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Various chimeric receptors have been developed and used for biological experiments. In the present study, we constructed three types of chimeric receptor activator of nuclear factor-kappa B (RANK) with the glutathione S-transferase (GST) protein in the extracellular domain, and stimulated them using newly synthesized chemical trimerizers with three glutathiones. Although this stimulation did not activate these proteins, we unexpectedly found that the chimera named RANK-GST-SC, in which GST replaced a major part of the RANK extracellular domain, activated nuclear factor-kappa B (NF-κB) signaling approximately sixfold more strongly than wild-type RANK without the ligand. The dimerization of extracellular GST is considered to function as a switch outside the cell, and signal transduction then occurs. GST has been widely employed as a tag for protein purification; GST-fusion protein can be conveniently captured by glutathione-conjugated beads and easily purified from impurity. The present study is a pioneering example of the novel utility of GST and provides information for the development of new chemical biology systems., (© 2021 John Wiley & Sons A/S.)

NF-κB was first identified in 1986 as a B cell-specific transcription factor inducing immunoglobulin κ light chain expression. Subsequent studies revealed that NF-κB plays important roles in development, organogenesis, immunity, inflammation, and neurological functions by spatiotemporally regulating cell proliferation, differentiation, and apoptosis in several cell types. Furthermore, studies on the signal pathways that activate NF-κB led to the discovery of TRAF family proteins with E3 ubiquitin ligase activity, which function downstream of the receptor. This discovery led to the proposal of an entirely new signaling mechanism concept, wherein K63-ubiquitin chains act as a scaffold for the signaling complex to activate downstream kinases. This concept has revolutionized ubiquitin studies by revealing the importance of the nonproteolytic functions of ubiquitin not only in NF-κB signaling but also in a variety of other biological systems. TRAF6 is the most diverged among the TRAF family proteins, and our studies uncovered its notable physiological and pathological functions.

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is crucial for the development of adult T-cell leukemia (ATL), a highly malignant CD4+ T cell neoplasm. Among the multiple aberrant Tax-induced effects on cellular processes, persistent activation of transcription factor NF-κB, which is activated only transiently upon physiological stimulation, is essential for leukemogenesis. We and others have shown that Tax induces activation of the IκB kinase (IKK) complex, which is a critical step in NF-κB activation, by generating Lys63-linked polyubiquitin chains. However, the molecular mechanism underlying Tax-induced IKK activation is controversial and not fully understood. Here, we demonstrate that Tax recruits linear (Met1-linked) ubiquitin chain assembly complex (LUBAC) to the IKK complex and that Tax fails to induce IKK activation in cells that lack LUBAC activity. Mass spectrometric analyses revealed that both Lys63-linked and Met1-linked polyubiquitin chains are associated with the IKK complex. Furthermore, treatment of the IKK-associated polyubiquitin chains with Met1-linked-chain-specific deubiquitinase (OTULIN) resulted in the reduction of high molecular weight polyubiquitin chains and the generation of short Lys63-linked ubiquitin chains, indicating that Tax can induce the generation of Lys63- and Met1-linked hybrid polyubiquitin chains. We also demonstrate that Tax induces formation of the active macromolecular IKK complex and that the blocking of Tax-induced polyubiquitin chain synthesis inhibited formation of the macromolecular complex. Taken together, these results lead us to propose a novel model in which the hybrid-chain-dependent oligomerization of the IKK complex triggered by Tax leads to trans-autophosphorylation-mediated IKK activation., Competing Interests: The authors have declared that no competing interests exist.

Nuclear factor-κB (NF-κB) is an inducible transcription factor activated by a variety of cytokines, and promotes the transcription of genes involved in cancer, inflammation, autoimmune disease, and viral infection, among others. Because of its involvement in numerous disease processes, considerable research has focused on NF-κB as a potential drug target. We previously reported that cupric ion (Cu(2+)) blocks NF-κB activation. However, Cu(2+) is unsuitable for drug applications. The copper complex of an artificial peptide HPH-Pep (HPH-Pep-Cu(2+)) was a promising alternative, but it did not easily cross the cell membrane. We report the development of a NF-κB inhibiting Cu(2+) complex with improved cell-penetrating activity arising from the coupling of a Tat peptide to HPH-Pep-Cu(2+).

Lactoferrin (LF) has been implicated in innate immunity. Here we reveal the signal transduction pathway responsible for human LF (hLF)-triggered nuclear factor-kappaB (NF-kappaB) activation. Endotoxin-depleted hLF induces NF-kappaB activation at physiologically relevant concentrations in the human monocytic leukemia cell line, THP-1, and in mouse embryonic fibroblasts (MEFs). In MEFs, in which both tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF5 are deficient, hLF causes NF-kappaB activation at a level comparable to that seen in wild-type MEFs, whereas TRAF6-deficient MEFs show significantly impaired NF-kappaB activation in response to hLF. TRAF6 is known to be indispensable in leading to NF-kappaB activation in myeloid differentiating factor 88 (MyD88)-dependent signaling pathways, while the role of TRAF6 in the MyD88-independent signaling pathway has not been clarified extensively. When we examined the hLF-dependent NF-kappaB activation in MyD88-deficient MEFs, delayed, but remarkable, NF-kappaB activation occurred as a result of the treatment of cells with hLF, indicating that both MyD88-dependent and MyD88-independent pathways are involved. Indeed, hLF fails to activate NF-kappaB in MEFs lacking Toll-like receptor 4 (TLR4), a unique TLR group member that triggers both MyD88-depependent and MyD88-independent signalings. Importantly, the carbohydrate chains from hLF are shown to be responsible for TLR4 activation. Furthermore, we show that lipopolysaccharide-induced cytokine and chemokine production is attenuated by intact hLF but not by the carbohydrate chains from hLF. Thus, we present a novel model concerning the biological function of hLF: hLF induces moderate activation of TLR4-mediated innate immunity through its carbohydrate chains; however, hLF suppresses endotoxemia by interfering with lipopolysaccharide-dependent TLR4 activation, probably through its polypeptide moiety.

The cytokine interleukin-1 (IL-1) mediates immune and inflammatory responses by activating the transcription factor nuclear factor kappaB (NF-kappaB). Although transforming growth factor-beta-activated kinase 1 (TAK1) and mitogen-activated protein kinase (MAPK) kinase kinase 3 (MEKK3) are both crucial for IL-1-dependent activation of NF-kappaB, their potential functional and physical interactions remain unclear. Here, we showed that TAK1-mediated activation of NF-kappaB required the transient formation of a signaling complex that included tumor necrosis factor receptor-associated factor 6 (TRAF6), MEKK3, and TAK1. Site-specific, lysine 63-linked polyubiquitination of TAK1 at lysine 209, likely catalyzed by TRAF6 and Ubc13, was required for the formation of this complex. After TAK1-mediated activation of NF-kappaB, TRAF6 subsequently activated NF-kappaB through MEKK3 independently of TAK1, thereby establishing continuous activation of NF-kappaB, which was required for the production of sufficient cytokines. Therefore, we propose that the cooperative activation of NF-kappaB by two mechanistically and temporally distinct MEKK3-dependent pathways that diverge at TRAF6 critically contributes to immune and inflammatory systems.

Background: In response to viral infection, the innate immune system recognizes viral nucleic acids and then induces production of proinflammatory cytokines and type I interferons (IFNs). Toll-like receptor 7 (TLR7) and TLR9 detect viral RNA and DNA, respectively, in endosomal compartments, leading to the activation of nuclear factor kappaB (NF-kappaB) and IFN regulatory factors (IRFs) in plasmacytoid dendritic cells. During such TLR signaling, TNF receptor-associated factor 6 (TRAF6) is essential for the activation of NF-kappaB and the production of type I IFN. In contrast, RIG-like helicases (RLHs), cytosolic RNA sensors, are indispensable for antiviral responses in conventional dendritic cells, macrophages, and fibroblasts. However, the contribution of TRAF6 to the detection of cytosolic viral nucleic acids has been controversial, and the involvement of TRAF6 in IRF activation has not been adequately addressed., Principal Findings: Here we first show that TRAF6 plays a critical role in RLH signaling. The absence of TRAF6 resulted in enhanced viral replication and a significant reduction in the production of IL-6 and type I IFNs after infection with RNA virus. Activation of NF-kappaB and IRF7, but not that of IRF3, was significantly impaired during RLH signaling in the absence of TRAF6. TGFbeta-activated kinase 1 (TAK1) and MEKK3, whose activation by TRAF6 during TLR signaling is involved in NF-kappaB activation, were not essential for RLH-mediated NF-kappaB activation. We also demonstrate that TRAF6-deficiency impaired cytosolic DNA-induced antiviral responses, and this impairment was due to defective activation of NF-kappaB and IRF7., Conclusions/significance: Thus, TRAF6 mediates antiviral responses triggered by cytosolic viral DNA and RNA in a way that differs from that associated with TLR signaling. Given its essential role in signaling by various receptors involved in the acquired immune system, TRAF6 represents a key molecule in innate and antigen-specific immune responses against viral infection.

Human T-cell leukemia virus type 1 (HTLV-1) Tax-induced activation of nuclear factor-kappaB (NFkappaB) is thought to play a critical role in T-cell transformation and onset of adult T-cell leukemia. However, the molecular mechanism of the Tax-induced NFkappaB activation remains unknown. One of the mitogen-activated protein kinase kinase kinses (MAP3Ks) members, TAK1, plays a critical role in cytokine-induced activation of NFkappaB, which involves lysine 63-linked (K63) polyubiquitination of NEMO, a noncatalytic subunit of the IkappaB kinase complex. Here we show that Tax induces K63 polyubiquitination of NEMO. However, TAK1 is dispensable for Tax-induced NFkappaB activation, and deubiquitination of the K63 polyubiquitin chain failed to block Tax-induced NFkappaB activation. In addition, silencing of other MAP3Ks, including MEKK1, MEKK3, NIK, and TPL-2, did not affect Tax-induced NFkappaB activation. These results strongly suggest that unlike cytokine signaling, Tax-induced NFkappaB activation does not involve K63 polyubiquitination-mediated MAP3K activation.

Nuclear factor-kappaBeta (NF-kappaB) binds specifically to NF-kappaB-binding sites (kappaB sites, 5'-GGGRNNYYCC-3'; R, purine; Y, pyrimidine; N, any nucleotide) present in enhancer regions of various genes. Binding of various cytokines, growth factors and pathogen-associated molecular patterns to specific receptors activates NF-kappaB and expression of genes that play critical roles in inflammation, innate and acquired immunity, bone remodeling and generation of skin appendices. Activation of NF-kappaB is also involved in cancer development and progression. NF-kappaB is activated in cells that become malignant tumors and in cells that are recruited to and constitute the tumor microenvironment. In the latter scenario, the TLR-TRAF6-NF-kB pathways seem to play major roles, and NF-kappaB activation results in production of cytokines, which in turn induce NF-kappaB activation in premalignant cells, leading to expression of genes involved abnormal growth and malignancy. Furthermore, NF-kappaB activation is involved in bone metastasis. Osteoclasts, whose generation requires the RANK-TRAF6-NF-kappaB pathways, release various growth factors stored in bone, which results in creation of microenvironment suitable for proliferation and colonization of cancer cells. Therefore, NF-kappaB and molecules involved its activation, such as TRAF6, are attractive targets for therapeutic strategies against cancer.