Gerhard Krumschnabel

Caspase-2 activation in the absence of PIDDosome formation

PIDD (p53-induced protein with a death domain [DD]), together with the bipartite adapter protein RAIDD (receptor-interacting protein-associated ICH-1/CED-3 homologous protein with a DD), is implicated in the activation of pro–caspase-2 in a high molecular weight complex called the PIDDosome during apoptosis induction after DNA damage. To investigate the role of PIDD in cell death initiation, we generated PIDD-deficient mice. Processing of caspase-2 is readily detected in the absence of PIDDosome formation in primary lymphocytes. Although caspase-2 processing is delayed in simian virus 40–immortalized pidd−/− mouse embryonic fibroblasts, it still depends on loss of mitochondrial integrity and effector caspase activation. Consistently, apoptosis occurs normally in all cell types analyzed, suggesting alternative biological roles for caspase-2 after DNA damage. Because loss of either PIDD or its adapter molecule RAIDD did not affect subcellular localization, nuclear translocation, or caspase-2 activation in high molecular weight complexes, we suggest that at least one alternative PIDDosome-independent mechanism of caspase-2 activation exists in mammals in response to DNA damage.

The enigma of caspase-2: the laymen's view.

Proteolysis of cellular substrates by caspases (cysteine-dependent aspartate-specific proteases) is one of the hallmarks of apoptotic cell death. Although the activation of apoptotic caspases is considered a ‘late-stage’ event in apoptosis signaling, past the commitment stage, one caspase family member, caspase-2, splits the cell death community into half – those searching for evidence of an apical initiator function of this molecule and those considering it as an amplifier of the apoptotic caspase cascade, at best, if relevant for apoptosis at all. This review screens past and present biochemical as well as genetic evidence for caspase-2 function in cell death signaling and beyond.

Apoptosis of leukocytes triggered by acute DNA damage promotes lymphoma formation

Apoptosis triggered by p53 upon DNA damage secures removal of cells with compromised genomes, and is thought to prevent tumorigenesis. In contrast, we provide evidence that p53-induced apoptosis can actively drive tumor formation. Mice defective in p53-induced apoptosis due to loss of its proapoptotic target gene, puma, resist gamma-irradiation (IR)-induced lymphomagenesis. In wild-type animals, repeated irradiation injury-induced expansion of hematopoietic stem/progenitor cells (HSCs) leads to lymphoma formation. Puma(-/-) HSCs, protected from IR-induced cell death, show reduced compensatory proliferation and replication stress-associated DNA damage, and fail to form thymic lymphomas, demonstrating that the maintenance of stem/progenitor cell homeostasis is critical to prevent IR-induced tumorigenesis.

Fish as model systems for the study of vertebrate apoptosis

Apoptosis is a process of pivotal importance for multi-cellular organisms and due to its implication in the development of cancer and degenerative disease it is intensively studied in humans and mammalian model systems. Invertebrate models of apoptosis have been well-studied, especially in C. elegans and D. melanogaster, but as these are evolutionarily distant from mammals the relevance of findings for human research is sometimes limited. Presently, a non-mammalian vertebrate model for studying apoptosis is missing. However, in the past few years an increasing number of studies on cell death in fish have been published and thus new model systems may emerge. This review aims at highlighting the most important of these findings, showing similarities and dissimilarities between fish and mammals, and will suggest topics for future research. In addition, the outstanding usefulness of fish as research models will be pointed out, hoping to spark future research on this exciting, often underrated group of vertebrates.

Coupling of Energy Supply and Energy Demand in Isolated Goldfish Hepatocytes

The allocation of metabolic energy under fluctuating conditions of supply and demand was studied in isolated goldfish hepatocytes. Inhibition of glycolytic ATP production resulted in a decrease in the activity of one of the major ATP consuming components of cellular energy budgets, the sodium pump ((Na⁺, K⁺)-ATPase), because of the removal of substrates for the trichloroacetic acid (TCA) cycle. This was confirmed through restoration of activity to control levels by adding pyruvate and malate to the suspension. Ouabain-sensitive oxygen consumption constituted a fairly constant fraction of 15% to 25% of total oxygen consumption, whereas the relative load exerted by the pump on anaerobic energy metabolism (on the basis of measurements of Rb⁺ flux and lactate production) was much higher, ranging from 90% to 50% during the experimental period. Moreover, when pump activity was inhibited by the addition of ouabain, oxidative energy output decreased immediately, whereas under the restricted conditions of glycolytic energy supply a corresponding ouabain-sensitive component of lactate production was not observed. Such an apparent mismatch between supply and demand ofATP is interpreted as reflecting the flexibility of energy allocation under energy-limiting conditions. When ATP production was reduced by inhibiting either glycolysis or oxidative phosphorylation, the concentration of ATP in the hepatocytes dropped within 30 min from the control steady state to a new, lower, steady state that was maintained for at least 60 min.

High-Resolution Respirometry for Simultaneous Measurement of Oxygen and Hydrogen Peroxide Fluxes in Permeabilized Cells, Tissue Homogenate and Isolated Mitochondria

Whereas mitochondria are well established as the source of ATP in oxidative phosphorylation (OXPHOS), it is debated if they are also the major cellular sources of reactive oxygen species (ROS). Here we describe the novel approach of combining high-resolution respirometry and fluorometric measurement of hydrogen peroxide (H2O2) production, applied to mitochondrial preparations (permeabilized cells, tissue homogenate, isolated mitochondria). The widely used H2O2 probe Amplex Red inhibited respiration in intact and permeabilized cells and should not be applied at concentrations above 10 µM. H2O2 fluxes were generally less than 1% of oxygen fluxes in physiological substrate and coupling states, specifically in permeabilized cells. H2O2 flux was consistently highest in the Complex II-linked LEAK state, reduced with CI&II-linked convergent electron flow and in mitochondria respiring at OXPHOS capacity, and were further diminished in noncoupled mitochondria respiring at electron transfer system capacity. Simultaneous measurement of mitochondrial respiration and H2O2 flux requires careful optimization of assay conditions and reveals information on mitochondrial function beyond separate analysis of ROS production.

PIDDosome-independent tumor suppression by Caspase-2

The PIDDosome, a multiprotein complex constituted of the ‘p53-induced protein with a death domain (PIDD), ‘receptor-interacting protein (RIP)-associated ICH-1/CED-3 homologous protein with a death domain’ (RAIDD) and pro-Caspase-2 has been defined as an activating platform for this apoptosis-related protease. PIDD has been implicated in p53-mediated cell death in response to DNA damage but also in DNA repair and nuclear factor kappa-light-chain enhancer (NF-κB) activation upon genotoxic stress, together with RIP-1 kinase and Nemo/IKKγ. As all these cellular responses are critical for tumor suppression and deregulated expression of individual PIDDosome components has been noted in human cancer, we investigated their role in oncogenesis induced by DNA damage or oncogenic stress in gene-ablated mice. We observed that Pidd or Caspase-2 failed to suppress lymphoma formation triggered by γ-irradiation or 3-methylcholanthrene-driven fibrosarcoma development. In contrast, Caspase-2 showed tumor suppressive capacity in response to aberrant c-Myc expression, which did not rely on PIDD, the BH3-only protein Bid (BH3 interacting domain death agonist) or the death receptor ligand Trail (TNF-related apoptosis-inducing ligand), but associated with reduced rates of p53 loss and increased extranodal dissemination of tumor cells. In contrast, Pidd deficiency associated with abnormal M-phase progression and delayed disease onset, indicating that both proteins are differentially engaged upon oncogenic stress triggered by c-Myc, leading to opposing effects on tumor-free survival.

Apoptosis and necroptosis are induced in rainbow trout cell lines exposed to cadmium.

Cadmium is an important environmental toxicant that can kill cells. A number of studies have implicated apoptosis as well as necrosis and, most recently, a form of programmed necrosis termed necroptosis in the process of cadmium-mediated toxicity, but the exact mechanism remains ill-defined and may depend on the affected cell type. This study investigated which mode of cell death may be responsible for cell death induction in cadmium-exposed trout cell lines from gill and liver and if this cell death was sensitive to inhibitors of necroptosis or apoptosis, respectively. It was observed that intermediate levels of cadmium that killed approximately 50% of the cells over 96-120h of exposure caused cell death that morphologically resembled apoptosis and was associated with an increase of apoptotic markers such as the number of cells with diminished DNA content (sub-G1 cells), condensed or fragmented nuclei, and elevation of caspase-3 activity. At the same time, however, cells also lost plasma membrane integrity, as indicated by uptake of propidium iodide, showed a decrease of ATP levels and mitochondrial membrane potential, and displayed cell swelling, signs associated with secondary necrosis, or equally possible, necroptotic cell death. Importantly, many of these alterations were at least partly inhibited by the necroptosis inhibitor necrostatin-1 and were to a lesser extent also sensitive to the pan-caspase inhibitor zVAD-fmk, indicating that multiple modes of cell death are concurrently induced in cadmium-exposed trout cells, including necroptosis and apoptosis. Cell death appeared to lack concurrent radical formation, consistent with genetically regulated necroptotic cell death, but was characterized by the rapid induction of DNA damage markers, and the early onset of disintegration of the Golgi complex. Comparative experiments evaluating copper-toxicity indicated that in comparison to cadmium much higher concentrations of this metal were required to induce cell death and that neither necrostatin-1 nor a pan-caspase inhibitor conferred protection, suggesting that additional modes of cell death can be triggered in response to poisoning with heavy metals.

BH3-only protein Bmf mediates apoptosis upon inhibition of CAP-dependent protein synthesis

Tight transcriptional regulation, alternative splicing and/or post-translational modifications of BH3-only proteins fine-tune their proapoptotic function. In this study, we characterize the gene locus of the BH3-only protein Bmf (Bcl-2-modifying factor) and describe the generation of two major isoforms from a common transcript in which initiation of protein synthesis involves leucine-coding CUG. BmfCUG and the originally described isoform, Bmf-short, display comparable binding affinities to prosurvival Bcl-2 family members, localize preferentially to the outer mitochondrial membrane and induce rapid Bcl-2-blockable apoptosis. Notably, endogenous Bmf expression is induced on forms of cell stress known to cause repression of the CAP-dependent translation machinery such as serum deprivation, hypoxia, inhibition of the PI3K/AKT pathway or mTOR, as well as direct pharmacological inhibition of the eukaryotic translation initiation factor eIF-4E. Knock down or deletion of Bmf reduces apoptosis under some of these conditions, demonstrating that Bmf can act as a sentinel for stress-impaired CAP-dependent protein translation machinery.

Caspase-2: killer, savior and safeguard—emerging versatile roles for an ill-defined caspase

Despite the early discovery of caspase-2, its physiological function has long remained an enigma. A number of recent publications now suggest not just one, but multiple functions, including roles in apoptosis, DNA repair and tumor suppression. How can one enzyme have so many talents? Considering the diversity of interaction partners and the specific mode of pro-apoptotic action proposed in these studies, caspase-2 might in fact represent a primordial protease serving numerous pathways, established before the advent of a more elaborate functionally diversified caspases system.