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1.
Arch Virol ; 164(11): 2823-2828, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31485748

RESUMO

A 278-bp region upstream of the beet curly top virus-SpCT (BCTV-SpCT) C2/C3 genes is necessary for promoter activity and exhibits significant sequence similarity to AL2/3 promoter sequences in tomato golden mosaic virus (TGMV). Maximal expression of the downstream C2/3 genes in BCTV-SpCT requires the presence of the C1 protein, which is supported by observations that mutation of the initiator codon for C1 results in decreased C2/C3 expression. This is similar to TGMV and cabbage leaf curl virus, where AL1 is required for maximal AL2/3 expression. Together, these data suggest a common strategy for complementary-sense gene regulation amongst curtoviruses and begomoviruses.


Assuntos
Begomovirus/genética , Geminiviridae/genética , Regulação Viral da Expressão Gênica/genética , Begomovirus/metabolismo , Sítios de Ligação/genética , Geminiviridae/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Virais/genética
2.
Mol Biol (Mosk) ; 53(4): 692-704, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31397443

RESUMO

miRNAs regulate the expression of many genes and are involved in the development of diseases. We studied miRNAs that interact partly or fully complementarily with the 5'UTR, CDS and 3'UTR of mRNAs of target genes. The MirTarget program used in this study allows for the discovery of miRNA binding sites (BS) in the entire nucleotide sequence of the mRNA and for determining the characteristics of the interactions of miRNAs with mRNAs. We identified five pairs of fully complementary BS for miR-127-5p and miR-127-3p, miR-136-5p and miR-136-3p, miR-431-5p and miR-431-3p, miR-432-5p and miR-432-3p, and miR-433-5p and miR-433-3p in the CDS of the human and animal mRNA of RTL1 gene. The fully complementary BS for miR-6720-5p, miR-6720-3p were identified in the CDS of the FOXF2 gene; BS for miR-3187-5p, miR-3187-3p were found in the CDS of the PLPPR3 gene; BS for miR-4665-5p, miR-4665-3p were found in the 5'UTR of the KIAA2026 gene; BS for miR-135a-5p, miR-135a-3p were found in the 3'UTR of the GLYCTK gene; BS for miR-7106-5p, miR-7106-3p were found in the 3'UTR of the CCDC42B gene. The miRNA-5p and miRNA-3p associated with the RTL1 gene have BS in the mRNAs of 32 target human genes. The miRNA-5p and miRNA-3p associated with the FOXF2, PLPPR3, KIAA2026, GLYCTK and CCDC42B genes have BS in the mRNAs of 27 target genes, involved in development of several diseases. Nucleotide sequences of miRNA-5p and miRNA-3p and BS are conserved over tens of millions of years of divergence of the studied animal species. Binding characteristics of miR-3120-3p and miR-3120-5p, miR-196b-3p and miR-196b-5p, miR-125a-3p and miR-125a-3p, let-7e-3p and let-7e-5p, miR-99b-3p in fully complementary BS of non-coding DMN3OS, HOXA10-AS, SPACA6P-AS genes have been established.


Assuntos
Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , Sítios de Ligação/genética , Humanos
3.
Comput Biol Chem ; 80: 270-277, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31054539

RESUMO

Panomycocin is a naturally produced potent antimycotic/antifungal protein secreted by the yeast Wickerhamomyces anomalus NCYC 434 with an exo-ß-1,3-glucanase activity. In this study the three dimensional structure of panomycocin was predicted and the computational site-directed mutagenesis was performed to enhance its thermal stability in liquid formulations over the body temperature for topical therapeutic applications. Homology modeling was performed with MODELLER and I-TASSER. Among the generated models, the model with the lowest energy and DOPE score was selected for further loop modeling. The loop model was optimized and the reliability of the model was confirmed with ERRAT, Verify 3D and Ramachandran plot values. Enhancement of the thermal stability of the model was done using contemporary servers and programs such as SPDBViewer, CNA, I-Mutant2.0, Eris, AUTO-MUTE and MUpro. In the region outside the binding site of the model Leu52 Arg, Phe223Arg and Gly254Arg were found to be the best thermostabilizing mutations with 6.26 K, 6.26 K and 8.27 K increases, respectively. In the binding site Glu186Arg was found to be the best thermostabilizer mutation with a 9.58 K temperature increase. The results obtained in this study led us to design a mutant panomycocin that can be used as a novel antimycotic/antifungal drug in a liquid formulation for topical applications over the normal body temperature.


Assuntos
Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Micotoxinas/química , Micotoxinas/genética , Pichia/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida/métodos , Mutação , Estabilidade Proteica , Estrutura Terciária de Proteína , Temperatura Ambiente
4.
Biochim Biophys Acta Rev Cancer ; 1872(1): 11-23, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31034924

RESUMO

The ubiquitous family of AP-1 dimeric transcription complexes is involved in virtually all cellular and physiological functions. It is paramount for cells to reprogram gene expression in response to cues of many sorts and is involved in many tumorigenic processes. How AP-1 controls gene transcription has largely remained elusive till recently. The advent of the "omics" technologies permitting genome-wide studies of transcription factors has however changed and improved our view of AP-1 mechanistical actions. If these studies confirm that AP-1 can sometimes act as a local transcriptional switch operating in the vicinity of transcription start sites (TSS), they strikingly indicate that AP-1 principally operates as a remote command binding to distal enhancers, placing chromatin architecture dynamics at the heart of its transcriptional actions. They also unveil novel constraints operating on AP-1, as well as novel mechanisms used to regulate gene expression via transcription-pioneering-, chromatin-remodeling- and chromatin accessibility maintenance effects.


Assuntos
Complexos Multiproteicos/genética , Fator de Transcrição AP-1/genética , Transcrição Genética , Ativação Transcricional/genética , Sítios de Ligação/genética , Núcleo Celular/genética , Montagem e Desmontagem da Cromatina/genética , Humanos , Complexos Multiproteicos/química , Fator de Transcrição AP-1/química , Sítio de Iniciação de Transcrição
5.
In Vitro Cell Dev Biol Anim ; 55(5): 323-330, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30945114

RESUMO

Insulin-like growth factor-1 (IGF-1) is a functional candidate gene for pig growth and development due to its crucial role in the growth axis of growth hormone-IGF-1. Considering that the 3' untranslated region (3'UTR) of gene may affect its expression, we analyzed the effect of a single-nucleotide polymorphism (SNP) (rs34142920, c.674C > T) on gene expression, cell proliferation, and apoptosis and the possible related molecular mechanisms in PK-15 cells. The SNP was found in the 3'UTR of IGF-1 in Bama Xiang pig in previous investigations. Results showed that the SNP was located at the target site binding to microRNA (miR-511). The 3'UTR of IGF-1 gene with C allele significantly downregulated the expression of IGF-1 gene compared with that of the gene with T allele by luciferase assay. miR-511 was transfected into porcine kidney cell line (PK-15 cells) to reveal its effects on cells and whether or not it targets IGF-1. The expression levels of IGF-1 at mRNA and protein levels were remarkably downregulated. miR-511 significantly inhibited cell proliferation and promoted cell apoptosis by downregulating the phosphorylation level of AKT and ERK1/2. This finding confirmed that miR-511 inhibits proliferation and promotes apoptosis by downregulating the IGF-1 in PK-15 cells.


Assuntos
Apoptose/genética , Proliferação de Células/genética , Fator de Crescimento Insulin-Like I/genética , MicroRNAs/genética , Regiões 3' não Traduzidas/genética , Animais , Sítios de Ligação/genética , Linhagem Celular , Regulação da Expressão Gênica/genética , Humanos , Rim/citologia , Rim/metabolismo , Luciferases/genética , Sistema de Sinalização das MAP Quinases/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Mensageiro/genética , Suínos , Transfecção
6.
Nat Genet ; 51(5): 777-785, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30988513

RESUMO

BMP/SMAD signaling is a crucial regulator of intestinal differentiation1-4. However, the molecular underpinnings of the BMP pathway in this context are unknown. Here, we characterize the mechanism by which BMP/SMAD signaling drives enterocyte differentiation. We establish that the transcription factor HNF4A acts redundantly with an intestine-restricted HNF4 paralog, HNF4G, to activate enhancer chromatin and upregulate the majority of transcripts enriched in the differentiated epithelium; cells fail to differentiate on double knockout of both HNF4 paralogs. Furthermore, we show that SMAD4 and HNF4 function via a reinforcing feed-forward loop, activating each other's expression and co-binding to regulatory elements of differentiation genes. This feed-forward regulatory module promotes and stabilizes enterocyte cell identity; disruption of the HNF4-SMAD4 module results in loss of enterocyte fate in favor of progenitor and secretory cell lineages. This intersection of signaling and transcriptional control provides a framework to understand regenerative tissue homeostasis, particularly in tissues with inherent cellular plasticity5.


Assuntos
Enterócitos/citologia , Enterócitos/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Proteína Smad4/metabolismo , Animais , Sítios de Ligação/genética , Células CACO-2 , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Elementos Facilitadores Genéticos , Fator 4 Nuclear de Hepatócito/deficiência , Fator 4 Nuclear de Hepatócito/genética , Humanos , Camundongos , Camundongos Knockout , Transdução de Sinais , Proteína Smad4/deficiência , Proteína Smad4/genética
7.
Nat Commun ; 10(1): 1669, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30971696

RESUMO

Fungal cell wall synthesis is achieved by a balance of glycosyltransferase, hydrolase and transglycosylase activities. Transglycosylases strengthen the cell wall by forming a rigid network of crosslinks through mechanisms that remain to be explored. Here we study the function of the Aspergillus fumigatus family of five Crh transglycosylases. Although crh genes are dispensable for cell viability, simultaneous deletion of all genes renders cells sensitive to cell wall interfering compounds. In vitro biochemical assays and localisation studies demonstrate that this family of enzymes functions redundantly as transglycosylases for both chitin-glucan and chitin-chitin cell wall crosslinks. To understand the molecular basis of this acceptor promiscuity, we solved the crystal structure of A. fumigatus Crh5 (AfCrh5) in complex with a chitooligosaccharide at the resolution of 2.8 Å, revealing an extensive elongated binding cleft for the donor (-4 to -1) substrate and a short acceptor (+1 to +2) binding site. Together with mutagenesis, the structure suggests a "hydrolysis product assisted" molecular mechanism favouring transglycosylation over hydrolysis.


Assuntos
Aspergillus fumigatus/metabolismo , Proteínas Fúngicas/metabolismo , Glicosiltransferases/metabolismo , Sítios de Ligação/genética , Parede Celular/metabolismo , Quitina/metabolismo , Cristalografia por Raios X , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Técnicas de Silenciamento de Genes , Glicosiltransferases/química , Glicosiltransferases/genética , Mutagênese Sítio-Dirigida , Domínios Proteicos/genética , Especificidade por Substrato , beta-Glucanas/metabolismo
8.
PLoS Genet ; 15(4): e1008026, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30933976

RESUMO

Phosphorothioate (PT) modifications of the DNA backbone, widespread in prokaryotes, are first identified in bacterial enteropathogens Escherichia coli B7A more than a decade ago. However, methods for high resolution mapping of PT modification level are still lacking. Here, we developed the PT-IC-seq technique, based on iodine-induced selective cleavage at PT sites and high-throughput next generation sequencing, as a mean to quantitatively characterizing the genomic landscape of PT modifications. Using PT-IC-seq we foud that most PT sites are partially modified at a lower PT frequency (< 5%) in E. coli B7A and Salmonella enterica serovar Cerro 87, and both show a heterogeneity pattern of PT modification similar to those of the typical methylation modification. Combining the iodine-induced cleavage and absolute quantification by droplet digital PCR, we developed the PT-IC-ddPCR technique to further measure the PT modification level. Consistent with the PT-IC-seq measurements, PT-IC-ddPCR analysis confirmed the lower PT frequency in E. coli B7A. Our study has demonstrated the heterogeneity of PT modification in the bacterial population and we also established general tools for rigorous mapping and characterization of PT modification events at whole genome level. We describe to our knowledge the first genome-wide quantitative characterization of PT landscape and provides appropriate strategies for further functional studies of PT modification.


Assuntos
DNA Bacteriano/química , DNA Bacteriano/metabolismo , Fosfatos/metabolismo , Sequência de Bases , Sítios de Ligação/genética , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Iodo , Reação em Cadeia da Polimerase , Salmonella enterica/genética , Salmonella enterica/metabolismo , Análise de Sequência de DNA , Espectrometria de Massas em Tandem
9.
PLoS Genet ; 15(4): e1008068, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30969965

RESUMO

The roles of histone demethylation in the regulation of plant flowering, disease resistance, rhythmical response, and seed germination have been elucidated recently; however, how histone demethylation affects leaf senescence remains largely unclear. In this study, we exploited yeast one-hybrid (Y1H) to screen for the upstream regulators of NONYELLOWING1 (NYE1), and identified RELATIVE OF EARLY FLOWERING6 (REF6), a histone H3 lysine 27 tri-methylation (H3K27me3) demethylase, as a putative binding protein of NYE1 promoter. By in vivo and in vitro analyses, we demonstrated that REF6 directly binds to the motif CTCGYTY in NYE1/2 promoters through its zinc finger domain and positively regulates their expression. Loss-of-function of REF6 delayed chlorophyll (Chl) degradation, whereas overexpression of REF6 accelerated Chl degradation. Subsequently, we revealed that REF6 positively regulates the general senescence process by directly up-regulating ETHYLENE INSENSITIVE 2 (EIN2), ORESARA1 (ORE1), NAC-LIKE, ACTIVATED BY AP3/PI (NAP), PYRUVATE ORTHOPHOSPHATE DIKINASE (PPDK), PHYTOALEXIN DEFICIENT 4 (PAD4), LIPOXYGENASE 1 (LOX1), NAC DOMAIN CONTAINING PROTEIN 3 (AtNAC3), and NAC TRANSCRIPTION FACTOR-LIKE 9 (NTL9), the key regulatory and functional genes predominantly involved in the regulation of developmental leaf senescence. Importantly, loss-of-function of REF6 increased H3K27me3 levels at all the target Senescence associated genes (SAGs). We therefore conclusively demonstrate that H3K27me3 methylation represents an epigenetic mechanism prohibiting the premature transcriptional activation of key developmentally up-regulated senescence regulatory as well as functional genes in Arabidopsis.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Arabidopsis/crescimento & desenvolvimento , Sítios de Ligação/genética , Clorofila/metabolismo , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Genes Reguladores , Modelos Genéticos , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas
10.
Nat Commun ; 10(1): 1832, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015432

RESUMO

Ceramides draw wide attention as tumor suppressor lipids that act directly on mitochondria to trigger apoptotic cell death. However, molecular details of the underlying mechanism are largely unknown. Using a photoactivatable ceramide probe, we here identify the voltage-dependent anion channels VDAC1 and VDAC2 as mitochondrial ceramide binding proteins. Coarse-grain molecular dynamics simulations reveal that both channels harbor a ceramide binding site on one side of the barrel wall. This site includes a membrane-buried glutamate that mediates direct contact with the ceramide head group. Substitution or chemical modification of this residue abolishes photolabeling of both channels with the ceramide probe. Unlike VDAC1 removal, loss of VDAC2 or replacing its membrane-facing glutamate with glutamine renders human colon cancer cells largely resistant to ceramide-induced apoptosis. Collectively, our data support a role of VDAC2 as direct effector of ceramide-mediated cell death, providing a molecular framework for how ceramides exert their anti-neoplastic activity.


Assuntos
Apoptose , Ceramidas/metabolismo , Mitocôndrias/fisiologia , Canal de Ânion 2 Dependente de Voltagem/metabolismo , Sítios de Ligação/genética , Ceramidas/química , Técnicas de Inativação de Genes , Ácido Glutâmico/química , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Simulação de Dinâmica Molecular , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/genética , Canal de Ânion 1 Dependente de Voltagem/isolamento & purificação , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Canal de Ânion 2 Dependente de Voltagem/química , Canal de Ânion 2 Dependente de Voltagem/genética , Canal de Ânion 2 Dependente de Voltagem/isolamento & purificação
11.
Cancer Sci ; 110(6): 1974-1986, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31012976

RESUMO

We previously found that circulating ß2 -glycoprotein I inhibits human endothelial cell migration, proliferation, and angiogenesis by diverse mechanisms. In the present study, we investigated the antitumor activities of ß2 -glycoprotein I using structure-function analysis and mapped the critical region within the ß2 -glycoprotein I peptide sequence that mediates anticancer effects. We constructed recombinant cDNA and purified different ß2 -glycoprotein I polypeptide domains using a baculovirus expression system. We found that purified ß2 -glycoprotein I, as well as recombinant ß2 -glycoprotein I full-length (D12345), polypeptide domains I-IV (D1234), and polypeptide domain I (D1) significantly inhibited melanoma cell migration, proliferation and invasion. Western blot analyses were used to determine the dysregulated expression of proteins essential for intracellular signaling pathways in B16-F10 treated with ß2 -glycoprotein I and variant recombinant polypeptides. Using a melanoma mouse model, we found that D1 polypeptide showed stronger potency in suppressing tumor growth. Structural analysis showed that fragments A and B within domain I would be the critical regions responsible for antitumor activity. Annexin A2 was identified as the counterpart molecule for ß2 -glycoprotein I by immunofluorescence and coimmunoprecipitation assays. Interaction between specific amino acids of ß2 -glycoprotein I D1 and annexin A2 was later evaluated by the molecular docking approach. Moreover, five amino acid residues were selected from fragments A and B for functional evaluation using site-directed mutagenesis, and P11A, M42A, and I55P mutations were shown to disrupt the anti-melanoma cell migration ability of ß2 -glycoprotein I. This is the first study to show the therapeutic potential of ß2 -glycoprotein I D1 in the treatment of melanoma progression.


Assuntos
Movimento Celular/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Peptídeos/farmacologia , beta 2-Glicoproteína I/química , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Linhagem Celular Tumoral , Masculino , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Peptídeos/química , Peptídeos/metabolismo , Domínios Proteicos , Homologia de Sequência de Aminoácidos , beta 2-Glicoproteína I/genética , beta 2-Glicoproteína I/metabolismo
12.
BMC Bioinformatics ; 20(Suppl 4): 119, 2019 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-30999858

RESUMO

BACKGROUND: The search for molecular biomarkers of early-onset colorectal cancer (CRC) is an important but still quite challenging and unsolved task. Detection of CpG methylation in human DNA obtained from blood or stool has been proposed as a promising approach to a noninvasive early diagnosis of CRC. Thousands of abnormally methylated CpG positions in CRC genomes are often located in non-coding parts of genes. Novel bioinformatic methods are thus urgently needed for multi-omics data analysis to reveal causative biomarkers with a potential driver role in early stages of cancer. METHODS: We have developed a method for finding potential causal relationships between epigenetic changes (DNA methylations) in gene regulatory regions that affect transcription factor binding sites (TFBS) and gene expression changes. This method also considers the topology of the involved signal transduction pathways and searches for positive feedback loops that may cause the carcinogenic aberrations in gene expression. We call this method "Walking pathways", since it searches for potential rewiring mechanisms in cancer pathways due to dynamic changes in the DNA methylation status of important gene regulatory regions ("epigenomic walking"). RESULTS: In this paper, we analysed an extensive collection of full genome gene-expression data (RNA-seq) and DNA methylation data of genomic CpG islands (using Illumina methylation arrays) generated from a sample of tumor and normal gut epithelial tissues of 300 patients with colorectal cancer (at different stages of the disease) (data generated in the EU-supported SysCol project). Identification of potential epigenetic biomarkers of DNA methylation was performed using the fully automatic multi-omics analysis web service "My Genome Enhancer" (MGE) (my-genome-enhancer.com). MGE uses the database on gene regulation TRANSFAC®, the signal transduction pathways database TRANSPATH®, and software that employs AI (artificial intelligence) methods for the analysis of cancer-specific enhancers. CONCLUSIONS: The identified biomarkers underwent experimental testing on an independent set of blood samples from patients with colorectal cancer. As a result, using advanced methods of statistics and machine learning, a minimum set of 6 biomarkers was selected, which together achieve the best cancer detection potential. The markers include hypermethylated positions in regulatory regions of the following genes: CALCA, ENO1, MYC, PDX1, TCF7, ZNF43.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Metilação de DNA/genética , Retroalimentação Fisiológica , Transdução de Sinais/genética , Sítios de Ligação/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/patologia , Ilhas de CpG/genética , Epigênese Genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Fatores de Transcrição/metabolismo
13.
Mar Drugs ; 17(3)2019 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-30823399

RESUMO

α-Conotoxin RgIA is a selective and potent competitive antagonist of rat α9α10 nicotinic acetylcholine receptors (nAChR), but it is much less potent towards human α9α10 nAChR. Furthermore, RgIA is susceptible to proteolytic degradation due to containing four arginine residues. These disadvantages greatly limit its use for clinical applications. The purpose of this research was to identify critical stereocenters of RgIA and discover more stable analogues, enhancing its bioavailability by using the d-amino acid scan method. The activity of each variant was investigated against rat and human α9α10 nAChRs, which were expressed in Xenopus oocytes. Experimental assays showed that 14 out of 15 analogues had a substantial reduction in potency towards rat α9α10 nAChR. Noticeably, analogue 13 retained full biological activity compared with RgIA. Meanwhile, two other analogues, 14 and 15, of which l-Args were substituted with d-Args, exhibited a significantly increased potency towards human α9α10 nAChR, although these analogues showed decreased activities against rat α9α10 nAChR. Additionally, these three analogues exhibited a high resistance against enzymatic degradation in human serum and simulated intestinal fluid (SIF). Collectively, our findings suggest that a d-amino acid scan is a useful strategy for investigating how the side-chain chirality of amino acids affects the structure and function of peptides and may facilitate the development of more stable analogues to increase therapeutic potential.


Assuntos
Substituição de Aminoácidos , Conotoxinas/química , Antagonistas Nicotínicos/química , Receptores Nicotínicos/metabolismo , Sequência de Aminoácidos/genética , Animais , Sítios de Ligação/genética , Conotoxinas/genética , Estabilidade Enzimática/genética , Humanos , Modelos Moleculares , Oócitos , Receptores Nicotínicos/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Xenopus
14.
Nat Biotechnol ; 37(4): 430-435, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30833658

RESUMO

Adenine base editors1 enable efficient targeted adenine-to-guanine single nucleotide conversions to induce or correct point mutations in human cells, animals, and plants1-4. Here we present a modified version of Digenome-seq, an in vitro method for identifying CRISPR (clustered regularly interspaced short palindromic repeats)-induced double-strand breaks using whole-genome sequencing5-8, to assess genome-wide target specificity of adenine base editors. To produce double-strand breaks at sites containing inosines, the products of adenine deamination, we treat human genomic DNA with an adenine base editor 7.10 protein-guide RNA complex and either endonuclease V or a combination of human alkyladenine DNA glycosylase and endonuclease VIII in vitro. Digenome-seq detects adenine base editor off-target sites with a substitution frequency of 0.1% or more. We show that adenine base editor 7.10, the cytosine base editor BE3, and unmodified CRISPR-associated protein 9 (Cas9) often recognize different off-target sites, highlighting the need for independent assessments of their genome-wide specificities6. Using targeted sequencing, we also show that use of preassembled adenine base editor ribonucleoproteins, modified guide RNAs5,8-11, and Sniper/Cas9 (ref. 12) reduces adenine base editor off-target activity in human cells.


Assuntos
Adenina/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , RNA Guia/genética , Aminoidrolases/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Biotecnologia , Proteína 9 Associada à CRISPR/metabolismo , Citosina Desaminase/metabolismo , Quebras de DNA de Cadeia Dupla , Células HEK293 , Humanos , RNA Guia/metabolismo , Sequenciamento Completo do Genoma
15.
Comput Biol Chem ; 80: 31-45, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30884445

RESUMO

BMPR1A (BMP type 1 receptor) is a transmembrane cell-surface receptor also known as ALK3 (activin-like kinases-3) encodes for a type I serine/threonine kinase receptor and a member of the transforming growth-factor ß-receptor (TGF-ß) super family. The BMPR1A has a significant interaction with BMP-2 for protein activity and also has a low affinity with growth and differentiation factor 5 (GDF5); positively regulates chondrocyte differentiation. The genetic variations can alter the structure and function of the BMPR1A gene that causes several diseases such as juvenile polyposis syndrome or hereditary cancer-predisposing syndrome. The current study was carried out to identify potential deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) in BMPR1A by implementing different computational algorithms such as SIFT, PolyPhen2, SNAP2, PROVEAN, PhD-SNP, SNPs&GO, nsSNPAnalyzer, and P-Mut. From 205 nsSNPs in BMPR1A, 7 nsSNPs (C76Y, C124R, C124Y, C376Y, R443C, R480W, and W487R) were predicted as deleterious in 8 prediction algorithms. The Consurf analysis showed that selected 7 nsSNPs were present in the highly conserved regions. Molecular dynamics simulation analysis also performed to explore conformational changes in the variant structure with respect to its native structure. According to the MDS result, all variants flexibility and rigidity were unbalanced, which may alter the structural and functional behavior of the native protein. Although, three nsSNPs i.e., C124R, C376Y, and R443C have already been reported in patients associated with JPS, but their structural and functional molecular studies remain uncharacterized. Therefore, the findings of this study can provide a better understanding of uncharacterized nsSNPS and to find their association with disease susceptibility and also facilitate to the researchers for designing or developing the target dependent drugs.


Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Polimorfismo de Nucleotídeo Único , Algoritmos , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação/genética , Biologia Computacional/métodos , Humanos , Ligações de Hidrogênio , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Conformação Proteica em alfa-Hélice/genética , Estabilidade Proteica , Software
16.
Nat Commun ; 10(1): 1189, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30867425

RESUMO

In jawed vertebrates (gnathostomes), Hox genes play an important role in patterning head and jaw formation, but mechanisms coupling Hox genes to neural crest (NC) are unknown. Here we use cross-species regulatory comparisons between gnathostomes and lamprey, a jawless extant vertebrate, to investigate conserved ancestral mechanisms regulating Hox2 genes in NC. Gnathostome Hoxa2 and Hoxb2 NC enhancers mediate equivalent NC expression in lamprey and gnathostomes, revealing ancient conservation of Hox upstream regulatory components in NC. In characterizing a lamprey hoxα2 NC/hindbrain enhancer, we identify essential Meis, Pbx, and Hox binding sites that are functionally conserved within Hoxa2/Hoxb2 NC enhancers. This suggests that the lamprey hoxα2 enhancer retains ancestral activity and that Hoxa2/Hoxb2 NC enhancers are ancient paralogues, which diverged in hindbrain and NC activities. This identifies an ancestral mechanism for Hox2 NC regulation involving a Hox-TALE regulatory circuit, potentiated by inputs from Meis and Pbx proteins and Hox auto-/cross-regulatory interactions.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genes Homeobox/fisiologia , Proteínas de Homeodomínio/metabolismo , Crista Neural/embriologia , Vertebrados/genética , Animais , Animais Geneticamente Modificados , Sítios de Ligação/genética , Linhagem Celular , Sequência Conservada/fisiologia , Elementos Facilitadores Genéticos/genética , Proteínas de Homeodomínio/genética , Lampreias , Camundongos , Células-Tronco Embrionárias Murinas , Crista Neural/metabolismo , Alinhamento de Sequência , Vertebrados/embriologia , Peixe-Zebra
17.
Gene ; 699: 1-7, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-30853631

RESUMO

Gonadotropin-releasing hormone (GnRH) plays an important role in regulating the activities of other components downstream of the hypothalamic-pituitary-gonadal (HPG) axis and maintaining the normal reproductive cycle of animals. However, the molecular mechanisms by which GnRH synthesis and secretion are regulated in sheep remains unclear. In this study, a series of eight recombinant vectors with deletion fragments were constructed and cotransfected with pGL3-Basic and pRL-SV40 into sheep hypothalamic neuronal cells. After treatment with 1 nM kisspeptin, the core promoter of the sheep GnRH gene was identified to be in the region of -1912 bp to -1461 bp by dual-luciferase reporter assay. Bioinformatics analysis showed that there was a binding site for the transcription factor Otx-2 in the core promoter region (-1786 to -1770 bp) that was highly conserved among different species. The expression patterns of Kiss-1, Otx-2 and GnRH in the sheep hypothalamus were the same, and the expression of Kiss-1, Otx-2 and GnRH was significantly higher in the breeding season than in nonbreeding season (P < 0.01). In addition, when hypothalamic neurons were cultured in vitro with kisspeptin, kisspeptin induced the expression of GnRH and Otx-2. In conclusion, these results provide evidence that the core promoter region (-1786 to -1770 bp) of the GnRH gene is involved in the regulation of hypothalamic activity by kisspeptin and that binding of the transcription factor Otx-2 mediates this activation.


Assuntos
Expressão Gênica/genética , Hormônio Liberador de Gonadotropina/genética , Kisspeptinas/genética , Regiões Promotoras Genéticas/genética , Ovinos/genética , Animais , Sítios de Ligação/genética , Cruzamento/métodos , Hipotálamo/fisiologia , Neurônios/fisiologia , Fatores de Transcrição Otx/genética , Reprodução/genética
18.
PLoS Comput Biol ; 15(3): e1006921, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30897079

RESUMO

ChIP-seq (Chromatin Immunoprecipitation followed by sequencing) is a high-throughput technique to identify genomic regions that are bound in vivo by a particular protein, e.g., a transcription factor (TF). Biological factors, such as chromatin state, indirect and cooperative binding, as well as experimental factors, such as antibody quality, cross-linking, and PCR biases, are known to affect the outcome of ChIP-seq experiments. However, the relative impact of these factors on inferences made from ChIP-seq data is not entirely clear. Here, via a detailed ChIP-seq simulation pipeline, ChIPulate, we assess the impact of various biological and experimental sources of variation on several outcomes of a ChIP-seq experiment, viz., the recoverability of the TF binding motif, accuracy of TF-DNA binding detection, the sensitivity of inferred TF-DNA binding strength, and number of replicates needed to confidently infer binding strength. We find that the TF motif can be recovered despite poor and non-uniform extraction and PCR amplification efficiencies. The recovery of the motif is, however, affected to a larger extent by the fraction of sites that are either cooperatively or indirectly bound. Importantly, our simulations reveal that the number of ChIP-seq replicates needed to accurately measure in vivo occupancy at high-affinity sites is larger than the recommended community standards. Our results establish statistical limits on the accuracy of inferences of protein-DNA binding from ChIP-seq and suggest that increasing the mean extraction efficiency, rather than amplification efficiency, would better improve sensitivity. The source code and instructions for running ChIPulate can be found at https://github.com/vishakad/chipulate.


Assuntos
Imunoprecipitação da Cromatina/métodos , Biologia Computacional/métodos , Análise de Sequência de DNA/métodos , Software , Fatores de Transcrição , Sítios de Ligação/genética , Simulação por Computador , DNA/química , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Ligação Proteica/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
19.
Enzyme Microb Technol ; 125: 29-36, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30885322

RESUMO

ChKRED20 is a robust NADH-dependent ketoreductase identified from the genome of Chryseobacterium sp. CA49 that can use 2-propanol as the ultimate reducing agent. The wild-type can reduce over 100 g/l ketones for some pharmaceutical relevant substrates, exhibiting a remarkable potential for industrial application. In this work, to overcome the limitation of ChKRED20 to aryl ketoesters, we first refined the X-ray crystal structure of ChKRED20/NAD+ complex at a resolution of 1.6 Å, and then performed three rounds of iterative saturation mutagenesis at critical amino acid sites to reshape the active cavity of the enzyme. For methyl 2-oxo-2-phenylacetate and ethyl 3-oxo-3-phenylpropanoate, several gain-of-activity mutants were achieved, and for ethyl 2-oxo-4-phenylbutanoate, improved mutants were achieved with kcat/Km increasing to 196-fold of the wild-type. All three substrates were completely reduced at 100 g/l loading catalyzed with selected ChKRED20 mutants, and deliver the corresponding chiral alcohols with >90% isolated yield and 97 - >99%ee.


Assuntos
Oxirredutases do Álcool/química , Oxirredutases do Álcool/metabolismo , Chryseobacterium/enzimologia , Cetonas/metabolismo , Oxirredutases do Álcool/genética , Álcoois/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Biocatálise , Chryseobacterium/genética , Cristalografia por Raios X , Mutação com Ganho de Função , Cetonas/química , Cinética , Simulação de Acoplamento Molecular , Estrutura Molecular , Mutagênese Sítio-Dirigida , Engenharia de Proteínas , Relação Estrutura-Atividade
20.
Genes Genomics ; 41(5): 557-566, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30796706

RESUMO

BACKGROUND: Inflammation in the central nervous system is closely associated with pathological neurodegenerative diseases as well as psychiatric disorders. Prolonged activation of microglia can produce many inflammatory mediators, which may result in pathological neurotoxic side effects. Interleukin (IL)-6 serves as a hallmark of the injured brain. OBJECTIVE: Whole grains are known to contain many bioactive components. However, little information is available about anti-neuroinflammatory effects of grains in the CNS. This study aims to investigate the effect of Hordeum vulgare ethanol extract (HVE) on the suppression of IL-6 expression in BV2 microglia. METHODS: Inhibitory effects of HVE on IL-6 expression were analyzed by immunoblot anaysis, immunofluoresce microscopic analysis, reverse transcription-polymerase chain reaction, and luciferase promoter reporter assay. RESULTS: HVE inhibited TNFα-induced phosphorylation of IKKα/ß, IκB, and p65/RelA NF-κB. TNFα-induced IL-6 mRNA expression and promoter activity were reduced by HVE. Point mutation of NF-κB-binding site within the IL-6 gene promoter abolished TNFα-induced reporter activity, whereas exogenous expression of p65 NF-κB enhanced IL-6 promoter activity. CONCLUSION: NF-κB-binding site within the IL-6 promoter region is a HVE target element involved in the inhibition of TNFα-induced IL-6 gene transcription. HVE inhibits TNFα-induced IL-6 expression via suppression of NF-κB signaling in BV2 microglial cells.


Assuntos
Hordeum/metabolismo , Interleucina-6/antagonistas & inibidores , Microglia/efeitos dos fármacos , Animais , Sítios de Ligação/genética , Linhagem Celular , Grão Comestível/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hordeum/fisiologia , Quinase I-kappa B/metabolismo , Proteínas I-kappa B/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Fosforilação/efeitos dos fármacos , Extratos Vegetais/farmacologia , Regiões Promotoras Genéticas/genética , Ratos , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/fisiologia
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