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Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria
Published on: September 17, 2013
Characterization of Tollip protein upon Lipopolysaccharide challenge
1Department of Medicine, Medical Center Blvd, 3045 Hanes Bldg., Wake Forest University School of Medicine, Winston Salem, NC 27157, USA.
Abstract:
Tollip protein serves as a suppressor of innate immunity signaling with unknown mechanism. In this report, we observed that Tollip preferentially bound with phosphatidylinositol-3-phosphate (PtdIns(3)P) and phosphatidylinositol-3,4,5-phosphate (PtdIns(3,4,5)P) in vitro. Mutation of lysine 150 to glutamic acid (Tollip(KE)) within the C2 domain abolished such binding, indicating that C2 domain is critically involved. We demonstrated that overexpression of Tollip inhibited NFkB reporter gene transcription. On the contrary, overexpression of Tollip(KE) mutant has no inhibitory effect on LPS-induced NFkB reporter activity. Tollip binding with 3'-phosphorylated phosphatidylinositides implies that PI3 kinase may regulate its function. We observed that Tollip-mediated inhibition could be alleviated by wortmannin. We also showed that pretreatment with wortmannin augmented LPS-induced endogenous IL-1beta gene expression in monocytic THP-1 cells. THP-1 cells with prolonged LPS treatment develop a state of hyporesponsiveness and no longer respond to further LPS challenge in terms of IL-1beta gene expression. Here we demonstrated that Tollip protein levels were increased following LPS treatment in THP-1 cells as well as in human primary blood mononuclear cells. Increased protein stability upon LPS challenge was likely the cause for the Tollip protein increase. Upon over expression with an enhanced green fluorescent tag, Tollip localized to Golgi apparatus. Our study provides yet another mechanism for suppressing excessive TLR signaling activation mediated by Tollip.
Insights
Tollip protein suppresses innate immunity by binding to specific phosphoinositides. This interaction, dependent on its C2 domain, regulates Toll-like receptor signaling and is modulated by PI3 kinase activity.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Tollip protein is a known suppressor of innate immunity signaling.
- The precise mechanism by which Tollip exerts its function remains largely unknown.
- Understanding Tollip's mechanism is crucial for modulating immune responses.
Purpose of the Study:
- To elucidate the molecular mechanism of Tollip's function in innate immunity.
- To investigate the binding partners and regulatory pathways of Tollip.
- To explore Tollip's role in Toll-like receptor (TLR) signaling.
Main Methods:
- In vitro binding assays with phosphoinositides.
- Site-directed mutagenesis of the Tollip C2 domain.
- Reporter gene assays for NF-kB activity.
- Pharmacological inhibition using wortmannin.
- Analysis of Tollip protein levels and localization in response to LPS stimulation.
Main Results:
- Tollip preferentially binds to phosphatidylinositol-3-phosphate (PtdIns(3)P) and phosphatidylinositol-3,4,5-phosphate (PtdIns(3,4,5)P) via its C2 domain.
- Tollip inhibits NF-kB reporter gene transcription, an effect dependent on its phosphoinositide-binding ability.
- Tollip-mediated inhibition is alleviated by wortmannin, suggesting PI3 kinase involvement.
- LPS treatment increases Tollip protein levels in THP-1 cells and primary blood mononuclear cells, likely due to enhanced protein stability.
- Overexpressed Tollip localizes to the Golgi apparatus.
Conclusions:
- Tollip's interaction with 3'-phosphorylated phosphatidylinositides is critical for its inhibitory function in innate immunity signaling.
- PI3 kinase activity and Tollip protein levels, regulated by LPS, are key factors in modulating TLR signaling suppression.
- This study reveals a novel mechanism for Tollip-mediated suppression of excessive TLR signaling.
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