Peroxynitrite nitration of Tyr 56 in Hsp90 induces PC12 cell death through P2X7R-dependent PTEN activation
Megan Jandy,
Asra Noor,
Pascal Nelson,
Cassandra N. Dennys,
Isabella M. Karabinas,
Jeanine C. Pestoni,
Gautam D. Singh,
Lam Luc,
Rachel Devyldere,
Nathalie Perdomo,
Catherine E. Mitchell,
Levi Adams,
Marisa A. Fuse,
Francine A. Mendoza,
Carrie L. Marean-Reardon,
Ryan A. Mehl,
Alvaro G. Estevez,
Maria Clara Franco
Affiliations
Megan Jandy
Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, 32826, USA
Asra Noor
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, 97331, USA
Pascal Nelson
Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, 32826, USA
Cassandra N. Dennys
Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, 32826, USA
Isabella M. Karabinas
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, 97331, USA
Jeanine C. Pestoni
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, 97331, USA
Gautam D. Singh
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, 97331, USA
Lam Luc
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, 97331, USA
Rachel Devyldere
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, 97331, USA
Nathalie Perdomo
Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, 32826, USA
Catherine E. Mitchell
Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, 32826, USA
Levi Adams
Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, 32826, USA
Marisa A. Fuse
Rollins College, Winter Park, FL, 32789, USA
Francine A. Mendoza
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, 97331, USA
Carrie L. Marean-Reardon
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, 97331, USA
Ryan A. Mehl
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, 97331, USA
Alvaro G. Estevez
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, 97331, USA
Maria Clara Franco
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, 97331, USA; Corresponding author. Department of Biochemistry and Biophysics, Oregon State University, ALS 2011, Corvallis, OR, 97331, USA.
The diffusion-limited reaction of nitric oxide (NO) and superoxide (O2−) 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 (Hsp90NY). Peroxynitrite, Hsp90NY, 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.
