RESUMEN
Gene knock outs (KOs) are efficiently engineered through CRISPR-Cas9-induced frameshift mutations. While the efficiency of DNA editing is readily verified by DNA sequencing, a systematic understanding of the efficiency of protein elimination has been lacking. Here we devised an experimental strategy combining RNA sequencing and triple-stage mass spectrometry to characterize 193 genetically verified deletions targeting 136 distinct genes generated by CRISPR-induced frameshifts in HAP1 cells. We observed residual protein expression for about one third of the quantified targets, at variable levels from low to original, and identified two causal mechanisms, translation reinitiation leading to N-terminally truncated target proteins or skipping of the edited exon leading to protein isoforms with internal sequence deletions. Detailed analysis of three truncated targets, BRD4, DNMT1 and NGLY1, revealed partial preservation of protein function. Our results imply that systematic characterization of residual protein expression or function in CRISPR-Cas9-generated KO lines is necessary for phenotype interpretation.
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Sistemas CRISPR-Cas/genética , Técnicas de Inactivación de Genes , Proteínas de Ciclo Celular/genética , ADN (Citosina-5-)-Metiltransferasa 1/genética , Exones , Humanos , Mutación , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/genética , Factores de Transcripción/genéticaRESUMEN
The jumonji (JMJ) family of histone demethylases are Fe2+- and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes. These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context. Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance, as well as in development, physiology and disease. However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX). The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Secuencia de Aminoácidos , Animales , Biocatálisis/efectos de los fármacos , Dominio Catalítico , Células Cultivadas , Inhibidores Enzimáticos/metabolismo , Evolución Molecular , Histonas/química , Histonas/metabolismo , Humanos , Concentración 50 Inhibidora , Histona Demetilasas con Dominio de Jumonji/química , Histona Demetilasas con Dominio de Jumonji/clasificación , Histona Demetilasas con Dominio de Jumonji/metabolismo , Lisina/metabolismo , Macrófagos/enzimología , Macrófagos/metabolismo , Metilación/efectos de los fármacos , Ratones , Modelos Moleculares , Especificidad por Sustrato , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Dysregulated proteostasis is a key feature of a variety of neurodegenerative disorders. In Alzheimer's disease (AD), progression of symptoms closely correlates with spatiotemporal progression of Tau aggregation, with "early" oligomeric Tau forms rather than mature neurofibrillary tangles (NFTs) considered to be pathogenetic culprits. The ubiquitin-proteasome system (UPS) controls degradation of soluble normal and abnormally folded cytosolic proteins. The UPS is affected in AD and is identified by genomewide association study (GWAS) as a risk pathway for AD. The UPS is determined by balanced regulation of ubiquitination and deubiquitination. In this work, we performed isobaric tags for relative and absolute quantitation (iTRAQ)-based Tau interactome mapping to gain unbiased insight into Tau pathophysiology and to identify novel Tau-directed therapeutic targets. Focusing on Tau deubiquitination, we here identify Otub1 as a Tau-deubiquitinating enzyme. Otub1 directly affected Lys48-linked Tau deubiquitination, impairing Tau degradation, dependent on its catalytically active cysteine, but independent of its noncanonical pathway modulated by its N-terminal domain in primary neurons. Otub1 strongly increased AT8-positive Tau and oligomeric Tau forms and increased Tau-seeded Tau aggregation in primary neurons. Finally, we demonstrated that expression of Otub1 but not its catalytically inactive form induced pathological Tau forms after 2 months in Tau transgenic mice in vivo, including AT8-positive Tau and oligomeric Tau forms. Taken together, we here identified Otub1 as a Tau deubiquitinase in vitro and in vivo, involved in formation of pathological Tau forms, including small soluble oligomeric forms. Otub1 and particularly Otub1 inhibitors, currently under development for cancer therapies, may therefore yield interesting novel therapeutic avenues for Tauopathies and AD.
Asunto(s)
Cisteína Endopeptidasas/genética , Enzimas Desubicuitinizantes/metabolismo , Ovillos Neurofibrilares/patología , Tauopatías/patología , Proteínas tau/metabolismo , Animales , Humanos , Ratones Transgénicos , Ovillos Neurofibrilares/metabolismo , Neuronas/metabolismo , Neuronas/patología , Tauopatías/metabolismo , Ubiquitina/metabolismo , Ubiquitinación/fisiologíaRESUMEN
PAD4 has been strongly implicated in the pathogenesis of autoimmune, cardiovascular and oncological diseases through clinical genetics and gene disruption in mice. New selective PAD4 inhibitors binding a calcium-deficient form of the PAD4 enzyme have validated the critical enzymatic role of human and mouse PAD4 in both histone citrullination and neutrophil extracellular trap formation for, to our knowledge, the first time. The therapeutic potential of PAD4 inhibitors can now be explored.
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Bencimidazoles/farmacología , Inhibidores Enzimáticos/farmacología , Hidrolasas/antagonistas & inhibidores , Neutrófilos/efectos de los fármacos , Animales , Bencimidazoles/síntesis química , Unión Competitiva , Calcio/metabolismo , Citrulina/metabolismo , Inhibidores Enzimáticos/síntesis química , Células HEK293 , Histonas/metabolismo , Humanos , Técnicas In Vitro , Ratones , Modelos Moleculares , Arginina Deiminasa Proteína-Tipo 4 , Desiminasas de la Arginina Proteica , Bibliotecas de Moléculas Pequeñas , Especificidad por SustratoRESUMEN
Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are driver mutations in acute myeloid leukemia (AML) and other cancers. We report the development of new allosteric inhibitors of mutant IDH1. Crystallographic and biochemical results demonstrated that compounds of this chemical series bind to an allosteric site and lock the enzyme in a catalytically inactive conformation, thereby enabling inhibition of different clinically relevant IDH1 mutants. Treatment of IDH1 mutant primary AML cells uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block and induction of granulocytic differentiation at the level of leukemic blasts and more immature stem-like cells, in vitro and in vivo. Molecularly, treatment with the inhibitors led to a reversal of the DNA cytosine hypermethylation patterns caused by mutant IDH1 in the cells of individuals with AML. Our study provides proof of concept for the molecular and biological activity of novel allosteric inhibitors for targeting different mutant forms of IDH1 in leukemia.
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Dihidropiridinas/farmacología , Inhibidores Enzimáticos/farmacología , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Pirazoles/farmacología , Regulación Alostérica , Sitio Alostérico , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Islas de CpG , Cristalografía por Rayos X , Citosina/química , Citosina/metabolismo , Metilación de ADN/efectos de los fármacos , Dihidropiridinas/química , Dihidropiridinas/farmacocinética , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Granulocitos/efectos de los fármacos , Granulocitos/enzimología , Granulocitos/patología , Humanos , Isocitrato Deshidrogenasa/química , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Cinética , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Masculino , Ratones , Modelos Moleculares , Mutación , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , Cultivo Primario de Células , Unión Proteica , Pirazoles/química , Pirazoles/farmacocinética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The search for novel therapeutic interventions for viral disease is a challenging pursuit, hallmarked by the paucity of antiviral agents currently prescribed. Targeting of viral proteins has the inextricable challenge of rise of resistance. Safe and effective vaccines are not possible for many viral pathogens. New approaches are required to address the unmet medical need in this area. We undertook a cell-based high-throughput screen to identify leads for development of drugs to treat respiratory syncytial virus (RSV), a serious pediatric pathogen. We identified compounds that are potent (nanomolar) inhibitors of RSV in vitro in HEp-2 cells and in primary human bronchial epithelial cells and were shown to act postentry. Interestingly, two scaffolds exhibited broad-spectrum activity among multiple RNA viruses. Using the chemical matter as a probe, we identified the targets and identified a common cellular pathway: the de novo pyrimidine biosynthesis pathway. Both targets were validated in vitro and showed no significant cell cytotoxicity except for activity against proliferative B- and T-type lymphoid cells. Corollary to this finding was to understand the consequences of inhibition of the target to the host. An in vivo assessment for antiviral efficacy failed to demonstrate reduced viral load, but revealed microscopic changes and a trend toward reduced pyrimidine pools and findings in histopathology. We present here a discovery program that includes screen, target identification, validation, and druggability that can be broadly applied to identify and interrogate other host factors for antiviral effect starting from chemical matter of unknown target/mechanism of action.
Asunto(s)
Antivirales , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Infecciones por Virus Sincitial Respiratorio/metabolismo , Virus Sincitiales Respiratorios/metabolismo , Animales , Antivirales/síntesis química , Antivirales/química , Antivirales/farmacología , Linfocitos B/metabolismo , Linfocitos B/patología , Linfocitos B/virología , Proliferación Celular/efectos de los fármacos , Chlorocebus aethiops , Perros , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Células Jurkat , Infecciones por Virus Sincitial Respiratorio/patología , Linfocitos T/metabolismo , Linfocitos T/patología , Linfocitos T/virología , Células VeroRESUMEN
BACKGROUND: The heat shock protein 90 (Hsp90) is required for the stability of many signalling kinases. As a target for cancer therapy it allows the simultaneous inhibition of several signalling pathways. However, its inhibition in healthy cells could also lead to severe side effects. This is the first comprehensive analysis of the response to Hsp90 inhibition at the kinome level. METHODS: We quantitatively profiled the effects of Hsp90 inhibition by geldanamycin on the kinome of one primary (Hs68) and three tumour cell lines (SW480, U2OS, A549) by affinity proteomics based on immobilized broad spectrum kinase inhibitors ("kinobeads"). To identify affected pathways we used the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway classification. We combined Hsp90 and proteasome inhibition to identify Hsp90 substrates in Hs68 and SW480 cells. The mutational status of kinases from the used cell lines was determined using next-generation sequencing. A mutation of Hsp90 candidate client RIPK2 was mapped onto its structure. RESULTS: We measured relative abundances of > 140 protein kinases from the four cell lines in response to geldanamycin treatment and identified many new potential Hsp90 substrates. These kinases represent diverse families and cellular functions, with a strong representation of pathways involved in tumour progression like the BMP, MAPK and TGF-beta signalling cascades. Co-treatment with the proteasome inhibitor MG132 enabled us to classify 64 kinases as true Hsp90 clients. Finally, mutations in 7 kinases correlate with an altered response to Hsp90 inhibition. Structural modelling of the candidate client RIPK2 suggests an impact of the mutation on a proposed Hsp90 binding domain. CONCLUSIONS: We propose a high confidence list of Hsp90 kinase clients, which provides new opportunities for targeted and combinatorial cancer treatment and diagnostic applications.
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Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/genética , Factor de Crecimiento Transformador beta/metabolismo , Benzoquinonas/farmacología , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/genética , Proteínas HSP90 de Choque Térmico/química , Humanos , Lactamas Macrocíclicas/farmacología , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteómica , Transducción de Señal/efectos de los fármacosRESUMEN
Signal transduction pathways are modular composites of functionally interdependent sets of proteins that act in a coordinated fashion to transform environmental information into a phenotypic response. The pro-inflammatory cytokine tumour necrosis factor (TNF)-alpha triggers a signalling cascade, converging on the activation of the transcription factor NF-kappa B, which forms the basis for numerous physiological and pathological processes. Here we report the mapping of a protein interaction network around 32 known and candidate TNF-alpha/NF-kappa B pathway components by using an integrated approach comprising tandem affinity purification, liquid-chromatography tandem mass spectrometry, network analysis and directed functional perturbation studies using RNA interference. We identified 221 molecular associations and 80 previously unknown interactors, including 10 new functional modulators of the pathway. This systems approach provides significant insight into the logic of the TNF-alpha/NF-kappa B pathway and is generally applicable to other pathways relevant to human disease.
Asunto(s)
Proteínas de Drosophila , FN-kappa B/metabolismo , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Chaperoninas , Cromatografía de Afinidad/métodos , Activación Enzimática , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Proteínas I-kappa B/aislamiento & purificación , Proteínas I-kappa B/metabolismo , MAP Quinasa Quinasa Quinasa 3 , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Sustancias Macromoleculares , Espectrometría de Masas/métodos , Modelos Biológicos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , FN-kappa B/genética , FN-kappa B/aislamiento & purificación , Proteoma/análisis , Interferencia de ARN , Receptores del Factor de Necrosis Tumoral/metabolismo , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/aislamiento & purificación , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Host factor pathways are known to be essential for hepatitis C virus (HCV) infection and replication in human liver cells. To search for novel host factor proteins required for HCV replication, we screened a subgenomic genotype 1b replicon cell line (Luc-1b) with a kinome and druggable collection of 20,779 siRNAs. We identified and validated several enzymes required for HCV replication, including class III phosphatidylinositol 4-kinases (PI4KA and PI4KB), carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and mevalonate (diphospho) decarboxylase. Knockdown of PI4KA could inhibit the replication and/or HCV RNA levels of the two subgenomic genotype 1b clones (SG-1b and Luc-1b), two subgenomic genotype 1a clones (SG-1a and Luc-1a), JFH-1 genotype 2a infectious virus (JFH1-2a), and the genomic genotype 1a (FL-1a) replicon. In contrast, PI4KB knockdown inhibited replication and/or HCV RNA levels of Luc-1b, SG-1b, and Luc-1a replicons. The small molecule inhibitor, PIK93, was found to block subgenomic genotype 1b (Luc-1b), subgenomic genotype 1a (Luc-1a), and genomic genotype 2a (JFH1-2a) infectious virus replication in the nanomolar range. PIK93 was characterized by using quantitative chemical proteomics and in vitro biochemical assays to demonstrate PIK93 is a bone fide PI4KA and PI4KB inhibitor. Our data demonstrate that genetic or pharmacological modulation of PI4KA and PI4KB inhibits multiple genotypes of HCV and represents a novel druggable class of therapeutic targets for HCV infection.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa/metabolismo , Hepacivirus/genética , Hepacivirus/metabolismo , Hígado/virología , Replicación Viral , 1-Fosfatidilinositol 4-Quinasa/química , Antivirales/farmacología , Unión Competitiva , Línea Celular , Silenciador del Gen , Genotipo , Humanos , Concentración 50 Inhibidora , Espectrometría de Masas/métodos , Proteómica/métodos , ARN Interferente Pequeño/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tiazoles/farmacologíaRESUMEN
CRISPR/Cas9-based gene knockouts (KOs) enable precise perturbation of target gene function in human cells, which is ideally assessed in an unbiased fashion by molecular omics readouts. Typically, this requires the lengthy process of isolating KO subclones. We show here that KO subclones are phenotypically heterogenous, regardless of the guide RNA used. We present an experimental strategy that avoids subcloning and achieves fast and efficient gene silencing on cell pools, based on the synergistic combination of two guide RNAs mapping at close (40-300 bp) genomic proximity. Our strategy results in better predictable indel generation with a low allelic heterogeneity, concomitant with low or undetectable residual target protein expression, as determined by MS3 mass spectrometry proteomics. Our method is compatible with nondividing primary cells and can also be used to study essential genes. It enables the generation of high confidence omics data which solely reflect the phenotype of the target ablation.
Asunto(s)
Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Técnicas de Inactivación de Genes/métodos , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , ADN/genética , Silenciador del Gen/fisiología , Células Hep G2 , Humanos , Mutación INDEL/genética , ARN Guía de Kinetoplastida/genéticaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Myofibroblasts are the key effector cells responsible for excessive extracellular matrix deposition in multiple fibrotic conditions, including idiopathic pulmonary fibrosis (IPF). The PI3K/Akt/mTOR axis has been implicated in fibrosis, with pan-PI3K/mTOR inhibition currently under clinical evaluation in IPF. Here we demonstrate that rapamycin-insensitive mTORC1 signaling via 4E-BP1 is a critical pathway for TGF-ß1 stimulated collagen synthesis in human lung fibroblasts, whereas canonical PI3K/Akt signaling is not required. The importance of mTORC1 signaling was confirmed by CRISPR-Cas9 gene editing in normal and IPF fibroblasts, as well as in lung cancer-associated fibroblasts, dermal fibroblasts and hepatic stellate cells. The inhibitory effect of ATP-competitive mTOR inhibition extended to other matrisome proteins implicated in the development of fibrosis and human disease relevance was demonstrated in live precision-cut IPF lung slices. Our data demonstrate that the mTORC1/4E-BP1 axis represents a critical signaling node during fibrogenesis with potential implications for the development of novel anti-fibrotic strategies.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Colágeno/biosíntesis , Fibroblastos/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Fosfoproteínas/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Proteínas de Ciclo Celular , Línea Celular , Humanos , Fibrosis Pulmonar Idiopática/etiología , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Sirolimus , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Abdominal pain and abnormal bowel habits represent major symptoms for irritable bowel syndrome (IBS) patients that are not adequately managed. Although the etiology of IBS is not completely understood, many of the functions of the gastrointestinal (GI) tract are regulated by the enteric nervous system (ENS). Inflammation or stress-induced expression of growth factors or cytokines may lead to hyperinnervation of visceral afferent neurons in GI tract and contribute to the pathophysiology of IBS. Rearranged during transfection (RET) is a neuronal growth factor receptor tyrosine kinase critical for the development of the ENS as exemplified by Hirschsprung patients who carry RET loss-of-function mutations and lack normal colonic innervation leading to colonic obstruction. Similarly, RET signaling in the adult ENS maintains neuronal function by contributing to synaptic formation, signal transmission, and neuronal plasticity. Inhibition of RET in the ENS represents a novel therapeutic strategy for the normalization of neuronal function and the symptoms of IBS patients. Herein, we describe our screening effort and subsequent structure-activity relationships (SARs) in optimizing potency, selectivity, and mutagenicity of the series, which led to the discovery of a first-in-class, gut-restricted RET kinase inhibitor, 2-(4-(4-ethoxy-6-oxo-1,6-dihydropyridin-3-yl)-2-fluorophenyl)-N-(5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-3-yl)acetamide (15, GSK3179106), as a clinical candidate for the treatment of IBS. GSK3179106 is a potent, selective, and gut-restricted pyridone hinge binder small molecule RET kinase inhibitor with a RET IC50 of 0.3 nM and is efficacious in vivo.
RESUMEN
Epithelial cells display apical-basal polarity, and the apical surface is segregated from the basolateral membranes by a barrier called the tight junction (TJ). TJs are constructed from transmembrane proteins that form cell-cell contacts-claudins, occludin, and junctional adhesion molecule (JAM)-plus peripheral proteins such as ZO-1. The Par proteins (partitioning-defective) Par3 and Par6, plus atypical protein kinase C (aPKC) function in the formation or maintenance of TJs and more generally in metazoan cell polarity establishment. Par6 contains a PDZ domain and a partial CRIB (Cdc42/Rac interactive binding) domain and binds the small GTPase Cdc42. Here, we show that Par6 inhibits TJ assembly in MDCK II epithelial cells after their disruption by Ca(2+) depletion but does not inhibit adherens junction (AJ) formation. Transepithelial resistance and paracellular diffusion assays confirmed that assembly of functional TJs is delayed by Par6 overexpression. Strikingly, the isolated, N-terminal fragment of PKCzeta, which binds Par6, also inhibits TJ assembly. Activated Cdc42 can disrupt TJs, but neither a dominant-negative Cdc42 mutant nor the CRIB domain of gammaPAK (p21-activated kinase), which inhibits Cdc42 function, observably inhibit TJ formation. These results suggest that Cdc42 and Par6 negatively regulate TJ assembly in mammalian epithelial cells.
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Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteínas/metabolismo , Uniones Estrechas/metabolismo , Uniones Adherentes/metabolismo , Uniones Adherentes/ultraestructura , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Línea Celular , Difusión , Perros , Ratones , Microscopía Electrónica , Datos de Secuencia Molecular , Isoformas de Proteínas/metabolismo , Proteína Quinasa C/metabolismo , Estructura Terciaria de Proteína , Proteínas/química , Proteínas/genética , Relación Estructura-Actividad , Factores de Tiempo , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Ubiquitin specific protease 7 (USP7) belongs to the family of deubiquitinating enzymes. Among other functions, USP7 is involved in the regulation of stress response pathways, epigenetic silencing and the progress of infections by DNA viruses. USP7 is a 130-kDa protein with a cysteine peptidase core, N- and C-terminal domains required for protein-protein interactions. In the present study, recombinant USP7 full length, along with several variants corresponding to domain deletions, were expressed in different hosts in order to analyze post-translational modifications, oligomerization state, enzymatic properties and subcellular localization patterns of the enzyme. USP7 is phosphorylated at S18 and S963, and ubiquitinated at K869 in mammalian cells. In in vitro activity assays, N- and C-terminal truncations affected the catalytic efficiency of the enzyme different. Both the protease core alone and in combination with the N-terminal domain are over 100-fold less active than the full length enzyme, whereas a construct including the C-terminal region displays a rather small decrease in catalytic efficiency. Limited proteolysis experiments revealed that USP7 variants containing the C-terminal domain interact more tightly with ubiquitin. Besides playing an important role in substrate recognition and processing, this region might be involved in enzyme dimerization. USP7 constructs lacking the N-terminal domain failed to localize in the cell nucleus, but no nuclear localization signal could be mapped within the enzyme's first 70 amino acids. Instead, the tumor necrosis factor receptor associated factor-like region (amino acids 70-205) was sufficient to achieve the nuclear localization of the enzyme, suggesting that interaction partners might be required for USP7 nuclear import.
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Núcleo Celular/metabolismo , Citosol/metabolismo , Procesamiento Proteico-Postraduccional , Ubiquitina Tiolesterasa/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico/genética , Línea Celular , Línea Celular Tumoral , Cromatografía en Gel , Dimerización , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Immunoblotting , Cinética , Espectrometría de Masas , Microscopía Confocal , Datos de Secuencia Molecular , Peso Molecular , Mutación , Transporte de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por Sustrato , Transfección , Ubiquitina Tiolesterasa/química , Ubiquitina Tiolesterasa/genética , Peptidasa Específica de Ubiquitina 7RESUMEN
BACKGROUND: The amino acid response (AAR) is an evolutionarily conserved protective mechanism activated by amino acid deficiency through a key kinase, general control nonderepressible 2. In addition to mobilizing amino acids, the AAR broadly affects gene and protein expression in a variety of pathways and elicits antifibrotic, autophagic, and anti-inflammatory activities. However, little is known regarding its role in cardiac stress. Our aim was to investigate the effects of halofuginone, a prolyl-tRNA synthetase inhibitor, on the AAR pathway in cardiac fibroblasts, cardiomyocytes, and in mouse models of cardiac stress and failure. METHODS AND RESULTS: Consistent with its ability to inhibit prolyl-tRNA synthetase, halofuginone elicited a general control nonderepressible 2-dependent activation of the AAR pathway in cardiac fibroblasts as evidenced by activation of known AAR target genes, broad regulation of the transcriptome and proteome, and reversal by l-proline supplementation. Halofuginone was examined in 3 mouse models of cardiac stress: angiotensin II/phenylephrine, transverse aortic constriction, and acute ischemia reperfusion injury. It activated the AAR pathway in the heart, improved survival, pulmonary congestion, left ventricle remodeling/fibrosis, and left ventricular function, and rescued ischemic myocardium. In human cardiac fibroblasts, halofuginone profoundly reduced collagen deposition in a general control nonderepressible 2-dependent manner and suppressed the extracellular matrix proteome. In human induced pluripotent stem cell-derived cardiomyocytes, halofuginone blocked gene expression associated with endothelin-1-mediated activation of pathologic hypertrophy and restored autophagy in a general control nonderepressible 2/eIF2α-dependent manner. CONCLUSIONS: Halofuginone activated the AAR pathway in the heart and attenuated the structural and functional effects of cardiac stress.
Asunto(s)
Aminoácidos/metabolismo , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de los fármacos , Insuficiencia Cardíaca/prevención & control , Miocitos Cardíacos/efectos de los fármacos , Piperidinas/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Quinazolinonas/farmacología , Estrés Fisiológico , Aminoácidos/deficiencia , Aminoacil-ARNt Sintetasas/antagonistas & inhibidores , Aminoacil-ARNt Sintetasas/metabolismo , Animales , Autofagia/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Humanos , Hipertrofia Ventricular Izquierda/metabolismo , Hipertrofia Ventricular Izquierda/patología , Hipertrofia Ventricular Izquierda/fisiopatología , Hipertrofia Ventricular Izquierda/prevención & control , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Tiempo , Función Ventricular Izquierda/efectos de los fármacos , Remodelación Ventricular/efectos de los fármacosRESUMEN
The 2-oxoglutarate-dependent dioxygenase target class comprises around 60 enzymes including several subfamilies with relevance to human disease, such as the prolyl hydroxylases and the Jumonji-type lysine demethylases. Current drug discovery approaches are largely based on small molecule inhibitors targeting the iron/2-oxoglutarate cofactor binding site. We have devised a chemoproteomics approach based on a combination of unselective active-site ligands tethered to beads, enabling affinity capturing of around 40 different dioxygenase enzymes from human cells. Mass-spectrometry-based quantification of bead-bound enzymes using a free-ligand competition-binding format enabled the comprehensive determination of affinities for the cosubstrate 2-oxoglutarate and for oncometabolites such as 2-hydroxyglutarate. We also profiled a set of representative drug-like inhibitor compounds. The results indicate that intracellular competition by endogenous cofactors and high active site similarity present substantial challenges for drug discovery for this target class.
Asunto(s)
Dioxigenasas/metabolismo , Ácidos Cetoglutáricos/metabolismo , ProteómicaRESUMEN
Following the discovery of cell penetrant pyridine-4-carboxylate inhibitors of the KDM4 (JMJD2) and KDM5 (JARID1) families of histone lysine demethylases (e.g., 1), further optimization led to the identification of non-carboxylate inhibitors derived from pyrido[3,4-d]pyrimidin-4(3H)-one. A number of exemplars such as compound 41 possess interesting activity profiles in KDM4C and KDM5C biochemical and target-specific, cellular mechanistic assays.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Histona Demetilasas/antagonistas & inhibidores , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Pirimidinonas/química , Pirimidinonas/farmacología , Línea Celular , Permeabilidad de la Membrana Celular , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacocinética , Histona Demetilasas/química , Histona Demetilasas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/química , Histona Demetilasas con Dominio de Jumonji/metabolismo , Modelos Moleculares , Simulación del Acoplamiento Molecular , Pirimidinonas/farmacocinética , Relación Estructura-ActividadRESUMEN
Optimization of KDM6B (JMJD3) HTS hit 12 led to the identification of 3-((furan-2-ylmethyl)amino)pyridine-4-carboxylic acid 34 and 3-(((3-methylthiophen-2-yl)methyl)amino)pyridine-4-carboxylic acid 39 that are inhibitors of the KDM4 (JMJD2) family of histone lysine demethylases. Compounds 34 and 39 possess activity, IC50 ≤ 100 nM, in KDM4 family biochemical (RFMS) assays with ≥ 50-fold selectivity against KDM6B and activity in a mechanistic KDM4C cell imaging assay (IC50 = 6-8 µM). Compounds 34 and 39 are also potent inhibitors of KDM5C (JARID1C) (RFMS IC50 = 100-125 nM).