- The C-terminus of p63 contains multiple regulatory elements with different functions (2010)
- The transcription factor p63 is expressed as at least six different isoforms, of which two have been assigned critical biological roles within ectodermal development and skin stem cell biology on the one hand and supervision of the genetic stability of oocytes on the other hand. These two isoforms contain a C-terminal inhibitory domain that negatively regulates their transcriptional activity. This inhibitory domain contains two individual components: one that uses an internal binding mechanism to interact with and mask the transactivation domain and one that is based on sumoylation. We have carried out an extensive alanine scanning study to identify critical regions within the inhibitory domain. These experiments show that a stretch of ~13 amino acids is crucial for the binding function. Further, investigation of transcriptional activity and the intracellular level of mutants that cannot be sumoylated suggests that sumoylation reduces the concentration of p63. We therefore propose that the inhibitory function of the C-terminal domain is in part due to direct inhibition of the transcriptional activity of the protein and in part due to indirect inhibition by controlling the concentration of p63. Keywords: p63, transcriptional regulation, auto-inhibition, sumoylation
- IAP-IAP complexes required for apoptosis resistance of C. trachomatis-infected cells (2006)
- Host cells infected with obligate intracellular bacteria Chlamydia trachomatis are profoundly resistant to diverse apoptotic stimuli. The molecular mechanisms underlying the block in apoptotic signaling of infected cells is not well understood. Here we investigated the molecular mechanism by which apoptosis induced via the tumor necrosis factor (TNF) receptor is prevented in infected epithelial cells. Infection with C. trachomatis leads to the up-regulation of cellular inhibitor of apoptosis (cIAP)-2, and interfering with cIAP-2 up-regulation sensitized infected cells for TNF-induced apoptosis. Interestingly, besides cIAP-2, cIAP-1 and X-linked IAP, although not differentially regulated by infection, are required to maintain apoptosis resistance in infected cells. We detected that IAPs are constitutively organized in heteromeric complexes and small interfering RNA-mediated silencing of one of these IAPs affects the stability of another IAP. In particular, the stability of cIAP-2 is modulated by the presence of X-linked IAP and their interaction is stabilized in infected cells. Our observations suggest that IAPs are functional and stable as heteromers, a thus far undiscovered mechanism of IAP regulation and its role in modulation of apoptosis.
- IAPs as E3 ligases of Rac1 : shaping the move (2012)
- Inhibitors of Apoptosis Proteins (IAPs) are well-studied E3 ubiquitin ligases predominantly known for regulation of apoptosis. We uncovered that IAPs can function as a direct E3 ubiquitin ligase of RhoGTPase Rac1. cIAP1 and XIAP directly conjugate polyubiquitin chains to Lysine 147 of activated Rac1 and target it for proteasomal degradation. Consistently, loss of these IAPs by various strategies led to stabilization of Rac1 and mesenchymal mode of migration in tumor cells. IAPs also regulate Rac1 degradation upon RhoGDI1 depletion and CNF1 toxin treatment. Our observations revealed an evolutionarily conserved role of IAPs in regulating Rac1 stability shedding light on to the mechanisms behind ubiquitination–dependent inactivation of Rac1 signaling.
- Neisseria meningitidis differentially controls host cell motility through PilC1 and PilC2 components of type IV pili (2009)
- Neisseria meningitidis is a strictly human pathogen that has two facets since asymptomatic carriage can unpredictably turn into fulminant forms of infection. Meningococcal pathogenesis relies on the ability of the bacteria to break host epithelial or endothelial cellular barriers. Highly restrictive, yet poorly understood, mechanisms allow meningococcal adhesion to cells of only human origin. Adhesion of encapsulated and virulent meningococci to human cells relies on the expression of bacterial type four pili (T4P) that trigger intense host cell signalling. Among the components of the meningococcal T4P, the concomitantly expressed PilC1 and PilC2 proteins regulate pili exposure at the bacterial surface, and until now, PilC1 was believed to be specifically responsible for T4P-mediated meningococcal adhesion to human cells. Contrary to previous reports, we show that, like PilC1, the meningococcal PilC2 component is capable of mediating adhesion to human ME180 epithelial cells, with cortical plaque formation and F-actin condensation. However, PilC1 and PilC2 promote different effects on infected cells. Cellular tracking analysis revealed that PilC1-expressing meningococci caused a severe reduction in the motility of infected cells, which was not the case when cells were infected with PilC2-expressing strains. The amount of both total and phosphorylated forms of EGFR was dramatically reduced in cells upon PilC1-mediated infection. In contrast, PilC2-mediated infection did not notably affect the EGFR pathway, and these specificities were shared among unrelated meningococcal strains. These results suggest that meningococci have evolved a highly discriminative tool for differential adhesion in specific microenvironments where different cell types are present. Moreover, the fine-tuning of cellular control through the combined action of two concomitantly expressed, but distinctly regulated, T4P-associated variants of the same molecule (i.e. PilC1 and PilC2) brings a new model to light for the analysis of the interplay between pathogenic bacteria and human host cells.
- Mcl-1 is a key regulator of apoptosis resistance in Chlamydia trachomatis-infected cells (2008)
- Chlamydia are obligate intracellular bacteria that cause variety of human diseases. Host cells infected with Chlamydia are protected against many different apoptotic stimuli. The induction of apoptosis resistance is thought to be an important immune escape mechanism allowing Chlamydia to replicate inside the host cell. Infection with C. trachomatis activates the Raf/MEK/ERK pathway and the PI3K/AKT pathway. Here we show that inhibition of these two pathways by chemical inhibitors sensitized C. trachomatis infected cells to granzyme B-mediated cell death. Infection leads to the Raf/MEK/ERK-mediated up-regulation and PI3K-dependent stabilization of the anti-apoptotic Bcl-2 family member Mcl-1. Consistently, interfering with Mcl-1 up-regulation sensitized infected cells for apoptosis induced via the TNF receptor, DNA damage, granzyme B and stress. Our data suggest that Mcl-1 up-regulation is primarily required to maintain apoptosis resistance in C. trachomatis-infected cells.
- Bacterial porin disrupts mitochondrial membrane potential and sensitizes host cells to apoptosis (2009)
- The bacterial PorB porin, an ATP-binding beta-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (delta psi m). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of beta-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of delta psi m. The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce delta psi m loss and apoptosis, demonstrating that dissipation of delta psi m is a requirement for cell death caused by neisserial infection.
- Bim and Bmf synergize to induce apoptosis in Neisseria gonorrhoeae infection (2009)
- Abstract: Bcl-2 family proteins including the pro-apoptotic BH3-only proteins are central regulators of apoptotic cell death. Here we show by a focused siRNA miniscreen that the synergistic action of the BH3-only proteins Bim and Bmf is required for apoptosis induced by infection with Neisseria gonorrhoeae (Ngo). While Bim and Bmf were associated with the cytoskeleton of healthy cells, they both were released upon Ngo infection. Loss of Bim and Bmf from the cytoskeleton fraction required the activation of Jun-N-terminal kinase-1 (JNK-1), which in turn depended on Rac-1. Depletion and inhibition of Rac-1, JNK-1, Bim, or Bmf prevented the activation of Bak and Bax and the subsequent activation of caspases. Apoptosis could be reconstituted in Bim-depleted and Bmf-depleted cells by additional silencing of antiapoptotic Mcl-1 and Bcl-XL, respectively. Our data indicate a synergistic role for both cytoskeletal-associated BH3-only proteins, Bim, and Bmf, in an apoptotic pathway leading to the clearance of Ngo-infected cells. Author Summary: A variety of physiological death signals, as well as pathological insults, trigger apoptosis, a genetically programmed form of cell death. Pathogens often induce host cell apoptosis to establish a successful infection. Neisseria gonorrhoeae (Ngo), the etiological agent of the sexually transmitted disease gonorrhoea, is a highly adapted obligate human-specific pathogen and has been shown to induce apoptosis in infected cells. Here we unveil the molecular mechanisms leading to apoptosis of infected cells. We show that Ngo-mediated apoptosis requires a special subset of proapoptotic proteins from the group of BH3-only proteins. BH3-only proteins act as stress sensors to translate toxic environmental signals to the initiation of apoptosis. In a siRNA-based miniscreen, we found Bim and Bmf, BH3-only proteins associated with the cytoskeleton, necessary to induce host cell apoptosis upon infection. Bim and Bmf inactivated different inhibitors of apoptosis and thereby induced cell death in response to infection. Our data unveil a novel pathway of infection-induced apoptosis that enhances our understanding of the mechanism by which BH3-only proteins control apoptotic cell death.
- Ubiquitylation in immune disorders and cancer: from molecular mechanisms to therapeutic implications (2012)
- Conjugation of ubiquitin to proteins (ubiquitylation) has emerged to be one of the most crucial post-translational modifications controlling virtually all cellular processes. What was once regarded as a mere signal for protein degradation has turned out to be a major regulator of molecular signalling networks. Deregulation of ubiquitin signalling is closely associated with various human pathologies. Here, we summarize the current knowledge of ubiquitin signalling in immune deficiencies and cancer as well as the available therapeutic strategies targeting the ubiquitin system in combating these pathogenic conditions.
- IAPs on the move: role of inhibitors of apoptosis proteins in cell migration (2013)
- Inhibitors of Apoptosis Proteins (IAPs) are a class of highly conserved proteins predominantly known for the regulation of caspases and immune signaling. However, recent evidence suggests a crucial role for these molecules in the regulation of tumor cell shape and migration by controlling MAPK, NF-κB and Rho GTPases. IAPs directly control Rho GTPases, thus regulating cell shape and migration. For instance, XIAP and cIAP1 function as the direct E3 ubiquitin ligases of Rac1 and target it for proteasomal degradation. IAPs are differentially expressed in tumor cells and have been targeted by several cancer therapeutic drugs that are currently in clinical trials. Here, we summarize the current knowledge on the role of IAPs in the regulation of cell migration and discuss the possible implications of these observations in regulating tumor cell metastases.