Many primary alkyl N-nitroso compounds, such as CH3N(H)NO, tend to be unstable with respect to hydrolysis to the alcohol. Those derived from secondary amines (e.g., (CH3)2NNO derived from dimethylamine) are more robust. It is these N-nitrosamines that are carcinogens in rodents.
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Nitric oxide (NO) is a signaling molecule regulating numerous cellular functions in development and disease. In the brain, neuronal injury or neuroinflammation can lead to microglial activation, which induces NO production. NO can react with critical cysteine thiols of target proteins forming S-nitroso-proteins. This modification, known as S-nitrosylation, is an evolutionarily conserved redox-based post-translational modification (PTM) of specific proteins analogous to phosphorylation. In this study, we describe a protocol for analyzing S-nitrosylation of proteins using a gel-based proteomic approach and use it to investigate the modes of action of a botanical compound found in green tea, epigallocatechin-3-gallate (EGCG), on protein S-nitrosylation after microglial activation.
A 100 mM stock solution of a physiological NO donor S-nitrosocysteine (SNOC) was freshly prepared. For in vitro S-nitrosylation, BV-2 cell lysates were treated with various amounts of SNOC (10, 20, 40, 80, or 200 μM) for 30 minutes at room temperature. For ex vivo S-nitrosylation, BV-2 cells were exposed to 20 μM SNOC in FBS-free DMEM and incubated at 37C for 30 minutes. For in vivo S-nitrosylation, BV-2 cells were starved for 4 hours after replenishment with FBS-free DMEM (without phenol red). The cells were then exposed to 100 ng/mL LPS for 20 hours to induce NO production. To examine the action of the green tea active component, 10 μM EGCG was added to the medium 1 hour prior to LPS exposure.
In this study, we developed a NitroDIGE method for the determination of redox-based protein S-nitrosylation. The NitroDIGE method uses the same blocking and reduction steps as the previously established BST, but the specific thiol linker Biotin-HPDP is replaced with the irreversible fluorescence-based thiol reactive reagents, maleimide-linked dyes (Cy3 and Cy5) [15], which label the nascent thiols reduced from S-nitrosocysteines by ascorbate (Figure 1A). More specifically, free thiols were blocked by methylthiolation with MMTS, and then the excessive un-labeled MMTS was removed by acetone precipitation. Nitrosothiols on SNO-proteins were selectively reduced with ascorbate and then reacted with the fluorescence (Cy3 or Cy5)-tagged thiol linkers, forming stable fluorescence-tagged complexes.
We then asked what underlying molecular and cellular functions are associated with the mode of action of EGCG in LPS-induced microglial activation. IPA annotation with the identified proteins responding to EGCG revealed that EGCG was significantly involved in free radical scavenging (9 proteins), PTM (15 proteins), protein folding (7 proteins), nucleic acid metabolism (17 proteins), and small molecule biochemistry (34 proteins) (see details in Table 2). Thus, these findings suggest that EGCG exhibited multi-modal action by alleviating NO production and further protein S-nitrosylation under microglial activation. 2ff7e9595c
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