Abstract
TNF receptor-associated factor 6 (TRAF6) is a key hub protein involved in Toll-like receptor-dependent inflammatory signaling pathway, and it recruits additional proteins to form multiprotein complexes capable of activating downstream NF-κB inflammatory signaling pathway. Ubiquitin-proteasome system (UPS) plays a crucial role in various protein degradations, such as TRAF6, leading to inhibitory effects on inflammatory response and immunologic function. However, whether ubiquitination-dependent TRAF6 degradation can be used as a novel anti-inflammatory drug target still remains to be explored. FMHM, a bioactive natural small molecule compound extracted from Chinese herbal medicine Radix Polygalae, suppressed acute inflammatory response by targeting ubiquitin protein and inducing UPS-dependent TRAF6 degradation mechanism. It was found that FMHM targeted ubiquitin protein via Lys48 site directly induced Lys48 residue-linked polyubiquitination. This promoted Lys48 residue-linked polyubiquitin chain formation on TRAF6, resulting in increased TRAF6 degradation via UPS and inactivation of downstream NF-κB inflammatory pathway. Consequently, FMHM down-regulated inflammatory mediator levels in circulation, protected multiple organs against inflammatory injury in vivo, and prolong the survival of endotoxemia mouse models. Therefore, FMHM can serve as a novel lead compound for the development of TRAF6 scavenging agent via ubiquitination-dependent mode, which represents a promising strategy for treating inflammatory diseases.
Highlights
Toll-like receptors (TLRs) in immune cells are critical for defense against invading pathogens; uncontrolled TLRs activation causes human inflammatory disorders and autoimmune diseases, such as endotoxemia, asthma, rheumatoid arthritis and multiple sclerosis[1,2,3]
Immunoblotting assay revealed that FMHM effectively inhibited some pivotal inflammatory protein expressions such as inducible nitric oxide synthase and cyclooxygenase (COX-2) (Fig. 1H)
Cell viability assay indicated that 5–20 μ M FMHM was not cytotoxic (Supplement Figure S1), excluding the possibility that the observed inflammatory inhibition effects might be resulted from FMHM-induced cytotoxicity
Summary
Toll-like receptors (TLRs) in immune cells are critical for defense against invading pathogens; uncontrolled TLRs activation causes human inflammatory disorders and autoimmune diseases, such as endotoxemia, asthma, rheumatoid arthritis and multiple sclerosis[1,2,3]. K63-linked ubiquitination of TRAF6 is essential for the activation of TLR4-dependent NF-κ B signaling through supporting an anchoring platform for corresponding downstream adaptor proteins such as TAK1 and NEMO, further leading to the phosphorylation and nuclear translocation of NF-κ B transcription factor[13]. Recently it is found that TRAF6 can be modified by K48-linked ubiquitination, which can promote TRAF6 degradation through proteasome and block the inflammatory signal transduction in TLR4 pathway[14,15,16,17]. The promotion of the assembly of K48-linked polyubiquitination chains on TRAF6 becomes a promising strategy to negatively regulate TLR4-mediated NF-κ B signaling pathway and suppress excessive inflammatory responses. Prior to the present study, it was unclear whether a natural small molecule could induce TRAF6 degradation through mediating protein post-translational modification via specific polyubiquitin chain editing. The present study explored the inflammation-regulating mechanism of FMHM by activity-based protein profiling technology (ABPP) and identified FMHM as a TRAF6 degrading agent via editing polyubiquitin chains
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