Xavier Saelens

Toxic proteins released from mitochondria in cell death

A plethora of apoptotic stimuli converge on the mitochondria and affect their membrane integrity. As a consequence, multiple death-promoting factors residing in the mitochondrial intermembrane space are liberated in the cytosol. Pro- and antiapoptotic Bcl-2 family proteins control the release of these mitochondrial proteins by inducing or preventing permeabilization of the outer mitochondrial membrane. Once released into the cytosol, these mitochondrial proteins activate both caspase-dependent and -independent cell death pathways. Cytochrome c was the first protein shown to be released from the mitochondria into the cytosol, where it induces apoptosome formation. Other released mitochondrial proteins include apoptosis-inducing factor (AIF) and endonuclease G, both of which contribute to apoptotic nuclear DNA damage in a caspase-independent way. Other examples are Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP-binding protein with low PI) and the serine protease HtrA2/OMI (high-temperature requirement protein A2), which both promote caspase activation and instigate caspase-independent cytotoxicity. The precise mode of action and importance of cytochrome c in apoptosis in mammalian cells has become clear through biochemical, structural and genetic studies. More recently identified factors, for example HtrA2/OMI and Smac/DIABLO, are still being studied intensively in order to delineate their functions in apoptosis. A better understanding of these functions may help to develop new strategies to treat cancer.

A UNIVERSAL INFLUENZA A VACCINE BASED ON THE EXTRACELLULAR DOMAIN OF THE M2 PROTEIN

The antigenic variation of influenza virus represents a major health problem. However, the extracellular domain of the minor, virus-coded M2 protein is nearly invariant in all influenza A strains. We genetically fused this M2 domain to the hepatitis B virus core (HBc) protein to create fusion gene coding for M2HBc; this gene was efficiently expressed in Escherichia coli. Intraperitoneal or intranasal administration of purified M2HBc particles to mice provided 90–100% protection against a lethal virus challenge. The protection was mediated by antibodies, as it was transferable by serum. The enhanced immunogenicity of the M2 extracellular domain exposed on HBc particles allows broad-spectrum, long-lasting protection against influenza A infections.

The role of mitochondrial factors in apoptosis: a Russian roulette with more than one bullet

Mitochondria are ‘life-essential’ organelles for the production of metabolic energy in the form of ATP. Paradoxically mitochondria also play a key role in controlling the pathways that lead to cell death. This latter role of mitochondria is more than just a ‘loss of function’ resulting in an energy deficit but is an active process involving different mitochondrial proteins. Cytochrome c was the first characterised mitochondrial factor shown to be released from the mitochondrial intermembrane space and to be actively implicated in apoptotic cell death. Since then, other mitochondrial proteins, such as AIF, Smac/DIABLO, endonuclease G and Omi/HtrA2, were found to undergo release during apoptosis and have been implicated in various aspects of the cell death process. Members of the Bcl-2 protein family control the integrity and response of mitochondria to apoptotic signals. The molecular mechanism by which mitochondrial intermembrane space proteins are released and the regulation of mitochondrial homeostasis by Bcl-2 proteins is still elusive. This review summarises and evaluates the current knowledge concerning the complex role of released mitochondrial proteins in the apoptotic process.

Mitochondrial intermembrane proteins in cell death.

Apoptosis is a form of programmed cell death important in the development and tissue homeostasis of multicellular organisms. Mitochondria have, next to their function in respiration, an important role in the apoptotic-signaling pathway. Malfunctioning at any level of the cell is eventually translated in the release of apoptogenic factors from the mitochondrial intermembrane space resulting in the organized demise of the cell. Some of these factors, such as AIF and endonuclease G, appear to be highly conserved during evolution. Other factors, like cytochrome c, have gained their apoptogenic function later during evolution. In this review, we focus on the role of cytochrome c, AIF, endonuclease G, Smac/DIABLO, Omi/HtrA2, Acyl-CoA-binding protein, and polypyrimidine tract-binding protein in the initiation and modulation of cell death in different model organisms. These mitochondrial factors may contribute to both caspase-dependent and caspase-independent processes in apoptotic cell death.

Universal Vaccine Based on Ectodomain of Matrix Protein 2 of Influenza A: Fc Receptors and Alveolar Macrophages Mediate Protection

The ectodomain of matrix protein 2 (M2e) of influenza A virus is an attractive target for a universal influenza A vaccine: the M2e sequence is highly conserved across influenza virus subtypes, and induced humoral anti-M2e immunity protects against a lethal influenza virus challenge in animal models. Clinical phase I studies with M2e vaccine candidates have been completed. However, the in vivo mechanism of immune protection induced by M2e-carrier vaccination is unclear. Using passive immunization experiments in wild-type, FcRγ−/−, FcγRI−/−, FcγRIII−/−, and (FcγRI, FcγRIII)−/− mice, we report in this study that Fc receptors are essential for anti-M2e IgG-mediated immune protection. M2e-specific IgG1 isotype Abs are shown to require functional FcγRIII for in vivo immune protection but other anti-M2e IgG isotypes can rescue FcγRIII−/− mice from a lethal challenge. Using a conditional cell depletion protocol, we also demonstrate that alveolar macrophages (AM) play a crucial role in humoral M2e-specific immune protection. Additionally, we show that adoptive transfer of wild-type AM into (FcγRI, FcγRIII)−/− mice restores protection by passively transferred anti-M2e IgG. We conclude that AM and Fc receptor-dependent elimination of influenza A virus-infected cells are essential for protection by anti-M2e IgG.

M2e-based universal influenza A vaccine

Human influenza causes substantial morbidity and mortality. Currently, licensed influenza vaccines offer satisfactory protection if they match the infecting strain, but they come with significant drawbacks. These vaccines are derived from prototype viruses, containing the hemagglutinin of influenza viruses that are likely to cause the next epidemic. Their usefulness against a future pandemic, however, remains problematic. A vaccine based on the ectodomain of influenza matrix protein 2 (M2e) could overcome these drawbacks. M2e is highly conserved in both human and avian influenza A viruses. The low immunogenicity against natural M2e can be overcome by fusing M2e to an appropriate carrier such as Hepatitis B virus-derived virus-like particles. Such chimeric particles can be produced in a simple and safe bacterial expression system, requiring minimal biocontainment, and can be obtained in a pure form. Experiments in animal models have demonstrated that M2e-based vaccines induce protection against a lethal challenge with various influenza A virus subtypes. Furthermore, the production and use of an effective M2e-vaccine could be implemented at any time regardless of seasonality, both in an epidemic as well as in a pandemic preparedness program. In animal models, M2e-vaccines administered parenterally or intranasally protect against disease and mortality following challenge with various influenza A strains. Adjuvants suitable for human use improve protection, which correlates with higher anti-M2e antibody responses of defined subtypes. Recently, Phase I clinical studies with M2e-vaccines have been completed, indicating their safety and immunogenicity. Further clinical development of this universal influenza A vaccine candidate is being pursued in order to validate its protective efficacy in humans.

Universal M2 ectodomain-based influenza A vaccines: preclinical and clinical developments

Influenza vaccines used today are strain specific and need to be adapted every year to try and match the antigenicity of the virus strains that are predicted to cause the next epidemic. The strain specificity of the next pandemic is unpredictable. An attractive alternative approach would be to use a vaccine that matches multiple influenza virus strains, including multiple subtypes. In this review, we focus on the development and clinical potential of a vaccine that is based on the conserved ectodomain of matrix protein 2 (M2) of influenza A virus. Since 1999, a number of studies have demonstrated protection against influenza A virus challenge in animal models using chemical or genetic M2 external domain (M2e) fusion constructs. More recently, Phase I clinical studies have been conducted with M2e vaccine candidates, demonstrating their safety and immunogenicity in humans. Ultimately, and possibly in the near future, efficacy studies in humans should provide proof that this novel vaccine concept can mitigate epidemic and even pandemic influenza A virus infections.

Tipping the balance between necrosis and apoptosis in human and murine cells treated with interferon and dsRNA.

Interferons enhance the cellular antiviral response by inducing expression of protective proteins. Many of these proteins are activated by dsRNA, a typical by-product of viral infection. Here we show that type-I and type-II interferons can sensitize cells to dsRNA-induced cytotoxicity. In caspase-8- or FADD-deficient Jurkat cells dsRNA induces necrosis, instead of apoptosis. In L929sA cells dsRNA-induced necrosis involves high reactive oxygen species production. The antioxidant butylated hydroxyanisole protects cells from necrosis, but shifts the response to apoptosis. Treatment with the caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp(OMe)-fluoromethylketone or overexpression of Bcl-2 prevent this shift and promote necrosis. Our results suggest that a single stimulus can initiate different death-signaling pathways, leading to either necrotic or apoptotic cell death. Inhibition of key events in these signaling pathways, such as caspase activation, cytochrome c release or mitochondrial reactive oxygen species production, tips the balance between necrosis and apoptosis, leading to dominance of one of these death programs.

An Influenza A Vaccine Based on Tetrameric Ectodomain of Matrix Protein 2

Matrix protein 2 (M2) of influenza A is a tetrameric type III membrane protein that functions as a proton-selective channel. The extracellular domain (M2e) has remained nearly invariable since the first human influenza strain was isolated in 1933. By linking a modified form of the leucine zipper of the yeast transcription factor GCN4 to M2e, we obtained a recombinant tetrameric protein, M2e-tGCN4. This protein mimics the quaternary structure of the ectodomain of the natural M2 protein. M2e-tGCN4 was purified, biochemically characterized, and used to immunize BALB/c mice. High M2e-specific serum IgG antibody titers were obtained following either intraperitoneal or intranasal administration. Immunized mice were protected fully against a potentially lethal influenza A virus challenge. Antibodies raised by M2e-tGCN4 immunization specifically bound to the surface of influenza-infected cells and to an M2-expressing cell line. Using a M2e peptide competition enzyme-linked immunosorbent assay with M2-expressing cells as target, we obtained evidence that M2e-tGCN4 induces antibodies that are specific for the native tetrameric M2 ectodomain. Therefore, fusion of an oligomerization domain to the extracellular part of a transmembrane protein allows it to mimic the natural quaternary structure and can promote the induction of oligomer-specific antibodies.

Differential signaling to apoptotic and necrotic cell death by Fas-associated death domain protein FADD.

Two general pathways for cell death have been defined, apoptosis and necrosis. Previous studies in Jurkat cells have demonstrated that the Fas-associated death domain (FADD) is required for Fas-mediated signaling to apoptosis and necrosis. Here we developed L929rTA cell lines that allow Tet-on inducible expression and FK506-binding protein (FKBP)-mediated dimerization of FADD, FADD-death effector domain (FADD-DED), or FADD-death domain (FADD-DD). We show that expression and dimerization of FADD leads to necrosis. However, pretreatment of the cells with the Hsp90 inhibitor geldanamycin, which leads to proteasome-mediated degradation of receptor interacting protein 1 (RIP1), reverts FKBP-FADD-induced necrosis to apoptosis. Expression and dimerization of FADD-DD mediates necrotic cell death. We found that FADD-DD is able to bind RIP1, another protein necessary for Fas-mediated necrosis. Expression and dimerization of FADD-DED initiates apoptosis. Remarkably, in the presence of caspase inhibitors, FADD-DED mediates necrotic cell death. Coimmunoprecipitation studies revealed that FADD-DED in the absence procaspase-8 C/A is also capable of recruiting RIP1. However, when procaspase-8 C/A and RIP1 are expressed simultaneously, FADD-DED preferentially recruits procaspase-8 C/A.