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1.
Pestic Biochem Physiol ; 159: 17-21, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400779

RESUMO

Capsella bursa-pastoris is a serious broadleaf weed in winter wheat fields in China. It has evolved high levels of resistance to acetolactate synthase (ALS) inhibiting herbicides and has caused substantial losses of wheat yield in recent years. We monitored the herbicide resistance of Capsella bursa-pastoris collected from 18 regions of Shandong Province in 2009, 2013 and 2017, respectively. Compared with the 2009 populations, the number of populations resistant to florasulam had increased in 2013 and 2017. Resistance to tribenuron-methyl increased in 2013, but decreased in 2017. The 2009 and 2013 populations developed resistance only to tribenuron-methyl, but some 2017 populations developed cross-resistance to imazethapyr and florasulam as well. Mutations in ALS (Pro-197-Thr/Ser/His/Arg/Leu/Gln) were identified in the 2009 and 2013 populations; however, two ALS mutations (Pro197 and/or Trp574) were identified in 2017 plants. Meanwhile, plants containing both point mutations (Pro197 + Trp574) were identified in the 2017 populations. This study demonstrated that target site gene mutations were the main reason for Capsella bursa-pastoris resistance to ALS-inhibiting herbicides. Although target-site mutation is the reason for resistance to ALS-inhibiting herbicides in Capsella bursa-pastoris, the resistance patterns and mutations identified have changed over time.


Assuntos
Acetolactato Sintase/genética , Capsella/efeitos dos fármacos , Resistência a Herbicidas/genética , Herbicidas/farmacologia , Proteínas de Plantas/genética , Sulfonatos de Arila/farmacologia , Capsella/enzimologia , Capsella/genética , Mutação/genética , Ácidos Nicotínicos/farmacologia , Mutação Puntual/genética , Pirimidinas/farmacologia , Sulfonamidas/farmacologia
2.
Pestic Biochem Physiol ; 158: 77-87, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31378364

RESUMO

Pyrethroid-resistance in onion thrips, Thrips tabaci, has been reported in many countries including Japan. Identifying factors of the resistance is important to correctly monitoring the resistance in field populations. To identify pyrethroid-resistance related genes in T. tabaci in Japan, we performed RNA-Seq analysis of seven T. tabaci strains including two pyrethroid-resistant and five pyrethroid-susceptible strains. We identified a pair of single point mutations, T929I and K1774N, introducing two amino acid mutations, in the voltage-gated sodium channel gene, a pyrethroid target gene, in the two resistant strains. The K1774N is a newly identified mutation located in the fourth repeat domain of the sodium channel. Genotyping analysis of field-collected populations showed that most of the T. tabaci individuals in resistant populations carried the mutation pair, indicating that the mutation pair is closely associated with pyrethroid-resistance in Japan. Another resistance-related mutation, M918L, was also identified in part of the resistant populations. Most of the individuals with the mutation pair were arrhenotokous while all individuals with the M918L single mutation were thelytokous. The result of differentially expressed gene analysis revealed a small number of up-regulated detoxification genes in each resistant strain which might be involved in resistance to pyrethroid. However, no up-regulated detoxification genes common to the two resistant strains were detected. Our results indicate that the mutation pair in the sodium channel gene is the most important target for monitoring pyrethroid-resistance in T. tabaci, and that pyrethroid-resistant arrhenotokous individuals with the mutation pair are likely to be widely distributed in Japan.


Assuntos
Piretrinas/farmacologia , Tisanópteros/efeitos dos fármacos , Tisanópteros/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Japão , Mutação/genética , Mutação Puntual/genética , Tisanópteros/genética , Canais de Sódio Disparados por Voltagem/genética
3.
Plant Dis ; 103(10): 2536-2540, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31424998

RESUMO

Fusarium head blight, also called scab, is caused by Fusarium graminearum and is one of the most important destructive diseases of wheat. The frequency of carbendazim resistance in 1,132 isolates of F. graminearum recovered from fields in different regions of Henan Province in 2016, 2017, and 2018 was determined. A total of 31 F. graminearum isolates resistant to carbendazim were detected, including 30 moderately resistant isolates and one highly resistant isolate. The frequency of resistance of F. graminearum isolates to carbendazim was 2.7%. The range of effective concentration (EC50) values of 1,101 sensitive isolates and 30 moderately resistant isolates was 0.08 to 0.98 µg ml-1 and 2.73 to 13.28 µg ml-1, respectively. The mean ± SD EC50 value was 0.55 ± 0.13 µg ml-1 and 5.61 ± 2.58 µg ml-1, respectively. The EC50 value of the highly resistant isolate was 21.12 µg ml-1. Point mutation types of the carbendazim-resistant isolates were characterized by cloning the ß2-tubulin gene of 31 resistant isolates. Three point mutation types at amino acids F167Y, E198Q, and E198L in the ß2-tubulin gene of resistant isolates were identified. Among 31 resistant isolates, the frequency of point mutation types in F167Y, E198Q, and E198L of the ß2-tubulin gene was 71.0, 25.8, and 3.2%, respectively. The data indicate that F. graminearum has developed resistance to carbendazim in Henan Province, and single point mutations at amino acid F167Y were the predominant type of mutation detected.


Assuntos
Benzimidazóis , Carbamatos , Farmacorresistência Fúngica , Fusarium , Triticum , Benzimidazóis/farmacologia , Carbamatos/farmacologia , Farmacorresistência Fúngica/genética , Fungicidas Industriais , Fusarium/efeitos dos fármacos , Fusarium/genética , Genes de Plantas/genética , Mutação Puntual , Triticum/microbiologia , Tubulina (Proteína)/genética
4.
Chem Commun (Camb) ; 55(72): 10677-10680, 2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31424057

RESUMO

Beacon-mediated Exponential Amplification Reaction (BEAR) enables isothermal, exponential signal amplification. BEAR uses only a single enzyme and a single primer. Detection of 0.2 amol of a mitochondrial DNA with a point mutation in less than an hour demonstrates an application of the BEAR technique for nucleic acid research.


Assuntos
Técnicas Biossensoriais , DNA Mitocondrial/análise , DNA Polimerase Dirigida por DNA/química , Técnicas de Amplificação de Ácido Nucleico , DNA Mitocondrial/genética , DNA Polimerase Dirigida por DNA/metabolismo , Mutação Puntual
5.
Biomed Khim ; 65(4): 263-276, 2019 Jun.
Artigo em Russo | MEDLINE | ID: mdl-31436168

RESUMO

Protein p53 is one of the most studied proteins. This attention is primarily due to its key role in the cellular mechanisms associated with carcinogenesis. Protein p53 is a transcription factor involved in a wide variety of processes: cell cycle regulation and apoptosis, signaling inside the cell, DNA repair, coordination of metabolic processes, regulation of cell interactions, etc. This multifunctionality is apparently determined by the fact that p53 is a vivid example of how the same protein can be represented by numerous proteoforms bearing completely different functional loads. By alternative splicing, using different promoters and translation initiation sites, the TP53 gene gives rise to at least 12 isoforms, which can additionally undergo numerous (>200) post-translational modifications. Proteoforms generated due to numerous point mutations in the TP53 gene are adding more complexity to this picture. The proteoforms produced are involved in various processes, such as the regulation of p53 transcriptional activity in response to various factors. This review is devoted to the description of the currently known p53 proteoforms, as well as their possible functionality.


Assuntos
Processamento Alternativo , Genes p53 , Proteína Supressora de Tumor p53/química , Humanos , Mutação Puntual , Isoformas de Proteínas/química , Processamento de Proteína Pós-Traducional , Relação Estrutura-Atividade
7.
BMC Bioinformatics ; 20(Suppl 14): 335, 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31266447

RESUMO

BACKGROUND: Predicting the effect of single point variations on protein stability constitutes a crucial step toward understanding the relationship between protein structure and function. To this end, several methods have been developed to predict changes in the Gibbs free energy of unfolding (∆∆G) between wild type and variant proteins, using sequence and structure information. Most of the available methods however do not exhibit the anti-symmetric prediction property, which guarantees that the predicted ∆∆G value for a variation is the exact opposite of that predicted for the reverse variation, i.e., ∆∆G(A → B) = -∆∆G(B → A), where A and B are amino acids. RESULTS: Here we introduce simple anti-symmetric features, based on evolutionary information, which are combined to define an untrained method, DDGun (DDG untrained). DDGun is a simple approach based on evolutionary information that predicts the ∆∆G for single and multiple variations from sequence and structure information (DDGun3D). Our method achieves remarkable performance without any training on the experimental datasets, reaching Pearson correlation coefficients between predicted and measured ∆∆G values of ~ 0.5 and ~ 0.4 for single and multiple site variations, respectively. Surprisingly, DDGun performances are comparable with those of state of the art methods. DDGun also naturally predicts multiple site variations, thereby defining a benchmark method for both single site and multiple site predictors. DDGun is anti-symmetric by construction predicting the value of the ∆∆G of a reciprocal variation as almost equal (depending on the sequence profile) to -∆∆G of the direct variation. This is a valuable property that is missing in the majority of the methods. CONCLUSIONS: Evolutionary information alone combined in an untrained method can achieve remarkably high performances in the prediction of ∆∆G upon protein mutation. Non-trained approaches like DDGun represent a valid benchmark both for scoring the predictive power of the individual features and for assessing the learning capability of supervised methods.


Assuntos
Algoritmos , Estabilidade Proteica , Proteínas/química , Sequência de Aminoácidos , Evolução Molecular , Humanos , Mutação Puntual , Proteínas/genética , Termodinâmica
8.
Gene ; 714: 143990, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31326550

RESUMO

BACKGROUND: Progressive cardiac conduction defect (PCCD), also known as Lenegre-Lev disease, is one of the most common heart conduction abnormalities. Previous studies have screened for known mutation sites that cause heart block in a 68-person family with a history of PCCD, revealed no mutations. OBJECTIVE: To screen pathogenic genes of the PCCD family and to study the function of the gene mutations related to heart block diseases. METHODS: Whole exome sequencing (WES) was performed on two PCCD patients and one non-PCCD family member to find the related pathogenic gene. After family co-segregation and preliminary functional analysis, we identified the mutant gene CLCA2. To study the function of this gene, we constructed mutant-gene mice using CRISPR-Cas9 technology, and electrocardiogram monitoring was performed after genotype verification. RESULTS: The CLCA2 c.G1725T mutation was identified and co-segregated with the phenotype. The analysis showed that the CLCA2 c.G1725T mutation is harmful and mainly affects protein glycosylation. Immunofluorescence staining revealed that CLCA2 was highly expressed in the sinoatrial node (SAN) tissues. Electrocardiogram monitoring of the mice revealed that CLCA2 point mutations induced mild conduction block and ectopic pacemakers. CONCLUSION: Our findings indicate that a novel heterozygous missense mutation c.G1725T of the CLCA2 gene may be associated with heart block disease and the mutation in this gene may lead to sinus node lesions and conduction blocking.


Assuntos
Canais de Cloreto/genética , Bloqueio Cardíaco/genética , Mutação de Sentido Incorreto/genética , Sequência de Aminoácidos , Animais , Eletrocardiografia/métodos , Feminino , Heterozigoto , Humanos , Masculino , Camundongos , Linhagem , Fenótipo , Mutação Puntual/genética , Nó Sinoatrial/patologia
9.
BMC Infect Dis ; 19(1): 616, 2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31299916

RESUMO

BACKGROUND: The point mutations in 23S rRNA gene of Mycoplasma pneumoniae (M. pneumoniae) can lead to high-level resistance to macrolides. This study aimed to evaluate allele-specific real-time PCR (ASPCR) to detect the resistance-related mutations located at positions A2063G and A2064G of 23S rRNA gene. METHODS: We detected 178 pharyngeal swab specimens and calculated the proportions of resistant and sensitive quasispecies using ASPCR assays. ASPCR assays can detect down to 10 copies of 23S rRNA gene and achieved sensitivities of < 0.1% for A2063G and A2064G. We also compared the findings of ASPCR with the results of nested PCR with sequencing. RESULTS: Of 178 samples, 164 were found to have M. pneumoniae including 90.85% (149/164) samples with macrolide-resistant M. pneumoniae (MRMP) quasispecies by ASPCR, while 153 were found to be M. pneumoniae-positive including 71.90% (110/153) samples with MRMP quasispecies by nested PCR with sequencing. Of the 164 M. pneumoniae-positive samples, 61.59% (101/164) had the mixed population of wild-type and mutant M. pneumoniae, and 56.44% (57/101) of the latter contained the mutations at low frequency (≤50%). CONCLUSION: ASPCR indicated that sensitive and resistant quasispecies coexisted in most of the M. pneumoniae positive samples. The ASPCR was a highly sensitive, accurate and rapid method for detecting the macrolide resistance-associated mutations and it could provide earlier and more drug-resistant information for M. pneumoniae research and the clinical therapy.


Assuntos
Farmacorresistência Bacteriana/genética , Mycoplasma pneumoniae/genética , Pneumonia por Mycoplasma/diagnóstico , RNA Ribossômico 23S/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Antibacterianos/uso terapêutico , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Feminino , Humanos , Macrolídeos/uso terapêutico , Mycoplasma pneumoniae/isolamento & purificação , Faringe/microbiologia , Pneumonia por Mycoplasma/tratamento farmacológico , Pneumonia por Mycoplasma/microbiologia , Mutação Puntual , RNA Ribossômico 23S/genética , Reprodutibilidade dos Testes
10.
Cancer Sci ; 110(9): 2973-2981, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31293054

RESUMO

Every year, approximately 1.2 million cases of colorectal carcinoma (CRC) are newly diagnosed worldwide. Although metastases to distant organs are often fatal complications of CRC, little information is known as to how such metastatic lesions are formed. To reveal the genetic profiles for CRC metastasis, we conducted whole-exome RNA sequencing on CRC tumors with liver metastasis (LM) (group A, n = 12) and clinical stage-matched larger tumors without LM (group B, n = 16). While the somatic mutation profiles were similar among the primary tumors and LM lesions in group A and the tumors in group B, the A-to-C nucleotide change in the context of "AAG" was only enriched in the LM regions in group A, suggesting the presence of a DNA damage process specific to metastasis. Genes already known to be associated with CRC were mutated in all groups at a similar frequency, but we detected somatic nonsynonymous mutations in a total of 707 genes in the LM regions, but not in the tumors without LM. Signaling pathways linked to such "LM-associated" genes were overrepresented for extracellular matrix-receptor interaction or focal adhesion. Further, fusions of the ADAP1 (ArfGAP with dual PH domain 1) were newly identified in our cohort (3 out of 28 patients), which activated ARF6, an ADAP1-substrate. Infrequently, mutated genes may play an important role in metastasis formation of CRC. Additionally, recurrent ADAP1 fusion genes were unexpectedly discovered. As these fusions activate small GTPase, further experiments are warranted to examine their contribution to CRC carcinogenesis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias Colorretais/genética , Fusão Gênica , Neoplasias Hepáticas/genética , Proteínas do Tecido Nervoso/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinogênese/genética , Neoplasias Colorretais/patologia , Análise Mutacional de DNA , Feminino , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Neoplasias Hepáticas/secundário , Masculino , Pessoa de Meia-Idade , Mutação Puntual , Sequenciamento Completo do Exoma
11.
Pestic Biochem Physiol ; 157: 80-87, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31153480

RESUMO

The European red mite Panonychus ulmi (Koch) is a major pest of apple trees worldwide and causes significant damage to apple orchards in Iran. Pyrethroid insecticides/acaricides, such as fenpropathrin and fenvalerate, are widely used to control P. ulmi, but their long-term use may lead to low efficacy. Earlier studies investigating pyrethroid resistance in closely related mites such as Tetranychus urticae revealed that pyrethroid resistance was associated with point mutations in the voltage-gated sodium channel gene (vgsc). The aim of this study was to investigate the biochemical and molecular mechanisms of fenpropathrin and fenvalerate resistance in Iranian populations of P. ulmi. Pyrethroid toxicity bioassays were carried out on different P. ulmi field populations. Marand (resistance ratio, RR = 149), Maraqeh (RR = 90) and Mianeh2 (RR = 71) populations exhibited high levels of resistance to fenpropathrin, compared to a susceptible field population (Shahin Dej). Resistance was also observed for fenvalerate with resistance ratio's ranging from 2- to 20-fold. Synergism experiments and enzyme activity assays predicted a minor role for classical detoxification enzymes. In contrast, two amino acid substitutions in the VGSC, L1024V and F1538I, that were previously shown to confer pyrethroid resistance, were detected in all three resistant P. ulmi populations and point towards target-site insensitivity as the most likely resistance mechanism. Furthermore, sequencing after cloning of vgsc fragments from single haploid males revealed the presence of multiple copies of vgsc in a highly resistant strain. The link between resistance mutations and vgsc copy number variation should be the subject of future study, as this might be used to develop molecular markers for monitoring pyrethroid resistance of P. ulmi in the field.


Assuntos
Mutação Puntual/genética , Piretrinas/farmacologia , Canais de Sódio Disparados por Voltagem/genética , Animais , Variações do Número de Cópias de DNA/genética , Duplicação Gênica/efeitos dos fármacos , Duplicação Gênica/genética , Resistência a Inseticidas/genética , Irã (Geográfico) , Ácaros , Canais de Sódio Disparados por Voltagem/metabolismo
12.
Anal Chim Acta ; 1075: 137-143, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31196419

RESUMO

Nucleic acid probes are very useful tools in biological and medical science. However, the essential sensing mechanism of nucleic acid probes was prone to the interference of surrounding sequences. Especially when the target sequences formed secondary structures such as hairpin or quadruplex, the nucleic acid probes were hindered from hybridizing with target strands, greatly disabled the function of probes. Herein, we have established an Open strand based strategy for eliminating the influence of secondary structures on the performance of nucleic acid probes. The strategy was general toward different lengths, secondary structures and sequences of the targeting strand, and we found that the improvement was higher when the secondary structure of the targeting strand was more complicated. Experiments on synthetic single stranded DNA and real clinical genomic DNA samples were conducted for low abundance mutation detection, and the limit of detection for TERT-C228T and BRCA2 rs80359065 mutations could be 0.02% and 0.05% respectively, demonstrating the clinical practicability of our proposed strategy in low abundance mutation detection.


Assuntos
Sondas de DNA/química , DNA de Cadeia Simples/análise , Proteína BRCA2/genética , Sondas de DNA/genética , DNA de Cadeia Simples/genética , Desoxirribonuclease IV (Fago T4-Induzido)/química , Feminino , Corantes Fluorescentes/química , Humanos , Limite de Detecção , Medições Luminescentes/métodos , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Neoplasias Ovarianas/genética , Mutação Puntual , Telomerase/genética
13.
Nat Commun ; 10(1): 2569, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189880

RESUMO

Synonymous mutations have been viewed as silent mutations, since they only affect the DNA and mRNA, but not the amino acid sequence of the resulting protein. Nonetheless, recent studies suggest their significant impact on splicing, RNA stability, RNA folding, translation or co-translational protein folding. Hence, we compile 659194 synonymous mutations found in human cancer and characterize their properties. We provide the user-friendly, comprehensive resource for synonymous mutations in cancer, SynMICdb ( http://SynMICdb.dkfz.de ), which also contains orthogonal information about gene annotation, recurrence, mutation loads, cancer association, conservation, alternative events, impact on mRNA structure and a SynMICdb score. Notably, synonymous and missense mutations are depleted at the 5'-end of the coding sequence as well as at the ends of internal exons independent of mutational signatures. For patient-derived synonymous mutations in the oncogene KRAS, we indicate that single point mutations can have a relevant impact on expression as well as on mRNA secondary structure.


Assuntos
Bases de Dados de Ácidos Nucleicos , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias/genética , Mutação Silenciosa/genética , Conjuntos de Dados como Assunto , Humanos , Mutação de Sentido Incorreto/genética , Mutação Puntual/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Dobramento de RNA/genética , Processamento de RNA/genética , RNA Mensageiro/química , RNA Mensageiro/genética
14.
Nat Commun ; 10(1): 2852, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253764

RESUMO

Cytosine base editors (CBEs) enable programmable C-to-T conversion without DNA double-stranded breaks and homology-directed repair in a variety of organisms, which exhibit great potential for agricultural and biomedical applications. However, all reported cases only involved C-to-T substitution at a single targeted genomic site. Whether C-to-T substitution is effective in multiple sites/loci has not been verified in large animals. Here, by using pigs, an important animal for agriculture and biomedicine, as the subjective animal, we showed that CBEs could efficiently induce C-to-T conversions at multiple sites/loci with the combination of three genes, including DMD, TYR, and LMNA, or RAG1, RAG2, and IL2RG, simultaneously, at the embryonic and cellular levels. CBEs also could disrupt genes (pol gene of porcine endogenous retrovirus) with dozens of copies by introducing multiple premature stop codons. With the CBEs, pigs carrying single gene or multiple gene point mutations were generated through embryo injection or nuclear transfer approach.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Mutação Puntual , Suínos/genética , Desaminase APOBEC-1 , Animais , Sequência de Bases , Proteína 9 Associada à CRISPR , DNA/genética , Técnicas de Cultura Embrionária , Embrião de Mamíferos , Genoma , Técnicas de Transferência Nuclear/veterinária , RNA Guia/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
15.
Chem Commun (Camb) ; 55(52): 7510-7513, 2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-31187817

RESUMO

Self-assembly of protein nanocages into two-dimensional superlattices can be achieved by disulfide-mediated modular interactions, which can be carried out by introducing single point mutation on the exterior surfaces of the protein nanocages nearby the symmetry rotation axes. As designed, the protein cages arrange in an on-axis alignment pattern.


Assuntos
Dissulfetos/química , Nanoestruturas/química , Proteínas/metabolismo , Cisteína/química , Ferritinas/genética , Ferritinas/metabolismo , Humanos , Oxirredução , Mutação Puntual , Proteínas/genética
16.
Genes Dev ; 33(13-14): 799-813, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171700

RESUMO

Mammalian development requires effective mechanisms to repress genes whose expression would generate inappropriately specified cells. The Polycomb-repressive complex 1 (PRC1) family complexes are central to maintaining this repression. These include a set of canonical PRC1 complexes, each of which contains four core proteins, including one from the CBX family. These complexes have been shown previously to reside in membraneless organelles called Polycomb bodies, leading to speculation that canonical PRC1 might be found in a separate phase from the rest of the nucleus. We show here that reconstituted PRC1 readily phase-separates into droplets in vitro at low concentrations and physiological salt conditions. This behavior is driven by the CBX2 subunit. Point mutations in an internal domain of Cbx2 eliminate phase separation. These same point mutations eliminate the formation of puncta in cells and have been shown previously to eliminate nucleosome compaction in vitro and generate axial patterning defects in mice. Thus, the domain of CBX2 that is important for phase separation is the same domain shown previously to be important for chromatin compaction and proper development, raising the possibility of a mechanistic or evolutionary link between these activities.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Complexo Repressor Polycomb 1/química , Animais , Linhagem Celular , Escherichia coli/genética , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Organelas/metabolismo , Mutação Puntual , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Domínios Proteicos , Células Sf9
17.
Phys Chem Chem Phys ; 21(22): 12021-12028, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31135801

RESUMO

PI3Kα is a principal Ras effector that phosphorylates PIP2 to PIP3 in the PI3K/Akt/mTOR pathway. How Ras activates PI3K has been unclear: is Ras' role confined to PI3K recruitment to the membrane or does Ras activation also involve allostery? Recently, we determined the mechanism of PI3Kα activation at the atomic level. We showed the vital role and significance of conformational change in PI3Kα activation. Here, by a 'best-match for hydrogen-bonding pair' (BMHP) computational protocol and molecular dynamics (MD) simulations, we model the atomic structure of KRas4B in complex with the Ras binding domain (RBD) of PI3Kα, striving to understand the mechanism of PI3Kα activation by Ras. Point mutations T208D, K210E, and K227E disrupt the KRas4B-RBD interface in the models, in line with the experiments. We identify allosteric signaling pathways connecting Ras to RBD in the p110α subunit. However, the observed weak allosteric signals coupled with the detailed mechanism of PI3Kα activation make us conclude that the dominant mechanistic role of Ras is likely to be recruitment and restriction of the PI3Kα population at the membrane. Thus, RTK recruits the PI3Kα to the membrane and activates it by relieving its autoinhibition exerted by the nSH2 domain, leading to exposure of the kinase domain, which permits PIP2 binding. Ras recruitment can shift the PI3Kα ensemble toward a population where the kinase domain surface and the active site position and orientation favor PIP2 insertion. This work helps elucidate Ras-mediated PI3K activation and explores the structural basis for Ras-PI3Kα drug discovery.


Assuntos
Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Regulação Alostérica , Classe I de Fosfatidilinositol 3-Quinases/química , Classe I de Fosfatidilinositol 3-Quinases/genética , Ativação Enzimática , Humanos , Simulação de Dinâmica Molecular , Mutação Puntual , Ligação Proteica , Conformação Proteica em Folha beta , Domínios Proteicos , Proteínas Proto-Oncogênicas p21(ras)/química
18.
Virol J ; 16(1): 59, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31046787

RESUMO

BACKGROUND: Much evidence has demonstrated the influence of Hepatitis B virus (HBV) mutations on the clinical course of HBV infection. As large (L) protein plays a crucial role for viral entry, we hypothesized that mutations in the pre-S1 promoter region might affect the expression of L protein and subsequently change the biological characters of virus. METHODS: Patients infected with genotype C HBV were enrolled for analysis. HBV DNA sequences were inserted into a TA cloning vector and analyzed. To evaluate the effects of mutations in the pre-S1 promoter region, promoter activity and the expression of mRNA and L protein were analyzed using HepG2 cells. RESULTS: In total, 35 patients were enrolled and 13 patients (37.1%) had a single base substitution in the pre-S1 promoter region; the most frequent substitution was a G-to-A substitution at the 2765th base (G2765A) in the Sp1 region. The HBV viral load showed a negative correlation with the substitution ratio of the Sp1 region or G2765A (r = - 0.493 and - 0.473, respectively). Among those with a viral load ≤5.0 log IU/ml, patients with the G2765A substitution showed a significantly lower HBV viral load than those with the wild-type sequence. HepG2 cells transfected with the G2765A substitution vector showed reduced luciferase activity of the pre-S1 promoter, as well as reduced expression of pre-S1 mRNA and L protein. Furthermore, the G2765A substitution greatly reduced the L protein expression level of vector-produced virus particles. CONCLUSION: G2765A substitution in the pre-S1 promoter reduced the expression of L protein and resulted in a low viral load and less severe disease in chronic HBV infections.


Assuntos
Regulação Viral da Expressão Gênica , Vírus da Hepatite B/genética , Mutação Puntual , Regiões Promotoras Genéticas , Proteínas do Envelope Viral/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Substituição de Aminoácidos , Feminino , Hepatite B Crônica/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Carga Viral , Adulto Jovem
19.
Mol Med Rep ; 19(6): 4906-4918, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059089

RESUMO

The six members of the interleukin (IL)­17 gene family (IL­17A­F) have been identified in various types of cancer. Although lung cancer is the leading cause of cancer­related death worldwide and IL­17A was found to play a critical role in lung cancer, there is little knowledge concerning the association between the other five members of the IL­17 family and lung cancer. The genetic mutations and expression of IL­17 family members were investigated using the Catalogue of Somatic Mutations in Cancer (COSMIC), Oncomine, and cBio Cancer Genomics Portal (cBioPortal) databases. Prognostic values and interaction networks of the members were assessed by the Kaplan­Meier plotter, Search Tool for the Retrieval of Interacting Genes (STRING) database and FunRich software. The results found that, across 5,238 lung cancer patients in the cBioPortal, the results of IL­17 family gene alteration frequencies and types showed that IL­17A, IL­25 and IL­17F exhibited higher alteration frequencies (2, 2.1 and 1.9%, respectively), and gene amplification accounted for the majority of changes. IL­17B, IL­17C and IL­17D exhibited lower alteration frequencies (0.8, 1.1 and 1.1%, respectively), and deep deletion accounted for the majority of changes. The rates of point mutations in IL­17A through IL­17F family genes in lung cancer were 0.66, 0.18, 0.13, 0.09, 0.27 and 0.44% in the COSMIC database. Within the Oncomine database, five datasets showed that IL­17D was significantly decreased in lung cancer, while no dataset showed a significant difference in the expression of IL­17A, IL­17B, IL­17C, IL­25 or IL17­F between lung cancer and normal controls. The frequencies of IL­17A, IL­17B and IL­17C mRNA upregulation in lung squamous cell carcinoma were lower than those in lung adenocarcinoma (2.7, 1.9 and 2.1%, respectively), whereas the frequencies of IL­17D, IL­25 and IL­17F mRNA upregulation were higher in lung squamous cell carcinoma than those in lung adenocarcinoma (3, 6 and 6%, respectively). IL­17A and IL­17B were unrelated to overall survival (p=0.11; P=0.17), whereas IL­17C, IL­17D, IL­25 and IL­17F influenced prognosis (P=0.0023, P=0.0059, P=0.039 and P=0.0017, respectively) according to the Kaplan­Meier plotter. Moreover, the expression level of IL­17C was the highest in lung tissues, and IL­17 family genes mainly participate in the 'IFN­Î³ pathway' according to the STRING database and Funrich software. In conclusion, we performed the first comprehensive investigation of the IL­17 gene family in lung cancer, including gene mutation, mRNA expression levels, prognostic values and network pathways. Our results revealed that IL­17 family gene mutation rates were in general low and that amplification and deep deletion were the main mutation type. The expression and function of IL­17A and IL­17B in lung cancer are still not fully elucidated and warrant research with larger sample sizes. IL­17D was significantly decreased in lung cancer and was correlated with better OS. Studies of IL­17C­F in lung cancer are limited. Further experimental studies on the association between IL­17D and lung cancer progression are needed to identify more effective therapeutic targets for lung cancer.


Assuntos
Interleucina-17/genética , Neoplasias Pulmonares/patologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Bases de Dados Factuais , Deleção de Genes , Frequência do Gene , Genótipo , Humanos , Interleucina-17/metabolismo , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Mutação Puntual , Prognóstico , Mapas de Interação de Proteínas , Regulação para Cima
20.
BMC Genomics ; 20(1): 417, 2019 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-31126231

RESUMO

BACKGROUND: Mutations in the transcription factor, KLF1, are common within certain populations of the world. Heterozygous missense mutations in KLF1 mostly lead to benign phenotypes, but a heterozygous mutation in a DNA-binding residue (E325K in human) results in severe Congenital Dyserythropoietic Anemia type IV (CDA IV); i.e. an autosomal-dominant disorder characterized by neonatal hemolysis. RESULTS: To investigate the biochemical and genetic mechanism of CDA IV, we generated murine erythroid cell lines that harbor tamoxifen-inducible (ER™) versions of wild type and mutant KLF1 on a Klf1-/- genetic background. Nuclear translocation of wild type KLF1 results in terminal erythroid differentiation, whereas mutant KLF1 results in hemolysis without differentiation. The E to K variant binds poorly to the canonical 9 bp recognition motif (NGG-GYG-KGG) genome-wide but binds at high affinity to a corrupted motif (NGG-GRG-KGG). We confirmed altered DNA-binding specificity by quantitative in vitro binding assays of recombinant zinc-finger domains. Our results are consistent with previously reported structural data of KLF-DNA interactions. We employed 4sU-RNA-seq to show that a corrupted transcriptome is a direct consequence of aberrant DNA binding. CONCLUSIONS: Since all KLF/SP family proteins bind DNA in an identical fashion, these results are likely to be generally applicable to mutations in all family members. Importantly, they explain how certain mutations in the DNA-binding domain of transcription factors can generate neomorphic functions that result in autosomal dominant disease.


Assuntos
Anemia Diseritropoética Congênita/genética , DNA/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Mutação Puntual , Animais , Linhagem Celular , DNA/química , Regulação da Expressão Gênica , Camundongos , Motivos de Nucleotídeos , Ligação Proteica , Transcrição Genética
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