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Functional characterization of two polymorphic variants of human cytochrome P450 1A1 using recombinant protein expression : 유전적 다형성에 의한 human cytochrome P450 1A1변이형의 기능 분석
DC Field | Value | Language |
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dc.contributor.advisor | 류덕영 | - |
dc.contributor.author | 이승헌 | - |
dc.date.accessioned | 2017-07-13T16:44:02Z | - |
dc.date.available | 2018-10-25 | - |
dc.date.issued | 2015-08 | - |
dc.identifier.other | 000000053354 | - |
dc.identifier.uri | https://hdl.handle.net/10371/120218 | - |
dc.description | 학위논문 (박사)-- 서울대학교 대학원 : 수의학과(수의병인생물학 및 예방수의학(환경위생학)전공), 2015. 8. 류덕영. | - |
dc.description.abstract | The cytochromes P450 (CYP) constitute a superfamily of heme-thiolate enzymes, which catalyze various phase I metabolism reactions, including hydroxylation of saturated carbon-hydrogen bonds, epoxidation of double bonds, oxidation of heteroatoms, dealkylation reactions, and oxidations of aromatics drugs. CYP1A1 is a member of the CYP1 family, which is expressed at high level in extrahepatic tissues and plays an important role in the metabolism of environmental chemicals and hormones.
Genetic polymorphism in the coding sequence of CYP gene influences the metabolism of substrates, bioavailability and the development of toxicity, including adverse drug effects and even cancer susceptibility. The genetic polymorphisms of CYP1A1 containing a G to C transition at nucleotide 184 (results in Ala62Pro substitution) and a G to A transition at nucleotide 134 (results in Gly45Asp substitution), which have been has been found limitedly in East Asian population. Previous case-control study suggested that the genetic polymorphism of Ala62Pro and Gly45Asp loci in CYP1A1 might be related to a higher incidence of lung cancer and uterine leiomyoma development, respectively. However, further studies are required to evaluate the effects of these polymorphisms on the metabolism of xenobiotics and their roles in carcinogenesis. In the present study, the CYP1A1 variant harboring Ala62Pro and Gly45Asp were characterized by using heterologous expression in Escherichia coli (E. coli ) and mammalian cells. E. coli and its membrane fraction expressing the variants (Ala62Pro and Gly45Asp), as well as the purified variant protein, had lower CYP (i.e., holoenzyme) contents than their wild-type (WT) equivalents. However, based on the immunoblot analysis, the total level of CYP1A1 apoprotein in E. coli expressing the variants were similar to that in cells expressing the WT. The membrane expressing the variants and the purified variant proteins had reduced heme contents compared with their WT equivalents. In addition, enhanced supplementation of a heme precursor, δ-aminolevulinic acid, during culture did not increase CYP content in E. coli expressing the variant, but did for the WT. E. coli membrane and mammalian microsomes expressing the variants, as well as purified variant proteins, had reduced ethoxyresorufin O-dealkylation activities benzo[a]pyrene hydroxylation activities, compared with the WT equivalents. These findings led us to hypothesize that the Ala62Pro and Gly45Asp substitution alters the ordered framework of the protein structure and causes a loss of heme incorporation. The elucidation of the protein structure responsible for CYP specificity is of great importance in understanding enzyme functions and mechanisms. Molecular modeling based on three-dimensional structure of CYP1A1(PDB code: 4I8V) suggested that, Ala62 and Gly45 residue are localized in an α-helix A and a proline-rich region at N-terminus of CYP1A1, respectively, and considered one of key residue composing the interaction networks responsible for heme binding. In support, the single amino acid substitution of those two and surrounding residues (interacting residues) dramatically decreased CYP content in E. coli expressing the variants. Taken together, our findings suggest the Ala62Pro and Gly45Asp substitution distorts the structural integrity of CYP1A1, and results in the formation of protein that has a decreased heme-binding affinity and holoenzyme with altered enzymatic characteristics. Thus, the G184C and G134A nucleotide polymorphism responsible for this amino acid variation may have a critical impact on affected individuals phenotype in metabolism. | - |
dc.description.tableofcontents | Table of contents
Abstract i Table of contents v Literature review 1 General introduction of cytochrome P450 1 Evolution the cytochrome P450 and nomenclature 1 Cytochrome P450 functions 3 General introduction of cytochrome P450 1A1 5 Identification of the cytochrome P450 1A1 gene 5 Cytochrome P450 1A1 expression 7 Subcellular localization of the cytochrome P450 1A1 8 Substrate specificity of the cytochrome P450 1A1 10 Cytochrome P450 1A1 structure 11 Polymorphisms of the cytochrome P450 1A1 14 Aim for study 16 Materials and methods 18 Chemicals 18 Bacterial constructs 18 Bacterial expression 19 CYP and heme contents 20 Immunoblots 21 Purification of His-tagged proteins 21 dALA supplementation 22 Molecular modeling 22 Sequence alignment 23 Site-directed mutagenesis 23 Expression in mammalian cells and microsomal preparations 23 7-Ethoxyresorufin O-dealkylation (EROD) 24 BaP hydroxylation 25 Statistics 26 Chapter I Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression 27 Abstract 27 Introduction 29 Results 33 Expression in E. coli 33 Enzyme activities of the E. coli membrane fractions and mammalian microsomes 34 Purification 34 EROD activities of the purified proteins 35 Effect of repeated dALA supplementation 36 Structural analysis of CYP1A1 37 Sequence alignment 38 Substitutions of CYP1A1 Ala62 and CYP1A2 Ala64 residues 39 Expression in mammalian cells 40 Substitution of residues in Regions A, B, C and D 41 Discussion 33 Figures and legends 33 Chapter II Characterization of the Gly45Asp polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression 68 Abstract 68 Introduction 70 Results 73 Expression in E. coli 73 Protein purification 74 Effect of dALA supplementation 75 Structural analysis of CYP1A1 75 Sequence alignment 76 Substitution of Gly45 77 Substitutions of Arg77 and PR region residues 78 Enzyme activities of E. coli membranes and mammalian microsomes 78 Enzyme activities of the purified proteins 79 Discussion 81 Figures and legends 85 References 103 Supplementary data 124 국문 초록 145 | - |
dc.format | application/pdf | - |
dc.format.extent | 29510824 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | CYP1A1 | - |
dc.subject | polymorphism | - |
dc.subject | α-helix | - |
dc.subject | proline-rich region | - |
dc.subject | heme | - |
dc.subject | enzyme activity | - |
dc.subject.ddc | 636 | - |
dc.title | Functional characterization of two polymorphic variants of human cytochrome P450 1A1 using recombinant protein expression | - |
dc.title.alternative | 유전적 다형성에 의한 human cytochrome P450 1A1변이형의 기능 분석 | - |
dc.type | Thesis | - |
dc.contributor.AlternativeAuthor | Seung Heon Lee | - |
dc.description.degree | Doctor | - |
dc.citation.pages | iv, 145 | - |
dc.contributor.affiliation | 수의과대학 수의학과 | - |
dc.date.awarded | 2015-08 | - |
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