HSP70 Binds to the Fast-twitch Skeletal Muscle Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA1a) and Prevents Thermal Inactivation

Abstract

This study examined whether HSP70 could bind to and protect against thermal inactivation of SERCA1a, the SERCA isoform expressed in adult fast-twitch skeletal muscle. Sarcoplasmic reticulum vesicles prepared from rat gastrocnemius muscle were incubated with purified HSP70 at both 37 and 41°C for either 30, 60, or 120 min. Maximal SERCA1a activity (mol/g protein/min) in the absence of HSP70 was reduced progressively with time, with greater reductions occurring at 41°C compared with 37°C. HSP70 protected against thermal inactivation of SERCA1a activity at 37°C but not at 41°C and only at 30 and 60 min but not at 120 min. HSP70 also protected against reductions in binding capacity for fluorescein isothiocyanate, a fluorescent probe that binds to Lys 515 in the nucleotide binding domain of SERCA, at 30 and 60 min but not at 120 min, an effect that was independent of temperature. HEK-293 cells were cotransfected with cDNAs encoding rabbit SERCA1a and human HSP-EYFP and subjected to 40°C for 1 h. Immunohistochemistry revealed nearly complete co-localization of SERCA1a with HSP70 under these conditions. Co-immunoprecipitation showed physical interaction between HSP70 and SERCA1a under all thermal conditions both in vitro and in HEK-293 cells. Modeling showed that the fluorescein isothiocyanate-binding site of intact SERCA1a in the E2 form lies in its close proximity to a potential interaction site between SERCA1a and HSP70. These results indicate that HSP70 can bind to SERCA1a and, depending on the severity of heat stress, protect SERCA1a function by stabilizing the nucleotide binding domain. Sarco(endo)plasmic reticulum Ca2ϩ -ATPases (SERCAs) 1 are 110-kDa integral membrane proteins that catalyze the ATPdependent transport of Ca 2ϩ from the cytosol to the lumen of the sarco(endo)plasmic reticulum (SR) (1). Two SERCA isoforms predominate in adult striated muscle, namely SERCA1a and SERCA2a. SERCA1a accounts for more than 99% of the SERCA isoforms expressed in adult fast-twitch skeletal muscle, whereas SERCA2a is the major isoform expressed in heart and slow-twitch skeletal muscle (2). In muscle, SERCAs perform at least two crucial functions obligatory for precise control of intracellular Ca 2ϩ . In conjunction with plasma membrane Ca 2ϩ -ATPases, SERCAs are responsible for setting resting cytoplasmic Ca 2ϩ concentrations, and during repetitive muscle contractions, they induce muscle relaxation through the rapid sequestration of large Ca 2ϩ loads from the cytoplasm into the lumen of the SR, thereby determining the size of the SR Ca 2ϩ

Protective effects of Hsp70 on the structure and function of SERCA2a expressed in HEK-293 cells during heat stress

“…The culture of HEK-293 cells and their cotransfection with cDNAs have been described in earlier publications (18,50). cDNA encoding human heat shock 70-kDa protein 1A (55) and rabbit SERCA2a (18) were used in these experiments.…”

“…To determine whether Hsp70 could bind to SERCA2a, coimmunoprecipitation of SERCA2a and Hsp70 was carried out as described previously (3,4,50) with modifications. Microsomes were diluted in ddH2O to a final concentration of 0.5 mg/ml in a total of 500 l. Purified SERCA2a protein (300 ng) and Hsp70 protein (3.0 g, SPP-758) were used as control.…”

“…We have shown that Hsp70 can directly bind to SERCA1a during heat stress in vitro and in cultured cells (50). Here we used coimmunoprecipitation to test for physical association between SERCA2a and Hsp70 following conditions of heat stress in all groups of HEK-293 cells (i.e., S2a/pMT2 and S2a/Hsp70 cultured at either 37 or 40°C for 1 h) (Fig.…”

“…For example, biological aging or exposure to peroxynitrite (ONOO Ϫ ) in vitro results in the selective nitration of SERCA2a at Tyr 294 -Tyr 295 , which are located in the transmembrane domain, and at Tyr 753 , which is located in the stalk region of the enzyme (44). In contrast, it appears that reactive oxygen species (ROS) directly inactivate SERCA1a by inducing structural alterations to the nucleotidebinding site as indicated by reductions in fluorescein isothiocyanate (FITC) binding (10,23,28,50,51). FITC is a fluorescent probe that covalently labels LYS-515 (33), which is located within the N domain close to the ATP-binding site (48).…”

“…The molecular basis for the cytoprotective effects of Hsp70 is related to the ability of Hsp70 to prevent protein aggregation and assist with refolding of denatured or partially folded proteins (20,54). In a recent study, we showed that Hsp70 can stabilize the N domain of SERCA1a and prevent thermal inactivation of SERCA1a in vitro (50). An earlier study showed that the induction of Hsp70 in rat myocardium by whole body hyperthermia is associated with increased sarcoplasmic reticulum (SR) Ca 2ϩ -pumping activity and protection of myocardial physiological performance against ischemia-reperfusion (32).…”