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dc.contributor.advisor우현애-
dc.contributor.author심주현-
dc.creator심주현-
dc.date.accessioned2017-09-01T18:31:32Z-
dc.date.available2017-09-01T18:31:32Z-
dc.date.issued2014-
dc.identifier.otherOAK-000000089481-
dc.identifier.urihttp://dspace.ewha.ac.kr/handle/2015.oak/211543-
dc.identifier.urihttp://dcollection.ewha.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000089481-
dc.description.abstractPeroxiredoxins (Prxs) are a family of peroxidases that reduces peroxides such as hydrogen peroxide with reactive cysteine residues in their active sites. Mammalian cells express six isoforms of Prx (Prx I to VI). These isoforms vary in subcellular localization, with Prx I, II, and VI being localized mainly in the cytosol, Prx III being restricted to mitochondria, and Prx IV being found predominantly in the endoplasmic reticulum (ER). Prx V gene contains two ATG initiation codons, giving a short (SPrx V) and long form of Prx V (LPrx V), which are proposed to be in cytosol, nucleus, mitochondria, and peroxisomes. In the first part of this study, I investigated the submitochondrial localization of Prx V and the effect of oxidative stress on the stability of mitochondrial Prx V. Mouse embryonic fibroblasts derived from Prx V knockout (KO) mice were used extensively in this study. I found the presence of Prx V in the intermembrane space (IMS) as well as the matrix of mitochondria. Unlike other Prxs (Prx I-IV) that easily undergo inactivation through the hyperoxidation of their active site cysteine, Prx V was resistant to hyperoxidation. In the cells lacking Prx V, hyperoxidation of Prx III was increased significantly in response to various oxidative stresses, though its concentration is much less than that of Prx III in the organelle, attesting the peroxidase role of Prx V in mitochondria. LPrx V is expressed as a precursor protein containing an 49-residue N-terminal mitochondria targeting sequence, of which the positively charged, N-terminal 41-residue sequence is cleaved by mitochondrial processing peptidase (MPP) located in the matrix, producing an intermediate Prx V (Octa-Prx V) with an 8-residue hydrophobic sequence. Evidence suggests that the 8-residue peptide is finally cleaved by mitochondrial intermediate peptidase (MIP) to produce mature Prx V. Given that MIP is sensitive to oxidative inactivation, Octa-Prx V accumulates temporarily in cells under oxidative stress as seen in cells lacking Prx III or Srx. Octa-Prx V with destabilizing amino acid residues at its N-terminus is degraded readily according to the N-end rule pathway of protein degradation, thus resulting in the decrease in the amount of Prx V in oxidatively stressed cells. In the second part, I investigated the potential role of Prx I against cisplatin-induced renal damage in mice. The anticancer drug cis-diamminedichloroplatinum (cisplatin) is a chemotherapeutic agent widely used for solid tumors. Nephrotoxicity of cisplatin is a serious dose-limiting side effect. Oxidative stress is a major cause of cisplatin-induced nephrotoxicity through damaging DNA, membranes and mtiochondria and increasing ER stress. Cisplatin is known to produce reactive oxygen species through Cytochrome P450 2E1 (CYP2E1) and localize at the surface of the ER where CYP2E1 is located. I found that among six Prx isoforms Prx I is selectively degraded in cisplatin-treated kidney around the time when renal functions are severely damaged. The Prx I degradation was blocked in mouse proximal tubular cells treated with 3-methyladenine, an autophagy inhibitor or in mouse embryonic fibroblast (MEF) cells lacking ATG7. Moreover, increased ROS on ER surface by overexpression of CYP2E1 further accelerated Prx I degradation. These results suggests that Prx I degradation is mediated largely through autophagy that is promoted by cisplatin-induced ER stress. Ablation of Prx I attenuated cisplatin-induced nephrotoxicity and significantly increased the abundance of markers for oxidative stress, ER stress and inflammation in kidney, indicating Prx I palys a protective role against cisplatin-induced nephrotoxicity.;Peroxiredoxins (Prxs)는 활성 부위의 반응성이 높은 cysteine 잔기를 이용하여 hydrogen peroxide와 같은 과산화물질을 환원시키는 peroxidase이다. 포유류에는 여섯 종류의 Prx (Prx I~VI)가 있다. 이 isoform들은 세포 내에서의 위치가 모두 다른데, Prx I, II, VI는 주로 세포 기질에 존재하고 Prx III는 미토콘드리아에만 존재하며 Prx IV는 주로 ER에서 발견되었다. Prx V 유전자는 두 개의 ATG 개시 코돈을 포함하고 있어서 짧고 (SPrx V) 긴 (LPrx V) 형태의 Prx V를 만든다. 그로 인해 Prx V는 세포 기질, 핵, 퍼옥시좀과 미토콘드리아에 존재하는 것으로 알려져 있다. 이 연구의 첫번째 파트에서 Prx V의 미토콘드리아 내부 위치와 산화적 스트레스가 미토콘드리아 Prx V의 안정성에 미치는 영향에 대해 연구하였다. Prx V 결핍 mouse에서 얻은 mouse embryonic fibroblast가 이 연구에서 사용되었다. Prx V가 미토콘드리아 내부에서 matrix 뿐만 아니라 intermembrane space (IMS)에도 존재하는 것을 발견하였다. 활성 부위의 cysteine이 쉽게 과산화되어 비활성화되는 다른 Prxs (Prx I~IV)와 다르게, Prx V는 쉽게 과산화되지 않는다. Prx V가 없는 세포에서 다양한 산화 스트레스에 의해 미토콘드리아에 있는 Prx III의 과산화가 증가하는 것을 보았다. Prx V가 미토콘드리아 내부에서 Prx III에 비해 훨씬 양이 적은 것을 고려했을 때, Prx V가 미토콘드리아에서 중요한 peroxidase로 작용한다는 것을 보여준다. LPrx V는 positive charge를 띄는 49개 잔기의 아미노 말단 미토콘드리아 표적 서열을 갖는 precursor protein이다. 이 미토콘드리아 표적 서열의 41개 잔기는 matrix에 존재하는 mitochondrial processing peptidase (MPP)에 의해 잘리고, 8개의 소수성 서열을 갖는 intermediate Prx V (Octa-Prx V)가 만들어진다. 여러 증거들을 통해 이 8개의 펩티드는 결국 mitochondrial intermediate peptidase (MIP)에 의해 제거되어 완전히 성숙한 Prx V가 생성되는 것으로 보인다. MIP가 산화적 비활성화에 민감하다는 것을 고려했을 때, 미토콘드리아 특이적인 Prx인 Prx III나 과산화되어 비활성화된 Prx를 재활성화하는 효소인 sulfiredoxin (Srx)가 없는 세포에서 Octa-Prx V는 산화적 스트레스에 의해 세포 내에 일시적으로 축적된다. 아미노 말단에 destabilizing 아미노산 잔기를 갖고 있는 Octa-Prx V는 단백질 분해 과정중 N-end rule pathway에 의해 빠르게 분해되고, 따라서 산화적으로 스트레스를 받은 세포 내에서 Prx V의 양이 줄어들게 된다. 이 연구의 두번째 파트에서 Prx I의 mouse에서의 cisplatin 유도 신장 독성에 대한 잠재적인 역할에 대해 연구했다. 항암제인 cis-diammine dichloroplatinum (cisplatin)은 다양한 solid tumor에 사용되는 화학요법제이다. Cisplatin의 신장 독성은 용량을 제한하는 심각한 부작용의 하나이다. 산화적 스트레스는 cisplatin에 의해 유도된 신장 독성에서 중요한 요인으로 DNA, 세포막, 미토콘드리아에 손상을 입히고 ER stress를 증가시킨다. Cisplatin은 Cytochrome P450 (CYP2E1)을 통해서 활성 산소종을 만드는 것으로 알려져 있고 CYP2E1이 위치한 ER 표면에서도 발견된다. 여섯 종류의 Prx isoform 중에서 Prx I이 cisplatin을 처리한 신장에서 신장기능이 심각하게 손상을 받을 때 선택적으로 분해되는 것을 보았다. Prx I의 분해는 마우스의 근위세뇨관 세포에서 autophagy의 저해제인 3-methyladenine을 처리했을 경우와, autophagy가 일어나지 않는 ATG7 KO MEF 세포에서 저해되었다. 게다가, CYP2E1을 과발현시켜서 ER 표면에서 활성산소 생성을 증가시켰을 때 Prx I의 분해가 촉진되었다. 이러한 결과는 Prx I의 분해가 많은 부분 cisplatin에 의한 ER stress에 의해 유도된 autophagy에 의해 일어난다는 것을 보여준다. Prx I의 결핍은 cisplatin에 의한 신장 독성을 더욱 악화시키고 산화 스트레스와 ER stress, 그리고 염증 반응의 표현형을 증가시켰다. 이는 Prx I이 cisplatin에 의한 신장 독성에 대하여 보호 기능이 있다는 것을 의미한다.-
dc.description.tableofcontentsAbstract 1 Introduction 3 Tabel 1. Six isoforms of Prxs from mammals. 5 Fig. 1. Reaction Mechanisms of Prxs. 6 Part I. Studies on the Subcellular Localization of Peroxiredoxin V and its Stability in Mitochondria and Antioxidant Function in Mitochondria 8 Introduction 9 Fig. 2. Two forms of PrxV. 11 Fig. 3. Overview of presequence processing and degradation enzymes in mitochondria. (from Ref. (32)) 12 Fig. 4. N-end rule pathway and mechanism of cleavage of presequence in mitochondria. (from ref. (42)) 13 Materials & Methods 15 1. Generation of PrxV KO mouse 15 2. Cell Culture 16 3. Cell viability assay 16 4. Subcellular fractionation 16 5. Submitochondrial fractionation 17 6. Immunofluorescence analysis 17 7. Structured illumination microscopy (SIM) 18 8. Peroxiredoxin activity assay 18 9. 2-dimensional gel electrophoresis 18 10. Immunoblot analysis 19 11. Purification of PrxV 19 12. Mass spectrometer (MS/MS and MALDI-TOF) 20 13. RT-PCR and quantitative real-time PCR 21 Table 2. Sequence of siRNAs 22 Table 3. Primers used for real-time PCR in part I 23 Results 24 1. Generation of PrxV KO mice 24 Fig.5.Gene targeting of murine Prx V 25 2. Distribution and Subcellular Localization of Prx V 26 Fig. 6. Tissue and subcellular distribution of Prx V in C57BL/6 mice. 27 Fig. 7. Subcellular distribution of Prx V in MEF cells. 28 3. Mitochondrial Prx V Resides in Mitochondrial Intermembrane Space as well as in Mitochondrial Matrix 30 Fig. 8. Submitochondrial localization of Prx V in HeLa cells and mouse hearts. 31 Fig. 9. Submitochondrial localization of Prx V in fractionated mitochondria from mouse tissues 33 Fig. 10. Submitochondrial localization of Prx V with confocal microscopy. 34 4. Prx V is more Resistant to Inactivation by Cysteine Sulfinic Acid Formation than other Prxs 35 Fig. 11. Prx V is highly resistant to inactivation via hyperoxidation. 36 5. Protective Effects of Mitochondrial Prx V against Oxidative Stresses 37 Fig. 12. Protective role of Prx V against oxidative stress and its effect on Prx III hyperoxidation. 39 Fig. 13. Increase of Prx III hyperoxidation by siRNA-mediated depletion of mitochondrial Prx V. 40 6. Identification of Imperfectly Processed LPrx V 41 Fig. 14. Identification of upper band of Prx V, Octa-Prx V, in mitochondria. 43 Fig. 15. Sequential cleavage of LPrx V by MPP and MIP and the effect of decreased MIP activity on the formation of Octa-Prx V 45 7. Effect of Prx III-deficiency on Complete Processing of LPrxV 46 Fig. 16. Increase of Octa-Prx V and decrease of total Prx V in Prx III-deficient mice. 48 Fig. 17. Decrease of mitochondrial Prx V in Prx III-deficient mice. 49 8. Octa-PrxV is increased when Hyperoxidation of Prx III is increased by Srxdeficiency 50 Fig. 18. Increase of Octa-Prx V in hearts of Srx-deficient mice. 51 Discussion 52 Fig. 19. Scheme for degradation of mitochondrial Prx V in Prx III-deficient mice. 55 References 57 Part II. Studies on the Function of Peroxiredoxin I in the Kidney of Cisplatin-injected Mice 64 Introduction 65 Fig. 20. Major Pathways in cisplatin-induced acute tubular cell injury. (from Ref.(40)) 66 Fig. 21. Effects of cisplatin on the endoplasmic reticulum. (from Ref. (31)) 68 Materials & Methods 69 1. Cisplatin-induced kidney failure model 69 2. Histology and Immunohistochemistry 69 3. Assessment of renal function 69 4. Immunoblot analysis 70 5. Cell Culture 70 6. Cell viability assay 70 7. Subcellular fractionation 71 8. RT-PCR and quantitative real-time PCR 71 Table. 3. Primers used for real-time PCR in part II 72 Results 73 1. PrxI is Selectively Decreased in Cisplatin-treated Kidney. 73 Fig. 22. Effect of cisplatin on the kidney of C57/BL6 mice. 74 Fig. 23. Selective decrease of Prx I in cisplatin-treated kidney. 75 Fig. 24. Nrf-2 dependent induction of Srx in the kidney of cisplatin-injected mice. 77 2. The Degradation of Prx I Occurs at the Late Stage of Cisplatin-induced Damage 78 Fig. 25. Expression of Prxs, Srx and autophagy-related genes in kidney of cisplatin-injected mice. 80 3. Cisplatin-induced Prx I Degradation is Mediated by Autophagy 81 Fig. 26. Inhibition of Prx I degradation by autophagy inhibitors in mouse proximal tubular cells. 82 Fig. 27. Effect of cisplatin on the degradation of Prx I in autophagy-deficient ATG7 KO MEF cells. 83 4. Degradation of Prx I is Accelerated in CYP2E1 Overexpressing Cells 84 Fig. 28. Effect of CYP2E1 overexpression on the degradation of Prx I by cisplatin. 85 Fig. 29. Inhibition of Prx I degradation by autophagy inhibitor in cisplatintreated CYP2E1 overexpressing HepG2 cells. 86 Fig. 30. Effect of the proteasomal degradation or secretion on the decrease of Prx I by cisplatin. 88 5. Prx I-deficient Mice were More Severely Damaged by Cisplatin 89 Fig. 31. Effect of Prx I ablation on oxidative kidney damage induced by cisplatin. 92 Fig. 32. Effect of Prx I ablation on renal functions and gene expression in kidney damage induced by cisplatin. 93 Fig. 33. Expression of Prxs, Srx and autophagy-related proteins of cisplatintreated kidney of Prx I WT and Prx I KO mice. 94 Discussion 95 Fig. 34. Model for protective effect of Prx I and its degradation in the kidney of cisplatin-injected mice. 98 References 99 국문초록 104-
dc.formatapplication/pdf-
dc.format.extent3837505 bytes-
dc.languageeng-
dc.publisher이화여자대학교 대학원-
dc.subject.ddc600-
dc.titleStudies on the Subcellular Localization of Peroxiredoxin V and its Stability and Antioxidant Function in Mitochondria and the Function of Peroxiredoxin I in the Kidney of Cisplatin-injected Mice-
dc.typeDoctoral Thesis-
dc.format.pageix, 103 p.-
dc.identifier.thesisdegreeDoctor-
dc.identifier.major대학원 약학과-
dc.date.awarded2014. 8-
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