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  • 學位論文

Hsp90等蛋白參與細胞內磷酸鈣沈澱調控機制之研究

Proteomic Identification of a Novel Hsp90-Containing Protein-Mineral Complex Which Can Be Induced in Cells in Response to Massive Calcium Influx

指導教授 : 張震東

摘要


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。

並列摘要


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.

並列關鍵字

Hsp90 fetuin-A calcium phosphate mineral precipitation membrane damage FMC PMC

參考文獻


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