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1.
Cell Death Differ ; 11(4): 424-38, 2004 Apr.
Article in English | MEDLINE | ID: mdl-14713958

ABSTRACT

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder resulting in selective neuronal loss and dysfunction in the striatum and cortex. The molecular pathways leading to the selectivity of neuronal cell death in HD are poorly understood. Proteolytic processing of full-length mutant huntingtin (Htt) and subsequent events may play an important role in the selective neuronal cell death found in this disease. Despite the identification of Htt as a substrate for caspases, it is not known which caspase(s) cleaves Htt in vivo or whether regional expression of caspases contribute to selective neuronal cells loss. Here, we evaluate whether specific caspases are involved in cell death induced by mutant Htt and if this correlates with our recent finding that Htt is cleaved in vivo at the caspase consensus site 552. We find that caspase-2 cleaves Htt selectively at amino acid 552. Further, Htt recruits caspase-2 into an apoptosome-like complex. Binding of caspase-2 to Htt is polyglutamine repeat-length dependent, and therefore may serve as a critical initiation step in HD cell death. This hypothesis is supported by the requirement of caspase-2 for the death of mouse primary striatal cells derived from HD transgenic mice expressing full-length Htt (YAC72). Expression of catalytically inactive (dominant-negative) forms of caspase-2, caspase-7, and to some extent caspase-6, reduced the cell death of YAC72 primary striatal cells, while the catalytically inactive forms of caspase-3, -8, and -9 did not. Histological analysis of post-mortem human brain tissue and YAC72 mice revealed activation of caspases and enhanced caspase-2 immunoreactivity in medium spiny neurons of the striatum and the cortical projection neurons when compared to controls. Further, upregulation of caspase-2 correlates directly with decreased levels of brain-derived neurotrophic factor in the cortex and striatum of 3-month YAC72 transgenic mice and therefore suggests that these changes are early events in HD pathogenesis. These data support the involvement of caspase-2 in the selective neuronal cell death associated with HD in the striatum and cortex.


Subject(s)
Caspases/metabolism , Huntington Disease/metabolism , Neurons/metabolism , Animals , Brain/metabolism , Brain/pathology , Caspase 2 , Caspase 3 , Caspase 6 , Caspase 7 , Cell Death/physiology , Cerebral Cortex/metabolism , Corpus Striatum/metabolism , Disease Models, Animal , Gene Expression Regulation/genetics , Humans , Huntingtin Protein , Huntington Disease/genetics , Huntington Disease/pathology , Mice , Mice, Transgenic/genetics , Mutation , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/pathology , Nuclear Proteins/genetics , Nuclear Proteins/metabolism
2.
Free Radic Biol Med ; 31(12): 1550-9, 2001 Dec 15.
Article in English | MEDLINE | ID: mdl-11744329

ABSTRACT

Bcl-2 is a gene family involved in the suppression of apoptosis in response to a wide range of cellular insults. Multiple papers have suggested a link between Bcl-2 and oxidative damage/antioxidant protection. We therefore examined parameters of antioxidant defense and oxidative damage in two different cell lines, NT-2/D1 (NT-2) and SK-N-MC, overexpressing Bcl-2 as compared with vector-only controls. Bcl-2 transfectants of both cell lines were more resistant to H(2)O(2) and showed increases in GSH level and Cu/Zn-superoxide dismutase (SOD1) activity, but not in Mn-superoxide dismutase, glutathione peroxidase, or glutathione reductase activities. Catalase activity was increased in SK-N-MC cells. Overexpression of Bcl-2 did not significantly decrease levels of oxidative DNA damage (measured as 8-hydroxyguanine) or lipid peroxidation, but it decreased levels of 3-nitrotyrosine in both cell lines and protein carbonyls in SK-N-MC cells only. It also increased proteasome activity in both cell lines. We conclude that Bcl-2 raises cellular antioxidant defense status, but this is not necessarily reflected in decreased levels of oxidative damage to DNA and lipids. The ability of Bcl-2 overexpression to decrease 3-nitrotyrosine levels suggests that it may decrease formation of peroxynitrite or other reactive nitrogen species; this was confirmed as decreased production of NO(2)(-)/NO(3)(-) in the transfected cells and a fall in the level of nNOS protein.


Subject(s)
Antioxidants/metabolism , Cysteine Endopeptidases/metabolism , Multienzyme Complexes/metabolism , Nitric Oxide/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Cell Survival/physiology , Enzyme Activation , Glutathione/metabolism , Humans , Male , Neoplasms, Nerve Tissue , Neuroblastoma , Nitric Oxide Synthase/metabolism , Oxidative Stress/physiology , Proteasome Endopeptidase Complex , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Reactive Nitrogen Species/metabolism , Superoxide Dismutase/metabolism , Superoxide Dismutase-1 , Teratocarcinoma , Testicular Neoplasms , Transfection , Tumor Cells, Cultured , Up-Regulation
3.
J Biol Chem ; 276(36): 33869-74, 2001 Sep 07.
Article in English | MEDLINE | ID: mdl-11448953

ABSTRACT

The endoplasmic reticulum (ER) is the site of assembly of polypeptide chains destined for secretion or routing into various subcellular compartments. It also regulates cellular responses to stress and intracellular Ca(2+) levels. A variety of toxic insults can result in ER stress that ultimately leads to apoptosis. Apoptosis is initiated by the activation of members of the caspase family and serves as a central mechanism in the cell death process. The present study was carried out to determine the role of caspases in triggering ER stress-induced cell death. Treatment of cells with ER stress inducers such as brefeldin-A or thapsigargin induces the expression of caspase-12 protein and also leads to translocation of cytosolic caspase-7 to the ER surface. Caspase-12, like most other members of the caspase family, requires cleavage of the prodomain to activate its proapoptotic form. Caspase-7 associates with caspase-12 and cleaves the prodomain to generate active caspase-12, resulting in increased cell death. We propose that any cellular insult that causes prolonged ER stress may induce apoptosis through caspase-7-mediated caspase-12 activation. The data underscore the involvement of ER and caspases associated with it in the ER stress-induced apoptotic process.


Subject(s)
Caspases/metabolism , Cell Death , Endoplasmic Reticulum/metabolism , Animals , Apoptosis , Blotting, Western , Brefeldin A/pharmacology , Calcium/metabolism , Caspase 12 , Caspase 7 , Caspase 9 , Caspases/biosynthesis , Catalysis , Cell Line , Cell-Free System , DNA, Complementary/metabolism , Enzyme Activation , Humans , Mice , Mutation , Plasmids/metabolism , Precipitin Tests , Protein Transport , Recombinant Proteins/metabolism , Stress, Physiological , Subcellular Fractions , Thapsigargin/pharmacology , Transfection
4.
Hum Mol Genet ; 10(15): 1531-8, 2001 Jul 15.
Article in English | MEDLINE | ID: mdl-11468270

ABSTRACT

Fourteen neurological diseases have been associated with the expansion of trinucleotide repeat regions. These diseases have been categorized into those that give rise to the translation of toxic polyglutamine proteins and those that are untranslated. Thus far, compelling evidence has not surfaced for the inclusion of a model in which a common mechanism may participate in the pathobiology of both translated and untranslated trinucleotide diseases. In these studies we show that a double-stranded RNA-binding protein, PKR, which has previously been linked to virally-induced and stress-mediated apoptosis, preferentially binds mutant huntingtin RNA transcripts immobilized on streptavidin columns that have been incubated with human brain extracts. These studies also show, by immunodetection in tissue slices, that PKR is present in its activated form in both human Huntington autopsy material and brain tissue derived from Huntington yeast artificial chromosome transgenic mice. The increased immunolocalization of the activated kinase is more pronounced in areas most affected by the disease and, coupled with the RNA binding results, suggests a role for PKR activation in the disease process.


Subject(s)
Huntington Disease/genetics , Huntington Disease/metabolism , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/metabolism , Nuclear Proteins/biosynthesis , Nuclear Proteins/metabolism , eIF-2 Kinase/metabolism , Animals , Biotinylation , Blotting, Western , Brain/metabolism , Chromosomes, Artificial, Yeast/metabolism , Cytoplasm/metabolism , Humans , Huntingtin Protein , Immunohistochemistry , Mice , Mice, Transgenic , Mutation , Phosphorylation , Protein Binding , RNA/metabolism , Streptavidin/metabolism
5.
J Biol Chem ; 276(5): 3149-57, 2001 Feb 02.
Article in English | MEDLINE | ID: mdl-11073962

ABSTRACT

We investigated the mechanism of lysosome-mediated cell death using purified recombinant pro-apoptotic proteins, and cell-free extracts from the human neuronal progenitor cell line NT2. Potential effectors were either isolated lysosomes or purified lysosomal proteases. Purified lysosomal cathepsins B, H, K, L, S, and X or an extract of mouse lysosomes did not directly activate either recombinant caspase zymogens or caspase zymogens present in an NT2 cytosolic extract to any significant extent. In contrast, a cathepsin L-related protease from the protozoan parasite Trypanosoma cruzi, cruzipain, showed a measurable caspase activation rate. This demonstrated that members of the papain family can directly activate caspases but that mammalian lysosomal members of this family may have been negatively selected for caspase activation to prevent inappropriate induction of apoptosis. Given the lack of evidence for a direct role in caspase activation by lysosomal proteases, we hypothesized that an indirect mode of caspase activation may involve the Bcl-2 family member Bid. In support of this, Bid was cleaved in the presence of lysosomal extracts, at a site six residues downstream from that seen for pathways involving capase 8. Incubation of mitochondria with Bid that had been cleaved by lysosomal extracts resulted in cytochrome c release. Thus, cleavage of Bid may represent a mechanism by which proteases that have leaked from the lysosomes can precipitate cytochrome c release and subsequent caspase activation. This is supported by the finding that cytosolic extracts from mice ablated in the bid gene are impaired in the ability to release cytochrome c in response to lysosome extracts. Together these data suggest that Bid represents a sensor that allows cells to initiate apoptosis in response to widespread adventitious proteolysis.


Subject(s)
Apoptosis/physiology , Endopeptidases/physiology , Lysosomes/enzymology , Animals , BH3 Interacting Domain Death Agonist Protein , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Caspase 3 , Caspase 7 , Caspases/metabolism , Cytosol/metabolism , Humans , Mice , Models, Molecular , Rats , Tumor Cells, Cultured
7.
J Biol Chem ; 275(21): 16127-33, 2000 May 26.
Article in English | MEDLINE | ID: mdl-10821864

ABSTRACT

Cytochrome c released from vertebrate mitochondria engages apoptosis by triggering caspase activation. We previously reported that, whereas cytochromes c from higher eukaryotes can activate caspases in Xenopus egg and mammalian cytosols, iso-1 and iso-2 cytochromes c from the yeast Saccharomyces cerevisiae cannot. Here we examine whether the inactivity of the yeast isoforms is related to a post-translational modification of lysine 72, N-epsilon-trimethylation. This modification was found to abrogate pro-apoptotic activity of metazoan cytochrome c expressed in yeast. However, iso-1 cytochrome c lacking the trimethylation modification also was devoid of pro-apoptotic activity. Thus, both lysine 72 trimethylation and other features of the iso-1 sequence preclude pro-apoptotic activity. Competition studies suggest that the lack of pro-apoptotic activity was associated with a low affinity for Apaf-1. As cytochromes c that lack apoptotic function still support respiration, different mechanisms appear to be involved in the two activities.


Subject(s)
Apoptosis , Cytochrome c Group/metabolism , Cytochromes c , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Animals , Horses , Lysine/analogs & derivatives , Lysine/metabolism , Methylation , Mitochondria/metabolism , Models, Molecular , Molecular Sequence Data , Oocytes , Peptide Hydrolases/metabolism , Protein Isoforms , Sequence Homology, Amino Acid , Xenopus
8.
Nat Med ; 6(4): 397-404, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10742146

ABSTRACT

The amyloid beta-protein precursor gives rise to the amyloid beta-protein, the principal constituent of senile plaques and a cytotoxic fragment involved in the pathogenesis of Alzheimer disease. Here we show that amyloid beta-protein precursor was proteolytically cleaved by caspases in the C terminus to generate a second unrelated peptide, called C31. The resultant C31 peptide was a potent inducer of apoptosis. Both caspase-cleaved amyloid beta-protein precursor and activated caspase-9 were present in brains of Alzheimer disease patients but not in control brains. These findings indicate the possibility that caspase cleavage of amyloid beta-protein precursor with the generation of C31 may be involved in the neuronal death associated with Alzheimer disease.


Subject(s)
Amyloid beta-Protein Precursor/metabolism , Caspases/metabolism , Peptides/metabolism , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amyloid beta-Protein Precursor/genetics , Animals , Apoptosis , Brain/enzymology , Brain/pathology , Caspase 9 , Caspases/genetics , Cell Line, Transformed , Enzyme Activation , Humans , Mice , Peptide Fragments/metabolism , Peptides/physiology , Substrate Specificity
9.
J Mol Neurosci ; 15(2): 71-83, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11220787

ABSTRACT

Both transgenic mouse and cell culture models of familial amyotrophic lateral sclerosis (FALS) support a gain-of-function effect for the mutations in copper-zinc superoxide dismutase (CuZnSOD) associated with FALS, but the nature of the function gained remains incompletely characterized. We previously reported an enhanced peroxidase activity for FALS-associated CuZnSOD mutants. Because one of the targets of such activity is CuZnSOD itself, we examined peroxide-mediated inactivation of wild-type and mutant CuZnSODs, and found that the mutants are more readily inactivated. Inactivation of the mutants was associated with fragmentation, which did not occur in the wild-type enzyme under these conditions. Furthermore, the reduction of the FALS-associated mutants by ascorbate was enhanced markedly when compared to the wild-type enzyme. The visible spectra of the mutants showed a consistent blue shift of the peak at 680 nm in the wild-type enzyme, suggesting an alteration in copper-site geometry. These results extend previous studies demonstrating enhanced peroxidase activity in the mutants, and suggest that the toxic function that leads to motor neuron degeneration may result from a loss of specificity of the redox reactions catalyzed by CuZnSOD.


Subject(s)
Motor Neuron Disease/enzymology , Motor Neuron Disease/genetics , Mutation , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Amino Acid Substitution , Humans , Kinetics , Oxidation-Reduction , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Spectrophotometry , Superoxide Dismutase/chemistry
10.
J Mol Neurosci ; 15(3): 215-29, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11303785

ABSTRACT

The mechanisms underlying neurotrophin dependence, and cellular dependent states in general, are unknown. We show that a 29 amino acid region in the intracellular domain of the common neurotrophin receptor, p75NTR, is required for the mediation of apoptosis by p75NTR. Furthermore, contrary to results obtained with Fas, monomeric p75NTR is required for apoptosis induction, whereas multimerization inhibits the pro-apoptotic effect. Within the 29-residue domain required for apoptosis induction by p75NTR, a 14-residue region is sufficient as a peptide inducer of apoptosis. This 14-residue peptide requires the positively charged carboxyterminal residues for its effect on cell death, and these same residues are required by the full-length p75NTR. These studies define a novel type of domain that mediates neurotrophin dependence, and suggest that other cellular dependent states may be mediated by proteins displaying similar domains.


Subject(s)
Apoptosis/genetics , Receptor, Nerve Growth Factor/chemistry , Receptor, Nerve Growth Factor/metabolism , Amino Acid Sequence/genetics , Animals , Cell-Free System/metabolism , Dimerization , Genetic Vectors/genetics , Humans , Mutation/genetics , Peptide Fragments/genetics , Plasmids/biosynthesis , Plasmids/genetics , Protein Structure, Tertiary/genetics , Receptor, Nerve Growth Factor/genetics , Recombinant Fusion Proteins/genetics , Transfection , Tumor Cells, Cultured/cytology , Tumor Cells, Cultured/metabolism
11.
Nat Med ; 5(9): 1032-8, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10470080

ABSTRACT

We have designed short peptides composed of two functional domains, one a tumor blood vessel 'homing' motif and the other a programmed cell death-inducing sequence, and synthesized them by simple peptide chemistry. The 'homing' domain was designed to guide the peptide to targeted cells and allow its internalization. The pro-apoptotic domain was designed to be nontoxic outside cells, but toxic when internalized into targeted cells by the disruption of mitochondrial membranes. Although our prototypes contain only 21 and 26 residues, they were selectively toxic to angiogenic endothelial cells and showed anti-cancer activity in mice. This approach may yield new therapeutic agents.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Breast Neoplasms/blood supply , Peptides/pharmacology , Protein Sorting Signals/physiology , Amino Acid Sequence , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Breast Neoplasms/ultrastructure , Cells, Cultured , Dose-Response Relationship, Drug , Drug Design , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Endothelium, Vascular/pathology , Endothelium, Vascular/ultrastructure , Female , Humans , Intracellular Membranes/drug effects , Intracellular Membranes/pathology , Intracellular Membranes/ultrastructure , Mice , Mice, Inbred BALB C , Mice, Nude , Mitochondria, Liver/drug effects , Mitochondria, Liver/pathology , Mitochondria, Liver/ultrastructure , Neoplasm Transplantation , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/pathology , Peptides/chemistry , Peptides/metabolism , Peptides/therapeutic use , Protein Sorting Signals/genetics , Rats , Transplantation, Heterologous , Tumor Cells, Cultured
12.
Proc Natl Acad Sci U S A ; 96(10): 5752-7, 1999 May 11.
Article in English | MEDLINE | ID: mdl-10318956

ABSTRACT

Caspase-9 is critical for cytochrome c (cyto-c)-dependent apoptosis and normal brain development. We determined that this apical protease in the cyto-c pathway for apoptosis resides inside mitochondria in several types of cells, including cardiomyocytes and many neurons. Caspase-9 is released from isolated mitochondria on treatment with Ca2+ or Bax, stimuli implicated in ischemic neuronal cell death that are known to induce cyto-c release from mitochondria. In neuronal cell culture models, apoptosis-inducing agents trigger translocation of caspase-9 from mitochondria to the nucleus, which is inhibitable by Bcl-2. Similarly, in an animal model of transient global cerebral ischemia, caspase-9 release from mitochondria and accumulation in nuclei was observed in hippocampal and other vulnerable neurons exhibiting early postischemic changes preceding apoptosis. Loss of mitochondrial barrier function during neuronal damage from ischemia or other insults therefore may play an important role in making certain caspases available to participate in apoptosis.


Subject(s)
Apoptosis , Brain Ischemia/metabolism , Brain/metabolism , Caspases/metabolism , Mitochondria/enzymology , Amino Acid Sequence , Animals , Calcium/pharmacology , Caspase 9 , Cell Nucleus/enzymology , Cytochrome c Group/metabolism , Enzyme Precursors/metabolism , Immunohistochemistry , Molecular Sequence Data , Myocardium/metabolism , Neurons/metabolism , PC12 Cells , Proto-Oncogene Proteins/pharmacology , Proto-Oncogene Proteins c-bcl-2/metabolism , Rats , Reperfusion Injury/enzymology , bcl-2-Associated X Protein
13.
J Biol Chem ; 274(13): 8730-6, 1999 Mar 26.
Article in English | MEDLINE | ID: mdl-10085113

ABSTRACT

Dentatorubropallidoluysian atrophy (DRPLA) is one of eight autosomal dominant neurodegenerative disorders characterized by an abnormal CAG repeat expansion which results in the expression of a protein with a polyglutamine stretch of excessive length. We have reported recently that four of the gene products (huntingtin, atrophin-1 (DRPLA), ataxin-3, and androgen receptor) associated with these open reading frame triplet repeat expansions are substrates for the cysteine protease cell death executioners, the caspases. This led us to hypothesize that caspase cleavage of these proteins may represent a common step in the pathogenesis of each of these four neurodegenerative diseases. Here we present evidence that caspase cleavage of atrophin-1 modulates cytotoxicity and aggregate formation. Cleavage of atrophin-1 at Asp109 by caspases is critical for cytotoxicity because a mutant atrophin-1 that is resistant to caspase cleavage is associated with significantly decreased toxicity. Further, the altered cellular localization within the nucleus and aggregate formation associated with the expanded form of atrophin-1 are completely suppressed by mutation of the caspase cleavage site at Asp109. These results provide support for the toxic fragment hypothesis whereby cleavage of atrophin-1 by caspases may be an important step in the pathogenesis of DRPLA. Therefore, inhibiting caspase cleavage of the polyglutamine-containing proteins may be a feasible therapeutic strategy to prevent cell death.


Subject(s)
Caspases/metabolism , Nerve Tissue Proteins/metabolism , Amino Acid Sequence , Apoptosis/genetics , Atrophy/genetics , Caspase 3 , Cell Line , Fluorescent Antibody Technique , Humans , Molecular Sequence Data , Mutation/genetics , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/toxicity , Peptides/genetics , Protein Conformation , Tamoxifen/pharmacology , Transfection , Trinucleotide Repeats/genetics
14.
J Neurochem ; 72(1): 185-95, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9886069

ABSTRACT

X-linked spinal and bulbar muscular atrophy (SBMA), Kennedy's disease, is a degenerative disease of the motor neurons that is associated with an increase in the number of CAG repeats encoding a polyglutamine stretch within the androgen receptor (AR). Recent work has demonstrated that the gene products associated with open reading frame triplet repeat expansions may be substrates for the cysteine protease cell death executioners, the caspases. However, the role that caspase cleavage plays in the cytotoxicity associated with expression of the disease-associated alleles is unknown. Here, we report the first conclusive evidence that caspase cleavage is a critical step in cytotoxicity; the expression of the AR with an expanded polyglutamine stretch enhances its ability to induce apoptosis when compared with the normal AR. The AR is cleaved by a caspase-3 subfamily protease at Asp146, and this cleavage is increased during apoptosis. Cleavage of the AR at Asp146 is critical for the induction of apoptosis by AR, as mutation of the cleavage site blocks the ability of the AR to induce cell death. Further, mutation of the caspase cleavage site at Asp146 blocks the ability of the SBMA AR to form perinuclear aggregates. These studies define a fundamental role for caspase cleavage in the induction of neural cell death by proteins displaying expanded polyglutamine tracts, and therefore suggest a strategy that may be useful to treat neurodegenerative diseases associated with polyglutamine repeat expansions.


Subject(s)
Caspases/metabolism , Muscular Atrophy, Spinal/enzymology , Neurons/enzymology , Receptors, Androgen/metabolism , Carcinogens/pharmacology , Caspases/chemistry , Catalytic Domain/genetics , Cell Death/physiology , Cell Nucleus/enzymology , Cells, Cultured , Cytotoxins/metabolism , Enzyme Activation/genetics , Fetus/cytology , Gene Expression , Kidney/cytology , Muscular Atrophy, Spinal/genetics , Mutagenesis/physiology , Neurons/chemistry , Neurons/cytology , Peptides/metabolism , Receptors, Androgen/genetics , Testosterone/pharmacology , Transfection , Trinucleotide Repeats
15.
J Biol Chem ; 273(42): 27084-90, 1998 Oct 16.
Article in English | MEDLINE | ID: mdl-9765224

ABSTRACT

The apoptotic signal triggered by ligation of members of the death receptor family is promoted by sequential activation of caspase zymogens. We show here that in a purified system, the initiator caspases-8 and -10 directly process the executioner pro-caspase-3 with activation rates (kcat/Km) of 8.7 x 10(5) and 2.8 x 10(5) M-1 s-1, respectively. These rates are of sufficient magnitude to indicate direct processing in vivo. Differentially processed forms of caspase-3 that accumulate during its activation have similar rates of activation, activities, and specificities. The pattern and rate of caspase-8 induced activation of pro-caspase-3 in cytosolic extracts was the same as in a purified system. Moreover, immunodepletion of a putative intermediary in the pathway to activation, pro-caspase-9, was without consequence. Taken together these data demonstrate that the initiator caspase-8 can directly activate pro-caspase-3 without the requirement for an accelerator. The in vitro data thus help to deconvolute previous in vivo transfection studies which have debated the role of a direct versus indirect transmission of the apoptotic signal generated by ligation of death receptors.


Subject(s)
Caspases/metabolism , Enzyme Precursors/metabolism , Apoptosis , Caspase 10 , Caspase 3 , Caspase 8 , Caspase 9 , Caspases/genetics , Cytosol/metabolism , Enzyme Activation , Enzyme Precursors/genetics , Granzymes , Kinetics , Models, Biological , Protein Processing, Post-Translational , Serine Endopeptidases/metabolism , Signal Transduction
16.
J Biol Chem ; 273(15): 9158-67, 1998 Apr 10.
Article in English | MEDLINE | ID: mdl-9535906

ABSTRACT

The neurodegenerative diseases Huntington disease, dentatorubropallidoluysian atrophy, spinocerebellar atrophy type 3, and spinal bulbar muscular atrophy are caused by expansion of a polyglutamine tract within their respective gene products. There is increasing evidence that generation of truncated proteins containing an expanded polyglutamine tract may be a key step in the pathogenesis of these disorders. We now report that, similar to huntingtin, atrophin-1, ataxin-3, and the androgen receptor are cleaved in apoptotic extracts. Furthermore, each of these proteins is cleaved by one or more purified caspases, cysteine proteases involved in apoptotic death. The CAG length does not modulate susceptibility to cleavage of any of the full-length proteins. Our results suggest that by generation of truncated polyglutamine-containing proteins, caspase cleavage may represent a common step in the pathogenesis of each of these neurodegenerative diseases.


Subject(s)
Caspases , Nerve Tissue Proteins/metabolism , Neurodegenerative Diseases/genetics , Nuclear Proteins/metabolism , Peptides , Serine Endopeptidases/metabolism , Trinucleotide Repeats , Amino Acid Sequence , Apoptosis , Ataxin-3 , Caspase 1 , Caspase 3 , Caspase 7 , Caspase 8 , Caspase 9 , Cysteine Endopeptidases/metabolism , Humans , Huntingtin Protein , Jurkat Cells , Kinetics , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Osteosarcoma , Receptors, Androgen/chemistry , Receptors, Androgen/metabolism , Repressor Proteins , Substrate Specificity , Tumor Cells, Cultured
17.
J Neurosci ; 17(16): 6165-78, 1997 Aug 15.
Article in English | MEDLINE | ID: mdl-9236228

ABSTRACT

Apoptosis is a fundamental process required for normal development of the nervous system and is triggered during neurodegenerative disease. To dissect the molecular events leading to neuronal cell death, we have developed a cell-free model of neuronal apoptosis. The model faithfully reproduces key elements of apoptosis, including chromatin condensation, DNA fragmentation, caspase activation/processing, and selective substrate cleavage. We report that cell-free apoptosis is activated in premitochondrial, mitochondrial, and postmitochondrial phases by tamoxifen, mastoparan, and cytochrome c, respectively, allowing a functional ordering of these proapoptotic modulators. Furthermore, this is the first report of mitochondrial-mediated activation of cell-free apoptosis in a cell extract. Although Bcl-2 blocks activation at the premitochondrial and mitochondrial levels, it does not affect the postmitochondrial level. The cell-free system described here provides a valuable tool to elucidate the molecular events leading to neuronal cell death.


Subject(s)
Apoptosis/physiology , Caspases , Cerebellum/cytology , Mitochondria/enzymology , Neurons/cytology , Adenosine Triphosphate/pharmacology , Animals , Apoptosis/drug effects , Atractyloside/pharmacology , Caspase 3 , Cell Extracts/pharmacology , Cell-Free System , Cells, Cultured , Cysteine Endopeptidases/metabolism , Cytochrome c Group/pharmacology , Enzyme Inhibitors/pharmacology , Enzyme Precursors/metabolism , Estrogen Antagonists/pharmacology , Intercellular Signaling Peptides and Proteins , Molecular Sequence Data , Neurons/enzymology , Neurons/ultrastructure , Peptides , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Rats , Rats, Sprague-Dawley , Sequence Homology, Amino Acid , Staurosporine/pharmacology , Tamoxifen/pharmacology , Wasp Venoms/pharmacology
19.
J Cell Biol ; 137(7): 1581-8, 1997 Jun 30.
Article in English | MEDLINE | ID: mdl-9199172

ABSTRACT

We expressed the human anti-apoptotic protein, Bcl-2, in Saccharomyces cerevisiae to investigate its effects on antioxidant protection and stationary phase survival. Yeast lacking copper-zinc superoxide dismutase (sod1Delta) show a profound defect in entry into and survival during stationary phase even under conditions optimal for survival of wild-type strains (incubation in water after stationary phase is reached). Expression of Bcl-2 in the sod1Delta strain caused a large improvement in viability at entry into stationary phase, as well as increased resistance to 100% oxygen and increased catalase activity. In addition, Bcl-2 expression reduced mutation frequency in both wild-type and sod1Delta strains. In another set of experiments, wild-type yeast incubated in expired minimal medium instead of water lost viability quickly; expression of Bcl-2 significantly delayed this stationary phase death. Our results demonstrate that Bcl-2 has activities in yeast that are similar to activities it is known to possess in mammalian cells: (a) stimulation of antioxidant protection and (b) delay of processes leading to cell death.


Subject(s)
Gene Expression Regulation, Fungal , Proto-Oncogene Proteins c-bcl-2/genetics , Saccharomyces cerevisiae/genetics , Superoxide Dismutase/genetics , Gene Transfer Techniques , Humans , Mutation , Oxidative Stress , Saccharomyces cerevisiae/growth & development
20.
Neurology ; 47(4 Suppl 2): S36-8; discussion S38-9, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8858049

ABSTRACT

Mutations in copper-zinc superoxide dismutase (CuZnSOD) that are associated with familial ALS (FALS) are dominant, gain-of-function mutations, but the nature of the function gained has not been identified. In addition to catalyzing the dismutation of superoxide, copper-zinc superoxide dismutase also displays peroxidase activity. Whereas mutants A4V and G93A retained superoxide dismutase activity, they demonstrated a markedly enhanced copper-dependent peroxidase activity in comparison with that of the wild type enzyme as detected by the spin trap 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) in electron paramagnetic resonance measurements. Two copper chelators, diethyldithiocarbamate and penicillamine, inhibited the mutants' peroxidase activity, but not that of the wild type enzyme, at stoichiometric concentrations; furthermore, these copper chelators enhanced neural survival in a cell-culture model of ALS but did not alter survival of cells expressing only wild type copper-zinc superoxide dismutase. These observations suggest that oxidative reactions catalyzed by mutant copper-zinc superoxide dismutases may initiate the neuropathologic changes of FALS.


Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Cell Death , Humans , Superoxide Dismutase/metabolism
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