Fu M H, Tupling A R
Dept. of Kinesiology, Univ. of Waterloo, Waterloo, ON, Canada N2L 3G1.
Am J Physiol Heart Circ Physiol. 2009 Apr;296(4):H1175-83. doi: 10.1152/ajpheart.01276.2008. Epub 2009 Feb 27.
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.
热休克蛋白70（Hsp70）可与肌浆网/内质网Ca²⁺-ATP酶（SERCA）1a发生物理相互作用，并防止其热失活，SERCA1a是在成年快肌骨骼肌中表达的SERCA亚型。本研究检测了Hsp70是否能与心脏中表达的SERCA亚型SERCA2a发生物理相互作用并防止其热失活。将编码人Hsp70和兔SERCA2a的cDNA共转染到人胚肾293（HEK-293）细胞中（S2a/Hsp70）。将共转染SERCA2a cDNA和pMT2的细胞（S2a/pMT2）用作对照。在收获细胞并分离微粒体之前，将一半细胞在40℃热休克1小时（HS），另一半细胞维持在37℃。蛋白质免疫印迹分析表明，Hsp70和SERCA2a共定位于微粒体部分。与S2a/pMT2相比，S2a/Hsp70中Hsp70的水平高约五倍（P<0.05），并且在所有细胞中热休克后Hsp70水平高约两倍（P<0.05）。免疫共沉淀表明Hsp70直接与SERCA2a结合。热休克后，S2a/pMT2中SERCA2a最大活性降低（约52%，P<0.05），而S2a/Hsp70中SERCA2a最大活性增加（约33%，P<0.05）。S2a/pMT2中SERCA2a的热失活与异硫氰酸荧光素（FITC）结合能力降低（约49%，P<0.05）以及SERCA2a中羰基（约42%，P<0.05）和硝基酪氨酸（约40%，P<0.05）水平升高有关。相比之下，在S2a/Hsp70中观察到的热休克诱导的SERCA2a最大活性增加与FITC结合能力无变化（P>0.05）以及与S2a/pMT2相比SERCA2a中羰基（约40%，P<0.05）和硝基酪氨酸（约23%，P<0.05）水平降低相对应。这些结果表明，在热休克期间Hsp70与SERCA2a形成保护性相互作用，实际上减少了SERCA2a的氧化和亚硝化，从而增加了其最大活性。
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