Objective： We have continued previous work in which we demonstrated that #117 and #372 amino acids contributed to the high activities of human CYP2A13 in catalyzing 4-methylnitrosamino-1-（3-pyridyl）-1-butanone（NNK...Objective： We have continued previous work in which we demonstrated that #117 and #372 amino acids contributed to the high activities of human CYP2A13 in catalyzing 4-methylnitrosamino-1-（3-pyridyl）-1-butanone（NNK） and aflatoxin BI（AFB1） carcinogenic activation. The present study was designed to identify other potential amino acid residues that contribute to the different catalytic characteristics of two CYP2A enzymes, CYP2A6 and CYP2A13, in nicotine metabolism and provide insights of the substrate and related amino acid residues interactions. Methods： A series of reciprocally substituted mutants of CYP2A6lle^300→ Phe, CYP2A6Gly^301aAla, CYP2A6Ser^369 → Gly, CYP2A13Phe^300→ Ile, CYP2A13Ala^301 → Gly and CYP2A13Gly^369 → Set were generated by site-directed mutagenesis/baculovirus-Sf9 insect cells expression. Comparative kinetic analysis of nicotine 5＇hydroxylatin by wild type and mutant CYP2A proteins was performed. Results：All amino acid residue substitutions at 300, 301 and 369 caused significant kinetic property changes in nicotine metabolism. While CYP2A6Ile^300→ Phe and CYP2A6Gly^301→Ala mutations had notable catalytic efficiency increases compared to that for the wild type CYP2A6, CYP2A13Phe^300→Ile and CYP2A13Ala^301→Gly replacement introduced remarkable catalytic efficiency decreases. In addition, all these catalytic efficiency alterations were caused by Vmax variations rather than Km changes. Substitution of #369 residue significantly affected both Km and Vmax values. CYP2A6Ser^369 → Gly increase the catalytic efficiency via a significant Km decrease versus Vmax enhancement, while the opposite effects were seen with CYP2A13Gly^369 → Ser. Conclusion：#300, #301 and #369 residues in human CYP2A6/13 play important roles in nicotine 5＇ -oxidation. Switching #300 or #301 residues did not affect the CYP2A protein affinities toward nicotine, although these amino acids are located in the active center. Set369 to Gly substitution indirectly affected nicotine binding by creating more space and conformational flexibility for the nearby residues, such as Leu^370 which is crucial for many hydroxylations.展开更多
Photodynamic therapy(PDT)is a novel approach for the anti-tumor treatment of photosensitive drugs and laser activation.In the study,5-aminolevulinic acid(ALA)was used as a photosensitizer to mediate PDT to inhibit the...Photodynamic therapy(PDT)is a novel approach for the anti-tumor treatment of photosensitive drugs and laser activation.In the study,5-aminolevulinic acid(ALA)was used as a photosensitizer to mediate PDT to inhibit the growth,metastasis and epithelial-mesenchymal transformation of melanoma cells.Firstly,the therapeutic effect of PDT was investigated through in vitro experiments.Human melanoma A375 cells cultured with ALA were treated with different light intensity,and the cell survival rates of A375 cells were measured by CCK8 method.Subsequently,the metastasis and invasion rates of A375 cells were detected by scratch test and Transwell test.Western blot was used to detect the expression of molecules related to epithelial mesenchymal transformation,such as E-cadherin,Snail,TGF-β,Smad2 and Smad3.In vitro results showed that A375 cells in the ALA-PDT treatment group showed weaker migration and invasion ability,while A375 cells in the control group showed higher migration and invasion rate(P<0.05).E-cadherin expression was significantly increased in the ALA-PDT treatment group(P<0.01),while expression of Snail,Smad2,Smad3 and TGF-βwere significantly decreased in the ALA-PDT treatment group(P<0.05).Therefore,ALA is a good photosensitizer that can inhibit the migration and invasion of melanoma,which also can inhibit the epithelial mesenchymal transformation of melanoma.展开更多
文摘Objective： We have continued previous work in which we demonstrated that #117 and #372 amino acids contributed to the high activities of human CYP2A13 in catalyzing 4-methylnitrosamino-1-（3-pyridyl）-1-butanone（NNK） and aflatoxin BI（AFB1） carcinogenic activation. The present study was designed to identify other potential amino acid residues that contribute to the different catalytic characteristics of two CYP2A enzymes, CYP2A6 and CYP2A13, in nicotine metabolism and provide insights of the substrate and related amino acid residues interactions. Methods： A series of reciprocally substituted mutants of CYP2A6lle^300→ Phe, CYP2A6Gly^301aAla, CYP2A6Ser^369 → Gly, CYP2A13Phe^300→ Ile, CYP2A13Ala^301 → Gly and CYP2A13Gly^369 → Set were generated by site-directed mutagenesis/baculovirus-Sf9 insect cells expression. Comparative kinetic analysis of nicotine 5＇hydroxylatin by wild type and mutant CYP2A proteins was performed. Results：All amino acid residue substitutions at 300, 301 and 369 caused significant kinetic property changes in nicotine metabolism. While CYP2A6Ile^300→ Phe and CYP2A6Gly^301→Ala mutations had notable catalytic efficiency increases compared to that for the wild type CYP2A6, CYP2A13Phe^300→Ile and CYP2A13Ala^301→Gly replacement introduced remarkable catalytic efficiency decreases. In addition, all these catalytic efficiency alterations were caused by Vmax variations rather than Km changes. Substitution of #369 residue significantly affected both Km and Vmax values. CYP2A6Ser^369 → Gly increase the catalytic efficiency via a significant Km decrease versus Vmax enhancement, while the opposite effects were seen with CYP2A13Gly^369 → Ser. Conclusion：#300, #301 and #369 residues in human CYP2A6/13 play important roles in nicotine 5＇ -oxidation. Switching #300 or #301 residues did not affect the CYP2A protein affinities toward nicotine, although these amino acids are located in the active center. Set369 to Gly substitution indirectly affected nicotine binding by creating more space and conformational flexibility for the nearby residues, such as Leu^370 which is crucial for many hydroxylations.
文摘Photodynamic therapy(PDT)is a novel approach for the anti-tumor treatment of photosensitive drugs and laser activation.In the study,5-aminolevulinic acid(ALA)was used as a photosensitizer to mediate PDT to inhibit the growth,metastasis and epithelial-mesenchymal transformation of melanoma cells.Firstly,the therapeutic effect of PDT was investigated through in vitro experiments.Human melanoma A375 cells cultured with ALA were treated with different light intensity,and the cell survival rates of A375 cells were measured by CCK8 method.Subsequently,the metastasis and invasion rates of A375 cells were detected by scratch test and Transwell test.Western blot was used to detect the expression of molecules related to epithelial mesenchymal transformation,such as E-cadherin,Snail,TGF-β,Smad2 and Smad3.In vitro results showed that A375 cells in the ALA-PDT treatment group showed weaker migration and invasion ability,while A375 cells in the control group showed higher migration and invasion rate(P<0.05).E-cadherin expression was significantly increased in the ALA-PDT treatment group(P<0.01),while expression of Snail,Smad2,Smad3 and TGF-βwere significantly decreased in the ALA-PDT treatment group(P<0.05).Therefore,ALA is a good photosensitizer that can inhibit the migration and invasion of melanoma,which also can inhibit the epithelial mesenchymal transformation of melanoma.
