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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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Identification of residues required for productive Taspase1 cleavage in living cells.A.–C. Nuclear translocation of the indicated biosensor cleavage site mutants (TS-Cl2+CSmut) was analyzed in HeLa transfectants coexpressing the indicated biosensors together with Tasp- or inactive TaspT234V-mCherry. At least 200 fluorescent living cells were inspected, and representative examples are shown. Whereas substitution of Leu2 with Ile did not affect cleavage (A.), exchange of Asp1 with Ala completely abrogated cleavage (B.) LMB treatment verified that nuclear import of the variants was not affected. (C.) Scale bars, 10 µm. D./E. Cleavage of indicated cleavage site mutants by Tasp- (D.) or inactive TaspT234V-mCh (E.) analyzed by immunoblot. Notably, D1′A, D3′A and D4′A mutants run lower in the gel, most likely due to the loss of the negative charge. Expression of Taspase1 proteins as well as of cleavage products in 293T cell lysates was visualized using α-GST or -Taspase1 Abs. GAPDH served as loading control.
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pone-0018253-g005: Identification of residues required for productive Taspase1 cleavage in living cells.A.–C. Nuclear translocation of the indicated biosensor cleavage site mutants (TS-Cl2+CSmut) was analyzed in HeLa transfectants coexpressing the indicated biosensors together with Tasp- or inactive TaspT234V-mCherry. At least 200 fluorescent living cells were inspected, and representative examples are shown. Whereas substitution of Leu2 with Ile did not affect cleavage (A.), exchange of Asp1 with Ala completely abrogated cleavage (B.) LMB treatment verified that nuclear import of the variants was not affected. (C.) Scale bars, 10 µm. D./E. Cleavage of indicated cleavage site mutants by Tasp- (D.) or inactive TaspT234V-mCh (E.) analyzed by immunoblot. Notably, D1′A, D3′A and D4′A mutants run lower in the gel, most likely due to the loss of the negative charge. Expression of Taspase1 proteins as well as of cleavage products in 293T cell lysates was visualized using α-GST or -Taspase1 Abs. GAPDH served as loading control.

Mentions: Next, to uncover the sequence and spatial requirements for Taspase1 processing in vivo, we performed Ala scan mutagenesis of the MLL cleavage site (CS2; aa 2713KISQLD↓GVDD2722) in the biosensor background. As depicted in Figure 5, coexpression of the TS-Cl2+ mutants (TS-Cl2+CSmut) with Tasp-mCh resulted in proteolytic cleavage and nuclear accumulation of only those biosensors in which non-essential residues were mutated. In contrast, changing critical aa into Ala almost completely prevented cleavage and nuclear accumulation of the autofluorescent proteins, leading to the following consensus sequence: K6I5S4Q3L2D1↓G1′V2′D3′D4′ (essential aa in bold; see Table 1 for summarized results of targets). Notably, even replacing critical residues by chemically similar aa could not rescue cleavage, exempt the exchange of Leu for the also hydrophobic aa Ile or Val (Figure 5A/B and Table 1). These results could be confirmed by immunoblot analysis (Figure 5D/E). Again, specificity of the assay was verified by cotransfection of inactive TaspT234V-mCh, which did neither result in cleavage nor nuclear accumulation of the biosensors (Suppl. Figure S2C). Nuclear accumulation of all the TS-Cl2+CSmut variants upon LMB treatment further excluded the formal possibility that mutagenesis had affected import of the biosensors (Figure 5C). Collectively, these results underline the reliability and practical advantages of our visual cell based assay to probe Taspase1 function in living cells.


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)

Identification of residues required for productive Taspase1 cleavage in living cells.A.–C. Nuclear translocation of the indicated biosensor cleavage site mutants (TS-Cl2+CSmut) was analyzed in HeLa transfectants coexpressing the indicated biosensors together with Tasp- or inactive TaspT234V-mCherry. At least 200 fluorescent living cells were inspected, and representative examples are shown. Whereas substitution of Leu2 with Ile did not affect cleavage (A.), exchange of Asp1 with Ala completely abrogated cleavage (B.) LMB treatment verified that nuclear import of the variants was not affected. (C.) Scale bars, 10 µm. D./E. Cleavage of indicated cleavage site mutants by Tasp- (D.) or inactive TaspT234V-mCh (E.) analyzed by immunoblot. Notably, D1′A, D3′A and D4′A mutants run lower in the gel, most likely due to the loss of the negative charge. Expression of Taspase1 proteins as well as of cleavage products in 293T cell lysates was visualized using α-GST or -Taspase1 Abs. GAPDH served as loading control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102056&req=5

pone-0018253-g005: Identification of residues required for productive Taspase1 cleavage in living cells.A.–C. Nuclear translocation of the indicated biosensor cleavage site mutants (TS-Cl2+CSmut) was analyzed in HeLa transfectants coexpressing the indicated biosensors together with Tasp- or inactive TaspT234V-mCherry. At least 200 fluorescent living cells were inspected, and representative examples are shown. Whereas substitution of Leu2 with Ile did not affect cleavage (A.), exchange of Asp1 with Ala completely abrogated cleavage (B.) LMB treatment verified that nuclear import of the variants was not affected. (C.) Scale bars, 10 µm. D./E. Cleavage of indicated cleavage site mutants by Tasp- (D.) or inactive TaspT234V-mCh (E.) analyzed by immunoblot. Notably, D1′A, D3′A and D4′A mutants run lower in the gel, most likely due to the loss of the negative charge. Expression of Taspase1 proteins as well as of cleavage products in 293T cell lysates was visualized using α-GST or -Taspase1 Abs. GAPDH served as loading control.
Mentions: Next, to uncover the sequence and spatial requirements for Taspase1 processing in vivo, we performed Ala scan mutagenesis of the MLL cleavage site (CS2; aa 2713KISQLD↓GVDD2722) in the biosensor background. As depicted in Figure 5, coexpression of the TS-Cl2+ mutants (TS-Cl2+CSmut) with Tasp-mCh resulted in proteolytic cleavage and nuclear accumulation of only those biosensors in which non-essential residues were mutated. In contrast, changing critical aa into Ala almost completely prevented cleavage and nuclear accumulation of the autofluorescent proteins, leading to the following consensus sequence: K6I5S4Q3L2D1↓G1′V2′D3′D4′ (essential aa in bold; see Table 1 for summarized results of targets). Notably, even replacing critical residues by chemically similar aa could not rescue cleavage, exempt the exchange of Leu for the also hydrophobic aa Ile or Val (Figure 5A/B and Table 1). These results could be confirmed by immunoblot analysis (Figure 5D/E). Again, specificity of the assay was verified by cotransfection of inactive TaspT234V-mCh, which did neither result in cleavage nor nuclear accumulation of the biosensors (Suppl. Figure S2C). Nuclear accumulation of all the TS-Cl2+CSmut variants upon LMB treatment further excluded the formal possibility that mutagenesis had affected import of the biosensors (Figure 5C). Collectively, these results underline the reliability and practical advantages of our visual cell based assay to probe Taspase1 function in living cells.

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