RESUMEN
The opportunistic fungal pathogen Candida glabrata is a frequent cause of candidiasis, causing infections ranging from superficial to life-threatening disseminated disease. The inherent tolerance of C. glabrata to azole drugs makes this pathogen a serious clinical threat. To identify novel genes implicated in antifungal drug tolerance, we have constructed a large-scale C. glabrata deletion library consisting of 619 unique, individually bar-coded mutant strains, each lacking one specific gene, all together representing almost 12% of the genome. Functional analysis of this library in a series of phenotypic and fitness assays identified numerous genes required for growth of C. glabrata under normal or specific stress conditions, as well as a number of novel genes involved in tolerance to clinically important antifungal drugs such as azoles and echinocandins. We identified 38 deletion strains displaying strongly increased susceptibility to caspofungin, 28 of which encoding proteins that have not previously been linked to echinocandin tolerance. Our results demonstrate the potential of the C. glabrata mutant collection as a valuable resource in functional genomics studies of this important fungal pathogen of humans, and to facilitate the identification of putative novel antifungal drug target and virulence genes.
Asunto(s)
Antifúngicos/farmacología , Azoles/farmacología , Candida glabrata/genética , Farmacorresistencia Fúngica/genética , Equinocandinas/farmacología , Biopelículas/crecimiento & desarrollo , Candida glabrata/crecimiento & desarrollo , Candidiasis/tratamiento farmacológico , Caspofungina , Pared Celular/efectos de los fármacos , Pared Celular/genética , Proteínas Fúngicas/genética , Eliminación de Gen , Técnicas de Inactivación de Genes , Biblioteca de Genes , Lipopéptidos , Pruebas de Sensibilidad Microbiana , Presión Osmótica , FenotipoRESUMEN
Fungal infections are a leading cause of morbidity and death for hospitalized patients, mainly because they remain difficult to diagnose and to treat. Diseases range from widespread superficial infections such as vulvovaginal infections to life-threatening systemic candidiasis. For systemic mycoses, only a restricted arsenal of antifungal agents is available. Commonly used classes of antifungal compounds include azoles, polyenes, and echinocandins. Due to emerging resistance to standard therapies, significant side effects, and high costs for several antifungals, there is a need for new antifungals in the clinic. In order to expand the arsenal of compounds with antifungal activity, we previously screened a compound library using a cell-based screening assay. A set of novel benzimidazole derivatives, including (S)-2-(1-aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole (EMC120B12), showed high antifungal activity against several species of pathogenic yeasts, including Candida glabrata and Candida krusei (species that are highly resistant to antifungals). In this study, comparative analysis of EMC120B12 versus fluconazole and nocodazole, using transcriptional profiling and sterol analysis, strongly suggested that EMC120B12 targets Erg11p in the ergosterol biosynthesis pathway and not microtubules, like other benzimidazoles. In addition to the marker sterol 14-methylergosta-8,24(28)-dien-3ß,6α-diol, indicating Erg11p inhibition, related sterols that were hitherto unknown accumulated in the cells during EMC120B12 treatment. The novel sterols have a 3ß,6α-diol structure. In addition to the identification of novel sterols, this is the first time that a benzimidazole structure has been shown to result in a block of the ergosterol pathway.
Asunto(s)
Antifúngicos/farmacología , Bencimidazoles/farmacología , Ergosterol/antagonistas & inhibidores , Fluconazol/farmacología , Nocodazol/farmacología , Esterol 14-Desmetilasa/metabolismo , Antifúngicos/química , Bencimidazoles/química , Candida/efectos de los fármacos , Candida/crecimiento & desarrollo , Candida/metabolismo , Candida albicans/efectos de los fármacos , Candida albicans/crecimiento & desarrollo , Candida albicans/metabolismo , Candida glabrata/efectos de los fármacos , Candida glabrata/crecimiento & desarrollo , Candida glabrata/metabolismo , Ergosterol/análogos & derivados , Ergosterol/biosíntesis , Ergosterol/aislamiento & purificación , Fluconazol/química , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/metabolismo , Ensayos Analíticos de Alto Rendimiento , Nocodazol/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Micro-organisms sense the availability of nutrients in their environment to control cellular behaviour and the expression of transporters and enzymes that are required for the utilization of these nutrients. In the pathogenic yeast Candida albicans, the preferred nitrogen source ammonium suppresses the switch from yeast to filamentous growth in response to certain stimuli, and it also represses the secretion of proteases, which are required for the utilization of proteins as an alternative nitrogen source. To investigate whether C. albicans senses the availability of ammonium in the extracellular environment or if ammonium uptake into the cell is required to regulate morphogenesis and gene expression, we compared the behaviour of wild-type cells and ammonium uptake-deficient mutants in the presence and absence of extracellular ammonium. Arginine-induced filamentous growth was suppressed by ammonium in the wild-type, but not in mutants lacking the ammonium permeases Mep1 and Mep2. Similarly, ammonium suppressed protease secretion and extracellular protein degradation in the wild-type, but not in mutants lacking the ammonium transporters. By comparing the gene expression profiles of C. albicans grown in the presence of low or high ammonium concentrations, we identified a set of genes whose expression is controlled by nitrogen availability. The repression of genes involved in the utilization of alternative nitrogen sources, which occurred under ammonium-replete conditions in the wild-type, was abrogated in mep1Δ mep2Δ mutants. These results demonstrate that C. albicans does not respond to the presence of sufficient amounts of the preferred nitrogen source ammonium by sensing its availability in the environment. Instead, ammonium has to be taken up into the cell to control morphogenesis, protease secretion and gene expression.
Asunto(s)
Compuestos de Amonio/metabolismo , Candida albicans/crecimiento & desarrollo , Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Péptido Hidrolasas/metabolismo , Arginina/metabolismo , Candida albicans/enzimología , Candida albicans/genética , Proteínas Fúngicas/genética , Péptido Hidrolasas/genética , Transporte de ProteínasRESUMEN
The majority of microorganisms persist in nature as surface-attached communities often surrounded by an extracellular matrix, called biofilms. Most natural biofilms are not formed by a single species but by multiple species. Microorganisms not only cooperate as in some multispecies biofilms but also compete for available nutrients. The Gram-negative bacterium Pseudomonas aeruginosa and the polymorphic fungus Candida albicans are two opportunistic pathogens that are often found coexisting in a human host. Several models of mixed biofilms have been reported for these organisms showing antagonistic behavior. To investigate the interaction of P. aeruginosa and C. albicans in more detail, we analyzed the secretome of single and mixed biofilms of both organisms using MALDI-TOF MS/MS at several time points. Overall 247 individual proteins were identified, 170 originated from P. aeruginosa and 77 from C. albicans. Only 39 of the 131 in mixed biofilms identified proteins were assigned to the fungus whereby the remaining 92 proteins belonged to P. aeruginosa. In single-species biofilms, both organisms showed a higher diversity of proteins with 73 being assigned to C. albicans and 154 to P. aeruginosa. Most interestingly, P. aeruginosa in the presence of C. albicans secreted 16 proteins in significantly higher amounts or exclusively among other virulence factors such as exotoxin A and iron acquisition systems. In addition, the high affinity iron-binding siderophore pyoverdine was identified in mixed biofilms but not in bacterial biofilms, indicating that P. aeruginosa increases its capability to sequester iron in competition with C. albicans. In contrast, C. albicans metabolism was significantly reduced, including a reduction in detectable iron acquisition proteins. The results obtained in this study show that microorganisms not only compete with the host for essential nutrients but also strongly with the present microflora in order to gain a competitive advantage.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Candida albicans/metabolismo , Proteoma/análisis , Pseudomonas aeruginosa/metabolismo , ADP Ribosa Transferasas/biosíntesis , Toxinas Bacterianas/biosíntesis , Recuento de Colonia Microbiana , Exotoxinas/biosíntesis , Hierro/metabolismo , Oligopéptidos/biosíntesis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de Virulencia/biosíntesis , Exotoxina A de Pseudomonas aeruginosaRESUMEN
Corynebacterium pseudotuberculosis (CPS) is an important bacterial animal pathogen. CPS causes chronic, debilitating and currently incurable infectious diseases affecting a wide range of livestock and wild herbivores including camelids worldwide. Belonging to the Corynebacterium diphtheriae complex, this pathogen can also infect humans. The classical characterization of CPS is typically based on the testing of nitrate reductase activity, separating the two biovars Equi and Ovis. However, more refined resolutions are required to unravel routes of infection. This was realized in our study by generating and analyzing whole genome sequencing (WGS) data. Using newly created core genome multilocus sequence typing (cgMLST) profiles we were the first to discover isolates grouping in a cluster adjacent to clusters formed by CPS biovar Equi isolates. This novel cluster includes CPS isolates from alpacas, llamas, camels and dromedaries, which are characterized by a lack of nitrate reductase activity as encountered in biovar Ovis. This is of special interest for molecular epidemiology. Nevertheless, these isolates bear the genes of the nitrate locus, which are characteristic of biovar Equi isolates. However, sequence analysis of the genes narG and narH of the nitrate locus revealed indels leading to frameshifts and inactivity of the enzymes involved in nitrate reduction. Interestingly, one CPS isolate originating from another lama with an insertion in the MFS transporter (narT) is adjacent to a cluster formed by ovine CPS isolates biovar Equi. Based on this knowledge, the combination of biochemical and PCR based molecular biological nitrate reductase detection can be used for a fast and uncomplicated classification of isolates in routine diagnostics in order to check the origin of camelid CPS isolates. Further analysis revealed that partial sequencing of the ABC transporter substrate binding protein (CP258_RS07935) is a powerful tool to assign the biovars and the novel genomovar.
Asunto(s)
Corynebacterium pseudotuberculosis , Variación Genética , Secuenciación Completa del Genoma , Corynebacterium pseudotuberculosis/genética , Corynebacterium pseudotuberculosis/aislamiento & purificación , Animales , Filogenia , Genoma Bacteriano , Tipificación de Secuencias Multilocus , Infecciones por Corynebacterium/microbiología , Infecciones por Corynebacterium/veterinariaRESUMEN
For successful elucidation of a food-borne infection chain, the availability of high-quality sequencing data from suspected microbial contaminants is a prerequisite. Commonly, those investigations are a joint effort undertaken by different laboratories and institutes. To analyze the extent of variability introduced by differing wet-lab procedures on the quality of the sequence data we conducted an interlaboratory study, involving four bacterial pathogens, which account for the majority of food-related bacterial infections: Campylobacter spp., Shiga toxin-producing Escherichia coli, Listeria monocytogenes, and Salmonella enterica. The participants, ranging from German federal research institutes, federal state laboratories to universities and companies, were asked to follow their routine in-house protocols for short-read sequencing of 10 cultures and one isolated bacterial DNA per species. Sequence and assembly quality were then analyzed centrally. Variations within isolate samples were detected with SNP and cgMLST calling. Overall, we found that the quality of Illumina raw sequence data was high with little overall variability, with one exception, attributed to a specific library preparation kit. The variability of Ion Torrent data was higher, independent of the investigated species. For cgMLST and SNP analysis results, we found that technological sequencing artefacts could be reduced by the use of filters, and that SNP analysis was more suited than cgMLST to compare data of different contributors. Regarding the four species, a minority of Campylobacter isolate data showed the in comparison highest divergence with regard to sequence type and cgMLST analysis. We additionally compared the assembler SPAdes and SKESA for their performance on the Illumina data sets of the different species and library preparation methods and found overall similar assembly quality metrics and cgMLST statistics.
RESUMEN
The cell wall of the human-pathogenic fungus Candida albicans is a robust but also dynamic structure which mediates adaptation to changing environmental conditions during infection. Sap9 and Sap10 are cell surface-associated proteases which function in C. albicans cell wall integrity and interaction with human epithelial cells and neutrophils. In this study, we have analyzed the enzymatic properties of Sap9 and Sap10 and investigated whether these proteases cleave proteins on the fungal cell surface. We show that Sap9 and Sap10, in contrast to other aspartic proteases, exhibit a near-neutral pH optimum of proteolytic activity and prefer the processing of peptides containing basic or dibasic residues. However, both proteases also cleaved at nonbasic sites, and not all tested peptides with dibasic residues were processed. By digesting isolated cell walls with Sap9 or Sap10, we identified the covalently linked cell wall proteins (CWPs) Cht2, Ywp1, Als2, Rhd3, Rbt5, Ecm33, and Pga4 as in vitro protease substrates. Proteolytic cleavage of the chitinase Cht2 and the glucan-cross-linking protein Pir1 by Sap9 was verified using hemagglutinin (HA) epitope-tagged versions of both proteins. Deletion of the SAP9 and SAP10 genes resulted in a reduction of cell-associated chitinase activity similar to that upon deletion of CHT2, suggesting a direct influence of Sap9 and Sap10 on Cht2 function. In contrast, cell surface changes elicited by SAP9 and SAP10 deletion had no major impact on the phagocytosis and killing of C. albicans by human macrophages. We propose that Sap9 and Sap10 influence distinct cell wall functions by proteolytic cleavage of covalently linked cell wall proteins.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Candida albicans/enzimología , Pared Celular/metabolismo , Proteínas Fúngicas/metabolismo , Secuencia de Aminoácidos , Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/genética , Candida albicans/patogenicidad , Quitinasas/química , Quitinasas/metabolismo , Simulación por Computador , Pruebas de Enzimas , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Humanos , Concentración de Iones de Hidrógeno , Macrófagos/microbiología , Macrófagos/fisiología , Modelos Moleculares , Mutación , Pepstatinas/farmacología , Fagocitosis , Proteoma/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Células Tumorales CultivadasRESUMEN
Infectious diseases have long been regarded as losing their threat to mankind. However, in the recent decades infectious diseases have been regaining grounds and are back in the focus of research. This is also due to the fact that medical progress has enabled us to treat and cure a much higher fraction of severe diseases or trauma, resulting in a significant proportion of temporarily or constantly immune-suppressed patients. Infectious diseases result from the interplay between pathogenic microorganisms and the hosts they infect, especially their defense systems. Consequently, immune-suppressed patients are at high risk to succumb from opportunistic infections, like Candida infections. To study the balance between host and C. albicans with regard to the establishment of disease or asymptomatic, commensal colonisation, we developed host-pathogen interaction systems to study both the adaptation of C. albicans to different epithelia as well as to investigate the sensors of the innate immune system, the pattern recognition receptors. These host-pathogen interaction systems, as well as some of the results gained are described in this review.
Asunto(s)
Candida albicans/inmunología , Candida albicans/patogenicidad , Pared Celular/metabolismo , Proteínas Fúngicas/metabolismo , Interacciones Huésped-Patógeno , Factores de Virulencia/metabolismo , Adhesión Celular , Células Epiteliales/microbiología , Humanos , Inmunidad InnataRESUMEN
Protein production using processed cell lysates is a core technology in synthetic biology and these systems are excellent to produce difficult toxins or membrane proteins. However, the composition of the central lysate of cell-free systems is still a "black box". Escherichia coli lysates are most productive for cell-free expression, yielding several mgs of protein per ml of reaction. Their preparation implies proteome fractionation, resulting in strongly biased and yet unknown lysate compositions. Many metabolic pathways are expected to be truncated or completely removed. The lack of knowledge of basic cell-free lysate proteomes is a major bottleneck for directed lysate engineering approaches as well as for assay design using non-purified reaction mixtures. This study is starting to close this gap by providing a blueprint of the S30 lysate proteome derived from the commonly used E. coli strain A19. S30 lysates are frequently used for cell-free protein production and represent the basis of most commercial E. coli cell-free expression systems. A fraction of 821 proteins was identified as the core proteome in S30 lysates, representing approximately a quarter of the known E. coli proteome. Its classification into functional groups relevant for transcription/translation, folding, stability and metabolic processes will build the framework for tailored cell-free reactions. As an example, we show that SOS response induction during cultivation results in tuned S30 lysate with better folding capacity, and improved solubility and activity of synthesized proteins. The presented data and protocols can serve as a platform for the generation of customized cell-free systems and product analysis.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Proteoma/química , Proteoma/metabolismo , Cromatografía Liquida , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/química , Espectrometría de Masas , Pliegue de Proteína , Solubilidad , Espectrometría de FluorescenciaRESUMEN
Inactivated vaccines are commonly produced by incubating pathogens with chemicals such as formaldehyde or ß-propiolactone. This is a time-consuming process, the inactivation efficiency displays high variability and extensive downstream procedures are often required. Moreover, application of chemicals alters the antigenic components of the viruses or bacteria, resulting in reduced antibody specificity and therefore stimulation of a less effective immune response. An alternative method for inactivation of pathogens is ionizing radiation. It acts very fast and predominantly damages nucleic acids, conserving most of the antigenic structures. However, currently used irradiation technologies (mostly gamma-rays and high energy electrons) require large and complex shielding constructions to protect the environment from radioactivity or X-rays generated during the process. This excludes them from direct integration into biological production facilities. Here, low-energy electron irradiation (LEEI) is presented as an alternative inactivation method for pathogens in liquid solutions. LEEI can be used in normal laboratories, including good manufacturing practice (GMP)- or high biosafety level (BSL)-environments, as only minor shielding is necessary. We show that LEEI efficiently inactivates different viruses (influenza A (H3N8), porcine reproductive and respiratory syndrome virus (PRRSV), equine herpesvirus 1 (EHV-1)) and bacteria (Escherichia coli) and maintains their antigenicity. Moreover, LEEI-inactivated influenza A viruses elicit protective immune responses in animals, as analyzed by virus neutralization assays and viral load determination upon challenge. These results have implications for novel ways of developing and manufacturing inactivated vaccines with improved efficacy.
Asunto(s)
Antígenos Bacterianos/efectos de la radiación , Antígenos Virales/efectos de la radiación , Desinfección/métodos , Escherichia coli/efectos de la radiación , Radiación Ionizante , Virus/efectos de la radiación , Antígenos Bacterianos/inmunología , Antígenos Virales/inmunología , Electrones , Escherichia coli/inmunología , Vacunas de Productos Inactivados/inmunología , Virus/inmunologíaRESUMEN
Studying infectious diseases requires suitable hosts for experimental in vivo infections. Recent years have seen the advent of many alternatives to murine infection models. However, the use of non-mammalian models is still controversial because it is often unclear how well findings from these systems predict virulence potential in humans or other mammals. Here, we compare the commonly used models, fruit fly and mouse (representing invertebrate and mammalian hosts), for their similarities and degree of correlation upon infection with a library of mutants of an important fungal pathogen, the yeast Candida glabrata. Using two indices, for fly survival time and for mouse fungal burden in specific organs, we show a good agreement between the models. We provide a suitable predictive model for estimating the virulence potential of C. glabrata mutants in the mouse from fly survival data. As examples, we found cell wall integrity mutants attenuated in flies, and mutants of a MAP kinase pathway had defective virulence in flies and reduced relative pathogen fitness in mice. In addition, mutants with strongly reduced in vitro growth generally, but not always, had reduced virulence in flies. Overall, we demonstrate that surveying Drosophila survival after infection is a suitable model to predict the outcome of murine infections, especially for severely attenuated C. glabrata mutants. Pre-screening of mutants in an invertebrate Drosophila model can, thus, provide a good estimate of the probability of finding a strain with reduced microbial burden in the mouse host.
Asunto(s)
Modelos Animales de Enfermedad , Drosophila melanogaster/microbiología , Animales , Candida glabrata/patogenicidad , Ontología de Genes , Humanos , Modelos Logísticos , Masculino , Ratones , Mutación/genética , Micosis/microbiología , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , VirulenciaRESUMEN
Since its development, microarray technology has evolved to a standard method in the biotechnological and medical field with a broad range of applications. Nevertheless, the underlying mechanism of the hybridization process of PCR-products to microarray capture probes is still not completely understood, and several observed phenomena cannot be explained with current models. We investigated the influence of several parameters on the hybridization reaction and identified ssDNA to play a major role in the process. An increase of the ssDNA content in a hybridization reaction strongly enhanced resulting signal intensities. A strong influence could also be observed when unlabeled ssDNA was added to the hybridization reaction. A reduction of the ssDNA content resulted in a massive decrease of the hybridization efficiency. According to these data, we developed a novel model for the hybridization mechanism. This model is based on the assumption that single stranded DNA is necessary as catalyst to induce the hybridization of dsDNA. The developed hybridization model is capable of giving explanations for several yet unresolved questions regarding the functionality of microarrays. Our findings not only deepen the understanding of the hybridization process, but also have immediate practical use in data interpretation and the development of new microarrays.
Asunto(s)
ADN de Cadena Simple/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa/métodos , Biocatálisis , Hibridación de Ácido NucleicoRESUMEN
The human fungal pathogen Candida glabrata is related to Saccharomyces cerevisiae but has developed high resistance against reactive oxygen species. We find that induction of conserved genes encoding antioxidant functions is dependent on the transcription factors CgYap1 and CgSkn7 which cooperate for promoter recognition. Superoxide stress resistance of C. glabrata is provided by superoxide dismutase CgSod1, which is not dependent on CgYap1/Skn7. Only double mutants lacking both CgSod1 and CgYap1 were efficiently killed by primary mouse macrophages. Our results suggest that in C. glabrata the regulation of key genes providing stress protection is adopted to meet a host-pathogen situation.
Asunto(s)
Adaptación Fisiológica/genética , Candida glabrata/metabolismo , Regulación Fúngica de la Expresión Génica , Estrés Oxidativo , Animales , Proteínas Fúngicas , Macrófagos , Ratones , Superóxido Dismutasa , Factores de TranscripciónRESUMEN
The SUN gene family has been defined in Saccharomyces cerevisiae and comprises a fungus-specific family of proteins which show high similarity in their C-terminal domains. Genes of this family are involved in different cellular processes, like DNA replication, aging, mitochondrial biogenesis, and cytokinesis. In Candida albicans the SUN family comprises two genes, SUN41 and SIM1. We demonstrate that C. albicans mutants lacking SUN41 show similar defects as found for S. cerevisiae, including defects in cytokinesis. In addition, the SUN41 mutant showed a higher sensitivity towards the cell wall-disturbing agent Congo red, whereas no difference was observed in the presence of calcofluor white. Compared to the wild type, SUN41 deletion strains exhibited a defect in biofilm formation, a reduced adherence on a Caco-2 cell monolayer, and were unable to form hyphae on solid medium under the conditions tested. Interestingly, Sun41p was found to be secreted in the medium of cells growing as blastospores as well as those forming hyphae. Our results support a function of SUN41p as a glycosidase involved in cytokinesis, cell wall biogenesis, adhesion to host tissue, and biofilm formation, indicating an important role in the host-pathogen interaction.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Candida albicans/citología , Candida albicans/enzimología , Pared Celular/enzimología , Proteínas Fúngicas/metabolismo , Glucosidasas/metabolismo , Morfogénesis , Secuencia de Aminoácidos , Células CACO-2 , Candida albicans/genética , Candida albicans/crecimiento & desarrollo , Adhesión Celular , Rojo Congo/metabolismo , Citocinesis , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Humanos , Hifa/enzimología , Datos de Secuencia Molecular , Fenotipo , Transporte de Proteínas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Geminin contributes to cell cycle regulation by a timely inhibition of Cdt1p, the loading factor required for the assembly of pre-replication complexes. Geminin is expressed during S and G2 phase of the HeLa cell cycle and phosphorylated soon after its synthesis. We show here that Geminin is an excellent substrate for protein kinase CK2 in vitro; and that the highly specific CK2 inhibitor tetrabromobenzotriazole (TBB) blocks the phosphorylation of Geminin in HeLa protein extracts and HeLa cells in vivo. The sites of CK2 phosphorylation are located in the carboxyterminal region of Geminin, which carries several consensus sequence motifs for CK2. We also show that a minor phosphorylating activity in protein extracts can be attributed to glycogen synthase kinase 3 (GSK3), which most likely targets a central peptide in Geminin. Treatment of HeLa cells with TBB does not interfere with the ability of Geminin to interact with the loading factor Cdt1.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Autorradiografía , Sitios de Unión , Quinasa de la Caseína II , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Secuencia de Consenso , Inhibidores Enzimáticos/farmacología , Geminina , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Células HeLa , Humanos , Indoles/farmacología , Maleimidas/farmacología , Datos de Secuencia Molecular , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fase S/efectos de los fármacos , Fase S/fisiología , Triazoles/farmacologíaRESUMEN
It has been described that the replication regulator protein geminin is rapidly degraded at the end of mitosis and newly expressed at the beginning of the next S phase in the metazoan cell cycle. We have performed experiments to investigate the synthesis of geminin in cycling human HeLa cells. The levels of geminin-mRNA vary only modestly during the cell cycle with a 2-3-fold higher mRNA level at the G1/S phase transition, whereas newly synthesized geminin can only be detected in post-G1 phases. Surprisingly, geminin, once synthesized, does not remain stable, but is turned over during S phase with a half-life of 3-4h. We also show that geminin becomes phosphorylated as S phase proceeds and identify by MALDI mass spectrometry two specific major phosphorylation sites.