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dc.contributor.author
Knauer, Shirley K.
dc.contributor.author
Fetz, Verena
dc.contributor.author
Rabenstein, Jens
dc.contributor.author
Friedl, Sandra
dc.contributor.author
Hofmann, Bettina
dc.contributor.author
Sabiani, Samaneh
dc.contributor.author
Schröder, Elisabeth
dc.contributor.author
Kunst, Lena
dc.contributor.author
Proschak, Eugen
dc.contributor.author
Thines, Eckhard
dc.contributor.author
Kindler, Thomas
dc.contributor.author
Schneider, Gisbert
dc.contributor.author
Marschalek, Rolf
dc.contributor.author
Stauber, Roland H.
dc.contributor.author
Bier, Carolin
dc.date.accessioned
2018-11-01T10:21:38Z
dc.date.available
2017-06-14T18:03:42Z
dc.date.available
2018-11-01T10:21:38Z
dc.date.issued
2011-05-25
dc.identifier.issn
1932-6203
dc.identifier.other
10.1371/journal.pone.0018253
en_US
dc.identifier.uri
http://hdl.handle.net/20.500.11850/159912
dc.identifier.doi
10.3929/ethz-b-000159912
dc.description.abstract
Background Threonine Aspartase 1 (Taspase1) mediates cleavage of the mixed lineage leukemia (MLL) protein and leukemia provoking MLL-fusions. In contrast to other proteases, the understanding of Taspase1's (patho)biological relevance and function is limited, since neither small molecule inhibitors nor cell based functional assays for Taspase1 are currently available. Methodology/Findings Efficient cell-based assays to probe Taspase1 function in vivo are presented here. These are composed of glutathione S-transferase, autofluorescent protein variants, Taspase1 cleavage sites and rational combinations of nuclear import and export signals. The biosensors localize predominantly to the cytoplasm, whereas expression of biologically active Taspase1 but not of inactive Taspase1 mutants or of the protease Caspase3 triggers their proteolytic cleavage and nuclear accumulation. Compared to in vitro assays using recombinant components the in vivo assay was highly efficient. Employing an optimized nuclear translocation algorithm, the triple-color assay could be adapted to a high-throughput microscopy platform (Z'factor = 0.63). Automated high-content data analysis was used to screen a focused compound library, selected by an in silico pharmacophor screening approach, as well as a collection of fungal extracts. Screening identified two compounds, N-[2-[(4-amino-6-oxo-3H-pyrimidin-2-yl)sulfanyl]ethyl]benzenesulfonamide and 2-benzyltriazole-4,5-dicarboxylic acid, which partially inhibited Taspase1 cleavage in living cells. Additionally, the assay was exploited to probe endogenous Taspase1 in solid tumor cell models and to identify an improved consensus sequence for efficient Taspase1 cleavage. This allowed the in silico identification of novel putative Taspase1 targets. Those include the FERM Domain-Containing Protein 4B, the Tyrosine-Protein Phosphatase Zeta, and DNA Polymerase Zeta. Cleavage site recognition and proteolytic processing of these substrates were verified in the context of the biosensor. Conclusions The assay not only allows to genetically probe Taspase1 structure function in vivo, but is also applicable for high-content screening to identify Taspase1 inhibitors. Such tools will provide novel insights into Taspase1's function and its potential therapeutic relevance.
en_US
dc.format
application/pdf
en_US
dc.language.iso
en
en_US
dc.publisher
Public Library of Science
en_US
dc.rights.uri
http://creativecommons.org/licenses/by/3.0/
dc.title
Bioassays to Monitor Taspase 1 Function for the Identification of Pharmacogenetic Inhibitors
en_US
dc.type
Journal Article
dc.rights.license
Creative Commons Attribution 3.0 Unported
ethz.journal.title
PLoS ONE
ethz.journal.volume
6
en_US
ethz.journal.issue
5
en_US
ethz.journal.abbreviated
PLoS ONE
ethz.pages.start
e18253
en_US
ethz.size
14 p.
en_US
ethz.version.deposit
publishedVersion
en_US
ethz.identifier.nebis
006206116
ethz.publication.place
Lawrence, KS, USA
en_US
ethz.publication.status
published
en_US
ethz.leitzahl
ETH Zürich::00002 - ETH Zürich::00012 - Lehre und Forschung::00007 - Departemente::02020 - Dep. Chemie und Angewandte Biowiss. / Dep. of Chemistry and Applied Biosc.::02534 - Institut für Pharmazeutische Wiss. / Institute of Pharmaceutical Sciences::03852 - Schneider, Gisbert / Schneider, Gisbert
en_US
ethz.leitzahl.certified
ETH Zürich::00002 - ETH Zürich::00012 - Lehre und Forschung::00007 - Departemente::02020 - Dep. Chemie und Angewandte Biowiss. / Dep. of Chemistry and Applied Biosc.::02534 - Institut für Pharmazeutische Wiss. / Institute of Pharmaceutical Sciences::03852 - Schneider, Gisbert / Schneider, Gisbert
ethz.date.deposited
2017-06-14T18:05:28Z
ethz.source
ECIT
ethz.identifier.importid
imp59364e6e7addf64634
ethz.ecitpid
pub:64650
ethz.eth
yes
en_US
ethz.availability
Open access
en_US
ethz.rosetta.installDate
2017-07-15T00:13:42Z
ethz.rosetta.lastUpdated
2018-11-01T10:21:44Z
ethz.rosetta.versionExported
true
ethz.COinS
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