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DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 張震東 | |
dc.contributor.author | Wen-Hsiung Ho | en |
dc.contributor.author | 何文雄 | zh_TW |
dc.date.accessioned | 2021-06-16T23:06:06Z | - |
dc.date.available | 2012-08-10 | |
dc.date.copyright | 2012-08-10 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-08-06 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64892 | - |
dc.description.abstract | 磷為生物有機分子的重要組成之一,生物體內舉凡結構性之細胞膜成分、生物能量供應之ATP及細胞遺傳物質之DNA、RNA等皆存在著磷酸分子;此外,磷酸溶液也是生物體內普遍存在的緩衝環境之一,生物體內大部分合成或代謝等酵素反應,皆需要在緩衝溶液中才得以完成。而鈣離子則是細胞內、外重要訊息傳遞分子之一,當生物體接受內、外部訊息時,鈣離子會由細胞外或細胞內儲存大量鈣離子的貯藏庫中被釋放至需要的地方,所以,細胞內會產生瞬間高濃度之鈣離子擾動,如此,局部大量的鈣離子必然會造成局部磷酸鈣沈澱的發生,然而,除了骨骼、牙齒等生物支持性構造需要藉由調控磷酸鈣沈積而形成結構性組織外,生物體細胞內若有不正常產生大量磷酸鈣沈澱,勢必對細胞造成傷害或誘導細胞的凋亡等反應。在細胞外,Fetuin-A 已被證實參與防止磷酸鈣沈澱發生重要調控因子之一,藉由Fetuin-A與磷酸鈣結合以防止磷酸鈣在細胞外的堆積。然而,在充滿著許多磷酸根溶液的細胞內,是否亦存在防止或抑制磷酸鈣沈澱產生的調節因子呢?
在本研究中,我們利用蛋白純化、質譜鑑定、抗體免疫分析、細胞培養實驗及電子顯微鏡等方法證明:細胞內確實存在一些調節蛋白,這些蛋白分子包含有Hsp90, annexin A5, calreticulin, tubulin-β, nucleolin, PLC-β1, CAND-1及GAPDH等,這群調節蛋白我們稱為PMC (protein mineral complex)蛋白,在這群結合蛋白中Hsp90佔主要成分,為瞭解Hsp90與磷酸鈣調節機制,我們進一步從豬腦、原核生物E.coli.或真核細胞SiHa cell中純化或表現Hsp90蛋白,實驗結果發現:Hsp90可直接與amorphous calcium phosphate (ACP) 及hydroxyapatite (HA) 結合,其與磷酸鈣結合不需要其他輔助蛋白的幫忙;當Hsp90與磷酸鈣結合時,會產生多單元體蛋白組成的構形改變,此一Hsp90與磷酸鈣結合會被ATP、ADP、GTP、EDTA及雙磷酸鹽類藥物等所抑制,從免疫螢光細胞實驗中,我們亦發現其他如annexin A5, calreticulin, tubulin-β, nucleolin, PLC-β1, CAND-1及GAPDH等次主要PMC蛋白在細胞中與磷酸鈣結合部位與Hsp90相同。 我們進一步以鈣離子專一性通透劑(A23187, ionomycin)、鈣離子抑制劑(EDTA, EGTA)或骨質疏鬆症治療藥劑(雙磷酸鹽類藥物)等進行細胞試驗時,也證明細胞內這群PMC蛋白與磷酸鈣結合機制的功能存在,推測這些蛋白分子可能依其位於細胞中不同胞器內參與不同磷酸鈣沈澱調節功能;而雙磷酸鹽類之骨質疏鬆症治療藥劑對腸道所產生不適之副作用,也可由其細胞毒性試驗中得到推論證明。 | zh_TW |
dc.description.abstract | Fetuin-A is known for limiting the expansion and formation of hydroxyapatite crystals from calcium phosphate aggregates in circulation by forming a soluble fetuin−mineral complex. This study was aimed to uncover potential proteins involved in the regulation of calcium phosphate precipitation within cells. We found that a novel protein-mineral complex (PMC) can be generated after introduction of calcium chloride and sodium phosphate into the porcine brain protein extract prepared in Tris-HCl buffer. Selectively enriched proteins in the pellet were confirmed by immunoblotting, including heat shock protein 90 (Hsp90), annexin A5, calreticulin, nucleolin, and other proteins. In addition, purified native Hsp90 directly bound both amorphous calcium phosphate and hydroxyapatite and underwent conformational changes and oligomerization in the presence of excess calcium and phosphate. The morphology of the PMC prepared from Hsp90, calcium, and phosphate was distinctly different from that of hydroxyapatite under transmission electron microscopy observation. When cultured SiHa cells were treated with a calcium ionophore or damaged by scratch to induce the massive calcium influx, a complex was formed and observed at discrete sites near the plasma membrane as revealed by antibodies against Hsp90, annexin A5, calreticulin, nucleolin, and other proteins. This complex could also be probed in situ with fetuin-A suggesting the existence of calcium phosphate aggregates in this complex. Inhibition of the complex formation by bisphosphonates hindered cell recovery from A23187 assault.
Our results show that following membrane damage amorphous calcium phosphate develops at sites near membrane rupture where saturated calcium phosphate concentration is achieved. As a result, Hsp90 and other proteins are recruited, and the cytosolic PMC is formed. Inhibition of the cytosolic PMC formation may in part contribute to the cellular toxicity and in vivo side effects of bisphosphonates, particularly in cells prone to membrane damage under physiological conditions such as gastrointestinal epithelial and oral cavity epithelial cells. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T23:06:06Z (GMT). No. of bitstreams: 1 ntu-101-D93B46007-1.pdf: 13703989 bytes, checksum: bc171d0f2ba791eaad8c72f9fbd7ce60 (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | 中文摘要 i
Abstract iii Lists of Tables and Figures vii Lists of Appendices viii Lists of Abbreviations ix 1. Introduction 1 1.1. The phosphate buffer system in cells 1 1.2. Regulation of calcium in cells 1 1.3. Formation and regulation of the calcium phosphate precipitation 3 1.4. Interaction of calcium and phosphate in cells 4 1.5. Function and regulation of fetuin and Fetuin Mineral Complex, FMC 5 1.6. Functions and regulations of heat shock protein 90 5 1.7. The effect of ionophore, A23187, in cells 7 1.8. Summary 7 1.9. Specific aims 9 2. Materials and methods 10 2.1. Materials 10 2.2. Methods 11 2.2.1. Purified preparation of protein extracts and purification of Hsp90 11 2.2.2. Preparation of the PMC 12 2.2.3. Binding of Hsp90 to the CHT hydroxyapatite column 13 2.2.4. SDS-PAGE and native gel electrophoresis 13 2.2.5. Two-dimensional gel electrophoresis 13 2.2.6. In-gel digestion 14 2.2.7. LC-nano-ESI-MS/MS analysis 14 2.2.8. Data analysis 15 2.2.9. Immunoblotting 16 2.2.10. Construction of human partial Hsp90α or full-length Hsp90β into expression vectors 16 2.2.11. Expression of recombinant human partial Hsp90α or full-length Hsp90β in E. coli BL21 17 2.2.12. Cell culture and transfection 18 2.2.13. Cell treatments and damage assay 18 2.2.14. Effect of hyperthermia on the PMC formation 19 2.2.15. MTT assay 19 2.2.16. Immunoflourescence staining assay 20 2.2.17. Differential scanning fluorimetric assay 20 2.2.18. pH drop assay 21 2.2.19. Transmission electron microscopy 21 3. Results 23 3.1. Composition of the PMC 23 3.2. Mineral requirement of the PMC 24 3.3. Hsp90 as a calcium phosphate binding protein 25 3.4. The conformational change of Hsp90 during the calcium phosphate binding 26 3.5. Hsp90 directly binds calcium phosphate 28 3.6. The effect of Hsp90 on transformation of ACP 29 3.7. ATP affects Hsp90 binding to calcium phosphate 30 3.8. Formation of the PMC within Cells 31 3.9. Address the Hsp90 binding calcium phosphate by Fetuin-A in cell 32 3.10. Cellular localization of the PMC proteins 33 3.11. Effect of heat shock on the PMC formation 35 3.12. Consequences of the PMC impairment 35 4. Discussion 38 5. Conclusions/ perspectives 44 6. References 74 | |
dc.language.iso | en | |
dc.title | Hsp90等蛋白參與細胞內磷酸鈣沈澱調控機制之研究 | zh_TW |
dc.title | Proteomic Identification of a Novel Hsp90-Containing Protein-Mineral Complex Which Can Be Induced in Cells in Response to Massive Calcium Influx | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 黃銓珍,陳宏文,李明亭,張茂山 | |
dc.subject.keyword | Hsp90,fetuin-A,磷酸鈣,蛋白質體學,蛋白質純化,質譜鑑定, | zh_TW |
dc.subject.keyword | Hsp90,fetuin-A,calcium phosphate,mineral precipitation,membrane damage,FMC,PMC, | en |
dc.relation.page | 109 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2012-08-06 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
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ntu-101-1.pdf 目前未授權公開取用 | 13.38 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。