Induction of Noxa-mediated apoptosis by modified vaccinia virus Ankara depends on viral recognition by cytosolic helicases, leading to IRF-3/IFN-ß-dependent induction of pro-apoptotic Noxa.

Viral infection is a stimulus for apoptosis, and in order to sustain viral replication many viruses are known to carry genes encoding apoptosis inhibitors. F1L, encoded by the orthopoxvirus modified vaccinia virus Ankara (MVA) has a Bcl-2-like structure. An MVA mutant lacking F1L (MVAΔF1L) induces apoptosis, indicating that MVA infection activates and F1L functions to inhibit the apoptotic pathway. In this study we investigated the events leading to apoptosis upon infection by MVAΔF1L. Apoptosis largely proceeded through the pro-apoptotic Bcl-2 family protein Bak with some contribution from Bax. Of the family of pro-apoptotic BH3-only proteins, only the loss of Noxa provided substantial protection, while the loss of Bim had a minor effect. In mice, MVA preferentially infected macrophages and DCs in vivo. In both cell types wt MVA induced apoptosis albeit more weakly than MVAΔF1L. The loss of Noxa had a significant protective effect in macrophages, DC and primary lymphocytes, and the combined loss of Bim and Noxa provided strong protection. Noxa protein was induced during infection, and the induction of Noxa protein and apoptosis induction required transcription factor IRF3 and type I interferon signalling. We further observed that helicases RIG-I and MDA5 and their signalling adapter MAVS contribute to Noxa induction and apoptosis in response to MVA infection. RNA isolated from MVA-infected cells induced Noxa expression and apoptosis when transfected in the absence of viral infection. We thus here describe a pathway leading from the detection of viral RNA during MVA infection by the cytosolic helicase-pathway, to the up-regulation of Noxa and apoptosis via IRF3 and type I IFN signalling.

Targeting of a chlamydial protease impedes intracellular bacterial growth.

Chlamydiae are obligate intracellular bacteria that propagate in a cytosolic vacuole. Recent work has shown that growth of Chlamydia induces the fragmentation of the Golgi apparatus (GA) into ministacks, which facilitates the acquisition of host lipids into the growing inclusion. GA fragmentation results from infection-associated cleavage of the integral GA protein, golgin-84. Golgin-84-cleavage, GA fragmentation and growth of Chlamydia trachomatis can be blocked by the peptide inhibitor WEHD-fmk. Here we identify the bacterial protease chlamydial protease-like activity factor (CPAF) as the factor mediating cleavage of golgin-84 and as the target of WEHD-fmk-inhibition. WEHD-fmk blocked cleavage of golgin-84 as well as cleavage of known CPAF targets during infection with C. trachomatis and C. pneumoniae. The same effect was seen when active CPAF was expressed in non-infected cells and in a cell-free system. Ectopic expression of active CPAF in non-infected cells was sufficient for GA fragmentation. GA fragmentation required the small GTPases Rab6 and Rab11 downstream of CPAF-activity. These results define CPAF as the first protein that is essential for replication of Chlamydia. We suggest that this role makes CPAF a potential anti-infective therapeutic target.

Hepatocarcinogenesis in non-cirrhotic liver is associated with a reduced number of clonal hepatocellular patches in non-tumorous liver parenchyma.

We investigated circumscribed cell proliferations in healthy livers in comparison to non-cirrhotic livers bearing hepatocellular carcinoma. Using histochemical staining for cytochrome c oxidase, the fourth complex of the respiratory chain, we visualized patch-forming descendents of regeneratively active liver cells. The clonal nature of these patches was verified by laser-capture microdissection and Sanger sequencing of the enzyme's core subunits in patches carrying marker mutations on the mtDNA. We demonstrate a highly significant increase of the patch size and also a highly significant increase in the number of patches carrying marker mutations between hepatocellular carcinoma-free and -bearing livers. Thus, the carcinoma-bearing livers accumulated more genetic damage on mtDNA than the control group. Furthermore, for the first time, we present evidence in hepatocellular carcinoma-bearing non-cirrhotic livers of a significantly reduced pool of regeneratively active liver cells that are genetically and functionally altered. The analogy to ageing-related changes is suggestive of premature ageing of stem cells in non-cirrhotic hepatocellular carcinoma-bearing liver as an early step to hepatocarcinogenesis.

The N-terminal domain of Tob55 has a receptor-like function in the biogenesis of mitochondrial beta-barrel proteins.

beta-Barrel proteins constitute a distinct class of mitochondrial outer membrane proteins. For import into mitochondria, their precursor forms engage the TOM complex. They are then relayed to the TOB complex, which mediates their insertion into the outer membrane. We studied the structure-function relationships of the core component of the TOB complex, Tob55. Tob55 precursors with deletions in the N-terminal domain were not affected in their targeting to and insertion into the mitochondrial outer membrane. Replacement of wild-type Tob55 by these deletion variants resulted in reduced growth of cells, and mitochondria isolated from such cells were impaired in their capacity to import beta-barrel precursors. The purified N-terminal domain was able to bind beta-barrel precursors in a specific manner. Collectively, these results demonstrate that the N-terminal domain of Tob55 recognizes precursors of beta-barrel proteins. This recognition may contribute to the coupling of the translocation of beta-barrel precursors across the TOM complex to their interaction with the TOB complex.

BimS-induced apoptosis requires mitochondrial localization but not interaction with anti-apoptotic Bcl-2 proteins.

Release of apoptogenic proteins such as cytochrome c from mitochondria is regulated by pro- and anti-apoptotic Bcl-2 family proteins, with pro-apoptotic BH3-only proteins activating Bax and Bak. Current models assume that apoptosis induction occurs via the binding and inactivation of anti-apoptotic Bcl-2 proteins by BH3-only proteins or by direct binding to Bax. Here, we analyze apoptosis induction by the BH3-only protein Bim(S). Regulated expression of Bim(S) in epithelial cells was followed by its rapid mitochondrial translocation and mitochondrial membrane insertion in the absence of detectable binding to anti-apoptotic Bcl-2 proteins. This caused mitochondrial recruitment and activation of Bax and apoptosis. Mutational analysis of Bim(S) showed that mitochondrial targeting, but not binding to Bcl-2 or Mcl-1, was required for apoptosis induction. In yeast, Bim(S) enhanced the killing activity of Bax in the absence of anti-apoptotic Bcl-2 proteins. Thus, cell death induction by a BH3-only protein can occur through a process that is independent of anti-apoptotic Bcl-2 proteins but requires mitochondrial targeting.

Cleavage of the NF-κB family protein p65/RelA by the chlamydial protease-like activity factor (CPAF) impairs proinflammatory signaling in cells infected with Chlamydiae.

Chlamydiae are obligate intracellular bacteria that frequently cause human disease. Chlamydiae replicate in a membranous vacuole in the cytoplasm termed inclusion but have the ability to transport proteins into the host cell cytosol. Chlamydial replication is associated with numerous changes of host cell functions, and these changes are often linked to proteolytic events. It has been shown earlier that the member of the NF-κB family of inflammation-associated transcription factors, p65/RelA, is cleaved during chlamydial infection, and a chlamydial protease has been implicated. We here provide evidence that the chlamydial protease chlamydial protease-like activity factor (CPAF) is responsible for degradation of p65/RelA during infection. This degradation was seen in human and in mouse cells infected with either Chlamydia trachomatis or Chlamydia pneumoniae where it correlated with the expression of CPAF and CPAF activity. Isolated expression of active C. trachomatis or C. pneumoniae CPAF in human or mouse cells yielded a p65 fragment of indistinguishable size from the one generated during infection. Expression of active CPAF in human cells caused a mild reduction in IκBα phosphorylation but a strong reduction in NF-κB reporter activity in response to interleukin-1ß. Infection with C. trachomatis likewise reduced this responsiveness. IL-1ß-dependent secretion of IL-8 was further reduced by CPAF expression. Secretion of CPAF is, thus, a mechanism that reduces host cell sensitivity to a proinflammatory stimulus, which may facilitate bacterial growth in vivo.

Evolutionary conservation of biogenesis of beta-barrel membrane proteins.

The outer membranes of mitochondria and chloroplasts are distinguished by the presence of beta-barrel membrane proteins. The outer membrane of Gram-negative bacteria also harbours beta-barrel proteins. In mitochondria these proteins fulfil a variety of functions such as transport of small molecules (porin/VDAC), translocation of proteins (Tom40) and regulation of mitochondrial morphology (Mdm10). These proteins are encoded by the nucleus, synthesized in the cytosol, targeted to mitochondria as chaperone-bound species, recognized by the translocase of the outer membrane, and then inserted into the outer membrane where they assemble into functional oligomers. Whereas some knowledge has been accumulated on the pathways of insertion of proteins that span cellular membranes with alpha-helical segments, very little is known about how beta-barrel proteins are integrated into lipid bilayers and assembled into oligomeric structures. Here we describe a protein complex that is essential for the topogenesis of mitochondrial outer membrane beta-barrel proteins (TOB). We present evidence that important elements of the topogenesis of beta-barrel membrane proteins have been conserved during the evolution of mitochondria from endosymbiotic bacterial ancestors.

Biogenesis of beta-barrel membrane proteins of mitochondria.

beta-Barrel membrane proteins have several important functions in outer membranes of Gram-negative bacteria and in the organelles of endosymbiotic origin, mitochondria and chloroplasts. The biogenesis of beta-barrel membrane proteins was, until recently, an unresolved process. A breakthrough was achieved when a specific pathway for the insertion of beta-barrel outer-membrane proteins was identified in both mitochondria and Gram-negative bacteria. The key component of this pathway is Tob55 (also known as Sam50) in mitochondria and Omp85 in bacteria, both beta-barrel membrane proteins themselves. Tob55 is part of the hetero-oligomeric TOB (topogenesis of mitochondrial outer-membrane beta-barrel proteins) or SAM (sorting and assembly of mitochondria) complex, which is present in the mitochondrial outer membrane. Tob55 belongs to an evolutionarily conserved protein family, the members of which are present in almost all eukaryotes and in Gram-negative bacteria and chloroplasts. Thus, is it emphasized that the insertion pathway of mitochondrial beta-barrel membrane proteins was conserved during evolution of mitochondria from endosymbiotic bacterial ancestors.

Chlamydia trachomatis can protect host cells against apoptosis in the absence of cellular Inhibitor of Apoptosis Proteins and Mcl-1.

Infection with Chlamydia protects mammalian host cells against apoptosis. Hypotheses have been proposed to explain this molecularly, including the up-regulation of host anti-apoptotic proteins such as cellular Inhibitor of Apoptosis Protein (IAP) 2 and the Bcl-2 protein Mcl-1. To test for the importance of these proteins, we used mouse embryonic fibroblasts from gene-targeted mice that were deficient in cIAP1, cIAP2, cIAP1/cIAP2, XIAP, or Mcl-1. Infection with Chlamydia trachomatis protected all cells equally well against apoptosis, which was induced either with tumour necrosis factor/cycloheximide (IAP-knock-out cells) or staurosporine (Mcl-1-knock-out). Therefore, these cellular anti-apoptotic proteins are not essential for apoptosis-protection by C. trachomatis.

Cytopathicity of Chlamydia is largely reproduced by expression of a single chlamydial protease.

Chlamydiae replicate in a vacuole within epithelial cells and commonly induce cell damage and a deleterious inflammatory response of unknown molecular pathogenesis. The chlamydial protease-like activity factor (CPAF) translocates from the vacuole to the cytosol, where it cleaves several cellular proteins. CPAF is synthesized as an inactive precursor that is processed and activated during infection. Here, we show that CPAF can be activated in uninfected cells by experimentally induced oligomerization, reminiscent of the activation mode of initiator caspases. CPAF activity induces proteolysis of cellular substrates including two novel targets, cyclin B1 and PARP, and indirectly results in the processing of pro-apoptotic BH3-only proteins. CPAF activation induces striking morphological changes in the cell and, later, cell death. Biochemical and ultrastructural analysis of the cell death pathway identify the mechanism of cell death as nonapoptotic. Active CPAF in uninfected human cells thus mimics many features of chlamydial infection, implicating CPAF as a major factor of chlamydial pathogenicity, Chlamydia-associated cell damage, and inflammation.

Therapeutic targets in the mitochondrial apoptotic pathway.

Every cell in the human body has most of the components of the apoptotic apparatus and is thus principally equipped to die by apoptosis. Situations of increased or decreased apoptosis contribute to many forms of human disease, making this pathway an attractive target of therapeutic intervention. The past few years have seen an enormous refinement in the understanding how apoptosis works on a molecular level and the role of mitochondria as a central element in apoptotic signal transduction has become obvious. Here, the authors consider the events that are critical in this mitochondrial pathway, in particular at mitochondria but also upstream and downstream. The authors' opinion is presented on the merits and feasibility of approaches that aim at treating disease by interfering with the mitochondrial apoptotic pathway.

Mitochondrial protein import: a matter of death?

Mitochondria play a central role not only in energy generation but also for apoptosis. A key step in mitochondrial apoptosis is the release of mitochondrial proteins, most importantly cytochrome c. This release is orchestrated by the pro- and anti-apoptotic members of the Bcl-2 protein family. The functions of these Bcl-2 family members are clear in terms of order and of principle: the pro-apoptotic BH3-only protein group contains the triggers, which cause the activation of the effectors Bax and Bak, while the anti-apoptotic Bcl-2-like proteins prevent this activation. However, the molecular details are still insufficiently clear and the proposed models have certain gaps and are partly contradictory. We have recently presented evidence that targeting to mitochondria of at least one BH3-only protein is essential for its pro-apoptotic functions. Here we discuss how this mechanism might fit into and expand existing models and speculate about the potential implications of this finding.

Characterization of host cell death induced by Chlamydia trachomatis.

Chlamydia are obligate intracellular bacteria that modulate apoptosis of the host cell. Strikingly, chlamydial infection has been reported both to inhibit and to induce apoptosis. Although the ability to inhibit apoptosis has been corroborated by the identification of cellular targets, confirmation of cell death induction has been complicated by a mixture of apoptotic features and atypical cell death during infection, as well as by differences in the experimental techniques used to measure cell death. Here we use a panel of well-established approaches in the study of apoptosis to define the form of cell death induced by Chlamydia trachomatis infection. Infected cells displayed apoptotic features such as nuclear condensation and fragmentation, as well as positive TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining. Fragmentation of genomic DNA occurred, but was atypical. Clear evidence against the activation of effector caspases was found. Nuclear changes were measured in fibroblasts lacking one or both of the effectors of mitochondrial apoptosis, Bax and Bak. A slight reduction in nuclear changes was observed in Bax-deficient cells and in Bax/Bak double-deficient cells. Most surprisingly, this reduction was almost complete in Bak-deficient cells. Finally, dying infected cells were efficiently taken up by professional phagocytes, suggesting that Chlamydia-induced host-cell death could play a role in the immune response. In conclusion, chlamydial infection can induce cell death. Although Chlamydia-induced cell death has certain morphological features of apoptosis, it does not result from activation of the apoptotic pathway.

Tob38, a novel essential component in the biogenesis of beta-barrel proteins of mitochondria.

Insertion of beta-barrel proteins into the outer membrane of mitochondria is mediated by the TOB complex. Known constituents of this complex are Tob55 and Mas37. We identified a novel component, Tob38. It is essential for viability of yeast and the function of the TOB complex. Tob38 is exposed on the surface of the mitochondrial outer membrane. It interacts with Mas37 and Tob55 and is associated with Tob55 even in the absence of Mas37. The Tob38-Tob55 core complex binds precursors of beta-barrel proteins and facilitates their insertion into the outer membrane. Depletion of Tob38 results in strongly reduced levels of Tob55 and Mas37 and the residual proteins no longer form a complex. Tob38-depleted mitochondria are deficient in the import of beta-barrel precursor proteins, but not of other outer membrane proteins or proteins of other mitochondrial subcompartments. We conclude that Tob38 has a crucial function in the biogenesis of beta-barrel proteins of mitochondria.

The protein import motor of mitochondria: a targeted molecular ratchet driving unfolding and translocation.

Unfolding and import of preproteins into mitochondria are facilitated by a molecular motor in which heat shock protein 70 (Hsp70) in the matrix plays an essential role. Here we present two different experimental approaches to analyze mechanisms underlying this function of Hsp70. First, preproteins containing stretches of glutamic acid (polyE) or glycine (polyG) repeats in front of folded domains were imported into mitochondria. This occurred although Hsp70 cannot pull on these stretches to unfold the folded domains, since it does not bind to polyE and polyG. Secondly, preproteins containing titin immunoglobulin (Ig)-like domains were imported into mitochondria, despite the fact that forces of >200 pN are required to mechanically unfold these domains. Since molecular motors generate forces of approximately 5 pN, Hsp70 could not promote unfolding of the Ig-like domains by mechanical pulling. Our observations suggest that Hsp70 acts as an element of a Brownian ratchet, which mediates unfolding and translocation of preproteins across the mitochondrial membranes.

Tim50, a novel component of the TIM23 preprotein translocase of mitochondria.

The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. We isolated the TIM23 complex of Neurospora crassa. Besides Tim23 and Tim17, it contained a novel component, referred to as Tim50. Tim50 spans the inner membrane with a single transmembrane segment and exposes a large hydrophilic domain in the intermembrane space. Tim50 is essential for viability of yeast. Mitochondria from cells depleted of Tim50 displayed strongly reduced import kinetics of preproteins using the TIM23 complex. Tim50 could be cross-linked to preproteins that were halted at the level of the translocase of the outer membrane (TOM complex) or spanning both TOM and TIM23 complexes. We suggest that Tim50 plays a crucial role in the transfer of preproteins from the TOM complex to the TIM23 complex through the intermembrane space.