. We will also study how CRM1 is degraded in response to inhibitors, understand how the oncogenic E571K mutation of CRM1 affects NES-binding and CRM1-mediated traffic, and study CRM1-mediated mRNA export. Finally, we will expand our study to the exportin Msn5, which binds intrinsically disordered and phosphorylated segments of multiple cargos, hence an excellent system to define a new NES class.