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. 2013 Mar 19;110(12):E1102-11.
doi: 10.1073/pnas.1215177110. Epub 2013 Mar 4.

Nitration of Hsp90 induces cell death

Maria Clara Franco  1 , Yaozu Ye, Christian A Refakis, Jessica L Feldman, Audrey L Stokes, Manuela Basso, Raquel M Melero Fernández de Mera, Nicklaus A Sparrow, Noel Y Calingasan, Mahmoud Kiaei, Timothy W Rhoads, Thong C Ma, Martin Grumet, Stephen Barnes, M Flint Beal, Joseph S Beckman, Ryan Mehl, Alvaro G Estévez
Affiliations

Nitration of Hsp90 induces cell death

Maria Clara Franco et al. Proc Natl Acad Sci U S A. .

Abstract

Oxidative stress is a widely recognized cause of cell death associated with neurodegeneration, inflammation, and aging. Tyrosine nitration in these conditions has been reported extensively, but whether tyrosine nitration is a marker or plays a role in the cell-death processes was unknown. Here, we show that nitration of a single tyrosine residue on a small proportion of 90-kDa heat-shock protein (Hsp90), is sufficient to induce motor neuron death by the P2X7 receptor-dependent activation of the Fas pathway. Nitrotyrosine at position 33 or 56 stimulates a toxic gain of function that turns Hsp90 into a toxic protein. Using an antibody that recognizes the nitrated Hsp90, we found immunoreactivity in motor neurons of patients with amyotrophic lateral sclerosis, in an animal model of amyotrophic lateral sclerosis, and after experimental spinal cord injury. Our findings reveal that cell death can be triggered by nitration of a single protein and highlight nitrated Hsp90 as a potential target for the development of effective therapies for a large number of pathologies.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Peroxynitrite-treated Hsp90 induces cell death. (A) Actin, tubulin, Hsp70, and Hsp90 (1 mg/mL) were treated with 0.5 mM peroxynitrite (ONOO−), decomposed peroxynitrite (reverse order addition, ROA), or 0.5 mM H2O2 and were delivered into PC12 cells and motor neurons using the permeating agent Chariot. PC12 cell viability was determined 24 h later using fluorescein diacetate/propidium iodide, and motor neuron survival was established by counting neurons with neurites longer than four soma diameters after 24 h in culture. (B) Uric acid (0.5 mM) was added to Hsp90 5 min before or after the addition of peroxynitrite, and the treated protein was added to PC12 cells or motor neurons in the presence of Chariot. Columns represent the mean ± SD of 4–10 experiments performed in quadruplicate. Statistical analyses were performed by ANOVA followed by Bonferroni multiple comparison test. *P < 0.05 versus Chariot, **P < 0.05 versus ONOO−. (C) Representative images of motor neurons 24 h after the intracellular delivery of either Hsp90 or peroxynitrite-treated Hsp90 (Hsp90+ONOO−) and in the presence of Chariot alone (control). The number of motor neurons per well was counted (A and B) as described in Materials and Methods. (D) Representative images of motor neurons 24 h after the intracellular delivery of either Hsp90 or peroxynitrite-treated Hsp90 (Hsp90+ONOO−) and in the presence of Chariot alone (control). The motor neurons were stained with calcein-AM, and the images were captured with a Runner HD (Trophos). The number of motor neurons per well in each condition was quantified using Tina software, as described in Materials and Methods. The results were similar to the quantitation showed in A.
Fig. 2.
Fig. 2.
Only a small percentage of total Hsp90 is endogenously nitrated. (A) The proportion of nitrated Hsp90 with respect to total Hsp90 in PC12 cells and motor neurons was determined by quantitative dot blot. For the standard curves, peroxynitrite-treated Hsp90 (NO2Hsp90, green) or untreated Hsp90 (red) purified from rat liver was loaded at the indicated amounts (upper and lower sections of the dot blot). Samples were collected from PC12 cells and motor neurons upon treatment as described in D and were loaded on the dot blot (middle section). Infrared detection allowed the merging of the two signals (yellow) (LiCor Biosciences). (B and C) Calibration curves were blotted for Hsp90 (B) and NO2Hsp90 (C) infrared signals. The graphs show the standard curves for Hsp90 and NO2Hsp90 from four independent experiments (R2 > 0.92 for all calibration curves). The intensity of the signal (expressed as arbitrary fluorescence units, AFU) was quantified as the integrated intensity of fluorescence for each dot in the dot blot using the Odyssey software. (D) The percentage of NO2Hsp90 versus total cytosolic Hsp90 was determined in trophic factor-deprived motor neurons (TFD), peroxynitrite-treated PC12 cells (ONOO−), and motor neurons and PC12 cells 2 h after the intracellular delivery of nitrated Hsp90 (ID) by interpolating the infrared signal of the samples on the corresponding standard curves. Columns represent the mean ± SD (n = 4, in triplicate).
Fig. 3.
Fig. 3.
Tyrosine nitration is necessary and sufficient for the conversion of Hsp90 to a toxic protein. (A) The number of nitrated tyrosine residues on Hsp90 was assayed by quantitative dot blot. Nitrated BSA (NO2BSA) was used as the standard for nitrotyrosine, and Hsp90 purified from rat liver was the standard for Hsp90. NO2BSA and Hsp90 were loaded at the indicated amounts. Purified Hsp90 was treated with peroxynitrite and loaded at different amounts in the linear range of detection (middle of the dot blot, Hsp90+ONOO−). The dot blot was probed with antibodies against Hsp90 (in red) and nitrotyrosine (in green). (B and C) The standard curve regressions for nitrotyrosine (B) and Hsp90 (C) were performed in duplicate and were repeated three times (R2 > 0.91 for all calibration curves). The molar ratio of nitrotyrosine to Hsp90 was 5.3 ± 0.5 nitrated residues per Hsp90 molecule. (D) Table showing the positions of the nitrated tyrosine residues on peroxynitrite-treated Hsp90 as determined by mass spectrometry analysis. (E) Model of the crystal structure of human Hsp90β [based on the yeast Hsp82 structure PDB 2CG9 (24)] showing the location of the tyrosine residues prone to nitration (red) including the two tyrosine residues located in the amino terminal domain (bright red). CD, carboxy-terminal domain; MD, middle domain; NTD, amino-terminal domain. (F) Treatment with peroxynitrite inhibits the intrinsic ATPase activity of Hsp90. Geldanamycin (5 µM), a specific inhibitor of Hsp90 ATPase activity, was used to verify Hsp90 ATP hydrolysis. Columns show the mean ± SD (n = 5). ***P < 0.05 versus ROA. (G) Treatment with peroxynitrite reduces 50% of Hsp90 activity to prevent heat-induced aggregation of citrate synthase (CS) at 43 °C.
Fig. 4.
Fig. 4.
Nitration of a single tyrosine residue of Hsp90 is sufficient to induce cell death. (A) The replacement of the tyrosine residues prone to nitration on Hsp90 (WT) by phenylalanine (5xPhe) prevented peroxynitrite-treated Hsp90 toxicity. (B) The presence of nitrotyrosine (3NT) in the recombinant proteins carrying a single nitrated residue was assayed by Western blot probed with antibodies against myc-tag (green) and nitrotyrosine (red). The merged image is shown in yellow. (C) Assay for the toxicity of Hsp90 with nitrotyrosine at position 33, 56, 276, 484, or 596 with (yellow bars) or without (blue bars) peroxynitrite treatment. Nitrotyrosine at positions 33 or 56 was sufficient to induce motor neuron death. (D and E) The relevance of tyrosine nitration was confirmed by directed mutagenesis of the relevant residues. Peroxynitrite-treated Hsp90 remained toxic with tyrosine only at position 33 or 56 and the other four residues replaced by phenylalanine [4xPhe(Y33) and 4xPhe(Y56)] (D), whereas replacement of residues 33 and 56 of Hsp90β by phenylalanine (Y33F + Y56F) prevented toxicity induced by peroxynitrite treatment (E). *P < 0.05 versus WT, **P < 0.05 versus WT + ONOO− by ANOVA followed by Bonferroni multiple comparison test.
Fig. 5.
Fig. 5.
Nitrated Hsp90 induces cell death through the activation of the P2X7 receptor that leads to exposure of FasL on the cellular membrane and the activation of the Fas pathway. (A) Incubation with the FasL decoy Fas:FC (1 μg/mL) for 24 h completely protected motor neurons from nitrated Hsp90-induced cell death. (B) Motor neurons were incubated for 24 h with inhibitors of caspase 8 (IEDT-fmk; 10 μM), 9 (LEHD-fmk; 20 μM), and 3 (DEVD-fmk; 20 μM) after the intracellular delivery of peroxynitrite-treated Hsp90. *P < 0.05 versus WT, **P < 0.05 versus WT + ONOO− by ANOVA followed by Bonferroni multiple comparison test. (C) Motor neurons show mobilization of FasL to the plasma membrane upon treatment with peroxynitrite-treated Hsp90. The motor neurons were incubated for 16 h after the intracellular delivery of unmodified (Hsp90) or peroxynitrite-treated Hsp90 (Hsp90 + ONOO−). The cells then were stained for FasL (green). The cell nuclei are stained in blue. (Upper) The white arrows in indicate the cells that are shown magnified in the lower panels. (Lower) The white arrows indicate the cellular location of FasL signal. See also Fig. S3. (D) The motor neurons were cultured in the presence of NEM (2 μM) or the intracellular calcium chelator BAPTA-AM (5 μM) for 24 h after the intracellular delivery of peroxynitrite-treated Hsp90. The inhibition of the last steps of exocytosis by NEM and the chelation of intracellular calcium by BAPTA-AM completely protected motor neurons from peroxynitrite-treated Hsp90-induced cell death. (E and F) Both the inhibition of the purinergic receptor P2X7 by BBG (10 μM) and KN-62 (1 μM) (E) and the receptor knockdown by lentiviral particles expressing P2X7 shRNA (F) prevented motor neuron death induced by peroxynitrite-treated Hsp90. Motor neurons were transduced with lentiviral particles expressing P2X7 or control shRNA for 84 h before the intracellular delivery of nitrated Hsp90. *P < 0.05 versus Chariot, **P < 0.05 versus WT + ONOO− by ANOVA followed by Bonferroni multiple comparison test. (G) Nitrated Hsp90 coimmunoprecipitated with the P2X7 receptor. Homogenates from PC12 were incubated for 1 h with Hsp90β-myc (WT) or with Hsp90β-myc nitrated at position 56 (3NT56), and the recombinant proteins were immunoprecipitated with an anti-myc antibody before SDS/PAGE. The membranes then were blotted for P2X7 (green) and the myc-tag (red). Brain homogenate was used as a positive control for P2X7.
Fig. 6.
Fig. 6.
The toxic form of nitrated Hsp90 is present in vivo in neuropathological conditions. (A) Hsp90, Hsp90 nitrated at tyrosine-56 (nitrated Hsp90), and nitrotyrosine immunoreactivity in adjacent sections (7 µm thick) of spinal cord from a human ALS patient. Black arrows show the immunoreactivity for Hsp90, nitrated Hsp90, and nitrotyrosine in motor neurons. The star indicates a blood vessel. (B) Intense immunoreactivity for nitrated Hsp90 is present in the spinal cord of sporadic ALS patient but not in a control patient. The lower panels at higher magnification show a motor neuron stained for nitrated Hsp90 in the ALS patient. (C) Intense nitrated Hsp90 immunoreactivity was found in the spinal cord of fully symptomatic ALS mutant G93A mice (110 d old) but not in age-matched control mice. Remaining large neurons in the anterior horn of G93A spinal cord sections were densely stained with the antibody recognizing nitrated Hsp90, compared with the control spinal cord sections. (D) Nitrated Hsp90 is present in the spinal cord after spinal cord injury. (Left) Immunoreactivity for nitrotyrosine and nitrated Hsp90 is detected in the spinal cord as early as 6 h after spinal cord contusion injury in rat. (Right) Nitrated Hsp90 is still present at the sections indicated in the figure 24 h after the spinal cord injury. MAP2 was used as a neuronal marker. The cell nuclei are stained in blue. As indicated by the arrows, the top section is rostral to the injury, the middle section corresponds to the place of contusion, and the bottom section is caudal from the injury site. Nitrated Hsp90 is present in all sections, is localized mostly in neuronal somas (upper right corners of nitrated Hsp90 panels), and is absent in uninjured control animals (Bottom Right).
Fig. 7.
Fig. 7.
Model for the induction of cell death by nitrated Hsp90 in motor neurons. Many types of stress, including trophic factor deprivation, exposure to nitric oxide when the cells also are expressing mutant SOD, and activation of the DAXX-mediated Fas pathway, result in motor neurons producing peroxynitrite before undergoing apoptosis. Nitration of one of two tyrosine residues on Hsp90 is sufficient to activate a toxic gain of function involving stimulation of the P2 P2X7 receptor. The influx of calcium through the receptor in turn mobilizes FasL to the plasma membrane and activates the FADD-mediated Fas pathway leading to cell death by apoptosis.
Fig. P1.
Fig. P1.
Induction of motor neuron death by nitrated Hsp90. Many types of stress, including deprivation of growth factors and exposure to nitric oxide in cells that express mutant SOD, result in the endogenous production of ONOO−. Nitration of either of two tyrosine residues on the prosurvival protein Hsp90 is sufficient to activate a gain of function that makes the protein toxic. The activation of the P2X7 receptor by nitrated Hsp90 results in an influx of calcium that mobilizes FasL to the plasma membrane and activates the FADD-mediated Fas pathway. These events culminate in cell death by apoptosis.
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