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Regulation of Weibel-Palade Body Exocytosis by alpha-Synuclein in Endothelial Cells

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dc.contributor.author김, 광수-
dc.date.accessioned2011-11-08T01:18:59Z-
dc.date.available2011-11-08T01:18:59Z-
dc.date.issued2011-
dc.identifier.urihttp://repository.ajou.ac.kr/handle/201003/4381-
dc.description.abstracta-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.-
dc.description.tableofcontentsⅠ. 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
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dc.language.isoen-
dc.titleRegulation of Weibel-Palade Body Exocytosis by alpha-Synuclein in Endothelial Cells-
dc.title.alternative혈관내피세포에서 알파시누클레인 (alpha-Synuclein)에 의한 Weibel-Palade Body의 exocytosis 조절-
dc.typeThesis-
dc.identifier.urlhttp://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000011658-
dc.subject.keyword혈관내피세포-
dc.subject.keywordWeibel-Palade Body-
dc.subject.keywordexocytosis-
dc.subject.keywordEndothelial cells-
dc.subject.keyword알파시누클레인-
dc.subject.keywordalpha-Synuclein-
dc.description.degreeDoctor-
dc.contributor.department대학원 의생명과학과-
dc.contributor.affiliatedAuthor김, 광수-
dc.date.awarded2011-
dc.type.localTheses-
dc.citation.date2011-
dc.embargo.liftdate9999-12-31-
dc.embargo.terms9999-12-31-
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