Objective:To investigate the multienzyine complex formation of human malaria parasite Plasmodium falciparum[P.falciparum)orotate phosphoribosyltransferase(OPRT)and orotidine5'-monophosphate decarboxylase(OMPDC),th...Objective:To investigate the multienzyine complex formation of human malaria parasite Plasmodium falciparum[P.falciparum)orotate phosphoribosyltransferase(OPRT)and orotidine5'-monophosphate decarboxylase(OMPDC),the fifth and sixth enzyme of the de novo pyrimidine biosynthetic palhway.Previously,we have clearly established that the two enzymes in the malaria parasite exist physically as a heterotetrameric(OPRT)_2(OMPDG)_2 complex containing two subunits each of OPRT and OMPDC.and that the complex have catalytic kinetic advantages over the monofunetional enzyme.Methods:Both enzymes were cloned and expressed as recombinant proteins.The protein-protein interaction in the enzyme complex was identified using bifunctionul chemical cross-linker,liquid chromatography-mass spectrometric analysis and homology modeling,Results:The unique insertions of low complexity region at the a 2 and a 5 helices of the parasite OMPDC,characterized by single amino acid repeat sequence which was not found in homologous proteins from other organisms,was located on the OPRT-OMPDC interface.The structural models for the protein-prolein interaction of the helerotetrameric(OPRT)_2(OMPDC)_2multienzyme complex were proposed.Conclusions:Based on the proteomic data and structural modeling,it is surmised that the human malaria parasite low complexity region is responsible for the OPRT-OMPDC interaction.The structural complex of the parasite enzymes,thus,represents an efficient functional kinetic advantage,which in line with co-localization principles of evolutional origin,and allosteric control in protein-protein-interactions.展开更多
Objective：To investigate the effect of Dan-gua Fang（丹瓜方） on adenosine 5’-monophosphate（AMP） activated protein kinase（AMPK） α expression in liver and subsequent improvement of glucose and lipid metabolism.M...Objective：To investigate the effect of Dan-gua Fang（丹瓜方） on adenosine 5’-monophosphate（AMP） activated protein kinase（AMPK） α expression in liver and subsequent improvement of glucose and lipid metabolism.Methods：Forty 13-week-old diabetic Goto-Kakizaki（GK） rats were randomly divided into model,Dan-gua Fang,metformin and simvastatin groups（n=10 for each）,and fed high-fat diet ad libitum.Ten Wistar rats were used as normal group and fed normal diet.After 24 weeks,liver expression of AMPK α mRNA was assessed by real-time PCR.AMPK α and phospho-AMPK α protein expression in liver was evaluated by Western blot.Liver histomorphology was carried out after hematoxylin-eosin staining,and blood glucose（BG）,glycosylated hemoglobin A1c（HbA1c）,food intake and body weight recorded.Results：Similar AMPK α mRNA levels were found in the Dan-gua Fang group and normal group,slightly higher than the values obtained for the remaining groups（P〈0.05）.AMPK α protein expression in the Dan-gua Fang group animals was similar to other diabetic rats,whereas phospho-AMPK α（Thr-172） protein levels were markedly higher than in the metformin group and simvastatin group（P〈0.05）,respectively.However,phosphor-AMPKa/AMPK α ratios were similar in all groups.Dan-gua Fang reduced fasting blood glucose with similar strength to metformin,and was superior in reducing cholesterol,triglycerides,high-density lipoprotein cholesterol as well as improving low-density lipoprotein cholesterol in comparison with simvastatin and metformin.Dan-gua Fang decreases plasma alanine aminotransferase（ALT） significantly.Conclusion：Dan-gua Fang,while treating phlegm-stasis,could decrease BG and lipid in type 2 diabetic GK rats fed with high-fat diet,and effectively protect liver histomorphology and function.This may be partly explained by increased AMPK expression in liver.Therefore,Dan-gua Fang might be an ideal drug for comprehensive Intervention for glucose and lipid metabolism disorders in type 2 diabetes mellitus.展开更多
基金supported in part by Faculty of Graduate School(to W.L)Faculty of Medicine(contract no. RAH/54(1) to J.K.),Chulalongkorn University
文摘Objective:To investigate the multienzyine complex formation of human malaria parasite Plasmodium falciparum[P.falciparum)orotate phosphoribosyltransferase(OPRT)and orotidine5'-monophosphate decarboxylase(OMPDC),the fifth and sixth enzyme of the de novo pyrimidine biosynthetic palhway.Previously,we have clearly established that the two enzymes in the malaria parasite exist physically as a heterotetrameric(OPRT)_2(OMPDG)_2 complex containing two subunits each of OPRT and OMPDC.and that the complex have catalytic kinetic advantages over the monofunetional enzyme.Methods:Both enzymes were cloned and expressed as recombinant proteins.The protein-protein interaction in the enzyme complex was identified using bifunctionul chemical cross-linker,liquid chromatography-mass spectrometric analysis and homology modeling,Results:The unique insertions of low complexity region at the a 2 and a 5 helices of the parasite OMPDC,characterized by single amino acid repeat sequence which was not found in homologous proteins from other organisms,was located on the OPRT-OMPDC interface.The structural models for the protein-prolein interaction of the helerotetrameric(OPRT)_2(OMPDC)_2multienzyme complex were proposed.Conclusions:Based on the proteomic data and structural modeling,it is surmised that the human malaria parasite low complexity region is responsible for the OPRT-OMPDC interaction.The structural complex of the parasite enzymes,thus,represents an efficient functional kinetic advantage,which in line with co-localization principles of evolutional origin,and allosteric control in protein-protein-interactions.
基金Supported by the National Natural Science Foundation of China(No.81173179)the Natural Science Foundation of Fujian Province(No.2011J01198)+5 种基金the Fujian Medical Innovation Project(No.2009-CX-19)the Research Foundation of Fujian Health Department(No.Zlcnfm02)the Fujian Provincial Department of Education Category A Projects(No.JA09131)the Fujian Health Department of Traditional Chinese Medicine Research(No.WZY0920)the CHEN Ke-ji Integrative Medicine Development Fund(No.CKJ2008047,CKJ2009004)the Integrative Medicine of Fujian Key Laboratory of Age-related Diseases Funded Projects(No.2008J1004-10)
文摘Objective：To investigate the effect of Dan-gua Fang（丹瓜方） on adenosine 5’-monophosphate（AMP） activated protein kinase（AMPK） α expression in liver and subsequent improvement of glucose and lipid metabolism.Methods：Forty 13-week-old diabetic Goto-Kakizaki（GK） rats were randomly divided into model,Dan-gua Fang,metformin and simvastatin groups（n=10 for each）,and fed high-fat diet ad libitum.Ten Wistar rats were used as normal group and fed normal diet.After 24 weeks,liver expression of AMPK α mRNA was assessed by real-time PCR.AMPK α and phospho-AMPK α protein expression in liver was evaluated by Western blot.Liver histomorphology was carried out after hematoxylin-eosin staining,and blood glucose（BG）,glycosylated hemoglobin A1c（HbA1c）,food intake and body weight recorded.Results：Similar AMPK α mRNA levels were found in the Dan-gua Fang group and normal group,slightly higher than the values obtained for the remaining groups（P〈0.05）.AMPK α protein expression in the Dan-gua Fang group animals was similar to other diabetic rats,whereas phospho-AMPK α（Thr-172） protein levels were markedly higher than in the metformin group and simvastatin group（P〈0.05）,respectively.However,phosphor-AMPKa/AMPK α ratios were similar in all groups.Dan-gua Fang reduced fasting blood glucose with similar strength to metformin,and was superior in reducing cholesterol,triglycerides,high-density lipoprotein cholesterol as well as improving low-density lipoprotein cholesterol in comparison with simvastatin and metformin.Dan-gua Fang decreases plasma alanine aminotransferase（ALT） significantly.Conclusion：Dan-gua Fang,while treating phlegm-stasis,could decrease BG and lipid in type 2 diabetic GK rats fed with high-fat diet,and effectively protect liver histomorphology and function.This may be partly explained by increased AMPK expression in liver.Therefore,Dan-gua Fang might be an ideal drug for comprehensive Intervention for glucose and lipid metabolism disorders in type 2 diabetes mellitus.
