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1.
J Biol Chem ; 299(6): 104797, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-37156397

RESUMEN

Coenzyme Q (CoQ) is an essential component of the electron transport system in aerobic organisms. CoQ10 has ten isoprene units in its quinone structure and is especially valuable as a food supplement. However, the CoQ biosynthetic pathway has not been fully elucidated, including synthesis of the p-hydroxybenzoic acid (PHB) precursor to form a quinone backbone. To identify the novel components of CoQ10 synthesis, we investigated CoQ10 production in 400 Schizosaccharomyces pombe gene-deleted strains in which individual mitochondrial proteins were lost. We found that deletion of coq11 (an S. cerevisiae COQ11 homolog) and a novel gene designated coq12 lowered CoQ levels to ∼4% of that of the WT strain. Addition of PHB or p-hydroxybenzaldehyde restored the CoQ content and growth and lowered hydrogen sulfide production of the Δcoq12 strain, but these compounds did not affect the Δcoq11 strain. The primary structure of Coq12 has a flavin reductase motif coupled with an NAD+ reductase domain. We determined that purified Coq12 protein from S. pombe displayed NAD+ reductase activity when incubated with ethanol-extracted substrate of S. pombe. Because purified Coq12 from Escherichia coli did not exhibit reductase activity under the same conditions, an extra protein is thought to be necessary for its activity. Analysis of Coq12-interacting proteins by LC-MS/MS revealed interactions with other Coq proteins, suggesting formation of a complex. Thus, our analysis indicates that Coq12 is required for PHB synthesis, and it has diverged among species.


Asunto(s)
NADH NADPH Oxidorreductasas , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Cromatografía Liquida , NAD/metabolismo , NADH NADPH Oxidorreductasas/química , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/aislamiento & purificación , NADH NADPH Oxidorreductasas/metabolismo , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/aislamiento & purificación , Proteínas de Schizosaccharomyces pombe/metabolismo , Espectrometría de Masas en Tándem , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo
2.
Open Biol ; 13(3): 220185, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36854376

RESUMEN

The Rho GTPase family proteins are key regulators of cytoskeletal dynamics. Deregulated activity of Rho GTPases is associated with cancers and neurodegenerative diseases, and their potential as drug targets has long been recognized. Using an economically effective drug screening workflow in fission yeast and human cells, we have identified a Rho GTPase inhibitor, O1. By a suppressor mutant screen in fission yeast, we find a point mutation in the rho1 gene that confers resistance to O1. Consistent with the idea that O1 is the direct inhibitor of Rho1, O1 reduced the cellular amount of activated, GTP-bound Rho1 in wild-type cells, but not in the O1-resistant mutant cells, in which the evolutionarily conserved Ala62 residue is mutated to Thr. Similarly, O1 inhibits activity of the human orthologue RhoA GTPase in tissue culture cells. Our studies illustrate the power of yeast phenotypic screens in the identification and characterization of drugs relevant to human cells and have identified a novel GTPase inhibitor for fission yeast and human cells.


Asunto(s)
Proteínas de Unión al GTP Monoméricas , Schizosaccharomyces , Proteína de Unión al GTP rhoA , Humanos , Citoesqueleto , Evaluación Preclínica de Medicamentos , Proteínas de Unión al GTP Monoméricas/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Schizosaccharomyces/enzimología
3.
Annu Rev Microbiol ; 71: 413-439, 2017 09 08.
Artículo en Inglés | MEDLINE | ID: mdl-28715960

RESUMEN

Histone-modifying enzymes are responsible for regulating transcription, recombination, DNA repair, DNA replication, chromatid cohesion, and chromosome segregation. Fungi are ideally suited for comparative chromatin biology because sequencing of numerous genomes from many clades is coupled to existing rich methodology that allows truly holistic approaches, integrating evolutionary biology with mechanistic molecular biology and ecology, promising applications in medicine or plant pathology. While genome information is rich, mechanistic studies on histone modifications are largely restricted to two yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, and one filamentous fungus, Neurospora crassa-three species that arguably are not representative of this diverse kingdom. Here, histone methylation serves as a paradigm to illustrate the roles chromatin modifications may play in more complex fungal life cycles. This review summarizes recent advances in our understanding of histone H3 methylation at two sites associated with active transcription, lysine 4 and lysine 36 (H3K4, H3K36); a site associated with the formation of constitutive heterochromatin, lysine 9 (H3K9); and a site associated with the formation of facultative heterochromatin, lysine 27 (H3K27). Special attention is paid to differences in how methylation marks interact in different taxa.


Asunto(s)
Histonas/metabolismo , Neurospora crassa/enzimología , Dominios PR-SET , Proteína Metiltransferasas/metabolismo , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/enzimología , Schizosaccharomyces/enzimología , Cromosomas Fúngicos/metabolismo , Heterocromatina/metabolismo , Metilación , Neurospora crassa/metabolismo , Proteína Metiltransferasas/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo
4.
RNA ; 22(4): 518-29, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26826132

RESUMEN

Dcp1/2 is the major eukaryotic RNA decapping complex, comprised of the enzyme Dcp2 and activator Dcp1, which removes the 5' m(7)G cap from mRNA, committing the transcript to degradation. Dcp1/2 activity is crucial for RNA quality control and turnover, and deregulation of these processes may lead to disease development. The molecular details of Dcp1/2 catalysis remain elusive, in part because both cap substrate (m(7)GpppN) and m(7)GDP product are bound by Dcp1/2 with weak (mM) affinity. In order to find inhibitors to use in elucidating the catalytic mechanism of Dcp2, we screened a small library of synthetic m(7)G nucleotides (cap analogs) bearing modifications in the oligophosphate chain. One of the most potent cap analogs, m(7)GpSpppSm(7)G, inhibited Dcp1/2 20 times more efficiently than m(7)GpppN or m(7)GDP. NMR experiments revealed that the compound interacts with specific surfaces of both regulatory and catalytic domains of Dcp2 with submillimolar affinities. Kinetics analysis revealed that m(7)GpSpppSm(7)G is a mixed inhibitor that competes for the Dcp2 active site with micromolar affinity. m(7)GpSpppSm(7)G-capped RNA undergoes rapid decapping, suggesting that the compound may act as a tightly bound cap mimic. Our identification of the first small molecule inhibitor of Dcp2 should be instrumental in future studies aimed at understanding the structural basis of RNA decapping and may provide insight toward the development of novel therapeutically relevant decapping inhibitors.


Asunto(s)
Análogos de Caperuza de ARN/química , Proteínas de Schizosaccharomyces pombe/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , División del ARN , ARN Mensajero/química , Schizosaccharomyces/enzimología
5.
Rev Iberoam Micol ; 31(1): 81-5, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24270073

RESUMEN

The enzyme 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR) catalyzes the conversion of HMG-Co-A into mevalonate. This step is the limiting point for the synthesis of cholesterol in mammals and ergosterol in fungi. We describe in this article the genome organization of HMGR coding genes and those deduced from different fungi, recount the evidence showing statins as HMGR inhibitors for ergosterol synthesis and its effect in yeast viability, and propose fungal HMGR (HMGRf) as a model to study the use of pharmaceutical compounds to inhibit cholesterol and ergosterol synthesis. Bibliographical search and bioinformatic analyses were performed and discussed. HMGRfs belong to the class I with a high homology in the catalytic region. The sterol biosynthetic pathway in humans and fungi share many enzymes in the initial steps (such as the HMGR enzyme), but in the last steps enzymes are different rendering the two final products: cholesterol in mammals and ergosterol in fungi. With regards to inhibitors such as statins and other compounds, these affect also fungal viability. Since HMGR from Schizosaccharomyces pombe and Ustilago maydis are very similar to the human HMGR in the catalytic regions, we propose that fungal enzymes can be used to test inhibitors for a potential use in humans. We consider that HMGRf is a good therapeutic target to design and test new antifungal compounds. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012).


Asunto(s)
Antifúngicos/farmacología , Evaluación Preclínica de Medicamentos/métodos , Proteínas Fúngicas/fisiología , Hongos/enzimología , Hidroximetilglutaril-CoA Reductasas/fisiología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Terapia Molecular Dirigida , Animales , Antifúngicos/uso terapéutico , Candida/enzimología , Colesterol/biosíntesis , Cristalografía por Rayos X , Ergosterol/biosíntesis , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/genética , Hongos/efectos de los fármacos , Genes Fúngicos , Humanos , Hidroximetilglutaril-CoA Reductasas/química , Hidroximetilglutaril-CoA Reductasas/efectos de los fármacos , Hidroximetilglutaril-CoA Reductasas/genética , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Micosis/tratamiento farmacológico , Micosis/microbiología , NADP/metabolismo , Roedores/metabolismo , Schizosaccharomyces/enzimología , Especificidad de la Especie , Ustilago/enzimología
6.
Structure ; 20(6): 977-86, 2012 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-22608966

RESUMEN

In eukaryotes, mRNA degradation begins with poly(A) tail removal, followed by decapping, and the mRNA body is degraded by exonucleases. In recent years, the major influence of 3'-end uridylation as a regulatory step within several RNA degradation pathways has generated significant attention toward the responsible enzymes, which are called poly(U) polymerases (PUPs). We determined the atomic structure of the Cid1 protein, the founding member of the PUP family, in its UTP-bound form, allowing unambiguous positioning of the UTP molecule. Our data also suggest that the RNA substrate accommodation and product translocation by the Cid1 protein rely on local and global movements of the enzyme. Supplemented by point mutations, the atomic model is used to propose a catalytic cycle. Our study underlines the Cid1 RNA binding properties, a feature with critical implications for miRNAs, histone mRNAs, and, more generally, cellular RNA degradation.


Asunto(s)
Nucleotidiltransferasas/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/enzimología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Dominio Catalítico , Secuencia Conservada , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , ARN de Hongos/química , Especificidad por Sustrato , Propiedades de Superficie , Uridina Trifosfato/química
7.
Handb Exp Pharmacol ; (204): 135-49, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21695638

RESUMEN

Fission yeast strains have been engineered so that their growth behavior reflects the activity of heterologous cyclic nucleotide phosphodiesterases (PDEs). These strains can be used in High-Throughput Screens (HTSs) for PDE inhibitors that possess "drug-like" characteristics, displaying activity in a growth stimulation assay over a 48-h period. Through three generations of development, a collection of strains expressing 10 of the 11 mammalian PDE families that is appropriate for small molecule inhibitor screening has been generated in our laboratory. Strains unable to synthesize cyclic nucleotides allow characterization of PDE activity in that the enzyme's potency is reflected in the amount of either cAMP or cGMP that must be added to the growth medium to stimulate cell growth. In the future, this system could be used to screen cDNA libraries for biological regulators of target PDEs and for the construction of strains that co-express PDEs and associated regulatory proteins to facilitate molecular and genetic studies of their functions and, in particular, to identify whether different PDE-partner protein complexes show distinct patterns of inhibitor sensitivity.


Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , Schizosaccharomyces/efectos de los fármacos , AMP Cíclico/fisiología , Schizosaccharomyces/enzimología
8.
Anal Biochem ; 410(1): 133-40, 2011 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-21073853

RESUMEN

Homocitrate synthase (HCS) catalyzes the first step of l-lysine biosynthesis in fungi by condensing acetyl-coenzyme A and 2-oxoglutarate to form 3R-homocitrate and coenzyme A. Due to its conservation in pathogenic fungi, HCS has been proposed as a candidate for antifungal drug design. Here we report the development and validation of a robust fluorescent assay for HCS that is amenable to high-throughput screening for inhibitors in vitro. Using this assay, Schizosaccharomyces pombe HCS was screened against a diverse library of approximately 41,000 small molecules. Following confirmation, counter screens, and dose-response analysis, we prioritized more than 100 compounds for further in vitro and in vivo analysis. This assay can be readily adapted to screen for small molecule modulators of other acyl-CoA-dependent acyltransferases or enzymes that generate a product with a free sulfhydryl group, including histone acetyltransferases, aminoglycoside N-acetyltransferases, thioesterases, and enzymes involved in lipid metabolism.


Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Pruebas de Enzimas/métodos , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Histona Acetiltransferasas/metabolismo , Oxo-Ácido-Liasas/antagonistas & inhibidores , Espectrometría de Fluorescencia/métodos , Acilcoenzima A/metabolismo , Quelantes/química , Quelantes/farmacología , Inhibidores Enzimáticos/química , Metales/química , Naftalenos/química , Oxo-Ácido-Liasas/química , Oxo-Ácido-Liasas/metabolismo , Pirroles/química , Reproducibilidad de los Resultados , Schizosaccharomyces/enzimología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Compuestos de Sulfhidrilo/química
9.
FEBS J ; 275(14): 3653-68, 2008 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-18540885

RESUMEN

Ubiquinone is an essential factor for the electron transfer system and is also a known lipid antioxidant. The length of the ubiquinone isoprenoid side-chain differs amongst living organisms, with six isoprene units in the budding yeast Saccharomyces cerevisiae, eight units in Escherichia coli and 10 units in the fission yeast Schizosaccharomyces pombe and in humans. The length of the ubiquinone isoprenoid is determined by the product generated by polyprenyl diphosphate synthases (poly-PDSs), which are classified into homodimer (i.e. octa-PDS IspB in E. coli) and heterotetramer [i.e. deca-PDSs Dps1 and D-less polyprenyl diphosphate synthase (Dlp1) in Sc. pombe and in humans] types. In this study, we characterized the hexa-PDS (Coq1) of S. cerevisiae to identify whether this enzyme was a homodimer (as in bacteria) or a heteromer (as in fission yeast). When COQ1 was expressed in an E. coli ispB disruptant, only hexa-PDS activity and ubiquinone-6 were detected, indicating that the expression of Coq1 alone results in bacterial enzyme-like functionality. However, when expressed in fission yeast Deltadps1 and Deltadlp1 strains, COQ1 restored growth on minimal medium in the Deltadlp1 but not Deltadps1 strain. Intriguingly, ubiquinone-9 and ubiquinone-10, but not ubiquinone-6, were identified and deca-PDS activity was detected in the COQ1-expressing Deltadlp1 strain. No enzymatic activity or ubiquinone was detected in the COQ1-expressing Deltadps1 strain. These results indicate that Coq1 partners with Dps1, but not with Dlp1, to be functional in fission yeast. Binding of Coq1 and Dps1 was demonstrated by coimmunoprecipitation, and the formation of a tetramer consisting of Coq1 and Dps1 was detected in Sc. pombe. Thus, Coq1 is functional when expressed alone in E. coli and in budding yeast, but is only functional as a partner with Dps1 in fission yeast. This unusual observation indicates that different folding processes or protein modifications in budding yeast/E. coli versus those in fission yeast might affect the formation of an active enzyme. These results provide important insights into the process of how PDSs have evolved from homo- to hetero-types.


Asunto(s)
Dimetilaliltranstransferasa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Secuencia de Aminoácidos , ADN Complementario/aislamiento & purificación , Dimetilaliltranstransferasa/química , Dimetilaliltranstransferasa/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutación , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Homología de Secuencia de Aminoácido , Ubiquinona/biosíntesis , Ubiquinona/química
10.
J Biomol Screen ; 13(1): 62-71, 2008 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-18227226

RESUMEN

Cyclic nucleotide phosphodiesterases (PDEs) comprise a superfamily of enzymes that serve as drug targets in many human diseases. There is a continuing need to identify high-specificity inhibitors that affect individual PDE families or even subtypes within a single family. The authors describe a fission yeast-based high-throughput screen to detect inhibitors of heterologously expressed adenosine 3',5'-cyclic monophosphate (cAMP) PDEs. The utility of this system is demonstrated by the construction and characterization of strains that express mammalian PDE2A, PDE4A, PDE4B, and PDE8A and respond appropriately to known PDE2A and PDE4 inhibitors. High-throughput screens of 2 bioactive compound libraries for PDE inhibitors using strains expressing PDE2A, PDE4A, PDE4B, and the yeast PDE Cgs2 identified known PDE inhibitors and members of compound classes associated with PDE inhibition. The authors verified that the furanocoumarin imperatorin is a PDE4 inhibitor based on its ability to produce a PDE4-specific elevation of cAMP levels. This platform can be used to identify PDE activators, as well as genes encoding PDE regulators, which could serve as targets for future drug screens.


Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos/métodos , Inhibidores de Fosfodiesterasa/farmacología , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/enzimología , 3',5'-AMP Cíclico Fosfodiesterasas/genética , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Animales , AMP Cíclico/metabolismo , Activación Enzimática/efectos de los fármacos , Genes Fúngicos , Genes Reporteros , Ratones , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Schizosaccharomyces/genética
11.
Eukaryot Cell ; 6(11): 2092-101, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17905925

RESUMEN

To investigate the contributions of phosphatidylserine to the growth and morphogenesis of the rod-shaped fission yeast Schizosaccharomyces pombe, we have characterized the single gene in this organism, pps1, encoding a predicted phosphatidylserine synthase. S. pombe pps1Delta mutants grow slowly in rich medium and are inviable in synthetic minimal medium. They do not produce detectable phosphatidylserine in vivo and possess negligible in vitro phosphatidylserine synthase activity, indicating that pps1 encodes the major phosphatidylserine synthase activity in S. pombe. Supplementation of growth medium with ethanolamine partially suppresses the growth-defective phenotype of pps1Delta cells, reflecting the likely importance of phosphatidylserine as a precursor for phosphatidylethanolamine in S. pombe. In medium lacking ethanolamine, pps1Delta mutants exhibit striking cell morphology, cytokinesis, actin cytoskeleton, and cell wall remodeling and integrity defects. Overexpression of pps1 likewise leads to defects in cell morphology and cytokinesis, thus implicating phosphatidylserine as a dosage-dependent regulator of these processes. During log-phase growth, green fluorescent protein-Pps1p fusion proteins are concentrated at the cell and nuclear peripheries as well as presumptive endoplasmic reticulum membranes, while in stationary-phase cells, they are redistributed to unusual cytoplasmic structures of unknown origin. Moreover, stationary-phase pps1Delta cultures retain very poor viability relative to wild-type S. pombe cells, even in medium containing ethanolamine, demonstrating a role for phosphatidylserine in the physiological adaptations required for stationary-phase survival. Our findings reveal novel cellular functions for phosphatidylserine and emphasize the usefulness of S. pombe as a model organism for elucidating potentially conserved biological and molecular functions of this phospholipid.


Asunto(s)
CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/genética , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/enzimología , Actinas/metabolismo , Secuencia de Aminoácidos , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/química , Pared Celular/efectos de los fármacos , Pared Celular/enzimología , Colina/farmacología , Clonación Molecular , Citocinesis/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Citoesqueleto/enzimología , Etanolamina/farmacología , Eliminación de Gen , Viabilidad Microbiana/efectos de los fármacos , Datos de Secuencia Molecular , Fenotipo , Transporte de Proteínas/efectos de los fármacos , Schizosaccharomyces/genética , Schizosaccharomyces/crecimiento & desarrollo , Proteínas de Schizosaccharomyces pombe/química , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/enzimología
12.
BMC Mol Biol ; 7: 43, 2006 Nov 28.
Artículo en Inglés | MEDLINE | ID: mdl-17132162

RESUMEN

BACKGROUND: Helicases play essential roles in many cellular processes including replication, transcription and translation. Most helicases translocate along one strand of the duplex while displacing the complementary strand (of either DNA or RNA). Thus, helicases have directionality. They move along nucleic acids in either the 3'--> 5' or 5'--> 3' direction. The directionality of helicases with low activity or of those that cannot initiate duplex unwinding from a substrate that contains only one single-stranded overhang region is difficult to determine. RESULTS: An improved assay to determine helicase directionality was developed that uses a substrate containing biotinylated oligonucleotides. As a proof of concept, it was shown that the substrates substantially improve helicase activity and directionality determination for several DNA helicases in comparison to more traditional substrates. In addition, a universal substrate that can be used to determine the directionality of both 3'--> 5' and 5'--> 3' helicases was developed. CONCLUSION: It is shown here that the use of a biotin-streptavidin complex as a helicase substrate improves helicase activity and the determination of helicase directionality. The method described is simpler that the currently available techniques.


Asunto(s)
Proteínas Bacterianas , Biotina/análogos & derivados , ADN Helicasas/metabolismo , Oligonucleótidos/metabolismo , Biotinilación , ADN Helicasas/química , Methanobacteriaceae/enzimología , Schizosaccharomyces/enzimología , Thermoplasma/enzimología
13.
Anal Biochem ; 338(1): 124-30, 2005 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-15707942

RESUMEN

A high-throughput screening method based on the competitive binding of a lumazine synthase inhibitor and riboflavin to the active site of Schizosaccharomyces pombe lumazine synthase was developed. This assay is sensitive, simple, and robust. During assay development, all of the known active inhibitors tested were positively identified. Preliminary high-throughput screening in 384-well format resulted in a Z factor of 0.7. The approach utilizes a thermodynamic assay to bypass the problems associated with the instabilities of both lumazine synthase substrates that complicate the use of a kinetic assay in a high-throughput format, and it removes the time element from the assay, thus simplifying the procedure.


Asunto(s)
Inhibidores Enzimáticos/aislamiento & purificación , Complejos Multienzimáticos/antagonistas & inhibidores , Riboflavina/química , Sitios de Unión , Evaluación Preclínica de Medicamentos , Ligandos , Schizosaccharomyces/enzimología , Espectrometría de Fluorescencia
14.
J Mol Biol ; 342(5): 1353-8, 2004 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-15364564

RESUMEN

The mitogen-activated protein kinase (MAPK) Byr2 and its activator Ste4 are involved in the mating pheromone response pathway of Schizosaccharomyces pombe and interact via their SAM domains. SAM domains can self-associate to form higher-order structures, including dimers, polymers and closed oligomers. Ste4-SAM is adjacent to a trimeric leucine zipper domain and we have shown previously that the two domains together (Ste4-LZ-SAM) bind to a monomeric Byr2-SAM with high affinity (Kd approximately 20 nM), forming a 3:1 complex. Here, we map the surfaces of Byr2-SAM and Ste4-SAM that is involved the interaction. A set of 38 mutants of Byr2-SAM and 33 mutants of Ste4-SAM were prepared, covering most of the protein surfaces. These mutants were purified and screened for binding, yielding a map of residues that are required for binding and a complementary map of residues that are not required. We find that the interface maps to regions of the SAM domains that are known to be important for the formation of SAM polymers. These results indicate that SAM domains can create a variety of oligomeric architectures utilizing common binding surfaces.


Asunto(s)
Subunidades beta de la Proteína de Unión al GTP/química , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Quinasas Quinasa Quinasa PAM/química , Quinasas Quinasa Quinasa PAM/metabolismo , Mutación/genética , S-Adenosilmetionina/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Sitios de Unión , Dimerización , Subunidades beta de la Proteína de Unión al GTP/genética , Quinasas Quinasa Quinasa PAM/genética , Unión Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe/genética , Resonancia por Plasmón de Superficie
15.
Appl Environ Microbiol ; 69(3): 1861-5, 2003 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-12620884

RESUMEN

The Saccharomyces cerevisiae PGU1 gene was successfully expressed in Schizosaccharomyces pombe. The optimum pH and temperature for the recombinant enzyme were 5 and 40 degrees C, respectively, these being around 0.5 U higher and 5 degrees C lower than those shown by the native enzyme. The K(m) value was about fourfold higher than that of the S. cerevisiae enzyme. The recombinant endopolygalacturonase was more efficient in reducing the viscosity of polygalacturonic acid and was also more stable at different pHs and temperatures than the native enzyme.


Asunto(s)
Poligalacturonasa/biosíntesis , Poligalacturonasa/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Medios de Cultivo , Industria de Alimentos , Concentración de Iones de Hidrógeno , Pectinas/metabolismo , Proteínas Recombinantes/biosíntesis , Saccharomyces cerevisiae/enzimología , Schizosaccharomyces/enzimología , Temperatura , Viscosidad
16.
Biochem J ; 364(Pt 3): 825-31, 2002 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-12049647

RESUMEN

In ureotelic animals, N-acetylglutamate (NAG) is an essential allosteric activator of carbamylphosphate synthetase I (CPSI), the first enzyme in the urea cycle. NAG synthase (NAGS; EC 2.3.1.1) catalyses the formation of NAG from glutamate and acetyl-CoA in liver and intestinal mitochondria. This enzyme is supposed to regulate ureagenesis by producing variable amounts of NAG, thus modulating CPSI activity. Moreover, inherited deficiencies in NAGS have been associated with hyperammonaemia, probably due to the loss of CPSI activity. Although the existence of the NAGS protein in mammals has been known for decades, the gene has remained elusive. We identified the mouse (Mus musculus) and human NAGS genes using their similarity to the respective Neurospora crassa gene. NAGS was cloned from a mouse liver cDNA library and was found to encode a 2.3 kb message, highly expressed in liver and small intestine with lower expression levels in kidney, spleen and testis. The deduced amino acid sequence contains a putative mitochondrial targeting signal at the N-terminus. The cDNA sequence complements an argA (NAGS)-deficient Escherichia coli strain, reversing its arginine auxotrophy. His-tagged versions of the pre-protein and two putative mature proteins were each overexpressed in E. coli, and purified to apparent homogeneity by using a nickel-affinity column. The pre-protein and the two putative mature proteins catalysed the NAGS reaction but one of the putative mature enzymes had significantly higher activity than the pre-protein. The addition of l-arginine increased the catalytic activity of the purified recombinant NAGS enzymes by approx. 2-6-fold.


Asunto(s)
Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Secuencia de Aminoácidos , N-Acetiltransferasa de Aminoácidos , Animales , Clonación Molecular , ADN Complementario , Escherichia coli/genética , Regulación Enzimológica de la Expresión Génica , Prueba de Complementación Genética , Cinética , Ratones , Datos de Secuencia Molecular , Neurospora crassa/enzimología , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Schizosaccharomyces/enzimología , Alineación de Secuencia , Homología de Secuencia de Aminoácido
17.
Biochim Biophys Acta ; 1517(1): 171-5, 2000 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-11118633

RESUMEN

The genomic DNA encoding thioltransferase was isolated from Schizosaccharomyces pombe using the polymerase chain reaction. The amplified DNA fragment was confirmed by Southern hybridization, completely digested with HindIII and BamHI, and then ligated into the yeast-Escherichia coli shuttle vector pRS316, which resulted in plasmid pEH1. The insert of plasmid pEH1 was transferred into the multi-copy vector YEp357 to generate plasmid pYEH1. The determined nucleotide sequence harbors an open reading frame consisting of four exons and three introns, which encodes a polypeptide of 101 amino acids with a molecular mass of 11261 Da. Thioltransferase activity was increased 1.6-fold in Saccharomyces cerevisiae containing plasmid pYEH1, and 1.8- and 2.7-fold in S. pombe containing plasmid pEH1 and pYEH1, respectively. The upstream sequence and the region encoding the N-terminal six amino acids were fused into promoterless beta-galactosidase gene of the shuttle vector YEp357R to generate the fusion plasmid pYEHR1. Synthesis of beta-galactosidase from the fusion plasmid was found to be enhanced by zinc and NO-generating S-nitroso-N-acetylpenicillamine.


Asunto(s)
Oxidorreductasas/genética , Penicilamina/análogos & derivados , Proteína Disulfuro Reductasa (Glutatión) , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Cloruros/farmacología , ADN Complementario/aislamiento & purificación , Galactosidasas/biosíntesis , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glutarredoxinas , Datos de Secuencia Molecular , Oxidorreductasas/biosíntesis , Penicilamina/farmacología , Plásmidos , Reacción en Cadena de la Polimerasa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/enzimología , Alineación de Secuencia , Compuestos de Zinc/farmacología
18.
J Bacteriol ; 182(24): 6933-9, 2000 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-11092853

RESUMEN

Ubiquinone is an essential component of the electron transfer system in both prokaryotes and eukaryotes and is synthesized from chorismate and polyprenyl diphosphate by eight steps. p-Hydroxybenzoate (PHB) polyprenyl diphosphate transferase catalyzes the condensation of PHB and polyprenyl diphosphate in ubiquinone biosynthesis. We isolated the gene (designated ppt1) encoding PHB polyprenyl diphosphate transferase from Schizosaccharomyces pombe and constructed a strain with a disrupted ppt1 gene. This strain could not grow on minimal medium supplemented with glucose. Expression of COQ2 from Saccharomyces cerevisiae in the defective S. pombe strain restored growth and enabled the cells to produce ubiquinone-10, indicating that COQ2 and ppt1 are functional homologs. The ppt1-deficient strain required supplementation with antioxidants, such as cysteine, glutathione, and alpha-tocopherol, to grow on minimal medium. This suggests that ubiquinone can act as an antioxidant, a premise supported by our observation that the ppt1-deficient strain is sensitive to H(2)O(2) and Cu(2+). Interestingly, we also found that the ppt1-deficient strain produced a significant amount of H(2)S, which suggests that oxidation of sulfide by ubiquinone may be an important pathway for sulfur metabolism in S. pombe. Ppt1-green fluorescent protein fusion proteins localized to the mitochondria, indicating that ubiquinone biosynthesis occurs in the mitochondria in S. pombe. Thus, analysis of the phenotypes of S. pombe strains deficient in ubiquinone production clearly demonstrates that ubiquinone has multiple functions in the cell apart from being an integral component of the electron transfer system.


Asunto(s)
Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Eliminación de Gen , Schizosaccharomyces/enzimología , Ubiquinona/metabolismo , Transferasas Alquil y Aril/química , Secuencia de Aminoácidos , Southern Blotting , Prueba de Complementación Genética , Sulfuro de Hidrógeno/metabolismo , Mitocondrias/enzimología , Datos de Secuencia Molecular , Fenotipo , Schizosaccharomyces/clasificación , Schizosaccharomyces/genética
19.
J Bacteriol ; 182(13): 3619-25, 2000 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-10850973

RESUMEN

Lithium is the drug of choice for the treatment of bipolar affective disorder. The identification of an in vivo target of lithium in fission yeast as a model organism may help in the understanding of lithium therapy. For this purpose, we have isolated genes whose overexpression improved cell growth under high LiCl concentrations. Overexpression of tol1(+), one of the isolated genes, increased the tolerance of wild-type yeast cells for LiCl but not for NaCl. tol1(+) encodes a member of the lithium-sensitive phosphomonoesterase protein family, and it exerts dual enzymatic activities, 3'(2'),5'-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase. tol1(+) gene-disrupted cells required high concentrations of sulfite in the medium for growth. Consistently, sulfite repressed the sulfate assimilation pathway in fission yeast. However, tol1(+) gene-disrupted cells could not fully recover from their growth defect and abnormal morphology even when the medium was supplemented with sulfite, suggesting the possible implication of inositol polyphosphate 1-phosphatase activity for cell growth and morphology. Given the remarkable functional conservation of the lithium-sensitive dual-specificity phosphomonoesterase between fission yeast and higher-eukaryotic cells during evolution, it may represent a likely in vivo target of lithium action across many species.


Asunto(s)
Litio/metabolismo , Nucleotidasas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/enzimología , Sulfitos/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Medios de Cultivo/farmacología , ADN de Hongos , Eliminación de Gen , Expresión Génica , Genes Fúngicos , Cloruro de Litio/farmacología , Datos de Secuencia Molecular , Nucleotidasas/genética , Monoéster Fosfórico Hidrolasas/genética , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/metabolismo , Homología de Secuencia de Aminoácido , Cloruro de Sodio/farmacología , Sulfatos/metabolismo
20.
Life Sci ; 65(4): 381-94, 1999.
Artículo en Inglés | MEDLINE | ID: mdl-10421424

RESUMEN

Hypothemycin was originally isolated as an antifungal metabolite of Hypomyces trichothecoides. Here we report that treatment on v-K-ras-transformed NIH3T3 cells (DT cells) with hypothemycin caused drastic decrease in amount of cyclin D1 protein with concomitant prolongation of G1 phase in their cell cycle. Analysis using hypothemycin-resistant mutant of Schizosaccharomyces pombe (S. pombe) was carried out to show that S. pombe rhp6+ (homologue of Saccharomyces cerevisiae RAD6) and mammalian ubiquitin-conjugating enzyme 2 (ubc2) are the targets of hypothemycin or its downstream molecules in ubiquitin-conjugation process. Furthermore, in the presence of lactacystin, a specific inhibitor for proteasome, hypothemycin greatly enhanced the accumulation of multi-ubiquitinated form of cyclin D1 in DT cells. Therefore, it is indicated that hypothemycin facilitates ubiquitinating process of cyclin D1. In terms of malignant phenotype, hypothemycin inhibited anchorage-independent growth and reverted the morphology of DT cells. On the contrary, their morphology still remained transformed in the additional presence of lactacystin. Our results suggest that cyclin D1 is a key molecule working downstream in ras-signaling and that the transformation can be inhibited by the compound which can activate ubiquitin-proteasome pathway including degradation of cyclin D1.


Asunto(s)
Antineoplásicos/farmacología , Transformación Celular Neoplásica/efectos de los fármacos , Ciclina D1/metabolismo , Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/metabolismo , Ubiquitinas/metabolismo , Células 3T3 , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Animales , Antineoplásicos/aislamiento & purificación , Western Blotting , Línea Celular Transformada , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , ADN Complementario/biosíntesis , ADN Complementario/aislamiento & purificación , Regulación hacia Abajo , Fase G1/efectos de los fármacos , Ratones , Hongos Mitospóricos/química , Complejo de la Endopetidasa Proteasomal , Proteínas Represoras/genética , Schizosaccharomyces/enzimología , Ubiquitinas/genética , Zearalenona/análogos & derivados
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