RESUMO
GLIS1 has multiple roles in embryonic development and in deriving induced pluripotent stem cells by aiding signaling pathways and chromatin assembly. An inexpensive and simple method to produce human GLIS1 protein from Escherichia coli (E. coli) is demonstrated in this study. Various parameters such as codon usage bias, E. coli strains, media, induction conditions (such as inducer concentration, cell density, time, and temperature), and genetic constructs were investigated to obtain soluble expression of human GLIS1 protein. Using identified expression conditions and an appropriate genetic construct, the human GLIS1 protein was homogeneously purified (purity > 90%) under native conditions. Importantly, the purified protein has upheld a stable secondary structure, as demonstrated by circular dichroism spectroscopy. To the best of our knowledge, this is the first study to report the ideal expression conditions of human GLIS1 protein in E. coli to achieve soluble expression and purification under native conditions, upholding its stable secondary structure post-purification. The biological activity of the purified GLIS1 fusion protein was further assessed in MDA-MB-231 cells. This biologically active human GLIS1 protein potentiates new avenues to understand its molecular mechanisms in different cellular functions in various cancers and in the generation of induced pluripotent stem cells.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Escherichia coli/genética , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Linhagem Celular Tumoral , Movimento Celular , Clonagem Molecular , Códon , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos , Humanos , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
RNA Polymerase II (Pol II) transcriptional recycling is a mechanism for which the required factors and contributions to overall gene expression levels are poorly understood. We describe an in vitro methodology facilitating unbiased identification of putative RNA Pol II transcriptional recycling factors and quantitative measurement of transcriptional output from recycled transcriptional components. Proof-of-principle experiments identified PAF1 complex components among recycling factors and detected defective transcriptional output from Pol II recycling following PAF1 depletion. Dynamic ChIP-seq confirmed PAF1 silencing triggered defective Pol II recycling in human cells. Prostate tumors exhibited enhanced transcriptional recycling, which was attenuated by antibody-based PAF1 depletion. These findings identify Pol II recycling as a potential target in cancer and demonstrate the applicability of in vitro and cellular transcription assays to characterize Pol II recycling in other disease states.
Assuntos
RNA Polimerase II/metabolismo , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Células HeLa , Humanos , Masculino , Neoplasias , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
A lot of researches have been focused on the evolution and function of MYB transcription factors (TFs). For revealing the formation of petunia flower color diversity, MYB gene family in petunia was identified and analyzed. In this study, a total of 155 MYB genes, including 40 1R-MYBs, 106 R2R3-MYBs, 7 R1R2R3-MYBs and 2 4R-MYBs, have been identified in the Petunia axillaris genome. Most R2R3 genes contain three exons and two introns, whereas the number of PaMYB introns varies from 0 to 12. The R2R3-MYB members could be divided into 28 subgroups. Analysis of gene structure and protein motifs revealed that members within the same subgroup presented similar exon/intron and motif organization, further supporting the results of phylogenetic analysis. Genes in subgroup 10, 11 and 21 were mainly expressed in petal, not in vegetative tissues. Genes in subgroup 9, 19, 25 and 27 expressed in all tissues, but the expression patterns of each gene were different. According to the promoter analysis, five R2R3-MYB and two MYB-related genes contained MBSI cis-element, which was involved in flavonoid biosynthetic regulation. PaMYB100/DPL has been reported to positively regulate to pigmentation. However, although PaMYB82, PaMYB68 and Pa1RMYB36 contained MBSI cis-element, their function in flavonoid biosynthesis has not been revealed. Consistent with existing knowledge, PaMYBs in subgroup 11 had similar function to AtMYBs in subgroup 6, genes in which played an important role in anthocyanin biosynthesis. In addition, PaMYB1 and PaMYB40 belonged to P9 (S7) and were potentially involved in regulation of flavonoid synthesis in petunia vegetative organs. This work provides a comprehensive understanding of the MYB gene family in petunia and lays a significant foundation for future studies on the function and evolution of MYB genes in petunia.
Assuntos
Antocianinas/biossíntese , Genes myb/genética , Petunia/genética , Fatores de Transcrição/genética , Antocianinas/genética , Proteínas de Arabidopsis/genética , Flores/genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta/genética , Família Multigênica/genética , Filogenia , Pigmentação/genética , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/classificação , Fatores de Transcrição/isolamento & purificaçãoRESUMO
The PLATZ (plant AT-rich protein and zinc-binding protein) transcription factor family is a class of plant-specific zinc-dependent DNA-binding proteins. PLATZ has essential roles in seed endosperm development, as well as promoting cell proliferation duration in the earlier stages of the crops. In the present study, 62 TaPLATZ genes were identified from the wheat genome, and they were unequally distributed on 15 chromosomes. According to the phylogenetic analysis, 62 TaPLATZ genes were classified into six groups, including two groups that were unique in wheat. Members in the same groups shared similar exon-intron structures. The polyploidization, together with genome duplication of wheat, plays a crucial role in the expansion of the TaPLATZs family. Transcriptome data indicated a distinct divergence expression pattern of TaPLATZ genes that could be clustered into four modules. The TaPLATZs in Module b possessed a seed-specific expression pattern and displayed obvious high expression in the earlier development stage of seeds. Subcellular localization data of TaPLATZs suggesting that they likely perform a function as a conventional transcription factor. This study provides insight into understanding the structure divergence, evolutionary features, expression profiles, and potential function of PLATZ in wheat.
Assuntos
Evolução Molecular , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Triticum/genética , Cromossomos de Plantas/genética , Regulação da Expressão Gênica de Plantas/genética , Genoma de Planta , Família Multigênica/genética , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Fatores de Transcrição/isolamento & purificação , Triticum/químicaRESUMO
The MYB transcription factors are involved in the regulation of plant secondary metabolism, cell development and morphogenesis, and stress response. Here, a full-length, 816-bp NtMYB4a cDNA, which encodes a protein comprising 271 amino acids, was isolated from tobacco leaves. Phylogenetic analysis revealed that NtMYB4a is most similar to Nicotiana. attenuata MYB4, followed by Eriobotrya japonica MYB4, and NtMYB4a clustered with transcriptional activators rather than repressors. Subcellular localization assays showed that NtMYB4 localized in the nucleus, membrane, and cytoplasm. Expression analyses revealed differential expression of NtMYB4a among different tissues and organs and between different developmental stages, with most expression occurring in the stems and leaves during the full-bloom stage. Moreover, NtMYB4a expression was induced by cold, NaCl, PEG, abscisic acid, methyl jasmonate, and dark stressors, and the expression patterns and maximum expression levels varied with the type of stress. Overexpression of NtMYB4a upregulated NtPAL, Nt4CL, NtCHS, NtCHI, NtF3H, NtDFR, NtANS, and NtUFGT, which resulted in increased anthocyanin content in the tobacco corolla and darker colors. However, CRISPR/Cas9-mediated knockout of NtMYB4a downregulated NtPAL, NtC4H, Nt4CL, NtCHS, NtCHI, NtF3H, NtANS, and NtUFGT, which resulted in reduced anthocyanin content, and lighter corolla colors. These results indicated that NtMYB4a positively regulates anthocyanin biosynthesis and is involved in abiotic stress responses in tobacco plants.
Assuntos
Nicotiana/metabolismo , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismo , Ácido Abscísico/metabolismo , Sequência de Aminoácidos , Antocianinas/biossíntese , Regulação da Expressão Gênica de Plantas/genética , Filogenia , Folhas de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Estresse Fisiológico/genética , Nicotiana/genética , Fatores de Transcrição/genética , Ativação Transcricional/genéticaRESUMO
Neurofibromatosis type 1 (NF1) displays overlapping phenotypes with other neurocutaneous diseases such as Legius Syndrome. Here, we present results obtained using a next generation sequencing (NGS) panel including NF1, NF2, SPRED1, SMARCB1, and LZTR1 genes on Ion Torrent. Together with NGS, the Multiplex Ligation-Dependent Probe Amplification Analysis (MLPA) method was performed to rule out large deletions/duplications in NF1 gene; we validated the MLPA/NGS approach using Sanger sequencing on DNA or RNA of both positive and negative samples. In our cohort, a pathogenic variant was found in 175 patients; the pathogenic variant was observed in NF1 gene in 168 cases. A SPRED1 pathogenic variant was also found in one child and in a one year old boy, both NF2 and LZTR1 pathogenic variants were observed; in addition, we identified five LZTR1 pathogenic variants in three children and two adults. Six NF1 pathogenic variants, that the NGS analysis failed to identify, were detected on RNA by Sanger. NGS allows the identification of novel mutations in five genes in the same sequencing run, permitting unambiguous recognition of disorders with overlapping phenotypes with NF1 and facilitating genetic counseling and a personalized follow-up.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neurofibromatose 1/genética , Neurofibromina 1/genética , Neurofibromina 2/genética , Fatores de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal/isolamento & purificação , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mutação/genética , Neurilemoma/diagnóstico , Neurilemoma/genética , Neurilemoma/patologia , Neurofibromatoses/diagnóstico , Neurofibromatoses/genética , Neurofibromatoses/patologia , Neurofibromatose 1/diagnóstico , Neurofibromatose 1/patologia , Neurofibromina 1/isolamento & purificação , Neurofibromina 2/isolamento & purificação , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Fatores de Transcrição/isolamento & purificação , Adulto JovemRESUMO
BRG1/SMARCA4 and its paralog BRM/SMARCA2 are the ATPase subunits of human SWI/SNF chromatin remodeling complexes. These multisubunit assemblies can act as either tumor suppressors or drivers of cancer, and inhibiting both BRG1 and BRM, is emerging as an effective therapeutic strategy in diverse cancers. BRG1 and BRM contain a BRK domain. The function of this domain is unknown, but it is often found in proteins involved in transcription and developmental signaling in higher eukaryotes, in particular in proteins that remodel chromatin. We report the NMR structure of the BRG1 BRK domain. It shows similarity to the glycine-tyrosine-phenylalanine (GYF) domain, an established protein-protein interaction module. Computational peptide-binding-site analysis of the BRK domain identifies a binding site that coincides with a highly conserved groove on the surface of the protein. This sets the scene for experiments to elucidate the role of this domain, and evaluate the potential of targeting it for cancer therapy.
Assuntos
Montagem e Desmontagem da Cromatina , DNA Helicases/química , Proteínas Nucleares/química , Fatores de Transcrição/química , Cromatina/química , Cromatina/metabolismo , DNA Helicases/genética , DNA Helicases/isolamento & purificação , Humanos , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Ligação Proteica , Conformação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Domínios de Homologia de srcRESUMO
Liver sinusoidal endothelial cells play a key role maintaining the hepatic homeostasis, the disruption of which is associated with such end-stage liver diseases as hepatocellular carcinoma and cirrhosis. In the present study we investigated the role of brahma-related gene 1 (BRG1), a chromatin remodeling protein, in regulating endothelial transcription and the implication in liver fibrosis. We report that endothelial-specific deletion of BRG1 in mice attenuated liver fibrosis induced by injection with thioacetamide (TAA). Coincidently, alleviation of liver fibrosis as a result of endothelial BRG1 deletion was accompanied by an up-regulation of eNOS activity and NO bioavailability. In cultured endothelial cells, exposure to lipopolysaccharide (LPS) suppressed eNOS activity whereas BRG1 depletion with small interfering RNA restored eNOS-dependent NO production. Further analysis revealed that BRG1 was recruited to the caveolin-1 (CAV1) promoter by Sp1 and activated transcription of CAV1, which in turn inhibited eNOS activity. Mechanistically, BRG1 interacted with the H3K4 trimethyltransferase MLL1 to modulate H3K4 trimethylation surrounding the CAV1 promoter thereby contributing to LPS-induced CAV1 activation. In conclusion, our data unveil a novel role for BRG1 in the regulation of endothelial function and liver fibrosis.
Assuntos
DNA Helicases/metabolismo , Células Endoteliais/metabolismo , Fibrose/metabolismo , Fígado/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , DNA Helicases/deficiência , DNA Helicases/isolamento & purificação , Fibrose/induzido quimicamente , Humanos , Fígado/efeitos dos fármacos , Camundongos , Óxido Nítrico/análise , Proteínas Nucleares/deficiência , Proteínas Nucleares/isolamento & purificação , Tioacetamida , Fatores de Transcrição/deficiência , Fatores de Transcrição/isolamento & purificaçãoRESUMO
In barley and other higher plants, phosphate homeostasis is maintained by a regulatory network involving the PHO2 (PHOSPHATE2) encoding ubiquitin-conjugating (UBC) E2 enzyme, the PHR1 (PHOSPHATE STARVATION RESPONSE 1) transcription factor (TF), IPS1 (INDUCED BYPHOSPHATESTARVATION1) RNA, and miR399. During phosphate ion (Pi) deprivation, PHR1 positively regulates MIR399 expression, after transcription and processing mature miR399 guides the Ago protein to the 5'-UTR of PHO2 transcripts. Non-coding IPS1 RNA is highly expressed during Pi starvation, and the sequestration of miR399 molecules protects PHO2 mRNA from complete degradation. Here, we reveal new cis- and trans-regulatory elements that are crucial for efficient PHO2 gene expression in barley. We found that the 5'-UTR of PHO2 contains two PHR1 binding sites (P1BSs) and one Pi-responsive PHO element. Using a yeast one-hybrid (Y1H) assay, we identified two candidate proteins that might mediate this transcriptional regulation: a barley PHR1 ortholog and a TF containing an uncharacterized MYB domain. Additional results classified this new potential TF as belonging to the APL (ALTERED PHLOEM DEVELOPMENT) protein family, and we observed its nuclear localization in barley protoplasts. Pi starvation induced the accumulation of barley APL transcripts in both the shoots and roots. Interestingly, the deletion of the P1BS motif from the first intron of the barley 5'-UTR led to a significant increase in the transcription of a downstream ß-glucuronidase (GUS) reporter gene in tobacco leaves. Our work extends the current knowledge about putative cis- and trans-regulatory elements that may affect the expression of the barley PHO2 gene.
Assuntos
Regiões 5' não Traduzidas/fisiologia , Hordeum/genética , Hordeum/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Regulação da Expressão Gênica de Plantas , Glucuronidase/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Fosfatos/metabolismo , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , RNA Mensageiro/metabolismo , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Background: In Saccharomyces cerevisiae, Msn2, which acts as a key transcription factor downstream the MAPKHOG cascade pathway, also regulates the expression of genes related to stress responses. However, little is known about the regulation mechanisms of the transcription factor in Setosphaeria turcica. Results: In this study, a zinc finger DNA-binding protein, designated as StMSN2, was cloned from S. turcica. Sequencing results showed that StMSN2 had a 1752 bp open reading frame (ORF), which was interrupted by an intron (135 bp) and encoded a putative 538-amino acid protein. Phylogenetic analysis further revealed that StMsn2 was more closely related to Msn2 of Aspergillus parasiticus. StMSN2 was cloned into the pET-28a vector with His (Histidine) tags and induced with 1 mM IPTG (isopropyl-ß-D-thiogalactoside) at 37°C. The recombinant His-tagged StMsn2 was purified, and a band of size approximately 58.8 kDa was obtained. The high specificity of the polyclonal antibody Msn2-2 was detected with the StMsn2 protein from S. turcica and prokaryotic expression system, respectively. Conclusions: A new gene, named StMSN2, with 1617 bp ORF was cloned from S. turcica and characterized using bioinformatics methods. StMsn2 was expressed and purified in a prokaryotic system. A polyclonal antibody, named Msn2-2, against StMsn2 with high specificity was identified.
Assuntos
Doenças das Plantas , Ascomicetos/genética , Ascomicetos/patogenicidade , Fatores de Transcrição/isolamento & purificação , Ascomicetos/metabolismo , Estresse Fisiológico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Transporte , Expressão Gênica , Western Blotting , Fases de Leitura Aberta , Dedos de Zinco , Clonagem Molecular , Zea mays , Escherichia coli , Helminthosporium , EpitoposRESUMO
The scaffolds of polyketides are constructed via assembly of extender units based on malonyl-CoA and its derivatives that are substituted at the C2-position with diverse chemical functionality. Subsequently, a transcription-factor-based biosensor for malonyl-CoA has proven to be a powerful tool for detecting malonyl-CoA, facilitating the dynamic regulation of malonyl-CoA biosynthesis and guiding high-throughput engineering of malonyl-CoA-dependent processes. Yet, a biosensor for the detection of malonyl-CoA derivatives has yet to be reported, severely restricting the application of high-throughput synthetic biology approaches to engineering extender unit biosynthesis and limiting the ability to dynamically regulate the biosynthesis of polyketide products that are dependent on such α-carboxyacyl-CoAs. Herein, the FapR biosensor was re-engineered and optimized for a range of mCoA concentrations across a panel of E. coli strains. The effector specificity of FapR was probed by cell-free transcription-translation, revealing that a variety of non-native and non-natural acyl-thioesters are FapR effectors. This FapR promiscuity proved sufficient for the detection of the polyketide extender unit methylmalonyl-CoA in E. coli, providing the first reported genetically encoded biosensor for this important metabolite. As such, the previously unknown broad effector promiscuity of FapR provides a platform to develop new tools and approaches that can be leveraged to overcome limitations of pathways that construct diverse α-carboxyacyl-CoAs and those that are dependent on them, including biofuels, antibiotics, anticancer drugs, and other value-added products.
Assuntos
Técnicas Biossensoriais/métodos , Malonil Coenzima A/análise , Policetídeo Sintases/metabolismo , Engenharia de Proteínas/métodos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Malonil Coenzima A/metabolismo , Redes e Vias Metabólicas , Policetídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Biologia Sintética , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismoRESUMO
Bromodomain PHD finger transcription factor (BPTF), a bromodomain-containing protein, plays a crucial role in the regulation of downstream gene expression through the specific recognition of lysine acetylation on bulk histones. The dysfunction of BPTF is closely involved with the development and progression of many human diseases, especially cancer. Therefore, BPTF bromodomain has become a promising drug target for epigenetic cancer therapy. However, unlike BET family inhibitors, few BPTF bromodomain inhibitors have been reported. In this study, by integrating docking-based virtual screening with biochemical analysis, we identified a novel selective BPTF bromodomain inhibitor DCB29 with the IC50 value of 13.2⯱â¯1.6⯵M by homogenous time-resolved fluorescence resonance energy transfer (HTRF) assays. The binding between DCB29 and BPTF was confirmed by NMR and SPR. Molecular docking disclosed that DCB29 occupied the pocket of acetylated H4 peptide substrate and provided detailed SAR explanations for its derivatives. Collectively, DCB29 presented great potential as a powerful tool for BPTF-related biological research and further medicinal chemistry optimization.
Assuntos
Álcoois/farmacologia , Benzamidas/farmacologia , Descoberta de Drogas , Fatores de Transcrição/antagonistas & inibidores , Álcoois/síntese química , Álcoois/química , Benzamidas/síntese química , Benzamidas/química , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Transferência Ressonante de Energia de Fluorescência , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Domínios Proteicos/efeitos dos fármacos , Relação Estrutura-Atividade , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismoRESUMO
BAF250a and BAF250b are subunits of the SWI/SNF chromatin-remodeling complex that recruit the complex to chromatin allowing transcriptional activation of several genes. Despite being the central subunits of the SWI/SNF complex, the structural and functional annotation of BAF250a/b remains poorly understood. BAF250a (nearly 2200 residues protein) harbors an N-terminal DNA binding ARID (~110 residues) and a C-terminal folded region (~250 residues) of unknown structure and function, recently annotated as BAF250_C. Using hydrophobic core analysis, fold prediction and comparative modeling, here we have defined a domain boundary and associate a ß-catenin like ARM-repeat fold to the C-terminus of BAF250a that encompass BAF250_C. The N-terminal DNA-binding ARID is found in diverse domain combinations in proteins imparting unique functions. We used a comparative sequence analysis based approach to study the ARIDs from diverse domain contexts and identified conserved residue positions that are important to preserve its core structure. Supporting this, mutation of one such conserved residue valine, at position 1067, to glycine, resulted in destabilization, loss of structural integrity and DNA binding affinity of ARID. Additionally, we identified a set of conserved and surface-exposed residues unique to the ARID when it co-occurs with the ARM repeat containing BAF250_C in BAF250a. Several of these residues are found mutated in somatic cancers. We predict that these residues in BAF250a may play important roles in mediating protein-DNA and protein-protein interactions in the BAF complex.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Complexos Multienzimáticos/genética , Proteínas Nucleares/genética , Domínios Proteicos/genética , Fatores de Transcrição/genética , Diferenciação Celular , Biologia Computacional , Proteínas de Ligação a DNA , Conjuntos de Dados como Assunto , Glicina/genética , Simulação de Dinâmica Molecular , Complexos Multienzimáticos/química , Mutagênese Sítio-Dirigida , Mutação , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/química , Proteínas Nucleares/isolamento & purificação , Ligação Proteica/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/isolamento & purificação , Valina/genéticaRESUMO
The human polybromo-1 protein (BAF180) is a known driver mutation in clear cell renal cell carcinoma, where it is mutated in approximately 40% of cases. BAF180 is the chromatin-targeting subunit of the PBAF complex. BAF180 has six bromodomains, two BAH domains, and one HMG box. Bromodomains are known to recognize acetylated-lysines on histones and play a role in nucleosome recognition. BAH domains are required for ubiquitination of PCNA, a key regulator of DNA damage. The putative HMG box, if functional, may be involved in DNA-binding. While the binding specificities of individual bromodomains have been studied by our lab and others, the results have failed to reach a consensus. The acetyl-histone binding features of the full-length protein is unknown and is the motivation for this work. The hypothetical HMG and BAH domains have not been studied and the actual function of these regions is currently unknown. Thus, the precise interactions of this large and complex protein are not well-studied. Advances in understanding this large protein have been hindered by the inability to express and purify recombinant full-length BAF180 protein. Currently, only phenomenological studies using BAF180 expressed in mammalian cells have been conducted. Here, we report the successful expression, purification of full-length biologically active BAF180 protein using the GAP promoter in the heterologous host Pichia pastoris. The ability to express full-length and mutated BAF180 will allow for biophysical binding studies. Knowledge of the binding interactions is critical for us to understand the role of BAF180 in cancer development and its progression.
Assuntos
Histonas/metabolismo , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Pichia/genética , Fatores de Transcrição/genética , Sítios de Ligação , Clonagem Molecular , Proteínas de Ligação a DNA , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histonas/química , Histonas/genética , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/isolamento & purificação , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Pichia/metabolismo , Regiões Promotoras Genéticas , Análise Serial de Proteínas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismoRESUMO
The aim of this study was to determine the anticancer potential of Leonurus sibiricus extract derived from in vitro transgenic roots transformed by Agrobacetrium rhizogenes with AtPAP1 transcriptional factor, and that of transformed roots without construct, on grade IV human glioma cells and the U87MG cell line, and attempt to characterize the mechanism involved in this process. The anticancer effect induced by the tested extracts was associated with DNA damage, PARP cleavage/increased H2A.X histone levels and UHRF-1/DNMT1 down-regulation of mRNA levels. Additionally, we demonstrated differences in the content of compounds in the tested extracts by HPLC analysis with ATPAP1 construct and without. Both the tested extracts showed anticancer properties and the better results were observed for AtPAP1 with transcriptional factor root extract; this effect could be ascribed to the presence of higher condensed phenolic acids such as neochlorogenic acid, chlorogenic acids, ferulic acid, caffeic acid and p-coumaric acid. Further studies with AtPAP1 (with the transcriptional factor from Arabidopisi thaliana) root extract which showed better activities in combination with anticancer drugs are needed.
Assuntos
Proteínas de Arabidopsis/toxicidade , Dano ao DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Leonurus , Extratos Vegetais/toxicidade , Raízes de Plantas , Fatores de Transcrição/toxicidade , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas de Arabidopsis/isolamento & purificação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Dano ao DNA/fisiologia , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Epigênese Genética/fisiologia , Humanos , Extratos Vegetais/isolamento & purificação , Fatores de Transcrição/isolamento & purificaçãoRESUMO
Lens epithelium-derived growth factor (LEDGF)/p75 is the dominant binding partner of HIV-1 integrase in human cells. The crystal structure of the HIV integrase-binding domain (IBD) of LEDGF has been determined in the absence of ligand. IBD was overexpressed in Escherichia coli, purified and crystallized by sitting-drop vapour diffusion. X-ray diffraction data were collected at Diamond Light Source to a resolution of 2.05â Å. The crystals belonged to space group P21, with eight polypeptide chains in the asymmetric unit arranged as an unusual octamer composed of four domain-swapped IBD dimers. IBD exists as a mixture of monomers and dimers in concentrated solutions, but the dimers are unlikely to be biologically relevant.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Integrase de HIV/química , Integrase de HIV/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/isolamento & purificação , Sequência de Aminoácidos , Domínio Catalítico , Cristalização , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , Fatores de Transcrição/isolamento & purificaçãoRESUMO
Chimeric transcription factor E2A-PBX1 induces the development of acute lymphoblastic B-cell leukemia in children. Using a transgenic mouse model, we previously demonstrated that homeobox (HOX) gene HOXA9 genetically interact with E2A-PBX1 gene in the development of B-cell leukemia in mice. HOXA9 itself is a potent oncogene resulting in myeloid leukemia when overexpressed, which is strongly accelerated by its collaborator Meis1. HOX, PBX1 and MEIS1 proteins have been shown to form hetero dimeric or trimeric complexes in different combinations. Cooperative interaction between PBX1 and HOX proteins enhances their DNA binding specificity, essential for HOX dependent developmental programs. PBX1 is retained in E2A-PBX1, and thus the strong transcriptional activator properties of E2A-PBX1 may lead to aberrant activation of normally repressed targets of HOX-PBX complexes. However, although there is evidence that E2A-PBX1 could bind to HOX and MEIS1 proteins it is still unclear whether such complexes are actually required for leukemic transformation or whether E2A-PBX1 and HOXA9 are each part of larger protein complexes acting in independent complementing oncogenic pathways. In this study we aim to search for other HOXA9 and E2A-PBX1 interacting proteins. To identify novel proteins interacting with human E2A-PBX1 or HOXA9 we used tandem affinity purification (TAP) of protein complexes from 697 pre-B leukemic and HeLa cell lines transduced to express E2A-PBX1 or HOXA9, respectively, with covalently attached FLAG/HA peptides. The protein composition of each complex was determined using tandem mass-spectrometry. In the E2A-PBX1 containing complex we identified lymphoid transcription factor IKAROS, chromatin remodeling factors of SWI/SNF family while multiple subunits of translation initiation factor eIF3, E3 ubiquitin ligase UBR5 emerged from the HOXA9 complex as potential critical protein partners. This is the first time the protein partners of either E2A-PBX1 or HOXA9 oncoproteins were identified using an unbiased biochemical approach. The identification of translation initiation factors associated with HOXA9 might indicate a novel function for HOX proteins independent of their transcriptional activity.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Homeodomínio/genética , Leucemia de Células B/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Regulação Leucêmica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/isolamento & purificação , Leucemia de Células B/patologia , Proteína Meis1 , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Ligação Proteica , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas/metabolismo , Espectrometria de Massas em Tandem , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/isolamento & purificaçãoRESUMO
Proper chromatin regulation is central to genome function and maintenance. The group III chromodomain-helicase-DNA-binding (CHD) family of ATP-dependent chromatin remodeling enzymes, comprising CHD6, CHD7, CHD8, and CHD9, has well-documented roles in transcription regulation, impacting both organism development and disease etiology. These four enzymes are similar in their constituent domains, but they fill surprisingly non-redundant roles in the cell, with deficiencies in individual enzymes leading to dissimilar disease states such as CHARGE syndrome or autism spectrum disorders. The mechanisms explaining their divergent, non-overlapping functions are unclear. In this study, we performed an in-depth biochemical analysis of purified CHD6, CHD7, and CHD8 and discovered distinct differences in chromatin remodeling specificities and activities among them. We report that CHD6 and CHD7 both bind with high affinity to short linker DNA, whereas CHD8 requires longer DNA for binding. As a result, CHD8 slides nucleosomes into positions with more flanking linker DNA than CHD7. Moreover, we found that, although CHD7 and CHD8 slide nucleosomes, CHD6 disrupts nucleosomes in a distinct non-sliding manner. The different activities of these enzymes likely lead to differences in chromatin structure and, thereby, transcriptional control, at the enhancer and promoter loci where these enzymes bind. Overall, our work provides a mechanistic basis for both the non-redundant roles and the diverse mutant disease states of these enzymes in vivo.
Assuntos
Trifosfato de Adenosina/metabolismo , Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Nucleossomos/enzimologia , Fatores de Transcrição/metabolismo , Animais , Transporte Biológico , DNA/química , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/isolamento & purificação , DNA Recombinante/química , DNA Recombinante/metabolismo , DNA Viral/química , DNA Viral/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Células HeLa , Humanos , Hidrólise , Cinética , Peso Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/isolamento & purificação , Nucleossomos/metabolismo , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificaçãoRESUMO
The histone demethylase PHF8 catalyzes demethylation of mono- and di-methylated Lys9 on histone H3 (H3K9me1/2), and is a transcriptional activator involved in the development and cancer. Affinity and specificity of PHF8 towards H3K9me2 is affected by interaction with both the catalytic domain and a PHD reader domain. The latter specifically recognizes tri-methylated Ly4 on histone H3. A fragment of the histone H3 tail with tri-methylated Lys4 was used as a template for the structure-based design of a cyclic, cell-penetrating peptide that exhibits micromolar binding affinity to PHF8 in biochemical assays. The inhibitor has significantly lower affinity towards KDM2 enzymes (the phylogenetically closest subfamily), and to KDM3 and KDM6 subfamilies. Selectivity is only marginal towards an enzyme from the KDM4 family, which shares histone tail specificity with PHF8. It is a substrate of KDM5B, thus implying that the free Nâ terminus is not part of the KDM5 enzyme substrate recognition machinery. The cyclic peptide's ability to penetrate cells is achieved by incorporation of a sequence derived from HIV Tat. The derived cyclic peptide can be used as a starting compound in the search for potent and selective PHF8 inhibitors.
Assuntos
Peptídeos Penetradores de Células/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Peptídeos Penetradores de Células/síntese química , Peptídeos Penetradores de Células/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Células HEK293 , Histona Desmetilases/isolamento & purificação , Histona Desmetilases/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismoRESUMO
Transcription factors (TFs) bind to specific double-stranded DNA (dsDNA) sequences in the regulatory regions of genes to regulate the process of gene transcription. Their expression levels sensitively reflect cell developmental situation and disease state. TFs have become potential diagnostic markers and therapeutic targets of cancers and some other diseases. Hence, high sensitive detection of TFs is of vital importance for early diagnosis of diseases and drugs development. The traditional exonucleases-assisted signal amplification methods suffered from the false positives caused by incomplete digestion of excess recognition probes. Herein, based on a new recognition way-colocalization recognition (CR)-activated dual signal amplification, an ultrasensitive fluorescent detection strategy for TFs was developed. TFs-induced the colocalization of three split recognition components resulted in noticeable increases of local effective concentrations and hybridization of three split components, which activated the subsequent cascade signal amplification including strand displacement amplification (SDA) and exponential rolling circle amplification (ERCA). This strategy eliminated the false positive influence and achieved ultra-high sensitivity towards the purified NF-κB p50 with detection limit of 2.0×10-13M. Moreover, NF-κB p50 can be detected in as low as 0.21ngµL-1 HeLa cell nuclear extracts. In addition, this proposed strategy could be used for the screening of NF-κB p50 activity inhibitors and potential anti-NF-κB p50 drugs. Finally, our proposed strategy offered a potential method for reliable detection of TFs in medical diagnosis and treatment research of cancers and other related diseases.