Toll-like receptors (TLRs) mediate responses to pathogenassociated molecules as part of the vertebrate innate immune response to infection. Receptor dimerization is coupled to downstream signal transduction by the recruitment of a postreceptor complex containing the adaptor protein MyD88 and the IRAK protein kinases. In this work, we show that the death domains of human MyD88 and IRAK-4 assemble into closed complexes having unusual stoichiometries of 7:4 and 8:4, the Myddosome. Formation of the Myddosome is likely to be a key event for TLR4 signaling in vivo as we show here that pathway activation requires that the receptors cluster into lipid rafts. Taken together, these findings indicate that TLR activation causes the formation of a highly oligomeric signaling platform analogous to the death-inducing signaling complex of the Fas receptor pathway.
Sp1-like proteins are defined by three highly homologous C 2 H 2 zinc finger motifs that bind GC-rich sequences found in the promoters of a large number of genes essential for mammalian cell homeostasis. Here we report that TIEG2, a transforming growth factor -inducible Sp1-like protein with antiproliferative functions, represses transcription through recruitment of the mSin3A-histone deacetylase complex. The interaction of TIEG2 with mSin3A is mediated by an alpha-helical repression motif (␣-HRM) located within the repression domain (R1) of TIEG2. This ␣-HRM specifically associates with the second paired amphipathic helix (PAH2) domain of mSin3A. Mutations in the TIEG2 ␣-HRM domain that disrupt its helical structure abolish its ability to both bind mSin3A and repress transcription. Interestingly, the ␣-HRM is conserved in both the TIEG (TIEG1 and TIEG2) and BTEB (BTEB1, BTEB3, and BTEB4) subfamilies of Sp1-like proteins. The ␣-HRM from these proteins also mediates direct interaction with mSin3A and represses transcription. Surprisingly, we found that the ␣-HRM of the Sp1-like proteins characterized here exhibits structural and functional resemblance to the Sin3A-interacting domain previously described for the basic helix-loop-helix protein Mad1. Thus, our study defines a mechanism of transcriptional repression via the interactions of the ␣-HRM with the Sin3-histone deacetylase complex that is utilized by at least five Sp1-like transcriptional factors. More importantly, we demonstrate that a helical repression motif which mediates Sin3 interaction is not an exclusive structural and functional characteristic of the Mad1 subfamily but rather has a wider functional impact on transcriptional repression than previously demonstrated.The Sp1-like family of transcription factors is characterized by the presence of three highly homologous C-terminal zinc finger motifs that are capable of binding GC-rich DNA sequences. These GC-rich motifs are present in the promoters of more than a thousand different gene products (10,17,21,23). Currently, the Sp1-like proteins identified contain at least 16 members that can be classified into several subgroups, including Sp (Sp1, Sp2, Sp3, and Sp4), BTEB (BTEB1), KLF (BKLF, BKLF3, EKLF, GKLF, BTEB2/IKLF, and LKLF), CPBP (CPBP and UKLF), TIEG (TIEG1 and TIEG2), and Ap-2rep. The detailed nomenclature and classification of these proteins can be found in several recent reviews (7,8,26,34). Several new members, including BTEB3 (J. Kaczynski et al., unpublished data), BTEB4 (A. Conley et al., unpublished data), Sp5 (14), and SP6/KLF14 (27), have recently been added to this growing family of proteins. Because many of the genes essential for the regulation of cell growth (6, 18, 28, 30), differentiation (2, 9), and apoptosis (21, 32) contain Sp1-like binding sites, it is not surprising that members of the Sp1 family are important regulators of mammalian cell homeostasis. Additionally, Sp1-like proteins are critical for normal development. Studies with animal models have shown that disruption o...
In Drosophila, the signaling pathway mediated by the Toll receptor is critical for the establishment of embryonic dorso-ventral pattern and for innate immune responses to bacterial and fungal pathogens. Toll is activated by high affinity binding of the cytokine Spä tzle, a dimeric ligand of the cystine knot family. In vertebrates, a related family of Toll-like receptors play a critical role in innate immune responses. Despite the importance of this family of receptors, little is known about the biochemical events that lead to receptor activation and signaling. Here, we show that Spä tzle binds to the Nterminal region of Toll and, using biophysical methods, that the binding is complex. The two binding events that cause formation of the cross-linked complex are nonequivalent: the first Toll ectodomain binds Spä tzle with an affinity 3-fold higher than the second molecule suggesting that pathway activation involves negative cooperativity. We further show that the Toll ectodomains are able to form low affinity dimers in solution and that juxtamembrane sequences of Toll are critical for the activation or derepression of the pathway. These results, taken together, suggest a mechanism of signal transduction that requires both ligand-receptor and receptor-receptor interactions.
RNase E is an essential endoribonuclease that plays a central role in the processing and degradation of RNA in Escherichia coli and other bacteria. Most endoribonucleases have been shown to act distributively; however, Feng et al. [(2002) Proc. Natl. Acad. Sci. U.S.A. 99, 14746-14751] have recently found that RNase E acts via a scanning mechanism. A structural explanation for the processivity of RNase E is provided here, with our finding that the conserved catalytic domain of E. coli RNase E forms a homotetramer. Nondissociating nanoflow-electrospray mass spectrometry suggests that the tetramer binds up to four molecules of a specific substrate RNA analogue. The tetrameric assembly of the N-terminal domain of RNase E is consistent with crystallographic analyses, which indicate that the tetramer possesses approximate D(2) dihedral symmetry. Using X-ray solution scattering data and symmetry restraints, a solution shape is calculated for the tetramer. This shape, together with limited proteolysis data, suggests that the S1-RNA binding domains of RNase E lie on the periphery of the tetramer. These observations have implications for the structure and function of the RNase E/RNase G ribonuclease family and for the assembly of the E. coli RNA degradosome, in which RNase E is the central component.
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