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J. Biol. Chem., Vol. 282, Issue 47, 34276-34287, November 23, 2007

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Myopathy-associated B-crystallin Mutants

ABNORMAL PHOSPHORYLATION, INTRACELLULAR LOCATION, AND INTERACTIONS WITH OTHER SMALL HEAT SHOCK PROTEINS^*

Stephanie Simon¹, Jean-Marc Fontaine, Jody L. Martin^¶, Xiankui Sun, Adam D. Hoppe^||, Michael J. Welsh, Rainer Benndorf, and Patrick Vicart

From the EA300 Stress et Pathologies du Cytosquelette, Université Paris 7, UFR de Biochimie, 75005 Paris, France, the Departments of Cell and Developmental Biology and ^||Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan 48109, and the ^¶Department of Medicine, Cardiovascular Institute, Loyola University Chicago, Maywood, Illinois 60153

Received for publication, April 18, 2007 , and in revised form, September 11, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Three mutations (R120G, Q151X, and 464delCT) in the small heat shock protein B-crystallin cause inherited myofibrillar myopathy. In an effort to elucidate the molecular basis for the associated myopathy, we have determined the following for these mutant B-crystallin proteins: (i) the formation of aggregates in transfected cells; (ii) the partition into different subcellular fractions; (iii) the phosphorylation status; and (iv) the ability to interact with themselves, with wild-typeB-crystallin, and with other small heat shock proteins that are abundant in muscles. We found that all three B-crystallin mutants have an increased tendency to form cytoplasmic aggregates in transfected cells and significantly increased levels of phosphorylation when compared with the wild-type protein. Although wild-type B-crystallin partitioned essentially into the cytosol and membranes/organelles fractions, mutant B-crystallin proteins partitioned additionally into the nuclear and cytoskeletal fractions. By using various protein interaction assays, including quantitative fluorescence resonance energy transfer measurements in live cells, we found abnormal interactions of the various B-crystallin mutants with wild-type B-crystallin, with themselves, and with the other small heat shock proteins Hsp20, Hsp22, and possibly with Hsp27. The collected data suggest that eachB-crystallin mutant has a unique pattern of abnormal interaction properties. These distinct properties of the B-crystallin mutants identified are likely to contribute to a better understanding of the gradual manifestation and clinical heterogeneity of the associated myopathy in patients.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
B-crystallin (BC)² is a ubiquitously occurring small heat shock protein (sHsp) with particularly high abundance in skeletal and cardiac muscles (1) in which it can be incorporated into the sarcomeric structure (2, 3). It is now well established that BC is a major player in the function of muscular tissues, for example it protects cardiomyocytes from adverse conditions such as ischemic stress (4).

BC and sHsps in general are widely believed to act as molecular chaperones, preventing the aggregation and precipitation of damaged or misfolded proteins in an ATP-independent way in stress conditions (5, 6). Consistent with this property, sHsps accumulate in human degenerative diseases, particularly in diseases involving abnormal protein aggregation (7). Usually, sHsps exist as polydisperse hetero-oligomers that change in size and/or organization when interacting with substrates or upon stress exposure (8–12). At the cellular level, sHsps protect cells from noxious conditions as diverse as toxicity promoted by aberrantly folded proteins, oxidative conditions, and proteasome inhibition. Distinct mechanisms have been proposed for the protective effects of sHsps, including chaperone-like activity, anti-apoptotic effects, or intracellular redox homeostasis (13–19). These distinct mechanisms are not exclusive and could occur concomitantly.

Mutations in sHsps are associated with the development of several degenerative diseases. A number of mutations in B-crystallin were identified that lead to the degeneration of distinct tissues, including the lens of the eye and/or cardiac and skeletal muscles (20–26). Which of the tissues actually is affected depends on the specific mutation, and currently it is not known what causes this clinical heterogeneity. In addition, two others sHsps, Hsp22 and Hsp27, have been associated with the human degenerative diseases Charcot-Marie-Tooth and distal hereditary motor neuron diseases (27–33). The first discovered sHsp mutation is the missense mutation R120G in BC (^R120GBC) (20), and to date this is the best studied mutation. The R120G mutation in BC results in dominant gain-of-function properties and causes a particular subtype of myofibrillar myopathy (desmin-related myopathy or B-crystallinopathy) with associated cardiac involvement and cataract formation. The ^R120GBC protein exhibits changes in its secondary, tertiary, and quaternary structural features (34–37). It is more polydisperse than the wild-type BC (^WTBC) (36) and is inherently unstable in solution (37). These structural disturbances correlate with a decreased in vitro chaperone-like activity (36). A recent study has established that ^R120GBC directly promotes the aggregation of the desmin filament network and that desmin networks are differently affected, depending on the cellular backgrounds (38). In various cell lines, independent of desmin levels, ^R120GBC aggregates in a time-dependent process starting with the formation of multiple foci of insoluble proteins in the cytoplasm. Subsequently, these foci coalesce into large amorphous perinuclear aggregates in a microtubular network-dependent manner. Expression of ^R120GBC leads to the formation of a cage of type III intermediate filament proteins such as vimentin, but also of a cage of type II intermediate filament proteins such as keratins, suggesting a general response of the intermediate filament networks to the aggregate formation. Nevertheless, these intermediate filament proteins have not been found as components of the aggregates by themselves, in contrast to desmin (13). Moreover, ^R120GBC and its pseudophosphorylated mutants are unable to confer resistance to differentiation-induced apoptosis during C2C12 myoblast differentiation because of their impaired capacity to inhibit the proteolytic activation of caspase-3 (39). Overexpression of ^R120GBC in cardiomyocytes of transgenic mice results in a 100% mortality by early adulthood in high expressing lines, whereas a modest expression level results in a strikingly similar phenotype to that observed in patients with ^R120GBC-associated cardiomyopathies (40). In these transgenic mice, the desmin network, myofibril alignment, mitochondrial-sarcomere architecture, mitochondrial function, and the ubiquitin/proteasome system (UPS) were significantly impaired (41, 42). In addition, a hypertrophic response occurred, and the apoptotic pathways were activated (41). Taken together, it appears that all the known protective functions of BC are impaired. The two other BC mutations that are associated with myofibrillar myopathy are the nonsense mutation Q151X (^Q151XBC) and the frameshift mutation 464delCT (⁴⁶⁴BC) (22). Both mutants caused the formation of cytoplasmic aggregates in skeletal muscles, without cardiac or eye lens involvement. No further information on these two mutants is available.

Recently, several studies have investigated the potential of sHsp overexpression for the treatment of degenerative diseases. Hsp22, Hsp27, and ^WTBC revealed a high capacity to dissociate the aggregates formed by ^R120GBC expression in several cell lines (13, 43–45). Nevertheless, recent studies in cardiomyocytes suggest that the resulting hetero-oligomers of ^WTBC/^R120GBC were more toxic for the cells than the homo-oligomers formed by the ^R120GBC alone (44). In contrast, both Hsp22 and Hsp25 (murine equivalent of Hsp27) co-expression with ^R120GBC rescued cell viability (45). So far, there is no explanation for this differential effect of BC and Hsp22 or Hsp27/Hsp25 to date.

In this study we have determined properties of the three mutants of BC (^MTTBC), ^R120GBC, ^Q151XBC, and of ⁴⁶⁴BC, that are associated with myofibrillar myopathy. We show that all three mutant proteins form abnormal cytoplasmic aggregates in both cardiomyocytes and in COS-7 cells. Moreover, ^MTTBC proteins expressed in COS-7 cells distribute into additional cell fractions as compared with ^WTBC. We also show that all three mutant proteins are hyperphosphorylated in all the three known phosphorylation sites (serine residues 19, 45, and 59) when expressed in COS-7 cells. We also investigated the interaction properties of these three ^MTTBC with themselves, with ^WTBC, and with Hsp20, Hsp22, and Hsp27. These sHsps are known as interaction partners of BC and are abundant in muscles (46–48). Using the yeast two-hybrid (TH) method, chemical cross-linking (CL), pulldown (PD) assays, and the quantitative fluorescence resonance energy transfer (qFRET) method in live mammalian cells, we show here that all three ^MTTBC proteins are able to interact with themselves, with ^WTBC, and with the other sHsps. We have identified a unique interaction pattern for each mutant with the other sHsps. It is expected that these identified abnormal properties of the three studied ^MTTBC forms will contribute to a better understanding of the molecular processes that lead to the associated diseases and to the design of therapeutic strategies.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Vector Constructs—TH vector constructs were made using the vectors pACT2 and pAS2 (Clontech). Cyan (CFP) and citrine (Cit) fluorescent fusion protein expression vectors were made using the vectors pECFP-N1, pECFP-C1 (both from Clontech), and pCit-N1 and pCit-C1 (49). Myc/His-tagged and untagged protein constructs were made using the vectors pcDNA3.1(+)/myc-His B and pcDNA3, respectively (both from Invitrogen). A list of all used constructs and more detailed cloning information is given in supplemental Table 1 (construct numbers as used in this study are given in square braquets). Additional information on the constructs used is also given in previous publications (20, 48, 50, 51).

Two-hybrid Method—Small scale sequential transformation of yeast strain AH109 was performed as described in the manufacturer's instructions (Clontech). Colonies were selected on -Trp, -Leu, -His medium for the phenotypes His+ (growth) and LacZ+ (blue color). The interaction assays were considered positive if both reporter genes were activated. For negative controls, yeast were transformed with each used vector alone and tested on -Trp, -Leu, and -His medium (not shown). Additionally, yeast were co-transformed with each vector and with the `empty' partner vector as indicated in the figure legends (C1–C11, cf. supplemental Figs. 1B, 2A, 3A, and 4A). In none of these controls were the reporter genes activated.

Cell Culture and Transfections—COS-7 cells were grown in DMEM (Invitrogen) supplemented with 10% fetal calf serum (FCS; Invitrogen) and penicillin/streptomycin (Invitrogen) in a 5% CO₂ humidified atmosphere. One day prior to transfections, cells were trypsinized and plated as specified below. Transfections were carried out using FuGENE 6 (Roche Diagnostics) with 0.75 µg (single construct) or 1.5 µg (two constructs) of vector DNA for CFP or Cit constructs or 2 µg of vector DNA for pcDNA3 or pcDNA3.1(+)/myc-His B constructs.

Cardiomyocyte Isolation, Culture, and Infection—Rat neonatal ventricular myocytes were isolated by standard batch collagenase digestion and subsequently preplated to remove the fibroblasts. Cardiomyocytes were then counted and plated on gelatin-coated dishes in serum-free PC-1 medium (BioWhittaker). Twenty four hours after plating, the cells were washed, and the medium was changed to DMEM/M199 (4:1) maintenance medium. The cells were then transduced with adenovirus expressing the appropriate CFP fusion at an multiplicity of infection of 50. Two days later the cells were fixed in 2% paraformaldehyde solution, and rhodamine-phalloidin was stained before imaging.

Immunofluorescence Microscopy—COS-7 cells were grown on coverslips, transfected, and 48 h later were washed twice with ice-cold PBS, fixed, and permeabilized at 4 °C for 5 min with cold methanol/acetone (7:3). Subsequently, cells were incubated for 1 h each at room temperature with primary polyclonal rabbit antibody (diluted 1:200 in PBS, 2% FCS) directed against the first 10 residues of BC (Abcam) and with secondary goat anti-rabbit antibody (diluted 1:1000 in PBS, 2% FCS) coupled to AlexaFluor 11034 (Invitrogen). The coverslips were mounted on slides using Mowiol (Sigma). Images were collected using a fluorescent microscope (Leitz) equipped with a digital camera ORCA-ER (Hamamatsu) and processed using the Simple PCI 6.0 software (Compix Inc. Imaging Systems).

Live Cell Imaging—COS-7 cells were grown in 6-well glass-bottom culture plates (MatTek Corp.). 48 h after transfection with the various CFP and Cit fusion protein vectors, cells were washed twice with PBS and kept in DMEM without phenol red (Invitrogen). For fluorescence microscopy an inverted epifluorescence microscope (Eclipse TE-2000 U; Nikon) was used equipped with a 100-watt mercury arc-lamp, exciter filters 430/25 and 500/20, a dichroic microscope filter 86002bs, and with a 505dcxr Dual View Micro Imager MSMI.DV.CC (Optical Insights) with the emission filters 470/30 and 535/30. Images were collected by a digital CoolSnap CCD camera (Photometrics) and processed using Metamorph image processing software version 6.2r5 (Molecular Devices).

For determination of the fraction of cells with aggregates, microscopic fields were selected randomly using a Plan fluor ELWD x40/0.6 objective lens (Nikon). At least 100 cells per sample group were included in these evaluations.

Quantitative Fluorescence Resonance Energy Transfer Measurements in Live Cells—The qFRET method was applied for quantification of apparent fluorescence resonance energy transfer efficiencies (AAFE) as indicators of protein interaction. The configuration of the microscope was as described above for live cell imaging using a Fluor ELWD x40/1.3 oil Dic H objective lens (Nikon). Maintenance and transfection of COS-7 cells with the various CFP and Cit fusion protein vectors was as described for live cell imaging. In each cell to be analyzed, three cytoplasmic areas without protein aggregates were selected for qFRET measurements. Images from at least 30 microscopic fields per sample group were acquired and background/shading-corrected prior to computation by the qFRET algorithm. The calculated output data were expressed as (E_A + E_D)/2 (E_A is apparent acceptor efficiency calculated from sensitized emission and dependent on the fraction of acceptor in complex; E_D is apparent donor efficiency calculated relative to donor fluorescence and dependent on the fraction of the donor in complex). More details concerning qFRET are given in earlier publications (49, 51). As negative controls, the cells were transfected with the "empty" CFP (peCFP-N1) and Cit (peCit-N1) vectors (Fig. 3D) or with the empty Cit (peCit-N1/C1) and the various CFP constructs (supplemental Figs. 2C, 3C, and 4D). AAFE values that were significantly different from the control signals indicated interaction. Quantitative data are expressed as the mean of AAFE values ±S.E. The data between groups were analyzed using one-way ANOVA. When overall significance was detected, a post hoc multiple group comparison was conducted using Tukey HSD adjustment. Differences between groups were considered statistically significant if p < 0.05.

Analysis of the Phosphorylation Status of B-crystallin—COS-7 cells were grown in 6-well plates. 48 h after transfection, cells were washed twice in ice-cold PBS and lysed using the ReadyPrep protein extraction kit (Bio-Rad) according to the manufacturer's instructions. One volume of buffer A (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 400 mM dithiothreitol, 0.01% bromphenol blue) was added, and the samples were boiled for 3 min followed by SDS-PAGE/Western blotting. Equal loading of the samples was verified by visualization of vimentin on the same blots using a monoclonal anti-vimentin antibody diluted to 1:2000 (Sigma). Phosphorylation of the three known serine phosphorylation sites (Ser-19, Ser-45, and Ser-59) of BC (52) was determined using phosphorylation site-specific polyclonal antibodies, diluted to 1:2000 (Stress-Gen). After electrotransfer of the proteins, the polyvinylidene difluoride membrane was blocked with bovine serum albumin free of IgG (Interchim). For immunodetection, secondary goat anti-mouse or anti-rabbit horseradish peroxidase-coupled secondary antibodies diluted to 1:10,000 (Pierce) were used. The degree of phosphorylation of the various BC species was quantified on scanned images using ImageJ software (53). The base-line signal was obtained from untransfected control cells.

Protein Fractionation—COS-7 cells were grown in 10-cm cell culture dishes and transfected with vectors coding for Myc/His-tagged BC species [5–7]. 48 h after transfection, 4 x 10⁶ cells were harvested, and the cell proteins were differentially extracted yielding the fractions of cytosolic proteins, membrane/organelle proteins, nuclear proteins, and cytoskeletal proteins using the ProteoExtract subcellular proteome extraction kit (Calbiochem) according to the manufacturer's instructions. Each extract was dosed using the Dc-Kit (Bio-Rad) prior to mixing with 1 volume of buffer A. Equal aliquots of protein extracts (15 µg of protein) were analyzed by SDS-PAGE/Western blotting. A polyclonal rabbit anti-Myc primary antibody diluted to 1:10,000 (Sigma) and a goat anti-rabbit horseradish peroxidase-coupled secondary antibody (Pierce) were used for immunodetection. Nontransfected cells were used as negative control.

Cross-linking—COS-7 cells were grown in 6-well plates and transfected with vectors coding for Myc/His-tagged BC species [5–8]. 48 h after transfection, cells were washed three times with ice-cold PBS and incubated for 30 min at room temperature with 0.5 mM of the homo-bifunctional amine-reactive cross-linker disuccinimidyl suberate (DSS; Pierce). The reaction was stopped by adding Tris-HCl, pH 7.5, to a final concentration of 15 mM and incubating for 15 min at room temperature. Cells were lysed by adding 1 volume of buffer A. After brief sonication, samples were boiled for 3 min and analyzed by SDS-PAGE/Western blotting. A polyclonal rabbit anti-Myc primary antibody (Sigma) and a goat anti-rabbit horseradish peroxidase-coupled secondary antibody (Pierce) were used for immunodetection as described previously.

Pulldown Assays—COS-7 cells were grown in 6-well plates, and singly or doubly transfected with vectors coding for Myc/His-tagged [5–9] and CFP-tagged BC or other sHsp species [10–13, 26, 28] as indicated in Fig. 3A and supplemental Figs. 1A, 2B, 3B, and 4B. 48 h after transfection, cells were collected, washed two times with ice-cold PBS, resuspended, lysed in ice-cold buffer B (50 mM NaH₂PO₄·NaOH, pH 8.0, 300 mM NaCl, 10 mM imidazole, 0.05% Tween 20), and briefly sonicated. An aliquot of the protein extracts was mixed with 1 volume of buffer A, boiled for 3 min, and analyzed by SDS-PAGE/Western blotting to determine the expression of the various transgenes. Equal loading was verified by visualization of vimentin on gels. To immobilize Myc/His-tagged proteins, protein extracts were incubated on a rotary shaker at 4 °C with pre-equilibrated nickel/nitrilotriacetic acid beads (nickel beads; Qiagen) for 24 h (48 h for binding of Myc/His-tagged Hsp22). The nickel beads were then washed with ice-cold buffer C (50 mM NaH₂PO₄·NaOH, pH 8.0, 300 mM NaCl, 20 mM imidazole, 0.05% Tween 20) and buffer D (50 mM NaH₂PO₄·NaOH, pH 8.0, 300 mM NaCl, 25 mM imidazole), and the proteins bound to the nickel beads were eluted using buffer A and analyzed by SDS-PAGE/Western blotting. Myc/His-tagged and CFP-tagged proteins were detected using polyclonal anti-Myc (Sigma) and anti-GFP (CliniSciences) primary antibodies both diluted to 1:10,000. The secondary antibody was goat anti-rabbit horseradish peroxidase-coupled antibodies (Pierce).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Intracellular Location and Phosphorylation Status of Myopathy-associated BC Mutants in COS-7 Cells—To determine possible differences between ^WTBC and myopathy-associated ^MTTBC species, we have investigated the intracellular location of untagged ^MTTBC species [2–4] expressed in COS-7 cells by indirect immunofluorescence microscopy and compared it with the location of ^WTBC [1]. Similarly to previous reports (13, 20), we found that 48 h after transfection ^R120GBC localized in cytoplasmic aggregates in about 30% of the transfected cells, whereas expression of ^WTBC resulted in cytoplasmic location without aggregate formation in essentially all cells (about 97%). Similarly, expression of ^Q151XBC and ⁴⁶⁴BC resulted in formation of cytoplasmic aggregates in about 30% of the transfected cells. Representative images of cells expressing ^WTBC without aggregates (panel a) and of cells expressing ^MTTBC without (panels b–d) and with aggregates (panels e–g) are shown in Fig. 1A. Occasionally, we also noted the presence of these BC species in the nuclei as can be seen for ^R120GBC and ^Q151XBC in Fig. 1A (panels b and c, respectively).

The altered intracellular location of the ^MTTBC species may be accompanied by altered partition of those proteins into subcellular fractions. Therefore, we determined the partition of two of these mutant proteins, ^R120GBC and ^Q151XBC, into the subcellular fractions of cytosol, membranes/organelles, nuclei, and cytoskeleton, using a method based on differential cell extraction. Myc/His-tagged ^WTBC [5] expressed in COS-7 cells partitioned almost completely into the cytosol and membranes/organelles fractions (Fig. 1B). In contrast, Myc/His-tagged ^R120GBC [6] and ^Q151XBC [7] both partitioned additionally into the nuclear and cytoskeletal fractions, with the strongest signals being obtained in the cytoskeletal fraction. As expected, no signals were observed in nontransfected control cells.

View larger version (28K): [in this window] [in a new window]   FIGURE 1. Intracellular location and phosphorylation status of MTTBC in COS-7 cells. A, images of cells without (panels a–d) and with protein aggregates (panels e–g). Cells were transfected with expression constructs coding for untagged WTBC (panel a), R120GBC (panels b and e), Q151XBC (panels c and f), and 464BC (panels d and g). 48 h later, cells were fixed and stained with polyclonal anti-BC antibody. Arrows indicate protein aggregates. B, distribution of Myc/His-tagged WTBC, R120GBC, Q151XBC into subcellular fractions. Cells were transfected with expression constructs coding Myc/His-tagged WTBC, R120GBC, and Q151XBC. The cytosolic, membranes/organelles, and nucleic and cytoskeletal fractions were obtained 48 h later. Equal aliquots of each fraction were analyzed by Western blotting using the polyclonal anti-Myc antibody. Untransfected cells were used as negative control. C, phosphorylation status of the various BC mutants. Cells were transfected with expression constructs coding for Myc-His-tagged WTBC, R120GBC, Q151XBC, and 464BC. 48 h later, cells were harvested and processed for SDS-PAGE/Western blotting using the phospho-specific anti-BC S19P, S45P, and S59P antibodies. Equal loading and protein expression was verified using monoclonal anti-vimentin (Vim) and polyclonal anti-Myc antibodies. Untransfected cells were used as negative control. D, quantification of the involvement of the single phosphorylation sites of the various BC mutants as shown in C. The Western blots were evaluated using ImageJ freeware. The base-line signal was obtained from blots of untransfected cells.

Intracellular Location of Cit- and CFP-tagged BC Mutants in Neonatal Cardiomyocytes and COS-7 Cells—Because all three ^MTTBC species which this study focuses on are associated with muscle disorders, we performed similar localization experiments in neonatal cardiomyocytes. The neonatal cardiomyocytes were refractory to transfection, and therefore the cardiomyocytes were infected with recombinant adenoviruses expressing the various fusion proteins. 48 hours after infection, 11% of the cells expressing CFP-tagged ^WTBC showed formation of cytoplasmic aggregates, thus defining the base-line level for this construct. Cells expressing ^R120GBC, ^Q151XBC, or ⁴⁶⁴BC formed cytoplasmic aggregates in significantly higher proportions of cells, 42, 21, and 20%, respectively. Selected images of cells without and with aggregates are shown in Fig. 2A (panels a–e and f–h, respectively). These data suggest that the altered properties of ^MTTBC species result in abnormally increased aggregate formation in neonatal cardiomyocytes, similar to what was observed in COS-7 cells. Because of the limitations related to the work with cardiomyocytes, we used the experimentally more feasible COS-7 cells to determine further properties of the ^MTTBC species. Because Cit- and CFP-tagged BC species were used in most of the assays shown below, we also determined the aggregate formation of these constructs after expression in COS-7 cells. Similar to what was observed in neonatal cardiomyocytes, a portion of COS-7 cells expressing the various ^WT/MTTBC species contained cytoplasmic aggregates, with ^MTTBC species exhibiting increased tendencies to form aggregates. For example, expression of CFP-tagged ^R120GBC [11], ^Q151XBC [12], and ⁴⁶⁴BC [13] resulted in 35, 25, and 33%, respectively, of cells containing aggregates, as compared with 15% for ^WTBC [10], which defines the base-line level for this group of constructs. Selected images of COS-7 cells without and with aggregates, expressing Cit-tagged ^WT/MTTBC species [18–21], are shown in Fig. 2B (panels a–e and f–h, respectively).

View larger version (57K): [in this window] [in a new window]   FIGURE 2. Fluorescence images of neonatal cardiomyocytes and COS-7 cells expressing Cit- or CFP-tagged MTTBC. A, images of freshly isolated neonatal cardiomyocytes cells expressing CFP-tagged BC species. Cells were infected with adenoviruses coding for GFP (panel a), WTBC-CFP (panel e), R120GBC-CFP (panels b and f), Q151XBC-CFP (panels c and g), or 464BC-CFP (panels d and h), and images were taken 48 h later. Images of cells without (panels a–e) and with protein aggregates (panels f–h) are shown. B, images of COS-7 cells expressing Cit-tagged BC species. Cells were transfected with expression constructs coding for Cit (panel a), WTBC-Cit (panel b), R120GBC-Cit (panels c and f), Q151XBC-Cit (panels d and g), or 464BC-Cit (panels e and h), and images were taken 48 h later. Images of cells without (panels a–e) and with protein aggregates (panels f–h) are shown. C, images of COS-7 co-expressing Cit-tagged R120GBC and the various CFP-tagged sHsps. Cells were co-transfected with R120GBC-Cit (panels a–e) and CFP (panel f), WTBC-CFP (panel g), Hsp20-CFP (panel h), Hsp22-CFP (panel i), or Hsp27-CFP (panel j). Arrows indicate protein aggregates.

Protein Interactions Involving Myopathy-associated Mutant and Wild-type B-Crystallin—We have determined the ability of ^MTTBC species to interact with ^WTBC and with themselves using different methods to assay protein/protein interactions including the PD, CL, TH, and qFRET methods. Previously, ^WTBC was shown to interact with itself (55–57), and this interaction served as a positive control in all assays.

For the PD assays, the various CFP- and Myc-His-tagged constructs were used with nickel beads. After controlling the proper functioning of the method (see supplemental Fig. 1A), PD assays were conducted to determine the ability of myopathy-associated ^MTTBC species to interact with ^WTBC and with themselves. Cells were doubly transfected with the various Myc/His-[5–8] and CFP-tagged [10–13] ^WT/MTTBC species as indicated in Fig. 3A. All Myc/His-tagged (Fig. 3A, row I) and CFP-tagged (row II) ^WT/MTTBC species were expressed in the COS-7 cells in similar amounts. After incubation of the cell extracts with the nickel beads, all Myc/His-tagged ^WT/MTTBC species did bind, as shown by SDS-PAGE/Western blotting after elution (Fig. 3A, row III). Analysis of the same fractions for the presence of the various CFP-tagged ^WT/MTTBC species revealed their presence (Fig. 3A, row IV). This co-elution of the CFP-tagged ^WT/MTTBC species (that do not bind to the nickel beads by themselves; see supplemental Fig. 1A) suggests interaction in all tested combinations. After elution (Fig. 3A, rows III and IV), the intensity of the obtained bands resulting from interactions for all ^MTTBC species with ^WTBC (lanes 2–4) or from all ^MTTBC proteins with themselves (lanes 5–7) was similar to that of the ^WTBC/^WTBC interaction (lane 1). Again, visualization of vimentin was used as loading control (Fig. 3A, row V). The data in Fig. 3A suggest that all myopathy-associated ^MTTBC forms interact both with ^WTBC and with themselves, with no obvious differences between any of the ^MTTBC forms and ^WTBC.

To verify the ability of the various ^MTTBC to form homodimers, we applied CL as an independent assay. COS-7 cells were transfected with Myc/His-tagged constructs [5–8] to express each of the ^WT/MTTBC species as indicated in Fig. 3B or were not transfected (control). Subsequently, cells were treated with the cross-linker DSS (Fig. 3B, right panel) or were not treated for control purposes (left panel). In the absence of DSS, only monomers of the ^WTBC or ^MTTBC were detected by SDS-PAGE/Western blotting. In contrast, after incubation with DSS, additional bands that correspond to homodimers were detected. No major differences between ^MTTBC forms and ^WTBC were found. Thus, all three ^MTTBC forms were able to form homodimers similarly as ^WTBC.

TH assays were applied as a further method to independently verify these interactions among the various ^WT/MTTBC species. In the negative TH controls, the reporter genes were not activated (see supplemental Fig. 1B) thus reducing the risk of false positive data. The data showed that all myopathy-associated ^MTTBC forms, in combination with themselves or with ^WTBC, activated both reporter genes to a similar extent (Fig. 3C). In summary, the TH data also suggested interaction of all myopathy-associated ^MTTBC forms with themselves and with ^WTBC, with no major differences between ^MTTBC forms and ^WTBC.

The qFRET method using CFP and Cit fusion proteins was developed to determine subtle changes in the stoichiometry of protein interactions (49), and by this method abnormal interactions involving mutant sHsps were identified that could not be revealed by other methods (51). Therefore, we applied this more sensitive method to determine possibly abnormal interactions involving ^MTTBC forms. Homodimer formation of ^WTBC essentially involves the C-terminal parts of both interacting molecules (55–57). To avoid possible steric hindrance in the C-terminal regions, the qFRET experiments were conducted using fusion protein constructs with the CFP and Cit tags being fused to the N termini of the ^WT/MTTBC forms [10–13, 18–21]. The determined AAFE values for all the tested interactions were significantly different from the negative control value using CFP/Cit (Fig. 3D). This indicated that all ^MTTBC and ^WTBC forms interacted with one another. For most of the interactions (^WTBC/^R120GBC, ^WTBC/^Q151XBC, ^R120GBC/^R120GBC, ^Q151XBC/^Q151XBC, and ⁴⁶⁴BC/⁴⁶⁴BC), the determined AAFE values were very similar to that of the ^WTBC/^WTBC interaction. Despite this high degree of similarity, some minor differences to the ^WTBC/^WTBC interaction were statistically significant as was revealed by one-way ANOVA followed by post hoc pairwise group comparisons (Fig. 3D). Interestingly, all ^MTTBC/^MTTBC interactions showed a small increase as compared with the ^WTBC/ ^WTBC interaction (11%). Only the ^WTBC/⁴⁶⁴BC interaction was moderately reduced (17%) and significantly different from all other tested interactions shown in Fig. 3D.

Taken together, the data resulting from all applied methods strongly suggest that the myopathy-associated ^MTTBC forms interact with themselves and with ^WTBC. The PD, CL, and TH assays did not reveal any differences in these interactions. Although the qFRET method, because of its greater sensitivity, revealed minor differences in most of these interactions as compared with the ^WTBC/^WTBC interaction, the data suggest that these mutations do not have major consequences for the interaction of BC with itself at the level of dimers. The only significant consequence was that the ^WTBC/⁴⁶⁴BC interaction was moderately decreased by this mutation.

Interaction of B-Crystallin Mutants with Hsp20—To determine potentially aberrant interaction properties of the myopathy-associated ^MTTBC species with Hsp20, we conducted TH, PD, and qFRET assays (see supplemental Fig. 2, A–C, respectively). The interaction between ^WTBC and Hsp20 has been reported previously (46, 47) and served as positive control.

The TH data confirmed the occurrence of the ^WTBC/Hsp20 interaction. Additionally, the results suggested interaction between both ^Q151XBC and ⁴⁶⁴BC with Hsp20 to similar degrees, whereas the interaction of ^R120GBC with Hsp20 apparently was decreased as suggested by the weak activation of both reporter genes. All negative TH controls (supplemental Fig. 1B, C1–C4, and supplemental Fig. 2A, C9) provided negative results as expected, thus rendering false-positive interaction data unlikely.

To confirm these results by an independent method, we conducted PD experiments. When CFP-tagged Hsp20 was co-expressed with any of the Myc/His-tagged ^WT/MTTBC species, Hsp20 was co-eluted from the nickel beads together with the tested ^WT/MTTBC species (supplemental Fig. 2B, row IV, lanes 2–5). Thus, the PD assays suggest interaction between the tested ^MTTBC species and Hsp20. The ^R120GBC/Hsp20 interaction appeared to be weaker when compared with the ^WTBC/Hsp20 interaction, which is in agreement with the TH data.

View larger version (28K): [in this window] [in a new window]   FIGURE 3. PD, CL, TH, and qFRET assays to determine interactions of MTTBC species with WTBC and themselves. A, PD assays to determine binding of each MTTBC with WTBC (lanes 2–4) and with themselves (lanes 5–7). COS-7 cells were doubly transfected with the various Myc- and CFP-tagged BC species as indicated and were harvested 48 h later. The expression of all transgenes in the cell extracts was verified using Myc- and GFP/CFP-specific antibodies as indicated (rows I and II). Myc/His-tagged BC species in cell extracts were bound to nickel beads, and after washing, bound proteins were eluted with buffer A and analyzed by SDS-PAGE/Western blotting, again using the Myc- and GFP/CFP-specific antibodies (rows III and IV). The results revealed that all probed CFP-taggedBC species that did not bind by themselves to the nickel beads were bound through interaction with the Myc/His-taggedBC species (rows III and IV). Vimentin was visualized by an anti-vimentin (anti-Vim) antibody to verify equal sample loading (row V). B, CL of WTBC and MTTBC species. COS-7 cells were transfected to express Myc/His-tagged WTBC, R120GBC, Q151XBC, and 464BC. 48 h later, and cells were resuspended in PBS and treated with 0.5 mM of DSS (right panel) or not (left panel) prior to analysis by SDS-PAGE/Western blotting using a polyclonal anti-Myc antibody. CL resulted in similar homodimer formation of WTBC and all MTTBC species. The position of molecular mass marker proteins (kDa) is indicated on the left. C, TH assays. Yeast was co-transformed with pairs of the various BC species as indicated. In all combinations, the BC species activated both reporter genes (His, growth; LacZ, -galactosidase resulting in blue color) indicating interaction. D, qFRET assays. The AAFE was determined in selected areas of doubly transfected COS-7 cells co-expressing CFP- and Cit-tagged BC species as indicated. COS-7 cells co-expressing CFP and Cit were used as negative control. #, indicates significant differences of the AAFE values from negative control. *, indicates significant differences of the AAFE values from the WTBC/WTBC interaction. All sample values were significantly different from this negative control thus indicating interaction. A minor increase in the interaction of MTTBC/MTTBC is observed, whereas the WTBC/464BC showed a moderate decrease as compared with WTBC/WTBC.

Taken together, the data collected from the TH, PD, and qFRET experiments showed that all myopathy-associated ^MTTBC forms are able to interact with Hsp20. By all three methods, the ^R120GBC/Hsp20 interaction was found to be greatly diminished. Additionally, the qFRET method revealed somewhat decreased interactions of ^Q151XBC and ⁴⁶⁴BC with Hsp20.

Interaction of B-Crystallin Mutants with Hsp22—We applied similar approaches to determine potentially aberrant interaction properties of the ^MTTBC with Hsp22, again using the TH, PD, and qFRET assays (see supplemental Fig. 3). The previously reported ^WTBC/Hsp22 interaction served as positive control (48). ^R120GBC was reported to have an increased interaction with Hsp22 in vivo (13).

The TH data confirmed the ^WTBC/Hsp22 interaction and also suggested interaction between all ^MTTBC [29–32] and Hsp22 [38] with no major apparent differences between the various ^WT/MTTBC species (supplemental Fig. 3A). As expected, all negative TH controls (supplemental Fig. 1B, C1–C4; Fig. 3A, C10) provided negative results.

Similarly as described above, we conducted PD assays to confirm these interactions (supplemental Fig. 3B). The ⁴⁶⁴BC/ Hsp22 interaction appeared to be unchanged as compared with the ^WTBC/Hsp22 interaction, whereas the ^R120GBC/Hsp22 and ^Q151XBC/Hsp22 interactions appeared to be increased or decreased, respectively.

For the qFRET analysis of the impact of the mutations on these interactions, CFP-tagged Hsp22 [27] and Cit-tagged ^WT/MTTBC species with the Cit moiety fused to the C termini of the ^WT/MTTBC species [22–25] were used. The determined AAFE values for all tested interactions were significantly different from the negative control value (CFP-tagged Hsp22/Cit alone) thus indicating interaction and confirming the results obtained by the TH and PD assays (supplemental Fig. 3C).

Additionally these data revealed distinct differences in the interaction of the various ^MTTBC with Hsp22. ^R120GBC showed a greatly increased (53%) interaction with Hsp22 and ^Q151XBC a greatly decreased (51%) interaction with Hsp22, as compared with ^WTBC, whereas the interaction of ⁴⁶⁴BC with Hsp22 remained essentially unchanged. One-way ANOVA and subsequent post hoc pairwise group comparisons indicated statistical significance of these differences, whereas the ⁴⁶⁴BC/Hsp22 and ^WTBC/Hsp22 interactions were not significantly different. Thus, all three ^MTTBC species had a different interaction stoichiometry with Hsp22. These different interactions are consistent with the data collected in the PD assays (supplemental Fig. 3B). We also conducted the complementary experiments using constructs with the Cit moiety fused to the N termini of the ^WT/MTTBC species [18–21]. The results were similar to those using C-terminal fusion constructs (data not shown). Collectively, the data collected from the TH, PD, and qFRET assays suggest that all myopathy-associated ^MTTBC forms were able to interact with Hsp22. By two methods it was demonstrated that ^R120GBC and ^Q151XBC had increased or decreased, respectively, interaction with Hsp22, whereas the interaction of ⁴⁶⁴BC with Hsp22 was not affected by this mutation.

Interaction of B-Crystallin Mutants with Hsp27—Finally, experiments were conducted to determine potentially aberrant interaction properties of the ^MTTBC with Hsp27 (see supplemental Fig. 4). Both ^WTBC and Hsp27 are interacting proteins (46, 47, 56), and this ^WTBC/Hsp27 interaction served as positive control.

The TH data confirmed the interaction between ^WTBC [29] and Hsp27 [39] (supplemental Fig. 4A). In addition, the TH results suggest interaction between all ^MTTBC species [30–32] and Hsp27 with no major differences in the interactions. Again, all negative TH controls (supplemental Fig. 1B, C1–C4; Fig. 4A, C11) provided negative results.

To confirm these interactions, corresponding PD experiments were conducted (supplemental Fig. 4B). PD assays confirmed interaction between all the tested ^MTTBC species and Hsp27. Those interactions appeared to be slightly increased when compared with the ^WTBC/Hsp27 interaction. Finally, qFRET experiments were conducted with the intent to quantify possible differences in binding of the various ^MTTBC species to Hsp27. Two series of experiments were performed using CFP-tagged Hsp27 [28], and Cit-tagged ^WT/MTTBC species with the Cit moiety fused to the C terminus of the ^WT/MTTBC species (vector group I, constructs 22–25) (supplemental Fig. 4C) or Cit-tagged ^WT/MTTBC species with the Cit moiety fused to the N terminus of the ^WT/MTTBC species (vector group II, constructs 18–21) (supplemental Fig. 4D). Using both vector groups, all determined AAFE values were significantly greater than the negative control (CFP-tagged HSP27/Cit alone). This confirmed that all ^WT/MTTBC species indeed did interact with Hsp27.

However, the results were not consistent using the constructs of vector groups I and II. When ^WT/MTTBC species of the vector group I were used, all ^MTTBC showed increased interaction with Hsp27 when compared with the ^WTBC/Hsp27 interaction (by 81, 58, and 60% increase for ^R120GBC, ^Q151XBC, and ⁴⁶⁴BC, respectively; see supplemental Fig. 4C). This set of data is consistent with the slight increase in interaction that was observed for all ^MTTBC forms by the pulldown assays (supplemental Fig. 4B). In contrast, when ^WT/MTTBC species of the vector group II were used, ^WTBC and ^R120GBC showed a similar interaction with Hsp27, whereas the ^Q151XBC and ⁴⁶⁴BC mutants showed moderately decreased interactions when compared with the ^WTBC/Hsp27 interaction (by 25 and 19% decrease for ^Q151XBC and ⁴⁶⁴BC, respectively; supplemental Fig. 4D). Using one-way ANOVA and subsequent post hoc pairwise group comparisons indicated statistical significance for the data obtained by both vector groups. This inconsistency was probably caused by steric hindrance involving the CFP and Cit tags. For that reason, no conclusion regarding possible differences between ^WTBC and ^MTTBC in the interaction with Hsp27 can be drawn at this time.

In summary, the data collected from the TH, PD, and qFRET assays showed that all myopathy-associated ^MTTBC forms are able to interact with Hsp27. Possible differences in the interactions between ^MTTBC forms and ^WTBC could not be unambiguously verified.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In this study, we focused our work to examine the interaction properties of the three known myofibrillar myopathy-associated ^MTTBC species ^R120GBC, ^Q151XBC, and ⁴⁶⁴BC. First, we examined the cellular localization of the mutant proteins in transfected cells. We established that all the three mutant proteins have an increased tendency to form cytoplasmic aggregates in both COS-7 cells and in cardiomyocytes. This result confirmed the tendency of myopathy-associated BC mutants to form aggregates in cultured cells in the presence or absence of desmin. This aggregate formation may result from misfolding of the mutant proteins, as was shown for ^R120GBC (37). No data are available about the stability of ^Q151XBC and ⁴⁶⁴BC; however, previous studies have established that the affected C-terminal arm of BC is particularly important for the stability and function of BC (4, 55, 57). This increased tendency to form aggregates may impair the cellular function of all ^MTTBC species, as was shown for the cytoprotective ability of ^R120GBC (34, 36, 38–40, 54, 58).

In cell fractionation experiments using transfected COS-7 cells, we demonstrated that ^R120GBC or ^Q151XBC associated with the nuclear and especially the cytoskeletal fractions, whereas ^WTBC did not. This finding strongly suggests that those ^MTTBC forms have an abnormally increased affinity for cytoskeletal components. For ^R120GBC, a previous study has demonstrated increased affinity for desmin intermediate filaments that are specifically expressed in muscle (38). Thus, ^Q151XBC and perhaps also ⁴⁶⁴BC, may share this enhanced desmin-binding property and thus lead to similar clinical phenotypes as the desmin-related myopathies.

The intracellular location of ^WTBC has been found to be related to its phosphorylation state (52, 54, 59–65). The only mutant studied to date, ^R120GBC, was seen to be hyperphosphorylated at all three phosphorylation sites in muscles of transgenic mice or at two phosphorylation sites when it was expressed in HeLa cells (54). Using COS-7 cells, we observed that all three myopathy-associated ^MTTBC forms indeed are hyperphosphorylated at all three phosphorylation sites as compared with ^WTBC. This BC hyperphosphorylation could be a specific feature of myopathies caused by mutation in BC gene as hyperphosphorylation of Hsp27 as been described in myopathies caused by desmin mutation (66). Interestingly, we observed a higher tendency to be phosphorylated in Ser-45 for ^R120GBC than for ^Q151XBC or ⁴⁶⁴BC mutants. Considering that ^R120GBC is also implied in cardiomyopathy development whereas ^Q151XBC or ⁴⁶⁴BC are only linked to myopathies, this result suggest a differential effect of Ser-45 phosphorylation in skeletal and cardiac muscles. Our findings reinforced the hypothesis suggesting that hyperphosphorylation of ^MTTBC may be part of the disease mechanism, as has been previously proposed for ^R120GBC (54).

Second, we examined if the ^MTTBC species have altered properties in regard to interactions with themselves, with ^WTBC, and with the other sHsps that are abundant in muscle. It is now well established that composition and size of the oligomeric structures formed by the various sHsps are important for both properties and functions of the sHsp complexes in cells (67). Indeed, the significance of protein-protein interactions is not only to form protein complexes for cellular functions but could also be implied in protein stability or solubility (19).

Our data show that the interactions of ^MTTBC with ^WTBC were not affected or were only slightly affected, with the exception of the ⁴⁶⁴BC/^WTBC interaction that was moderately decreased. A minor increase was also observed in the interaction of the ^MTTBC species with themselves. Recent studies showed that despite its capacity to reduce the formation of ^R120GBC aggregates, ^WTBC is unable to reduce the cytotoxicity induced by expression of the mutated protein (44, 45). Our results showed that the effect on aggregation of ^WTBC is probably because of a passive mechanism (there is not an active recruitment of ^WTBC) leading to the formation of more soluble heterocomplexes of ^WTBC/^MTTBC than homocomplexes of ^MTTBC without a real chaperone mechanism involved.

Our data also show that the interactions of all ^MTTBC species with Hsp20 were decreased as compared with the ^WTBC/Hsp20 interaction. It is interesting to note that Hsp20, which is known as an sHsp with relatively low chaperone function, exhibits only decreased interaction with all ^MTTBCs (68). We measured a major decrease in ^R120GBC/Hsp20 interaction and moderate decreases in the ^Q151XBC/Hsp20 and ⁴⁶⁴BC/Hsp20 interactions. Considering that ^R120GBC induces myopathy with cardiac involvement, whereas ^Q151XBC and ⁴⁶⁴BC result in myopathy without cardiac involvement (20, 22), and that Hsp20 is involved in cardiac protection (69), our data suggest that decreased ^R120GBC/Hsp20 interaction may play a role in the manifestation of cardiac involvement of the disease.

Our data also reveal distinct differences in the interaction of the various ^MTTBC species with Hsp22. ^R120GBC and ^Q151XBC showed greatly increased or decreased, respectively, interactions with Hsp22 as compared with the ^WTBC/Hsp22 interaction, whereas the interaction between ⁴⁶⁴BC and Hsp22 remained essentially unchanged. This increased ^R120GBC/Hsp22 interaction confirms the results of Chavez Zobel et al. (13). Recently, several studies have suggested that Hsp22 overexpression may be an efficient therapeutic tool to restore the functions of ^R120GBC (13, 45). Following this argument, the decreased or unchanged interactions of ^Q151XBC and ⁴⁶⁴BC, respectively, with Hsp22 would not support the idea that overexpression of Hsp22 might be a useful method to neutralize the adverse effects of these two ^MTTBC species in myofibrillar myopathies.

Our results collected by various methods concerning the interaction of the different ^MTTBC species with Hsp27 confirm the interaction between both proteins. However, the qFRET data were inconsistent with respect to possibly increased or decreased interactions of ^MTTBC species as compared with ^WTBC, depending on the constructs used. Although the qFRET experiments using C-terminally tagged ^WT/MTTBC species showed a pronounced increase in interactions for all three ^MTTBC species, the experiments using N-terminally tagged ^WT/MTTBC species showed unchanged or decreased interactions resulting from the mutations. Consistent with this latter observation is that the N-terminally tagged ^MTTBC species as used in the PD assays also indicated slightly increased interactions. Although the reason for this vector-dependent inconsistency is not known, steric hindrance of the N-terminally tagged ^MTTBC species is a plausible explanation.

In summary, this study for the first time characterizes the myopathy-associated mutants ^Q151XBC and ⁴⁶⁴BC and compares these mutants with the previously characterized ^R120GBC. We established that these three myopathy-associated ^MTTBC proteins show both abnormal protein aggregation and abnormal subcellular localization. Moreover, we show that all three mutants are hyperphosphorylated at the three serine phosphorylation sites. These data reinforce the emerging concept of a key role of sHsp phosphorylation in the development of degenerative diseases (39, 54, 62, 66).

Modifications in the interaction level between sHsps caused