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In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity
Published on: January 29, 2018
Modulation of inducible nitric oxide synthase expression by sumoylation
Candan A Akar1, Douglas L Feinstein
1Department of Anesthesiology, University of Illinois, Chicago, IL 60612, USA. candan@uic.edu
Journal of Neuroinflammation
|March 28, 2009
Summary
Inflammation reduces SUMOylation in astrocytes, impacting nitric oxide synthase type 2 (NOS2) expression. Noradrenaline (NA) counteracts this, suggesting SUMOylation and C/EBPbeta modification as therapeutic targets for neurological diseases.
Area of Science:
- Neuroscience
- Molecular Biology
- Immunology
Background:
- Inflammatory induction of nitric oxide synthase type 2 (NOS2) in astrocytes is transcriptionally inhibited by noradrenaline (NA).
- The precise transcription factors modulated by NA and SUMOylation in this process remain unclear.
- SUMOylation, a post-translational modification, influences transcription factor activity, including C/EBPbeta, crucial for NOS2 expression.
Purpose of the Study:
- To investigate the impact of inflammation and NA on SUMOylation-related genes (SRGs) in astrocytes.
- To determine if SUMO-1 regulates NOS2 expression through interaction with C/EBPbeta.
Main Methods:
- Primary astrocytes were treated with lipopolysaccharide (LPS) to induce inflammation and NOS2 expression.
- mRNA levels of SUMO-1, Ubc9, and SENP1 were quantified using QPCR.
- Functional roles of SUMOylation were assessed via reporter gene assays, chromatin immunoprecipitation (ChIP), and co-immunoprecipitation with FRET analysis.
Main Results:
- LPS decreased SRG mRNA levels in astrocytes; NA attenuated these reductions and increased SUMO-1.
- Over-expression of SRGs modulated NOS2 promoter activity, with SUMO-1 uniquely affecting NFkappaB reporter activity.
- ChIP and FRET assays confirmed interactions between SUMO-1, C/EBPbeta, and the NOS2 promoter, modulated by LPS and NA.
Conclusions:
- SUMOylation significantly regulates NOS2 expression in astrocytes.
- Modification of the transcription factor C/EBPbeta by SUMO-1 is a likely mechanism.
- Targeting the SUMOylation pathway presents a potential therapeutic strategy for neurological conditions characterized by inflammatory NOS2 expression.
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