TNF-RII and c-IAP1 mediate ubiquitination and degradation of TRAF2

Tumour necrosis factor-alpha (TNF-alpha) is a proinflammatory mediator that exerts its biological functions by binding two TNF receptors (TNF-RI and TNF-RII), which initiate biological responses by interacting with adaptor and signalling proteins. Among the signalling components that associate with TNF receptors are members of the TNF-R-associated factor (TRAF) family. TRAF2 is required for TNF-alpha-mediated activation of c-Jun N-terminal kinase (JNK), contributes to activation of NF-kappaB, and mediates anti-apoptotic signals,. TNF-RI and TNF-RII signalling complexes also contain the anti-apoptotic ('inhibitor of apoptosis') molecules c-IAP1 and c-IAP2 (refs 5, 6), which also have RING domain-dependent ubiquitin protein ligase (E3) activity. The function of IAPs in TNF-R signalling is unknown. Here we show that binding of TNF-alpha to TNF-RII induces ubiquitination and proteasomal degradation of TRAF2. Although c-IAP1 bound TRAF2 and TRAF1 in vitro, it ubiquitinated only TRAF2. Expression of wild-type c-IAP1, but not an E3-defective mutant, resulted in TRAF2 ubiquitination and degradation. Moreover, E3-defective c-IAP1 prevented TNF-alpha-induced TRAF2 degradation and inhibited apoptosis. These findings identify a physiologic role for c-IAP1 and define a mechanism by which TNF-RII-regulated ubiquitin protein ligase activity can potentiate TNF-induced apoptosis.     

Distinct molecular mechanism for initiating TRAF6 signalling

Tumour-necrosis factor (TNF) receptor-associated factor 6 (TRAF6) is the only TRAF family member that participates in signal transduction of both the TNF receptor (TNFR) superfamily and the interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) superfamily; it is important for adaptive immunity, innate immunity and bone homeostasis. Here we report crystal structures of TRAF6, alone and in complex with TRAF6-binding peptides from CD40 and TRANCE-R (also known as RANK), members of the TNFR superfamily, to gain insight into the mechanism by which TRAF6 mediates several signalling cascades. A 40 degrees difference in the directions of the bound peptides in TRAF6 and TRAF2 shows that there are marked structural differences between receptor recognition by TRAF6 and other TRAFs. The structural determinant of the petide TRAF6 interaction reveals a Pro-X-Glu-X-X-(aromatic/acidic residue) TRAF6-binding motif, which is present not only in CD40 and TRANCE-R but also in the three IRAK adapter kinases for IL-1R/TLR signalling. Cell-permeable peptides with the TRAF6-binding motif inhibit TRAF6 signalling, which indicates their potential as therapeutic modulators. Our studies identify a universal mechanism by which TRAF6 regulates several signalling cascades in adaptive immunity, innate immunity and bone homeostasis.     

TAK1 is a ubiquitin-dependent kinase of MKK and IKK.

TRAF6 is a signal transducer that activates IkappaB kinase (IKK) and Jun amino-terminal kinase (JNK) in response to pro-inflammatory mediators such as interleukin-1 (IL-1) and lipopolysaccharides (LPS). IKK activation by TRAF6 requires two intermediary factors, TRAF6-regulated IKK activator 1 (TRIKA1) and TRIKA2 (ref. 5). TRIKA1 is a dimeric ubiquitin-conjugating enzyme complex composed of Ubc13 and Uev1A (or the functionally equivalent Mms2). This Ubc complex, together with TRAF6, catalyses the formation of a Lys 63 (K63)-linked polyubiquitin chain that mediates IKK activation through a unique proteasome-independent mechanism. Here we report the purification and identification of TRIKA2, which is composed of TAK1, TAB1 and TAB2, a protein kinase complex previously implicated in IKK activation through an unknown mechanism. We find that the TAK1 kinase complex phosphorylates and activates IKK in a manner that depends on TRAF6 and Ubc13-Uev1A. Moreover, the activity of TAK1 to phosphorylate MKK6, which activates the JNK-p38 kinase pathway, is directly regulated by K63-linked polyubiquitination. We also provide evidence that TRAF6 is conjugated by the K63 polyubiquitin chains. These results indicate that ubiquitination has an important regulatory role in stress response pathways, including those of IKK and JNK.     

Critical role of TRAF3 in the Toll-like receptor-dependent and -independent antiviral response

Type I interferon (IFN) production is a critical component of the innate defence against viral infections. Viral products induce strong type I IFN responses through the activation of Toll-like receptors (TLRs) and intracellular cytoplasmic receptors such as protein kinase R (PKR). Here we demonstrate that cells lacking TRAF3, a member of the TNF receptor-associated factor family, are defective in type I IFN responses activated by several different TLRs. Furthermore, we show that TRAF3 associates with the TLR adaptors TRIF and IRAK1, as well as downstream IRF3/7 kinases TBK1 and IKK-epsilon, suggesting that TRAF3 serves as a critical link between TLR adaptors and downstream regulatory kinases important for IRF activation. In addition to TLR stimulation, we also show that TRAF3-deficient fibroblasts are defective in their type I IFN response to direct infection with vesicular stomatitis virus, indicating that TRAF3 is also an important component of TLR-independent viral recognition pathways. Our data demonstrate that TRAF3 is a major regulator of type I IFN production and the innate antiviral response.     

Specificity in Toll-like receptor signalling through distinct effector functions of TRAF3 and TRAF6

Toll-like receptors (TLRs) are activated by pathogen-associated molecular patterns to induce innate immune responses and production of pro-inflammatory cytokines, interferons and anti-inflammatory cytokines. TLRs activate downstream effectors through adaptors that contain Toll/interleukin-1 receptor (TIR) domains, but the mechanisms accounting for diversification of TLR effector functions are unclear. To dissect biochemically TLR signalling, we established a system for isolating signalling complexes assembled by dimerized adaptors. Using MyD88 as a prototypical adaptor, we identified TNF receptor-associated factor 3 (TRAF3) as a new component of TIR signalling complexes that is recruited along with TRAF6. Using myeloid cells from TRAF3- and TRAF6-deficient mice, we show that TRAF3 is essential for the induction of type I interferons (IFN) and the anti-inflammatory cytokine interleukin-10 (IL-10), but is dispensable for expression of pro-inflammatory cytokines. In fact, TRAF3-deficient cells overproduce pro-inflammatory cytokines owing to defective IL-10 production. Despite their structural similarity, the functions of TRAF3 and TRAF6 are largely distinct. TRAF3 is also recruited to the adaptor TRIF (Toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta) and is required for marshalling the protein kinase TBK1 (also called NAK) into TIR signalling complexes, thereby explaining its unique role in activation of the IFN response.     

Anti-inflammatory cyclopentenone prostaglandins are direct inhibitors of IkappaB kinase

NF-kappaB is a critical activator of genes involved in inflammation and immunity. Pro-inflammatory cytokines activate the IkappaB kinase (IKK) complex that phosphorylates the NF-kappaB inhibitors, triggering their conjugation with ubiquitin and subsequent degradation. Freed NF-kappaB dimers translocate to the nucleus and induce target genes, including the one for cyclo-oxygenase 2 (COX2), which catalyses the synthesis of pro-inflammatory prostaglandins, in particular PGE. At late stages of inflammatory episodes, however, COX2 directs the synthesis of anti-inflammatory cyclopentenone prostaglandins, suggesting a role for these molecules in the resolution of inflammation. Cyclopentenone prostaglandins have been suggested to exert anti-inflammatory activity through the activation of peroxisome proliferator-activated receptor-gamma. Here we demonstrate a novel mechanism of antiinflammatory activity which is based on the direct inhibition and modification of the IKKbeta subunit of IKK. As IKKbeta is responsible for the activation of NF-kappaB by pro-inflammatory stimuli, our findings explain how cyclopentenone prostaglandins function and can be used to improve the utility of COX2 inhibitors.     

Molecular and biological characterization of a murine ligand for CD40.

The CD40 surface molecule is a 277-amino-acid glycoprotein expressed on B lymphocytes, epithelial cells and some carcinoma cell lines. Monoclonal antibodies against CD40 mediate a variety of effects on B lymphocytes, including induction of intercellular adhesion, short- and long-term proliferation, differentiation and enhanced tyrosine phosphorylation of proteins. In addition, germinal centre centrocytes are prevented from undergoing apoptosis by activation through CD40 and receptor for antigen. These data indicate that CD40 could be a receptor for an unknown ligand with important functions in B-cell development and activation. This hypothesis is strengthened by the homology of the extracellular region of the CD40 molecule with a family of cell-surface glycoproteins that includes the receptors for nerve growth factor and tumour necrosis factor. Here we report the cloning of a ligand for CD40 that is expressed on the cell surface of activated T cells and mediates B-cell proliferation in the absence of co-stimulus, as well as IgE production in the presence of interleukin-4.     

Structural basis for self-association and receptor recognition of human TRAF2

Tumour necrosis factor (TNF)-receptor-associated factors (TRAFs) form a family of cytoplasmic adapter proteins that mediate signal transduction from many members of the TNF-receptor superfamily and the interleukin-1 receptor. They are important in the regulation of cell survival and cell death. The carboxy-terminal region of TRAFs (the TRAF domain) is required for self-association and interaction with receptors. The domain contains a predicted coiled-coil region that is followed by a highly conserved TRAF-C domain. Here we report the crystal structure of the TRAF domain of human TRAF2, both alone and in complex with a peptide from TNF receptor-2 (TNF-R2). The structures reveal a trimeric self-association of the TRAF domain, which we confirm by studies in solution. The TRAF-C domain forms a new, eight-stranded antiparallel beta-sandwich structure. The TNF-R2 peptide binds to a conserved shallow surface depression on one TRAF-C domain and does not contact the other protomers of the trimer. The nature of the interaction indicates that an SXXE motif may be a TRAF2-binding consensus sequence. The trimeric structure of the TRAF domain provides an avidity-based explanation for the dependence of TRAF recruitment on the oligomerization of the receptors by their trimeric extracellular ligands.     

HIV-1 Nef intersects the macrophage CD40L signalling pathway to promote resting-cell infection

All primate lentiviruses (HIV-1, HIV-2, SIV) encode Nef proteins, which are important for viral replication and pathogenicity in vivo. It is not known how Nef regulates these processes. It has been suggested that Nef protects infected cells from apoptosis and recognition by cytotoxic T lymphocytes. Other studies suggest that Nef influences the activation state of the infected cell, thereby enhancing the ability of that cell to support viral replication. Here we show that macrophages that express Nef or are stimulated through the CD40 receptor release a paracrine factor that renders T lymphocytes permissive to HIV-1 infection. This activity requires the upregulation of B-cell receptors involved in the alternative pathway of T-lymphocyte stimulation. T lymphocytes stimulated through this pathway become susceptible to viral infection without progressing through the cell cycle. We identify two proteins, soluble CD23 and soluble ICAM, that are induced from macrophages by Nef and CD40L, and which mediate their effects on lymphocyte permissivity. Our results reveal a mechanism by which Nef expands the cellular reservoir of HIV-1 by permitting the infection of resting T lymphocytes.