혈관내피세포에서 알파시누클레인 (alpha-Synuclein)에 의한 Weibel-Palade Body의 exocytosis 조절
Regulation of Weibel-Palade Body Exocytosis by alpha-Synuclein in Endothelial Cells
- 발행기관 아주대학교
- 지도교수 주일로, 박상면
- 발행년도 2011
- 학위수여년월 2011. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000011658
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
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.
more목차
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
