No abstract

Interleukin-1β (IL-1β) is a potent inflammatory cytokine that is usually cleaved and activated by inflammasome-associated caspase-1. To determine whether IL-1β activation is regulated by inhibitor of apoptosis (IAP) proteins, we treated macrophages with an IAP-antagonist "Smac mimetic" compound or genetically deleted the genes that encode the three IAP family members cIAP1, cIAP2, and XIAP. After Toll-like receptor priming, IAP inhibition triggered cleavage of IL-1β that was mediated not only by the NLRP3-caspase-1 inflammasome, but also by caspase-8 in a caspase-1-independent manner. In the absence of IAPs, rapid and full generation of active IL-1β by the NLRP3-caspase-1 inflammasome, or by caspase-8, required the kinase RIP3 and reactive oxygen species production. These results demonstrate that activation of the cell death-inducing ripoptosome platform and RIP3 can generate bioactive IL-1β and implicate them as additional targets for the treatment of pathological IL-1-driven inflammatory responses.

Integral proteins in the outer membrane of mitochondria control all aspects of organelle biogenesis, being required for protein import, mitochondrial fission, and, in metazoans, mitochondrial aspects of programmed cell death. How these integral proteins are assembled in the outer membrane had been unclear. In bacteria, Omp85 is an essential component of the protein insertion machinery, and we show that members of the Omp85 protein family are also found in eukaryotes ranging from plants to humans. In eukaryotes, Omp85 is present in the mitochondrial outer membrane. The gene encoding Omp85 is essential for cell viability in yeast, and conditional omp85 mutants have defects that arise from compromised insertion of integral proteins like voltage-dependent anion channel (VDAC) and components of the translocase in the outer membrane of mitochondria (TOM) complex into the mitochondrial outer membrane.

Tumor necrosis factor (TNF) receptor-associated factor-2 (TRAF2) binds to cIAP1 and cIAP2 (cIAP1/2) and recruits them to the cytoplasmic domain of several members of the TNF receptor (TNFR) superfamily, including the TNF-TNFR1 ligand-receptor complex. Here, we define a cIAP1/2-interacting motif (CIM) within the TRAF-N domain of TRAF2, and we use TRAF2 CIM mutants to determine the role of TRAF2 and cIAP1/2 individually, and the TRAF2-cIAP1/2 interaction, in TNFR1-dependent signaling. We show that both the TRAF2 RING domain and the TRAF2 CIM are required to regulate NF-Binducing kinase stability and suppress constitutive noncanonical NF-B activation. Conversely, following TNFR1 stimulation, cells bearing a CIM-mutated TRAF2 showed reduced canonical NF-B activation and TNF-induced RIPK1 ubiquitylation. Remarkably, the RING domain of TRAF2 was dispensable for these functions. However, like the TRAF2 CIM, the RING domain of TRAF2 was required for protection against TNF-induced apoptosis. These results show that TRAF2 has anti-apoptotic signaling roles in addition to promoting NF-B signaling and that efficient activation of NF-B by TNFR1 requires the recruitment of cIAP1/2 by TRAF2. The inhibitor of apoptosis (IAP)7 family is composed of baculoviral IAP repeat-containing proteins, several of which also bear a RING domain that is capable of acting as a ubiquitin E3 ligase (1). cIAP1 and cIAP2 (cIAP1/2) are two RING-containing IAPs whose amplification or genetic mutation has been associated with cancers and may promote tumor cell survival (2-8). These highly conserved IAPs were initially identified as components of a TRAF2-containing complex bound to the cytoplasmic domain of TNFR2 (9), and they have subsequently been implicated in the regulation of signaling by several more receptors of the TNF superfamily (10 -21). Although the BIR1 domain of cIAP1/2 mediates binding to TRAF2 (17,22,23), the complementary binding region of TRAF2 is unknown, and the relative roles of TRAF2 and cIAP1/2 E3 ligase activity in TNF superfamily signaling remain unclear.Genetic deletion of TRAF2 in the mouse results in early postnatal lethality that is caused by increased NF-B-mediated TNF production and increased cellular sensitivity to TNF killing (12,24,25). Most TRAF family members, including TRAF2, bear RING E3 ubiquitin ligase domains that are believed to catalyze 27). One of the proposed targets of TRAF2 RING E3 ligase activity is RIPK1 (28, 29), which is modified with Lys-63-linked polyubiquitin chains upon TNFR1 activation. This modified TNFR1-complexed RIPK1 has been proposed to serve as a platform for the recruitment and/or activation of TAB2-TAB3-TAK1 and NEMO-IKK␣-IKK␤ kinase complexes (30 -32), which target the NF-B inhibitor IB for Lys-48-linked polyubiquitin-mediated proteasomal degradation, to initiate canonical p65-dependent NF-B gene transcription.As in the case of TRAF2, genetic deletion of cIAP1, or IAP antagonist-induced loss of cIAP1/2, also sensitizes cells to TNF killing (16,19,(33)(34)(35)(36). Also like TRAF2, both ...

SummaryOmp85 is a protein found in Gram-negative bacteria where it serves to integrate proteins into the bacterial outer membrane. Members of the Omp85 family of proteins are defined by the presence of two domains: an N-terminal, periplasmic domain rich in POTRA repeats and a C-terminal beta-barrel domain embedded in the outer membrane. The widespread distribution of Omp85 family members together with their fundamental role in outer membrane assembly suggests the ancestral Omp85 arose early in the evolution of prokaryotic cells. Mitochondria, derived from an ancestral bacterial endosymbiont, also use a member of the Omp85 family to assemble proteins in their outer membranes. More distant relationships are seen between the Omp85 family and both the core proteins in two-partner secretion systems and the Toc75 family of protein translocases found in plastid outer envelopes. Aspects of the ancestry and molecular architecture of the Omp85 family of proteins is providing insight into the mechanism by which proteins might be integrated and assembled into bacterial outer membranes.