SummaryFADD is a common adaptor shared by several death-receptors (DRs) for signaling apoptosis through recruitment and activation of caspase 81-3. DRs are essential for immune homeostasis, but dispensable during embryogenesis. Surprisingly, FADD−/− mice die in utero4-5 and conditional deletion of FADD leads to impaired lymphocyte proliferation6-7. How FADD regulates embryogenesis and lymphocyte responses has been a long standing enigma. FADD could directly bind to RIP1, a serine/threonine kinase which mediates both necrosis and NF-κB activation. Here we show that FADD−/− embryos contain elevated levels of RIP1 and exhibit massive necrosis. To investigate potential in vivo functional interaction between RIP1 and FADD, null alleles of RIP1 were crossed into FADD−/− mice. Strikingly, RIP1 deficiency allowed normal embryogenesis of FADD−/− mice. Conversely, the developmental defect of RIP1−/− lymphocytes was partially corrected by FADD deletion. Furthermore, RIP1 deficiency fully restored normal proliferation in FADD−/− T cells but not in FADD−/− B cells. FADD−/−RIP1−/− double knockout (DKO) T cells are resistant to death induced by Fas or TNFα and display reduced NF-κB activity. Therefore, our data demonstrate an unexpected cell type-specific interplay between FADD and RIP1, which is critical for the regulation of apoptosis and necrosis during embryogenesis and lymphocyte function.

TLR2 promotes NLRP3 inflammasome activation via an early MyD88-IRAK1-dependent pathway that provides a priming signal (signal 1) necessary for activation of the inflammasome by a second potassium-depleting signal (signal 2). Here we show that TLR3 binding to dsRNA promotes post-translational inflammasome activation through intermediate and late TRIF/RIPK1/FADD-dependent pathways. Both pathways require the scaffolding but not the catalytic function of caspase-8 or RIPK1. Only the late pathway requires kinase competent RIPK3 and MLKL function. Mechanistically, FADD/caspase-8 scaffolding function provides a post-translational signal 1 in the intermediate pathway, whereas in the late pathway it helps the oligomerization of RIPK3, which together with MLKL provides both signal 1 and 2 for inflammasome assembly. Cytoplasmic dsRNA activates NLRP3 independent of TRIF, RIPK1, RIPK3 or mitochondrial DRP1, but requires FADD/caspase-8 in wildtype macrophages to remove RIPK3 inhibition. Our study provides a comprehensive analysis of pathways that lead to NLRP3 inflammasome activation in response to dsRNA.

The Bacilus subtilis spore coat is composed of at least 15 polypeptides plus an insoluble protein fraction arranged in three morphological layers. The insoluble fraction accounts for about 30% of the coat protein and is resistant to solubilization by a variety of reagents, implying extensive cross-linking. A dodecapeptide was purified from this fraction by formic acid hydrolysis and reverse-phase high-performance liquid chromatography. This peptide was sequenced, and a gene designated cotX was cloned by reverse genetics. The cotX gene encoding the dodecapeptide at its amino end was clustered with four other genes designated cotV, cotW, cotY, and cotZ. These genes were mapped to 1070 between thiB and metA on the B. subtilis chromosome. The deduced amino acid sequences of the cotY and cotZ genes are very similar. Both proteins are cysteine rich, and CotY antigen was present in spore coat extracts as disulfide cross-linked multimers. There was little CotX antigen in the spore coat soluble fraction, and deletion of this gene resulted in a 30%o reduction in the spore coat insoluble fraction. Spores produced by strains with deletions of the cotX, cotYZ, or cotXYZ genes were heat and lysozyme resistant but readily clumped and responded more rapidly to germinants than did spores from the wild type. In electron micrographs, there was a less densely staining outer coat in spores produced by the cotX null mutant, and those produced by a strain with a deletion of the cotXYZ genes had an incomplete outer coat. These proteins, as part of the coat insoluble fraction, appear to be localized to the outer coat and influence spore hydrophobicity as well as the accessibility of germinants.