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1.
Nat Commun ; 12(1): 879, 2021 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-33563986

RESUMEN

Salmonella Typhimurium establishes systemic infection by replicating in host macrophages. Here we show that macrophages infected with S. Typhimurium exhibit upregulated glycolysis and decreased serine synthesis, leading to accumulation of glycolytic intermediates. The effects on serine synthesis are mediated by bacterial protein SopE2, a type III secretion system (T3SS) effector encoded in pathogenicity island SPI-1. The changes in host metabolism promote intracellular replication of S. Typhimurium via two mechanisms: decreased glucose levels lead to upregulated bacterial uptake of 2- and 3-phosphoglycerate and phosphoenolpyruvate (carbon sources), while increased pyruvate and lactate levels induce upregulation of another pathogenicity island, SPI-2, known to encode virulence factors. Pharmacological or genetic inhibition of host glycolysis, activation of host serine synthesis, or deletion of either the bacterial transport or signal sensor systems for those host glycolytic intermediates impairs S. Typhimurium replication or virulence.


Asunto(s)
Proteínas Bacterianas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Macrófagos/metabolismo , Salmonella typhimurium/crecimiento & desarrollo , Salmonella typhimurium/patogenicidad , Sistemas de Secreción Tipo III/metabolismo , Animales , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Islas Genómicas , Glucosa/metabolismo , Ácidos Glicéricos/metabolismo , Glucólisis , Factores de Intercambio de Guanina Nucleótido/genética , Macrófagos/microbiología , Ratones , Células RAW 264.7 , Salmonella typhimurium/metabolismo , Serina/biosíntesis , Transducción de Señal , Sistemas de Secreción Tipo III/genética , Virulencia
2.
Nat Commun ; 12(1): 1032, 2021 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-33589587

RESUMEN

Pulmonary alveolar proteinosis (PAP) is a devastating lung disease caused by abnormal surfactant homeostasis, with a prevalence of 6-7 cases per million population worldwide. While mutations causing hereditary PAP have been reported, the genetic basis contributing to autoimmune PAP (aPAP) has not been thoroughly investigated. Here, we conducted a genome-wide association study of aPAP in 198 patients and 395 control participants of Japanese ancestry. The common genetic variant, rs138024423 at 6p21, in the major-histocompatibility-complex (MHC) region was significantly associated with disease risk (Odds ratio [OR] = 5.2; P = 2.4 × 10-12). HLA fine-mapping revealed that the common HLA class II allele, HLA-DRB1*08:03, strongly drove this signal (OR = 4.8; P = 4.8 × 10-12), followed by an additional independent risk allele at HLA-DPß1 amino acid position 8 (OR = 0.28; P = 3.4 × 10-7). HLA-DRB1*08:03 was also associated with an increased level of anti-GM-CSF antibody, a key driver of the disease (ß = 0.32; P = 0.035). Our study demonstrated a heritable component of aPAP, suggesting an underlying genetic predisposition toward an abnormal antibody production.


Asunto(s)
Autoanticuerpos/genética , Enfermedades Autoinmunes/genética , Predisposición Genética a la Enfermedad , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Cadenas HLA-DRB1/genética , Proteinosis Alveolar Pulmonar/genética , Adulto , Anciano , Alelos , Grupo de Ascendencia Continental Asiática , Autoanticuerpos/biosíntesis , Enfermedades Autoinmunes/etnología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Estudios de Casos y Controles , Cromosomas Humanos Par 6 , Femenino , Expresión Génica , Frecuencia de los Genes , Estudio de Asociación del Genoma Completo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Cadenas HLA-DRB1/inmunología , Humanos , Japón , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Isoformas de Proteínas/genética , Proteinosis Alveolar Pulmonar/etnología , Proteinosis Alveolar Pulmonar/inmunología , Proteinosis Alveolar Pulmonar/patología , Surfactantes Pulmonares/inmunología , Surfactantes Pulmonares/metabolismo , Riesgo
3.
Medicine (Baltimore) ; 100(4): e24125, 2021 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-33530204

RESUMEN

BACKGROUND: In the present study, we aimed to detect the expression of CXCL2 in epithelial ovarian cancer (OC) and explore its clinical significance. METHODS: TCGA (The Cancer Genome Atlas) database was adopted to assess the significance of CXCL2. Tissue microarray and immunohistochemical staining were used to detect the expression of CXCL2 in epithelial OC, and its correlation with clinicopathological features and prognosis was statistically analyzed. RESULTS: CXCL2 was highly expressed in epithelial OC tissues compared with the adjacent tissues. Such up-regulation of CXCL2 was significantly correlated with tumor differentiation (P = .001), tumor stage (P = .01), tumor location (unilateral or bilateral) (P = .003), and metastasis (P = .003). Kaplan-Meier and Cox proportional hazards regression analyses showed that high expression of CXCL2 was not an independent predictor of poor prognosis in epithelial OC. CONCLUSIONS: Collectively, the high expression of CXCL2 might be related to the invasion and metastasis of epithelial OC.


Asunto(s)
Quimiocina CXCL2/biosíntesis , Neoplasias Ováricas/patología , Factores de Edad , Biomarcadores de Tumor , Antígeno Ca-125/sangre , Epitelio/metabolismo , Femenino , Humanos , Estimación de Kaplan-Meier , Metástasis de la Neoplasia , Estadificación de Neoplasias , Pronóstico , Modelos de Riesgos Proporcionales , Análisis de Matrices Tisulares
4.
Nat Commun ; 12(1): 857, 2021 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-33558498

RESUMEN

Bacteria often live in diverse communities where the spatial arrangement of strains and species is considered critical for their ecology. However, a test of this hypothesis requires manipulation at the fine scales at which spatial structure naturally occurs. Here we develop a droplet-based printing method to arrange bacterial genotypes across a sub-millimetre array. We print strains of the gut bacterium Escherichia coli that naturally compete with one another using protein toxins. Our experiments reveal that toxin-producing strains largely eliminate susceptible non-producers when genotypes are well-mixed. However, printing strains side-by-side creates an ecological refuge where susceptible strains can persist in large numbers. Moving to competitions between toxin producers reveals that spatial structure can make the difference between one strain winning and mutual destruction. Finally, we print different potential barriers between competing strains to understand how ecological refuges form, which shows that cells closest to a toxin producer mop up the toxin and protect their clonemates. Our work provides a method to generate customised bacterial communities with defined spatial distributions, and reveals that micron-scale changes in these distributions can drive major shifts in ecology.


Asunto(s)
Escherichia coli/citología , Impresión Tridimensional , Colicinas/biosíntesis , Escherichia coli/genética , Genotipo , Microbiota
5.
Nat Commun ; 12(1): 867, 2021 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-33558520

RESUMEN

Statins are effective cholesterol-lowering drugs. Lovastatin, one of the precursors of statins, is formed from dihydromonacolin L (DML), which is synthesized by lovastatin nonaketide synthase (LovB), with the assistance of a separate trans-acting enoyl reductase (LovC). A full DML synthesis comprises 8 polyketide synthetic cycles with about 35 steps. The assembling of the LovB-LovC complex, and the structural basis for the iterative and yet permutative functions of the megasynthase have remained a mystery. Here, we present the cryo-EM structures of the LovB-LovC complex at 3.60 Å and the core LovB at 2.91 Å resolution. The domain organization of LovB is an X-shaped face-to-face dimer containing eight connected domains. The binding of LovC laterally to the malonyl-acetyl transferase domain allows the completion of a L-shaped catalytic chamber consisting of six active domains. This architecture and the structural details of the megasynthase provide the basis for the processing of the intermediates by the individual catalytic domains. The detailed architectural model provides structural insights that may enable the re-engineering of the megasynthase for the generation of new statins.


Asunto(s)
Lovastatina/biosíntesis , Lovastatina/química , Biocatálisis , Modelos Moleculares , Naftalenos/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Sintasas Poliquetidas/química , Sintasas Poliquetidas/metabolismo , Sintasas Poliquetidas/ultraestructura , Dominios Proteicos , Especificidad por Sustrato
6.
Nat Commun ; 12(1): 818, 2021 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-33547293

RESUMEN

Venoms have evolved over a hundred times in animals. Venom toxins are thought to evolve mostly by recruitment of endogenous proteins with physiological functions. Here we report phylogenetic analyses of venom proteome-annotated venom gland transcriptome data, assisted by genomic analyses, to show that centipede venoms have recruited at least five gene families from bacterial and fungal donors, involving at least eight horizontal gene transfer events. These results establish centipedes as currently the only known animals with venoms used in predation and defence that contain multiple gene families derived from horizontal gene transfer. The results also provide the first evidence for the implication of horizontal gene transfer in the evolutionary origin of venom in an animal lineage. Three of the bacterial gene families encode virulence factors, suggesting that horizontal gene transfer can provide a fast track channel for the evolution of novelty by the exaptation of bacterial weapons into animal venoms.


Asunto(s)
Proteínas de Artrópodos/genética , Venenos de Artrópodos/genética , Transferencia de Gen Horizontal , Genes Bacterianos , Genes Fúngicos , Animales , Proteínas de Artrópodos/biosíntesis , Proteínas de Artrópodos/clasificación , Venenos de Artrópodos/biosíntesis , Venenos de Artrópodos/clasificación , /microbiología , Expresión Génica , Filogenia , Proteómica/métodos , Transcriptoma
7.
J Vis Exp ; (167)2021 01 16.
Artículo en Inglés | MEDLINE | ID: mdl-33522504

RESUMEN

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing need in developing efficient methods for recombinant antibody production. As of 2019, there were more than 70 FDA-approved monoclonal antibodies, and there is exponential growth potential. Despite their promise, limiting factors for widespread use are manufacturing costs and complexity. Potentially, plants offer low-cost, safe, and easily scalable protein manufacturing strategies. Plants like Nicotiana benthamiana not only can correctly fold and assemble complex mammalian proteins but also can add critical post-translational modifications similar to those offered by mammalian cell cultures. In this work, by using native GFP and an acid-stable variant of green fluorescent protein (GFP) fused to human monoclonal antibodies, we were able to visualize the entire transient antibody expression and purification process from N. benthamiana plants. Depending on the experiment's purpose, native GFP fusion can ensure easier visualization during the expression phase in the plants, while acid-stable GFP fusion allows for visualization during downstream processing. This scalable and straightforward procedure can be performed by a single researcher to produce milligram quantities of highly pure antibody or antibody fusion proteins in a matter of days using only a few small plants. Such a technique can be extended to the visualization of any type of antibody purification process and potentially many other proteins, both in plant and other expression systems. Moreover, these techniques can benefit virtual instructions and be executed in a teaching laboratory by undergraduate students possessing minimal prior experience with molecular biology techniques, providing a foundation for project-based exploration with real-world applications.


Asunto(s)
Inmunoglobulina G/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Tabaco/genética , Agrobacterium tumefaciens/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Secuencia de Bases , Cromatografía , Electroforesis en Gel de Poliacrilamida , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Kanamicina/farmacología , Hojas de la Planta/microbiología , Plantas Modificadas Genéticamente , Tabaco/crecimiento & desarrollo , Tabaco/microbiología , Rayos Ultravioleta
8.
Clin Appl Thromb Hemost ; 27: 1076029620977702, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33539214

RESUMEN

The SARS-CoV-2 pandemic has focused attention on prevention, restriction and treatment methods that are acceptable worldwide. This means that they should be simple and inexpensive. This review examines the possible role of glycosaminoglycan (GAG) antithrombotics in the treatment of COVID-19. The pathophysiology of this disease reveals a complex interplay between the hemostatic and immune systems that can be readily disrupted by SARS-CoV-2. Some of the GAG antithrombotics also possess immune-modulatory actions and since they are relatively inexpensive they could play an important role in the management of COVID-19 and its complications.


Asunto(s)
/tratamiento farmacológico , Fibrinolíticos/uso terapéutico , Heparina/uso terapéutico , Autoanticuerpos/biosíntesis , /fisiopatología , Endotelio Vascular/inmunología , Endotelio Vascular/fisiopatología , Endotelio Vascular/virología , Femenino , Glicosaminoglicanos/uso terapéutico , Hemorragia/etiología , Trastornos Hemostáticos/tratamiento farmacológico , Trastornos Hemostáticos/etiología , Trastornos Hemostáticos/fisiopatología , Humanos , Factores Inmunológicos/uso terapéutico , Inflamación/tratamiento farmacológico , Inflamación/etiología , Inflamación/fisiopatología , Masculino , Pandemias , Factores de Riesgo , Trombina/biosíntesis , Trombosis/etiología
9.
Arch Virol ; 166(3): 755-766, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-33420627

RESUMEN

MicroRNAs (miRNAs) are important host molecules involved in human immunodeficiency virus type 1 (HIV-1) infection. Antiretroviral therapy (ART) can affect the miRNA expression profile, but differentially expressed miRNAs still remain to be identified. In this study, we used gene chips to analyze miRNA expression profiles in peripheral blood mononuclear cells from ART-naive HIV-1 patients and those receiving ART, as well as from uninfected individuals. We measured differences in miRNA expression by quantitative polymerase chain reaction (qPCR) in an expanded sample. We found significant differences in the expression of has-miR-191-5p among the three groups (P < 0.05). Furthermore, we showed that hsa-miR-191-5p has an inhibitory effect on HIV-1 replication in cell models in vitro. We identified CCR1 and NUP50 as target molecules of hsa-miR-191-5p and found that hsa-miR-191-5p inhibits the expression of CCR1 and NUP50. Knockdown of NUP50 resulted in significant inhibition of HIV-1 replication. In summary, our research shows that hsa-miR-191-5p expression is reduced in HIV-1-infected patients and acts an inhibitor of HIV-1 infection via a mechanism that may involve targeted repression of NUP50 expression.


Asunto(s)
Regulación de la Expresión Génica/genética , VIH-1/metabolismo , MicroARNs/genética , Proteínas de Complejo Poro Nuclear/biosíntesis , Proteínas Nucleares/biosíntesis , Receptores CCR1/biosíntesis , Adulto , Línea Celular , Regulación hacia Abajo , Femenino , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Células Jurkat , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa , Replicación Viral/genética , Adulto Joven
10.
Nat Metab ; 3(1): 43-58, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33432202

RESUMEN

The mammalian liver is a central hub for systemic metabolic homeostasis. Liver tissue is spatially structured, with hepatocytes operating in repeating lobules, and sub-lobule zones performing distinct functions. The liver is also subject to extensive temporal regulation, orchestrated by the interplay of the circadian clock, systemic signals and feeding rhythms. However, liver zonation has previously been analysed as a static phenomenon, and liver chronobiology has been analysed at tissue-level resolution. Here, we use single-cell RNA-seq to investigate the interplay between gene regulation in space and time. Using mixed-effect models of messenger RNA expression and smFISH validations, we find that many genes in the liver are both zonated and rhythmic, and most of them show multiplicative space-time effects. Such dually regulated genes cover not only key hepatic functions such as lipid, carbohydrate and amino acid metabolism, but also previously unassociated processes involving protein chaperones. Our data also suggest that rhythmic and localized expression of Wnt targets could be explained by rhythmically expressed Wnt ligands from non-parenchymal cells near the central vein. Core circadian clock genes are expressed in a non-zonated manner, indicating that the liver clock is robust to zonation. Together, our scRNA-seq analysis reveals how liver function is compartmentalized spatio-temporally at the sub-lobular scale.


Asunto(s)
Relojes Circadianos/genética , Expresión Génica/fisiología , Hígado/metabolismo , Periodicidad , Algoritmos , Aminoácidos/metabolismo , Animales , Metabolismo de los Hidratos de Carbono/genética , Perfilación de la Expresión Génica , Hepatocitos/metabolismo , Metabolismo de los Lípidos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Chaperonas Moleculares/metabolismo , Proteínas Circadianas Period/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Vía de Señalización Wnt/genética
11.
Nat Metab ; 3(1): 75-89, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33462516

RESUMEN

NADPH has long been recognized as a key cofactor for antioxidant defence and reductive biosynthesis. Here we report a metabolism-independent function of NADPH in modulating epigenetic status and transcription. We find that the reduction of cellular NADPH levels, achieved by silencing malic enzyme or glucose-6-phosphate dehydrogenase, impairs global histone acetylation and transcription in both adipocytes and tumour cells. These effects can be reversed by supplementation with exogenous NADPH or by inhibition of histone deacetylase 3 (HDAC3). Mechanistically, NADPH directly interacts with HDAC3 and interrupts the association between HDAC3 and its co-activator nuclear receptor corepressor 2 (Ncor2; SMRT) or Ncor1, thereby impairing HDAC3 activation. Interestingly, NADPH and the inositol tetraphosphate molecule Ins(1,4,5,6)P4 appear to bind to the same domains on HDAC3, with NADPH having a higher affinity towards HDAC3 than Ins(1,4,5,6)P4. Thus, while Ins(1,4,5,6)P4 promotes formation of the HDAC3-Ncor complex, NADPH inhibits it. Collectively, our findings uncover a previously unidentified and metabolism-independent role of NADPH in controlling epigenetic change and gene expression by acting as an endogenous inhibitor of HDAC3.


Asunto(s)
Epigénesis Genética/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , NADP/farmacología , Co-Represor 1 de Receptor Nuclear/metabolismo , Co-Represor 2 de Receptor Nuclear/metabolismo , Acetilación , Animales , Línea Celular Tumoral , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Histona Desacetilasas/biosíntesis , Histona Desacetilasas/genética , Histonas/metabolismo , Humanos , Fosfatos de Inositol/farmacología , Malato Deshidrogenasa/metabolismo , Ratones , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Células 3T3 NIH , Co-Represor 1 de Receptor Nuclear/biosíntesis , Co-Represor 1 de Receptor Nuclear/genética , Co-Represor 2 de Receptor Nuclear/biosíntesis , Co-Represor 2 de Receptor Nuclear/genética
12.
Commun Biol ; 4(1): 129, 2021 01 29.
Artículo en Inglés | MEDLINE | ID: mdl-33514825

RESUMEN

Development of antibody protection during SARS-CoV-2 infection is a pressing question for public health and for vaccine development. We developed highly sensitive SARS-CoV-2-specific antibody and neutralization assays. SARS-CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n = 115) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. SARS-CoV-2 neutralization was determined in COVID-19 and convalescent plasma at up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which were also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Interestingly, some COVID-19 patients also possessed NAbs against SARS-CoV Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines.


Asunto(s)
Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/química , /química , Glicoproteína de la Espiga del Coronavirus/química , Adulto , /inmunología , Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , /virología , Convalecencia , /metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Epítopos/química , Epítopos/inmunología , Epítopos/metabolismo , Femenino , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Sueros Inmunes/química , Inmunidad Humoral , Lentivirus/genética , Lentivirus/inmunología , Masculino , Persona de Mediana Edad , Pruebas de Neutralización , Fosfoproteínas/química , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Unión Proteica , Receptores Virales/química , Receptores Virales/inmunología , Receptores Virales/metabolismo , /patogenicidad , Índice de Severidad de la Enfermedad , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Análisis de Supervivencia
13.
Nucleic Acids Res ; 49(2): 1065-1074, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33398328

RESUMEN

Terminal deoxynucleotidyl transferase (TdT) enzyme plays an integral part in the V(D)J recombination, allowing for the huge diversity in expression of immunoglobulins and T-cell receptors within lymphocytes, through their unique ability to incorporate single nucleotides into oligonucleotides without the need of a template. The role played by TdT in lymphocytes precursors found in early vertebrates is not known. In this paper, we demonstrated a new screening method that utilises TdT to form libraries of variable sized (vsDNA) libraries of polynucleotides that displayed binding towards protein targets. The extent of binding and size distribution of each vsDNA library towards their respective protein target can be controlled through the alteration of different reaction conditions such as time of reaction, nucleotide ratio and initiator concentration raising the possibility for the rational design of aptamers prior to screening. The new approach, allows for the screening of aptamers based on size as well as sequence in a single round, which minimises PCR bias. We converted the protein bound sequences to dsDNA using rapid amplification of variable ends assays (RAVE) and sequenced them using next generation sequencing. The resultant aptamers demonstrated low nanomolar binding and high selectivity towards their respective targets.


Asunto(s)
Aptámeros de Nucleótidos/metabolismo , ADN Nucleotidilexotransferasa/fisiología , Evaluación Preclínica de Medicamentos/métodos , Aptámeros de Nucleótidos/biosíntesis , Aptámeros de Nucleótidos/aislamiento & purificación , Sitios de Unión , ADN/metabolismo , ADN de Cadena Simple/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Lactoferrina/metabolismo , Técnicas de Amplificación de Ácido Nucleico , Unión Proteica , Especificidad por Sustrato , Trombina/metabolismo , Recombinación V(D)J
14.
Nucleic Acids Res ; 49(2): 1075-1093, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33398350

RESUMEN

Defects in the posttranscriptional modifications of mitochondrial tRNAs have been linked to human diseases, but their pathophysiology remains elusive. In this report, we investigated the molecular mechanism underlying a deafness-associated tRNAIle 4295A>G mutation affecting a highly conserved adenosine at position 37, 3' adjacent to the tRNA's anticodon. Primer extension and methylation activity assays revealed that the m.4295A>G mutation introduced a tRNA methyltransferase 5 (TRMT5)-catalyzed m1G37 modification of tRNAIle. Molecular dynamics simulations suggested that the m.4295A>G mutation affected tRNAIle structure and function, supported by increased melting temperature, conformational changes and instability of mutated tRNA. An in vitro processing experiment revealed that the m.4295A>G mutation reduced the 5' end processing efficiency of tRNAIle precursors, catalyzed by RNase P. We demonstrated that cybrid cell lines carrying the m.4295A>G mutation exhibited significant alterations in aminoacylation and steady-state levels of tRNAIle. The aberrant tRNA metabolism resulted in the impairment of mitochondrial translation, respiratory deficiency, decreasing membrane potentials and ATP production, increasing production of reactive oxygen species and promoting autophagy. These demonstrated the pleiotropic effects of m.4295A>G mutation on tRNAIle and mitochondrial functions. Our findings highlighted the essential role of deficient posttranscriptional modifications in the structure and function of tRNA and their pathogenic consequence of deafness.


Asunto(s)
Pérdida Auditiva Sensorineural/genética , Mutación Puntual , ARN de Transferencia de Isoleucina/genética , Adenosina Trifosfato/biosíntesis , Adulto , Proteínas Arqueales/metabolismo , Autofagia , Secuencia de Bases , Línea Celular , ADN Mitocondrial/genética , Grupos Étnicos/genética , Femenino , Pleiotropía Genética , Pérdida Auditiva Sensorineural/etnología , Humanos , Isoleucina/metabolismo , Masculino , Herencia Materna , Potencial de la Membrana Mitocondrial , Methanocaldococcus/enzimología , Metilación , Persona de Mediana Edad , Mitocondrias/metabolismo , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Fosforilación Oxidativa , Linaje , Biosíntesis de Proteínas , Procesamiento Postranscripcional del ARN , Proteínas Recombinantes/metabolismo , Aminoacilación de ARN de Transferencia , Adulto Joven , ARNt Metiltransferasas/metabolismo
15.
Nucleic Acids Res ; 49(2): 1133-1151, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33406240

RESUMEN

Alternative splicing generates multiple transcript and protein isoforms from a single gene and controls transcript intracellular localization and stability by coupling to mRNA export and nonsense-mediated mRNA decay (NMD). RNA interference (RNAi) is a potent mechanism to modulate gene expression. However, its interactions with alternative splicing are poorly understood. We used artificial microRNAs (amiRNAs, also termed shRNAmiR) to knockdown all splice variants of selected target genes in Arabidopsis thaliana. We found that splice variants, which vary by their protein-coding capacity, subcellular localization and sensitivity to NMD, are affected differentially by an amiRNA, although all of them contain the target site. Particular transcript isoforms escape amiRNA-mediated degradation due to their nuclear localization. The nuclear and NMD-sensitive isoforms mask RNAi action in alternatively spliced genes. Interestingly, Arabidopsis SPL genes, which undergo alternative splicing and are targets of miR156, are regulated in the same manner. Moreover, similar results were obtained in mammalian cells using siRNAs, indicating cross-kingdom conservation of these interactions among RNAi and splicing isoforms. Furthermore, we report that amiRNA can trigger artificial alternative splicing, thus expanding the RNAi functional repertoire. Our findings unveil novel interactions between different post-transcriptional processes in defining transcript fates and regulating gene expression.


Asunto(s)
Empalme Alternativo/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/genética , Técnicas de Silenciamiento del Gen , Degradación de ARNm Mediada por Codón sin Sentido , Isoformas de Proteínas/genética , Interferencia de ARN , Precursores del ARN/metabolismo , ARN de Planta/metabolismo , Proteínas de Arabidopsis/biosíntesis , Exones , Genes de Plantas , Células HeLa , Humanos , MicroARNs/genética , Plantas Modificadas Genéticamente , Isoformas de Proteínas/biosíntesis , Protoplastos/metabolismo , Precursores del ARN/genética , Procesamiento Postranscripcional del ARN , ARN de Planta/genética , Factores de Empalme Serina-Arginina/biosíntesis , Factores de Empalme Serina-Arginina/genética , Transcripción Genética , Transfección
16.
Nucleic Acids Res ; 49(2): 818-831, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33410890

RESUMEN

Codon usage bias is a universal feature of all genomes. Although codon usage has been shown to regulate mRNA and protein levels by influencing mRNA decay and transcription in eukaryotes, little or no genome-wide correlations between codon usage and mRNA levels are detected in mammalian cells, raising doubt on the significance of codon usage effect on gene expression. Here we show that gene-specific regulation reduces the genome-wide codon usage and mRNA correlations: Constitutively expressed genes exhibit much higher genome-wide correlations than differentially expressed genes from fungi to human cells. Using Drosophila S2 cells as a model system, we showed that the effect of codon usage on mRNA expression level is promoter-dependent. Regions downstream of the core promoters of differentially expressed genes can repress the codon usage effects on mRNA expression. An element in the Hsp70 promoter was identified to be necessary and sufficient for this inhibitory effect. The promoter-dependent codon usage effects on mRNA levels are regulated at the transcriptional level through modulation of histone modifications, nucleosome densities and premature termination. Together, our results demonstrate that promoters play a major role in determining whether codon usage influences gene expression and further establish the transcription-dependent codon usage effects on gene expression.


Asunto(s)
Uso de Codones , Drosophila melanogaster/genética , Regulación de la Expresión Génica/genética , Regiones Promotoras Genéticas/genética , Acetilación , Animales , Composición de Base , Línea Celular , Cromatina/genética , Cromatina/ultraestructura , Codón sin Sentido , Proteínas de Drosophila/fisiología , Drosophila melanogaster/citología , Genes Reporteros , Código de Histonas , Humanos , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Ratones , Neurospora crassa/genética , Nucleosomas/metabolismo , Procesamiento Proteico-Postraduccional , Estabilidad del ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Especificidad de la Especie
17.
Nucleic Acids Res ; 49(2): 847-863, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33410915

RESUMEN

Well-differentiated liposarcoma (WDLPS) is a malignant neoplasia hard to diagnose and treat. Its main molecular signature is amplification of the MDM2-containing genomic region. The MDM2 oncogene is the master regulator of p53: its overexpression enhances p53 degradation and inhibits apoptosis, leading to the tumoral phenotype. Here, we show that the MDM2 inducible promoter G-rich region folds into stable G-quadruplexes both in vitro and in vivo and it is specifically recognized by cellular helicases. Cell treatment with G-quadruplex-ligands reduces MDM2 expression and p53 degradation, thus stimulating cancer cell cycle arrest and apoptosis. Structural characterization of the MDM2 G-quadruplex revealed an extraordinarily stable, unique four-tetrad antiparallel dynamic conformation, amenable to selective targeting. These data indicate the feasibility of an out-of-the-box G-quadruplex-targeting approach to defeat WDLPS and all tumours where restoration of wild-type p53 is sought. They also point to G-quadruplex-dependent genomic instability as possible cause of MDM2 expansion and WDLPS tumorigenesis.


Asunto(s)
G-Cuádruplex , Regulación Neoplásica de la Expresión Génica/genética , Liposarcoma/terapia , Terapia Molecular Dirigida , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Neoplasias de los Tejidos Blandos/terapia , Apoptosis , Ciclo Celular , Línea Celular Tumoral , Simulación por Computador , Humanos , Ligandos , Modelos Genéticos , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Mapeo de Interacción de Proteínas , Proteolisis , Proteínas Proto-Oncogénicas c-mdm2/biosíntesis , Proteína p53 Supresora de Tumor/metabolismo
18.
Medicine (Baltimore) ; 100(2): e23904, 2021 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-33466133

RESUMEN

BACKGROUND: Traditional Chinese medicine (TCM) has been widely applied as promising adjunctive drugs for ovarian carcinoma (OC) in China and other Asian countries. However, its exact clinical efficacy and safety is still not well investigated. In this study, we aimed to summarize the efficacy of TCM on survival, quality of life (QoL), and immune function in patients with OC through the meta-analysis. METHODS: Relevant clinical trials of TCM for the treatment OC patients will be searched in Cochrane Library, Web of Science, Google Scholar, PubMed, Medline, Embase, China Scientific Journal Database, China National Knowledge Infrastructure, Chinese Biomedical Literature Database, and Wanfang Database from their inception to November 2020. Two researchers will perform data extraction and risk of bias assessment independently. The clinical outcomes, including overall survival (OS), QoL, immune function, tumor markers, and adverse events, were systematically evaluated by using Review Manager 5.3 and Stata 14.0 statistical software. RESULTS: The results of this study will provide high-quality evidence for the effect of TCM on survival, QoL and immune function in patients with OC. CONCLUSION: The conclusions of this meta-analysis will be published in a peer-reviewed journal, and draw an objective conclusion of the efficacy of TCM on survival, QoL, and immune function in patients with OC. TRIAL REGISTRATION NUMBER: INPLASY2020110104.


Asunto(s)
Medicina China Tradicional/métodos , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/terapia , Calidad de Vida , Biomarcadores de Tumor , Ensayos Clínicos como Asunto , Citocinas/biosíntesis , Medicamentos Herbarios Chinos/uso terapéutico , Femenino , Humanos , Linfocitos/metabolismo , Medicina China Tradicional/efectos adversos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/mortalidad , Proyectos de Investigación
19.
Med Hypotheses ; 147: 110475, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33421689

RESUMEN

Coagulopathy has recently been recognized as a recurring complication of COVID-19, most typically associated with critical illness. There are epidemiological, mechanistic and transcriptomic evidence that link Selenium with SARS-CoV-2's intracellular latency. Taking into consideration the vital role of selenoproteins in maintaining an adequate immune response, endothelial homeostasis and a non-prothrombotic platelet activation status, we propose that impairment in selenocysteine synthesis, via perturbations in the aforementioned physiological functions, potentially constitutes a mechanism of coagulopathy in COVID 19 patients other than those developed in critical illness.


Asunto(s)
Trastornos de la Coagulación Sanguínea/complicaciones , /patogenicidad , Selenocisteína/biosíntesis , Trastornos de la Coagulación Sanguínea/virología , Plaquetas/metabolismo , Enfermedad Crítica , Endotelio Vascular/metabolismo , Homeostasis , Humanos , Sistema Inmunológico , Inflamación , Modelos Teóricos , Estrés Oxidativo , Activación Plaquetaria , Selenio/química , Selenocisteína/química , Transcriptoma
20.
Viruses ; 13(1)2021 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-33435207

RESUMEN

The structures of the four N-linked glycans from the prototype chlorovirus PBCV-1 major capsid protein do not resemble any other glycans in the three domains of life. All known chloroviruses and antigenic variants (or mutants) share a unique conserved central glycan core consisting of five sugars, except for antigenic mutant virus P1L6, which has four of the five sugars. A combination of genetic and structural analyses indicates that the protein coded by PBCV-1 gene a111/114r, conserved in all chloroviruses, is a glycosyltransferase with three putative domains of approximately 300 amino acids each. Here, in addition to in silico sequence analysis and protein modeling, we measured the hydrolytic activity of protein A111/114R. The results suggest that domain 1 is a galactosyltransferase, domain 2 is a xylosyltransferase and domain 3 is a fucosyltransferase. Thus, A111/114R is the protein likely responsible for the attachment of three of the five conserved residues of the core region of this complex glycan, and, if biochemically corroborated, it would be the second three-domain protein coded by PBCV-1 that is involved in glycan synthesis. Importantly, these findings provide additional support that the chloroviruses do not use the canonical host endoplasmic reticulum-Golgi glycosylation pathway to glycosylate their glycoproteins; instead, they perform glycosylation independent of cellular organelles using virus-encoded enzymes.


Asunto(s)
Glicosiltransferasas/metabolismo , Phycodnaviridae/fisiología , Polisacáridos/biosíntesis , Dominios Proteicos , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Glicosiltransferasas/química , Enlace de Hidrógeno , Hidrólisis , Modelos Moleculares , Conformación Proteica , Relación Estructura-Actividad , Proteínas Virales/química
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