RESUMO
Glioblastoma, the most common primary malignant brain tumor, harbors a small population of tumor initiating cells (glioblastoma stem cells) that have many properties similar to neural stem cells. To investigate common regulatory networks in both neural and glioblastoma stem cells, we subjected both cell types to in-vitro differentiation conditions and measured global gene-expression changes using gene expression microarrays. Analysis of enriched transcription factor DNA-binding sites in the promoters of differentially expressed genes was used to reconstruct regulatory networks involved in differentiation. Computational predictions, which were biochemically validated, show an extensive overlap of regulatory circuitry between cell types including a network centered on the transcription factor KLF4. We further demonstrate that EGR1, a transcription factor previously shown to be downstream of the MAPK pathway, regulates KLF4 expression and that KLF4 in turn transcriptionally activates NOTCH as well as SOX2. These results demonstrate how known genomic alterations in glioma that induce constitutive activation of MAPK are transcriptionally linked to master regulators essential for neural stem cell identify.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neurais/metabolismo , Animais , Sítios de Ligação , Biomarcadores , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Biologia Computacional/métodos , Progressão da Doença , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Glioblastoma/patologia , Humanos , Fator 4 Semelhante a Kruppel , Camundongos , Gradação de Tumores , Ligação Proteica , Transdução de Sinais , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
Many current therapies target G protein coupled receptors (GPCR), transporters, or ion channels. In addition to directly targeting these proteins, disrupting the protein-protein interactions that localize or regulate their function could enhance selectivity and provide unique pharmacologic actions. Regulators of G protein signaling (RGS) proteins, especially RGS4, play significant roles in epilepsy and Parkinson's disease. Thiadiazolidinone (TDZD) inhibitors of RGS4 are nanomolar potency blockers of the biochemical actions of RGS4 in vitro. Here, we demonstrate the substantial selectivity (8- to >5000-fold) of CCG-203769 for RGS4 over other RGS proteins. It is also 300-fold selective for RGS4 over GSK-3ß, another target of this class of chemical scaffolds. It does not inhibit the cysteine protease papain at 100 µM. CCG-203769 enhances Gαq-dependent cellular Ca(2+) signaling in an RGS4-dependent manner. TDZD inhibitors also enhance Gαi-dependent δ-OR inhibition of cAMP production in SH-SY-5Y cells, which express endogenous receptors and RGS4. Importantly, CCG-203769 potentiates the known RGS4 mechanism of Gαi-dependent muscarinic bradycardia in vivo. Furthermore, it reverses raclopride-induced akinesia and bradykinesia in mice, a model of some aspects of the movement disorder in Parkinson's disease. A broad assessment of compound effects revealed minimal off-target effects at concentrations necessary for cellular RGS4 inhibition. These results expand our understanding of the mechanism and specificity of TDZD RGS inhibitors and support the potential for therapeutic targeting of RGS proteins in Parkinson's disease and other neural disorders.
Assuntos
Antiparkinsonianos/farmacologia , Proteínas RGS/antagonistas & inibidores , Animais , Bradicardia/tratamento farmacológico , Bradicardia/fisiopatologia , Cálcio/metabolismo , Carbacol/farmacologia , Linhagem Celular Tumoral , Agonistas Colinérgicos/farmacologia , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Papaína/metabolismo , Transtornos Parkinsonianos/tratamento farmacológico , Transtornos Parkinsonianos/fisiopatologia , Proteínas RGS/metabolismo , Racloprida , Ratos Sprague-DawleyRESUMO
The serum response factor (SRF) binds to coactivators, such as myocardin-related transcription factor-A (MRTF-A), and mediates gene transcription elicited by diverse signaling pathways. SRF/MRTF-A-dependent gene transcription is activated when nuclear MRTF-A levels increase, enabling the formation of transcriptionally active SRF/MRTF-A complexes. The level of nuclear MRTF-A is regulated by nuclear G-actin, which binds to MRTF-A and promotes its nuclear export. However, pathways that regulate nuclear actin levels are poorly understood. Here, we show that MICAL-2, an atypical actin-regulatory protein, mediates SRF/MRTF-A-dependent gene transcription elicited by nerve growth factor and serum. MICAL-2 induces redox-dependent depolymerization of nuclear actin, which decreases nuclear G-actin and increases MRTF-A in the nucleus. Furthermore, we show that MICAL-2 is a target of CCG-1423, a small molecule inhibitor of SRF/MRTF-A-dependent transcription that exhibits efficacy in various preclinical disease models. These data identify redox modification of nuclear actin as a regulatory switch that mediates SRF/MRTF-A-dependent gene transcription.
Assuntos
Núcleo Celular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Oxirredutases/metabolismo , Fator de Resposta Sérica/metabolismo , Transdução de Sinais , Actinas/metabolismo , Sequência de Aminoácidos , Anilidas/farmacologia , Animais , Benzamidas/farmacologia , Linhagem Celular , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Proteínas dos Microfilamentos/análise , Proteínas dos Microfilamentos/genética , Oxigenases de Função Mista/análise , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Fator de Crescimento Neural/metabolismo , Neuritos/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Oxirredução , Oxirredutases/análise , Oxirredutases/genética , Ratos , Alinhamento de Sequência , Transativadores , Transcrição Gênica , Peixe-ZebraRESUMO
Regulators of G protein signaling (RGS) proteins are key players in regulating signaling via G protein-coupled receptors. RGS proteins directly bind to the Gα-subunits of activated heterotrimeric G-proteins, and accelerate the rate of GTP hydrolysis, thereby rapidly deactivating G-proteins. Using atomistic simulations and NMR spectroscopy, we have studied in molecular detail the mechanism of action of CCG-50014, a potent small molecule inhibitor of RGS4 that covalently binds to cysteine residues on RGS4. We apply temperature-accelerated molecular dynamics (TAMD) to carry out enhanced conformational sampling of apo RGS4 structures, and consistently find that the α5-α6 helix pair of RGS4 can spontaneously span open-like conformations, allowing binding of CCG-50014 to the buried side-chain of Cys95. Both NMR experiments and MD simulations reveal chemical shift perturbations in residues in the vicinity of inhibitor binding site as well as in the RGS4-Gα binding interface. Consistent with a loss of G-protein binding, GAP activity, and allosteric mechanism of action of CCG-50014, our simulations of the RGS4-Gα complex in the presence of inhibitor suggest a relatively unstable protein-protein interaction. These results have potential implications for understanding how the conformational dynamics among RGS proteins may play a key role in the sensitivity of inhibitors.
Assuntos
Cisteína/química , Simulação de Dinâmica Molecular , Proteínas RGS/química , Bibliotecas de Moléculas Pequenas/química , Tiazolidinedionas/química , Regulação Alostérica , Sítio Alostérico , Cisteína/metabolismo , Humanos , Cinética , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas RGS/antagonistas & inibidores , Proteínas RGS/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Tiazolidinedionas/metabolismoRESUMO
Regulator of G-protein signaling (RGS) proteins potently suppress G-protein coupled receptor (GPCR) signal transduction by accelerating GTP hydrolysis on activated heterotrimeric G-protein α subunits. RGS4 is enriched in the CNS and is proposed as a therapeutic target for treatment of neuropathological states including epilepsy and Parkinson's disease. Therefore, identification of novel RGS4 inhibitors is of interest. An HEK293-FlpIn cell-line stably expressing M3-muscarinic receptor with doxycycline-regulated RGS4 expression was employed to identify compounds that inhibit RGS4-mediated suppression of M3-muscarinic receptor signaling. Over 300,000 compounds were screened for an ability to enhance Gαq-mediated calcium signaling in the presence of RGS4. Compounds that modulated the calcium response in a counter-screen in the absence of RGS4 were not pursued. Of the 1365 RGS4-dependent primary screen hits, thirteen compounds directly target the RGS-G-protein interaction in purified systems. All thirteen compounds lose activity against an RGS4 mutant lacking cysteines, indicating that covalent modification of free thiol groups on RGS4 is a common mechanism. Four compounds produce >85% inhibition of RGS4-G-protein binding at 100µM, yet are >50% reversible within a ten-minute time frame. The four reversible compounds significantly alter the thermal melting temperature of RGS4, but not G-protein, indicating that inhibition is occurring through interaction with the RGS protein. The HEK cell-line employed for this study provides a powerful tool for efficiently identifying RGS-specific modulators within the context of a GPCR signaling pathway. As a result, several new reversible, cell-active RGS4 inhibitors have been identified for use in future biological studies.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Proteínas de Ligação ao GTP/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Cálcio/metabolismo , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Proteínas de Ligação ao GTP/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Proteínas RGS/antagonistas & inibidores , Proteínas RGS/metabolismo , Receptor Muscarínico M3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/químicaRESUMO
The duration and amplitude of G-protein-coupled receptor (GPCR) signaling is controlled by regulator of G-protein signaling (RGS) proteins. The 20 RGS family members act as GTPase accelerating proteins through their interaction with the Gα subunit of the Gαßγ heterotrimer. Their influence over GPCR signaling has attracted many to these proteins as advantageous therapeutic targets. The nature of the RGS structure has proven to be difficult to target with small molecules using traditional high-throughput screening methods. This chapter describes NMR methods for studying small molecule interactions on RGS4. These methods can detect ligand binding without the requirement for an effect on protein function. Furthermore, the sensitivity of NMR permits detection of weaker protein-ligand interactions, such as those found with smaller fragment compounds. Fragment-based screening may be path forward to identifying a number of active small molecules toward RGS proteins. Methods and considerations for running a fragment-based screen on RGS4 using NMR are outlined in this section.
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas RGS/metabolismo , Escherichia coli/genética , Expressão Gênica , Humanos , Marcação por Isótopo/métodos , Ligantes , Mutação , Isótopos de Nitrogênio , Ligação Proteica , Teoria Quântica , Proteínas RGS/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , SolubilidadeRESUMO
Degradation by the proteasome typically requires substrate ubiquitination. Two ubiquitin receptors exist in the proteasome, S5a/Rpn10 and Rpn13. Whereas Rpn13 has only one ubiquitin-binding surface, S5a binds ubiquitin with two independent ubiquitin-interacting motifs (UIMs). Here, we use nuclear magnetic resonance (NMR) and analytical ultracentrifugation to define at atomic level resolution how S5a binds K48-linked diubiquitin, in which K48 of one ubiquitin subunit (the "proximal" one) is covalently bonded to G76 of the other (the "distal" subunit). We demonstrate that S5a's UIMs bind the two subunits simultaneously with a preference for UIM2 binding to the proximal subunit while UIM1 binds to the distal one. In addition, NMR experiments reveal that Rpn13 and S5a bind K48-linked diubiquitin simultaneously with subunit specificity, and a model structure of S5a and Rpn13 bound to K48-linked polyubiquitin is provided. Altogether, our data demonstrate that S5a is highly adaptive and cooperative toward binding ubiquitin chains.