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N Zamzami

Publications and source records attributed to N Zamzami.

At least 19 recordsLinked to original sources
PubMed2006

GAPDH, a novel regulator of the pro-apoptotic mitochondrial membrane permeabilization.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a pleiotropic enzyme that is overexpressed in apoptosis and in several human chronic pathologies. Here, we report that the protein accumulates in mitochondria during apoptosis, and induces the pro-apoptotic mitochondrial membrane permeabilization, a decisive event of the intrinsic pathway of apoptosis. GAPDH was localized by immunogold labeling and identified by matrix-assisted laser desorption/ionization-time of flight and nano liquid chromatography mass spectroscopy/mass spectroscopy in the mitochondrion of various tissues and origins. In isolated mitochondria, GAPDH can be imported and interact with the voltage-dependent anion channel (VDAC1), but not the adenine nucleotide translocase (ANT). The protein mediates a cyclosporin A-inhibitable permeability transition, characterized by a loss of the inner transmembrane potential, matrix swelling, permeabilization of the inner mitochondrial membrane and the release of two pro-apoptotic proteins, cytochrome c and apoptosis-inducing factor (AIF). This novel function of GAPDH might have implications for the understanding of mitochondrial biology, oncogenesis and apoptosis.

Amino Acid Sequence↗
PubMed2006

Mitochondria as therapeutic targets for cancer chemotherapy.

Mitochondria are vital for cellular bioenergetics and play a central role in determining the point-of-no-return of the apoptotic process. As a consequence, mitochondria exert a dual function in carcinogenesis. Cancer-associated changes in cellular metabolism (the Warburg effect) influence mitochondrial function, and the invalidation of apoptosis is linked to an inhibition of mitochondrial outer membrane permeabilization (MOMP). On theoretical grounds, it is tempting to develop specific therapeutic interventions that target the mitochondrial Achilles' heel, rendering cancer cells metabolically unviable or subverting endogenous MOMP inhibitors. A variety of experimental therapeutic agents can directly target mitochondria, causing apoptosis induction. This applies to a heterogeneous collection of chemically unrelated compounds including positively charged alpha-helical peptides, agents designed to mimic the Bcl-2 homology domain 3 of Bcl-2-like proteins, ampholytic cations, metals and steroid-like compounds. Such MOMP inducers or facilitators can induce apoptosis by themselves (monotherapy) or facilitate apoptosis induction in combination therapies, bypassing chemoresistance against DNA-damaging agents. In addition, it is possible to design molecules that neutralize inhibitor of apoptosis proteins (IAPs) or heat shock protein 70 (HSP70). Such IAP or HSP70 inhibitors can mimic the action of mitochondrion-derived mediators (Smac/DIABLO, that is, second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with a low isoelectric point, in the case of IAPs; AIF, that is apoptosis-inducing factor, in the case of HSP70) and exert potent chemosensitizing effects.

Animals↗
PubMed2003

Cytofluorometric quantitation of apoptosis-driven inner mitochondrial membrane permeabilization.

The mitochondrial matrix can be specifically labeled by loading cells with calcein and simultaneous quenching of the non-mitochondrial calcein fluorescence with cobalt (Co2+). Positive staining of mitochondria thus requires that the inner mitochondrial membrane functions as a barrier separating calcein (within the matrix) from Co2+ (outside of the matrix). Upon induction of apoptosis, such calcein/Co2+ -labeled cells, demonstrate a decrease in the overall calcein fluorescence resulting from inner mitochondrial membrane permeabilization. This decrease can be quantified by cytofluorometry and can be dissociated from other apoptosis-associated mitochondrial perturbations such as the loss of the mitochondrial transmembrane potential (delta phi m), the local overproduction of re-active oxygen species, and the mitochondrial release of cytochrome c. In some paradigms of apoptosis the loss of calcein/Co2+ (CC) staining can be dissociated from the delta phi m loss, both of which may occur in a caspase-dependent or caspase-independent fashion, depending on the apoptosis inducer. Importantly, inner membrane permeabilization to CC may occur without a permanent delta phi m dissipation in apoptosis, suggesting that transient permeabilization events could participate at the apoptotic cascade. Altogether, our data demonstrate that inner mitochondrial membrane permeabilization constitutes an early event in the apoptotic cascade.

Apoptosis↗
PubMed2002

Pre-processed caspase-9 contained in mitochondria participates in apoptosis.

As shown here, mitochondria purified from different organs (liver, brain, kidney, spleen and heart) contain both pro-caspase-9 and the processed, mature form of caspase-9. Purified liver mitochondria release mature caspase-9 upon induction of permeability transition in vitro. This is accompanied by a discrete increase in the enzymatic cleavage of pro-caspase-9 substrates. We found that SHEP neuroblastoma cells constitutively contain pre-processed caspase-9 in their mitochondria, using a combination of subcellular fractionation and immunofluorescence with an antibody specific for the processed caspase. This is a cell type-specific phenomenon since HeLa cells mitochondria mainly contain pro-caspase-9 and comparatively little processed caspase-9. Upon introduction of apoptosis, mitochondrial pro-caspase-9 translocates to the cytosol and to the nucleus. This phenomenon is inhibited by transfection with Bcl-2. In synthesis, we report the unexpected finding that mitochondria can contain a pre-processed caspase isoform in non-apoptotic cells. Bcl-2-mediated regulation of mitochondrial membrane permeabilization may contribute to apoptosis control by preventing mitochondrial, pre-processed caspase-9 from interacting with its cytosolic activators.

Apoptosis↗
PubMed2001

Endoplasmic reticulum localized Bcl-2 prevents apoptosis when redistribution of cytochrome c is a late event.

The disruption of mitochondrial function is a key component of apoptosis in most cell types. Localization of Bcl-2 to the outer mitochondrial and endoplasmic reticulum membranes is consistent with a role in the inhibition of many forms of apoptosis. In Rat-1 cells, a Bcl-2 mutant targeted exclusively to the endoplasmic reticulum (Bcl-cb5) was effective at inhibiting apoptosis induced by serum starvation/myc, or ceramide but not apoptosis induced by etoposide. The former conditions cause a decrease in mitochondrial transmembrane potential (Deltapsi(m)) as an early event that precedes the release of cytochrome c from mitochondria. By contrast, when cells are exposed to etoposide, a situation in which cytochrome c release and membrane localization of the pro-apoptotic protein Bax precede loss of Deltapsi(m), wild type Bcl-2 but not Bcl-cb5 prevents apoptosis. Therefore, Bcl-2 functions in spatially distinct pathways of apoptosis distinguished by the order of cytochrome c release and loss of Deltapsi(m).

Amino Acid Chloromethyl Ketones↗
PubMed2001

Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death.

Programmed cell death is a fundamental requirement for embryogenesis, organ metamorphosis and tissue homeostasis. In mammals, release of mitochondrial cytochrome c leads to the cytosolic assembly of the apoptosome-a caspase activation complex involving Apaf1 and caspase-9 that induces hallmarks of apoptosis. There are, however, mitochondrially regulated cell death pathways that are independent of Apaf1/caspase-9. We have previously cloned a molecule associated with programmed cell death called apoptosis-inducing factor (AIF). Like cytochrome c, AIF is localized to mitochondria and released in response to death stimuli. Here we show that genetic inactivation of AIF renders embryonic stem cells resistant to cell death after serum deprivation. Moreover, AIF is essential for programmed cell death during cavitation of embryoid bodies-the very first wave of cell death indispensable for mouse morphogenesis. AIF-dependent cell death displays structural features of apoptosis, and can be genetically uncoupled from Apaf1 and caspase-9 expression. Our data provide genetic evidence for a caspase-independent pathway of programmed cell death that controls early morphogenesis.

Animals↗
PubMed2001

Control of mitochondrial membrane permeabilization by adenine nucleotide translocator interacting with HIV-1 viral protein rR and Bcl-2.

Viral protein R (Vpr), an apoptogenic accessory protein encoded by HIV-1, induces mitochondrial membrane permeabilization (MMP) via a specific interaction with the permeability transition pore complex, which comprises the voltage-dependent anion channel (VDAC) in the outer membrane (OM) and the adenine nucleotide translocator (ANT) in the inner membrane. Here, we demonstrate that a synthetic Vpr-derived peptide (Vpr52-96) specifically binds to the intermembrane face of the ANT with an affinity in the nanomolar range. Taking advantage of this specific interaction, we determined the role of ANT in the control of MMP. In planar lipid bilayers, Vpr52-96 and purified ANT cooperatively form large conductance channels. This cooperative channel formation relies on a direct protein-protein interaction since it is abolished by the addition of a peptide corresponding to the Vpr binding site of ANT. When added to isolated mitochondria, Vpr52-96 uncouples the respiratory chain and induces a rapid inner MMP to protons and NADH. This inner MMP precedes outer MMP to cytochrome c. Vpr52-96-induced matrix swelling and inner MMP both are prevented by preincubation of purified mitochondria with recombinant Bcl-2 protein. In contrast to König's polyanion (PA10), a specific inhibitor of the VDAC, Bcl-2 fails to prevent Vpr52-96 from crossing the mitochondrial OM. Rather, Bcl-2 reduces the ANT-Vpr interaction, as determined by affinity purification and plasmon resonance studies. Concomitantly, Bcl-2 suppresses channel formation by the ANT-Vpr complex in synthetic membranes. In conclusion, both Vpr and Bcl-2 modulate MMP through a direct interaction with ANT.

Amino Acid Sequence↗
PubMed2001

The mitochondrion in apoptosis: how Pandora's box opens.

There is widespread agreement that mitochondria have a function in apoptosis, but the mechanisms behind their involvement remain controversial. Here we suggest that opening of a multiprotein complex called the mitochondrial permeability transition pore complex is sufficient (and, usually, necessary) for triggering apoptosis.

Animals↗
PubMed2001

Heat-shock protein 70 antagonizes apoptosis-inducing factor.

Heat-shock protein 70 (Hsp70) has been reported to block apoptosis by binding apoptosis protease activating factor-1 (Apaf-1), thereby preventing constitution of the apoptosome, the Apaf-1/cytochrome c/caspase-9 activation complex [1,2]. Here we show that overexpression of Hsp70 protects Apaf-1-/- cells against death induced by serum withdrawal, indicating that Apaf-1 is not the only target of the anti-apoptotic action of Hsp70. We investigated the effect of Hsp70 on apoptosis mediated by the caspase-independent death effector apoptosis inducing factor (AIF), which is a mitochondrial intermembrane flavoprotein [3,4]. In a cell-free system, Hsp70 prevented the AIF-induced chromatin condensation of purified nuclei. Hsp70 specifically interacted with AIF, as shown by ligand blots and co-immunoprecipitation. Cells overexpressing Hsp70 were protected against the apoptogenic effects of AIF targeted to the extramitochondrial compartment. In contrast, an anti-sense Hsp70 complementary DNA, which reduced the expression of endogenous Hsp70, increased sensitivity to the lethal effect of AIF. The ATP-binding domain of Hsp70 seemed to be dispensable for inhibiting cell death induced by serum withdrawal, AIF binding and AIF inhibition, although it was required for Apaf-1 binding. Together, our data indicate that Hsp70 can inhibit apoptosis by interfering with target proteins other than Apaf-1, one of which is AIF.

Animals↗
PubMed2001

Dominant cell death induction by extramitochondrially targeted apoptosis-inducing factor.

The complete AIF cDNA comprising the amino-terminal mitochondrial localization sequence (MLS) and the oxidoreductase domain has been fused in its carboxyl terminus to enhanced green fluorescent protein (GFP), thereby engineering an AIF-GFP fusion protein that is selectively targeted to the mitochondrial intermembrane space. Upon induction of apoptosis, the AIF-GFP protein translocates together with cytochrome c (Cyt-c) to the extramitochondrial compartment. Microinjection of recombinant AIF leads to the release of AIF-GFP and Cyt-c-GFP, indicating that ectopic AIF can favor permeabilization of the outer mitochondrial membrane. These mitochondrial effects of AIF are caspase independent, whereas the Cyt-c-microinjection induced translocation of AIF-GFP and Cyt-c-GFP is suppressed by the pan-caspase inhibitor Z-VAD.fmk. Upon prolonged culture, transfection-enforced overexpression of AIF results in spontaneous translocation of AIF-GFP from mitochondria, nuclear chromatin condensation, and cell death. These effects are caspase independent and do not rely on the oxidoreductase function of AIF. Spontaneous AIF-GFP translocation and subsequent nuclear apoptosis can be retarded by overexpression of a Bcl-2 protein selectively targeted to mitochondria, but not by a Bcl-2 protein targeted to the endoplasmic reticulum. Overexpression of a mutant AIF protein in which the MLS has been deleted (AIF Delta 1-100) results in the primary cytosolic accumulation of AIF. AIF Delta 1-100-induced cell death is suppressed by neither Z-VAD.fmk or by Bcl-2. Thus, extramitochondrially targeted AIF is a dominant cell death inducer.

Active Transport, Cell Nucleus↗
PubMed2000

Bid acts on the permeability transition pore complex to induce apoptosis.

Similar to most if not all pro-apoptotic members of the Bcl-2 family, Bid (and its truncated product t-Bid) triggers cell death via mitochondrial membrane permeabilization (MMP). This effect can be monitored in intact cells, upon microinjection of recombinant Bid protein into the cytoplasm, as well as in purified mitochondria, upon addition of Bid protein. Here we show that Bid-induced MMP can be inhibited, both in cells and in the cell-free system, by three pharmacological inhibitors of the permeability transition pore complex (PTPC), namely cyclosporin A, N-methyl-4-Val-cyclosporin A, and bongkrekic acid (a ligand of the adenine nucleotide translocase, ANT, one of the PTPC components). Bid effects on synthetic membranes were studied either in proteoliposomes or in synthetic bilayers subjected to electrophysiological measurements. Full length Bid preferentially permeabilizes membranes and induces the formation of large conductance channels at neutral pH, when added to liposomes or bilayers containing both purified ANT and Bax, yet has no or little effect combined with ANT or Bax alone. t-Bid acts on membranes containing ANT alone with the same efficiency as on those containing both ANT and Bax. These results suggest that the proapoptotic effects of Bid are mediated, at least in part, by its functional interaction with ANT, one of the major components of PTPC.

Animals↗
PubMed2000

Apoptosis control in syncytia induced by the HIV type 1-envelope glycoprotein complex: role of mitochondria and caspases.

Syncytia arising from the fusion of cells expressing a lymphotropic HIV type 1-encoded envelope glycoprotein complex (Env) with cells expressing the CD4/CXC chemokine receptor 4 complex spontaneously undergo cell death. Here we show that this process is accompanied by caspase activation and signs of mitochondrial membrane permeabilization (MMP), including the release of intermembrane proteins such as cytochrome c (Cyt-c) and apoptosis-inducing factor (AIF) from mitochondria. In Env-induced syncytia, caspase inhibition did not suppress AIF- and Cyt-c translocation, yet it prevented all signs of nuclear apoptosis. Translocation of Bax to mitochondria led to MMP, which was inhibited by microinjected Bcl-2 protein or bcl-2 transfection. Bcl-2 also prevented the subsequent nuclear chromatin condensation and DNA fragmentation. The release of AIF occurred before that of Cyt-c and before caspase activation. Microinjection of AIF into syncytia sufficed to trigger rapid, caspase-independent Cyt-c release. Neutralization of endogenous AIF by injection of an antibody prevented all signs of spontaneous apoptosis occurring in syncytia, including the Cyt-c release and nuclear apoptosis. In contrast, Cyt-c neutralization only prevented nuclear apoptosis, and did not affect AIF release. Our results establish that the following molecular sequence governs apoptosis of Env-induced syncytia: Bax-mediated/Bcl-2-inhibited MMP --> AIF release --> Cyt-c release --> caspase activation --> nuclear apoptosis.

Apoptosis↗
PubMed2000

Two distinct pathways leading to nuclear apoptosis.

Apaf-1(-/-) or caspase-3(-/-) cells treated with a variety of apoptosis inducers manifest apoptosis-associated alterations including the translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, large scale DNA fragmentation, and initial chromatin condensation (stage I). However, when compared with normal control cells, Apaf-1(-/-) or caspase-3(-/-) cells fail to exhibit oligonucleosomal chromatin digestion and a more advanced pattern of chromatin condensation (stage II). Microinjection of such cells with recombinant AIF only causes peripheral chromatin condensation (stage I), whereas microinjection with activated caspase-3 or its downstream target caspase-activated DNAse (CAD) causes a more pronounced type of chromatin condensation (stage II). Similarly, when added to purified HeLa nuclei, AIF causes stage I chromatin condensation and large-scale DNA fragmentation, whereas CAD induces stage II chromatin condensation and oligonucleosomal DNA degradation. Furthermore, in a cell-free system, concomitant neutralization of AIF and CAD is required to suppress the nuclear DNA loss caused by cytoplasmic extracts from apoptotic wild-type cells. In contrast, AIF depletion alone suffices to suppress the nuclear DNA loss contained in extracts from apoptotic Apaf-1(-/-) or caspase-3(-/-) cells. As a result, at least two redundant parallel pathways may lead to chromatin processing during apoptosis. One of these pathways involves Apaf-1 and caspases, as well as CAD, and leads to oligonucleosomal DNA fragmentation and advanced chromatin condensation. The other pathway, which is caspase-independent, involves AIF and leads to large-scale DNA fragmentation and peripheral chromatin condensation.

Animals↗
PubMed2000

Apoptosis-inducing factor (AIF): a ubiquitous mitochondrial oxidoreductase involved in apoptosis.

Apoptosis-inducing factor (AIF) is encoded by one single gene located on the X chromosome. AIF is ubiquitously expressed, both in normal tissues and in a variety of cancer cell lines. The AIF precursor is synthesized in the cytosol and is imported into mitochondria. The mature AIF protein, a flavoprotein (prosthetic group: flavine adenine dinucleotide) with significant homology to plant ascorbate reductases and bacterial NADH oxidases, is normally confined to the mitochondrial intermembrane space. In a variety of different apoptosis-inducing conditions, AIF translocates through the outer mitochondrial membrane to the cytosol and to the nucleus. Ectopic (extra-mitochondrial) AIF induces nuclear chromatin condensation, as well as large scale ( approximately 50 kb) DNA fragmentation. Thus, similar to cytochrome c, AIF is a phylogenetically old, bifunctional protein with an electron acceptor/donor (oxidoreductase) function and a second apoptogenic function. In contrast to cytochrome c, however, AIF acts in a caspase-independent fashion. The molecular mechanisms via which AIF induces apoptosis are discussed.

Animals↗
PubMed2000

Bcl-2 and Bax regulate the channel activity of the mitochondrial adenine nucleotide translocator.

Bcl-2 family protein including anti-apoptotic (Bcl-2) or pro-apoptotic (Bax) members can form ion channels when incorporated into synthetic lipid bilayers. This contrasts with the observation that Bcl-2 stabilizes the mitochondrial membrane barrier function and inhibits the permeability transition pore complex (PTPC). Here we provide experimental data which may explain this apparent paradox. Bax and adenine nucleotide translocator (ANT), the most abundant inner mitochondrial membrane protein, can interact in artificial lipid bilayers to yield an efficient composite channel whose electrophysiological properties differ quantitatively and qualitatively from the channels formed by Bax or ANT alone. The formation of this composite channel can be observed in conditions in which Bax protein alone has no detectable channel activity. Cooperative channel formation by Bax and ANT is stimulated by the ANT ligand atractyloside (Atr) but inhibited by ATP, indicating that it depends on the conformation of ANT. In contrast to the combination of Bax and ANT, ANT does not form active channels when incorporated into membranes with Bcl-2. Rather, ANT and Bcl-2 exhibit mutual inhibition of channel formation. Bcl-2 prevents channel formation by Atr-treated ANT and neutralizes the cooperation between Bax and ANT. Our data are compatible with a ménage à trois model of mitochondrial apoptosis regulation in which ANT, the likely pore forming protein within the PTPC, interacts with Bax or Bcl-2 which influence its pore forming potential in opposing manners.

Animals↗