Tripartite motif-containing protein 32 regulates Ca2+ movement in skeletal muscle.

Mutations in tripartite motif-containing protein 32 (TRIM32), especially in NHL repeats, have been found in skeletal muscle in patients with type 2H limb-girdle muscular dystrophy (LGMD2H). However, the roles of the NHL repeats of TRIM32 in skeletal muscle functions have not been well addressed. In the present study, to examine the functional role(s) of the TRIM32 NHL repeats in skeletal muscle, TRIM32-binding proteins in skeletal muscle were first searched using a binding assay and MALDI-TOF/TOF. Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a (SERCA1a) was found to be a TRIM32-binding protein. Next, a deletion mutant of TRIM32 missing the NHL repeats (NHL-Del) was expressed in mouse primary skeletal myotubes during myoblast differentiation into myotubes. Ca2+ movement in the myotubes was examined using single-cell Ca2+ imaging. Unlike wild-type (WT) TRIM32, NHL-Del did not enhance the amount of Ca2+ release from the sarcoplasmic reticulum (SR), Ca2+ release for excitation-contraction (EC) coupling, or extracellular Ca2+ entry via store-operated Ca2+ entry (SOCE). In addition, even compared with the vector control, NHL-Del resulted in reduced SOCE due to reduced expression of extracellular Ca2+ entry channels. Transmission electron microscopy (TEM) observation of the myotubes revealed that NHL-Del induced the formation of abnormal vacuoles and tubular structures in the cytosol. Therefore, by binding to SERCA1a via its NHL repeats, TRIM32 may participate in the regulation of Ca2+ movement for skeletal muscle contraction and the formation of cellular vacuoles and tubular structures in skeletal muscle. Functional defects in TRIM32 due to mutations in NHL repeats may be pathogenic toward LGMD2H.

Corrigendum: Deciphering the mechanism of inhibition of SERCA1a by sarcolipin using molecular simulations.

[This corrects the article DOI: 10.3389/fmolb.2020.606254.].

Effect of Stem Cells, Ascorbic Acid and SERCA1a Gene Transfected Stem Cells in Experimentally Induced Type I Diabetic Myopathy.

BACKGROUND AND OBJECTIVES: Sarco/endoplasmic reticulum Ca2＋-ATPase (SERCA) inhibition was proved in streptozotocin (STZ)-diabetic rats. The present study aimed at investigating and comparing the therapeutic effect of bone marrow mesenchymal stem cells (BMMSCs), BMMSCs combined with ascorbic acid (AA) and SERCA1a gene transfected BMMSCs in induced type I diabetic myopathy of male albino rat. METHODS AND RESULTS: 54 rats were divided into donor group of 6 rats for isolation, propagation and characterization of BMMSCs and SERCA1a transfected BMMSCs, groups I∼V 48 rats. Group I of 8 control rats, group II (Diabetic) of 10 rats given STZ 50 mg/kg intraperitoneal, group III (BMMSCs) of 10 rats given STZ and BMMSCs intravenous (IV), group IV (BMMSCs and AA) of 10 rats given STZ, BMMSCs IV and AA 500 mg/kg and group V (SERCA 1a transfected BMMSCs) of 10 rats given STZ and SERCA1a transfected BMMSCs IV. The rats were sacrificed after 8 weeks. Gastrocnemius specimens were subjected to biochemical, histological, morphometric and statistical studies. Diabetic rats revealed inflammatory and degenerative muscle changes, a significant increase in blood glucose level, mean DNA fragmentation and mean MDA values and a significant decrease in mean GSH and catalase values, area of pale nuclei, area% of CD105 and CD34 ＋ve cells, SERCA1a protein and gene values. The morphological changes regressed by therapy. In group III significant decrease in DNA fragmentation and MDA, significant increase in GSH and catalase, significant increase in the mean area of pale nuclei, area % of CD105 and CD34 ＋ve cells versus diabetic group. In group IV, same findings as group III versus diabetic and BMMSCs groups. In group V, same findings as group IV versus diabetic and treated groups. Western blot and PCR proved a mean value of SERCA1a protein and gene comparable to the control group. Mean calcium concentration values revealed a significant increase in the diabetic group, in BMMSCs and AA group versus control and SERCA1a group. CONCLUSIONS: SERCA1a transfected BMMSCs proved a definite therapeutic effect, more remarkable than BMMSCs combined with AA. This effect was evidenced histologically and confirmed by significant changes in the biochemical tests indicating oxidative stress, muscle calcium concentration, morphometric parameters and PCR values of SERCA1a.

Structural Changes of Sarco/Endoplasmic Reticulum Ca2+-ATPase Induced by Rutin Arachidonate: A Molecular Dynamics Study.

Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) maintains the level of calcium concentration in cells by pumping calcium ions from the cytoplasm to the lumen while undergoing substantial conformational changes, which can be stabilized or prevented by various compounds. Here we attempted to clarify the molecular mechanism of action of new inhibitor rutin arachidonate, one of the series of the acylated rutin derivatives. We performed molecular dynamics simulations of SERCA1a protein bound to rutin arachidonate positioned in a pure dipalmitoylphosphatidylcholine bilayer membrane. Our study predicted the molecular basis for the binding of rutin arachidonate towards SERCA1a in the vicinity of the binding site of calcium ions and near the location of the well-known inhibitor thapsigargin. The stable hydrogen bond between Glu771 and rutin arachidonate plays a key role in the binding. SERCA1a is kept in the E2 conformation preventing the formation of important salt bridges between the side chains of several residues, primarily Glu90 and Lys297. All in all, the structural changes induced by the binding of rutin arachidonate to SERCA1a may shift proton balance near the titrable residues Glu771 and Glu309 into neutral species, hence preventing the binding of calcium ions to the transmembrane binding sites and thus affecting calcium homeostasis. Our results could lead towards the design of new types of inhibitors, potential drug candidates for cancer treatment, which could be anchored to the transmembrane region of SERCA1a by a lipophilic fatty acid group.

Deciphering the Mechanism of Inhibition of SERCA1a by Sarcolipin Using Molecular Simulations.

SERCA1a is an ATPase calcium pump that transports Ca2+ from the cytoplasm to the sarco/endoplasmic reticulum lumen. Sarcolipin (SLN), a transmembrane peptide, regulates the activity of SERCA1a by decreasing its Ca2+ transport rate, but its mechanism of action is still not well-understood. To decipher this mechanism, we have performed normal mode analysis in the all-atom model, with the SERCA1a-SLN complex, or the isolated SERCA1a, embedded in an explicit membrane. The comparison of the results allowed us to provide an explanation at the atomic level for the action of SLN that is in good agreement with experimental observations. In our analyses, the presence of SLN locally perturbs the TM6 transmembrane helix and as a consequence modifies the position of D800, one of the key metal-chelating residues. Additionally, it reduces the flexibility of the gating residues, V304, and E309 in TM4, at the entrance of the Ca2+ binding sites, which would decrease the affinity for Ca2+. Unexpectedly, SLN has also an effect on the ATP binding site more than 35 Å away, due to the straightening of TM5, a long helix considered as the spine of the protein. The straightening of TM5 modifies the structure of the P-N linker that sits above it, and which comprises the 351DKTG354 conserved motif, resulting in an increase of the distance between ATP and the phosphorylation site. As a consequence, the turn-over rate could be affected. All this gives SERCA1a the propensity to go toward a Ca2+ low-affinity E2-like state in the presence of SLN and toward a Ca2+ high-affinity E1-like state in the absence of SLN. In addition to a general mechanism of inhibition of SERCA1a regulatory peptides, this study also provides an insight into the conformational transition between the E2 and E1 states.

Screening of Detergents for Stabilization of Functional Membrane Proteins.

Membrane protein studies usually require use of detergents to extract and isolate proteins from membranes and manipulate them in a soluble context for their functional or structural characterization. However, solubilization with detergent may interfere with MP stability and may directly affect MP function or structure. Moreover, detergent properties can be affected such as critical micellar concentration (CMC) can be affected by the experimental conditions. Consequently, the experimenter must pay attention to both the protein and the behavior of the detergent. This article provides a convenient protocol for estimating the CMC of detergents in given experimental conditions. Then, it presents two protocols aimed at monitoring the function of a membrane protein in the presence of detergent. Such experiments may help to test various detergents for their inactivating or stabilizing effects on long incubation times, ranging from few hours to some days. © 2018 by John Wiley & Sons, Inc.

A robust method to screen detergents for membrane protein stabilization, revisited.

This report is a follow up of our previous paper (Lund, Orlowski, de Foresta, Champeil, le Maire and Møller (1989), J Biol Chem 264:4907-4915) showing that solubilization in detergent of a membrane protein may interfere with its long-term stability, and proposing a protocol to reveal the kinetics of such irreversible inactivation. We here clarify the fact that when various detergents are tested for their effects, special attention has of course to be paid to their critical micelle concentration. We also investigate the effects of a few more detergents, some of which have been recently advertised in the literature, and emphasize the role of lipids together with detergents. Among these detergents, lauryl maltose neopentyl glycol (LMNG) exerts a remarkable ability, even higher than that of ß-dodecylmaltoside (DDM), to protect our test enzyme, the paradigmatic P-type ATPase SERCA1a from sarcoplasmic reticulum. Performing such experiments for one's favourite protein probably remains useful in pre-screening assays testing various detergents.

Functional analysis of SERCA1b, a highly expressed SERCA1 variant in myotonic dystrophy type 1 muscle.

Myotonic dystrophy type 1 (DM1) is a genetic disorder in which multiple genes are aberrantly spliced. Sarco/endoplasmic reticulum Ca(2+)-ATPase 1 (SERCA1) is one of these genes, and it encodes a P-type ATPase. SERCA1 transports Ca(2+) from the cytosol to the lumen, and is involved in muscular relaxation. It has two splice variants (SERCA1a and SERCA1b) that differ in the last eight amino acids, and the contribution of these variants to DM1 pathology is unclear. Here, we show that SERCA1b protein is highly expressed in DM1 muscle tissue, mainly localised at fast twitch fibres. Additionally, when SERCA1a and SERCA1b were overexpressed in cells, we found that the ATPase and Ca(2+) uptake activity of SERCA1a was almost double that of SERCA1b. Although the affinity for both ATP and Ca(2+) was similar between the two variants, SERCA1b was more sensitive to the inner microsomal environment. Thus, we hypothesise that aberrant expression of SERCA1b in DM1 patients is the cause of abnormal intracellular Ca(2+) homeostasis.