Abstract
BackgroundToll-like receptors (TLRs) play a pivotal role in the defense against invading pathogens by detecting pathogen-associated molecular patterns (PAMPs). TLR4 recognizes lipopolysaccharides (LPS) in the cell walls of Gram-negative bacteria, resulting in the induction and secretion of proinflammatory cytokines such as TNF-α and IL-6. The WW domain containing E3 ubiquitin protein ligase 1 (WWP1) regulates a variety of cellular biological processes. Here, we investigated whether WWP1 acts as an E3 ubiquitin ligase in TLR-mediated inflammation.Methodology/ResultsKnocking down WWP1 enhanced the TNF-α and IL-6 production induced by LPS, and over-expression of WWP1 inhibited the TNF-α and IL-6 production induced by LPS, but not by TNF-α. WWP1 also inhibited the IκB-α, NF-κB, and MAPK activation stimulated by LPS. Additionally, WWP1 could degrade TRAF6, but not IRAK1, in the proteasome pathway, and knocking down WWP1 reduced the LPS-induced K48-linked, but not K63-linked, polyubiquitination of endogenous TRAF6.Conclusions/SignificanceWe identified WWP1 as an important negative regulator of TLR4-mediated TNF-α and IL-6 production. We also showed that WWP1 functions as an E3 ligase when cells are stimulated with LPS by binding to TRAF6 and promoting K48-linked polyubiquitination. This results in the proteasomal degradation of TRAF6.
Highlights
Toll-like receptors (TLRs) play a pivotal role in defense against invading pathogens through their detection of pathogen-associated molecular patterns (PAMPs) [1,2,3]
WWP1 was weakly associated with TNF receptor-associated factor 6 (TRAF6) in untreated RAW264.7 cells, and this level of association was significantly higher at 30 minute and 60 minute time points post-LPS treatment (Figure 4C), and with the time going on, this interaction ability was weaken especially in 12 h (S4).These results suggest that LPS promotes the endogenous association of WWP1 with TRAF6, but not IRAK1
The specific responses initiated by individual TLRs depends on the recruitment of a single or specific combination of TIR-domain-containing adaptor protein(s) (e.g., myeloid differentiation factor 88 (MyD88), TRIAP, TIR domain-containing adaptor-inducing IFN-b (TRIF), or TRAM) [1,2]
Summary
Toll-like receptors (TLRs) play a pivotal role in defense against invading pathogens through their detection of pathogen-associated molecular patterns (PAMPs) [1,2,3]. TLR4 signaling consists of two distinct pathways: the myeloid differentiation factor 88 (MyD88)-dependent pathway and the TIR domain-containing adaptor-inducing IFN-b (TRIF)dependent pathway [7].The recognition of LPS by TLR4 recruits adaptors, including MyD88, IL-1 receptor-associated kinases (IRAK), and TNF receptor-associated factor 6 (TRAF6) [8], to the infection site which triggers the activation of a number of intracellular signaling cascades. These signaling cascades include the NF-kB and mitogen-activated protein (MAP) kinase (ERK, JNK and P38) pathways [9]. We investigated whether WWP1 acts as an E3 ubiquitin ligase in TLR-mediated inflammation
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