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1.
Arch Virol ; 164(11): 2793-2797, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31440811

RESUMO

The DC-SIGN glycoprotein is responsible for the initial adhesion of dengue virus (DENV) to immune cells by the carbohydrate recognition domain (CRD). There are thirteen soluble and membrane-bound DC-SIGN isoforms, but the role of soluble isoforms in the DENV internalization process is not known. Five isoforms with an altered or absent CRD were identified, and three different soluble isoforms were used to confirm the interactions with mannose residues. The results show the loss of binding ability of one soluble isoform and binding ability of two of them. All of them will be used to verify their role in the DENV internalization process.


Assuntos
Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Vírus da Dengue/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Manose/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Ligação Viral , Internalização do Vírus , Sequência de Aminoácidos , Sequência de Bases , Dengue/virologia , Vírus da Dengue/genética , Ligantes , Ligação Proteica/genética , Isoformas de Proteínas/genética
2.
Nat Commun ; 10(1): 2682, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31213602

RESUMO

RNA-protein complexes play essential regulatory roles at nearly all levels of gene expression. Using in vivo crosslinking and RNA capture, we report a comprehensive RNA-protein interactome in a metazoan at four levels of resolution: single amino acids, domains, proteins and multisubunit complexes. We devise CAPRI, a method to map RNA-binding domains (RBDs) by simultaneous identification of RNA interacting crosslinked peptides and peptides adjacent to such crosslinked sites. CAPRI identifies more than 3000 RNA proximal peptides in Drosophila and human proteins with more than 45% of them forming new interaction interfaces. The comparison of orthologous proteins enables the identification of evolutionary conserved RBDs in globular domains and intrinsically disordered regions (IDRs). By comparing the sequences of IDRs through evolution, we classify them based on the type of motif, accumulation of tandem repeats, conservation of amino acid composition and high sequence divergence.


Assuntos
Evolução Molecular , Proteômica/métodos , Motivos de Ligação ao RNA/genética , Proteínas de Ligação a RNA/genética , RNA/metabolismo , Sequência de Aminoácidos/genética , Animais , Linhagem Celular , Sequência Conservada/genética , Reagentes para Ligações Cruzadas/química , Drosophila , Humanos , Peptídeos/química , Peptídeos/genética , Ligação Proteica/genética , Proteoma/genética , RNA/química , Proteínas de Ligação a RNA/química
3.
Nat Commun ; 10(1): 2607, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197133

RESUMO

Inhibiting the RAS oncogenic protein has largely been through targeting the switch regions that interact with signalling effector proteins. Here, we report designed ankyrin repeat proteins (DARPins) macromolecules that specifically inhibit the KRAS isoform by binding to an allosteric site encompassing the region around KRAS-specific residue histidine 95 at the helix α3/loop 7/helix α4 interface. We show that these DARPins specifically inhibit KRAS/effector interactions and the dependent downstream signalling pathways in cancer cells. Binding by the DARPins at that region influences KRAS/effector interactions in different ways, including KRAS nucleotide exchange and inhibiting KRAS dimerization at the plasma membrane. These results highlight the importance of targeting the α3/loop 7/α4 interface, a previously untargeted site in RAS, for specifically inhibiting KRAS function.


Assuntos
Sítio Alostérico/efeitos dos fármacos , Antineoplásicos/farmacologia , Desenho de Drogas , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Repetição de Anquirina , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Histidina/metabolismo , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias/genética , Neoplasias/patologia , Biblioteca de Peptídeos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais/efeitos dos fármacos
4.
Mol Plant Microbe Interact ; 32(9): 1121-1133, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31039081

RESUMO

ChiIV3, a chitinase of pepper (Capsicum annuum), stimulates cell death in pepper plants. However, there are only scarce reports on its role in resistance against bacterial wilt disease such as that caused by Ralstonia solanacearum and their transcriptional regulation. In this study, the silencing of ChiIV3 in pepper plants significantly reduced the resistance to R. solanacearum. The transcript of ChiIV3 was induced by R. solanacearum inoculation (RSI) as well as exogenous application of methyl jasmonate and abscisic acid. The bioinformatics analysis revealed that the ChiIV3 promoter consists of multiple stress-related cis elements, including six W-boxes and one MYB1AT. With the 5' deletion assay in the ChiIV3 promoter, the W4-box located from -640 to -635 bp was identified as the cis element that is required for the response to RSI. In addition, the W4-box element was shown to be essential for the binding of the ChiIV3 promoter by the WRKY40 transcription factor, which is known to positively regulate the defense response to R. solanacearum. Site-directed mutagenesis in the W4-box sequence impaired the binding of WRKY40 to the ChiIV3 promoter. Subsequently, the transcription of ChiIV3 decreased in WRKY40-silenced pepper plants. These results demonstrated that the expression of the defense gene ChiIV3 is controlled through multiple modes of regulation, and WRKY40 directly binds to the W4-box element of the ChiIV3 promoter region for its transcriptional regulation.


Assuntos
Capsicum , Quitinases , Resistência à Doença , Ralstonia solanacearum , Fatores de Transcrição , Capsicum/enzimologia , Capsicum/genética , Capsicum/microbiologia , Quitinases/genética , Quitinases/metabolismo , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Humanos , Mutagênese Sítio-Dirigida , Doenças das Plantas/microbiologia , Proteínas de Plantas , Ligação Proteica/genética , Ralstonia solanacearum/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
Nat Commun ; 10(1): 1855, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015431

RESUMO

DHX36 is a DEAH-box helicase that resolves parallel G-quadruplex structures formed in DNA and RNA. The recent co-crystal structure of DHX36 bound G4-DNA revealed an intimate contact, but did not address the role of ATP hydrolysis in G4 resolving activity. Here, we demonstrate that unlike on G4-DNA, DHX36 displays ATP-independent unfolding of G4-RNA followed by ATP-dependent refolding, generating a highly asymmetric pattern of activity. Interestingly, DHX36 refolds G4-RNA in several steps, reflecting the discrete steps in forming the G4 structure. We show that the ATP-dependent activity of DHX36 arises from the RNA tail rather than the G4. Mutations that perturb G4 contact result in quick dissociation of the protein from RNA upon ATP hydrolysis, while mutations that interfere with binding the RNA tail induce dysregulated activity. We propose that the ATP-dependent activity of DHX36 may be useful for dynamically resolving various G4-RNA structures in cells.


Assuntos
Trifosfato de Adenosina/metabolismo , RNA Helicases DEAD-box/metabolismo , Quadruplex G , Dobramento de RNA , RNA/metabolismo , RNA Helicases DEAD-box/genética , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Microscopia de Fluorescência/métodos , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica/genética , RNA/química , Imagem Individual de Molécula/métodos
6.
Nat Commun ; 10(1): 1884, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015495

RESUMO

DNA methyltransferases (DNMTs) deposit DNA methylation, which regulates gene expression and is essential for mammalian development. Histone post-translational modifications modulate the recruitment and activity of DNMTs. The PWWP domains of DNMT3A and DNMT3B are posited to interact with histone 3 lysine 36 trimethylation (H3K36me3); however, the functionality of this interaction for DNMT3A remains untested in vivo. Here we present a mouse model carrying a D329A point mutation in the DNMT3A PWWP domain. The mutation causes dominant postnatal growth retardation. At the molecular level, it results in progressive DNA hypermethylation across domains marked by H3K27me3 and bivalent chromatin, and de-repression of developmental regulatory genes in adult hypothalamus. Evaluation of non-CpG methylation, a marker of de novo methylation, further demonstrates the altered recruitment and activity of DNMT3AD329A at bivalent domains. This work provides key molecular insights into the function of the DNMT3A-PWWP domain and role of DNMT3A in regulating postnatal growth.


Assuntos
Cromatina/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Transtornos do Crescimento/genética , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Feminino , Mutação com Ganho de Função/fisiologia , Transtornos do Crescimento/patologia , Histonas/metabolismo , Humanos , Hipotálamo/metabolismo , Hipotálamo/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação Puntual/fisiologia , Ligação Proteica/genética , Domínios Proteicos/genética , Processamento de Proteína Pós-Traducional/fisiologia
7.
Genes Dev ; 33(7-8): 418-435, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30819820

RESUMO

The RNA polymerase II (RNAPII) C-terminal domain kinase, CDK12, regulates genome stability, expression of DNA repair genes, and cancer cell resistance to chemotherapy and immunotherapy. In addition to its role in mRNA biosynthesis of DNA repair genes, we show here that CDK12 phosphorylates the mRNA 5' cap-binding repressor, 4E-BP1, to promote translation of mTORC1-dependent mRNAs. In particular, we found that phosphorylation of 4E-BP1 by mTORC1 (T37 and T46) facilitates subsequent CDK12 phosphorylation at two Ser-Pro sites (S65 and T70) that control the exchange of 4E-BP1 with eIF4G at the 5' cap of CHK1 and other target mRNAs. RNA immunoprecipitation coupled with deep sequencing (RIP-seq) revealed that CDK12 regulates release of 4E-BP1, and binding of eIF4G, to many mTORC1 target mRNAs, including those needed for MYC transformation. Genome-wide ribosome profiling (Ribo-seq) further identified specific CDK12 "translation-only" target mRNAs, including many mTORC1 target mRNAs as well as many subunits of mitotic and centromere/centrosome complexes. Accordingly, confocal imaging analyses revealed severe chromosome misalignment, bridging, and segregation defects in cells deprived of CDK12 or CCNK. We conclude that the nuclear RNAPII-CTD kinase CDK12 cooperates with mTORC1, and controls a specialized translation network that is essential for mitotic chromosome stability.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Quinase 1 do Ponto de Checagem/genética , Quinases Ciclina-Dependentes/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Instabilidade Genômica/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosfoproteínas/metabolismo , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Ciclinas/metabolismo , Fator de Iniciação 4G em Eucariotos/metabolismo , Humanos , Mitose/genética , Fosforilação/genética , Ligação Proteica/genética
8.
Nat Commun ; 10(1): 1231, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30874556

RESUMO

The Mycobacterium tuberculosis kinase PknB is essential for growth and survival of the pathogen in vitro and in vivo. Here we report the results of our efforts to elucidate the mechanism of regulation of PknB activity. The specific residues in the PknB extracytoplasmic domain that are essential for ligand interaction and survival of the bacterium are identified. The extracytoplasmic domain interacts with mDAP-containing LipidII, and this is abolished upon mutation of the ligand-interacting residues. Abrogation of ligand-binding or sequestration of the ligand leads to aberrant localization of PknB. Contrary to the prevailing hypothesis, abrogation of ligand-binding is linked to activation loop hyperphosphorylation, and indiscriminate hyperphosphorylation of PknB substrates as well as other proteins, ultimately causing loss of homeostasis and cell death. We propose that the ligand-kinase interaction directs the appropriate localization of the kinase, coupled to stringently controlled activation of PknB, and consequently the downstream processes thereof.


Assuntos
Mycobacterium tuberculosis/fisiologia , Fosforilação/fisiologia , Domínios Proteicos/genética , Proteínas Serina-Treonina Quinases/metabolismo , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Homeostase/fisiologia , Ligantes , Mutação , Ligação Proteica/genética , Proteínas Serina-Treonina Quinases/genética , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismo
9.
PLoS Comput Biol ; 15(3): e1006921, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30897079

RESUMO

ChIP-seq (Chromatin Immunoprecipitation followed by sequencing) is a high-throughput technique to identify genomic regions that are bound in vivo by a particular protein, e.g., a transcription factor (TF). Biological factors, such as chromatin state, indirect and cooperative binding, as well as experimental factors, such as antibody quality, cross-linking, and PCR biases, are known to affect the outcome of ChIP-seq experiments. However, the relative impact of these factors on inferences made from ChIP-seq data is not entirely clear. Here, via a detailed ChIP-seq simulation pipeline, ChIPulate, we assess the impact of various biological and experimental sources of variation on several outcomes of a ChIP-seq experiment, viz., the recoverability of the TF binding motif, accuracy of TF-DNA binding detection, the sensitivity of inferred TF-DNA binding strength, and number of replicates needed to confidently infer binding strength. We find that the TF motif can be recovered despite poor and non-uniform extraction and PCR amplification efficiencies. The recovery of the motif is, however, affected to a larger extent by the fraction of sites that are either cooperatively or indirectly bound. Importantly, our simulations reveal that the number of ChIP-seq replicates needed to accurately measure in vivo occupancy at high-affinity sites is larger than the recommended community standards. Our results establish statistical limits on the accuracy of inferences of protein-DNA binding from ChIP-seq and suggest that increasing the mean extraction efficiency, rather than amplification efficiency, would better improve sensitivity. The source code and instructions for running ChIPulate can be found at https://github.com/vishakad/chipulate.


Assuntos
Imunoprecipitação da Cromatina/métodos , Biologia Computacional/métodos , Análise de Sequência de DNA/métodos , Software , Fatores de Transcrição , Sítios de Ligação/genética , Simulação por Computador , DNA/química , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Ligação Proteica/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
Int J Mol Sci ; 20(6)2019 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-30884815

RESUMO

The primary cilium is a solitary, nonmotile and transitory appendage that is present in virtually all mammalian cells. Our knowledge of its ultrastructure and function is the result of more than fifty years of research that has dramatically changed our perspectives on the primary cilium. The mutual regulation between ciliogenesis and the cell cycle is now well-recognized, as well as the function of the primary cilium as a cellular "antenna" for perceiving external stimuli, such as light, odorants, and fluids. By displaying receptors and signaling molecules, the primary cilium is also a key coordinator of signaling pathways that converts extracellular cues into cellular responses. Given its critical tasks, any defects in primary cilium formation or function lead to a wide spectrum of diseases collectively called "ciliopathies". An emerging role of primary cilium is in the regulation of cancer development. In this review, we seek to describe the current knowledge about the influence of the primary cilium in cancer progression, with a focus on some of the events that cancers need to face to sustain survival and growth in hypoxic microenvironment: the cancer hallmarks.


Assuntos
Autofagia/genética , Biomarcadores Tumorais/genética , Cílios/ultraestrutura , Neoplasias/ultraestrutura , Ciclo Celular , Cílios/genética , Humanos , Neoplasias/genética , Neoplasias/patologia , Ligação Proteica/genética , Transdução de Sinais/genética , Microambiente Tumoral/genética
11.
J Exp Clin Cancer Res ; 38(1): 114, 2019 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-30841931

RESUMO

BACKGROUND: Mast cells are being increasingly recognized as critical components in the tumor microenvironment. Protein Kinase D (PKD) is essential for the progression of prostate cancer, but its role in prostate cancer microenvironment remains poorly understood. METHODS: The expression of PKD, mast cells and microvessel density were examined by IHC. The clinical significance was determined by statistical analyses. The biological function of PKD and the underlying mechanisms were investigated using in vitro and in vivo models. RESULTS: PKD2/3 contributed to MCs recruitment and tumor angiogenesis in the prostate cancer microenvironment. Clinical data showed that increased activation of PKD at Ser744/748 in prostate cancer was correlated with mast cell infiltration and microvascular density. PKD2/3 silencing of prostate cancer cells markedly decreased MCs migration and tube formation of HUVEC cells. Moreover, PKD2/3 depletion not only reduced SCF, CCL5 and CCL11 expression in prostate cancer cells but also inhibited angiogenic factors in MCs. Conversely, exogenous SCF, CCL5 and CCL11 reversed the effect on MCs migration inhibited by PKD2/3 silencing. Mechanistically, PKD2/3 interacted with Erk1/2 and activated Erk1/2 or NF-κB signaling pathway, leading to AP-1 or NF-κB binding to the promoter of scf, ccl5 and ccl11. Finally, PKD-specific inhibitor significantly reduced tumor volume and tumor growth in mice bearing RM-1 prostate cancer cells, which was attributed to attenuation of mast cell recruitment and tumor angiogenesis. CONCLUSIONS: These results demonstrate a novel PKDs function that contributes to tumor angiogenesis and progression through mast cells recruitment in prostate cancer microenvironment.


Assuntos
Proteínas Angiogênicas/genética , Neovascularização Patológica/genética , Neoplasias da Próstata/genética , Proteína Quinase C/genética , Proteínas Angiogênicas/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Quimiocina CCL11/genética , Quimiocina CCL5/genética , Regulação Neoplásica da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Mastócitos/metabolismo , Mastócitos/patologia , Camundongos , Neovascularização Patológica/patologia , Fosforilação , Regiões Promotoras Genéticas , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/patologia , Ligação Proteica/genética , Proteína Quinase C/antagonistas & inibidores , Fator de Células-Tronco/genética , Fator de Transcrição AP-1/genética , Fator de Transcrição RelA/genética , Microambiente Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Proc Natl Acad Sci U S A ; 116(8): 3294-3299, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30718391

RESUMO

The parathyroid hormone (PTH) and its related peptide (PTHrP) activate PTH receptor (PTHR) signaling, but only the PTH sustains GS-mediated adenosine 3',5'-cyclic monophosphate (cAMP) production after PTHR internalization into early endosomes. The mechanism of this unexpected behavior for a G-protein-coupled receptor is not fully understood. Here, we show that extracellular Ca2+ acts as a positive allosteric modulator of PTHR signaling that regulates sustained cAMP production. Equilibrium and kinetic studies of ligand-binding and receptor activation reveal that Ca2+ prolongs the residence time of ligands on the receptor, thus, increasing both the duration of the receptor activation and the cAMP signaling. We further find that Ca2+ allostery in the PTHR is strongly affected by the point mutation recently identified in the PTH (PTHR25C) as a new cause of hypocalcemia in humans. Using high-resolution and mass accuracy mass spectrometry approaches, we identified acidic clusters in the receptor's first extracellular loop as key determinants for Ca2+ allosterism and endosomal cAMP signaling. These findings coupled to defective Ca2+ allostery and cAMP signaling in the PTHR by hypocalcemia-causing PTHR25C suggest that Ca2+ allostery in PTHR signaling may be involved in primary signaling processes regulating calcium homeostasis.


Assuntos
AMP Cíclico/genética , Hipocalcemia/genética , Hormônio Paratireóideo/genética , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Regulação Alostérica/genética , Animais , Células COS , Sinalização do Cálcio/genética , Cercopithecus aethiops , AMP Cíclico/metabolismo , Humanos , Hipocalcemia/metabolismo , Hipocalcemia/patologia , Cinética , Ligantes , Hormônio Paratireóideo/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/genética , Mutação Puntual/genética , Ligação Proteica/genética , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo
13.
Cancer Biother Radiopharm ; 34(4): 252-257, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30724592

RESUMO

Background: Neuroblastoma (NB) is one of the most aggressive and common solid tumors in pediatrics. Development of effective new therapeutics for NB is in progress to help reduce mortality and morbidity of the disease, particularly in relapsed patients. The tumor suppressor protein p53 plays a critical role in multiple signaling pathways to maintain cellular hemostasis. Dysregulation of p53 protein and/or molecular aberrations have been associated with multiple human malignancies. p53 stability and protein activity is negatively regulated by the E3 ubiquitin ligase (MDM2). Thus, targeting p53-MDM2 protein-protein interaction is a feasible and promising therapeutic strategy to restore the physiological function of p53 in cancer cells. RG7112 is a highly potent and selective small molecule inhibitor, which target a unique structure located within p53 binding motif of MDM2. Methods: The efficacy of RG7112 in vitro using NB cell lines was examined. Two wild-type (WT)-p53 NB cell lines IMR5 and LAN-5, a mutant p53 cell line SK-N-BE(2), and a WT-p53/p14 deleted cell line SH-EP were employed. Results: Data showed that RG7112 significantly reduced cellular viability of IMR5 (IC50, 562 nM) and LAN-5 (IC50, 430 nM), but not SK-N-BE(2) and SH-EP cells. Further, RG7112 restores p53 and p21 protein levels in IMR5 and LAN-5 in a dose-dependent manner. RG7112 induces cell cycle arresting (60% G1 arresting) in WT-p53 cells (IMR5), but no pronounced effect observed in SK-N-BE(2). In this study, 15 different drugs in combination with RG7112 in IMR5 cell line and identified venetoclax (Bcl-2/Bcl-xL inhibitor) as a promising candidate were evaluated. Conclusions: Taken together, these findings provide initial proof-of-concept data for further investigations of RG7112 in selected subgroups of NB patients.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Imidazolinas/farmacologia , Neuroblastoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Motivos de Aminoácidos/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Humanos , Imidazolinas/uso terapêutico , Concentração Inibidora 50 , Neuroblastoma/genética , Neuroblastoma/patologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Proteína Supressora de Tumor p53/genética
14.
Cell Mol Life Sci ; 76(10): 2015-2030, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30725116

RESUMO

Women with triple-negative breast cancer (TNBC) are generally treated by chemotherapy but their responsiveness may be blunted by DNA double-strand break (DSB) repair. We previously reported that IGFBP-3 forms nuclear complexes with EGFR and DNA-dependent protein kinase (DNA-PKcs) to modulate DSB repair by non-homologous end-joining (NHEJ) in TNBC cells. To discover IGFBP-3 binding partners involved in chemoresistance through stimulation of DSB repair, we analyzed the IGFBP-3 interactome by LC-MS/MS and confirmed interactions by coimmunoprecipitation and proximity ligation assay. Functional effects were demonstrated by DNA end-joining in vitro and measurement of γH2AX foci. In response to 20 µM etoposide, the DNA/RNA-binding protein, non-POU domain-containing octamer-binding protein (NONO) and its dimerization partner splicing factor, proline/glutamine-rich (SFPQ) formed complexes with IGFBP-3, demonstrated in basal-like TNBC cell lines HCC1806 and MDA-MB-468. NONO binding to IGFBP-3 was also shown in a cell-free biochemical assay. IGFBP-3 complexes with NONO and SFPQ were blocked by inhibiting EGFR with gefitinib or DNA-PKcs with NU7026, and by the PARP inhibitors veliparib and olaparib, which also reduced DNA end-joining activity and delayed the resolution of the γH2AX signal (i.e. inhibited DNA DSB repair). Downregulation of the long noncoding RNA in NHEJ pathway 1 (LINP1) by siRNA also blocked IGFBP-3 interaction with NONO-SFPQ. These findings suggest a PARP-dependent role for NONO and SFPQ in IGFBP-3-dependent DSB repair and the involvement of LINP1 in the complex formation. We propose that targeting of the DNA repair function of IGFBP-3 may enhance chemosensitivity in basal-like TNBC, thus improving patient outcomes.


Assuntos
Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Fatores de Transcrição de Octâmero/metabolismo , Fator de Processamento Associado a PTB/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteínas Associadas à Matriz Nuclear/genética , Fatores de Transcrição de Octâmero/genética , Fator de Processamento Associado a PTB/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Interferência de RNA , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia
15.
Cell Mol Life Sci ; 76(9): 1807-1819, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30788513

RESUMO

Through their ability to edit 6-O-sulfation pattern of Heparan sulfate (HS) polysaccharides, Sulf extracellular endosulfatases have emerged as critical regulators of many biological processes, including tumor progression. However, study of Sulfs remains extremely intricate and progress in characterizing their functional and structural features has been hampered by limited access to recombinant enzyme. In this study, we unlock this critical bottleneck, by reporting an efficient expression and purification system of recombinant HSulf-2 in mammalian HEK293 cells. This novel source of enzyme enabled us to investigate the way the enzyme domain organization dictates its functional properties. By generating mutants, we confirmed previous studies that HSulf-2 catalytic (CAT) domain was sufficient to elicit arylsulfatase activity and that its hydrophilic (HD) domain was necessary for the enzyme 6-O-endosulfatase activity. However, we demonstrated for the first time that high-affinity binding of HS substrates occurred through the coordinated action of both domains, and we identified and characterized 2 novel HS binding sites within the CAT domain. Altogether, our findings contribute to better understand the molecular mechanism governing HSulf-2 substrate recognition and processing. Furthermore, access to purified recombinant protein opens new perspectives for the resolution of HSulf structure and molecular features, as well as for the development of Sulf-specific inhibitors.


Assuntos
Domínio Catalítico/genética , Heparitina Sulfato/química , Sulfotransferases/genética , Sulfotransferases/metabolismo , Sítios de Ligação/genética , Linhagem Celular , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Ligação Proteica/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato/genética , Sulfotransferases/biossíntese
16.
Virus Genes ; 55(3): 267-273, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30796742

RESUMO

The helicase eIF4A is part of the cellular eIF4F translation initiation complex. The main functions of eIF4A are to remove secondary complex structures within the 5'-untranslated region and to displace proteins attached to mRNA. As intracellular parasites, viruses regulate the processes involved in protein synthesis, and different mechanisms related to controlling translation factors, such as eIF4A, have been found. The inhibitors of this factor are currently known; these substances could be used in the near future as part of antiviral pharmacological therapies in instances of replication cycles in which eIF4A is required. In this review, the particularities of how some viruses make use of this initiation factor to synthesize their proteins are discussed.


Assuntos
Fator de Iniciação 4A em Eucariotos/genética , Biossíntese de Proteínas , Viroses/genética , Regiões 5' não Traduzidas/genética , Humanos , Ligação Proteica/genética , RNA Mensageiro/genética , Viroses/virologia
17.
Nat Genet ; 51(2): 217-223, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30617255

RESUMO

R-loops are DNA-RNA hybrids enriched at CpG islands (CGIs) that can regulate chromatin states1-8. How R-loops are recognized and interpreted by specific epigenetic readers is unknown. Here we show that GADD45A (growth arrest and DNA damage protein 45A) binds directly to R-loops and mediates local DNA demethylation by recruiting TET1 (ten-eleven translocation 1). Studying the tumor suppressor TCF21 (ref. 9), we find that antisense long noncoding (lncRNA) TARID (TCF21 antisense RNA inducing promoter demethylation) forms an R-loop at the TCF21 promoter. Binding of GADD45A to the R-loop triggers local DNA demethylation and TCF21 expression. TARID transcription, R-loop formation, DNA demethylation, and TCF21 expression proceed sequentially during the cell cycle. Oxidized DNA demethylation intermediates are enriched at genomic R-loops and their levels increase upon RNase H1 depletion. Genomic profiling in embryonic stem cells identifies thousands of R-loop-dependent TET1 binding sites at CGIs. We propose that GADD45A is an epigenetic R-loop reader that recruits the demethylation machinery to promoter CGIs.


Assuntos
Proteínas de Ciclo Celular/genética , Ilhas de CpG/genética , Oxigenases de Função Mista/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , Animais , Ciclo Celular/genética , Linhagem Celular , Cromatina/genética , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Epigênese Genética/genética , Epigenômica/métodos , Células HEK293 , Humanos , Camundongos , Ligação Proteica/genética , RNA Longo não Codificante/genética , Ribonuclease H/genética , Transcrição Genética/genética
18.
Nucleic Acids Res ; 47(4): 1896-1907, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30629181

RESUMO

Telomeres are nucleoprotein structures at the ends of linear chromosomes and present an essential feature for genome integrity. Vertebrate telomeres usually consist of hexameric TTAGGG repeats, however, in cells that use the alternative lengthening of telomeres (ALT) mechanism, variant repeat sequences are interspersed throughout telomeres. Previously, it was shown that NR2C/F transcription factors bind to TCAGGG variant repeats and contribute to telomere maintenance in ALT cells. While specific binders to other variant repeat sequences have been lacking to date, we here identify ZBTB10 as the first TTGGGG-binding protein and demonstrate direct binding via the two zinc fingers with affinity in the nanomolar range. Concomitantly, ZBTB10 co-localizes with a subset of telomeres in ALT-positive U2OS cells and interacts with TRF2/RAP1 via the N-terminal region of TRF2. Our data establishes ZBTB10 as a novel variant repeat binding protein at ALT telomeres.


Assuntos
Proteínas Repressoras/genética , Homeostase do Telômero/genética , Telômero/genética , Proteína 2 de Ligação a Repetições Teloméricas/genética , Sítios de Ligação/genética , Cromossomos/genética , Proteínas de Ligação a DNA/genética , Genoma/genética , Humanos , Ligação Proteica/genética , Sequências Repetitivas de Ácido Nucleico/genética , Proteínas de Ligação a Telômeros/genética
19.
Biosci Rep ; 39(1)2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30622148

RESUMO

Deviant levels of available heme and related molecules can result from pathological situations such as impaired heme biosynthesis or increased hemolysis as a consequence of vascular trauma or bacterial infections. Heme-related biological processes are affected by these situations, and it is essential to fully understand the underlying mechanisms. While heme has long been known as an important prosthetic group of various proteins, its function as a regulatory and signaling molecule is poorly understood. Diseases such as porphyria are caused by impaired heme metabolism, and heme itself might be used as a drug in order to downregulate its own biosynthesis. In addition, heme-driven side effects and symptoms emerging from heme-related pathological conditions are not fully comprehended and thus impede adequate medical treatment. Several heme-regulated proteins have been identified in the past decades, however, the molecular basis of transient heme-protein interactions remains to be explored. Herein, we summarize the results of an in-depth analysis of heme binding to proteins, which revealed specific binding modes and affinities depending on the amino acid sequence. Evaluating the binding behavior of a plethora of heme-peptide complexes resulted in the implementation of a prediction tool (SeqD-HBM) for heme-binding motifs, which eventually led and will perspectively lead to the identification and verification of so far unknown heme-regulated proteins. This systematic approach resulted in a broader picture of the alternative functions of heme as a regulator of proteins. However, knowledge on heme regulation of proteins is still a bottomless barrel that leaves much scope for future research and development.


Assuntos
Heme/genética , Hemeproteínas/genética , Complexos Multiproteicos/genética , Peptídeos/genética , Sequência de Aminoácidos , Bases de Dados Genéticas , Heme/metabolismo , Hemeproteínas/metabolismo , Humanos , Complexos Multiproteicos/química , Peptídeos/química , Ligação Proteica/genética
20.
Nucleic Acids Res ; 47(7): 3550-3567, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30698745

RESUMO

Activation of the checkpoint protein Tel1 requires the Mre11-Rad50-Xrs2 (MRX) complex, which recruits Tel1 at DNA double-strand breaks (DSBs) through direct interaction between Tel1 and Xrs2. However, in vitro Tel1 activation by MRX requires ATP binding to Rad50, suggesting a role also for the MR subcomplex in Tel1 activation. Here we describe two separation-of-functions alleles, mre11-S499P and rad50-A78T, which we show to specifically affect Tel1 activation without impairing MRX functions in DSB repair. Both Mre11-S499P and Rad50-A78T reduce Tel1-MRX interaction leading to poor Tel1 association at DSBs and consequent loss of Tel1 activation. The Mre11-S499P variant reduces Mre11-Rad50 interaction, suggesting an important role for MR complex formation in Tel1 activation. Molecular dynamics simulations show that the wild type MR subcomplex bound to ATP lingers in a tightly 'closed' conformation, while ADP presence leads to the destabilization of Rad50 dimer and of Mre11-Rad50 association, both events being required for MR conformational transition to an open state. By contrast, MRA78T undertakes complex opening even if Rad50 is bound to ATP, indicating that defective Tel1 activation caused by MRA78T results from destabilization of the ATP-bound conformational state.


Assuntos
Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Ativação Transcricional/genética , Trifosfato de Adenosina/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Dano ao DNA/genética , Reparo do DNA/genética , DNA Fúngico/genética , Conformação Molecular , Complexos Multiproteicos/genética , Ligação Proteica/genética , Multimerização Proteica/genética , Saccharomyces cerevisiae/genética , Transdução de Sinais/genética
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