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1.
J Phys Chem Lett ; 11(3): 864-868, 2020 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-31940206

RESUMO

The transcriptional adaptor zinc-binding 1 (TAZ1) domain of the transcriptional coactivator CBP/P300 and two disordered peptides, HIF-1α and CITED2, form a delicate protein switch that regulates cellular hypoxic response. In hypoxia, HIF-1α binds TAZ1 to control the transcription of adaptive genes critical for the recovery from hypoxic stress. CITED2 acts as the negative feedback regulator to rapidly displace HIF-1α and efficiently attenuate the hypoxic response. Though CITED2 and HIF-1α have the same dissociation constant (Kd = 10 nM) in their binary complexes with TAZ1, CITED2 is much more competitive than HIF-1α upon binding the same target TAZ1 in ternary ( Berlow et al. Nature 2017 , 543 , 447 - 451 ). Here we demonstrate that a simple coarse-grained model can recapitulate this negative allosteric effect and provide detailed physical insights into the displacement mechanism. We find that long-range electrostatic forces are essential for the efficient displacement of HIF-1α by CITED2. The strong electrostatic interactions between CITED2 and TAZ1, along with the unique binding mode, make CITED2 much more competitive than HIF-1α in binding TAZ1.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Proteínas Repressoras/química , Transativadores/química , Fatores de Transcrição de p300-CBP/química , Regulação Alostérica , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Eletricidade Estática
2.
Emerg Microbes Infect ; 9(1): 5-19, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31859607

RESUMO

Transition metals participate in numerous enzymatic reactions and they are essential for survival in all living organisms. For this reason, bacterial pathogens have evolved dedicated machineries to effectively compete with their hosts and scavenge metals at the site of infection. In this study, we investigated the mechanisms controlling metal acquisition in the emerging human pathogen Mycoplasma genitalium. We observed a robust transcriptional response to metal starvation, and many genes coding for predicted lipoproteins and ABC-transporters were significantly up-regulated. Transcriptional analysis of a mutant strain lacking a metalloregulator of the Fur family revealed the activation of a full operon encoding a putative metal transporter system and a gene coding for a Histidine-rich lipoprotein (Hrl). We recognized a conserved sequence with dyad symmetry within the promoter region of the Fur-regulated genes. Mutagenesis of the predicted Fur operator within the hrl promoter abrogated Fur- and metal-dependent expression of a reporter gene. Metal starvation still impelled a strong transcriptional response in the fur mutant, demonstrating the existence of Fur-independent regulatory pathways controlling metal homeostasis. Finally, analysis of metal accumulation in the wild-type strain and the fur mutant by ICP-MS revealed an important role of Fur in nickel acquisition.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Ferro/metabolismo , Mycoplasma genitalium/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Genética , 2,2'-Dipiridil/farmacologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Homeostase , Modelos Moleculares , Mycoplasma genitalium/genética , Regiões Promotoras Genéticas , Proteômica , Proteínas Repressoras/química , Transcrição Genética/efeitos dos fármacos
3.
PLoS Comput Biol ; 15(12): e1007562, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31860667

RESUMO

Pseudomonas aeruginosa, a main cause of human infection, can gain resistance to the antibiotic aztreonam through a mutation in NalD, a transcriptional repressor of cellular efflux. Here we combine computational analysis of clinical isolates, transcriptomics, metabolic modeling and experimental validation to find a strong association between NalD mutations and resistance to aztreonam-as well as resistance to other antibiotics-across P. aeruginosa isolated from different patients. A detailed analysis of one patient's timeline shows how this mutation can emerge in vivo and drive rapid evolution of resistance while the patient received cancer treatment, a bone marrow transplantation, and antibiotics up to the point of causing the patient's death. Transcriptomics analysis confirmed the primary mechanism of NalD action-a loss-of-function mutation that caused constitutive overexpression of the MexAB-OprM efflux system-which lead to aztreonam resistance but, surprisingly, had no fitness cost in the absence of the antibiotic. We constrained a genome-scale metabolic model using the transcriptomics data to investigate changes beyond the primary mechanism of resistance, including adaptations in major metabolic pathways and membrane transport concurrent with aztreonam resistance, which may explain the lack of a fitness cost. We propose that metabolic adaptations may allow resistance mutations to endure in the absence of antibiotics and could be targeted by future therapies against antibiotic resistant pathogens.


Assuntos
Farmacorresistência Bacteriana/genética , Mutação com Perda de Função , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Antibacterianos/farmacologia , Aztreonam/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biologia Computacional , Perfilação da Expressão Gênica , Genes Bacterianos , Humanos , Redes e Vias Metabólicas , Modelos Biológicos , Modelos Moleculares , Filogenia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Análise de Sistemas
4.
BMC Plant Biol ; 19(1): 395, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31510917

RESUMO

BACKGROUND: Leaf morphology and spikelet number are two important traits associated with grain yield. To understand how genes coordinating with sink and sources of cereal crops is important for grain yield improvement guidance. Although many researches focus on leaf morphology or grain number in rice, the regulating molecular mechanisms are still unclear. RESULTS: In this study, we identified a prohibitin complex 2α subunit, NAL8, that contributes to multiple developmental process and is required for normal leaf width and spikelet number at the reproductive stage in rice. These results were consistent with the ubiquitous expression pattern of NAL8 gene. We used genetic complementation, CRISPR/Cas9 gene editing system, RNAi gene silenced system and overexpressing system to generate transgenic plants for confirming the fuctions of NAL8. Mutation of NAL8 causes a reduction in the number of plastoglobules and shrunken thylakoids in chloroplasts, resulting in reduced cell division. In addition, the auxin levels in nal8 mutants are higher than in TQ, while the cytokinin levels are lower than in TQ. Moreover, RNA-sequencing and proteomics analysis shows that NAL8 is involved in multiple hormone signaling pathways as well as photosynthesis in chloroplasts and respiration in mitochondria. CONCLUSIONS: Our findings provide new insights into the way that NAL8 functions as a molecular chaperone in regulating plant leaf morphology and spikelet number through its effects on mitochondria and chloroplasts associated with cell division.


Assuntos
Oryza/genética , Proteínas de Plantas/genética , Proteínas Repressoras/genética , Sequência de Aminoácidos , Cloroplastos/fisiologia , Inflorescência/genética , Inflorescência/crescimento & desenvolvimento , Mitocôndrias/fisiologia , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Alinhamento de Sequência
5.
Int J Mol Sci ; 20(15)2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31390828

RESUMO

Protein arginine methyltransferase 1 (PRMT1) can catalyze protein arginine methylation by transferring the methyl group from S-adenosyl-L-methionine (SAM) to the guanidyl nitrogen atom of protein arginine, which influences a variety of biological processes. The dysregulation of PRMT1 is involved in a diverse range of diseases, including cancer. Therefore, there is an urgent need to develop novel and potent PRMT1 inhibitors. In the current manuscript, a series of 1-substituted 1H-tetrazole derivatives were designed and synthesized by targeting at the substrate arginine-binding site on PRMT1, and five compounds demonstrated significant inhibitory effects against PRMT1. The most potent PRMT1 inhibitor, compound 9a, displayed non-competitive pattern with respect to either SAM or substrate arginine, and showed the strong selectivity to PRMT1 compared to PRMT5, which belongs to the type II PRMT family. It was observed that the compound 9a inhibited the functions of PRMT1 and relative factors within this pathway, and down-regulated the canonical Wnt/ß-catenin signaling pathway. The binding of compound 9a to PRMT1 was carefully analyzed by using molecular dynamic simulations and binding free energy calculations. These studies demonstrate that 9a was a potent PRMT1 inhibitor, which could be used as lead compound for further drug discovery.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/química , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/química , Tetrazóis/química , Tetrazóis/farmacologia , Sítios de Ligação , Relação Dose-Resposta a Droga , Humanos , Metilação , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade
6.
Nucleic Acids Res ; 47(16): 8913-8925, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31392336

RESUMO

The development of synthetic biological systems requires modular biomolecular components to flexibly alter response pathways. In previous studies, we have established a module-swapping design principle to engineer allosteric response and DNA recognition properties among regulators in the LacI family, in which the engineered regulators served as effective components for implementing new cellular behavior. Here we introduced this protein engineering strategy to two regulators in the TetR family: TetR (UniProt Accession ID: P04483) and MphR (Q9EVJ6). The TetR DNA-binding module and the MphR ligand-binding module were used to create the TetR-MphR. This resulting hybrid regulator possesses DNA-binding properties of TetR and ligand response properties of MphR, which is able to control gene expression in response to a molecular signal in cells. Furthermore, we studied molecular interactions between the TetR DNA-binding module and MphR ligand-binding module by using mutant analysis. Together, we demonstrated that TetR family regulators contain discrete and functional modules that can be used to build biological components with novel properties. This work highlights the utility of rational design as a means of creating modular parts for cell engineering and introduces new possibilities in rewiring cellular response pathways.


Assuntos
DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Repressoras/química , Fatores de Transcrição/química , Regulação Alostérica , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
Nucleic Acids Res ; 47(18): 9696-9707, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31400115

RESUMO

Ubiquitous Structural Maintenance of Chromosomes (SMC) complexes use a proteinaceous ring-shaped architecture to organize and individualize chromosomes, thereby facilitating chromosome segregation. They utilize cycles of adenosine triphosphate (ATP) binding and hydrolysis to transport themselves rapidly with respect to DNA, a process requiring protein conformational changes and multiple DNA contact sites. By analysing changes in the architecture and stoichiometry of the Escherichia coli SMC complex, MukBEF, as a function of nucleotide binding to MukB and subsequent ATP hydrolysis, we demonstrate directly the formation of dimer of MukBEF dimer complexes, dependent on dimeric MukF kleisin. Using truncated and full length MukB, in combination with MukEF, we show that engagement of the MukB ATPase heads on nucleotide binding directs the formation of dimers of heads-engaged dimer complexes. Complex formation requires functional interactions between the C- and N-terminal domains of MukF with the MukB head and neck, respectively, and MukE, which organizes the complexes by stabilizing binding of MukB heads to MukF. In the absence of head engagement, a MukF dimer bound by MukE forms complexes containing only a dimer of MukB. Finally, we demonstrate that cells expressing MukBEF complexes in which MukF is monomeric are Muk-, with the complexes failing to associate with chromosomes.


Assuntos
Proteínas Cromossômicas não Histona/química , Proteínas de Escherichia coli/genética , Proteínas Repressoras/genética , Proteínas Cromossômicas não Histona/genética , Cromossomos/química , Cromossomos/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Ligação Proteica , Proteínas Repressoras/química
8.
BMC Mol Cell Biol ; 20(1): 30, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387520

RESUMO

BACKGROUND: Several human cancers, especially cervical cancer are caused by the infection of high risk strains of human papillomaviruses (HPV), notably HPV16. It is implicated that the oncoprotein E6 expressed from HPV, is inhibiting the apoptotic pathway by binding to adaptor molecule FADD (Fas-associated death domain). Inhibiting E6 interactions with FADD could provide a promising treatment for cervical cancer. There are few small molecules reported to inhibit such interactions. However, the FADD binding site information on the HPV E6 is not currently available. This binding site information may provide an opportunity to design new small molecule inhibitors to treat E6 mediated cancers. In this study we report the possible binding pocket on HPV16 E6 oncoprotein by using activity data of reported inhibitors through a stepwise molecular modeling approach. RESULTS: Blind docking and removing duplicates followed by visual inspection to determine ligand-receptor interactions provided 68 possible binding sites on the E6 protein. Individual docking of all known inhibitors lead to the identification of 28 pockets having some kind of correlation with their activity data. It was also observed that several of these pockets overlapped with each other, having some amino acids in common. Amino acids Leu50 and Cys51 were identified as key E6 residues for high affinity ligand binding which are seen in most of these pockets. In most cases, ligands demonstrated a hydrogen bond interaction with Cys51. Ala61, Arg131 and Gln107 were also frequently observed showing interactions among these pockets. A few amino acids unique to each ligand were also identified representing additional interactions at the receptor site. CONCLUSIONS: After determining receptor-ligand interactions between E6 oncoprotein and the six known inhibitors, the amino acids Cys51, Leu50, Arg102, Arg131, Leu67, Val62, and Gln107 were identified to have importance in E6 inhibition. It was generally observed that Leu50 and Cys51 are necessary for high binding affinity with Cys51 being essential for hydrogen bonding. This study identified a potential binding pocket for the E6 inhibitors. Identification of the ligand binding pocket helps to design novel inhibitors of HPV16 E6 oncoprotein as a promising treatment for cervical cancer.


Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Proteínas Oncogênicas Virais/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Aminoácidos/metabolismo , Sítios de Ligação , Flavonóis/química , Flavonóis/farmacologia , Concentração Inibidora 50 , Ligantes , Simulação de Acoplamento Molecular , Proteínas Oncogênicas/química , Proteínas Oncogênicas Virais/química , Proteínas Repressoras/química
9.
Biomol NMR Assign ; 13(2): 357-360, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31388821

RESUMO

TGIF1 is an essential regulator of cell differentiation in various biological processes, and is associated with holoprosencephaly and many cancers. The C-terminal domain of TGIF1 that was originally defined as repressive domain 2 can interact with a variety of proteins, such as transcription factor Smad2 and co-repressor Sin3A, to mediate the regulative roles of TGIF1 in diverse cell signaling pathways. However, the recognition mechanism of TGIF1 C-terminal domain for different interacting proteins remains unknown. Here, we report the nearly complete 1H, 13C, and 15N backbone and side chain resonance assignments of TGIF1 C-terminal domain (residues 256-375), laying a foundation for further research on the structure-function relationship of TGIF1.


Assuntos
Proteínas de Homeodomínio/química , Ressonância Magnética Nuclear Biomolecular , Proteínas Repressoras/química , Proteínas de Homeodomínio/metabolismo , Humanos , Fosforilação , Domínios Proteicos , Proteínas Repressoras/metabolismo
10.
Redox Biol ; 26: 101293, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31421411

RESUMO

Sulfane sulfur species including hydrogen polysulfide and organic persulfide are newly recognized normal cellular components, and they participate in signaling and protect cells from oxidative stress. Their production has been extensively studied, but their removal is less characterized. Herein, we showed that sulfane sulfur at high levels was toxic to Escherichia coli under both anaerobic and aerobic conditions. OxyR, a well-known regulator against H2O2, also sensed sulfane sulfur, as revealed via mutational analysis, constructed gene circuits, and in vitro gene expression. Hydrogen polysulfide modified OxyR at Cys199 to form a persulfide OxyR C199-SSH, and the modified OxyR activated the expression of thioredoxin 2 and glutaredoxin 1. The two enzymes are known to reduce sulfane sulfur to hydrogen sulfide. Bioinformatics analysis indicated that OxyR homologs are widely present in bacteria, including obligate anaerobic bacteria. Thus, the OxyR sensing of sulfane sulfur may represent a preserved mechanism for bacteria to deal with sulfane sulfur stress.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas Repressoras/genética , Enxofre/metabolismo , Cromatografia Líquida , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/metabolismo , Mutação , Oxirredução , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Enxofre/farmacologia , Espectrometria de Massas em Tandem , Tiorredoxinas/metabolismo , Ativação Transcricional
11.
Nat Commun ; 10(1): 3414, 2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31363087

RESUMO

Despite the vast number of modification sites mapped within mRNAs, known examples of consequential mRNA modifications remain rare. Here, we provide multiple lines of evidence to show that Ime4p, an N6-methyladenosine (m6A) methyltransferase required for meiosis in yeast, acts by methylating a site in the 3' UTR of the mRNA encoding Rme1p, a transcriptional repressor of meiosis. Consistent with this mechanism, genetic analyses reveal that IME4 functions upstream of RME1. Transcriptome-wide, RME1 is the primary message that displays both increased methylation and reduced expression in an Ime4p-dependent manner. In yeast strains for which IME4 is dispensable for meiosis, a natural polymorphism in the RME1 promoter reduces RME1 transcription, obviating the requirement for methylation. Mutation of a single m6A site in the RME1 3' UTR increases Rme1p repressor production and reduces meiotic efficiency. These results reveal the molecular and physiological consequences of a modification in the 3' UTR of an mRNA.


Assuntos
Regiões 3' não Traduzidas , Adenosina/análogos & derivados , RNA Mensageiro/genética , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Adenosina/metabolismo , Regulação Fúngica da Expressão Gênica , Meiose , Metilação , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
12.
EMBO J ; 38(18): e101220, 2019 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-31403225

RESUMO

Krüppel-associated box (KRAB)-containing zinc finger proteins (KZFPs) are encoded in the hundreds by the genomes of higher vertebrates, and many act with the heterochromatin-inducing KAP1 as repressors of transposable elements (TEs) during early embryogenesis. Yet, their widespread expression in adult tissues and enrichment at other genetic loci indicate additional roles. Here, we characterized the protein interactome of 101 of the ~350 human KZFPs. Consistent with their targeting of TEs, most KZFPs conserved up to placental mammals essentially recruit KAP1 and associated effectors. In contrast, a subset of more ancient KZFPs rather interacts with factors related to functions such as genome architecture or RNA processing. Nevertheless, KZFPs from coelacanth, our most distant KZFP-encoding relative, bind the cognate KAP1. These results support a hypothetical model whereby KZFPs first emerged as TE-controlling repressors, were continuously renewed by turnover of their hosts' TE loads, and occasionally produced derivatives that escaped this evolutionary flushing by development and exaptation of novel functions.


Assuntos
Placenta/metabolismo , Proteínas Repressoras/metabolismo , Proteína 28 com Motivo Tripartido/metabolismo , Animais , Elementos de DNA Transponíveis , Evolução Molecular , Feminino , Proteínas de Peixes/metabolismo , Peixes/metabolismo , Células HEK293 , Humanos , Gravidez , Mapas de Interação de Proteínas , Proteínas Repressoras/química , Dedos de Zinco
13.
BMC Mol Cell Biol ; 20(1): 35, 2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31426742

RESUMO

BACKGROUND: HPV16 infection is one of the main risk factors involved in the development of cervical cancer, mainly due to the high oncogenic potential of the viral proteins E6 and E7, which are involved in the different processes of malignant transformation. There is a broad spectrum of intratypical variation of E6, which is reflected in its high diversity, biological behavior, global distribution and risk of causing cervical cancer. Experimental studies have shown that the intratypical variants of the protein E6 from the European variants (E-G350, E-A176/G350, E-C188/G350) and Asian-American variants (AAa and AAc), are capable of inducing the differential expression of genes involved in the development of cervical cancer. RESULTS: An in silico analysis was performed to characterize the molecular effects of these variations using the structure of the HPV16 E6 oncoprotein (PDB: 4XR8; chain H) as a template. In particular, we evaluated the 3D structures of the intratypical variants by structural alignment, ERRAT, Ramachandran plots and prediction of protein disorder, which was further validated by molecular dynamics simulations. Our results, in general, showed no significant changes in the protein 3D structure. However, we observed subtle changes in protein physicochemical features and structural disorder in the N- and C-termini. CONCLUSIONS: Our results showed that mutations in the viral oncogene E6 of six high-risk HPV16 variants are effectively neutral and do not cause significant structural changes except slight variations of structural disorder. As structural disorder is involved in rewiring protein-protein interactions, these results suggest a differential pattern of interaction of E6 with the target protein P53 and possibly different patterns of tumor aggressiveness associated with certain types of variants of the E6 oncoprotein.


Assuntos
Simulação por Computador , Variação Genética , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Proteínas Repressoras/química , Proteínas Repressoras/genética , Sequência de Aminoácidos , Humanos , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína
14.
Oxid Med Cell Longev ; 2019: 7318796, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31428229

RESUMO

Ankrd2 (ankyrin repeats containing domain 2) or Arpp (ankyrin repeat, PEST sequence, and proline-rich region) is a member of the muscle ankyrin repeat protein family. Ankrd2 is mostly expressed in skeletal muscle, where it plays an intriguing role in the transcriptional response to stress induced by mechanical stimulation as well as by cellular reactive oxygen species. Our studies in myoblasts from Emery-Dreifuss muscular dystrophy 2, a LMNA-linked disease affecting skeletal and cardiac muscles, demonstrated that Ankrd2 is a lamin A-binding protein and that mutated lamins found in Emery-Dreifuss muscular dystrophy change the dynamics of Ankrd2 nuclear import, thus affecting oxidative stress response. In this review, besides describing the latest advances related to Ankrd2 studies, including novel discoveries on Ankrd2 isoform-specific functions, we report the main findings on the relationship of Ankrd2 with A-type lamins and discuss known and potential mechanisms involving defective Ankrd2-lamin A interplay in the pathogenesis of muscular laminopathies.


Assuntos
Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Emery-Dreifuss/patologia , Proteínas Nucleares/metabolismo , Estresse Oxidativo , Proteínas Repressoras/metabolismo , Humanos , Lamina Tipo A/metabolismo , Mecanotransdução Celular , Proteínas Musculares/química , Distrofia Muscular de Emery-Dreifuss/metabolismo , Miocárdio/metabolismo , Proteínas Nucleares/química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/química
15.
Talanta ; 204: 569-575, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31357335

RESUMO

Generation of a combinatorial gradient for multiple chemicals is essential for studies of biochemical stimuli, chemoattraction, protein crystallization and others. While currently available platforms require complex design/settings to obtain a double-gradient chemical matrix, we herein report for the first time a simple triple-gradient matrix (TGM) device for efficient screening of chemical space. The TGM device is composed of two glass slides and works following the concept of SlipChip. The device utilizes XYZ space to distribute three chemicals and establishes a chemical gradient matrix within 5 min. The established matrix contains 24 or 104 screening conditions depending on the device used, which covers a concentration range of [0.117-1, 0.117-1 and 0.686-1] and [0.0830-1, 0.0830-1, 0.686-1] respectively for the three chemicals. With the triple gradients built simultaneously, this TGM device provides order-of-magnitude improvement in screening efficiency over existing single- or double-gradient generators. As a proof of concept, we applied the device to screen the crystallization conditions for two model proteins of lysozyme and trypsin and confirmed the crystal structures using X-ray diffraction. Furthermore, we successfully obtained the crystallization condition of adhesin competence repressor, a protein that senses the alterations in intracellular zinc concentrations. We expect the TGM system to be widely used as an analytical platform for material synthesis and chemical screening beyond for protein crystallization.


Assuntos
Proteínas de Bactérias/química , Dispositivos Lab-On-A-Chip , Muramidase/química , Proteínas Repressoras/química , Tripsina/química , Animais , Bovinos , Galinhas , Cristalização , Fluoresceína/química , Corantes Fluorescentes/química , Indóis/química , Estudo de Prova de Conceito , Rodaminas/química , Difração de Raios X
16.
Cell Prolif ; 52(5): e12615, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31310044

RESUMO

OBJECTIVES: It has been widely reported that long non-coding RNAs (lncRNAs) can participate in multiple biological processes of human cancers. lncRNA HLA complex group 11 (HCG11) has been reported in human cancers as a tumour suppressor. This study focused on investigating the function and mechanism of HCG11 in glioma. MATERIALS AND METHODS: Based on The Cancer Genome Atlas (TCGA) data set and qRT-PCR analysis, the expression pattern of HCG11 was identified in glioma samples. The mechanism associated with HCG11 downregulation was determined by mechanism experiments. Gain-of-function assays were conducted for the identification of HCG11 function in glioma progression. Mechanism investigation based on the luciferase reporter assay, RIP assay and pull-down assay was used to explore the downstream molecular mechanism of HCG11. The role of molecular pathway in the progression of glioma was analysed in accordance with the rescue assays. RESULTS: HCG11 was expressed at low level in glioma samples compared with normal samples. FOXP1 could bind with HCG11 and transcriptionally inactivated HCG11. Overexpression of HCG11 efficiently suppressed cell proliferation, induced cell cycle arrest and promoted cell apoptosis. HCG11 was predominantly enriched in the cytoplasm of glioma cells and acted as a competing endogenous RNAs (ceRNAs) by sponging micro-496 to upregulate cytoplasmic polyadenylation element binding protein 3 (CPEB3). CEPB3 and miR-496 involved in HCG11-mediated glioma progression. CONCLUSIONS: HCG11 inhibited glioma progression by regulating miR-496/CPEB3 axis.


Assuntos
Glioma/patologia , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Antagomirs/metabolismo , Apoptose , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Movimento Celular , Proliferação de Células , Progressão da Doença , Fatores de Transcrição Forkhead/química , Fatores de Transcrição Forkhead/metabolismo , Glioma/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Interferência de RNA , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo
17.
Artigo em Inglês | MEDLINE | ID: mdl-31312617

RESUMO

Porphyromonas gingivalis, a keystone pathogen of chronic periodontitis, uses ferric uptake regulator homolog (PgFur) to regulate production of virulence factors. This study aimed to characterize PgFur protein in regard to its structure-function relationship. We experimentally identified the 5' mRNA sequence encoding the 171-amino-acid-long PgFur protein in the A7436 strain and examined this PgFur version as a full-length protein. PgFur protein did not bind to the canonical Escherichia coli Fur box, but the wild-type phenotype of the mutant Δpgfur strain was restored partially when expression of the ecfur gene was induced from the native pgfur promoter. The full-length PgFur protein contained one zinc atom per protein monomer, but did not bind iron, manganese, or heme. Single cysteine substitutions of CXXC motifs resulted in phenotypes similar to the mutant Δpgfur strain. The modified proteins were produced in E. coli at significantly lower levels, were highly unstable, and did not bind zinc. The pgfur gene was expressed at the highest levels in bacteria cultured for 24 h in the absence of iron and heme or at higher levels in bacteria cultured for 10 h in the presence of protoporphyrin IX source. No influence of high availability of Fe2+, Zn2+, or Mn2+ on pgfur gene expression was observed. Two chromosomal mutant strains producing protein lacking 4 (pgfurΔ4aa) or 13 (pgfurΔ13aa) C-terminal amino acid residues were examined in regard to importance of the C-terminal lysine-rich region. The pgfurΔ13aa strain showed a phenotype typical for the mutant Δpgfur strain, but both the wild-type PgFur protein and its truncated version bound zinc with similar ability. The Δpgfur mutant strain produced higher amounts of HmuY protein compared with the wild-type strain, suggesting compromised regulation of its expression. Potential PgFur ligands, Fe2+, Mn2+, Zn2+, PPIX, or serum components, did not influence HmuY production in the Δpgfur mutant strain. The mutant pgfurΔ4aa and pgfurΔ13aa strains exhibited affected HmuY protein production. PgFur, regardless of the presence of the C-terminal lysine-rich region, bound to the hmu operon promoter. Our data suggest that cooperation of PgFur with partners/cofactors and/or protein/DNA modifications would be required to accomplish its role played in an in vivo multilayer regulatory network.


Assuntos
Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/metabolismo , Proteínas Repressoras/classificação , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Regulação Bacteriana da Expressão Gênica , Heme/metabolismo , Ferro/metabolismo , Manganês/metabolismo , Modelos Moleculares , Filogenia , Conformação Proteica , Protoporfirinas , Proteínas Recombinantes , Proteínas Repressoras/química , Análise de Sequência de Proteína , Zinco/metabolismo
18.
Molecules ; 24(12)2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31234337

RESUMO

Buruli ulcer is a neglected tropical disease caused by the bacterium Mycobacterium ulcerans. Its virulence is attributed to the dermo-necrotic polyketide toxin mycolactone, whose synthesis is regressed when its iron acquisition system regulated by the iron-dependent regulator (ideR) is deactivated. Interfering with the activation mechanism of ideR to inhibit the toxin's synthesis could serve as a possible cure for Buruli ulcer. The three-dimensional structure of the ideR for Mycobacterium ulcerans was generated using homology modeling. A library of 832 African natural products (AfroDB), as well as five known anti-mycobacterial compounds were docked against the metal binding site of the ideR. The area under the curve (AUC) values greater than 0.7 were obtained for the computed Receiver Operating Characteristics (ROC) curves, validating the docking protocol. The identified top hits were pharmacologically profiled using Absorption, Distribution, Metabolism, Elimination and Toxicity (ADMET) predictions and their binding mechanisms were characterized. Four compounds with ZINC IDs ZINC000018185774, ZINC000095485921, ZINC000014417338 and ZINC000005357841 emerged as leads with binding energies of -7.7 kcal/mol, -7.6 kcal/mol, -8.0 kcal/mol and -7.4 kcal/mol, respectively. Induced Fit Docking (IFD) was also performed to account for the protein's flexibility upon ligand binding and to estimate the best plausible conformation of the complexes. Results obtained from the IFD were consistent with that of the molecular docking with the lead compounds forming interactions with known essential residues and some novel critical residues Thr14, Arg33 and Asp17. A hundred nanoseconds molecular dynamic simulations of the unbound ideR and its complexes with the respective lead compounds revealed changes in the ideR's conformations induced by ZINC000018185774. Comparison of the lead compounds to reported potent inhibitors by docking them against the DNA-binding domain of the protein also showed the lead compounds to have very close binding affinities to those of the potent inhibitors. Interestingly, structurally similar compounds to ZINC000018185774 and ZINC000014417338, as well as analogues of ZINC000095485921, including quercetin are reported to possess anti-mycobacterial activity. Also, ZINC000005357841 was predicted to possess anti-inflammatory and anti-oxidative activities, which are relevant in Buruli ulcer and iron acquisition mechanisms, respectively. The leads are molecular templates which may serve as essential scaffolds for the design of future anti-mycobacterium ulcerans agents.


Assuntos
Proteínas de Bactérias/química , Produtos Biológicos/química , Úlcera de Buruli/tratamento farmacológico , Mycobacterium ulcerans/química , Proteínas Repressoras/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Sítios de Ligação/efeitos dos fármacos , Úlcera de Buruli/microbiologia , Biologia Computacional , Humanos , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Mycobacterium ulcerans/efeitos dos fármacos , Mycobacterium ulcerans/patogenicidade , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética
19.
Blood ; 134(6): 548-560, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31217189

RESUMO

The presence of FMS-like receptor tyrosine kinase-3 internal tandem duplication (FLT3-ITD) mutations in patients with acute myeloid leukemia (AML) is associated with poor clinical outcome. FLT3 tyrosine kinase inhibitors (TKIs), although effective in kinase ablation, do not eliminate primitive FLT3-ITD+ leukemia cells, which are potential sources of relapse. Thus, understanding the mechanisms underlying FLT3-ITD+ AML cell persistence is essential to devise future AML therapies. Here, we show that expression of protein arginine methyltransferase 1 (PRMT1), the primary type I arginine methyltransferase, is increased significantly in AML cells relative to normal hematopoietic cells. Genome-wide analysis, coimmunoprecipitation assay, and PRMT1-knockout mouse studies indicate that PRMT1 preferentially cooperates with FLT3-ITD, contributing to AML maintenance. Genetic or pharmacological inhibition of PRMT1 markedly blocked FLT3-ITD+ AML cell maintenance. Mechanistically, PRMT1 catalyzed FLT3-ITD protein methylation at arginine 972/973, and PRMT1 promoted leukemia cell growth in an FLT3 methylation-dependent manner. Moreover, the effects of FLT3-ITD methylation in AML cells were partially due to cross talk with FLT3-ITD phosphorylation at tyrosine 969. Importantly, FLT3 methylation persisted in FLT3-ITD+ AML cells following kinase inhibition, indicating that methylation occurs independently of kinase activity. Finally, in patient-derived xenograft and murine AML models, combined administration of AC220 with a type I PRMT inhibitor (MS023) enhanced elimination of FLT3-ITD+ AML cells relative to AC220 treatment alone. Our study demonstrates that PRMT1-mediated FLT3 methylation promotes AML maintenance and suggests that combining PRMT1 inhibition with FLT3 TKI treatment could be a promising approach to eliminate FLT3-ITD+ AML cells.


Assuntos
Arginina/metabolismo , Duplicação Gênica , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo , Animais , Biomarcadores Tumorais , Catálise , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Metilação , Camundongos , Camundongos Knockout , Modelos Moleculares , Prognóstico , Ligação Proteica , Conformação Proteica , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/química , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/química , Ensaios Antitumorais Modelo de Xenoenxerto , Tirosina Quinase 3 Semelhante a fms/química
20.
Int J Mol Sci ; 20(12)2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-31212749

RESUMO

Hac1p is a key transcription factor regulating the unfolded protein response (UPR) induced by abnormal accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) in Saccharomyces cerevisiae. The accumulation of unfolded/misfolded proteins is sensed by protein Ire1p, which then undergoes trans-autophosphorylation and oligomerization into discrete foci on the ER membrane. HAC1 pre-mRNA, which is exported to the cytoplasm but is blocked from translation by its intron sequence looping back to its 5'UTR to form base-pair interaction, is transported to the Ire1p foci to be spliced, guided by a cis-acting bipartite element at its 3'UTR (3'BE). Spliced HAC1 mRNA can be efficiently translated. The resulting Hac1p enters the nucleus and activates, together with coactivators, a large number of genes encoding proteins such as protein chaperones to restore and maintain ER homeostasis and secretary protein quality control. This review details the translation regulation of Hac1p production, mediated by the nonconventional splicing, in the broad context of translation control and summarizes the evolution and diversification of the UPR signaling pathway among fungal, metazoan and plant lineages.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Regulação Fúngica da Expressão Gênica , Processamento de RNA , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina Básica/química , Fatores de Transcrição de Zíper de Leucina Básica/genética , Íntrons , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Ligação Proteica , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Resposta a Proteínas não Dobradas
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