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1.
Int J Mol Sci ; 22(6)2021 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-33803916

RESUMEN

The glycine conjugation pathway in humans is involved in the metabolism of natural substrates and the detoxification of xenobiotics. The interactions between the various substrates in this pathway and their competition for the pathway enzymes are currently unknown. The pathway consists of a mitochondrial xenobiotic/medium-chain fatty acid: coenzyme A (CoA) ligase (ACSM2B) and glycine N-acyltransferase (GLYAT). The catalytic mechanism and substrate specificity of both of these enzymes have not been thoroughly characterised. In this study, the level of evolutionary conservation of GLYAT missense variants and haplotypes were analysed. From these data, haplotype variants were selected (156Asn > Ser, [17Ser > Thr,156Asn > Ser] and [156Asn > Ser,199Arg > Cys]) in order to characterise the kinetic mechanism of the enzyme over a wide range of substrate concentrations. The 156Asn > Ser haplotype has the highest frequency and the highest relative enzyme activity in all populations studied, and hence was used as the reference in this study. Cooperative substrate binding was observed, and the kinetic data were fitted to a two-substrate Hill equation. The coding region of the GLYAT gene was found to be highly conserved and the rare 156Asn > Ser,199Arg > Cys variant negatively affected the relative enzyme activity. Even though the 156Asn > Ser,199Arg > Cys variant had a higher affinity for benzoyl-CoA (s0.5,benz = 61.2 µM), kcat was reduced to 9.8% of the most abundant haplotype 156Asn > Ser (s0.5,benz = 96.6 µM), while the activity of 17Ser > Thr,156Asn > Ser (s0.5,benz = 118 µM) was 73% of 156Asn > Ser. The in vitro kinetic analyses of the effect of the 156Asn > Ser,199Arg > Cys variant on human GLYAT enzyme activity indicated that individuals with this haplotype might have a decreased ability to metabolise benzoate when compared to individuals with the 156Asn > Ser variant. Furthermore, the accumulation of acyl-CoA intermediates can inhibit ACSM2B leading to a reduction in mitochondrial energy production.


Asunto(s)
Acilcoenzima A/metabolismo , Aciltransferasas/genética , Aciltransferasas/metabolismo , Glicina/metabolismo , Mutación/genética , Animales , Secuencia Conservada/genética , Frecuencia de los Genes/genética , Humanos , Cinética , Filogenia
2.
Nat Commun ; 12(1): 2076, 2021 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-33824317

RESUMEN

Knowledge of genomic features specific to the human lineage may provide insights into brain-related diseases. We leverage high-depth whole genome sequencing data to generate a combined annotation identifying regions simultaneously depleted for genetic variation (constrained regions) and poorly conserved across primates. We propose that these constrained, non-conserved regions (CNCRs) have been subject to human-specific purifying selection and are enriched for brain-specific elements. We find that CNCRs are depleted from protein-coding genes but enriched within lncRNAs. We demonstrate that per-SNP heritability of a range of brain-relevant phenotypes are enriched within CNCRs. We find that genes implicated in neurological diseases have high CNCR density, including APOE, highlighting an unannotated intron-3 retention event. Using human brain RNA-sequencing data, we show the intron-3-retaining transcript to be more abundant in Alzheimer's disease with more severe tau and amyloid pathological burden. Thus, we demonstrate potential association of human-lineage-specific sequences in brain development and neurological disease.


Asunto(s)
Apolipoproteínas E/genética , Genoma Humano , Enfermedades Neurodegenerativas/genética , Filogenia , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Encéfalo/patología , Cromosomas Humanos Par 19/genética , Secuencia Conservada/genética , ADN Intergénico/genética , Ontología de Genes , Humanos , Intrones/genética , Desequilibrio de Ligamiento/genética , Anotación de Secuencia Molecular , Fenotipo , Polimorfismo de Nucleótido Simple/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Regresión
3.
Int J Mol Sci ; 22(4)2021 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-33670757

RESUMEN

The GATA proteins, functioning as transcription factors (TFs), are involved in multiple plant physiological and biochemical processes. In this study, 28 GATA TFs of Brachypodium distachyon (BdGATA) were systematically characterized via whole-genome analysis. BdGATA genes unevenly distribute on five chromosomes of B. distachyon and undergo purifying selection during the evolution process. The putative cis-acting regulatory elements and gene interaction network of BdGATA were found to be associated with hormones and defense responses. Noticeably, the expression profiles measured by quantitative real-time PCR indicated that BdGATA genes were sensitive to methyl jasmonate (MeJA) and salicylic acid (SA) treatment, and 10 of them responded to invasion of the fungal pathogen Magnaporthe oryzae, which causes rice blast disease. Genome-wide characterization, evolution, and expression profile analysis of BdGATA genes can open new avenues for uncovering the functions of the GATA genes family in plants and further improve the knowledge of cellular signaling in plant defense.


Asunto(s)
Brachypodium/genética , Evolución Molecular , Factores de Transcripción GATA/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Secuencias de Aminoácidos , Ascomicetos/efectos de los fármacos , Ascomicetos/fisiología , Brachypodium/efectos de los fármacos , Cromosomas de las Plantas/genética , Secuencia Conservada/genética , Factores de Transcripción GATA/química , Factores de Transcripción GATA/metabolismo , Duplicación de Gen , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Genes de Plantas , MicroARNs/genética , MicroARNs/metabolismo , Filogenia , Reguladores del Crecimiento de las Plantas/farmacología , Sintenía/genética
4.
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
5.
Plant Mol Biol ; 106(1-2): 207-220, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33738679

RESUMEN

KEY MESSAGE: The genome-wide allele-specific expression in F1 hybrids from the cross of tropical and temperate lotus unveils how cis-regulatory divergences affect genes in key pathways related to ecotypic divergence. Genetic variation, particularly cis-regulatory variation, plays a crucial role in phenotypic variation and adaptive evolution in plants. Temperate and tropical lotus, the two ecotypes of Nelumbo nucifera, show distinction in the degree of rhizome enlargement, which is associated with winter dormancy. To understand the roles of genome-wide cis-regulatory divergences on adaptive evolution of temperate and tropical lotus (Nelumbo nucifera), here we performed allele-specific expression (ASE) analyses on the tissues including flowers, leaves and rhizome from F1 hybrids of tropical and temperate lotus. For all investigated tissues in F1s, about 36% of genes showed ASE and about 3% of genes showed strong consistent ASE. Most of ASEs were biased towards the tropical parent in all surveyed samples, indicating that the tropical genome might be dominant over the temperate genome in gene expression of tissues from their F1 hybrids. We found that promoter sequences with similar allelic expression are more conserved than genes with significant or conditional ASE, suggesting the cis-regulatory sequence divergence underlie the allelic expression bias. We further uncovered biased genes being related to phenotypic differentiation between two lotus ecotypes, especially metabolic and phytohormone-related pathways in the rhizome. Overall, our study provides a global landscape of cis-regulatory variations between two lotus ecotypes and highlights their roles in rhizome growth variation for the climatic adaptation.


Asunto(s)
Alelos , Cruzamientos Genéticos , Regulación de la Expresión Génica de las Plantas , Hibridación Genética , Nelumbo/genética , Clima Tropical , Secuencia Conservada/genética , Genoma de Planta , Especificidad de Órganos/genética , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas/genética , Mapas de Interacción de Proteínas/genética , RNA-Seq , Rizoma/genética
6.
Nat Commun ; 12(1): 1361, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33649327

RESUMEN

Sperm contributes diverse RNAs to the zygote. While sperm small RNAs have been shown to impact offspring phenotypes, our knowledge of the sperm transcriptome, especially the composition of long RNAs, has been limited by the lack of sensitive, high-throughput experimental techniques that can distinguish intact RNAs from fragmented RNAs, known to abound in sperm. Here, we integrate single-molecule long-read sequencing with short-read sequencing to detect sperm intact RNAs (spiRNAs). We identify 3440 spiRNA species in mice and 4100 in humans. The spiRNA profile consists of both mRNAs and long non-coding RNAs, is evolutionarily conserved between mice and humans, and displays an enrichment in mRNAs encoding for ribosome. In sum, we characterize the landscape of intact long RNAs in sperm, paving the way for future studies on their biogenesis and functions. Our experimental and bioinformatics approaches can be applied to other tissues and organisms to detect intact transcripts.


Asunto(s)
Secuencia Conservada/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN/genética , Imagen Individual de Molécula , Espermatozoides/metabolismo , Animales , Evolución Molecular , Ontología de Genes , Humanos , Masculino , Ratones Endogámicos C57BL , ARN/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Testículo/metabolismo , Transcriptoma/genética
7.
Nat Commun ; 12(1): 604, 2021 01 27.
Artículo en Inglés | MEDLINE | ID: mdl-33504782

RESUMEN

De novo gene origination has been recently established as an important mechanism for the formation of new genes. In organisms with a large genome, intergenic and intronic regions provide plenty of raw material for new transcriptional events to occur, but little is know about how de novo transcripts originate in more densely-packed genomes. Here, we identify 213 de novo originated transcripts in Saccharomyces cerevisiae using deep transcriptomics and genomic synteny information from multiple yeast species grown in two different conditions. We find that about half of the de novo transcripts are expressed from regions which already harbor other genes in the opposite orientation; these transcripts show similar expression changes in response to stress as their overlapping counterparts, and some appear to translate small proteins. Thus, a large fraction of de novo genes in yeast are likely to co-evolve with already existing genes.


Asunto(s)
Genes Fúngicos , Saccharomyces cerevisiae/genética , Transcriptoma/genética , Secuencia Conservada/genética , Regulación Fúngica de la Expresión Génica , Redes Reguladoras de Genes , Sistemas de Lectura Abierta/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
Nat Commun ; 12(1): 567, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33495464

RESUMEN

The regulatory elements controlling gene expression during acute inflammation are not fully elucidated. Here we report the identification of a set of NF-κB-bound elements and common chromatin landscapes underlying the acute inflammatory response across cell-types and mammalian species. Using primary vascular endothelial cells (human/mouse/bovine) treated with the pro-inflammatory cytokine, Tumor Necrosis Factor-α, we identify extensive (~30%) conserved orthologous binding of NF-κB to accessible, as well as nucleosome-occluded chromatin. Regions with the highest NF-κB occupancy pre-stimulation show dramatic increases in NF-κB binding and chromatin accessibility post-stimulation. These 'pre-bound' regions are typically conserved (~56%), contain multiple NF-κB motifs, are utilized by diverse cell types, and overlap rare non-coding mutations and common genetic variation associated with both inflammatory and cardiovascular phenotypes. Genetic ablation of conserved, 'pre-bound' NF-κB regions within the super-enhancer associated with the chemokine-encoding CCL2 gene and elsewhere supports the functional relevance of these elements.


Asunto(s)
Cromatina/genética , Células Endoteliales/metabolismo , Regulación de la Expresión Génica/genética , Inflamación/genética , FN-kappa B/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Enfermedad Aguda , Animales , Sitios de Unión/genética , Bovinos , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Cromatina/metabolismo , Secuencia Conservada/genética , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inflamación/metabolismo , Inflamación/patología , Lógica , Ratones , Modelos Genéticos , Unión Proteica , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/farmacología
9.
Plant Sci ; 303: 110753, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33487341

RESUMEN

Dynein light chain (DLC) proteins are an important component of dynein complexes, which are widely distributed in plants and animals and involved in a variety of cellular processes. The functions of DLC genes in plant chilling stress remain unclear. In this study, we isolated a DLC gene from tomato, designated SlLC6D. Promoter analysis revealed many cis-elements involved in abiotic stress in the SlLC6D promoter. Expression of SlLC6D was induced by heat and salt stress, and inhibited by polyethylene glycol and chilling stress. Knockdown of SlLC6D in tomato exhibited low relative electrolyte leakage, malondialdehyde content, and reactive oxygen species (ROS) accumulation under chilling stress. The content of proline and activities of superoxide dismutase and peroxidase in knockdown lines were higher than in the wild type and overexpression lines during chilling stress. The high transcript abundances of three cold-responsive genes were detected in knockdown lines in response to chilling stress. Seedling growth of knockdown lines was significantly higher than that of the wild type and overexpression lines under chilling stress. These results suggest that SlLC6D is a negative regulator of chilling stress tolerance, possibly by regulating ROS contents and the ICE1-CBF-COR pathway.


Asunto(s)
Dineínas/genética , Genes de Plantas/genética , Lycopersicon esculentum/genética , Proteínas de Plantas/genética , Respuesta al Choque por Frío , Secuencia Conservada/genética , Dineínas/metabolismo , Dineínas/fisiología , Genes de Plantas/fisiología , Lycopersicon esculentum/metabolismo , Lycopersicon esculentum/fisiología , Malondialdehído/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiología , Prolina/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa
10.
Plant Sci ; 303: 110766, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33487351

RESUMEN

UV RESISTANCE LOCUS 8 (UVR8) is a photoreceptor that regulates UV-B photomorphogenesis in plants. UV-B photon perception promotes UVR8 homodimer dissociation into monomer, which is reverted to homodimer post UV-B, forming a complete photocycle. UVR8 monomer interacts with CONSTITUTIVELY PHOTOMORPHOGENEIC 1 (COP1) to initiate UV-B signaling. The function and mechanism of Arabidopsis UVR8 (AtUVR8) are extensively investigated, however, little is known about UVR8 and its signaling mechanisms in other plant species. Tomato is a widely used model plant for horticulture research. In this report we tested whether an ortholog of AtUVR8 in Tomato (SIUVR8) can complement Arabidopsis uvr8 mutant and whether the above-mentioned key signaling mechanisms of UVR8 are conserved. Heterologous expressed SIUVR8 in an Arabidopsis uvr8 null mutant rescued the uvr8 mutant in the tested UV-B responses including hypocotyl elongation, UV-B target gene expression and anthocyanin accumulation, demonstrating that the SIUVR8 is a putative UV-B photoreceptor. Moreover, in response to UV-B, SIUVR8 forms a protein complex with Arabidopsis COP1 in plants, suggesting conserved signaling mechanism. SIUVR8 exhibits similar photocycle as AtUVR8 in plants, which highlights conserved photoreceptor activation and inactivation mechanisms.


Asunto(s)
Lycopersicon esculentum/genética , Fotorreceptores de Plantas/genética , Proteínas de Plantas/genética , Antocianinas/metabolismo , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/fisiología , Secuencia Conservada/genética , Luz , Lycopersicon esculentum/metabolismo , Fotorreceptores de Plantas/metabolismo , Fotorreceptores de Plantas/fisiología , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiología , Plantas Modificadas Genéticamente , Reacción en Cadena en Tiempo Real de la Polimerasa , Técnicas del Sistema de Dos Híbridos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/fisiología
11.
Int J Biol Macromol ; 170: 343-353, 2021 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-33383075

RESUMEN

Homologous proteins differ in their amino acid sequences at several positions. Generally, conserved sites are recognized as not suitable for amino acid substitution, and thus in evolutionary protein engineering, non-conserved sites are often selected as mutation sites. However, there have also been reports of possible mutations in conserved sites. In this study, we explored mutable conserved sites and immutable non-conserved sites by testing random mutations of two thermostable proteins, an esterase from Sulfolobus tokodaii (Sto-Est) and a subtilisin from Thermococcus kodakarensis (Tko-Sub). The subtilisin domain of Tko-Sub needs Ca2+ ions and the propeptide domain for stability, folding and maturation. The results from the two proteins showed that about one-third of the mutable sites were detected in conserved sites and some non-conserved sites lost enzymatic activity at high temperatures due to mutation. Of the conserved sites in Sto-Est, the sites on the loop, on the surface, and far from the active site are more resistant to mutation. In Tko-Sub, the sites flanking Ca2+-binding sites and propeptide were undesirable for mutation. The results presented here serve as an index for selecting mutation sites and contribute to the expansion of available sequence range by introducing mutations at conserved sites.


Asunto(s)
Esterasas/genética , Subtilisina/genética , Secuencia de Aminoácidos/genética , Sustitución de Aminoácidos/genética , Sitios de Unión/genética , Dominio Catalítico/genética , Secuencia Conservada/genética , Modelos Moleculares , Mutación/genética , Homología de Secuencia de Ácido Nucleico , Sulfolobus/genética , Thermococcus/genética
12.
Gene ; 764: 145078, 2021 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-32858175

RESUMEN

In maize, eat rot and stalk rot caused by Fusarium verticillioides and Fusarium graminearum lead to contamination of moldy grains to produce mycotoxins. Identification of resistance genes against these pathogens for maize breeding is an effective way for disease control. Several 2-oxoglutarate-dependent dioxygenase (2OGD) proteins have been found to confer resistance to different pathogens in diverse plant species. However, little is known about the 2OGD superfamily in maize. Here, we identified 103 putative 2OGD genes in maize from a genome-wide analysis, and divided them into three classes - DOXA, DOXB, and DOXC. We further comprehensively investigated their gene structure, chromosome distribution, phylogenetic tree, gene-function enrichment, and expression profiles among different tissues. The genes encoding three 2OGD proteins, ACO, F3H, and NCS involved in ethylene biosynthesis, flavonoids biosynthesis, and alkaloids biosynthesis pathways, respectively, were identified to be induced by F. verticillioides and F. graminearum. The promoters of the three genes contain the binding sites for the transcription factor ZmDOF and ZmHSF, which are also induced by the two pathogens. The results imply that the three 2OGDs and the two transcription factors might be involved in the resistance to the two pathogens. This study provided a comprehensive understanding of the 2OGD superfamily in maize and laid the foundation for the further functional analysis of their roles in maize resistance to eat rot and stalk rot.


Asunto(s)
Dioxigenasas/genética , Fusarium/inmunología , Proteínas de Plantas/genética , Zea mays/fisiología , Secuencia de Bases/genética , Sitios de Unión/genética , Cromosomas de las Plantas/genética , Coenzimas/metabolismo , Secuencia Conservada/genética , Dioxigenasas/inmunología , Dioxigenasas/metabolismo , Resistencia a la Enfermedad/genética , Evolución Molecular , Fusarium/patogenicidad , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/fisiología , Estudio de Asociación del Genoma Completo , Ácidos Cetoglutáricos/metabolismo , Filogenia , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/inmunología , Proteínas de Plantas/metabolismo , Tallos de la Planta/enzimología , Tallos de la Planta/crecimiento & desarrollo , Tallos de la Planta/microbiología , Regiones Promotoras Genéticas/genética , RNA-Seq , Factores de Transcripción/metabolismo , Zea mays/microbiología
13.
Elife ; 92020 12 30.
Artículo en Inglés | MEDLINE | ID: mdl-33377866

RESUMEN

Accurate chromosome segregation requires kinetochores on duplicated chromatids to biorient by attaching to dynamic microtubules from opposite spindle poles, which exerts forces to bring kinetochores under tension. However, kinetochores initially bind to microtubules indiscriminately, resulting in errors that must be corrected. While the Aurora B protein kinase destabilizes low-tension attachments by phosphorylating kinetochores, low-tension attachments are intrinsically less stable than those under higher tension in vitro independent of Aurora activity. Intrinsic tension-sensitive behavior requires the microtubule regulator Stu2 (budding yeast Dis1/XMAP215 ortholog), which we demonstrate here is likely a conserved function for the TOG protein family. The human TOG protein, chTOG, localizes to kinetochores independent of microtubules by interacting with Hec1. We identify a chTOG mutant that regulates microtubule dynamics but accumulates erroneous kinetochore-microtubule attachments that are not destabilized by Aurora B. Thus, TOG proteins confer a unique, intrinsic error correction activity to kinetochores that ensures accurate chromosome segregation.


Asunto(s)
Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/fisiología , Segregación Cromosómica , Secuencia Conservada/genética , Células HCT116 , Células HeLa , Humanos , Inmunoprecipitación , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitosis/genética , Mutación/genética , Saccharomyces cerevisiae
14.
Nat Commun ; 11(1): 5535, 2020 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-33139697

RESUMEN

The ASCC3 subunit of the activating signal co-integrator complex is a dual-cassette Ski2-like nucleic acid helicase that provides single-stranded DNA for alkylation damage repair by the α-ketoglutarate-dependent dioxygenase AlkBH3. Other ASCC components integrate ASCC3/AlkBH3 into a complex DNA repair pathway. We mapped and structurally analyzed interacting ASCC2 and ASCC3 regions. The ASCC3 fragment comprises a central helical domain and terminal, extended arms that clasp the compact ASCC2 unit. ASCC2-ASCC3 interfaces are evolutionarily highly conserved and comprise a large number of residues affected by somatic cancer mutations. We quantified contributions of protein regions to the ASCC2-ASCC3 interaction, observing that changes found in cancers lead to reduced ASCC2-ASCC3 affinity. Functional dissection of ASCC3 revealed similar organization and regulation as in the spliceosomal RNA helicase Brr2. Our results delineate functional regions in an important DNA repair complex and suggest possible molecular disease principles.


Asunto(s)
ADN Helicasas/genética , Reparación del ADN , Neoplasias/genética , Proteínas Nucleares/genética , Secuencia de Aminoácidos , Secuencia Conservada/genética , ADN Helicasas/aislamiento & purificación , ADN Helicasas/metabolismo , Células HEK293 , Humanos , Mutación , Proteínas Nucleares/aislamiento & purificación , Proteínas Nucleares/metabolismo , Unión Proteica/genética , Conformación Proteica en Hélice alfa/genética , Dominios Proteicos/genética , ARN Helicasas/genética , ARN Helicasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/genética , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Empalmosomas/metabolismo
15.
Nat Commun ; 11(1): 5560, 2020 11 03.
Artículo en Inglés | MEDLINE | ID: mdl-33144558

RESUMEN

Cancers result from a set of genetic and epigenetic alterations. Most known oncogenes were identified by gain-of-function mutations in cancer, yet little is known about their epigenetic features. Through integrative analysis of 11,596 epigenomic profiles and mutations from >8200 tumor-normal pairs, we discover broad genic repression domains (BGRD) on chromatin as an epigenetic signature for oncogenes. A BGRD is a widespread enrichment domain of the repressive histone modification H3K27me3 and is further enriched with multiple other repressive marks including H3K9me3, H3K9me2, and H3K27me2. Further, BGRD displays widespread enrichment of repressed cis-regulatory elements. Shortening of BGRDs is linked to derepression of transcription. BGRDs at oncogenes tend to be conserved across normal cell types. Putative tumor-promoting genes and lncRNAs defined using BGRDs are experimentally verified as required for cancer phenotypes. Therefore, BGRDs play key roles in epigenetic regulation of cancer and provide a direction for mutation-independent discovery of oncogenes.


Asunto(s)
Cromatina/genética , Silenciador del Gen , Oncogenes , Neoplasias de la Mama/genética , Linfocitos T CD4-Positivos/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Secuencia Conservada/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilación , Motivos de Nucleótidos/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Elongación de la Transcripción Genética , Iniciación de la Transcripción Genética
16.
PLoS Genet ; 16(9): e1009027, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32966296

RESUMEN

The availability of genomes for many species has advanced our understanding of the non-protein-coding fraction of the genome. Comparative genomics has proven itself to be an invaluable approach for the systematic, genome-wide identification of conserved non-protein-coding elements (CNEs). However, for many non-mammalian model species, including chicken, our capability to interpret the functional importance of variants overlapping CNEs has been limited by current genomic annotations, which rely on a single information type (e.g. conservation). We here studied CNEs in chicken using a combination of population genomics and comparative genomics. To investigate the functional importance of variants found in CNEs we develop a ch(icken) Combined Annotation-Dependent Depletion (chCADD) model, a variant effect prediction tool first introduced for humans and later on for mouse and pig. We show that 73 Mb of the chicken genome has been conserved across more than 280 million years of vertebrate evolution. The vast majority of the conserved elements are in non-protein-coding regions, which display SNP densities and allele frequency distributions characteristic of genomic regions constrained by purifying selection. By annotating SNPs with the chCADD score we are able to pinpoint specific subregions of the CNEs to be of higher functional importance, as supported by SNPs found in these subregions are associated with known disease genes in humans, mice, and rats. Taken together, our findings indicate that CNEs harbor variants of functional significance that should be object of further investigation along with protein-coding mutations. We therefore anticipate chCADD to be of great use to the scientific community and breeding companies in future functional studies in chicken.


Asunto(s)
Pollos/genética , ADN Intergénico/genética , Genómica/métodos , Alelos , Animales , Secuencia Conservada/genética , ADN Intergénico/metabolismo , Evolución Molecular , Frecuencia de los Genes/genética , Variación Genética/genética , Genoma/genética , Intrones/genética , Metagenómica/métodos , Polimorfismo de Nucleótido Simple/genética , Análisis de Secuencia/métodos
17.
Proc Natl Acad Sci U S A ; 117(40): 24920-24928, 2020 10 06.
Artículo en Inglés | MEDLINE | ID: mdl-32958636

RESUMEN

Australian funnel-web spiders are infamous for causing human fatalities, which are induced by venom peptides known as δ-hexatoxins (δ-HXTXs). Humans and other primates did not feature in the prey or predator spectrum during evolution of these spiders, and consequently the primate lethality of δ-HXTXs remains enigmatic. Funnel-web envenomations are mostly inflicted by male spiders that wander from their burrow in search of females during the mating season, which suggests a role for δ-HXTXs in self-defense since male spiders rarely feed during this period. Although 35 species of Australian funnel-web spiders have been described, only nine δ-HXTXs from four species have been characterized, resulting in a lack of understanding of the ecological roles and molecular evolution of δ-HXTXs. Here, by profiling venom-gland transcriptomes of 10 funnel-web species, we report 22 δ-HXTXs. Phylogenetic and evolutionary assessments reveal a remarkable sequence conservation of δ-HXTXs despite their deep evolutionary origin within funnel-web spiders, consistent with a defensive role. We demonstrate that δ-HXTX-Ar1a, the lethal toxin from the Sydney funnel-web spider Atrax robustus, induces pain in mice by inhibiting inactivation of voltage-gated sodium (NaV) channels involved in nociceptive signaling. δ-HXTX-Ar1a also inhibited inactivation of cockroach NaV channels and was insecticidal to sheep blowflies. Considering their algogenic effects in mice, potent insecticidal effects, and high levels of sequence conservation, we propose that the δ-HXTXs were repurposed from an initial insecticidal predatory function to a role in defending against nonhuman vertebrate predators by male spiders, with their lethal effects on humans being an unfortunate evolutionary coincidence.


Asunto(s)
Evolución Molecular , Neurotoxinas/genética , Poliaminas/química , Arañas/genética , Secuencia de Aminoácidos/genética , Animales , Australia , Secuencia Conservada/genética , Femenino , Humanos , Masculino , Ratones , Neurotoxinas/química , Neurotoxinas/metabolismo , Péptidos/genética , Filogenia , Poliaminas/metabolismo , Conducta Sexual Animal/fisiología , Venenos de Araña/genética , Arañas/patogenicidad , Transcriptoma/genética , Vertebrados/genética , Vertebrados/fisiología
18.
Gene ; 760: 145020, 2020 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-32755656

RESUMEN

Conserved sequences across species have always provided valuable insights to improve our understanding on the human genome's entity and the interplay among different loci. Lymphoma/leukemia related factor (LRF) is encoded by ZBTB7A gene and belongs to an evolutionarily conserved family of transcription factors, implicated in vital cellular functions. The present data, demonstrating the wide-spread and the high overlap of the LRF/ZBTB7A recognition sites with genomic segments identified as CpG islands in the human genome, suggest that its binding capacity strongly depends on a specific sequence-encoded feature within CpGs. We have previously shown that de-methylation of the CpG island 326 lying in the ZBTB7A gene promoter is associated with impaired pharmacological induction of fetal hemoglobin in ß-type hemoglobinopathies patients. Within this context we aimed to investigate the extent of the LRF/ZBTB7A conservation among primates and mouse genome, focusing our interest also on the CpG island flanking the gene's promoter region, in an effort to further establish its epigenetic regulatory role in human hematopoiesis and pharmacological involvement in hematopoietic disorders. Comparative analysis of the human ZBTB7A nucleotide and amino acid sequences and orthologous sequences among non-human primates and mouse, exhibited high conservation scores. Pathway analysis, clearly indicated that LRF/ZBTB7A influences conserved cellular processes. These data in conjunction with the high levels of expression foremost in hematopoietic tissues, highlighted LRF/ZBTB7A as an essential factor operating indisputably during hematopoiesis.


Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Enfermedades Hematológicas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Sitios de Unión/genética , Diferenciación Celular/genética , Línea Celular Tumoral , Secuencia Conservada/genética , Islas de CpG/genética , Bases de Datos Genéticas , Hemoglobina Fetal/genética , Hematopoyesis/genética , Humanos , Ratones , Primates/genética , Regiones Promotoras Genéticas/genética
19.
Comput Biol Med ; 125: 103963, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32828990

RESUMEN

Knowledge management tools that assist in systematic review and exploration of scientific knowledge generally are of obvious potential importance in evidence based medicine in general, but also to the design of therapeutics based on the protein subsequences and fold motifs of virus proteins as considered here. Rapid access to bundles (clusters) of related elements of knowledge gathered from diverse sources on the Internet and from growing knowledge repositories seem particularly helpful when exploring less obvious therapeutic targets in viruses (for which knowledge new to the researcher is important), and when using the following concept. Subsequences of amino acid residue sequences of proteins that are conserved across strains and species are (a) more likely to be important targets and (b) less likely to exhibit escape mutations that would make them resistant to vaccines and therapeutic agents. However, the terms "conserved" and even "highly conserved" used by authors are matters of degree, depending on how distant from SARS-CoV-2 they wished to go in comparing other sequences. The binding site to the human ACE2 protein as virus receptor and human antibody CR3022 binding site on the spike glycoprotein are rather variable by the criteria used in the present and preceding studies. To look for more strongly conserved targets, open reading frames of SARS-CoV-2 were examined for extremely highly conserved regions, meaning recognizable across many viruses and organisms. Most prominent is a motif found in SARS-CoV-2 non-structural protein 3 (Nsp3). It relates to a fold called type called the macro domain and has remarkably wide distribution across organisms including humans with significant homologies involving three especially conserved subsequences (a) VVVNAANVYLKHGGGVAGALNK, (b) LHVVGPNVNKG, and (c) PLLSAGIFG. Careful study of the variations of these and of the more variable sequences between and around them might provide a finer "scalpel" to ensure inhibition of a vital function of the virus without impairing the functions of related host macro domains.


Asunto(s)
Inteligencia Artificial , Biología Computacional/métodos , Secuencia Conservada/genética , Infecciones por Coronavirus , Pandemias , Neumonía Viral , Proteínas no Estructurales Virales , Secuencia de Aminoácidos , Betacoronavirus , Sitios de Unión , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/virología , Desarrollo de Medicamentos , Humanos , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/virología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética
20.
Proc Natl Acad Sci U S A ; 117(36): 22462-22472, 2020 09 08.
Artículo en Inglés | MEDLINE | ID: mdl-32839311

RESUMEN

Huntingtin-interacting protein family members are evolutionarily conserved from yeast to humans, and they are known to be key factors in clathrin-mediated endocytosis. Here we identified the Caenorhabditis elegans protein huntingtin-interacting protein-related 1 (HIPR-1) as a host factor essential for Orsay virus infection of C. elegans Ablation of HIPR-1 resulted in a greater than 10,000-fold reduction in viral RNA, which could be rescued by ectopic expression of HIPR-1. Viral RNA replication from an endogenous transgene replicon system was not affected by lack of HIPR-1, suggesting that HIPR-1 plays a role during an early, prereplication virus life-cycle stage. Ectopic expression of HIPR-1 mutants demonstrated that neither the clathrin light chain-binding domain nor the clathrin heavy chain-binding motif were needed for virus infection, whereas the inositol phospholipid-binding and F-actin-binding domains were essential. In human cell culture, deletion of the human HIP orthologs HIP1 and HIP1R led to decreased infection by Coxsackie B3 virus. Finally, ectopic expression of a chimeric HIPR-1 harboring the human HIP1 ANTH (AP180 N-terminal homology) domain rescued Orsay infection in C. elegans, demonstrating conservation of its function through evolution. Collectively, these findings further our knowledge of cellular factors impacting viral infection in C. elegans and humans.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Unión al ADN/metabolismo , Interacciones Huésped-Patógeno , Proteínas de Microfilamentos/metabolismo , Células A549 , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/virología , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiología , Secuencia Conservada/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Enterovirus Humano B/patogenicidad , Enterovirus Humano B/fisiología , Femenino , Técnicas de Silenciamiento del Gen , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/fisiología , Humanos , Masculino , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/fisiología , Nodaviridae/patogenicidad , Nodaviridae/fisiología , Dominios Proteicos/genética , Replicación Viral
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