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1.
Fish Shellfish Immunol ; 98: 130-137, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31904541

RESUMO

Transcription factor activator protein 1 (AP1) plays an irreplaceable role in the response to a variety of external stimulants, such as cellar stress, bacterial and viral infections, and inflammatory cytokines. In this study, we identified a novel AP1 gene from Macrobrachium nipponense and named it MnAP1, which has a full length of 1747 bp contains an 882 bp open reading frame, and encodes a protein with 293 amino acids. The MnAP1 protein contains Pfam and bZIP domains. MnAP1 is widely distributed in hemocytes, heart, hepatopancreas, gill, stomach, and intestinal tissues. The expression levels of MnAP1 in the gills and stomach were significantly upregulated after Vibrio parahaemolyticus and Staphylococcus aureus attacks. We studied the relationship between MnAP1 and the transcripts of antimicrobial peptides (AMPs) in gills through RNA interference. Interestingly, the regulatory effects of MnAP1 on the expression of different AMPs were different. We found that the expression levels of crustins, including Cru1, Cru3, and Cru4 in the gills were evidently decreased, whereas the synthesis of Cru5 and anti-lipopolysaccharide factors (ALF3 and ALF4) were obviously increased. We further explored the effect of MnAP1 on the expression of transcription factor relish from M. nipponense. The result showed that the knockdown of MnAP1 can remarkably upregulate the expression of MnRelish. Relish as a member of the nuclear factor κB family that regulates the expression of AMPs in the innate immunity of crustacean. Hence, we also detected the expression levels of Cru5, ALF3, and ALF4 in the gills of MnRelish-silenced prawns. The Data showed that the expression levels of these three AMPs were evidently reduced after MnRelish silencing. Our results indicated that MnAP1 plays a positive role in regulating the expression of AMPs, promotes the JNK/AP1 signaling pathway, and exerts a negative regulatory effect on the synthesis of AMPs by inhibiting the transcription of NF-κB factor in the innate immunity of M. nipponense.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Palaemonidae/genética , Fator de Transcrição AP-1/genética , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/imunologia , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Palaemonidae/imunologia , Filogenia , Alinhamento de Sequência , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/imunologia
2.
Biochemistry ; 59(4): 530-540, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31804811

RESUMO

Basic leucine-zipper (bZIP) proteins represent difficult, yet compelling, oncogenic targets since numerous cell-signaling cascades converge upon them, where they function to modulate the transcription of specific gene targets. bZIPs are widely recognized as important regulators of cellular processes that include cell proliferation, apoptosis, and differentiation. Once such validated transcriptional regulator, activator protein-1, is typically composed of heterodimers of Fos and Jun family members, with cFos-cJun being the best described. It has been shown to be key in the progression and development of a number of different diseases. As a proof-of-principle for our approach, we describe the first use of a novel combined in silico/in cellulo peptide-library screening platform that facilitates the derivation of a sequence that displays high selectivity for cJun relative to cFos, while also avoiding homodimerization. In particular, >60 million peptides were computationally screened and all potential on/off targets ranked according to predicted stability, leading to a reduced size library that was further refined by intracellular selection. The derived sequence is predicted to have limited cross-talk with a second previously derived peptide antagonist that is selective for cFos in the presence of cJun. The study provides new insight into the use of multistate screening with the ability to combine computational and intracellular approaches in evolving multiple cocompatible peptides that are capable of satisfying conflicting design requirements.


Assuntos
Biologia Computacional/métodos , Proteínas Proto-Oncogênicas c-fos/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-jun/antagonistas & inibidores , Sequência de Aminoácidos , Fatores de Transcrição de Zíper de Leucina Básica/química , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proliferação de Células , Simulação por Computador , Dimerização , Genes fos/fisiologia , Genes jun/fisiologia , Humanos , Oncogenes , Biblioteca de Peptídeos , Peptídeos/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/metabolismo
3.
J Chem Inf Model ; 59(12): 5276-5280, 2019 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-31774677

RESUMO

Transcription factor activator protein-1 (AP-1) binds to cognate DNA and regulates gene expression. In recent decades, small-molecule inhibitors have been developed for therapeutic applications that block AP-1 binding to DNA. However, the mechanism by which small molecules inhibit AP-1-DNA binding remains elusive. Here, computational studies identified a drug-binding site on the AP-1 Fos/Jun apo structure. Induced fit docking of known inhibitors, together with metadynamics simulations to identify the most plausible binding pose, showed a consensus mode of AP-1/inhibitor interaction. The in silico binding mode of the inhibitors suggests a mechanism of AP-1-DNA binding inhibition, where the inhibitors block the base-contacting residues, preclude access of DNA, and prohibit conformational changes of AP-1 upon DNA binding.


Assuntos
DNA/metabolismo , Simulação de Acoplamento Molecular , Bibliotecas de Moléculas Pequenas/farmacologia , Fator de Transcrição AP-1/metabolismo , DNA/química , Conformação de Ácido Nucleico , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Bibliotecas de Moléculas Pequenas/metabolismo , Termodinâmica , Fator de Transcrição AP-1/química
4.
Cell Rep ; 29(3): 560-572.e4, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31618627

RESUMO

DNA double-strand breaks (DSBs) are deleterious and tumorigenic but could also be essential for DNA-based processes. Yet the landscape of physiological DSBs and their role and repair are still elusive. Here, we mapped DSBs at high resolution in cancer and non-tumorigenic cells and found a transcription-coupled repair mechanism at oncogenic super-enhancers. At these super-enhancers the transcription factor TEAD4, together with various transcription factors and co-factors, co-localizes with the repair factor RAD51 of the homologous recombination pathway. Depletion of TEAD4 or RAD51 increases DSBs at RAD51/TEAD4 common binding sites within super-enhancers and decreases expression of related genes, which are mostly oncogenes. Co-localization of RAD51 with transcription factors at super-enhancers occurs in various cell types, suggesting a broad phenomenon. Together, our findings uncover a coupling between transcription and repair mechanisms at oncogenic super-enhancers, to control the hyper-transcription of multiple cancer drivers.


Assuntos
Reparo do DNA , Rad51 Recombinase/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Replicação do DNA , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Estradiol/farmacologia , Humanos , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Rad51 Recombinase/antagonistas & inibidores , Rad51 Recombinase/genética , Radiação Ionizante , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
Fish Shellfish Immunol ; 93: 597-611, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31400511

RESUMO

The transcription factor, activator protein-1 (AP-1), is a dimeric protein and a downstream member of the mitogen-activated protein kinase (MAPK) signaling pathway. It regulates a wide array of functions including, cell proliferation, survival, differentiation, response to UV-irradiation, immune responses, and inflammatory conditions. AP-1 belongs to the basic leucine zipper (bZIP) protein family, which consists of members from Jun, Fos, Maf, and ATF subfamilies. In the present study, c-Jun and c-Fos homologs were identified from a transcriptome database of Liza haematocheila and designated as Lhc-Jun and Lhc-Fos. In both sequences, the signature bZIP domain was identified and also the DNA binding sites, dimerization sites, as well as the phosphorylation sites, were found to be highly conserved through evolution. Tissue distribution analysis revealed that both Lhc-Jun and Lhc-Fos transcripts were ubiquitously expressed in all examined tissues of healthy mullets. In order to determine the transcriptional modulations of Lhc-Jun and Lhc-Fos, challenge experiments were carried out using LPS, poly I:C, and L. garvieae. The qRT-PCR analysis revealed significant upregulation of Lhc-Jun and Lhc-Fos in blood, gill, liver, and spleen. This is the first study that explores the correlation between UV-irradiation and AP-1 ortholog expression in teleosts. Also, this is the first time that the functional characterization of the teleost c-Fos ortholog has been carried out. Sub-cellular localization of Lhc-Jun and Lhc-Fos was observed in the nucleus. AP-1-Luc reporter assays revealed significant higher luciferase activities in both Lhc-Jun and Lhc-Fos proteins compared to mock controls. These results strongly suggest that Lhc-Jun and Lhc-Fos might play a significant role in Liza haematocheila immunity by regulating AP-1 promoter sequences in immune and stress-related genes.


Assuntos
Doenças dos Peixes/imunologia , Peixes/genética , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Infecções por Bactérias Gram-Positivas/imunologia , Infecções por Bactérias Gram-Positivas/veterinária , Lactococcus/fisiologia , Lipopolissacarídeos/farmacologia , Filogenia , Poli I-C/farmacologia , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/imunologia , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/imunologia , Alinhamento de Sequência/veterinária , Fator de Transcrição AP-1/química
6.
Methods Mol Biol ; 1965: 375-388, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31069687

RESUMO

The electrophoretic mobility shift assay (EMSA) is a sensitive and relatively straightforward methodology used to detect sequence-specific DNA-protein interactions. It is the fundamental procedure of several variants that allow qualitative and quantitative assessments of protein-nucleic acid complexes. Classically, nuclear proteins and DNA are combined, and the resulting mixture is electrophoretically separated in polyacrylamide or agarose gel under native conditions. The distribution within the gel is generally detected with autoradiography of the 32P-labelled DNA. The underlying principle is that nucleic acid with protein bound to it will migrate more slowly through a gel matrix than the free nucleic acid. In this chapter, a representative protocol is described that addresses specific challenges of using whole embryos as the nuclear protein source, and the most common and informative EMSA variant, the "super-shift", is also presented. The important points are underscored, and approaches for troubleshooting are explained. References are provided for alternative methods and extensions of the basic protocol.


Assuntos
DNA/metabolismo , Embrião de Mamíferos/citologia , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/metabolismo , Animais , DNA/química , Ensaio de Desvio de Mobilidade Eletroforética , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica , Radioisótopos de Fósforo/química , Ligação Proteica , Ratos
7.
Biochim Biophys Acta Rev Cancer ; 1872(1): 11-23, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31034924

RESUMO

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


Assuntos
Complexos Multiproteicos/genética , Fator de Transcrição AP-1/genética , Transcrição Genética , Ativação Transcricional/genética , Sítios de Ligação/genética , Núcleo Celular/genética , Montagem e Desmontagem da Cromatina/genética , Humanos , Complexos Multiproteicos/química , Fator de Transcrição AP-1/química , Sítio de Iniciação de Transcrição
8.
Nucleic Acids Res ; 47(4): 1774-1785, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30566668

RESUMO

CCAAT/enhancer binding proteins (C/EBPs) regulate gene expression in a variety of cells/tissues/organs, during a range of developmental stages, under both physiological and pathological conditions. C/EBP-related transcription factors have a consensus binding specificity of 5'-TTG-CG-CAA-3', with a central CpG/CpG and two outer CpA/TpG dinucleotides. Methylation of the CpG and CpA sites generates a DNA element with every pyrimidine having a methyl group in the 5-carbon position (thymine or 5-methylcytosine (5mC)). To understand the effects of both CpG and CpA modification on a centrally-important transcription factor, we show that C/EBPß binds the methylated 8-bp element with modestly-increased (2.4-fold) binding affinity relative to the unmodified cognate sequence, while cytosine hydroxymethylation (particularly at the CpA sites) substantially decreased binding affinity (36-fold). The structure of C/EBPß DNA binding domain in complex with methylated DNA revealed that the methyl groups of the 5mCpA/TpG make van der Waals contacts with Val285 in C/EBPß. Arg289 recognizes the central 5mCpG by forming a methyl-Arg-G triad, and its conformation is constrained by Val285 and the 5mCpG methyl group. We substituted Val285 with Ala (V285A) in an Ala-Val dipeptide, to mimic the conserved Ala-Ala in many members of the basic leucine-zipper family of transcription factors, important in gene regulation, cell proliferation and oncogenesis. The V285A variant demonstrated a 90-fold binding preference for methylated DNA (particularly 5mCpA methylation) over the unmodified sequence. The smaller side chain of Ala285 permits Arg289 to adopt two alternative conformations, to interact in a similar fashion with either the central 5mCpG or the TpG of the opposite strand. Significantly, the best-studied cis-regulatory elements in RNA polymerase II promoters and enhancers have variable sequences corresponding to the central CpG or reduced to a single G:C base pair, but retain a conserved outer CpA sequence. Our analyses suggest an important modification-dependent CpA recognition by basic leucine-zipper transcription factors.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/química , Metilação de DNA/genética , Proteínas de Ligação a DNA/química , DNA/genética , 5-Metilcitosina/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/genética , Sequência Conservada/genética , Ilhas de CpG/genética , Cristalografia por Raios X , Citosina/metabolismo , Proteínas de Ligação a DNA/genética , Elementos E-Box/genética , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Conformação Proteica , Timina/metabolismo , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/genética
9.
Sci Rep ; 8(1): 11409, 2018 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-30061607

RESUMO

Ixodes scapularis ticks transmit several pathogens to humans including rickettsial bacterium, Anaplasma phagocytophilum. Here, we report that A. phagocytophilum uses tick transcriptional activator protein-1 (AP-1) as a molecular switch in the regulation of arthropod antifreeze gene, iafgp. RNAi-mediated silencing of ap-1 expression significantly affected iafgp gene expression and A. phagocytophilum burden in ticks upon acquisition from the murine host. Gel shift assays provide evidence that both the bacterium and AP-1 influences iafgp promoter and expression. The luciferase assays revealed that a region of approximately 700 bp upstream of the antifreeze gene is sufficient for AP-1 binding to promote iafgp gene expression. Furthermore, survival assays revealed that AP-1-deficient ticks were more susceptible to cold in comparison to the mock controls. In addition, this study also indicates arthropod AP-1 as a global regulator for some of the tick genes critical for A. phagocytophilum survival in the vector. In summary, our study defines a novel mode of arthropod signaling for the survival of both rickettsial pathogen and its medically important vector in the cold.


Assuntos
Adaptação Fisiológica , Anaplasma phagocytophilum/fisiologia , Temperatura Baixa , Ixodes/metabolismo , Fator de Transcrição AP-1/metabolismo , Sequência de Aminoácidos , Animais , Pareamento de Bases/genética , Sequência de Bases , Feminino , Regulação da Expressão Gênica , Genoma de Inseto , Ixodes/genética , Ixodes/microbiologia , Larva/microbiologia , Camundongos , Modelos Biológicos , Fases de Leitura Aberta/genética , Filogenia , Regiões Promotoras Genéticas/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/genética
10.
Nucleic Acids Res ; 45(19): 11425-11436, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-28981703

RESUMO

The transcription factor, activator protein-1 (AP-1), binds to cognate DNA under redox control; yet, the underlying mechanism has remained enigmatic. A series of crystal structures of the AP-1 FosB/JunD bZIP domains reveal ordered DNA-binding regions in both FosB and JunD even in absence DNA. However, while JunD is competent to bind DNA, the FosB bZIP domain must undergo a large conformational rearrangement that is controlled by a 'redox switch' centered on an inter-molecular disulfide bond. Solution studies confirm that FosB/JunD cannot undergo structural transition and bind DNA when the redox-switch is in the 'OFF' state, and show that the mid-point redox potential of the redox switch affords it sensitivity to cellular redox homeostasis. The molecular and structural studies presented here thus reveal the mechanism underlying redox-regulation of AP-1 Fos/Jun transcription factors and provide structural insight for therapeutic interventions targeting AP-1 proteins.


Assuntos
DNA/química , Domínios Proteicos , Proteínas Proto-Oncogênicas c-jun/química , Fator de Transcrição AP-1/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Moleculares , Oxirredução , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/química , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Homologia de Sequência de Aminoácidos , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo
11.
Sci Rep ; 7(1): 7148, 2017 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-28769048

RESUMO

AP-1 is a clathrin adaptor recruited to the trans-Golgi Network where it can interact with specific signals found in the cytosolic tail of cargo proteins to incorporate them into clathrin-coated vesicles for trafficking. The small G protein Arf1 regulates the spatiotemporal recruitment of AP-1 and also drives a conformational change favoring an interaction with cargo proteins. A recent crystal structure and in vitro experiments highlighted potential residues mediating the AP-1/Arf1 interaction and the unlocking of the complex. We have used bioluminescence resonance energy transfer (BRET) to study the Arf1/AP-1 interaction and AP-1 conformational changes in vivo. We identified novel residues required for this interaction in addition to those predicted in the crystal structure. We also studied the conformational changes in AP-1 driven by Arf1 in live cells and found that opening of the complex is prerequisite for oligomerization. Using Arf1 knockout cells generated by CRISPR/Cas9, we demonstrated that residue 172 in Arf1 is necessary for AP-1 activation and is required for the efficient sorting of the lysosomal protein prosaposin. We have used BRET to study the in vivo activation of AP-1. The advantages of BRET include expressing full-length proteins in their native environment that have been fully post-translationally modified.


Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator 1 de Ribosilação do ADP/química , Fator 1 de Ribosilação do ADP/genética , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Linhagem Celular , Vesículas Revestidas por Clatrina/metabolismo , Expressão Gênica , Genes Reporter , Células HEK293 , Humanos , Mutação , Ligação Proteica , Multimerização Proteica , Transporte Proteico , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/genética , Rede trans-Golgi/metabolismo
12.
Nucleic Acids Res ; 45(14): 8596-8608, 2017 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-28591827

RESUMO

The glucocorticoid receptor (GR) is a ligand-regulated transcription factor that controls the expression of extensive gene networks, driving both up- and down-regulation. GR utilizes multiple DNA-binding-dependent and -independent mechanisms to achieve context-specific transcriptional outcomes. The DNA-binding-independent mechanism involves tethering of GR to the pro-inflammatory transcription factor activator protein-1 (AP-1) through protein-protein interactions. This mechanism has served as the predominant model of GR-mediated transrepression of inflammatory genes. However, ChIP-seq data have consistently shown GR to occupy AP-1 response elements (TREs), even in the absence of AP-1. Therefore, the current model is insufficient to explain GR action at these sites. Here, we show that GR regulates a subset of inflammatory genes in a DNA-binding-dependent manner. Using structural biology and biochemical approaches, we show that GR binds directly to TREs via sequence-specific contacts to a GR-binding sequence (GBS) half-site found embedded within the TRE motif. Furthermore, we show that GR-mediated transrepression observed at TRE sites to be DNA-binding-dependent. This represents a paradigm shift in the field, showing that GR uses multiple mechanisms to suppress inflammatory gene expression. This work further expands our understanding of this complex multifaceted transcription factor.


Assuntos
Regulação da Expressão Gênica , Inflamação/genética , Receptores de Glucocorticoides/genética , Elementos de Resposta/genética , Fator de Transcrição AP-1/genética , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular Tumoral , Cristalografia por Raios X , DNA/química , DNA/genética , DNA/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/metabolismo , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/metabolismo
13.
Nucleic Acids Res ; 45(5): 2503-2515, 2017 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-28158710

RESUMO

T: Activator protein 1 (AP-1) is a transcription factor that recognizes two versions of a 7-base pair response element, either 5΄- GAG CA-3΄ or 5΄- GAG CA-3΄ (where M = 5-methylcytosine). These two elements share the feature that 5-methylcytosine and thymine both have a methyl group in the same position, 5-carbon of the pyrimidine, so each of them has two methyl groups at nucleotide positions 1 and 5 from the 5΄ end, resulting in four methyl groups symmetrically positioned in duplex DNA. Epstein-Barr Virus Zta is a key transcriptional regulator of the viral lytic cycle that is homologous to AP-1. Zta recognizes several methylated Zta-response elements, including meZRE1 (5΄- GAG C A-3΄) and meZRE2 (5΄- GAG G A-3΄), where a methylated cytosine occupies one of the inner thymine residues corresponding to the AP-1 element, resulting in the four spatially equivalent methyl groups. Here, we study how AP-1 and Zta recognize these methyl groups within their cognate response elements. These methyl groups are in van der Waals contact with a conserved di-alanine in AP-1 dimer (Ala265 and Ala266 in Jun), or with the corresponding Zta residues Ala185 and Ser186 (via its side chain carbon Cß atom). Furthermore, the two ZRE elements differ at base pair 6 (C:G versus G:C), forming a pseudo-symmetric sequence (meZRE1) or an asymmetric sequence (meZRE2). In vitro DNA binding assays suggest that Zta has high affinity for all four sequences examined, whereas AP-1 has considerably reduced affinity for the asymmetric sequence (meZRE2). We ascribe this difference to Zta Ser186 (a unique residue for Zta) whose side chain hydroxyl oxygen atom interacts with the two half sites differently, whereas the corresponding Ala266 of AP-1 Jun protein lacks such flexibility. Our analyses demonstrate a novel mechanism of 5mC/T recognition in a methylation-dependent, spatial and sequence-specific approach by basic leucine-zipper transcriptional factors.


Assuntos
Metilação de DNA , Proteínas Proto-Oncogênicas c-jun/química , Elementos de Resposta , Transativadores/química , 5-Metilcitosina/química , Pareamento de Bases , Sítios de Ligação , DNA/química , DNA/metabolismo , Humanos , Ligação Proteica , Multimerização Proteica , Proteínas Proto-Oncogênicas c-jun/metabolismo , Timina/química , Transativadores/metabolismo , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/metabolismo
14.
Oncotarget ; 8(1): 883-899, 2017 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-27903990

RESUMO

Increasing clinical and experimental studies have demonstrated that refractory chronic inflammation will result in malignant tumor and anti-angiogenic therapy may be an effective way to thwart the progression. Baicalein, one of the major active flavanoids found in Scutellaria baicalensis Georgi, has been exhibited potent anti-inflammation and anti-tumor effects by reducing angiogenesis. However, the exact mechanism of baicalein on endothelial cells in inflammatory microenvironment was not clear yet. Here, we investigated the anti-angiogenic effect of baicalein by incubating human umbilical vein endothelial cells (HUVECs) with THP-1 conditioned medium in vitro. The tube formation of HUVECs and microvessel outgrowth of rat aorta were attenuated, as well as the number of newly formed blood vessels in chicken chorioallantoic membrane (CAM) was reduced by baicalein. This anti-angiogenic effect was mainly on account of the inhibited motility, migration and invasion of HUVECs. In addition, mechanistic studies showed that baicalein could bind to AP-1 directly and the expression of c-Jun and c-Fos in HUVECs was reduced, accompanied by their increased proteasomal degradation. Besides, baicalein suppressed the nuclear translation, heterodimer formation and DNA binding affinity of c-Jun and c-Fos. What's more, the anti-angiogenic effect of baicalein was further confirmed by matrigel plug assay in vivo. Taken together, our study demonstrated that baicalein could exert its anti-angiogenic effect in the inflammation microenvironment via inhibiting the transcriptional activity of AP-1, which suggested that baicalein might be an alternative treatment against refractory chronic inflammation.


Assuntos
Flavanonas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neovascularização Patológica/genética , Fator de Transcrição AP-1/genética , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Embrião de Galinha , Flavanonas/química , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Neovascularização Patológica/metabolismo , Ligação Proteica , Ratos , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/metabolismo
15.
J Biol Chem ; 291(29): 14963-72, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27226616

RESUMO

Nearly all members of the inwardly rectifying potassium (Kir) channel family share a cytoplasmic domain structure that serves as an unusual AP-1 clathrin adaptor-dependent Golgi export signal in one Kir channel, Kir2.1 (KCNJ2), raising the question whether Kir channels share a common Golgi export mechanism. Here we explore this idea, focusing on two structurally and functionally divergent Kir family members, Kir2.3 (KCNJ4) and Kir4.1/5.1 (KCNJ10/16), which have ∼50% amino identity. We found that Golgi export of both channels is blocked upon siRNA-mediated knockdown of the AP-1 γ subunit, as predicted for the common AP-1-dependent trafficking process. A comprehensive mutagenic analysis, guided by homology mapping in atomic resolution models of Kir2.1, Kir2.3, and Kir4.1/5.1, identified a common structure that serves as a recognition site for AP-1 binding and governs Golgi export. Larger than realized from previous studies with Kir2.1, the signal is created by a patch of residues distributed at the confluence of cytoplasmic N and C termini. The signal involves a stretch of hydrophobic residues from the C-terminal region that form a hydrophobic cleft, an adjacent cluster of basic residues within the N terminus, and a potential network of salt bridges that join the N- and C-terminal poles together. Because patch formation and AP-1 binding are dependent on proper folding of the cytoplasmic domains, the signal provides a common quality control mechanism at the Golgi for Kir channels. These findings identify a new proteostatic mechanism that couples protein folding of channels to forward trafficking in the secretory pathway.


Assuntos
Complexo de Golgi/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/química , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Fator de Transcrição AP-1/metabolismo , Complexo 1 de Proteínas Adaptadoras/química , Complexo 1 de Proteínas Adaptadoras/genética , Complexo 1 de Proteínas Adaptadoras/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Humanos , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Canais de Potássio Corretores do Fluxo de Internalização/genética , Conformação Proteica , Sinais Direcionadores de Proteínas/genética , Transporte Proteico , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/genética
16.
Nucleic Acids Res ; 44(6): e51, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-26635393

RESUMO

Protein binding to DNA is a fundamental process in gene regulation. Methodologies such as ChIP-Seq and mapping of DNase I hypersensitive sites provide global information on this regulation in vivo In vitro methodologies provide valuable complementary information on protein-DNA specificities. However, current methods still do not measure absolute binding affinities. There is a real need for large-scale quantitative protein-DNA affinity measurements. We developed QPID, a microfluidic application for measuring protein-DNA affinities. A single run is equivalent to 4096 gel-shift experiments. Using QPID, we characterized the different affinities of ATF1, c-Jun, c-Fos and AP-1 to the CRE consensus motif and CRE half-site in two different genomic sequences on a single device. We discovered that binding of ATF1, but not of AP-1, to the CRE half-site is highly affected by its genomic context. This effect was highly correlated with ATF1 ChIP-seq and PBM experiments. Next, we characterized the affinities of ATF1 and ATF3 to 128 genomic CRE and CRE half-site sequences. Our affinity measurements explained that in vivo binding differences between ATF1 and ATF3 to CRE and CRE half-sites are partially mediated by differences in the minor groove width. We believe that QPID would become a central tool for quantitative characterization of biophysical aspects affecting protein-DNA binding.


Assuntos
Fator 1 Ativador da Transcrição/química , Proteínas Quinases JNK Ativadas por Mitógeno/química , Microfluídica/métodos , Proteínas Proto-Oncogênicas c-fos/química , Elementos de Resposta , Fator de Transcrição AP-1/química , Fator 1 Ativador da Transcrição/genética , Fator 1 Ativador da Transcrição/metabolismo , Sítios de Ligação , DNA/química , DNA/genética , DNA/metabolismo , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Cinética , Microfluídica/instrumentação , Dados de Sequência Molecular , Motivos de Nucleotídeos , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo
17.
Sci Rep ; 5: 14408, 2015 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-26404494

RESUMO

Activator protein-1 (AP-1) is an important bZIP transcription factor that regulates a series of physiological processes by specifically activating transcription of several genes, and one of its well-chartered functions in mammals is participating in bone mineralization. We isolated and cloned the complete cDNA of a Jun/AP-1 homolog from Pinctada fucata and called it Pf-AP-1. Pf-AP-1 had a highly conserved bZIP region and phosphorylation sites compared with those from mammals. A tissue distribution analysis showed that Pf-AP-1 was ubiquitously expressed in P. fucata and the mRNA level of Pf-AP-1 is extremely high in mantle. Pf-AP-1 expression was positively associated with multiple biomineral proteins in the mantle. The luciferase reporter assay in a mammalian cell line showed that Pf-AP-1 significantly up-regulates the transcriptional activity of the promoters of KRMP, Pearlin, and Prisilkin39. Inhibiting the activity of Pf-AP-1 depressed the expression of multiple matrix proteins. Pf-AP-1 showed a unique expression pattern during shell regeneration and pearl sac development, which was similar to the pattern observed for biomineral proteins. These results suggest that the Pf-AP-1 AP-1 homolog is an important transcription factor that regulates transcription of several biomineral proteins simultaneously and plays a role in P. fucata biomineralization, particularly during pearl and shell formation.


Assuntos
Calcificação Fisiológica/genética , Regulação da Expressão Gênica , Pinctada/genética , Pinctada/metabolismo , Fator de Transcrição AP-1/metabolismo , Ativação Transcricional , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Regulação da Expressão Gênica no Desenvolvimento , Dados de Sequência Molecular , Fases de Leitura Aberta , Especificidade de Órgãos/genética , Filogenia , Transporte Proteico , RNA Mensageiro/genética , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/genética , Transcriptoma
18.
Fish Shellfish Immunol ; 45(2): 927-32, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26093208

RESUMO

The transcription factor activator protein-1 (AP-1) is an important gene expression regulator with typical Jun and region-leucine zipper (bZIP) domains and can respond to a plethora of physiological and pathological stimulus. In this study, we identified a novel AP-1 gene in Apostichopus japonicus by transcriptome sequencing and RACE approaches (designated as AjAP-1). The full-length of AjAP-1 was of 2944 bp including a 5' untranslated region (UTR) of 201 bp, a 3' UTR of 1753 bp and a putative open reading frame of 990 bp encoding a polypeptide of 329 amino acid residues. Two representative domains of Jun and bZIP as well as two nuclear localization signals (NLSs) were also detected in deduced amino acid of AjAP-1. Spatial distribution expression indicated that AjAP-1 was ubiquitously expressed in all examined tissues with predominant expression in the body wall, moderate in the tube feet, respiratory tree and colemocytes and slightly weak in the intestine and longitudinal muscle. Time-course expression analysis in intestine and coelomocytes revealed that AjAP-1 both reached its peak expression at 4 h after Vibrio splendidus challenge with a 2.6 and 8.2-fold increase compared to their control groups, respectively. Taken together, all these results suggested that AjAP-1 was a novel immune factor and might be involved in the processes of anti-bacteria response in sea cucumber.


Assuntos
Regulação da Expressão Gênica , Stichopus/genética , Stichopus/imunologia , Fator de Transcrição AP-1/genética , Vibrio/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/microbiologia , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Stichopus/metabolismo , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/metabolismo
19.
PLoS One ; 10(4): e0123070, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25875593

RESUMO

We collected mobility and interaction maps of c-Fos-eGFP and c-Jun-mRFP1 transcription factors within living cell nuclei. c-Fos dimerizes with c-Jun to form the transcription activator protein-1 (AP-1) which binds to the specific recognition site. To monitor this process, we used fluorescence cross-correlation spectroscopy on a single plane illumination microscope (SPIM-FCCS), which provides diffusion coefficient and protein-protein interaction data in the whole image plane simultaneously, instead of just one point on conventional confocal FCS. We find a strong correlation between diffusional mobility and interaction: regions of strong interaction show slow mobility. Controls containing either an eGFP-mRFP dimer, separately expressing eGFP and mRPF, or c-Fos-eGFP and c-Jun-mRFP1 mutants lacking dimerization and DNA-binding domains, showed no such correlation. These results extend our earlier findings from confocal FCCS to include spatial information.


Assuntos
Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Sítios de Ligação , Núcleo Celular/metabolismo , DNA/química , DNA/metabolismo , Dimerização , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Mutagênese , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas c-fos/química , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-jun/química , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo
20.
Int J Mol Sci ; 15(8): 13802-16, 2014 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-25110868

RESUMO

S100A12 is involved in the inflammatory response and is considered an important marker for many inflammatory diseases in humans. Our previous studies indicated that the S100A12 gene was abundant in the immune tissues of pigs and was significantly upregulated during infection with Haemophilus parasuis (HPS) or porcine circovirus type 2 (PCV2). In this study, the mechanism of transcriptional regulation of S100A12 was investigated in pigs. Our results showed that S100A12, CCAAT/enhancer-binding protein beta (C/EBPß) and activator protein-1 (AP-1) genes were up-regulated in PK-15 (ATCC, CCL-33) cells when treated with LPS or Poly I: C. Additionally, the promoter activity and expression level of the S100A12 gene were significantly upregulated when C/EBPß or AP-1 were overexpressed. We utilized electromobility shift assays (EMSA) to confirm that C/EBPß and AP-1 could directly bind the S100A12 gene promoter. We also found that the transcriptional activity and expression levels of C/EBPß and AP-1 could positively regulate each other. Furthermore, the promoter activity of the S100A12 gene was higher when C/EBPß and AP-1 were cotransfected than when they were transfected individually. We concluded that the S100A12 gene was cooperatively and positively regulated by C/EBPß and AP-1 in pigs. Our study offers new insight into the transcriptional regulation of the S100A12 gene.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas S100/genética , Fator de Transcrição AP-1/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Proteína beta Intensificadora de Ligação a CCAAT/química , Proteína beta Intensificadora de Ligação a CCAAT/genética , Linhagem Celular , Inflamação/genética , Inflamação/patologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lipopolissacarídeos/toxicidade , Dados de Sequência Molecular , Poli I-C/farmacologia , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas S100/metabolismo , Suínos , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/genética , Ativação Transcricional , Regulação para Cima/efeitos dos fármacos
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