RESUMEN
Availability of protein structural data is accelerating at an astounding rate, facilitating in silico biochemical and biophysical analyses that require visualization methods. In particular, increased accessibility of representatives within respective protein families is empowering investigators to perform structural model comparisons that provide both functional and evolutionary insights at much more refined levels than previously possible. Numerous software platforms, including several free and open source versions, are available for users to interrogate protein structural models. In this article, three structural alignment protocols are described using freely available software to investigate aspects of protein structure evolution at quaternary, tertiary, and domain levels, respectively. Mapping distinct subunit interfaces and active site positioning within the PfpI/DJ-1 protein superfamily reveals quaternary structure that can have a prominent role in determination of distinct enzyme activities. In contrast, cytochrome c proteins are under strong evolutionary constraints due to their critical role in energy generation, and as a result, structural conservation is observed. However, substitutions within these conserved folds occur in distinct species, presumably to influence interactions with protein complexes involved in electron transport. Lastly, evolution of distinct allosteric mechanisms within winged helix-turn-helix transcriptional regulators, as well as protein dynamics, are revealed through visualization of metal- and redox-responsive DNA-binding proteins. The software platforms used in these protocols are Swiss-PDBViewer and PyMOL. Swiss-PDBViewer is an easy to implement, end-user software that is excellent for entry into protein visualization methods. PyMOL is also easy to implement, but offers greater depth for advanced investigations and visualizations, as well as the ability to capture protein structure conformational changes. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Exploring quaternary structure evolution with Swiss-PDBViewer Alternate Protocol: Exploring tertiary structure evolution with Swiss-PDBViewer Basic Protocol 2: Visualizing allostery using PyMOL.
Asunto(s)
Proteínas , Programas Informáticos , Simulación por Computador , HumanosRESUMEN
Widely distributed among prokaryotes, short chain fatty acid kinases provide a path for fatty acid entry into central metabolic pathways. These enzymes catalyze the reversible, ATP-dependent synthesis of acyl-phosphates, which leads to the production of acyl-CoA derivatives by a coordinate acyltransferase. To date, characterized representatives of short chain fatty acid kinases exhibit relatively narrow substrate specificity. In this work, biochemical characterization of a predicted acetate kinase from Rhodobacter sphaeroides reveals a novel enzyme with broad substrate specificity for primary fatty acids of varying lengths (C2--C8).
Asunto(s)
Acetato Quinasa/metabolismo , Rhodobacter sphaeroides/enzimología , Acilcoenzima A/metabolismo , Aciltransferasas/metabolismo , Ácidos Grasos/metabolismo , Especificidad por SustratoRESUMEN
Short and branched chain fatty acid kinases participate in both bacterial anabolic and catabolic processes, including fermentation, through the reversible, ATP-dependent synthesis of acyl phosphates. This study reports biochemical properties of a predicted butyrate kinase from Desulfovibrio vulgaris str. Hildenborough (DvBuk) expressed heterologously and purified from Escherichia coli. Gel filtration chromatography indicates purified DvBuk is active as a dimer. The optimum temperature and pH for DvBuk activity is 44°C and 7.5, respectively. The enzyme displays enhanced thermal stability in the presence of substrates as observed for similar enzymes. Measurement of kcat and KM for various substrates reveals DvBuk exhibits the highest catalytic efficiencies for butyrate, valerate and isobutyrate. In particular, these measurements reveal this enzyme's apparent high affinity for C4 fatty acids relative to other butyrate kinases. These results have implications on structure and function relationships within the ASKHA superfamily of phosphotransferases, particularly regarding the acyl binding pocket, as well as potential physiological roles for this enzyme in Desulfovibrio vulgaris str. Hildenborough.
Asunto(s)
Desulfovibrio vulgaris/enzimología , Fosfotransferasas (aceptor de Grupo Carboxilo)/metabolismo , Proteínas Recombinantes/metabolismo , Cromatografía en Gel , Desulfovibrio vulgaris/genética , Estabilidad de Enzimas , Escherichia coli/genética , Concentración de Iones de Hidrógeno , Fosfotransferasas (aceptor de Grupo Carboxilo)/genética , Fosfotransferasas (aceptor de Grupo Carboxilo)/aislamiento & purificación , Proteínas Recombinantes/genética , Relación Estructura-Actividad , TemperaturaRESUMEN
The genome sequence of the aceticlastic methanoarchaeon Methanosaeta concilii GP6, comprised of a 3,008,626-bp chromosome and an 18,019-bp episome, has been determined and exhibits considerable differences in gene content from that of Methanosaeta thermophila.
Asunto(s)
Genoma Arqueal , Metano/metabolismo , Methanosarcinaceae/genética , Methanosarcinaceae/metabolismo , Secuencia de Bases , ADN de Archaea/genética , Methanosarcinaceae/clasificación , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
Microbial genome sequencing projects have revealed an apparently wide distribution of SmtB/ArsR metal-responsive transcriptional regulators among prokaryotes. Using a position-dependent weight matrix approach, prokaryotic genome sequences were screened for SmtB/ArsR DNA binding sites using data derived from intergenic sequences upstream of orthologous genes encoding these regulators. Sixty SmtB/ArsR operators linked to metal detoxification genes, including nine among various archaeal species, are predicted among 230 annotated and draft prokaryotic genome sequences. Independent multiple sequence alignments of putative operator sites and corresponding winged helix-turn-helix motifs define sequence signatures for the DNA binding activity of this SmtB/ArsR subfamily. Prediction of an archaeal SmtB/ArsR based upon these signature sequences is confirmed using purified Methanosarcina acetivorans C2A protein and electrophoretic mobility shift assays. Tools used in this study have been incorporated into a web application, the Prokaryotic InterGenic Exploration Database (PIGED; http://bioinformatics.uwp.edu/~PIGED/home.htm), facilitating comparable studies. Use of this tool and establishment of orthology based on DNA binding signatures holds promise for deciphering potential cellular roles of various archaeal winged helix-turn-helix transcriptional regulators.
Asunto(s)
Archaea/genética , Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Genes Arqueales , Proteínas Represoras/genética , Transactivadores/genética , Proteínas Bacterianas/análisis , Sitios de Unión , ADN de Archaea/metabolismo , Proteínas de Unión al ADN/análisis , Bases de Datos como Asunto , Filogenia , Células Procariotas/metabolismo , Proteínas Represoras/análisis , Transactivadores/análisis , Factores de Transcripción Winged-Helix/fisiologíaRESUMEN
Prophage loci often remain under-annotated or even unrecognized in prokaryotic genome sequencing projects. A PHP application, Prophage Finder, has been developed and implemented to predict prophage loci, based upon clusters of phage-related gene products encoded within DNA sequences. This application provides results detailing several facets of these clusters to facilitate rapid prediction and analysis of prophage sequences. Prophage Finder was tested using previously annotated prokaryotic genomic sequences with manually curated prophage loci as benchmarks. Additional analyses from Prophage Finder searches of several draft prokaryotic genome sequences are available through the Web site (http://bioinformatics.uwp.edu/~phage/DOEResults.php) to illustrate the potential of this application.
Asunto(s)
ADN Viral/química , Genoma Viral , Profagos/genética , Bacterias/genética , Secuencia de Bases , Simulación por Computador , ADN Viral/genética , Hongos/genética , Modelos Genéticos , Reproducibilidad de los ResultadosRESUMEN
Quantitative gene expression data are often normalized to the expression levels of control or so-called "housekeeping" genes. An inherent assumption in the use of housekeeping genes is that expression of the genes remains constant in the cells or tissues under investigation. Although exceptions to this assumption are well documented, housekeeping genes are of value in fully characterized systems. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the most commonly used housekeeping genes used in comparisons of gene expression data. To investigate the value of GAPDH as a housekeeping gene in human tissues, the expression of GAPDH mRNA was measured in a panel of 72 different pathologically normal human tissue types. Measurements were obtained from 371,088 multiplexed, quantitative real-time RT-PCRs with specific target genes. Significant differences in the expression levels of GAPDH mRNA were observed between tissue types and between donors of the same tissue. A 15-fold difference in GAPDH mRNA copy numbers was observed between the highest and lowest expressing tissue types, skeletal muscle and breast, respectively. No specific effect of either age or gender was observed on GAPDH mRNA expression. These data provide an extensive analysis of GAPDH mRNA expression in human tissues and confirm previous reports of the marked variability of GAPDH expression between tissue types. These data establish comparative levels of expression and can be used to add value to gene expression data in which GAPDH is used as the internal control.
Asunto(s)
Gliceraldehído-3-Fosfato Deshidrogenasas/genética , ARN Mensajero/genética , Actinas/genética , Cadáver , Femenino , Regulación Enzimológica de la Expresión Génica , Humanos , Masculino , Especificidad de Órganos , ARN Mensajero/aislamiento & purificación , Receptores de Transferrina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción GenéticaRESUMEN
Vectors based on lentiviruses are opening up new approaches for the treatment of neurodegenerative diseases. Currently, the equine infectious anaemia virus (EIAV) vector is one of the most attractive gene delivery systems with respect to neuronal tropism. The aim was to validate EIAV-lentiviral vectors as a gene delivery system for neurotrophic factor genes in an animal model of Parkinson's disease. EIAV carrying the glial cell line-derived neurotrophic factor (GDNF) gene was unilaterally injected into rat striatum and above the substantia nigra (SN). One week later, the rats received a 6-OHDA lesion into the ipsilateral striatum. GDNF delivery led to extensive expression of GDNF protein within the striatum. In addition, near complete protection against dopaminergic cell death was observed in the GDNF-treated group.
Asunto(s)
Terapia Genética/métodos , Vectores Genéticos/uso terapéutico , Factores de Crecimiento Nervioso/biosíntesis , Fármacos Neuroprotectores/uso terapéutico , Enfermedad de Parkinson/prevención & control , Animales , Femenino , Vectores Genéticos/genética , Factor Neurotrófico Derivado de la Línea Celular Glial , Virus de la Anemia Infecciosa Equina/genética , Factores de Crecimiento Nervioso/genética , Enfermedad de Parkinson/genética , Ratas , Ratas Sprague-DawleyRESUMEN
Parkinson's disease (PD) is a neurodegenerative disorder characterized by the selective loss of dopaminergic neurons in the substantia nigra. This loss leads to complete dopamine depletion in the striatum and severe motor impairment. It has been demonstrated previously that a lentiviral vector system based on equine infectious anemia virus (EIAV) gives rise to highly efficient and sustained transduction of neurons in the rat brain. Therefore, a dopamine replacement strategy using EIAV has been investigated as a treatment in the 6-hydroxydopamine (6-OHDA) animal model of PD. A self-inactivating EIAV minimal lentiviral vector that expresses tyrosine hydroxylase (TH), aromatic amino acid dopa decarboxylase (AADC), and GTP cyclohydrolase 1 (CH1) in a single transcription unit has been generated. In cultured striatal neurons transduced with this vector, TH, AADC, and CH1 proteins can all be detected. After stereotactic delivery into the dopamine-denervated striatum of the 6-OHDA-lesioned rat, sustained expression of each enzyme and effective production of catecholamines were detected, resulting in significant reduction of apomorphine-induced motor asymmetry compared with control animals (p < 0.003). Expression of each enzyme in the striatum was observed for up to 5 months after injection. These data indicate that the delivery of three catecholaminergic synthetic enzymes by a single lentiviral vector can achieve functional improvement and thus open the potential for the use of this vector for gene therapy of late-stage PD patients.
Asunto(s)
Cuerpo Estriado/efectos de los fármacos , Dopamina/biosíntesis , Vectores Genéticos/administración & dosificación , Trastornos Parkinsonianos/terapia , Animales , Descarboxilasas de Aminoácido-L-Aromático/administración & dosificación , Descarboxilasas de Aminoácido-L-Aromático/biosíntesis , Descarboxilasas de Aminoácido-L-Aromático/genética , Catecolaminas/metabolismo , Células Cultivadas , Cuerpo Estriado/patología , Cuerpo Estriado/fisiopatología , Modelos Animales de Enfermedad , GTP Ciclohidrolasa/administración & dosificación , GTP Ciclohidrolasa/biosíntesis , GTP Ciclohidrolasa/genética , Expresión Génica/efectos de los fármacos , Técnicas de Transferencia de Gen , Genes/genética , Terapia Genética/métodos , Vectores Genéticos/genética , Humanos , Riñón/citología , Riñón/metabolismo , Lentivirus/genética , Masculino , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Oxidopamina , Trastornos Parkinsonianos/inducido químicamente , Trastornos Parkinsonianos/fisiopatología , Ratas , Ratas Wistar , Recuperación de la Función/efectos de los fármacos , Transgenes , Resultado del Tratamiento , Tirosina 3-Monooxigenasa/administración & dosificación , Tirosina 3-Monooxigenasa/biosíntesis , Tirosina 3-Monooxigenasa/genéticaRESUMEN
Retinoic acid, acting through the nuclear retinoic acid receptor beta2 (RARbeta2), stimulates neurite outgrowth from peripheral nervous system tissue that has the capacity to regenerate neurites, namely, embryonic and adult dorsal root ganglia. Similarly, in central nervous system tissue that can regenerate, namely, embryonic mouse spinal cord, retinoic acid also stimulates neurite outgrowth and RARbeta2 is upregulated. By contrast, in the adult mouse spinal cord, which cannot regenerate, no such upregulation of RARbeta2 by retinoic acid is observed and no neurites are extended in vitro. To test our hypothesis that the upregulation of RARbeta2 is crucial to neurite regeneration, we have transduced adult mouse or rat spinal cord in vitro with a minimal equine infectious anaemia virus vector expressing RARbeta2. After transduction, prolific neurite outgrowth occurs. Outgrowth does not occur when the cord is transduced with a different isoform of RARbeta nor does it occur following treatment with nerve growth factor. These data demonstrate that RARbeta2 is involved in neurite outgrowth, at least in vitro, and that this gene may in the future be of some therapeutic use.
Asunto(s)
Diferenciación Celular/genética , Factores de Crecimiento Nervioso/metabolismo , Regeneración Nerviosa/genética , Neuritas/metabolismo , Receptores de Ácido Retinoico/metabolismo , Médula Espinal/crecimiento & desarrollo , Tretinoina/metabolismo , Envejecimiento/genética , Envejecimiento/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Feto , Regulación del Desarrollo de la Expresión Génica/genética , Vectores Genéticos/genética , Vectores Genéticos/uso terapéutico , Inmunohistoquímica , Lentivirus/genética , Ratones , Factores de Crecimiento Nervioso/farmacología , Neuritas/efectos de los fármacos , Neuritas/ultraestructura , Proteínas de Neurofilamentos/metabolismo , Ratas , Receptores de Ácido Retinoico/efectos de los fármacos , Receptores de Ácido Retinoico/genética , Médula Espinal/citología , Médula Espinal/metabolismo , Transducción Genética , Tretinoina/farmacologíaRESUMEN
Methanogenesis, the biological production of methane, plays a pivotal role in the global carbon cycle and contributes significantly to global warming. The majority of methane in nature is derived from acetate. Here we report the complete genome sequence of an acetate-utilizing methanogen, Methanosarcina acetivorans C2A. Methanosarcineae are the most metabolically diverse methanogens, thrive in a broad range of environments, and are unique among the Archaea in forming complex multicellular structures. This diversity is reflected in the genome of M. acetivorans. At 5,751,492 base pairs it is by far the largest known archaeal genome. The 4524 open reading frames code for a strikingly wide and unanticipated variety of metabolic and cellular capabilities. The presence of novel methyltransferases indicates the likelihood of undiscovered natural energy sources for methanogenesis, whereas the presence of single-subunit carbon monoxide dehydrogenases raises the possibility of nonmethanogenic growth. Although motility has not been observed in any Methanosarcineae, a flagellin gene cluster and two complete chemotaxis gene clusters were identified. The availability of genetic methods, coupled with its physiological and metabolic diversity, makes M. acetivorans a powerful model organism for the study of archaeal biology. [Sequence, data, annotations and analyses are available at http://www-genome.wi.mit.edu/.]
Asunto(s)
Variación Genética , Genoma Arqueal , Methanosarcina/genética , Proteínas Arqueales/genética , Proteínas Arqueales/fisiología , Monóxido de Carbono/metabolismo , Movimiento Celular/genética , Movimiento Celular/fisiología , Euryarchaeota/metabolismo , Regulación de la Expresión Génica Arqueal/fisiología , Hidrógeno/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Methanosarcina/fisiología , Datos de Secuencia Molecular , Familia de Multigenes/genética , Familia de Multigenes/fisiología , Fijación del Nitrógeno/genética , Fijación del Nitrógeno/fisiología , Oxígeno/metabolismo , Polisacáridos/biosíntesis , Polisacáridos/genética , Biosíntesis de Proteínas/fisiología , Origen de Réplica/genética , Origen de Réplica/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Transcripción GenéticaRESUMEN
The presence of a glutathione-dependent pathway for formaldehyde oxidation in the facultative phototroph Rhodobacter sphaeroides has allowed the identification of gene products that contribute to formaldehyde metabolism. Mutants lacking the glutathione-dependent formaldehyde dehydrogenase (GSH-FDH) are sensitive to metabolic sources of formaldehyde, like methanol. This growth phenotype is correlated with a defect in formaldehyde oxidation. Additional methanol-sensitive mutants were isolated that contained Tn5 insertions in pntA, which encodes the alpha subunit of the membrane-bound pyridine nucleotide transhydrogenase. Mutants lacking transhydrogenase activity have phenotypic and physiological characteristics that are different from those that lack GSH-FDH activity. For example, cells lacking transhydrogenase activity can utilize methanol as a sole carbon source in the absence of oxygen and do not display a formaldehyde oxidation defect, as determined by whole-cell (13)C-nuclear magnetic resonance. Since transhydrogenase can be a major source of NADPH, loss of this enzyme could result in a requirement for another source for this compound. Evidence supporting this hypothesis includes increased specific activities of other NADPH-producing enzymes and the finding that glucose utilization by the Entner-Doudoroff pathway restores aerobic methanol resistance to cells lacking transhydrogenase activity. Mutants lacking transhydrogenase activity also have higher levels of glutathione disulfide under aerobic conditions, so it is consistent that this strain has increased sensitivity to oxidative stress agents like diamide, which are known to alter the oxidation reduction state of the glutathione pool. A model will be presented to explain the role of transhydrogenase under aerobic conditions when cells need glutathione both for GSH-FDH activity and to repair oxidatively damaged proteins.