Peroxynitrite nitration of Tyr 56 in Hsp90 induces PC12 cell death through P2X7R-dependent PTEN activation

Abstract

The diffusion-limited reaction of nitric oxide (NO) and superoxide (O 2 − ) produces peroxynitrite (ONOO − ), a biological oxidant that has been implicated in a number of pathological conditions, including neurodegenerative disorders. We previously reported that incubation of PC12 cells with peroxynitrite triggers apoptosis by simultaneously inhibiting the PI3K/Akt survival pathway, and activating the p38 and JNK MAP kinase pathways. We also reported that peroxynitrite-treated Heat Shock Protein 90 (Hsp90) stimulates PC12 cell death. Here, we show that nitrated Hsp90 mediates peroxynitrite-induced apoptosis by regulating specific signaling pathways triggered by activation of the purine receptor P2X7 (P2X7R) and downstream activation of PTEN. Intracellular delivery of peroxynitrite-treated Hsp90 was sufficient to stimulate PC12 cell death. In contrast, intracellular delivery of peroxynitrite-treated Hsp90 in which the five tyrosine (Tyr) residues susceptible to nitration were replaced by nitration-resistant phenylalanine had no effect on PC12 cell survival. Further, only nitration of Hsp90 at Tyr 56 was necessary and sufficient to stimulate PC12 cell apoptosis, and incubation of PC12 cells with peroxynitrite resulted in Hsp90 nitration at Tyr 56. Inhibition of P2X7R or downstream inhibition of PTEN prevented PC12 cell death stimulated by both incubation with peroxynitrite and nitrated Hsp90 (Hsp90 NY ). Peroxynitrite, Hsp90 NY , and P2X7R activation all increased p38 and JNK MAP kinases activity, while inhibiting the Akt survival pathway. These results suggest that, in undifferentiated PC12 cells, peroxynitrite triggers apoptosis via nitration of Hsp90 at Tyr 56, which in turn activates P2X7R and PTEN. These results contrast with observations in motor neurons where the nitration of either Tyr 33 or Tyr 56 in Hsp90 stimulates apoptosis, suggesting that the targets of peroxynitrite may be different in different cell types. However, uncovering the pathways through which peroxynitrite triggers cell death in neurodegenerative conditions will provide new potential targets for therapeutic treatment.

Interplay of carbon dioxide and peroxide metabolism in mammalian cells

“…Both tyrosine nitration yields and regioselectivity are affected by the presence of CO 2 ( 151 , 152 , 153 ). As biologically relevant examples, protein tyrosine nitration in HSP90 (heat shock protein 90) ( 154 , 155 ) and NGF (nerve growth factor) ( 156 ) triggers cell death pathways, causes PP2A (protein serine phosphatase) inactivation and enhanced cellular phosphorylation ( 157 , 158 ), affects the ability of the cytokine CCL2 (C–C motif chemokine ligand 2) to exert its normal chemoattractant activity for immune cells ( 159 ), and disrupts insulin receptor substrate−1 (IRS−1)–dependent signaling ( 160 , 161 ). In these contexts, information on how CO 2 levels modulate tyrosine nitration and signaling events is basically lacking.…”

Protein Tyrosine Amination: Detection, Imaging, and Chemoproteomic Profiling with Synthetic Probes

“…Furthermore, SALc-FL was employed to detect PTN in brain samples from a mouse model of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis . The oxidative injury in human multiple sclerosis lesions has been extensively documented, and PTN has been reported to cause toxic gain-of-function in neuronal cells . After sodium dithionite reduction, SALc-FL successfully detected 3NT-containing proteins in EAE mouse brain tissue (Figure d).…”

“…29 The oxidative injury in human multiple sclerosis lesions has been extensively documented, 30 and PTN has been reported to cause toxic gain-of-function in neuronal cells. 31 After sodium dithionite reduction, SALc-FL successfully detected 3NTcontaining proteins in EAE mouse brain tissue (Figure 3d). These results demonstrate that SALc-FL can be widely applied to pure proteins, cell lysates, and brain tissues for the detection of PTA and PTN, with good sample compatibility and high efficiency.…”

Unveiling the human nitroproteome: Protein tyrosine nitration in cell signaling and cancer

“…Site-directed mutagenesis of Y33 and Y56 on HSP90 to phenylalanine protects PC12 cells from peroxynitrite-induced death ( 171 ). Mechanistically, nitration of Y33 decreases mitochondrial activity, whereas nitration at Y56 induces cell death in a P2X7R-PTEN dependent manner ( 84 , 172 ). Increased levels of nitrated Y56 on HSP90 have been shown in both mouse models of amyotrophic lateral sclerosis and spinal cord trauma as well in motor neurons from amyotrophic lateral sclerosis patients, suggesting that therapies targeting nitrated HSP90 merit investigation.…”