Limits...
Identification Of Small Molecule TRABID Deubiquitinase Inhibitors By Computation-Based Virtual Screen.

Shi T, Bao J, Wang NX, Zheng J, Wu D - BMC Chem Biol (2012)

Bottom Line: However, these inhibitors failed to show inhibitory effects on β-catenin-mediated gene transcription.In addition, expression of TRABID shRNAs, wildtype TRABID, or the DUB activity-deficient mutant showed little effects on β-catenin-mediated gene transcription.TRABID may not be a critical component in canonical Wnt/β-catenin signal transduction or that a minute amount of this protein is sufficient for its role in regulating Wnt activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510, USA. jie.zheng@stjude.org.

ABSTRACT

Background: Wnt/β-catenin-mediated gene transcription plays important roles in a wide range of biological and pathophysiological processes including tumorigenesis where β-catenin-mediated transcription activity frequently elevates. TRABID, a deubiquitinase, was shown to have a positive Wnt/β-catenin-mediated gene transcription and hence holds a promise as a putative anti-cancer target.

Results: In this study, we used a combination of structure based virtual screening and an in vitro deubiquitinase (DUB) assay to identify several small molecules that inhibit TRABID DUB activity. However, these inhibitors failed to show inhibitory effects on β-catenin-mediated gene transcription. In addition, expression of TRABID shRNAs, wildtype TRABID, or the DUB activity-deficient mutant showed little effects on β-catenin-mediated gene transcription.

Conclusions: TRABID may not be a critical component in canonical Wnt/β-catenin signal transduction or that a minute amount of this protein is sufficient for its role in regulating Wnt activity.

No MeSH data available.


Related in: MedlinePlus

TRABID DUB activity assays and structural modeling of TRABID OTU domain. A) Sequence alignment of TRABID OTU domain and A20 OTU domain, the identical (yellow) and homologous (green) pairs are highlighted. B,C) Cleavage of K63 Chains by Recombinant TRABID. Hexa-K63 ubiquitin (0.01 ug/ul) were incubated with recombinant full length TRABID protein prepared from bacteria (B) or Flag-TRABID or Flag-TRABID-C443S precipitated by anti-FLAG agarose from transfected HEK293T cells (C) for 3 hrs in the DUB reaction buffer. Samples were analyzed by Western under a non-reducing condition using an anti-ubiquitin antibody. D-F) Modeled structure of TRABID OTU domain based on the crystal structure of A20 OTU domain. The modeled catalytic site of TRABID OTU domain is shown in D. The detected potential pocket of TRABID OTU domain is shown in E and the corresponding pocket in A20 is shown in F for comparison. The modeled Cys443 is highlighted in sphere representation in E and F.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3475094&req=5

Figure 1: TRABID DUB activity assays and structural modeling of TRABID OTU domain. A) Sequence alignment of TRABID OTU domain and A20 OTU domain, the identical (yellow) and homologous (green) pairs are highlighted. B,C) Cleavage of K63 Chains by Recombinant TRABID. Hexa-K63 ubiquitin (0.01 ug/ul) were incubated with recombinant full length TRABID protein prepared from bacteria (B) or Flag-TRABID or Flag-TRABID-C443S precipitated by anti-FLAG agarose from transfected HEK293T cells (C) for 3 hrs in the DUB reaction buffer. Samples were analyzed by Western under a non-reducing condition using an anti-ubiquitin antibody. D-F) Modeled structure of TRABID OTU domain based on the crystal structure of A20 OTU domain. The modeled catalytic site of TRABID OTU domain is shown in D. The detected potential pocket of TRABID OTU domain is shown in E and the corresponding pocket in A20 is shown in F for comparison. The modeled Cys443 is highlighted in sphere representation in E and F.

Mentions: To screen for compounds that can inhibit TRABID DUB activity in vitro, we established an assay to measure the DUB activity of TRABID. Consistent with the previous report by Tran et al. [11], we observed that recombinant TRABID proteins purified from an E coli expression system or pulled down from HEK293 cells overexpressing Flag-TRABID by an Flag antibody were able to specifically cleave hexa-K63 (Figure 1B,C), but not penta-K48 (data not shown), ubiquitin chains. We also tested a TRABID DUB-deficient mutant containing a substitution mutation (C443A), a residue located in the OTU domain and critical for its catalytic activity [11]. The mutation abrogated the ability of TRABID to cleave the Hexa-K63 ubiquitin substrate (Figure 1C), confirming the importance of this residue for the DUB activity. Homology modeling of TRABID OTU domain based on the crystal structure of A20 OTU domain [17] revealed that the common cysteine catalytic site was well conserved in TRABID. This site is characterized by a cysteine residue that forms an electrostatic network with a histidine and a residue with an acidic side chain. In TRABID, these 3 residues are C443, H596 and D410, respectively, based on the homology model (Figure 1D).


Identification Of Small Molecule TRABID Deubiquitinase Inhibitors By Computation-Based Virtual Screen.

Shi T, Bao J, Wang NX, Zheng J, Wu D - BMC Chem Biol (2012)

TRABID DUB activity assays and structural modeling of TRABID OTU domain. A) Sequence alignment of TRABID OTU domain and A20 OTU domain, the identical (yellow) and homologous (green) pairs are highlighted. B,C) Cleavage of K63 Chains by Recombinant TRABID. Hexa-K63 ubiquitin (0.01 ug/ul) were incubated with recombinant full length TRABID protein prepared from bacteria (B) or Flag-TRABID or Flag-TRABID-C443S precipitated by anti-FLAG agarose from transfected HEK293T cells (C) for 3 hrs in the DUB reaction buffer. Samples were analyzed by Western under a non-reducing condition using an anti-ubiquitin antibody. D-F) Modeled structure of TRABID OTU domain based on the crystal structure of A20 OTU domain. The modeled catalytic site of TRABID OTU domain is shown in D. The detected potential pocket of TRABID OTU domain is shown in E and the corresponding pocket in A20 is shown in F for comparison. The modeled Cys443 is highlighted in sphere representation in E and F.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475094&req=5

Figure 1: TRABID DUB activity assays and structural modeling of TRABID OTU domain. A) Sequence alignment of TRABID OTU domain and A20 OTU domain, the identical (yellow) and homologous (green) pairs are highlighted. B,C) Cleavage of K63 Chains by Recombinant TRABID. Hexa-K63 ubiquitin (0.01 ug/ul) were incubated with recombinant full length TRABID protein prepared from bacteria (B) or Flag-TRABID or Flag-TRABID-C443S precipitated by anti-FLAG agarose from transfected HEK293T cells (C) for 3 hrs in the DUB reaction buffer. Samples were analyzed by Western under a non-reducing condition using an anti-ubiquitin antibody. D-F) Modeled structure of TRABID OTU domain based on the crystal structure of A20 OTU domain. The modeled catalytic site of TRABID OTU domain is shown in D. The detected potential pocket of TRABID OTU domain is shown in E and the corresponding pocket in A20 is shown in F for comparison. The modeled Cys443 is highlighted in sphere representation in E and F.
Mentions: To screen for compounds that can inhibit TRABID DUB activity in vitro, we established an assay to measure the DUB activity of TRABID. Consistent with the previous report by Tran et al. [11], we observed that recombinant TRABID proteins purified from an E coli expression system or pulled down from HEK293 cells overexpressing Flag-TRABID by an Flag antibody were able to specifically cleave hexa-K63 (Figure 1B,C), but not penta-K48 (data not shown), ubiquitin chains. We also tested a TRABID DUB-deficient mutant containing a substitution mutation (C443A), a residue located in the OTU domain and critical for its catalytic activity [11]. The mutation abrogated the ability of TRABID to cleave the Hexa-K63 ubiquitin substrate (Figure 1C), confirming the importance of this residue for the DUB activity. Homology modeling of TRABID OTU domain based on the crystal structure of A20 OTU domain [17] revealed that the common cysteine catalytic site was well conserved in TRABID. This site is characterized by a cysteine residue that forms an electrostatic network with a histidine and a residue with an acidic side chain. In TRABID, these 3 residues are C443, H596 and D410, respectively, based on the homology model (Figure 1D).

Bottom Line: However, these inhibitors failed to show inhibitory effects on β-catenin-mediated gene transcription.In addition, expression of TRABID shRNAs, wildtype TRABID, or the DUB activity-deficient mutant showed little effects on β-catenin-mediated gene transcription.TRABID may not be a critical component in canonical Wnt/β-catenin signal transduction or that a minute amount of this protein is sufficient for its role in regulating Wnt activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510, USA. jie.zheng@stjude.org.

ABSTRACT

Background: Wnt/β-catenin-mediated gene transcription plays important roles in a wide range of biological and pathophysiological processes including tumorigenesis where β-catenin-mediated transcription activity frequently elevates. TRABID, a deubiquitinase, was shown to have a positive Wnt/β-catenin-mediated gene transcription and hence holds a promise as a putative anti-cancer target.

Results: In this study, we used a combination of structure based virtual screening and an in vitro deubiquitinase (DUB) assay to identify several small molecules that inhibit TRABID DUB activity. However, these inhibitors failed to show inhibitory effects on β-catenin-mediated gene transcription. In addition, expression of TRABID shRNAs, wildtype TRABID, or the DUB activity-deficient mutant showed little effects on β-catenin-mediated gene transcription.

Conclusions: TRABID may not be a critical component in canonical Wnt/β-catenin signal transduction or that a minute amount of this protein is sufficient for its role in regulating Wnt activity.

No MeSH data available.


Related in: MedlinePlus