Mechanism Study On Substrate-specific Recognition Of IKKβ Mediated By USP16 In Regulating Autoimmune Inflammation

The classic NF-κB pathway plays a crucial role in various immune responses and inflammatory diseases,its essential functions in innate immune responses mediated by macrophages and neutrophils are well known.When stimulated by LPS,TNF-α,or IL-1β,IKKβ,the key kinase in classic NF-κB pathway,selectively recognizes and activates its downstream substrates,including p105,p65,and IκBα,to promote the nuclear translocation of p65 and p50 and the transcription of NF-κB targeting genes,thereby regulating the expression of pro-inflammatory cytokines and leading to tissue inflammation.Although it has been reported that NF-κB essential modifier(NEMO)selectively recruits and activates IκBα as an adaptor but has no effect on the phosphorylation of p105,the underlying mechanism that determines the selective substrate recognition and activation of IKKβ remains unclear.Additionally,researches revealed that abnormal expression of IKKβ or p105 in macrophages is closely related to the onset of inflammatory bowel disease(IBD).Therefore,we speculate that the selective recognition and activation of p105 by IKKβ plays an important role in the onset of IBD.In this study,we found that IKKβ ubiquitination on lysine-238 was substantially increased during inflammation.Using mass spectrometry,we identified a deubiquitinase,ubiquitin carboxyl-terminal hydrolase 16(USP16),that specifically bond IKKβ and IKKα but not NEMO.Our recent study demonstrated that USP16 specifically removes the K29-linked polyubiquitin chains of calcineurin A to regulate the recruitment and activation of its substrate NFAT in activated T cells,thereby affecting the maintenance of peripheral T cells as well as the development of autoimmune symptoms.In this study,we further elucidated the function of USP16 in non-proliferative immune cells.Using the truncations of USP16 and IKKβ,we demonstrated in vitro that USP16 competed with NEMO for binding to IKKβ.Subsequent co-immunoprecipitation results confirmed that,in macrophages,USP16 functioned as an essential regulator of the IKKβ ubiquitination that selectively controlled p105 phosphorylation without affecting p65 or IκBα phosphorylation.Both q PCR and ELISA showed that the deficiency of USP16 significantly inhibited the function of macrophages to secrete pro-inflammatory cytokines and chemokines.Furthermore,public datasets and biopsy samples from IBD patients also revealed high level expression of USP16 in colonic macrophages of patients with IBD,and myeloidconditional USP16-knockout mice exhibited reduced severity of colitis and colitisassociated colorectal cancer.In summary,our study revealed that USP16 mediated-IKKβ ubiquitination contributed to the selective recognition of IKKβ substrate p105 and promoted NF-κB signaling activation,which subsequently induced the development of colitis-associated colorectal cancer.Our data establish USP16 and USP16-mediated IKKβdeubiquitination as a novel regulatory mechanism of NF-κB signaling and intestinal tumorigenesis.We also provide a new theoretical basis for IBD pathogenesis and targeted precision intervention therapy.