Fortilin binds Ca2+ and blocks Ca2+-dependent apoptosisin vivo
| Autor: | Jui Yoa Chang, Potchanapond Graidist, Amornrat Phongdara, Akiko Nakatomi, Michio Yazawa, Kenichi Fujise, Curtis Chun Jen Lin, Moltira Tonganunt |
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| Rok vydání: | 2007 |
| Předmět: |
Intracellular Fluid
Programmed cell death Thapsigargin Cations Divalent Down-Regulation Apoptosis Plasma protein binding Biology Biochemistry Protein Structure Secondary Cell Line Mice chemistry.chemical_compound Biomarkers Tumor Animals Molecular Biology Tumor Protein Translationally-Controlled 1 Cell Biology Molecular biology Cell biology Cytosol chemistry Cell culture Calcium Signal transduction Intracellular Protein Binding Signal Transduction Research Article |
| Zdroj: | Biochemical Journal. 408:181-191 |
| ISSN: | 1470-8728 0264-6021 |
| Popis: | Fortilin, a 172-amino-acid polypeptide present both in the cytosol and nucleus, possesses potent anti-apoptotic activity. Although fortilin is known to bind Ca2+, the biochemistry and biological significance of such an interaction remains unknown. In the present study we report that fortilin must bind Ca2+ in order to protect cells against Ca2+-dependent apoptosis. Using a standard Ca2+-overlay assay, we first validated that full-length fortilin binds Ca2+ and showed that the N-terminus (amino acids 1–72) is required for its Ca2+-binding. We then used flow dialysis and CD spectropolarimetry assays to demonstrate that fortilin binds Ca2+ with a dissociation constant (Kd) of approx. 10 μM and that the binding of fortilin to Ca2+ induces a significant change in the secondary structure of fortilin. In order to evaluate the impact of the binding of fortilin to Ca2+in vivo, we measured intracellular Ca2+ levels upon thapsigargin challenge and found that the lack of fortilin in the cell results in the exaggerated elevation of intracellular Ca2+ in the cell. We then tested various point mutants of fortilin for their Ca2+ binding and identified fortilin(E58A/E60A) to be a double-point mutant of fortilin lacking the ability of Ca2+-binding. We then found that wild-type fortilin, but not fortilin(E58A/E60A), protected cells against thapsigargin-induced apoptosis, suggesting that the binding of fortilin to Ca2+ is required for fortilin to protect cells against Ca2+-dependent apoptosis. Together, these results suggest that fortilin is an intracellular Ca2+ scavenger, protecting cells against Ca2+-dependent apoptosis by binding and sequestering Ca2+ from the downstream Ca2+-dependent apoptotic pathways. |
| Databáze: | OpenAIRE |
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