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Janendra K. Batra - One of the best experts on this subject based on the ideXlab platform.
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Ribotoxin restrictocin manifests anti-HIV-1 activity through its specific ribonuclease activity.
International Journal of Biological Macromolecules, 2015Co-Authors: Santosh Kumar Yadav, Janendra K. BatraAbstract:Abstract Restrictocin, a highly specific ribonuclease produced by Aspergillus restrictus , cleaves a single phosphodiester bond in a universally conserved stem and loop structure termed sarcin/ricin loop within the large ribosomal RNA of all organisms. In the current study, we demonstrate restrictocin to manifest anti-HIV-1 activity in two model cell systems. Using two mutants of restrictocin, we further show that the anti-HIV-1 activity of restrictocin is due to its specific ribonucleolytic activity. The study suggests that restrictocin is able to recognize region(s) within HIV-1 genome as its target. Restrictocin appears to have potential as a therapeutic antiviral agent against HIV-1.
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Localization of the catalytic activity in restrictocin molecule by deletion mutagenesis.
FEBS Journal, 2000Co-Authors: Surendra K. Nayak, Shveta, Janendra K. BatraAbstract:Restrictocin, produced by the fungus Aspergillus restrictus, is a highly specific ribonucleolytic toxin which cleaves a single phosphodiester bond between G4325 and A4326 in the 28S rRNA. It is a nonglycosylated, single-chain, basic protein of 149 amino acids. The putative catalytic site of restrictocin includes Tyr47, His49, Glu95, Arg120 and His136. To map the catalytic activity in the restrictocin molecule, and to study the role of N- and C-terminus in its activity, we have systematically deleted amino-acid residues from both the termini. Three N-terminal deletions removing 8, 15 and 30 amino acids, and three C-terminal deletions lacking 4, 6, and 11 amino acids were constructed. The deletion mutants were expressed in Escherichia coli, purified to homogeneity and functionally characterized. Removal of eight N-terminal or four C-terminal amino acids rendered restrictocin partially inactive, whereas any further deletions from either end resulted in the complete inactivation of the toxin. The study demonstrates that intact N- and C-termini are required for the optimum functional activity of restrictocin.
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Role of Individual Cysteine Residues and Disulfide Bonds in the Structure and Function of Aspergillus Ribonucleolytic Toxin Restrictocin
Biochemistry, 1999Co-Authors: Surendra K. Nayak, Dharmendar Rathore, Janendra K. BatraAbstract:Restrictocin, produced by the fungus Aspergillus restrictus, belongs to the group of ribonucleolytic toxins called ribotoxins. It specifically cleaves a single phosphodiester bond in a conserved stem and loop structure in the 28S rRNA of large ribosomal subunit and potently inhibits eukaryotic protein synthesis. Restrictocin contains 149 amino acid residues and includes four cysteines at positions 5, 75, 131, and 147. These cysteine residues are involved in the formation of two disulfide bonds, one between Cys 5 and Cys 147 and another between Cys 75 and Cys 131. In the current study, all four cysteine residues were changed to alanine individually and in different combinations by site-directed mutagenesis so as to remove one or both the disulfides. The mutants were expressed and purified from Escherichia coli. Removal of any cysteine or any one of the disulfide bonds individually did not affect the ability of the toxin to specifically cleave the 28S rRNA or to inhibit protein synthesis in vitro. However, the toxin without both disulfide bonds completely lost both ribonucleolytic and protein synthesis inhibition activities. The active mutants, containing only one disulfide bond, exhibited relatively high susceptibility to trypsin digestion. Thus, none of the four cysteine residues is directly involved in restrictocin catalysis; however, the presence of any one of the two disulfide bonds is absolutely essential and sufficient to maintain the enzymatically active conformation of restrictocin. For maintenance of the unique stability displayed by the native toxin, both disulfide bonds are required.
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A single amino acid substitution in ribonucleolytic toxin restrictocin abolishes its specific substrate recognition activity.
Biochemistry, 1997Co-Authors: Surendra K. Nayak, Janendra K. BatraAbstract:: Restrictocin is a small basic protein produced by the fungus Aspergillus restrictus. It potently inhibits protein synthesis in eukaryotic cells by specifically cleaving a single phosphodiester bond in 28S rRNA. A histidine residue at position 49 in restrictocin has been implicated in its active site. A mutant of restrictocin in which the histidine at position 49 was changed to an alanine was constructed by site-directed mutagenesis, and the protein was expressed in Escherichia coli. The mutant and the wild type proteins were found to be structurally identical. Unlike restrictocin, the restrictocin H49A mutant did not cleave the ribosomal RNA specifically at the target phosphodiester bond; instead, it extensively degraded the RNA substrate with altered specificity. The mutant exhibited a high ribonuclease activity compared to restrictocin on yeast tRNA, and poly(U) and poly(C). The mutant also poorly inhibited protein synthesis in eukaryotic cells as well as in a cell free system. We therefore propose that histidine 49 of restrictocin is not involved per se in the enzymatic activity; however, it does play a crucial role in the specific recognition of the target sequence by restrictocin.
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Cytotoxic activity of ribonucleolytic toxin restrictocin-based chimeric toxins targeted to epidermal growth factor receptor
FEBS Letters, 1997Co-Authors: Dharmendar Rathore, Janendra K. BatraAbstract:Targeted toxins represent a new approach to specific cytocidal therapy. The ribonucleolytic protein toxin restrictocin is a potent protein synthesis inhibitor produced by the fungus Aspergillus restrictus. In the present study we have constructed two restrictocin based chimeric toxins where human transforming growth factor alpha (TGFα) has been used as a ligand. TGFα is a single chain polypeptide, which binds to epidermal growth factor receptor (EGFR) and causes proliferation in a large number of cancers. The ligand has been separately fused either at the amino terminus or carboxyl terminus of restrictocin, giving rise to TGFα-restrictocin and restrictocin-TGFα respectively. The fusion proteins were overexpressed in Escherichia coli and purified from inclusion bodies by a denaturation-renaturation protocol. Both the chimeric toxins actively inhibited eukaryotic protein synthesis in a cell free in vitro translation assay system. These chimeric toxins selectively killed human epidermal growth factor receptor positive target cells in culture. Among the two proteins, restrictocin-TGFα was more active than TGFα-restrictocin on all the cell lines studied.
William R Kenealy - One of the best experts on this subject based on the ideXlab platform.
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The effect of fungal ribosome inactivating proteins upon feeding choice in C. freemani, and indications of a mutualistic relationship with A. restrictus. Environmental Mycology
Mycopathologia, 2001Co-Authors: Tristan Brandhorst, Patrick F. Dowd, William R KenealyAbstract:Carpophilus freemani beetles' feeding on the fungus Aspergillus nidulans was substantially inhibited when A. nidulans was transformed and induced to secrete the ribosome inactivating protein, restrictocin (genetic source: Aspergillus restrictus ). No inhibition of feeding was observed when A. nidulans was transformed and induced to produce an inactive form of restrictocin with a single amino-acid substitution in the active site. Similarly, there was no inhibition of feeding upon transgenic strains when the production of restrictocin was not induced. Feeding inhibition of C. freemani by restrictocin requires that the ribonuclease be active and is not due to other characteristics of the protein or the transgenic host fungus.
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The ribosome-inactivating protein restrictocin deters insect feeding on Aspergillus restrictus
Microbiology, 1996Co-Authors: Tristan T Brandhorst, Patrick F. Dowd, William R KenealyAbstract:The fungus-feeding beetle, Carpophilus freemani, consumed equal quantities of young mycelia, fewer phialides bearing mature spores and much fewer phialides bearing developing spores of Aspergillus restrictus compared to those of Aspergillus nidulans when tested in diet choice assays. The degree to which specific fungal structures were consumed was inversely related to the localization of high levels of restrictocin, a ribosome-inactivating protein, to those structures. Pure restrictocin added to the insect diet at 1000 p.p.m. killed 38.5% of C. freemani larvae and 62.5% of Spodoptera frugiperda larvae in 48 h, but did not affect C. freemani adults or Helicoverpa zea larvae over the same interval. In diet choice assays, 1000 p.p.m. of restrictocin deterred feeding by adult C. freemani and Sitophilus zeamais compared to control diets. Thus, restrictocin production and localization may have a natural defensive role against insect feeding at times critical to spore formation by A. restrictus, and may have potential as an insect control agent.
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production and localization of restrictocin in Aspergillus restrictus
Microbiology, 1992Co-Authors: Tristan T Brandhorst, William R KenealyAbstract:The production and secretion of restrictocin (a cytotoxin that cleaves ribosomal RNA) by cultures of the fungus Aspergillus restrictus was investigated. Previous studies have indicated that restrictocin production in liquid culture coincides with the appearance of differentiated cell structures. A study of the correlation between the appearance of differentiated structures and restrictocin production was conducted with A. restrictus grown on agar medium. Restrictocin was found to be associated with the cell mass of the agar-grown culture (in contrast to liquid cultures), and was first observed when aerial hyphae emerged. Restrictocin levels increased until the time of conidiation, after which they fell off sharply. No restrictocin could be found in the agar medium. The presence of restrictocin upon and within various cell structures was determined by immunofluorescent laser microscopy. This study showed that restrictocin became localized to the conidiophores and phialides during the process of conidiation. Prior to this, restrictocin was found within the hyphae in localized concentrations that may correspond to secretory vesicles.
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regulation of restrictocin production in Aspergillus restrictus
Microbiology, 1992Co-Authors: Rong Yang, William R KenealyAbstract:The production of restrictocin (a cytotoxin that specifically cleaves ribosomal RNA) by cultures of Aspergillus restrictus grown in liquid medium was investigated. The function of restrictocin, the method of its accumulation and the mode of resistance to restrictocin in A. restrictus are unknown. Previous studies have indicated that restrictocin accumulates in the medium with culture age. These observations have been extended in this study by cloning the cDNA of the res gene and using this cDNA clone to probe the onset of messenger RNA synthesis in the cells. The results of the Northern analysis were compared to the production and accumulation of restrictocin and morphological differentiation of the cells in culture. Restrictocin was found in the medium at the same time that mRNA was detected in the cells. This suggests that the leader sequence encoded by the cDNA provides an efficient secretion system for the protein. Both the protein and the mRNA were detected coincident with the formation of differentiated cell structures. These structures develop into conidiophores with one layer of sterigmata and conidia forming from the sterigmata. These results suggest that restrictocin is either involved in the process of conidiation or is coordinately regulated with differentiation leading to conidiation.
Dharmendar Rathore - One of the best experts on this subject based on the ideXlab platform.
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Role of Individual Cysteine Residues and Disulfide Bonds in the Structure and Function of Aspergillus Ribonucleolytic Toxin Restrictocin
Biochemistry, 1999Co-Authors: Surendra K. Nayak, Dharmendar Rathore, Janendra K. BatraAbstract:Restrictocin, produced by the fungus Aspergillus restrictus, belongs to the group of ribonucleolytic toxins called ribotoxins. It specifically cleaves a single phosphodiester bond in a conserved stem and loop structure in the 28S rRNA of large ribosomal subunit and potently inhibits eukaryotic protein synthesis. Restrictocin contains 149 amino acid residues and includes four cysteines at positions 5, 75, 131, and 147. These cysteine residues are involved in the formation of two disulfide bonds, one between Cys 5 and Cys 147 and another between Cys 75 and Cys 131. In the current study, all four cysteine residues were changed to alanine individually and in different combinations by site-directed mutagenesis so as to remove one or both the disulfides. The mutants were expressed and purified from Escherichia coli. Removal of any cysteine or any one of the disulfide bonds individually did not affect the ability of the toxin to specifically cleave the 28S rRNA or to inhibit protein synthesis in vitro. However, the toxin without both disulfide bonds completely lost both ribonucleolytic and protein synthesis inhibition activities. The active mutants, containing only one disulfide bond, exhibited relatively high susceptibility to trypsin digestion. Thus, none of the four cysteine residues is directly involved in restrictocin catalysis; however, the presence of any one of the two disulfide bonds is absolutely essential and sufficient to maintain the enzymatically active conformation of restrictocin. For maintenance of the unique stability displayed by the native toxin, both disulfide bonds are required.
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Cytotoxic activity of ribonucleolytic toxin restrictocin-based chimeric toxins targeted to epidermal growth factor receptor
FEBS Letters, 1997Co-Authors: Dharmendar Rathore, Janendra K. BatraAbstract:Targeted toxins represent a new approach to specific cytocidal therapy. The ribonucleolytic protein toxin restrictocin is a potent protein synthesis inhibitor produced by the fungus Aspergillus restrictus. In the present study we have constructed two restrictocin based chimeric toxins where human transforming growth factor alpha (TGFα) has been used as a ligand. TGFα is a single chain polypeptide, which binds to epidermal growth factor receptor (EGFR) and causes proliferation in a large number of cancers. The ligand has been separately fused either at the amino terminus or carboxyl terminus of restrictocin, giving rise to TGFα-restrictocin and restrictocin-TGFα respectively. The fusion proteins were overexpressed in Escherichia coli and purified from inclusion bodies by a denaturation-renaturation protocol. Both the chimeric toxins actively inhibited eukaryotic protein synthesis in a cell free in vitro translation assay system. These chimeric toxins selectively killed human epidermal growth factor receptor positive target cells in culture. Among the two proteins, restrictocin-TGFα was more active than TGFα-restrictocin on all the cell lines studied.
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Expression of ribonucleolytic toxin restrictocin in Escherichia coli: Purification and characterization
FEBS Letters, 1996Co-Authors: Dharmendar Rathore, S. K. Nayak, Janendra K. BatraAbstract:Restrictocin is a toxin produced by the fungus Aspergillus restrictus. The DNA coding for restrictocin was isolated from the host by polymerase chain reaction and cloned into a T7 promoter-based expression vector. The protein was overproduced in Escherichia coli and remained insoluble in the cell in the form of inclusion bodies. Recombinant restrictocin was purified in large amounts, by a simple denaturation-renaturation protocol involving a redox system, with typical yields of 45 mg/l of original culture, Restrictocin could be secreted into the bacterial medium using ompA, pelB and LTB signal sequences. Among the three signal sequences, ompA was found to be the most efficient in secreting the recombinant protein. The protein secreted into the extracellular medium was properly processed as evident by the amino-terminal sequencing. Recombinant restrictocin was readily purified to homogeneity from either the medium or inclusion bodies by simple chromatographic techniques and was found to be functionally as active as the native fungal protein in inhibiting the eukaryotic translation.
Julian Davies - One of the best experts on this subject based on the ideXlab platform.
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isolation and nucleotide sequence of the Aspergillus restrictus gene coding for the ribonucleolytic toxin restrictocin and its expression in Aspergillus nidulans the leader sequence protects producing strains from suicide
Nucleic Acids Research, 1991Co-Authors: Bernard Lamy, Julian DaviesAbstract:Abstract We describe the cloning and characterization of the gene coding for the ribotoxin restrictocin, from Aspergillus restrictus (gene res, EMBL accession Number X56176). This toxin is a potent inhibitor of protein synthesis in eucaryotes and is of potential interest as a component of immunotoxins. To analyze the mechanism of self-protection in the producing organism, the res gene was cloned into the vector pFB39 and introduced into Aspergillus nidulans. The secretion of active restrictocin from transformants suggests that the pro-toxin is not an active nuclease but is activated during the process of secretion.
Andrew Yarwood - One of the best experts on this subject based on the ideXlab platform.
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from colony to rodlet a six meter long portrait of the xerophilic fungus Aspergillus restrictus decorates the hall of the westerdijk institute
Fungal Biology, 2020Co-Authors: Jan Dijksterhuis, Wim Van Egmond, Andrew YarwoodAbstract:Abstract The extreme xerophilic fungus Aspergillus restrictus is used as a model for a large artwork existing out of five microscopic pictures in total measuring 80 cm by 624 cm. The artwork is printed on aluminium and located at the entrance of the Westerdijk Institute, Utrecht, The Netherlands. The first picture is made from a colony of the fungus, which has a dimension of 1 cm and the last picture shows details of ornamentation on conidia and phialides of the fungus. The first two pictures of the artwork are made using a unique method of light microscopy in which many hundreds of pictures are made at different focal depths resulting in high detail and resolution of the pictures. For three other pictures, cryo-electron scanning microscopy was used including both a conventional system for lower magnification and a field emission scanning electron microscope for high resolution micrographs. The range of magnification is, at real size, between 78 and 63,000 times. When the observer passes the artwork it acts like a virtual microscope, just by walking past it you zoom-in to the smallest possible details. This coherent increase of magnification of one fungus, with very high quality light- and electron microscopy micrographs, shows different layers of fungal organization and emergent properties. These include the occurrence of secondary outcrops of hyphae and conidiophores in a colony; the formation of a stipe on a thin aerial hyphae; the presence and formation of characteristic structures on stipes, vesicles and phialides and a continuous zone between the forming conidia and phialides.
