Activation of GluR6-containing Kainate Receptors Induces Ubiquitin-dependent Bcl-2 Degradation via Denitrosylation in the Rat Hippocampus after Kainate Treatment

Abstract

We previously showed that Bcl-2 (B-cell lymphoma 2) is down-regulated in a kainate (KA)-induced rat epileptic seizure model. The underlying mechanism had remained largely unknown, but we here report for the first time that denitrosylation and ubiquitination are involved. Our results show that the S-nitrosylation levels of Bcl-2 are down-regulated after KA injection and that the GluR6 (glutamate receptor 6) antagonist NS102 can inhibit the denitrosylation of Bcl-2. Moreover, the ubiquitin-dependent degradation of Bcl-2 was found to be promoted after KA treatment, which could be suppressed by the proteasome inhibitor MG132 and the NO donors, sodium nitroprusside and S-nitrosoglutathione. In addition, experiments based on siRNA transfections were performed in the human SH-SY5Y neuroblastoma cell line to verify that the stability of Bcl-2 is causal to neuronal survival. At the same time, it was found that the exogenous NO donor GSNO could protect neurons when Bcl-2 is targeted. Subsequently, these mechanisms were morphologically validated by immunohistochemistry, cresyl violet staining, and in situ TUNEL staining to analyze the expression of Bcl-2 as well as the survival of CA1 and CA3/DG pyramidal neurons. NS102, GSNO, sodium nitroprusside, and MG132 contribute to the survival of CA1 and CA3/DG pyramidal neurons by attenuating Bcl-2 denitrosylation. Taken together, our data reveal that Bcl-2 ubiquitin-dependent degradation is induced by Bcl-2 denitrosylation during neuronal apoptosis after KA treatment.

Distinction of thioredoxin transnitrosylation and denitrosylation target proteins by the ICAT quantitative approach

“…Dynamic regulation of protein nitrosylation has been shown in SH-SY5Y and other neuronal cells. For example, increased denitrosylation of anti-apoptotic Bcl-2 was previously reported in this neuroblastoma cell line upon kainic acid treatment—a model for studying epileptic seizure [24]. In this study, we demonstrate the comparable specificity of biotin-HPDP and ICAT reagents to label SNO-Cys sites, and the advantage of ICAT to accurately quantify the extent of S-nitrosylation within specific peptides.…”

Cuprous oxide nanoparticles inhibit the growth and metastasis of melanoma by targeting mitochondria

“…To evaluate Z-VAD-FMK effects, cells in the log growth phase were seeded onto a 96-well culture plate at 1000 cells per well and incubated at 37 °C in a CO 2 incubator for 24 h until the cells adhered to the plate. Before serial dilutions of CONPs were added, the Z-VAD-FMK was added in the medium at the final concentration of 20  μ M. 49 After 24 h, and 48 h, cell viability was measured using the MTT assay as we did before.…”

N-Methyl-D-Aspartate Receptor-Dependent Denitrosylation of Neuronal Nitric Oxide Synthase Increase the Enzyme Activity

“…The negative control used was a UUCUCCGAACGUGUCACGUTT sense strand. SH-SY5Y cells were transfected with siRNAs and harvested as described previously [25].…”