19 results on '"Volohonsky G"'
Search Results
2. Kinetics of Plasmodium midgut invasion in Anopheles mosquitoes.
- Author
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Volohonsky G, Paul-Gilloteaux P, Štáfková J, Soichot J, Salamero J, and Levashina EA
- Subjects
- Animals, Anopheles growth & development, Female, Malaria parasitology, Mice, Species Specificity, Anopheles parasitology, Digestive System parasitology, Host-Parasite Interactions, Malaria transmission, Mosquito Vectors pathogenicity, Plasmodium berghei physiology
- Abstract
Malaria-causing Plasmodium parasites traverse the mosquito midgut cells to establish infection at the basal side of the midgut. This dynamic process is a determinant of mosquito vector competence, yet the kinetics of the parasite migration is not well understood. Here we used transgenic mosquitoes of two Anopheles species and a Plasmodium berghei fluorescence reporter line to track parasite passage through the mosquito tissues at high spatial resolution. We provide new quantitative insight into malaria parasite invasion in African and Indian Anopheles species and propose that the mosquito complement-like system contributes to the species-specific dynamics of Plasmodium invasion., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2020
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3. Transgenic Expression of the Anti-parasitic Factor TEP1 in the Malaria Mosquito Anopheles gambiae.
- Author
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Volohonsky G, Hopp AK, Saenger M, Soichot J, Scholze H, Boch J, Blandin SA, and Marois E
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- Animals, Animals, Genetically Modified, Blotting, Western, Immunohistochemistry, Plasmodium berghei, Polymerase Chain Reaction, Anopheles genetics, Insect Proteins genetics, Insect Vectors genetics, Malaria parasitology, Pest Control, Biological methods
- Abstract
Mosquitoes genetically engineered to be resistant to Plasmodium parasites represent a promising novel approach in the fight against malaria. The insect immune system itself is a source of anti-parasitic genes potentially exploitable for transgenic designs. The Anopheles gambiae thioester containing protein 1 (TEP1) is a potent anti-parasitic protein. TEP1 is secreted and circulates in the mosquito hemolymph, where its activated cleaved form binds and eliminates malaria parasites. Here we investigated whether TEP1 can be used to create malaria resistant mosquitoes. Using a GFP reporter transgene, we determined that the fat body is the main site of TEP1 expression. We generated transgenic mosquitoes that express TEP1r, a potent refractory allele of TEP1, in the fat body and examined the activity of the transgenic protein in wild-type or TEP1 mutant genetic backgrounds. Transgenic TEP1r rescued loss-of-function mutations, but did not increase parasite resistance in the presence of a wild-type susceptible allele. Consistent with previous reports, TEP1 protein expressed from the transgene in the fat body was taken up by hemocytes upon a challenge with injected bacteria. Furthermore, although maturation of transgenic TEP1 into the cleaved form was impaired in one of the TEP1 mutant lines, it was still sufficient to reduce parasite numbers and induce parasite melanization. We also report here the first use of Transcription Activator Like Effectors (TALEs) in Anopheles gambiae to stimulate expression of endogenous TEP1. We found that artificial elevation of TEP1 expression remains moderate in vivo and that enhancement of endogenous TEP1 expression did not result in increased resistance to Plasmodium. Taken together, our results reveal the difficulty of artificially influencing TEP1-mediated Plasmodium resistance, and contribute to further our understanding of the molecular mechanisms underlying mosquito resistance to Plasmodium parasites., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2017
- Full Text
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4. Tools for Anopheles gambiae Transgenesis.
- Author
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Volohonsky G, Terenzi O, Soichot J, Naujoks DA, Nolan T, Windbichler N, Kapps D, Smidler AL, Vittu A, Costa G, Steinert S, Levashina EA, Blandin SA, and Marois E
- Subjects
- Animals, Animals, Genetically Modified, Cloning, Molecular, Codon genetics, DNA Transposable Elements genetics, Embryo, Nonmammalian metabolism, Flow Cytometry, Genes, Insect, Genes, Reporter, Genetic Vectors metabolism, Integrases metabolism, Larva genetics, Microinjections, Mosaicism, Ovum metabolism, Plasmids genetics, Promoter Regions, Genetic genetics, Transgenes, Transposases metabolism, Anopheles genetics, Gene Transfer Techniques
- Abstract
Transgenesis is an essential tool to investigate gene function and to introduce desired characters in laboratory organisms. Setting-up transgenesis in non-model organisms is challenging due to the diversity of biological life traits and due to knowledge gaps in genomic information. Some procedures will be broadly applicable to many organisms, and others have to be specifically developed for the target species. Transgenesis in disease vector mosquitoes has existed since the 2000s but has remained limited by the delicate biology of these insects. Here, we report a compilation of the transgenesis tools that we have designed for the malaria vector Anopheles gambiae, including new docking strains, convenient transgenesis plasmids, a puromycin resistance selection marker, mosquitoes expressing cre recombinase, and various reporter lines defining the activity of cloned promoters. This toolbox contributed to rendering transgenesis routine in this species and is now enabling the development of increasingly refined genetic manipulations such as targeted mutagenesis. Some of the reagents and procedures reported here are easily transferable to other nonmodel species, including other disease vector or agricultural pest insects., (Copyright © 2015 Volohonsky et al.)
- Published
- 2015
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5. A heterodimeric complex of the LRR proteins LRIM1 and APL1C regulates complement-like immunity in Anopheles gambiae.
- Author
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Baxter RH, Steinert S, Chelliah Y, Volohonsky G, Levashina EA, and Deisenhofer J
- Subjects
- Animals, Crystallography, X-Ray, Cysteine metabolism, Hemolymph immunology, Insect Proteins chemistry, Insect Proteins genetics, Protein Conformation, Protein Multimerization, Protein Stability, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Anopheles immunology, Complement System Proteins metabolism, Insect Proteins metabolism
- Abstract
The leucine-rich repeat (LRR) proteins LRIM1 and APL1C control the function of the complement-like protein TEP1 in Anopheles mosquitoes. The molecular structure of LRIM1 and APL1C and the basis of their interaction with TEP1 represent a new type of innate immune complex. The LRIM1/APL1C complex specifically binds and solubilizes a cleaved form of TEP1 without an intact thioester bond. The LRIM1 and APL1C LRR domains have a large radius of curvature, glycosylated concave face, and a novel C-terminal capping motif. The LRIM1/APL1C complex is a heterodimer with a single intermolecular disulfide bond. The structure of the LRIM1/APL1C heterodimer reveals an interface between the two LRR domains and an extensive C-terminal coiled-coil domain. We propose that a cleaved form of TEP1 may act as a convertase for activation of other TEP1 molecules and that the LRIM1/APL1C heterodimer regulates formation of this TEP1 convertase.
- Published
- 2010
- Full Text
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6. Focusing on complement in the antiparasitic defense of mosquitoes.
- Author
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Volohonsky G, Steinert S, and Levashina EA
- Subjects
- Animals, Gene Expression Regulation immunology, Insect Proteins genetics, Insect Proteins metabolism, Protein Binding, Anopheles immunology, Plasmodium physiology
- Abstract
Malaria is an infectious disease caused by Plasmodium and transmitted to humans by the Anopheles mosquitoes. The mosquito immune system predominantly targets Plasmodium at the ookinete stage, and efficiently eliminates the majority of invading parasites. Identification of the components of the mosquito complement system now provides new focus for studies on the activation and control of this pathway, whose manipulation is expected to block malaria transmission at the vector level., (Copyright 2009 Elsevier Ltd. All rights reserved.)
- Published
- 2010
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7. Post-transcriptional repression of the Drosophila midkine and pleiotrophin homolog miple by HOW is essential for correct mesoderm spreading.
- Author
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Toledano-Katchalski H, Nir R, Volohonsky G, and Volk T
- Subjects
- 3' Untranslated Regions, Animals, Animals, Genetically Modified, Cytokines genetics, Drosophila genetics, Drosophila metabolism, Drosophila Proteins deficiency, Drosophila Proteins genetics, Gene Expression, Genes, Insect, MAP Kinase Signaling System, Mesoderm embryology, Mesoderm metabolism, Midkine, Nuclear Proteins deficiency, Nuclear Proteins genetics, Phenotype, Protein-Tyrosine Kinases genetics, Protein-Tyrosine Kinases metabolism, RNA Processing, Post-Transcriptional, RNA, Messenger genetics, RNA, Messenger metabolism, RNA-Binding Proteins genetics, Receptors, Fibroblast Growth Factor genetics, Receptors, Fibroblast Growth Factor metabolism, Cytokines metabolism, Drosophila embryology, Drosophila Proteins metabolism, Nuclear Proteins metabolism, RNA-Binding Proteins metabolism
- Abstract
The even spreading of mesoderm cells in the Drosophila embryo is essential for its proper patterning by ectodermally derived signals. In how germline clone embryos, defects in mesoderm spreading lead to a partial loss of dorsal mesoderm derivatives. HOW is an RNA-binding protein that is thought to regulate diverse mRNA targets. To identify direct HOW targets, we implemented a series of selection methods on mRNAs whose levels were elevated in how germline clone embryos during the stage of mesoderm spreading. Four mRNAs were found to be specifically elevated in the mesoderm of how germline clone embryos, and to exhibit specific binding to HOW via their 3' UTRs. Importantly, overexpression of three of these genes phenocopied the mesoderm-spreading phenotype of how germline clone embryos. Further analysis showed that overexpressing one of these genes, miple (a Drosophila midkine and pleiotrophin heparin-binding growth factor), in the mesoderm led to abnormal scattered MAPK activation, a phenotype that might explain the abnormal mesoderm spreading. In addition, the number of EVE-positive cells, which are responsive to receptor tyrosine kinase (RTK) signaling, was increased following Miple overexpression in the mesoderm and appeared to be dependent on Heartless function. In summary, our analysis suggests that HOW downregulates the levels of a number of mRNA species in the mesoderm in order to enable proper mesoderm spreading during early embryogenesis.
- Published
- 2007
- Full Text
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8. Muscle-dependent maturation of tendon cells is induced by post-transcriptional regulation of stripeA.
- Author
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Volohonsky G, Edenfeld G, Klämbt C, and Volk T
- Subjects
- Alternative Splicing, Animals, Base Sequence, Body Patterning, DNA, Complementary genetics, DNA-Binding Proteins metabolism, Drosophila metabolism, Drosophila Proteins metabolism, Genes, Insect, Muscles embryology, Muscles metabolism, Mutation, Nuclear Proteins genetics, Nuclear Proteins metabolism, Protein Isoforms genetics, Protein Isoforms metabolism, RNA Processing, Post-Transcriptional, RNA, Messenger genetics, RNA, Messenger metabolism, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, Tendons embryology, Tendons metabolism, Transcription Factors metabolism, DNA-Binding Proteins genetics, Drosophila embryology, Drosophila genetics, Drosophila Proteins genetics, Transcription Factors genetics
- Abstract
Terminal differentiation of single cells selected from a group of equivalent precursors may be random, or may be regulated by external signals. In the Drosophila embryo, maturation of a single tendon cell from a field of competent precursors is triggered by muscle-dependent signaling. The transcription factor Stripe was reported to induce both the precursor cell phenotype, as well as the terminal differentiation of muscle-bound tendons. The mechanism by which Stripe activates these distinct differentiation programs remained unclear. Here, we demonstrate that each differentiation state is associated with a distinct Stripe isoform and that the Stripe isoforms direct different transcriptional outputs. Importantly, the transition to the mature differentiation state is triggered post-transcriptionally by enhanced production of the stripeA splice variant, which is typical of the tendon mature state. This elevation is mediated by the RNA-binding protein How(S), with levels sensitive to muscle-dependent signals. In how mutant embryos the expression of StripeA is significantly reduced, while overexpression of How(S) enhances StripeA protein as well as mRNA levels in embryos. Analysis of the expression of a stripeA minigene in S-2 cells suggests that this elevation may be due to enhanced splicing of stripeA. Consistently, stripeA mRNA is specifically reduced in embryos mutant for the splicing factor Crn, which physically interacts with How(S). Thus, we demonstrate a mechanism by which tendon cell terminal differentiation is maintained and reinforced by the approaching muscle.
- Published
- 2007
- Full Text
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9. The splicing factor crooked neck associates with the RNA-binding protein HOW to control glial cell maturation in Drosophila.
- Author
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Edenfeld G, Volohonsky G, Krukkert K, Naffin E, Lammel U, Grimm A, Engelen D, Reuveny A, Volk T, and Klämbt C
- Subjects
- Animals, Animals, Genetically Modified, Cell Differentiation physiology, Cell Growth Processes, Cell Line, Cell Nucleus metabolism, Drosophila, Drosophila Proteins genetics, Embryo, Nonmammalian, Female, Genes, Insect physiology, Green Fluorescent Proteins metabolism, Male, Microscopy, Electron, Transmission methods, Mutation physiology, Neuroglia ultrastructure, Nuclear Proteins genetics, RNA Splicing physiology, RNA-Binding Proteins genetics, Transfection methods, Wings, Animal growth & development, Cell Movement physiology, Drosophila Proteins physiology, Neuroglia physiology, Nuclear Proteins physiology, RNA-Binding Proteins physiology
- Abstract
In both vertebrates and invertebrates, glial cells wrap axonal processes to ensure electrical conductance. Here we report that Crooked neck (Crn), the Drosophila homolog of the yeast Clf1p splicing factor, is directing peripheral glial cell maturation. We show that crooked neck is expressed and required in glial cells to control migration and axonal wrapping. Within the cytoplasm, Crn interacts with the RNA-binding protein HOW and then translocates to the nucleus where the Crn/HOW complex controls glial differentiation by facilitating splicing of specific target genes. By using a GFP-exon trap approach, we identified some of the in vivo target genes that encode proteins localized in autocellular septate junctions. In conclusion, here we show that glial cell differentiation is controlled by a cytoplasmic assembly of splicing components, which upon translocation to the nucleus promote the splicing of genes involved in the assembly of cellular junctions.
- Published
- 2006
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10. Cell divisions in the drosophila embryonic mesoderm are repressed via posttranscriptional regulation of string/cdc25 by HOW.
- Author
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Nabel-Rosen H, Toledano-Katchalski H, Volohonsky G, and Volk T
- Subjects
- Animals, Blotting, Western, Cell Cycle Proteins, Cells, Cultured, Drosophila metabolism, Gastrula metabolism, Immunohistochemistry, Protein Binding, Reverse Transcriptase Polymerase Chain Reaction, Cell Division physiology, Drosophila embryology, Drosophila Proteins metabolism, Mesoderm physiology, Nuclear Proteins metabolism, Protein Tyrosine Phosphatases metabolism, RNA Interference physiology, RNA-Binding Proteins metabolism
- Abstract
Background: Cell-cycle progression is tightly regulated during embryonic development. In the Drosophila early embryo, the levels of String/Cdc25 define the precise timing and sites of cell divisions. However, cell-cycle progression is arrested in the mesoderm of gastrulating embryos despite a positive transcriptional string/cdc25 activation provided by the mesoderm-specific action of Twist. Whereas String/Cdc25 is negatively regulated by Tribbles in the mesoderm at these embryonic stages, the factor(s) controlling string/cdc25 mRNA levels has yet to be elucidated., Results: Here, we show that the repressor isoform of the Drosophila RNA binding protein Held Out Wing [HOW(L)] is required to inhibit mesodermal cell division during gastrulation. Embryos mutant for how exhibited an excess of cell divisions, leading to delayed mesoderm invagination. The levels of the mitotic activator string/cdc25 mRNA in these embryos were significantly elevated. Protein-RNA precipitation experiments show that HOW(L) binds string/cdc25 mRNA. Overexpression of HOW(L) in Schneider cells reduces specifically the steady-state mRNA levels of a gfp reporter fused to string/cdc25 untranslated region (3'UTR)., Conclusions: Our results suggest that in wild-type embryos, string/cdc25 mRNA levels are downregulated by the repressor isoform HOW(L), which binds directly to string/cdc25 mRNA and regulates its degradation. Thus, we are proposing a novel posttranscriptional mechanism controlling cell-cycle progression in the Drosophila embryo.
- Published
- 2005
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11. EGFR signaling attenuates Groucho-dependent repression to antagonize Notch transcriptional output.
- Author
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Hasson P, Egoz N, Winkler C, Volohonsky G, Jia S, Dinur T, Volk T, Courey AJ, and Paroush Z
- Subjects
- Animals, Basic Helix-Loop-Helix Transcription Factors, Drosophila Proteins metabolism, Drosophila melanogaster metabolism, Immunohistochemistry, Receptors, Notch, Signal Transduction physiology, Wings, Animal abnormalities, Wings, Animal growth & development, ras Proteins metabolism, DNA-Binding Proteins metabolism, ErbB Receptors metabolism, Gene Expression Regulation physiology, Membrane Proteins metabolism, Repressor Proteins metabolism
- Abstract
Crosstalk between signaling pathways is crucial for the generation of complex and varied transcriptional networks. Antagonism between the EGF-receptor (EGFR) and Notch pathways in particular is well documented, although the underlying mechanism is poorly understood. The global corepressor Groucho (Gro) and its transducin-like Enhancer-of-split (TLE) mammalian homologs mediate repression by a myriad of repressors, including effectors of the Notch, Wnt (Wg) and TGF-beta (Dpp) signaling cascades. Given that there are genetic interactions between gro and components of the EGFR pathway (ref. 9 and P.H. et al., unpublished results), we tested whether Gro is at a crossroad between this and other pathways. Here we show that phosphorylation of Gro in response to MAPK activation weakens its repressor capacity, attenuating Gro-dependent transcriptional silencing by the Enhancer-of-split proteins, effectors of the Notch cascade. Thus, Gro is a new junction between signaling pathways, enabling EGFR signaling to antagonize transcriptional output by Notch and potentially other Gro-dependent pathways.
- Published
- 2005
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12. The role of gamma-glutamyl transpeptidase in the biosynthesis of glutathione.
- Author
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Stark AA, Porat N, Volohonsky G, Komlosh A, Bluvshtein E, Tubi C, and Steinberg P
- Subjects
- Animals, Carcinogens, Dipeptides metabolism, Humans, Inactivation, Metabolic, Lipid Peroxidation, Mutagenesis, Neoplasms enzymology, Glutathione biosynthesis, gamma-Glutamyltransferase metabolism
- Published
- 2003
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13. Nuclear localization of non-receptor protein tyrosine phosphatase epsilon is regulated by its unique N-terminal domain.
- Author
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Kraut J, Volohonsky G, Toledano-Katchalski H, and Elson A
- Subjects
- Active Transport, Cell Nucleus genetics, Amino Acid Sequence physiology, Animals, Cell Nucleus ultrastructure, Eukaryotic Cells cytology, Gene Expression Regulation, Enzymologic physiology, Green Fluorescent Proteins, Luminescent Proteins, Mice, Mutation genetics, Oxidative Stress genetics, Protein Isoforms genetics, Protein Isoforms metabolism, Protein Structure, Tertiary physiology, Protein Tyrosine Phosphatases genetics, Cell Compartmentation physiology, Cell Nucleus enzymology, Eukaryotic Cells enzymology, Protein Tyrosine Phosphatases metabolism
- Abstract
Precise subcellular localization is an important factor in regulation of the functions of protein tyrosine phosphatases. The non-receptor form of protein tyrosine phosphatase epsilon (cyt-PTP(epsilon)) can be found in cell nuclei, among other cellular locations, while p67 PTP(epsilon), a naturally occurring isoform which lacks the 27 N terminal residues of cyt-PTP(epsilon), is exclusively cytosolic. Using deletion and scanning mutagenesis we report that the first 10 amino acid residues of cyt-PTP(epsilon), in particular residues R4, K5, and R9, are critical components for its nuclear localization. We also establish that increased oxidative stress enhances accumulation of cyt-PTP(epsilon) in cell nuclei. Of the four known protein forms of PTP(epsilon), cyt-PTP(epsilon) is the only one which includes the extreme N-terminal sequence containing R4, K5, and R9. The role of the unique N terminus of cyt-PTP(epsilon) is therefore to regulate its subcellular localization. The existence of naturally occurring forms of PTP(epsilon) which lack this sequence and which are generated by translational and posttranslational mechanisms, suggests that nuclear localization of cyt-PTP(epsilon) can be actively regulated by cells.
- Published
- 2002
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14. A spectrophotometric assay of gamma-glutamylcysteine synthetase and glutathione synthetase in crude extracts from tissues and cultured mammalian cells.
- Author
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Volohonsky G, Tuby CN, Porat N, Wellman-Rousseau M, Visvikis A, Leroy P, Rashi S, Steinberg P, and Stark AA
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- Animals, Mice, Rats, Rats, Sprague-Dawley, Spectrophotometry methods, Tumor Cells, Cultured, 3T3 Cells enzymology, Aminoacyltransferases analysis, Glutathione Synthase analysis, Kidney enzymology, Liver enzymology
- Abstract
An assay of gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GS) in crude extracts of cultured cells and tissues is described. It represents a novel combination of known methods, and is based on the formation of glutathione (GSH) from cysteine, glutamate and glycine in the presence of rat kidney GS for the assay of gamma-GCS, or from gamma-glutamylcysteine and glycine for the assay of GS. GSH is then quantified by the Tietze recycling method. Assay mixtures contain the gamma-glutamyl transpeptidase (GGT) inhibitor acivicin in order to prevent the degradation of gamma-glutamylcysteine and of the accumulating GSH, and dithiothreitol in order to prevent the oxidation of cysteine and gamma-glutamylcysteine. gamma-GCS and GS levels determined by this method are comparable to those determined by others. The method is suitable for the rapid determination of gamma-GCS GS in GGT-containing tissues and for the studies of induction of gamma-GCS and GS in tissue cultures.
- Published
- 2002
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15. gamma-Glutamyl transpeptidase and glutathione biosynthesis in non-tumorigenic and tumorigenic rat liver oval cell lines.
- Author
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Komlosh A, Volohonsky G, Porat N, Tuby C, Bluvshtein E, Steinberg P, Oesch F, and Stark AA
- Subjects
- Aminopeptidases antagonists & inhibitors, Aminopeptidases metabolism, Animals, Cell Division drug effects, Cell Line, Cystine metabolism, Enzyme Inhibitors pharmacology, Glutamic Acid metabolism, Glutamine metabolism, Glutathione metabolism, Glutathione pharmacology, Kinetics, Liver drug effects, Liver enzymology, Rats, Tumor Cells, Cultured, gamma-Glutamyltransferase antagonists & inhibitors, Glutathione biosynthesis, Liver cytology, gamma-Glutamyltransferase metabolism
- Abstract
Glutathione synthesis and growth properties were studied in the gamma-glutamyl transpeptidase(GGT)-negative, non-tumorigenic rat liver oval cell line OC/CDE22, and in its GGT-positive, tumorigenic counterpart line M22. gamma-Glutamylcysteine synthetase (GGCS) activities were comparable. Growth rates of M22 cells exceeded those of OC/CDE22 cells at non-limiting and limiting exogenous cysteine concentrations. A monoclonal antibody (Ab 5F10) that inhibits the transpeptidatic but not the hydrolytic activity of GGT did not affect the growth rates of OC/CDE22, and decreased those of M22 to the OC/CDE22 level. In GSH-depleted M22, but not in OC/CDE22 cells, the rate and extent of GSH repletion with exogenous cysteine and glutamine exceeded those obtained with exogenous cysteine and glutamate. With Ab 5F10, repletion with cysteine/glutamine was similar to that obtained with cysteine/glutamate. Repletion with exogenous GSH occurred only in M22 cells, and was abolished by the GGT inhibitor acivicin. Repletion with gamma-glutamylcysteine (GGC) in OC/CDE22 was resistant to acivicin whereas that in M22 was inhibited by acivicin. Repletion with exogenous GSH or cysteinylglycine (CG) required aminopeptidase activity and was lower than that obtained with cysteine. Unless reduced, CG disulfide did not support GSH repletion. The findings are compatible with the notions that (i) GGT-catalyzed transpeptidation was largely responsible for the growth advantage of M22 cells at limiting cysteine concentration, and for their high GSH content via the formation of GGC from a gamma-glutamyl donor (glutamine) and cyst(e)ine, and (ii) aminopeptidase/dipeptidase activity is rate-limiting in GSH repletion when GSH or CG serve as cysteine sources.
- Published
- 2002
- Full Text
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16. Gamma-glutamyl transpeptidase and glutathione biosynthesis in non-tumorigenic and tumorigenic rat liver oval cell lines.
- Author
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Komlosh A, Volohonsky G, Porat N, Tuby C, Bluvshtein E, Steinberg P, Oesch F, and Stark AA
- Subjects
- Aminopeptidases antagonists & inhibitors, Aminopeptidases metabolism, Animals, Cell Division drug effects, Cell Line, Cystine metabolism, Enzyme Inhibitors pharmacology, Glutamic Acid metabolism, Glutamine metabolism, Glutathione metabolism, Glutathione pharmacology, Kinetics, Liver drug effects, Liver enzymology, Rats, Tumor Cells, Cultured, gamma-Glutamyltransferase antagonists & inhibitors, Glutathione biosynthesis, Liver cytology, gamma-Glutamyltransferase metabolism
- Abstract
Glutathione synthesis and growth properties were studied in the gamma-glutamyl transpeptidase(GGT)-negative, non-tumorigenic rat liver oval cell line OC/CDE22, and in its GGT-positive, tumorigenic counterpart line M22. gamma-Glutamylcysteine synthetase (GGCS) activities were comparable. Growth rates of M22 cells exceeded those of OC/CDE22 cells at non-limiting and limiting exogenous cysteine concentrations. A monoclonal antibody (Ab 5F10) that inhibits the transpeptidatic but not the hydrolytic activity of GGT did not affect the growth rates of OC/CDE22, and decreased those of M22 to the OC/CDE22 level. In GSH-depleted M22, but not in OC/CDE22 cells, the rate and extent of GSH repletion with exogenous cysteine and glutamine exceeded those obtained with exogenous cysteine and glutamate. With Ab 5F10, repletion with cysteine/glutamine was similar to that obtained with cysteine/glutamate. Repletion with exogenous GSH occurred only in M22 cells, and was abolished by the GGT inhibitor acivicin. Repletion with gamma-glutamylcysteine (GGC) in OC/CDE22 was resistant to acivicin whereas that in M22 was inhibited by acivicin. Repletion with exogenous GSH or cysteinylglycine (CG) required aminopeptidase activity and was lower than that obtained with cysteine. Unless reduced, CG disulfide did not support GSH repletion. The findings are compatible with the notions that (i) GGT-catalyzed transpeptidation was largely responsible for the growth advantage of M22 cells at limiting cysteine concentration, and for their high GSH content via the formation of GGC from a gamma-glutamyl donor (glutamine) and cyst(e)ine, and (ii) aminopeptidase/dipeptidase activity is rate-limiting in GSH repletion when GSH or CG serve as cysteine sources.
- Published
- 2001
- Full Text
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17. Regulation of protein-tyrosine phosphatases alpha and epsilon by calpain-mediated proteolytic cleavage.
- Author
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Gil-Henn H, Volohonsky G, and Elson A
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cytoplasm enzymology, DNA Primers, Humans, Hydrolysis, Protein Transport, Sequence Homology, Amino Acid, Calpain metabolism, Isoenzymes metabolism, Protein Tyrosine Phosphatases metabolism
- Abstract
The precise subcellular localization of non-receptor tyrosine phosphatases is a major factor in regulating their physiological functions. We have previously shown that cellular processing of protein-tyrosine phosphatase epsilon (PTP epsilon) generates a physiologically distinct, cytoplasmic form of this protein, p65 PTP epsilon. Here we describe a novel protein form of the related receptor-type tyrosine phosphatase alpha (RPTP alpha), p66 PTP alpha, which is detected in nearly all cell types where RPTP alpha is expressed. Both p66 PTP alpha and p65 PTP epsilon are produced by calpain-mediated proteolytic cleavage in vivo. Cleavage is inhibited in living cells by a variety of calpain inhibitors, can be induced in primary cortical neurons treated with calcium chloride, and is observed in lysates of brain or of cultured cells following addition of purified calpain. Cleavage occurs within the intracellular juxtamembrane domain of RPTP alpha, releasing the phosphatase catalytic domains from their membranal anchors and translocating them to the cytoplasm. Translocation reduces the ability of PTPalpha to act on membrane-associated substrates, as it loses its ability to dephosphorylate Src at its C-terminal regulatory site, and its ability to dephosphorylate the Kv2.1 voltage-gated potassium channel is severely impaired. In all, the data indicate that control of phosphatase function via post-translational processing occurs also among receptor-type phosphatases, and demonstrate the molecular complexity of regulating these parameters within the PTP alpha/PTP epsilon phosphatase subfamily.
- Published
- 2001
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18. Generation of novel cytoplasmic forms of protein tyrosine phosphatase epsilon by proteolytic processing and translational control.
- Author
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Gil-Henn H, Volohonsky G, Toledano-Katchalski H, Gandre S, and Elson A
- Subjects
- 3T3 Cells, Amino Acid Sequence, Animals, Cell Line, Codon, Cysteine Proteinase Inhibitors pharmacology, Cytoplasm drug effects, GRB2 Adaptor Protein, Humans, Leupeptins pharmacology, Mice, Molecular Sequence Data, Phosphorylation, Potassium Channels metabolism, Protein Biosynthesis, Proteins metabolism, Receptor-Like Protein Tyrosine Phosphatases, Class 4, src-Family Kinases metabolism, Adaptor Proteins, Signal Transducing, Cytoplasm metabolism, Protein Processing, Post-Translational, Protein Tyrosine Phosphatases genetics, Protein Tyrosine Phosphatases metabolism
- Abstract
Two protein forms of tyrosine phosphatase epsilon (PTPepsilon) are known - receptor-like (tm-PTPepsilon) and non receptor-like (cyt-PTPepsilon), with each form possessing unique tissue-specific expression patterns, subcellular localization, and physiological functions. We describe two additional forms of PTPepsilon protein - p67 and p65. p67 is produced by initiation of translation at an internal initiation codon of PTPepsilon mRNA molecules, while p65 is produced by specific proteolytic cleavage of larger PTPepsilon proteins. Cleavage is inhibited by MG132, but is proteasome-independent. In contrast with full-length tm-PTPepsilon and cyt-PTPepsilon, p67 and p65 are exclusively cytoplasmic, are not phosphorylated by Neu, and do not associate with Grb2 in unstimulated cells. p67 and p65 are catalytically active and can reduce Src-mediated phosphorylation of the Kv2.1 voltage-gated potassium channel, albeit with reduced efficiency which most likely results from their cytoplasmic localization. We also show that full-length cyt-PTPepsilon protein can be found at the cell membrane and in the nucleus and that it is the first 27 residues of cyt-PTPepsilon which determine this localization. p67 and p65 provide mechanisms for removing PTPepsilon activity from the cell membrane, possibly serving to down-regulate PTPepsilon activity there. PTPepsilon emerges as a family of four related proteins whose expression, subcellular localization and most likely physiological roles are subject to complex regulation at the transcriptional, translational and post-translational levels.
- Published
- 2000
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19. Inhibition of the hydrolytic and transpeptidase activities of rat kidney gamma-glutamyl transpeptidase by specific monoclonal antibodies.
- Author
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Bluvshtein E, Glass GA, Volohonsky G, Yaakubowitz M, Harness E, Smorodinsky NI, Seidel A, Frank H, Steinberg P, and Stark AA
- Subjects
- Animals, Antibodies, Monoclonal isolation & purification, Carbohydrates immunology, Female, Glutaminase antagonists & inhibitors, Humans, Hydrolysis, Isoenzymes antagonists & inhibitors, Isoenzymes immunology, Mice, Mice, Inbred BALB C, Rats, Rats, Wistar, gamma-Glutamyltransferase immunology, Antibodies, Monoclonal immunology, Kidney enzymology, gamma-Glutamyltransferase antagonists & inhibitors
- Abstract
Monoclonal antibodies (mAb) against the native form of rat kidney gamma-glutamyl transpeptidase (GGT) were isolated by screening hybridomas with rat kidney brush-border membrane vesicles. They were directed against protein rather than sugar epitopes in that each recognized all GGT isoforms. All of them inhibited partially the enzyme activity of GGT. They were specific in that they inhibited the rat enzyme, but not the mouse or human enzyme. Kinetic analyses were carried out with free GGT and GGT-mAb complexes with d-gamma-glutamyl-p-nitroanilide in the presence or absence of maleate, or in the presence or absence of alanine, cysteine, cystine or glycylglycine as gamma-glutamyl acceptors. mAbs 2A10 and 2E9 inhibited the hydrolytic and glutaminase activities of GGT and had little effect on the transpeptidation activity of the enzyme, whereas mAbs 4D7 and 5F10 inhibited transpeptidation, but not hydrolytic or glutaminase activities. mAb 5F10 mimicked the effect of maleate on GGT, in that it inhibited transpeptidation, enhanced the glutaminase activity and increased the affinity of the donor site of GGT for acivicin. Such mAbs may be useful for long-term studies in tissue cultures and in vivo, and for the identification of GGT epitopes that are important for the hydrolytic and transpeptidase activities.
- Published
- 1999
- Full Text
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