RESUMO
The YAP-TEAD transcriptional complex is responsible for the expression of genes that regulate cancer cell growth and proliferation. Dysregulation of the Hippo pathway due to overexpression of TEAD has been reported in a wide range of cancers. Inhibition of TEAD represses the expression of associated genes, demonstrating the value of this transcription factor for the development of novel anti-cancer therapies. We report herein the design, synthesis and biological evaluation of LM98, a flufenamic acid analogue. LM98 shows strong affinity to TEAD, inhibits its autopalmitoylation and reduces the YAP-TEAD transcriptional activity. Binding of LM98 to TEAD was supported by 19 F-NMR studies while co-crystallization experiments confirmed that LM98 is anchored within the palmitic acid pocket of TEAD. LM98 reduces the expression of CTGF and Cyr61, inhibits MDA-MB-231 breast cancer cell migration and arrests cell cycling in the S phase during cell division.
Assuntos
Antineoplásicos/farmacologia , Ácido Flufenâmico/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição de Domínio TEA/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Ácido Flufenâmico/química , Humanos , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Fatores de Transcrição de Domínio TEA/metabolismo , Células Tumorais CultivadasRESUMO
Remodelin is a putative small molecule inhibitor of the RNA acetyltransferase NAT10 which has shown preclinical efficacy in models of the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS). Here we evaluate remodelin's assay interference characteristics and effects on NAT10-catalyzed RNA cytidine acetylation. We find the remodelin chemotype constitutes a cryptic assay interference compound, which does not react with small molecule thiols but demonstrates protein reactivity in ALARM NMR and proteome-wide affinity profiling assays. Biophysical analyses find no direct evidence for interaction of remodelin with the NAT10 acetyltransferase active site. Cellular studies verify that N4-acetylcytidine (ac4C) is a nonredundant target of NAT10 activity in human cell lines and find that this RNA modification is not affected by remodelin treatment in several orthogonal assays. These studies display the potential for remodelin's chemotype to interact with multiple protein targets in cells and indicate remodelin should not be applied as a specific chemical inhibitor of NAT10-catalyzed RNA acetylation.
RESUMO
The cyclic guanosine-3',5'-monophosphate (cGMP)-dependent protein kinase (PKG) was identified >25 y ago; however, efforts to obtain a structure of the entire PKG enzyme or catalytic domain from any species have failed. In malaria parasites, cooperative activation of PKG triggers crucial developmental transitions throughout the complex life cycle. We have determined the cGMP-free crystallographic structures of PKG from Plasmodium falciparum and Plasmodium vivax, revealing how key structural components, including an N-terminal autoinhibitory segment (AIS), four predicted cyclic nucleotide-binding domains (CNBs), and a kinase domain (KD), are arranged when the enzyme is inactive. The four CNBs and the KD are in a pentagonal configuration, with the AIS docked in the substrate site of the KD in a swapped-domain dimeric arrangement. We show that although the protein is predominantly a monomer (the dimer is unlikely to be representative of the physiological form), the binding of the AIS is necessary to keep Plasmodium PKG inactive. A major feature is a helix serving the dual role of the N-terminal helix of the KD as well as the capping helix of the neighboring CNB. A network of connecting helices between neighboring CNBs contributes to maintaining the kinase in its inactive conformation. We propose a scheme in which cooperative binding of cGMP, beginning at the CNB closest to the KD, transmits conformational changes around the pentagonal molecule in a structural relay mechanism, enabling PKG to orchestrate rapid, highly regulated developmental switches in response to dynamic modulation of cGMP levels in the parasite.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/química , Malária/genética , Plasmodium falciparum/química , Conformação Proteica , Sequência de Aminoácidos/genética , Animais , Sítios de Ligação/genética , Domínio Catalítico/genética , Cristalografia por Raios X , GMP Cíclico/química , Proteínas Quinases Dependentes de GMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/ultraestrutura , Humanos , Cinética , Malária/parasitologia , Plasmodium falciparum/patogenicidade , Plasmodium falciparum/ultraestrutura , Ligação ProteicaRESUMO
Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is a multidomain protein that plays a critical role in maintaining DNA methylation patterns through concurrent recognition of hemimethylated DNA and histone marks by various domains, and recruitment of DNA methyltransferase 1 (DNMT1). UHRF1 is overexpressed in various cancers, including breast cancer. The tandem tudor domain (TTD) of UHRF1 specifically and tightly binds to histone H3 di- or trimethylated at lysine 9 (H3K9me2 or H3K9me3, respectively), and this binding is essential for UHRF1 function. We developed an H3K9me3 peptide displacement assay, which was used to screen a library of 44,000 compounds for small molecules that disrupt the UHRF1-H3K9me3 interaction. This screen resulted in the identification of NV01, which bound to UHRF1-TTD with a Kd value of 5 µM. The structure of UHRF1-TTD in complex with NV01 confirmed binding to the H3K9me3-binding pocket. Limited structure-based optimization of NV01 led to the discovery of NV03 (Kd of 2.4 µM). These well-characterized small-molecule antagonists of the UHRF1-H3K9me2/3 interaction could be valuable starting chemical matter for developing more potent and cell-active probes toward further characterizing UHRF1 function, with possible applications as anticancer therapeutics.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/química , Descoberta de Drogas/métodos , Histonas/química , Ligação Proteica/efeitos dos fármacos , Domínio Tudor , Sítios de Ligação , Bioensaio/métodos , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Histonas/metabolismo , Humanos , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Mutagênese Sítio-Dirigida , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , Ubiquitina-Proteína LigasesRESUMO
UHRF1 is a key mediator of inheritance of epigenetic DNA methylation patterns during cell division and is a putative target for cancer therapy. Recent studies indicate that interdomain interactions critically influence UHRF1's chromatin-binding properties, including allosteric regulation of its histone binding. Here, using an integrative approach that combines small angle X-ray scattering, NMR spectroscopy, and molecular dynamics simulations, we characterized the dynamics of the tandem tudor domain-plant homeodomain (TTD-PHD) histone reader module, including its 20-residue interdomain linker. We found that the apo TTD-PHD module in solution comprises a dynamic ensemble of conformers, approximately half of which are compact conformations, with the linker lying in the TTD peptide-binding groove. These compact conformations are amenable to cooperative, high-affinity histone binding. In the remaining conformations, the linker position was in flux, and the reader adopted both extended and compact states. Using a small-molecule fragment screening approach, we identified a compound, 4-benzylpiperidine-1-carboximidamide, that binds to the TTD groove, competes with linker binding, and promotes open TTD-PHD conformations that are less efficient at H3K9me3 binding. Our work reveals a mechanism by which the dynamic TTD-PHD module can be allosterically targeted with small molecules to modulate its histone reader function for therapeutic or experimental purposes.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/química , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Regulação Alostérica , Cristalografia por Raios X , Epigênese Genética , Histonas/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Espalhamento a Baixo Ângulo , Ubiquitina-Proteína Ligases , Difração de Raios XRESUMO
Polycomb repressive complex 2 (PRC2) is a regulator of epigenetic states required for development and homeostasis. PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), which leads to gene silencing, and is dysregulated in many cancers. The embryonic ectoderm development (EED) protein is an essential subunit of PRC2 that has both a scaffolding function and an H3K27me3-binding function. Here we report the identification of A-395, a potent antagonist of the H3K27me3 binding functions of EED. Structural studies demonstrate that A-395 binds to EED in the H3K27me3-binding pocket, thereby preventing allosteric activation of the catalytic activity of PRC2. Phenotypic effects observed in vitro and in vivo are similar to those of known PRC2 enzymatic inhibitors; however, A-395 retains potent activity against cell lines resistant to the catalytic inhibitors. A-395 represents a first-in-class antagonist of PRC2 protein-protein interactions (PPI) for use as a chemical probe to investigate the roles of EED-containing protein complexes.
Assuntos
Antineoplásicos/farmacologia , Indanos/farmacologia , Complexo Repressor Polycomb 2/antagonistas & inibidores , Sulfonamidas/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Indanos/química , Modelos Moleculares , Estrutura Molecular , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/metabolismo , Ligação Proteica/efeitos dos fármacos , Relação Estrutura-Atividade , Sulfonamidas/química , Células Tumorais CultivadasRESUMO
Well-characterized selective inhibitors of protein arginine methyltransferases (PRMTs) are invaluable chemical tools for testing biological and therapeutic hypotheses. Based on 4, a fragment-like inhibitor of type I PRMTs, we conducted structure-activity relationship (SAR) studies and explored three regions of this scaffold. The studies led to the discovery of a potent, selective, and cell-active dual inhibitor of PRMT4 and PRMT6, 17 (MS049). As compared to 4, 17 displayed much improved potency for PRMT4 and PRMT6 in both biochemical and cellular assays. It was selective for PRMT4 and PRMT6 over other PRMTs and a broad range of other epigenetic modifiers and nonepigenetic targets. We also developed 46 (MS049N), which was inactive in biochemical and cellular assays, as a negative control for chemical biology studies. Considering possible overlapping substrate specificity of PRMTs, 17 and 46 are valuable chemical tools for dissecting specific biological functions and dysregulation of PRMT4 and PRMT6 in health and disease.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Arginina , Cristalografia por Raios X , Descoberta de Drogas , Células HEK293 , Humanos , Modelos Moleculares , Proteínas Nucleares/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismoRESUMO
WD repeat-containing protein 5 (WDR5) is an important component of the multiprotein complex essential for activating mixed-lineage leukemia 1 (MLL1). Rearrangement of the MLL1 gene is associated with onset and progression of acute myeloid and lymphoblastic leukemias, and targeting the WDR5-MLL1 interaction may result in new cancer therapeutics. Our previous work showed that binding of small molecule ligands to WDR5 can modulate its interaction with MLL1, suppressing MLL1 methyltransferase activity. Initial structure-activity relationship studies identified N-(2-(4-methylpiperazin-1-yl)-5-substituted-phenyl) benzamides as potent and selective antagonists of this protein-protein interaction. Guided by crystal structure data and supported by in silico library design, we optimized the scaffold by varying the C-1 benzamide and C-5 substituents. This allowed us to develop the first highly potent (Kdisp < 100 nM) small molecule antagonists of the WDR5-MLL1 interaction and demonstrate that N-(4-(4-methylpiperazin-1-yl)-3'-(morpholinomethyl)-[1,1'-biphenyl]-3-yl)-6-oxo-4-(trifluoromethyl)-1,6-dihydropyridine-3-carboxamide 16d (OICR-9429) is a potent and selective chemical probe suitable to help dissect the biological role of WDR5.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Compostos de Bifenilo/síntese química , Compostos de Bifenilo/farmacologia , Di-Hidropiridinas/síntese química , Di-Hidropiridinas/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/efeitos dos fármacos , Leucemia/tratamento farmacológico , Proteína de Leucina Linfoide-Mieloide/antagonistas & inibidores , Animais , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Desenho de Fármacos , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos SCID , Modelos Moleculares , Simulação de Acoplamento Molecular , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , Difração de Raios XRESUMO
We report the design and characterization of UNC3866, a potent antagonist of the methyllysine (Kme) reading function of the Polycomb CBX and CDY families of chromodomains. Polycomb CBX proteins regulate gene expression by targeting Polycomb repressive complex 1 (PRC1) to sites of H3K27me3 via their chromodomains. UNC3866 binds the chromodomains of CBX4 and CBX7 most potently, with a K(d) of â¼100 nM for each, and is 6- to 18-fold selective as compared to seven other CBX and CDY chromodomains while being highly selective over >250 other protein targets. X-ray crystallography revealed that UNC3866's interactions with the CBX chromodomains closely mimic those of the methylated H3 tail. UNC4195, a biotinylated derivative of UNC3866, was used to demonstrate that UNC3866 engages intact PRC1 and that EED incorporation into PRC1 is isoform dependent in PC3 prostate cancer cells. Finally, UNC3866 inhibits PC3 cell proliferation, consistent with the known ability of CBX7 overexpression to confer a growth advantage, whereas UNC4219, a methylated negative control compound, has negligible effects.
Assuntos
Oligopeptídeos/farmacologia , Complexo Repressor Polycomb 1/antagonistas & inibidores , Complexo Repressor Polycomb 1/genética , Animais , Disponibilidade Biológica , Biotinilação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Regulação da Expressão Gênica/genética , Humanos , Isomerismo , Ligases , Masculino , Metilação , Camundongos , Modelos Moleculares , Complexo Repressor Polycomb 1/biossíntese , Complexo Repressor Polycomb 1/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Especificidade por Substrato , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Protein methyltransferases (PMTs) are a promising target class in oncology and other disease areas. They are composed of SET domain methyltransferases and structurally unrelated Rossman-fold enzymes that include protein arginine methyltransferases (PRMTs). In the absence of a well-defined medicinal chemistry tool-kit focused on PMTs, most current inhibitors were identified by screening large and diverse libraries of leadlike molecules. So far, no successful fragment-based approach was reported against this target class. Here, by deconstructing potent PRMT inhibitors, we find that chemical moieties occupying the substrate arginine-binding site can act as efficient fragment inhibitors. Screening a fragment library against PRMT6 produced numerous hits, including a 300 nM inhibitor (ligand efficiency of 0.56) that decreased global histone 3 arginine 2 methylation in cells, and can serve as a warhead for the development of PRMT chemical probes.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteínas Repressoras/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Relação Estrutura-AtividadeRESUMO
Protein arginine methyltransferases (PRMTs) play a crucial role in a variety of biological processes. Overexpression of PRMTs has been implicated in various human diseases including cancer. Consequently, selective small-molecule inhibitors of PRMTs have been pursued by both academia and the pharmaceutical industry as chemical tools for testing biological and therapeutic hypotheses. PRMTs are divided into three categories: type I PRMTs which catalyze mono- and asymmetric dimethylation of arginine residues, type II PRMTs which catalyze mono- and symmetric dimethylation of arginine residues, and type III PRMT which catalyzes only monomethylation of arginine residues. Here, we report the discovery of a potent, selective, and cell-active inhibitor of human type I PRMTs, MS023, and characterization of this inhibitor in a battery of biochemical, biophysical, and cellular assays. MS023 displayed high potency for type I PRMTs including PRMT1, -3, -4, -6, and -8 but was completely inactive against type II and type III PRMTs, protein lysine methyltransferases and DNA methyltransferases. A crystal structure of PRMT6 in complex with MS023 revealed that MS023 binds the substrate binding site. MS023 potently decreased cellular levels of histone arginine asymmetric dimethylation. It also reduced global levels of arginine asymmetric dimethylation and concurrently increased levels of arginine monomethylation and symmetric dimethylation in cells. We also developed MS094, a close analog of MS023, which was inactive in biochemical and cellular assays, as a negative control for chemical biology studies. MS023 and MS094 are useful chemical tools for investigating the role of type I PRMTs in health and disease.
Assuntos
Antineoplásicos/farmacologia , Etanolaminas/farmacologia , Etilenodiaminas/farmacologia , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Pirróis/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Etanolaminas/química , Etilenodiaminas/química , Humanos , Modelos Moleculares , Estrutura Molecular , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Pirróis/químicaRESUMO
The CEBPA gene is mutated in 9% of patients with acute myeloid leukemia (AML). Selective expression of a short (30-kDa) CCAAT-enhancer binding protein-α (C/EBPα) translational isoform, termed p30, represents the most common type of CEBPA mutation in AML. The molecular mechanisms underlying p30-mediated transformation remain incompletely understood. We show that C/EBPα p30, but not the normal p42 isoform, preferentially interacts with Wdr5, a key component of SET/MLL (SET-domain/mixed-lineage leukemia) histone-methyltransferase complexes. Accordingly, p30-bound genomic regions were enriched for MLL-dependent H3K4me3 marks. The p30-dependent increase in self-renewal and inhibition of myeloid differentiation required Wdr5, as downregulation of the latter inhibited proliferation and restored differentiation in p30-dependent AML models. OICR-9429 is a new small-molecule antagonist of the Wdr5-MLL interaction. This compound selectively inhibited proliferation and induced differentiation in p30-expressing human AML cells. Our data reveal the mechanism of p30-dependent transformation and establish the essential p30 cofactor Wdr5 as a therapeutic target in CEBPA-mutant AML.
Assuntos
Antineoplásicos/farmacologia , Compostos de Bifenilo/farmacologia , Di-Hidropiridinas/farmacologia , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Leucemia Mieloide Aguda/metabolismo , Proteína de Leucina Linfoide-Mieloide/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Sequência de Aminoácidos , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Terapia de Alvo Molecular , Mutação , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Transdução de Sinais , Células Tumorais CultivadasRESUMO
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
Pathogens can interfere with vital biological processes of their host by mimicking host proteins. The NS1 protein of the influenza A H3N2 subtype possesses a histone H3K4-like sequence at its carboxyl terminus and has been reported to use this mimic to hijack host proteins. However, this mimic lacks a free N-terminus that is essential for binding to many known H3K4 readers. Here we show that the double chromodomains of CHD1 adopt an 'open pocket' to interact with the free N-terminal amine of H3K4, and the open pocket permits the NS1 mimic to bind in a distinct conformation. We also explored the possibility that NS1 hijacks other cellular proteins and found that the NS1 mimic has access to only a subset of chromatin-associated factors, such as WDR5. Moreover, methylation of the NS1 mimic can not be reversed by the H3K4 demethylase LSD1. Overall, we thus conclude that the NS1 mimic is an imperfect histone mimic.
Assuntos
Histonas/metabolismo , Interações Hospedeiro-Patógeno , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Calorimetria , Cristalografia por Raios X , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histona Desmetilases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Vírus da Influenza A Subtipo H3N2/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Espectrometria de Massas , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
PRDM9 (PR domain-containing protein 9) is a meiosis-specific protein that trimethylates H3K4 and controls the activation of recombination hot spots. It is an essential enzyme in the progression of early meiotic prophase. Disruption of the PRDM9 gene results in sterility in mice. In human, several PRDM9 SNPs have been implicated in sterility as well. Here we report on kinetic studies of H3K4 methylation by PRDM9 in vitro indicating that PRDM9 is a highly active histone methyltransferase catalyzing mono-, di-, and trimethylation of the H3K4 mark. Screening for other potential histone marks, we identified H3K36 as a second histone residue that could also be mono-, di-, and trimethylated by PRDM9 as efficiently as H3K4. Overexpression of PRDM9 in HEK293 cells also resulted in a significant increase in trimethylated H3K36 and H3K4 further confirming our in vitro observations. Our findings indicate that PRDM9 may play critical roles through H3K36 trimethylation in cells.
Assuntos
Metilação de DNA , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Calorimetria , Histonas/química , Humanos , Cinética , Espectrometria de Massas , Especificidade por SubstratoRESUMO
Polycomb repressive complex 2 (PRC2) is an important regulator of cellular differentiation and cell type identity. Overexpression or activating mutations of EZH2, the catalytic component of the PRC2 complex, are linked to hyper-trimethylation of lysine 27 of histone H3 (H3K27me3) in many cancers. Potent EZH2 inhibitors that reduce levels of H3K27me3 kill mutant lymphoma cells and are efficacious in a mouse xenograft model of malignant rhabdoid tumors. Unlike most SET domain methyltransferases, EZH2 requires PRC2 components, SUZ12 and EED, for activity, but the mechanism by which catalysis is promoted in the PRC2 complex is unknown. We solved the 2.0 Å crystal structure of the EZH2 methyltransferase domain revealing that most of the canonical structural features of SET domain methyltransferase structures are conserved. The site of methyl transfer is in a catalytically competent state, and the structure clarifies the structural mechanism underlying oncogenic hyper-trimethylation of H3K27 in tumors harboring mutations at Y641 or A677. On the other hand, the I-SET and post-SET domains occupy atypical positions relative to the core SET domain resulting in incomplete formation of the cofactor binding site and occlusion of the substrate binding groove. A novel CXC domain N-terminal to the SET domain may contribute to the apparent inactive conformation. We propose that protein interactions within the PRC2 complex modulate the trajectory of the post-SET and I-SET domains of EZH2 in favor of a catalytically competent conformation.