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D W Nicholson

Publications and source records attributed to D W Nicholson.

At least 55 records · Page 3Linked to original sources
PubMed1999

Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner.

Exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homologue, Apaf-1, thereby triggering Apaf-1-mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that caspase-9 selectively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 did not. Depletion of caspase-9 from cell extracts abrogated cytochrome c-inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9.

Animals↗
PubMed1999

A yeast artificial chromosome-based physical map of the juvenile amyotrophic lateral sclerosis (ALS2) critical region on human chromosome 2q33-q34.

The autosomal recessive form of juvenile amyotrophic lateral sclerosis (ALS2; RFALS Type 3) has previously been mapped to the 8-cM interval flanked by D2S115 and D2S155 on human chromosome 2q33-q34. We have established a yeast artificial chromosome (YAC) contig spanning an approximately 8-Mb region of the ALS2 candidate region and mapped 52 transcribed DNA sequences including 13 known genes and 39 expressed sequenced tags within this YAC contig. The establishment of a YAC contig and transcript map that spans the region containing the ALS2 mutation is an essential step in the identification of the ALS2 gene.

Amyotrophic Lateral Sclerosis↗
PubMed1999

Purification and catalytic properties of human caspase family members.

Members of the caspase family of cysteine proteases are known to be key mediators of mammalian inflammation and apoptosis. To better understand the catalytic properties of these enzymes, and to facilitate the identification of selective inhibitors, we have systematically purified and biochemically characterized ten homologues of human origin (caspases 1 - 10). The method used for production of most of these enzymes involves folding of active enzymes from their constituent subunits which are expressed separately in E. coli, followed by ion exchange chromatography. In cases where it was not possible to use this method (caspase-6 and -10), the enzymes were instead expressed as soluble proteins in E. coli, and partially purified by ion exchange chromatography. Based on the optimal tetrapeptide recognition motif for each enzyme, substrates with the general structure Ac-XEXD-AMC were used to develop continuous fluorometric assays. In some cases, enzymes with virtually identical tetrapeptide specificities have kcat/Km values for fluorogenic substrates that differ by more than 1000-fold. Using these assays, we have investigated the effects of a variety of environmental factors (e.g. pH, NaCl, Ca2+) on the activities of these enzymes. Some of these variables have a profound effect on the rate of catalysis, a finding that may have important biological implications.

Apoptosis↗
PubMed1999

Caspase structure, proteolytic substrates, and function during apoptotic cell death.

Caspases play an essential role during apoptotic cell death. These enzymes define a new class of cysteine proteases and comprise a multi-gene family with more than a dozen distinct mammalian family members. The discrete and highly limited subset of cellular polypeptides that are cleaved by these proteases is sufficient to account for the majority of cellular and morphological events that occur during cell death. In some cases, caspases also play a contributory role in escalating the propensity for apoptosis, and in doing so may exacerbate disease pathogenesis.

Amino Acid Sequence↗
PubMed1998

E1A-induced processing of procaspase-8 can occur independently of FADD and is inhibited by Bcl-2.

Expression of the 243-residue form of the adenovirus E1A protein in the absence of other viral proteins triggers apoptosis by a pathway that requires p53. This pathway includes processing and activation of initiator procaspase-8, redistribution of cytochrome c, and activation of procaspase-3. Bcl-2 functions at or upstream of procaspase-8 processing to inhibit all of these events and prevent cell death. This contrasts with the anti-apoptotic influence of Bcl-2 family proteins in the cell death pathway induced by Fas ligand or tumor necrosis factor (TNF), in which Bcl-2 typically acts downstream of Fas/TNFR1-mediated activation of caspase-8. Moreover, E1A induces procaspase-8 processing and cell death in cells deleted of FADD, an adaptor protein critical for Fas/TNFR1 activation of caspase-8. The results indicate that E1A is capable of activating caspase-8 by a Bcl-2-inhibitable pathway that does not involve autocrine stimulation of FADD-dependent death receptor pathways.

Adenovirus E1A Proteins↗
PubMed1998

Inhibition of human caspases by peptide-based and macromolecular inhibitors.

Studies with peptide-based and macromolecular inhibitors of the caspase family of cysteine proteases have helped to define a central role for these enzymes in inflammation and mammalian apoptosis. A clear interpretation of these studies has been compromised by an incomplete understanding of the selectivity of these molecules. Here we describe the selectivity of several peptide-based inhibitors and the coxpox serpin CrmA against 10 human caspases. The peptide aldehydes that were examined (Ac-WEHD-CHO, Ac-DEVD-CHO, Ac-YVAD-CHO, t-butoxycarbonyl-IETD-CHO, and t-butoxycarbonyl-AEVD-CHO) included several that contain the optimal tetrapeptide recognition motif for various caspases. These aldehydes display a wide range of selectivities and potencies against these enzymes, with dissociation constants ranging from 75 pM to >10 microM. The halomethyl ketone benzyloxycarbonyl-VAD fluoromethyl ketone is a broad specificity irreversible caspase inhibitor, with second-order inactivation rates that range from 2.9 x 10(2) M-1 s-1 for caspase-2 to 2.8 x 10(5) M-1 s-1 for caspase-1. The results obtained with peptide-based inhibitors are in accord with those predicted from the substrate specificity studies described earlier. The cowpox serpin CrmA is a potent (Ki < 20 nM) and selective inhibitor of Group I caspases (caspase-1, -4, and -5) and most Group III caspases (caspase-8, -9, and -10), suggesting that this virus facilitates infection through inhibition of both apoptosis and the host inflammatory response.

Aldehydes↗
PubMed1998

Caspases cleave focal adhesion kinase during apoptosis to generate a FRNK-like polypeptide.

Focal adhesion kinase (Fak) is a non-receptor protein-tyrosine kinase that stimulates cell spreading and motility by promoting the formation of contact sites between the cell and the extracellular matrix (focal adhesions). It suppresses apoptosis by transducing survival signals that emanate from focal adhesions via the clustering of transmembrane integrins by components of the extracellular matrix. We demonstrate that Fak is cleaved by caspases at two distinct sites during apoptosis. The sites were mapped to DQTD772, which was preferentially cleaved by caspase-3, and VSWD704, which was preferentially cleaved by caspase-6 and cytotoxic T lymphocyte-derived granzyme B. The cleavage of Fak during apoptosis separates the tyrosine kinase domain from the focal adhesion targeting (FAT) domain. The carboxyl-terminal fragments that are generated suppress phosphorylation of endogenous Fak and thus resemble a natural variant of Fak, FRNK, that inhibits Fak activity by preventing the localization of Fak to focal adhesions. The cleavage of Fak by caspases may thus play an important role in the execution of the suicide program by disabling the anti-apoptotic function of Fak. Interestingly, rodent Fak lacks an optimal caspase-3 consensus cleavage site although it is cleaved in murine cells undergoing apoptosis at an upstream site. This appears to be the first example of a caspase substrate where the cleavage sites are not conserved between species.

Amino Acid Sequence↗
PubMed1998

Caspase cleavage of gene products associated with triplet expansion disorders generates truncated fragments containing the polyglutamine tract.

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.

Amino Acid Sequence↗
PubMed1998

The caspase-3 precursor has a cytosolic and mitochondrial distribution: implications for apoptotic signaling.

Caspase-3-mediated proteolysis is a critical element of the apoptotic process. Recent studies have demonstrated a central role for mitochondrial proteins (e.g., Bcl-2 and cytochrome c) in the activation of caspase-3, by a process that involves interaction of several protein molecules. Using antibodies that specifically recognize the precursor form of caspase-3, we demonstrate that the caspase-3 proenzyme has a mitochondrial and cytosolic distribution in nonapoptotic cells. The mitochondrial caspase-3 precursor is contained in the intermembrane space. Delivery of a variety of apoptotic stimuli is accompanied by loss of mitochondrial caspase-3 precursor staining and appearance of caspase-3 proteolytic activity. We propose that the mitochondrial subpopulation of caspase-3 precursor molecules is coupled to a distinct subset of apoptotic signaling pathways that are Bcl-2 sensitive and that are transduced through multiple mitochondrion-specific protein interactions.

Apoptosis↗
PubMed1998

Involvement of CPP32/Caspase-3 in c-Myc-induced apoptosis.

c-Myc is a transcriptional activator implicated in the control of cell proliferation, differentiation and transformation, but is also involved in the regulation of programmed cell death, apoptosis. Despite intensive research, the molecular mechanisms by which c-Myc triggers and executes cell death remain still elusive. Here, we made use of Rat 1A MycER cells expressing a conditionally active c-Myc protein and tested first the hypothesis that ornithine decarboxylase (ODC), which is a transcriptional target of c-Myc, were a mediator of c-Myc-induced apoptosis. However, our results show that the activity of ODC is not required for the c-Myc-mediated apoptosis to occur in these cells. We also found that the expression of p53, p21waf1/cip1, Bcl-2, Bax, Bcl-xL, Bad and cyclins D1, E, A and B did not show any significant changes following c-Myc induction. But, our studies revealed that the c-Myc induced apoptosis is associated with a specific cleavage of poly(ADPribose) polymerase (PARP), suggesting that a cysteine protease of the ICE/CED-3 family is involved. Moreover, we found that the cysteine protease CPP32/Caspase-3, which is known to cleave PARP, is processed from its inactive form to an active protease composed of 17 and 12 kDa subunits; whilst Ich-1/Caspase-2 belonging to another subset of this protease family was not processed/ activated following c-Myc activation. The activation of CPP32 and apoptotic cell death were inhibited by addition of Z-VAD-fmk, a universal inhibitor of ICE-like proteases. Further, a selective inhibitor of CPP32-like proteases (Z-DEVD-fmk) partly inhibited apoptosis. These results provide evidence that the ICE/CED3-family proteases, CPP32 and likely others, play a critical role in the execution of a nuclear proto-oncogene, c-Myc-induced apoptosis.

Animals↗
PubMed1998

Co-localization of the cysteine protease caspase-3 with apoptotic myocytes after in vivo myocardial ischemia and reperfusion in the rat.

The aim of our study was to characterize the temporal relationship of apoptosis to regional myocardial ischemia and reperfusion and we aimed to determine the effect of ischemia and reperfusion on the distribution of the pro-apoptotic cysteine protease caspase-3 (CPP 32, apopain, Yama) in an in vivo rat model. Male Sprague-Dawley rats (250-400 g) were anesthetized with sodium pentobarbital (65 mg/kg, i.p.), the left external carotid artery was isolated to monitor arterial pressure and a left thoracotomy was performed. Regional myocardial ischemia was induced by occluding the left main coronary artery for 45 min. The heart was reperfused for 0, 60, 120 or 180 min. TUNEL staining of formalin-fixed, paraffin-embedded left ventricle, and DNA fragmentation analysis, showed that apoptosis occurred during 45 min of ischemia alone, but further developed during the 3-h reperfusion period. Immunohistochemical analysis of ischemic/reperfused left ventricle showed caspase-3 levels were substantially elevated and localized in the ischemic/reperfused region, and that caspase-3 co-localized to TUNEL positive myocytes. Therefore, regional myocardial ischemia serves as a stimulus for myocyte apoptosis, and this form of cell death progresses time-dependently after the onset of reperfusion. Our studies implicate caspase-3 to be involved in apoptotic cell death in ischemic/reperfused rat heart.

Animals↗
PubMed1998

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.

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.

Alternative Splicing↗
PubMed1997

p28 Bap31, a Bcl-2/Bcl-XL- and procaspase-8-associated protein in the endoplasmic reticulum.

We have identified a human Bcl-2-interacting protein, p28 Bap31. It is a 28-kD (p28) polytopic integral protein of the endoplasmic reticulum whose COOH-terminal cytosolic region contains overlapping predicted leucine zipper and weak death effector homology domains, flanked on either side by identical caspase recognition sites. In cotransfected 293T cells, p28 is part of a complex that includes Bcl-2/Bcl-XL and procaspase-8 (pro-FLICE). Bax, a pro-apoptotic member of the Bcl-2 family, does not associate with the complex; however, it prevents Bcl-2 from doing so. In the absence (but not presence) of elevated Bcl-2 levels, apoptotic signaling by adenovirus E1A oncoproteins promote cleavage of p28 at the two caspase recognition sites. Purified caspase-8 (FLICE/MACH/Mch5) and caspase-1(ICE), but not caspase-3 (CPP32/apopain/ Yama), efficiently catalyze this reaction in vitro. The resulting NH2-terminal p20 fragment induces apoptosis when expressed ectopically in otherwise normal cells. Taken together, the results suggest that p28 Bap31 is part of a complex in the endoplasmic reticulum that mechanically bridges an apoptosis-initiating caspase, like procaspase-8, with the anti-apoptotic regulator Bcl-2 or Bcl-XL. This raises the possibility that the p28 complex contributes to the regulation of procaspase-8 or a related caspase in response to E1A, dependent on the status of the Bcl-2 setpoint within the complex.

Amino Acid Sequence↗
PubMed1997

A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis.

There is compelling evidence that members of the caspase (interleukin-1beta converting enzyme/CED-3) family of cysteine proteases and the cytotoxic lymphocyte-derived serine protease granzyme B play essential roles in mammalian apoptosis. Here we use a novel method employing a positional scanning substrate combinatorial library to rigorously define their individual specificities. The results divide these proteases into three distinct groups and suggest that several have redundant functions. The specificity of caspases 2, 3, and 7 and Caenorhabditis elegans CED-3 (DEXD) suggests that all of these enzymes function to incapacitate essential homeostatic pathways during the effector phase of apoptosis. In contrast, the optimal sequence for caspases 6, 8, and 9 and granzyme B ((I/L/V)EXD) resembles activation sites in effector caspase proenzymes, consistent with a role for these enzymes as upstream components in a proteolytic cascade that amplifies the death signal.

Amino Acid Sequence↗
PubMed1997

Intact cell evidence for the early synthesis, and subsequent late apopain-mediated suppression, of poly(ADP-ribose) during apoptosis.

Poly(ADP-ribose) polymerase (PARP), which is catalytically activated by DNA strand breaks, has been implicated in apoptosis, or programmed cell death. A protease (CPP32) responsible for the cleavage of PARP and necessary for apoptosis was recently purified and characterized. The coordinated sequence of events related to PARP activation and cleavage in apoptosis has now been examined in individual cells. Apoptosis was studied in a human osteosarcoma cell line that undergoes a slow (8 to 10 days), spontaneous, and reproducible death program in culture. Changes in the abundance of intact PARP, poly(ADP-ribose) (PAR), and a proteolytic cleavage product of PARP that contains the DNA-binding domain were examined during apoptosis in the context of individual, whole cells by immunofluorescence with specific antibodies. The synthesis of PAR from NAD increased early, within 2 days of cell plating for apoptosis, prior to the appearance of internucleosomal DNA cleavage and before the cells become irreversibly committed to apoptosis, since replating yields viable, nonapoptotic cells. Strong expression of full-length PARP was also detected, by immunofluorescence as well as by Western analysis, during this same time period. However, after approximately 4 days in culture, the abundance of both full-length PARP and PAR decreased markedly. After 6 days, a proteolytic cleavage product containing the DNA-binding domain of PARP was detected immunocytochemically and confirmed by Western analysis, both in the nuclei and in the cytoplasm of cells. A recombinant peptide spanning the DNA-binding domain of PARP was expressed, purified, and biotinylated, and was then used as a probe for DNA strand breaks. Fluorescence microscopy with this probe revealed extensive DNA fragmentation during the later stages of apoptosis. This is the first report, using individual, intact cells, demonstrating that poly(ADP-ribosyl)ation of nuclear proteins occurs prior to the commitment to apoptosis, that inactivation and cleavage of PARP begin shortly thereafter, and that very little PAR per se is present during the later stages of apoptosis, despite the presence of a very large number of DNA strand breaks. These results suggest a negative regulatory role for PARP during apoptosis, which in turn may reflect the requirement for adequate NAD and ATP during the later stages of programmed cell death.

Apoptosis↗
PubMed1997

Actin cleavage by CPP-32/apopain during the development of apoptosis.

Interleukin-1beta-converting enzyme (ICE)/ced-3 family proteases play key roles in apoptosis. However, cellular substrates for ICE family proteases involved in apoptosis are not well understood. We previously showed that actin is cleaved in vitro by an ICE family protease, distinct from ICE itself, which is activated during VP-16-induced apoptosis. In this report, we demonstrate that the actin-cleaving ICE-family protease in the apoptotic cell extract is the activated CPP-32/apopain. CPP-32 effectively cleaves actin protein to 15 kDa and 31 kDa fragments. Studies with an antibody raised against Gly-Gln-Val-Ile-Thr peptide, the N-terminal sequence of the cleaved 15 kDa actin fragment, showed that actin is also cleaved in vivo during the development of apoptosis. Moreover, Benzyloxycarbonyl-Glu-Val-Asp-CH2OC(O)-2,6,-dichlorobenzene (Z-EVD-CH2-DCB), a selective inhibitor of CPP-32(-like) protease, efficiently inhibited the cleavage of actin and the apoptosis of VP-16-treated U937 cells. Our present results indicate that actin is the substrate of CPP-32/apopain(-like) protease both in vitro and in vivo and suggest the role of actin in the control of cell growth and apoptosis.

Actins↗