ABSTRACT
The transgalactosylase activity of ß-galactosidase produces galacto-oligosaccharides (GOSs) with prebiotic effects similar to those of major oligosaccharides in human milk. ß-Galactosidases from Bacillus circulans ATCC 31382 are important enzymes in industrial-scale GOS production. Here, we show the high GOS yield of ß-galactosidase II from B. circulans (ß-Gal-II, Lactazyme-B), compared to other commercial enzymes. We also determine the crystal structure of the five conserved domains of ß-Gal-II in an apo-form and complexed with galactose and an acceptor sugar, showing the heterogeneous mode of transgalactosylation by the enzyme. Truncation studies of the five conserved domains reveal that all five domains are essential for enzyme catalysis, while some truncated constructs were still expressed as soluble proteins. Structural comparison of ß-Gal-II with other ß-galactosidase homologues suggests that the GOS linkage preference of the enzyme might be quite different from other enzymes. The structural information on ß-Gal-II might provide molecular insights into the transgalactosylation process of the ß-galactosidases in GOS production.
Subject(s)
Lactose , Oligosaccharides , Bacillus/chemistry , Bacillus/enzymology , Galactose , Models, Structural , beta-Galactosidase/geneticsABSTRACT
Multiple transcriptional regulators play important roles in the coordination of developmental processes, including asexual and sexual development, and secondary metabolism in the filamentous fungus Aspergillus nidulans. In the present study, we characterized a novel putative C2H2-type transcription factor (TF), RocA, in relation to development and secondary metabolism. Deletion of rocA increased conidiation and caused defective sexual development. In contrast, the overexpression of rocA exerted opposite effects on both phenotypes. Additionally, nullifying rocA resulted in enhanced brlA expression and reduced nsdC expression, whereas its overexpression exerted the opposite effects. These results suggest that RocA functions as a negative regulator of asexual development by repressing the expression of brlA encoding a key asexual development activator, but as a positive regulator of sexual development by enhancing the expression of nsdC encoding a pivotal sexual development activator. Deletion of rocA increased the production of sterigmatocystin (ST), as well as the expression of its biosynthetic genes, aflR and stcU. Additionally, the expression of the biosynthetic genes for penicillin (PN), ipnA and acvA, and for terrequinone (TQ), tdiB and tdiE, was increased by rocA deletion. Thus, it appears that RocA functions as a negative transcriptional modulator of the secondary metabolic genes involved in ST, PN, and TQ biosynthesis. Taken together, we propose that RocA is a novel transcriptional regulator that may act either positively or negatively at multiple target genes necessary for asexual and sexual development and secondary metabolism.
Subject(s)
Aspergillus nidulans/genetics , Aspergillus nidulans/metabolism , Gene Expression Regulation, Fungal/genetics , Secondary Metabolism/genetics , Trans-Activators/genetics , Fungal Proteins/genetics , Indoles/metabolism , Penicillins/biosynthesis , Secondary Metabolism/physiology , Sterigmatocystin/biosynthesis , Transcription, Genetic/geneticsABSTRACT
The yeast Saccharomyces cerevisiae has two isoforms of NADP+-dependent glutamate dehydrogenase (Gdh1 and Gdh3) that catalyze the synthesis of glutamate from α-ketoglutarate and NH4+. In the present study, we confirmed that Gdh3, but not Gdh1, mainly contributes to the oxidative stress resistance of stationary-phase cells and found evidence suggesting that the insignificance of Gdh1 to stress resistance is possibly resulted from conditional and reversible aggregation of Gdh1 into punctuate foci initiated in parallel with post-diauxic growth. Altered localization to the mitochondria or peroxisomes prevented Gdh1, which was originally localized in the cytoplasm, from stationary phase-specific aggregation, suggesting that some cytosolic factors are involved in the process of Gdh1 aggregation. Glucose starvation triggered the transition of the soluble form of Gdh1 into the insoluble aggregate form, which could be redissolved by replenishing glucose, without any requirement for protein synthesis. Mutational analysis showed that the N-terminal proximal region of Gdh1 (NTP1, aa 21-26, TLFEQH) is essential for glucose starvation-induced aggregation. We also found that the substitution of NTP1 with the corresponding region of Gdh3 (NTP3) significantly increased the contribution of the mutant Gdh1 to the stress resistance of stationary-phase cells. Thus, this suggests that NTP1 is responsible for the negligible role of Gdh1 in maintaining the oxidative stress resistance of stationary-phase cells and the stationary phase-specific stresssensitive phenotype of the mutants lacking Gdh3.
Subject(s)
Glutamate Dehydrogenase (NADP+)/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Gene Expression Regulation, Fungal , Glucose/metabolism , Glutamate Dehydrogenase (NADP+)/genetics , NADP/metabolism , Oxidative Stress , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Recently there has been a growing interest in three-dimensional (3D) cell culture systems for drug discovery and development. These 3D culture systems better represent the in vivo cellular environment compared to two-dimensional (2D) cell culture, thereby providing more physiologically reliable information on drug screening and testing. Here we present the quantitative profiling of a drug-induced proteome in 2D- and 3D-cultured colorectal cancer SW480 cells using 2D nanoflow liquid chromatography-tandem mass spectrometry (2D-nLC-MS/MS) integrated with isobaric tags for relative and absolute quantitation (iTRAQ). We identified a total of 4854 shared proteins between 2D- and 3D-cultured SW480 cells and 136/247 differentially expressed proteins (up/down-regulated in 3D compared to 2D). These up/down-regulated proteins were mainly involved in energy metabolism, cell growth, and cell-cell interactions. We also investigated the XAV939 (tankyrase inhibitor)-induced proteome to reveal factors involved in the 3D culture-selective growth inhibitory effect of XAV939 on SW480 cells. We identified novel XAV939-induced proteins, including gelsolin (a possible tumor suppressor) and lactate dehydrogenase A (a key enzyme of glycolysis), which were differentially expressed between 2D- and 3D-cultured SW480 cells. These results provide a promising informative protein dataset to determine the effect of XAV939 on the expression levels of proteins involved in SW480 cell growth.
Subject(s)
Cell Culture Techniques/methods , Colorectal Neoplasms/metabolism , Heterocyclic Compounds, 3-Ring/pharmacology , Proteomics/methods , Cell Communication/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Gelsolin/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Humans , L-Lactate Dehydrogenase/metabolism , Proteome/drug effects , Spheroids, CellularABSTRACT
The filamentous fungus Aspergillus nidulans primarily reproduces by forming asexual spores called conidia and produces the mycotoxin sterigmatocystin (ST), the penultimate precursor of aflatoxins. It has been known that asexual development and ST production are tightly co-regulated by various regulatory inputs. Here, we report that the novel regulator AslA with a C2H2 domain oppositely regulates development and ST biosynthesis. Nullifying aslA resulted in defective conidiation and reduced expression of brlA encoding a key activator of asexual development, which indicates that AslA functions as an upstream activator of brlA expression. aslA deletion additionally caused enhanced ST production and expression of aflR encoding a transcriptional activator for ST biosynthetic genes, suggesting that AslA functions as an upstream negative regulator of aflR. Cellular and molecular studies showed that AslA has a trans-activation domain and is localized in the nuclei of vegetative and developing cells but not in spores, indicating that AslA is likely a transcription factor. Introduction of the aslA homologs from distantly-related aspergilli complemented the defects caused by aslA null mutation in A. nidulans, implying a functional conservancy of AslA. We propose that AslA is a novel regulator that may act at the split control point of the developmental and metabolic pathways.
ABSTRACT
In fungi and plants, vacuoles function as a storage and sequestration vessel for a wide variety of ions and are responsible for cytosolic ion homeostasis and responses to ionic shock. In the filamentous fungus Aspergillus nidulans, however, little is known about the molecular genetic mechanisms of vacuolar biogenesis and function. In the present study, we analyzed the function of the aslA gene (AN5583) encoding a novel C2H2-type zinc finger transcription factor (TF) in relation to K(+) stress resistance, vacuolar morphology, and vacuolar transporters. The mutant lacking aslA showed increased mycelial growth and decreased branching at high K(+) concentrations. Deletion of aslA also caused elevated K(+) stress-inducible expression of the genes, nhxA (AN2288), vnxA (AN6986), and vcxA (AN0471), encoding putative endosomal and vacuolar cation/H(+) exchangers, as well as cpyA and vpsA genes encoding the proteins involved in vacuolar biogenesis. Interestingly, vacuolar fragmentation induced by K(+) stress was alleviated by aslA deletion, resulting in persistence of unfragmented vacuoles. In the presence of bafilomycin, an inhibitor of vacuolar H(+)-ATPase, the mutant phenotype was suppressed in terms of growth rates and vacuolar morphology. These results together suggest that the C2H2-type zinc finger TF AslA attenuates the K(+) stress-inducible expression of the genes encoding the ion pumps involved in vacuolar sequestration of K(+) ions powered by vacuolar H(+)-ATPase, as well as the proteins that function in vacuolar biogenesis.
Subject(s)
Aspergillus nidulans/genetics , Aspergillus nidulans/physiology , Fungal Proteins/genetics , Gene Expression Regulation, Fungal , Potassium/pharmacology , Transcription Factors/genetics , Vacuoles/physiology , Zinc Fingers , Amino Acid Sequence , Enzyme Inhibitors , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Macrolides/pharmacology , Molecular Sequence Data , Mutation , Phenotype , Potassium/metabolism , Sequence Alignment , Sequence Deletion , Stress, Physiological , Transcription Factors/chemistry , Transcription Factors/metabolism , Vacuoles/ultrastructureABSTRACT
Tacrolimus (FK506) is an important macrocyclic polyketide showing antifungal and immunosuppressive activities, as well as neuroregenerative properties. Tacrolimus biosynthetic machinery should incorporate the shikimate-derived 4,5-dihydroxycyclohex-1-enecarboxylic acid (DHCHC) as a biosynthetic starter unit into the biosynthetic line of tacrolimus. fkbO is a homologue of rapK encoding chorismatase related to the biosynthesis of starter unit DHCHC from chorismate in the rapamycin biosynthetic gene cluster. FkbO and RapK are good targets for mutational biosynthesis to produce novel analogues of tacrolimus, ascomycin, and rapamycin, which could be important drugs for clinical application in the treatment of cancer and immune and neurodegenerative diseases. To make novel tacrolimus analogues, we prepared an fkbO in-frame deletion mutant, Streptomyces sp. GT110507, from a tacrolimus high producer. We scrutinized the cyclic carboxylic acids that were possibly incorporated instead of DHCHC by precursor-directed mutasynthesis using Streptomyces sp. GT110507 to lead tacrolimus analogues. Among them, trans-4-hydroxycyclohexanecarboxylic acid and 3-hydroxybenzoic acid were successfully incorporated into the tacrolimus backbone, which led to the production of 31-desmethoxytacrolimus and TC-225, respectively. Especially, adding of trans-4-hydroxycyclohexanecarboxylic acid produced a high amount (55 mg/L) of 31-desmethoxytacrolimus. Interestingly, in the rapK mutant, it has been reported that the incorporation of cyclohexanecarboxylic acid (CHC) led to 39-desmethoxy rapamycin. However, in Streptomyces sp. GT110507, CHC is not successfully incorporated. This discrepancy should reflect the differences in the DHCHC biosynthesis mechanism and/or substrate specificity of starter unit loading machineries (FkbP and RapP) of tacrolimus and rapamycin.
Subject(s)
Biosynthetic Pathways/genetics , Metabolic Engineering/methods , Mutation , Sequence Deletion , Streptomyces/genetics , Streptomyces/metabolism , Tacrolimus/metabolism , Tacrolimus/analogs & derivativesABSTRACT
The distribution characteristics of Enterococcus spp., which are indicators of fecal pollution, were investigated at 33 sites within the 3 major water systems of Korea. Enterococci were detected at concentrations ranging from 1 to 37 CFU/100mL in 41 of 132 samples (31.1%) from the 3 major water systems. The overall average detected concentration was 1.2 CFU/100mL, while the average concentration for all detection sites was 5.3 CFU/100mL. After optimized multiplex polymerase chain reaction (PCR) was performed with newly developed VanA, VanB, VanC-1, and VanC-2/3 primers, concentrations of vancomycin-resistant Enterococcus spp. (VRE) ranging from 1 to 23 CFU/100mL were detected in 17 of 132 samples (12.9%). Of 216 individual enterococcal colonies, 64 (29.6%) displayed the VanC genotype. The results of a susceptibility test to vancomycin showed that the range of the minimal inhibitory concentration (MIC), an indicator of bacterial resistance, was 4 to 24µg/mL, with the average MIC at 9.2±4.5µg/mL. Of the bacterial isolates, 1 colony with the VanC-1 genotype was identified as E. gallinarum by 16S rDNA sequencing, whereas the other 63 colonies had the VanC-2/3 genotype and were identified as E. casseliflavus. Although these results imply that the major head bays of Korea are not contaminated with the highly vancomycin-resistant VanA- or VanB-type VREs, the misuse of antibiotics should be prohibited to minimize the presence of VREs and to maintain a safe water supply for protecting the health of the general population. Based on the study results, we also recommend the implementation of a continuous, broad-spectrum inspection program for Enterococcus spp. and VRE contamination in the major head bays. Furthermore, the multiplex PCR method described in this study can be used effectively for this purpose.
Subject(s)
Enterococcus/genetics , Environmental Monitoring/methods , Multiplex Polymerase Chain Reaction , Vancomycin Resistance/genetics , Bacterial Load , Bacterial Proteins/genetics , DNA, Bacterial/genetics , Enterococcus/isolation & purification , Genotype , Republic of Korea , Rivers/microbiology , Water Pollutants/isolation & purificationABSTRACT
Glutamate metabolism is linked to a number of fundamental metabolic pathways such as amino acid metabolism, the TCA cycle, and glutathione (GSH) synthesis. In the yeast Saccharomyces cerevisiae, glutamate is synthesized from α-ketoglutarate by two NADP(+)-dependent glutamate dehydrogenases (NADP-GDH) encoded by GDH1 and GDH3. Here, we report the relationship between the function of the NADP-GDH and stress-induced apoptosis. Gdh3-null cells showed accelerated chronological aging and hypersusceptibility to thermal and oxidative stress during stationary phase. Upon exposure to oxidative stress, Gdh3-null strains displayed a rapid loss in viability associated with typical apoptotic hallmarks, i.e. reactive oxygen species accumulation, nuclear fragmentation, DNA breakage, and phosphatidylserine translocation. In addition, Gdh3-null cells, but not Gdh1-null cells, had a higher tendency toward GSH depletion and subsequent reactive oxygen species accumulation than did WT cells. GSH depletion was rescued by exogenous GSH or glutamate. The hypersusceptibility of stationary phase Gdh3-null cells to stress-induced apoptosis was suppressed by deletion of GDH2. Promoter swapping and site-directed mutagenesis of GDH1 and GDH3 indicated that the necessity of GDH3 for the resistance to stress-induced apoptosis and chronological aging is due to the stationary phase-specific expression of GDH3 and concurrent degradation of Gdh1 in which the Lys-426 residue plays an essential role.
Subject(s)
Apoptosis , Glutamate Dehydrogenase (NADP+)/metabolism , Oxidative Stress , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/growth & development , Gene Expression Regulation, Fungal , Glutamate Dehydrogenase (NADP+)/genetics , Glutamic Acid/metabolism , Mutagenesis, Site-Directed , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Gaf1 is the first GATA family zinc-finger transcription factor identified in Schizosaccharomyces pombe. Here, we report that Gaf1 functions as a negatively acting transcription factor of ste11(+), delaying the entrance of cells exposed to transient nitrogen starvation into the meiotic cycle. gaf1Δ strains exhibited accelerated G(1)-arrest upon nitrogen starvation. Moreover, gaf1Δ mutation caused increased mating and sporulation frequency under both nitrogen-starved and unstarved conditions, while overexpression of gaf1(+) led to a significant impairment of sporulation. By microarray analysis, we found that approximately 63% (116 genes) of the 183 genes up-regulated in unstarved gaf1Δ cells were nitrogen starvation-responsive genes, and furthermore that 25 genes among the genes up-regulated by gaf1Δ mutation are Ste11 targets (e.g., gpa1(+), ste4(+), spk1(+), ste11(+), and mei2(+)). The phenotype caused by gaf1Δ mutation was masked by ste11Δ mutation, indicating that ste11(+) is epistatic to gaf1(+) with respect to sporulation efficiency, and accordingly that gaf1(+) functions upstream of ste11(+) in the signaling pathway governing sexual development. gaf1Δ strains showed accelerated ste11(+) expression under nitrogen starvation and increased ste11(+) expression even under normal conditions. Electrophoretic mobility shift assay analysis demonstrated that Gaf1 specifically binds to the canonical GATA motif (5'-HGATAR-3') spanning from -371 to -366 in ste11(+) promoter. Consequently, Gaf1 provides the prime example for negative regulation of ste11(+) transcription through direct binding to a cis-acting motif of its promoter.
Subject(s)
GATA Transcription Factors/metabolism , Gene Expression Regulation, Fungal , Nitrogen/metabolism , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Trans-Activators/metabolism , Transcription Factors/genetics , Base Sequence , Binding Sites/genetics , Down-Regulation , Epistasis, Genetic , G1 Phase Cell Cycle Checkpoints/genetics , Gene Deletion , Gene Expression Profiling , Mutation , Nucleotide Motifs , Pheromones/genetics , Promoter Regions, Genetic , Schizosaccharomyces/physiology , Spores, Fungal/genetics , Trans-Activators/geneticsABSTRACT
In Saccharomyces cerevisiae, the accepted theory is that due to TCA cycle dysfunction, the Δcit1 mutant lacking the mitochondrial enzyme citrate synthase (Cit1) cannot grow on acetate, regardless of the presence of the peroxisomal isoenzyme (Cit2). In this study, we re-evaluated the roles of Cit1 and Cit2 in acetate utilization and examined the pathway of acetate metabolism by analysing mutants defective in TCA or glyoxylate cycle enzymes. Although Δcit1 cells showed significantly reduced growth on rich acetate medium (YPA), they exhibited growth similar to Δcit2 and the wild-type cells on minimal acetate medium (YNBA). Impaired acetate utilization by Δcit1Δcit2 cells on YNBA was restored by ectopic expression of either Cit2 or its cytoplasmically localized variants. Deletion of any of the genes for the enzymes solely involved in the TCA cycle (IDH1, KGD1 and LSC1), except for SDH1, caused little defect in acetate utilization on YNBA but resulted in significant growth impairment on YPA. In contrast, cells lacking any of the genes involved in the glyoxylate cycle (ACO1, FUM1, MLS1, ICL1 and MDH2) did not grow on either YNBA or YPA. Deletion of SFC1 encoding the succinate-fumarate carrier also caused similar growth defects on YNBA. Our results suggest that in S. cerevisiae the glyoxylate cycle functions as a competent metabolic pathway for acetate utilization on YNBA, while both the TCA and glyoxylate cycles are essential for growth on YPA.
Subject(s)
Acetates/metabolism , Energy Metabolism , Glyoxylates/metabolism , Metabolic Networks and Pathways/genetics , Saccharomyces cerevisiae/metabolism , Culture Media/chemistry , Gene Deletion , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Chives have been used both as food and as medicine. Previously, two fibrinolytic enzymes, ATFE-I (90 kDa) and ATFE-II (55 kDa), were identified in chives (Allium tuberosum), a perennial herb. In the present work, ATFE-II was purified by ion-exchange chromatography followed by gel filtration. In addition, the enzyme properties of ATFE-I and ATFE-II were compared. The molecular mass and isoelectric point (pI value) of ATFE-II were 55 kDa and pI 4.0, respectively, as revealed using one- or two-dimensional fibrin zymography. ATFE-II was optimally active at pH 7.0 and 45°C. ATFE-II degraded the Aα-chain of human fibrinogen but did not hydrolyze the Bß-chain or the γ-chain, indicating that the enzyme is an α-fibrinogenase. The proteolytic activity of ATFE-II was completely inhibited by 1 mM leupeptin, indicating that the enzyme belongs to the cysteine protease class. ATFE-II was also inhibited by 1 mM Fe²(+). ATFE-II exhibited high specificity for MeO-Suc-Arg-Pro-Tyr-p-nitroaniline (S-2586), a synthetic chromogenic substrate of chymotrypsin. Thus proteolytic enzymes from A. tuberosum may be useful as thrombolytic agents.
Subject(s)
Chive/enzymology , Cysteine Endopeptidases , Drug Discovery , Fibrinolysis , Fibrinolytic Agents , Plant Components, Aerial/enzymology , Plant Proteins , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/isolation & purification , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Ferrous Compounds/pharmacology , Fibrinogen/metabolism , Fibrinolytic Agents/chemistry , Fibrinolytic Agents/isolation & purification , Fibrinolytic Agents/metabolism , Hydrogen-Ion Concentration , Hydrolysis , Isoelectric Point , Isoenzymes/antagonists & inhibitors , Isoenzymes/chemistry , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Leupeptins/pharmacology , Molecular Weight , Oligopeptides/metabolism , Plant Proteins/antagonists & inhibitors , Plant Proteins/chemistry , Plant Proteins/isolation & purification , Plant Proteins/metabolism , Substrate Specificity , Temperature , Thrombolytic Therapy , Thrombosis/drug therapyABSTRACT
As an extension of our previous studies on the mitochondrial citrate synthase of Aspergillus nidulans and cloning of its coding gene (citA), we analyzed differential expression of citA in response to the progress of development and change of carbon source. The cDNA consisted of 1,700 nucleotides and was predicted to encode a 474-amino acid protein. By comparing the cDNA sequence with the corresponding genomic sequence, we confirmed that citA gene contains 7 introns and that its transcription starts at position -26 (26-nucleotide upstream from the initiation codon). Four putative CreA binding motifs and three putative stress-response elements (STREs) were found within the 1.45-kb citA promoter region. The mode of citA expression was examined by both Northern blot and confocal microscopy using green fluorescent protein (sGFP) as a vital reporter. During vegetative growth and asexual development, the expression of citA was ubiquitous throughout the whole fungal body including mycelia and conidiophores. During sexual development, the expression of citA was quite strong in cleistothecial shells, but significantly weak in the content of cleistothecia including ascospores. Acetate showed a strong inductive effect on citA expression, which is subjected to carbon catabolite repression (CCR) caused by glucose. The recombinant fusion protein CitA(40)::sGFP (sGFP containing the 40-amino acid N-terminal segment of CitA) was localized into mitochondria, which supports that a mitochondrial targeting signal is included within the 40-amino acid N-terminal segment of CitA.
Subject(s)
Aspergillus nidulans/growth & development , Aspergillus nidulans/physiology , Carbon/metabolism , Citrate (si)-Synthase/biosynthesis , Fungal Proteins/biosynthesis , Gene Expression Profiling , Gene Expression Regulation, Fungal , Acetates/metabolism , Amino Acid Sequence , Aspergillus nidulans/genetics , Base Sequence , Binding Sites , Citrate (si)-Synthase/genetics , DNA, Complementary/genetics , DNA, Fungal/genetics , Fungal Proteins/genetics , Fungal Proteins/metabolism , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Introns , Microscopy, Confocal , Microscopy, Fluorescence , Mitochondria/chemistry , Molecular Sequence Data , Promoter Regions, Genetic , Protein Binding , Protein Sorting Signals , Repressor Proteins/metabolism , Transcription Initiation SiteABSTRACT
The ability to sense and adapt to a hostile host environment is a crucial element for virulence of pathogenic fungi, including Cryptococcus neoformans. These cellular responses are evoked by diverse signaling cascades, including the stress-activated HOG pathway. Despite previous analysis of central components of the HOG pathway, its downstream signaling network is poorly characterized in C. neoformans. Here we performed comparative transcriptome analysis with HOG signaling mutants to explore stress-regulated genes and their correlation with the HOG pathway in C. neoformans. In this study, we not only provide important insights into remodeling patterns of global gene expression for counteracting external stresses but also elucidate novel characteristics of the HOG pathway in C. neoformans. First, inhibition of the HOG pathway increases expression of ergosterol biosynthesis genes and cellular ergosterol content, conferring a striking synergistic antifungal activity with amphotericin B and providing an excellent opportunity to develop a novel therapeutic method for treatment of cryptococcosis. Second, a number of cadmium-sensitive genes are differentially regulated by the HOG pathway, and their mutation causes resistance to cadmium. Finally, we have discovered novel stress defense and HOG-dependent genes, which encode a sodium/potassium efflux pump, protein kinase, multidrug transporter system, and elements of the ubiquitin-dependent system.
Subject(s)
Cryptococcus neoformans/metabolism , Fungal Proteins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Signal Transduction , Transcription, Genetic , Cryptococcus neoformans/genetics , Fungal Proteins/genetics , Gene Expression Regulation, Fungal , Mitogen-Activated Protein Kinases/geneticsABSTRACT
A novel fibrinolytic enzyme (AJ) was purified from Staphylococcus sp. strain AJ screened from Korean salt-fermented Anchovy-jeot. Relative molecular weight of AJ was determined as 26 kDa by using SDS-PAGE and fibrin zymography. Based on a 2D gel, AJ was found to consist of three active isoforms (pI 5.5-6.0) with the same N-terminal amino acid sequence. AJ exhibited optimum pH and temperature at 2.5-3.0 and 85 degrees C, respectively. AJ kept 85% of the initial activity after heating at 100 degrees C for 20 min on the zymogram gel. The Michaelis constant (K (m)) and K (cat) values of AJ towards alpha-casein were 0.38 mM and 19.73 s(-1), respectively. AJ cleaved the A alpha-chain of fibrinogen but did not affect the B beta- and gamma-chains, indicating that it is an alpha-fibrinogenase. The fibrinolytic activity was inhibited by diisopropyl fluorophosphate, indicating AJ is a serine protease. Interestingly, AJ was very stable at acidic condition, SDS, and heat (100 degrees C), whereas it was easily degraded at neutral and alkaline conditions. In particular, AJ formed an active homo-dimer in the pH range from 7.0 to 8.0. To our knowledge, a similar combination of acid and heat stability has not yet been reported for other fibrinolytic enzymes.
Subject(s)
Bacterial Proteins , Enzyme Stability , Fibrin/metabolism , Fishes/microbiology , Hot Temperature , Sodium Chloride , Staphylococcus/enzymology , Amino Acid Sequence , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Fermentation , Fibrinolysis , Food Microbiology , Humans , Hydrogen-Ion Concentration , Korea , Molecular Sequence Data , Sequence Analysis, DNA , Staphylococcus/genetics , Staphylococcus/isolation & purificationABSTRACT
Abnormal phenotypes resulting from haploinsufficiency (HI) are due to the loss of one allele. Recent studies in budding yeast have shown that HI originates from insufficient protein levels or from a stoichiometric imbalance between subunits of protein complexes. In humans, however, HI often involves transcription factors. Therefore, the species differences in HI and the molecular mechanisms of species-specific HI remain under investigation. In this study, HI in fission yeast was systematically surveyed. HI in fission yeast affected genes related to signaling and to basic cellular processes, as observed in budding yeast. These results suggest that there are species differences in HI and that the HI that occurs in fission yeast is intermediate to and HI in budding yeast and humans.
Subject(s)
Gene Deletion , Genome, Fungal , Schizosaccharomyces/genetics , Alleles , Fungal Proteins/genetics , Gene Dosage , Genes, Fungal , Phenotype , Schizosaccharomyces/growth & development , Species SpecificityABSTRACT
Hydrazinocurcumin (HC), a synthetic derivative of curcumin, has been reported to inhibit angiogenesis via unknown mechanisms. Understanding the molecular mechanisms of the drug's action is important for the development of improved compounds with better pharmacological properties. A genomewide drug-induced haploinsufficiency screening of fission yeast gene deletion mutants has been applied to identify drug targets of HC. As a first step, the 50% inhibition concentration (IC50) of HC was determined to be 2.2 microM. The initial screening of 4,158 mutants in 384-well plates using robotics was performed at concentrations of 2, 3, and 4 microM. A second screening was performed to detect sensitivity to HC on the plates. The first screening revealed 178 candidates, and the second screening resulted in 13 candidates, following the elimination of 165 false positives. Final filtering of the condition-dependent haploinsufficient genes gave eight target genes. Analysis of the specific targets of HC has shown that they are related to septum formation and the general transcription processes, which may be related to histone acetyl transferase. The target mutants showed 65% growth inhibition in response to HC compared with wild-type controls, as shown by liquid culture assay.
Subject(s)
Curcumin/analogs & derivatives , Gene Deletion , Genome, Fungal , Hydrazines/pharmacology , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces/drug effects , Curcumin/pharmacology , Drug Evaluation, Preclinical , Haploidy , Heterozygote , Inhibitory Concentration 50 , Schizosaccharomyces/genetics , Schizosaccharomyces/growth & development , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/antagonists & inhibitors , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
Hepatitis B virus X protein (HBx) is essential for hepatitis B virus infection and exerts a pleiotropic effect on various cellular machineries. HBx has been also demonstrated as an indirect transcriptional transactivator of various different viral and cellular promoters. In addition, HBx is involved in the development of various liver diseases including hepatocellular carcinoma. However the mechanism of HBx in hepatocellular carcinogenesis remains largely unknown. In this study, to identify possible new cellular proteins interacting with HBx, we carried out yeast two-hybrid assay. We obtained several possible cellular partners including VBP1, a binding factor for VHL tumor suppressor protein. The direct physical interaction between HBx and VBP1 in vitro and in vivo was confirmed by immunoprecipitation assay. In addition, we found that VBP1 facilitates HBx-induced NFkappaB activation and cell proliferation. These results implicate the important role of HBx in the development of hepatocellular carcinoma through its interaction with VBP1.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Carrier Proteins/metabolism , Hepatitis B virus/metabolism , NF-kappa B/metabolism , Trans-Activators/metabolism , Blotting, Western , Carcinoma, Hepatocellular/pathology , Cells, Cultured , Cytoskeletal Proteins , DNA Primers , Hepatitis B virus/genetics , Humans , Immunoprecipitation , Kidney/metabolism , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Molecular Chaperones , NF-kappa B/genetics , Plasmids , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcriptional Activation , Two-Hybrid System Techniques , Viral Regulatory and Accessory ProteinsABSTRACT
In Saccharomyces cerevisiae, the initial reaction of the tricarboxylic acid cycle is catalyzed by the mitochondrial citrate synthase Cit1. The function of Cit1 has previously been studied mainly in terms of acetate utilization and metabolon construction. Here, we report the relationship between the function of Cit1 and apoptosis. Yeast cells with cit1 deletion showed a temperature-sensitive growth phenotype, and they displayed a rapid loss in viability associated with typical apoptotic hallmarks, i.e., reactive oxygen species (ROS) accumulation and nuclear fragmentation, DNA breakage, and phosphatidylserine translocation, when exposed to heat stress. On long-term cultivation, cit1 null strains showed increased potentials for both aging-induced apoptosis and adaptive regrowth. Activation of the metacaspase Yca1 was detected during heat- or aging-induced apoptosis in cit1 null strains, and accordingly, deletion of YCA1 suppressed the apoptotic phenotype caused by cit1 null mutation. Cells with cit1 deletion showed higher tendency toward glutathione (GSH) depletion and subsequent ROS accumulation than the wild type, which was rescued by exogenous GSH, glutamate, or glutathione disulfide (GSSG). These results led us to conclude that GSH deficiency in cit1 null cells is caused by an insufficient supply of glutamate necessary for biosynthesis of GSH rather than the depletion of reducing power required for reduction of GSSG to GSH.