The discovery of Toll-like receptors (TLRs) as components that recognize conserved structures in pathogens has greatly advanced understanding of how the body senses pathogen invasion, triggers innate immune responses and primes antigen-specific adaptive immunity. Although TLRs are critical for host defense, it has become apparent that loss of negative regulation of TLR signaling, as well as recognition of self molecules by TLRs, are strongly associated with the pathogenesis of inflammatory and autoimmune diseases. Furthermore, it is now clear that the interaction between TLRs and recently identified cytosolic innate immune sensors is crucial for mounting effective immune responses. Here we describe the recent advances that have been made by research into the role of TLR biology in host defense and disease.

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The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors

The posttranslational modification with ubiquitin, a process referred to as ubiquitylation, controls almost every process in cells. Ubiquitin can be attached to substrate proteins as a single moiety or in the form of polymeric chains in which successive ubiquitin molecules are connected through specific isopeptide bonds. Reminiscent of a code, the various ubiquitin modifications adopt distinct conformations and lead to different outcomes in cells. Here, we discuss the structure, assembly, and function of this ubiquitin code.

The Ubiquitin Code

The NF-κB family of transcription factors has an essential role in inflammation and innate immunity. Furthermore, NF-κB is increasingly recognized as a crucial player in many steps of cancer initiation and progression. During these latter processes NF-κB cooperates with multiple other signaling molecules and pathways. Prominent nodes of crosstalk are mediated by other transcription factors such as STAT3 and p53 or the ETS related gene ERG. These transcription factors either directly interact with NF-κB subunits or affect NF-κB target genes. Crosstalk can also occur through different kinases, such as GSK3-β, p38, or PI3K, which modulate NF-κB transcriptional activity or affect upstream signaling pathways. Other classes of molecules that act as nodes of crosstalk are reactive oxygen species and miRNAs. In this review, we provide an overview of the most relevant modes of crosstalk and cooperativity between NF-κB and other signaling molecules during inflammation and cancer.

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The complexity of NF-κB signaling in inflammation and cancer

E3 ligases confer specificity to ubiquitination by recognizing target substrates and mediating transfer of ubiquitin from an E2 ubiquitinconjugating enzyme to substrate. The activity of most E3s is specified by a RING domain, which binds to an E2∼ubiquitin thioester and activates discharge of its ubiquitin cargo. E2-E3 complexes can either monoubiquitinate a substrate lysine or synthesize polyubiquitin chains assembled via different lysine residues of ubiquitin. These modifications can have diverse effects on the substrate, ranging from proteasome-dependent proteolysis to modulation of protein function, structure, assembly, and/or localization. Not surprisingly, RING E3mediated ubiquitination can be regulated in a number of ways. RING-based E3s are specified by over 600 human genes, surpassing the 518 protein kinase genes. Accordingly, RING E3s have been linked to the control of many cellular processes and to multiple human diseases. Despite their critical importance, our knowledge of the physiological partners, biological functions, substrates, and mechanism of action for most RING E3s remains at a rudimentary stage.

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RING Domain E3 Ubiquitin Ligases

For a long time, apoptosis was considered the sole form of programmed cell death during development, homeostasis and disease, whereas necrosis was regarded as an unregulated and uncontrollable process. Evidence now reveals that necrosis can also occur in a regulated manner. The initiation of programmed necrosis, 'necroptosis', by death receptors (such as tumour necrosis factor receptor 1) requires the kinase activity of receptor-interacting protein 1 (RIP1; also known as RIPK1) and RIP3 (also known as RIPK3), and its execution involves the active disintegration of mitochondrial, lysosomal and plasma membranes. Necroptosis participates in the pathogenesis of diseases, including ischaemic injury, neurodegeneration and viral infection, thereby representing an attractive target for the avoidance of unwarranted cell death.

Molecular mechanisms of necroptosis: an ordered cellular explosion.

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.

Involvement of linear polyubiquitylation of NEMO in NF-kappaB activation.

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.

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Michiko Kato

Papers

Involvement of linear polyubiquitylation of NEMO in NF-kappaB activation.

A ubiquitin ligase complex assembles linear polyubiquitin chains

Involvement of Heme Regulatory Motif in Heme-Mediated Ubiquitination and Degradation of IRP2

The COP9/Signalosome Increases the Efficiency of von Hippel-Lindau Protein Ubiquitin Ligase-mediated Hypoxia-inducible Factor-α Ubiquitination