Activation of nuclear factor-kappaB (NF-kappaB), a key mediator of inducible transcription in immunity, requires binding of NF-kappaB essential modulator (NEMO) to ubiquitinated substrates. Here, we report that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO selectively binds linear (head-to-tail) ubiquitin chains. Crystal structures of the UBAN motif revealed a parallel coiled-coil dimer that formed a heterotetrameric complex with two linear diubiquitin molecules. The UBAN dimer contacted all four ubiquitin moieties, and the integrity of each binding site was required for efficient NF-kappaB activation. Binding occurred via a surface on the proximal ubiquitin moiety and the canonical Ile44 surface on the distal one, thereby providing specificity for linear chain recognition. Residues of NEMO involved in binding linear ubiquitin chains are required for NF-kappaB activation by TNF-alpha and other agonists, providing an explanation for the detrimental effect of NEMO mutations in patients suffering from X-linked ectodermal dysplasia and immunodeficiency.

N uclear factor B (NF-B) is a family of transcription factors that play essential roles in many biological phenomena, including inflammatory responses, cell survival, and innate and acquired immune responses (1). Because aberrant activation of NF-B signaling is associated with many pathological conditions, such as autoinflammatory diseases and malignancies (2, 3), signalinduced activation of NF-B has been studied extensively (4). In resting cells, inactive NF-B resides in the cytoplasm bound to its inhibitor proteins, the inhibitors of B (IBs). Stimulation by inflammatory cytokines activates the IB kinase (IKK) complex, composed of IKK1, IKK2, and NF-B essential modulator (NEMO). Following phosphorylation by activated IKK, IBs are degraded by the proteasome, leading to the release of NF-B, which then translocates to the nucleus to induce transcription of its target genes (5).The ubiquitin (Ub) conjugation system is deeply involved in the regulation of NF-B pathway (6). Recent studies showed that the linear ubiquitin chain assembly complex (LUBAC) ligase, which specifically generates linear polyubiquitin chains, is involved in NF-B activation (7,8). LUBAC is composed of three subunits: HOIP, HOIL-1L, and SHARPIN. Patients lacking HOIL-1L and mice lacking SHARPIN exhibit immunodeficiency and chronic inflammation, demonstrating the physiological significance of LUBAC-mediated linear polyubiquitination (9-12). In cells from mice lacking HOIL-1L or SHARPIN, the level of the residual LUBAC complex (consisting of the remaining two components) is reduced, and tumor necrosis factor alpha (TNF-␣)-induced NF-B activation is sharply attenuated (9-12). Although NEMO is a target of linear polyubiquitination by LUBAC, it is not yet clear how linear polyubiquitination of NEMO triggers IKK activation.In this study, using an in vitro LUBAC-mediated IKK activation assay, we found that linear diubiquitin conjugation to NEMO potently induces IKK activation. We then dissected the molecular mechanism underlying linear polyubiquitination of NEMO by LUBAC and found that the NPL4 zinc finger 1 (NZF1) domain of HOIP is responsible for recognition of a region in the coiled-coil 2 and leucine zipper (CoZi) domains of NEMO. Mutational analyses based on a cocrystal structure of HOIP NZF1 and NEMO CoZi revealed that HOIP NZF1 binds to NEMO and ubiquitin simultaneously and that both interactions are involved in linear polyubiquitination of NEMO, IKK activation, and subsequent activation of NF-B. Finally, we showed that homodimerization of IKK2 is involved in linear ubiquitin chain-mediated IKK activation. Taken together, our results suggest that recognition of linear polyubiquitins conjugated to NEMO, possibly by NEMO in another IKK complex, triggers activation of IKK2 by trans autophosphorylation. MATERIALS AND METHODS RT-PCR and plasmids.The open reading frames (ORFs) of mouse HOIP and NEMO were amplified by reverse transcription-PCR (RT-PCR) of total RNA from C57BL/6 mouse liver. Other cDNAs used in this study were described previously (8, 1...

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, one of the most common human genetic enzymopathies, is caused by over 160 different point mutations and contributes to the severity of many acute and chronic diseases associated with oxidative stress, including hemolytic anemia and bilirubin-induced neurological damage particularly in newborns. As no medications are available to treat G6PD deficiency, here we seek to identify a small molecule that corrects it. Crystallographic study and mutagenesis analysis identify the structural and functional defect of one common mutant (Canton, R459L). Using high-throughput screening, we subsequently identify AG1, a small molecule that increases the activity of the wild-type, the Canton mutant and several other common G6PD mutants. AG1 reduces oxidative stress in cells and zebrafish. Furthermore, AG1 decreases chloroquine- or diamide-induced oxidative stress in human erythrocytes. Our study suggests that a pharmacological agent, of which AG1 may be a lead, will likely alleviate the challenges associated with G6PD deficiency.