Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.652
Filtrar
2.
Int J Mol Sci ; 22(7)2021 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-33807484

RESUMEN

Transcription factors play a crucial role in regulating biological processes such as cell growth, differentiation, organ development and cellular signaling. Within this group, proteins equipped with zinc finger motifs (ZFs) represent the largest family of sequence-specific DNA-binding transcription regulators. Numerous studies have proven the fundamental role of BCL11B for a variety of tissues and organs such as central nervous system, T cells, skin, teeth, and mammary glands. In a previous work we identified a novel atypical zinc finger domain (CCHC-ZF) which serves as a dimerization interface of BCL11B. This domain and formation of the dimer were shown to be critically important for efficient regulation of the BCL11B target genes and could therefore represent a promising target for novel drug therapies. Here, we report the structural basis for BCL11B-BCL11B interaction mediated by the N-terminal ZF domain. By combining structure prediction algorithms, enhanced sampling molecular dynamics and fluorescence resonance energy transfer (FRET) approaches, we identified amino acid residues indispensable for the formation of the single ZF domain and directly involved in forming the dimer interface. These findings not only provide deep insight into how BCL11B acquires its active structure but also represent an important step towards rational design or selection of potential inhibitors.


Asunto(s)
Proteínas Represoras/metabolismo , Proteínas Represoras/ultraestructura , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/ultraestructura , Secuencia de Aminoácidos/genética , Proteínas de Unión al ADN/metabolismo , Dimerización , Transferencia Resonante de Energía de Fluorescencia/métodos , Células HEK293 , Humanos , Simulación de Dinámica Molecular , Proteínas Represoras/genética , Factores de Transcripción/metabolismo , Dedos de Zinc/genética
3.
Nat Commun ; 12(1): 1852, 2021 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-33767175

RESUMEN

TEM-1 ß-lactamase degrades ß-lactam antibiotics with a strong preference for penicillins. Sequence reconstruction studies indicate that it evolved from ancestral enzymes that degraded a variety of ß-lactam antibiotics with moderate efficiency. This generalist to specialist conversion involved more than 100 mutational changes, but conserved fold and catalytic residues, suggesting a role for dynamics in enzyme evolution. Here, we develop a conformational dynamics computational approach to rationally mold a protein flexibility profile on the basis of a hinge-shift mechanism. By deliberately weighting and altering the conformational dynamics of a putative Precambrian ß-lactamase, we engineer enzyme specificity that mimics the modern TEM-1 ß-lactamase with only 21 amino acid replacements. Our conformational dynamics design thus re-enacts the evolutionary process and provides a rational allosteric approach for manipulating function while conserving the enzyme active site.


Asunto(s)
beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Secuencia de Aminoácidos/genética , Dominio Catalítico/genética , Biología Computacional , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Evolución Molecular , Simulación de Dinámica Molecular , Penicilinas/metabolismo , Conformación Proteica , Especificidad por Sustrato
4.
Nat Commun ; 12(1): 1605, 2021 03 11.
Artículo en Inglés | MEDLINE | ID: mdl-33707415

RESUMEN

Deep learning algorithms have been utilized to achieve enhanced performance in pattern-recognition tasks. The ability to learn complex patterns in data has tremendous implications in immunogenomics. T-cell receptor (TCR) sequencing assesses the diversity of the adaptive immune system and allows for modeling its sequence determinants of antigenicity. We present DeepTCR, a suite of unsupervised and supervised deep learning methods able to model highly complex TCR sequencing data by learning a joint representation of a TCR by its CDR3 sequences and V/D/J gene usage. We demonstrate the utility of deep learning to provide an improved 'featurization' of the TCR across multiple human and murine datasets, including improved classification of antigen-specific TCRs and extraction of antigen-specific TCRs from noisy single-cell RNA-Seq and T-cell culture-based assays. Our results highlight the flexibility and capacity for deep neural networks to extract meaningful information from complex immunogenomic data for both descriptive and predictive purposes.


Asunto(s)
Algoritmos , Aprendizaje Profundo , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Secuencia de Aminoácidos/genética , Animales , Bases de Datos Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Ratones , Redes Neurales de la Computación , RNA-Seq/métodos
5.
Molecules ; 26(5)2021 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-33652602

RESUMEN

Hepatitis B virus (HBV) is a circular, and partially double-stranded DNA virus. Upon infection, the viral genome is translocated into the cell nucleus, generating the covalently closed circular DNA (cccDNA) intermediate, and forming a mini chromosome. HBV HBx is a small protein displaying multiple roles in HBV-infected cells, and in different subcellular locations. In the nucleus, the HBx protein is required to initiate and maintain viral transcription from the viral mini chromosome. In contrast, HBx also functions in the cytoplasm, where it is able to alter multiple cellular functions such as mitochondria metabolism, apoptosis and signal transduction pathways. It has been reported that in cultured cells, at low expression levels, the HBx protein is localized in the nucleus, whereas at high expression levels, it accumulates in the cytoplasm. This dynamic subcellular distribution of HBx might be essential to exert its multiple roles during viral infection. However, the mechanism that regulates different subcellular localizations of the HBx protein is unknown. We have previously taken a bioinformatics approach to investigate whether HBx might be regulated via post-translational modification, and we have proposed that the multiple nucleocytoplasmic functions of HBx might be regulated by an evolutionarily conserved mechanism via phosphorylation. In the current study, phylogenetically conserved amino acids of HBx with a high potential of phosphorylation were targeted for site-directed mutagenesis. Two conserved serine (Ser25 and Ser41), and one conserved threonine (Thr81) amino acids were replaced by either alanine or aspartic acid residues to simulate an unphosphorylated or phosphorylated state, respectively. Human hepatoma cells were transfected with increasing amounts of the HBx DNA constructs, and the cells were analyzed by fluorescence microscopy. Together, our results show that the nucleocytoplasmic distribution of the HBx protein could be regulated by phosphorylation since some of the modified proteins were mainly confined to distinct subcellular compartments. Remarkably, both HBx Ser41A, and HBx Thr81D proteins were predominantly localized within the nuclear compartment throughout the different expression levels of HBx mutants.


Asunto(s)
Carcinoma Hepatocelular/genética , Hepatitis B/genética , Neoplasias Hepáticas/genética , Transactivadores/genética , Proteínas Reguladoras y Accesorias Virales/genética , Secuencia de Aminoácidos/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Secuencia Conservada/genética , Regulación Viral de la Expresión Génica/genética , Genoma Viral/genética , Células Hep G2 , Hepatitis B/patología , Hepatitis B/virología , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/patogenicidad , Humanos , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Fosforilación/genética , Filogenia
6.
Molecules ; 26(5)2021 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-33652901

RESUMEN

Slow-channel congenital myasthenic syndromes (SCCMSs) are rare genetic diseases caused by mutations in muscle nicotinic acetylcholine receptor (nAChR) subunits. Most of the known SCCMS-associated mutations localize at the transmembrane region near the ion pore. Only two SCCMS point mutations are at the extracellular domains near the acetylcholine binding site, α1(G153S) being one of them. In this work, a combination of molecular dynamics, targeted mutagenesis, fluorescent Ca2+ imaging and patch-clamp electrophysiology has been applied to G153S mutant muscle nAChR to investigate the role of hydrogen bonds formed by Ser 153 with C-loop residues near the acetylcholine-binding site. Introduction of L199T mutation to the C-loop in the vicinity of Ser 153 changed hydrogen bonds distribution, decreased acetylcholine potency (EC50 2607 vs. 146 nM) of the double mutant and decay kinetics of acetylcholine-evoked cytoplasmic Ca2+ rise (τ 14.2 ± 0.3 vs. 34.0 ± 0.4 s). These results shed light on molecular mechanisms of nAChR activation-desensitization and on the involvement of such mechanisms in channelopathy genesis.


Asunto(s)
Acetilcolina/genética , Secuencia de Aminoácidos/genética , Síndromes Miasténicos Congénitos/genética , Receptores Nicotínicos/genética , Acetilcolina/metabolismo , Sitios de Unión/genética , Calcio/metabolismo , Humanos , Cinética , Síndromes Miasténicos Congénitos/patología , Técnicas de Placa-Clamp , Mutación Puntual/genética , Unión Proteica/genética
7.
J Steroid Biochem Mol Biol ; 208: 105834, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33548461

RESUMEN

Androgens are critical for male sex differentiation. Their actions are mediated by the androgen receptor (AR). Mutations disrupting AR function result in the androgen insensitivity syndrome (AIS). In this study, we identified in a patient with complete AIS, a novel AR mutation p.R856L. To investigate the functional properties of p.R856L, we performed functional studies. In comparison, we have characterized two already described mutations: p.R856H and p.R856C. We used a model composed of two different promoters fused to a reporter gene, two cell lines, and showed that all mutations were able to transactivate the (ARE)2-TATA promoter expressed in CHO cells more highly. Moreover, we confirmed the pathogenicity of the p.R856L and p.R856C mutations, and their associations with complete AIS. In contrast, the p.R856H mutation, which is associated with a spectrum of AIS phenotypes, showed less severe transcriptional constraints. Altogether, our studies allowed us to better characterize arginine residue at p.R856 position.


Asunto(s)
Síndrome de Resistencia Androgénica/genética , Andrógenos/genética , Receptores Androgénicos/genética , Diferenciación Sexual/genética , Secuencia de Aminoácidos/genética , Síndrome de Resistencia Androgénica/patología , Andrógenos/metabolismo , Animales , Arginina/genética , Cricetinae , Cricetulus , Humanos , Ligandos , Masculino , Mutación/genética , Dominios Proteicos/genética
8.
Molecules ; 26(3)2021 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-33572696

RESUMEN

Inducible lysine decarboxylases (LDCs) are essential in various cellular processes of microorganisms and plants, especially under acid stress, which induces the expression of genes encoding LDCs. In this study, a novel Serratia marcesenes LDC (SmcadA) was successfully expressed in E. coli, purified and characterized. The protein had an optimal pH of 6 and a temperature of 40 °C and phylogenetic analysis to determine the evolution of SmcadA, which revealed a close relation to Enterobacteriaceae, Klebsiella sp., among others. The molecular weight of SmcadA was approximately 75 kDa after observation on SDS-PAGE and structural modeling showed the protein as a decamer, comprised of five interlinked dimers. The biocatalytic activity of the purified wild-type SmcadA (WT) was improved through site directed mutations and the results showed that the Arg595Lys mutant had the highest specific activity of 286.55 U/mg, while the Ser512Ala variant and wild-type SmcadA had 215.72 and 179.01 U/mg, respectively. Furthermore, molecular dynamics simulations revealed that interactions through hydrogen bonds between the protein residues and cofactor pyridoxal-5-phosphate (PLP) are vital for biocatalysis. Molecular Dynamics (MD) simulations also indicated that mutations conferred structural changes on protein residues and PLP hence altered the interacting residues with the cofactor, subsequently influencing substrate bioconversion. Moreover, the temperature also induced changes in orientation of cofactor PLP and amino acid residues. This work therefore demonstrates the successful expression and characterization of the purified novel lysine decarboxylase from Serratia marcesenes and provided insight into the mechanism of protein-cofactor interactions, highlighting the role of protein-ligand interactions in altering cofactor and binding site residue conformations, thus contributing to improved biocatalysis.


Asunto(s)
Carboxiliasas/química , Conformación Proteica , Serratia marcescens/enzimología , Secuencia de Aminoácidos/genética , Sitios de Unión/genética , Biocatálisis , Carboxiliasas/genética , Carboxiliasas/ultraestructura , Dominio Catalítico/genética , Escherichia coli/genética , Simulación de Dinámica Molecular , Serratia marcescens/química , Serratia marcescens/ultraestructura , Especificidad por Sustrato
9.
Molecules ; 26(3)2021 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-33572971

RESUMEN

Understanding protein stability is critical for the application of enzymes in biotechnological processes. The structural basis for the stability of thermally adapted chitinases has not yet been examined. In this study, the amino acid sequences and X-ray structures of psychrophilic, mesophilic, and hyperthermophilic chitinases were analyzed using computational and molecular dynamics (MD) simulation methods. From the findings, the key features associated with higher stability in mesophilic and thermophilic chitinases were fewer and/or shorter loops, oligomerization, and less flexible surface regions. No consistent trends were observed between stability and amino acid composition, structural features, or electrostatic interactions. Instead, unique elements affecting stability were identified in different chitinases. Notably, hyperthermostable chitinase had a much shorter surface loop compared to psychrophilic and mesophilic homologs, implying that the extended floppy surface region in cold-adapted and mesophilic chitinases may have acted as a "weak link" from where unfolding was initiated. MD simulations confirmed that the prevalence and flexibility of the loops adjacent to the active site were greater in low-temperature-adapted chitinases and may have led to the occlusion of the active site at higher temperatures compared to their thermostable homologs. Following this, loop "hot spots" for stabilizing and destabilizing mutations were also identified. This information is not only useful for the elucidation of the structure-stability relationship, but will be crucial for designing and engineering chitinases to have enhanced thermoactivity and to withstand harsh industrial processing conditions.


Asunto(s)
Quitinasas/química , Estabilidad de Enzimas/genética , Extremófilos/química , Conformación Proteica , Secuencia de Aminoácidos/genética , Dominio Catalítico/genética , Quitinasas/genética , Quitinasas/ultraestructura , Biología Computacional , Extremófilos/enzimología , Extremófilos/genética , Calor , Simulación de Dinámica Molecular , Estabilidad Proteica
10.
Molecules ; 26(3)2021 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-33573083

RESUMEN

Collagen contains hydroxyproline (Hyp), which is a unique amino acid. Three collagen-derived small peptides (Gly-Pro-Hyp, Pro-Hyp, and Gly-Hyp) interacting across a lipid bilayer (POPC model membrane) for cellular uptakes of these collagen-derived small peptides were studied using accelerated molecular dynamics simulation. The ligands were investigated for their binding modes, hydrogen bonds in each coordinate frame, and mean square displacement (MSD) in the Z direction. The lipid bilayers were evaluated for mass and electron density profiles of the lipid molecules, surface area of the head groups, and root mean square deviation (RMSD). The simulation results show that hydrogen bonding between the small collagen peptides and plasma membrane plays a significant role in their internalization. The translocation of the small collagen peptides across the cell membranes was shown. Pro-Hyp laterally condensed the membrane, resulting in an increase in the bilayer thickness and rigidity. Perception regarding molecular behaviors of collagen-derived peptides within the cell membrane, including their interactions, provides the novel design of specific bioactive collagen peptides for their applications.


Asunto(s)
Colágeno/química , Membrana Dobles de Lípidos/química , Péptidos/química , Secuencia de Aminoácidos/genética , Transporte Biológico/genética , Dicroismo Circular , Colágeno/genética , Simulación por Computador , Dipéptidos/química , Dipéptidos/genética , Enlace de Hidrógeno/efectos de los fármacos , Hidroxiprolina/química , Péptidos/genética , Unión Proteica/genética , Conformación Proteica
11.
Molecules ; 26(3)2021 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-33573096

RESUMEN

Phosphorylation represents one of the most important modifications of amino acids, peptides, and proteins. By modifying the latter, it is useful in improving the functional properties of foods. Although all these substances are broadly annotated in internet databases, there is no unified code for their annotation. The present publication aims to describe a simple code for the annotation of phosphopeptide sequences. The proposed code describes the location of phosphate residues in amino acid side chains (including new rules of atom numbering in amino acids) and the diversity of phosphate residues (e.g., di- and triphosphate residues and phosphate amidation). This article also includes translating the proposed biological code into SMILES, being the most commonly used chemical code. Finally, it discusses possible errors associated with applying the proposed code and in the resulting SMILES representations of phosphopeptides. The proposed code can be extended to describe other modifications in the future.


Asunto(s)
Secuencia de Aminoácidos/genética , Aminoácidos/genética , Anotación de Secuencia Molecular , Proteínas/genética , Aminoácidos/clasificación , Código Genético/genética , Humanos , Fosfopéptidos/clasificación , Fosfopéptidos/genética , Fosforilación/genética , Proteínas/clasificación
12.
Nat Chem Biol ; 17(4): 428-437, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33542533

RESUMEN

Tryptophan C-mannosylation is an unusual co-translational protein modification performed by metazoans and apicomplexan protists. The prevalence and biological functions of this modification are poorly understood, with progress in the field hampered by a dearth of convenient tools for installing and detecting the modification. Here, we engineer a yeast system to produce a diverse array of proteins with and without tryptophan C-mannosylation and interrogate the modification's influence on protein stability and function. This system also enabled mutagenesis studies to identify residues of the glycosyltransferase and its protein substrates that are crucial for catalysis. The collection of modified proteins accrued during this work facilitated the generation and thorough characterization of monoclonal antibodies against tryptophan C-mannosylation. These antibodies empowered proteomic analyses of the brain C-glycome by enriching for peptides possessing tryptophan C-mannosylation. This study revealed many new modification sites on proteins throughout the secretory pathway with both conventional and non-canonical consensus sequences.


Asunto(s)
Manosa/química , Ingeniería de Proteínas/métodos , Triptófano/metabolismo , Secuencia de Aminoácidos/genética , Anticuerpos/inmunología , Glicosilación , Glicosiltransferasas/metabolismo , Manosa/metabolismo , Péptidos/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Estabilidad Proteica , Proteómica/métodos , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Triptófano/química
13.
Nat Commun ; 12(1): 380, 2021 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-33452262

RESUMEN

Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds. Here, we present the exceptional properties of a putative ancestor of bacterial and eukaryotic family-1 glycosidases. The ancestral protein shares the TIM-barrel fold with its modern descendants but displays large regions with greatly enhanced conformational flexibility. Yet, the barrel core remains comparatively rigid and the ancestral glycosidase activity is stable, with an optimum temperature within the experimental range for thermophilic family-1 glycosidases. None of the ∼5500 reported crystallographic structures of ∼1400 modern glycosidases show a bound porphyrin. Remarkably, the ancestral glycosidase binds heme tightly and stoichiometrically at a well-defined buried site. Heme binding rigidifies this TIM-barrel and allosterically enhances catalysis. Our work demonstrates the capability of ancestral protein reconstructions to reveal valuable but unexpected biomolecular features when sampling distant sequence space. The potential of the ancestral glycosidase as a scaffold for custom catalysis and biosensor engineering is discussed.


Asunto(s)
Bacterias/enzimología , Eucariontes/enzimología , Glicósido Hidrolasas/metabolismo , Hemo/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos/genética , Bacterias/genética , Cristalografía por Rayos X , Eucariontes/genética , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/ultraestructura , Simulación de Dinámica Molecular , Filogenia , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido
14.
J Med Microbiol ; 70(2)2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33404383

RESUMEN

Introduction. Moraxella bovoculi is frequently isolated from the eyes of cattle with infectious bovine keratoconjunctivitis (IBK; pinkeye). As with M. bovis, which has been causally linked to IBK, M. bovoculi expresses an RTX (repeats in the structural toxin) cytotoxin that is related to M. bovis cytotoxin. Pilin, another pathogenic factor in M. bovis, is required for corneal attachment. Seven antigenically distinct pilin serogroups have been described in M. bovis.Hypothesis/Gap Statement. Multiple different serogroups exist amongst type IV pilin encoded by M. bovis, however, it is not known whether M. bovoculi exhibits a similar degree of diversity in type IV pilin that it encodes.Aim. This study was done to characterize a structural pilin (PilA) encoded by M. bovoculi isolated from cases of IBK to determine if diversity exists amongst PilA sequences.Methodology. Ninety-four isolates of M. bovoculi collected between 2002 and 2017 from 23 counties throughout California and from five counties in four other Western states were evaluated.Results. DNA sequencing and determination of deduced amino acid sequences revealed ten (designated groups A through J) unique PilA sequences that were ~96.1-99.3 % identical. Pilin groups A and C matched previously reported putative PilA sequences from M. bovoculi isolated from IBK-affected cattle in the USA (Virginia, Nebraska, and Kansas) and Asia (Kazakhstan). The ten pilin sequences identified were only ~74-76 % identical to deduced amino acid sequences of putative pilin proteins identified from the previously reported whole-genome sequences of M. bovoculi derived from deep nasopharyngeal swabs of IBK-asymptomatic cattle.Conclusions. Compared to the diversity reported between structural pilin proteins amongst different serogroups of M. bovis, M. bovoculi PilA from geographically diverse isolates derived from IBK-affected cattle are more conserved.


Asunto(s)
Proteínas Fimbrias/genética , Fimbrias Bacterianas/genética , Queratoconjuntivitis/veterinaria , Moraxella/patogenicidad , Infecciones por Moraxellaceae/veterinaria , Secuencia de Aminoácidos/genética , Animales , Bovinos , Enfermedades de los Bovinos/microbiología , Proteínas Fimbrias/metabolismo , Variación Genética/genética , Genoma Bacteriano/genética , Queratoconjuntivitis/microbiología , Moraxella/genética , Moraxella/aislamiento & purificación , Infecciones por Moraxellaceae/diagnóstico
15.
Gene ; 777: 145459, 2021 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-33515726

RESUMEN

Enterococcal plasmids have generated renewed interest for their indispensable role in pathogenesis and dissemination of multidrug-resistance. Recently, a novel plasmid pSM409 (4303-bp, GC% = 33.6%), devoid of antibiotic-resistance and virulence genes, has been identified in Enterococcus faecium RME, isolated from raw milk by us. pSM409 contains six open reading frames encoding a replication initiator protein (RepB) and five accessory proteins: antitoxin epsilon, bacteriocin immunity protein, HsdS, and two hypothetical proteins. Comparative sequence analysis of pSM409 reveals a mosaic pattern of similarity with different loci obtained from different theta plasmids, which dictates the plasmid to be heterogeneous or mosaic, possibly due to recombination. The pSM409 comprised of a typical theta-type origin of replication with four and a half direct repeats (iterons) of 22 nucleotides. The pSM409-RepB shared 76-82% homology with the RepB of reported theta plasmids from different genera, with dissimilarities mostly in its DNA-binding and C-terminal domain. The RepB sequence-based phylogenetic tree revealed its distinct position relative to the reported ones. The RepB grouped in the same clade has identical DNA-binding domains and their cognate iterons, possibly due to their sequence-specific interaction to initiate plasmid replication. Comparative analysis of the pSM409-iteron reveals that the repeats markedly differed from their closest homologues. This clade-specific relationship provides a new concept of classifying theta plasmids. The theta-type replicon identified in pSM409 has been found to be unique to E. faecium RME, prompting us to further investigate its utility as a vector for genetic manipulation of enterococci for health and industry.


Asunto(s)
Enterococcus faecium/genética , Leche/microbiología , Plásmidos/genética , Secuencia de Aminoácidos/genética , Animales , Secuencia de Bases/genética , Bovinos , Replicación del ADN/genética , ADN Bacteriano/genética , Enterococcus faecium/aislamiento & purificación , Sistemas de Lectura Abierta/genética , Secuencias Repetitivas de Ácidos Nucleicos , Replicón/genética
16.
Proc Natl Acad Sci U S A ; 118(1)2021 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-33443166

RESUMEN

Fusion-associated small transmembrane (FAST) proteins are a diverse family of nonstructural viral proteins. Once expressed on the plasma membrane of infected cells, they drive fusion with neighboring cells, increasing viral spread and pathogenicity. Unlike viral fusogens with tall ectodomains that pull two membranes together through conformational changes, FAST proteins have short fusogenic ectodomains that cannot bridge the intermembrane gap between neighboring cells. One orthoreovirus FAST protein, p14, has been shown to hijack the actin cytoskeleton to drive cell-cell fusion, but the actin adaptor-binding motif identified in p14 is not found in any other FAST protein. Here, we report that an evolutionarily divergent FAST protein, p22 from aquareovirus, also hijacks the actin cytoskeleton but does so through different adaptor proteins, Intersectin-1 and Cdc42, that trigger N-WASP-mediated branched actin assembly. We show that despite using different pathways, the cytoplasmic tail of p22 can replace that of p14 to create a potent chimeric fusogen, suggesting they are modular and play similar functional roles. When we directly couple p22 with the parallel filament nucleator formin instead of the branched actin nucleation promoting factor N-WASP, its ability to drive fusion is maintained, suggesting that localized mechanical pressure on the plasma membrane coupled to a membrane-disruptive ectodomain is sufficient to drive cell-cell fusion. This work points to a common biophysical strategy used by FAST proteins to push rather than pull membranes together to drive fusion, one that may be harnessed by other short fusogens responsible for physiological cell-cell fusion.


Asunto(s)
Actinas/metabolismo , Proteínas de la Fusión de la Membrana/metabolismo , Fusión de Membrana/fisiología , Citoesqueleto de Actina/metabolismo , Secuencia de Aminoácidos/genética , Animales , Evolución Biológica , Fusión Celular/métodos , Línea Celular , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Evolución Molecular , Humanos , Orthoreovirus/genética , Unión Proteica/genética , Reoviridae/genética , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/metabolismo , Proteínas no Estructurales Virales/metabolismo , Internalización del Virus
17.
PLoS Genet ; 17(1): e1009305, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33465083

RESUMEN

Many genes are regulated by two or more enhancers that drive similar expression patterns. Evolutionary theory suggests that these seemingly redundant enhancers must have functionally important differences. In the simple ascidian chordate Ciona, the transcription factor Brachyury is induced exclusively in the presumptive notochord downstream of lineage specific regulators and FGF-responsive Ets family transcription factors. Here we exploit the ability to finely titrate FGF signaling activity via the MAPK pathway using the MEK inhibitor U0126 to quantify the dependence of transcription driven by different Brachyury reporter constructs on this direct upstream regulator. We find that the more powerful promoter-adjacent proximal enhancer and a weaker distal enhancer have fundamentally different dose-response relationships to MAPK inhibition. The Distal enhancer is more sensitive to MAPK inhibition but shows a less cooperative response, whereas the Proximal enhancer is less sensitive and more cooperative. A longer construct containing both enhancers has a complex dose-response curve that supports the idea that the proximal and distal enhancers are moderately super-additive. We show that the overall expression loss from intermediate doses of U0126 is not only a function of the fraction of cells expressing these reporters, but also involves graded decreases in expression at the single-cell level. Expression of the endogenous gene shows a comparable dose-response relationship to the full length reporter, and we find that different notochord founder cells are differentially sensitive to MAPK inhibition. Together, these results indicate that although the two Brachyury enhancers have qualitatively similar expression patterns, they respond to FGF in quantitatively different ways and act together to drive high levels of Brachyury expression with a characteristic input/output relationship. This indicates that they are fundamentally not equivalent genetic elements.


Asunto(s)
Ciona intestinalis/genética , Elementos de Facilitación Genéticos/genética , Proteínas Fetales/genética , Factores de Crecimiento de Fibroblastos/genética , Proteínas de Dominio T Box/genética , Secuencia de Aminoácidos/genética , Animales , Ciona intestinalis/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica/genética , Sistema de Señalización de MAP Quinasas/genética , Notocorda/crecimiento & desarrollo , Notocorda/metabolismo , Regiones Promotoras Genéticas/genética , Factores de Transcripción/genética
18.
Proc Natl Acad Sci U S A ; 118(2)2021 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-33443157

RESUMEN

The sex-determining region on the Y chromosome (SRY) is thought to be the central genetic element of male sex development in mammals. Pathogenic modifications within the SRY gene are associated with a male-to-female sex reversal syndrome in humans and other mammalian species, including rabbits and mice. However, the underlying mechanisms are largely unknown. To understand the biological function of the SRY gene, a site-directed mutational analysis is required to investigate associated phenotypic changes at the molecular, cellular, and morphological level. Here, we successfully generated a knockout of the porcine SRY gene by microinjection of two CRISPR-Cas ribonucleoproteins, targeting the centrally located "high mobility group" (HMG), followed by a frameshift mutation of the downstream SRY sequence. This resulted in the development of genetically male (XY) pigs with complete external and internal female genitalia, which, however, were significantly smaller than in 9-mo-old age-matched control females. Quantitative digital PCR analysis revealed a duplication of the SRY locus in Landrace pigs similar to the known palindromic duplication in Duroc breeds. Our study demonstrates the central role of the HMG domain in the SRY gene in male porcine sex determination. This proof-of-principle study could assist in solving the problem of sex preference in agriculture to improve animal welfare. Moreover, it establishes a large animal model that is more comparable to humans with regard to genetics, physiology, and anatomy, which is pivotal for longitudinal studies to unravel mammalian sex determination and relevant for the development of new interventions for human sex development disorders.


Asunto(s)
Procesos de Determinación del Sexo/genética , Proteína de la Región Y Determinante del Sexo/genética , Proteína de la Región Y Determinante del Sexo/metabolismo , Secuencia de Aminoácidos/genética , Animales , Proteínas de Unión al ADN/genética , Trastornos del Desarrollo Sexual/genética , Mutación del Sistema de Lectura/genética , Genes sry/genética , Dominios HMG-Box/genética , Masculino , Mutación/genética , Proteínas Nucleares/genética , Prueba de Estudio Conceptual , Dominios Proteicos/genética , Porcinos/genética , Factores de Transcripción/genética , Cromosoma Y/genética
19.
Arch Virol ; 166(3): 779-788, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-33433693

RESUMEN

Ungulate protoparvovirus 1, also known as porcine parvovirus 1 (PPV1), is considered to be one of the major causes of reproductive failure in pig breeding herds. Other parvoviruses have also been identified in pigs, including ungulate tetraparvovirus 3, or PPV2, ungulate tetraparvovirus 2, or PPV3, and ungulate copiparvovirus 2, or PPV4, but their significance for pigs is unknown. In the present study, the prevalence of PPV1-4 was investigated using a total of 231 lung and serum samples collected from slaughterhouses in 13 provinces throughout Vietnam. The overall prevalence was 54.5% (126/231) for PPV1, 28.0% (65/231) for PPV2, 17.7% (41/231) for PPV3, and 7.8% (18/231) for PPV4. While PPV1 and PPV2 were found in 11 provinces, PPV4 was detected in only three provinces. Co-circulation of PPV1, PPV2 and PPV3 was frequently observed, with PPV1/PPV2 coinfection predominating, with 20.8% (48/231). All four PPVs were detected together in only one sample from Thua Thien Hue. Three nearly complete PPV4 genome sequences of 5,453 nt were determined and deposited in the GenBank database. Alignment and comparison of the three genome sequences showed 99.5-99.6% nucleotide sequence identity, and the deduced amino acid sequences of open reading frames 1-3 were 99.6-99.9% identical to each other, 98.9-99.3% identical to those of other Vietnamese strains and 99.4-99.7% identical to those of Chinese strains). Phylogenetic analysis further confirmed a close relationship between Vietnamese and Chinese PPV4 strains. These results are the first to report the prevalence of PPV1, PPV2, PPV3, and PPV4 and nearly complete genomic sequences of PPV4 in pigs from slaughterhouses in Vietnam.


Asunto(s)
Infecciones por Parvoviridae/epidemiología , Parvovirinae/clasificación , Parvovirinae/genética , Enfermedades de los Porcinos/epidemiología , Mataderos , Secuencia de Aminoácidos/genética , Animales , ADN Viral/genética , Genoma/genética , Genoma Viral/genética , Infecciones por Parvoviridae/patología , Parvovirinae/aislamiento & purificación , Análisis de Secuencia de ADN , Sus scrofa/virología , Porcinos , Enfermedades de los Porcinos/virología , Vietnam/epidemiología
20.
Arch Virol ; 166(3): 885-890, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-33454861

RESUMEN

African swine fever (ASF) is a highly infectious disease of pigs caused by African swine fever virus (ASFV). In order to identify potential genetic variations among ASFV strains circulating in Vietnam, 26 ASFV isolates from organs and blood samples collected from domestic pigs from 23 different provinces of northern, central and southern Vietnam during 2019-2020 ASF outbreaks were genetically characterized. Nucleotide sequences were determined for a portion of the B646L (p72) gene, the complete E183L (p54) gene, the variable region of EP402R (CD2v), the central variable region (CVR) of pB602L, and a tandem repeat sequence (TRS) between the I73R and I329L genes. Analysis of the partial B646L (p72) and EP402R (CD2v) gene sequences and the full-length E183L (p54) gene sequence showed that all 26 ASFV isolates belonged to genotype II and serotype VIII and that they were identical to the strain Georgia/2007/1 and all ASFV strains sequenced in China. The TRS between the I73R and I329L genes contained a 10-nucleotide insertion that was observed in the Chinese ASFV strain CN201801 isolated from domestic pigs in 2018, but not in the Georgia/2007/1 and China/Jilin/2018/boar strains isolated from wild boar in China. This is the first intra-epidemic genome analysis reported for the ASFV strains circulating in Vietnam.


Asunto(s)
Virus de la Fiebre Porcina Africana/genética , Fiebre Porcina Africana/epidemiología , Variación Genética/genética , Genoma Viral/genética , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Secuencia de Aminoácidos/genética , Animales , ADN Viral/genética , Mutagénesis Insercional/genética , Análisis de Secuencia de ADN , Sus scrofa/virología , Porcinos , Secuencias Repetidas en Tándem/genética , Vietnam/epidemiología
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...