Ian E. Gentle

The Omp85 family of proteins is essential for outer membrane biogenesis in mitochondria and bacteria

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.

Molecular architecture and function of the Omp85 family of proteins

Omp85 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.

TRAF2 Must Bind to Cellular Inhibitors of Apoptosis for Tumor Necrosis Factor (TNF) to Efficiently Activate NF-κB and to Prevent TNF-induced Apoptosis

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-κB-inducing kinase stability and suppress constitutive noncanonical NF-κB activation. Conversely, following TNFR1 stimulation, cells bearing a CIM-mutated TRAF2 showed reduced canonical NF-κB 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-κB signaling and that efficient activation of NF-κB by TNFR1 requires the recruitment of cIAP1/2 by TRAF2.

In TNF-stimulated cells, RIPK1 promotes cell survival by stabilizing TRAF2 and cIAP1, which limits induction of non-canonical NF-κB and activation of caspase-8.

RIPK1 is involved in signaling from TNF and TLR family receptors. After receptor ligation, RIPK1 not only modulates activation of both canonical and NIK-dependent NF-κB, but also regulates caspase-8 activation and cell death. Although overexpression of RIPK1 can cause caspase-8-dependent cell death, when RIPK1−/− cells are exposed to TNF and low doses of cycloheximide, they die more readily than wild-type cells, indicating RIPK1 has pro-survival as well as pro-apoptotic activities (1, 2). To determine how RIPK1 promotes cell survival, we compared wild-type and RIPK1−/− cells treated with TNF. Although TRAF2 levels remained constant in TNF-treated wild-type cells, TNF stimulation of RIPK1−/− cells caused TRAF2 and cIAP1 to be rapidly degraded by the proteasome, which led to an increase in NIK levels. This resulted in processing of p100 NF-κB2 to p52, a decrease in levels of cFLIPL, and activation of caspase-8, culminating in cell death. Therefore, the pro-survival effect of RIPK1 is mediated by stabilization of TRAF2 and cIAP1.

Cell wall integrity is linked to mitochondria and phospholipid homeostasis in Candida albicans through the activity of the post‐transcriptional regulator Ccr4‐Pop2

The cell wall is essential for viability of fungi and is an effective drug target in pathogens such as Candida albicans. The contribution of post‐transcriptional gene regulators to cell wall integrity in C. albicans is unknown. We show that the C. albicans Ccr4‐Pop2 mRNA deadenylase, a regulator of mRNA stability and translation, is required for cell wall integrity. The ccr4/pop2 mutants display reduced wall β‐glucans and sensitivity to the echinocandin caspofungin. Moreover, the deadenylase mutants are compromised for filamentation and virulence. We demonstrate that defective cell walls in the ccr4/pop2 mutants are linked to dysfunctional mitochondria and phospholipid imbalance. To further understand mitochondrial function in cell wall integrity, we screened a Saccharomyces cerevisiae collection of mitochondrial mutants. We identify several mitochondrial proteins required for caspofungin tolerance and find a connection between mitochondrial phospholipid homeostasis and caspofungin sensitivity. We focus on the mitochondrial outer membrane SAM complex subunit Sam37, demonstrating that it is required for both trafficking of phospholipids between the ER and mitochondria and cell wall integrity. Moreover, in C. albicans also Sam37 is essential for caspofungin tolerance. Our study provides the basis for an integrative view of mitochondrial function in fungal cell wall biogenesis and resistance to echinocandin antifungal drugs.

Integral membrane proteins in the mitochondrial outer membrane of Saccharomyces cerevisiae.

Mitochondria evolved from a bacterial endosymbiont ancestor in which the integral outer membrane proteins would have been β‐barrel structured within the plane of the membrane. Initial proteomics on the outer membrane from yeast mitochondria suggest that while most of the protein components are integral in the membrane, most of these mitochondrial proteins behave as if they have α‐helical transmembrane domains, rather than β‐barrels. These proteins are usually predicted to have a single α‐helical transmembrane segment at either the N‐ or C‐terminus, however, more complex topologies are also seen. We purified the novel outer membrane protein Om14 and show it is encoded in the gene YBR230c. Protein sequencing revealed an intron is spliced from the transcript, and both transcription from the YBR230c gene and steady‐state level of the Om14 protein is dramatically less in cells grown on glucose than in cells grown on nonfermentable carbon sources. Hydropathy predictions together with data from limited protease digestion show three α‐helical transmembrane segments in Om14. The α‐helical outer membrane proteins provide functions derived after the endosymbiotic event, and require the translocase in the outer mitochondrial membrane complex for insertion into the outer membrane.

Conserved substrate binding by chaperones in the bacterial periplasm and the mitochondrial intermembrane space.

Mitochondria were derived from intracellular bacteria and the mitochondrial intermembrane space is topologically equivalent to the bacterial periplasm. Both compartments contain ATP-independent chaperones involved in the transport of hydrophobic membrane proteins. The mitochondrial TIM (translocase of the mitochondrial inner membrane) 10 complex and the periplasmic chaperone SurA were examined in terms of evolutionary relation, structural similarity, substrate binding specificity and their function in transporting polypeptides for insertion into membranes. The two chaperones are evolutionarily unrelated; structurally, they are also distinct both in their characteristics, as determined by SAXS (small-angle X-ray scattering), and in pairwise structural comparison using the distance matrix alignment (DALILite server). Despite their structural differences, SurA and the TIM10 complex share a common binding specificity in Pepscan assays of substrate proteins. Comprehensive analysis of the binding on a total of 1407 immobilized 13-mer peptides revealed that the TIM10 complex, like SurA, does not bind hydrophobic peptides generally, but that both chaperones display selectivity for peptides rich in aromatic residues and with net positive charge. This common binding specificity was not sufficient for SurA to completely replace TIM10 in yeast cells in vivo. In yeast cells lacking TIM10, when SurA is targeted to the intermembrane space of mitochondria, it binds translocating substrate proteins, but fails to completely transfer the substrate to the translocase in the mitochondrial inner membrane. We suggest that SurA was incapable of presenting substrates effectively to the primitive TOM (translocase of the mitochondrial outer membrane) and TIM complexes in early mitochondria, and was replaced by the more effective small Tim chaperone.

Inhibitors of apoptosis proteins (IAPs) are required for effective T-cell expansion/survival during antiviral immunity in mice

Inhibitors of apoptosis proteins (IAPs) were originally described as regulating apoptosis by direct binding to caspases. More recently, IAPs have been identified as important modulators of canonical and noncanonical nuclear factor κB signaling via their ubiquitin-E3 ligase activity. IAPs are therefore, not only gatekeepers of cell death, but are probably also involved in the regulation of inflammation, as well as innate and adaptive immunity. In this study, we analyzed the role of IAPs in T-cell immunity during lymphocytic choriomeningitis virus (LCMV) infection by pharmacological targeting with an IAP antagonist/second mitochondria-derived activator of caspase-mimetic. Expansion of virus-specific CD8 T cells was drastically reduced in LCMV-infected mice exposed to IAP antagonists. Accordingly, virus control was substantially impaired, indicated by high virus titres in the spleen and the spread of LCMV to peripheral organs. The profound negative effect of IAP antagonists on T-cell immunity was partially linked to tumor necrosis factor-mediated cell death of activated T cells and required inhibition of X-linked inhibitor of apoptosis, as well as cellular IAP-1. Thus, IAPs play an important role in T-cell expansion and survival in the context of a highly inflammatory environment such as a virus infection, indicating that IAP antagonists may interfere with immune responses.

TAK1 is required for survival of mouse fibroblasts treated with TRAIL, and does so by NF-κB dependent induction of cFLIPL

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known as a “death ligand”—a member of the TNF superfamily that binds to receptors bearing death domains. As well as causing apoptosis of certain types of tumor cells, TRAIL can activate both NF-κB and JNK signalling pathways. To determine the role of TGF-β-Activated Kinase-1 (TAK1) in TRAIL signalling, we analyzed the effects of adding TRAIL to mouse embryonic fibroblasts (MEFs) derived from TAK1 conditional knockout mice. TAK1−/− MEFs were significantly more sensitive to killing by TRAIL than wild-type MEFs, and failed to activate NF-κB or JNK. Overexpression of IKK2-EE, a constitutive activator of NF-κB, protected TAK1−/− MEFs against TRAIL killing, suggesting that TAK1 activation of NF-κB is critical for the viability of cells treated with TRAIL. Consistent with this model, TRAIL failed to induce the survival genes cIAP2 and cFlipL in the absence of TAK1, whereas activation of NF-κB by IKK2-EE restored the levels of both proteins. Moreover, ectopic expression of cFlipL, but not cIAP2, in TAK1−/− MEFs strongly inhibited TRAIL-induced cell death. These results indicate that cells that survive TRAIL treatment may do so by activation of a TAK1–NF-κB pathway that drives expression of cFlipL, and suggest that TAK1 may be a good target for overcoming TRAIL resistance.

Cycloheximide Can Induce Bax/Bak Dependent Myeloid Cell Death Independently of Multiple BH3-Only Proteins

Apoptosis mediated by Bax or Bak is usually thought to be triggered by BH3-only members of the Bcl-2 protein family. BH3-only proteins can directly bind to and activate Bax or Bak, or indirectly activate them by binding to anti-apoptotic Bcl-2 family members, thereby relieving their inhibition of Bax and Bak. Here we describe a third way of activation of Bax/Bak dependent apoptosis that does not require triggering by multiple BH3-only proteins. In factor dependent myeloid (FDM) cell lines, cycloheximide induced apoptosis by a Bax/Bak dependent mechanism, because Bax-/-Bak-/- lines were profoundly resistant, whereas FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Addition of cycloheximide led to the rapid loss of Mcl-1 but did not affect the expression of other Bcl-2 family proteins. In support of these findings, similar results were observed by treating FDM cells with the CDK inhibitor, roscovitine. Roscovitine reduced Mcl-1 abundance and caused Bax/Bak dependent cell death, yet FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Therefore Bax/Bak dependent apoptosis can be regulated by the abundance of anti-apoptotic Bcl-2 family members such as Mcl-1, independently of several known BH3-only proteins.