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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.
Anticancer Res ; 39(8): 4129-4136, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31366497

RESUMO

BACKGROUND/AIM: 5-Aza-2-deoxycytidine (5-Aza-CdR) enhances the sensitivity to 5-fluorouracil (5-FU), but the molecular mechanism is not fully understood. The aim of this study was to investigate the molecular mechanism that enhances the sensitivity to 5-FU treated with 5-Aza-CdR via thymidine phosphorylase (TP). MATERIALS AND METHODS: The sensitivity to drugs was determined on several cancer cell lines by the MTT assay. Protein and mRNA levels were examined by immunoblot and RT-PCR, respectively. Gene silencing, binding of Sp1 to DNA and methylation of DNA was performed by siRNA, ChIP assay and sodium bisulfate genomic sequencing, respectively. RESULTS: Sp1-binding sites in the TP promoter were methylated in epidermoid carcinoma. 5-Aza-CdR demethylated Sp1-binding sites and enhanced sensitivity to 5-FU. CONCLUSION: Demethylation of Sp1-binding sites by 5-Aza-CdR was a key factor enhancing 5-FU sensitivity, which may enable more effective treatments for cancer patients with the combination of 5-Aza-CdR and 5-FU.


Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Metilação de DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Fator de Transcrição Sp1/genética , Timidina Fosforilase/genética , Sítios de Ligação/efeitos dos fármacos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Decitabina/metabolismo , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/genética , Timidina Fosforilase/química
3.
J Chem Theory Comput ; 15(9): 4974-4981, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31402652

RESUMO

Predicting the costructure of small-molecule ligands and their respective target proteins has been a long-standing problem in drug discovery. For weak binding compounds typically identified in fragment-based screening (FBS) campaigns, determination of the correct binding site and correct binding mode is usually done experimentally via X-ray crystallography. For many targets of pharmaceutical interest, however, establishing an X-ray system which allows for sufficient throughput to support a drug discovery project is not possible. In this case, exploration of fragment hits becomes a very laborious and consequently slow process with the generation of protein/ligand cocrystal structures as the bottleneck of the entire process. In this work, we introduce a computational method which is able to reliably predict binding sites and binding modes of fragment-like small molecules using solely the structure of the apoprotein and the ligand's chemical structure as input information. The method is based on molecular dynamics simulations and Markov-state models and can be run as a fully automated protocol requiring minimal human intervention. We describe the application of the method to a representative subset of different target classes and fragments from historical FBS efforts at Boehringer Ingelheim and discuss its potential integration into the overall fragment-based drug discovery workflow.


Assuntos
Cadeias de Markov , Simulação de Dinâmica Molecular , Proteínas/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Ligantes
4.
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
5.
Sheng Wu Gong Cheng Xue Bao ; 35(8): 1469-1477, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31441618

RESUMO

The aim of this study was to screen the active regions and transcription factor binding sites in the promoter of the CBD103 gene related to Arctic fox coat color, and to provide a basis for revealing the molecular genetic mechanism of CBD103 gene regulating the coat color formation. The 5'-flanking region fragment 2 123 bp of Arctic fox CBD103 gene was cloned, and 4 truncated promoter reporter vectors of different lengths were constructed. The promoter activity was detected by the dual-luciferase reporter assay system. Point mutations were performed on the 3 predicted specificity protein 1 (Sp1) transcription factor binding sites in the highest promoter active region, and 3 mutant vectors were constructed. The activity was then detected by the dual-luciferase reporter assay system. The results showed that the region 1 656 (-1 604/+51) had the highest activity in the 4 truncated promoters of different lengths, and the promoter activity of the three mutant vectors constructed in this region were significantly lower than that of the wild type (fragment 1 656). The region of -1 604 /+51 was the core promoter region of CBD103 gene in Arctic fox and -1 552/-1 564, -1 439/-1 454 and -329/-339 regions were positive regulatory regions. This study successfully obtained the core promoter region and positive regulation regions of the Arctic fox CBD103 gene, which laid a foundation for further study on the molecular genetic mechanism of this gene regulating Arctic fox coat color.


Assuntos
Regiões Promotoras Genéticas , Animais , Sítios de Ligação , Raposas , Luciferases , Fator de Transcrição Sp1 , beta-Defensinas
6.
Inorg Chem ; 58(17): 11782-11792, 2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31433630

RESUMO

Reproduction of the dominant vector of Zika and dengue diseases, Aedes aegypti mosquito, is controlled by an active heterodimer complex composed of the 20-hydroxyecdysone receptor (EcR) and ultraspiracle protein. Although A. aegypti EcR shares the structural and functional organization with other nuclear receptors, its C-terminus has an additional long F domain (AaFEcR). Recently, we showed that the full length AaFEcR is intrinsically disordered with the ability to specifically bind divalent metal ions. Here, we describe the details of the exhaustive structural and thermodynamic properties of Zn2+- and Cu2+-complexes with the AaFEcR domain, based on peptide models of its two putative metal binding sites (Ac-HGPHPHPHG-NH2 and Ac-QQLTPNQQQHQQQHSQLQQVHANGS-NH2). Unexpectedly, only in the presence of increasing concentrations of Cu2+ ions, the Ac-HGPHPHPHG-NH2 peptide gained a metal ion-induced poly-l-proline type II helical structure, which is unique for members of the family of nuclear receptors.


Assuntos
Aedes/efeitos dos fármacos , Antivirais/farmacologia , Cobre/farmacologia , Compostos Organometálicos/farmacologia , Peptídeos/farmacologia , Receptores de Esteroides/antagonistas & inibidores , Animais , Antivirais/química , Sítios de Ligação/efeitos dos fármacos , Cobre/química , Dengue/tratamento farmacológico , Dengue/metabolismo , Estrutura Molecular , Compostos Organometálicos/química , Peptídeos/química , Receptores de Esteroides/metabolismo , Termodinâmica , Infecção por Zika virus/tratamento farmacológico , Infecção por Zika virus/metabolismo
7.
Yi Chuan ; 41(8): 677-685, 2019 Aug 20.
Artigo em Chinês | MEDLINE | ID: mdl-31447419

RESUMO

MicroRNAs (miRNAs) compose a class of non-coding transcripts with a mean length of 22 nucleotides, and play critical roles in regulating gene expression in the process of development, proliferation and differentiation of neurons. Recent genome-wide association studies (GWAS) find most of schizophrenia-associated single nucleotide polymorphisms (SNPs) locating in the non-coding regions, providing functional implications of miRNAs in the development of schizophrenia. In this review, we highlight the interplays between GWAS-SNPs and miRNAs in four perspectives: SNP in miRNA gene; miRNA located in the host gene; SNP located in the miRNA's seed sequence; SNP located in the miRNA's binding site. We also speculate on the future research on the role of miRNA in the development of schizophrenia.


Assuntos
MicroRNAs/genética , Polimorfismo de Nucleotídeo Único , Esquizofrenia/genética , Sítios de Ligação , Estudo de Associação Genômica Ampla , Humanos
8.
J Agric Food Chem ; 67(35): 9727-9737, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31398034

RESUMO

The present study aimed to search for chicken abdominal fat deposition-related polymorphisms within RB1 and to provide functional evidence for significantly associated genetic variants. Association analyses showed that 11 single nucleotide polymorphisms (SNPs) in intron 17 of RB1, were significantly associated with both abdominal fat weight (P < 0.05) and abdominal fat percentage (P < 0.05). Functional analysis revealed that the A allele of g.32828A>G repressed the transcriptional efficiency of RB1 in vitro, through binding nuclear factor-kappa B (NF-KB) and SRY-related HMG box protein 2 (SOX2). Furthermore, RB1 mRNA expression levels in the abdominal fat tissue of individuals with the A/A genotype of g.32828A>G were lower than those of individuals with the G/G genotype. Collectively, we propose that the intronic SNP g.32828A>G of RB1 is an obesity-associated variant that directly affects binding with NF-KB and SOX2, leading to changes in RB1 expression which in turn may influence chicken abdominal fat deposition.


Assuntos
Adiposidade , Proteínas Aviárias/metabolismo , Galinhas/metabolismo , NF-kappa B/metabolismo , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição SOX/metabolismo , Gordura Abdominal/metabolismo , Alelos , Animais , Proteínas Aviárias/genética , Sítios de Ligação , Galinhas/genética , Galinhas/crescimento & desenvolvimento , Íntrons , NF-kappa B/genética , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Fatores de Transcrição SOX/genética
9.
Chem Pharm Bull (Tokyo) ; 67(7): 609-619, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31257315

RESUMO

To develop potent ligands for the vitamin D receptor (VDR), we designed and synthesized a series of vitamin D analogues with and without 22-alkyl substituents. These analogues exhibited agonistic, partial agonistic, or antagonistic activity. To elucidate the mechanism of action of the analogues, we conducted crystal structure analyses of the ligand-binding domain (LBD) of VDR complexed with the analogues. The VDR-LBD/agonist complex exhibited precise interactions, which clearly explained VDR agonism. The VDR-LBD/partial agonist complex showed two conformers (agonist and antagonist binding conformers) in a single crystal, demonstrating that partial agonism could be explained by the sum of the agonistic and antagonistic activities. Antagonist binding to the VDR-LBD structure was elucidated using both crystal structure analysis and in-solution structural analyses with the small-angle X-ray scattering (SAXS)-molecular dynamics (MD) and hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) methods. Several antagonist-binding structures were detected. We found that the antagonist binding structures differed depending on the structure of the antagonist itself, and those structures clearly explained the VDR antagonism. Furthermore, the apo VDR-LBD structure without the ligand in the ligand-binding pocket was revealed and found to have an entrance to accommodate the ligand. Thus we elucidated the mechanisms of action of agonists, partial agonists, and antagonists based on structural changes (differences) in the receptor protein induced by ligand binding.


Assuntos
Ligantes , Receptores de Calcitriol/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Receptores de Calcitriol/agonistas , Receptores de Calcitriol/antagonistas & inibidores , Vitamina D/análogos & derivados , Vitamina D/metabolismo
10.
J Enzyme Inhib Med Chem ; 34(1): 1218-1225, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31286785

RESUMO

WaterLOGSY is a sensitive ligand-observed NMR experiment for detection of interaction between a ligand and a protein and is now well-established as a screening technique for fragment-based lead discovery. Here we develop and assess a protocol to derive ligand epitope mapping from WaterLOGSY data and demonstrate its general applicability in studies of fragment-sized ligands binding to six different proteins (glycogen phosphorylase, protein peroxiredoxin 5, Bcl-xL, Mcl-1, HSP90, and human serum albumin). We compare the WaterLOGSY results to those obtained from the more widely used saturation transfer difference experiments and to the 3D structures of the complexes when available. In addition, we evaluate the impact of ligand labile protons on the WaterLOGSY data. Our results demonstrate that the WaterLOGSY experiment can be used as an additional confirmation of the binding mode of a ligand to a protein.


Assuntos
Descoberta de Drogas/métodos , Espectroscopia de Ressonância Magnética/métodos , Sítios de Ligação , Proteínas/química
11.
J Agric Food Chem ; 67(32): 8896-8904, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31339308

RESUMO

The mosquito Aedes aegypti is associated with the spread of many viral diseases in humans, including Dengue virus (DENVs), Yellow fever virus (YFV), Zika virus (ZIKV), and Chikungunya virus (CHIKV). Bacillus thuringiensis (Bt) is widely used as a biopesticide, which produces Cry toxins for mosquito control. The Cry toxins bind mainly to important receptors, including alkaline phosphatase (ALP) and aminopeptidase-N (APN). This work investigated the function of a C-type lectin, CTLGA9, in A. aegypti in response to Cry toxins. Our results showed by far-western blot and ELISA methods that the CTLTGA9 protein interacted with brush border membrane vesicles (BBMVs) of A. aegypti larvae and with ALP1, APN, and Cry11Aa proteins. Furthermore, molecular docking showed overlapping binding sites in ALP1 and APN for binding to Cry11Aa and CTLGA9. The toxicity assays further demonstrated that CTLGA9 inhibited the larvicidal activity of Cry toxins. According to the results of molecular docking, CTLGA9 may compete with Cry11Aa for binding to ALP1 and APN receptors and thus decreases the mosquitocidal toxicity of Cry11Aa. Our results provide further insights into better understanding the mechanism of Cry toxins and help improve the Cry toxicity for mosquito control.


Assuntos
Aedes/efeitos dos fármacos , Aedes/metabolismo , Proteínas de Bactérias/toxicidade , Endotoxinas/toxicidade , Proteínas Hemolisinas/toxicidade , Proteínas de Insetos/metabolismo , Aedes/química , Aedes/genética , Animais , Proteínas de Bactérias/química , Sítios de Ligação , Endotoxinas/química , Proteínas Hemolisinas/química , Proteínas de Insetos/química , Proteínas de Insetos/genética , Larva/efeitos dos fármacos , Larva/genética , Larva/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica/efeitos dos fármacos
12.
J Agric Food Chem ; 67(31): 8527-8535, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31298526

RESUMO

l-Valine belongs to the branched-chain amino acids (BCAAs) and is an essential amino acid that is crucial for all living organisms. l-Valine is industrially produced by the nonpathogenic bacterium Corynebacterium glutamicum and is synthesized by the BCAA biosynthetic pathway. Ketol-acid reductoisomerase (KARI) is the second enzyme in the BCAA pathway and catalyzes the conversion of (S)-2-acetolactate into (R)-2,3-dihydroxy-isovalerate, or the conversion of (S)-2-aceto-2-hydroxybutyrate into (R)-2,3-dihydroxy-3-methylvalerate. To elucidate the enzymatic properties of KARI from C. glutamicum (CgKARI), we successfully produced CgKARI protein and determined its crystal structure in complex with NADP+ and two Mg2+ ions. Based on the complex structure, docking simulations, and site-directed mutagenesis experiments, we revealed that CgKARI belongs to Class I KARI and identified key residues involved in stabilization of the substrate, metal ions, and cofactor. Furthermore, we confirmed the difference in the binding of metal ions that depended on the conformational change.


Assuntos
Proteínas de Bactérias/química , Corynebacterium glutamicum/enzimologia , Cetol-Ácido Redutoisomerase/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico , Corynebacterium glutamicum/química , Corynebacterium glutamicum/genética , Cristalografia por Raios X , Cetol-Ácido Redutoisomerase/genética , Cetol-Ácido Redutoisomerase/metabolismo , Metais/química , Metais/metabolismo , Simulação de Acoplamento Molecular , NADP/química , NADP/metabolismo
13.
Yi Chuan ; 41(6): 509-523, 2019 Jun 20.
Artigo em Chinês | MEDLINE | ID: mdl-31257199

RESUMO

UDP-glucuronosyltransferases (UGTs) are an important family of phase 2 drug-metabolizing enzymes that catalyze the glucuronidation of numerous endogenous or exogenous small compounds. The aberrant expression of UGT isoforms causes many diseases, such as hyperbilirubinemia and affect drug efficacy or toxicity. Understanding mechanisms of UGT gene regulation will provide scientific foundations for disease prevention and personalized or precision medicine. Vertebrate UGT family genes can be divided into UGT1 and UGT2 subfamilies. Similar to the protocadherin, immunoglobulin, and T-cell receptor gene clusters and different from the UGT2 gene cluster, the UGT1 gene cluster is organized into variable and constant regions. The UGT1 variable region contains a tandem array of variable exons, each of which can be alternatively spliced to a single set of 4 downstream constant exons, generating at least nine UGT1 mRNAs that could be translated into different UGT1 glucuronyltransferase isoforms. Our previous work reveals that the relative orientations and locations of CTCF binding sites play a key role in the three-dimensional organization of the mammalian genomes in cell nuclei. Thus in order to study the transcriptional mechanisms of UGT1 gene cluster, the distributions and orientations of CTCF binding sites (CBSs) are analyzed and compared between human and mouse UGT1 gene clusters. We find that the CBSs in the UGT1 gene cluster are not conserved between human and mouse species. We show that CTCF and cohesin regulate the transcription of the UGT1 gene cluster by knocking down the CTCF or the cohesin subunit SMC3 in the human A549 cell line. By using CRISPR DNA-fragment editing, we deleted and inverted hCBS1. By RNA-seq experiments, we find that hCBS1 deletion results in a significant decrease of levels of the UGT1A6, UGT1A7, and UGT1A9 gene expression and that hCBS1 inversion results in a significant decrease of levels of the UGT1A7 gene expression. Our data suggest that the CTCF binding site hCBS1 plays an important regulatory role in the regulation of UGT1 gene expression, providing an experimental basis for further mechanistic studies of the 3D genome regulation of the UGT1 gene cluster.


Assuntos
Fator de Ligação a CCCTC/metabolismo , Regulação da Expressão Gênica , Glucuronosiltransferase/genética , Família Multigênica , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/metabolismo , Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Éxons , Humanos , Camundongos , RNA Mensageiro
14.
Nat Commun ; 10(1): 2917, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266949

RESUMO

Novel antibacterial agents are needed to address the emergence of global antibiotic resistance. MraY is a promising candidate for antibiotic development because it is the target of five classes of naturally occurring nucleoside inhibitors with potent antibacterial activity. Although these natural products share a common uridine moiety, their core structures vary substantially and they exhibit different activity profiles. An incomplete understanding of the structural and mechanistic basis of MraY inhibition has hindered the translation of these compounds to the clinic. Here we present crystal structures of MraY in complex with representative members of the liposidomycin/caprazamycin, capuramycin, and mureidomycin classes of nucleoside inhibitors. Our structures reveal cryptic druggable hot spots in the shallow inhibitor binding site of MraY that were not previously appreciated. Structural analyses of nucleoside inhibitor binding provide insights into the chemical logic of MraY inhibition, which can guide novel approaches to MraY-targeted antibiotic design.


Assuntos
Antibacterianos/química , Bactérias/enzimologia , Proteínas de Bactérias/química , Produtos Biológicos/química , Inibidores Enzimáticos/química , Nucleosídeos/antagonistas & inibidores , Transferases/química , Aminoglicosídeos/química , Arginina/análogos & derivados , Arginina/química , Bactérias/química , Bactérias/genética , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Transferases/antagonistas & inibidores , Transferases/genética , Transferases/metabolismo
15.
Chem Biol Interact ; 310: 108757, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31323226

RESUMO

Fucoxanthin and fucosterol are archetypal lipid components of edible brown algae that provide several health benefits. Lately, their protective role in Aß1-42-induced cognitive dysfunction in animal models has been reported (Alghazwi et al., 2019; Oh et al., 2018). However, their role in the aminergic system and as a prime treatment approach for multifactorial neurodegenerative diseases still requires exploration. The main aims of the present study are to characterize the role of fucoxanthin and fucosterol in the aminergic pathway via in vitro human monoamine oxidase (hMAO) inhibition and cell-based functional G-protein coupled receptor (GPCR) assays and to underline their possible mechanisms of action via in silico molecular docking studies. Fucoxanthin displayed weak inhibition with IC50 values of 197.41 ±â€¯2.20 and 211.12 ±â€¯1.17 µM over two isoenzymes hMAO-A and hMAO-B, respectively. Fucosterol remained inactive up to 500 µM. In functional assay results, fucoxanthin showed a concentration-dependent agonist effect on dopamine D3 and D4 receptors. The half maximal effective concentration (EC50) of fucoxanthin for dopamine D3 and D4 receptors was 16.87 ±â€¯3.41 and 81.87 ±â€¯6.11 µM, respectively. For dopamine as a reference agonist, the EC50 values for these two receptors were 3.7 and 24 nM, respectively. Fucosterol showed no agonist activity on any of the tested receptors. Similarly, fucoxanthin showed a mild antagonist effect on dopamine D1 and tachykinin (NK1) receptor with inhibition of control agonist response by approximately 40% at 100 µM. Fucosterol displayed mild antagonist effects only on dopamine D1 and D4 receptors. In silico studies revealed potential mechanisms by which fucoxanthin binds to dopamine receptors to exert its agonist effects, including low binding energy and H-bond interactions with Ser196 and Thr115 at the D3 receptor and with Ser196 and Asp115 at the D4 receptor. Our results collectively suggest that fucoxanthin is a potential D3/D4 agonist for the management of neurodegenerative diseases, such as Parkinson's disease.


Assuntos
Doença de Parkinson/tratamento farmacológico , Receptores de Dopamina D3/agonistas , Receptores de Dopamina D4/agonistas , Xantofilas/farmacologia , Sítios de Ligação , Aminas Biogênicas , Humanos , Simulação de Acoplamento Molecular , Doenças Neurodegenerativas/tratamento farmacológico , Neurotransmissores , Feófitas/química , Ligação Proteica , Xantofilas/uso terapêutico
16.
Gene ; 711: 143953, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31269463

RESUMO

Both SMAD4 and miR-126* have been proven to be involved in granulosa cell (GC) apoptosis and even follicular atresia, through commonly regulating follicle-stimulating hormone receptor (FSHR), the FSH-specific transmembrane receptor of GCs. However, the regulatory relationship between them in GCs is still unknown. In this study, we report that SMAD4 suppresses the expression of miR-126* and impairs its function in GCs of the porcine ovary by acting as a transcription factor. A classic SMAD4-binding element (SBE) site was found in the promoter of miR-126* by using in silico methods. Luciferase assay, qRT-PCR, and ChIP assay proved that SMAD4 serves as a transcriptional repressor and directly binds to SBE site within miR-126* gene promoter, which further reduces miR-126* gene expression and inhibits its transcriptional activity in GCs. Furthermore, SMAD4 also controls miR-126*-mediated expression of FSHR (a direct target of miR-126* in GCs). In addition, we prove that SMAD4 induces CYP19A1 expression (encodes aromatase, the key enzyme for oestrogen biosynthesis) and inhibits GC apoptosis through the miR-126*/FSHR axis. Taken together, our findings not only established a direct link between SMAD4 and miRNA-126*, two key factors of GC apoptosis, but also revealed an important way in which the SMAD4 regulates GC function, the miRNA-126*/FSHR axis.


Assuntos
Células da Granulosa/citologia , MicroRNAs/química , MicroRNAs/genética , Proteína Smad4/metabolismo , Animais , Apoptose , Aromatase/metabolismo , Sítios de Ligação , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Células da Granulosa/metabolismo , Regiões Promotoras Genéticas , Receptores do FSH/genética , Suínos
17.
Adv Exp Med Biol ; 1162: 39-50, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31332733

RESUMO

Cannabinoids have been widely used for recreational and medicinal purposes. The increasing legalization of cannabinoid use and the growing success in Medicinal Chemistry of cannabinoids have fueled recent interest in cannabinoid-sensing sites in receptor proteins. Here, we review structural data from high-resolution cryo-EM and crystallography studies that depict phytocannabinoid, endocannabinoid, and synthetic cannabinoid molecules bound to various proteins. The latter include antigen-binding fragment (Fab), cellular retinol binding protein 2 (CRBP2), fatty acid-binding protein 5 (FABP5), peroxisome proliferator-activated receptor γ (PPAR γ), and cannabinoid receptor types 1 and 2 (CB1 and CB2). Cannabinoid-protein complexes reveal the complex design of cannabinoid binding sites that are usually presented by conventional ligand-binding pockets on respective proteins. However, subtle differences in cannabinoid interaction with amino acids within the binding pocket often result in diverse consequences for protein function. The rapid increase in available structural data on cannabinoid-protein interactions will ultimately direct drug design efforts toward rendering highly potent cannabinoid-related pharmacotherapies that are devoid of side effects.


Assuntos
Canabinoides/química , Endocanabinoides/química , Sítios de Ligação , Proteínas de Ligação a Ácido Graxo/química , Humanos , Fragmentos Fab das Imunoglobulinas/química , PPAR gama/química , Mapeamento de Interação de Proteínas , Receptores de Canabinoides/química , Proteínas Celulares de Ligação ao Retinol/química
18.
BMC Bioinformatics ; 20(1): 400, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31319797

RESUMO

BACKGROUND: The CATH database provides a hierarchical classification of protein domain structures including a sub-classification of superfamilies into functional families (FunFams). We analyzed the similarity of binding site annotations in these FunFams and incorporated FunFams into the prediction of protein binding residues. RESULTS: FunFam members agreed, on average, in 36.9 ± 0.6% of their binding residue annotations. This constituted a 6.7-fold increase over randomly grouped proteins and a 1.2-fold increase (1.1-fold on the same dataset) over proteins with the same enzymatic function (identical Enzyme Commission, EC, number). Mapping de novo binding residue prediction methods (BindPredict-CCS, BindPredict-CC) onto FunFam resulted in consensus predictions for those residues that were aligned and predicted alike (binding/non-binding) within a FunFam. This simple consensus increased the F1-score (for binding) 1.5-fold over the original prediction method. Variation of the threshold for how many proteins in the consensus prediction had to agree provided a convenient control of accuracy/precision and coverage/recall, e.g. reaching a precision as high as 60.8 ± 0.4% for a stringent threshold. CONCLUSIONS: The FunFams outperformed even the carefully curated EC numbers in terms of agreement of binding site residues. Additionally, we assume that our proof-of-principle through the prediction of protein binding residues will be relevant for many other solutions profiting from FunFams to infer functional information at the residue level.


Assuntos
Domínios Proteicos , Proteínas/química , Sítios de Ligação , Bases de Dados de Proteínas , Ligação Proteica , Proteínas/classificação , Proteínas/metabolismo
19.
Molecules ; 24(13)2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31288414

RESUMO

Due to their sedentary lifestyle, plants are constantly exposed to different stress stimuli. Stress comes in variety of forms where factors like radiation, free radicals, "replication errors, polymerase slippage", and chemical mutagens result in genotoxic or cytotoxic damage. In order to face "the base oxidation or DNA replication stress", plants have developed many sophisticated mechanisms. One of them is the DNA mismatch repair (MMR) pathway. The main part of the MMR is the MutS homologue (MSH) protein family. The genome of Arabidopsis thaliana encodes at least seven homologues of the MSH family: AtMSH1, AtMSH2, AtMSH3, AtMSH4, AtMSH5, AtMSH6, and AtMSH7. Despite their importance, the functions of AtMSH homologs have not been investigated. In this work, bioinformatics tools were used to obtain a better understanding of MSH-mediated DNA repair mechanisms in Arabidopsis thaliana and to understand the additional biological roles of AtMSH family members. In silico analysis, including phylogeny tracking, prediction of 3D structure, interactome analysis, and docking site prediction, suggested interactions with proteins were important for physiological development of A. thaliana. The MSH homologs extensively interacted with both TIL1 and TIL2 (DNA polymerase epsilon catalytic subunit), proteins involved in cell fate determination during plant embryogenesis and involved in flowering time repression. Additionally, interactions with the RECQ protein family (helicase enzymes) and proteins of nucleotide excision repair pathway were detected. Taken together, the results presented here confirm the important role of AtMSH proteins in mismatch repair and suggest important new physiological roles.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Simulação de Acoplamento Molecular , Proteínas MutS/metabolismo , Proteínas de Arabidopsis/química , Sítios de Ligação , Simulação por Computador , Dano ao DNA , Reparo de Erro de Pareamento de DNA , Reparo do DNA , Replicação do DNA , Proteínas MutS/química , Conformação Proteica
20.
Chem Biol Interact ; 311: 108746, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31301288

RESUMO

Utilizing food additives at their optimized concentration is believed to be relatively safe, but their combinatorial effects remain largely unexplored. The influence of mixed food additives on the macromolecules may be altered by synergistic or antagonistic effects. It is previously shown that curcumin enhances the catalase activity by affecting its structural pocket in the active site. The aim of this study was to investigate the combination effects of food colorants sunset yellow FCF (SNY) and curcumin on the activation and/or inactivation of catalase activity using multispectral (fluorescence, FTIR, and UV-vis) analysis and simultaneous docking simulations. Kinetic studies demonstrated that SNY could significantly decrease catalase activity through a non-competitive inhibition mechanism. Fluorescence data indicated that SNY reduces intrinsic emission of catalase via a static quenching mechanism. Thermodynamic and molecular docking investigations suggested that catalase has one binding site for SNY, and hydrogen binding plays a main role in the binding reaction of catalase -SNY complex. Molecular dynamic simulation data indicated that the curcumin binding to the cavity, in the middle of the catalase helical domain, facilitates SNY binding to the enzyme pocket. For this purpose, the equilibrium dialysis system was used to study the stability and reversibility of SNY-catalase in the absence or presence of curcumin. The obtained data indicated that the binding of SNY-catalase is reversible and the stability of the complex is time-dependent. However, curcumin could make the complex more stable enhancing the SNY inhibition of catalase activity.


Assuntos
Compostos Azo/química , Catalase/metabolismo , Curcumina/química , Corantes de Alimentos/química , Compostos Azo/metabolismo , Sítios de Ligação , Catalase/antagonistas & inibidores , Domínio Catalítico , Curcumina/metabolismo , Eritrócitos/enzimologia , Corantes de Alimentos/metabolismo , Humanos , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Termodinâmica
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