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Bioassays to monitor Taspase1 function for the identification of pharmacogenetic inhibitors.

Knauer SK, Fetz V, Rabenstein J, Friedl S, Hofmann B, Sabiani S, Schröder E, Kunst L, Proschak E, Thines E, Kindler T, Schneider G, Marschalek R, Stauber RH, Bier C - PLoS ONE (2011)

Bottom Line: 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.Such tools will provide novel insights into Taspase1's function and its potential therapeutic relevance.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Biology, Centre for Medical Biotechnology (ZMB), University Duisburg-Essen, Essen, Germany.

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.

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Related in: MedlinePlus

Biosensor-based probing of Taspase1 function in vivo.A./B. Cleavage of the biosensor correlated with endogenous Taspase1 levels in adherent tumor cell lines. A. Indicated cell lines were transfected with equal amounts of TS-Cl2+ expression plasmid. 24 h later, localization of the biosensor was analyzed in at least 200 fluorescent cells displaying similar fluorescence intensity. Representative examples are shown. Cleavage-induced nuclear translocation differed significantly among tested cell lines. B. Endogenous Taspase1 levels were analysed by immunoblot using α-Tasp and -GAPDH Abs. C. Biosensor-based analysis of the proteolytic activity of Taspase1 variants in HeLa transfectants. Coexpression of TaspT234A- or TaspT234V-GFP fusion did not result in cleavage and nuclear accumulation of TS-Cl2+R. TaspD233A-GFP displayed a reduced enzymatic activity compared to wt Taspase1-GFP. Scale bars, 10 µm. Dashed lines mark nuclear/cytoplasmic cell boundaries obtained from the corresponding phase contrast images.
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pone-0018253-g004: Biosensor-based probing of Taspase1 function in vivo.A./B. Cleavage of the biosensor correlated with endogenous Taspase1 levels in adherent tumor cell lines. A. Indicated cell lines were transfected with equal amounts of TS-Cl2+ expression plasmid. 24 h later, localization of the biosensor was analyzed in at least 200 fluorescent cells displaying similar fluorescence intensity. Representative examples are shown. Cleavage-induced nuclear translocation differed significantly among tested cell lines. B. Endogenous Taspase1 levels were analysed by immunoblot using α-Tasp and -GAPDH Abs. C. Biosensor-based analysis of the proteolytic activity of Taspase1 variants in HeLa transfectants. Coexpression of TaspT234A- or TaspT234V-GFP fusion did not result in cleavage and nuclear accumulation of TS-Cl2+R. TaspD233A-GFP displayed a reduced enzymatic activity compared to wt Taspase1-GFP. Scale bars, 10 µm. Dashed lines mark nuclear/cytoplasmic cell boundaries obtained from the corresponding phase contrast images.

Mentions: Compounds and extracts were tested in 293T cells, which not express detectable levels of endogenous Taspase1 (Figure 4B). Cells coexpressing TS-Cl2+ and Tasp-mCh or TS-Cl2+R and Tasp-GFP (Figure 3C) were challenged in 96-well plates and analyzed 48 h after transfection to ensure that lack of inhibition is not due to slow intracellular entry rates of the substances. Although the majority of substances did not significantly affect Taspase1's trans cleavage activity at a concentration of 50 µM, we identified two compounds, N-[2-[(4-amino-6-oxo-3H-pyrimidin-2-yl)sulfanyl]ethyl]benzenesulfonamide (CHC-A4) and 2-benzyltriazole-4,5-dicarboxylic acid (DHC-C1), partially inhibiting biosensor translocation (Figure 3E). In contrast, the tested fungal extracts did not show detectable inhibition in our assay, although we observed cytotoxicity for some extracts (data not shown). As we previously identified transport inhibitors by chemicogenomic screens [20], we first verified that CHC-A4 and DHC-C1 did not affect nuclear import of the biosensor rather than cleavage. Treatment with LMB resulted in nuclear accumulation of TS-Cl2+R or TS-Cl2+ even in the presence of the inhibitors, excluding interference with nuclear import (Suppl. Figure S2C and data not shown). Taspase1 inhibition could be confirmed in other cell lines using a compound concentration of 50 µM, although no inhibition was detectable at a concentration of 5 µM (Figure 3D and data not shown). Factors contributing to the weak inhibitory activity observed may be compound instability and/or their inefficient cell entry. Hence, to circumvent these limitations, we directly microinjected both compounds into TS-Cl2+R/Tasp-GFP expressing transfectants. Compared to adding the compounds directly to the cell culture medium, cytoplasmic injection of both compounds resulted in improved Taspase1 inhibition reducing nuclear translocation of the biosensor in the majority of cells (Figure 3F). The coinjected fluorescent Ab allowed to select only healthy cells for the analysis showing no signs of damage due to the microinjection procedure. In order to allow a comparison of both experimental approaches, the cells were inspected after 48 h. The reason why inhibition did not occur in all injected cells is not known, indicating that rational chemical modification of the primary hits is required to improve their activity.


Bioassays to monitor Taspase1 function for the identification of pharmacogenetic inhibitors.

Knauer SK, Fetz V, Rabenstein J, Friedl S, Hofmann B, Sabiani S, Schröder E, Kunst L, Proschak E, Thines E, Kindler T, Schneider G, Marschalek R, Stauber RH, Bier C - PLoS ONE (2011)

Biosensor-based probing of Taspase1 function in vivo.A./B. Cleavage of the biosensor correlated with endogenous Taspase1 levels in adherent tumor cell lines. A. Indicated cell lines were transfected with equal amounts of TS-Cl2+ expression plasmid. 24 h later, localization of the biosensor was analyzed in at least 200 fluorescent cells displaying similar fluorescence intensity. Representative examples are shown. Cleavage-induced nuclear translocation differed significantly among tested cell lines. B. Endogenous Taspase1 levels were analysed by immunoblot using α-Tasp and -GAPDH Abs. C. Biosensor-based analysis of the proteolytic activity of Taspase1 variants in HeLa transfectants. Coexpression of TaspT234A- or TaspT234V-GFP fusion did not result in cleavage and nuclear accumulation of TS-Cl2+R. TaspD233A-GFP displayed a reduced enzymatic activity compared to wt Taspase1-GFP. Scale bars, 10 µm. Dashed lines mark nuclear/cytoplasmic cell boundaries obtained from the corresponding phase contrast images.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102056&req=5

pone-0018253-g004: Biosensor-based probing of Taspase1 function in vivo.A./B. Cleavage of the biosensor correlated with endogenous Taspase1 levels in adherent tumor cell lines. A. Indicated cell lines were transfected with equal amounts of TS-Cl2+ expression plasmid. 24 h later, localization of the biosensor was analyzed in at least 200 fluorescent cells displaying similar fluorescence intensity. Representative examples are shown. Cleavage-induced nuclear translocation differed significantly among tested cell lines. B. Endogenous Taspase1 levels were analysed by immunoblot using α-Tasp and -GAPDH Abs. C. Biosensor-based analysis of the proteolytic activity of Taspase1 variants in HeLa transfectants. Coexpression of TaspT234A- or TaspT234V-GFP fusion did not result in cleavage and nuclear accumulation of TS-Cl2+R. TaspD233A-GFP displayed a reduced enzymatic activity compared to wt Taspase1-GFP. Scale bars, 10 µm. Dashed lines mark nuclear/cytoplasmic cell boundaries obtained from the corresponding phase contrast images.
Mentions: Compounds and extracts were tested in 293T cells, which not express detectable levels of endogenous Taspase1 (Figure 4B). Cells coexpressing TS-Cl2+ and Tasp-mCh or TS-Cl2+R and Tasp-GFP (Figure 3C) were challenged in 96-well plates and analyzed 48 h after transfection to ensure that lack of inhibition is not due to slow intracellular entry rates of the substances. Although the majority of substances did not significantly affect Taspase1's trans cleavage activity at a concentration of 50 µM, we identified two compounds, N-[2-[(4-amino-6-oxo-3H-pyrimidin-2-yl)sulfanyl]ethyl]benzenesulfonamide (CHC-A4) and 2-benzyltriazole-4,5-dicarboxylic acid (DHC-C1), partially inhibiting biosensor translocation (Figure 3E). In contrast, the tested fungal extracts did not show detectable inhibition in our assay, although we observed cytotoxicity for some extracts (data not shown). As we previously identified transport inhibitors by chemicogenomic screens [20], we first verified that CHC-A4 and DHC-C1 did not affect nuclear import of the biosensor rather than cleavage. Treatment with LMB resulted in nuclear accumulation of TS-Cl2+R or TS-Cl2+ even in the presence of the inhibitors, excluding interference with nuclear import (Suppl. Figure S2C and data not shown). Taspase1 inhibition could be confirmed in other cell lines using a compound concentration of 50 µM, although no inhibition was detectable at a concentration of 5 µM (Figure 3D and data not shown). Factors contributing to the weak inhibitory activity observed may be compound instability and/or their inefficient cell entry. Hence, to circumvent these limitations, we directly microinjected both compounds into TS-Cl2+R/Tasp-GFP expressing transfectants. Compared to adding the compounds directly to the cell culture medium, cytoplasmic injection of both compounds resulted in improved Taspase1 inhibition reducing nuclear translocation of the biosensor in the majority of cells (Figure 3F). The coinjected fluorescent Ab allowed to select only healthy cells for the analysis showing no signs of damage due to the microinjection procedure. In order to allow a comparison of both experimental approaches, the cells were inspected after 48 h. The reason why inhibition did not occur in all injected cells is not known, indicating that rational chemical modification of the primary hits is required to improve their activity.

Bottom Line: 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.Such tools will provide novel insights into Taspase1's function and its potential therapeutic relevance.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Biology, Centre for Medical Biotechnology (ZMB), University Duisburg-Essen, Essen, Germany.

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.

Show MeSH
Related in: MedlinePlus