We have previously introduced the use of permeabilized fission yeast cells(enzyme bags)that recombinantly express full-length CYPs for drug metabolism studies.Such enzyme bags are cells with pores that function as enz...We have previously introduced the use of permeabilized fission yeast cells(enzyme bags)that recombinantly express full-length CYPs for drug metabolism studies.Such enzyme bags are cells with pores that function as enzymes in situ.They can easily be prepared without a need for ultracentrifugation and may be used in similar protocols as microsomes.In this study we report the preparation of enzyme bag cocktails that permit the testing of multiple CYPs in a single enzyme bag reaction.Moreover,we established a convenient testing scheme that permits a rapid screen of all human CYPs for activity towards any given candidate substrate.An important aspect of this approach is the reduction of individual CYP test assays.If a cocktail containing many CYPs tests negative,it follows that all CYPs included in that cocktail need not be tested individually,thus saving time and resources.The new protocol was validated using two probe substrates.展开更多
Gene circuits allow cells to carry out complex functions such as the precise regulation of biological metabolic processes.In this study,we combined,in the yeast S.cerevisiae,genetic regulatory elements with the enzyma...Gene circuits allow cells to carry out complex functions such as the precise regulation of biological metabolic processes.In this study,we combined,in the yeast S.cerevisiae,genetic regulatory elements with the enzymatic reactions of the human CYP2C9 and its redox partner CPR on luciferin substrates and diclofenac.S.cerevisiae cells were permeabilized and used as enzyme bags in order to host these metabolic reactions.We engineered three different(genetic)-enzymatic basic Boolean gates(YES,NOT,and N-IMPLY).In the YES and N-IMPLY gates,human CYP2C9 was expressed under the galactose-inducible GAL1 promoter.The carbon monoxide releasing molecule CORM-401 was used as an input in the NOT and N-IMPLY gates to impair CYP2C9 activity through inhibition of the Fe+2-heme prosthetic group in the active site of the human enzyme.Our study provides a new approach in designing synthetic bio-circuits and optimizing experimental conditions to favor the heterologous expression of human drug metabolic enzymes over their endogenous counterparts.This new approach will help study precise metabolic attributes of human P450s.展开更多
Autoantibodies （aAbs） are generated by the immune system in response to a number of stimuli, one of which is believed to be aberrant protein targeting to the plasma surface. In this study, the presence of human cyto...Autoantibodies （aAbs） are generated by the immune system in response to a number of stimuli, one of which is believed to be aberrant protein targeting to the plasma surface. In this study, the presence of human cytochrome P450 enzyme CYP4Z1 on the outer surface of the plasma membrane of MCF-7 breast cancer cells is demonstrated by immunofluorescence. Moreover, anti- CYP4Z1 aAbs could clearly be detected in breast cancer patient sera by both immunoprecipitation and western blot analysis, while weak or no signals were obtained when testing controls.展开更多
文摘We have previously introduced the use of permeabilized fission yeast cells(enzyme bags)that recombinantly express full-length CYPs for drug metabolism studies.Such enzyme bags are cells with pores that function as enzymes in situ.They can easily be prepared without a need for ultracentrifugation and may be used in similar protocols as microsomes.In this study we report the preparation of enzyme bag cocktails that permit the testing of multiple CYPs in a single enzyme bag reaction.Moreover,we established a convenient testing scheme that permits a rapid screen of all human CYPs for activity towards any given candidate substrate.An important aspect of this approach is the reduction of individual CYP test assays.If a cocktail containing many CYPs tests negative,it follows that all CYPs included in that cocktail need not be tested individually,thus saving time and resources.The new protocol was validated using two probe substrates.
文摘Gene circuits allow cells to carry out complex functions such as the precise regulation of biological metabolic processes.In this study,we combined,in the yeast S.cerevisiae,genetic regulatory elements with the enzymatic reactions of the human CYP2C9 and its redox partner CPR on luciferin substrates and diclofenac.S.cerevisiae cells were permeabilized and used as enzyme bags in order to host these metabolic reactions.We engineered three different(genetic)-enzymatic basic Boolean gates(YES,NOT,and N-IMPLY).In the YES and N-IMPLY gates,human CYP2C9 was expressed under the galactose-inducible GAL1 promoter.The carbon monoxide releasing molecule CORM-401 was used as an input in the NOT and N-IMPLY gates to impair CYP2C9 activity through inhibition of the Fe+2-heme prosthetic group in the active site of the human enzyme.Our study provides a new approach in designing synthetic bio-circuits and optimizing experimental conditions to favor the heterologous expression of human drug metabolic enzymes over their endogenous counterparts.This new approach will help study precise metabolic attributes of human P450s.
文摘Autoantibodies （aAbs） are generated by the immune system in response to a number of stimuli, one of which is believed to be aberrant protein targeting to the plasma surface. In this study, the presence of human cytochrome P450 enzyme CYP4Z1 on the outer surface of the plasma membrane of MCF-7 breast cancer cells is demonstrated by immunofluorescence. Moreover, anti- CYP4Z1 aAbs could clearly be detected in breast cancer patient sera by both immunoprecipitation and western blot analysis, while weak or no signals were obtained when testing controls.
