RESUMO
Protein phosphorylation is a prevalent and ubiquitous mechanism of regulation. Kinases are popular drug targets, but identifying selective phosphatase inhibitors has been challenging. Here, we used surface plasmon resonance to design a method to enable target-based discovery of selective serine/threonine phosphatase inhibitors. The method targeted a regulatory subunit of protein phosphatase 1, PPP1R15B (R15B), a negative regulator of proteostasis. This yielded Raphin1, a selective inhibitor of R15B. In cells, Raphin1 caused a rapid and transient accumulation of its phosphorylated substrate, resulting in a transient attenuation of protein synthesis. In vitro, Raphin1 inhibits the recombinant R15B-PP1c holoenzyme, but not the closely related R15A-PP1c, by interfering with substrate recruitment. Raphin1 was orally bioavailable, crossed the blood-brain barrier, and demonstrated efficacy in a mouse model of Huntington's disease. This identifies R15B as a druggable target and provides a platform for target-based discovery of inhibitors of serine/threonine phosphatases.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Guanidinas/farmacologia , Proteína Fosfatase 1/antagonistas & inibidores , Animais , Peso Corporal , Modelos Animais de Doenças , Descoberta de Drogas , Feminino , Guanidinas/química , Células HeLa , Humanos , Doença de Huntington/metabolismo , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteína Fosfatase 1/metabolismo , Subunidades Proteicas/antagonistas & inibidores , Proteostase , Proteínas Recombinantes/farmacologia , Ressonância de Plasmônio de SuperfícieRESUMO
The proteasome is essential for the selective degradation of most cellular proteins. To survive overwhelming demands on the proteasome arising during environmental stresses, cells increase proteasome abundance. Proteasome assembly is known to be complex. How stressed cells overcome this vital challenge is unknown. In an unbiased suppressor screen aimed at rescuing the defects of a yeast Rpt6 thermosensitive proteasome mutant, we identified a protein, hereafter named Adc17, as it functions as an ATPase dedicated chaperone. Adc17 interacts with the amino terminus of Rpt6 to assist formation of the Rpt6-Rpt3 ATPase pair, an early step in proteasome assembly. Adc17 is important for cell fitness, and its absence aggravates proteasome defects. The abundance of Adc17 increases upon proteasome stresses, and its function is crucial to maintain homeostatic proteasome levels. Thus, cells have mechanisms to adjust proteasome assembly when demands increase, and Adc17 is a critical effector of this process.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas Fúngicas/metabolismo , Chaperonas Moleculares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Leveduras/metabolismo , Dicroísmo Circular , Proteínas Fúngicas/genética , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/genética , Alinhamento de Sequência , Estresse Fisiológico/fisiologia , Leveduras/genéticaRESUMO
The retinoid X receptors (RXRs) are ligand-activated transcription factors which heterodimerize with a number of nuclear hormone receptors, thereby controlling a variety of (patho)-physiological processes. Although synthetic RXR ligands are developed for the treatment of various diseases, endogenous ligand(s) for these receptors have not been conclusively identified. We show here that mice lacking cellular retinol binding protein (Rbp1-/-) display memory deficits reflecting compromised RXR signaling. Using HPLC-MS and chemical synthesis we identified in Rbp1-/- mice reduced levels of 9-cis-13,14-dihydroretinoic acid (9CDHRA), which acts as an RXR ligand since it binds and transactivates RXR in various assays. 9CDHRA rescues the Rbp1-/- phenotype similarly to a synthetic RXR ligand and displays similar transcriptional activity in cultured human dendritic cells. High endogenous levels of 9CDHRA in mice indicate physiological relevance of these data and that 9CDHRA acts as an endogenous RXR ligand.
Assuntos
Transtornos da Memória/genética , Receptores X de Retinoides/metabolismo , Tretinoína/análogos & derivados , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Humanos , Ligantes , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Receptores X de Retinoides/química , Receptores X de Retinoides/genética , Proteínas Celulares de Ligação ao Retinol/genética , Proteínas Celulares de Ligação ao Retinol/metabolismo , Tretinoína/metabolismoRESUMO
Phosphorylation of the translation initiation factor eIF2α is an adaptive signaling event that is essential for cell and organismal survival from yeast to humans. It is central to the integrated stress response (ISR) that maintains cellular homeostasis in the face of threats ranging from viral infection, amino acid, oxygen, and heme deprivation to the accumulation of misfolded proteins in the endoplasmic reticulum. Phosphorylation of eIF2α has broad physiological, pathological, and therapeutic relevance. However, despite more than two decades of research and growing pharmacological interest, phosphorylation of eIF2α remains difficult to detect and quantify, because of its transient nature and because substoichiometric amounts of this modification are sufficient to profoundly reshape cellular physiology. This review aims to provide a roadmap for facilitating a robust evaluation of eIF2α phosphorylation and its downstream consequences in cells and organisms.
Assuntos
Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Fosforilação , Proteína Fosfatase 1/metabolismo , Proteínas/metabolismoRESUMO
9-cis-13,14-dihydroretinoic acid (9CDHRA), acts as an endogenous ligand of the retinoid X receptors (RXRs), and is an active form of a suggested new vitamin, vitamin A5/X. Nutritional-relevance of this pathway as well as its detailed role in vertebrate physiology, remain largely unknown. Since recent GWAS data and experimental studies associated RXR-mediated signaling with depression, we explored here the relevance of RXR and vitamin A5/X-mediated signaling in the control of stress adaptation and depressive-like behaviors in mice. We found that compromised availability of 9CDHRA in Rbp1-/- mice was associated with increased despair in the forced swim and anhedonia in the sucrose preference test. 9CDHRA similarly to synthetic RXR agonist, BMS649, normalized despair behaviors in Rbp1-/- but not Rxrγ-/- mice, supporting involvement of RXR signaling in anti-despair activity of these ligands. Importantly, similarly to BMS649, the 9CDHRA and its nutritional-precursor, 9-cis-13,14-dihydroretinol (vitamin A5/X alcohol), prevented development of depressive-like behaviors in mice exposed to chronic social defeat stress, revealing the beneficial role of RXRs and its endogenous ligand in stress adaptation process. These data point to the need for relevant nutritional, biochemical and pharmacological studies of this signaling pathway in human, both in physiological conditions and in pathologies of stress-related disorders.
RESUMO
Retinoic acid (RA) signaling through retinoic acid receptors (RARs), known for its multiple developmental functions, emerged more recently as an important regulator of adult brain physiology. How RAR-mediated regulation is achieved is poorly known, partly due to the paucity of information on critical target genes in the brain. Also, it is not clear how reduced RA signaling may contribute to pathophysiology of diverse neuropsychiatric disorders. We report the first genome-wide analysis of RAR transcriptional targets in the brain. Using chromatin immunoprecipitation followed by high-throughput sequencing and transcriptomic analysis of RARß-null mutant mice, we identified genomic targets of RARß in the striatum. Characterization of RARß transcriptional targets in the mouse striatum points to mechanisms through which RAR may control brain functions and display neuroprotective activity. Namely, our data indicate with statistical significance (FDR 0.1) a strong contribution of RARß in controlling neurotransmission, energy metabolism, and transcription, with a particular involvement of G-protein coupled receptor (p = 5.0e-5), cAMP (p = 4.5e-4), and calcium signaling (p = 3.4e-3). Many identified RARß target genes related to these pathways have been implicated in Alzheimer's, Parkinson's, and Huntington's disease (HD), raising the possibility that compromised RA signaling in the striatum may be a mechanistic link explaining the similar affective and cognitive symptoms in these diseases. The RARß transcriptional targets were particularly enriched for transcripts affected in HD. Using the R6/2 transgenic mouse model of HD, we show that partial sequestration of RARß in huntingtin protein aggregates may account for reduced RA signaling reported in HD.
Assuntos
Estudo de Associação Genômica Ampla , Doença de Huntington/genética , Doenças Neurodegenerativas/genética , Receptores do Ácido Retinoico/metabolismo , Transdução de Sinais , Transcrição Gênica , Tretinoína/metabolismo , Animais , Sítios de Ligação , DNA/metabolismo , Proteína Huntingtina/metabolismo , Doença de Huntington/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neostriado/metabolismo , Neostriado/patologia , Doenças Neurodegenerativas/patologia , Agregados Proteicos , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Elementos de Resposta/genéticaRESUMO
Protein phosphorylation regulates virtually all biological processes. Although protein kinases are popular drug targets, targeting protein phosphatases remains a challenge. Here, we describe Sephin1 (selective inhibitor of a holophosphatase), a small molecule that safely and selectively inhibited a regulatory subunit of protein phosphatase 1 in vivo. Sephin1 selectively bound and inhibited the stress-induced PPP1R15A, but not the related and constitutive PPP1R15B, to prolong the benefit of an adaptive phospho-signaling pathway, protecting cells from otherwise lethal protein misfolding stress. In vivo, Sephin1 safely prevented the motor, morphological, and molecular defects of two otherwise unrelated protein-misfolding diseases in mice, Charcot-Marie-Tooth 1B, and amyotrophic lateral sclerosis. Thus, regulatory subunits of phosphatases are drug targets, a property exploited here to safely prevent two protein misfolding diseases.
Assuntos
Inibidores Enzimáticos/farmacologia , Guanabenzo/análogos & derivados , Proteína Fosfatase 1/antagonistas & inibidores , Deficiências na Proteostase/tratamento farmacológico , Deficiências na Proteostase/prevenção & controle , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Células Cultivadas , Doença de Charcot-Marie-Tooth/tratamento farmacológico , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/toxicidade , Guanabenzo/síntese química , Guanabenzo/metabolismo , Guanabenzo/farmacologia , Guanabenzo/toxicidade , Células HeLa , Humanos , Camundongos , Camundongos Transgênicos , Terapia de Alvo Molecular , Fosforilação , Dobramento de Proteína , Transdução de SinaisRESUMO
Abnormal signaling by retinoids or n-3 polyunsaturated fatty acids has been implicated in clinical depression. The converging point in activities of these two classes of molecules is transcriptional activation of retinoid X receptors (Rxr). We show here that ablation of Rxrgamma in mice leads to depressive-like behaviors including increased despair and anhedonia, which were accompanied by reduced expression of dopamine D2 receptor in the shell of nucleus accumbens (NAc) and altered serotonin signaling. While abnormal serotonin signaling is not sufficient to generate the depressive behaviors, increasing D2r expression by chronic fluoxetine (Prozac) treatment or adenoassociated virus type2 (AAV2) mediated expression of Rxrgamma or D2r in the NAc of Rxrgamma(-/-) mice normalizes depressive-like behaviors in Rxrgamma(-/-) animals. Conversely, NAc infusion of raclopride, a D2r antagonist prevents AAV2-Rxrgamma-mediated rescue of despair behaviors in Rxrgamma(-/-) mice. Combined, our data argue that control of NAc D2r expression is critical for Rxrgamma-mediated modulation of affective behaviors.