0 5 5Recognition of pathogen-associated molecules in microbes by TLRs leads to activation of transcription factors such as NF-κB that promote increased transcription of proinflammatory cytokines and interferons 1 . All mammalian TLRs, with the exception of TLR3, use the adaptor MyD88 as the receptor-proximal signaling molecule to trigger downstream activation of NF-κB 2 . The association of MyD88 with TLRs facilitates recruitment of members of the IRAK family of kinases that in turn activate the E3 ubiquitin ligase TRAF6 (refs. 3-5). The formation of polyubiquitin chains by TRAF6 serves to bring TAK1 into close proximity with its substrates, including IκB kinases (IKKs). The TAK1-induced phosphorylation and activation of IKKα and IKKβ promotes IKK-induced phosphorylation of IκB proteins 6 that normally sequester NF-κB in an inactive form in the cytoplasm. Phosphorylated forms of IκB are subject to polyubiquitination and subsequently proteasome-dependent degradation, thus liberating NF-κB to translocate to the nucleus and transcriptionally upregulate the expression of a plethora of genes 7 . Most TLRs use this MyD88-dependent pathway to activate NF-κB, but TLR4 can additionally deploy another adaptor protein, TRIF, to trigger a MyD88-independent pathway that also activates NF-κB 8 . Among TLRs, TLR3 uses TRIF as its exclusive receptor-proximal adaptor protein. TRIF interacts with RIP1 kinase to trigger downstream IKK-mediated activation of NF-κB 9,10 . TRAF6 has been reported to associate with TRIF and mediate activation of NF-κB 11-13 , but other studies had concluded that TRAF6 is dispensable for TLR3 signaling 14,15 . Such discrepancies in relation to the role of TRAF6 in TRIF signaling may be due to cell-specific roles for TRAF6 and/or functional redundancy of TRAF6 with other members of the TRAF family 11 . In addition to activation of NF-κB, TRIF can also trigger activation of interferon-regulatory factor (IRF) transcription factors. Thus, TRIF forms a complex with the kinases TBK1 and IKKi (also known as IKKε) and both kinases can catalyze phosphorylation and activation of IRF3 and IRF7, leading to their nuclear translocation and induction of type I interferons 1,16 . The latter are key antiviral molecules that block viral replication 17,18 .It is clear from the above that ubiquitination is important in TLR signal transduction. Additionally, there is an emerging appreciation of the roles of the E3 ubiquitin ligase family of Pellino proteins in TLR signaling. The mammalian Pellino family consists of four members: Pellino1, Pellino2 and splice variants of Pellino3 termed Pellino3 long (Pellino3L; also known as Pellino3a) and Pellino3 short (Pellino3S; also known as Pellino3b) 19,20 . Each Pellino family member contains an N-terminal forkhead-associated (FHA) domain that recognizes phosphothreonine residues and mediates association with IRAKs 21 , and a C-terminal RING-like domain that confers E3 ubiquitin ligase activity and an ability to catalyze lysine 63 (Lys63)-linked polyubiquitination of IRAKs [22][23]...

Tumour necrosis factor-a (TNF) can activate NF-kB to induce pro-inflammatory genes but can also stimulate the caspase cascade to promote apoptosis. Here we show that deficiency of the ubiquitin E3 ligase, Pellino3, sensitizes cells to TNF-induced apoptosis without inhibiting the NF-kB pathway. Suppressed expression of Pellino3 leads to enhanced formation of the death-induced signalling complex, complex II, in response to TNF. We show that Pellino3 targets RIP1, in a TNF-dependent manner, to inhibit TNF-induced complex II formation and caspase 8-mediated cleavage of RIP1 in response to TNF/cycloheximide co-stimulation. Pellino3-deficient mice also show increased sensitivity to TNF-induced apoptosis and greatly increased lethality in response to TNF administration. These findings define Pellino3 as a novel regulator of TNF signalling and an important determining factor in dictating whether TNF induces cell survival or death.

The use of biological entities in diagnostics has recently become widespread. Such devices are called biosensors and consist of a detector and a reporter component, of which one is a biological moiety. Many biological molecules have been incorporated into biosensors with examples being, Abs, enzymes, DNA, and more recently microorganisms. In this chapter we discuss the advantages and disadvantages of using live organisms for biosensors. In particular we discuss the suitability of yeast for biosensor development using mammalian GPCR's. In addition we also discuss the potential use of these biosensors in high‐throughput screening.

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