Apoptosis inducing factor (AIF): a phylogenetically old, caspase-independent effector of cell death.
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Although much emphasis has been laid on the role of caspase in cell death, recent data indicate that, in many instances, mammalian cell death is caspase-independent. Thus, in many examples of mammalian cell death the 'decision' between death and life is upstream or independent of caspase activation. Similarly, it is unclear whether PCD of plants and fungi involves the activation of caspase-like enzymes, and no caspase-like gene has thus far been cloned in these phyla. Apoptosis inducing factor (AIF) is a new mammalian, caspase-independent death effector which, upon apoptosis induction, translocates from its normal localization, the mitochondrial intermembrane space, to the nucleus. Once in the nucleus, AIF causes chromatin condensation and large scale DNA fragmentation to fragments of approximately 50 kbp. The AIF cDNA from mouse and man codes for a protein which possesses three domains (i) an amino-terminal presequence which is removed upon import into the intermembrane space of mitochondria; (ii) a spacer sequence of approximately 27 amino acids; and (iii) a carboxyterminal 484 amino acid oxidoreductase domain with strong homology to oxidoreductases from other vertebrates (X. laevis), non-vertebrate animals (C. elegans, D. melanogaster), plants, fungi, eubacteria, and archaebacteria. Functionally important amino acids involved in the interaction with the prosthetic groups flavin adenine nucleotide and nicotinamide adenine nucleotide are strongly conserved between AIF and bacterial oxidoreductase. Several eukaryotes possess a similar domain organisation in their AIF homologs, making them candidates to be mitochondrial oxidoreductases as well as caspase-independent death effectors. The phylogenetic implications of these findings are discussed. (+info)
Molecular cloning and functional analysis of the mouse homologue of the KILLER/DR5 tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor.
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Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors are members of the tumor necrosis factor superfamily. TRAIL selectively kills cancer cells but not normal cells. We report here the cloning of the mouse homologue of the TRAIL receptor KILLER/DR5 (MK). The cDNA of MK is 1146 bp in length and encodes a protein of 381 amino acids. MK contains an extracellular cysteine-rich domain, a transmembrane domain, and a cytoplasmic death-domain characteristic of Fas, tumor necrosis factor, and human TRAIL receptors. MK is highly homologous and binds TRAIL with similar affinity as human DR4 and KILLER/DR5. MK induces apoptosis in mouse and human cells and inhibits colony growth of NIH3T3 cells. Expression of MK is p53-dependent and up-regulated by tumor suppressor p53 and by DNA damaging agents in mouse cells undergoing apoptosis. This is the first report describing a mouse TRAIL receptor gene and also demonstrating that the p53-dependent regulation of KILLER/DR5-mediated apoptosis is conserved between human and mouse. (+info)
Enhancer-blocking activity within the DNase I hypersensitive site 2 to 6 region between the TCR alpha and Dad1 genes.
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Although tightly linked, the TCR alpha and delta genes are expressed specifically in T lymphocytes, whereas the Dad1 gene is ubiquitously expressed. Between TCR alpha and Dad1 are eight DNase I hypersensitive sites (HS). HS1 colocalizes with the TCR alpha enhancer (Ealpha) and is T cell-specific; HS2, -3, -4, -5, and -6 map downstream of HS1 and are tissue-nonspecific. The region spanning HS2-6 was reported to display chromatin-opening activity and to confer copy number-dependent and integration site-independent transgene expression in transgenic mice. Here, we demonstrate that HS2-6 also displays enhancer-blocking activity, as it can block an enhancer from activating a promoter when located between the two in a chromatin-integrated context, and can do so without repressing either the enhancer or the promoter. Multiple enhancer-blocking elements are arrayed across HS2-6. We show that HS2-6 by itself does not activate transcription in chromatin context, but can synergize with an enhancer when located upstream of an enhancer and promoter. We propose that HS2-6 primarily functions as an insulator or boundary element that may be critical for the autonomous regulation of the TCR alpha and Dad1 genes. (+info)
Inactivation of the inhibitory kappaB protein kinase/nuclear factor kappaB pathway by Par-4 expression potentiates tumor necrosis factor alpha-induced apoptosis.
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Par-4 is a novel protein identified in cells undergoing apoptosis. The ability of Par-4 to promote apoptotic cell death is dependent on the binding and inactivation of the atypical protein kinases C (PKCs). This subfamily of kinases has been reported to control nuclear factor kappaB (NF-kappaB) through the regulation of the IkappaB kinase activity. NF-kappaB activation by tumor necrosis factor alpha (TNFalpha) provides a survival signal that impairs the TNFalpha-induced apoptotic response. We show here that expression of Par-4 inhibits the TNFalpha-induced nuclear translocation of p65 as well as the kappaB-dependent promoter activity. Interestingly, Par-4 expression blocks inhibitory kappaB protein (IkappaB) kinase activity, which leads to the inhibition of IkappaB phosphorylation and degradation, in a manner that is dependent on its ability to inhibit lambda/iotaPKC. Of potential functional relevance, the expression of Par-4 allows TNFalpha to induce apoptosis in NIH-3T3 cells. In addition, the down-regulation of Par-4 levels by oncogenic Ras sensitizes cells to TNFalpha-induced NF-kappaB activation. (+info)
Evidence that Par-4 participates in the pathogenesis of HIV encephalitis.
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Progressive neuronal degeneration in brain regions involved in learning and memory processes is a common occurrence in patients infected with human immunodeficiency virus type 1 (HIV-1). We now report that levels of Par-4, a protein recently linked to neuronal apoptosis in Alzheimer's disease, are increased in neurons in hippocampus of human patients with HIV encephalitis and in monkeys infected with a chimeric strain of HIV-1 and simian immunodeficiency virus. Par-4 levels increased rapidly in cultured hippocampal neurons following exposure to the neurotoxic HIV-1 protein Tat, and treatment of the cultures with a Par-4 antisense oligonucleotide protected the neurons against Tat-induced apoptosis. Additional findings show that Par-4 participates at an early stage of Tat-induced neuronal apoptosis before caspase activation, oxidative stress, and mitochondrial dysfunction. Our data suggest that Par-4 may be a mediator of neuronal apoptosis in HIV encephalitis and that therapeutic approaches targeting the Par-4 apoptotic cascade may prove beneficial in preventing neuronal degeneration and associated dementia in patients infected with HIV-1. (+info)
IFN-gamma mediates a novel antiviral activity through dynamic modulation of TRAIL and TRAIL receptor expression.
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TNF-related apoptosis-inducing ligand (TRAIL) is able to kill many transformed cells of diverse tissue types. We show that TRAIL is inducible by IFN-gamma, by TNF-alpha, and by infection with human CMV, and has potent antiviral activity in vitro. CMV infection and IFN-gamma also reciprocally modulate TRAIL receptor (TRAIL-R) expression. CMV infection increased the expression of TRAIL-R1 and -R2, whereas IFN-gamma down-regulated the expression of TRAIL-Rs on uninfected fibroblasts. Moreover, IFN-gamma significantly decreased the basal level of NF-kappaB activation, a known survival factor that inhibits apoptosis. Thus, TRAIL selectively kills virus-infected cells while leaving uninfected cells intact, and IFN-gamma potentiates these effects by dynamic modulation of TRAIL and TRAIL-R expression and by sensitizing cells to apoptosis. The regulation of TRAIL and TRAIL-R expression may represent a general mechanism that contributes to the control of TRAIL-mediated apoptosis. (+info)
DAP-kinase participates in TNF-alpha- and Fas-induced apoptosis and its function requires the death domain.
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Death-associated protein (DAP)-kinase is a calcium/calmodulin regulated serine/threonine kinase that carries ankyrin repeats, a death domain, and is localized to the cytoskeleton. Here, we report that this kinase is involved in tumor necrosis factor (TNF)-alpha and Fas-induced apoptosis. Expression of DAP-kinase antisense RNA protected cells from killing by anti-Fas/APO-1 agonistic antibodies. Deletion of the death domain abrogated the apoptotic functions of the kinase, thus, documenting for the first time the importance of this protein domain. Overexpression of a fragment encompassing the death domain of DAP-kinase acted as a specific dominant negative mutant that protected cells from TNF-alpha, Fas, and FADD/MORT1-induced cell death. DAP-kinase apoptotic function was blocked by bcl-2 as well as by crmA and p35 inhibitors of caspases, but not by the dominant negative mutants of FADD/MORT1 or of caspase 8. Thus, it functions downstream to the receptor complex and upstream to other caspases. The multidomain structure of this serine/threonine kinase, combined with its involvement in cell death induced by several different triggers, place DAP-kinase at one of the central molecular pathways leading to apoptosis. (+info)
Functional interaction of DFF35 and DFF45 with caspase-activated DNA fragmentation nuclease DFF40.
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DNA fragmentation factor (DFF) functions downstream of caspase-3 and directly triggers DNA fragmentation during apoptosis. Here we described the identification and characterization of DFF35, an isoform of DFF45 comprised of 268 amino acids. Functional assays have shown that only DFF45, not DFF35, can assist in the synthesis of highly active DFF40. Using the deletion mutants, we mapped the function domains of DFF35/45 and demonstrated that the intact structure/conformation of DFF45 is essential for it to function as a chaperone and assist in the synthesis of active DFF40. Whereas the amino acid residues 101-180 of DFF35/45 mediate its binding to DFF40, the amino acid residues 23-100, which is homologous between DFF35/45 and DFF40, may function to inhibit the activity of DFF40. In contrast to DFF45, DFF35 cannot work as a chaperone, but it can bind to DFF40 more strongly than DFF45 and can inhibit its nuclease activity. These findings suggest that DFF35 may function in vivo as an important alternative mechanism to inhibit the activity of DFF40 and further, that the inhibitory effects of both DFF35 and DFF45 on DFF40 can put the death machinery under strict control. (+info)