RNF149 negatively regulates LPS/TLR4 signal transduction by ubiquitination-mediated CD63 degradation

Area of Science:

• Immunology
• Molecular Biology
• Cell Signaling

Background:

• Lipopolysaccharide (LPS) and Toll-like receptor 4 (TLR4) signaling are crucial in innate immunity and inflammation.
• Tetraspanin CD63 is implicated in various cellular processes, including immune responses.
• The role of RNF149 in TLR4 signaling remains largely uncharacterized.

Purpose of the Study:

• To elucidate the functional relationship between RNF149 and CD63 in LPS/TLR4 signal transduction.
• To determine how RNF149 and CD63 modulate TLR4 pathway activation.
• To investigate the molecular mechanisms underlying RNF149-mediated regulation of CD63.

Main Methods:

• Enzyme-linked immunosorbent assay (ELISA) for TNF-α.
• Flow cytometry for TLR4 distribution.
• Real-time PCR for gene expression.
• Western blotting for protein detection.
• Immunoprecipitation and confocal microscopy for protein interactions.
• Bioinformatic analysis of transcriptome array data (GSE7547).

Main Results:

• RNF149 directly interacts with CD63 and targets it for degradation via ubiquitination at lysine 29.
• CD63 promotes LPS/TLR4 signaling, while RNF149 inhibits it.
• RNF149 and CD63 both interact with TLR4.
• CD63 interacts with Myd88 but not TIRAP, IRAK4, or TRAF6.
• CD63 knockdown does not affect TLR4 cell surface distribution.

Conclusions:

• RNF149 acts as a negative regulator of LPS/TLR4 signaling by inducing CD63 ubiquitination and degradation.
• The RNF149-CD63 axis represents a novel regulatory mechanism in TLR4-mediated inflammatory responses.