RESUMEN
Infections caused by Mycobacterium abscessus are difficult to treat due to its intrinsic resistance to most antibiotics. Formation of biofilms and the capacity of M. abscessus to survive inside host phagocytes further complicate eradication. Herein, we explored whether addition of a carbamate-linked group at the C25 position of rifamycin SV blocks enzymatic inactivation by ArrMab, an ADP-ribosyltransferase conferring resistance to rifampicin (RMP). Unlike RMP, 5j, a benzyl piperidine rifamycin derivative with a morpholino substituted C3 position and a naphthoquinone core, is not modified by purified ArrMab. Additionally, we show that the ArrMab D82 residue is essential for catalytic activity. Thermal profiling of ArrMab in the presence of 5j, RMP, or rifabutin shows that 5j does not bind to ArrMab. We found that the activity of 5j is comparable to amikacin against M. abscessus planktonic cultures and pellicles. Critically, 5j also exerts potent antimicrobial activity against M. abscessus in human macrophages and shows synergistic activity with amikacin and azithromycin.
RESUMEN
As the scientific community strives to make published results more transparent and reliable, it has become obvious that poor data reproducibility can often be attributed to insufficient quality control of experimental reagents. In this context, proteins and peptides reagents require much stricter quality controls than those routinely performed on them in a significant proportion of research laboratories. Members of the ARBRE-MOBIEU and the P4EU networks have combined their expertise to generate guidelines for the evaluation of purified proteins used in life sciences and medical trials. These networks, representing more than 150 laboratories specialized in protein production and/or protein molecular biophysics, have implemented such guidelines in their respective laboratories. Over a one-year period, the network members evaluated the contribution these guidelines made toward obtaining more productive, robust and reproducible research by correlating the applied quality controls to given samples with the reliability and reproducibility of the scientific data obtained using these samples in follow-up experiments. The results indicate that QC guideline implementation facilitates the optimization of the protein purification process and improves the reliability of downstream experiments. It seems, therefore, that investing in protein QC might be advantageous to all the stakeholders in life sciences (researchers, editors, and funding agencies alike), because this practice improves data veracity and minimizes loss of valuable time and resources. In the light of these conclusions, the network members suggest that the implementation of these simple QC guidelines should become minimal reporting practice in the publication of data derived from the use of protein and peptide reagents.
Asunto(s)
Exactitud de los Datos , Control de Calidad , Reproducibilidad de los ResultadosRESUMEN
Structural and biophysical characterization of molecular mechanisms of disease-causing pathogens, such as Mycobacterium tuberculosis, often requires recombinant expression of large amounts highly pure protein. For the production of mycobacterial proteins, overexpression in the fast-growing and non-pathogenic species Mycobacterium smegmatis has several benefits over the standard Escherichia coli expression strains. However, unlike for E. coli, the range of expression vectors currently available is limited. Here we describe the development of the pMy vector series, a set of expression plasmids for recombinant production of single proteins and protein complexes in M. smegmatis. By incorporating an alternative selection marker, we show that these plasmids can also be used for co-expression studies. All vectors in the pMy vector series are available in the Addgene repository (www.addgene.com).
Asunto(s)
Clonación Molecular , Vectores Genéticos , Mycobacterium smegmatis , Plásmidos , Escherichia coli/genética , Escherichia coli/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Plásmidos/genética , Plásmidos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
Toxin-antitoxin (TA) systems regulate fundamental cellular processes in bacteria and represent potential therapeutic targets. We report a new RES-Xre TA system in multiple human pathogens, including Mycobacterium tuberculosis. The toxin, MbcT, is bactericidal unless neutralized by its antitoxin MbcA. To investigate the mechanism, we solved the 1.8 Å-resolution crystal structure of the MbcTA complex. We found that MbcT resembles secreted NAD+-dependent bacterial exotoxins, such as diphtheria toxin. Indeed, MbcT catalyzes NAD+ degradation in vitro and in vivo. Unexpectedly, the reaction is stimulated by inorganic phosphate, and our data reveal that MbcT is a NAD+ phosphorylase. In the absence of MbcA, MbcT triggers rapid M. tuberculosis cell death, which reduces mycobacterial survival in macrophages and prolongs the survival of infected mice. Our study expands the molecular activities employed by bacterial TA modules and uncovers a new class of enzymes that could be exploited to treat tuberculosis and other infectious diseases.
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Antitoxinas/metabolismo , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Macrófagos/microbiología , Mycobacterium tuberculosis/enzimología , Fosforilasas/metabolismo , Sistemas Toxina-Antitoxina , Tuberculosis/microbiología , Animales , Antibióticos Antituberculosos/farmacología , Antitoxinas/química , Antitoxinas/genética , Carga Bacteriana , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Interacciones Huésped-Patógeno , Humanos , Cinética , Macrófagos/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones SCID , Ratones Transgénicos , Viabilidad Microbiana , Modelos Moleculares , Mycobacterium smegmatis/enzimología , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/patogenicidad , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , NAD/metabolismo , Fosforilasas/química , Fosforilasas/genética , Conformación Proteica , Sistemas Toxina-Antitoxina/genética , Tuberculosis/tratamiento farmacológicoRESUMEN
Type VII secretion systems (ESX) are responsible for transport of multiple proteins in mycobacteria. How different ESX systems achieve specific secretion of cognate substrates remains elusive. In the ESX systems, the cytoplasmic chaperone EspG forms complexes with heterodimeric PE-PPE substrates that are secreted from the cells or remain associated with the cell surface. Here we report the crystal structure of the EspG1 chaperone from the ESX-1 system determined using a fusion strategy with T4 lysozyme. EspG1 adopts a quasi 2-fold symmetric structure that consists of a central ß-sheet and two α-helical bundles. In addition, we describe the structures of EspG3 chaperones from four different crystal forms. Alternate conformations of the putative PE-PPE binding site are revealed by comparison of the available EspG3 structures. Analysis of EspG1, EspG3, and EspG5 chaperones using small-angle X-ray scattering reveals that EspG1 and EspG3 chaperones form dimers in solution, which we observed in several of our crystal forms. Finally, we propose a model of the ESX-3 specific EspG3-PE5-PPE4 complex based on the small-angle X-ray scattering analysis.
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Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Chaperonas Moleculares/metabolismo , Mycobacterium tuberculosis/metabolismo , Sistemas de Secreción Tipo VII/metabolismo , Factores de Virulencia/metabolismo , Secuencia de Aminoácidos , Sitios de Unión/fisiología , Conformación Proteica , Conformación Proteica en Hélice alfa/fisiología , Conformación Proteica en Lámina beta/fisiologíaRESUMEN
Mycobacteria are characterized by their impermeable outer membrane, which is rich in mycolic acids1. To transport substrates across this complex cell envelope, mycobacteria rely on type VII (also known as ESX) secretion systems2. In Mycobacterium tuberculosis, these ESX systems are essential for growth and full virulence and therefore represent an attractive target for anti-tuberculosis drugs3. However, the molecular details underlying type VII secretion are largely unknown, due to a lack of structural information. Here, we report the molecular architecture of the ESX-5 membrane complex from Mycobacterium xenopi determined at 13â Å resolution by electron microscopy. The four core proteins of the ESX-5 complex (EccB5, EccC5, EccD5 and EccE5) assemble with equimolar stoichiometry into an oligomeric assembly that displays six-fold symmetry. This membrane-associated complex seems to be embedded exclusively in the inner membrane, which indicates that additional components are required to translocate substrates across the mycobacterial outer membrane. Furthermore, the extended cytosolic domains of the EccC ATPase, which interact with secretion effectors, are highly flexible, suggesting an as yet unseen mode of substrate interaction. Comparison of our results with known structures of other bacterial secretion systems demonstrates that the architecture of type VII secretion system is fundamentally different, suggesting an alternative secretion mechanism.
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Membrana Celular/metabolismo , Mycobacterium tuberculosis/química , Sistemas de Secreción Tipo VII/química , Sistemas de Secreción Tipo VII/genética , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Membrana Celular/química , Pared Celular/metabolismo , Tomografía con Microscopio Electrónico , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Sistemas de Secreción Tipo VII/ultraestructuraRESUMEN
PURPOSE: Tuberculosis is still a major threat to global health. New tools and strategies to produce disease-related proteins are quintessential for the development of novel vaccines and diagnostic markers. EXPERIMENTAL DESIGN: To obtain recombinant proteins from Mycobacterium tuberculosis (Mtb) for use in clinical applications, a standardized procedure was developed that includes subcloning, protein expression in Mycobacterium smegmatis and protein purification using chromatography. The potential for the different protein targets to serve as diagnostic markers for tuberculosis was established using multiplex immunoassays. RESULTS: Twelve soluble proteins from Mtb, including one protein complex, were purified to near-homogeneity following recombinant expression in M. smegmatis. Protein purity was assessed both by size exclusion chromatography and MS. Multiplex serological testing of the final protein preparations showed that all but one protein displayed a clear antibody response in serum samples from 278 tuberculosis patients. CONCLUSION AND CLINICAL RELEVANCE: The established workflow comprises a simple, cost-effective, and scalable pipeline for production of soluble proteins from Mtb and can be used to prioritize immunogenic proteins suitable for use as diagnostic markers.
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Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteómica/normas , Anticuerpos Antibacterianos/sangre , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Clonación Molecular , Regulación Bacteriana de la Expresión Génica , Humanos , Espectrometría de Masas , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/fisiología , Estándares de Referencia , Solubilidad , Tuberculosis/sangreRESUMEN
A variety of methods to create specific constructs for protein expression, in a broad range of organisms, are available nowadays. Restriction enzyme-free, ligation-independent and recombinase-based cloning methods have enabled high-throughput protein expression for structural and functional studies. These methods are also instrumental for modification of target genes including gene truncations, site-specific mutagenesis and domain swapping. Here, we describe the most common cloning techniques that are currently at hand for recombinant protein expression studies, including a brief overview of techniques associated with co-expression experiments. We also provide an inventory of many of the available reagents for the various cloning methods, and an overview for some computational tools that can help with the design of expression constructs.
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Clonación Molecular/métodos , Proteínas Recombinantes/genética , Animales , Enzimas de Restricción del ADN/metabolismo , Expresión Génica , Humanos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/metabolismo , Eliminación de SecuenciaRESUMEN
Gene synthesis enables the exploitation of the degeneracy of the genetic code to boost expression of recombinant protein targets for structural studies. This has created new opportunities to obtain structural information on proteins that are normally present in low abundance. Unfortunately, synthetic gene expression occasionally leads to insoluble or misfolded proteins. This could be remedied by recent insights in the effect of codon usage on translation initiation and elongation. In this review, we discuss the interplay between optimal gene and vector design to enhance expression in a particular host and highlight the benefits and potential pitfalls associated with protein expression from synthetic genes.
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Ingeniería Genética/métodos , Proteínas Recombinantes/genética , Codón/genética , Vectores Genéticos/genética , Pliegue de Proteína , ARN Mensajero/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/químicaRESUMEN
UNLABELLED: Regulatory pathways that drive early hematogenous dissemination of tumor cells are insufficiently defined. Here, we used the presence of disseminated tumor cells (DTC) in the bone marrow to define patients with early disseminated breast cancer and identified low retinoic acid-induced 2 (RAI2) expression to be significantly associated with DTC status. Low RAI2 expression was also shown to be an independent poor prognostic factor in 10 different cancer datasets. Depletion of RAI2 protein in luminal breast cancer cell lines resulted in dedifferentiation marked by downregulation of ERα, FOXA1, and GATA3, together with increased invasiveness and activation of AKT signaling. Functional analysis of the previously uncharacterized RAI2 protein revealed molecular interaction with CtBP transcriptional regulators and an overlapping function in controlling the expression of a number of key target genes involved in breast cancer. These results suggest that RAI2 is a new metastasis-associated protein that sustains differentiation of luminal breast epithelial cells. SIGNIFICANCE: We identified downregulation of RAI2 as a novel metastasis-associated genetic alteration especially associated with early occurring bone metastasis in ERα-positive breast tumors. We specified the role of the RAI2 protein to function as a transcriptional regulator that controls the expression of several key regulators of breast epithelial integrity and cancer.
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Médula Ósea/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Células Neoplásicas Circulantes/patología , Proteínas/genética , Oxidorreductasas de Alcohol/metabolismo , Secuencia de Aminoácidos , Neoplasias de la Mama/mortalidad , Línea Celular Tumoral , Análisis por Conglomerados , Proteínas de Unión al ADN/metabolismo , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular , Datos de Secuencia Molecular , Pronóstico , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas/química , Proteínas/metabolismo , Reproducibilidad de los Resultados , Alineación de Secuencia , TranscriptomaRESUMEN
The growth or virulence of Mycobacterium tuberculosis bacilli depends on homologous type VII secretion systems, ESX-1, ESX-3 and ESX-5, which export a number of protein effectors across membranes to the bacterial surface and environment. PE and PPE proteins represent two large families of highly polymorphic proteins that are secreted by these ESX systems. Recently, it was shown that these proteins require system-specific cytoplasmic chaperones for secretion. Here, we report the crystal structure of M. tuberculosisâ ESX-5-secreted PE25-PPE41 heterodimer in complex with the cytoplasmic chaperone EspG(5). EspG(5) represents a novel fold that is unrelated to previously characterized secretion chaperones. Functional analysis of the EspG(5) -binding region uncovered a hydrophobic patch on PPE41 that promotes dimer aggregation, and the chaperone effectively abolishes this process. We show that PPE41 contains a characteristic chaperone-binding sequence, the hh motif, which is highly conserved among ESX-1-, ESX-3- and ESX-5-specific PPE proteins. Disrupting the interaction between EspG(5) and three different PPE target proteins by introducing different point mutations generally affected protein secretion. We further demonstrate that the EspG(5) chaperone plays an important role in the ESX secretion mechanism by keeping aggregation-prone PE-PPE proteins in their soluble state.
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Proteínas Bacterianas/química , Sistemas de Secreción Bacterianos , Chaperonas Moleculares/química , Mycobacterium tuberculosis/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Análisis Mutacional de ADN , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Mycobacterium tuberculosis/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Transporte de ProteínasRESUMEN
Mycobacteria use the dedicated type VII protein secretion systems ESX-1 and ESX-5 to secrete virulence factors across their highly hydrophobic cell envelope. The substrates of these systems include the large mycobacterial PE and PPE protein families, which are named after their characteristic Pro-Glu and Pro-Pro-Glu motifs. Pathogenic mycobacteria secrete large numbers of PE/PPE proteins via the major export pathway, ESX-5. In addition, a few PE/PPE proteins have been shown to be exported by ESX-1. It is not known how ESX-1 and ESX-5 recognize their cognate PE/PPE substrates. In this work, we investigated the function of the cytosolic protein EspG(5), which is essential for ESX-5-mediated secretion in Mycobacterium marinum, but for which the role in secretion is not known. By performing protein co-purifications, we show that EspG(5) interacts with several PPE proteins and a PE/PPE complex that is secreted by ESX-5, but not with the unrelated ESX-5 substrate EsxN or with PE/PPE proteins secreted by ESX-1. Conversely, the ESX-1 paralogue EspG(1) interacted with a PE/PPE couple secreted by ESX-1, but not with PE/PPE substrates of ESX-5. Furthermore, structural analysis of the complex formed by EspG(5) and PE/PPE indicates that these proteins interact in a 1:1:1 ratio. In conclusion, our study shows that EspG(5) and EspG(1) interact specifically with PE/PPE proteins that are secreted via their own ESX systems and suggests that EspG proteins are specific chaperones for the type VII pathway.
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Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/química , Chaperonas Moleculares/química , Mycobacterium marinum/enzimología , Clonación Molecular , Citosol/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Prueba de Complementación Genética , Espectrometría de Masas/métodos , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Mycobacterium marinum/metabolismo , Níquel/química , Plásmidos/metabolismo , Estructura Terciaria de Proteína , Vías Secretoras , Espectrometría de Masas en Tándem/métodosRESUMEN
CONTEXT: Autosomal dominant inactivating sprouty-related EVH1 domain-containing protein 1 (SPRED1) mutations have recently been described in individuals presenting mainly with café au lait macules (CALMs), axillary freckling, and macrocephaly. The extent of the clinical spectrum of this new disorder needs further delineation. OBJECTIVE: To determine the frequency, mutational spectrum, and phenotype of neurofibromatosis type 1-like syndrome (NFLS) in a large cohort of patients. DESIGN, SETTING, AND PARTICIPANTS: In a cross-sectional study, 23 unrelated probands carrying a SPRED1 mutation identified through clinical testing participated with their families in a genotype-phenotype study (2007-2008). In a second cross-sectional study, 1318 unrelated anonymous samples collected in 2003-2007 from patients with a broad range of signs typically found in neurofibromatosis type 1 (NF1) but no detectable NF1 germline mutation underwent SPRED1 mutation analysis. MAIN OUTCOME MEASURES: Comparison of aggregated clinical features in patients with or without a SPRED1 or NF1 mutation. Functional assays were used to evaluate the pathogenicity of missense mutations. RESULTS: Among 42 SPRED1-positive individuals from the clinical cohort, 20 (48%; 95% confidence interval [CI], 32%-64%) fulfilled National Institutes of Health (NIH) NF1 diagnostic criteria based on the presence of more than 5 CALMs with or without freckling or an NF1-compatible family history. None of the 42 SPRED1-positive individuals (0%; 95% CI, 0%-7%) had discrete cutaneous or plexiform neurofibromas, typical NF1 osseous lesions, or symptomatic optic pathway gliomas. In the anonymous cohort of 1318 individuals, 34 different SPRED1 mutations in 43 probands were identified: 27 pathogenic mutations in 34 probands and 7 probable nonpathogenic missense mutations in 9 probands. Of 94 probands with familial CALMs with or without freckling and no other NF1 features, 69 (73%; 95% CI, 63%-80%) had an NF1 mutation and 18 (19%; 95% CI, 12%-29%) had a pathogenic SPRED1 mutation. In the anonymous cohort, 1.9% (95% CI, 1.2%-2.9%) of individuals with the clinical diagnosis of NF1 according to the NIH criteria had NFLS. CONCLUSIONS: A high SPRED1 mutation detection rate was found in NF1 mutation-negative families with an autosomal dominant phenotype of CALMs with or without freckling and no other NF1 features. Among individuals in this study, NFLS was not associated with the peripheral and central nervous system tumors seen in NF1.
Asunto(s)
Manchas Café con Leche/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Neurofibromatosis 1/diagnóstico , Neurofibromatosis 1/genética , Proteínas Adaptadoras Transductoras de Señales , Adolescente , Adulto , Niño , Preescolar , Estudios Transversales , Análisis Mutacional de ADN , Femenino , Genes de Neurofibromatosis 1 , Estudios de Asociación Genética , Pruebas Genéticas , Humanos , Lactante , Masculino , Persona de Mediana Edad , Mutación , Mutación Missense , Fenotipo , Síndrome , Adulto JovenRESUMEN
Loss-of-function mutations in the NF1 tumor suppressor result in deregulated Ras signaling and drive tumorigenesis in the familial cancer syndrome neurofibromatosis type I. However, the extent to which NF1 inactivation promotes sporadic tumorigenesis is unknown. Here we report that NF1 is inactivated in sporadic gliomas via two mechanisms: excessive proteasomal degradation and genetic loss. NF1 protein destabilization is triggered by the hyperactivation of protein kinase C (PKC) and confers sensitivity to PKC inhibitors. However, complete genetic loss, which only occurs when p53 is inactivated, mediates sensitivity to mTOR inhibitors. These studies reveal an expanding role for NF1 inactivation in sporadic gliomagenesis and illustrate how different mechanisms of inactivation are utilized in genetically distinct tumors, which consequently impacts therapeutic sensitivity.
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Genes Supresores de Tumor , Glioblastoma/genética , Glioma/genética , Mutación , Neurofibromatosis 1/genética , Neurofibromina 1/antagonistas & inhibidores , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Células 3T3 , Animales , Regulación hacia Abajo , Genes p53 , Genes ras , Glioblastoma/enzimología , Glioma/enzimología , Ratones , Neurofibromatosis 1/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
The brain-specific synaptic guanosine triphosphatase (GTPase)-activating protein (SynGAP) is important in synaptic plasticity. It shows dual specificity for the small guanine nucleotide-binding proteins Rap and Ras. Here, we show that RapGAP activity of SynGAP requires its C2 domain. In contrast to the isolated GAP domain, which does not show any detectable RapGAP activity, a fragment comprising the C2 and GAP domains (C2-GAP) stimulates the intrinsic GTPase reaction of Rap by approximately 1 x 10(4). The C2-GAP crystal structure, complemented by modelling and biochemical analyses, favours a concerted movement of the C2 domain towards the switch II region of Rap to assist in GTPase stimulation. Our data support a catalytic mechanism similar to that of canonical RasGAPs and distinct from the canonical RapGAPs. SynGAP presents the first example, to our knowledge, of a GAP that uses a second domain for catalytic activity, thus pointing to a new function of C2 domains.
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Encéfalo/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Modelos Moleculares , Estructura Terciaria de Proteína , Proteínas de Unión al GTP rap/metabolismo , Catálisis , Cristalización , Proteínas Activadoras de GTPasa/genética , Humanos , Cinética , Proteínas Activadoras de ras GTPasaRESUMEN
Costello syndrome is a mental retardation syndrome characterized by high birth weight, postnatal growth retardation, coarse face, loose skin, cardiovascular problems, and tumor predisposition. De novo heterozygous missense mutations in HRAS codon 12 and 13 disturbing the intrinsic GTP hydrolysis cause Costello syndrome. We report a patient with typical Costello syndrome and a novel heterozygous missense mutation in codon 117 (c.350A>G, p.Lys117Arg) of the HRAS gene, resulting in constitutive activation of the RAS/MAPK pathway similar to the typical p.Gly12Ser and p.Gly12Ala mutations. Recombinant HRAS p.Lys117Arg demonstrates normal intrinsic GTP hydrolysis and responsiveness to GTPase-activating proteins, but the nucleotide dissociation rate is increased 80-fold. Consistent with the biochemical data, the crystal structure of the p.Lys117Arg mutant indicates an altered interaction pattern of the side chain that is associated with unfavorable nucleotide binding properties. Together, these data show that a RAS mutation that only perturbs guanine nucleotide binding has similar functional consequences as mutations that impair GTP hydrolysis and causes human disease.
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Anomalías Múltiples/genética , Arginina/genética , Lisina/genética , Mutación/genética , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/genética , Secuencia de Bases , Línea Celular , Proliferación Celular , Niño , Preescolar , Codón , Análisis Mutacional de ADN , Guanosina Difosfato/metabolismo , Humanos , Hidrólisis , Lactante , Recién Nacido , Datos de Secuencia Molecular , Proteínas Mutantes/química , SíndromeRESUMEN
Bacteriocin LlpA, produced by Pseudomonas sp. strain BW11M1, is a peculiar antibacterial protein due to its homology to mannose-binding lectins mostly found in monocots (A. H. A. Parret, G. Schoofs, P. Proost, and R. De Mot, J. Bacteriol. 185:897-908, 2003). Biocontrol strain Pseudomonas fluorescens Pf-5 contains two llpA-like genes, named llpA1(Pf-5) and llpA2(Pf-5). Recombinant Escherichia coli cells expressing llpA1(Pf-5) or llpA2(Pf-5) acquired bacteriocin activity and secreted a 31-kDa protein cross-reacting with LlpA(BW11M1) antibodies. Antibacterial activity of the recombinant proteins was evidenced by gel overlay assays. Analysis of the antimicrobial spectrum indicated that LlpA1(Pf-5) and LlpA2(Pf-5) are able to inhibit P. fluorescens strains, as well as the related mushroom pathogen Pseudomonas tolaasii. LlpA-type bacteriocins are characterized by a domain structure consisting of tandem monocot mannose-binding lectin (MMBL) domains. Molecular phylogeny of these MMBL domains suggests that the individual MMBL domains within an LlpA protein have evolved separately toward a specific, as yet unknown, function or, alternatively, were acquired from different ancestral sources. Our observations are consistent with earlier observations, which hinted that MMBL-like bacteriocins represent a new family of antibacterial proteins, probably with a novel mode of action.
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Bacteriocinas/metabolismo , Lectinas de Unión a Manosa/química , Control Biológico de Vectores , Enfermedades de las Plantas/microbiología , Pseudomonas fluorescens/metabolismo , Pseudomonas/crecimiento & desarrollo , Secuencia de Aminoácidos , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Bacteriocinas/química , Bacteriocinas/genética , Bacteriocinas/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Pseudomonas/efectos de los fármacos , Pseudomonas fluorescens/efectos de los fármacos , Pseudomonas fluorescens/genética , Pseudomonas fluorescens/crecimiento & desarrolloRESUMEN
Pseudomonas sp. BW11M1 produces a novel type of bacteriocin that inhibits the growth of Pseudomonas putida GR12-2R3 and some phytopathogenic fluorescent Pseudomonas. A collection of mutants was screened for altered bacteriocin production phenotypes. Strongly reduced bacteriocin production was found to be caused by inactivation of the recA gene or the spoT gene. Conversely, in a recJ mutant, the bacteriocin was constitutively overproduced. The same phenotype was observed for a mutant hit in a gene of unknown function. The predicted gene product belongs to a distinct subgroup of prokaryotic helicase-like proteins within the SWI/SNF family of regulatory proteins. One mutant that also exhibited a bacteriocin overproducer phenotype was deficient in the production of the peptidoglycan-associated lipoprotein OprL. This study shows that various environmental stress response pathways are involved in controlling expression of the Pseudomonas sp. BW11M1 bacteriocin.
Asunto(s)
Bacteriocinas/metabolismo , Regulación Bacteriana de la Expresión Génica , Pseudomonas putida/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Bacteriocinas/química , Bacteriocinas/aislamiento & purificación , Proteínas de Escherichia coli , Lipoproteínas/genética , Mutación , Peptidoglicano , Pseudomonas putida/genéticaRESUMEN
LlpA is a bacteriocin produced by Pseudomonas sp. BW11M1 that shows remarkable similarity to a family of mannose-binding plant lectins. A His-tagged version of LlpA was recombinantly produced in Escherichia coli and purified to homogeneity. Single crystals were grown by vapour diffusion and belong to space group P2(1)2(1)2, with unit-cell parameters a = 150.5, b = 154.5, c = 34.2 A. The crystals diffract to at least 2.2 A using synchrotron radiation.