Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.22.56 (caspase-3)
35,750 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe the characterization and purification of a protease that cleaves sterol regulatory element-binding protein-1 (SREBP-1) and SREBP-2 in vitro. Cleavage occurs between the basic helix-loop-helix-leucine zipper and the first transmembrane domain of each SREBP. This is the region in which the SREBPs are cleaved physiologically by a sterol-regulated protease that releases an NH2-terminal fragment that activates transcription of the genes for the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl CoA synthase. The cleavage enzyme, designated SREBP cleavage activity (SCA), belongs to a new class of cysteine proteases of the interleukin-1 beta-converting enzyme (ICE) family, all of which cleave at aspartic acid residues. Like ICE, SCA was inactive in cytosol, and it was activated in vitro by incubation at 30 degrees C. SCA was resistant to inhibitors of serine, aspartyl, and metalloproteases, but it was sensitive to N-ethylmaleimide. The enzyme cleaved SREBP-1 and SREBP-2 between the Asp and Ser of a conserved sequence (S/DEPDSP). The activity was blocked by a tetrapeptide aldehyde, Ac-Asp-Glu-Ala-Asp-aldehyde (Ac-DEAD-CHO). A purified preparation of SCA from hamster liver contained a prominent 20-kDa polypeptide that could be labeled with [14C]iodoacetic acid. Labeling was blocked by Ac-DEAD-CHO. Partial amino acid sequence of this polypeptide revealed that it was the hamster equivalent of human CPP32, a putative protease whose cDNA was recently identified by virtue of sequence homology to ICE. CPP32 and ICE have been implicated in apoptosis in animal cells. Whether SCA/CPP32 participates in vivo in the sterol-regulated activation of SREBP, or whether it activates SREBPs during apoptosis, remains to be determined.
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PMID:Purification of an interleukin-1 beta converting enzyme-related cysteine protease that cleaves sterol regulatory element-binding proteins between the leucine zipper and transmembrane domains. 762 13

Cloning of interleukin-1 beta converting enzyme (ICE) and Caenorhabditis elegans death protein CED-3 revealed the structural and functional homology between these two proteases. It also suggested the involvement of ICE-like cysteine proteases in apoptosis. Several CED-3- and ICE-like cysteine proteases have been described, including Nedd2/Ich-1, CPP32 beta, Tx, ICErel3, and Mch2. We have previously described a mouse ortholog of cysteine protease CPP32 beta that shares strong homology with ICE and CED-3. Here, we describe the cloning of mouse and human Casp7, another member of this family of cysteine proteases. Mouse Casp7 encodes a putative 340-amino-acid polypeptide that contains all the known conserved residues required for protease function, including the QACRG sequence, aspartic acid residues for internal cleavage sites, and the residues required for substrate binding. Three RNA variants of human Casp7 were also cloned. Amino acid sequence analysis indicated that Casp7 shared high homology with CPP32 beta/Casp3 and Mch2/Casp6. Northern blot analysis demonstrated that a 2.6-kb Casp7 mRNA was expressed in various tissues except brain. Mouse interspecific backcross mapping allowed localization of Casp7 to the distal region of mouse chromosome 19, linked to Mxi1, Adra2a, and Aop1.
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PMID:Identification and mapping of Casp7, a cysteine protease resembling CPP32 beta, interleukin-1 beta converting enzyme, and CED-3. 907 Sep 23

PS2, the chromosome 1 familial Alzheimer's disease gene, has been shown to be involved in programmed cell death by three complementary experimental approaches. Reduction of PS2 protein levels by antisense RNA protects from apoptosis, whereas overexpression of an Alzheimer's PS2 mutant increases cell death induced by several stimuli. In addition, ALG-3, a truncated PS2 cDNA, encodes an artificial COOH-terminal PS2 segment that dominantly inhibits apoptosis. Here we describe a physiological COOH-terminal PS2 polypeptide (PS2s, Met298-Ile448) generated by both an alternative PS2 transcript and proteolytic cleavage. We find that PS2s protects transfected cells from Fas- and tumor necrosis factor alpha (TNFalpha)-induced apoptosis. Furthermore, a similar anti-apoptotic COOH-terminal PS2 polypeptide (PS2Ccas) is generated by caspase-3 cleavage at Asp329. These results suggest that caspase-3 not only activates pro-apoptotic substrates but also generates a negative feedback signal in which PS2Ccas antagonizes the progression of cell death. Thus, whereas PS2 is required for apoptosis, PS2s and PS2Ccas oppose this process, and the balance between PS2 and these COOH-terminal fragments may dictate the cell fate.
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PMID:Generation of anti-apoptotic presenilin-2 polypeptides by alternative transcription, proteolysis, and caspase-3 cleavage. 935 87

Interleukin-16, a proinflammatory cytokine produced in CD8(+) lymphocytes, is synthesized as a precursor protein (pro-IL-16). It is postulated that the C-terminal region of pro-IL-16 is cleaved, releasing bioactive IL-16. To characterize IL-16 cleavage, we transfected COS cells with a cDNA encoding a approximately 50-kDa form of pro-IL-16. Transfected COS cells released a approximately 20-kDa IL-16 cleavage product shown to consist of the 121 C-terminal residues of pro-IL-16 by immunoblotting and amino acid sequencing. Cleaved IL-16, but not pro-IL-16, exhibited lymphocyte chemoattractant activity. A C-terminal approximately 20-kDa IL-16 polypeptide was also released when pro-IL-16 was treated with concanavalin A-stimulated CD8(+) lymphocyte lysate. Cleavage occurred after an Asp, suggesting involvement of a caspase (interleukin-1beta-converting enzyme/CED-3) family protease. Using recombinant caspases and granzyme B, we determined that pro-IL-16 cleavage is mediated only by caspase-3. Relevance to pro-IL-16 processing in primary lymphocytes was supported by identifying the p20 subunit of activated caspase-3 in stimulated CD8(+) lymphocytes and by inhibition of CD8(+) lymphocyte lysate-mediated cleavage with Ac-DEVD-CHO. Pro-IL-16 is a substrate for caspase-3, and cleavage by this enzyme releases biologically active IL-16 from its inactive precursor.
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PMID:Processing and activation of pro-interleukin-16 by caspase-3. 942 80

Removal of nerve growth factor (NGF) from sympathetic neurons initiates a neuronal death program and apoptosis. We show that pituitary adenylyl cyclase-activating polypeptide (PACAP) prevents apoptosis in NGF-deprived sympathetic neurons. PACAP (100 nM) added to culture medium at the time of plating failed to support neuronal survival. However, in neurons grown for 2 days with NGF and then deprived of NGF, PACAP prevented cell death for the next 24-48 h. Uptake of [3H]norepinephrine ([3H]NE) was used as an index of survival and decreased >50% in NGF-deprived cultures within 24 h. PACAP (1-100 nM) restored [3H]NE uptake to 92 +/- 8% of that of NGF-supported controls. Depolarization-induced [3H]NE release in neurons rescued by PACAP was the same as that in NGF-supported neurons. PACAP rescue was not mimicked by forskolin or 8-bromo-cyclic AMP and was not blocked by the protein kinase A inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate. Mobilization of phosphatidylinositol by muscarine failed to support NGF-deprived neurons. Thus, PACAP may use novel signaling to promote survival of sympathetic neurons. The apoptosis-associated caspase CPP32 activity increased approximately fourfold during 6 h of NGF withdrawal (145 +/- 40 versus 38 +/- 17 nmol of substrate cleaved/min/mg of protein) and returned to even below the control level in NGF-deprived, PACAP-rescued cultures (14 +/- 7 nmol/min/mg of protein). Readdition of NGF or PACAP to NGF-deprived cultures reversed CPP32 activation, and this was blocked by lactacystin, a potent and specific inhibitor of the 20S proteasome, suggesting that NGF and PACAP target CPP32 for destruction by the proteasome. As PACAP is a preganglionic neurotransmitter in autonomic ganglia, we propose a novel function for this transmitter as an apoptotic rescuer of sympathetic neurons when the supply of NGF is compromised.
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PMID:Pituitary adenylyl cyclase-activating polypeptide and nerve growth factor use the proteasome to rescue nerve growth factor-deprived sympathetic neurons cultured from chick embryos. 979 12

Apoptotic cell suicide initiated by ligation of CD95 (Fas/APO-1) occurs through recruitment, oligomerization and autocatalytic activation of the cysteine protease, caspase-8 (MACH, FLICE, Mch5). An endogenous mammalian regulator of this process, named Usurpin, has been identified (aliases for Usurpin include CASH, Casper, CLARP, FLAME-1, FLIP, I-FLICE and MRIT). This protein is ubiquitously expressed and exists as at least three isoforms arising by alternative mRNA splicing. The Usurpin gene is comprised of 13 exons and is clustered within approximately 200 Kb with the caspase-8 and -10 genes on human chromosome 2q33-34. The Usurpin polypeptide has features in common with pro-caspase-8 and -10, including tandem 'death effector domains' on the N-terminus of a large subunit/small subunit caspase-like domain, but it lacks key residues that are necessary for caspase proteolytic activity, including the His and Cys which form the catalytic substrates diad, and residues that stabilize the P1 aspartic acid in substrates. Retro-mutation of these residues to functional caspase counterparts failed to restore proteolytic activity, indicating that other determinants also ensure the absence of catalytic potential. Usurpin heterodimerized with pro-caspase-8 in vitro and precluded pro-caspase-8 recruitment by the FADD/MORT1 adapter protein. Cell death induced by CD95 (Fas/APO-1) ligation was attenuated in cells transfected with Usurpin. In vivo, a Usurpin deficit was found in cardiac infarcts where TUNEL-positive myocytes and active caspase-3 expression were prominent following ischemia/reperfusion injury. In contrast, abundant Usurpin expression (and a caspase-3 deficit) occurred in surrounding unaffected cardiac tissue, suggesting reciprocal regulation of these pro- and anti-apoptotic molecules in vivo. Usurpin thus appears to be an endogenous modulator of apoptosis sensitivity in mammalian cells, including the susceptibility of cardiac myocytes to apoptotic death following ischemia/ reperfusion injury.
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PMID:Cell death attenuation by 'Usurpin', a mammalian DED-caspase homologue that precludes caspase-8 recruitment and activation by the CD-95 (Fas, APO-1) receptor complex. 1020 Apr 73

Activation of a member of the caspase family of cysteine proteases is thought to be required for the execution of apoptosis in neurons and other cell types. We describe here an antibody (Ab127) reactive with a neoantigenic site on caspase substrate proteins degraded during apoptosis, and its characterization as a biochemical and histochemical probe for apoptosis-associated proteolysis in growth factor-deprived neural cells in vitro and the developing postnatal rat brain. Neuronally differentiated PC12 cells became strongly Ab127 immunoreactive only during apoptosis following nerve growth factor withdrawal. Apoptosis-associated caspase proteolysis was detectable on western blots as markedly increased immunoreactivity of a approximately 46,000 mol. wt polypeptide, a product also generated by caspase-3 treatment of cell-free extracts. In the postnatal rat brain, intense immunoreactivity indicative of caspase activation was exhibited by small proportions of neurons and glia in distinct regional and temporal patterns. The degenerating nature of these cells was confirmed by their argyrophilia, cytoplasmic immunoreactivity for c-jun and fragmented processes. Combined immunofluorescence and Hoechst 33342 staining demonstrated that cells immunopositive for caspase activation have apoptotic nuclear morphologies. Caspase proteolysis was observed throughout the neuraxis in a minority of progenitor cells in germinal zones, postmitotic neurons in the parenchyma, and glia in the corpus callosum and other white matter tracts, but was observed rarely in the adult brain. These data characterize a new approach for evaluating apoptosis in physiological and pathological neurodegeneration, and demonstrate that caspase-associated apoptosis is a widespread mechanism for the programmed death of neurons and glia in the postnatal rat brain.
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PMID:Immunolocalization of caspase proteolysis in situ: evidence for widespread caspase-mediated apoptosis of neurons and glia in the postnatal rat brain. 1068 85

GRP94 is a 94-kDa chaperone glycoprotein with Ca(2+)-binding properties. We report here that during apoptosis induced by the topoisomerase II inhibitor etoposide, a fraction of GRP94 associated with the endoplasmic reticulum membrane undergoes specific proteolytic cleavage, coinciding with the activation of the caspase CPP32 and initiation of DNA fragmentation. In vivo, inhibitors of caspases able to block etoposide-induced apoptosis can only partially protect GRP94 from proteolytic cleavage, whereas complete inhibition is observed with calpain inhibitor I but not with the proteasome inhibitor. In vitro, GRP94 is not a substrate for CPP32; rather, it can be completely cleaved by calpain, a Ca(2+)-regulated protease. The cleavage of GRP94 by calpain is Ca(2+)-dependent and generates a discrete polypeptide of 80 kDa. In contrast, calpain has no effect on other stress proteins such as GRP78 or HSP70. Further, immunohistochemical staining reveals specific co-localization of GRP94 with calpain in the perinuclear region following etoposide treatment. We further showed that reduction of GRP94 by antisense decreased cell viability in etoposide-treated Jurkat cells. Our studies provide new evidence that the cytoprotective GRP94, as in the case of the antiapoptotic protein Bcl-2, can be targets of proteolytic cleavage themselves during the apoptotic process.
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PMID:The endoplasmic reticulum chaperone glycoprotein GRP94 with Ca(2+)-binding and antiapoptotic properties is a novel proteolytic target of calpain during etoposide-induced apoptosis. 1049 10

Induction of apoptosis results in inhibition of the rate of overall protein synthesis in a variety of cell types. We have shown previously that polypeptide chain initiation factor eIF4GI is rapidly cleaved by caspase-3, whereas other components of the eIF4F complex are much more stable during apoptosis in BJAB and Jurkat cells. We have now extended our analysis to other factors involved in the initiation of protein synthesis and we report here that eIF4B, the p35 subunit of eIF3, and minor proportions of the alpha subunit of eIF2 and the eIF4E-binding protein 4E-BP1 are also cleaved to give rise to discrete fragments. These cleavages occur with delayed kinetics relative to that seen for eIF4GI, and eIF2beta and eIF2gamma levels also decrease at a relatively late stage of apoptosis. In contrast, the second form of eIF4G described recently, eIF4GII, is cleaved as rapidly as eIF4GI under the same conditions. Purified recombinant caspase-3 is able to degrade eIF4B and eIF3(p35) in vitro, producing fragments of the same sizes as those seen in intact cells. Induction of apoptosis also results in a biphasic change in the association of 4E-BP1 with eIF4E. Thus the progress of apoptosis is characterized by a complex programme of changes in several initiation factors, including the specific fragmentation or complete degradation of some and alterations in the association status of others. These events are likely to contribute to the inhibition of protein synthesis seen under these conditions.
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PMID:Changes in integrity and association of eukaryotic protein synthesis initiation factors during apoptosis. 1067 17

We have previously reported the activation of procalpain mu (precursor for low-calcium-requiring calpain) in apoptotic cells using a cleavage-site-directed antibody specific to active calpain [Kikuchi, H. and Imajoh-Ohmi, S. (1995) Cell Death Differ. 2, 195-199]. In this study, calpastatin, the endogenous inhibitor protein for calpain, was cleaved to a 90-kDa polypeptide during apoptosis in human Jurkat T cells. The limited proteolysis of calpastatin preceded the autolytic activation of procalpain. Inhibitors for caspases rescued the cells from apoptosis and simultaneously inhibited the cleavage of calpastatin. The full-length recombinant calpastatin was also cleaved by caspase-3 or caspase-7 at Asp-233 into the same size fragment. Cys-241 was also targeted by these caspases in vitro but not in apoptotic cells. Caspase-digested calpastatin lost its amino-terminal inhibitory unit, and inhibited three moles of calpain per mole. Our findings suggest that caspases trigger the decontrol of calpain activity suppression by degrading calpastatin.
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PMID:Caspases cleave the amino-terminal calpain inhibitory unit of calpastatin during apoptosis in human Jurkat T cells. 1073 97


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