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1.
Cell Death Differ ; 13(7): 1128-37, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16282981

ABSTRACT

Although the role of the b-cell lymphoma (Bcl)-2 family of apoptosis inhibitors is well documented in tumor cells and tissue morphogenesis, their role during the early development of vertebrates is unknown. Here, we characterize Nrz, a new Bcl-2-related inhibitor of apoptosis in zebrafish. Nrz is a mitochondrial protein, antagonizing the death-accelerator Bax. The nrz gene is mainly expressed during gastrulation and somitogenesis. The knockdown of nrz with antisense morpholinos leads to alterations of the somites, correlated with an increase in apoptosis. In addition, earlier during development, in the zebrafish gastrula, nrz knockdown results in an increase of snail-1 expression at the margin and frequent gastrulation arrest at the shield stage, independently of apoptosis. Together these data suggest that Nrz, in addition to its effect on apoptosis, contributes to cell movements during gastrulation by negatively regulating the expression of Snail-1, a transcription factor that controls cell adhesion.


Subject(s)
Apoptosis/physiology , Gastrula/physiology , Proto-Oncogene Proteins/physiology , Somites/physiology , Zebrafish Proteins/physiology , Zebrafish/genetics , Amino Acid Sequence , Animals , Blotting, Western , COS Cells , Chlorocebus aethiops , Cloning, Molecular , Gastrula/cytology , Gastrula/metabolism , Gene Expression Regulation, Developmental , In Situ Hybridization , Microscopy, Confocal , Molecular Sequence Data , Oligonucleotides, Antisense/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins c-bcl-2/physiology , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Snail Family Transcription Factors , Somites/cytology , Somites/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription Factors/physiology , Zebrafish/embryology , Zebrafish/physiology , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism
2.
Cell Death Differ ; 9(11): 1212-9, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12404120

ABSTRACT

Previous biochemical studies suggested that HIV-1-encoded Vpr may kill cells through an effect on the adenine nucleotide translocase (ANT), thereby causing mitochondrial membrane permeabilization (MMP). Here, we show that Vpr fails to activate caspases in conditions in which it induces cell killing. The knock-out of essential caspase-activators (Apaf-1 or caspase-9) or the knock-out of a mitochondrial caspase-independent death effector (AIF) does not abolish Vpr-mediated killing. In contrast, the cytotoxic effects of Vpr are reduced by transfection-enforced overexpression of two MMP-inhibitors, namely the endogenous protein Bcl-2 or the cytomegalovirus-encoded ANT-targeted protein vMIA. Vpr, which can elicit MMP through a direct effect on mitochondria, and HIV-1-Env, which causes MMP through an indirect pathway, exhibit additive (but not synergic) cytotoxic effects. In conclusion, it appears that Vpr induces apoptosis through a caspase-independent mitochondrial pathway.


Subject(s)
Apoptosis/physiology , Gene Products, vpr/metabolism , HIV-1/metabolism , Mitochondria/metabolism , Viral Proteins , Apoptosis Inducing Factor , Caspases/metabolism , Cell Line , Cytomegalovirus/metabolism , Flavoproteins/physiology , Gene Products, env/metabolism , Humans , Immediate-Early Proteins/metabolism , Matrix Metalloproteinases/metabolism , Membrane Proteins/physiology , Proto-Oncogene Proteins c-bcl-2/metabolism , Stem Cells , vpr Gene Products, Human Immunodeficiency Virus
4.
Nat Cell Biol ; 3(11): E255-63, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11715037

ABSTRACT

Nuclear DNA damage and ligation of plasma-membrane death receptors have long been recognized as initial triggers of apoptosis that induce mitochondrial membrane permeabilization (MMP) and/or the direct activation of caspases. Accumulating evidence suggests that other organelles, including the endoplasmic reticulum (ER), lysosomes and the Golgi apparatus, are also major points of integration of pro-apoptotic signalling or damage sensing. Each organelle possesses sensors that detect specific alterations, locally activates signal transduction pathways and emits signals that ensure inter-organellar cross-talk. The ER senses local stress through chaperones, Ca2+-binding proteins and Ca2+ release channels, which might transmit ER Ca2+ responses to mitochondria. The ER also contains several Bcl-2-binding proteins, and Bcl-2 has been reported to exert part of its cytoprotective effect within the ER. Upon membrane destabilization, lysosomes release cathepsins that are endowed with the capacity of triggering MMP. The Golgi apparatus constitutes a privileged site for the generation of the pro-apoptotic mediator ganglioside GD3, facilitates local caspase-2 activation and might serve as a storage organelle for latent death receptors. Intriguingly, most organelle-specific death responses finally lead to either MMP or caspase activation, both of which might function as central integrators of the death pathway, thereby streamlining lysosome-, Golgi- or ER-elicited responses into a common pathway.


Subject(s)
Apoptosis , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Lysosomes/metabolism , Mitochondria/metabolism , Animals , Cell Nucleus , DNA Damage , Endosomes/metabolism , Organelles
5.
J Exp Med ; 194(8): 1097-110, 2001 Oct 15.
Article in English | MEDLINE | ID: mdl-11602639

ABSTRACT

Syncytia arising from the fusion of cells expressing a lymphotropic human immunodeficiency virus (HIV)-1-encoded envelope glycoprotein complex (Env) gene with cells expressing the CD4/CXCR4 complex undergo apoptosis through a mitochondrion-controlled pathway initiated by the upregulation of Bax. In syncytial apoptosis, phosphorylation of p53 on serine 15 (p53S15) precedes Bax upregulation, the apoptosis-linked conformational change of Bax, the insertion of Bax in mitochondrial membranes, subsequent release of cytochrome c, caspase activation, and apoptosis. p53S15 phosphorylation also occurs in vivo, in HIV-1(+) donors, where it can be detected in preapoptotic and apoptotic syncytia in lymph nodes, as well as in peripheral blood mononuclear cells, correlating with viral load. Syncytium-induced p53S15 phosphorylation is mediated by the upregulation/activation of mammalian target of rapamycin (mTOR), also called FKBP12-rapamycin-associated protein (FRAP), which coimmunoprecipitates with p53. Inhibition of mTOR/FRAP by rapamycin reduces apoptosis in several paradigms of syncytium-dependent death, including in primary CD4(+) lymphoblasts infected by HIV-1. Concomitantly, rapamycin inhibits p53S15 phosphorylation, mitochondrial translocation of Bax, loss of the mitochondrial transmembrane potential, mitochondrial release of cytochrome c, and nuclear chromatin condensation. Transfection with dominant negative p53 has a similar antiapoptotic action as rapamycin, upstream of the Bax upregulation/translocation. In summary, we demonstrate that phosphorylation of p53S15 by mTOR/FRAP plays a critical role in syncytial apoptosis driven by HIV-1 Env.


Subject(s)
Apoptosis/immunology , Carrier Proteins , HIV Envelope Protein gp120/immunology , HIV Envelope Protein gp41/immunology , HIV-1/immunology , Immunophilins/immunology , Phosphotransferases (Alcohol Group Acceptor) , Tumor Suppressor Protein p53/immunology , Animals , Giant Cells , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp41/genetics , HeLa Cells , Humans , Mammals , Phosphorylation , Serine/metabolism , TOR Serine-Threonine Kinases , Transcriptional Activation , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
6.
Oncogene ; 20(32): 4305-16, 2001 Jul 19.
Article in English | MEDLINE | ID: mdl-11466611

ABSTRACT

Nitric oxide (NO), peroxynitrite, and 4-hydroxynonenal (HNE) may be involved in the pathological demise of cells via apoptosis. Apoptosis induced by these agents is inhibited by Bcl-2, suggesting the involvement of mitochondria in the death pathway. In vitro, NO, peroxynitrite and HNE can cause direct permeabilization of mitochondrial membranes, and this effect is inhibited by cyclosporin A, indicating involvement of the permeability transition pore complex (PTPC) in the permeabilization event. NO, peroxynitrite and HNE also permeabilize proteoliposomes containing the adenine nucleotide translocator (ANT), one of the key components of the PTPC, yet have no or little effects on protein-free control liposomes. ANT-dependent, NO-, peroxynitrite- or HNE-induced permeabilization is at least partially inhibited by recombinant Bcl-2 protein, as well as the antioxidants trolox and butylated hydroxytoluene. In vitro, none of the tested agents (NO, peroxynitrite, HNE, and tert-butylhydroperoxide) causes preferential carbonylation HNE adduction, or nitrotyrosylation of ANT. However, all these agents induced ANT to undergo thiol oxidation/derivatization. Peroxynitrite and HNE also caused significant lipid peroxidation, which was antagonized by butylated hydroxytoluene but not by recombinant Bcl-2. Transfection-enforced expression of vMIA, a viral apoptosis inhibitor specifically targeted to ANT, largely reduces the mitochondrial and nuclear signs of apoptosis induced by NO, peroxynitrite and HNE in intact cells. Taken together these data suggest that NO, peroxynitrite, and HNE may directly act on ANT to induce mitochondrial membrane permeabilization and apoptosis.


Subject(s)
Aldehydes/pharmacology , Apoptosis , Ion Channels , Mitochondrial ADP, ATP Translocases/metabolism , Nitrates/pharmacology , Nitric Oxide/metabolism , Oxidants/pharmacology , Animals , Cell Nucleus/ultrastructure , HeLa Cells , Humans , Inhibitor of Apoptosis Proteins , Intracellular Membranes/metabolism , Jurkat Cells , Lipid Peroxidation , Membrane Proteins/physiology , Mice , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins , Mitochondrial Permeability Transition Pore , Permeability , Proteins/physiology , Proteolipids/metabolism , Proto-Oncogene Proteins c-bcl-2/physiology
7.
Nature ; 410(6828): 549-54, 2001 Mar 29.
Article in English | MEDLINE | ID: mdl-11279485

ABSTRACT

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.


Subject(s)
Apoptosis/physiology , Flavoproteins/physiology , Membrane Proteins/physiology , Mitochondria/physiology , Animals , Apoptosis Inducing Factor , Apoptotic Protease-Activating Factor 1 , Caspase 9 , Caspases/metabolism , Cell Differentiation , Chimera , Embryo, Mammalian/cytology , Embryonic and Fetal Development/physiology , Female , Flavoproteins/genetics , Gene Targeting , Male , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Morphogenesis , Proteins/physiology , Recombination, Genetic , Stem Cells
8.
J Exp Med ; 193(4): 509-19, 2001 Feb 19.
Article in English | MEDLINE | ID: mdl-11181702

ABSTRACT

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.


Subject(s)
Gene Products, vpr/pharmacology , Intracellular Membranes/metabolism , Mitochondria/metabolism , Mitochondrial ADP, ATP Translocases/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Amino Acid Sequence , HIV-1 , Ion Channels/metabolism , Liposomes , Models, Biological , Models, Molecular , Molecular Sequence Data , Oxygen Consumption , Peptide Fragments/pharmacology , Permeability , Protein Binding , Surface Plasmon Resonance , vpr Gene Products, Human Immunodeficiency Virus
9.
Bioessays ; 23(2): 111-5, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11169582

ABSTRACT

One of the near-to-invariant hallmarks of early apoptosis (programmed cell death) is mitochondrial membrane permeabilization (MMP). It appears that mitochondria fulfill a dual role during the apoptotic process. On the one hand, they integrate multiple different pro-apoptotic signal transducing cascades into a common pathway initiated by MMP. On the other hand, they coordinate the catabolic reactions accompanying late apoptosis by releasing soluble proteins that are normally sequestered within the intermembrane space. In a recent study, Li et al. described a nuclear transcription factor (Nur77/TR1/NGFI-B) that can translocate to mitochondrial membranes to induce MMP. Moreover, two groups identified a novel intermembrane protein (Smac/DIABLO) that specifically neutralizes the inhibitor of apoptosis (IAP) proteins, thereby facilitating the activation of caspases, a class of proteases activated during apoptosis. These findings refine our knowledge how MMP connects to the cellular suicide machinery.


Subject(s)
Apoptosis , Mitochondria/physiology , Animals , Humans , Organelles
10.
Exp Cell Res ; 261(1): 119-26, 2000 Nov 25.
Article in English | MEDLINE | ID: mdl-11082282

ABSTRACT

Cells stably transfected with a lymphotropic HIV-1 Env gene form syncytia when cocultured with CD4(+)CXCR4(+) cells. Heterokaryons then spontaneously undergo apoptosis, while manifesting signs of mitochondrial membrane pemeabilization as well as nuclear chromatin condensation. Modulation of cellular geometry was achieved by growing syncytia on self-assembled monolayers of terminally substituted alkanethiolates designed to control the adhesive properties of the substrates. Spreading of syncytia, induced by culturing them on small circular adhesive islets (diameter 5 microm), placed at a distance that cells can bridge (10 microm), inhibited spontaneous and staurosporin-induced signs of apoptosis, both at the mitochondrial and at the nuclear levels, and allowed for the generation of larger syncytia. Transient cell spreading conferred a memory of apoptosis inhibition which was conserved upon adoption of a conventional cell shape. Limiting syncytium size by culturing them on square-shaped planar adhesive islands of defined size (400 to 2500 microm(2)), separated by nonadhesive regions, enhanced the rate of apoptotic cell death, as indicated by an accelerated permeabilization of the outer mitochondrial membrane, loss of the mitochondrial inner transmembrane potential, and an increased frequency of nuclear apoptosis. In conclusion, external constraints on syncytial size and shape strongly modulate their propensity to undergo apoptosis.


Subject(s)
Apoptosis/physiology , Gene Products, env/physiology , Giant Cells/virology , HIV-1/physiology , CD4 Antigens/physiology , Cell Adhesion , Cell Nucleus/ultrastructure , Cell Size/physiology , Culture Techniques/methods , Gene Products, env/genetics , Giant Cells/cytology , Giant Cells/physiology , HeLa Cells , Humans , Mitochondria/ultrastructure , Recombinant Proteins/metabolism , Transfection
11.
J Exp Med ; 192(8): 1081-92, 2000 Oct 16.
Article in English | MEDLINE | ID: mdl-11034598

ABSTRACT

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.


Subject(s)
Apoptosis/physiology , CD4 Antigens/physiology , Caspases/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Gene Products, env/metabolism , Giant Cells/virology , HIV-1/physiology , Mitochondria/physiology , CD4-Positive T-Lymphocytes/physiology , Cell Fusion , Coculture Techniques , Genes, env , Giant Cells/cytology , Giant Cells/physiology , HeLa Cells , Humans , Intracellular Membranes/physiology , Kinetics , Permeability , Proto-Oncogene Proteins c-bcl-2/metabolism , Transfection
12.
J Exp Med ; 192(4): 571-80, 2000 Aug 21.
Article in English | MEDLINE | ID: mdl-10952727

ABSTRACT

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.


Subject(s)
Apoptosis/physiology , Caspases/metabolism , Cell Nucleus/metabolism , Flavoproteins/metabolism , Membrane Proteins/metabolism , Mitochondria/metabolism , Proteins/metabolism , Animals , Antineoplastic Agents/pharmacology , Apoptosis Inducing Factor , Apoptotic Protease-Activating Factor 1 , Arsenites/pharmacology , Caspase 3 , Caspases/genetics , Cells, Cultured , Cisplatin/pharmacology , Cytochrome c Group/genetics , Cytochrome c Group/metabolism , DNA Fragmentation , Deoxyribonucleases/genetics , Deoxyribonucleases/metabolism , Etoposide/pharmacology , Fibroblasts/metabolism , Fibroblasts/ultrastructure , Flavoproteins/genetics , Fluorescent Dyes/metabolism , HeLa Cells , Humans , Membrane Proteins/genetics , Mice , Mice, Knockout , Microinjections , Proteins/genetics , Recombinant Proteins/metabolism
13.
Pathol Biol (Paris) ; 48(3): 271-9, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10858959

ABSTRACT

The question as to whether apoptosis (programmed cell death) is controlled by one or few checkpoints is still unresolved. A growing body of evidence suggests that (one of) the decisive event(s) of cell death consists in the permeabilization of mitochondrial membranes. Indeed, multiple pro-apopotic signal transduction pathways converge on the proteins of the Bcl-2/Bax family which, in concert with the so-called permeability transition pore complex (PTPC), regulate mitochondrial membrane barrier function. Mitochondrial permeabilization causes the release of soluble intermembrane proteins, some of which are involved in the activation of apoptotic proteases and nucleases. Thus, the putative checkpoint determining the death/life decision is clearly different from the known checkpoints of cell cycle progression. Prominent oncogenes (e.g., c-Myc, Ras, Raf, Bcl-2) and tumor suppressor genes (e.g., p53, Bax) have been shown to modulate apoptosis via a direct or indirect effect on mitochondrial membranes. All these oncoproteins and tumor suppressor proteins may simultaneously influence the cell cycle and the propensity to undergo apoptosis. Several cell cycle regulatory proteins (e.g., cyclins, cdk, etc.) can induce or inhibit apoptosis via yet unknown pathways.


Subject(s)
Apoptosis , Cell Cycle/physiology , Homeostasis , Animals , Cell Cycle Proteins/physiology , Humans , Oncogenes
14.
FASEB J ; 14(5): 729-39, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10744629

ABSTRACT

Apoptosis inducing factor (AIF) is a novel apoptotic effector protein that induces chromatin condensation and large-scale ( approximately 50 kbp) DNA fragmentation when added to purified nuclei in vitro. Confocal and electron microscopy reveal that, in normal cells, AIF is strictly confined to mitochondria and thus colocalizes with heat shock protein 60 (hsp60). On induction of apoptosis by staurosporin, c-Myc, etoposide, or ceramide, AIF (but not hsp60) translocates to the nucleus. This suggests that only the outer mitochondrial membrane (which retains AIF in the intermembrane space) but not the inner membrane (which retains hsp60 in the matrix) becomes protein permeable. The mitochondrio-nuclear redistribution of AIF is prevented by a Bcl-2 protein specifically targeted to mitochondrial membranes. The pan-caspase inhibitor Z-VAD. fmk does not prevent the staurosporin-induced translocation of AIF, although it does inhibit oligonucleosomal DNA fragmentation and arrests chromatin condensation at an early stage. ATP depletion is sufficient to cause AIF translocation to the nucleus, and this phenomenon is accelerated by the apoptosis inducer staurosporin. However, in conditions in which both glycolytic and respiratory ATP generation is inhibited, cells fail to manifest any sign of chromatin condensation and advanced DNA fragmentation, thus manifesting a 'necrotic' phenotype. Both in the presence of Z-VAD. fmk and in conditions of ATP depletion, AIF translocation correlates with the appearance of large-scale DNA fragmentation. Altogether, these data are compatible with the hypothesis that AIF is a caspase-independent mitochondrial death effector responsible for partial chromatinolysis.


Subject(s)
Apoptosis/physiology , Flavoproteins/metabolism , Membrane Proteins/metabolism , Necrosis , Adenosine Triphosphate/metabolism , Animals , Apoptosis/drug effects , Apoptosis Inducing Factor , Biological Transport, Active , Caspases/metabolism , Cell Line , Cell Nucleus/metabolism , Chromatin/metabolism , Cytochrome c Group/metabolism , DNA Damage , Humans , Microscopy, Confocal , Microscopy, Electron , Mitochondria/metabolism , Models, Biological , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Rats , Staurosporine/pharmacology
15.
Nat Cell Biol ; 2(4): E63-4, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10783253

ABSTRACT

Apoptotic DNA degradation has been thought to be a cell-autonomous process. Recent evidence suggests that heterophagic recognition and engulfment of dying cells by non-apoptotic cells may be critical for the activation and/or action of apoptogenic DNases.


Subject(s)
Apoptosis/genetics , DNA Fragmentation/physiology , Thymus Gland/cytology , Animals , In Situ Nick-End Labeling
16.
J Exp Med ; 191(1): 33-46, 2000 Jan 03.
Article in English | MEDLINE | ID: mdl-10620603

ABSTRACT

Viral protein R (Vpr) encoded by HIV-1 is a facultative inducer of apoptosis. When added to intact cells or purified mitochondria, micromolar and submicromolar doses of synthetic Vpr cause a rapid dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), as well as the mitochondrial release of apoptogenic proteins such as cytochrome c or apoptosis inducing factor. The same structural motifs relevant for cell killing are responsible for the mitochondriotoxic effects of Vpr. Both mitochondrial and cytotoxic Vpr effects are prevented by Bcl-2, an inhibitor of the permeability transition pore complex (PTPC). Coincubation of purified organelles revealed that nuclear apoptosis is only induced by Vpr when mitochondria are present yet can be abolished by PTPC inhibitors. Vpr favors the permeabilization of artificial membranes containing the purified PTPC or defined PTPC components such as the adenine nucleotide translocator (ANT) combined with Bax. Again, this effect is prevented by addition of recombinant Bcl-2. The Vpr COOH terminus binds purified ANT, as well as a molecular complex containing ANT and the voltage-dependent anion channel (VDAC), another PTPC component. Yeast strains lacking ANT or VDAC are less susceptible to Vpr-induced killing than control cells yet recover Vpr sensitivity when retransfected with yeast ANT or human VDAC. Hence, Vpr induces apoptosis via a direct effect on the mitochondrial PTPC.


Subject(s)
Apoptosis , Gene Products, vpr/physiology , HIV-1/physiology , Mitochondria/physiology , Cell-Free System , Gene Products, vpr/chemistry , Humans , Jurkat Cells , Permeability , Proto-Oncogene Proteins c-bcl-2/physiology , vpr Gene Products, Human Immunodeficiency Virus
18.
Ann N Y Acad Sci ; 926: 149-64, 2000.
Article in English | MEDLINE | ID: mdl-11193032

ABSTRACT

In most examples of physiological or pathological cell death, mitochondrial membrane permeabilization (MMP) constitutes an early critical event of the lethal process. Signs of MMP that precede nuclear apoptosis include the translocation of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria to an extra-mitochondrial localization, as well as the dissipation of the mitochondrial transmembrane potential. MMP also occurs in HIV-1-induced apoptosis. Different HIV-1 encoded proteins (Env, Vpr, Tat, PR) can directly or indirectly trigger MMP, thereby causing cell death. The gp120/gp41 Env complex constitutes an example for an indirect MMP inducer. Env expressed on the plasma membrane of HIV-1 infected (or Env-transfected) cells mediates cell fusion with CD4/CXCR4-expressing uninfected cells. After a cell type-dependent latency period, syncytia then undergo MMP and apoptosis. Vpr exemplifies a direct MMP inducer. Vpr binds to the adenine nucleotide translocator (ANT), a mitochondrial inner membrane protein which also interacts with apoptosis-regulatory proteins from the Bcl-2/Bax family. Binding of Vpr to ANT favors formation of a non-specific pore leading to MMP. The structural motifs of the Vpr protein involved in MMP are conserved among most pathogenic HIV-1 isolates and determine the cytotoxic effect of Vpr. These data suggest the possibility that viruses employ multiple strategies to regulate host cell apoptosis by targeting mitochondria.


Subject(s)
Apoptosis/physiology , Cytochrome c Group/metabolism , HIV Infections/metabolism , HIV-1/metabolism , Mitochondria/metabolism , Retroviridae Proteins/metabolism , Animals , Apoptosis Inducing Factor , Cell Line , Flavoproteins/metabolism , HIV Infections/pathology , HIV-1/genetics , Humans , Lymphocytes/metabolism , Membrane Proteins/metabolism , Protein Structure, Tertiary
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