Abstract
This study aims to investigate the role of RNF149 and tetraspanin CD63 in lipopolysaccharide/Toll-like receptor 4 (LPS/TLR4) signal transduction. TNF-α was assessed using enzyme-linked immunosorbent assay. The distribution of TLR4 was examined through flow cytometry after CD63 knockdown. Real-time polymerase chain reaction was used to analyze the expression of the target genes RNF149 and CD63 under different conditions. Western blotting was employed to detect gene expression, while immunoprecipitation and confocal microscopy were used to evaluate protein interactions. Transcriptome array data from stimulated monocytes (GSE7547) was obtained from GEO and subjected to bioinformatic analysis. It is suggested that CD63 may serve as a substrate of RNF149, with RNF149 capable of directly interacting with CD63. RNF149 degrades CD63 through covalent modification of CD63 at lysine 29 of the ubiquitin monomer, leading to the formation of a multiubiquitin chain. Both RNF149 and CD63 interact with TLR4, with CD63 promoting LPS/TLR4 signaling and RNF149 inhibits it. CD63 does not impact the distribution of TLR4 on the cell surface and does not directly interact with TIRAP, IRAK4, or TRAF6, but does interact with Myd88.RNF149 plays a negative regulatory role in LPS/TLR4 signal transduction by mediating ubiquitination-induced CD63 degradation.