The prostaglandin 15-deoxy- Δ12,14 -PGJ2 inhibits CRM1 mediated protein export. Analysis of nuclear import of human telomerase reverse transcriptase
by Cornelia Frohnert
Date of Examination:2013-08-15
Date of issue:2014-02-07
Advisor:Prof. Dr. Ralph Kehlenbach
Referee:Prof. Dr. Ralph Kehlenbach
Referee:Prof. Dr. Ralf Ficner
Persistent Address:
http://dx.doi.org/10.53846/goediss-4344
Files in this item
Name:Dissertation Cornelia Frohnert_The prostagla...pdf
Size:2.78Mb
Format:PDF
The following license files are associated with this item:
Abstract
English
Nuclear import and export of macromoloecules are essential processes in eukaryotic cells. In consequence, different mechanisms of transport developed in the cell, resulting in the involvement of different monodirectional or bidirectional transport receptors, which recognize specific sequences or regions in their cargos. CRM1, the major cellular export receptor can be specifically inhibited by the fungal compound leptomycin B (LMB). The inhibitor binds to a sensitive cysteine residue (at position 528) in CRM1 resulting in a blockade of the NES recognition site (Kudo et al., 1999; Sun et al., 2013). The question arose whether endogenous substances exist, which also have the ability to inhibit CRM1 mediated protein export in a similar way. The prostaglandin 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) was found to exhibit a partly similar structure like LMB and CRM1 was identified as a target of 15d-PGJ2. Mass spectrometry analysis identified 15d-PGJ2 to modify CRM1 at the same cysteine residue like LMB. After incubation of cells with 15d-PGJ2, inhibition of CRM1- dependent export was observed. Also kinetic measurements of CRM1 mediated protein export in living cells showed a clear decrease in export capability of endogenous CRM1 after incubation of cells with 15d-PGJ2. Rescue experiments using an LMB insensitive CRM1 with a mutation at cysteine 528 also suggested that 15d-PGJ2 modifies CRM1 at the same residue as LMB (Hilliard et al., 2010). As a conclusion we present a CRM1 inhibitor which can be produced endogenously by cells e.g. during an inflammatory response. In contrast to export, import of proteins is largely mediated by import receptors from the importin-β superfamily. Import receptors recognize their cargos at specific sequences and some of these sequences are already mapped and classified as nuclear localization signals (NLS). For some of import receptors, no specific consensus sequence for cargo recognition could be identified so far. In consequence, the import pathways of many nuclear proteins still remain elusive. In this work we gained further insights into the nuclear import of the protein subunit of telomerase reverse transcriptase (TERT), which catalyzes the de novo synthesis of telomeric chromosome ends. Previous studies identified the cytoplasmic filament nucleoporin Nup358 to be involved in TERT nuclear import (Hutten, 2007). After depletion of Nup358, TERT showed an import defect, resulting in a cytoplasmic or equal cellular localization instead of a nuclear localization. In this work, we were able to identify a zinc finger region in Nup358 to play a crucial role in TERT nuclear import. Using mutagenesis, we discovered two independent NLS sites in the TERT protein, which contribute to nuclear import of TERT. By siRNA mediated depletion of major eukaryotic import receptors we were able to identify importin-7 as a responsible import receptor of TERT. FLIP experiments, measuring the import kinetics of TERT showed a clear decrease in TERT nuclear import upon depletion of importin-7. Rescue experiments using a siRNA resistant mutant of importin-7 confirmed these results. Together, our findings provide further detailed insights into the nuclear import pathways of TERT pointing to important roles of Nup358 and importin-7.
Keywords: inhibition of CRM1; import of human telomerase reverse transcriptase; import of hTERT; prostaglandin