MABS451 Sigma-AldrichAnti-Linear Ubiquitin Antibody, clone LUB9

Linear ubiquitin chains generated by the linear ubiquitin chain assembly complex (LUBAC) play an important role in NF-κB activation. However, the regulation of linear ubiquitin chain generation by LUBAC is not well characterized. Here, we identified two deubiquitinating enzymes (DUBs), ovarian tumor DUB with linear linkage specificity (OTULIN/Gumby/FAM105B) and cylindromatosis (CYLD) that can cleave linear polyubiquitin chains and interact with LUBAC via the N-terminal PNGase/UBA or UBX (PUB) domain of HOIP, a catalytic subunit of LUBAC. HOIP interacts with both CYLD and OTULIN even in unstimulated cells. The interaction of CYLD and OTULIN with HOIP synergistically suppresses LUBAC-mediated linear polyubiquitination and NF-κB activation. Moreover, introduction of a HOIP mutant unable to bind either deubiquitinase into HOIP-null cells augments the activation of NF-κB by TNF-α stimulation. Thus, the interactions between these two deubiquitinases and the LUBAC ubiquitin ligase are involved in controlling the extent of TNF-α-induced NF-κB activation in cells by fine-tuning the generation of linear ubiquitin chains by LUBAC. The interaction of HOIP with OTULIN is also involved in OTULIN suppressing the canonical Wnt signaling pathway activation by LUBAC. Our observations provide molecular insights into the roles of ligase-deubiquitinase interactions in regulating molecular events resulting from linear ubiquitin conjugation.

The linear ubiquitin chain assembly complex (LUBAC) ubiquitin ligase complex, composed of HOIL-1L-interacting protein (HOIP), heme-oxidized IRP2 ubiquitin ligase-1L (HOIL-1L), and SHANK-associated RH domain protein, specifically generates linear polyubiquitin chains and is involved in NF-κB activation. Lack of SHANK-associated RH domain protein, which drastically reduces the amount of HOIP and HOIL-1L, causes chronic proliferative dermatitis (cpdm) in mice. Impaired NF-κB activation and augmented apoptosis have been implicated in the pathogenesis of cpdm in mice. In this study, we found that IFN-γ increased the amount of LUBAC by inducing HOIP and HOIL-1L mRNA transcription and enhanced the signal-induced NF-κB activation in embryonic fibroblasts, keratinocytes, and bone marrow-derived macrophages from wild-type and/or cpdm mice; however, IFN-γ failed to augment NF-κB activation in mouse embryonic fibroblasts lacking linear polyubiquitination activity of LUBAC. Moreover, s.c. injection of IFN-γ for 3 wk into the skin of cpdm mice increased the amount of HOIP, suppressed apoptosis, and ameliorated the dermatitis. Inhibition of keratinocyte apoptosis by IFN-γ injection suppressed neutrophil, macrophage, and mast cell infiltration and the amount of TNF-α in the skin of cpdm mice. Similarly, IFN-α also enhanced the amount of HOIP as well as NF-κB activation, inhibited apoptosis, and ameliorated cpdm dermatitis. These results indicate that the IFNs enhance NF-κB activation and ameliorate cpdm dermatitis by augmenting expression of HOIP and HOIL-1L and linear polyubiquitination activity of LUBAC.

DNA damage-induced activation of the transcription factor NF-κB plays an important role in the cellular response to genotoxic stress. However, uncontrolled NF-κB activation upon DNA damage may lead to deleterious consequences. Although the mechanisms mediating genotoxic NF-κB activation have been elucidated, how this signalling is terminated remains poorly understood. Here, we show that the CCCH-type zinc finger-containing protein MCPIP1 (monocyte chemotactic protein-1-induced protein-1; also known as ZC3H12A) is induced upon genotoxic treatment in an NF-κB-dependent manner. MCPIP1 upregulation reduces NEMO linear ubiquitylation, resulting in decreased activation of IKK and NF-κB. NEMO ubiquitylation is decreased through the deubiquitinase USP10, which interacts with NEMO via MCPIP1 upon genotoxic stress. USP10 association with NEMO leads to removal of NEMO-attached linear polyubiquitin chains and subsequent inhibition of the genotoxic NF-κB signalling cascade. Consistently, USP10 is required for MCPIP1-mediated inhibition of genotoxic NF-κB activation and promotion of apoptosis. Thus, by mediating USP10-dependent deubiquitination of NEMO, MCPIP1 induction serves as a negative feedback mechanism for attenuating genotoxic NF-κB activation.

The linear ubiquitin chain assembly complex (LUBAC) plays a crucial role in activating the canonical NF-κB pathway, which is important for B-cell development and function. Here, we describe a mouse model (B-HOIP(Δlinear)) in which the linear polyubiquitination activity of LUBAC is specifically ablated in B cells. Canonical NF-κB and ERK activation, mediated by the tumour necrosis factor (TNF) receptor superfamily receptors CD40 and TACI, was impaired in B cells from B-HOIP(Δlinear) mice due to defective activation of the IKK complex; however, B-cell receptor (BCR)-mediated activation of the NF-κB and ERK pathways was unaffected. B-HOIP(Δlinear) mice show impaired B1-cell development and defective antibody responses to thymus-dependent and thymus-independent II antigens. Taken together, these data suggest that LUBAC-mediated linear polyubiquitination is essential for B-cell development and activation, possibly via canonical NF-κB and ERK activation induced by the TNF receptor superfamily, but not by the BCR.