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
Bernis, Cyril; Swift-Taylor, Beth; Nord, Matthew et al. (2014) Transportin acts to regulate mitotic assembly events by target binding rather than Ran sequestration. Mol Biol Cell 25:992-1009
Fung, Ho Yee Joyce; Chook, Yuh Min (2014) Atomic basis of CRM1-cargo recognition, release and inhibition. Semin Cancer Biol 27:52-61
Sun, Qingxiang; Carrasco, Yazmin P; Hu, Youcai et al. (2013) Nuclear export inhibition through covalent conjugation and hydrolysis of Leptomycin B by CRM1. Proc Natl Acad Sci U S A 110:1303-8
Etchin, J; Sun, Q; Kentsis, A et al. (2013) Antileukemic activity of nuclear export inhibitors that spare normal hematopoietic cells. Leukemia 27:66-74
Soniat, Michael; Sampathkumar, Parthasarathy; Collett, Garen et al. (2013) Crystal structure of human Karyopherin *2 bound to the PY-NLS of Saccharomyces cerevisiae Nab2. J Struct Funct Genomics 14:31-5
Zhang, Zi Chao; Satterly, Neal; Fontoura, Beatriz M A et al. (2011) Evolutionary development of redundant nuclear localization signals in the mRNA export factor NXF1. Mol Biol Cell 22:4657-68
Chook, Yuh Min; Suel, Katherine E (2011) Nuclear import by karyopherin-*s: recognition and inhibition. Biochim Biophys Acta 1813:1593-606
Xu, Darui; Farmer, Alicia; Chook, Yuh Min (2010) Recognition of nuclear targeting signals by Karyopherin-ýý proteins. Curr Opin Struct Biol 20:782-90
Suel, Katherine E; Chook, Yuh Min (2009) Kap104p imports the PY-NLS-containing transcription factor Tfg2p into the nucleus. J Biol Chem 284:15416-24
Dong, Xiuhua; Biswas, Anindita; Suel, Katherine E et al. (2009) Structural basis for leucine-rich nuclear export signal recognition by CRM1. Nature 458:1136-41

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