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PubMed1998

Cathepsin B-mediated activation of the proinflammatory caspase-11.

Members of the caspase (CASP) family of cysteine proteases can be subdivided in proapoptotic caspases and proinflammatory caspases. Whereas the apical activation pathways for the caspases that are involved in the execution of the apoptotic process are beginning to be understood, the pathways that lead to the activation of proinflammatory caspases are still largely unknown. Analysis of subcellular fractions for their ability to process and activate several caspases in vitro led to the identification of lysosomes as the source for a protease that could proteolytically activate the proinflammatory CASP-11. Although this lysosomal activity was sensitive to caspase inhibitors, affinity purification with the biotinylated broad spectrum caspase inhibitor z-VAD.fmk revealed the CASP-11 activating protease as cathepsin B. Activation of CASP-11 by cathepsin B as well as its sensitivity to several caspase inhibitors was further confirmed with purified proteases. Similar to the role of mitochondrial factors in the activation of proapoptotic caspases, our results suggest a potential role for lysosomes and cathepsin B as activators of specific proinflammatory caspases. In addition, the aspecific inhibition of cathepsin B by so-called specific caspase inhibitors implicates that results obtained with these inhibitors should be interpreted with care.

Animals↗

PubMed2005

Impact of interleukin-12, oxidative burst, and iNOS on the survival of murine fecal peritonitis.

Abdominal sepsis due to secondary fecal peritonitis following anastomosis insufficiency is a rare but life threatening complication of colorectal surgery. The induction of IFN-gamma by IL-12 is believed to play a key role in sepsis as it promotes antibacterial effector mechanisms such as oxidative burst or nitric oxide induction. The impact of gene deficiency for IL-12 (IL-12p40 KO), oxidative burst (p47(phox) KO), or NO induction (iNOS KO) on the outcome of fecal peritonitis was characterized using the murine Colon Ascendens Stent Peritonitis model (CASP). In the IL-12p40 KO model, 3 and 12 h after surgery, serum cytokine levels of IL-1beta, TNF, IL-18, and IL-10 were analyzed. Expression of IL-1beta, IL-10, IP-10, and MIP-1alpha was measured in lung and liver by RNAse Protection Assay. IL-12p40 and iNOS-deficient mice exhibited a significantly higher susceptibility to CASP as compared to the controls, whereas no significant difference was observed in p47(phox) KO mice. Absence of IL-12 resulted in delayed expression of proinflammatory cytokines and chemokines in both the liver and the lung, and was associated with significant reduction of IL-1beta levels in the serum 12 h after CASP. IL-12 and iNOS possess protective functions in fecal murine peritonitis. Surprisingly, no significant contribution of oxidative burst to the immune response was observed. Overall, these findings suggest that IL-12 deficiency causes a profound delay of the immune response after polymicrobial challenge resulting in significantly increased susceptibility in the CASP model.

Animals↗

PubMed1989

Complete primary structure of calcium-dependent serine proteinase capable of degrading extracellular matrix proteins.

A novel calcium-dependent serine proteinase (CASP) secreted from malignant hamster embryo fibroblast Ni 12C2 degrades extracellular matrix proteins. A complementary DNA encoding CASP has been isolated with the use of oligonucleotide probes synthesized based on partial amino acid sequences of CASP. The complete amino acid sequences of CASP revealed that it has a active site at the C-terminal side. Glu rich and proEGF homologous sites are found at the N-terminal site suggesting that it is structurally similar to blood coagulation factors such as IX, X and an anticoagulation factor, protein C.

Amino Acid Sequence↗

PubMed2002

Differential influence of etoposide on two caspase-2 mRNA isoforms in leukemic cells.

Etoposide (VP-16) is an anticancer agent that induces apoptosis in human leukemic cell lines such as U937 and HL60. We performed RNase protection assays, with two distinct cRNA panels covering most of caspase and BCL-2-related genes, using total RNA from cell lines exposed to various concentrations of the drug. Our results show that VP-16 down-regulates expression of most surveyed genes with the noticeable exception of casp-2S mRNA that is up regulated whereas casp-2L mRNA is decreased. Since these mRNAs are produced by the alternative splicing of exon 9, we devised a reverse transcriptase-polymerase chain reaction method using primers from exons 8 and 10 to demonstrate that VP-16 stimulates the production of exon 9-containing sequences, irrespective of active transcription. However, this effect is specific of the 3'-end of the CASP-2 gene since no difference in the relative amounts of the 5'-end of the mRNA species is detected. Nevertheless, the level of full-length casp-2L mRNA together with that of procaspase-2L protein, which is pro-apoptotic, are decreased under VP-16 treatment, suggesting that an early cell response to treatment by cytotoxic agents is to down-regulate expression of selected pro-apoptotic proteins.

Alternative Splicing↗

PubMed2000

Caspase-9 regulates cisplatin-induced apoptosis in human head and neck squamous cell carcinoma cells.

The aim of this study was to understand the molecular requirements for cisplatin-induced apoptosis in human head and neck squamous cell carcinoma (HNSCC) cell death. Cisplatin induced apoptosis in HNSCC cell lines, HSC-2, HSC-3 and HSC-4 in a dose-dependent manner. However, cisplatin did not induce the expression of Fas-ligand mRNA or upregulation of Fas protein. By caspase activation assays, cisplatin induced Caspase-3 (Casp-3), -8 and -9 activation. In all three lines tested, the use of a specific inhibitor of Casp-9 almost completely blocked cisplatin-induced apoptosis, while the use of Casp-3 and -8 inhibitors resulted in a partial blockade of cisplatin-induced apoptosis. Our results strongly suggest that Casp-9-dependent apoptosis plays an important role in cisplatin-induced HNSCC apoptosis.

Apoptosis↗

PubMed1998

Analysis of apoptosis and expression of bcl-2 gene family members in the human and baboon ovary.

Recent data support a role for apoptosis, under tight regulatory control by bcl-2, oxidative stress response, tumor suppressor, and CASP gene family members, in mediating granulosa cell demise during follicular atresia in the rodent and avian ovary. Herein we evaluated the occurrence of apoptosis in the human and baboon ovary relative to follicular health status, and analyzed expression of several cell death genes in these tissues. In situlocalization of DNA strand breaks in fixed human and baboon ovarian tissue sections indicated that apoptosis was essentially restricted to granulosa cells of atretic antral follicles. Biochemical analysis of DNA oligonucleosomes in individual follicles isolated from baboon ovaries during the ovulatory phase revealed the presence of apoptotic DNA fragments in subordinate but not dominant follicles, thus substantiating the in situ labeling studies. Messenger RNA transcripts encoded by the bax death susceptibility gene, the bcl-xlong survival gene, the bcl-xshort pro-apoptosis gene, the p53 tumor suppressor gene, and two members of the CASP gene family (CASP-2/Ich-1, CASP-3/CPP32), were detected by Northern blot analysis of total RNA prepared either from human ovaries or from Percoll-purified granulosa-lutein cells obtained from patients undergoing assisted reproductive technologies. Lastly, immunohistochemical localization of the BAX death-susceptibility protein in the human ovary revealed abundant expression in granulosa cells of early atretic follicles, whereas BAX protein was extremely low or non-detectable in healthy or grossly-atretic follicles. We conclude that apoptosis occurs during, and is probably responsible for, folicular atresia in the human and baboon ovary. Moreover, apoptosis in the human ovary is likely controlled by altered expression of the same cohort of cell death regulatory factors recently implicated as primary determinants of apoptosis induction or suppression in the rodent ovary.

Adult↗

PubMed2003

The human caspase-2 gene: alternative promoters, pre-mRNA splicing and AUG usage direct isoform-specific expression.

Caspases have been shown to play important roles in apoptotic cell death, cytokine maturation and cell differentiation. However, the transcriptional regulation of the corresponding CASP genes remains poorly known. We describe a 5.1 kb fragment located upstream of the first translated exon in the human CASP-2 gene, which is known to encode caspase-2L and -2S protein isoforms. Transient transfection experiments, together with transcription start site mapping and transcript analysis, demonstrate that each caspase mRNA is initiated from separate promoter regions, and produced from alternative splicing events in these regions. The CASP-2L promoter is much stronger than the CASP-2S promoter, in good agreement with the respective transcript levels of the two caspases. In addition, several in-frame translational start sites can be identified for each isoform, one of which is common to both, present in the second common exon, and used efficiently. Surprisingly, the short isoform may also be initiated at a downstream AUG codon within the same exon. Thus, promoter strength, alternative transcriptional initiation and 5'-splicing events regulate the expression of the main caspase-2 isoforms that may be translated from alternative translation initiation codons.

5' Flanking Region↗

PubMed1997

CASH, a novel caspase homologue with death effector domains.

CASP-8 and CASP-10, members of a cysteine protease family that participates in apoptosis, interact with MORT1/FADD, an adapter protein in the CD120a (p55 tumor necrosis factor receptor), and CD95 (Fas/Apo-1) death-inducing signaling pathways, through a shared N-terminal sequence motif, the death effector domain. We report cloning of two splice variants of a novel protein, CASH, that contain two N-terminal death effector domains and can bind through them to each other, to MORT1/FADD, to CASP-8, and to CASP-10. The unique C-terminal part of the longer variant shows marked sequence homology to the caspase protease region yet lacks several of the conserved caspase active site residues, suggesting that it is devoid of cysteine protease activity. Overexpression of the short CASH splice variant strongly inhibited cytotoxicity induction by CD120a and CD95. Expression of the longer variant, while inhibiting cytotoxicity in HeLa cells, had a marked cytocidal effect in 293 cells that could be shown to involve its protease homology region. The findings suggest that CASH acts as an attenuator and/or initiator in CD95 and CD120a signaling for cell death.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Regulation of the expression of caspase-9 by the transcription factor activator protein-4 in glucocorticoid-induced apoptosis.

Caspase-9 (Casp-9) induces death signals by triggering other types of caspase activation, and its expression greatly influences the onset of apoptosis. During the isolation of apoptosis-related genes involved in glucocorticoid (GC)-induced cell death in murine thymic lymphomas, we found that the antisense gene of the transcription factor activator protein-4 (AP-4) inhibited dexamethasone-induced apoptosis. Western blot analysis revealed that the expression of Bcl-xL, Bax, and Apaf-1 was not affected in cells transfected with sense or antisense AP-4 genes. In contrast, both the expression and activation of Casp-9 were inhibited in the antisense AP-4 transfectants. We isolated the 2.4-kb 5'-flanking region of Casp-9, and the promoter activity was investigated. We found the AP-4-binding sites at -1.55 and -1.38 kb to be responsible for the promoter activity. Furthermore, a negative cis-element was expected to exist between bases -1140 and -944. When the cells were treated with dexamethasone, a rapid down-regulation of AP-4 and Casp-9 was observed whether the cells were GC-sensitive lymphomas or GC-insensitive L929 fibroblast cells. In addition, L929 cells pretreated with dexamethasone were found to be resistant to subsequent treatment with etoposide, an apoptosis-inducing reagent. GC has a two-sided effect on apoptosis, i.e. a pro-apoptotic effect on certain cell types and a prosurvival effect on other cell types. Our findings will explain, at least in part, this effect.

Animals↗

PubMed2006

Crystal structure of TTHA0252 from Thermus thermophilus HB8, a RNA degradation protein of the metallo-beta-lactamase superfamily.

In bacterial RNA metabolism, mRNA degradation is an important process for gene expression. Recently, a novel ribonuclease (RNase), belonging to the beta-CASP family within the metallo-beta-lactamase superfamily, was identified as a functional homologue of RNase E, a major component for mRNA degradation in Escherichia coli. Here, we have determined the crystal structure of TTHA0252 from Thermus thermophilus HB8, which represents the first report of the tertiary structure of a beta-CASP family protein. TTHA0252 comprises two separate domains: a metallo-beta-lactamase domain and a "clamp" domain. The active site of the enzyme is located in a cleft between the two domains, which includes two zinc ions coordinated by seven conserved residues. Although this configuration is similar to those of other beta-lactamases, TTHA0252 has one conserved His residue characteristic of the beta-CASP family as a ligand. We also detected nuclease activity of TTHA0252 against rRNAs of T. thermophilus. Our results reveal structural and functional aspects of novel RNase E-like enzymes with a beta-CASP fold.

Amino Acid Sequence↗

PubMed1999

Human immunodeficiency virus type 1 Tat induces apoptosis and increases sensitivity to apoptotic signals by up-regulating FLICE/caspase-8.

Apoptosis contributes to the loss of CD4 cells during human immunodeficiency virus type 1 (HIV-1) infection. Although the product of the env gene, gp160/gp120, is known to play a role in cell death mediated by HIV-1, the role of other HIV-1 genes in the process is unclear. We found that HIV-1 lacking the env gene (HIVDeltaenv) still induced apoptosis in T-cell lines and primary CD4 T cells. The ability to induce apoptosis was attributable to Tat, a viral regulatory protein. Tat induction of apoptosis was separate from the transactivation function of Tat, required expression of the second exon of Tat, and was associated with the increased expression and activity of caspase-8 (casp-8), a signaling molecule in apoptotic pathways. Moreover, induction of apoptosis could be prevented by treating cells with an inhibitor of casp-8. In addition, we show that HIV-1Deltaenv infection and Tat expression increased the sensitivity of cells to Fas-mediated apoptosis, an apoptotic pathway that signals via casp-8. The up-regulation of casp-8 by HIV-1 Tat expression may contribute to the increased apoptosis and sensitivity to apoptotic signals observed in the cells of HIV-1-infected persons.

Apoptosis↗

PubMed1991

Chloroaluminum sulfonated phthalocyanine versus dihematoporphyrin ether: early vascular events in the rat window chamber.

The development of a simple and well-tolerated rat window chamber has allowed direct comparison of the vascular effects of two photosensitizers, chloroaluminum sulfonated phthalocyanine (CASP) and dihematoporphyrin ether (DHE). CASP and DHE were given 4 days after the implantation of the window chamber. Photodynamic therapy (PDT) with CASP was performed 24 hours after intravenous injection (10 mg/kg) with light at 675 nm (power density 200 mW/cm2, incident energy 100 J). DHE was given in a similar fashion (5 mg/kg intraperitoneally; light at 630 nm with matching power density and energy settings 24 hours after injection). Using videomicroscopic and integrating sphere measurements, marked differences were noted in the vascular effects of these photosensitizers. DHE caused immediate hemorrhage and disruption of the postcapillary venules, while CASP induced vascular spasm starting 4 hours after the completion of PDT. Forty-eight hours after PDT, both systems demonstrated a loss of chamber-induced neovascularization.

Animals↗

PubMed2002

Stent placement for the treatment of occlusive atherosclerotic carotid artery disease in patients with concomitant coronary artery disease.

OBJECT: The authors report their experience with carotid artery stent placement (CASP) in patients with concomitant carotid artery (CA) and coronary artery (CorA) diseases. METHODS: In a review of 320 consecutive patients who underwent CASP, the authors identified 49 with severe CorA disease in addition to significant CA stenosis, who had undergone CASP before planned CorA bypass grafting (CorABG). The average age of these 49 patients was 68 years. In 39 patients (80%) the New York Heart Association functional classification grade was IV and in 10 the grade was III. In 26 patients 50% or greater stenosis of the left main CorA was found. Seventeen patients (35%) suffered from either significant hemodynamic contralateral CA stenosis (> 60% stenosis; eight patients) or contralateral CA occlusion (nine patients). Sixteen patients (33%) had symptomatic CA disease. No cerebrovascular events occurred during CorABG. Four patients (8%) died of cardiac arrest and one patient (2%) suffered a major stroke within 30 days after the CorABG procedure. No patient experienced clinically significant recurrent CA stenosis during the study period (average clinical follow-up period 27 months). CONCLUSIONS: Carotid artery stent placement should be considered as an alternative for the management of concomitant CA and CorA diseases. These preliminary results support the feasibility and durability of CASP in the population studied.

Aged↗

PubMed2000

Identification of a caspase-2 isoform that behaves as an endogenous inhibitor of the caspase cascade.

Procaspase-2 is one of the aspartate-specific cysteine proteases that are activated in response to various apoptotic stimuli. Two isoforms of human procaspase-2 have been described initially. Overexpression of the long isoform (caspase-2L) promotes cell death whereas the short isoform (caspase-2S) antagonizes some apoptotic pathways. In the present study, we identified two additional CASP-2 mRNAs, designated CASP-2L-Pro and CASP-2s-Pro. The proteins encoded by these isoforms corresponded to the prodomain of procaspase-2L and -2S, in which the last alpha-helix of their caspase recruitment domains was deleted. Caspase-2L-Pro mRNA and protein were detected in a series of human tissues and cell lines. Yeast 2-hybrid assays and immunoprecipitation studies indicated that caspase-2L-Pro can interact with procaspase-2L and the adaptor protein RAIDD/CRADD, but not with FADD/MORT1 or APAF-1 adaptor proteins. The addition of recombinant caspase-2L-Pro negatively interfered with cytochrome c/dATP-mediated activation of the caspase cascade in a cell-free system. In transient expression studies of human B lymphoma Namalwa cells, overexpression of caspase-2L-Pro weakly induced apoptosis, which was prevented by a D83A/E87A double mutation. In stable selected CASP-2L-Pro-transfected Namalwa cells, overexpression of caspase-2L-Pro delayed apoptotic DNA fragmentation induced by death receptor agonists (anti-Fas antibodies, tumor necrosis factor-alpha) and DNA topoisomerase I- (camptothecin) and II- (etoposide) inhibitors, and prevented etoposide-induced activation of the caspase cascade. These inhibitory effects were not observed in stable transfected cells expressing the D83A/E87A double mutant. Altogether, these data indicated that the caspase-2L-Pro isoform functions as an endogenous apoptosis inhibitory protein that antagonizes caspase activation and cell death.

Amino Acid Sequence↗

PubMed2006

Metronidazole improves intestinal microcirculation in septic rats independently of bacterial burden.

To explore the effects of metronidazole (Me) on intestinal microcirculation in septic rats, intravital microscopy (IVM) following 16 hours of colon ascendens stent peritonitis (CASP model) was used. Four groups of animals were studied: control group (sham operation) and CASP group, each with and without Me treatment (10 mg/kg i.v.). In order to investigate the substance-specific effects of Me independently of the antibacterial effects within a pathologically altered microcirculation, a second experimental series with lipopolysaccharide challenge (LPS model) was carried out. The LPS model consisted of the four groups (control animals and LPS animals (15 mg/kg i.v. LPS from E. coli) with and without Me). IVM in the LPS experiments was performed following a two hour observation period. Me treated CASP or LPS animals, as compared with untreated, demonstrated significant improvement of functional capillary density (FCD) of the intestinal wall. The increase in the number of leukocytes firmly adhered to the endothelium (leukocyte sticking) in the untreated CASP or LPS animals within the V1 venules of the intestinal submucosal layer, was significantly reduced in the Me treated animals. In conclusion, Me exerts beneficial anti-bacterial and anti-inflammatory effects within the septic microcirculation.

Animals↗

PubMed2004

Modeling structurally variable regions in homologous proteins with rosetta.

A major limitation of current comparative modeling methods is the accuracy with which regions that are structurally divergent from homologues of known structure can be modeled. Because structural differences between homologous proteins are responsible for variations in protein function and specificity, the ability to model these differences has important functional consequences. Although existing methods can provide reasonably accurate models of short loop regions, modeling longer structurally divergent regions is an unsolved problem. Here we describe a method based on the de novo structure prediction algorithm, Rosetta, for predicting conformations of structurally divergent regions in comparative models. Initial conformations for short segments are selected from the protein structure database, whereas longer segments are built up by using three- and nine-residue fragments drawn from the database and combined by using the Rosetta algorithm. A gap closure term in the potential in combination with modified Newton's method for gradient descent minimization is used to ensure continuity of the peptide backbone. Conformations of variable regions are refined in the context of a fixed template structure using Monte Carlo minimization together with rapid repacking of side-chains to iteratively optimize backbone torsion angles and side-chain rotamers. For short loops, mean accuracies of 0.69, 1.45, and 3.62 A are obtained for 4, 8, and 12 residue loops, respectively. In addition, the method can provide reasonable models of conformations of longer protein segments: predicted conformations of 3A root-mean-square deviation or better were obtained for 5 of 10 examples of segments ranging from 13 to 34 residues. In combination with a sequence alignment algorithm, this method generates complete, ungapped models of protein structures, including regions both similar to and divergent from a homologous structure. This combined method was used to make predictions for 28 protein domains in the Critical Assessment of Protein Structure 4 (CASP 4) and 59 domains in CASP 5, where the method ranked highly among comparative modeling and fold recognition methods. Model accuracy in these blind predictions is dominated by alignment quality, but in the context of accurate alignments, long protein segments can be accurately modeled. Notably, the method correctly predicted the local structure of a 39-residue insertion into a TIM barrel in CASP 5 target T0186.

Algorithms↗

PubMed2004

A structural pattern-based method for protein fold recognition.

A method (SPREK) was developed to evaluate the register of a sequence on a structure based on the matching of structural patterns against a library derived from the protein structure databank. The scores obtained were normalized against random background distributions derived from sequence shuffling and permutation methods. 'Random' structures were also used to evaluate the effectiveness of the method. These were generated by a simple random-walk and a more sophisticated structure prediction method that produced protein-like folds. For comparison with other methods, the performance of the method was assessed using collections of models including decoys and models from the CASP-5 exercise. The performance of SPREK on the decoy models was equivalent to (and sometimes better than) those obtained with more complex approaches. An exception was the two smallest proteins, for which SPREK did not perform well due to a lack of patterns. Using the best parameter combination from trials on decoy models, the CASP models of intermediate difficulty were evaluated by SPREK and the quality of the top scoring model was evaluated by its CASP ranking. Of the 14 targets in this class, half lie in the top 10% (out of around 140 models for each target). The two worst rankings resulted from the selection by our method of a well-packed model that was based on the wrong fold. Of the other poor rankings, one was the smallest protein and the others were the four largest (all over 250 residues).

Algorithms↗

PubMed2005

System for accepting server predictions in CASP6.

We describe the new CASP system for collecting and verifying predictions generated by servers. The system was developed to ensure reliable execution of the server assessment part of CASP, with particular emphasis on data consistency. Following the principle that predictions should not be modified by anyone but their authors and to allow a later meaningful assessment, submissions are now verified for correctness of format and contents within the strict 48 hour CASP deadlines for this type of submission. This article also provides an overview of the rules governing server participation in CASP6 and some statistics pertaining to servers in CASP6.

Automation↗