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Query: UNIPROT:P06889 (Mol)
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In Neurospora crassa, expression of the laccase gene is induced by treatment with the protein synthesis inhibitor cycloheximide (CHX). This expression is mediated by CPC1, which acts as a general transcriptional activator when mycelia are treated with CHX or starved for any one of the amino acids. A laccase-derepressed mutant, lah-1, shows pleiotropic deficiencies in growth, hyphal morphology, CHX sensitivity, and production of protoperithecia. Moreover, in the lah-1 mutant, transcript levels of CHX-inducible genes, including lacc, tub-2, tef-1, and amino acid biosynthetic genes such as cpc-1, trp-3, and arg-12, are increased without exposure to CHX. All of the defects exhibited in the lah-1 mutant are suppressed by a mutation in the cpc-1 locus. These findings suggest that the cpc-1 mutation is epistatic to the lah-1 mutation and that the pleiotropic defects in the lah-1 mutant are attributable to constitutive expression of CPCI. These conclusions are supported by a developmental Northern blot analysis of the CHX-inducible genes. Based on these results, the lah-1 gene product appears to regulate expression of the cpc-1 gene negatively. Expression of the CHX-inducible genes was induced by CHX treatment in the lah-1 cpc-1 mutant, as well as in the cpc-1 mutant. This observation indicates that LAH1 is not a component of CHX-responsive pathway itself.
Mol Gen Genet 1998 Jun
PMID:Pleiotropic deficiencies of the laccase-derepressed mutant lah-1 are caused by constitutively increased expression of the cross-pathway control gene cpc-1 in Neurospora crassa. 967 Oct 30

Four closely related cDNA clones encoding laccase isoenzymes from xylem tissues of yellow-poplar (Ltlacc2.1-4) were identified and sequenced. The inferred yellow-poplar laccase gene products were highly related to one another (79-91% at the amino acid level) and showed significant similarity to other blue copper oxidases, especially with respect to the copper-binding domains. The encoded proteins had N-terminal signal sequences and 17-19 potential N-linked glycosylation sites. The mature proteins were predicted to have molecular masses of ca. 61 kDa (unglycosylated) and high isoelectric points (pI 9.3-9.5). The canonical copper ligands were conserved, with the exception of a Leu residue associated with the axial position of the Type-1 cupric ion. The residue at this position has been proposed to influence the redox potential of Type-1 cupric ions. Northern blot analysis revealed that the yellow-poplar laccase genes are differentially expressed in xylem tissues. The genes were verified as encoding active laccases by heterologous expression in tobacco cells and demonstration of laccase activity in extracts from transformed tobacco cell lines.
Plant Mol Biol 1999 May
PMID:Characterization and heterologous expression of laccase cDNAs from xylem tissues of yellow-poplar (Liriodendron tulipifera). 1039 42

The human pathogenic fungus Cryptococcus neoformans secretes a phospholipase enzyme that demonstrates phospholipase B (PLB), lysophospholipase hydrolase and lysophospholipase transacylase activities. This enzyme has been postulated to be a cryptococcal virulence factor. We cloned a phospholipase-encoding gene (PLB1) from C. neoformans and constructed plb1 mutants using targeted gene disruption. All three enzyme activities were markedly reduced in the mutants compared with the wild-type parent. The plb1 strains did not have any defects in the known cryptococcal virulence phenotypes of growth at 37 degrees C, capsule formation, laccase activity and urease activity. The plb1 strains were reconstituted using the wild-type locus and this resulted in restoration of all extracellular PLB activities. In vivo testing demonstrated that the plb1 strain was significantly less virulent than the control strains in both the mouse inhalational model and the rabbit meningitis model. We also found that the plb1 strain exhibited a growth defect in a macrophage-like cell line. These data demonstrate that secretory phospholipase is a virulence factor for C. neoformans.
Mol Microbiol 2001 Jan
PMID:Extracellular phospholipase activity is a virulence factor for Cryptococcus neoformans. 1112 98

Double-stranded RNAs (dsRNAs) are widespread in plant pathogenic fungi, but their functions in fungal hosts remain mostly unclear, with a few exceptions. We analyzed dsRNAs from Nectria radicicola, the causal fungus of ginseng root rot. Four distinct sizes of dsRNAs, 6.0, 5.0, 2.5, and 1.5 kbp, were detected in 24 out of the 81 strains tested. Curing tests of individual dsRNAs suggested that the presence of 6.0-kbp dsRNA was associated with high levels of virulence, sporulation, laccase activity, and pigmentation in this fungus. The 6.0-kbp dsRNA-cured strains completely lost virulence-related phenotypes. This 6.0-kbp dsRNA was reintroduced by hyphal anastomosis to a dsRNA-cured strain marked with hygromycin resistance, which resulted in the restoration of virulence-related phenotypes. These results strongly suggest that 6.0-kbp dsRNA up regulates fungal virulence in N. radicicola. Sequencing of several cDNA clones derived from 6.0-kbp dsRNA revealed the presence of a RNA-dependent RNA polymerase (RDRP) gene. Phylogenetic analysis showed that this gene is closely related to those of plant cryptic viruses. Biochemical analyses suggested that the 6.0-kbp dsRNA may regulate fungal virulence through signal-transduction pathways involving cyclic AMP-dependent protein kinase and protein kinase C.
Mol Plant Microbe Interact 2001 Apr
PMID:A viral double-stranded RNA up regulates the fungal virulence of Nectria radicicola. 1131 Jul 37

Acidification of vesicular compartments plays an important role in a number of cellular transport processes, including protein secretion, metal cofactor insertion, glycosylation and pH stability. In the present study, we identify and characterize a component of the vesicular proton pump, Vph1p, to determine its role in the virulence of the AIDS-related fungal pathogen Cryptococcus neoformans. Insertional mutagenesis and plasmid rescue were used to identify the VPH1 gene by screening for mutants defective in laccase activity. Disruption of VPH1 resulted in defects in three virulence factors (capsule production, laccase and urease expression), as well as a growth defect at 37 degrees C, but only a small growth reduction at 30 degrees C. These effects were duplicated by the vacuolar (H+)-ATPase inhibitor bafilomycin A1. Furthermore, the vph1 insertional mutant was also avirulent in a mouse meningo-encephalitis model. Complementation of the insertional mutant with wild-type VPH1 resulted in a recovery of virulence factor expression, normal growth at 37 degrees C and restoration of full virulence. These studies establish the importance of the VPH1 gene and vesicular acidification in the virulence of C. neoformans.
Mol Microbiol 2001 Nov
PMID:Multiple virulence factors of Cryptococcus neoformans are dependent on VPH1. 1173 51

The grapevine (Vitis) secondary metabolite resveratrol is considered a phytoalexin, which protects the plant from Botrytis cinerea infection. Laccase activity displayed by the fungus is assumed to detoxify resveratrol and to facilitate colonization of grape. We initiated a functional molecular genetic analysis of B. cinerea laccases by characterizing laccase genes and evaluating the phenotype of targeted gene replacement mutants. Two different laccase genes from B. cinerea were characterized, Bclcc1 and Bclcc2. Only Bclcc2 was strongly expressed in liquid cultures in the presence of either resveratrol or tannins. This suggested that Bclcc2, but not Bclcc1, plays an active role in the oxidation of both resveratrol and tannins. Gene replacement mutants in the Bclcc1 and Bclcc2 gene were made to perform a functional analysis. Only Bclcc2 replacement mutants were incapable of converting both resveratrol and tannins. When grown on resveratrol, both the wild type and the Bclcc1 replacement mutant showed inhibited growth, whereas Bclcc2 replacement mutants were unaffected. Thus, contrary to the current theory, BcLCC2 does not detoxify resveratrol but, rather, converts it into compounds that are more toxic for the fungus itself. The Bclcc2 gene was expressed during infection of B. cinerea on a resveratrol-producing host plant, but Bclcc2 replacement mutants were as virulent as the wild-type strain on various hosts. The activation of a plant secondary metabolite by a pathogen introduces a new dimension to plant-pathogen interactions and the phytoalexin concept.
Mol Microbiol 2002 Feb
PMID:Resveratrol acts as a natural profungicide and induces self-intoxication by a specific laccase. 1192 39

Low temperature electron paramagnetic resonance (EPR) spectroscopy with frequencies between 95 and 345 GHz and magnetic fields up to 12 T have been used to study radicals and metal sites in proteins and small inorganic model complexes. We have studied radicals, Fe, Cu and Mn containing proteins. For S = 1/2 systems, the high frequency method can resolve the g-value anisotropy. It was used in mouse ribonucleotide reductase (RNR) to show the presence of a hydrogen bond to the tyrosyl radical oxygen. At 285 GHz the type 2 Cu(II) signal in the complex enzyme laccase is clearly resolved from the Hg(II) containing laccase peroxide adduct. For simple metal sites, the systems over S = 1/2 can be described by the spin Hamiltonian: H(S) = BgS + D[Sz2 - S(S + 1)/3 + E/D (Sx2 - Sy2)]. From the high frequency EPR the D-value can be determined directly by, (I) shifts of g(eff) for half-integer spin systems with large D-values as observed at 345 GHz on an Fe(II)-NO-EDTA complex, which is best described as S = 3/2 system with D = 11.5 cm(-1), E = 0.1 cm(-1) and gx = gy = gz = 2.0; (II) measuring the outermost signal, for systems with small D values, distant of (2S - 1) x absolute value(D) from the center of the spectrum as observed in S= 5/2 Fe(III)-EDTA. In Mn(II) substituted mouse RNR R2 protein the weakly interacting Mn(II) at X-band could be observed as decoupled Mn(II) at 285 GHz.
Spectrochim Acta A Mol Biomol Spectrosc 2002 Apr
PMID:The use of high field/frequency EPR in studies of radical and metal sites in proteins and small inorganic models. 1199 59

The crystal structures of the Met148Leu and Ser86Asp mutants of rusticyanin are presented at 1.82 and 1.65 A resolution, respectively. Both of these structures have two molecules in the asymmetric unit compared to the one present in the crystal form of the native protein. This provides an opportunity to investigate intramolecular electron transfer pathways in rusticyanin. The redox potential of the Met148Leu mutant ( approximately 800 mV) is elevated compared to that of the native protein ( approximately 670 mV at pH 3.2) while that of the Ser86Asp mutant ( approximately 623 mV at pH 3.2) is decreased. The effect of the Ser86Asp mutation on the hydrogen bonding near the type 1 Cu site is discussed and hence its role in determining acid stability is examined. The type 1 Cu site of Met148Leu mimics the structural and biochemical characteristics of those found in domain II of ceruloplasmin and fungal laccase. Moreover, the native rusticyanin's cupredoxin core and the type 1 Cu site closely resemble those found in ascorbate oxidase and nitrite reductase. Structure based phylogenetic trees have been re-examined in view of the additional structural data on rusticyanin and fungal laccase. We confirm that rusticyanin is in the same class as nitrite reductase domain 2, laccase domain 3 and ceruloplasmin domains 2, 4 and 6.
J Mol Biol 2002 Jul 05
PMID:Crystal structures of the Met148Leu and Ser86Asp mutants of rusticyanin from Thiobacillus ferrooxidans: insights into the structural relationship with the cupredoxins and the multi copper proteins. 1207 84

Previous studies have shown that a Deltavph1 Cryptococcus neoformans mutant defective in vesicular acidification lacked several important virulence factors including a copper-containing laccase and was avirulent in a mouse model. In the present studies, we characterized laccase transcription and protein production to obtain insights into the mechanism of the vph1 mutation in this pathogen. Although transcription and protein expression were somewhat reduced, laccase protein was found to be successfully translated and correctly targeted to the cell wall in the Deltavph1 mutant as shown by Western blot and immuno-electron microscopy, despite a complete lack of laccase activity. Laccase activity was substantially restored in metabolically active Deltavph1 cells at 30 degrees C by addition of 100 micro M copper sulphate. This restoration by copper was found to occur through both transcriptional and post-translational mechanisms. Laccase transcriptional induction by copper was found to be dependent on enhancer region II within the 5'-untranslated region of CNLAC1. Copper was also found to restore partial activity to Deltavph1 cells at 0 degrees C, suggesting that cell wall laccase was expressed in the mutant as an apo-enzyme. Apo-laccase restoration by copper was found to be facilitated by an acidic environment, consistent with a role for the vacuolar (H+)-ATPase proton pump in copper assembly of laccase in C. neoformans.
Mol Microbiol 2003 Feb
PMID:Copper-mediated reversal of defective laccase in a Deltavph1 avirulent mutant of Cryptococcus neoformans. 1258 55

The pathogenic yeast Cryptococcus neoformans (Cn) var. gattii causes meningoencephalitis in healthy individuals, unlike the better known Cn varieties grubii and neoformans, which are common in immunocompromised individuals. The virulence determinants and mechanisms of host predilection are poorly defined for var. gattii. The present study focused on the characterization of a Cu,Zn superoxide dismutase (SOD1) gene knock-out mutant constructed by developing a DNA transformation system. The sod1 mutant was highly sensitive to the redox cycling agent menadione, and showed fragmentation of the large vacuole in the cytoplasm, but no other defects were seen in growth, capsule synthesis, mating, sporulation, stationary phase survival or auxotrophies for sulphur-containing amino acids. The sod1 mutant was markedly attenuated in virulence in a mouse model, and it was significantly susceptible to in vitro killing by human neutrophils (PMNs). The deletion of SOD1 also resulted in defects in the expression of a number of virulence factors, i.e. laccase, urease and phospholipase. Complementation of the sod1 mutant with SOD1 resulted in recovery of virulence factor expression and menadione resistance, and in restoration of virulence. Overall, these results suggest that the antioxidant function of Cu,Zn SOD is critical for the pathogenesis of the fungus, but is dispensable in its saprobic life. This report constitutes the first instance in which superoxide dismutase has been directly implicated in the virulence of a fungal pathogen.
Mol Microbiol 2003 Mar
PMID:Characterization of Cu,Zn superoxide dismutase (SOD1) gene knock-out mutant of Cryptococcus neoformans var. gattii: role in biology and virulence. 1262 21


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