Hsp90 is a highly conserved and abundant heat shock protein (HSP) involved in a myriad of cellular processes. The abundance of Hsp90 accounts for ~1–3% of the total cytosolic proteins [12
]. It is required to facilitate protein folding, stabilize client proteins and protect proteins from ubiquitin-dependent proteasomal degradation [13
]. Geldanamycin binds to the ATP-binding site of Hsp90 to abolish the interaction between Hsp90 and its client proteins [14
]. GA abolishes TNF-induced IKKβ activation and IκBα degradation, thus blocking NF-κB activation [15
]. GA treatment also causes proteasomal degradation of IKKβ [16
], leading to altered migration of the IKK complex [11
NleH1 and NleH2 share 84% amino acid sequence identity, but have differential abilities to regulate the NF-κB pathway [17
]. NleH1, but not NleH2, inhibits the phosphorylation of RPS3 by IKKβ without affecting the kinetics of IκBα δεγραδατιον (degradation) [17
]. However, when IKKβ is overexpressed, both NleH1 and NleH2 were shown to attenuate NF-κB activation [18
]. Though our results indicated that both NleH1 and NleH2 interact with Hsp90, because only NleH1 inhibits RPS3 nuclear translocation, we thus only focused on NleH1 for these functional studies.
IKKβ is crucial for regulating NF-κB signaling and plays an important role in tumorigenesis, as evidenced by the fact that deletion of IKKβ in a colitis-associated cancer model leads to decreased tumor incidence [19
] and that T-cell restricted constitutively active IKKβ improves tumor control [20
]. Hsp90 activity is required for IKKβ biosynthesis and activation [16
] and the formation of a Cdc37-Hsp90 complex is important for stabilizing the IKK complex [10
Here we present evidence that Hsp90 also interacts with the EHEC T3SS effector NleH1. NleH1 only restricts a subset of the IKKβ substrates, most importantly RPS3 [4
]. Hsp90 binds directly to RPS3 and this Hsp90-RPS3 interaction was previously shown to protect RPS3 from proteasome-dependent degradation [21
]. Our studies now identify an additional role for Hsp90 in regulating RPS3 nuclear translocation. We determined that GA inhibited both TNF-mediated p65 nuclear translocation and RPS3 nuclear translocation, indicating that Hsp90 is an important cofactor in regulating host inflammatory responses to bacterial virulence proteins. We also identified a direct interaction between NleH1 and IKKβ and identified Hsp90 as a cofactor in this complex. Importantly, inhibiting Hsp90 had a significant impact on wild-type EHEC adherence to Caco-2 cells, but did not impact the adherence of EHEC ΔnleH1
. Thus, there may be a functional link between Hsp90 and NleH1 that impacts EHEC virulence and warrants further investigation.
4. Materials and Methods
Cloning, Chemicals, and Antibodies
. The strains and plasmids used in this study are listed in Table 1
. All chemicals were used according to manufacturer’s recommendations and were obtained from Sigma, except for the following: Nickel-nitrilotriacetic acid (Ni-NTA) agarose beads (Qiagen, Hilden, Germany), Glutathione sepharose 4B GST-tagged protein purification resin (GE healthcare Life Sciences, Marlborough, MA, USA), Polyjet DNA In Vitro Transfection Reagent (SignaGen Laboratories, Rockville, MD, USA), TNF-α (Cell Signaling, Danvers, MA, USA). Antibodies were obtained from the following resources: anti-FLAG, anti-HA, Sigma; anti-IκBα, Cell Signaling; anti-β-tubulin, anti-β-actin, anti-His, Santa Cruz Biotechnology; anti-PARP, BD Transduction Laboratories; anti-RPS3, Proteintech Group.
Protein purification. IKKβ was cloned into pET28a, and NleH1, NleH2, and NleB1 were cloned into pET42a. Recombinant proteins were expressed in E. coli BL21(DE3) cells. Bacterial cultures were grown to an OD600 of 0.6, and isopropyl β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 0.6 mM. After 4 h of additional growth, cells were pelleted using centrifugation, and lysed in 50 mM sodium phosphate, pH 8.0, supplemented with 0.5 mg/mL lysozyme and halt proteinase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA). Lysates were incubated on ice for 30 min with occasional shaking, after which an equal volume of 50 mM sodium phosphate, pH 8.0, 1 M NaCl, 8 mM imidazole, 20% glycerol, 1% sarkosyl was added, followed by further incubation for 30 min. Lysates were sonicated, clarified by centrifugation, and the supernatants were applied to nickel-nitrilotriacetic acid beads (Qiagen) with end-to-end rotation for 2 h at 4 °C. After washing with 50 mM sodium phosphate, pH 8.0, 600 mM NaCl, 60 mM imidazole, 10% glycerol, proteins were eluted in 50 mM sodium phosphate, pH 8.0, 600 mM NaCl, 250 mM imidazole, 20% glycerol. Samples were resuspended in 2× SDS sample buffer, heated for 5 min at 95 °C, and analyzed using 10% SDS-PAGE.
Cell culture and transfection. HEK293 cells were maintained at 37 °C, 5% CO2 in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (100 U/mL). Cells were seeded in a 6-well plates 18–24 h prior to transfection. Media was replaced with 1 mL complete DMEM per well 1 h prior to transfection. DNA was transfected into cells using Polyjet DNA transfection reagent (SignaGen Laboratories). After 24 h of incubation at 37 °C, the cells were harvested.
Co-immunoprecipitation assay. Transfected HEK293 cells were washed once using pre-chilled 1× PBS. Washed cells were scraped into pre-chilled 1× PBS, pooled, centrifuged at 12,000× g for 5 min. Supernatants were disposed, and cells were lysed in 50 mM Tris-HCl, pH 7.4, 0.15 mM NaCl, 1 mM EDTA, 1% Triton X-100, supplemented with halt protease inhibitor cocktail (Thermo Fisher). Samples were incubated on ice for 30 min, with occasional shaking, and lysates were collected by centrifugation at 12,000× g for 10 min at 4 °C. Anti-FLAG M2 Affinity Gel was incubated with cell lysates for 45 min at 4 °C. The mixture was pelleted by centrifugation at 7000× g for 45 s at 4 °C, and washed 3 times with 50 mM Tris-HCl, 250 mM NaCl, pH 7.4. Samples were resuspended in 2× SDS sample buffer, heated for 5 min at 95 °C, and analyzed using 10% SDS-PAGE.
Pulldown assays. GST-tagged NleH1 and NleH2 (10 µM) were immobilized on glutathione sepharose 4B beads (GE Healthcare) in 20 mM Tris-HCl, pH 7.9, 0.1 M NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM DTT, 0.2 mM PMSF, 20% glycerol, 0.1% Nonidet P-40, supplemented with 0.33 U/µL of DNase I and RNase A. After overnight incubation at 4 °C, the beads were incubated with His-tagged purified IKK-β proteins (10 µM) for 1 h at 4 °C. The beads were then washed 3 times with 20 mM Tris-HCl, pH 7.9, 1 M NaCl, 1 mM EDTA, 1 mM DTT, 0.2 mM PMSF, 20% glycerol, 0.1% Nonidet P-40. Proteins were eluted with 10 mM reduced glutathione and analyzed using 10% SDS-PAGE.
Mass Spectrometry and Protein Identification. For mass spectrometry analysis, 500 mg of HEK293T cells was collected and lysed in 50 mM Tris-HCl, pH 7.4, 0.15 mM NaCl, 1 mM EDTA, 1% Triton X-100, supplemented with halt protease inhibitor cocktail (Thermo Fisher) for 30 min on ice, with occasional shaking. Cell lysates were collected by centrifugation at 12,000× g for 10 min at 4 °C. Washed beads were incubated with cell lysates for 45 min at 4 °C. The mixture was pelleted by centrifugation at 7000× g for 45 s at 4 °C, and washed 3 times with 50 mM Tris-HCl, 250 mM NaCl, pH 7.4. The beads were resuspended in 2× SDS sample buffer, boiled and subjected to SDS-PAGE and an aliquot used for Western blotting analysis. The gel was stained with GelCode Blue Stain Reagent (Pierce Manufacturing, Bradenton, Fl, USA) overnight and destained with ddH2O. The stained bands of the gels were cut for in-gel tryptic digestion and introduced into an LTQ-FT tandem mass spectrometer (ThermoFinnigan). Mass spectra were acquired in the positive ion mode.
RNA interference and transfection. siRNAs targeting Hsp90, as well as a negative control siRNA, were obtained from Santa Cruz Biotechnology. Transient transfection of 25 pmol siRNA into HEK293 cells was performed using Lipofectamine RNAiMAX (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions.
. Nuclear and cytosolic protein extracts were obtained as described previously [22
]. HEK293 cells were transfected with NleH1-HA and after 36 h, TNF-α was added at 50 ng/mL for 30 min to promote RPS3 nuclear translocation. Cells were harvested and resuspended in the buffer, nuclear and cytosolic protein extracts were prepared using the NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher). Data were analyzed by Western blotting for nuclear RPS3. Poly(ADP-ribose) polymerase and β-tubulin were used to normalize the protein concentrations of nuclear and cytoplasmic fractions, respectively.
EHEC adherence. Caco-2 cells (ATCC HTB37) were grown at 37 °C/5% CO2 in DMEM (Gibco™, Carlsbad, CA, USA) and treated with 1 μM GA for 20 h. Caco-2 cells were infected with WT or ΔnleH1 EHEC for 3 h at a multiplicity of infection (MOI) of 100. Non-adherent bacteria were removed from the cells by washing with PBS. Cells and adherent EHEC were scraped into 1% Triton X-100 in PBS, and serial dilutions were plated onto LB agar plates.
Statistics. Protein abundance was quantified using Li-COR Image Studio software. RPS3 and p65 nuclear abundance was analyzed statistically using either one-way analysis of variance (ANOVA) or t-tests. p values < 0.05 were considered significant.