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dc.description.abstractNm23 is a housekeeping enzyme having NDP kinase activity, and recently identified having many cellular functions such as metastasis suppressor, transactivation activity on c-myc and DNase. However, the regulatory mechanism of Nm23 was not well understood.In this study, I have examined protein modifications and interacting properties of Nm23 in response to oxidative stress.I have identified the formation of the intra-and inter-disulfide bonds of Nm23 in response to H_(2)O_(2) treatment in vivo and in vitro.The cross-linking sites of Nm23 were mapped using Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS).To investigate how the cross-linking of Nm23 is regulated, I examined the interacting proteins of Nm23 in response to oxidative stress.Thioredoxin reductase 1 (TR1) was identified as an interacting protein with only oxidized Nm23.It turns out that Nm23 is oxidized by reactive oxygen species and reversibly reduced by NADPH-TR1-Thioredoxin oxidoreduction shuttle system.This suggests that Nm23 can be a key molecule regulated by oxidative stress.-
dc.description.tableofcontents1. INTRODUCTION = 1 2. MATERIALS & METHODS = 5 2.1. Materials & Instruments = 5 2.2. Plasmids = 6 2.3. Protein Purification = 6 2.4. NDP kinase Assay = 7 2.5. TR assay = 7 2.6. Cell cultures = 7 2.7. Transient Transfection and H_(2)O_(2) Treatment = 7 2.8. Antibody Immobilization = 8 2.9. Immunoprecipitation and Immnoblotting = 9 2.10. In-gel Digestion & Mass Spectrometric Analysis of proteins = 9 2.11. DNA binding and Gel mobility shift Assay = 10 3. RESULTS = 11 3.1. Oxidative modification of Nm23-H1 after exposure to hydrogen peroxide = 11 3.2. Identification of Nm23-H1 interacting protein upon exposure to hydrogen peroxide = 21 3.3. Oxidized Nm23-H1 is a substrate of thioredoxin = 25 3.3.1. Oxidized Nm23-H1 is a substrate of thioredoxin = 25 3.3.2. Cysteine 4 of Nm23-H1 might be involved in the catalytic site of reduced Trx 1 = 31 3.4. Redox regulation of the NDP kinase activity of Nm23-H1 by thioredoxin = 33 3.5. Oxidative modification of Nm23-H2 in response to oxidizing agent and redox regulation of the DNA binding activity of Nm23-H2 on c-myc = 38 4. DISCUSSION = 44 REFERENCES = 49 Acknowledgement = 49-
dc.format.extent7037799 bytes-
dc.publisher이화여자대학교 대학원-
dc.titleOxidative modifications of Nm23 regulate cellular functions-
dc.typeMaster's Thesis-
dc.format.pagevi, 52 p.-
dc.identifier.major대학원 분자생명과학부- 2-
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