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Claude P Selitrennikoff - One of the best experts on this subject based on the ideXlab platform.

  • Cryptococcus neoformans Resistance to Echinocandins: (1,3)β-Glucan Synthase Activity Is Sensitive to Echinocandins
    Antimicrobial agents and chemotherapy, 2005
    Co-Authors: Marybeth A. Maligie, Claude P Selitrennikoff
    Abstract:

    (1,3)β-d-Glucan Synthase (EC 2.4.1.34. UDP-glucose: 1,3-β-d-Glucan 3-β-glucosyltransferase) uses UDP-glucose as substrate and catalyzes the polymerization of glucose ([1,3]-β-linkages) to form the major carbohydrate component of the fungal cell wall. We have optimized in vitro assay conditions for (1,3)β-Glucan Synthase activity from Cryptococcus neoformans. Cells lysed in 50 mM Tris, pH 7.75, containing 20% glycerol, 2 mM NaF, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM MgCl2, 0.1% protease and phosphatase inhibitor cocktails, and 60 μM GTPγS produced maximum specific activity in vitro. We tested in vitro C. neoformans (1,3)β-Glucan Synthase activity against the (1,3)β-Glucan Synthase inhibitors, caspofungin and cilofungin, and have determined that (1,3)β-Glucan Synthase activity is very sensitive (apparent Ki of 0.17 ± 0.02 μM and 22 ± 5.7 μM, respectively) to these echinocandins. Taken together with high MICs for C. neoformans (caspofungin MIC, 16 μg/ml; cilofungin MIC, 64 μg/ml), our results indicate that C. neoformans is resistant to caspofungin and cilofungin by a mechanism(s) unrelated to (1,3)β-Glucan Synthase resistance.

  • Neurospora crassa FKS Protein Binds to the (1,3)β-Glucan Synthase Substrate, UDP-Glucose
    Current microbiology, 2003
    Co-Authors: Rebecca Schimoler-o'rourke, Samantha Renault, Claude P Selitrennikoff
    Abstract:

    The essential fungal cell-wall polymer (1,3)beta-Glucan is synthesized by the enzyme (1,3)beta-Glucan Synthase. This enzyme, which is the target of the echinocandin and pneumocandin families of fungicidal antibiotics, is a complex composed of at least two proteins, Rho1p and Fks1p. Homologs of the yeast FKS1 gene have been discovered in numerous fungi, and existing evidence points to, but has not yet proved, Fks1p being the catalytic subunit of (1,3)beta-Glucan Synthase. We have purified (1,3)beta-Glucan Synthase from Neurospora crassa approximately 400-fold enrichment and labeled the substrate-binding protein by using a UDP-glucose analog, 5-azido-[beta-(32)P]-UDP-glucose. UDP-glucose-binding proteins were photo-crosslinked to the substrate analog and identified from SDS-PAGE gels by Quadrupole time-of-flight mass spectrometry by sequencing the tryptic peptides. Two plasma membrane proteins were labeled FKS and H(+)-ATPase. These results suggest that FKS appears to be the substrate-binding subunit of (1,3)beta-Glucan Synthase.

  • characterization and optimization of in vitro assay conditions for 1 3 beta Glucan Synthase activity from aspergillus fumigatus and candida albicans for enzyme inhibition screening
    The Journal of Antibiotics, 1998
    Co-Authors: Rebecca L Wood, Tamara K Miller, Amy E Wright, Peter J Mccarthy, Cathy S Taft, Shirley A Pomponi, Claude P Selitrennikoff
    Abstract:

    (1,3)Beta-D-Glucan Synthase (E.C.2.4.1.34. UDP-glucose: 1,3-beta-D-Glucan 3-beta-glucosyl transferase) catalyzes the polymerization of glucose ([1-3]-beta-linkages) using UDP-glucose as substrate. We have determined optimal in vitro conditions for the assay of (1,3)beta-Glucan Synthase activity from Aspergillus fumigatus and Candida albicans. These included lysis of cells in the following for C. albicans, 100 mM HEPES, pH 8.0, 10 microM guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), 2 mM ethylenediaminetetraacetic acid (EDTA), disodium salt, 5 mM NaF, 250 mM sucrose, and 10 mM NaH2PO4; and for A. fumigatus, 50 mM HEPES, 10mM EDTA, 750 mM sucrose, 10 mM NaH2PO4, 100 mM cellobiose and 50 microM GTPgammaS. Resulting low-speed supernatants were used as enzyme sources to determine the optimal in vitro assay conditions. We have characterized the resulting enzyme activities and tested the optimized assays with known (1,3)beta-Glucan Synthase inhibitors including cilofungin, papulacandin, aculeacin A, and echinocandin B. We have used both optimized assays to screen > 1000 extracts of marine macroorganisms and, using bioassay-guided purification, have identified (1,3)beta-Glucan Synthase inhibitors.

  • Inhibition of Neurospora crassa growth by a Glucan Synthase-1 antisense construct
    Current microbiology, 1997
    Co-Authors: S. Tentler, Cathy S Taft, J. Palas, Carol S. Enderlin, J. Campbell, T.k. Miller, R.l. Wood, Claude P Selitrennikoff
    Abstract:

    We have used the filamentous fungus, Neurospora crassa, as a model system to test the concept that antisense targeting of the cell-wall assembly enzyme, (1,3)beta-Glucan Synthase [E.C. 2.4.1.34; UDPglucose: 1,3-beta-D-Glucan 3-beta-D-glucosyltransferase], leads to a corresponding decrease in growth of the organism. Previously, our laboratory isolated a gene (Glucan Synthase-1, gs-1) that is required for (1,3)beta-Glucan Synthase activity. Wild-type cells were transformed with DNA vectors encoding various RNAs complementary to the gs-1 messenger RNA (antisense RNA) cloned downstream from an inducible promoter (quinic acid-2[qa-2p]). Stable transformants, expressing a partially inverted antisense message of gs-1 (pMYX107), exhibited dramatic reduction ingrowth compared with empty vector controls. Hyphal measurements of these transformants grown on race tubes indicated that all of the transformants showed various degrees of inhibition. Microscopic observations of transformants revealed shorter hyphal lengths when grown under conditions expressing antisense. Further characterization revealed that the specific activities of (1,3)beta-Glucan Synthase were decreased by as much as 63% relative to empty vector controls. Together, these observations suggest that antisense against (1,3)beta-Glucan Synthase led to a reduction in enzyme levels that resulted in altered cell-wall morphology and inhibition of growth. It is possible that antisense oligonucleotides against gs-1 may be useful antifungal agents.

  • Antifungal drugs : (1,3) β-Glucan Synthase inhibitors
    1995
    Co-Authors: Claude P Selitrennikoff
    Abstract:

    This text brings together recent research findings concerning (1,3) beta-Glucan Synthase and inhibitors of the enzyme. Glucan Synthase inhibitors act on Pneumocepts, which could be very important for the treatment of AIDS. In addition, a number of other unexplored cell-wall targets are presented. The book also provides an overview of a key enzyme involved in fungal growth and its inhibitors.

Yoshikazu Ohya - One of the best experts on this subject based on the ideXlab platform.

  • Homologous subunits of 1,3-beta-Glucan Synthase are important for spore wall assembly in Saccharomyces cerevisiae.
    Eukaryotic cell, 2006
    Co-Authors: Satoru Ishihara, Jean-paul Latgé, Aiko Hirata, Satoru Nogami, Anne Beauvais, Yoshikazu Ohya
    Abstract:

    During sporulation in Saccharomyces cerevisiae, the four haploid nuclei are encapsulated within multilayered spore walls. Glucan, the major constituent of the spore wall, is synthesized by 1,3-beta-Glucan Synthase, which is composed of a putative catalytic subunit encoded by FKS1 and FKS2. Although another homolog, encoded by FKS3, was identified by homology searching, its function is unknown. In this report, we show that FKS2 and FKS3 are required for spore wall assembly. The ascospores of fks2 and fks3 mutants were enveloped by an abnormal spore wall with reduced resistance to diethyl ether, elevated temperatures, and ethanol. However, deletion of the FKS1 gene did not result in a defective spore wall. The construction of fusion genes that expressed Fks1p and Fks2p under the control of the FKS2 promoter revealed that asci transformed with FKS2p-driven Fks1p and Fks2p were resistant to elevated temperatures, which suggests that the expression of FKS2 plays an important role in spore wall assembly. The expression of FKS1p-driven Fks3p during vegetative growth did not affect 1,3-beta-Glucan Synthase activity in vitro but effectively suppressed the growth defect of the temperature-sensitive fks1 mutant by stabilizing Rho1p, which is a regulatory subunit of Glucan Synthase. Based on these results, we propose that FKS2 encodes the primary 1,3-beta-Glucan Synthase in sporulation and that FKS3 is required for normal spore wall formation because it affects the upstream regulation of 1,3-beta-Glucan Synthase.

  • piperazine propanol derivative as a novel antifungal targeting 1 3 β d Glucan Synthase
    Biological & Pharmaceutical Bulletin, 2005
    Co-Authors: Yoshikazu Ohya, Osamu Kondoh, Yukiko Inagaki, Hiroshi Fukuda, Eisaku Chugai Seiyaku Kabushiki Kaiha Mizuguchi, Mikio Arisawa
    Abstract:

    1,3-β-D-Glucan Synthase, which synthesizes a main component of fungal cell wall, is one of the promising targets for antifungal agents. In order to identify novel chemical classes of 1,3-β-D-Glucan Synthase inhibitors, we screened a chemical library monitoring inhibition of the Candida albicans 1,3-β-D-Glucan Synthase activity. The piperazine propanol derivative GSI578 [(2,6-difluoro-phenyl)-carbamic acid 3-(4-benzothiazol-2-yl-piperazine-1-yl)-propyl ester] was identified as a potent inhibitor against 1,3-β-D-Glucan Synthase with an IC50 value of 0.16 μM. GSI578 exhibited in vitro antifungal activity against pathogenic fungi including C. albicans and Aspergillus fumigatus. Temperature-sensitive mutations of the FKS1 gene in the Δfks2 background of Saccharomyces cerevisiae, where FKS1 and FKS2 encode putative catalytic subunits of 1,3-β-D-Glucan Synthase, altered sensitivity to GSI578. This suggests that the antifungal activity of the piperazine propanol derivative has an effect on 1,3-β-D-Glucan Synthase inhibition. Results of our initial evaluation suggest that the piperazine propanol derivative is a novel chemical structure of the class of antifungals which inhibit fungal cell growth by inhibiting fungal 1,3-β-D-Glucan Synthase.

  • Movement of yeast 1,3-β-Glucan Synthase is essential for uniform cell wall synthesis
    Genes to cells : devoted to molecular & cellular mechanisms, 2002
    Co-Authors: Takahiko Utsugi, Aiko Hirata, Masayo Minemura, Mitsuhiro Abe, Daisuke Watanabe, Yoshikazu Ohya
    Abstract:

    Background : The cell wall has an important role in maintaining cell shape. In the budding yeast Saccharomyces cerevisiae , the major filamentous component of the cell wall responsible for its rigidity is 1,3- β Glucan and is synthesized by 1,3- β β β -Glucan Synthase (GS), localized on the plasma membrane.

  • Prenylation of Rho1p is required for activation of yeast 1,3-β-Glucan Synthase
    The Journal of biological chemistry, 1999
    Co-Authors: Shunsuke B. Inoue, Hiroshi Qadota, Mikio Arisawa, Takahide Watanabe, Yoshikazu Ohya
    Abstract:

    Abstract One of the essential protein substrates of geranylgeranyl transferase type I in the budding yeastSaccharomyces cerevisiae is a rho-type GTPase, Rho1p, which is a regulatory subunit of 1,3-β-Glucan Synthase. Previous studies have indicated that modification of Rho1p is significantly reduced in a mutant of the β subunit of geranylgeranyl transferase type I calledcal1-1. Here we present genetic and biochemical evidence showing that modification of Rho1p is required for activity of 1,3-β-Glucan Synthase. The 1,3-β-Glucan Synthase activity of thecal1-1 membrane was significantly reduced compared with that of the wild-type membrane. The impaired activity was partly due to the reduced amount of Fks1p, a putative catalytic subunit of 1,3-β-Glucan Synthase, but also partly due to reduced affinity between unmodified Rho1p and Fks1p. GlutathioneS-transferase (GST)-Rho1 proteins with or without the C-terminal motif required for the modification were purified and used to analyze the interaction. The modified form of GST-Rho1p was specifically able to restore the 1,3-β-Glucan Synthase of therho1-3 membrane. Gel overlay analysis indicated that an unmodified form of GST-Rho1p fails to interact with Fks1p. These results indicated that the geranylgeranylation of Rho1p is a prerequisite to the assembly and activation of 1,3-β-Glucan Synthasein vitro. Increased cytoplasmic levels of divalent cations such as Ca2+ restored both Rho1p modification and the 1,3-β-Glucan Synthase activity of cal1-1, suggesting that cytoplasmic levels of the divalent cations affect geranylgeranyl transferase type I activity in vivo.

  • Identification of yeast Rho1p GTPase as a regulatory subunit of 1,3-beta-Glucan Synthase.
    Science (New York N.Y.), 1996
    Co-Authors: Hiroshi Qadota, Christophe P. Python, Shunsuke B. Inoue, Mikio Arisawa, Yasuhiro Anraku, Yi Zheng, Takahide Watanabe, David E. Levin, Yoshikazu Ohya
    Abstract:

    1,3-β-D-Glucan Synthase [also known as β(1→3)Glucan Synthase] is a multi-enzyme complex that catalyzes the synthesis of 1,3-β-linked Glucan, a major structural component of the yeast cell wall. Temperature-sensitive mutants in the essential Rho-type guanosine triphosphatase (GTPase), Rho1p, displayed thermolabile Glucan Synthase activity, which was restored by the addition of recombinant Rho1p. Glucan Synthase from mutants expressing constitutively active Rho1p did not require exogenous guanosine triphosphate for activity. Rho1p copurified with β(1→3)Glucan Synthase and associated with the Fks1p subunit of this complex in vivo. Both proteins were localized predominantly at sites of cell wall remodeling. Therefore, it appears that Rho1p is a regulatory subunit of β(1→3)Glucan Synthase.

Takahide Watanabe - One of the best experts on this subject based on the ideXlab platform.

  • differential sensitivity between fks1p and fks2p against a novel β 1 3 Glucan Synthase inhibitor aerothricin1
    Journal of Biological Chemistry, 2002
    Co-Authors: Osamu Kondoh, Mikio Arisawa, Tsuyoshi Takasuka, Yuko Aoki, Takahide Watanabe
    Abstract:

    Fks1p and Fks2p are catalytic subunits of beta-1,3-Glucan Synthase, which synthesize beta-1,3-Glucan, a main component of the cell wall in Saccharomyces cerevisiae. Although Fks1p and Fks2p are highly homologous, sharing 88.1% identity, it has been shown that Fks2p is more sensitive than Fks1p to one of echinocandin derivatives, which inhibits beta-1,3-Glucan Synthase activity. Here we show a similar differential sensitivity between Fks1p and Fks2p to a novel beta-1,3-Glucan Synthase inhibitor, aerothricin3 [corrected]. To investigate the molecular mechanism of this differential sensitivity, we constructed a series of chimeric genes of FKSs and examined their sensitivity to aerothricin3 [corrected]. As a result, it was shown that a region around the fourth extracellular domain of Fks2p, containing 10 different amino acid residues from those of Fks1p, provided Fks1p aerothricin3 [corrected] sensitivity when the region was replaced with a corresponding region of Fks1p. In order to identify essential amino acid residues responsible for the sensitivity, each of the 10 non-conserved amino acids of Fks1p was substituted into the corresponding amino acid of Fks2p by site-directed mutagenesis. Surprisingly, only one amino acid substitution of Fks1p (K1336I) conferred Fks1p hypersensitivity to aerothricin3 [corrected]. On the other hand, reverse substitution of the corresponding amino acid of Fks2p (I1355K) resulted in loss of hypersensitivity to aerothricin3 [corrected]. These results suggest that the 1355th isoleucine of Fks2p plays a key role in aerothricin3 [corrected] sensitivity.

  • Prenylation of Rho1p is required for activation of yeast 1,3-β-Glucan Synthase
    The Journal of biological chemistry, 1999
    Co-Authors: Shunsuke B. Inoue, Hiroshi Qadota, Mikio Arisawa, Takahide Watanabe, Yoshikazu Ohya
    Abstract:

    Abstract One of the essential protein substrates of geranylgeranyl transferase type I in the budding yeastSaccharomyces cerevisiae is a rho-type GTPase, Rho1p, which is a regulatory subunit of 1,3-β-Glucan Synthase. Previous studies have indicated that modification of Rho1p is significantly reduced in a mutant of the β subunit of geranylgeranyl transferase type I calledcal1-1. Here we present genetic and biochemical evidence showing that modification of Rho1p is required for activity of 1,3-β-Glucan Synthase. The 1,3-β-Glucan Synthase activity of thecal1-1 membrane was significantly reduced compared with that of the wild-type membrane. The impaired activity was partly due to the reduced amount of Fks1p, a putative catalytic subunit of 1,3-β-Glucan Synthase, but also partly due to reduced affinity between unmodified Rho1p and Fks1p. GlutathioneS-transferase (GST)-Rho1 proteins with or without the C-terminal motif required for the modification were purified and used to analyze the interaction. The modified form of GST-Rho1p was specifically able to restore the 1,3-β-Glucan Synthase of therho1-3 membrane. Gel overlay analysis indicated that an unmodified form of GST-Rho1p fails to interact with Fks1p. These results indicated that the geranylgeranylation of Rho1p is a prerequisite to the assembly and activation of 1,3-β-Glucan Synthasein vitro. Increased cytoplasmic levels of divalent cations such as Ca2+ restored both Rho1p modification and the 1,3-β-Glucan Synthase activity of cal1-1, suggesting that cytoplasmic levels of the divalent cations affect geranylgeranyl transferase type I activity in vivo.

  • Identification of yeast Rho1p GTPase as a regulatory subunit of 1,3-beta-Glucan Synthase.
    Science (New York N.Y.), 1996
    Co-Authors: Hiroshi Qadota, Christophe P. Python, Shunsuke B. Inoue, Mikio Arisawa, Yasuhiro Anraku, Yi Zheng, Takahide Watanabe, David E. Levin, Yoshikazu Ohya
    Abstract:

    1,3-β-D-Glucan Synthase [also known as β(1→3)Glucan Synthase] is a multi-enzyme complex that catalyzes the synthesis of 1,3-β-linked Glucan, a major structural component of the yeast cell wall. Temperature-sensitive mutants in the essential Rho-type guanosine triphosphatase (GTPase), Rho1p, displayed thermolabile Glucan Synthase activity, which was restored by the addition of recombinant Rho1p. Glucan Synthase from mutants expressing constitutively active Rho1p did not require exogenous guanosine triphosphate for activity. Rho1p copurified with β(1→3)Glucan Synthase and associated with the Fks1p subunit of this complex in vivo. Both proteins were localized predominantly at sites of cell wall remodeling. Therefore, it appears that Rho1p is a regulatory subunit of β(1→3)Glucan Synthase.

  • Characterization and gene cloning of 1,3-beta-D-Glucan Synthase from Saccharomyces cerevisiae.
    European journal of biochemistry, 1995
    Co-Authors: Shunsuke B. Inoue, Mikio Arisawa, Yasuhiro Furuichi, Noriko Takewakt, Tsuyoshi Takasuka, Toshiyuki Mio, Miki Adachi, Yukako Fujii, Chikara Miyamoto, Takahide Watanabe
    Abstract:

    1,3-β-D-Glucan Synthase of Saccharomyces cerevisiae was solubilized and purified up to 700-fold by product entrapment. The specific activity of the partially purified enzyme was around 4 μmol glucose incorporated · min−1· mg protein−1. In SDS/PAGE, enrichment of a 200-kDa protein was clearly observed in parallel with the increase in specific activity. mAbs that could immunoprecipitate the 1,3-β-D-Glucan Synthase activity were isolated, and some of them also recognized this 200-kDa protein in the Western blot. Internal amino acid sequences of this 200-kDa protein were determined after lysyl endopeptidase digestion. With the information of these amino acid sequences, we cloned two genes, GSC1 and GSC2 (Glucan Synthase of S. cerevisiae 1 and 2), which are very similar to each other (88% at the amino acid level); hydropathy profiles of both proteins suggest that these genes encode integral membrane proteins which can be assumed to have approximately 16 transmembrane domains. Disruption of each gene was not lethal, but disruption of both genes was lethal. The 1,3-β-D-Glucan Synthase activities of membrane and partially purified enzyme of gsc1:: URA3 cells were significantly lower than those of the wild-type and gsc2::LEU2 cells.

Jean-paul Latgé - One of the best experts on this subject based on the ideXlab platform.

  • Homologous subunits of 1,3-beta-Glucan Synthase are important for spore wall assembly in Saccharomyces cerevisiae.
    Eukaryotic cell, 2006
    Co-Authors: Satoru Ishihara, Jean-paul Latgé, Aiko Hirata, Satoru Nogami, Anne Beauvais, Yoshikazu Ohya
    Abstract:

    During sporulation in Saccharomyces cerevisiae, the four haploid nuclei are encapsulated within multilayered spore walls. Glucan, the major constituent of the spore wall, is synthesized by 1,3-beta-Glucan Synthase, which is composed of a putative catalytic subunit encoded by FKS1 and FKS2. Although another homolog, encoded by FKS3, was identified by homology searching, its function is unknown. In this report, we show that FKS2 and FKS3 are required for spore wall assembly. The ascospores of fks2 and fks3 mutants were enveloped by an abnormal spore wall with reduced resistance to diethyl ether, elevated temperatures, and ethanol. However, deletion of the FKS1 gene did not result in a defective spore wall. The construction of fusion genes that expressed Fks1p and Fks2p under the control of the FKS2 promoter revealed that asci transformed with FKS2p-driven Fks1p and Fks2p were resistant to elevated temperatures, which suggests that the expression of FKS2 plays an important role in spore wall assembly. The expression of FKS1p-driven Fks3p during vegetative growth did not affect 1,3-beta-Glucan Synthase activity in vitro but effectively suppressed the growth defect of the temperature-sensitive fks1 mutant by stabilizing Rho1p, which is a regulatory subunit of Glucan Synthase. Based on these results, we propose that FKS2 encodes the primary 1,3-beta-Glucan Synthase in sporulation and that FKS3 is required for normal spore wall formation because it affects the upstream regulation of 1,3-beta-Glucan Synthase.

  • Glucan Synthase Complex of Aspergillus fumigatus
    Journal of Bacteriology, 2001
    Co-Authors: A. Beauvais, J.-m. Bruneau, M. J. Buitrago, Raymond Legrand, Jean-paul Latgé
    Abstract:

    The Glucan Synthase complex of the human pathogenic mold Aspergillus fumigatus has been investigated. The genes encoding the putative catalytic subunit Fks1p and four Rho proteins of A. fumigatus were cloned and sequenced. Sequence analysis showed that AfFks1p was a transmembrane protein very similar to other Fksp proteins in yeasts and in Aspergillus nidulans. Heterologous expression of the conserved internal hydrophilic domain of AfFks1p was achieved in Escherichia coli. Anti-Fks1p antibodies labeled the apex of the germ tube, as did aniline blue fluorochrome, which was specific for β(1–3) Glucans, showing that AfFks1p colocalized with the newly synthesized β(1–3) Glucans. AfRHO1, the most homologous gene to RHO1 of Saccharomyces cerevisiae, was studied for the first time in a filamentous fungus. AfRho proteins have GTP binding and hydrolysis consensus sequences identical to those of yeast Rho proteins and have a slightly modified geranylation site in AfRho1p and AfRho3p. Purification of the Glucan Synthase complex by product entrapment led to the enrichment of four proteins: Fks1p, Rho1p, a 100-kDa protein homologous to a membrane H+-ATPase, and a 160-kDa protein which was labeled by an anti-β(1–3) Glucan antibody and was homologous to ABC bacterial β(1–2) Glucan transporters.

  • Glucan Synthase complex of aspergillus fumigatus
    Journal of Bacteriology, 2001
    Co-Authors: A. Beauvais, J.-m. Bruneau, M. J. Buitrago, Raymond Legrand, P C Mol, Jean-paul Latgé
    Abstract:

    The Glucan Synthase complex of the human pathogenic mold Aspergillus fumigatus has been investigated. The genes encoding the putative catalytic subunit Fks1p and four Rho proteins of A. fumigatus were cloned and sequenced. Sequence analysis showed that AfFks1p was a transmembrane protein very similar to other Fksp proteins in yeasts and in Aspergillus nidulans. Heterologous expression of the conserved internal hydrophilic domain of AfFks1p was achieved in Escherichia coli. Anti-Fks1p antibodies labeled the apex of the germ tube, as did aniline blue fluorochrome, which was specific for beta(1-3) Glucans, showing that AfFks1p colocalized with the newly synthesized beta(1-3) Glucans. AfRHO1, the most homologous gene to RHO1 of Saccharomyces cerevisiae, was studied for the first time in a filamentous fungus. AfRho proteins have GTP binding and hydrolysis consensus sequences identical to those of yeast Rho proteins and have a slightly modified geranylation site in AfRho1p and AfRho3p. Purification of the Glucan Synthase complex by product entrapment led to the enrichment of four proteins: Fks1p, Rho1p, a 100-kDa protein homologous to a membrane H(+)-ATPase, and a 160-kDa protein which was labeled by an anti-beta(1-3) Glucan antibody and was homologous to ABC bacterial beta(1-2) Glucan transporters.

  • Unsaturated fatty acids are the active molecules of a Glucan-Synthase-inhibitory fraction isolated from entomophthoralean protoplasts
    Microbiology, 1995
    Co-Authors: Joanna K. Mackichan, Jean-paul Latgé, Lene Thomsen, James L. Kerwin, Anne Beauvais
    Abstract:

    Summary: A few entomophthoralean species are able to multiply in a protoplast form. The polysaccharide Synthases which synthesize the cell wall are inactivated in this form. An inhibitor of one of the key enzymes of wall synthesis, Glucan Synthase, was isolated from entomophthoralean protoplasts, using silica column chromatography and HPLC. Thin-layer and gas chromatography revealed free fatty acids in the inhibitory fractions. These fatty acids, including long-chain unsaturated fatty acids, were shown to be responsible for the inhibition of Glucan Synthase. The fatty acids were generated during incubation of a protoplast homogenate for 36 h at 37 °C and were shown to be non-competitive and non-specific inhibitors of Glucan Synthase.

  • Characterization of the 1,3-β-Glucan Synthase of Aspergillus fumigatus
    Journal of general microbiology, 1993
    Co-Authors: Anne Beauvais, R. Drake, M. Diaquin, Jean-paul Latgé
    Abstract:

    SUMMARY: 1,3-β-Glucan Synthase activity has been detected in a membrane fraction extracted from the mycelium of the filamentous fungus Aspergillus fumigatus. The enzyme was solubilized by CHAPS and stabilized by filtration on a Bio-gel P30 column. Highest activity was obtained in the early exponential phase of growth. Four factors - GTP, NaF, sucrose and EDTA - added during the extraction procedure, were essential for optimal 1,3-β-Glucan Synthase activity. The soluble enzyme preparation was photolabelled with 5-azido-[32P]UDP-glucose and 5-125IASA-UDP-glucose which bind covalently to the enzyme after UV irradiation. These UDP-glucose substrate analogues were competitive inhibitors of the enzyme with a K i of 1.42 mM and 0.3 mM for 5-azido-UDP-glucose and 5-ASA-UDP-glucose, respectively (K m for UDP-glucose = 1.9 mM). Potential UDP-glucose-binding polypeptides were identified with molecular masses of 31, 50 and 115 kDa.

A. Beauvais - One of the best experts on this subject based on the ideXlab platform.

  • Glucan Synthase Complex of Aspergillus fumigatus
    Journal of Bacteriology, 2001
    Co-Authors: A. Beauvais, J.-m. Bruneau, M. J. Buitrago, Raymond Legrand, Jean-paul Latgé
    Abstract:

    The Glucan Synthase complex of the human pathogenic mold Aspergillus fumigatus has been investigated. The genes encoding the putative catalytic subunit Fks1p and four Rho proteins of A. fumigatus were cloned and sequenced. Sequence analysis showed that AfFks1p was a transmembrane protein very similar to other Fksp proteins in yeasts and in Aspergillus nidulans. Heterologous expression of the conserved internal hydrophilic domain of AfFks1p was achieved in Escherichia coli. Anti-Fks1p antibodies labeled the apex of the germ tube, as did aniline blue fluorochrome, which was specific for β(1–3) Glucans, showing that AfFks1p colocalized with the newly synthesized β(1–3) Glucans. AfRHO1, the most homologous gene to RHO1 of Saccharomyces cerevisiae, was studied for the first time in a filamentous fungus. AfRho proteins have GTP binding and hydrolysis consensus sequences identical to those of yeast Rho proteins and have a slightly modified geranylation site in AfRho1p and AfRho3p. Purification of the Glucan Synthase complex by product entrapment led to the enrichment of four proteins: Fks1p, Rho1p, a 100-kDa protein homologous to a membrane H+-ATPase, and a 160-kDa protein which was labeled by an anti-β(1–3) Glucan antibody and was homologous to ABC bacterial β(1–2) Glucan transporters.

  • Glucan Synthase complex of aspergillus fumigatus
    Journal of Bacteriology, 2001
    Co-Authors: A. Beauvais, J.-m. Bruneau, M. J. Buitrago, Raymond Legrand, P C Mol, Jean-paul Latgé
    Abstract:

    The Glucan Synthase complex of the human pathogenic mold Aspergillus fumigatus has been investigated. The genes encoding the putative catalytic subunit Fks1p and four Rho proteins of A. fumigatus were cloned and sequenced. Sequence analysis showed that AfFks1p was a transmembrane protein very similar to other Fksp proteins in yeasts and in Aspergillus nidulans. Heterologous expression of the conserved internal hydrophilic domain of AfFks1p was achieved in Escherichia coli. Anti-Fks1p antibodies labeled the apex of the germ tube, as did aniline blue fluorochrome, which was specific for beta(1-3) Glucans, showing that AfFks1p colocalized with the newly synthesized beta(1-3) Glucans. AfRHO1, the most homologous gene to RHO1 of Saccharomyces cerevisiae, was studied for the first time in a filamentous fungus. AfRho proteins have GTP binding and hydrolysis consensus sequences identical to those of yeast Rho proteins and have a slightly modified geranylation site in AfRho1p and AfRho3p. Purification of the Glucan Synthase complex by product entrapment led to the enrichment of four proteins: Fks1p, Rho1p, a 100-kDa protein homologous to a membrane H(+)-ATPase, and a 160-kDa protein which was labeled by an anti-beta(1-3) Glucan antibody and was homologous to ABC bacterial beta(1-2) Glucan transporters.

  • Unsaturated fatty acids are the active molecules of a Glucan-Synthase-inhibitory fraction isolated from entomophthoralean protoplasts.
    Microbiology (Reading England), 1995
    Co-Authors: Joanna K. Mackichan, Lene Thomsen, J Kerwin, J P Latgé, A. Beauvais
    Abstract:

    A few entomophthoralean species are able to multiply in a protoplast form. The polysaccharide Synthases which synthesize the cell wall are inactivated in this form. An inhibitor of one of the key enzymes of wall synthesis, Glucan Synthase, was isolated from entomophthoralean protoplasts, using silica column chromatography and HPLC. Thin-layer and gas chromatography revealed free fatty acids in the inhibitory fractions. These fatty acids, including long-chain unsaturated fatty acids, were shown to be responsible for the inhibition of Glucan Synthase. The fatty acids were generated during incubation of a protoplast homogenate for 36 h at 37 degrees C and were shown to be non-competitive and non-specific inhibitors of Glucan Synthase.

  • Characterization of the 1,3=P=Glucan Synthase of Aspergillus jiumigatus
    1993
    Co-Authors: A. Beauvais, R. Drake, M. Diaquin
    Abstract:

    1,3-P-Glucan Synthase activity has been detected in a membrane fraction extracted from the mycelium of the filamentous fungus AspergiZZus fumigatus. The enzyme was solubilized by CHAPS and stabilized by filtration on a Bio-gel P30 column. Highest activity was obtained in the early exponential phase of growth. Four factors - GTP, NaF, sucrose and EDTA - added during the extraction procedure, were essential for optimal 1,3-P-Glucan Synthase activity. The soluble enzyme preparation was photolabelled with 5-azido-(32P]UDP-glucose and 5-12'IASA-UDPglucose which bind covalently to the enzyme after UV irradiation. These UDP-glucose substrate analogues were competitive inhibitors of the enzyme with a Ki of 1-42 mM and 0.3 mM for 5-azido-UDP-glucose and 5-ASA-UDPglucose, respectively (K, for UDP-glucose = 1.9 mM). Potential UDP-glucose-binding polypeptides were identified with molecular masses of 31, 50 and 115 kDa.