| | Gentamicin inhibits HSP70-assisted protein folding by interfering with substrate recognitionEdited by Miguel De la Rosa Received 29 September 2009; received in revised form 24 November 2009; accepted 10 December 2009. published online 22 December 2009. Abstract We previously reported that gentamicin (GM) specifically binds to heat-shock protein with subunit molecular masses of 70 kDa (HSP70). In the present study, we have investigated the effects of GM binding on HSP70-assisted protein folding in vitro. The C-terminal, and not the N-terminal of HSP70 was found to bind to GM. GM significantly suppressed refolding of firefly luciferase in the presence of HSP70 and HSP40, although the ATPase activity of HSP70 was unaffected by GM. A surface plasmon resonance analysis revealed that GM specifically interferes with the binding of HSP70 to a model peptide that mimics the exposed hydrophobic surface of the folding intermediates. These results indicated that GM inhibits the chaperone activity of HSP70 and may suppress protein folding via inhibition of HSP70 in vivo. 1. Introduction  Aminoglycoside antibiotics are widely used drugs for the treatment of severe Gram-negative bacterial infections, and gentamicin (GM) is an aminoglycoside antibiotic frequently used for this purpose. However, aminoglycosides have many nephrotoxic side effects [1]. GM induces a complex phenomenon characterized by an increase in the blood urea nitrogen and serum creatinine concentration, leading to severe proximal renal tubular necrosis [2], [3]. Thus, its clinical use is often limited by the nephrotoxicity. Although the role of reactive oxygen metabolites in GM nephropathy has been suggested [4], molecular mechanisms by which GM disturb renal functions is poorly understood. We previously reported that heat-shock protein with subunit molecular masses of 70kDa (HSP70), a molecular chaperone required for protein folding, was induced in injured tubular epithelial rat kidneys with GM-induced acute renal failure [3]. We also found that GM co-localizes with HSP70 in the lysosome of GM-treated rat kidneys in vivo and HSP70 can be purified from kidney extract using a GM-affinity column in vitro [5]. Aggregation prevention activity of HSP70 was suppressed in the presence of GM, and this drug induced a conformational change in the HSP70. These observations suggest that GM may inhibit the activities of HSP70 in the kidney. However, whether GM affect the HSP70-assisted protein folding is unknown. Moreover, little is known about how GM affects the HSP70 activities by binding. The HSP70 molecule consists of two domains, i.e., a highly conserved amino-terminal nucleotide-binding domain (NBD) of 45kDa and a carboxyl-terminal substrate binding domain (SBD) of 25kDa. The SBD is further divided into a β-sandwich subdomain (β-PDB) and an α-helical subdomain (α-PDB) [6], [7], [8]. NBD and SBD are connected by a flexible linker region that contributes to allosteric regulation of NBD and SBD [9], [10]. The NBD has two structurally similar lobes (I and II), and each of them are further divided into two small subdomains (IA, IB, IIA and IIB). These subdomains are separated by a deep cleft that captures ATP and cations (one Mg2+ and two K+ ions) contacting the four subdomains [6]. The peptide-binding moiety is composed of two β-sheets with loops in the β-PDB and two helices (helices A and B) in the α-PDB [7], [11]. Helix B works as the “lid”, which closes the cavity through a salt bridge and two hydrogens bound to a loop of β-PDB. The ATP-bound form of HSP70 rapidly binds and releases a peptide [12], [13]. The binding of ATP to the NBD causes a conformational change, which results in structural alterations in the carboxyl-terminal domain leading to substrate release [14], [15]. In contrast, the ADP-bound form of HSP70 slowly binds the peptide but the binding is more stable. The ATP dependent HSP70 chaperone activity is tightly regulated by the various co-chaperones including J-domain proteins (e.g., HSP40) and nucleotide exchange factors [16], [17], [18], [19]. In the present study, we investigated how GM affects the HSP70 activities in vitro and found that GM inhibits the HSP70-assisted protein folding by blocking substrate binding through specific binding to the peptide-binding domain. We discuss the role of the GM–HSP70 interactions during the GM-induced perturbation of cellular activities including the chaperone-assisted protein folding. 2. Materials and methods 2.1. Expression vectors Human HSP40 (DNAJB1, Accession No. NM_006145) cDNA was obtained from the Kazusa DNA Research Institute, Japan, and amplified by a polymerase chain reaction using oligonucleotides (5′-CTCGAGTATTGGAAGAACCTGCTCAAGTA-3′ and 5′-CATATGGGTAAAGACTACCAGACG-3′) as primers. The amplified HSP40 cDNA was subcloned into the NdeI–XhoI sites of the pCR2.1 (Life Technologies Corporation, CA, USA) and pET29a (MERCK, Darmstadt, Germany) vectors. 2.2. Protein purification HSP40 was expressed in Eschericha coli BL21 (DE3) pLysS cells as a C-terminally 6×his-tagged protein. Cells were grown in LB medium containing kanamycin and chloramphenicol at 37°C, and expression of the 6×his-tagged HSP40 was induced with 0.1mM isopropyl-1-thio-β-d-galactopyranoside. Cells were resuspended in 10mM Tris–HCl (pH 7.4) and lysed by sonication on ice. The supernatant was recovered after centrifugation at 20000rpm for 10min at 4°C, and applied to a Ni-NTA affinity column (GE Healthcare, Uppsala, Sweden) equilibrated with buffer A (0.3MNaCl and 10mM Tris–HCl, pH 7.4) supplemented with 20mM imidazole. After washing with 50mM imidazole in buffer A, proteins were eluted with a linear gradient of 100–500mM imidazole in buffer A. The HSP40 peak fractions were pooled and loaded onto a hydroxyapatite HTP column (Bio-Rad, CA, USA) equilibrated with 100mM potassium phosphate buffer (pH 7.0). After washing, the proteins were eluted with a linear gradient of 100–500mM potassium phosphate (pH 7.0). After concentration by ultrafiltration, the HSP40 peak fractions were applied onto a Superdex 200 HR column (GE Healthcare) equilibrated with buffer B (5% glycerol, 0.1M NaCl in 25mM HEPES–KOH, pH 7.4). The HSP40 fractions were dialyzed against buffer C (5% glycerol, 1mM DTT in 25mM HEPES–KOH, pH 7.4) and stored at −80°C. HSP70 was purified from porcine brains as follows. The porcine brains were homogenized in 3 volumes of 1mM PMSF, 5mM EDTA, and 15mM β-mercaptoethanol in 10mM Tris–HCl (pH 7.4) on ice and centrifuged at 13000rpm for 30min at 4°C. The supernatants were fractionated by ammonium sulfate precipitation and the 45–90% ammonium sulfate fractions were collected. After overnight dialysis against 10mM Tris–HCl (pH 7.4), the proteins were applied onto a Q-sepharose column (GE Healthcare) equilibrated with 10mM Tris–HCl (pH 7.4). After washing with 0.1M NaCl in 10mM Tris–HCl (pH 7.4), the proteins were eluted with 0.3M NaCl in 10mM Tris–HCl (pH 7.4). After the addition of 50mM KCl and 5mM MgCl2 (final concentrations), the HSP70 peak fractions were load onto an ATP-sepharose column (Sigma, St. Louis, MO, USA) equilibrated with buffer D (0.3M NaCl, 5mM MgCl2, 50mM KCl and 10mM Tris–HCl, pH 7.4). After washing in buffer D, the column was further washed in buffer E (5mM MgCl2, 50mM KCl and 100mM Tris–HCl, pH 7.4). The proteins were eluted with a linear gradient of 0–20mM ATP in buffer E. After concentration by ultrafiltration, the HSP70 peak fractions were separated on a Superdex 200 column (GE Healthcare) equilibrated with buffer B. The HSP70 fractions were dialyzed against buffer C and stored at −80°C. The amino-terminal and carboxyl-terminal domains of HSP70 (HSP70N and HSP70C, respectively) were purified as previously described [20]. 2.4. Surface plasmon resonance (SPR) assay 2.4.2. Measurement of HSP70 interaction with immobilized peptide An HSP70-binding model peptide (CKETAAAKFERQHMDSSTS), which consists of the amino-terminal 1–18 residues of RNase A [21], [22] and an additional cysteine for immobilization on the sensor chip, was purchased from Biosynthesis, USA. Ethylenediamine was coupled on the surface of the C1 sensor chip (GE Healthcare) by subsequent injection of a 0.1M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/0.05M N-hydroxysuccinimide solution and 0.1M ethylenediamine (70μl each). Carboxyl groups on the sensor surface were blocked with 1M monoethanolamine, and NHS-PEG6-maleimide (Pierce, Rockford, USA), a spacer for coupling with the cysteine residue, was introduced using a 5mM solution. After the model peptide (1mM) was injected using the running buffer, the free maleimide residues are blocked with 1M l-cysteine. The peptides immobilized on the surface of the sensor chip were ∼210 RU. For the binding experiments, HSP70 (1μM) solutions containing 0–10mM GM were loaded onto the sensor chip. Regeneration of the sensor chip surface was achieved by a 30-s pulse (5μl) of 10mM NaOH and a 1-min pulse (10μl) of 0.5M NaCl in running buffer. 2.6. Luciferase refolding assay Recombinant firefly luciferase (6.25μM) (Promega, Madison, USA) was incubated for 30min at 30°C in denaturing buffer (6M guanidine–HCl, 5mM MgCl2, and 50mM KCl in 25mM HEPES–KOH, pH 7.4). For refolding, the denatured luciferase was diluted 125-fold into renaturation buffer (2mM DTT, 2mM ATP, 5mM MgCl2, and 50mM KCl in 25mM HEPES–KOH, pH 7.4) in the presence of 1μM HSP40 and 2μM HSP70. The refolding reaction mixture was incubated at 30°C up to 70min in the presence or absence of GM. At each sampling time point, aliquots (1μl) were mixed with 1mg/ml BSA/25mM HEPES–KOH, pH 7.4 (19μl) and the luciferase activity was determined using luciferase assay reagent (Promega) and Infinite M200 (TECAN, Mannedorf, Switzerland). The refolding activity was evaluated by the percent luciferase activity versus the native enzyme. 3. Results 3.2. GM inhibits HSP70-assisted protein folding We previously indicated that GM diminishes the aggregation prevention activity of HSP70 using rhodanase as a substrate [5]. To examine whether GM affects the protein folding by HSP70, we analyzed the refolding of firefly luciferase as a model substrate. Luciferase was denatured using 6M guanidine–HCl, and refolding was initiated by dilution in renaturation buffer containing HSP70 purified from the porcine brain. The spontaneous refolding was 5.4±2.3% and the refolding rate in the presence of HSP70 was 5.4±1.4% after incubation for 70min (Fig. 2A), indicating that HSP70 alone has no effect on the luciferase refolding. Although the refolding rate slightly increased to 12.4±1.9% in the presence of HSP40, it was significantly increased to 44.8±5.2% in the simultaneous presence of HSP70 and HSP40. These results indicate that HSP70 and HSP40 are both required for the luciferase refolding because they work as a chaperone system, in good agreement with previous reports [24]. In the presence of HSP70 and HSP40, the refolding of luciferase was strongly suppressed by GM in a concentration-dependent manner; 25.8±3.8% and 10.5±1.4% in the presence of 5mM and 10mM GM, respectively (Fig. 2B). In contrast, the inhibitory effect by GM on the luciferase refolding was very low in the presence of HSP40 alone; the refolding rate was 9.8±1.8% and 5.0±1.0% in the presence of 5mM and 10mM GM, respectively (Fig. 2C). In addition, GM at 10mM had no effect on the enzymatic activity of the native luciferase (data not shown). These results indicate that GM inhibited protein refolding assisted by the HSP40–HSP70 chaperone system, probably by direct binding to the HSP70C domain. We also analyzed the ATPase activity of HSP70 in the presence of GM, because the binding of substrates to HSP70 is known to stimulate the ATPase activity [23], [25]. However, the ATPase activity of HSP70 was unaffected in the presence of GM. Thus, although this drug binds to the substrate binding domain of HSP70, this binding does not affect the activity of the ATPase domain (Fig. 3). The GM and substrate polypeptides may differ in their ability to induce a conformational change in HSP70. Taken together, these observations suggest substrate recognition, but not the ATPase activity, of HSP70 is affected by the GM binding to a carboxyl-terminal region. 3.3. GM inhibits HSP70–substrate peptide interaction, but not HSP70–HSP40 interaction GM inhibited the refolding of luciferase by the HSP40–HSP70 chaperone system (Fig. 2B), and there were two possibilities explaining this result; GM inhibited the HSP70–substrate interaction or HSP70–HSP40 interaction. We first tested whether GM affects HSP70–substate interaction by measuring aggregation prevention activity of HSP70 using luciferase as a substrate. Luciferase was aggregated at 42°C, but the aggregation was completely inhibited by the presence of HSP70 (Fig. 4A). Aggregation prevention activity of HSP70 was diminished by GM in a concentration-dependent manner (Fig. 4B), and IC50 of the GM activity was calculated to be 140±7μM (Fig. 4C). These results strongly suggested that GM specifically inhibited substrate recognition of HSP70. Next, we examined the effects of GM on the HSP70 binding to the substrate by SPR using a model peptide containing the amino-terminal 1–18 residues of RNase A, because this peptide is known to bind to HSP70 [21], [25]. This model peptide was first immobilized on the surface of a sensor chip, then HSP70 solutions containing 0–10mM GM were injected. A sensorgram for the HSP70-model peptide system in the absence of GM is shown in Fig. 5A, which gives a KD of 2.26×10−8M. The KD value represents a strong interaction between HSP70 and the model peptide. In the presence of GM, the SPR signals significantly decreased with the change in the GM concentration. This result indicates that GM blocks the binding of HSP70 to the immobilized model peptide. Finally, we investigated the effects of GM on the HSP40–HSP70 interaction using an HSP40-immobilized chip. Since the HSP40–HSP70 interaction is known to be ATP dependent [28], an SPR assay was performed in the presence and absence of ATP. No significant HSP40–HSP70 interaction was detected in the absence of the ATP, regardless of the GM addition (Fig. 5B and unpublished data). In the presence of ATP, HSP70 significantly interacted with HSP40 in the absence of GM and KD was estimated to be 2.13±0.24×10−6M. In the presence of 10mM GM, the KD was 2.79±0.84×10−6, indicating that GM has a slight or no effect on the HSP40–HSP70 interaction (Fig. 5B and C, Supplementary Fig. 1 and Table 1). Taken together, these observations indicate that GM inhibits the chaperone activity of HSP70 by interfering with the substrate recognition. 4. Discussion In the present study, we demonstrated that GM strongly suppresses protein folding by the HSP40–HSP70 chaperone system without affecting the ATPase activity (Fig. 2, Fig. 3). Moreover, GM significantly inhibited the aggregation prevention activity of HSP70 (IC50=140±7μM), although the effect on the HSP40–HSP70 interaction was very low (Fig. 4, Fig. 5). These observations indicate that GM significantly inhibits the protein folding activity of HSP70 by modulating substrate recognition. We also found that GM binds to the carboxyl-terminal peptide-binding domain, but not to the amino-terminal nucleotide-binding domain of HSP70 (Fig. 1A and B). GM inhibited binding of HSP70 to a model peptide (Fig. 5A). These results suggest that GM has an inhibitory effect on peptide recognition by the carboxyl-terminal peptide-binding domain. However, KD value of HSP70–GM interaction was estimated to be 3.6×10−3M (Fig. 1C and D), indicating a weak or transient interaction. Since IC50 for the inhibition of HSP70-dependent aggregation prevention was significantly lower than the KD value, the weak or transient interaction may induce structural alteration of HSP70 rather than direct competition to substrate peptides. It may also be possible that GM interferes HSP70–substrate binding by interacting with HSP70 and substrate proteins. The peptide-binding domain in the carboxyl-terminal region of HSP70 is known to recognize the hydrophobic core of folding intermediates or the unstructured backbone of the non-native protein [26], [27]. These interactions stimulate productive folding of newly-synthesized polypeptides or refolding of denatured proteins by preventing any non-essential interaction of hydrophobic cores in a protein and aggregation of partially folded proteins. In our previously study, binding of GM to HSP70 resulted in a conformational change of HSP70 [5]. However, the ATPase activity of HSP70, which is known to be stimulated by the presence of the substrate protein, was unaffected by GM. Thus, the conformational change may be limited in the carboxyl-terminal region and the effect of the conformational change may not be transferred to the amino-terminal region. Although a number of inhibitors have been reported for HSP90, including geldanamycin derivatives, novobiocin and cisplatin [29], [30], [31], there are only a few inhibitors of HSP70. We previously reported that geranly-geranyl-acetone (GGA) inhibits the aggregation prevention activity of HSP70 by binding to the carboxyl-terminal region [20]. Liebscher et al. reported that N-α-[tetradecanoyl-(4-aminomethylbenzoyl)]-l-asparagine, a fatty acylated benzamido derivative, prevents bacteria growth by inhibiting the protein folding activity of the bacterial homologue of HSP70, DnaK [32]. As this substance competed with the substrate peptides for the DnaK binding, its binding site appears to be the substrate recognition domain at the carboxyl-terminus. An hydrophobic interaction is probably important to the binding of GGA and the fatty acylated benzamido derivative to HSP70/DnaK, because these substances have a hydrophobic straight carbon chain and the inhibitory effect of the latter is increased with extension of the carbon chain. In contrast, GM has no hydrophobic straight carbon chain. Thus, GM may differ from the other two substances in the mode of interaction with HSP70 at least in part, although these three substances block binding of the substrate proteins to the carboxyl-terminal region of HSP70/DnaK. GM is administered at 4–5mg/kg every 36–48h on clinical use (extended interval method), because concentration of GM in blood decreases 2.5mg/kg every 12h. The peak concentration and trough concentration of GM in blood using the extended interval method are 5–12μg/ml (approx. 11–26μM) and under 2μg/ml (approx. 4μM), respectively [33]. Most of administered GM (approx. 70–90%) is excretes into urine, and specifically accumulates in the cortex of kidney via megalin receptor within 30min after GM administration. The accumulated GM persists in the kidney for a long time (half-life>100h) [34], [35]. GM is highly accumulated in lysosomes (approx. 90%), providing a very high concentration (30mM or more) [36]. Approximately 10% of GM localizes with cytoplasm, nuclear, mitochondria and endoplasmic reticulum [37]. Concentration of GM to lysosome is thought to cause destruction of this organelle [34], [38], [39], and it probably increases GM concentration in the cytosol. Thus, the GM concentration in the cytosol of kidney cells in patients can be comparable to the GM concentration to inhibit 50% of HSP70-dependent aggregation prevention activity (140μM). The HSP70 family chaperones are highly conserved proteins that play essential roles in the protein metabolism and quality control by controlling the protein folding, translocation, degradation and gene expression [26], [40]. Thus, inhibition of the HSP70 activities, including folding of the newly-synthesized proteins and refolding of misfolded proteins by GM may result in perturbation of the cellular activities essential for cell survival. Since GM co-localizes with HSP70 in the enlarged lysosomes of the rat kidneys with GM-induced acute tubular injury in vivo [5], GM may affect the HSP70 activity required for kidney cells and cause nephrotoxicity via the HSP70 inhibition. Appendix A. Supplementary data Table S1. The kinetic parameter of HSP40–HSP70 interaction in the presence or absence of GM. 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a Department of Life Science, Faculty of Engineering and Resource Science, Akita University, 1-1 Tegata Gakuen Town, Akita City 010-8502, Japan b Department of Gastroenterology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-Ku, Tokyo, Japan c Third Department of Internal Medicine, Akita University School of Medicine, Akita 010-8543, Japan  Corresponding author. Fax: +81 18 883 3041.
PII: S0014-5793(09)01067-9 doi:10.1016/j.febslet.2009.12.021 © 2009 Federation of European Biochemical Societies. Published by Elsevier BV. All rights reserved. | |
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