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.

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM069909-09
Application #
8214560
Study Section
Nuclear Dynamics and Transport (NDT)
Program Officer
Ainsztein, Alexandra M
Project Start
2004-02-01
Project End
2013-01-31
Budget Start
2012-02-01
Budget End
2013-01-31
Support Year
9
Fiscal Year
2012
Total Cost
$307,751
Indirect Cost
$111,731
Name
University of Texas Sw Medical Center Dallas
Department
Pharmacology
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
Fu, Szu-Chin; Fung, Ho Yee Joyce; Ca?atay, Tolga et al. (2018) Correlation of CRM1-NES affinity with nuclear export activity. Mol Biol Cell 29:2037-2044
Ça?atay, Tolga; Chook, Yuh Min (2018) Karyopherins in cancer. Curr Opin Cell Biol 52:30-42
Pinarbasi, Emile S; Ca?atay, Tolga; Fung, Ho Yee Joyce et al. (2018) Active nuclear import and passive nuclear export are the primary determinants of TDP-43 localization. Sci Rep 8:7083
Yoshizawa, Takuya; Ali, Rustam; Jiou, Jenny et al. (2018) Nuclear Import Receptor Inhibits Phase Separation of FUS through Binding to Multiple Sites. Cell 173:693-705.e22
Guo, Lin; Kim, Hong Joo; Wang, Hejia et al. (2018) Nuclear-Import Receptors Reverse Aberrant Phase Transitions of RNA-Binding Proteins with Prion-like Domains. Cell 173:677-692.e20
Fung, Ho Yee Joyce; Fu, Szu-Chin; Chook, Yuh Min (2017) Nuclear export receptor CRM1 recognizes diverse conformations in nuclear export signals. Elife 6:
Hing, Z A; Fung, H Y J; Ranganathan, P et al. (2016) Next-generation XPO1 inhibitor shows improved efficacy and in vivo tolerability in hematological malignancies. Leukemia 30:2364-2372
Volpon, Laurent; Culjkovic-Kraljacic, Biljana; Osborne, Michael J et al. (2016) Importin 8 mediates m7G cap-sensitive nuclear import of the eukaryotic translation initiation factor eIF4E. Proc Natl Acad Sci U S A 113:5263-8
Soniat, Michael; Chook, Yuh Min (2016) Karyopherin-?2 Recognition of a PY-NLS Variant that Lacks the Proline-Tyrosine Motif. Structure 24:1802-1809
Soniat, Michael; Ca?atay, Tolga; Chook, Yuh Min (2016) Recognition Elements in the Histone H3 and H4 Tails for Seven Different Importins. J Biol Chem 291:21171-21183

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