NitroDIGE analysis reveals inhibition of protein S-nitrosylation by epigallocatechin gallates in lipopolysaccharide-stimulated microglial cells.
BACKGROUND
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.


RESULTS
To globally and quantitatively analyze NO-induced protein S-nitrosylation, the sensitive gel-based proteomic method, termed NitroDIGE, was developed by combining two-dimensional differential in-gel electrophoresis (2-D DIGE) with the modified biotin switch technique (BST) using fluorescence-tagged CyDye™ thiol reactive agents to label S-nitrosothiols. The NitroDIGE method showed high specificity and sensitivity in detecting S-nitrosylated proteins (SNO-proteins). Using this approach, we identified a subset of SNO-proteins ex vivo by exposing immortalized murine BV-2 microglial cells to a physiological NO donor, or in vivo by exposing BV-2 cells to endotoxin lipopolysaccharides (LPS) to induce a proinflammatory response. Moreover, EGCG was shown to attenuate S-nitrosylation of proteins after LPS-induced activation of microglial cells primarily by modulation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated oxidative stress response.


CONCLUSIONS
These results demonstrate that NitroDIGE is an effective proteomic strategy for "top-down" quantitative analysis of protein S-nitrosylation in multi-group samples in response to nitrosative stress due to excessive generation of NO in cells. Using this approach, we have revealed the ability of EGCG to down-regulate protein S-nitrosylation in LPS-stimulated BV-2 microglial cells, consistent with its known antioxidant effects.
Zhe
Z
Qu
Fanjun
F
Meng
Hui
H
Zhou
Jilong
J
Li
Quanhui
Q
Wang
Fan
F
Wei
Jianlin
J
Cheng
C Michael
CM
Greenlief
Dennis B
DB
Lubahn
Grace Y
GY
Sun
Siqi
S
Liu
Department of Pathology & Anatomical Sciences, University of Missouri School of Medicine, Columbia, MO 65212, USA. guze@health.missouri.edu.
Zezong
Z
Gu
D000818:Animals; D002392:Catechin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002461:Cell Line, Transformed; D002853:Chromatography, Liquid; D003545:Cysteine / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D017628:Microglia / Q000187:drug effects; D018696:Neuroprotective Agents / Q000494:pharmacology*; D019001:Nitric Oxide Synthase; D054464:Peroxiredoxins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D040901:Proteomics; D026403:S-Nitrosothiols / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013482:Superoxide Dismutase / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D043222:Ubiquitin Thiolesterase / Q000378:metabolism; D000099156:Superoxide Dismutase 2
D002392:Catechin; D003545:Cysteine; D008070:Lipopolysaccharides; D018696:Neuroprotective Agents; D019001:Nitric Oxide Synthase; D054464:Peroxiredoxins; D026403:S-Nitrosothiols; D013482:Superoxide Dismutase; D043222:Ubiquitin Thiolesterase; D000099156:Superoxide Dismutase 2; C469268:Usp14 protein, mouse; C045651:epigallocatechin gallate; C489899:Prdx1 protein, mouse