Identification of tyrosine-nitrated proteins in HT22 hippocampal cells during glutamate-induced oxidative stress

Abstract

Objectives: Nitration of tyrosine residues in protein is a post-translational modification, which occurs under oxidative stress, and is associated with several neurodegenerative diseases. To understand the role of nitrated proteins in oxidative stress-induced cell death, we identified nitrated proteins and checked correlation of their nitration in glutamate-induced HT22 cell death.

Materials and methods: Nitrated proteins were detected by western blotting using an anti-nitrotyrosine antibody, extracted from matching reference 2-dimensional electrophoresis gels, and identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Results: Glutamate treatment induced apoptosis in HT22 cells, while reactive oxygen species (ROS) inhibitor or neuronal nitric oxide synthase (nNOS) inhibitor blocked glutamate-induced HT22 cell death. Nitration levels of 13 proteins were increased after glutamate stimulation; six of them were involved in regulation of energy production and two were related to apoptosis. The other nitrated proteins were associated with calcium signal modulation, ER dysfunction, or were of unknown function.

Conclusions: The 13 tyrosine-nitrated proteins were detected in these glutamate-treated HT22 cells. Results demonstrated that cell death, ROS accumulation and nNOS expression were related to nitration of protein tyrosine in the glutamate-stimulated cells.

Figure 1

Effects of glutamate treatment on HT22 cell viability. Cells were grown on 96‐well plates (1 × 10⁴ cells per well) for 24 h, pre‐treated with N‐acetyl cysteine (NAC, 50 μm) for 30 min and then treated with 5 mm glutamate. Cell viability at each time point was measured using the MTT assay (a). Morphology of glutamate‐stimulated cells, as visualized by light microscopy at 200× magnification (b). Cells were pre‐treated with N‐acetyl cysteine (50 μm) for 30 min and then treated with 5 mm glutamate for 12 h. DNA was extracted and DNA fragments were analysed by 2% agarose gel electrophoresis (c).

Figure 2

nNOS expression was increased in glutamate‐stimulated HT22 cell death. Cells were subjected to time‐dependent treatment with 5 mm glutamate. NOS isoforms were determined by western blot analysis using anti‐iNOS, anti‐eNOS, or anti‐nNOS antibody (a). Cells were pre‐treated with nNOS inhibitor (7‐nitroindazole, 100 μm) for 30 min and then treated with 5 mm glutamate. At each time point, cell viability was measured by MTT assay. * and ** represent statistically significant differences relative to untreated cells (*P < 0.05; **P < 0.01). Data are mean ± SD of four independent experiments (b). Cells were pre‐treated with nNOS inhibitor (100 μm) for 30 min and then treated with 5 mm glutamate for 12 h. Morphology of glutamate‐treated cells, as visualized by light microscopy at 200× magnification (c).

Figure 3

Tyrosine‐nitrated proteins were identified by 2‐DE gels and MALDI‐TOF MS in glutamate‐stimulated HT22 cells. Proteomics analyses were performed by treating cells with 5 mm glutamate for 6 h and applying cell lysates (500 μg) to first dimension of pH 3–10 non‐linear IPG strips (18 cm) and second dimension of 12% SDS–PAGE gels, which were visualized by Coomassie blue staining. Representative 2‐DE protein profiles of normal (a) and 5 mm glutamate‐stimulated cells (b). Cell lysates were run on to 2‐DE gels, and transferred to nitrocellulose membranes. Tyrosine‐nitrated protein levels of normal (c) and 5 mm glutamate‐stimulated cells (d) was determined using western blot analysis with anti‐nitrotyrosine monoclonal antibody. Circled spots indicate proteins that were more highly expressed in glutamate‐stimulated cells than in normal cells.

Figure 4

Pre‐treatment of inhibitors of ROS (NAC) and nNOS (7‐NI) decrease tyrosine nitration levels of proteins identified. Cells were pre‐treated with NAC (50 μm) or 7‐nitroindazole (100 μm) for 30 min, followed by treatment with 5 mm glutamate for 6 h. Total cell lysates were immunoprecipitated with antibodies against calmodulin‐4 (a), HSP90 (b), ATP synthase α‐chain (c), glutamate dehydrogenase (d), tubulin (e) and acyl‐CoA dehydrogenase (f), then analysed by western blot analysis with anti‐nitrotyrosine monoclonal antibody to check levels of protein nitration. Nitrated proteins were quantified by densitometric analysis using image analysis software, ImageQuant v5.2 (Amersham Biosciences). * and ** represent statistically significant differences relative to untreated cells (*P < 0.05; **P < 0.01). Data are given as mean ± SD of three independent experiments.