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dc.description.abstractPeroxiredoxin(Prx)은 H₂O₂와 alkyl hydroxide를 물과 알코올로 환원시키는 역할을 하는 peroxidase로, NADPH와 thioredoxin reductase를 포함하는 thioredoxin system으로부터 reducing equivalents를 받아 활성을 나타낸다. Prx의 N-terminus에는 H₂O₂에 의해 선택적으로 산화되는 cysteine 잔기가 존재하며, 이것은 6개의 mammalian Prx에서 공통적으로 발견된다. Prx의 N-terminal cysteine은 catalytic cycle에서는 H₂O₂에 의해 cysteine sulfenic acid로 산화되어 homodimer를 이루고 있는 다른 subunit의 C-terminal cysteine과 disulfide bond를 형성하지만, H₂O₂에 의해 cysteine sulfinic acid로 한 단계 더 산화되는 경우에는 본래의 peroxidase 활성을 잃어버리게 된다. 그러나 최근에 sulfinic Prx를 환원시키는 역할을 하는 Sulfiredoxin(Srx)이라는 효소가 보고되면서 cysteine sulfinic acid로 산화되는 반응이 가역적인 과정임이 밝혀지게 되었다. Srx에 의해 cysteine sulfenic acid로 환원된 Prx는 다시 catalytic cycle을 돌며 peroxidase의 기능을 할 수 있게 된다. 이러한 Prx의 특성에 근거하여 hyperoxidation된 Prx I이 Srx에 의해 reactivation되지 않는 경우 세포 내에서 어떠한 현상이 관찰되는지를 알아보기 위한 실험을 진행하였다. 그 첫 번째 과정으로 우선 Prx의 peroxidase 활성은 그대로 나타내고 있지만 H₂O₂에 의해 hyperoxidation 되었을 때 Srx에 의한 reactivation은 일어나지 않는 Prx mutant를 screening하는 과정을 거쳤다. 그 중 몇 개의 Prx mutants를 선택하여 정제한 뒤 Prx activity assay를 통해 wild type Prx I과 비슷한 정도의 peroxidase activity를 나타내는 mutants를 다시 구분하였다. 그리고 나서 H₂O₂를 이용하여 선택된 Prx mutants를 hyperoxidation시킨 뒤 Prx activity assay를 통해 Srx에 의해 regeneration되지 않는 Prx I mutant L46R을 최종적으로 선별하게 되었다. 다음 단계로 Prx I adenovirus를 이용하여 Prx I-knockout MEF cell에 Prx I L46R을 발현시킨 뒤 세포 내에서 sulfinic Prx I L46R이 regeneration 되는지 관찰하였다. In vitro 결과와는 달리 cell 안에서는 L46R도 wild type처럼 regeneration되는 것처럼 보였으나, 2 dimensional electrophoresis를 이용해 선별한 Prx I L46R이 세포 내에서 얼마나 regeneration되는지 확인한 뒤 Prx I의 dysfunction과 관련된 실험을 진행하려고 한다.;Peroxiredoxin(Prx) is the peroxidase which reduce H₂O₂and alkyl hydroxide to H₂O and alcohol, and they act as a peroxidase by obtaining reducing equivalents from thioredoxin system including NADPH and thioredoxin reductase. Prxs have N-terminal conserved cysteines which are selectively oxidized by H₂O₂, and these conserved cysteines were found in all 6 isoforms of mammalian Prxs. In catalytic cycle, N-terminal conserved cysteines of 2-Cys Prxs form disulfide bond with C-terminal cysteine in other subunit of homodimer as they are oxidized to cysteine sulfenic acid by H₂O₂. Sulfenic Prxs can be further oxidized to sulfinic Prxs by H₂O₂, which lose peroxidase activity. Recently it has been revealed that oxidation to cysteine sulfinic acid is a reversible process and enzymes such as Sulfiredoxin(Srx) participate in Prx reactivation mechanism. Prxs reduced to cysteine sulfenic acid by Srx can be restored to catalytic cycle and act as a peroxidase. Based on these characters of Prx, we performed several experiments to investigate the cellular effects when hyperoxidized Prxs can not be reactivated by Srx. First we did screening of some Prx mutants which had peroxidase activity and whose cysteine sulfinic acid were not reactivated by Srx. Among them we selected several Prxs for purification and classified some Prx I mutants which have similar peroxidase activity with wild type Prx I through Prx activity assay. After the hyperoxidation of selected Prx I mutants with H₂O₂, we finally chose Prx I L46R which was not regenerated by Srx through Prx activity assay. In the next step, we expressed Prx I L46R in Prx I-knockout MEF cells with Prx I adenovirus and investigated whether sulfiic Prx I L46R would be regenerated by Srx. It seemed that sulfinic Prx I L46R was reactivated in Prx I -/- MEF cells, additional experiment using 2 dimensional electrophoresis is needed to confirm regeneration of Prx I L46R.-
dc.description.tableofcontentsAbstract = v 1. INTRODUCTION = 1 2. MATERIALS AND METHODS = 3 2.1. Materials = 3 2.2. Site-directed mutagenesis = 3 2.3. Purification of GST-rSrx and rSrx = 4 2.4. GST-pull down assay = 4 2.5. Purification of recombinant Prx I proteins = 5 2.6. Assay of Prx I activity = 6 2.7. Oxidation of Prx I proteins = 6 2.8. Assay of Srx acivity = 7 2.9. Cell culture = 7 2.10. Reduction of sulfinic Prx I in Prx I -/- MEF cells = 7 3. RESULTS = 11 3.1 Preparation of Prx I mutants = 11 3.2 Interaction between Prx I mutants and GST-rSrx = 12 3.3 Regeneration of Prx I mutants by Srx = 16 3.4 Regeneration of Prx I mutants in MEF Prx I -/- cells = 21 4. DICUSSION = 23 5. REFERENCES = 25 논문개요 = 28-
dc.format.extent440137 bytes-
dc.publisher이화여자대학교 대학원-
dc.titleThe screening of Peroxiredoxin I mutants that can not interact with Sulfiredoxin-
dc.typeMaster's Thesis-
dc.format.page29 p.-
dc.identifier.major대학원 분자생명과학부- 2-
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