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Inhibition of NF-kappa B by S-nitrosylation
1Department of Medicine and Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710, USA.
Biochemistry
|May 1, 2001
Summary
This study demonstrates that S-nitrosylation directly modifies the transcription factor NF-kappaB (nuclear factor kappa B) in cells. This post-translational modification inhibits NF-kappaB DNA binding, revealing a key mechanism for redox regulation of gene transcription.
Area of Science:
- Molecular Biology
- Cellular Signaling
- Biochemistry
Background:
- Redox regulation of transcription factors is not fully understood, with uncertainty regarding direct modifications versus indirect mechanisms.
- Demonstrating in vivo redox-related modifications of transcription factors has been a significant challenge.
- The transcription factor NF-kappaB (nuclear factor kappa B) is known to be redox-sensitive, but its regulation by nitric oxide has been attributed to indirect pathways.
Purpose of the Study:
- To investigate whether cellular NF-kappaB activity is directly regulated by S-nitrosylation.
- To determine if S-nitrosylation of NF-kappaB subunits occurs in vivo and affects DNA binding.
- To elucidate the physiological role of S-nitrosylation in NF-kappaB-mediated gene transcription.
Main Methods:
- Treatment of human respiratory cells and murine macrophages with S-nitrosocysteine and cytokine-activated NOS2.
- Assessing NF-kappaB inhibition and reversal using the denitrosylating agent dithiothreitol.
- In vitro studies with recombinant p50 to examine S-nitrosylation, DNA binding, and the effect of dithiothreitol.
- Detection and modulation of S-nitrosylated p50 levels in cells treated with TNFalpha.
Main Results:
- Cellular NF-kappaB activity was inhibited by S-nitrosocysteine and NOS2, with inhibition reversed by dithiothreitol.
- Nitric oxide bioactivity did not alter TNFalpha-induced IkappaBalpha degradation or p65 nuclear translocation.
- In vitro, S-nitrosylation of recombinant p50 inhibited its DNA binding, an effect reversible by dithiothreitol.
- Increased S-nitrosylated p50 was detected in cells and modulated by TNFalpha.
Conclusions:
- S-nitrosylation directly inhibits the DNA-binding activity of the p50 subunit of NF-kappaB.
- This S-nitrosylation occurs in vivo and is modulated by cellular signaling pathways.
- S-nitrosylation of p50 represents a physiological mechanism for regulating NF-kappaB transcriptional activity.