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1.
Biochim Biophys Acta Gene Regul Mech ; 1867(3): 195050, 2024 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-39029558

RESUMO

Armadillo repeat-containing proteins (ARMCs) are a large family found throughout eukaryotes, which play prominent roles in cell adhesion, signaling and cytoskeletal regulation. The ARMC6 protein is highly conserved in primates, including humans, but to date does not have a clear function beyond initial hints of a link to cancer and telomerase activity. We report here in vitro experiments showing ARMC6 binding to DNA promoter sequences from several cancer-related genes (e.g., EGFR, VEGF and c-MYC), and also to the telomeric RNA repeat (TERRA). ARMC6 binding activity appears to recognize G-quadruplex motifs, which are being increasingly implicated as structure-based protein binding sites in chromosome maintenance and repair. In vivo investigation of ARMC6 function revealed that when this protein is overexpressed in human cell lines, there is different expression of genes connected with oncogenic pathways and those implicated in downstream non-canonical telomerase pathways (e.g., VEGF, hTERT, c-MYC, ESM1, MMP3). ARMC6 is already known to interact with human shelterin protein TRF2 and telomerase. The protein binds G-quadruplex structures and does so preferentially to RNA over DNA. As such, this protein may be an example of how a non-canonical nucleic acid structural motif allows mediation between gene regulation and telomeric chromatin rearrangement pathways.

2.
Nucleic Acids Res ; 51(1): 420-433, 2023 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-36546771

RESUMO

In contrast to the catalytic subunit of telomerase, its RNA subunit (TR) is highly divergent in size, sequence and biogenesis pathways across eukaryotes. Current views on TR evolution assume a common origin of TRs transcribed with RNA polymerase II in Opisthokonta (the supergroup including Animalia and Fungi) and Trypanosomida on one hand, and TRs transcribed with RNA polymerase III under the control of type 3 promoter, found in TSAR and Archaeplastida supergroups (including e.g. ciliates and Viridiplantae taxa, respectively). Here, we focus on unknown TRs in one of the largest Animalia order - Hymenoptera (Arthropoda) with more than 300 available representative genomes. Using a combination of bioinformatic and experimental approaches, we identify their TRs. In contrast to the presumed type of TRs (H/ACA box snoRNAs transcribed with RNA Polymerase II) corresponding to their phylogenetic position, we find here short TRs of the snRNA type, likely transcribed with RNA polymerase III under the control of the type 3 promoter. The newly described insect TRs thus question the hitherto assumed monophyletic origin of TRs across Animalia and point to an evolutionary switch in TR type and biogenesis that was associated with the divergence of Arthropods.


Assuntos
Himenópteros , Telomerase , Animais , Telomerase/genética , Telomerase/metabolismo , Himenópteros/genética , Filogenia , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , Conformação de Ácido Nucleico , RNA/genética , Plantas/genética , Eucariotos/genética
3.
Molecules ; 24(11)2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31159174

RESUMO

Expansions of trinucleotide repeats (TNRs) are associated with genetic disorders such as Friedreich's ataxia. The tumor suppressor p53 is a central regulator of cell fate in response to different types of insults. Sequence and structure-selective modes of DNA recognition are among the main attributes of p53 protein. The focus of this work was analysis of the p53 structure-selective recognition of TNRs associated with human neurodegenerative diseases. Here, we studied binding of full length p53 and several deletion variants to TNRs folded into DNA hairpins or loops. We demonstrate that p53 binds to all studied non-B DNA structures, with a preference for non-B DNA structures formed by pyrimidine (Py) rich strands. Using deletion mutants, we determined the C-terminal DNA binding domain of p53 to be crucial for recognition of such non-B DNA structures. We also observed that p53 in vitro prefers binding to the Py-rich strand over the purine (Pu) rich strand in non-B DNA substrates formed by sequence derived from the first intron of the frataxin gene. The binding of p53 to this region was confirmed using chromatin immunoprecipitation in human Friedreich's ataxia fibroblast and adenocarcinoma cells. Altogether these observations provide further evidence that p53 binds to TNRs' non-B DNA structures.


Assuntos
DNA/química , DNA/metabolismo , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Conformação de Ácido Nucleico , Expansão das Repetições de Trinucleotídeos , Repetições de Trinucleotídeos , Proteína Supressora de Tumor p53/metabolismo , Expressão Gênica , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Pirimidinas , Proteínas Recombinantes , Proteína Supressora de Tumor p53/química
4.
PLoS One ; 11(12): e0167439, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27907175

RESUMO

Triplex DNA is implicated in a wide range of biological activities, including regulation of gene expression and genomic instability leading to cancer. The tumor suppressor p53 is a central regulator of cell fate in response to different type of insults. Sequence and structure specific modes of DNA recognition are core attributes of the p53 protein. The focus of this work is the structure-specific binding of p53 to DNA containing triplex-forming sequences in vitro and in cells and the effect on p53-driven transcription. This is the first DNA binding study of full-length p53 and its deletion variants to both intermolecular and intramolecular T.A.T triplexes. We demonstrate that the interaction of p53 with intermolecular T.A.T triplex is comparable to the recognition of CTG-hairpin non-B DNA structure. Using deletion mutants we determined the C-terminal DNA binding domain of p53 to be crucial for triplex recognition. Furthermore, strong p53 recognition of intramolecular T.A.T triplexes (H-DNA), stabilized by negative superhelicity in plasmid DNA, was detected by competition and immunoprecipitation experiments, and visualized by AFM. Moreover, chromatin immunoprecipitation revealed p53 binding T.A.T forming sequence in vivo. Enhanced reporter transactivation by p53 on insertion of triplex forming sequence into plasmid with p53 consensus sequence was observed by luciferase reporter assays. In-silico scan of human regulatory regions for the simultaneous presence of both consensus sequence and T.A.T motifs identified a set of candidate p53 target genes and p53-dependent activation of several of them (ABCG5, ENOX1, INSR, MCC, NFAT5) was confirmed by RT-qPCR. Our results show that T.A.T triplex comprises a new class of p53 binding sites targeted by p53 in a DNA structure-dependent mode in vitro and in cells. The contribution of p53 DNA structure-dependent binding to the regulation of transcription is discussed.


Assuntos
Proteínas de Ligação a DNA/genética , DNA/genética , Ativação Transcricional/genética , Proteína Supressora de Tumor p53/genética , Sítios de Ligação , DNA/química , Proteínas de Ligação a DNA/química , Humanos , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , Plasmídeos/genética , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico/genética , Deleção de Sequência/genética , Proteína Supressora de Tumor p53/química
5.
Biosci Rep ; 36(5)2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27634752

RESUMO

G-quadruplexes are four-stranded nucleic acid structures that are implicated in the regulation of transcription, translation and replication. Genome regions enriched in putative G-quadruplex motifs include telomeres and gene promoters. Tumour suppressor p53 plays a critical role in regulatory pathways leading to cell cycle arrest, DNA repair and apoptosis. In addition to transcriptional regulation mediated via sequence-specific DNA binding, p53 can selectively bind various non-B DNA structures. In the present study, wild-type p53 (wtp53) binding to G-quadruplex formed by MYC promoter nuclease hypersensitive element (NHE) III1 region was investigated. Wtp53 binding to MYC G-quadruplex is comparable to interaction with specific p53 consensus sequence (p53CON). Apart from the full-length wtp53, its isolated C-terminal region (aa 320-393) as well, is capable of high-affinity MYC G-quadruplex binding, suggesting its critical role in this type of interaction. Moreover, wtp53 binds to MYC promoter region containing putative G-quadruplex motif in two wtp53-expressing cell lines. The results suggest that wtp53 binding to G-quadruplexes can take part in transcriptional regulation of its target genes.


Assuntos
Proteínas de Ligação a DNA/genética , Quadruplex G , Proteínas Proto-Oncogênicas c-myc/genética , Proteína Supressora de Tumor p53/genética , Dicroísmo Circular , DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Células HCT116 , Humanos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína Supressora de Tumor p53/metabolismo
6.
Biochimie ; 128-129: 83-91, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27422117

RESUMO

The tumor suppressor protein p53 is a key factor in genome stability and one of the most studied of DNA binding proteins. This is the first study on the interaction of wild-type p53 with guanine quadruplexes formed by the human telomere sequence. Using electromobility shift assay and ELISA, we show that p53 binding to telomeric G-quadruplexes increases with the number of telomeric repeats. Further, p53 strongly favors G-quadruplexes folded in potassium over those formed in sodium, thus indicating the telomeric G-quadruplex conformational selectivity of p53. The presence of the quadruplex-stabilizing ligand, N-methyl mesoporphyrin IX (NMM), increases p53 recognition of G-quadruplexes in potassium. Using deletion mutants and selective p53 core domain oxidation, both p53 DNA binding domains are shown to be crucial for telomeric G-quadruplex recognition.


Assuntos
DNA/química , Quadruplex G , Telômero/química , Proteína Supressora de Tumor p53/química , Sequência de Bases , Sítios de Ligação/genética , Ligação Competitiva , Dicroísmo Circular , DNA/genética , DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Humanos , Mesoporfirinas/química , Mutação , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Potássio/química , Ligação Proteica , Sequências de Repetição em Tandem/genética , Telômero/genética , Telômero/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
7.
Chemistry ; 21(45): 16091-102, 2015 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-26377361

RESUMO

N-(3-Azidopropyl)vinylsulfonamide was developed as a new bifunctional bioconjugation reagent suitable for the cross-linking of biomolecules through copper(I)-catalyzed azide-alkyne cycloaddition and thiol Michael addition reactions under biorthogonal conditions. The reagent is easily clicked to an acetylene-containing DNA or protein and then reacts with cysteine-containing peptides or proteins to form covalent cross-links. Several examples of bioconjugations of ethynyl- or octadiynyl-modified DNA with peptides, p53 protein, or alkyne-modified human carbonic anhydrase with peptides are given.


Assuntos
Alcinos/química , Azidas/química , Anidrases Carbônicas/química , DNA/química , Indicadores e Reagentes/química , Peptídeos/química , Compostos de Sulfidrila/química , Sulfonamidas/química , Compostos de Vinila/química , Fenômenos Biológicos , Anidrases Carbônicas/metabolismo , Catálise , Química Click , Cobre/química , Reação de Cicloadição , DNA/metabolismo , Humanos , Peptídeos/metabolismo
8.
Biochem Biophys Res Commun ; 456(1): 29-34, 2015 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-25446071

RESUMO

Site-specific DNA recognition and binding activity belong to common attributes of all three members of tumor suppressor p53 family proteins: p53, p63 and p73. It was previously shown that heavy metals can affect p53 conformation, sequence-specific binding and suppress p53 response to DNA damage. Here we report for the first time that cadmium, nickel and cobalt, which have already been shown to disturb various DNA repair mechanisms, can also influence p63 and p73 sequence-specific DNA binding activity and transactivation of p53 family target genes. Based on results of electrophoretic mobility shift assay and luciferase reporter assay, we conclude that cadmium inhibits sequence-specific binding of all three core domains to p53 consensus sequences and abolishes transactivation of several promoters (e.g. BAX and MDM2) by 50µM concentrations. In the presence of specific DNA, all p53 family core domains were partially protected against loss of DNA binding activity due to cadmium treatment. Effective cadmium concentration to abolish DNA-protein interactions was about two times higher for p63 and p73 proteins than for p53. Furthermore, we detected partial reversibility of cadmium inhibition for all p53 family members by EDTA. DTT was able to reverse cadmium inhibition only for p53 and p73. Nickel and cobalt abolished DNA-p53 interaction at sub-millimolar concentrations while inhibition of p63 and p73 DNA binding was observed at millimolar concentrations. In summary, cadmium strongly inhibits p53, p63 and p73 DNA binding in vitro and in cells in comparison to nickel and cobalt. The role of cadmium inhibition of p53 tumor suppressor family in carcinogenesis is discussed.


Assuntos
Cádmio/química , Cobalto/química , Proteínas de Ligação a DNA/química , DNA/química , Proteínas de Membrana/química , Níquel/química , Proteínas Nucleares/química , Proteínas Supressoras de Tumor/química , Linhagem Celular Tumoral , Ditiotreitol/química , Ácido Edético/química , Humanos , Metais/química , Metais Pesados/química , Ligação Proteica , Estrutura Terciária de Proteína , Ativação Transcricional , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/química
9.
PLoS One ; 8(3): e59567, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23555710

RESUMO

Hot spot mutant p53 (mutp53) proteins exert oncogenic gain-of-function activities. Binding of mutp53 to DNA is assumed to be involved in mutp53-mediated repression or activation of several mutp53 target genes. To investigate the importance of DNA topology on mutp53-DNA recognition in vitro and in cells, we analyzed the interaction of seven hot spot mutp53 proteins with topologically different DNA substrates (supercoiled, linear and relaxed) containing and/or lacking mutp53 binding sites (mutp53BS) using a variety of electrophoresis and immunoprecipitation based techniques. All seven hot spot mutp53 proteins (R175H, G245S, R248W, R249S, R273C, R273H and R282W) were found to have retained the ability of wild-type p53 to preferentially bind circular DNA at native negative superhelix density, while linear or relaxed circular DNA was a poor substrate. The preference of mutp53 proteins for supercoiled DNA (supercoil-selective binding) was further substantiated by competition experiments with linear DNA or relaxed DNA in vitro and ex vivo. Using chromatin immunoprecipitation, the preferential binding of mutp53 to a sc mutp53BS was detected also in cells. Furthermore, we have shown by luciferase reporter assay that the DNA topology influences p53 regulation of BAX and MSP/MST1 promoters. Possible modes of mutp53 binding to topologically constrained DNA substrates and their biological consequences are discussed.


Assuntos
DNA Super-Helicoidal/metabolismo , Proteínas Mutantes/metabolismo , Mutação , Proteína Supressora de Tumor p53/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , DNA Super-Helicoidal/química , Regulação da Expressão Gênica/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Especificidade por Substrato , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2/genética
10.
Biochem Biophys Res Commun ; 433(4): 445-9, 2013 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-23501101

RESUMO

Cysteine oxidation and covalent modification of redox sensitive transcription factors including p53 are known, among others, as important events in cell response to oxidative stress. All p53 family proteins p53, p63 and p73 act as stress-responsive transcription factors. Oxidation of p53 central DNA binding domain destroys its structure and abolishes its sequence-specific binding by affecting zinc ion coordination at the protein-DNA interface. Proteins p63 and p73 can bind the same response elements as p53 but exhibit distinct functions. Moreover, all three proteins contain highly conserved cysteines in central DNA binding domain suitable for possible redox modulation. In this work we report for the first time the redox sensitivity of p63 and p73 core domains to a thiol oxidizing agent azodicarboxylic acid bis[dimethylamide] (diamide). Oxidation of both p63 and p73 abolished sequence-specific binding to p53 consensus sequence, depending on the agent concentration. In the presence of specific DNA all p53 family core domains were partially protected against loss of DNA binding activity due to diamide treatment. Furthermore, we detected conditional reversibility of core domain oxidation for all p53 family members and a role of zinc ions in this process. We showed that p63 and p73 proteins had greater ability to resist the diamide oxidation in comparison with p53. Our results show p63 and p73 as redox sensitive proteins with possible functionality in response of p53 family proteins to oxidative stress.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas Nucleares/química , Proteína Supressora de Tumor p53/química , Proteínas Supressoras de Tumor/química , Sequência de Bases , Cisteína/química , DNA/química , Proteínas de Ligação a DNA/síntese química , Diamida/química , Ditiotreitol/química , Ácido Edético/química , Eletroforese em Gel de Ágar , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Proteínas Nucleares/síntese química , Oxirredução , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/síntese química , Proteínas Supressoras de Tumor/síntese química , Zinco/química
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