A novel protein, MUDENG, induces cell death in cytotoxic T cells

A screening system comprised of a randomized hybrid-ribozyme library has previously been used to identify pro-death genes in Fas-mediated apoptosis, and short sequence information of candidate genes from this system was previously reported by Kawasaki and Taira [H. Kawasaki, K. Taira, A functional gene discovery in the Fas-mediated pathway to apoptosis by analysis of transiently expressed randomized hybrid-ribozyme libraries, Nucleic Acids Res. 30 (2002) 3609-3614]. In this study, we have cloned the full-length of the candidate’s open reading frames and found that one of the candidates, referred to as MUDENG (Mu-2 related death-inducing gene), which is composed of 490 amino acids that contain the adaptin domain found in the μ2 subunit of APs related to clathrin-mediated endocytosis, is able to induce cell death by itself. Ectopic expression of MUDENG induced cell death in Jurkat T cells and HeLa cells. In addition, when MUDENG expression was evaluated by immnuohistochemical staining, it was found in most tissues, including the intestine and testis. Furthermore, MUDENG appears to be evolutionary conserved from mammals to amphibians, suggesting that it may have a common role in cell death. Taken together, these results suggest that MUDENG is likely to play an important role in cell death in various tissues.     

A Bacillus thuringiensis delta-endotoxin induces programmed cell death in mosquito larvae

We present evidence that a delta-endotoxin isolated from Bacillus thuringiensis subsp.israelensis induces programmed cell death in polytene midgut cells of Culex pipiens larvae. After exposure to toxin, polytene nuclei in the anterior region of the larval midgut undergo many of the morphological and physiological changes which are characteristic of apoptosis, including the ability to stain with the vital dye, acridine orange, and fragmentation of nuclear DNA as demonstrated by agarose gel electrophoresis and in situ TUNEL labeling. The temporal sequence of toxin ingestion, acridine orange staining and larval death suggests a cause and effect relationship between programmed cell death and larval death. Amino sugars that interfere with toxicity also interfere with the time course of acridine orange staining of larval polytene nuclei. The toxin first causes programmed cell death of anterior midgut and gastric caeca cells and, subsequently, posterior midgut cells. This pattern is similar to the temporal sequence of larval polytene cell death that occurs during metamorphosis. From the size and distribution of the nuclei that are stained with acridine orange, it appears that only polytene midgut cells are affected by toxin and that the diploid regenerative cell are not affected.     

A novel Bacillus thuringiensis gene encoding a Spodoptera exigua-specific crystal protein

Only one of the four lepidoptera-specific crystal protein subclasses (CryIC) Bacillus thuringiensis was previously shown to be highly toxic against several Spodoptera species. By using a cryIC-derived nucleotide probe, DNA from 25 different strains of B. thuringiensis was screened for the presence of homologous sequences. A putative crystal protein gene, considerably different from the cryIC gene subclass, was identified in the DNA of strain 4F1 (serotype kenyae) and cloned in Escherichia coli. Its nucleotide sequence was determined and appeared to contain several features typical for a crystal protein gene. Furthermore, the region coding for the N-terminal part of the putative toxic fragment showed extensive homology to subclass cryIA sequences derived from gene BtII, whereas the region coding for the C-terminal part appeared to be highly homologous to the cryIC gene BtVI. With an anti-crystal protein antiserum, a polypeptide of the expected size could be demonstrated in Western immunoblots, onto which a lysate of E. coli cells harboring the putative gene, now designated as BtXI, had been transferred. Cells expressing the gene appeared to be equally toxic against larvae of Spodoptera exigua as recombinant cells expressing the BtVI (cryIC)-encoded crystal protein. However, no toxicity against larvae of Heliothis virescens, Mamestra brassicae, or Pieris brassicae could be demonstrated. The nucleotide sequence analysis and the toxicity studies showed that this novel crystal protein gene falls into a new cryl gene subclass. We propose that this subclass be referred to as cryIE.     

Prolonged activation of ERK1,2 induces FADD-independent caspase 8 activation and cell death

Prolonged ERK/MAPK activation has been implicated in neuronal cell death in vitro and in vivo. We found that HEK293 cells, recently reported to express neuronal markers, are exquisitely sensitive to long term ERK stimulation. Activation of an inducible form of Raf-1 (Raf-1:ER) in HEK293 cells induced massive apoptosis characterized by DNA degradation, loss of plasma membrane integrity and PARP cleavage. Cell death required MEK activity and protein synthesis and occurred via the death receptor pathway independently of the mitochondrial pathway. Accordingly, prolonged ERK stimulation activated caspase 8 and strongly potentiated Fas signaling. The death receptor adaptator FADD was found to be rapidly induced upon ERK activation. However using RNA interference and ectopic expression, we demonstrated that neither FADD nor Fas were necessary for caspase 8 activation and cell death. These findings reveal that prolonged ERK/MAPK stimulation results in caspase 8 activation and cell death. This work was supported by grant from Association pour la Recherche sur le Cancer (CNRS6543/ARC). S. Cagnol is supported by a fellowship from the Ligue Nationale contre le Cancer.     

A Bacillus thuringiensis crystal protein with selective cytocidal action to human cells

Bacillus thuringiensis crystal proteins, well known to be toxic to certain insects but not pathogenic to mammals, are used as insecticidal proteins in agriculture and forest management. We here identified a crystal protein that is non-insecticidal and non-hemolytic but has strong cytocidal activity against various human cells with a markedly divergent target specificity, e.g. highly cytotoxic to HepG2 and Jurkat and less cytotoxic to the normal hepatocyte (HC) and HeLa. In slices of liver and colon cancer tissues, the toxin protein preferentially killed the cancer cells, leaving other cells unaffected. The cytocidal effect of the protein is non-apoptotic with swelling and fragmentation of the susceptible cells, although the apoptotic process does occur when the cell damage proceeded slowly. The amino acid sequence deduced from the nucleotide sequence of the cloned gene of the protein has little sequence homology with the insecticidal crystal proteins of B. thuringiensis. These observations raise the presence of a new group of the B. thuringiensis toxin and the possibility of new applications for the protein in the medical field.     

The antiangiogenic factor 16K PRL induces programmed cell death in endothelial cells by caspase activation

We asked whether the antiangiogenic action of 16K human PRL (hPRL), in addition to blocking mitogen-induced vascular endothelial cell proliferation, involved activation of programmed cell death. Treatment with recombinant 16K hPRL increased DNA fragmentation in cultured bovine brain capillary endothelial (BBE) and human umbilical vein endothelial (HUVE) cells in a time- and dose-dependent fashion, independent of the serum concentration. The activation of apoptosis by 16K hPRL was specific for endothelial cells, and the activity of the peptide could be inhibited by heat denaturation, trypsin digestion, and immunoneutralization, but not by treatment with the endotoxin blocker, polymyxin-B. 16K hPRL-induced apoptosis was correlated with the rapid activation of caspases 1 and 3 and was blocked by pharmacological inhibition of caspase activity. Caspase activation was followed by inactivation of two caspase substrates, poly(ADP-ribose) polymerase (PARP) and the inhibitor of caspase-activated deoxyribonuclease (DNase) (ICAD). Furthermore, 16K hPRL increased the conversion of Bcl-X to its proapoptotic form, suggesting that the Bcl-2 protein family may also be involved in 16K hPRL-induced apoptosis. These findings support the hypothesis that the antiangiogenic action of 16K hPRL includes the activation of programmed cell death of vascular endothelial cells.     

Molecular characterization of two novel crystal protein genes from Bacillus thuringiensis subsp. thompsoni.

Two genes encoding the predominant polypeptides of Bacillus thuringiensis subsp. thompsoni cuboidal crystals were cloned in Escherichia coli and sequenced. The polypeptides have electrophoretic mobilities of 40 and 34 kDa, with the deduced amino acid sequences predicting molecular masses of 35,384 and 37,505 Da, respectively. No statistically significant similarities were detected between the 40- or 34-kDa crystal protein and any other characterized B. thuringiensis crystal protein, nor were they detected between the 40- and 34-kDa crystal proteins. A 100-MDa plasmid carries both crystal protein genes, which appear to be part of an operon, with the 40-kDa gene 64 nucleotides upstream of the 34-kDa gene. Both crystal proteins are synthesized in approximately the same amounts. Even though small compared with other crystal proteins, the 34-kDa crystal protein has insecticidal activity against lepidopteran larvae (Manduca sexta). The 40-kDa polypeptide appears to have no insecticidal activity, but it could have a role in crystal structure.     

Hydrogen peroxide induces caspase activation and programmed cell death in the amitochondrial Tritrichomonas foetus

Tritrichomonas foetus is an amitochondrial parasite protist which lacks typical eukaryote organelles such as mitochondria and peroxisomes, but possesses the hydrogenosome, a double-membrane-bound organelle that produces ATP. The cell death of amitochondrial organisms is poorly studied. In the present work, the cytotoxic effects of hydrogen peroxide on T. foetus and its participation on cell death were analyzed. We took advantage of several microscopy techniques, including videomicroscopy, light microscopy immunocytochemistry for detection of caspase activation, and scanning and transmission electron microscopy. We report here that in T. foetus: (1) H₂O₂ leads to loss of motility and induces cell death, (2) the dying cells exhibit some characteristics similar to those found during the death of other organisms, and (3) a caspase-like protein seems to be activated during the death process. Thus, we propose that, although T. foetus does not present mitochondria nor any known pathways of cell death, it is likely that it bears mechanisms of cell demise. T. foetus exhibits morphological and physiological alterations in response to H₂O₂ treatment. The hydrogenosome, a unique organelle which is supposed to share a common ancestral origin with mitochondria and has an important role in oxidative responses in trichomonads, is a candidate for participating in this event.Abbreviations TUNEL Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick-end labeling - PARP Poly (ADP-ribose) polymerase - DAPI 4,6-Diamidino-2-phenylindole dihydrochloride     

Cloning of a novel crystal protein gene cry 1K from Bacillus thuringiensis subsp. morrisoni

Abstract In Escherichia coli with group II capsules, the synthesis of capsular polysaccharide and its cellular expression are encoded by the kps gene cluster, which is composed of three regions. The central region 2 encodes proteins involved in polysaccharide synthesis, and the flanking regions 1 and 3 direct the translocation of the finished polysaccharide across the cytoplasmic membrane and its surface expression. The kps genes of E. coli with the group II capsular K5 polysaccharide, have been cloned and sequenced. Region 1 contains the kps E, D, U, C and S genes. In this communication we describe the overexpression of the kps D and kps U genes as well as the isolation of the KpsU protein from the recombinant bacteria by chloroform treatment. The purified KpsU protein exhibited CMP-Kdo-synthetase activity. The N-terminal sequence and two internal peptide sequences of the isolated protein are in agreement with that previously predicted from the DNA sequence of the kps U gene. The kinetic data of the CMP-Kdo-synthetase participating in K5 capsule expression (K-CMP-Kdo-synthetase) differ from those described for the CMP-Kdo-synthetase, participating in lipopolysaccharide synthesis (L-CMP-Kdo-synthetase).     

A strategy for shuffling numerous Bacillus thuringiensis crystal protein domains

Bacillus thuringiensis that produce Cry1Ba are toxic to Lucilia cuprina Wiedemann blow fly maggots in vivo, and when applied in quantity to sheep fleece, provide up to 6 wk protection against flystrike in the field. These strains also are toxic to Epiphyas postvittana (Walker) light brown apple moth caterpillars. B. thuringiensis expressing Cry1Db are toxic only to E. postvittana. When Cry1Ba and Cry1Db proteins are expressed within Escherichia coli, the recombinant bacteria have the same toxicity profile as the wild-type B. thuringiensis strain. In an effort to develop a Cry protein with improved blow fly toxicity, three different internal regions of Cry1Ba coding DNA, encoding all or part of domains I, II and III respectively were systematically exchanged with the corresponding region from a pool of other Cry protein coding DNAs. The chimeric products were then expressed in recombinant E. coli, and the resulting bacteria assayed for toxicity on L. cuprina and E. postvittana. Clones having insecticide bioactivity were characterized to identify the source of the replacement Cry domain. Despite successfully expressing a large number and variety of chimeric proteins within E. coli, many with measurable insecticidal activity, none of the chimeras had greater potency against L. cuprina than the wild-type Cry1Ba. Chimeric replacements involving domains I and II were rarely active, whereas a much higher proportion of domain III chimeras had some bioactivity. We conclude that shuffling of Cry coding regions through joining at the major conserved sequence motifs is an effective means for the production of a diverse number of chimeric Cry proteins but that such toxins with enhanced bioactive properties will be rare or nonexistent.     

Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai.

Bacillus thuringiensis subsp. aizawai EG6346, a novel grain dust isolate, was analyzed by Southern blot hybridization for its insecticidal crystal protein (ICP) gene profile. Strain EG6346 lacks previously characterized cryIA ICP genes yet does possess novel cryI-related gene sequences. A recombinant genomic plasmid library was constructed for strain EG6346 in Escherichia coli. One recombinant plasmid, pEG640, isolated from the library contained a novel ICP gene on a 5.7-kb Sau3A insert. The sequence of this gene, designated cryIF, was related to, but distinct from, the published sequences for other cryI genes. A second novel cryI-related sequence was also located on pEG640, approximately 500 bp downstream from cryIF. Introduction of cryIF into a Cry- B. thuringiensis recipient strain via electroporation enabled sufficient production of CryIF protein for quantitative bioassay analyses of insecticidal specificity. The CryIF crystal protein was selectively toxic to a subset of lepidopteran insects tested, including the larvae of Ostrinia nubilalis and Spodoptera exigua.     

Structure and function of the Bacillus thuringiensis protein crystal

The structural chemistry of the Bacillus thuringiensis parasporal protein crystal is discussed in terms of purification techniques, removal of contaminating proteases, crystal subunit size, crystal shape, interchain crosslinks, the ultimate toxin, and lysinoalanine. The alkaline pH cleavage of disulfide bonds is stressed in relationship to this role in crystal solubilization and toxin formation. The future implication s of plasmid-coded crystal formation and B. thuringiensis var. israelensis (effective against mosquitoes and black flies) are also discussed.     

Cry29A and Cry30A: two novel delta-endotoxins isolated from Bacillus thuringiensis serovar medellin

Two novel crystal protein genes from a highly mosquitocidal Bacillus thuringiensis serovar medellin strain were cloned and sequenced. The corresponding proteins, Cry29A and Cry30A, were nontoxic when tested individually against the mosquito species bioassayed (Aedes aegypti, Culex pipiens and Anopheles stephensi). However, Cry29A synergized the toxicity of Cry11Bb against Aedes aegypti by a four-fold factor.     

A novel influenza A virus mitochondrial protein that induces cell death.

While searching for alternative reading-frame peptides encoded by influenza A virus that are recognized by CD8+ T cells, we found an abundant immunogenic peptide encoded by the +1 reading frame of PB1. This peptide derives from a novel conserved 87-residue protein, PB1-F2, which has several unusual features compared with other influenza gene products in addition to its mode of translation. These include its absence from some animal (particularly swine) influenza virus isolates, variable expression in individual infected cells, rapid proteasome-dependent degradation and mitochondrial localization. Exposure of cells to a synthetic version of PB1-F2 induces apoptosis, and influenza viruses with targeted mutations that interfere with PB1-F2 expression induce less extensive apoptosis in human monocytic cells than those with intact PB1-F2. We propose that PB1-F2 functions to kill host immune cells responding to influenza virus infection.     

Isolation and characterization of a novel Bacillus thuringiensis strain expressing a novel crystal protein with cytocidal activity against human cancer cells

AIMS: To characterize a novel, unusual, Bacillus thuringiensis strain, to clone its Cry gene and determine the spectrum of action of the encoded Cry protein. METHODS AND RESULTS: The B. thuringiensis strain, referred to as M15, was isolated from dead two-spotted spider mites (Tetranychus urticae Koch; Arthropoda: Arachnida: Tetranychidae). It is an autoagglutination-positive strain and is therefore non-serotypeable. A sporulated culture produces a roughly spherical parasporal inclusion body, the crystal, tightly coupled to the spore. Although the crystal appears to be composed of at least two major polypeptides of 86 and 79 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Southern hybridization indicates that the corresponding crystal protein gene is likely present in only one copy. The crystal protein gene was cloned and, based on nucleotide sequence homology with an orthologous cry31Aa1 gene, assigned the name cry31Aa2. Although initially isolated from spider mites, B. thuringiensis M15 is non-toxic to spider mites and it does not produce the wide spectrum beta-exotoxin. Assays on mammalian cells, however, reveal that Cry31Aa2, when cleaved with trypsin, is cytocidal to some human cancer cells but not to normal human cells. No cytocidal activity was induced after protease treatment of Cry31Aa2 with either chymotrypsin or proteinase K. Trypsin, chymotrypsin and proteinase K cleavage sites were determined. CONCLUSIONS: The B. thuringiensis strain M15 exhibits specific cytocidal activities against some human cancer cells. Significance and Impact of the Study: This study raises questions as to the actual role of this bacterial strain and its crystal protein in the environment. It may be possible to further develop the Cry31Aa2 protein to target specific human cancer cells.