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1.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202734

RESUMO

The electrochemical behavior and the interaction of the immunosuppressive drug azathioprine (AZA) with deoxyribonucleic acid (DNA) were investigated using voltammetric techniques, mass spectrometry (MS), and scanning electron microscopy (SEM). The redox mechanism of AZA on glassy carbon (GC) was investigated using cyclic and differential pulse (DP) voltammetry. It was proven that the electroactive center of AZA is the nitro group and its reduction mechanism is a diffusion-controlled process, which occurs in consecutive steps with formation of electroactive products and involves the transfer of electrons and protons. A redox mechanism was proposed and the interaction of AZA with DNA was also investigated. Morphological characterization of the DNA film on the electrode surface before and after interaction with AZA was performed using scanning electron microscopy. An electrochemical DNA biosensor was employed to study the interactions between AZA and DNA with different concentrations, incubation times, and applied potential values. It was shown that the reduction of AZA molecules bound to the DNA layer induces structural changes of the DNA double strands and oxidative damage, which were recognized through the occurrence of the 8-oxo-deoxyguanosine oxidation peak. Mass spectrometry investigation of the DNA film before and after interaction with AZA also demonstrated the formation of AZA adducts with purine bases.


Assuntos
Azatioprina/química , Azatioprina/metabolismo , DNA/química , DNA/metabolismo , Oxirredução , Algoritmos , Azatioprina/farmacologia , Técnicas Biossensoriais , Fenômenos Químicos , Substâncias Macromoleculares/química , Substâncias Macromoleculares/ultraestrutura , Espectrometria de Massas , Modelos Teóricos
2.
Artigo em Inglês | MEDLINE | ID: mdl-34199549

RESUMO

Since varicocele is so common in infertile men, this study intends to analyse the relationships between varicocele and conventional semen characteristics, sperm nuclear DNA dispersion and oxidation-reduction potential (ORP) in semen. Varicocele-positive and varicocele-negative infertile men (study groups) showed significantly lower standard sperm parameters and higher sperm DNA fragmentation (SDF) and ORP in semen than healthy volunteers and subjects with proven fertility (control groups). A lower proportion of low SDF levels (0-15% SDF) and higher incidence of high SDF levels (>30% SDF), as well as a higher prevalence of high ORP values (>1.37 mV/106 sperm/mL), were found in the study groups vs. the control groups. Moreover, infertile men had significantly lower odds ratios (ORs) for low SDF levels and significantly higher ORs for high SDF levels and high ORP. SDF and ORP were negatively correlated with sperm number, morphology, motility and vitality. Furthermore, a significant positive correlation was found between SDF and ORP. The obtained results suggest that disorders of spermatogenesis may occur in varicocele-related infertility. These abnormalities are manifested not only by reduced standard semen parameters but also by decreased sperm DNA integrity and simultaneously increased oxidative stress in semen.


Assuntos
Infertilidade Masculina , Varicocele , DNA/metabolismo , Humanos , Infertilidade Masculina/genética , Masculino , Oxirredução , Sêmen , Análise do Sêmen , Contagem de Espermatozoides , Motilidade Espermática , Espermatozoides/metabolismo , Varicocele/metabolismo
3.
Molecules ; 26(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34204901

RESUMO

The topological properties of DNA molecules, supercoiling, knotting, and catenation, are intimately connected with essential biological processes, such as gene expression, replication, recombination, and chromosome segregation. Non-trivial DNA topologies present challenges to the molecular machines that process and maintain genomic information, for example, by creating unwanted DNA entanglements. At the same time, topological distortion can facilitate DNA-sequence recognition through localized duplex unwinding and longer-range loop-mediated interactions between the DNA sequences. Topoisomerases are a special class of essential enzymes that homeostatically manage DNA topology through the passage of DNA strands. The activities of these enzymes are generally investigated using circular DNA as a model system, in which case it is possible to directly assay the formation and relaxation of DNA supercoils and the formation/resolution of knots and catenanes. Some topoisomerases use ATP as an energy cofactor, whereas others act in an ATP-independent manner. The free energy of ATP hydrolysis can be used to drive negative and positive supercoiling or to specifically relax DNA topologies to levels below those that are expected at thermodynamic equilibrium. The latter activity, which is known as topology simplification, is thus far exclusively associated with type-II topoisomerases and it can be understood through insight into the detailed non-equilibrium behavior of type-II enzymes. We use a non-equilibrium topological-network approach, which stands in contrast to the equilibrium models that are conventionally used in the DNA-topology field, to gain insights into the rates that govern individual transitions between topological states. We anticipate that our quantitative approach will stimulate experimental work and the theoretical/computational modeling of topoisomerases and similar enzyme systems.


Assuntos
DNA Topoisomerases/metabolismo , DNA/química , DNA/metabolismo , Trifosfato de Adenosina/metabolismo , DNA Topoisomerases/química , Hidrólise , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica
4.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203807

RESUMO

Genome editing using CRISPR-Cas9 nucleases is based on the repair of the DNA double-strand break (DSB). In eukaryotic cells, DSBs are rejoined through homology-directed repair (HDR), non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) pathways. Among these, it is thought that the NHEJ pathway is dominant and occurs throughout a cell cycle. NHEJ-based DSB repair is known to be error-prone; however, there are few studies that delve into it deeply in endogenous genes. Here, we quantify the degree of NHEJ-based DSB repair accuracy (termed NHEJ accuracy) in human-originated cells by incorporating exogenous DNA oligonucleotides. Through an analysis of joined sequences between the exogenous DNA and the endogenous target after DSBs occur, we determined that the average value of NHEJ accuracy is approximately 75% in maximum in HEK 293T cells. In a deep analysis, we found that NHEJ accuracy is sequence-dependent and the value at the DSB end proximal to a protospacer adjacent motif (PAM) is relatively lower than that at the DSB end distal to the PAM. In addition, we observed a negative correlation between the insertion mutation ratio and the degree of NHEJ accuracy. Our findings would broaden the understanding of Cas9-mediated genome editing.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Clivagem do DNA , Reparo do DNA por Junção de Extremidades/genética , Sequência de Bases , DNA/metabolismo , Células HEK293 , Células HeLa , Humanos , Mutação/genética , Oligonucleotídeos/metabolismo , RNA Guia/genética , Deleção de Sequência/genética
5.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34204001

RESUMO

Radiodynamic therapy (RDT) is a recent extension of conventional photodynamic therapy, in which visible/near infrared light irradiation is replaced by a well-tolerated dose of high-energy X-rays. This enables greater tissue penetration to allow non-invasive treatment of large, deep-seated tumors. We report here the design and testing of a drug delivery system for RDT that is intended to enhance intra- or peri-nuclear localization of the photosensitizer, leading to DNA damage and resulting clonogenic cell kill. This comprises a photosensitizer (Verteporfin, VP) incorporated into poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) that are surface-functionalized with a cell-penetrating HIV trans-activator of transcription (TAT) peptide. In addition to a series of physical and photophysical characterization studies, cytotoxicity tests in pancreatic (PANC-1) cancer cells in vitro under 4 Gy X-ray exposure from a clinical 6 MV linear accelerator (LINAC) showed that TAT targeting of the nanoparticles markedly enhances the effectiveness of RDT treatment, particularly when assessed by a clonogenic, i.e., DNA damage-mediated, cell kill.


Assuntos
Composição de Medicamentos , Produtos do Gene tat/química , Nanopartículas/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Verteporfina/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Clonais , DNA/metabolismo , Endocitose/efeitos dos fármacos , Humanos , Lipídeos de Membrana/metabolismo , Nanopartículas/ultraestrutura , Oxigênio Singlete/metabolismo
6.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204214

RESUMO

Curaxins and especially the second-generation derivative curaxin CBL0137 have important antitumor activities in multiple cancers such as glioblastoma, melanoma and others. Although most of the authors suggest that their mechanism of action comes from the activation of p53 and inactivation of NF-kB by targeting FACT, there is evidence supporting the involvement of DNA binding in their antitumor activity. In this work, the DNA binding properties of curaxin CBL0137 with model quadruplex DNA oligomers were studied by 1H NMR, CD, fluorescence and molecular modeling. We provided molecular details of the interaction of curaxin with two G-quadruplex structures, the single repeat of human telomere d(TTAGGGT)4 and the c-myc promoter Pu22 sequence. We also performed 1H and 31P NMR experiments were also performed in order to investigate the interaction with duplex DNA models. Our data support the hypothesis that the interaction of curaxin with G-quadruplex may provide a novel insight into the DNA-binding properties of CBL0137, and it will be helpful for the design of novel selective DNA-targeting curaxin analogues.


Assuntos
Carbazóis/química , DNA/química , Quadruplex G , Substâncias Macromoleculares/química , Carbazóis/farmacologia , DNA/metabolismo , Quadruplex G/efeitos dos fármacos , Humanos , Substâncias Macromoleculares/metabolismo , Espectroscopia de Ressonância Magnética , Conformação Molecular , Estrutura Molecular , Relação Estrutura-Atividade , Telômero/genética , Telômero/metabolismo
7.
Molecules ; 26(12)2021 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-34205449

RESUMO

Restriction endonucleases (REs) are intra-bacterial scissors that are considered tools in the fight against foreign genetic material. SspI and BsmAI, examined in this study, cleave dsDNA at their site of recognition or within a short distance of it. Both enzymes are representatives of type II REs, which have played an extremely important role in research on the genetics of organisms and molecular biology. Therefore, the study of agents affecting their activity has become highly important. Ionizing radiation may damage basic cellular mechanisms by inducing lesions in the genome, with 5',8-cyclo-2'-deoxypurines (cdPus) as a model example. Since cdPus may become components of clustered DNA lesions (CDLs), which are unfavorable for DNA repair pathways, their impact on other cellular mechanisms is worthy of attention. This study investigated the influence of cdPus on the elements of the bacterial restriction-modification system. In this study, it was shown that cdPus present in DNA affect the activity of REs. SspI was blocked by any cdPu lesion present at the enzyme's recognition site. When lesions were placed near the recognition sequence, the SspI was inhibited up to 46%. Moreover, (5'S)-5',8-cyclo-2'-deoxyadenosine (ScdA) present in the oligonucleotide sequence lowered BsmAI activity more than (5'R)-5',8-cyclo-2'-deoxyadenosine (RcdA). Interestingly, in the case of 5',8-cyclo-2'-deoxyguanosine (cdG), both 5'S and 5'R diastereomers inhibited BsmAI activity (up to 55% more than cdA). The inhibition was weaker when cdG was present at the recognition site rather than the cleavage site.


Assuntos
Enzimas de Restrição do DNA/metabolismo , DNA/metabolismo , Desoxiadenosinas/metabolismo , Desoxiguanosina/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Animais , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Humanos , Oligonucleotídeos/metabolismo
8.
Bioinformatics ; 37(Suppl_1): i367-i375, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34252930

RESUMO

MOTIVATION: High-throughput chromatin immunoprecipitation (ChIP) sequencing-based assays capture genomic regions associated with the profiled transcription factor (TF). ChIP-exo is a modified protocol, which uses lambda exonuclease to digest DNA close to the TF-DNA complex, in order to improve on the positional resolution of the TF-DNA contact. Because the digestion occurs in the 5'-3' orientation, the protocol produces directional footprints close to the complex, on both sides of the double stranded DNA. Like all ChIP-based methods, ChIP-exo reports a mixture of different regions associated with the TF: those bound directly to the TF as well as via intermediaries. However, the distribution of footprints are likely to be indicative of the complex forming at the DNA. RESULTS: We present ExoDiversity, which uses a model-based framework to learn a joint distribution over footprints and motifs, thus resolving the mixture of ChIP-exo footprints into diverse binding modes. It uses no prior motif or TF information and automatically learns the number of different modes from the data. We show its application on a wide range of TFs and organisms/cell-types. Because its goal is to explain the complete set of reported regions, it is able to identify co-factor TF motifs that appear in a small fraction of the dataset. Further, ExoDiversity discovers small nucleotide variations within and outside canonical motifs, which co-occur with variations in footprints, suggesting that the TF-DNA structural configuration at those regions is likely to be different. Finally, we show that detected modes have specific DNA shape features and conservation signals, giving insights into the structure and function of the putative TF-DNA complexes. AVAILABILITY AND IMPLEMENTATION: The code for ExoDiversity is available on https://github.com/NarlikarLab/exoDIVERSITY. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
DNA , Exonucleases , Sítios de Ligação , Imunoprecipitação da Cromatina , DNA/metabolismo , Pegada de DNA , Ligação Proteica , Análise de Sequência de DNA
9.
Anticancer Res ; 41(6): 2859-2866, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34083276

RESUMO

BACKGROUND/AIM: Genetic manipulation of stem cells using non-viral vectors is still limited due to low transfection efficiency. We investigated whether the DNA-binding cell-permeation peptides (CPP) can enhance the transfection efficiency of non-viral vectors in adipose tissue-derived mesenchymal stem cells (ASCs) and whether ASCs over-expressing TRAIL through CPP can inhibit the growth of glioma U251MG cells in vitro and in vivo. MATERIALS AND METHODS: ASCs were genetically engineered to over-express TRAIL by using CPP, pCMV3-TRAIL and lipid-based transfection reagents (X-tremeGENE). RESULTS: The transfection efficiency of ASCs increased by approximately 7% using CPP; 53.9% of ASCs were transfected and TRAIL expression in ASCs increased by approximately 3 times compared to X-tremeGENE alone. ASCs over-expressing TRAIL using CPP inhibited growth of glioma U251MG cells both in vitro and in the U251MG xenograft model. CONCLUSION: CPP can be used as an enhancer for genetically manipulating ASCs and tumor treatment.


Assuntos
Tecido Adiposo/citologia , Neoplasias Encefálicas/patologia , Peptídeos Penetradores de Células/metabolismo , DNA/metabolismo , Glioma/patologia , Células-Tronco Mesenquimais/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Nus , Ligação Proteica , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Molecules ; 26(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069054

RESUMO

The DACHPtCl2 compound (trans-(R,R)-1,2-diaminocyclohexanedichloroplatinum(II)) is a potent anticancer drug with a broad spectrum of activity and is less toxic than oxaliplatin (trans-l-diaminocyclohexane oxalate platinum II), with which it shares the active metal fragment DACHPt. Nevertheless, due to poor water solubility, its use as a chemotherapeutic drug is limited. Here, DACHPtCl2 was conjugated, in a bidentate form, with half-generation PAMAM dendrimers (G0.5-G3.5) with carboxylate end-groups, and the resulting conjugates were evaluated against various types of cancer cell lines. In this way, we aimed at increasing the solubility and availability at the target site of DACHPt while potentially reducing the adverse side effects. DNA binding assays showed a hyperchromic effect compatible with DNA helix's disruption upon the interaction of the metallodendrimers and/or the released active metallic fragments with DNA. Furthermore, the prepared DACHPt metallodendrimers presented cytotoxicity in a wide set of cancer cell lines used (the relative potency regarding oxaliplatin was in general high) and were not hemotoxic. Importantly, their selectivity for A2780 and CACO-2 cancer cells with respect to non-cancer cells was particularly high. Subsequently, the anticancer drug 5-FU was loaded in a selected metallodendrimer (the G2.5COO(DACHPt)16) to investigate a possible synergistic effect between the two drugs carried by the same dendrimer scaffold and tested for cytotoxicity in A2780cisR and CACO-2 cancer cell lines. This combination resulted in IC50 values much lower than the IC50 for 5-FU but higher than those found for the metallodendrimers without 5-FU. It seems, thus, that the metallic fragment-induced cytotoxicity dominates over the cytotoxicity of 5-FU in the set of considered cell lines.


Assuntos
Antineoplásicos/farmacologia , Dendrímeros/química , Fluoruracila/farmacologia , Compostos Organoplatínicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Linhagem Celular Tumoral , DNA/metabolismo , Dendrímeros/síntese química , Liberação Controlada de Fármacos , Fluoruracila/síntese química , Fluoruracila/química , Humanos , Concentração Inibidora 50 , Compostos Organoplatínicos/síntese química , Compostos Organoplatínicos/química , Espectroscopia de Prótons por Ressonância Magnética , Espectrofotometria Ultravioleta , Eletricidade Estática , Termodinâmica
11.
Molecules ; 26(9)2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064380

RESUMO

The interest in the introduction of the oxime group in molecules aiming to improve their biological effects is increasing. This work aimed to develop new steroidal oximes of the estrane series with potential antitumor interest. For this, several oximes were synthesized by reaction of hydroxylamine with the 17-ketone of estrone derivatives. Then, their cytotoxicity was evaluated in six cell lines. An estrogenicity assay, a cell cycle distribution analysis and a fluorescence microscopy study with Hoechst 3358 staining were performed with the most promising compound. In addition, molecular docking studies against estrogen receptor α, steroid sulfatase, 17ß-hydroxysteroid dehydrogenase type 1 and ß-tubulin were also accomplished. The 2-nitroestrone oxime showed higher cytotoxicity than the parent compound on MCF-7 cancer cells. Furthermore, the oximes bearing halogen groups in A-ring evidenced selectivity for HepaRG cells. Remarkably, the Δ9,11-estrone oxime was the most cytotoxic and arrested LNCaP cells in the G2/M phase. Fluorescence microscopy studies showed the presence of condensed DNA typical of prophase and condensed and fragmented nuclei characteristic of apoptosis. However, this oxime promoted the proliferation of T47-D cells. Interestingly, molecular docking studies estimated a strong interaction between Δ9,11-estrone oxime and estrogen receptor α and ß-tubulin, which may account for the described effects.


Assuntos
Simulação de Acoplamento Molecular , Oximas/síntese química , Oximas/farmacologia , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA/metabolismo , Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Estrona/síntese química , Estrona/química , Estrona/farmacologia , Fluoruracila/farmacologia , Humanos , Concentração Inibidora 50 , Oximas/química
12.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070855

RESUMO

Lens epithelium-derived growth factor splice variant of 75 kDa (LEDGF/p75) plays an important role in cancer, but its DNA-damage repair (DDR)-related implications are still not completely understood. Different LEDGF model cell lines were generated: a complete knock-out of LEDGF (KO) and re-expression of LEDGF/p75 or LEDGF/p52 using CRISPR/Cas9 technology. Their proliferation and migration capacity as well as their chemosensitivity were determined, which was followed by investigation of the DDR signaling pathways by Western blot and immunofluorescence. LEDGF-deficient cells exhibited a decreased proliferation and migration as well as an increased sensitivity toward etoposide. Moreover, LEDGF-depleted cells showed a significant reduction in the recruitment of downstream DDR-related proteins such as replication protein A 32 kDa subunit (RPA32) after exposure to etoposide. The re-expression of LEDGF/p75 rescued all knock-out effects. Surprisingly, untreated LEDGF KO cells showed an increased amount of DNA fragmentation combined with an increased formation of γH2AX and BRCA1. In contrast, the protein levels of ubiquitin-conjugating enzyme UBC13 and nuclear proteasome activator PA28γ were substantially reduced upon LEDGF KO. This study provides for the first time an insight that LEDGF is not only involved in the recruitment of CtIP but has also an effect on the ubiquitin-dependent regulation of DDR signaling molecules and highlights the role of LEDGF/p75 in homology-directed DNA repair.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , DNA/genética , Regulação da Expressão Gênica , Reparo de DNA por Recombinação , Fatores de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Antineoplásicos Fitogênicos/farmacologia , Autoantígenos/genética , Autoantígenos/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , DNA/metabolismo , Dano ao DNA , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Etoposídeo/farmacologia , Técnicas de Inativação de Genes , Histonas/genética , Histonas/metabolismo , Humanos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Transdução de Sinais , Fatores de Transcrição/deficiência , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
13.
BMC Genomics ; 22(1): 482, 2021 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-34174819

RESUMO

BACKGROUND: Transcription factors (TFs) bind specifically to TF binding sites (TFBSs) at cis-regulatory regions to control transcription. It is critical to locate these TF-DNA interactions to understand transcriptional regulation. Efforts to predict bona fide TFBSs benefit from the availability of experimental data mapping DNA binding regions of TFs (chromatin immunoprecipitation followed by sequencing - ChIP-seq). RESULTS: In this study, we processed ~ 10,000 public ChIP-seq datasets from nine species to provide high-quality TFBS predictions. After quality control, it culminated with the prediction of ~ 56 million TFBSs with experimental and computational support for direct TF-DNA interactions for 644 TFs in > 1000 cell lines and tissues. These TFBSs were used to predict > 197,000 cis-regulatory modules representing clusters of binding events in the corresponding genomes. The high-quality of the TFBSs was reinforced by their evolutionary conservation, enrichment at active cis-regulatory regions, and capacity to predict combinatorial binding of TFs. Further, we confirmed that the cell type and tissue specificity of enhancer activity was correlated with the number of TFs with binding sites predicted in these regions. All the data is provided to the community through the UniBind database that can be accessed through its web-interface ( https://unibind.uio.no/ ), a dedicated RESTful API, and as genomic tracks. Finally, we provide an enrichment tool, available as a web-service and an R package, for users to find TFs with enriched TFBSs in a set of provided genomic regions. CONCLUSIONS: UniBind is the first resource of its kind, providing the largest collection of high-confidence direct TF-DNA interactions in nine species.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , DNA , Sítios de Ligação , Imunoprecipitação da Cromatina , Biologia Computacional , DNA/metabolismo , Ligação Proteica
14.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069086

RESUMO

Hydrogen sulfide (H2S) is an endogenously produced molecule with anti-inflammatory and cytoprotective properties. We aimed to investigate for the first time if a novel, esterase-sensitive H2S-prodrug, BW-HS-101 with the ability to release H2S in a controllable manner, prevents gastric mucosa against acetylsalicylic acid-induced gastropathy on microscopic and molecular levels. Wistar rats were pretreated intragastrically with vehicle, BW-HS-101 (0.5-50 µmol/kg) or its analogue without the ability to release H2S, BW-iHS-101 prior to ASA administration (125 mg/kg, intragastrically). BW-HS-101 was administered alone or in combination with nitroarginine (L-NNA, 20 mg/kg, intraperitoneally) or zinc protoporphyrin IX (10 mg/kg, intraperitoneally). Gastroprotective effects of BW-HS-101 were additionally evaluated against necrotic damage induced by intragastrical administration of 75% ethanol. Gastric mucosal damage was assessed microscopically, and gastric blood flow was determined by laser flowmetry. Gastric mucosal DNA oxidation and PGE2 concentration were assessed by ELISA. Serum and/or gastric protein concentrations of IL-1α, IL-1ß, IL-2, IL-4, IL-6, IL-10, IL-13, VEGF, GM-CSF, IFN-γ, TNF-α, and EGF were determined by a microbeads/fluorescent-based multiplex assay. Changes in gastric mucosal iNOS, HMOX-1, SOCS3, IL1-R1, IL1-R2, TNF-R2, COX-1, and COX-2 mRNA were assessed by real-time PCR. BW-HS-101 or BW-iHS-101 applied at a dose of 50 µmol/kg protected gastric mucosa against ASA-induced gastric damage and prevented a decrease in the gastric blood flow level. H2S prodrug decreased DNA oxidation, systemic and gastric mucosal inflammation with accompanied upregulation of SOCS3, and EGF and HMOX-1 expression. Pharmacological inhibition of nitric oxide (NO) synthase but not carbon monoxide (CO)/heme oxygenase (HMOX) activity by L-NNA or ZnPP, respectively, reversed the gastroprotective effect of BW-HS-101. BW-HS-101 also protected against ethanol-induced gastric injury formation. We conclude that BW-HS-101, due to its ability to release H2S in a controllable manner, prevents gastric mucosa against drugs-induced gastropathy, inflammation and DNA oxidation, and upregulate gastric microcirculation. Gastroprotective effects of this H2S prodrug involves endogenous NO but not CO activity and could be mediated by cytoprotective and anti-inflammatory SOCS3 and EGF pathways.


Assuntos
Mucosa Gástrica/efeitos dos fármacos , Sulfeto de Hidrogênio/farmacocinética , Substâncias Protetoras/farmacologia , Animais , Anti-Inflamatórios não Esteroides/efeitos adversos , Aspirina/efeitos adversos , DNA/metabolismo , Liberação Controlada de Fármacos , Etanol/toxicidade , Mucosa Gástrica/irrigação sanguínea , Mucosa Gástrica/patologia , Gastrite/induzido quimicamente , Gastrite/tratamento farmacológico , Gastrite/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Óxido Nítrico/metabolismo , Nitroarginina/administração & dosagem , Nitroarginina/farmacologia , Pró-Fármacos/farmacocinética , Prostaglandina-Endoperóxido Sintases/metabolismo , Prostaglandinas/metabolismo , Substâncias Protetoras/administração & dosagem , Protoporfirinas/administração & dosagem , Protoporfirinas/farmacologia , Ratos Wistar
15.
Science ; 372(6548): 1349-1353, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34140389

RESUMO

The epigenome of macrophages can be reprogrammed by extracellular cues, but the extent to which different stimuli achieve this is unclear. Nuclear factor κB (NF-κB) is a transcription factor that is activated by all pathogen-associated stimuli and can reprogram the epigenome by activating latent enhancers. However, we show that NF-κB does so only in response to a subset of stimuli. This stimulus specificity depends on the temporal dynamics of NF-κB activity, in particular whether it is oscillatory or non-oscillatory. Non-oscillatory NF-κB opens chromatin by sustained disruption of nucleosomal histone-DNA interactions, enabling activation of latent enhancers that modulate expression of immune response genes. Thus, temporal dynamics can determine a transcription factor's capacity to reprogram the epigenome in a stimulus-specific manner.


Assuntos
Epigenoma , Macrófagos/metabolismo , NF-kappa B/metabolismo , Fator de Transcrição RelA/metabolismo , Animais , Núcleo Celular/metabolismo , Cromatina/metabolismo , DNA/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Histonas/metabolismo , Sistema de Sinalização das MAP Quinases , Macrófagos/imunologia , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Inibidor de NF-kappaB alfa/genética , Inibidor de NF-kappaB alfa/metabolismo , Nucleossomos/metabolismo , Transdução de Sinais , Transcrição Genética
16.
Nat Commun ; 12(1): 3479, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108479

RESUMO

Human PARP2/ARTD2 is an ADP-ribosyltransferase which, when activated by 5'-phosphorylated DNA ends, catalyses poly-ADP-ribosylation of itself, other proteins and DNA. In this study, a crystal structure of PARP2 in complex with an activating 5'-phosphorylated DNA shows that the WGR domain bridges the dsDNA gap and joins the DNA ends. This DNA binding results in major conformational changes, including reorganization of helical fragments, in the PARP2 regulatory domain. A comparison of PARP1 and PARP2 crystal structures reveals how binding to a DNA damage site leads to formation of a catalytically competent conformation. In this conformation, PARP2 is capable of binding substrate NAD+ and histone PARylation factor 1 that changes PARP2 residue specificity from glutamate to serine when initiating DNA repair processes. The structure also reveals how the conformational changes in the autoinhibitory regulatory domain would promote the flexibility needed by the enzyme to reach the target macromolecule for ADP-ribosylation.


Assuntos
Dano ao DNA , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas de Transporte/metabolismo , DNA/química , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Ativação Enzimática , Humanos , NAD/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli ADP Ribosilação , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Desdobramento de Proteína
17.
Nat Commun ; 12(1): 3476, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108490

RESUMO

Cas12i is a newly identified member of the functionally diverse type V CRISPR-Cas effectors. Although Cas12i has the potential to serve as genome-editing tool, its structural and functional characteristics need to be investigated in more detail before effective application. Here we report the crystal structures of the Cas12i1 R-loop complexes before and after target DNA cleavage to elucidate the mechanisms underlying target DNA duplex unwinding, R-loop formation and cis cleavage. The structure of the R-loop complex after target DNA cleavage also provides information regarding trans cleavage. Besides, we report a crystal structure of the Cas12i1 binary complex interacting with a pseudo target oligonucleotide, which mimics target interrogation. Upon target DNA duplex binding, the Cas12i1 PAM-interacting cleft undergoes a remarkable open-to-closed adjustment. Notably, a zipper motif in the Helical-I domain facilitates unzipping of the target DNA duplex. Formation of the 19-bp crRNA-target DNA strand heteroduplex in the R-loop complexes triggers a conformational rearrangement and unleashes the DNase activity. This study provides valuable insights for developing Cas12i1 into a reliable genome-editing tool.


Assuntos
Proteínas Associadas a CRISPR/química , Clivagem do DNA , Endonucleases/química , Estruturas R-Loop , Pareamento de Bases , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Domínio Catalítico , DNA/química , DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Ativação Enzimática , Magnésio/química , Modelos Biológicos , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de Proteína , RNA Guia/química , RNA Guia/metabolismo , Temperatura
18.
ACS Appl Mater Interfaces ; 13(27): 31485-31494, 2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34184527

RESUMO

Owing to their favorable design flexibility and eminent signal amplification ability, DNA nanomachine-supported biosensors have provided an attractive avenue for intracellular fluorescence imaging, especially for DNA walkers. However, this promising option not only suffers from poor controllability but also needs to be supplied with additional driving forces on account of the frequent employment of metal ion-dependent DNAzymes. Aiming at overcoming these obstacles, we introduce some fruitful solutions. On one hand, innovative light-activated walking behavior induced by a photocleavage mode is established on the surfaces of gold nanoparticles, and such a photoselective sensing system can be perfectly prevented from pre-activating during the intracellular delivery process and made to achieve target identification only under irradiation using a moderate ultraviolet light source. On the other hand, this light-switchable sensing frame is encapsulated within a dissociable metal-organic framework (ZIF-8) to facilitate endocytosis and ensure sufficient internal cofactors (Zn2+) to realize a self-driven pattern in the acidic environment of the cell lysosome. Based on the abovementioned efforts, the newly constructed autonomous three-dimensional DNA walkers present satisfactory sensitivity (a limit of detection of down to 19.4 pM) and specificity (even distinguishing single-base changes) toward a model biomarker (microRNA-21). More importantly, the sensing method allows determination of the variations in targets in living cancer cells with exceptional precision and efficiency, offering a powerful assay platform for intracellular imaging.


Assuntos
DNA/química , DNA/metabolismo , Luz , MicroRNAs/metabolismo , Nanoestruturas/química , Nanotecnologia/métodos , Imagem Óptica/métodos , Sobrevivência Celular , Células HeLa , Humanos , Células MCF-7 , Estruturas Metalorgânicas/química
19.
J R Soc Interface ; 18(179): 20210206, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34062105

RESUMO

The origin of an ordered genetic response of a complex and noisy biological cell is intimately related to the detailed mechanism of protein-DNA interactions present in a wide variety of gene regulatory (GR) systems. However, the quantitative prediction of genetic response and the correlation between the mechanism and the response curve is poorly understood. Here, we report in silico binding studies of GR systems to show that the transcription factor (TF) binds to multiple DNA sites with high cooperativity spreads from specific binding sites into adjacent non-specific DNA and bends the DNA. Our analysis is not limited only to the isolated model system but also can be applied to a system containing multiple interacting genes. The controlling role of TF oligomerization, TF-ligand interactions, and DNA looping for gene expression has been also characterized. The predictions are validated against detailed grand canonical Monte Carlo simulations and published data for the lac operon system. Overall, our study reveals that the expression of target genes can be quantitatively controlled by modulating TF-ligand interactions and the bending energy of DNA.


Assuntos
Redes Reguladoras de Genes , Fatores de Transcrição , Sítios de Ligação , DNA/metabolismo , Regulação da Expressão Gênica , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
Nat Commun ; 12(1): 3621, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131149

RESUMO

Chromatin structure and accessibility, and combinatorial binding of transcription factors to regulatory elements in genomic DNA control transcription. Genetic variations in genes encoding histones, epigenetics-related enzymes or modifiers affect chromatin structure/dynamics and result in alterations in gene expression contributing to cancer development or progression. Gliomas are brain tumors frequently associated with epigenetics-related gene deregulation. We perform whole-genome mapping of chromatin accessibility, histone modifications, DNA methylation patterns and transcriptome analysis simultaneously in multiple tumor samples to unravel epigenetic dysfunctions driving gliomagenesis. Based on the results of the integrative analysis of the acquired profiles, we create an atlas of active enhancers and promoters in benign and malignant gliomas. We explore these elements and intersect with Hi-C data to uncover molecular mechanisms instructing gene expression in gliomas.


Assuntos
Cromatina , Glioma/genética , Sequências Reguladoras de Ácido Nucleico , Sítios de Ligação , Neoplasias Encefálicas/genética , Imunoprecipitação da Cromatina , DNA/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Epigênese Genética , Epigenômica , Proteína Forkhead Box M1 , Expressão Gênica , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioblastoma , Código das Histonas , Histonas , Humanos , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo
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