X-linked inhibitor of apoptosis protein (XIAP) is a client of heat shock protein 70 (Hsp70) and a biomarker of its inhibition

Hsp70 inhibition results in rapid degradation of XIAP

Chemical inhibitors of Hsp70 have been reported to enhance turnover of a number of proteins, including IAP-1, XIAP, Raf-1, tau, androgen receptor and others (35, 40, 46). However, it is not clear whether any of them might be relatively selective for Hsp70 compared with Hsp90. To address this question, we first examined the levels of XIAP, c-IAP1, and Raf-1 after treatment with either Hsp70 or Hsp90 inhibitors. These studies employed MDA-MB-231 breast cancer cells because growth of these cells had previously been shown to be sensitive to both Hsp70 and Hsp90 inhibitors (34). Moreover, we initially focused on XIAP and c-IAP1, rather than other putative clients, based on serendipitous observations made during recent studies of necroptosis (59). Finally, to provide greater confidence in the results, we used two structurally distinct inhibitors of each chaperone. For Hsp70, we used PES and JG-98, and we used AUY-922 and 17-DMAG as Hsp90 inhibitors (see Fig. 1A). In the first experiments, MTT assays were used to confirm that all of the inhibitors have anti-proliferative activity at the expected EC₅₀ values (Fig. 1B). These experiments allowed us to use each of the compounds at a concentration that ensured maximal activity (10 μm for JG-98, AUY-922, and 17-DMAG and 30 μm for PES). Next, we characterized the kinetics of cell death by performing MTT assays at 24, 48, and 72 h after treatment. We found that both JG-98 and PES caused relatively rapid responses, with 50% cell proliferation lost by ∼10 h and more than 80% by 24 h (Fig. 1C). In contrast, the response to AUY-922 and 17-DMAG took a longer period of time, with 72+ h required to reduce growth by 80%. The results of these kinetic experiments were important in our search for Hsp70 clients because we were most interested in those clients that are degraded prior to extensive loss of cell viability. More explicitly, we considered it likely that non-specific client degradation, triggered by downstream caspase activation and/or other proteolytic events, might be confounding at later times; whereas the bona fide clients should be direct physical interaction partners.

With these criteria in mind, we performed Western blottings of the candidate proteins at 0, 1, 3, 6, 12, and 24 h after treatment. From these experiments, we confirmed (47) that Raf-1 is a selective client of Hsp90 (Fig. 1D). Specifically, the levels of Raf-1 were significantly (>95%) decreased by 12 h after treatment with either 17-DMAG or AUY-922. In contrast, treatment with these inhibitors tended to have a less pronounced effect on cIAP-1 and XIAP levels. In contrast, treatment with either of the Hsp70 inhibitors JG-98 or PES caused a dramatic loss of cIAP-1 and XIAP at relatively early time points, although Raf-1 was largely spared (Fig. 1D). This effect was particularly strong in the first 6 h after treatment, when c-IAP1 and XIAP levels were reduced ∼75% and Raf-1 levels were only reduced ∼20%. Thus, XIAP and c-IAP1 seemed to be relatively selective clients of Hsp70 but not Hsp90.

To better understand the mechanism of IAP loss in response to Hsp70 inhibitors, we first tested whether activation of caspase activity might be involved by blotting the JG-98-treated MDA-MB-231 cell lysates for cleaved caspase-3 and poly(ADP-ribose) polymerase (PARP). Interestingly, cleaved caspase-3 and PARP only emerged 12–24 h after treatment (Fig. 1D), which is substantially after the levels of XIAP and c-IAP1 were reduced. This finding is consistent with a recent result, in which removal of the RING domain of XIAP was found to block turnover by JG-98 (59). Thus, inhibition of Hsp70 seems to initiate the normal turnover of IAPs rather than triggering loss through caspase activation.

Hsp70 and XIAP form a complex in vitro and in cells

We next wanted to determine whether Hsp70 physically interacts with the IAPs. In these studies, we focused on XIAP (Fig. 2A) as a representative member of the IAP family because of a wealth of available structural data (44). Using co-immunoprecipitations, we found that endogenous XIAP was bound to Hsp70 in MDA-MB-231 cell lysate (Fig. 2B). To understand whether this interaction might be direct, we first used a computational algorithm to predict possible Hsp70-binding sites in full-length human XIAP (18). This approach suggested that XIAP contains nine putative binding sites, all but two of which are located within the BIR2 and BIR3 domains (see Fig. 2A). To test this prediction, the region of XIAP(120–356) corresponding to the BIR2 and BIR3 domains was purified, and its binding to recombinant human Hsp70 was measured by ELISA. We found that Hsp70 bound XIAP(120–356) with a K_D = 260 ± 20 nm (Fig. 2C). Consistent with binding in the BIR2 and BIR3 domains, addition of a Smac mimetic (48) blocked interactions with Hsp70 (Fig. 2D). Also, addition of JG-98 (10 μm) weakened the apparent affinity of Hsp70 for XIAP(120–356) (see Fig. 2D). Thus, XIAP appears to be a direct client of Hsp70 in vitro and in cells, and its interaction with Hsp70 is mediated by the BIR2/3 domains.

We next wondered why Hsp90 inhibitors might have a less pronounced effect on XIAP stability. One possibility is that Hsp90 might not bind to this client. Hsp90 and Hsp70 are known to bind similar or even overlapping sites in other clients. To understand whether this might be the case for XIAP, we tested the ability of recombinant human Hsp90α to compete with Hsp70 for binding to XIAP(120–356) in the ELISA. Strikingly, Hsp90α was a relatively strong competitor, with an IC₅₀ of 227 ± 138 nm (Fig. 2E). Similarly, the highly conserved Hsp70 family member, Hsc70 (HSPA8), also competed with an IC₅₀ of 302 ± 196 nm. Thus, Hsp90 binds XIAP, although inhibitors do not seem to trigger degradation.

To further explore the interaction of XIAP with Hsp70, we created point mutations to disrupt each of the seven putative binding sites in XIAP(120–356) (Fig. 3A). Other than the possible exception of L231S, each of the mutants was folded to the same extent as wildtype, as judged by circular dichroism (CD) (Fig. 3B). In the ELISA platform, we found that Y190E, L207S, L307S, and L331S all had weaker affinity for Hsp70, with dissociation constants ranging from 2-fold to greater than 10-fold higher than the wildtype protein (Fig. 3C). To test whether this difference in affinity would translate into resistance to Hsp70 inhibitors, we expressed full-length XIAP (XIAP_WT, XIAP_Y190E, and XIAP_L207S) in HeLa cells and measured protein stability after treatment with JG-98. Consistent with the model, we found that XIAP_Y190E was difficult to overexpress, likely because of reduced Hsp70 binding, and that its levels were not sensitive to JG-98 (Fig. 3D). The other mutant, XIAP_L207S was not as dramatically affected, suggesting that some sites might be more functionally important for stability than others.

The ELISA results suggested that Hsp70 may bind to multiple sites on XIAP(120–356) because none of the individual mutations were sufficient to completely block the interaction. To examine this possibility, we incubated Hsp70 with XIAP(120–356) and analyzed the complex by size-exclusion chromatography and multiangle light scattering (SEC-MALS). Consistent with the other binding studies, we found that Hsp70 and XIAP(120–356) formed a stable, multimeric complex (Fig. 3E). Moreover, based on the apparent molecular mass of ∼240 kDa, we estimate that approximately three Hsp70s may be bound.

Interaction with XIAP involves non-canonical interactions with Hsp70

Next, we asked whether XIAP(120–356) binds to Hsp70 in the chaperone's canonical SBD. As a first test, we titrated a known SBD ligand, the NRLLLTG peptide, into the ELISA experiment. Surprisingly, we found that NRLLLTG was not able to compete with XIAP(120–356) for binding to Hsp70 (Fig. 4A), suggesting that the XIAP(120–356) was not binding the canonical cleft. A growing number of non-canonical interactions with Hsp70 have been discovered (49, 50), and these interactions often do not fit the normal profile for nucleotide dependence. In other words, the interaction is not always tighter in the ADP-bound state (20). Similarly, we found that the affinity of XIAP(120–356) for Hsp70 did not follow the canonical behavior; its affinity for ADP-bound or apo-Hsp70 was 1.5-fold worse than in the ATP-bound state Hsp70 (Fig. 4B). This result was consistent with our observation that the apparent affinity of Hsp70 for XIAP(120–356) was slightly weakened, not strengthened, by addition of JG-98 (see Fig. 2D), as this compound is known to stabilize the ADP-bound state (51). Some non-canonical interactions with Hsp70 are reported to involve contacts outside the SBD (49). To probe whether XIAP(120–356) might share this feature, we purified truncated mutants of Hsc70 that are composed of either the NBD (Hsc70_NBD) or SBD (Hsc70_SBD) alone and tested them for the ability to compete with full-length Hsp70 for binding to XIAP(120–356). Unlike what would be expected for a strictly canonical interaction, we found that both Hsc70_NBD and Hsc70_SBD competed with full-length Hsp70 for binding to XIAP(120–356) (Fig. 4C), although the Hsc70_NBD interaction seemed to be significantly weaker. Finally, we titrated NRLLLTG or XIAP(120–356) into samples of ¹⁵N-labeled Hsc70_SBD (residues 395-508, lacking the C-terminal subdomain). This NMR-based assay has previously (50) been used to measure binding of canonical clients, such as NRLLLTG, to the hydrophobic cleft, and it is based on the appearance of characteristic cross-peaks upon “rigidification” of the β-subdomain. Consistent with previous observations, we found that adding NRLLLTG to Hsc70_SBD caused the appearance of ∼ 15 new cross-peaks (Fig. 4D). Because the peptide is not labeled, these cross-peaks are likely due to “rigidification” of the SBD after binding to the canonical hydrophobic cleft. Consistent with the ELISA studies, XIAP(120–356) failed to produce these characteristic peaks, again suggesting that it is a non-canonical client. Together, these results all suggest that XIAP makes non-canonical contacts with Hsp70 and that the NBD is involved.

Stability of IAPs is responsive to inhibitors of Hsp70 but not Hsp90

Our experiments in MDA-MB-231 cells showed that Hsp70 inhibitors induce degradation of XIAP and c-IAP1. Thus, we envisioned that IAPs might be useful surrogates for Hsp70 target engagement, similar to how Raf-1 is used to monitor pharmacological inhibition of Hsp90 (11). However, we wanted to test this relationship in additional cell lines. Similar to what we found in MDA-MB-231 cells, we found that the IAPs in HeLa and MCF7 cells were selectively degraded in response to JG-98, but not 17-DMAG, at early time points (∼6 h) (Fig. 5A). Next, we wanted to test whether IAP stability was sensitive to Hsp70 inhibition in normal healthy cells. This experiment was important because clients often depend on chaperone only in a cancer cell-specific context (4). Consistent with this idea, JG-98 did not reduce levels of XIAP or c-IAP1 in IMR90 normal human lung fibroblasts (Fig. 5B). Finally, we wanted to understand whether IAPs might be surrogate biomarkers of Hsp70 engagement in vivo. Accordingly, we established MCF7 tumor xenografts in mice and treated with JG-98, using a previously reported dosing scheme (35). In this model, daily i.p. injections of JG-98 (7 mg/kg/day) begin to show a therapeutic effect on tumor volume 4–6 days after the initiation of treatment. When we isolated tumors from these treated animals, we found that the lysates had significantly lower levels of XIAP and c-IAP1 (Fig. 5C) when compared with those from mock-treated animals. Together, these results suggest that XIAP and c-IAP1 are candidate biomarkers of Hsp70 in cells and animals.

Reagents and general methods

Antibodies used are as follows: XIAP (Enzo Life Sciences ADI-AAM-050-E); c-IAP1 (Enzo Life Sciences ALX-803-335-C100); β-actin (AnaSpec AS-54591); FLAG (Sigma F1804); Hsp70 (Santa Cruz Biotechnology sc-137239 and sc-33575); Raf-1 (Santa Cruz Biotechnology sc-133); goat anti-mouse HRP (AnaSpec 28173); goat anti-rabbit HRP (AnaSpec 28177); and goat anti-rat HRP (Santa Cruz Biotechnology sc-2006). JG-98 was synthesized according to previously described methods (34), and PES, AUY-922, and 17-DMAG were purchased from Millipore, Selleckchem, and LC Laboratories, respectively. All other biological reagents were purchased from Sigma unless otherwise noted. All spectroscopic measurements were obtained with a SpectraMax M5 microplate reader (Molecular Devices).

Plasmids and site-directed mutagenesis

XIAP mutants were prepared using the QuikChange site-directed mutagenesis kit (Stratagene). The following mutants were engineered into the human XIAP(120–356) gene in the pet28a vector: L141S, Y190E, L207S, L231S, I276T, L307S, and L331S. Wildtype and mutant XIAP(120–356) constructs all contained additional C202A/C213G mutations for stability. N-terminally FLAG-tagged, full-length XIAP in pCMV6 was obtained from GeneArt (Invitrogen).

Protein expression and purification

All His-tagged Hsp70 proteins (HSPA1A, HSPA8, Hsc70_NBD(1–383), and Hsc70_SBD(395–509)) were purified as described previously (57) using batch purification with Ni-NTA resin (Novagen) and subsequent cleavage of the His tag with tobacco etch virus protease. Hsp70, Hsc70, and Hsc70_NBD were further purified using an ATP column, whereas Hsc70_SBD underwent gel-filtration chromatography on a Superdex 75 16/60 column (GE Healthcare). Hsc70 (HSPA8) domain truncations were used in Fig. 4, A and C, because we have found that they are more stable than the corresponding Hsp70 (HSPA1A) domains. WT His-tagged XIAP(120–356) and its mutants were batch-purified with Ni-NTA resin and eluted with 400 mm imidazole. DTT was added to 10 mm, and XIAP(120–356) was further purified by gel-filtration chromatography on a Superdex 75 16/60 column (GE Healthcare) in 20 mm Tris buffer containing 200 mm NaCl, 50 μm zinc acetate, and 1 mm DTT, pH 7.5. Fractions containing XIAP(120–356), as assessed by SDS-PAGE, were pooled and concentrated, and DTT was added to 10 mm before storing at −80 °C. The bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific, Inc.) was used to determined protein concentration, and protein purities were estimated at >90% by SDS-PAGE and Q-TOF LC-MS (Agilent).

Tissue culture, viability assays, and transfections

MCF-7 and HeLa cells (ATCC) were maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. MDA-MB-231 cells (ATCC) were maintained in DMEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and non-essential amino acids. Cells were used at low passage, typically less than 20. No further validation of the cell lines was performed. If indicated, cell viability was determined using the MTT assay as described previously (34). XIAP pCMV6 plasmids were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

Western blotting

Cell extracts were prepared in chilled RIPA buffer (50 mm Tris, pH 8, 150 mm NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) unless otherwise indicated. Protein concentration was determined by the BCA assay, and 20 μg of total protein was separated by SDS-PAGE on 10% Mini-PROTEAN TGX gel (Bio-Rad) and transferred to PVDF membrane (Thermo Fisher Scientific Inc). Membranes were blocked with 5% milk in TBS, 0.05% Tween for 1 h at room temperature, incubated with primary antibodies overnight at 4 °C, washed with TBS, 0.05% Tween, and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Membranes were developed using chemiluminescence (ECL Prime, GE Healthcare). Xenograft experiments were carried out as described (35, 58).

Co-immunoprecipitation

Cell extracts were prepared in chilled lysis buffer (50 mm Tris, pH 8, 150 mm NaCl, 1 mm ATP, 10 mm KCl, 5 mm Mg(OAc)₂, 1% Nonidet P-40) supplemented with protease inhibitor mixture (Roche Applied Science). The total protein concentration was adjusted to 5 mg of protein in 1 ml of cell extract. PureProteome protein G magnetic beads (Millipore) were incubated with 6 μg of the appropriate antibody or non-specific mouse IgG (Santa Cruz Biotechnology) for 30 min at room temperature with mixing, followed by antibody cross-linking with bis(sulfosuccinimidyl) suberate (Thermo Fisher Scientific, Inc.) for 1 h at room temperature with mixing. The cross-linking reaction was quenched with 1 m Tris, pH 7.5, for 1 h at room temperature with mixing. Meanwhile, equal 100-μl samples of cell lysate were pre-cleared by incubation with 50 μl of protein G beads for 1 h at room temperature with mixing. Protein complexes were immunoprecipitated by incubation of the pre-cleared lysate (1 mg of total protein per immunoprecipitation) with 50 μl of antibody–cross-linked protein G beads for 1 h at room temperature with mixing. The immunocomplexes were washed three times with 500 μl of wash buffer (PBS, pH 7.4, 0.1% Tween 20) and eluted with 0.1 m glycine, pH 2.6. Proteins were visualized by Western blotting.

ELISA

XIAP(120–356) was non-covalently immobilized in the wells of a clear, flat-bottom 96-well plate (Thermo Fisher Scientific, Inc.) by incubating 100 μl of 100 nm XIAP(120–356) in immobilization buffer (20 mm MES, pH 5.2) overnight at 37 °C. XIAP(120–356) was removed from the wells, and the wells were washed with 3× 150 μl of TBS supplemented with 0.05% Tween (TBS-T). Each wash was incubated, with gentle rocking, for 3 min at room temperature. Following washing, 30 μl of Hsp70 was added at the indicated concentrations in binding buffer (25 mm HEPES, pH 7.4, 40 mm KCl, 8 mm MgCl₂, 100 mm NaCl, 0.01% Tween), supplemented with 1 mm nucleotide and 1 mm DTT. The plates were incubated at room temperature for 24 h with gentle rocking. Solutions of Hsp70 were removed, and each well was washed as before and blocked with 100 μl of 5% milk in TBS-T for 5 min at room temperature. The plates were developed using 50 μl each of Hsp70 primary antibody (1:5000 in TBS-T) and an HRP-conjugated secondary antibody (1:5000 in TBS-T) and washed with TBS-T between each 1-h incubation at room temperature. Binding was detected using the TMB substrate kit (Cell Signaling Technology), and the absorbance was read at 450 nm. Data were analyzed using GraphPad Prism software and fit to the Langmuir binding isotherm (Y = B_maxX/[K_D + X]).

Size-exclusion chromatography and multiangle light scattering

XIAP(120–356) and Hsp70 were subjected to size-exclusion chromatography using a KW-803 silica resin column (Shodex Group) with an AKTA micro-HPLC (GE Healthcare) in 20 mm Tris buffer, pH 7.5, containing 200 mm NaCl, 5 mm MgCl₂, 10 mm KCl, 50 μm zinc acetate, 1 mm DTT. Separated samples were then analyzed for light scattering using a DAWN HELEOS II MALS detector and for protein concentration using the change in refractive index measured by a Optilab rEX. Molecular weights of species contained in the SEC peaks were calculated using ASTRA VI software (Wyatt Technology).

Circular dichroism

CD spectra of XIAP(120–356) and mutants were acquired on a J-715 spectropolarimeter (Jasco Inc.) using a 1 mm pathlength quartz cuvette, subtracting the CD signal acquired for buffer alone (10 mm sodium phosphate, pH 7.6, 100 mm NaF, 50 μm zinc acetate, 0.5 mm DTT). Data were converted to mean residue ellipticity (degrees cm⁻¹ dmol⁻¹) according to the equation Θ = Ψ/(1000 nlc), where Ψ is the CD signal in degrees; n is the number of amides; l is the path length in centimeters; and c is the concentration in decimoles/ml. Each spectrum reported is the average of six scans.

NMR spectroscopy

NMR-based binding studies were performed as described previously (50). Briefly, ¹³C-¹⁵N Hsc70 SBD (residues 395–508) was expressed in BL21 cells in M9 minimal media supplemented with ¹⁵NH₄Cl, purified, and refolded from 4 m GdHCl on a Ni-NTA column. This protein was dialyzed into 25 mm Tris, 50 mm NaCl, 1 mm EDTA, 2 mm DTT, 0.02% sodium azide, 5% D₂O, protease inhibitors, pH 7.2. Then, ¹⁵N⁻¹H TROSY-HSQC spectra with either NRLLLTG or XIAP(120–356) were recorded at 30 °C for 1 h, using a Bruker 600 MHz NMR spectrometer with cryoprobe.