Myd99 overexpression functions upstream of Drosomycin (Drs). S2 cells were cotransfected with a Myd88 expression vector and reporter plasmids. Overexpression of Myd88 is sufficient to trigger a marked (nearly 40-fold) induction of the Drs promoter. In contrast, the promoter of AttA, the gene encoding the antibacterial peptide Attacin A, was increased only threefold. Overexpression of the isolated TIR domain is sufficient to trigger induction, whereas the NH2-terminal death domain is not active. Double-stranded RNA interference (RNAi) technique was used to abolish expression of Myd88 in S2 cells. RNAi-mediated inhibition of Myd88 expression results in a marked and specific reduction of the Toll-mediated induction of the Drs promoter but does not affect the LPS-mediated induction of the AttA promoter. These results indicated that Myd88 participates in the Toll pathway in S2 cells and is not required for the response to LPS treatment (Tauszig-Delamasure, 2002).
Micro-RNAs are a class of small non-coding regulatory RNAs that impair translation by imperfect base pairing to mRNAs. For analysis of their cellular function, different miRNA-specific DNA antisense oligonucleotides were injected into Drosophila embryos. In four cases severe interference with normal development was observed; in another case, this had a moderate impact and six other oligonucleotides did not cause detectable phenotypes. The miR-13a DNA antisense oligonucleotide was used as a PCR primer on a cDNA library template. In this experimental way nine Drosophila genes were identifed, each characterized by 3' untranslated region motifs that allow imperfect duplex formation with miR-13 or related miRNAs. These genes, which include Sos and Myd88, represent putative targets for miRNA regulation. Mutagenesis of the target motif of two genes followed by transfection in Drosophila Schneider 2 (S2) cells and subsequent reporter gene analysis confirms the hypothesis that the binding potential of miR-13 is inversely correlated with gene expression (Boutla, 2003).
Drosophila Myd88 is an adapter in the Toll signaling pathway that associates with both the Toll receptor and the downstream kinase Pelle. Expression of Myd88 in S2 cells strongly induces activity of a Drosomycin reporter gene, whereas a dominant-negative version of Myd88 potently inhibits Toll-mediated signaling. Myd88 associates with the death domain-containing adapter Drosophila Fas-associated death domain-containing protein (FADD), which in turn interacts with the apical caspase Dredd. This pathway links a cell surface receptor to an apical caspase in invertebrate cells and therefore suggests that the Toll-mediated pathway of caspase activation may be the evolutionary ancestor of the death receptor-mediated pathway for apoptosis induction in mammals (Horng, 2001).
A BLAST search of the Drosophila genome identified the sequence encoding Myd88, a Drosophila homolog of human MyD88. Similar to its human homolog, Drosophila Myd88 contains an N-terminal death domain, an intermediate domain, and a TIR domain. However, unlike human MyD88, Drosophila Myd88 contains an additional 81 amino acids preceding the death domain and a 162-aa-long C-terminal region following the TIR domain (Horng, 2001).
Transfection of Myd88 into Drosophila S2 cells potently induces a Drosomycin reporter gene but not an Attacin reporter gene. This preferential ability to induce an antifungal gene is similar to that of Toll 10b, a constitutively active form of Toll, and suggests that Myd88 may be a component of the Toll-Tube-Pelle-Cactus-Dif signaling pathway. Previous studies have demonstrated that Toll-mediated Drosomycin induction requires the nuclear translocation of Dif. Dif is normally retained in the cytoplasm by the IkappaB inhibitor Cactus and is released only in response to signal-dependent degradation of Cactus. To test whether Myd88-mediated Drosomycin induction also depends on Cactus degradation, a Cactus mutant was constructed that contains mutations of the conserved serine residues that, in mammalian IkappaB, are the targets of signal-dependent phosphorylation. A Cactus mutant inhibits Drosomycin induction by Myd88 and, as expected, by Toll. This result indicates that, similar to Toll, Myd88 regulates Drosomycin induction through the Cactus-dependent pathway (Horng, 2001).
For further analyses, various deletion mutants of Myd88 were generated. Two of the deletion mutants, one containing the TIR domain and the C-terminal domain (amino acids 237-537) and another containing the intermediate, TIR, and C-terminal domains (amino acids 176-537), activate the Drosomycin reporter weakly (10-fold) in comparison to full length Myd88, indicating that the intact protein is required for optimal activity. However, the fact that these truncation mutants can still induce signaling is surprising, since they lack the death domain that mediates interactions with downstream signaling components. Moreover, similar analyses of human MyD88 have shown that a combination of the death domain and the intermediate domain is sufficient to induce signaling activity comparable to that of the wild-type protein. An equivalent truncation of Myd88 (amino acids 1-237) retains no residual activity despite being well expressed, suggesting that there are some differences in domain function between human and Drosophila Myd88 proteins (Horng, 2001).
To determine whether Myd88 is a component of the Toll signaling pathway, attempts were made to identify a deletion mutant that would have dominant-negative activity. Therefore, three Myd88 deletion mutants that do not activate the Drosomycin reporter were tested for their ability to inhibit Toll-mediated Drosomycin induction. The strongest inhibitor was the death domain- and middle domain-containing construct (amino acids 1-237), which at low concentrations potently inhibits Toll-mediated Drosomycin induction in a dose-dependent manner (Horng, 2001).
To order Myd88 in the pathway with respect to Pelle, Myd88 was tested for its ability to be inhibited by PelleN, a dominant-negative form of Pelle that consists of the N-terminal death domain-containing region of Pelle. Myd88, like Toll, is strongly inhibited by PelleN. Myd88, however, does not inhibit Pelle, demonstrating that, similar to the mammalian pathway, Myd88 functions upstream of Pelle (Horng, 2001).
To further establish Myd88 as a component of the Toll pathway, whether Myd88 interacts with Toll was tested by coimmunoprecipitation assays. The TIR domain-containing Myd88 construct is detected in anti-Toll immunoprecipitates. Interestingly, when cotransfected with Toll 10b, Myd88 reproducibly appears as two distinct bands -- a slower migrating upper band that may correspond to phosphorylated Myd88 construct and a faster migrating lower band. The predominant form of Myd88 detected in immunoprecipitates is the faster migrating species. Myd88 therefore associates with Toll, presumably through TIR domains, and is a component of the active receptor complex (Horng, 2001).
Because human MyD88 associates with IRAK through death domains, a likely immediate downstream target of MyD88 is the IRAK homolog Pelle. Interaction between the death domain-containing Drosophila Myd88 construct (amino acids 1-237) and Pelle was examined. Myd88 is detected in Pelle immunoprecipitates, indicating that Myd88 interacts with Pelle, presumably through their death domains (Horng, 2001).
These results therefore demonstrate that Myd88 is an adaptor in the Toll signaling pathway downstream of the receptor and upstream of Pelle. From genetic analyses, the adaptor protein Tube has also been implicated to be downstream of Toll and upstream of Pelle in the Toll signaling pathway. The death domain of Tube also interacts with Pelle. Because Tube and Myd88 also contain death domains that could potentially mediate their interaction, tests were performed for association between these two proteins in immunoprecipitation assays; Tube and Myd88 do indeed interact. Therefore, Myd88 and Tube both function as adaptors downstream of Toll, exist in the same active complex along with Pelle, and are probably both involved in the recruitment and/or activation of Pelle. Understanding functional differences between these two adapters will require further analysis (Horng, 2001).
To identify other potential downstream targets of Myd88, a search of the Drosophila genome was performed for other sequences that encode death domain-containing proteins that may interact with Myd88. One such sequence encodes a protein with a death domain as well as a death effector domain and appears to be a homolog of mammalian FADD. This cDNA has been identified and named FADD (Hu, 2000). Whether FADD can interact with Myd88 was tested. Lysates from S2 cells transfected with Myd88 were incubated with anti-Flag beads to immunoprecipitate FADD, and immunoprecipitates were blotted with anti-V5 antibody to look for associated Myd88. A strong band corresponding to Myd88 was observed, indicating that Myd88 can interact with FADD through death domains. Overexpression of FADD in S2 cells, however, does not lead to activation of either the Drosomycin or Attacin reporters (Horng, 2001).
Mammalian FADD is recruited to the tumor necrosis factor receptor complex through homophilic death domain interactions with the adapter TNFR-associated death domain-containing protein (TRADD). In turn, FADD recruits procaspase-8 through homophilic death effector domain associations. It is speculated that Drosophila FADD may likewise recruit a Drosophila caspase to the Toll receptor complex. A potential candidate caspase is Dredd, an apical caspase with a long prodomain shown to be essential for induction of antibacterial genes. Indeed, analysis of immunoprecipitated lysates from cells cotransfected with Drosophila FADD, and either full length Dredd or the death effector domain of Dredd showed strong association of Dredd with FADD. A second study (Hu, 2000) has also shown interaction of dFADD with Dredd (Horng, 2001).
Thus Drosophila Myd88 is an adapter in the Toll signaling pathway. Myd88 associates with both Toll and Pelle and functions upstream of Pelle. Tube is known from genetic studies to be an adapter in the Toll pathway that functions upstream of Pelle. Why Toll should signal through Myd88 and Tube, two receptor-proximal adapters with seemingly similar functions, is not yet clear. Myd88 associates with the receptor Toll as well as the downstream adapter FADD, which in turn interacts with the apical caspase Dredd. Because caspases are essential executioners of the apoptotic machinery in organisms from nematodes to mammals, and because Dredd has been shown to be involved in apoptosis during Drosophila development, it is possible that Toll-1 or some of the other eight Tolls that exist in Drosophila may induce apoptosis (or another Dredd-dependent pathway) through the Myd88/dFADD/Dredd pathway in a cell-type specific and/or developmental stage-specific manner. The pathway comprised of Toll, Myd88, dFADD, and Dredd would be the first description of a pathway in invertebrates that links a cell surface receptor to an apical caspase. Such a pathway, if it exists, would enable extracellular stimuli, perhaps ligands secreted by other cells during development or pathogen-derived products during infection, to instruct invertebrate cells to undergo cell death. In addition, the Toll/Myd88/dFADD/Dredd pathway is remarkably similar to that activated by the receptors of the tumor necrosis factor receptor (TNFR) superfamily in mammals, in which FADD-mediated recruitment of caspase-8 leads to induction of apoptosis. Since the Drosophila genome does not encode any cell surface receptors homologous to TNFRs, it appears that the Toll/Myd88/dFADD/Dredd pathway is the evolutionary ancestor of the mammalian death receptor pathways. This possibility is further supported by the recent finding that human TLR2 can induce apoptosis through the Myd88/FADD/Caspase-8 pathway (Horng, 2001).
Myd88 specifically associates with Toll in S2 cells The interaction of tagged versions of Myd88 and Toll was examined; Myd88 associates with Toll in transfected S2 cells. Higher expression of the V5-tagged Myd88 protein is reproducibly seen in cells cotransfected with the Toll expression vector; this might reflect stabilization of Myd88 by interaction with Toll. Similar experiments with truncated versions of Myd88 indicate that the interaction with the intracytoplasmic domain of Toll is mediated by the TIR domain of Myd88. However, no association of Myd88 was detected with the related receptors 18-wheeler, Toll-5, Toll-6, Toll-7 or Toll-8, showing that the interaction with Toll is specific. Myd88 is associated with the IRAK-related kinase Pelle in transfected S2 cells (Tauszig-Delamasure, 2002).
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