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.

There is evidence that protein S-nitrosothiols (PrSNOs) accumulate in inflammatory demyelinating disorders like multiple sclerosis and experimental allergic encephalomyelitis. However, very little is known regarding the mechanism by which PrSNOs are formed in target cells. The present study compares the ability of potential intercellular mediators of nitrosative damage including S-nitrosoglutathione (GSNO), S-nitrosocysteine and N 2 O 3 to induce protein S-nitros(yl)ation in the spinal cord, a CNS region that is commonly affected in multiple sclerosis and experimental allergic encephalomyelitis. The results clearly demonstrate that while all three NO-donors cause S-nitrosation of proteins in cell-free systems, only GSNO is a viable S-nitrosating agent in rat spinal cord slices. Generation of PrSNOs with GSNO occurs by S-transnitrosa-tion as the process was not inhibited by either the NO-scavenger rutin or the N 2 O 3-scavenger azide. Contrary to other cell types, nerve cells incorporate intact GSNO and neither functional L-amino acid transporters nor cell-surface thiols are required. We also found that there is a restricted number of proteins available for S-nitrosation, even at high, non-physiological concentrations of GSNO. These proteins are highly concentrated in mitochondria and mitochondria-rich subcellu-lar compartments. This study is relevant to those CNS disorders characterized by excessive nitric oxide production.

Nitric oxide (NO) is a ubiquitous signaling molecule involved in a wide variety of cellular physiological processes. In thyroid cells, NO-synthase III-endogenously produced NO reduces TSH-stimulated thyroid-specific gene expression, suggesting a potential autocrine role of NO in modulating thyroid function. Further studies indicate that NO induces thyroid dedifferentiation, because NO donors repress TSH-stimulated iodide (I(-)) uptake. Here, we investigated the molecular mechanism underlying the NO-inhibited Na(+)/I(-) symporter (NIS)-mediated I(-) uptake in thyroid cells. We showed that NO donors reduce I(-) uptake in a concentration-dependent manner, which correlates with decreased NIS protein expression. NO-reduced I(-) uptake results from transcriptional repression of NIS gene rather than posttranslational modifications reducing functional NIS expression at the plasma membrane. We observed that NO donors repress TSH-induced NIS gene expression by reducing the transcriptional activity of the nuclear factor-κB subunit p65. NO-promoted p65 S-nitrosylation reduces p65-mediated transactivation of the NIS promoter in response to TSH stimulation. Overall, our data are consistent with the notion that NO plays a role as an inhibitory signal to counterbalance TSH-stimulated nuclear factor-κB activation, thus modulating thyroid hormone biosynthesis.

Background: Glycogenin autoglucosylation, required to prime glycogen glucopolymerization, can be produced by the monomeric and dimeric forms of the enzyme. Results: Glycogenin intramonomer glucosylation produced full autoglucopolymerization, and intrasubunit glucosylation was necessary to complete dimer autoglucosylation. Conclusion: Glycogenin dimerization is not required for full autoglucosylation. Significance: De novo glycogen biosynthesis can be sustained by monomeric glycogenin.

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