Bioassays to monitor taspase1 function for the identification of pharmacogenetic inhibitors

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)biolog
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]benzenesulfonamideand 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.
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Author:Shirley Knauer, Verena Fetz, Jens Rabenstein, Sandra Friedl, Bettina Hofmann, Samaneh Sabiani, Elisabeth Schröder, Lena Kunst, Eugen Proschak, Eckhard Thines, Thomas Kindler, Gisbert Schneider, Rolf Marschalek, Roland Stauber, Carolin Bier
URN:urn:nbn:de:hebis:30-115096
DOI:http://dx.doi.org/10.1371/journal.pone.0018253
ISSN:1932-6203
Parent Title (English):PLoS ONE
Parent Title (English):PLoS one
Publisher:PLoS
Place of publication:Lawrence, Kan.
Document Type:Article
Language:English
Date of Publication (online):2011/09/08
Year of first Publication:2011
Publishing Institution:Univ.-Bibliothek Frankfurt am Main
Release Date:2011/09/08
Volume:6
Issue:5 (e18253)
Pagenumber:14
First Page:1
Last Page:14
Note:
Copyright: © 2011 Knauer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
HeBIS PPN:276737326
Institutes:Biochemie und Chemie
Pharmazie
Zentrum für Arzneimittelforschung, Entwicklung und Sicherheit
Dewey Decimal Classification:570 Biowissenschaften; Biologie
Sammlungen:Universitätspublikationen
Sondersammelgebiets-Volltexte
Licence (German):License LogoCreative Commons - Namensnennung 3.0

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