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Aspergillus restrictus

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Janendra K. Batra – 1st expert on this subject based on the ideXlab platform

  • Ribotoxin restrictocin manifests anti-HIV-1 activity through its specific ribonuclease activity.
    International Journal of Biological Macromolecules, 2015
    Co-Authors: Santosh Kumar Yadav, Janendra K. Batra

    Abstract:

    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.

  • Localization of the catalytic activity in restrictocin molecule by deletion mutagenesis.
    FEBS Journal, 2000
    Co-Authors: Surendra K. Nayak, Shveta, Janendra K. Batra

    Abstract:

    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.

  • Role of Individual Cysteine Residues and Disulfide Bonds in the Structure and Function of Aspergillus Ribonucleolytic Toxin Restrictocin
    Biochemistry, 1999
    Co-Authors: Surendra K. Nayak, Dharmendar Rathore, Janendra K. Batra

    Abstract:

    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.

William R Kenealy – 2nd expert on this subject based on the ideXlab platform

  • 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, 2001
    Co-Authors: Tristan Brandhorst, Patrick F. Dowd, William R Kenealy

    Abstract:

    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.

  • The ribosome-inactivating protein restrictocin deters insect feeding on Aspergillus restrictus
    Microbiology, 1996
    Co-Authors: Tristan T Brandhorst, Patrick F. Dowd, William R Kenealy

    Abstract:

    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.

  • production and localization of restrictocin in Aspergillus restrictus
    Microbiology, 1992
    Co-Authors: Tristan T Brandhorst, William R Kenealy

    Abstract:

    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.

Dharmendar Rathore – 3rd expert on this subject based on the ideXlab platform

  • Role of Individual Cysteine Residues and Disulfide Bonds in the Structure and Function of Aspergillus Ribonucleolytic Toxin Restrictocin
    Biochemistry, 1999
    Co-Authors: Surendra K. Nayak, Dharmendar Rathore, Janendra K. Batra

    Abstract:

    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.

  • Cytotoxic activity of ribonucleolytic toxin restrictocin-based chimeric toxins targeted to epidermal growth factor receptor
    FEBS Letters, 1997
    Co-Authors: Dharmendar Rathore, Janendra K. Batra

    Abstract:

    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.

  • Expression of ribonucleolytic toxin restrictocin in Escherichia coli: Purification and characterization
    FEBS Letters, 1996
    Co-Authors: Dharmendar Rathore, S. K. Nayak, Janendra K. Batra

    Abstract:

    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.