| Influence of active nuclear transport on caspase-3 localization and apoptotic chromatin changes | |
Abstract/OtherAbstract
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The subcellular localization of caspase-3 in a healthy cell was determined to be mainly cytosolic. In response to apoptotic stimuli, the apoptotic protease enters the nucleus. The mechanism employed remains the subject of controversy. In the presented study, the subcellular localization and the mechanism of nuclear entry of caspase-3 was studied employing a microinjection model. For this purpose, recombinant caspase-3 was overexpressed in E. coli and isolated in high purity. Fusion of GFP did not affect the the proteolytic properties of the enzyme as demonstrated by strong DEVD-afc cleavage by the recombinant protease. Injection of recombinant caspase-3 elicited the morphological features associated with apoptosis, accompanied by rapid equilibration of the apoptotic effector across the nuclear envelope. Both the inhibition of caspase-9 and caspase-3 activity failed to result in retention of the protease in the cytoplasm. Additionally, the rearrangement of the catalytic subunits following proteolytic activation of caspase-3 did not influence its cellular distribution. At the same time, the significantly smaller 40 kDa dextran was excluded from the nuclear compartment. Therefore, passive diffusion can be ruled out as the mode for caspase-3 entry into the nucleus. The observed accumulation of caspase-3 in the nuclear compartment relied on active transport processes, as demonstrated by the requirement for interaction with nucleoporins and energy dependence. The hypothetical import factors utilized by caspase-3 do not belong to the family of importins or transportins, as inhibition of these pathways failed to block caspase-3 accumulation in the nucleoplasm. Under conditions of import inhibition nuclear apoptosis is altered, as demonstrated by impaired chromatin condensation and DNA cleavage. The presence of caspase-3 was not required in the nucleus for the induction of these morphological features, indicating that the effectors are imported via an importin β dependent pathway from the cytosol following initiation of apoptosis. Inhibition of the importin β pathway sensitized HeLa cells to apoptosis, probably by preventing NF-κB dependent activation of transcription. Therefore, active transport processes are indispensable for mediating cellular resistance in response to TNFα ligation, as well as the proper execution of nuclear apoptosis. |
Authors
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Tautorat, Verena |
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Contributors
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Publication Detail
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Publisher : Universität Konstanz, Fachbereich Biologie. Fachbereich Biologie Type : Thesis.Doctoral Format : application/pdf |
Date Detail
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2004 |
Subject
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Apoptosis, Caspase-3, Chromatinkondensation, Kerntransport, DNA Fragmentierung, caspase-3, chromatin condensation, nuclear transport, DNA fragmentation, Life sciences |
Coverage
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Relation
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Source
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Copyright Information
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http://kops.ub.uni-konstanz.de/doku/urheberrecht.php |
Other Details
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Languages : eng |
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