Journal of the American Chemical Society, Mar 14, 2016
In viral proteins, labile Zn-sites, where Zn2+ is crucial for maintaining the native protein stru... more In viral proteins, labile Zn-sites, where Zn2+ is crucial for maintaining the native protein structure but the Zn-bound cysteines are reactive, are promising drug targets. Here, we aim to (i) identify labile Zn-sites in viral proteins using guidelines established from our previous work and (ii) assess if clinically safe Zn-ejecting agents could eject Zn2+ from the predicted target site and thus inhibit viral replication. As proof-of-concept, we identified a labile Zn-site in the hepatitis C virus (HCV) NS5A protein and showed that the anti-alcoholism drug, disulfiram, could inhibit HCV replication to a similar extent as the clinically used antiviral agent, ribavirin. The discovery of a novel viral target and a new role for disulfiram in inhibiting HCV replication will enhance the therapeutic armamentarium against HCV. The strategy presented can also be applied to identify labile sites in other bacterial or viral proteins that can be targeted by disulfiram or other clinically safe Zn-ejectors.
In viral proteins, labile Zn-sites, where Zn2+ is crucial for maintaining the native protein stru... more In viral proteins, labile Zn-sites, where Zn2+ is crucial for maintaining the native protein structure but the Zn-bound cysteines are reactive, are promising drug targets. Here, we aim to (i) identify labile Zn-sites in viral proteins using guidelines established from our previous work and (ii) assess if clinically safe Zn-ejecting agents could eject Zn2+ from the predicted target site and thus inhibit viral replication. As proof-of-concept, we identified a labile Zn-site in the hepatitis C virus (HCV) NS5A protein and showed that the anti-alcoholism drug, disulfiram, could inhibit HCV replication to a similar extent as the clinically used antiviral agent, ribavirin. The discovery of a novel viral target and a new role for disulfiram in inhibiting HCV replication will enhance the therapeutic armamentarium against HCV. The strategy presented can also be applied to identify labile sites in other bacterial or viral proteins that can be targeted by disulfiram or other clinically safe Zn...
Protein science : a publication of the Protein Society, Jan 12, 2015
RNase T is a classical member of the DEDDh family of exonucleases with a unique sequence preferen... more RNase T is a classical member of the DEDDh family of exonucleases with a unique sequence preference in that its 3'-to-5' exonuclease activity is blocked by a 3'-terminal dinucleotide CC in digesting both single-stranded RNA and DNA. Our previous crystal structure analysis of RNase T-DNA complexes show that four phenylalanine residues, F29, F77, F124, and F146, stack with the two 3'-terminal nucleobases. To elucidate if the π-π stacking interactions between aromatic residues and nucleobases play a critical role in sequence-specific protein-nucleic acid recognition, here we mutated two to four of the phenylalanine residues in RNase T to tryptophan (W mutants) and tyrosine (Y mutants). The Escherichia coli strains expressing either the W mutants or the Y mutants had slow growth phenotypes, suggesting that all of these mutants could not fully substitute the function of the wild-type RNase T in vivo. DNA digestion assays revealed W mutants shared similar sequence specific...
TatD is an evolutionarily conserved protein with thousands of homologues in all kingdoms of life.... more TatD is an evolutionarily conserved protein with thousands of homologues in all kingdoms of life. It has been suggested that TatD participates in DNA fragmentation during apoptosis in eukaryotic cells. However, the cellular functions and biochemical properties of TatD in bacterial and non-apoptotic eukaryotic cells remain elusive. Here we show that Escherichia coli TatD is a Mg(2+)-dependent 3'-5' exonuclease that prefers to digest single-stranded DNA and RNA. TatD-knockout cells are less resistant to the DNA damaging agent hydrogen peroxide, and TatD can remove damaged deaminated nucleotides from a DNA chain, suggesting that it may play a role in the H2O2-induced DNA repair. The crystal structure of the apo-form TatD and TatD bound to a single-stranded three-nucleotide DNA was determined by X-ray diffraction methods at a resolution of 2.0 and 2.9 Å, respectively. TatD has a TIM-barrel fold and the single-stranded DNA is bound at the loop region on the top of the barrel. Mut...
Journal of the American Chemical Society, Mar 14, 2016
In viral proteins, labile Zn-sites, where Zn2+ is crucial for maintaining the native protein stru... more In viral proteins, labile Zn-sites, where Zn2+ is crucial for maintaining the native protein structure but the Zn-bound cysteines are reactive, are promising drug targets. Here, we aim to (i) identify labile Zn-sites in viral proteins using guidelines established from our previous work and (ii) assess if clinically safe Zn-ejecting agents could eject Zn2+ from the predicted target site and thus inhibit viral replication. As proof-of-concept, we identified a labile Zn-site in the hepatitis C virus (HCV) NS5A protein and showed that the anti-alcoholism drug, disulfiram, could inhibit HCV replication to a similar extent as the clinically used antiviral agent, ribavirin. The discovery of a novel viral target and a new role for disulfiram in inhibiting HCV replication will enhance the therapeutic armamentarium against HCV. The strategy presented can also be applied to identify labile sites in other bacterial or viral proteins that can be targeted by disulfiram or other clinically safe Zn-ejectors.
In viral proteins, labile Zn-sites, where Zn2+ is crucial for maintaining the native protein stru... more In viral proteins, labile Zn-sites, where Zn2+ is crucial for maintaining the native protein structure but the Zn-bound cysteines are reactive, are promising drug targets. Here, we aim to (i) identify labile Zn-sites in viral proteins using guidelines established from our previous work and (ii) assess if clinically safe Zn-ejecting agents could eject Zn2+ from the predicted target site and thus inhibit viral replication. As proof-of-concept, we identified a labile Zn-site in the hepatitis C virus (HCV) NS5A protein and showed that the anti-alcoholism drug, disulfiram, could inhibit HCV replication to a similar extent as the clinically used antiviral agent, ribavirin. The discovery of a novel viral target and a new role for disulfiram in inhibiting HCV replication will enhance the therapeutic armamentarium against HCV. The strategy presented can also be applied to identify labile sites in other bacterial or viral proteins that can be targeted by disulfiram or other clinically safe Zn...
Protein science : a publication of the Protein Society, Jan 12, 2015
RNase T is a classical member of the DEDDh family of exonucleases with a unique sequence preferen... more RNase T is a classical member of the DEDDh family of exonucleases with a unique sequence preference in that its 3'-to-5' exonuclease activity is blocked by a 3'-terminal dinucleotide CC in digesting both single-stranded RNA and DNA. Our previous crystal structure analysis of RNase T-DNA complexes show that four phenylalanine residues, F29, F77, F124, and F146, stack with the two 3'-terminal nucleobases. To elucidate if the π-π stacking interactions between aromatic residues and nucleobases play a critical role in sequence-specific protein-nucleic acid recognition, here we mutated two to four of the phenylalanine residues in RNase T to tryptophan (W mutants) and tyrosine (Y mutants). The Escherichia coli strains expressing either the W mutants or the Y mutants had slow growth phenotypes, suggesting that all of these mutants could not fully substitute the function of the wild-type RNase T in vivo. DNA digestion assays revealed W mutants shared similar sequence specific...
TatD is an evolutionarily conserved protein with thousands of homologues in all kingdoms of life.... more TatD is an evolutionarily conserved protein with thousands of homologues in all kingdoms of life. It has been suggested that TatD participates in DNA fragmentation during apoptosis in eukaryotic cells. However, the cellular functions and biochemical properties of TatD in bacterial and non-apoptotic eukaryotic cells remain elusive. Here we show that Escherichia coli TatD is a Mg(2+)-dependent 3'-5' exonuclease that prefers to digest single-stranded DNA and RNA. TatD-knockout cells are less resistant to the DNA damaging agent hydrogen peroxide, and TatD can remove damaged deaminated nucleotides from a DNA chain, suggesting that it may play a role in the H2O2-induced DNA repair. The crystal structure of the apo-form TatD and TatD bound to a single-stranded three-nucleotide DNA was determined by X-ray diffraction methods at a resolution of 2.0 and 2.9 Å, respectively. TatD has a TIM-barrel fold and the single-stranded DNA is bound at the loop region on the top of the barrel. Mut...
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Papers by Yulander Duh