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Effect of Oxidative Stress on the Sulfiredoxin-depleted Red Blood Cells

Title
Effect of Oxidative Stress on the Sulfiredoxin-depleted Red Blood Cells
Authors
김유진
Issue Date
2011
Department/Major
대학원 생명·약학부생명과학전공
Publisher
이화여자대학교 대학원
Degree
Master
Advisors
이서구
Abstract
적혈구는 산소를 운반하는 역할을 하는데, 이는 산소와 결합할 수 있는 헤모글로빈이 적혈구내에 풍부하게 존재하기 때문에 가능하다. 전체 헤모글로빈의 3%가 자연적으로 산화되어 메티모글로빈이 되며, 이로 인해 발생된 ROS가 세포에 해를 입히기 때문에 적혈구는 ROS를 방어하기 위한 다양한 종류의 항산화 효소를 지니고 있다. 적혈구내의 항산화 효소들 중 하나인 Prx II 는 적혈구 내에서 세 번째로 많은 단백질이다. 일반적으로 Peroxiredoxin (Prx) 는 일차 산화되어 homodimer를 이루며, 더 산화될 경우 Sulfinic form 이 되어 peroxidase 기능을 잃는다고 알려져 있다. Prx 가 Homodimer 를 이룰 경우는 Thioredoxin (Trx) 라는 효소를 통해 환원되며, Sulfinic form 이 되었을 때는 Sulfiredoxin (Srx) 에 의해 환원된다. 이러한 환원시스템이 있는데도, 적혈구 내에서 Prx II 는 ROS의 비효소적 제거단백질로 생각된다. 적혈구내에 많은 양을 가지고 있기에 한 번만 반응하기에도 충분하며, Thioredoxin Reductase 양이 적기 때문이다. 우리는 이 논문에서, Srx 가 결핍된 쥐들을 사용하여 Prx II 가 효소적 특성을 가지고 있다는 것을 보여주고 있다. Srx 가 결핍된 쥐의 적혈구에서 Prx II 의 과산화 형태가 이미 형성되어 있고, ROS를 발생시키는 물질을 처리할 경우 Srx 단백질을 가지고 있는 적혈구 내에서도 Prx II 가 과산화되며, 시간이 지나면 다시 원상태로 돌아오는 것을 관찰할 수 있었다. 즉 이러한 실험결과들을 통해, 적혈구 내에서 Prx II 가 과산화될 수 있으며 이것을 환원시키는 역할을 Sulfiredoxin 이 한다는 것을 알 수 있다.;Physiological role of RBCs as oxygen transports results in a unique cellular environment rich in oxygen and heme-containing hemoglobin (Hb), which induces constant oxidative stress in red blood cells (RBCs). In RBCs, 3% of total Hb is autoxidized to metHb each day. RBCs are equipped with various antioxidant enzymes to protect cells from damage by reactive oxygen species (ROS) generated as a result of autoxidation of Hb. Peroxiredoxin (Prx) II is the principal enzyme responsible for peroxide elimination in RBCs. Prx contain two conserved cysteine residues, Cys-52 (designated the peroxidase cysteine, CP) and Cys-172 (resolving cysteine, CR). In Prx II, which are homodimers, CP-SH of one subunit is selectively oxidized by peroxides to CP-SOH, which then reacts with CR-SH of the other subunit to form intermolecular disulfide bond. Reduction of the disulfide intermediate is mediated by thioredoxin (Trx) using NADPH as a reducing power. However, the CP-SOH intermediate generated during catalysis is occasionally to undergoes further oxidation to sulfinic acid (CP-SO2H), leading to inactivation of peroxidase function. This CP-SO2H of Prx II are selectively reduced to CP-SH by sulfiredoxin (Srx) in the presence of ATP, which reactivate the peroxidase function of Prx II. In spite of this reactivation system by Srx, Prx II has been thought as noncatalytic scavenger of hydrogen peroxide in RBCs because of their high cellular concentration, accumulation of disulfide-linked dimer of the catalytic cycle, and the limited capacity of the Trx system in RBCs We have now showed the catalytic properties of Prx II by comparing hyperoxidation level of Prx II in wild type RBCs to that of RBCs in Srx knockout mice. Sulfinic form of Prx II (Prx-SO2H) was really generated in RBCs by glucose oxidase, and it was regenerated to reduced form of Prx II (Prx II-SH) by Srx. We also observed that Prx II in Srx-depleted RBCs were more severely hyperoxidized by hydrogen peroxide than Srx-containing normal RBCs. These results demonstrate that Prx II in RBCs functions as a catalytic scavenger of hydrogen peroxide and its hyperoxidized sulfinic cysteine was regenerated by Srx, which is important to sustain peroxidase activity of Prx II and protect RBCs from oxidative stress.
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