Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 42.797
Filtrar
1.
Int J Mol Sci ; 22(12)2021 Jun 09.
Artículo en Inglés | MEDLINE | ID: mdl-34207526

RESUMEN

Mutations in the PSEN1 gene are the most common cause of autosomal dominant Alzheimer's disease, and are characterized by a high phenotype variability. This study describes a five-generation family, with a prevalent late-onset of the disease and a high frequency of depression, in which a new missense mutation (c.789T > G, p.Cys263Trp) in exon 8 of the PSEN1 gene was found. Only the proband presented an early onset at the age of 45 with attention deficit, followed by spatial disorientation, psychiatric symptoms and parkinsonian signs. The other two cases had a late onset of the disease and a typical presentation with memory loss. Both were characterized by a high level of anxiety and depression. The disease course was different with signs of Lewy body dementia for the proband's mother, and pyramidal involvement and a shorter disease duration for the proband's maternal aunt. The other eight cases with late-onset dementia and three cases with a long history of depression have been reported in the family pedigree, underlying the high phenotype variability of PSEN1 mutations.


Asunto(s)
Enfermedad de Alzheimer/genética , Familia , Mutación Missense , Linaje , Presenilina-1/genética , Edad de Inicio , Sustitución de Aminoácidos , Femenino , Humanos , Italia , Masculino , Persona de Mediana Edad
2.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-34204499

RESUMEN

BACKGROUND: Brugada syndrome (BrS) is an autosomal dominantly inherited cardiac disease characterized by "coved type" ST-segment elevation in the right precordial leads, high susceptibility to ventricular arrhythmia and a family history of sudden cardiac death. The SCN5A gene, encoding for the cardiac voltage-gated sodium channel Nav1.5, accounts for ~20-30% of BrS cases and is considered clinically relevant. METHODS: Here, we describe the clinical findings of two Italian families affected by BrS and provide the functional characterization of two novel SCN5A mutations, the missense variant Pro1310Leu and the in-frame insertion Gly1687_Ile1688insGlyArg. RESULTS: Despite being clinically different, both patients have a family history of sudden cardiac death and had history of arrhythmic events. The Pro1310Leu mutation significantly reduced peak sodium current density without affecting channel membrane localization. Changes in the gating properties of expressed Pro1310Leu channel likely account for the loss-of-function phenotype. On the other hand, Gly1687_Ile1688insGlyArg channel, identified in a female patient, yielded a nearly undetectable sodium current. Following mexiletine incubation, the Gly1687_Ile1688insGlyArg channel showed detectable, albeit very small, currents and biophysical properties similar to those of the Nav1.5 wild-type channel. CONCLUSIONS: Overall, our results suggest that the degree of loss-of-function shown by the two Nav1.5 mutant channels correlates with the aggressive clinical phenotype of the two probands. This genotype-phenotype correlation is fundamental to set out appropriate therapeutical intervention.


Asunto(s)
Síndrome de Brugada/diagnóstico , Síndrome de Brugada/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Mutación , Canal de Sodio Activado por Voltaje NAV1.5/genética , Potenciales de Acción , Anciano , Anciano de 80 o más Años , Alelos , Sustitución de Aminoácidos , Electrocardiografía , Femenino , Estudios de Asociación Genética/métodos , Genotipo , Humanos , Italia , Masculino , Modelos Biológicos , Modelos Moleculares , Canal de Sodio Activado por Voltaje NAV1.5/química , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Linaje , Fenotipo , Conformación Proteica , Transporte de Proteínas
3.
Cell Rep ; 36(2): 109364, 2021 07 13.
Artículo en Inglés | MEDLINE | ID: mdl-34214467

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) variants govern transmissibility, responsiveness to vaccination, and disease severity. In a screen for new models of SARS-CoV-2 infection, we identify human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of angiotensin-converting enzyme 2 (ACE2) expression. Remarkably, H522 infection requires the E484D S variant; viruses expressing wild-type S are not infectious. Anti-S monoclonal antibodies differentially neutralize SARS-CoV-2 E484D S in H522 cells as compared to ACE2-expressing cells. Sera from vaccinated individuals block this alternative entry mechanism, whereas convalescent sera are less effective. Although the H522 receptor remains unknown, depletion of surface heparan sulfates block H522 infection. Temporally resolved transcriptomic and proteomic profiling reveal alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type I interferon signaling. These findings establish an alternative SARS-CoV-2 host cell receptor for the E484D SARS-CoV-2 variant, which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.


Asunto(s)
COVID-19/inmunología , COVID-19/metabolismo , Receptores Virales , Glicoproteína de la Espiga del Coronavirus/metabolismo , Sustitución de Aminoácidos , Enzima Convertidora de Angiotensina 2 , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Ciclo Celular , Línea Celular Tumoral , Chlorocebus aethiops , Perfilación de la Expresión Génica , Heparitina Sulfato/metabolismo , Humanos , Interferón Tipo I/metabolismo , Helicasa Inducida por Interferón IFIH1/metabolismo , Modelos Biológicos , Unión Proteica , Dominios Proteicos , Proteómica , Receptores Virales/metabolismo , SARS-CoV-2 , Serina Endopeptidasas/metabolismo , Transducción de Señal , Glicoproteína de la Espiga del Coronavirus/genética , Células Vero , Internalización del Virus , Replicación Viral
4.
Int J Mol Sci ; 22(10)2021 May 18.
Artículo en Inglés | MEDLINE | ID: mdl-34070125

RESUMEN

The neuropeptide substance P (SP) mediates neurogenic inflammation and pain and contributes to atopic dermatitis in mice through the activation of mast cells (MCs) via Mas-related G protein-coupled receptor (GPCR)-B2 (MrgprB2, human ortholog MRGPRX2). In addition to G proteins, certain MRGPRX2 agonists activate an additional signaling pathway that involves the recruitment of ß-arrestins, which contributes to receptor internalization and desensitization (balanced agonists). We found that SP caused ß-arrestin recruitment, MRGPRX2 internalization, and desensitization. These responses were independent of G proteins, indicating that SP serves as a balanced agonist for MRGPRX2. A tyrosine residue in the highly conserved NPxxY motif contributes to the activation and internalization of many GPCRs. We have previously shown that Tyr279 of MRGPRX2 is essential for G protein-mediated signaling and degranulation. To assess its role in ß-arrestin-mediated MRGPRX2 regulation, we replaced Tyr279 in the NPxxY motif of MRGPRX2 with Ala (Y279A). Surprisingly, we found that, unlike the wild-type receptor, Y279A mutant of MRGPRX2 was resistant to SP-induced ß-arrestin recruitment and internalization. This study reveals the novel findings that activation of MRGPRX2 by SP is regulated by ß-arrestins and that a highly conserved tyrosine residue within MRGPRX2's NPxxY motif contributes to both G protein- and ß-arrestin-mediated responses.


Asunto(s)
Proteínas del Tejido Nervioso/agonistas , Receptores Acoplados a Proteínas G/agonistas , Receptores de Neuropéptido/agonistas , Sustancia P/metabolismo , beta-Arrestinas/metabolismo , Sustitución de Aminoácidos , Animales , Degranulación de la Célula , Línea Celular , Femenino , Humanos , Masculino , Mastocitos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Neuroinmunomodulación/fisiología , Ratas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropéptido/química , Receptores de Neuropéptido/genética , Tirosina/química , Arrestina beta 2/deficiencia , Arrestina beta 2/genética , Arrestina beta 2/metabolismo
5.
Int J Mol Sci ; 22(11)2021 May 26.
Artículo en Inglés | MEDLINE | ID: mdl-34073489

RESUMEN

Alpha-1-antitrypsin (AAT) deficiency causes pulmonary disease due to decreased levels of circulating AAT and consequently unbalanced protease activity in the lungs. Deposition of specific AAT variants, such as the common Z AAT, within hepatocytes may also result in liver disease. These deposits are comprised of ordered polymers of AAT formed by an inter-molecular domain swap. The discovery and characterization of rare variants of AAT and other serpins have historically played a crucial role in the dissection of the structural mechanisms leading to AAT polymer formation. Here, we report a severely deficient shutter region variant, Bologna AAT (N186Y), which was identified in five unrelated subjects with different geographical origins. We characterized the new variant by expression in cellular models in comparison with known polymerogenic AAT variants. Bologna AAT showed secretion deficiency and intracellular accumulation as detergent-insoluble polymers. Extracellular polymers were detected in both the culture media of cells expressing Bologna AAT and in the plasma of a patient homozygous for this variant. Structural modelling revealed that the mutation disrupts the hydrogen bonding network in the AAT shutter region. These data support a crucial coordinating role for asparagine 186 and the importance of this network in promoting formation of the native structure.


Asunto(s)
Mutación Missense , Deficiencia de alfa 1-Antitripsina/metabolismo , alfa 1-Antitripsina , Sustitución de Aminoácidos , Células HEK293 , Humanos , Dominios Proteicos , alfa 1-Antitripsina/biosíntesis , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/patología
6.
Int J Mol Sci ; 22(11)2021 May 26.
Artículo en Inglés | MEDLINE | ID: mdl-34073630

RESUMEN

Motor neuron diseases (MNDs) are neurodegenerative disorders characterized by upper and/or lower MN loss. MNDs include amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and spinal and bulbar muscular atrophy (SBMA). Despite variability in onset, progression, and genetics, they share a common skeletal muscle involvement, suggesting that it could be a primary site for MND pathogenesis. Due to the key role of muscle-specific microRNAs (myomiRs) in skeletal muscle development, by real-time PCR we investigated the expression of miR-206, miR-133a, miR-133b, and miR-1, and their target genes, in G93A-SOD1 ALS, Δ7SMA, and KI-SBMA mouse muscle during disease progression. Further, we analyzed their expression in serum of SOD1-mutated ALS, SMA, and SBMA patients, to demonstrate myomiR role as noninvasive biomarkers. Our data showed a dysregulation of myomiRs and their targets, in ALS, SMA, and SBMA mice, revealing a common pathogenic feature associated with muscle impairment. A similar myomiR signature was observed in patients' sera. In particular, an up-regulation of miR-206 was identified in both mouse muscle and serum of human patients. Our overall findings highlight the role of myomiRs as promising biomarkers in ALS, SMA, and SBMA. Further investigations are needed to explore the potential of myomiRs as therapeutic targets for MND treatment.


Asunto(s)
Esclerosis Amiotrófica Lateral , Atrofia Bulboespinal Ligada al X , MicroARNs , Mutación Missense , Superóxido Dismutasa-1 , Superóxido Dismutasa , Sustitución de Aminoácidos , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Atrofia Bulboespinal Ligada al X/genética , Atrofia Bulboespinal Ligada al X/metabolismo , Humanos , Ratones , Ratones Transgénicos , MicroARNs/genética , MicroARNs/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo
7.
Int J Mol Sci ; 22(11)2021 May 23.
Artículo en Inglés | MEDLINE | ID: mdl-34071043

RESUMEN

A de novo missense variant in Rag GTPase protein C (RagCS75Y) was recently identified in a syndromic dilated cardiomyopathy (DCM) patient. However, its pathogenicity and the related therapeutic strategy remain unclear. We generated a zebrafish RragcS56Y (corresponding to human RagCS75Y) knock-in (KI) line via TALEN technology. The KI fish manifested cardiomyopathy-like phenotypes and poor survival. Overexpression of RagCS75Y via adenovirus infection also led to increased cell size and fetal gene reprogramming in neonatal rat ventricle cardiomyocytes (NRVCMs), indicating a conserved mechanism. Further characterization identified aberrant mammalian target of rapamycin complex 1 (mTORC1) and transcription factor EB (TFEB) signaling, as well as metabolic abnormalities including dysregulated autophagy. However, mTOR inhibition failed to ameliorate cardiac phenotypes in the RagCS75Y cardiomyopathy models, concomitant with a failure to promote TFEB nuclear translocation. This observation was at least partially explained by increased and mTOR-independent physical interaction between RagCS75Y and TFEB in the cytosol. Importantly, TFEB overexpression resulted in more nuclear TFEB and rescued cardiomyopathy phenotypes. These findings suggest that S75Y is a pathogenic gain-of-function mutation in RagC that leads to cardiomyopathy. A primary pathological step of RagCS75Y cardiomyopathy is defective mTOR-TFEB signaling, which can be corrected by TFEB overexpression, but not mTOR inhibition.


Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/fisiología , Cardiomiopatía Dilatada/genética , Mutación con Ganancia de Función , Proteínas de Unión al GTP Monoméricas/genética , Mutación Missense , Mutación Puntual , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Transporte Activo de Núcleo Celular , Sustitución de Aminoácidos , Animales , Autofagia , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/biosíntesis , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Cardiomiopatía Dilatada/terapia , Células Cultivadas , Técnicas de Sustitución del Gen , Técnicas de Inactivación de Genes , Ventrículos Cardíacos/citología , Humanos , Ratones , Proteínas de Unión al GTP Monoméricas/fisiología , Miocitos Cardíacos/metabolismo , Fenotipo , Ratas Wistar , Proteínas Recombinantes/metabolismo , Transducción de Señal , Nucleasas de los Efectores Tipo Activadores de la Transcripción , Pez Cebra , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/fisiología
8.
Science ; 372(6547): 1220-1224, 2021 06 11.
Artículo en Inglés | MEDLINE | ID: mdl-34112695

RESUMEN

Viruses are ubiquitous pathogens of global impact. Prompted by the hypothesis that their earliest progenitors recruited host proteins for virion formation, we have used stringent laboratory evolution to convert a bacterial enzyme that lacks affinity for nucleic acids into an artificial nucleocapsid that efficiently packages and protects multiple copies of its own encoding messenger RNA. Revealing remarkable convergence on the molecular hallmarks of natural viruses, the accompanying changes reorganized the protein building blocks into an interlaced 240-subunit icosahedral capsid that is impermeable to nucleases, and emergence of a robust RNA stem-loop packaging cassette ensured high encapsidation yields and specificity. In addition to evincing a plausible evolutionary pathway for primordial viruses, these findings highlight practical strategies for developing nonviral carriers for diverse vaccine and delivery applications.


Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cápside/metabolismo , Evolución Molecular Dirigida , ARN Mensajero/metabolismo , Sustitución de Aminoácidos , Aquifex/enzimología , Proteínas Bacterianas/química , Cápside/química , Microscopía por Crioelectrón , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Nucleocápside/química , Nucleocápside/genética , Nucleocápside/metabolismo , Dominios Proteicos , Estructura Secundaria de Proteína , Subunidades de Proteína , ARN Mensajero/química , ARN Mensajero/genética , Ribonucleasas/metabolismo
9.
Int J Mol Sci ; 22(11)2021 May 27.
Artículo en Inglés | MEDLINE | ID: mdl-34072007

RESUMEN

Adrenergic receptor ß3 (ADRß3) is a member of the rhodopsin-like G protein-coupled receptor family. The binding of the ligand to ADRß3 activates adenylate cyclase and increases cAMP in the cells. ADRß3 is highly expressed in white and brown adipocytes and controls key regulatory pathways of lipid metabolism. Trp64Arg (W64R) polymorphism in the ADRß3 is associated with the early development of type 2 diabetes mellitus, lower resting metabolic rate, abdominal obesity, and insulin resistance. It is unclear how the substitution of W64R affects the functioning of ADRß3. This study was initiated to functionally characterize this obesity-linked variant of ADRß3. We evaluated in detail the expression, subcellular distribution, and post-activation behavior of the WT and W64R ADRß3 using single cell quantitative fluorescence microscopy. When expressed in HEK 293 cells, ADRß3 shows a typical distribution displayed by other GPCRs with a predominant localization at the cell surface. Unlike adrenergic receptor ß2 (ADRß2), agonist-induced desensitization of ADRß3 does not involve loss of cell surface expression. WT and W64R variant of ADRß3 displayed comparable biochemical properties, and there was no significant impact of the substitution of tryptophan with arginine on the expression, cellular distribution, signaling, and post-activation behavior of ADRß3. The obesity-linked W64R variant of ADRß3 is indistinguishable from the WT ADRß3 in terms of expression, cellular distribution, signaling, and post-activation behavior.


Asunto(s)
Predisposición Genética a la Enfermedad , Variación Genética , Obesidad/genética , Obesidad/metabolismo , Receptores Adrenérgicos beta 3/genética , Receptores Adrenérgicos beta 3/metabolismo , Agonistas de Receptores Adrenérgicos beta 3/farmacología , Alelos , Sustitución de Aminoácidos , Línea Celular , Expresión Génica , Estudios de Asociación Genética , Humanos , Mutación , Polimorfismo Genético , Transporte de Proteínas
10.
Int J Food Microbiol ; 351: 109269, 2021 Aug 02.
Artículo en Inglés | MEDLINE | ID: mdl-34102570

RESUMEN

Microbial population heterogeneity contributes to differences in stress response between individual cells in a population, and can lead to the selection of genetically stable variants with increased stress resistance. We previously provided evidence that the multiple-stress resistant Listeria monocytogenes LO28 variant 15, carries a point mutation in the rpsU gene, resulting in an arginine-proline substitution in ribosomal protein RpsU (RpsU17Arg-Pro). Here, we investigated the trade-off between general stress sigma factor SigB-mediated stress resistance and fitness in variant 15 using experimental evolution. By selecting for higher fitness in two parallel evolving cultures, we identified two evolved variants: 15EV1 and 15EV2. Whole genome sequencing and SNP analysis showed that both parallel lines mutated in the same codon in rpsU as the original mutation resulting in RpsU17Pro-His (15EV1) and RpsU17Pro-Thr (15EV2). Using a combined phenotyping and proteomics approach, we assessed the resistance of the evolved variants to both heat and acid stress, and found that in both lines reversion to WT-like fitness also resulted in WT-like stress sensitivity. Proteome analysis of L. monocytogenes LO28 WT, variant 15, 15EV1, and 15EV2 revealed high level expression of SigB regulon members only in variant 15, whereas protein profiles of both evolved variants were highly similar to that of the LO28 WT. Experiments with constructed RpsU17Arg-Pro mutants in L. monocytogenes LO28 and EGDe, and RpsU17Arg-His and RpsU17Arg-Thr in LO28, confirmed that single amino acid substitutions in RpsU enable switching between multiple-stress resistant and high fitness states in L. monocytogenes.


Asunto(s)
Adaptación Fisiológica/genética , Proteínas Bacterianas/genética , Listeria monocytogenes/fisiología , Proteínas Ribosómicas/genética , Ácidos/metabolismo , Sustitución de Aminoácidos , Proteínas Bacterianas/metabolismo , Evolución Molecular Dirigida , Genoma Bacteriano/genética , Calor , Listeria monocytogenes/genética , Mutación , Proteoma/metabolismo , Proteínas Ribosómicas/metabolismo , Factor sigma/genética , Factor sigma/metabolismo
11.
Genes (Basel) ; 12(6)2021 05 27.
Artículo en Inglés | MEDLINE | ID: mdl-34072181

RESUMEN

The genomic diversity of SARS-CoV-2 has been a focus during the ongoing COVID-19 pandemic. Here, we analyzed the distribution and character of emerging mutations in a data set comprising more than 95,000 virus genomes covering eight major SARS-CoV-2 lineages in the GISAID database, including genotypes arising during COVID-19 therapy. Globally, the C>U transitions and G>U transversions were the most represented mutations, accounting for the majority of single-nucleotide variations. Mutational spectra were not influenced by the time the virus had been circulating in its host or medical treatment. At the amino acid level, we observed about a 2-fold excess of substitutions in favor of hydrophobic amino acids over the reverse. However, most mutations constituting variants of interests of the S-protein (spike) lead to hydrophilic amino acids, counteracting the global trend. The C>U and G>U substitutions altered codons towards increased amino acid hydrophobicity values in more than 80% of cases. The bias is explained by the existing differences in the codon composition for amino acids bearing contrasting biochemical properties. Mutation asymmetries apparently influence the biochemical features of SARS CoV-2 proteins, which may impact protein-protein interactions, fusion of viral and cellular membranes, and virion assembly.


Asunto(s)
COVID-19/virología , Genoma Viral , Interacciones Hidrofóbicas e Hidrofílicas , Mutación , SARS-CoV-2/genética , Proteínas Virales/química , Proteínas Virales/genética , Desaminasas APOBEC , Alelos , Sustitución de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Evolución Molecular , Variación Genética , Genotipo , Interacciones Huésped-Patógeno , Humanos , Filogenia , Polimorfismo de Nucleótido Simple , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética
12.
Sci Rep ; 11(1): 12740, 2021 06 17.
Artículo en Inglés | MEDLINE | ID: mdl-34140558

RESUMEN

The SARS-CoV-2 variants replacing the first wave strain pose an increased threat by their potential ability to escape pre-existing humoral protection. An angiotensin converting enzyme 2 (ACE2) decoy that competes with endogenous ACE2 for binding of the SARS-CoV-2 spike receptor binding domain (S RBD) and inhibits infection may offer a therapeutic option with sustained efficacy against variants. Here, we used Molecular Dynamics (MD) simulation to predict ACE2 sequence substitutions that might increase its affinity for S RBD and screened candidate ACE2 decoys in vitro. The lead ACE2(T27Y/H34A)-IgG1FC fusion protein with enhanced S RBD affinity shows greater live SARS-CoV-2 virus neutralization capability than wild type ACE2. MD simulation was used to predict the effects of S RBD variant mutations on decoy affinity that was then confirmed by testing of an ACE2 Triple Decoy that included an additional enzyme activity-deactivating H374N substitution against mutated S RBD. The ACE2 Triple Decoy maintains high affinity for mutated S RBD, displays enhanced affinity for S RBD N501Y or L452R, and has the highest affinity for S RBD with both E484K and N501Y mutations, making it a viable therapeutic option for the prevention or treatment of SARS-CoV-2 infection with a high likelihood of efficacy against variants.


Asunto(s)
Sustitución de Aminoácidos , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/metabolismo , Antivirales/farmacología , COVID-19/metabolismo , Descubrimiento de Drogas/métodos , Simulación de Dinámica Molecular , SARS-CoV-2/metabolismo , Transducción de Señal/efectos de los fármacos , Secuencia de Aminoácidos , COVID-19/virología , Humanos , Mutación , Unión Proteica/efectos de los fármacos , Dominios Proteicos/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del Virus/efectos de los fármacos
13.
Arch Virol ; 166(8): 2273-2278, 2021 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-34059971

RESUMEN

Feline panleukopenia virus (FPV) is a highly contagious infectious pathogen of cats globally. However, there is no information on the molecular identification and characterization of FPV in Bangladesh. Here, 8.16% (8/98) and 18.37% (18/98) of diarrheic cats tested positive for FPV by an immunochromatography (IC) test and PCR, respectively. The IC test showed 44.44% sensitivity and 100% specificity in comparison with PCR. Our newly sequenced Bangladeshi FPV strain (MN826076) showed the highest (99.71%) sequence identity to strains from the United Arab Emirates (UAE). Strain MN826076 contained two characteristic amino acid variations in VP2 identifying it as an FPV strain: valine at position 103 and aspartic acid at position 323. Phylogenetically, the VP2 of strain MN826076 was found to be closely related to 19 FPV strains, sharing the same clade.


Asunto(s)
Diarrea/veterinaria , Diarrea/virología , Virus de la Panleucopenia Felina/clasificación , Panleucopenia Felina/diagnóstico , Sustitución de Aminoácidos , Animales , Bangladesh , Proteínas de la Cápside/genética , Gatos , China , Cromatografía de Afinidad , Virus de la Panleucopenia Felina/genética , Virus de la Panleucopenia Felina/aislamiento & purificación , Filogenia , Filogeografía , Portugal , Sensibilidad y Especificidad , Tailandia , Emiratos Árabes Unidos
14.
Int J Mol Sci ; 22(10)2021 May 16.
Artículo en Inglés | MEDLINE | ID: mdl-34065754

RESUMEN

Cyanobacteriochromes (CBCRs) are promising optogenetic tools for their diverse absorption properties with a single compact cofactor-binding domain. We previously uncovered the ultrafast reversible photoswitching dynamics of a red/green photoreceptor AnPixJg2, which binds phycocyanobilin (PCB) that is unavailable in mammalian cells. Biliverdin (BV) is a mammalian cofactor with a similar structure to PCB but exhibits redder absorption. To improve the AnPixJg2 feasibility in mammalian applications, AnPixJg2_BV4 with only four mutations has been engineered to incorporate BV. Herein, we implemented femtosecond transient absorption (fs-TA) and ground state femtosecond stimulated Raman spectroscopy (GS-FSRS) to uncover transient electronic dynamics on molecular time scales and key structural motions responsible for the photoconversion of AnPixJg2_BV4 with PCB (Bpcb) and BV (Bbv) cofactors in comparison with the parent AnPixJg2 (Apcb). Bpcb adopts the same photoconversion scheme as Apcb, while BV4 mutations create a less bulky environment around the cofactor D ring that promotes a faster twist. The engineered Bbv employs a reversible clockwise/counterclockwise photoswitching that requires a two-step twist on ~5 and 35 picosecond (ps) time scales. The primary forward Pfr → Po transition displays equal amplitude weights between the two processes before reaching a conical intersection. In contrast, the primary reverse Po → Pfr transition shows a 2:1 weight ratio of the ~35 ps over 5 ps component, implying notable changes to the D-ring-twisting pathway. Moreover, we performed pre-resonance GS-FSRS and quantum calculations to identify the Bbv vibrational marker bands at ~659,797, and 1225 cm-1. These modes reveal a stronger H-bonding network around the BV cofactor A ring with BV4 mutations, corroborating the D-ring-dominant reversible photoswitching pathway in the excited state. Implementation of BV4 mutations in other PCB-binding GAF domains like AnPixJg4, AM1_1870g3, and NpF2164g5 could promote similar efficient reversible photoswitching for more directional bioimaging and optogenetic applications, and inspire other bioengineering advances.


Asunto(s)
Biliverdina/química , Cianobacterias/genética , Fotorreceptores Microbianos/química , Fitocromo/química , Sustitución de Aminoácidos , Biliverdina/genética , Sitios de Unión , Cianobacterias/metabolismo , Electrónica , Cinética , Procesos Fotoquímicos , Fotorreceptores Microbianos/genética , Fitocromo/genética , Ingeniería de Proteínas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Análisis Espectral , Espectrometría Raman , Tiempo , Factores de Tiempo
15.
Sci Rep ; 11(1): 13120, 2021 06 23.
Artículo en Inglés | MEDLINE | ID: mdl-34162970

RESUMEN

In December 2019, a novel coronavirus, termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the cause of pneumonia with severe respiratory distress and outbreaks in Wuhan, China. The rapid and global spread of SARS-CoV-2 resulted in the coronavirus 2019 (COVID-19) pandemic. Earlier during the pandemic, there were limited genetic viral variations. As millions of people became infected, multiple single amino acid substitutions emerged. Many of these substitutions have no consequences. However, some of the new variants show a greater infection rate, more severe disease, and reduced sensitivity to current prophylaxes and treatments. Of particular importance in SARS-CoV-2 transmission are mutations that occur in the Spike (S) protein, the protein on the viral outer envelope that binds to the human angiotensin-converting enzyme receptor (hACE2). Here, we conducted a comprehensive analysis of 441,168 individual virus sequences isolated from humans throughout the world. From the individual sequences, we identified 3540 unique amino acid substitutions in the S protein. Analysis of these different variants in the S protein pinpointed important functional and structural sites in the protein. This information may guide the development of effective vaccines and therapeutics to help arrest the spread of the COVID-19 pandemic.


Asunto(s)
Variación Genética , Glicoproteína de la Espiga del Coronavirus/genética , Sustitución de Aminoácidos , Furina/metabolismo , Glicosilación , Humanos , Modelos Moleculares , Profilaxis Pre-Exposición , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/aislamiento & purificación
16.
Int J Mol Sci ; 22(10)2021 May 12.
Artículo en Inglés | MEDLINE | ID: mdl-34065855

RESUMEN

To identify the physiological factors that limit the growth of Escherichia coli K-12 strains synthesizing minimal lipopolysaccharide (LPS), we describe the first construction of strains devoid of the entire waa locus and concomitantly lacking all three acyltransferases (LpxL/LpxM/LpxP), synthesizing minimal lipid IVA derivatives with a restricted ability to grow at around 21 °C. Suppressors restoring growth up to 37 °C of Δ(gmhD-waaA) identified two independent single-amino-acid substitutions-P50S and R310S-in the LPS flippase MsbA. Interestingly, the cardiolipin synthase-encoding gene clsA was found to be essential for the growth of ΔlpxLMP, ΔlpxL, ΔwaaA, and Δ(gmhD-waaA) bacteria, with a conditional lethal phenotype of Δ(clsA lpxM), which could be overcome by suppressor mutations in MsbA. Suppressor mutations basS A20D or basR G53V, causing a constitutive incorporation of phosphoethanolamine (P-EtN) in the lipid A, could abolish the Ca++ sensitivity of Δ(waaC eptB), thereby compensating for P-EtN absence on the second Kdo. A single-amino-acid OppA S273G substitution is shown to overcome the synthetic lethality of Δ(waaC surA) bacteria, consistent with the chaperone-like function of the OppA oligopeptide-binding protein. Furthermore, overexpression of GcvB sRNA was found to repress the accumulation of LpxC and suppress the lethality of LapAB absence. Thus, this study identifies new and limiting factors in regulating LPS biosynthesis.


Asunto(s)
Escherichia coli K12/crecimiento & desarrollo , Genes Esenciales , Lipopolisacáridos/biosíntesis , Lipopolisacáridos/genética , Transportadoras de Casetes de Unión a ATP/genética , Aciltransferasas/genética , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Cardiolipinas/genética , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/genética , Lipoproteínas/genética , Proteínas de la Membrana/genética , Mutaciones Letales Sintéticas , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética
17.
Hematol Oncol ; 39 Suppl 1: 15-23, 2021 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-34105821

RESUMEN

Children with Langerhnans cell histiocytosis (LCH) develop granulomatous lesions with characteristic clonal CD207+ dendritic cells that can arise as single lesions or life-threatening disseminated disease. Despite the wide range of clinical presentations, LCH lesions are histologically indistinguishable based on severity of disease, and uncertain classification as an immune versus neoplastic disorder has historically challenged the development of optimal clinical strategies for patients with LCH. Recently, activating somatic mutations in MAPK pathway genes, most notably BRAFV600E, have been discovered in almost all cases of LCH. Further, the stage of myeloid differentiation in which the mutation arises defines the extent of disease and risk of developing LCH-associated neurodegeneration. MAPK activation in LCH precursor cells drives myeloid differentiation, inhibits migration, and inhibits apoptosis, resulting in accumulation of resilient pathologic dendritic cells that recruit and activate T cells. Recurrent somatic mutations in MAPK pathway genes have also been identified in related histiocytic disorders: juvenile xanthogranuloma, Erdheim-Chester disease, and Rosai-Dorfman disease. New insights into pathogenesis support reclassification of these conditions as a myeloid neoplastic disorders. Continued research will uncover opportunities to identify novel targets and inform personalized therapeutic strategies based on cell of origin, somatic mutation, inherited risk factors, and residual disease.


Asunto(s)
Diferenciación Celular/inmunología , Movimiento Celular/inmunología , Células Dendríticas , Histiocitosis de Células de Langerhans , Medicina de Precisión , Linfocitos T , Sustitución de Aminoácidos , Diferenciación Celular/genética , Movimiento Celular/genética , Células Dendríticas/inmunología , Células Dendríticas/patología , Histiocitosis de Células de Langerhans/genética , Histiocitosis de Células de Langerhans/inmunología , Histiocitosis de Células de Langerhans/patología , Histiocitosis de Células de Langerhans/terapia , Humanos , Sistema de Señalización de MAP Quinasas/genética , Sistema de Señalización de MAP Quinasas/inmunología , Mutación Missense , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/inmunología , Linfocitos T/inmunología , Linfocitos T/patología
18.
Nat Commun ; 12(1): 2949, 2021 05 19.
Artículo en Inglés | MEDLINE | ID: mdl-34011959

RESUMEN

The antibiotic trimethoprim (TMP) is used to treat a variety of Escherichia coli infections, but its efficacy is limited by the rapid emergence of TMP-resistant bacteria. Previous laboratory evolution experiments have identified resistance-conferring mutations in the gene encoding the TMP target, bacterial dihydrofolate reductase (DHFR), in particular mutation L28R. Here, we show that 4'-desmethyltrimethoprim (4'-DTMP) inhibits both DHFR and its L28R variant, and selects against the emergence of TMP-resistant bacteria that carry the L28R mutation in laboratory experiments. Furthermore, antibiotic-sensitive E. coli populations acquire antibiotic resistance at a substantially slower rate when grown in the presence of 4'-DTMP than in the presence of TMP. We find that 4'-DTMP impedes evolution of resistance by selecting against resistant genotypes with the L28R mutation and diverting genetic trajectories to other resistance-conferring DHFR mutations with catalytic deficiencies. Our results demonstrate how a detailed characterization of resistance-conferring mutations in a target enzyme can help identify potential drugs against antibiotic-resistant bacteria, which may ultimately increase long-term efficacy of antimicrobial therapies by modulating evolutionary trajectories that lead to resistance.


Asunto(s)
Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/microbiología , Resistencia al Trimetoprim/genética , Trimetoprim/análogos & derivados , Sustitución de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacología , Cristalografía por Rayos X , Evolución Molecular Dirigida , Diseño de Fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Antagonistas del Ácido Fólico/química , Antagonistas del Ácido Fólico/farmacología , Genes Bacterianos , Genotipo , Humanos , Modelos Moleculares , Mutación , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/genética , Trimetoprim/química , Trimetoprim/farmacología
19.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artículo en Inglés | MEDLINE | ID: mdl-33946750

RESUMEN

Genetic testing in Brugada syndrome (BrS) is still not considered to be useful for clinical management of patients in the majority of cases, due to the current lack of understanding about the effect of specific variants. Additionally, family history of sudden death is generally not considered useful for arrhythmic risk stratification. We sought to demonstrate the usefulness of genetic testing and family history in diagnosis and risk stratification. The family history was collected for a proband who presented with a personal history of aborted cardiac arrest and in whom a novel variant in the SCN5A gene was found. Living family members underwent ajmaline testing, electrophysiological study, and genetic testing to determine genotype-phenotype segregation, if any. Patch-clamp experiments on transfected human embryonic kidney 293 cells enabled the functional characterization of the SCN5A novel variant in vitro. In this study, we provide crucial human data on the novel heterozygous variant NM_198056.2:c.5000T>A (p.Val1667Asp) in the SCN5A gene, and demonstrate its segregation with a severe form of BrS and multiple sudden deaths. Functional data revealed a loss of function of the protein affected by the variant. These results provide the first disease association with this variant and demonstrate the usefulness of genetic testing for diagnosis and risk stratification in certain patients. This study also demonstrates the usefulness of collecting the family history, which can assist in understanding the severity of the disease in certain situations and confirm the importance of the functional studies to distinguish between pathogenic mutations and harmless genetic variants.


Asunto(s)
Síndrome de Brugada/genética , Mutación Missense , Canal de Sodio Activado por Voltaje NAV1.5/genética , Adolescente , Adulto , Anciano , Ajmalina/farmacología , Sustitución de Aminoácidos , Síndrome de Brugada/complicaciones , Síndrome de Brugada/metabolismo , Muerte Súbita Cardíaca/etiología , Electrocardiografía , Femenino , Pruebas Genéticas , Células HEK293 , Heterocigoto , Humanos , Mutación con Pérdida de Función , Masculino , Persona de Mediana Edad , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Técnicas de Placa-Clamp , Linaje , Polimorfismo de Nucleótido Simple , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
20.
J Med Chem ; 64(10): 6696-6705, 2021 05 27.
Artículo en Inglés | MEDLINE | ID: mdl-33974425

RESUMEN

Innate defense regulator (IDR) peptides show promise as immunomodulatory therapeutics. However, there is limited understanding of the relationship of IDR peptide sequence and/or structure with its immunomodulatory activity. We previously reported that an IDR peptide, IDR-1002, reduces airway hyperresponsiveness (AHR) and inflammation in a house dust mite (HDM)-challenged murine model of airway inflammation. Here, we examined the sequence-to-function relationship of IDR-1002 in HDM-challenged mice and human bronchial epithelial cells (HBEC). We demonstrated that the tryptophan (W8) in the central hydrophobic region of IDR-1002 is required for the peptide to (i) suppress the pro-inflammatory cytokine IL-33, and induce anti-inflammatory mediators IL-1RA and stanniocalcin-1 in HBEC, and (ii) reduce IL-33 abundance, and eosinophil and neutrophil infiltration, in the lungs of HDM-challenged mice, without affecting the capacity to improve AHR, suggesting multimodal activity in vivo. Findings from this study can be used to design IDR peptides with targeted impact on immunomodulation and pathophysiology in respiratory diseases.


Asunto(s)
Antiinflamatorios/farmacología , Péptidos Catiónicos Antimicrobianos/química , Inmunomodulación/efectos de los fármacos , Triptófano/química , Sustitución de Aminoácidos , Animales , Antiinflamatorios/química , Antiinflamatorios/uso terapéutico , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Asma/tratamiento farmacológico , Modelos Animales de Enfermedad , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Interleucina-33/metabolismo , Ratones , Ratones Endogámicos BALB C , Infiltración Neutrófila/efectos de los fármacos , Análisis de Componente Principal , Estructura Secundaria de Proteína , Pyroglyphidae/patogenicidad , Triptófano/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...