Inhibition of Ubiquitin Proteasome System Rescues the Defective Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA1) Protein Causing Chianina Cattle Pseudomyotonia

Elisa Bianchini,Stefania Testoni,Arcangelo Gentile,Tito Calì,Denis Ottolini,Antonello Villa,Marisa Brini,Romeo Betto,Francesco Mascarello,Poul Nissen,Dorianna Sandonà,Roberta Sacchetto

doi:10.1074/jbc.m114.576157

Abstract

A missense mutation in ATP2A1 gene, encoding sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) protein, causes Chianina cattle congenital pseudomyotonia, an exercise-induced impairment of muscle relaxation. Skeletal muscles of affected cattle are characterized by a selective reduction of SERCA1 in sarcoplasmic reticulum membranes. In this study, we provide evidence that the ubiquitin proteasome system is involved in the reduced density of mutated SERCA1. The treatment with MG132, an inhibitor of ubiquitin proteasome system, rescues the expression level and membrane localization of the SERCA1 mutant in a heterologous cellular model. Cells co-transfected with the Ca(2+)-sensitive probe aequorin show that the rescued SERCA1 mutant exhibits the same ability of wild type to maintain Ca(2+) homeostasis within cells. These data have been confirmed by those obtained ex vivo on adult skeletal muscle fibers from a biopsy from a pseudomyotonia-affected subject. Our data show that the mutation generates a protein most likely corrupted in proper folding but not in catalytic activity. Rescue of mutated SERCA1 to sarcoplasmic reticulum membrane can re-establish resting cytosolic Ca(2+) concentration and prevent the appearance of pathological signs of cattle pseudomyotonia.

Highlights

Human Brody disease and cattle pseudomyotonia are due to mutations in SERCA1
Because the mutation does not affect the transcription of ATP2A1 gene, we put forward the hypothesis that the reduction of mutated SERCA1 protein was due to degradation by the ubiquitin-proteasome system (UPS)
In this study, using a combined experimental approach involving both a heterologous HEK293 cellular model overexpressing the R164H mutant of SERCA1 and biopsies from cattle pathological muscle, we provide unequivocal evidence that the UPS is involved in degradation of mutated SERCA1 protein in PMT-affected Chianina muscles

Summary

Background

Human Brody disease and cattle pseudomyotonia are due to mutations in SERCA1. Results: Cattle pseudomyotonia SERCA1, functional, is prematurely degraded by the ubiquitin-proteasome system; proteasome inhibition restores calcium homeostasis in a cellular model and in muscle fibers from affected cattle. A missense mutation in ATP2A1 gene, encoding sarco(endo)plasmic reticulum Ca2؉-ATPase (SERCA1) protein, causes Chianina cattle congenital pseudomyotonia, an exercise-induced impairment of muscle relaxation. (In these cases, the disease was called “congenital muscular dystonia1” [9] The relevance of these animal models resides in the similarity of the clinical phenotype to that of human Brody disease [10], a rare inherited disorder of skeletal muscle due to SR Ca2ϩATPase deficiency, resulting from a defect of ATP2A1 gene [11]. The muscular stiffness observed in Brody disease patients is currently thought to be due to a deficiency of SERCA1 protein at SR membranes, which causes a reduced uptake of Ca2ϩ into the lumen of SR after sustained exercise [12].

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION