2026-09-22T09:43:57Zhttps://repository.ajou.ac.kr/oai/request

oai:repository.ajou.ac.kr:201003/43812024-10-11T02:17:39Zcom_201003_14372com_201003_14368col_201003_14374

Regulation of Weibel-Palade Body Exocytosis by alpha-Synuclein in Endothelial Cells 혈관내피세포에서 알파시누클레인 (alpha-Synuclein)에 의한 Weibel-Palade Body의 exocytosis 조절 김, 광수 주, 일로 박, 상면 대학원 의생명과학과 105432 김, 광수 혈관내피세포 Weibel-Palade Body exocytosis Endothelial cells 알파시누클레인 alpha-Synuclein a-Synuclein (a-syn) is a small presynaptic protein implicated in the pathogenesis of Parkinson’s disease. Although gathering evidence has been proposed for its physiological roles, the precise roles of a-synuclein and mechanisms remain incompletely understood. a-Synuclein is not only expressed in neuron, but also in vascular endothelium. Endothelial cells contain intracellular granules called Weibel-Palade bodies (WPBs) that contain a number of chemokines, adhesive molecules and inflammatory cytokines. This study explored whether the exocytosis of WPB is regulated by a-synuclein. Results showed that PMA-, thrombin- or forskolin-induced VWF release or translocation of P-selectin from Human Umbilical Vein Endothelial Cells (HUVECs) were inhibited by overexpression of a- and b-synuclein, but not g-synuclein. The overexpression of three point mutants (A30P, A53T and E46K) found in familial Parkinson’s disease inhibited WPB exocytosis similar to that of wild-type a-synuclein. Results also showed that the negative regulation of WPB exocytosis required the N-terminus or NAC region of a-synuclein, but not C-terminal acidic tail, and that a-synuclein affected WPB exocytosis through interference with RalA activation by enhancing the interaction of RalGDS/b-arrestin complexes. Immuno-EM analysis revealed that a-synuclein was localized close to WPB. These findings imply that a-synuclein plays as a negative regulator in WPB exocytosis in endothelial cells. Ⅰ. INTRODUCTION 1 Ⅱ. MATERIALS AND METHODS 5 A. MATERIALS 5 1. Reagents and Antibodies 5 2. Constructs 5 B. METHODS 6 1. Cell Culture and Transfection 6 2. RT-PCR analysis 6 3. Determination of VWF by ELISA 7 4. Leukocyte Adhesion Assay 8 5. Ral Activation Assay 8 6. Confocal Microscopy 9 7. Immuno-Electron Microscopy 9 8. Western Blot and Immunoprecipitation 10 Ⅲ. RESULTS 12 1. Three different kinds of secretagogues induce vWF secretion in HUVECs in a time dependent manners 12 2. -Syn overexpression inhibits three secretagogues-induced VWF secretion in HUVECs 14 3. WPB exocytosis is inhibited by -syn and -syn, but not by -syn overexpression 19 4. -Syn mutants (A30P, A53T and E46K) inhibit WPB exocytosis similar to that of wild-type -syn 19 5. N-terminal or NAC region of a-syn is necessary for the inhibition of PMA-induced VWF release in HUVECs 23 6. Secretagogues-induced RalA activation is suppressed by -syn overexpression 28 7. The interaction between RalGDS and -arrestin is enhanced by -syn overexpression 31 8. -Syn is localized close to WPB in HUVECs 35 9. Exogenously added recombinant -syn also inhibits VWF release in HUVECs 38 Ⅳ. DISCUSSION 41 Ⅴ. CONCLUSION 47 REFERENCES 48 국문 요약 59 Doctor 2011-11-08T01:18:59Z 2011-11-08T01:18:59Z 2011 2011 Thesis Theses http://repository.ajou.ac.kr/handle/201003/4381 http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000011658 000000011658 en