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

• Main Authors: Wen-Hsiung Ho, 何文雄
• Other Authors: 張震東
• Format: Others
• Language: en_US
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/36247906639653164795

博士 === 國立臺灣大學 === 生化科學研究所 === 100 === 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.