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Mycobacterium bovis BCG is the only vaccine against tuberculosis. The variable forms of cultivation throughout the years, before seed-lots were developed, allowed in vitro evolution of the original strain, generating a family of vaccines with different phenotypic and genotypic characteristics. Molecular studies revealed regions of difference (RDs) in the genomes of the various BCG strains. This work aims to characterize the gene pair rv3407-rv3408 (vapB47-vapC47), coding for a toxin-antitoxin system of the VapBC family, and to evaluate possible transcriptional effects due to the adjacent BCG Moreau-specific genomic deletion RD16. We show that these genes are co-transcribed in BCG strains Moreau and Pasteur, and that the inactivation of an upstream transcriptional repressor (Rv3405c) due to RD16 has a polar effect, leading to increased vapBC47 expression. Furthermore, we detect VapB47 DNA binding in vitro, dependent on a 5' vapB47 sequence that contributes to a palindrome, spanning the promoter and coding region. Our data shed light on the regulation of VapBC systems and on the impact of the BCG Moreau RD16 deletion in the expression of adjacent genes, contributing to a better understanding of BCG Moreau physiology.
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Autoimmunity may have its origins of early repertoire selection in developmental B cells. Such a primary repertoire is probably shaped by selecting B cells that can efficiently perform productive signaling, stimulated by self-antigens in the bone marrow, such as DNA. In support of that idea, we previously found a V segment from VH10 family that can form antibodies that bind to DNA independent of CDR3 usage. In this paper we designed four antibody fragments in a novel single-chain pre-BCR (scpre-BCR) format containing germinal V gene segments from families known to bind DNA (VH10) or not (VH4) connected to a murine surrogate light chain (SLC), lacking the highly charged unique region (UR), by a hydrophilic peptide linker. We also tested the influence of CDR2 on DNA reactivity by shuffling the CDR2 loop. The scpre-BCRs were expressed in bacteria. VH10 bearing scpre-BCR could bind DNA, while scpre-BCR carrying the VH4 segment did not. The CDR2 loop shuffling hampered VH10 reactivity while displaying a gain-of-function in the nonbinding VH4 germline. We modeled the binding sites demonstrating the conservation of a positivity charged pocket in the VH10 CDR2 as the possible cross-reactive structural element. We presented evidence of DNA reactivity hardwired in a V gene, suggesting a structural mechanism for innate autoreactivity. Therefore, while autoreactivity to DNA can lead to autoimmunity, efficiently signaling for B cell development is likely a trade-off mechanism leading to the selection of potentially autoreactive repertoires.
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Región Variable de Inmunoglobulina/genética , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/inmunología , Secuencia de Aminoácidos/genética , Animales , Anticuerpos Antinucleares/genética , Arginina/genética , Arginina/metabolismo , Autoantígenos/genética , Autoinmunidad/inmunología , Secuencia de Bases/genética , ADN/inmunología , Células Germinativas/inmunología , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Región Variable de Inmunoglobulina/metabolismo , Región Variable de Inmunoglobulina/ultraestructura , Ratones , Anticuerpos de Dominio Único/ultraestructura , Relación Estructura-ActividadRESUMEN
Organic hydroperoxide resistance (Ohr) enzymes are unique Cys-based, lipoyl-dependent peroxidases. Here, we investigated the involvement of Ohr in bacterial responses toward distinct hydroperoxides. In silico results indicated that fatty acid (but not cholesterol) hydroperoxides docked well into the active site of Ohr from Xylella fastidiosa and were efficiently reduced by the recombinant enzyme as assessed by a lipoamide-lipoamide dehydrogenase-coupled assay. Indeed, the rate constants between Ohr and several fatty acid hydroperoxides were in the 107-108 M-1â s-1 range as determined by a competition assay developed here. Reduction of peroxynitrite by Ohr was also determined to be in the order of 107 M-1â s-1 at pH 7.4 through two independent competition assays. A similar trend was observed when studying the sensitivities of a ∆ohr mutant of Pseudomonas aeruginosa toward different hydroperoxides. Fatty acid hydroperoxides, which are readily solubilized by bacterial surfactants, killed the ∆ohr strain most efficiently. In contrast, both wild-type and mutant strains deficient for peroxiredoxins and glutathione peroxidases were equally sensitive to fatty acid hydroperoxides. Ohr also appeared to play a central role in the peroxynitrite response, because the ∆ohr mutant was more sensitive than wild type to 3-morpholinosydnonimine hydrochloride (SIN-1 , a peroxynitrite generator). In the case of H2O2 insult, cells treated with 3-amino-1,2,4-triazole (a catalase inhibitor) were the most sensitive. Furthermore, fatty acid hydroperoxide and SIN-1 both induced Ohr expression in the wild-type strain. In conclusion, Ohr plays a central role in modulating the levels of fatty acid hydroperoxides and peroxynitrite, both of which are involved in host-pathogen interactions.
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Proteínas Bacterianas/química , Escherichia coli/fisiología , Ácidos Grasos/química , Peróxido de Hidrógeno/química , Nitratos/química , Pseudomonas aeruginosa/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Ácidos Grasos/metabolismo , Interacciones Huésped-Patógeno , Peróxido de Hidrógeno/metabolismo , Simulación del Acoplamiento Molecular , Nitratos/metabolismoRESUMEN
Natural inhibitors of proteases have been classified into different families, among them is the Bowman-Birk Inhibitor (BBI) family. Members of BBI have two structurally reactive loops that simultaneously inhibit trypsin and chymotrypsin. Here, we have investigated the binding of bovine trypsin by a cyclic nonapeptide, named PTRY9 (CTKSIPPQC), derived of the black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) from Vigna unguiculata seeds. This peptide was synthetically produced with the disulfide bond restraining its conformation to mimic the reactive loop that inhibits trypsin. PTRY9 complexed to pancreatic bovine trypsin was crystallized in orthorhombic and trigonal space groups, P212121 and P3221, with maximum resolutions of 1.15 and 1.61â¯Å, respectively. The structures presented refinement parameters of Rworkâ¯=â¯14.52 % and Rfreeâ¯=â¯15.59 %; Rworkâ¯=â¯15.60 % and Rfreeâ¯=â¯18.78 %, and different surface area between the peptide and the enzyme of 1024â¯Å2 and 1070â¯Å2, respectively. The binding site of the PTRY9 is similar to that found for BTCI as shown by a r.m.s.d. of 0.358â¯Å between the superimposed structures and the electrostatic complementary pattern at the enzyme-peptide interface. Additionally, enzyme inhibition assays show that the affinity of trypsin for PTRY9 is smaller than that for BTCI. In vitro assays revealed that, like BTCI, this synthetic peptide is not cytotoxic for normal mammary epithelial MCF-10A cells, but exerts cytotoxic effects on MDA.MB.231 invasive human breast cancer cells.
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Oligopéptidos/química , Semillas/química , Inhibidor de la Tripsina de Soja de Bowman-Birk/química , Tripsina/química , Vigna/embriología , Línea Celular Tumoral , Cristalografía por Rayos X , HumanosRESUMEN
Persisters are individual bacterial cells that exhibit a phenotype characterized by slow growth, low metabolic rate and multidrug tolerance. The processes that drive cells into a persistence state constitute an active but incipient research field, and structural data regarding its components are scarce. The molecular targets of many therapeutic drugs are involved in cell wall synthesis and cell division, and these cellular processes are down-regulated in persister cells, consequently these cells are more likely to survive antibiotic treatment. Toxin-antitoxin systems were shown to have a leading role in the formation of persisters, and several pathogenic bacteria display a wide array of these systems. The Mycobacterium tuberculosis H37Rv genome presents 88 toxin-antitoxin loci, of which 47 code for members of the VapBC protein family. To date, only four crystal structures of Mycobacterium tuberculosis VapBC complexes are available, and all of them present the toxin bound to and inhibited by the antitoxin. We present the 1.31 Å resolution structure of VapC21, the first structure of a Mycobacterium tuberculosis VapC toxin in the absence of its cognate inhibitory antitoxin. Our data show that VapC21 is a dimer in solution, with conserved active site architecture and an extensive antitoxin binding groove. Additionally, the strategy used to mutate a putative catalytic residue allowing the expression and purification of soluble VapC21 will pave the way for the resolution of more toxin structures in the absence of antitoxins. Taken together, our findings represent an important step in unraveling the molecular mechanisms related to persistence, which will contribute for the design of faster and more efficient therapeutic approaches for the treatment of tuberculosis, particularly for infections with multidrug-resistant strains.
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Proteínas Bacterianas/química , Mycobacterium tuberculosis/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Secuencia Conservada , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Electricidad EstáticaRESUMEN
Biotechnological applications of phytocystatins have garnered significant interest due to their potential applications in crop protection and improve crop resistance to abiotic stress factors. Cof1 and Wal1 are phytocystatins derived from Coffea arabica and Juglans regia, respectively. These plants hold significant economic value due to coffee's global demand and the walnut tree's production of valuable timber and widely consumed walnuts with culinary and nutritional benefits. The study involved the heterologous expression in E. coli Lemo 21(DE3), purification by immobilized metal ion affinity and size exclusion chromatography, and biophysical characterization of both phytocystatins, focusing on isolating and interconverting their monomers and dimers. The crystal structure of the domain-swapped dimer of Wal1 was determined revealing two domain-swapped dimers in the asymmetric unit, an arrangement reminiscent of the human cystatin C structure. Alphafold models of monomers and Alphafold-Multimer models of domain-swapped dimers of Cof1 and Wal1 were analyzed in the context of the crystal structure. The methodology and data presented here contribute to a deeper understanding of the oligomerization mechanisms of phytocystatins and their potential biotechnological applications in agriculture.
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Juglans , Humanos , Juglans/genética , Árboles , Escherichia coli/genéticaRESUMEN
Humicola grisea var. thermoidea is an aerobic and thermophilic fungus that secretes the GH11 xylanase HXYN2 in the presence of sugarcane bagasse. In this study, HXYN2 was expressed in Pichia pastoris and characterized biochemically and structurally in the presence of beechwood xylan substrate and ferulic acid (FA). HXYN2 is a thermally stable protein, as indicated by circular dichroism, with greater activity in the range of 40-50 °C and pH 5.0-9.0, with optimal temperature and pH of 50 °C and 6.0, respectively. FA resulted in a 75% increase in enzyme activity and a 2.5-fold increase in catalytic velocity, catalytic efficiency, and catalytic rate constant (kcat), with no alteration in enzyme affinity for the substrate. Fluorescence quenching indicated that FA forms a complex with HXYN2 interacting with solvent-exposed tryptophan residues. The binding constants ranged from moderate (pH 7.0 and 9.0) to strong (pH 4.0) affinity. Isothermal titration calorimetry, structural models and molecular docking suggested that hydrogen bonds and hydrophobic interactions occur in the aglycone region inducing conformational changes in the active site driven by initial and final enthalpy- and entropy processes, respectively. These results indicate a potential for biotechnological application for HXYN2, such as in the bioconversion of plant residues rich in ferulic acid.
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Celulosa , Saccharum , Ascomicetos , Catálisis , Ácidos Cumáricos , Simulación del Acoplamiento MolecularRESUMEN
Phytocystatins are a family of plant cysteine-protease inhibitors of great interest due to their biotechnological application in culture improvement. It was shown that their expression in plants increases resistance to herbivory by insects and improves tolerance to both biotic and abiotic stress factors. In this work, owing to the economical relevance of the source organism, a phytocystatin from hop (Humulus lupulus), Hop1, was produced by heterologous expression in E. coli Lemo21 (DE3) cultivated in auto-inducing ZYM-5052 medium and purified by immobilized metal ion affinity and size exclusion chromatography. Thermal denaturation assays by circular dichroism showed that Hop1 exhibited high melting temperatures ranging from 82 °C to 85 °C and high thermal stability at a wide pH range, with ΔG25's higher than 12 kcal/mol. At 20 °C and pH 7.6, the dimeric conformation of the protein is favored according to size exclusion chromatography and analytical ultracentrifugation data, although monomers and higher order oligomers could still be detected in a lesser extent. The crystal structure of Hop1 was solved in the space groups P 2 21 21 and C 2 2 21 at resolutions of 1.80 Å and 1.68 Å, respectively. In both models, Hop1 is folded as a domain-swapped dimer where the first inhibitory loop undergoes a significant structural change and interacts with their equivalent from the other monomer forming a long antiparallel beta strand, leading to loss of inhibitory activity.
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Cistatinas/química , Inhibidores de Cisteína Proteinasa/química , Humulus/química , Proteínas de Plantas/química , Clonación Molecular , Cristalografía por Rayos X , Cistatinas/genética , Cistatinas/metabolismo , Inhibidores de Cisteína Proteinasa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Calor , Concentración de Iones de Hidrógeno , Modelos Moleculares , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TermodinámicaRESUMEN
Alzheimer's disease is an ultimately fatal neurodegenerative disease, and BACE-1 has become an attractive validated target for its therapy, with more than a hundred crystal structures deposited in the PDB. In the present study, we present a new methodology that integrates ligand-based methods with structural information derived from the receptor. 128 BACE-1 inhibitors recently disclosed by GlaxoSmithKline R&D were selected specifically because the crystal structures of 9 of these compounds complexed to BACE-1, as well as five closely related analogs, have been made available. A new fragment-guided approach was designed to incorporate this wealth of structural information into a CoMFA study, and the methodology was systematically compared to other popular approaches, such as docking, for generating a molecular alignment. The influence of the partial charges calculation method was also analyzed. Several consistent and predictive models are reported, including one with r (2) = 0.88, q (2) = 0.69 and r (pred) (2) = 0.72. The models obtained with the new methodology performed consistently better than those obtained by other methodologies, particularly in terms of external predictive power. The visual analyses of the contour maps in the context of the enzyme drew attention to a number of possible opportunities for the development of analogs with improved potency. These results suggest that 3D-QSAR studies may benefit from the additional structural information added by the presented methodology.
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Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Diseño de Fármacos , Relación Estructura-Actividad Cuantitativa , Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/química , Ácido Aspártico Endopeptidasas/química , Ligandos , Modelos Moleculares , Conformación Molecular , Unión Proteica , Conformación ProteicaRESUMEN
Human septins 3, 9 and 12 are the only members of a specific subgroup of septins that display several unusual features, including the absence of a C-terminal coiled coil. This particular subgroup (the SEPT3 septins) are present in rod-like octameric protofilaments but are lacking in similar hexameric assemblies, which only contain representatives of the three remaining subgroups. Both hexamers and octamers can self-assemble into mixed filaments by end-to-end association, implying that the SEPT3 septins may facilitate polymerization but not necessarily function. These filaments frequently associate into higher order complexes which associate with biological membranes, triggering a wide range of cellular events. In the present work, a complete compendium of crystal structures for the GTP-binding domains of all of the SEPT3 subgroup members when bound to either GDP or to a GTP analogue is provided. The structures reveal a unique degree of plasticity at one of the filamentous interfaces (dubbed NC). Specifically, structures of the GDP and GTPγS complexes of SEPT9 reveal a squeezing mechanism at the NC interface which would expel a polybasic region from its binding site and render it free to interact with negatively charged membranes. On the other hand, a polyacidic region associated with helix α5', the orientation of which is particular to this subgroup, provides a safe haven for the polybasic region when retracted within the interface. Together, these results suggest a mechanism which couples GTP binding and hydrolysis to membrane association and implies a unique role for the SEPT3 subgroup in this process. These observations can be accounted for by constellations of specific amino-acid residues that are found only in this subgroup and by the absence of the C-terminal coiled coil. Such conclusions can only be reached owing to the completeness of the structural studies presented here.
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Septins are able to polymerize into long apolar filaments and have long been considered to be a component of the cytoskeleton alongside intermediate filaments (which are also apolar in nature), microtubules and actin filaments (which are not). Their central guanosine triphosphate (GTP)-binding domain, which is essential for stabilizing the filament itself, is flanked by N- and C-terminal domains for which no direct structural information is yet available. In most cases, physiological filaments are built from a number of different septin monomers, and in the case of mammalian septins this is most commonly either three or four. Comprehending the structural basis for the spontaneous assembly of such filaments requires a deeper understanding of the interfaces between individual GTP-binding domains than is currently available. Nevertheless, in this review we will summarize the considerable progress which has been made over the course of the last 10 years. We will provide a brief description of each structure determined to date and comment on how it has added to the body of knowledge which is rapidly growing. Rather than simply repeat data which have already been described in the literature, as far as is possible we will try to take advantage of the full set of information now available (mostly derived from human septins) and draw the reader's attention to some of the details of the structures themselves and the filaments they form which have not be commented on previously. An additional aim is to clarify some misconceptions.
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Two cysteine proteinase inhibitors from cowpea, VuCys1 and VuCys2, were produced in E. coli ArcticExpress (DE3). The recombinant products strongly inhibited papain and chymopapain as well as the midgut proteases from Callosobruchus maculatus larvae, a bruchid that uses cysteine proteases as major digestive enzymes. Heat treatment at 100°C for up to 60min or incubation at various pH values caused little reduction in the papain inhibitory activity of both inhibitors. Moreover, minor conformational variations, as probed by circular dichroism spectroscopy, were observed after VuCys1 and VuCys2 were subjected to these treatments. The crystal structure of VuCys1 was determined at a resolution of 1.95Å, revealing a domain-swapped dimer in the asymmetric unit. However, the two lobes of the domain-swapped dimer are positioned closer to each other in VuCys1 in comparison to other similar cystatin structures. Moreover, some polar residues from opposite lobes recruit water molecules, forming a hydrogen bond network that mediates contacts between the lobes, thus generating an extended open interface. Due to the closer distance between the lobes, a small hydrophobic core is also formed, further stabilizing the folded domain-swapped dimer. These structural features might account for the extraordinary thermal and pH stability of VuCys1.
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Cistatinas/química , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/metabolismo , Escherichia coli/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Temperatura , Secuencia de Aminoácidos , Clonación Molecular , Cristalografía por Rayos X , Inhibidores de Cisteína Proteinasa/aislamiento & purificación , Estabilidad de Enzimas , Expresión Génica , Modelos Moleculares , Proteínas de Plantas/aislamiento & purificación , Dominios Proteicos , Análisis de Secuencia , Agua/químicaRESUMEN
Septins form a conserved family of filament forming GTP binding proteins found in a wide range of eukaryotic cells. They share a common structural architecture consisting of an N-terminal domain, a central GTP binding domain and a C-terminal domain, which is often predicted to adopt a coiled-coil conformation, at least in part. The crystal structure of the human SEPT2/SEPT6/SEPT7 heterocomplex has revealed the importance of the GTP binding domain in filament formation, but surprisingly no electron density was observed for the C-terminal domains and their function remains obscure. The dearth of structural information concerning the C-terminal region has motivated the present study in which the putative C-terminal domains of human SEPT2, SEPT6 and SEPT7 were expressed in E. coli and purified to homogeneity. The thermal stability and secondary structure content of the domains were studied by circular dichroism spectroscopy, and homo- and hetero-interactions were investigated by size exclusion chromatography, chemical cross-linking, analytical ultracentrifugation and surface plasmon resonance. Our results show that SEPT6-C and SEPT7-C are able to form both homo- and heterodimers with a high α-helical content in solution. The heterodimer is elongated and considerably more stable than the homodimers, with a K(D) of 15.8 nM. On the other hand, the homodimer SEPT2-C has a much lower affinity, with a K(D) of 4 µM, and a moderate α-helical content. Our findings present the first direct experimental evidence toward better understanding the biophysical properties and coiled-coil pairings of such domains and their potential role in filament assembly and stability.