Flagellin, the structural component of bacterial flagella, is secreted by pathogenic and commensal bacteria. Flagellin activates proinflammatory gene expression in intestinal epithelia. However, only flagellin that contacts basolateral epithelial surfaces is proinflammatory; apical flagellin has no effect. Pathogenic Salmonella, but not commensal Escherichia coli, translocate flagellin across epithelia, thus activating epithelial proinflammatory gene expression. Investigating how epithelia detect flagellin revealed that cell surface expression of Toll-like receptor 5 (TLR5) conferred NF-κB gene expression in response to flagellin. The response depended on both extracellular leucine-rich repeats and intracellular Toll/IL-1R homology region of TLR5 as well as the adaptor protein MyD88. Furthermore, immunolocalization and cell surface-selective biotinylation revealed that TLR5 is expressed exclusively on the basolateral surface of intestinal epithelia, thus providing a molecular basis for the polarity of this innate immune response. Thus, detection of flagellin by basolateral TLR5 mediates epithelial-driven inflammatory responses to Salmonella.

Inhibition of DNA synthesis induces transcription of DNA damage-inducible genes and prevents mitotic entry through the action of the S phase checkpoint. We have isolated a mutant, dun2, defective for both of these responses. DUN2 is identical to POL2, encoding DNA polymerase epsilon (pol epsilon). Unlike sad1 mutants defective for multiple cell cycle checkpoints, pol2 mutants are defective only for the S phase checkpoint and the activation of DUN1 kinase necessary for the transcriptional response to damage. Interallelic complementation and mutation analysis indicate that pol epsilon contains two separable essential domains, an N-terminal polymerase domain and a C-terminal checkpoint domain unique to epsilon polymerases. We propose that DNA pol epsilon acts as a sensor of DNA replication that coordinates the transcriptional and cell cycle responses to replication blocks.

RIP3 is a novel gene product containing a N-terminal kinase domain that shares extensive homology with the corresponding domain in RIP (receptor-interacting protein) and RIP2. Unlike RIP, which has a C-terminal death domain, and RIP2, which has a C-terminal caspase activation and recruitment domain, RIP3 has a unique C terminus. RIP3 binds RIP through its unique C-terminal segment and by virtue of this interaction is recruited to the tumor necrosis factor (TNF) receptor-1 signaling complex. Previous studies have shown that RIP mediates TNF-induced activation of the anti-apoptotic NF-B pathway. RIP3, however, attenuates both RIP and TNF receptor-1-induced NF-B activation. Overexpression studies revealed RIP3 to be a potent inducer of apoptosis, capable of selectively binding to large prodomain initiator caspases.Tumor necrosis factor receptor-1 mediates both the proinflammatory and pro-apoptotic effects of the pleiotropic cytokine TNF (1, 2). 1 The proinflammatory effects are mediated by activation of the transcription factor NF-B. Recently it has been shown that NF-B activation results in both the transcriptional activation of proinflammatory genes, including , and in activation of a cell survival pathway mediated at least in part by induction of anti-apoptotic IAP family members (6 -8). Therefore, quite paradoxically, two diametrically opposed pathways emanate from TNFR-1: a cell death pathway and a cell survival pathway mediated by activation of NF-B.The intracellular segment of TNFR-1 contains a 70-amino acid homophilic interaction domain, dubbed the "death domain," which is required for both signaling and NF-B activation. Upon activation of TNFR-1, a multi-component signaling complex is assembled by a series of homophilic interactions (1). Initially, the death domain-containing platform adapter molecule TRADD is recruited to TNFR-1 by virtue of a homophilic death domain interaction (9). TRADD in turn binds the death adapter molecule FADD (10, 11), which interacts with the zymogen form of the initiator death protease caspase-8 (12). Subsequent activation of caspase-8 by an induced proximity mechanism leads to amplification of the death signal through proteolytic activation of downstream caspase zymogens (13). Studies done with FADD-deficient embryonic fibroblasts suggest that this is the major but not the only pro-apoptotic pathway engaged by TNFR-1, because instead of being completely resistant to TNF-induced apoptosis, 30% of FADD-null cells are still sensitive (14,15). Taken together, these studies suggest that there exists a subsidiary FADD-independent TNFR-1-initiated death pathway. Earlier biochemical studies indicated that the TNFR-1-associated adapter molecule RAIDD might fulfill this function by recruiting caspase-2 to the receptor signaling complex (16). However, caspase-2 null cells do not show any loss of sensitivity to TNF-induced cytotoxicity (17); therefore, the physiological significance of this interaction remains unclear. Initial biochemical studies also suggested a role for the TRADD-bindin...

Toll-like receptors (TLRs) play a fundamental role in the recognition of bacteria and viruses. TLR3 is activated by viral dsRNA and polyinosinic-polycytidylic acid (poly(I:C)), a synthetic mimetic of viral RNA. We show that NK cells, known for their capacity to eliminate virally infected cells, express TLR3 and up-regulate TLR3 mRNA upon poly(I:C) stimulation. Treatment of highly purified NK cells with poly(I:C) significantly augments NK cell-mediated cytotoxicity. Poly(I:C) stimulation also leads to up-regulation of activation marker CD69 on NK cells. Furthermore, NK cells respond to poly(I:C) by producing proinflammatory cytokines like IL-6 and IL-8, as well as the antiviral cytokine IFN-γ. The induction of cytokine production by NK cells was preceded by activation of NF-κB. We conclude that the ability of NK cells to directly recognize and respond to viral products is important in mounting effective antiviral responses.

Ribonucleotide reductase (RNR) catalyzes the rate limiting step in the production of deoxyribonucleotides needed for DNA synthesis. In addition to the well documented allosteric regulation, the synthesis of the enzyme is also tightly regulated at the level of transcription. mRNAs for both subunits are cell cycle regulated and inducible by DNA damage in all organisms examined, including E. coli, S. cerevisiae and H. sapiens. This DNA damage regulation is thought to provide a metabolic state that facilitates DNA replicational repair processes. S. cerevisiae also encodes a second large subunit gene, RNR3, that is expressed only in the presence of DNA damage. Genetic analysis of the DNA damage response in S. cerevisiae has shown that RNR expression is under both positive and negative control. Among mutants constitutive for RNR expression are the general transcriptional repression genes, SSN6 and TUP1. Mutations in POL1 and POL3 also activate RNR expression, indicating that the DNA damage sensory network may respond directly to blocks in DNA synthesis. A protein kinase, Dun1, has been identified that controls inducibility of RNR1, RNR2 and RNR3 in response to DNA damage and replication blocks. This result suggests that the RNR genes in S. cerevisiae form a regulon that is coordinately regulated by protein phosphorylation in response to DNA damage.