Inhibition of Ubiquitin Proteasome System Rescues theDefective Sarco(endo)plasmic Reticulum Ca2�-ATPase(SERCA1) Protein Causing Chianina Cattle Pseudomyotonia*�

Received for publication, April 23, 2014, and in revised form, September 5, 2014 Published, JBC Papers in Press, October 6, 2014, DOI 10.1074/jbc.M114.576157

Elisa Bianchini‡, Stefania Testoni†§, Arcangelo Gentile¶, Tito Calì�, Denis Ottolini�, Antonello Villa**, Marisa Brini�,Romeo Betto‡‡, Francesco Mascarello§§, Poul Nissen¶¶, Dorianna Sandonà‡1, and Roberta Sacchetto§§2

From the Departments of ‡Biomedical Sciences and �Biology, University of Padova, 35131 Padova, Italy, the Departments of§Animal Medicine Production and Health and §§Comparative Biomedicine and Food Science, University of Padova,35020 Legnaro(Padova), Italy, the ¶Department of Veterinary Medical Sciences, University of Bologna, 40064 Bologna, Italy, the **ConsorzioM.I.A., University of Milano Bicocca, 20900 Monza, Italy, the ‡‡Neuroscience Institute, Consiglio Nazionale delle Ricerche Padova,35131 Padova, Italy, and the ¶¶Department of Molecular Biology and Genetics, Centre for Membrane Pumps in Cells and Disease,Aarhus University, 8000 Aarhus, Denmark

Background: Human Brody disease and cattle pseudomyotonia are due to mutations in SERCA1.Results: Cattle pseudomyotonia SERCA1, although 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.Conclusion: Functional rescue of mutated SERCA1 is feasible by preventing its degradation.Significance: The data suggest a therapeutic approach against Brody disease.

A missense mutation in ATP2A1 gene, encoding sarco(endo)-plasmic reticulum Ca2�-ATPase (SERCA1) protein, causesChianina cattle congenital pseudomyotonia, an exercise-in-duced impairment of muscle relaxation. Skeletal muscles ofaffected cattle are characterized by a selective reduction ofSERCA1 in sarcoplasmic reticulum membranes. In this study,we provide evidence that the ubiquitin proteasome system isinvolved in the reduced density of mutated SERCA1. The treat-ment with MG132, an inhibitor of ubiquitin proteasome system,rescues the expression level and membrane localization of theSERCA1 mutant in a heterologous cellular model. Cellsco-transfected with the Ca2�-sensitive probe aequorin showthat the rescued SERCA1 mutant exhibits the same ability ofwild type to maintain Ca2� homeostasis within cells. These datahave been confirmed by those obtained ex vivo on adult skeletalmuscle fibers from a biopsy from a pseudomyotonia-affectedsubject. Our data show that the mutation generates a proteinmost likely corrupted in proper folding but not in catalyticactivity. Rescue of mutated SERCA1 to sarcoplasmic reticulummembrane can re-establish resting cytosolic Ca2� concentra-tion and prevent the appearance of pathological signs of cattlepseudomyotonia.

Cattle “congenital pseudomyotonia” (PMT)3 is a musculardisorder described for the first time in the Italian Chianinabreed by Testoni et al. (1), characterized by an impairment ofmuscle relaxation induced by exercise. When animals are stim-ulated to perform intense muscular activities, muscles becomestiff and freeze up temporarily, inducing a rigid gait. If the exer-cise is prolonged, the sustained contraction immobilizes theaffected animal, which eventually falls down. After a few sec-onds at rest, the stiffness disappears, and the animal regains theability to stand up and move. By DNA sequencing of affectedChianina cattle, we provided evidence of a missense mutationin exon 6 of ATP2A1 gene, encoding sarco(endo)plasmic retic-ulum Ca2�-ATPase (SERCA) isoform 1 (2). SERCA, the mainprotein component of the non-junctional sarcoplasmic reticu-lum (SR) (3), is a key participant in the Ca2� homeostasis inskeletal muscle fibers, being responsible for pumping Ca2�

from cytosol back into SR lumen, thus initiating relaxation. Inskeletal muscle fibers, Ca2�-activating muscle contraction isreleased from the SR lumen into the cytosol via Ca2� releasechannel localized at the terminal cisternae of SR. At the end ofthe contraction cycle, SERCA allows relaxation by removingCa2� from the cytosol to restore resting Ca2� concentration.Three SERCA isoforms, products of different genes, areexpressed in striated muscles in a tissue- and stage of develop-ment-specific fashion. SERCA1 isoform is expressed in fast-twitch (type 2) skeletal muscle of mammalians (4).

The mutation underlying Chianina cattle PMT replaces anArg at position 164 by His (R164H), in a highly conservedregion of the Actuator (A) domain of SERCA1 protein (5). This

* This work was supported by grants from the University of Padova (ProgettoAteneo 2009 CPDA091413) (to F. M.) and the Italian Telethon Foundation(Project GEP12058) (to D. S.).

� This article was selected as a Paper of the Week.† This paper is dedicated to the memory of our colleague Prof. Stefania Tes-

toni (deceased November 17, 2012).1 To whom correspondence may be addressed: Dept. of Biomedical Sci-

ences, University of Padova Via Ugo Bassi 58/B, 35131 Padova, Italy.Tel.: 39-49-8276028; Fax: 39-49-8276049; E-mail: dorianna.sandona@unipd.it.

2 To whom correspondence may be addressed: Dept. of Comparative Bio-medicine and Food Science, University of Padova, viale dell’Università 16,35020 Legnaro (Padova), Italy. Tel.: 39-49-8272653; Fax: 39-49-8272796;E-mail: roberta.sacchetto@unipd.it.

3 The abbreviations used are: PMT, pseudomyotonia; SERCA, sarco(endo)-plasmic reticulum Ca2�-ATPase; SR, sarcoplasmic reticulum; ER, endoplas-mic reticulum; UPS, ubiquitin-proteasome system; TRITC, tetramethylrho-damine isothiocyanate; cytAEQ, cytosolic Ca2� probe aequorin; DMSO,dimethyl sulfoxide.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 48, pp. 33073–33082, November 28, 2014© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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mutation does not affect the expression of ATP2A1 gene asSERCA1 mRNA levels found in affected animals are compara-ble with mRNA expression in normal samples (6). However,Chianina pathological muscles are characterized by a striking,selective reduction in the expression level of SERCA1 protein(6). Although present at low levels, the R164H SERCA1 variantmaintained the basic intrinsic properties of WT SERCA1, nota-bly the Ca2�-dependent ATPase activity. Therefore, we con-cluded that the decrease in SR Ca2�-ATPase activity found inaffected animals was mainly due to reduction of SR SERCA1protein content (6). The consequent reduction in pumping effi-ciency of SR is likely responsible for muscle stiffness as theabnormally low rate of Ca2� removal from the cytosol supportsan elevated cytoplasmic Ca2� concentration, thereby triggeringcontractures.

More recently, cattle PMT associated with ATP2A1 genemutations different from that of Chianina breed has beendescribed in Romagnola breed (7), as a single case in a Dutchimproved Red and White cross-breed calf (8), and in the BelgianBlue breed. (In these cases, the disease was called “congenitalmuscular dystonia1” (9).

The relevance of these animal models resides in the similarityof the clinical phenotype to that of human Brody disease (10),a rare inherited disorder of skeletal muscle due to SR Ca2�-ATPase deficiency, resulting from a defect of ATP2A1 gene(11). Clinical key features are exercise-induced muscle stiffnessand delayed muscle relaxation after repetitive contraction. Themuscular stiffness observed in Brody disease patients is cur-rently thought to be due to a deficiency of SERCA1 protein atSR membranes, which causes a reduced uptake of Ca2� into thelumen of SR after sustained exercise (12). Like cattle congenitalPMT, Brody disease is genetically heterogeneous (13). There-fore, based on clinical presentation and genetic and biochemi-cal findings, it is possible to deem Chianina cattle congenitalPMT as a true counterpart of human Brody disease. Thus,Chianina PMT is a very useful, although unconventional, modelfor the study of myopathy in human Brody disease and for thedevelopment of innovative therapeutic approaches. The obser-vation that in cattle SERCA1 mRNA levels in diseased musclesare normal while protein levels are markedly reduced suggestedto us that the R164H mutation could cause SERCA1 misfoldingand accelerated removal by either the ubiquitin-proteasomesystem (UPS) or the autophagic-lysosomal pathway.

In this study, we have investigated the possible involvementof the UPS in the reduced levels of mutated SERCA1 in SR fromChianina PMT muscles. Our results provide strong support tothis interpretation.

EXPERIMENTAL PROCEDURES

SERCA1 Construct and Site-directed Mutagenesis—The orig-inal full-length rabbit neonatal SERCA1 cDNA clone was a kindgift of Prof. D. MacLennan (14). To obtain the adult full-lengthSERCA1 isoform, in which the C-terminal extension of the neo-natal form has been removed, a PCR was carried out with thefollowing primers: forward, 5�-CAAGAAGCTTGCGCAATG-GAG, and reverse, 5�-CTTTCGAATTCTTAACCTTCCAG-GTAGTTCC. The forward primer contains a HindIII restric-tion site (in bold in the primer sequence) upstream to the ATG

codon to facilitate cloning. The reverse primer permits us tochange the triplet coding for Glu994 into a Gly (in italics in theprimer sequence); the following triplet is in a STOP codon(underlined), and it introduces an EcoRI restriction site (inbold) to facilitate cloning. After restriction digestion, theamplified fragment was cloned in pcDNA3.1 expression vec-tor. The R164H SERCA1 mutant was obtained by site-directedmutagenesis according to the manufacturer’s instructions(Stratagene) (15). The constructs were verified by sequencing.

Cell Culture, Transfection, and Treatment with ProteasomeInhibitors—HEK293 cells were counted using the automatedcell counter Scepter (Merck Millipore) and then were seededand grown in DMEM high glucose medium supplemented with10% FCS. Cells were transfected using the TransIT-293 trans-fection reagent (Mirus Bio), according to manufacturer’sinstructions. Sixteen hours after transfection, cells were incu-bated for 8 h with the proteasome inhibitors MG132 (Sigma)(10 �M final concentration), lactacystin (Sigma) (20 �M finalconcentration), or bortezomib (Sigma) (0.5 �M final concentra-tion), and then dissolved in DMSO.

SERCA1 Half-life Determination—HEK293 cells were trans-fected with WT and R164H SERCA1 mutant cDNAs. Twelvehours after transfection, cells were incubated for 4 and 8 h withcycloheximide (Sigma) (40 �g/ml, final concentration).

Muscle Biopsies—Biopsy of semimembranosus muscle, a rep-resentative fast-twitch skeletal muscle (6), was collected fromPMT-affected Chianina animal under local anesthesia duringthe course of routine clinical investigation, in compliance withItalian law. Biopsy was divided in small bundles of fibers, placedin dishes containing Ham’s F14 supplemented with 15% FCS,50 �g/liter FGF, 10 mg/liter insulin, and 10 �g/liter EGF, andincubated for 24 h with the proteasome inhibitor MG132 (20�M) (16). Control muscle samples were collected from healthyChianina cattle euthanized at a slaughterhouse.

Transmission Electron Microscopy Analysis—Immediatelyafter collection, muscle samples were fixed with 4% paraformal-dehyde and 2% glutaraldehyde in 0.12 M phosphate buffer andpost-fixed with 1% OsO4 in cacodylate buffer, dehydrated inethanol, and embedded in epoxy resin. Ultrathin sections,obtained with an Ultracut E Reichert-Jung ultramicrotome,were doubly stained with uranyl acetate and lead citrate andexamined by a CM 10 Philips transmission electron microscope(FEI, Eindhoven, the Netherlands).

Immunocytochemical Analysis—HEK293 cells grown on13-mm glass coverslips, were washed with PBS, pH 7.4, andfixed for 10 min in 4% paraformaldehyde and then washed withPBS. The cells were permeabilized in 0.5% Triton X-100 in PBSand incubated with the following primary antibodies: mousemonoclonal antibody to SERCA1 (Biomol) (recognizing anepitope between amino acid residues 506 and C terminus ofrabbit fast-twitch SR Ca2�-ATPase) (dilution 1:500); and rabbitpolyclonal antibody to calreticulin (Affinity Bioreagents) (dilu-tion 1:100). Cells were then incubated with the appropriate sec-ondary antibody conjugated with TRITC or FITC (Dako). Con-focal microscopy was performed using a Leica TCS-SP2confocal laser scanning microscope.

Preparative Procedures—At the end of the incubation time,transfected HEK293 cells treated with MG132, lactacystin,

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bortezomib, and vehicle, or with cycloheximide, were washedtwice with PBS. Total cell lysate was obtained by solubilizationin 5% sodium deoxycholate containing protease inhibitors(Roche Applied Science). The isolation of microsomal fractionfrom HEK293 cells was carried out according to Ref. 14, fromfive 10-cm Petri dishes for each sample. The crude membranefraction, enriched in content of SR membranes, was isolated bydifferential centrifugations from muscle biopsy, as describedpreviously (6). Protein concentration was determined by themethod of Lowry et al. (17).

Gel Electrophoresis Immunoblotting and Biochemical As-say—SDS-PAGE and immunoblotting were carried out asdescribed (6). The blots were probed with mouse monoclonalantibodies to: SERCA1 (1:5000); �-actin (1:30000) (Sigma); andmono- and polyubiquitinated conjugates (1:1000) (Enzo LifeScience). Densitometric scanning of protein bands was carriedout using the ImageQuant 350 (GE Healthcare). ATPase activ-ity of the microsomal fractions (20 �g/ml) was measured byspectrophotometric determination of NADH oxidation cou-pled to an ATP-regenerating system, as described previously(6).

Immunoprecipitation—HEK293 cells were washed with PBSand subsequently lysed in radioimmunoprecipitation assay sol-ubilization buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1%Nonided P-40, 0.5% sodium deoxycholate, 1% Triton X-100, 1mM EDTA) supplemented with 2 mM N-ethyl-maleimide(Sigma) and protease inhibitors. A soluble supernatant and apellet were collected after spinning for 30 min at 10000 � g, at4 °C. The lysate was incubated overnight while tumbling at 4 °Cwith the specific antibody to SERCA1. The following day, pro-tein G-magnetic beads (Millipore) were added, and the mixturewas incubated for 1 h in the same condition. Beads were recov-ered and extensively washed with the lysis buffer and finallyaspirated to dryness.

Aequorin Ca2� Measurements—HEK293 cells co-trans-fected with the cytosolic Ca2� probe aequorin (cytAEQ) andwith empty, WT, and R164H SERCA1-expressing vectors ina 1:1 ratio were preincubated for 1–3 h with 5 �M coelen-terazine. Measurements and calibration of aequorin signalwere performed as described previously (18). Data are reportedas means � S.D. Statistical differences were evaluated byStudent’s two-tailed t test for unpaired samples. A p value of�0.05 was considered statistically significant.

RESULTS

Expression of WT and R164H Mutant SERCA1 Protein inHEK293 Cell—To develop a cellular model suitable for investi-gating the role of UPS in PMT, HEK293 cells were transfectedwith cDNA encoding WT (WT-HEK293) or R164H mutant(R164H-HEK293) SERCA1. Heterologous cellular systemswere previously successfully used for the investigation ofSERCA1 protein that displayed the same features as thosefound in the muscle SR (12, 14).

Immunofluorescence analysis showed that in WT-HEK293,SERCA1 localized to the endoplasmic reticulum (ER) as shownby the clear co-localization with calreticulin (Fig. 1A), a wellknown ER marker (19). The R164H SERCA1 mutant was alsocorrectly targeted to ER (Fig. 1A) (see merging with calreticu-

lin), but the expression level was much lower than that of WTSERCA1.

The reduction of SERCA1 immunoreactivity was fully con-firmed by immunoblot analysis of total cell lysate with the anti-body to SERCA1 (Fig. 1B). On the other hand, blocking of pro-tein neo-synthesis with cycloheximide showed that the stabilityof SERCA1 mutant was strongly reduced as compared with WT(Fig. 1C). Altogether, these results indicate that this hetero-logous expression system faithfully mimics the pattern ofSERCA1 expression observed in intact muscle fibers fromPMT-affected Chianina cattle (6).

Proteasome Inhibition with MG132 Lactacystin and Bort-ezomib Prevents Degradation of R164H SERCA1 Protein inVitro—To test the role of UPS in causing a reduced expressionof R164H SERCA1 mutant, WT-HEK293 and R164H-HEK293were incubated with MG132, a well recognized proteasomeinhibitor. As control, cells were transfected with the empty vec-tor. Although in WT-HEK293 the immunostaining pattern ofSERCA1 was slightly positively affected by MG132 treatment,cells expressing R164H SERCA1 mutant exhibited a strikingincrease of immunoreactivity to SERCA1 after treatment withMG132 (Fig. 2). As expected, no signal of SERCA1 was detectedin both treated and untreated cells transfected with the emptyvector.

Results obtained by immunofluorescence were paralleled bythose obtained by immunoblot analysis of total cell lysate withthe same antibody (Fig. 3A). The expression levels of theSERCA1 mutant protein were significantly increased inMG132-treated cells. The graph in the lower part of Fig. 3Ashows the average values of SERCA1 expression determined bydensitometric analyses of independent immunoblotting exper-iments. Values, depicted as the percentage of SERCA1 proteincontent in untreated cells expressing the WT form, showed thatafter MG132 treatment, the WT protein expression was virtu-ally unaffected, whereas that of the R164H mutant increased,reaching levels comparable with those of WT.

To strengthen the hypothesis that UPS is involved in thereduced expression of R164H SERCA1 mutant, WT-HEK293and R164H-HEK293 were incubated with other highly specificproteasome inhibitors, lactacystin and bortezomib (Fig. 3B).Immunoblot analysis of total cell lysate with antibodies toSERCA1 confirmed that the expression level of the mutantincreased after treatment with the different inhibitors. Takentogether, these results strongly support the hypothesis thatSERCA1 mutant protein is selectively degraded by the UPS.

Further evidence in favor of this suggestion was gained byassaying the degree of ubiquitination of the SERCA1 mutant.The first step of protein degradation by UPS is the conjugationof target protein with multiple copies of the small protein ubiq-uitin (20). MG132 inhibits UPS by covalently binding to theactive site of the �-subunits of 20 S proteasome (21). Thisinhibitory effect occurs downstream of the ubiquitination step,allowing the accumulation of polyubiquitinated forms of theprotein destined to degradation. Therefore, the treatment withMG132 should allow the recovery of mutated SERCA1 in apolyubiquitinated form. To test this hypothesis, WT-HEK293and R164H-HEK293 cells, treated or not with MG132, werelysed in mild conditions to permit the subsequent immunopre-

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cipitation assay. Under the lysis conditions used, SERCA1 wasnot totally solubilized, but was partially retained in the insolu-ble fraction obtained after cell lysate centrifugation (Fig. 4A,lanes 6 –10). Soluble supernatants (Fig. 4A, lanes 1–5) wereused as input to immunoprecipitate SERCA1 with the specificantibody. Recovered immunocomplexes were then subjected toimmunoblot analysis with anti-ubiquitin antibody (Fig. 4B).The antibody recognized trace amounts of ubiquitin linked toWT SERCA1, which slightly increased after proteasomal inhi-bition. On the other hand, although a remarkable amount ofmutant SERCA1 was retained in the insoluble fraction (Fig. 4A),immunoprecipitates from R164H-HEK293 treated withMG132 showed a large increase in polyubiquitination of R164HSERCA1 mutant, as hypothesized.

Qualitative Recovery of R164H Mutant SERCA1 Protein afterProteasome Inhibition in Vitro—These results clearly demon-strate that blockade of UPS activity in R164H-HEK293 causesaccumulation of SERCA1 protein. In a previous work, using SR

membrane from PMT-affected Chianina muscles, we demon-strated that the SERCA1 mutant displayed functional proper-ties indistinguishable from those of WT SERCA1 (6). There-fore, we hypothesized that R164H SERCA1 mutant rescued bytreatment with MG132 should exhibit functional propertiessimilar to those of WT SERCA1. To test this prediction, Ca2�-ATPase activity was measured, using microsomal membranefractions isolated from transfected HEK293 cells (14).

As shown in Fig. 5A, microsomes obtained from R164H-HEK293 displayed a Ca2�-ATPase activity much lower thanthat of microsomes from WT-HEK293. Incubation with pro-teasome inhibitor led to an increase in Ca2�-ATPase activity,which correlated with the increased expression of mutantSERCA1 within ER membranes (Fig. 5B).

To further investigate the functional effect of rescuingmutated SERCA1 by proteasome inhibitor treatment, the abil-ity of either WT or R164H SERCA1 to counteract cytosolicCa2� transients induced by cell stimulation was monitored in

FIGURE 1. Transfection of HEK293 cells with cDNA encoding WT or R164H mutant SERCA1. A, cellular localization of WT and mutated SERCA1 proteins inHEK293 cells. Transfected cells were immunolabeled with monoclonal antibodies to SERCA1 and subsequently with antibodies to calreticulin, a molecularmarker of ER. Cells were then incubated with TRITC-conjugated (red fluorescence) anti-mouse and FITC-conjugated (green fluorescence) anti-rabbit secondaryantibodies. Simultaneous visualization of the two fluorochromes (yellow signal) shows that both WT and R164H mutant SERCA1 proteins are correctly targetedto ER, where co-localize with calreticulin. All panels are the same magnification (scale bar, 10 �m). B, expression level analysis of WT and mutated SERCA1proteins in transfected HEK293 cells. Total cell lysates from HEK293 cells transfected with either WT or R164H mutant SERCA1 cDNAs or with the empty vectorwere obtained by solubilization with 5% sodium deoxycholate. An equal quantity of total lysate from each sample was separated by SDS-PAGE and blottedonto nitrocellulose. The blot was incubated with antibodies specific for SERCA1 and the 43-kDa �-actin, used as loading control. In HEK293 cells transfectedwith the empty vector, SERCA1 antibody was unable to detect the protein. C, protein turnover of WT and mutated SERCA1. HEK293 cells transfected with WT(circle) or R164H mutant (triangle) SERCA1 cDNA were treated with cycloheximide (CHX), to inhibit protein synthesis, and then harvested at the indicated timepoints. An equal quantity of total cell lysate was separated by SDS-PAGE and blotted onto nitrocellulose. The blots were incubated with antibodies specific forSERCA1, and the 43-kDa �-actin and densitometric analysis was performed. Quantification data represent the ratio relative to time 0. The graph shows theaverage values (� S.D.) of at least three independent experiments.

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HEK293 cells, before and after MG132 treatment. Cells wereco-transfected with the cytosolic Ca2�-sensitive photoproteinaequorin (18) and then stimulated with carbachol, an inositol-1,4,5-generating agonist that induces an increase in the cyto-plasmic Ca2�concentration by causing Ca2� release from theER as well as Ca2� influx across the plasma membrane. Fig. 6Ashows the Ca2� response in untreated cells. The lower peakamplitude and the faster decay kinetics in WT-HEK293 clearlyreflected the greater density of the Ca2� pump as comparedwith native HEK293 cells (CytAEQ). It is noteworthy that theheight of the Ca2� transient peak was not affected by the over-expression of R164H SERCA1, but the kinetics of the decliningphase of the Ca2� traces in the initial phase immediately afterthe peak (Fig. 6A) was faster than in native HEK293, and verysimilar to that of WT-HEK293. Because the declining phase ofthe Ca2� transients is an important parameter of Ca2� han-dling, its shape confirms that the mutated SERCA1 pump isactive. The R164H SERCA1-overexpressing cells are less effi-cient with respect to WT-HEK293 in counteracting the peak ofthe Ca2� concentration transient induced by stimulation, butthey restore Ca2� basal levels faster than native HEK.

These data confirm that the introduction of R164H mutationdoes not abolish pump activity, in agreement with the experi-ments where the Ca2�-ATPase activity was measured in micro-somes (Fig. 5A), and suggest that impaired ability to counteractthe peak transient depends on the lower amount of SERCApump expression, as demonstrated by immunoblotting data(Fig. 5B). Thus, to verify whether the recovery of SERCA levelscould re-establish Ca2� extrusion ability in intact cells, cytoso-

lic Ca2� transients were monitored in cells incubated withMG132 (Fig. 6, C and D). The traces clearly show that the Ca2�-pumping ability of R164H-HEK293 is indistinguishable fromthat of WT-HEK293, and is drastically increased with respect tonative HEK293. Taken together, these results demonstrate that

FIGURE 2. Restoration of expression level of R164H SERCA1 after treat-ment with the proteasome inhibitor MG132. HEK293 cells transfected withWT, R164H mutant SERCA1 cDNAs, or the empty vector were treated withMG132 (10 �M) or its vehicle DMSO. At the end of the incubation time, cellswere immunostained with SERCA1 primary antibody and TRITC-conjugatedsecondary antibody. Cells expressing R164H SERCA1 showed a strikingincrease of immunoreactivity after incubation with MG132. No effects wereobserved by the incubation with the vehicle alone. No signal was detected incells transfected with the empty vector (the same fields collected in transmis-sion light are shown in the inset). All panels are the same magnification (scalebar, 10.5 �m).

FIGURE 3. Immunodetection of WT and R164H mutated SERCA1 proteinsafter incubation with proteasome inhibitors MG132, bortezomib, andlactacystin. HEK293 cells were transfected with WT, R164H mutant SERCA1cDNAs, or the empty vector, when indicated. A, Western blot and densitomet-ric analysis of SERCA1 protein. Cells were treated with the proteasome inhib-itor MG132 (�) or its vehicle DMSO (�). An equal quantity of protein fromtotal cell lysates, obtained by solubilization with 5% sodium deoxycholate,was separated by SDS-PAGE and subjected to immunoblot analysis with anti-bodies specific to SERCA1 and 43-kDa �-actin, used as loading control. Thegraph shows quantification of protein bands performed by densitometricanalysis on Western blots. Data (means � S.D. from six independent experi-ments) are reported as the percentage of WT SERCA1 expressed in HEK293cells treated with the vehicle alone. ***, p � 0.001. B, Western blot of SERCA1protein. Cells were treated with the proteasome inhibitors bortezomib,MG132, and lactacystin or with their vehicle DMSO. An equal quantity of pro-tein from total cell lysates obtained by solubilization with 5% sodium deoxy-cholate was separated by SDS-PAGE and subjected to immunoblot analysiswith antibodies specific to SERCA1 and 43-kDa �-actin, used as loadingcontrol.

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the physiological Ca2� homeostasis in cells expressing mutatedSERCA1 can be restored by blocking the UPS.

Electron Microscopy of PMT-affected Muscles—We have pre-viously demonstrated by immunofluorescence and immuno-blotting methods that reduction of mutant SERCA1 in SRmembrane of PMT-affected cattle was selective, as both longi-tudinal SR and junctional SR were not depleted of their mainprotein components (6). Ultrastructural studies of affected cowsample and control sample confirmed that the general struc-ture of muscle fibers was normal (Fig. 7). Triads, although infre-quent in both control and pathological muscles, were normal insize and distribution.

Quantitative and Qualitative Rescue of R164H SERCA1 Pro-tein after Proteasome Inhibition in ex Vivo Experiments—Res-cue of R164H SERCA1 by MG132 was further investigated inadult muscle fibers from PMT-affected Chianina cow. Smallbundles of fibers from freshly isolated biopsy were maintainedunder tissue culture conditions as explant, in the absence andin the presence of MG132 (16). At the end of the incubationperiod, a crude post-mitochondrial membrane fraction,enriched in content of SR membranes, was isolated (6). Fig. 8Ashows the immunoblot analysis with antibody to SERCA1 ofsubfractions obtained with this method. In agreement with invitro results obtained with transfected cells, in these ex vivoexperiments, the treatment with MG132 also efficientlyincreased R164H SERCA1 mutant expression. The rescuedprotein was detected only in the SR microsomal fraction, beingalmost totally absent from both the soluble cytoplasm and the

myofibrillar fraction, in agreement with previous results (6, 15).As anticipated, the SR-enriched membrane fraction obtainedfrom MG132-treated muscle fibers displayed a large increase inmaximal Ca2�-ATPase activity, measured at optimum pCa(Fig. 8B).

DISCUSSION

In a previous work, we reported that a missense mutation inATP2A1 gene, leading to an R164H substitution in the SERCA1protein (2), underlies Chianina cattle PMT. The skeletal mus-cles of affected cattle are characterized by a selective reductionin the level of R164H SERCA1 protein in SR membranes (6).Because the mutation does not affect the transcription ofATP2A1 gene, we put forward the hypothesis that the reduc-tion of mutated SERCA1 protein was due to degradation by theUPS. In this study, using a combined experimental approachinvolving both a heterologous HEK293 cellular model overex-pressing the R164H mutant of SERCA1 and biopsies from cattlepathological muscle, we provide unequivocal evidence that theUPS is involved in degradation of mutated SERCA1 protein inPMT-affected Chianina muscles.

FIGURE 4. Polyubiquitination of WT and R164H mutated SERCA1 pro-teins. HEK293 cells transfected with either WT or R164H mutant SERCA1cDNA or the empty vector (e.v) were treated with the proteasome inhibitorMG132 (�) or its vehicle DMSO (�). A, solubilization of SERCA1 protein fromtransfected HEK293 cells. Transfected cells were lysed with radioimmunopre-cipitation assay buffer (see “Experimental Procedures”) and centrifuged toobtain a soluble fraction (lanes 1–5) and a pellet (lanes 6 –10). An equal quan-tity of protein or equal volume from solubilized supernatants and insolublepellets, respectively, was separated by SDS-PAGE and probed with antibodiesto SERCA1 and 43-kDa �-actin, used as loading control. B, ubiquitination sta-tus of WT and mutated SERCA1. From 200 �g of solubilized proteins (input forimmunoprecipitation), SERCA1 was immunoprecipitated with the specificantibody. Immunocomplexes were resolved by SDS-PAGE. The ubiquitin-conjugated SERCA1 was tested with anti-ubiquitin (Ub) antibodies. The arrowindicates the IgG whole molecules used not fully denatured.

FIGURE 5. Ca2�-ATPase activity and expression levels of WT and R164HSERCA1 in microsomes isolated from HEK293 cells. HEK293 cells weretransfected with WT or R164H SERCA1 cDNA or with the empty vector. R164H-HEK293 cells were treated with either the proteasome inhibitor MG132 (�)dissolved in DMSO or the vehicle alone (�). A microsomal membrane fractionwas isolated as described (14). A, Ca2�-ATPase activity of the microsomalmembrane fraction was determined by a spectrophotometric enzyme-cou-pled assay (6). The values are normalized to that of WT SERCA1 (100%) and arethe means � S.D. of three independent experiments. B, microsomal fractionswere separated by SDS-PAGE and blotted onto nitrocellulose. Expression ofCa2�-ATPase protein was detected by using an immunoblot procedure withantibody to SERCA1. The lower panel shows Ponceau Red staining of themembrane, used as loading control.

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By means of the widely used proteasome inhibitors MG132,lactacystin, and bortezomib, known to block UPS downstreamto the ubiquitination step, we show that R164H mutant under-goes ubiquitination and that, after blocking proteasome,R164H SERCA1 protein accumulates in HEK293 cells at levelsalmost comparable with those of cells expressing WT SERCA1.Furthermore, we show that the R164H mutation does not inter-fere with the proper localization of SERCA1 pump because therescued protein is correctly targeted to ER. These conclusionsare further strengthened by results obtained with adult skeletalmuscle fibers from a biopsy from a PMT subject, demonstrating

that ex vivo treatment with MG132 leads to the selective accu-mulation of rescued R164H SERCA1 mutant in SR membranes.

In previous work, in which functional properties of R164Hbovine SERCA1 protein were compared with those of rabbitand human homologs (22), we hypothesized that the SERCA1protein, although mutated, was fully functionally active andthat the prolonged Ca2� transients, responsible for muscle con-tractures in PMT-affected cattle, were likely due to the reducedamount of SERCA1 protein pumps in the SR membrane. Insupport of this hypothesis, Ca2�-ATPase activity in micro-somes isolated from transfected HEK293 cells, after treatment

FIGURE 6. Monitoring of cytosolic Ca2� concentration in HEK293 cells. The plasmid expressing the cytosolic Ca2�-sensitive photoprotein aequorin wasco-transfected in HEK293 cells with either WT or R164H mutant (R164H) SERCA1 cDNAs or the empty vector (CytAEQ). A and B, co-transfected cells were treatedwith the vehicle alone (DMSO). C and D, co-transfected cells were treated with the proteasome inhibitor MG132 dissolved in DMSO (indicated as �MG132).Cytoplasmic Ca2� transients (A and C) were measured after agonist stimulation (500 �M carbachol). The histograms in B and D show the means � S.D. of Ca2�

transient peaks from six independent experiments.

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with MG132, increased in parallel with the increase in ERR164H protein content. Furthermore, results obtained withHEK293 cells co-expressing the Ca2�-sensitive photoproteinaequorin clearly indicated that, after proteasomal inhibition,the rescued mutated SERCA1 was fully capable of maintainingcytoplasmic Ca2� levels similar to those of cells expressing theWT protein.

Taken together, our results show that the R164H SERCA1 isdegraded by UPS. The likely reason for this is that the aminoacid substitution leads to a misfolded protein. It is well knownthat cells have developed a variety of quality control systems toprevent the accumulation of abnormally folded or damaged andpotentially noxious proteins (23). In this respect, the UPS isthought to be mainly involved in the removal of misfoldedmutants (24), even when the conformational changes do notaffect the catalytic properties. Because the SR Ca2�-ATPase is akey element in the regulation of Ca2� homeostasis in skeletalmuscle fibers, it is conceivable that all minimal folding altera-tions might be recognized by the UPS and the defective enzymemight thus be diverted to degradation. Therefore, the inflexiblerules of this system might prove to be fatal for the mutatedprotein, even in the absence of important modifications of theenzymatic activity, as it seems to be our case.

The crystal structure of rabbit SERCA1a isoform was solvedby Toyoshima et al. (5), and the subsequent crystallographicanalysis provided the detailed characterization of the three-di-mensional structure of the rabbit enzyme. SERCA1a proteinconsists of three cytoplasmic domains, called A, N, and P, and10 transmembrane helices. The P and N domains contain thephosphorylation site and residues for adenosine binding,respectively, whereas the A domain, the smallest of the three, ishighly mobile and rotates during active transport to associatewith the P domain (25, 26).

Recently, we provided the crystal structure of the bovineSERCA1, crystallized in the E1 conformation (Protein DataBank (PDB) accession code 3TLM) (22). The bovine molecularmodel has proved to be very similar to that of the rabbit enzyme(25, 26), as expected by the high degree of similarity of theamino acid sequence. The R164H substitution (Fig. 9) occurs inthe A domain (27) at the N terminus of SERCA1 protein. Adetail of the bovine SERCA1 structure centered on the Arg164

(22) has shown that the long side chain of positively chargedArg could be counterbalanced by the negatively charged sidechain of Glu2 and in addition could form extensive interactionswith four surrounding residues: Ile165, Thr191, Lys7, and His5.So it is conceivable that Arg164, acting as an anchor point, couldbe central to proper folding. The substitution of the side chainof the Arg with the imidazole ring of the His residue, which issmaller and which at physiologic pH can be neutral, coulddestabilize interactions with surrounding residues. The resul-tant protein, although functional, could be affected in the properfolding at its N terminus, a region described as critical for thecorrect folding and for maintenance of the three-dimensionalstructure of the protein (28). In a study on the possible role ofthe N-terminal region of Ca2�-ATPase SERCA1, it has beendemonstrated that a small structural defect in this domaincould be fundamental in inducing an accelerated degradation(28).

Our results, showing that R164H mutation generates aSERCA1 protein most likely corrupted in proper folding, allowclassification of cattle PMT into the family of unfolded proteindiseases, an extremely heterogeneous and growing group ofgenetic disorders in which the pathogenic hallmark is the pre-mature degradation or aggregation of the misfolded gene prod-uct (29). Moreover, the relevance of these experiments tohuman pathology is due to the similarity between Chianinacattle PMT and human Brody disease, a rare genetic disorderdue to a defect of ATP2A1 gene. Pseudomyotonia is the mostimportant clinical feature, and a diminished SR Ca2�-ATPaseactivity is the major biochemical finding (30). However, themechanism underlying this functional impairment is not fullyclarified. At least in some patients, it is clear that ATP2A1mutations lead to a reduced expression of SERCA1 protein (31).This subgroup of patients is very similar biochemically andphenotypically to PMT-affected Chianina cattle.

At present, no specific therapy exists for human Brody dis-ease. The main finding of this study is that proteasomal inhibi-tion functionally rescues the mutated SERCA1 in a cell modelas well as in muscle fibers from PMT-affected animals. This is avery important result because it paves the way to a possibletherapeutical approach in vivo. This perspective seems to beeven more plausible because we confirm here, using electronmicroscopy, that the ultrastructure of SR is well preserved inmuscle specimens from PMT-affected animals, in full agree-ment with findings in muscle fibers from human Brody patients(31). Our results suggest that acting on the degradative pathwayto avoid the premature disposal of mutated SERCA1, mighthave beneficial effects at least in the specific population ofpatients in which a reduced expression of SERCA1 has beendocumented.

FIGURE 7. Ultrastructural studies of bovine skeletal muscle. Electronmicroscopy of semimembranosus muscle biopsies from PMT-affected andcontrol unaffected Chianina cattle is shown. Scale bar, 500 nm.

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Acknowledgments—We thank Prof. David H. MacLennan for the gen-erous gift of SERCA1 cDNA and are deeply indebted to Prof. ErnestoDamiani for writing help and advice. We thank Prof. Francesco DiVirgilio for critical reading of the manuscript. We thank Rizzato’sFarm.

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

Chianina Cattle Pseudomyotonia -ATPase (SERCA1) Protein Causing2+Sarco(endo)plasmic Reticulum Ca

Inhibition of Ubiquitin Proteasome System Rescues the Defective

doi: 10.1074/jbc.M114.576157 originally published online October 6, 20142014, 289:33073-33082.J. Biol. Chem. 

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