[Show abstract][Hide abstract] ABSTRACT: The caspase family of proteases cleaves large number of proteins resulting in major morphological and biochemical changes during apoptosis. Yet, only a few of these proteins have been reported to selectively cleaved by caspase-2. Numerous observations link caspase-2 to the disruption of the cytoskeleton, although it remains elusive whether any of the cytoskeleton proteins serve as bona fide substrates for caspase-2. Here, we undertook an unbiased proteomic approach to address this question. By differential proteome analysis using two-dimensional gel electrophoresis, we identified four cytoskeleton proteins that were degraded upon treatment with active recombinant caspase-2 in vitro. These proteins were degraded in a caspase-2-dependent manner during apoptosis induced by DNA damage, cytoskeleton disruption or endoplasmic reticulum stress. Hence, degradation of these cytoskeleton proteins was blunted by siRNA targeting of caspase-2 and when caspase-2 activity was pharmacologically inhibited. However, none of these proteins was cleaved directly by caspase-2. Instead, we provide evidence that in cells exposed to apoptotic stimuli, caspase-2 probed these proteins for proteasomal degradation. Taken together, our results depict a new role for caspase-2 in the regulation of the level of cytoskeleton proteins during apoptosis.
Cell Death & Disease 12/2013; 4:e940. · 5.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although reactive oxygen species (ROS) are well established mediators of oxidative damage and cell demise, the mechanisms by which they trigger specific cell death modalities and the temporal/spatial requirements underlying this phenomenon are largely unknown. Yet, it is well established that most anticancer therapies depend on ROS production for efficient tumor eradication. Using several non-small cell lung cancer cell lines, we have dissected how the site of ROS production and accumulation in various cell compartments affect cell fate. We demonstrate that high levels of exogenously generated H(2)O(2)induce extensive DNA damage, ATP depletion and severe cytotoxicity. While these effects were independent of caspase activity, they could - at least in part - be prevented by RIP1 kinase inhibition. In contrast, low levels of exogenously produced H(2)O(2) triggered a modest drop in ATP level, delayed toxicity, G2/M arrest and cell senescence. Mitochondrially-produced H(2)O(2) induced a reversible ATP drop without affecting cell viability. Instead, the cells accumulated in the G1/S phase of the cell cycle and became senescent. Concomitant inhibition of glycolysis was found to markedly sensitize cells to death in the presence of otherwise nontoxic concentrations of H(2)O(2,) presumably by the inhibition of ATP restoring mechanisms. Combined, our data provide evidence that ROS might dictate different cellular consequences depending on their overall concentration at steady state levels and on their site of generation.
Free Radical Biology and Medicine 01/2013; · 5.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mitochondria are emerging as intriguing targets for anti-cancer agents. We tested here a novel approach, whereby the mitochondrially targeted delivery of anti-cancer drugs is enhanced by the addition of a triphenylphosphonium group (TPP(+)). A mitochondrially targeted analog of vitamin E succinate (MitoVES), modified by tagging the parental compound with TPP(+), induced considerably more robust apoptosis in cancer cells with a 1-2 log gain in anti-cancer activity compared to the unmodified counterpart, while maintaining selectivity for malignant cells. This is because MitoVES associates with mitochondria and causes fast generation of reactive oxygen species that then trigger mitochondria-dependent apoptosis, involving transcriptional modulation of the Bcl-2 family proteins. MitoVES proved superior in suppression of experimental tumors compared to the untargeted analog. We propose that mitochondrially targeted delivery of anti-cancer agents offers a new paradigm for increasing the efficacy of compounds with anti-cancer activity.
Free Radical Biology and Medicine 03/2011; 50(11):1546-55. · 5.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Release of mitochondrial proteins such as cytochrome c, AIF, Smac/Diablo etc., plays a crucial role in apoptosis induction. A redox-silent analog of vitamin E, alpha-tocopheryl succinate (alpha-TOS), was shown to stimulate cytochrome c release via production of reactive oxygen species (ROS) and Bax-mediated permeabilization of the outer mitochondrial membrane. Here we show that alpha-TOS facilitates mitochondrial permeability transition (MPT) in isolated rat liver mitochondria, Tet21N neuroblastoma cells and Jurkat T-lymphocytes. In particular, in addition to ROS production, alpha-TOS stimulates rapid Ca(2+) entry into the cells with subsequent accumulation of Ca(2+) in mitochondria-a prerequisite step for MPT induction. Alteration of mitochondrial Ca(2+) buffering capacity was observed as early as 8 hr after incubation with alpha-TOS, when no activation of Bax was yet detected. Ca(2+) accumulation in mitochondria was important for apoptosis progression, since inhibition of mitochondrial Ca(2+) uptake significantly mitigated the apoptotic response. Importantly, Ca(2+)-induced mitochondrial destabilization might cooperate with Bax-mediated mitochondrial outer membrane permeabilization to induce cytochrome c release from mitochondria.
International Journal of Cancer 10/2010; 127(8):1823-32. · 6.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cellular calcium uptake is a controlled physiological process mediated by multiple ion channels. The exposure of cells to either one of the protein kinase C (PKC) inhibitors, staurosporine (STS) or PKC412, can trigger Ca²(+) influx leading to cell death. The precise molecular mechanisms regulating these events remain elusive. In this study, we report that the PKC inhibitors induce a prolonged Ca²(+) import through hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) in lung carcinoma cells and in primary culture of cortical neurons, sufficient to trigger apoptosis-inducing factor (AIF)-mediated apoptosis. Downregulation of HCN2 prevented the drug-induced Ca²(+) increase and subsequent apoptosis. Importantly, the PKC inhibitors did not cause Ca²(+) entry into HEK293 cells, which do not express the HCN channels. However, introduction of HCN2 sensitized them to STS/PKC412-induced apoptosis. Mutagenesis of putative PKC phosphorylation sites within the C-terminal domain of HCN2 revealed that dephosphorylation of Thr⁵⁴⁹ was critical for the prolonged Ca²(+) entry required for AIF-mediated apoptosis. Our findings demonstrate a novel role for the HCN2 channel by providing evidence that it can act as an upstream regulator of cell death triggered by PKC inhibitors.
The EMBO Journal 10/2010; 29(22):3869-78. · 10.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Apoptosis might proceed through the activation of both caspase-dependent and -independent pathways. Apoptosis-inducing factor (AIF) was discovered as the first protein that mediated caspase-independent cell death. Initially, it was regarded as a soluble protein residing in the intermembrane space of mitochondria, from where it could be exported to the nucleus to participate in large-scale DNA fragmentation and chromatin condensation. However, later it was demonstrated that AIF is N-terminally anchored to the inner mitochondrial membrane. Hence, AIF must be liberated from its membrane anchor prior to being released into the cytosol. The current knowledge about the molecular mechanisms regulating the processing and release of AIF from the mitochondria will be summarized and discussed in this review.
Biochemical and Biophysical Research Communications 05/2010; 396(1):95-100. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although processing of mitochondrial apoptosis-inducing factor (AIF) is essential for its function during apoptosis in most cell types, the detailed mechanisms of AIF cleavage remain elusive. Recent findings indicate that the proteolytic process is Ca(2+)-dependent and that it is mediated by a calpain located in the mitochondrial intermembrane space. We can now report that, in addition to a sustained intracellular Ca(2+) elevation, enhanced formation of reactive oxygen species (ROS) is a prerequisite step for AIF to be cleaved and released from mitochondria in staurosporine-treated cells. These events occurred independent of the redox state of the mitochondria and were not influenced by binding of pyridine nucleotides to AIF. Chelation of cytosolic Ca(2+) by BAPTA/AM suppressed the elevation of both Ca(2+) and ROS, suggesting that the Ca(2+) rise was the most upstream signal required for AIF processing. We could further show that the stimulated ROS production leads to oxidative modification (carbonylation) of AIF, which markedly increases its rate of cleavage by calpain. Accordingly, pretreatment of the cells with antioxidants blocked AIF carbonylation, as well as its subsequent cleavage and release from the mitochondria. Combined, our data provide evidence that ROS-mediated, posttranslational modification of AIF is critical for its cleavage by calpain and thus for AIF-mediated cell death.
Free Radical Biology and Medicine 03/2010; 48(6):791-7. · 5.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The release of pro-apoptotic proteins from the mitochondria is a key event in cell death signaling that is regulated by Bcl-2 family proteins. For example, cleavage of the BH3-only protein, Bid, by multiple proteases leads to the formation of truncated Bid that, in turn, promotes the insertion/oligomerization of Bax into the mitochondrial outer membrane, resulting in pore formation and the release of proteins residing in the intermembrane space. Bax, a monomeric protein in the cytosol is targeted to the mitochondria by a yet unknown mechanism. Several proteins of the outer mitochondrial membrane have been proposed to act as receptors for Bax, among them the voltage-dependent anion channel, VDAC, and the mitochondrial protein translocase of the outer membrane, the TOM complex. Alternatively, the unique mitochondrial phospholipid, cardiolipin, has been ascribed a similar function. Here, we review recent work on the mechanisms of activation and the targeting of Bax to the mitochondria and discuss the advantages and limitations of the methods used to study this process.
Cell death and differentiation 07/2009; 16(8):1075-82. · 8.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Apoptosis-inducing factor (AIF), a flavoprotein with NADH oxidase activity anchored to the mitochondrial inner membrane, is known to be involved in complex I maintenance. During apoptosis, AIF can be released from mitochondria and translocate to the nucleus, where it participates in chromatin condensation and large-scale DNA fragmentation. The mechanism of AIF release is not fully understood. Here, we show that a prolonged ( approximately 10 min) increase in intracellular Ca(2+) level is a prerequisite step for AIF processing and release during cell death. In contrast, a transient ATP-induced Ca(2+) increase, followed by rapid normalization of the Ca(2+) level, was not sufficient to trigger the proteolysis of AIF. Hence, import of extracellular Ca(2+) into staurosporine-treated cells caused the activation of a calpain, located in the intermembrane space of mitochondria. The activated calpain, in turn, cleaved membrane-bound AIF, and the soluble fragment was released from the mitochondria upon outer membrane permeabilization through Bax/Bak-mediated pores or by the induction of Ca(2+)-dependent mitochondrial permeability transition. Inhibition of calpain, or chelation of Ca(2+), but not the suppression of caspase activity, prevented processing and release of AIF. Combined, these results provide novel insights into the mechanism of AIF release during cell death.
Cell death and differentiation 10/2008; 15(12):1857-64. · 8.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytochrome c release from mitochondria is a key event in apoptosis signaling that is regulated by Bcl-2 family proteins. Cleavage of the BH3-only protein Bid by multiple proteases leads to the formation of truncated Bid (tBid), which, in turn, promotes the oligomerization/insertion of Bax into the mitochondrial outer membrane and the resultant release of proteins residing in the intermembrane space. Bax, a monomeric protein in the cytosol, is targeted by a yet unknown mechanism to the mitochondria. Several hypotheses have been put forward to explain this targeting specificity. Using mitochondria isolated from different mutants of the yeast Saccharomyces cerevisiae and recombinant proteins, we have now investigated components of the mitochondrial outer membrane that might be required for tBid/Bax-induced cytochrome c release. Here, we show that the protein translocase of the outer mitochondrial membrane is required for Bax insertion and cytochrome c release.
Journal of Biological Chemistry 10/2007; 282(38):27633-9. · 4.60 Impact Factor