Nuclear factor-kappaB (NF-kappaB) is a key transcription factor in inflammatory, anti-apoptotic and immune processes. The ubiquitin pathway is crucial in regulating the NF-kappaB pathway. We have found that the LUBAC ligase complex, composed of the two RING finger proteins HOIL-1L and HOIP, conjugates a head-to-tail-linked linear polyubiquitin chain to substrates. Here, we demonstrate that LUBAC activates the canonical NF-kappaB pathway by binding to NEMO (NF-kappaB essential modulator, also called IKKgamma) and conjugates linear polyubiquitin chains onto specific Lys residues in the CC2-LZ domain of NEMO in a Ubc13-independent manner. Moreover, in HOIL-1 knockout mice and cells derived from these mice, NF-kappaB signalling induced by pro-inflammatory cytokines such as TNF-alpha and IL-1beta was suppressed, resulting in enhanced TNF-alpha-induced apoptosis in hepatocytes of HOIL-1 knockout mice. These results indicate that LUBAC is involved in the physiological regulation of the canonical NF-kappaB activation pathway through linear polyubiquitylation of NEMO.
The ubiquitin system plays important roles in the regulation of numerous cellular processes by conjugating ubiquitin to target proteins. In most cases, conjugation of polyubiquitin to target proteins regulates their function. In the polyubiquitin chains reported to date, ubiquitin monomers are linked via isopeptide bonds between an internal Lys and a C‐terminal Gly. Here, we report that a protein complex consisting of two RING finger proteins, HOIL‐1L and HOIP, exhibits ubiquitin polymerization activity by recognizing ubiquitin moieties of proteins. The polyubiquitin chain generated by the complex is not formed by Lys linkages, but by linkages between the C‐ and N‐termini of ubiquitin, indicating that the ligase complex possesses a unique feature to assemble a novel head‐to‐tail linear polyubiquitin chain. Moreover, the complex regulates the stability of Ub‐GFP (a GFP fusion protein with an N‐terminal ubiquitin). The linear polyubiquitin chain generated post‐translationally may function as a new modulator of proteins.
LUBAC (linear ubiquitin chain assembly complex) activates the canonical NF-jB pathway through linear polyubiquitination of NEMO (NF-jB essential modulator, also known as IKKc) and RIP1. However, the regulatory mechanism of LUBAC-mediated NF-jB activation remains elusive. Here, we show that A20 suppresses LUBAC-mediated NF-jB activation by binding linear polyubiquitin via the C-terminal seventh zinc finger (ZF7), whereas CYLD suppresses it through deubiquitinase (DUB) activity. We determined the crystal structures of A20 ZF7 in complex with linear diubiquitin at 1.70-1.98 Å resolutions. The crystal structures revealed that A20 ZF7 simultaneously recognizes the Met1-linked proximal and distal ubiquitins, and that genetic mutations associated with B cell lymphomas map to the ubiquitin-binding sites. Our functional analysis indicated that the binding of A20 ZF7 to linear polyubiquitin contributes to the recruitment of A20 into a TNF receptor (TNFR) signalling complex containing LUBAC and IjB kinase (IKK), which results in NF-jB suppression. These findings provide new insight into the regulation of immune and inflammatory responses.
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