Inhibition and conformational change of SERCA3b induced by Bcl-2
| Autor: | Christian Schöneich, Asha Hewarathna, Elena S. Dremina |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Gene isoform Conformational change SERCA Protein Conformation Biophysics Biology Biochemistry Article Sarcoplasmic Reticulum Calcium-Transporting ATPases Analytical Chemistry 03 medical and health sciences chemistry.chemical_compound Microsomes Humans Immunoprecipitation Fluorescein isothiocyanate Molecular Biology Sequence Deletion HEK 293 cells Molecular biology In vitro Blot HEK293 Cells 030104 developmental biology Proto-Oncogene Proteins c-bcl-2 chemistry Microsome |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 1865:121-131 |
| ISSN: | 1570-9639 |
| DOI: | 10.1016/j.bbapap.2016.09.004 |
| Popis: | An interaction of Bcl-2 with SERCA had been documented in vitro using the SERCA1a isoform isolated from rat skeletal muscle [Dremina, E. S., Sharov, V. S., Kumar, K., Azidi, A., Michaelis, E. K., Schoneich, C. (2004) Biochem. J. 383 (361–370)]. Here, we demonstrate the interaction of Bcl-2 with the SERCA3b isoform both in vitro and in cell culture. In vitro, the interaction of Bcl-2 with SERCA3b was studied using Bcl-2∆21, a truncated form of human Bcl-2, and microsomes isolated from SERCA3b-overexpressing HEK-293 cells. For these experiments, SERCA3b was quantified by a combination of amino acid analysis and Western blotting. We observed that Bcl-2∆21 both inactivates SERCA3b and co-immunoprecipitates with SERCA3b. The incubation with Bcl-2∆21 changes the distribution of SERCA3b during sucrose density gradient centrifugation, likely as the result of Bcl-2∆21-induced conformational change of SERCA3b. When SERCA3b-overexpressing HEK-293 cells were co-transfected with Bcl-2, Bcl-2-dependent SERCA3b inactivation was observed. In these cells, Bcl-2 interaction with SERCA3b was demonstrated by co-immunoprecipitation. Furthermore, overexpression of Bcl-2 reduced fluorescein isothiocyanate (FITC) labeling of SERCA3b. Together, our data provide evidence for the interaction of Bcl-2 with SERCA3b in vitro and in cell culture, and for Bcl-2-dependent conformational and functional changes of SERCA3b. |
| Databáze: | OpenAIRE |
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