Phosphoproteomic analysis of neuronal cell death by glutamate‐induced oxidative stress

Abstract

Oxidative stress is one of the major causes of neuronal cell death in disorders such as perinatal hypoxia and ischemia. Protein phosphorylation is the most significant PTM of proteins and plays an important role in stress-induced signal transduction. Thus, the analysis of alternative protein phosphorylation states which occur during oxidative stress-induced cell death could provide valuable information regarding cell death. In this study, a reference phosphoproteome map of the mouse hippocampal cell line HT22 was constructed based on 125 spots that were identified by MALDI-TOF or LC-ESI-Q-TOF-MS analysis. In addition, proteins of HT22 cells at various stages of oxidative stress-induced cell death were separated by 2-DE and alterations in phosphoproteins were detected by Pro-Q Diamond staining. A total of 17 spots showing significant quantitative changes and seven newly appearing spots were identified after glutamate treatment. Splicing factor 2, peroxiredoxin 2, S100 calcium binding protein A11, and purine nucleoside phosphorylase were identified as up- or down-regulated proteins. CDC25A, caspase-8, and cyp51 protein appeared during oxidative stress-induced cell death. The data in this study from phosphoproteomic analysis provide a valuable resource for the understanding of HT22 cell death mechanisms mediated by oxidative stress.

Proteomic analysis of oxidative stress-induced neuronal cell death by using two-dimensional fluorescence difference gel electrophoresis

“…It is noteworthy that a similar nonreceptor-mediated glutamate oxidative toxicity has been reported in immature cortical neurons, implying that the mechanisms responsible for glutamate toxicity in HT22 cells may apply to some in vivo cases (10). Therefore, the oxidative pathway of glutamate toxicity serves as a model to study the response of neuronal cells to oxidative stress (7,8).…”

“…Trypsin in 50 mM ammonium bicarbonate (20 μg/μl) was added to each gel piece and incubated at 37˚C for 2 h. Peptides were then extracted using a mixture containing 20 μl of 0.1% v/v trifluoroacetic acid (TFA) and 70% ACN. The peptide solution was either manually or automatically desalted and concentrated using ZipTips™ (Millipore, Bedford, MA) (8,12), and spotted onto an Axima Maldi target plate. The peptide mass spectra of tryptic peptides were generated by using an Axima CFR+ matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS; Shimadzu Biotech, Japan).…”

“…The HT22 mouse hippocampal cell line that phenotypically resembles neuronal precursor cells has been used in model studies of glutamateinduced oxidative toxicity (6)(7)(8). Since this cell line lacks functional ionotropic glutamate receptors, the effect of glutamate on these cells is not due to receptor-initiated excitotoxicity (9), but is related to the glutamate/cystine antiporter, which is required for the delivery of cystine into neuronal cells.…”

Clear cell carcinoma of the ovary: Potential pathogenic mechanisms (Review)

“…The role of nucleoside phosphorylase (NP) is to cleave inosine, deoxyinosine, guanosine, and deoxyguanosine to their corresponding base and sugar 1-phosphate. Data exist suggesting the possibility of a connection between NP and glutamateinduced cell death through oxidative stress-related signaling cascade (68).…”

Comparative proteomic analysis of peripheral blood mononuclear cells from atopic dermatitis patients and healthy donors

“…As a typical proteomic analysis, two-dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS) has been used widely in research (11)(12)(13). 2-DE can be used to detect differences in protein expression levels in cell states between healthy and diseased cells, and is also a promising tool for use in identifying disease markers and candidates for therapeutic intervention (8,14).…”