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1 Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russian Federation2 Maturation des ARN et Enzymologie Moléculaire UMR 7567 CNRS-UHP B.P. 239, Faculté des Sciences Université Henry Poincaré Nancy I, 54506, Vandoeuvre-lès-Nancy Cedex, France
Reprint requests to: Vladimir I. Muronetz, Department of Biochemistry of Animal Cell, Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russian Federation; e-mail: vimuronets{at}belozersky.msu.ru; fax: +(095) 939-31-81.
(RECEIVED November 3, 2004; FINAL REVISION December 15, 2004; ACCEPTED December 31, 2004)
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Keywords: GAPDH; GroEL; oligomeric proteins; refolding; denaturation; immobilization; chemical modification; protein–protein interactions
Abbreviations: DAB, 3,5'-diaminobenzidine tetrahydrochloride • DTNB, 5,5'-dithiobis(2-nitrobenzoate) • DSC, differential scanning calorimetry • DTT, dithiothreitol • GAPDH, glyceraldehyde-3-phosphate dehydrogenase • TBS, Tris-buffered saline
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041211205.
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Introduction |
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-galactosidase, GroEL-mediated refolding does not even require ATP for substrate release from the chaperone (Ayling and Baneyx 1996). The interactions of GroEL with a broad spectrum of unfolded and partially unfolded proteins are well studied. GroEL helps them acquire their native conformation by interacting with folding intermediates, which possess a high degree of exposed hydrophobic surface (Lin et al. 1995). Association with GroEL prevents aggregation of nonnative proteins and allows them to fold in the central cavity of chaperonin in the absence of intermolecular interactions. GroEL can also unfold kinetically trapped folding intermediates, thus giving them a chance to fold correctly (Todd et al. 1994; Shtilerman et al. 1999). On the other hand, it is well known that some abnormal polypeptides can never reach their native conformation and fail to assemble into oligomeric proteins or multiprotein complexes even in the presence of molecular chaperones. These so-called misfolded proteins can be generated because of mutations or errors during translation and by chemical modifications or structural damage during their lifetime. The cell quality controls used to recognize and eliminate such proteins and the potential risks of their accumulation in large quantities are broadly discussed in the literature (Wickner et al. 1999; Dobson 2004). However, the possible interaction of misfolded proteins with molecular chaperones is not well studied.
In this study we analyzed the interaction of the chaperonin GroEL with misfolded forms of the tetrameric enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which were generated by chemical modification of SH-groups by 5,5'-dithiobis(2-nitrobenzoate) or site-directed mutagenesis (misfolded O-P-type and O-R-type dimers) (Roitel et al. 1999). Previously, we demonstrated that GroEL has a high affinity for immobilized monomers and dimers of wild-type GAPDH but does not interact with the native tetramer (Bulatnikov et al. 1999). Moreover, it was shown that GroEL assisted refolding of the urea-denatured wild-type GAPDH from Bacillus stearothermophilus. An elegant study by Li et al. (1998) showed that GroEL can also bind to folding intermediates of partially denatured rabbit GAPDH and suppress its aggregation. We suggested that the generation of abnormal variants of GAPDH, which have impaired tertiary and oligomeric structure but at the same time remain stable in neutral buffer conditions, could make them good model substrates to study the interactions of chaperonin GroEL with misfolded proteins. Indeed, we demonstrated that chemically modified GAPDH and O-R-type mutant dimers of GAPDH can not only form stable complexes with GroEL but inhibit chaperonin-assisted re-folding of the urea-denatured wild-type enzyme.
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Results |
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In this study, we used GroEL purified from E. coli transformed with plasmid pOF39, which encodes the wild-type chaperonin. GroEL was previously characterized by Bulatnikov et al. (1999) and possessed ATPase activity, and it effectively assisted refolding and reactivation of chemically denatured wild-type GAPDH from B. stearothermophilus.
Preparation and characterization of GAPDH from rabbit muscle modified by DTNB (GAPDHDTNB)
We used chemical modification of cysteine residues to generate misfolded forms of GAPDH, since these residues can be easily modified during disulfide exchange reactions and oxidation not only in vitro but also within the cell. For example, SH-groups of GAPDH are targets for S-glutathiolation during cardiac oxidative stress (Eaton et al. 2002). This type of GAPDH oxidation is associated with a loss in reduced cysteine status that correlates with the inactivation of this enzyme. GAPDH contains four cysteines per subunit, which are buried in the core of the protein or located in the intersubunit contacts. Thus, we suggested that their conjugation with bulky molecules such as 5,5'-dithio-bis(2-nitro-benzoate) might impair the quaternary and tertiary structure of the enzyme.
It was shown that 60-min incubation of rabbit muscle GAPDH with DTNB (molar ratio 1:10, counted per cysteine residues of GAPDH) resulted in modification of all cysteine residues of the protein (15.2 ± 0.2 mol per tetramer), complete loss of catalytic activity, and significant changes in physico-chemical properties and oligomeric structure. According to ultracentrifugation analysis, GAPDHDTNB constituted a mixture of dimers and tetramers in a ratio of ~1:1 (sedimentation coefficients 4.3 ± 0.6 S and 6.38 ± 0.5 S, respectively). The decrease in the stability of GAPDHDTNB was confirmed by differential scanning calorimetry. Thus, modification of GAPDH resulted in a shift of Tmax (maximum of the thermal transition peak) by 7°C toward lower temperatures. In addition, the 2.5-fold decrease in calorimetric enthalpy and broadening of the partial heat capacity profile suggested a decrease in cooperativity of the transition after chemical modification of GAPDH (Fig. 1
). At the same time, circular dichroism spectral data indicated that the secondary structure of the modified enzyme was not changed (data not shown). In view of the significant change in spatial structure and activity of the enzyme after chemical modification, we considered it a misfolded form of GAPDH. This misfolded variant was used in subsequent experiments to analyze its effect on chaperone-assisted reactivation of the urea-denatured wild-type GAPDH.
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Interaction of GroEL with DTNB-modified GAPDH
First, we studied the interaction of immobilized GAPDHDTNB with chaperonin using SDS-PAGE analysis (Fig. 2). Soluble GroEL (1.4 µM) was incubated with immobilized GAPDHDTNB (0.7 µM), immobilized active tetramers of GAPDH (1.0 µM), and control (blank) Sepharose, followed by extensive washing with the binding buffer (50 mM Tris/ HCl, pH 7.2, 0.1 mM EDTA). Proteins bound to the matrix were eluted with 2x SDS-PAGE sample buffer and analyzed by SDS-PAGE. No protein was detected in the eluate from control (blank) Sepharose, indicating that GroEL did not bind nonspecifically to the matrix (Fig. 2, lane 1). In the case of immobilized tetrameric GAPDH, a minor protein band with apparent molecular weight of 60 kDa corresponding to a monomer of GroEL was detected in the eluate (lane 2). The amount of GroEL bound to the matrix greatly increased in the case of immobilized GAPDHDTNB (lane 3). A protein band with an apparent molecular weight of 36 kDa corresponding to isolated polypeptide chains of GAPDH was identified in samples of immobilized tetramers and monomers (lanes 2 and 3, respectively). The presence of trace amounts of these polypeptides in the eluates from immobilized monomers of GAPDH may be accounted for by a weak dissociation of covalently bound protein from the matrix under elution conditions. Importantly, washing of the complex of immobilized GAPDHDTNB-GroEL with the buffer containing 5 mM Mg-ATP (normally leading to dissociation of polypeptides from chaperone) did not result in a decrease of GroEL bound to the immobilized enzyme (data not shown). Traces of chaperonin bound to immobilized native tetramer of GAPDH may be explained by small amounts of nonnative forms present in the enzyme coupled to the matrix (immobilized dimers and monomers).
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).
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Characterization of mutant dimeric GAPDH from B. stearothermophilus
To get a better understanding of the interaction of GroEL with misfolded forms of GAPDH, we used previously described O-P- and O-R-dimers of GAPDH from B. stearothermophilus with mutations in the area of interdimer contacts. O-R-type mutant dimers were produced by incubation of the mutated catalytically active tetrameric GAPDH (mutants D282G, Y283V, and Y283V/W84F) in the presence of 0.16 M KCl at 4°C. Under these conditions, tetrameric mutant GAPDH dissociated into dimers and lost its enzymatic activity. Since dissociation into dimers is a very slow process (k < 0.05 h < –1), the probability of the formation of the original tetramers from dimers is very low (Roitel et al. 2003). Despite the ability of O-R-dimers to bind cofactor NAD+ in both active centers, this binding was not cooperative (Roitel et al. 1999, 2003). Mutant O-P-dimers of GAPDH from B. stearothermophilus with amino acid substitutions Y46G/S48G and Y46G/R52G were directly purified in dimeric form and were catalytically inactive. However, only O-P-type dimers possessed a positive cooperativity in the binding of cofactor NAD+ (Roitel et al. 2003). These two types of GAPDH dimers were strictly different in their thermodynamic parameters (Roitel et al. 2002). Recent data using small-angle X-ray scattering confirmed that dimers with mutation D282G are O-R-type dimers, and dimers with mutations Y46G/S48G and Y46G/S52G are O-P-type dimers (Roitel et al. 2003).
Interaction of GroEL with mutant O-R-dimers of GAPDH from B. stearothermophilus
SDS-PAGE analysis of the complex formation between GroEL and O-R-dimers Y283V (Fig. 4A) revealed that incubation of immobilized GroEL with soluble Y283V mutants resulted in binding of the dimers to immobilized GroEL (lane 1). Remarkably, the amount of O-R-dimers bound to chaperonin did not change after washing of the complex with the binding buffer containing 5 mM Mg-ATP (lane 2), as shown previously for GAPDHDTNB (see above). Under these conditions, O-R-dimers Y283V did not bind to control Sepharose (lane 5). Similar results were obtained using D282G and Y283V/W84F dimers (data not shown).
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). Staining of the mixture of GroEL and O-R-dimers with antibodies against chaperonin revealed a new protein band with electrophoretic mobility lower than that of intact GroEL (lanes 2 and 3, respectively). This protein band was absent in samples from isolated O-R-dimers (lane 1) and may constitute a complex between chaperonin and mutant dimers, which was stable during electrophoresis under nondenaturing conditions. The same results were obtained for O-R-dimers D282G (data not shown). We confirmed the existence of the stable complex between GroEL and O-R-dimers using a co-immunoprecipitation approach: antibodies against GroEL coprecipitated O-R-dimers and vice versa (data not shown).
Using immobilized GroEL, we determined the parameters of chaperonin binding to soluble mutant O-R-dimers. The Kd calculated for the complex between GroEL and Y283V mutant dimers was 0.9 ± 0.21 µM, and the stoichiometry was ~1.0 (one GAPDH dimer per GroEL oligomer) (Fig. 5). The Kd values for D282G and Y283V/W84F dimers were 0.85 ± 0.15 and 0.8 ± 0.2 µM, respectively (data not shown).
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Influence of misfolded forms of GAPDH on GroEL-assisted folding of wild-type GAPDH from B. stearothermophilus
To study the effect of GAPDHDTNB and mutant O-R- and O-P-dimers on the chaperone function of GroEL, we used a model system of reactivation of GAPDH from B. stearothermophilus after its denaturation in 8 M urea.
It was previously reported that GroEL considerably increases the yield of GAPDH refolding compared to its spontaneous reactivation (Bulatnikov et al. 1999). As shown in Figure 6A, spontaneous refolding of denatured GAPDH after dilution resulted in 20%–25% recovery of initial enzymatic activity, whereas addition of GroEL and Mg-ATP increased the yield of reactivated enzyme to 50%–55%. Interestingly, D282G mutant O-R-dimers completely inhibited the GroEL-mediated folding, but had no effect on the spontaneous reactivation of GAPDH. The GroEL-assisted reactivation of denatured GAPDH was completely inhibited when the molar ratio of GroEL:denatured GAPDH mono-mers:mutant dimers was 2:1:1. At the same time, addition of O-P-dimers affected neither chaperonin-dependent nor spontaneous reactivation of GAPDH (data not shown).
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, preincubation of GAPDHDTNB with this antibody completely abolished its inhibitory action on GroEL-assisted refolding of GAPDH from B. stearothermophilus. |
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A distinguishing feature of the complexes of chemically modified GAPDH and O-R-dimers with chaperonin is their stability in the presence of Mg-ATP. It is possible that ATP binding induces conformational changes leading to the discharge of bound polypeptide in both types of complexes (GroEL-unfolded protein and GroEL-misfolded protein). However, since misfolded protein fails to reach the native state, and constantly exposes its hydrophobic surfaces, it could promptly rebind to the chaperonin. It is possible that repeated association-dissociation of GroEL and misfolded protein, accompanied by ATP binding and hydrolysis, might occur even without exclusion of the latter from the central cavity of GroEL. Indeed, when bound to misfolded protein, chaperonin cannot function properly since its poly-peptide-binding sites on the apical domain and/or central cavity are constantly occupied. In this study, we have provided direct evidence that some misfolded forms of GAPDH inhibit GroEL-assisted refolding of the urea-denatured wild-type enzyme.
We have shown that chemical modification of GAPDH renders it unable to fold into its native structure. Similar modifications of cellular proteins may be induced by a variety of damaging factors, particularly during oxidative stress (Ciolino and Levine 1997; Tamarit et al. 1998; Dukan et al. 2000). As more mistakes occur in protein folding, these errors would lead to further errors (especially of proteins involved in the protein synthetic pathway) (Levine et al. 1999). This may result in the permanent accumulation of mutated proteins, which cannot adopt a native structure and perform their functions correctly. Our experiments with dimeric forms of mutant GAPDHs—D282G, Y283V, and Y283V/W84F—provide evidence of such a possibility.
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Antibodies
Polyclonal anti-GroEL and anti-GAPDH antibodies were generated by immunization of rabbits with the wild-type GroEL or GAPDH from B. stearothermophilus. GroEL- and GAPDH-specific antibodies were purified using affinity chromatography on immobilized GroEL or dimeric GAPDH from B. stearothermophilus. Monoclonal antibodies 6G7 against nonnative rabbit GAPDH were kindly provided by A. Katrukha (School of Biology, Moscow State University). Goat anti-rabbit and anti-mouse HRP-conjugated IgG were from Pierce.
Protein purification
E. coli strain W3110 expressing the plasmid pOF39, which encodes wild-type GroEL, was kindly provided by Prof. V. Mesyanzhinov (Bakh Institute of Biochemistry, Russian Academy of Sciences). GroEL was purified as described by Corrales and Fersht (1996). According to analytical centrifugation analysis, the apparent molecular weight of purified GroEL was 840 kDa. GAPDH was isolated from rabbit muscle by the method of Scopes and Stoter (1982) with an additional purification step using gel filtration on Sephadex G-100 (superfine) to remove the traces of myoglobin. The wild type, O-R-type mutant dimers D282G, Y283V, and Y283V/W84F and O-P-type mutant dimers Y46G/S48G and Y46G/R52G of GAPDH from B. stearothermophilus were expressed in E. coli and purified as described (Roitel et al. 2002). The proteins were isolated as holoenzyme in the case of wild-type GAPDH from B. stearothermophilus (A280/A260 = 1.05 ± 0.05) and as apoenzyme in the case of dimeric mutant GAPDH (A280/A260 = 1.8 ± 0.1). Protein concentration was determined at 280 nm using A0.1% = 1.0 for holoenzyme of rabbit muscle GAPDH, A0.1% = 0.92 for holoenzyme of GAPDH B. stearothermophilus, A0.1% = 0.83 for apoenzyme of GAPDH from B. stearothermophilus, and A0.1% = 0.2 for GroEL. GroEL, O-R-, and O-P-dimers of GAPDH and denatured GAPDH were considered as tetradecamers, dimers, and monomers, respectively. Concentration of proteins covalently bound to Sepharose was determined by the modified Bradford’s method (Asryants et al. 1985).
GAPDH enzyme activity assay
GAPDH activity was measured by following the increase in the absorbance at 340 nm due to formation of NADH. The reaction was carried out at 25°C and was initiated by the addition of 0.5–2.0 µg of the enzyme to the reaction mixture containing 50 mM glycine, 50 mM potassium phosphate, pH 10.0, 5 mM EDTA, 1.5 mM NAD+, and 2 mM glyceraldehyde-3-phosphate.
Differential scanning calorimetry
Differential scanning calorimetry measurements were performed using a DASM-4 microcalorimeter (Biopribor Poushchino) with 0.45-mL cell volume at scan rate 1°C/min in 50 mM Tris-HCl, pH 7.2, 0.1 mM EDTA. The final protein concentration was 8.3 µM. The values of Tmax and calorimetric enthalpies (
Hcal) were determined using original software.
Immobilization of proteins
Immobilization of GroEL and GAPDH on CNBr-activated Sepharose was performed as previously described (Cherednikova et al. 1980). In the case of GroEL, 30 mg of CNBr per mL of the gel was used to activate Sepharose. This ensured the coupling of GroEL tetradecamer to Sepharose via two subunits, which was confirmed by determination of the matrix-bound protein after treatment of the immobilized chaperonin with 8 M urea for 24 h with subsequent washing of the gel. In the case of immobilized GAPDH, 5 mg of CNBr per mL of gel was used to obtain the enzyme bound to Sepharose via a single subunit.
Modification of GAPDH from rabbit muscle by 5,5'-dithiobis(2-nitrobenzoic acid)
The soluble modified GAPDHDTNB was prepared by incubation of 25 µM rabbit muscle GAPDH (calculated per monomer) in the presence of 1 mM DTNB (Habeeb 1972). The excess of DTNB was removed by gel-filtration on Sephadex G-50. To modify the immobilized GAPDH, it was incubated in the presence of 0.7 mM DTNB followed by extensive washing of the matrix with the re-folding buffer (without DTT).
Denaturation of GAPDH
GAPDH was denatured by incubation of the holoenzyme (17–25 µM) in 50 mM Tris-HCl, pH 7.2, 8 M urea, 0.1 mM EDTA at 20°C overnight. Under these conditions the denaturation of GAPDH was complete based on total loss of the enzymatic activity and change in the spectrum of circular dichroism.
Refolding of GAPDH
The refolding of urea-denatured GAPDH was initiated by 50- to 70-fold dilution of the denatured enzyme with the refolding buffer (50 mM Tris-HCl, pH 7.2, 5 mM KCl, 0.1 mM EDTA, and 1 mM DTT) containing 3 mM Mg-ATP at 40°C. GroEL or dimeric GAPDH mutants were added to the reactivation mixture as specified. During the time of reactivation, aliquots containing ~1.0 µg of GAPDH were analyzed for enzyme activity. The yield of re-folding was calculated as a percentage of the activity of native GAPDH treated in the same way. GroEL-assisted reactivation was initiated by addition of 3 mM Mg-ATP 1 min after dilution of denatured GAPDH in the presence of GroEL.
Titration of immobilized GroEL with soluble mutant dimers of GAPDH from B. stearothermophilus
Titration of immobilized GroEL with soluble mutant dimers of GAPDH was performed as follows. Different concentrations of the dimers were added to the samples containing 0.1 mL of gel with 80 µg of immobilized GroEL so that the total volume of each sample was constant (0.2 mL). After 30 min of incubation with mixing, the gel was precipitated by centrifugation and the protein concentration was determined in supernatant. Samples containing Sepharose gel without immobilized protein were used as controls. The exclusion volume of the immobilized enzyme suspension was determined as 0.2 ± 0.01 mL/mL of the packed gel. The data obtained were analyzed and fit by nonlinear regression.
SDS-PAGE analysis
Proteins covalently bound to Sepharose and proteins bound to immobilized GroEL or immobilized GAPDHDTNB were analyzed using SDS-PAGE, which was performed according to Laemmli (1970). Samples for SDS-PAGE were prepared as follows. A portion of Sepharose-gel was mixed with an equal volume of 125 mM Tris-HCl, pH 6.8, 10% -mercaptoethanol, 20% glycerol, 2% SDS, 0.006% bromophenol blue. After 5 min of incubation at 95°C, the Sepharose gel was precipitated by centrifugation, and aliquots from the supernatant were run on the gel.
Nondenaturing gel electrophoresis
Polyacrylamide gel electrophoresis under nondenaturing conditions (Medvedeva et al. 1996) was used to investigate the interaction of chaperonin GroEL with mutant dimeric GAPDH from B. stearothermophilus. Mutant dimeric GAPDH (4.5 µM, final concentration) was titrated by GroEL in the refolding buffer (0.85–7 µM, final concentration). After 30 min of incubation at 37°C, samples were run on 12.5% gels. Electrophoresis was performed in 80 mM Tris-glycine, pH 8.3.
Western blotting
Proteins were separated by SDS-PAGE or native gel electrophoresis and then transferred to nitrocellulose membrane in the transfer buffer containing 25 mM Tris, 192 mM glycine, 10% methanol, and 0.05% SDS (in the case of SDS-PAGE) (Towbin et al. 1979). The membrane was incubated overnight in TBS (50 mM Tris-HCl, pH7.2, 0.14 M NaCl) containing 0.05% Tween, 2% nonfat dry milk and anti-GroEL or anti-GAPDH from B. stearothermophilus antibodies. After washing, the membrane was incubated with goat anti-mouse or goat anti-rabbit IgG conjugated to horseradish peroxidase. Protein bands were visualized in TBS containing 0.06 mg/mL DAB, 0.03% H2O2, and 0.03% NiCl2.
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Acknowledgments |
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References |
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), 0.35 µM O-R-dimers D282G (
). (B) Effect of GAPDHDTNB on GroEL-assisted refolding of the urea-denatured wild-type GAPDH from B. stearothermophilus. Spontaneous reactivation (black bars) and GroEL-assisted reactivation (white bars) of urea-denatured GAPDH after dilution to 0.35 µM in the refolding buffer (first set of bars), in the presence of 0.35 µM GAPDHDTNB (second set of bars), or in the presence of 0.35 µM GAPDHDTNB and antibodies 6G7 (third set of bars).