RESUMO
SET domain containing (lysine methyltransferase) 7 (SETD7) is implicated in multiple signaling and disease related pathways with a broad diversity of reported substrates. Here, we report the discovery of (R)-PFI-2-a first-in-class, potent (Ki (app) = 0.33 nM), selective, and cell-active inhibitor of the methyltransferase activity of human SETD7-and its 500-fold less active enantiomer, (S)-PFI-2. (R)-PFI-2 exhibits an unusual cofactor-dependent and substrate-competitive inhibitory mechanism by occupying the substrate peptide binding groove of SETD7, including the catalytic lysine-binding channel, and by making direct contact with the donor methyl group of the cofactor, S-adenosylmethionine. Chemoproteomics experiments using a biotinylated derivative of (R)-PFI-2 demonstrated dose-dependent competition for binding to endogenous SETD7 in MCF7 cells pretreated with (R)-PFI-2. In murine embryonic fibroblasts, (R)-PFI-2 treatment phenocopied the effects of Setd7 deficiency on Hippo pathway signaling, via modulation of the transcriptional coactivator Yes-associated protein (YAP) and regulation of YAP target genes. In confluent MCF7 cells, (R)-PFI-2 rapidly altered YAP localization, suggesting continuous and dynamic regulation of YAP by the methyltransferase activity of SETD7. These data establish (R)-PFI-2 and related compounds as a valuable tool-kit for the study of the diverse roles of SETD7 in cells and further validate protein methyltransferases as a druggable target class.
Assuntos
Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Pirrolidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Tetra-Hidroisoquinolinas/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Fibroblastos/efeitos dos fármacos , Via de Sinalização Hippo , Histona-Lisina N-Metiltransferase/genética , Humanos , Células MCF-7 , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Mutação , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Pirrolidinas/química , Relação Estrutura-Atividade , Sulfonamidas/química , Tetra-Hidroisoquinolinas/química , Fatores de Transcrição , Proteínas de Sinalização YAPRESUMO
The RNA polymerase II (RNAPII) C-terminal domain (CTD) heptapeptide repeats (1-YSPTSPS-7) undergo dynamic phosphorylation and dephosphorylation during the transcription cycle to recruit factors that regulate transcription, RNA processing and chromatin modification. We show here that RPRD1A and RPRD1B form homodimers and heterodimers through their coiled-coil domains and interact preferentially via CTD-interaction domains (CIDs) with RNAPII CTD repeats phosphorylated at S2 and S7. Crystal structures of the RPRD1A, RPRD1B and RPRD2 CIDs, alone and in complex with RNAPII CTD phosphoisoforms, elucidate the molecular basis of CTD recognition. In an example of cross-talk between different CTD modifications, our data also indicate that RPRD1A and RPRD1B associate directly with RPAP2 phosphatase and, by interacting with CTD repeats where phospho-S2 and/or phospho-S7 bracket a phospho-S5 residue, serve as CTD scaffolds to coordinate the dephosphorylation of phospho-S5 by RPAP2.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Neoplasias/química , Processamento de Proteína Pós-Traducional , RNA Polimerase II/química , Proteínas Repressoras/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cristalografia por Raios X , Células HEK293 , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , RNA Polimerase II/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Serina/químicaRESUMO
Phosphatidylinositol 3,4,5-triphosphate (PIP3) plays a key role in neuronal polarization and axon formation. PIP3-containing vesicles are transported to axon tips by the kinesin KIF13B via an adaptor protein, centaurin α1 (CENTA1). KIF13B interacts with CENTA1 through its forkhead-associated (FHA) domain. We solved the crystal structures of CENTA1 in ligand-free, KIF13B-FHA domain-bound, and PIP3 head group (IP4)-bound conformations, and the CENTA1/KIF13B-FHA/IP4 ternary complex. The first pleckstrin homology (PH) domain of CENTA1 specifically binds to PIP3, while the second binds to both PIP3 and phosphatidylinositol 3,4-biphosphate (PI(3,4)P(2)). The FHA domain of KIF13B interacts with the PH1 domain of one CENTA1 molecule and the ArfGAP domain of a second CENTA1 molecule in a threonine phosphorylation-independent fashion. We propose that full-length KIF13B and CENTA1 form heterotetramers that can bind four phosphoinositide molecules in the vesicle and transport it along the microtubule.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Axônios/metabolismo , Cinesinas/química , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Neurônios/citologia , Fosfatos de Fosfatidilinositol/metabolismo , Conformação Proteica , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transporte Biológico/fisiologia , Calorimetria , Cromatografia de Afinidade , Cromatografia em Gel , Clonagem Molecular , Biologia Computacional , Cristalografia , Eletroforese em Gel de Poliacrilamida , Glutationa Transferase , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Modelos Químicos , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Vesículas Transportadoras/metabolismoRESUMO
Protein stabilization upon ligand binding has frequently been used to identify ligands for soluble proteins. Methods such as differential scanning fluorimetry (DSF) and differential static light scattering (DSLS) have been employed in the 384-well format and have been useful in identifying ligands that promote crystallization and 3D structure determination of proteins. However, finding a generic method that is applicable to membrane proteins has been a challenge as the high hydrophobicity of membrane proteins and the presence of detergents essential for their solubilization interfere with fluorescence-based detections. Here the authors used MsbA (an adenosine triphosphate binding cassette transporter), CorA (a Mg(++) channel), and CpxA (a histidine kinase) as model proteins and show that DSLS is not sensitive to the presence of detergents or protein hydrophobicity and can be used to monitor thermodenaturation of membrane proteins, assess their stability, and detect ligand binding in a 384-well format.
Assuntos
Bioensaio/métodos , Luz , Proteínas de Membrana/metabolismo , Espalhamento de Radiação , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Detergentes/farmacologia , Proteínas de Escherichia coli/metabolismo , Glucosídeos/metabolismo , Ligantes , Proteínas Mutantes/metabolismo , Desnaturação Proteica/efeitos dos fármacos , Proteínas Quinases/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas/análise , TemperaturaRESUMO
The histone code comprises many posttranslational modifications that occur mainly in histone tail peptides. The identity and location of these marks are read by a variety of histone-binding proteins that are emerging as important regulators of cellular differentiation and development and are increasingly being implicated in numerous disease states. The authors describe the development of the first high-throughput screening assay for the discovery of inhibitors of methyl-lysine binding proteins that will be used to initiate a full-scale discovery effort for this broad target class. They focus on the development of an AlphaScreen-based assay for malignant brain tumor (MBT) domain-containing proteins, which bind to the lower methylation states of lysine residues present in histone tail peptides. This assay takes advantage of the avidity of the AlphaScreen beads to clear the hurdle to assay development presented by the low micromolar binding constants of the histone binding proteins for their cognate peptides. The assay is applicable to other families of methyl-lysine binding proteins, and it has the potential to be used in screening efforts toward the discovery of novel small molecules with utility as research tools for cellular reprogramming and ultimately drug discovery.
Assuntos
Bioensaio/métodos , Epigênese Genética , Lisina/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/farmacologia , Automação , Ligação Competitiva/efeitos dos fármacos , Soluções Tampão , Epigênese Genética/efeitos dos fármacos , Histonas/metabolismo , Ligantes , Metilação/efeitos dos fármacos , Miniaturização , Proteínas de Neoplasias/metabolismo , Projetos Piloto , Polissorbatos , Ligação Proteica/efeitos dos fármacos , Cloreto de Sódio , Fatores de Tempo , TitulometriaRESUMO
The significant increase in the demand for purified protein for crystallization and structural studies has made necessary the development of multi-sample methods for identifying solution conditions that affect protein stability and aggregation. Conditions that stabilize proteins can improve protein purification and crystallization. These methods can be used to identify small molecule compounds or inhibitors that interact with the purified proteins, and might serve as starting points for drug discovery. In this article three methods for measuring protein stability and aggregation are described and discussed: differential scanning fluorimetry (DSF), differential static light scattering (DSLS), and isothermal denaturation (ITD).
Assuntos
Bioquímica/métodos , Estabilidade Proteica , Humanos , Luz , Desnaturação Proteica , Estrutura Quaternária de Proteína , Espalhamento de RadiaçãoRESUMO
Many diseases in humans are caused by mutations that decrease the stability of specific proteins or increase their susceptibility to aggregation. Consequently, the availability of high-throughput methods for assessing protein stability and aggregation properties under physiological conditions (e.g., 37 degrees C) is necessary to analyze physicochemical properties under conditions that are closer to in vivo models. Therefore, the authors have explored the use of isothermal denaturation (ITD) in a 384-well format to evaluate the reproducibility of the method in assessing the stability of proteins at temperatures below the melting temperature and detecting the binding of ligands. Under the conditions tested, the authors were able to assess the stability of citrate synthase and malate dehydrogenase at different constant temperatures and detect the binding of oxaloacetate and nicotinamide adenine dinucleotide to these 2 enzymes, respectively, using the 384-well format. The ITD experiments detected ligand binding to these proteins at about 4 times lower concentration compared with techniques that measure changes in melting temperature. The data show that ITD can be applied to screen libraries of a relatively large number of compounds or detect small stability differences between protein variants.
Assuntos
Desnaturação Proteica , Proteínas/química , Fluorescência , Ligantes , Proteínas/metabolismoRESUMO
Rapid identification of small molecules that interact with protein targets using a generic screening method greatly facilitates the development of therapeutic agents. The authors describe a novel method for performing homogeneous biophysical assays in a high-throughput format. The use of light scattering as a method to evaluate protein stability during thermal denaturation in a 384-well format yields a robust assay with a low frequency of false positives. This novel method leads to the identification of interacting small molecules without the addition of extraneous fluorescent probes. The analysis and interpretation of data is rapid, with sensitivity for protein stability comparable to differential scanning calorimetry. The authors propose potential uses in drug discovery, structural genomics, and functional genomics as a method to evaluate small-molecule interactions, identify natural cofactors that stabilize target proteins, and identify natural substrates and products for previously uncharacterized protein targets.