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dc.description.abstractReceptor-mediated endocytosis (RME) is well known as a process whereby internalization of proteins and lipids from the plasma membrane into internal membrane compartments in eukaryotic cells. RME should be well controlled to maintain the activity of signaling cascades, cell metabolism and cell homeostasis. Classical view concerning a role of RME in response to extracellular stimulations is a negative regulatory system of ligand activation to receptor at the plasma membrane by a degradation of ligand-receptor complex at lysosome. Recently, several studies showed that endocytosis and vesicle trafficking are important pathway to control the signaling transduction and cellular response from the plasma membrane into the cytosol. In the first part of this study, I investigated the roles of reactive oxygen species (ROS) which regulate the signaling cascades, especially at early endosomes. Before the sorting events, an appropriate recruitment of adaptor and effector proteins is a critical for determination of the fate of early endosomes. ROS, especially hydrogen peroxide (H2O2) are known to be signaling mediator during signal transduction from plasma membrane to cytosol. But molecular way of H2O2 regulation on endocytosis has not been studied, intensively. Therefore, I focused on two well-known early endosome binding proteins, Phosphatidylinositol 3-phosphate (PtdIns(3)P) and Adaptor protein containing PH domain, PTB domain and Leucine zipper motif 1 (APPL1) to figure out the molecular mechanism of endocytosis regulation by H2O2. Because these proteins are considered as important molecules as a second messenger in determining the signaling cascades. Here, using fluorescent APPL1, PtdIns(3)P binding probe and confocal imaging, I firstly showed that endosomal PtdIns(3)P levels were transiently decreased in cells exposed to a medium with H2O2 which was produced by glucose oxidase at the presence of high glucose. This result indicates that H2O2 can change the function of early endosome. To investigate whether the decrease of endosomal PtdIns(3)P level is related to conversion of early endosome into late endosome during RME, I monitored the level of endosomal APPL1, an effector of rab5 and precursor of classical PtdIns(3)P positive endosomes. APPL1 plays a role of platform for the assembly of signaling complexes and regulation of signaling cascades, including the MAPK and Akt pathways. Confocal imaging showed that both the number of APPL1 positive endosomes and the amount of APPL1 on endosome were increased by local H2O2 generated in cells during growth factor stimulations. Intriguingly, phosphorylation of Akt in endosome also increased in cells under the oxidative stress. Here, I propose that H2O2 molecules selectively act as a positive regulator for Akt activation in early endosome through the increase of APPL1 recruitment and reduction of endosomal PtdIns(3)P level by mechanism distinct from regulation of PtdIns(3,4,5)P3 level in plasma membrane. In the second part, I investigated generation of endosomal H2O2 monitoring probe, HyPer-Endo which can detect the dynamics of H2O2 amount at early endosomes. HyPer is recently generated new type of H2O2-selective probe which designed as a genetical ratiometric biosensor.(1) As I mentioned, localized ROS especially H2O2, play an important role as a second messenger that generated from intracellular organism in normal cellular processes. Redoxosomes, previously suggested theory, are redox-active signaling endosomes that redox-active signaling endosomes can include NADPH oxidase complex components which has a capable of transmitting ROS signals from the endosome interior to redox-sensitive effectors on the endosomal surface.(2) Here I firstly present an essential tool, HyPer-Endo, for visualizing only H2O2 which is transiently accumulated at early endosomes under the growth factor treatment conditions. HyPer-Endo has a possiblity to monitor the dynamics of endosomal H2O2. And I also studied about various redox-dependent signaling with endosomal targeting catalase, Cat-Endo which can remove the endosomal H2O2 in diverse extracellular stimuli conditions. Thus, localized H2O2 is tightly regulated in response to growth factor stimulation to maintain the cellular homeostasis.;세포 내 신호전달체계는 주로 세포막에서 이루어지는 것으로 일반적으로 알려져 왔으나 최근 여러 다양한 병증 및 신호, 환경 등에 더욱 효과적으로 대응할 수 있는 다양한 방법이 사실은 존재하고 있다는 사실이 대두되고 있다. 세포 내 흡입(Endocytosis)은 세포 내 단순한 물질 이동 뿐만 아니라 세포 내 신호를 핵으로 효과적으로 전달하기 위해 사용된다는 사실이 최근 여러 논문을 통해 명확해지고 있다. 이번 연구에서는 초기 엔도좀 (early endosome)에서 신호연계단백질 (adaptor protein)로 작용하여 여러 신호전달에 관여하는 단백질들이 결합되어 세포신호전달에 중요한 조절인자로 작용하는 APPL1 단백질이 세포 내 특정장소에 일시적으로 증가되어 2차 전달자 (Second messenger)로 작용한다고 알려져 있는 활성산소 (ROS) 중 가정 안정한 상태인 과산화수소 (H2O2) 에 의해서는 어떻게 변화하는지 알아보는 실험을 진행하였다. 공초점 현미경을 통한 이미징 실험으로 APPL1 단백질이 초기엔도좀에서 상피세포성장인자(EGF)에 의해 급격하게 증가하고 활성산소 발생인자인 Nox를 Nox저해제인 DPI를 처리해 감소시켰더니 증가현상이 확연히 감소되었다. 이를 통해 APPL1이 세포성장인자를 통해 부수적으로 생성되는 과산화수소에 의해 초기 엔도좀에서 증가한다는 사실을 밝혔다. 이 뿐만 아니라 활성산소는 APPL1과 결합해있는 엔도좀의 Akt 활성도 직접적으로 조절하며, 이 조절에 관여하는 Akt 활성은 세포 생존에 관여하는 ser473 잔기에 특이적으로 작용한다는 사실을 알게 되었다. APPL1 엔도좀 뿐만 아니라 초기엔도좀에 존재하고 APPL1 이 사라진 직후 그 자리에 결합하며 신호전달인자로 작용한다고 알려져 있는 phosphoinositides 중 PtdIns(3)P는 활성산소에 의해 그 양이 감소함을 관찰함으로써 두 엔도좀 간의 직접적인 변화를 활성산소가 정확히 어떠한 메커니즘으로 조절하는지는 알 수 없지만, 세포내 흡입과정 초기 엔도좀 형성 및 전체적인 조절과정에 과산화수소가 관여함을 알 수 있었다. 그리고 직접적으로 엔도좀에 관여하는 활성산소 중 과산화수소만을 선택적, 가역적으로 측정할 수 있는 HyPer 형광탐침과 엔도좀 특이적으로 과산화수소를 제거하는 Catalase를 만듦으로 인해 엔도좀에서 짧은 시간 증가한 과산화수소가 세포신호전달에 중요한 역할을 한다는 redoxosome 이론을뒷받침할 수 있는 훌륭한 재료를 제공하였다는데 큰 의미를 가진다.-
dc.description.tableofcontentsI.Abstract 1 II.Introduction 4 1. The Roles of ROS 4 A. ROS regulation system in mammalian cells 4 B. NADPH oxidase as a ROS source 7 2. Endocytosis progression 10 A. Receptor-mediated endocytosis at early endosome 10 B. Rab GTPase 20 III. Material and Methods 23 A. Chemicals and antibodies 23 B. Cell culture and transfection reagent 23 C. Plasmids and siRNAs 24 D. Biotinyl-cysteine labeling assay 25 E. Immunoblot and immunofluorescence 25 F. Acceptor bleaching imaging assay 27 G. Endocytosis analysis 27 H. Phagocytosis analysis 28 I. Statistical analysis 28 Part 1. Positive role of endosomal H2O2 on Akt ser473 phosphorylation through the recruitment of APPL1 29 II-1. Introduction 30 3. APPL1 protein 30 4. Phosphoinositides 33 5. Akt signaling 37 IV-1.The specific aim of the thesis 40 V-1.Result 42 1. Recruitment APPL1 into early endosomes increases under the ROS accumulated conditions. 42 2. APPL1 recruitment and endosomal Akt phosp-horylationintol early endosome are increased, transiently in physiological condition. 47 3. Hydrogen peroxide reduces PtdIn(3)P level in early endosomes. 56 4. Occurrence rate of endocytosis and MTM1, 3-phosphatase, were not influenced by extracellular H2O2. 60 5. Enhancement of APPL1 according to ROS regulates Akt signaling in specific substrate dependent manner. 64 PART 2. Development of biosensor for endosomal H2O2 HyPer-Endo 66 II-2.Introduction 67 6. Redoxosome 67 IV-2.The specific aim of the thesis 70 V-2.Results 71 1. Construction of early endosome targeting HyPer and Catalase. 71 2. Hyper-Endo can measure the H2O2 under the physiological condition. 79 3. Endosomal H2O2 on signaling process at cell survival. 82 4. Endosomal H2O2 complement a TNF mediated signaling. 87 VI.Discussion 94 VII.References 100 VIII.국문초록 110-
dc.format.extent6741773 bytes-
dc.publisher이화여자대학교 대학원-
dc.titlePositive role of endosomal H₂O₂on Akt ser473 phosphorylation through the recruitment of APPL1 and Development of biosensor for endosomal H₂O₂HyPer-Endo-
dc.typeDoctoral Thesis-
dc.format.pagevii, 111 p.-
dc.identifier.major대학원 생명과학과- 2-
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