Intracellular glutathione mediates the denitrosylation of protein nitrosothiols in the rat spinal cord

Abstract

Protein S-nitrosothiols (PrSNOs) have been implicated in the pathophysiology of neuroinflammatory and neurodegenerative disorders. Although the metabolically instability of PrSNOs is well known, there is little understanding of the factors involved in the cleavage of S-NO linkage in intact cells. To address this issue, we conducted chase experiments in spinal cord slices incubated with S-nitrosoglutathione (GSNO). The results show that removal of GSNO leads to a rapid disappearance of PrSNOs (t1/2 ~ 2h), which is greatly accelerated when glutathione (GSH) levels are raised with the permeable analogue GSH ethyl ester. Moreover, PrSNOs are stable in the presence of the GSH depletor diethyl maleate, indicating that GSH is critical for protein denitrosylation. Inhibition of GSH-dependent enzymes (glutathione S-transferase, glutathione peroxidase and glutaredoxin) and enzymes that could mediate denitrosylation (alcohol dehydrogense-III, thioredoxin and protein disulfide isomerase) do not alter the rate of PrSNO decomposition. These findings and the lack of protein glutathionylation during the chase indicate that most proteins are denitrosylated via rapid transnitrosylation with GSH. The differences in the denitrosylation rate of individual proteins suggest the existence of additional structural factors in this process. This study is relevant to our recent discovery that PrSNOs accumulate in the CNS of patients with multiple sclerosis.

Mechanisms of Altered Redox Regulation in Neurodegenerative Diseases—Focus on S-Glutathionylation

“…In the case of the protein-SNO reaction with GSH, two different outcomes are depicted, namely denitrosylation or S-glutathionylation. Both types of reactions have been reported (153,190) and further study is necessary to understand the molecular basis for why one or the other reaction is favored with particular proteins and conditions (64). Glutaredoxin and thioredoxin function to repair the oxidized sulfyhydryls and prevent irreversible modification, aggregation, and degradation.…”

Selective Vulnerability of Synaptic Signaling and Metabolism to Nitrosative Stress

“…In cultured cells, exposure to authentic NO, nitric oxide donors, or other RNS was found to inhibit GAPDH activity (75,218,266). The mechanisms of inhibition somewhat differ depending on the type of RNS used in experimental settings.…”

“…These effects are likely mediated by the nitrosative changes in microtubules and microtubuleassociated proteins, such as nitration of a-tubulin and tau (43,44,79). Furthermore, a-and b-tubulins are robustly and persistently S-nitrosylated in vitro and in vivo (20,218). However, the functional significance of S-nitrosylation of cytoskeletal proteins has not been thoroughly tested.…”

SNO-ing at the Nociceptive Synapse?

“…The latter constitutes a system together with GSH that governs the reversible S-nitrosylation of multiple SNO proteins, including G-protein receptor kinase, ␤-arrestin, and NF-B ). In vitro experiments with spinal cord slices revealed that SNO proteins are primarily denitrosylated via rapid transnitrosylation with GSH, suggesting that this system may be particularly important for SNO-mediated signaling in the nociceptive system (Romero and Bizzozero, 2009). …”