SUMMARY RIG-I detects invading viral RNA and activates the transcription factors NF-κB and IRF3 through the mitochondrial protein MAVS. Here we show that RNA bearing 5′-triphosphate strongly activates the RIG-I–IRF3 signaling cascade in a reconstituted system composed of RIG-I, mitochondria and cytosol. Activation of RIG-I requires not only RNA, but also polyubiquitin chains linked through lysine-63 (K63) of ubiquitin. RIG-I binds specifically to K63 polyubiquitin chains through its tandem CARD domains in a manner that depends on RNA and ATP. Mutations in the CARD domains that abrogate ubiquitin binding also impair RIG-I activation. Remarkably, unanchored K63 ubiquitin chains, which are not conjugated to any target protein, potently activate RIG-I. These ubiquitin chains function as an endogenous ligand of RIG-I in human cells. Our results delineate the mechanism of RIG-I activation, identify CARD domains as a new ubiquitin sensor, and demonstrate that unanchored K63 polyubiquitin chains are signaling molecules in antiviral innate immunity.
TRAF6 is a ubiquitin ligase essential for the activation of NF-κB and MAP kinases in multiple signaling pathways including those emanating from the interleukin-1 and Toll-like receptors (IL-1R/TLR)1-3. TRAF6 functions together with a ubiquitin-conjugating enzyme complex consisting of Ubc13 and Uev1A to catalyze Lys-63 (K63)-linked polyubiquitination, which activates the TAK1 kinase complex4,5. TAK1 in turn phosphorylates and activates IκB kinase (IKK), leading to activation of NF-κB. Although several proteins are known to be polyubiquitinated in the IL-1R/TLR pathways, it is not clear whether ubiquitination of any of these proteins is important for TAK1 or IKK activation. Herein, we reconstituted TAK1 activation in vitro using purified proteins and found that free K63 polyubiquitin chains, which are not conjugated to any target protein, directly activated TAK1 through binding to the ubiquitin receptor TAB2. This binding leads to autophosphorylation and activation of TAK1. We also found that unanchored polyubiquitin chains synthesized by TRAF6 and Ubc5 activated the IKK complex. Disassembly of the polyubiquitin chains by deubiquitination enzymes prevented TAK1 and IKK activation. These results indicate that unanchored polyubiquitin chains directly activate TAK1 and IKK, suggesting a novel mechanism of protein kinase regulation.
Interferon regulatory factor 4 (IRF4) and IRF8 regulate B, T, macrophage, and dendritic cell differentiation. They are recruited to cis-regulatory Ets-IRF composite elements by PU.1 or Spi-B. How these IRFs target genes in most T cells is enigmatic given the absence of specific Ets partners. Chromatin immunoprecipitation sequencing in T helper 17 (TH17) cells reveals that IRF4 targets sequences enriched for activating protein 1 (AP-1)–IRF composite elements (AICEs) that are co-bound by BATF, an AP-1 factor required for TH17, B, and dendritic cell differentiation. IRF4 and BATF bind cooperatively to structurally divergent AICEs to promote gene activation and TH17 differentiation. The AICE motif directs assembly of IRF4 or IRF8 with BATF heterodimers and is also used in TH2, B, and dendritic cells. This genomic regulatory element and cognate factors appear to have evolved to integrate diverse immunomodulatory signals.
Leptin is a hormone produced by the adipose tissue that acts in the brain, stimulating white fat breakdown. We find that the lipolytic effect of leptin is mediated through the action of sympathetic nerve fibers that innervate the adipose tissue. Using intravital two-photon microscopy, we observe that sympathetic nerve fibers establish neuro-adipose junctions, directly "enveloping" adipocytes. Local optogenetic stimulation of sympathetic inputs induces a local lipolytic response and depletion of white adipose mass. Conversely, genetic ablation of sympathetic inputs onto fat pads blocks leptin-stimulated phosphorylation of hormone-sensitive lipase and consequent lipolysis, as do knockouts of dopamine β-hydroxylase, an enzyme required for catecholamine synthesis. Thus, neuro-adipose junctions are necessary and sufficient for the induction of lipolysis in white adipose tissue and are an efferent effector of leptin action. Direct activation of sympathetic inputs to adipose tissues may represent an alternative approach to induce fat loss, circumventing central leptin resistance. PAPERCLIP.
Lysine-63 (K63)-linked polyubiquitination has emerged as a mechanism regulating diverse cellular functions, including activation of the protein kinase IKK in the NF-κB pathways. However, genetic evidence for a key role of K63 polyubiquitination in IKK activation is lacking. Here we devise a tetracycline-inducible RNAi strategy to replace endogenous ubiquitin with a K63R mutant in a human cell line. We demonstrate that K63 of ubiquitin and the catalytic activity of Ubc13, an E2 that catalyzes K63 polyubiquitination, are required for IKK activation by IL-1β, but surprisingly, not by TNFα. We further show that IKK activation by TNFα requires Ubc5, which functions with the E3 cIAP1 to catalyze polyubiquitination of RIP1 not restricted to K63 of ubiquitin. These results indicate that distinct ubiquitin-dependent mechanisms are employed for IKK activation by different pathways. The ubiquitin replacement methodology described here provides a means to investigate the function of polyubiquitin topology in various cellular processes.
Interleukin 22 (IL-22), which is produced by cells of the T(H)17 subset of helper T cells and other leukocytes, not only enhances proinflammatory innate defense mechanisms in epithelial cells but also provides crucial protection to tissues from damage caused by inflammation and infection. In T(H)17 cells, transforming growth factor-β (TGF-β) regulates IL-22 and IL-17 differently. IL-6 alone induces T cells to produce only IL-22, whereas the combination of IL-6 and high concentrations of TGF-β results in the production of IL-17 but not IL-22 by T cells. Here we identify the transcription factor c-Maf, which is induced by TGF-β, as a downstream repressor of Il22. We found that c-Maf bound to the Il22 promoter and was both necessary and sufficient for the TGF-β-dependent suppression of IL-22 production in T(H)17 cells.
SUMMARY The mitochondrial antiviral signaling protein (MAVS; also known as IPS-1, VISA and CARDIF) is essential for innate immune response against RNA viruses. MAVS transduces signals from the cytosolic RIG-I-like receptors, which bind to viral RNAs, but how MAVS activates downstream transcription factors such as IRF3 to induce type-I interferons is not well understood. We have established a cell-free system in which mitochondria derived from virus-infected cells activates IRF3 in the cytosol. Fractionation of the cytosol led to the identification of Ubc5 as a ubiquitin-conjugating enzyme (E2) required for IRF3 activation. Using an inducible RNAi strategy, we demonstrate that catalytically active Ubc5 is required for IRF3 activation by viral infection. The activation of IRF3 also requires two ubiquitin-binding domains of NEMO. Furthermore, we show that replacement of endogenous ubiquitin with its K63R mutant abolishes viral activation of IRF3, demonstrating that K63 polyubiquitination plays a key role in IRF3 activation.
Efferent signals from the central nervous system represent a key layer of regulation of white adipose tissue (WAT). However, the mechanism by which efferent neural signals control WAT metabolism remains to be better understood. Here, we exploit the volume fluorescence-imaging technique to visualize the neural arborizations in mouse inguinal WAT at single-fiber resolution. The imaging reveals a dense network of sympathetic arborizations that had been previously undetected by conventional methods, with sympathetic fibers being in close apposition to > 90% of adipocytes. We demonstrate that these sympathetic fibers originate from the celiac ganglia, which are activated by cold challenge. Sympathetic-specific deletion of TrkA receptor or pharmacologic ablation by 6-hydroxydopamine abolishes these intra-adipose arborizations and, as a result, cold-induced beiging of inguinal WAT. Furthermore, we find that local sympathetic arborizations function through beta-adrenergic receptors in this beiging process. These findings uncover an essential link connecting efferent neural signals with metabolism of individual adipocytes.
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