Protective effects of Hsp70 on the structure and function of SERCA2a expressed in HEK-293 cells during heat stress.
Heat shock protein 70 (Hsp70) can physically interact with and prevent thermal inactivation of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) 1a, the SERCA isoform expressed in adult fast-twitch skeletal muscle. This study examined whether Hsp70 could physically interact with and prevent thermal inactivation of SERCA2a, the SERCA isoform expressed in heart. HEK-293 cells were cotransfected with cDNAs encoding human Hsp70 and rabbit SERCA2a (S2a/Hsp70). Cells cotransfected with SERCA2a cDNA and pMT2 (S2a/pMT2) were used as control. One-half of the cells was heat shocked at 40 degrees C for 1 h (HS), and one-half was maintained at 37 degrees C before harvesting the cells and isolating microsomes. Western blot analysis showed that Hsp70 and SERCA2a were colocalized in the microsomal fraction. The levels of Hsp70 were approximately fivefold higher (P < 0.05) in S2a/Hsp70 compared with S2a/pMT2 and approximately twofold higher (P < 0.05) following HS in all cells. Coimmunoprecipitation demonstrated that Hsp70 directly binds to SERCA2a. Following HS, maximal SERCA2a activity was reduced ( approximately 52%, P < 0.05) in S2a/pMT2 but was increased ( approximately 33%, P < 0.05) in S2a/Hsp70. Thermal inactivation of SERCA2a in S2a/pMT2 was associated with decreased ( approximately 49%, P < 0.05) binding capacity for fluorescein isothiocyanate (FITC) and increased carbonyl ( approximately 42%, P < 0.05) and nitrotyrosine ( approximately 40%, P < 0.05) levels in SERCA2a. By contrast, the HS-induced increase in maximal SERCA2a activity observed in S2a/Hsp70 corresponded with no change (P > 0.05) in FITC-binding capacity and reductions in carbonyl ( approximately 40%, P < 0.05) and nitrotyrosine ( approximately 23%, P < 0.05) levels in SERCA2a compared with S2a/pMT2. These results show that Hsp70 forms a protective interaction with SERCA2a during HS actually reducing oxidation and nitrosylation of SERCA2a thus increasing its maximal activity.
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Dept. of Kinesiology, Univ. of Waterloo, Waterloo, ON, Canada N2L 3G1.
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D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D007668:Kidney / Q000166:cytology* / Q000196:embryology / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D050050:Protein Carbonylation / Q000502:physiology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism*
D018840:HSP70 Heat-Shock Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium