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1.
Nat Commun ; 12(1): 2176, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846315

RESUMO

The hexosamine pathway (HP) is a key anabolic pathway whose product uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor for glycosylation processes in mammals. It modulates the ER stress response and HP activation extends lifespan in Caenorhabditis elegans. The highly conserved glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the rate-limiting HP enzyme. GFAT-1 activity is modulated by UDP-GlcNAc feedback inhibition and via phosphorylation by protein kinase A (PKA). Molecular consequences of GFAT-1 phosphorylation, however, remain poorly understood. Here, we identify the GFAT-1 R203H substitution that elevates UDP-GlcNAc levels in C. elegans. In human GFAT-1, the R203H substitution interferes with UDP-GlcNAc inhibition and with PKA-mediated Ser205 phosphorylation. Our data indicate that phosphorylation affects the interactions of the two GFAT-1 domains to control catalytic activity. Notably, Ser205 phosphorylation has two discernible effects: it lowers baseline GFAT-1 activity and abolishes UDP-GlcNAc feedback inhibition. PKA controls the HP by uncoupling the metabolic feedback loop of GFAT-1.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Retroalimentação Fisiológica , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Hexosaminas/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Estresse do Retículo Endoplasmático , Mutação com Ganho de Função , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/química , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Cinética , Fosforilação , Ligação Proteica , Domínios Proteicos , Serina/genética , Uridina Difosfato N-Acetilglicosamina/metabolismo
2.
PLoS Genet ; 17(1): e1008711, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33493156

RESUMO

The rate of evolution differs between protein sites and changes with time. However, the link between these two phenomena remains poorly understood. Here, we design a phylogenetic approach for distinguishing pairs of amino acid sites that evolve concordantly, i.e., such that substitutions at one site trigger subsequent substitutions at the other; and also pairs of sites that evolve discordantly, so that substitutions at one site impede subsequent substitutions at the other. We distinguish groups of amino acid sites that undergo coordinated evolution and evolve discordantly from other such groups. In mitochondrion-encoded proteins of metazoans and fungi, we show that concordantly evolving sites are clustered in protein structures. By analysing the phylogenetic patterns of substitutions at concordantly and discordantly evolving site pairs, we find that concordant evolution has two distinct causes: epistatic interactions between amino acid substitutions and episodes of selection independently affecting substitutions at different sites. The rate of substitutions at concordantly evolving groups of protein sites changes in the course of evolution, indicating episodes of selection limited to some of the lineages. The phylogenetic positions of these changes are consistent between proteins, suggesting common selective forces underlying them.


Assuntos
Epistasia Genética , Evolução Molecular , Proteínas Mitocondriais/genética , Seleção Genética , Substituição de Aminoácidos/genética , Aminoácidos/genética , Animais , Fungos/genética , Genoma Mitocondrial/genética , Mitocôndrias/genética , Filogenia , Conformação Proteica , Mapas de Interação de Proteínas/genética
3.
PLoS One ; 16(1): e0244948, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33395407

RESUMO

For the last couple of decades, there has been a significant growth in sequencing data, leading to an extraordinary increase in the number of gene variants. This places a challenge on the bioinformatics research community to develop and improve computational tools for functional annotation of new variants. Genes coding for epigenetic regulators have important roles in cancer pathogenesis and mutations in these genes show great potential as clinical biomarkers, especially in hematologic malignancies. Therefore, we developed a model that specifically focuses on these genes, with an assumption that it would outperform general models in predicting the functional effects of amino acid substitutions. EpiMut is a standalone software that implements a sequence based alignment-free method. We applied a two-step approach for generating sequence based features, relying on the biophysical and biochemical indices of amino acids and the Fourier Transform as a sequence transformation method. For each gene in the dataset, the machine learning algorithm-Naïve Bayes was used for building a model for prediction of the neutral or disease-related status of variants. EpiMut outperformed state-of-the-art tools used for comparison, PolyPhen-2, SIFT and SNAP2. Additionally, EpiMut showed the highest performance on the subset of variants positioned outside conserved functional domains of analysed proteins, which represents an important group of cancer-related variants. These results imply that EpiMut can be applied as a first choice tool in research of the impact of gene variants in epigenetic regulators, especially in the light of the biomarker role in hematologic malignancies. EpiMut is freely available at https://www.vin.bg.ac.rs/180/tools/epimut.php.


Assuntos
Substituição de Aminoácidos/genética , Epigênese Genética , Neoplasias Hematológicas/genética , Algoritmos , Sequência de Bases/genética , Epigênese Genética/genética , Humanos , Aprendizado de Máquina , Modelos Estatísticos , Alinhamento de Sequência , Software
4.
Hum Genet ; 140(5): 805-812, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33502607

RESUMO

The interpretation of human genetic variation is one of the greatest challenges of modern genetics. New approaches are urgently needed to prioritize variants, especially those that are rare or lack a definitive clinical interpretation. We examined 10,136,597 human missense genetic variants from GnomAD, ClinVar and UniProt. We were able to perform large-scale atom-based mapping and phenotype interpretation of 3,960,015 of these variants onto 18,874 experimental and 84,818 in house predicted three-dimensional coordinates of the human proteome. We demonstrate that 14% of amino acid substitutions from the GnomAD database that could be structurally analysed are predicted to affect protein structure (n = 568,548, of which 566,439 rare or extremely rare) and may, therefore, have a yet unknown disease-causing effect. The same is true for 19.0% (n = 6266) of variants of unknown clinical significance or conflicting interpretation reported in the ClinVar database. The results of the structural analysis are available in the dedicated web catalogue Missense3D-DB ( http://missense3d.bc.ic.ac.uk/ ). For each of the 4 M variants, the results of the structural analysis are presented in a friendly concise format that can be included in clinical genetic reports. A detailed report of the structural analysis is also available for the non-experts in structural biology. Population frequency and predictions from SIFT and PolyPhen are included for a more comprehensive variant interpretation. This is the first large-scale atom-based structural interpretation of human genetic variation and offers geneticists and the biomedical community a new approach to genetic variant interpretation.


Assuntos
Mapeamento Cromossômico/métodos , Biologia Computacional/métodos , Bases de Dados Genéticas , Mutação de Sentido Incorreto/genética , Substituição de Aminoácidos/genética , Frequência do Gene/genética , Humanos , Conformação Proteica , Proteoma/genética
5.
Int J Biol Macromol ; 170: 343-353, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33383075

RESUMO

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.


Assuntos
Esterases/genética , Subtilisina/genética , Sequência de Aminoácidos/genética , Substituição de Aminoácidos/genética , Sítios de Ligação/genética , Domínio Catalítico/genética , Sequência Conservada/genética , Modelos Moleculares , Mutação/genética , Homologia de Sequência do Ácido Nucleico , Sulfolobus/genética , Thermococcus/genética
6.
Nucleic Acids Res ; 48(18): 10280-10296, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32955564

RESUMO

In translation initiation, AUG recognition triggers rearrangement of the 48S preinitiation complex (PIC) from an open conformation to a closed state with more tightly-bound Met-tRNAi. Cryo-EM structures have revealed interactions unique to the closed complex between arginines R55/R57 of eIF2α with mRNA, including the -3 nucleotide of the 'Kozak' context. We found that R55/R57 substitutions reduced recognition of a UUG start codon at HIS4 in Sui- cells (Ssu- phenotype); and in vitro, R55G-R57E accelerated dissociation of the eIF2·GTP·Met-tRNAi ternary complex (TC) from reconstituted PICs with a UUG start codon, indicating destabilization of the closed complex. R55/R57 substitutions also decreased usage of poor-context AUGs in SUI1 and GCN4 mRNAs in vivo. In contrast, eIF2α-R53 interacts with the rRNA backbone only in the open complex, and the R53E substitution enhanced initiation at a UUG codon (Sui- phenotype) and poor-context AUGs, while reducing the rate of TC loading (Gcd- phenotype) in vivo. Consistently, R53E slowed TC binding to the PIC while decreasing TC dissociation at UUG codons in vitro, indicating destabilization of the open complex. Thus, distinct interactions of eIF2α with rRNA or mRNA stabilize first the open, and then closed, conformation of the PIC to influence the accuracy of initiation in vivo.


Assuntos
Arginina/análogos & derivados , Fator de Iniciação 2 em Eucariotos/genética , RNA Mensageiro/genética , Substituição de Aminoácidos/genética , Arginina/genética , Códon de Iniciação/genética , Humanos , Complexos Multiproteicos/genética , Iniciação Traducional da Cadeia Peptídica , Subunidades Ribossômicas Menores de Eucariotos/genética , Saccharomyces cerevisiae/genética
7.
Proc Natl Acad Sci U S A ; 117(38): 23807-23814, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32873642

RESUMO

Avian-origin influenza viruses overcome the bottleneck of the interspecies barrier and infect humans through the evolution of variants toward more efficient replication in mammals. The dynamic adaptation of the genetic substitutions and the correlation with the virulence of avian-origin influenza virus in patients remain largely elusive. Here, based on the one-health approach, we retrieved the original virus-positive samples from patients with H7N9 and their surrounding poultry/environment. The specimens were directly deep sequenced, and the subsequent big data were integrated with the clinical manifestations. Unlike poultry/environment-derived samples with the consistent dominance of avian signature 627E of H7N9 polymerase basic protein 2 (PB2), patient specimens had diverse ratios of mammalian signature 627K, indicating the rapid dynamics of H7N9 adaptation in patients during the infection process. In contrast, both human- and poultry/environment-related viruses had constant dominance of avian signature PB2-701D. The intrahost dynamic adaptation was confirmed by the gradual replacement of 627E by 627K in H7N9 in the longitudinally collected specimens from one patient. These results suggest that host adaptation for better virus replication to new hosts, termed "genetic tuning," actually occurred in H7N9-infected patients in vivo. Notably, our findings also demonstrate the correlation between rapid host adaptation of H7N9 PB2-E627K and the fatal outcome and disease severity in humans. The feature of H7N9 genetic tuning in vivo and its correlation with the disease severity emphasize the importance of testing for the evolution of this avian-origin virus during the course of infection.


Assuntos
Adaptação Biológica/genética , Substituição de Aminoácidos/genética , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Influenza Humana/virologia , Proteínas Virais/genética , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno , Humanos , RNA Viral/genética , Análise de Sequência de RNA , Replicação Viral/genética
8.
Proc Natl Acad Sci U S A ; 117(37): 23165-23173, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32868448

RESUMO

To engineer Mo-dependent nitrogenase function in plants, expression of the structural proteins NifD and NifK will be an absolute requirement. Although mitochondria have been established as a suitable eukaryotic environment for biosynthesis of oxygen-sensitive enzymes such as NifH, expression of NifD in this organelle has proven difficult due to cryptic NifD degradation. Here, we describe a solution to this problem. Using molecular and proteomic methods, we found NifD degradation to be a consequence of mitochondrial endoprotease activity at a specific motif within NifD. Focusing on this functionally sensitive region, we designed NifD variants comprising between one and three amino acid substitutions and distinguished several that were resistant to degradation when expressed in both plant and yeast mitochondria. Nitrogenase activity assays of these resistant variants in Escherichia coli identified a subset that retained function, including a single amino acid variant (Y100Q). We found that other naturally occurring NifD proteins containing alternate amino acids at the Y100 position were also less susceptible to degradation. The Y100Q variant also enabled expression of a NifD(Y100Q)-linker-NifK translational polyprotein in plant mitochondria, confirmed by identification of the polyprotein in the soluble fraction of plant extracts. The NifD(Y100Q)-linker-NifK retained function in bacterial nitrogenase assays, demonstrating that this polyprotein permits expression of NifD and NifK in a defined stoichiometry supportive of activity. Our results exemplify how protein design can overcome impediments encountered when expressing synthetic proteins in novel environments. Specifically, these findings outline our progress toward the assembly of the catalytic unit of nitrogenase within mitochondria.


Assuntos
Genes Bacterianos/genética , Mitocôndrias/genética , Mitocôndrias/fisiologia , Proteínas de Plantas/genética , Plantas/genética , Substituição de Aminoácidos/genética , Escherichia coli/genética , Fixação de Nitrogênio/genética , Nitrogenase/genética , Poliproteínas/genética , Proteômica/instrumentação
9.
PLoS One ; 15(8): e0237744, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841246

RESUMO

Both the Mediterranean (MED) species of the Bemisia tabaci whitefly complex and the greenhouse whitefly (Trialeurodes vaporariorum, TV) are important agricultural pests. The two species of whiteflies differ in many aspects such as morphology, geographical distribution, host plant range, plant virus transmission, and resistance to insecticides. However, the molecular basis underlying their differences remains largely unknown. In this study, we analyzed the genetic divergences between the transcriptomes of MED and TV. In total, 2,944 pairs of orthologous genes were identified. The average identity of amino acid sequences between the two species is 93.6%. The average nonsynonymous (Ka) and synonymous (Ks) substitution rates and the ratio of Ka/Ks of the orthologous genes are 0.0389, 2.23 and 0.0204, respectively. The low average Ka/Ks ratio indicates that orthologous genes tend to be under strong purified selection. The most divergent gene classes are related to the metabolisms of xenobiotics, cofactors, vitamins and amino acids, and this divergence may underlie the different biological characteristics between the two species of whiteflies. Genes of differential expression between the two species are enriched in carbohydrate metabolism and regulation of autophagy. These findings provide molecular clues to uncover the biological and molecular differences between the two species of whiteflies.


Assuntos
Produção Agrícola , Genes de Insetos/genética , Especiação Genética , Hemípteros/genética , Proteínas de Insetos/genética , Sequência de Aminoácidos/genética , Substituição de Aminoácidos/genética , Aminoácidos/metabolismo , Animais , Hemípteros/metabolismo , Proteínas de Insetos/metabolismo , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Região do Mediterrâneo , Anotação de Sequência Molecular , RNA-Seq , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Vitaminas/metabolismo , Xenobióticos/metabolismo
10.
PLoS One ; 15(8): e0238121, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32845893

RESUMO

Variants implicated in childhood epilepsy have been identified in all four voltage-gated sodium channels that initiate action potentials in the central nervous system. Previous research has focused on the functional effects of particular variants within the most studied of these channels (NaV1.1, NaV1.2 and NaV1.6); however, there have been few comparative studies across channels to infer the impact of mutations in patients with epilepsy. Here we compare patterns of variation in patient and public databases to test the hypothesis that regions of known functional significance within voltage-gated sodium (NaV) channels have an increased burden of deleterious variants. We assessed mutational burden in different regions of the Nav channels by (1) performing Fisher exact tests on odds ratios to infer excess variants in domains, segments, and loops of each channel in patient databases versus public "control" databases, and (2) comparing the cumulative distribution of variant sites along DNA sequences of each gene in patient and public databases (i.e., independent of protein structure). Patient variant density was concordant among channels in regions known to play a role in channel function, with statistically significant higher patient variant density in S4-S6 and DIII-DIV and an excess of public variants in SI-S3, DI-DII, DII-DIII. On the other hand, channel-specific patterns of patient burden were found in the NaV1.6 inactivation gate and NaV1.1 S5-S6 linkers, while NaV1.2 and NaV1.6 S4-S5 linkers and S5 segments shared patient variant patterns that contrasted with those in NaV1.1. These different patterns may reflect different roles played by the NaV1.6 inactivation gate in action potential propagation, and by NaV1.1 S5-S6 linkers in loss of function and haploinsufficiency. Interestingly, NaV1.2 and NaV1.6 both lack amino acid substitutions over significantly long stretches in both the patient and public databases suggesting that new mutations in these regions may cause embryonic lethality or a non-epileptic disease phenotype.


Assuntos
Epilepsia/patologia , Ativação do Canal Iônico/genética , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Substituição de Aminoácidos/genética , Sequência de Bases , Encéfalo/fisiologia , Epilepsia/genética , Variação Genética/genética , Humanos , Potenciais da Membrana/genética , Mutação/genética , Técnicas de Patch-Clamp , Análise de Sequência de DNA
11.
Proc Natl Acad Sci U S A ; 117(34): 20926-20931, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32747571

RESUMO

The circadian clock of cyanobacteria consists of only three clock proteins, KaiA, KaiB, and KaiC, which generate a circadian rhythm of KaiC phosphorylation in vitro. The adenosine triphosphatase (ATPase) activity of KaiC is the source of the 24-h period and temperature compensation. Although numerous circadian mutants of KaiC have been identified, the tuning mechanism of the 24-h period remains unclear. Here, we show that the circadian period of in vitro phosphorylation rhythm of mutants at position 402 of KaiC changed dramatically, from 15 h (0.6 d) to 158 h (6.6 d). The ATPase activities of mutants at position 402 of KaiC, without KaiA and KaiB, correlated with the frequencies (1/period), indicating that KaiC structure was the source of extra period change. Despite the wide-range tunability, temperature compensation of both the circadian period and the KaiC ATPase activity of mutants at position 402 of KaiC were nearly intact. We also found that in vivo and in vitro circadian periods and the KaiC ATPase activity of mutants at position 402 of KaiC showed a correlation with the side-chain volume of the amino acid at position 402 of KaiC. Our results indicate that residue 402 is a key position of determining the circadian period of cyanobacteria, and it is possible to dramatically alter the period of KaiC while maintaining temperature compensation.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/metabolismo , Ritmo Circadiano/genética , Adenosina Trifosfatases/metabolismo , Substituição de Aminoácidos/genética , Relógios Circadianos/genética , Cianobactérias/genética , Cianobactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Mutação/genética , Fosforilação , Synechococcus/genética , Synechococcus/metabolismo
12.
BMC Bioinformatics ; 21(1): 311, 2020 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-32677889

RESUMO

BACKGROUND: Polyploid organisms such as wheat complicate even the simplest of procedures in molecular biology. Whilst knowledge of genomic sequences in crops is increasing rapidly, the scientific community is still a long way from producing a full pan-genome for every species. Polymerase chain reaction and Sanger sequencing therefore remain widely used as methods for characterizing gene sequences in many varieties of crops. High sequence similarity between genomes in polyploids means that if primers are not homeologue-specific via the incorporation of a SNP at the 3' tail, sequences other than the target sequence will also be amplified. Current consensus for gene cloning in wheat is to manually perform many steps in a long bioinformatics pipeline. RESULTS: Here we present AutoCloner ( www.autocloner.com ), a fully automated pipeline for crop gene cloning that includes a free-to-use web interface for users. AutoCloner takes a sequence of interest from the user and performs a basic local alignment search tool (BLAST) search against the genome assembly for their particular polyploid crop. Homologous sequences are then compiled with the input sequence into a multiple sequence alignment which is mined for single-nucleotide polymorphisms (SNPs). Various combinations of potential primers that cover the entire gene of interest are then created and evaluated by Primer3; the set of primers with the highest score, as well as all possible primers at every SNP location, are then returned to the user for polymerase chain reaction (PCR). We have successfully used AutoCloner to clone various genes of interest in the Apogee wheat variety, which has no current genome sequence. In addition, we have successfully run the pipeline on ~ 80,000 high-confidence gene models from a wheat genome assembly. CONCLUSION: AutoCloner is the first tool to fully-automate primer design for gene cloning in polyploids, where previously the consensus within the wheat community was to perform this process manually. The web interface for AutoCloner provides a simple and effective polyploid primer-design method for gene cloning, with no need for researchers to download software or input any other details other than their sequence of interest.


Assuntos
Clonagem Molecular , Biologia Computacional/métodos , Primers do DNA/metabolismo , Poliploidia , Homologia de Sequência , Software , Triticum/genética , Substituição de Aminoácidos/genética , Sequência de Bases , Primers do DNA/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único/genética
13.
BMC Infect Dis ; 20(1): 513, 2020 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-32677899

RESUMO

BACKGROUND: Imported falciparum malaria from Africa has become a key public health challenge in Guizhou Province since 2012. Understanding the polymorphisms of molecular markers of drug resistance can guide selection of antimalarial drugs for the treatment of malaria. This study was aimed to analyze the polymorphisms of pfcrt, pfmdr1, and K13-propeller among imported falciparum malaria cases in Guizhou Province, China. METHOD: Fifty-five imported falciparum malaria cases in Guizhou Province during 2012-2016 were included in this study. Their demographic information and filter paper blood samples were collected. Genomic DNA of Plasmodium falciparum was extracted from the blood samples, and polymorphisms of pfcrt, pfmdr1, and K13-propeller were analyzed with nested PCR amplification followed by sequencing. Data were analyzed with the SPSS17.0 software. RESULTS: The prevalence of pfcrt K76T, pfmdr1 N86Y, and pfmdr1 Y184F mutation was 56.6, 22.2, and 72.2%, respectively, in imported falciparum malaria cases in Guizhou Province. We detected two mutant haplotypes of pfcrt, IET and MNT, with IET being more commonly found (54.7%), and five mutant haplotypes of pfmdr1, of which NFD was the most frequent (53.7%). There were totally 10 combined haplotypes of pfcrt and pfmdr1, of which the haplotype IETNFD possessed a predominance of 28.8%. In addition, three nonsynonymous mutations (S459T, C469F, and V692L) and two synonymous mutations (R471R and V589V) were detected in K13-propeller, all having prevalence less than 6.0%. In particular, a candidate K13 resistance mutation, C469F, was identified for the first time from Democratic Republic of the Congo with the prevalence of 2.0%. CONCLUSIONS: The high prevalence of IET haplotype of pfcrt and NFD haplotype of pfmdr1 suggests the presence of chloroquine, artemether/lumefantrine, and dihydroartemisinin/piperaquine resistance in these cases. Therefore cautions should be made to artemisinin therapy for P. falciparum in Africa. Continuous monitoring of anti-malarial drug efficacy in imported malaria cases is helpful for optimizing antimalarial drug therapy in Guizhou Province, China.


Assuntos
Doenças Transmissíveis Importadas/parasitologia , Resistência a Medicamentos/genética , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Polimorfismo Genético , Proteínas de Protozoários/genética , Adulto , África/epidemiologia , Substituição de Aminoácidos/genética , Antimaláricos/uso terapêutico , China/epidemiologia , Estudos de Coortes , Feminino , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologia , Masculino , Proteínas de Membrana Transportadoras/genética , Pessoa de Meia-Idade , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutação , Plasmodium falciparum/isolamento & purificação , Doença Relacionada a Viagens
14.
PLoS Genet ; 16(7): e1008901, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32645003

RESUMO

The RNA exosome is an evolutionarily-conserved ribonuclease complex critically important for precise processing and/or complete degradation of a variety of cellular RNAs. The recent discovery that mutations in genes encoding structural RNA exosome subunits cause tissue-specific diseases makes defining the role of this complex within specific tissues critically important. Mutations in the RNA exosome component 3 (EXOSC3) gene cause Pontocerebellar Hypoplasia Type 1b (PCH1b), an autosomal recessive neurologic disorder. The majority of disease-linked mutations are missense mutations that alter evolutionarily-conserved regions of EXOSC3. The tissue-specific defects caused by these amino acid changes in EXOSC3 are challenging to understand based on current models of RNA exosome function with only limited analysis of the complex in any multicellular model in vivo. The goal of this study is to provide insight into how mutations in EXOSC3 impact the function of the RNA exosome. To assess the tissue-specific roles and requirements for the Drosophila ortholog of EXOSC3 termed Rrp40, we utilized tissue-specific RNAi drivers. Depletion of Rrp40 in different tissues reveals a general requirement for Rrp40 in the development of many tissues including the brain, but also highlight an age-dependent requirement for Rrp40 in neurons. To assess the functional consequences of the specific amino acid substitutions in EXOSC3 that cause PCH1b, we used CRISPR/Cas9 gene editing technology to generate flies that model this RNA exosome-linked disease. These flies show reduced viability; however, the surviving animals exhibit a spectrum of behavioral and morphological phenotypes. RNA-seq analysis of these Drosophila Rrp40 mutants reveals increases in the steady-state levels of specific mRNAs and ncRNAs, some of which are central to neuronal function. In particular, Arc1 mRNA, which encodes a key regulator of synaptic plasticity, is increased in the Drosophila Rrp40 mutants. Taken together, this study defines a requirement for the RNA exosome in specific tissues/cell types and provides insight into how defects in RNA exosome function caused by specific amino acid substitutions that occur in PCH1b can contribute to neuronal dysfunction.


Assuntos
Doenças Cerebelares/genética , Proteínas do Citoesqueleto/genética , Drosophila melanogaster/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Proteínas de Ligação a RNA/genética , Substituição de Aminoácidos/genética , Animais , Sistemas CRISPR-Cas/genética , Doenças Cerebelares/patologia , Cerebelo/metabolismo , Cerebelo/patologia , Modelos Animais de Doenças , Exossomos/genética , Humanos , Mutação/genética , Neurônios/patologia , RNA/genética
15.
Hum Genet ; 139(12): 1513-1529, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32529326

RESUMO

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder caused by the loss of function of a set of imprinted genes on chromosome 15q11-15q13. One of these genes, NDN, encodes necdin, a protein that is important for neuronal differentiation and survival. Loss of Ndn in mice causes defects in the formation and function of the nervous system. Necdin is a member of the melanoma-associated antigen gene (MAGE) protein family. The functions of MAGE proteins depend highly on their interactions with other proteins, and in particular MAGE proteins interact with E3 ubiquitin ligases and deubiquitinases to form MAGE-RING E3 ligase-deubiquitinase complexes. Here, we used proximity-dependent biotin identification (BioID) and mass spectrometry (MS) to determine the network of protein-protein interactions (interactome) of the necdin protein. This process yielded novel as well as known necdin-proximate proteins that cluster into a protein network. Next, we used BioID-MS to define the interactomes of necdin proteins carrying coding variants. Variant necdin proteins had interactomes that were distinct from wildtype necdin. BioID-MS is not only a useful tool to identify protein-protein interactions, but also to analyze the effects of variants of unknown significance on the interactomes of proteins involved in genetic disease.


Assuntos
Substituição de Aminoácidos/genética , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Mapas de Interação de Proteínas/genética , Ubiquitina-Proteína Ligases/genética , Animais , Biotinilação/genética , Diferenciação Celular/genética , Enzimas Desubiquitinantes/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Espectrometria de Massas/métodos , Camundongos , Mutação/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/ultraestrutura , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/patologia , Neurônios/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/ultraestrutura , Proteínas de Ligação a Poli(A)/química , Proteínas de Ligação a Poli(A)/genética , Síndrome de Prader-Willi/genética , Conformação Proteica , Relação Estrutura-Atividade , Ubiquitina-Proteína Ligases/química
16.
BMC Evol Biol ; 20(1): 57, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32429841

RESUMO

BACKGROUND: Amino acid substitution models play an important role in inferring phylogenies from proteins. Although different amino acid substitution models have been proposed, only a few were estimated from mitochondrial protein sequences for specific taxa such as the mtArt model for Arthropoda. The increasing of mitochondrial genome data from broad Orthoptera taxa provides an opportunity to estimate the Orthoptera-specific mitochondrial amino acid empirical model. RESULTS: We sequenced complete mitochondrial genomes of 54 Orthoptera species, and estimated an amino acid substitution model (named mtOrt) by maximum likelihood method based on the 283 complete mitochondrial genomes available currently. The results indicated that there are obvious differences between mtOrt and the existing models, and the new model can better fit the Orthoptera mitochondrial protein datasets. Moreover, topologies of trees constructed using mtOrt and existing models are frequently different. MtOrt does indeed have an impact on likelihood improvement as well as tree topologies. The comparisons between the topologies of trees constructed using mtOrt and existing models show that the new model outperforms the existing models in inferring phylogenies from Orthoptera mitochondrial protein data. CONCLUSIONS: The new mitochondrial amino acid substitution model of Orthoptera shows obvious differences from the existing models, and outperforms the existing models in inferring phylogenies from Orthoptera mitochondrial protein sequences.


Assuntos
Substituição de Aminoácidos/genética , Mitocôndrias/genética , Modelos Genéticos , Ortópteros/genética , Software , Sequência de Aminoácidos , Animais , Intervalos de Confiança , Bases de Dados Genéticas , Genoma Mitocondrial , Funções Verossimilhança , Ortópteros/classificação , Filogenia
17.
BMC Evol Biol ; 20(1): 56, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32414322

RESUMO

BACKGROUND: Amylase gene clusters have been implicated in adaptive copy number changes in response to the amount of starch in the diet of humans and mammals. However, this interpretation has been questioned for humans and for mammals there is a paucity of information from natural populations. RESULTS: Using optical mapping and genome read information, we show here that the amylase cluster in natural house mouse populations is indeed copy-number variable for Amy2b paralogous gene copies (called Amy2a1 - Amy2a5), but a direct connection to starch diet is not evident. However, we find that the amylase cluster was subject to introgression of haplotypes between Mus musculus sub-species. A very recent introgression can be traced in the Western European populations and this leads also to the rescue of an Amy2b pseudogene. Some populations and inbred lines derived from the Western house mouse (Mus musculus domesticus) harbor a copy of the pancreatic amylase (Amy2b) with a stop codon in the first exon, making it non-functional. But populations in France harbor a haplotype introgressed from the Eastern house mouse (M. m. musculus) with an intact reading frame. Detailed analysis of phylogenetic patterns along the amylase cluster suggest an additional history of previous introgressions. CONCLUSIONS: Our results show that the amylase gene cluster is a hotspot of introgression in the mouse genome, making it an evolutionary active region beyond the previously observed copy number changes.


Assuntos
Amilases/genética , Família Multigênica , Pseudogenes , Substituição de Aminoácidos/genética , Animais , Sequência de Bases , Genoma , Haplótipos/genética , Camundongos , Filogenia , Alinhamento de Sequência
18.
Tunis Med ; 98(2): 161-163, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32395807

RESUMO

We report the case of a 23-year-old woman with a not yet described (to the best of our knowledge) association of left ventricle non-compaction with both atrial and ventricular defects. Family genetic survey concluded to, a probably sporadic, E101K gene mutation.


Assuntos
Cardiopatias Congênitas/diagnóstico , Comunicação Interatrial/diagnóstico , Ventrículos do Coração/anormalidades , Substituição de Aminoácidos/genética , Proteínas Associadas à Distrofina/genética , Feminino , Ácido Glutâmico/genética , Cardiopatias Congênitas/genética , Comunicação Interatrial/complicações , Comunicação Interatrial/genética , Humanos , Lisina/genética , Mutação de Sentido Incorreto , Neuropeptídeos/genética , Adulto Jovem
19.
Arch Virol ; 165(8): 1739-1748, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32409874

RESUMO

In Korea, dengue infection has been frequently reported in travelers to tropical and subtropical countries. Global warming increases the probability of autochthonous dengue outbreaks in Korea. In this report, the molecular and evolutionary properties of four dengue virus (DENV) type 2 isolates from Korean overseas travelers were examined. Three of these isolates were classified as Cosmopolitan genotypes and further divided into sublineages 1 (43,253, 43,254) and 2 (43,248), while the other isolate (KBPV-VR29) was related to American genotypes. The variable amino acid motifs related to virulence and replication were identified in the structural and non-structural proteins. A negative selection mechanism was clearly verified in all of the DENV proteins. Potential recombination events were identified in the NS5 protein of the XSBN10 strain. The substitution rate (5.32 × 10-4 substitutions per site) and the time of the most recent common ancestor (TMRCA) for each evolutionary group were determined by the Bayesian skyline coalescent method. This study shows that DENV type 2 strains with distinct phylogenetic, evolutionary, and virulence characteristics have been introduced into Korea by overseas travelers and have the potential to trigger autochthonous dengue outbreaks.


Assuntos
Vírus da Dengue/genética , Vírus da Dengue/isolamento & purificação , Substituição de Aminoácidos/genética , Dengue/virologia , Surtos de Doenças , Evolução Molecular , Genoma Viral/genética , Genótipo , Humanos , Filogenia , RNA Viral/genética , República da Coreia , Sorogrupo , Proteínas Virais/genética , Virulência/genética , Replicação Viral/genética
20.
Viruses ; 12(5)2020 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-32370153

RESUMO

The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a lethal zoonotic pathogen circulating in the Arabian Peninsula since 2012. There is no vaccine for MERS and anti-viral treatment is generally not applicable. We investigated the evolution of the MERS-CoV spike gene sequences and changes in viral loads over time from patients in Saudi Arabia from 2105-2017. All the MERS-CoV strains belonged to lineage 5, and showed high sequence homology (99.9%) to 2017 strains. Recombination analysis showed a potential recombination event in study strains from patients in Saudi Arabia. The spike gene showed eight amino acid substitutions, especially between the A1 and B5 lineage, and contained positively selected codon 1020. We also determined that the viral loads were significantly (p < 0.001) higher in fatal cases, and virus shedding was prolonged in some fatal cases beyond 21 days. The viral concentration peaked during the first week of illness, and the lower respiratory specimens had higher levels of MERS-CoV RNA. The presence of the diversifying selection and the topologies with the structural mapping of residues under purifying selection suggested that codon 1020 might have a role in the evolution of spike gene during the divergence of different lineages. This study will im-prove our understanding of the evolution of MERS-CoV, and also highlights the need for enhanced surveillance in humans and dromedaries. The presence of amino acid changes at the N-terminal domain and structural mapping of residues under positive selection at heptad repeat 1 provides better insight into the adaptive evolution of the spike gene and might have a potential role in virus-host tropism and pathogenesis.


Assuntos
Substituição de Aminoácidos/genética , Infecções por Coronavirus/patologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Glicoproteína da Espícula de Coronavírus/genética , Adulto , Idoso , Sequência de Aminoácidos , Animais , Sequência de Bases , Camelus/virologia , Dipeptidil Peptidase 4/metabolismo , Evolução Molecular , Feminino , Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Domínios Proteicos/genética , RNA Viral/genética , Receptores Virais/genética , Recombinação Genética/genética , Arábia Saudita , Análise de Sequência de RNA , Homologia de Sequência , Carga Viral , Tropismo Viral/genética
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