초록

기존 유효 cytokine타켓으로 검증된 ARS단백질복합체들에 대한 구조적 정보를 TEM 이미징과 Single particle analysis를 활용하여 분자에서 규명한다. Extracellular exosome vesicle들의 endocytosis를 통한 세포 내 유입과정을 subcellular level에서 확인하기 위해, isolated된 ARS associated vesicle들의 세포유입경로를 Electron tomography와 Immuno-EM기법을 통해 규명한다.

(출처 : 보고서 요약서 3p)

Abstract ▼

□ Purpose & Contents
This proposal is aimed at elucidating regulatory mechanisms on the intracellular/extracellular secretion of ARS cytokines via electron microscopy. Firstly, the ARS complex will be determined structurally. Secondly, Immuno-EM will be applied to trafficking pathway of ARS vesic

□ Purpose & Contents
This proposal is aimed at elucidating regulatory mechanisms on the intracellular/extracellular secretion of ARS cytokines via electron microscopy. Firstly, the ARS complex will be determined structurally. Secondly, Immuno-EM will be applied to trafficking pathway of ARS vesicles containing target cytokines from intracellular origin to extracellular exertion and their pathway during endocytosis. Thirdly, visualized cellular events of interest will be reconstructed into 3D density maps through electron tomography in order to observe their detailed structural information. Fourthly, conventional EM and cryo-EM applications will be used to identify the localization and protein-protein interactions of target ARS cytokine proteins. Through carrying out this proposal, we will achieve insights into how ARS cytokines can be regulated during the exocytosis and endocytosis as well as how their multi-functional roles are related with biological events. The final goal will be used as a fundamental studies which can contribute to understand overall regulatory mechanisms of ARS cytokines secretion and to aid on therapeutics developments in related human diseases.
· Determining structural features and protein interactions from identified ARS proteins To identify structural characterization of particular ARS sub-complex as therapeutic target and their multi-functional roles, single particle analysis followed by cryo-EM and conventional EM techniques will be performed on structural determination of intermolecular interactions. The ultimate goal will be structural determination of MSC complex through high resolution cryo-electron microscopy.
· Identifying structural features of exosome(-like vesicles) associated with ARS proteins and their intracellular/extracellular trafficking pathways to demonstrate extra-physiological impacts of the ARS at subcellular level
- To provide a structural indicator, electron microscopy will be carried out to identify target exosome (-like vesicle) associated with known ARS proteins.
- To determine trafficking pathway of target exosome (-like) vesicle at intracellular level, time resolved immuno EM studies using target cells treated with particular environment factors such as glucose deprivation.
- To elucidate the endocytosis pathway of extracellular (-like) vesicle at subcellular level, isolated exosomes potentially interacting to target cells will be used. This In vitro study will be carried out by electron tomography and sectional studies of the target cell.
- To build up structural database of 3D density maps reconstructed ultracellular structures at particular pathway of interest, electron tomography will be adopted in this study. This application will provide suggestive evidence to elucidate and comparatively analysis biological meaning of extra-role of the ARS proteins. In addition, chemical and physical effects will be employed into this studies to interpret physiological impacts of the ARS network.

□ Results
There are two major development done for the 1st year of this research project
- Newly developed technical methods of specimen preparation for Immuno-EM of isolated exosome: feasible to visualize localization of ARS cytokines within exosome even from low concentration(amount) of isolated exosome. With using the technical approaches from this study structural features of exosome and microvesicles are identified as analytical plateform.
- Negative staining followed by single particle analysis were done to determine 3D structure of MSC sub-complex

□ Expected Contribution
● Academic achievements
The findings of the structure of ARS sub-complex (and MSC) intracellular/extracellular secretion of ARS cytokines and establishment of each multi-functional role of ARS cytokines proteins provide the understandings of various ARS network in biological system. The final goals taken from these studies including addressed studies using chemical and physical stress will be imaged and these will ultimately facilitate access to choose therapeutic targets for diagnostics and drug development.
● Application fields and Scopes
A more detailed knowledge on multi-functional roles of ARS network in biological system through this project will be transferred into pipeline of drug development for treatment of related human diseases as well as cancer. In summary, this proposal will shed promising lights into reinterpretation of additional functional roles of ARS proteins.

(출처 : SUMMARY 5p)

목차 Contents

• 표지 ... 1
• 제 출 문 ... 2
• 보고서 요약서 ... 3
• 요약문 ... 4
• SUMMARY ... 5
• CONTENTS ... 6
• 목차 ... 7
• 제1장 연구개발과제의 개요 ... 8
• 1. 연구개발 목적 ... 8
• 2. 연구개발의 필요성 ... 8
• 3. 연구개발 범위 ... 8
• 제2장. 연구수행내용 및 성과 ... 9
• 1. ARS cytokine associated exosome의 구조적 특성 및 세포내/외 전달경로 규명 ... 9
• 2. ARS cytokine 발굴 단백질들의 기능적 분자구조규명 ... 15
• 제3장. 목표 달성도 및 관련 분야 기여도 ... 35
• 1. 목표 ... 35
• 2. 목표 달성여부 ... 35
• 3. 관련분야 기여도 ... 36
• 4. 목표 미달성 시 원인(사유) 및 차후대책(후속연구의 필요성 등) ... 36
• 제4장. 연구개발성과의 활용 계획 ... 37
• 제5장. 연구개발성과의 보안등급 ... 37
• 제6장. 국가과학기술종합정보시스템에 등록한 연구시설·장비 현황 ... 37
• 제7장. 연구개발과제 수행에 따른 연구실 등의 안전 조치 이행 실적 ... 38
• 제8장. 연구개발과제의 대표 연구 실적 ... 39
• 제9장. 참고문헌 ... 40
• 끝페이지 ... 42