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1.
Cell ; 167(3): 789-802.e12, 2016 Oct 20.
Artículo en Inglés | MEDLINE | ID: mdl-27768897

RESUMEN

Two complementary approaches were used in search of the intracellular targets of the toxic PR poly-dipeptide encoded by the repeat sequences expanded in the C9orf72 form of amyotrophic lateral sclerosis. The top categories of PRn-bound proteins include constituents of non-membrane invested cellular organelles and intermediate filaments. PRn targets are enriched for the inclusion of low complexity (LC) sequences. Evidence is presented indicating that LC sequences represent the direct target of PRn binding and that interaction between the PRn poly-dipeptide and LC domains is polymer-dependent. These studies indicate that PRn-mediated toxicity may result from broad impediments to the dynamics of cell structure and information flow from gene to message to protein.


Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Dipéptidos/metabolismo , Demencia Frontotemporal/metabolismo , Péptidos/metabolismo , Proteínas/metabolismo , Esclerosis Amiotrófica Lateral/genética , Proteína C9orf72 , Expansión de las Repeticiones de ADN , Dipéptidos/química , Dipéptidos/genética , Demencia Frontotemporal/genética , Células HeLa , Humanos , Proteínas de Filamentos Intermediarios/metabolismo , Péptidos/química , Péptidos/genética , Dominios Proteicos , Proteínas/genética
2.
Cell ; 155(5): 1049-1060, 2013 Nov 21.
Artículo en Inglés | MEDLINE | ID: mdl-24267890

RESUMEN

The low-complexity (LC) domains of the products of the fused in sarcoma (FUS), Ewings sarcoma (EWS), and TAF15 genes are translocated onto a variety of different DNA-binding domains and thereby assist in driving the formation of cancerous cells. In the context of the translocated fusion proteins, these LC sequences function as transcriptional activation domains. Here, we show that polymeric fibers formed from these LC domains directly bind the C-terminal domain (CTD) of RNA polymerase II in a manner reversible by phosphorylation of the iterated, heptad repeats of the CTD. Mutational analysis indicates that the degree of binding between the CTD and the LC domain polymers correlates with the strength of transcriptional activation. These studies offer a simple means of conceptualizing how RNA polymerase II is recruited to active genes in its unphosphorylated state and released for elongation following phosphorylation of the CTD.


Asunto(s)
ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , Activación Transcripcional , Células HeLa , Humanos , Hidrogeles/química , Hidrogeles/metabolismo , Repeticiones de Microsatélite , Fosforilación , Polimerizacion , Estructura Terciaria de Proteína , Proteína EWS de Unión a ARN/metabolismo , Proteína FUS de Unión a ARN/química , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Factores Asociados con la Proteína de Unión a TATA/química , Factores Asociados con la Proteína de Unión a TATA/metabolismo
3.
Proc Natl Acad Sci U S A ; 119(11): e2113813119, 2022 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-35259014

RESUMEN

SignificanceThe GGGGCC hexanucleotide repeat expansion in the chromosome 9 open reading frame 72 (C9orf72) gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS). Despite myriad studies on the toxic effects of poly-dipeptides produced from the C9orf72 repeats, the mechanisms underlying the selective hyperexcitability of motor cortex that characterizes the early stages of C9orf72 ALS patients remain elusive. Here, we show that the proline-arginine poly-dipeptides cause hyperexcitability in cortical motor neurons by increasing persistent sodium currents conducted by the Nav1.2/ß4 sodium channel complex, which is highly expressed in the motor cortex. These findings provide the basis for understanding how the C9orf72 mutation causes motor neuron hyperactivation that can lead to the motor neuron death in C9orf72 ALS.


Asunto(s)
Esclerosis Amiotrófica Lateral/etiología , Esclerosis Amiotrófica Lateral/metabolismo , Proteína C9orf72/genética , Dipéptidos/genética , Hipercinesia/genética , Neuronas Motoras/metabolismo , Esclerosis Amiotrófica Lateral/patología , Arginina , Corteza Cerebral/metabolismo , Corteza Cerebral/fisiopatología , Dipéptidos/metabolismo , Susceptibilidad a Enfermedades , Potenciales Evocados Motores , Predisposición Genética a la Enfermedad , Humanos , Fenotipo , Prolina , Sodio/metabolismo
4.
Proc Natl Acad Sci U S A ; 118(50)2021 12 14.
Artículo en Inglés | MEDLINE | ID: mdl-34873036

RESUMEN

Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of RNA-binding proteins that control key events in RNA biogenesis under both normal and diseased cellular conditions. The low-complexity (LC) domain of hnRNPs can become liquid-like droplets or reversible amyloid-like polymers by phase separation. Yet, whether phase separation of the LC domains contributes to physiological functions of hnRNPs remains unclear. hnRNPH1 contains two LC domains, LC1 and LC2. Here, we show that reversible phase separation of the LC1 domain is critical for both interaction with different kinds of RNA-binding proteins and control of the alternative-splicing activity of hnRNPH1. Interestingly, although not required for phase separation, the LC2 domain contributes to the robust transcriptional activation of hnRNPH1 when fused to the DNA-binding domain, as found recently in acute lymphoblastic leukemia. Our data suggest that the ability of the LC1 domain to phase-separate into reversible polymers or liquid-like droplets is essential for function of hnRNPH1 as an alternative RNA-splicing regulator, whereas the LC2 domain may contribute to the aberrant transcriptional activity responsible for cancer transformation.


Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/metabolismo , Empalme del ARN/fisiología , Anticuerpos , Clonación Molecular , Regulación de la Expresión Génica , Células HEK293 , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/genética , Humanos , Dominios Proteicos , Proteínas Recombinantes
5.
J Cell Physiol ; 237(7): 3069-3079, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35580065

RESUMEN

Recent studies have shown that protein arginine methyltransferase 1 (PRMT1) is highly expressed in the human heart, and loss of PRMT1 contributes to cardiac remodeling in the heart failure. However, the functional importance of PRMT1 in cardiac ion channels remains uncertain. The slow activating delayed rectifier K+ (IKs ) channel is a cardiac K+ channel composed of KCNQ1 and KCNE1 subunits and is a new therapeutic target for treating lethal arrhythmias in many cardiac pathologies, especially heart failure. Here, we demonstrate that PRMT1 is a critical regulator of the IKs channel and cardiac rhythm. In the guinea pig ventricular myocytes, treatment with furamidine, a PRMT1-specific inhibitor, prolonged the action potential duration (APD). We further show that this APD prolongation was attributable to IKs reduction. In HEK293T cells expressing human KCNQ1 and KCNE1, inhibiting PRMT1 via furamidine reduced IKs and concurrently decreased the arginine methylation of KCNQ1, a pore-forming α-subunit. Evidence presented here indicates that furamidine decreased IKs mainly by lowering the affinity of IKs channels for the membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2 ), which is crucial for pore opening. Finally, applying exogenous PIP2 to cardiomyocytes prevented the furamidine-induced IKs reduction and APD prolongation. Taken together, these results indicate that PRMT1 positively regulated IKs activity through channel-PIP2 interaction, thereby restricting excessive cardiac action potential.


Asunto(s)
Insuficiencia Cardíaca , Canal de Potasio KCNQ1 , Fosfatos de Fosfatidilinositol/metabolismo , Potenciales de Acción , Animales , Cobayas , Células HEK293 , Insuficiencia Cardíaca/metabolismo , Humanos , Canal de Potasio KCNQ1/genética , Canal de Potasio KCNQ1/metabolismo , Miocitos Cardíacos/metabolismo , Canales de Potasio con Entrada de Voltaje , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/metabolismo
6.
Biochem J ; 478(4): 799-810, 2021 02 26.
Artículo en Inglés | MEDLINE | ID: mdl-33522570

RESUMEN

A key nucleolar protein, fibrillarin, has emerged as an important pharmacological target as its aberrant expression and localization are related to tumorigenesis, chemoresistance and poor survival in breast cancer patients. Fibrillarin contains a N-terminal low complexity sequence (LC) domain with a skewed amino acid distribution, which is known to undergo a phase transition to liquid-like droplets. However, the underlying mechanism of the phase transition of the fibrillarin LC domain and its physiological function are still elusive. In this study, we show that the localization of fibrillarin and its association with RNA binding proteins is regulated by this phase transition. Phenylalanine-to-serine substitutions of the phenylalanine:glycine repeats in the fibrillarin LC domain impede its phase transition into liquid-like droplets, as well as the hydrogel-like state composed of polymers, and also its incorporation into hydrogel or liquid-like droplets composed of wild-type LC domains. When expressed in cultured cells, fibrillarin containing the mutant LC domain fails to localize to the dense fibrillar component of nucleoli in the same way as intact fibrillarin. Moreover, the phase transition of the fibrillarin LC domain is required for the interaction of fibrillarin with other RNA binding proteins, such as FUS, TAF15, DDX5 and DHX9. Taken together, the results suggest that the phenylalanine residues in the LC domain are critical for the phase transition of fibrillarin, which in turn regulates the sub-nucleolar localization of fibrillarin and its interaction with RNA binding proteins, providing a useful framework for regulating the function of fibrillarin.


Asunto(s)
Proteínas Cromosómicas no Histona/química , Sustitución de Aminoácidos , Nucléolo Celular/metabolismo , Proteínas Cromosómicas no Histona/antagonistas & inhibidores , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , Hidrogeles , Mutación Missense , Transición de Fase , Fenilalanina/química , Mutación Puntual , Unión Proteica , Dominios Proteicos , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/metabolismo
7.
PLoS Genet ; 12(1): e1005773, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26726767

RESUMEN

Pathogen expulsion from the gut is an important defense strategy against infection, but little is known about how interaction between the intestinal microbiome and host immunity modulates defecation. In Drosophila melanogaster, dual oxidase (Duox) kills pathogenic microbes by generating the microbicidal reactive oxygen species (ROS), hypochlorous acid (HOCl) in response to bacterially excreted uracil. The physiological function of enzymatically generated HOCl in the gut is, however, unknown aside from its anti-microbial activity. Drosophila TRPA1 is an evolutionarily conserved receptor for reactive chemicals like HOCl, but a role for this molecule in mediating responses to gut microbial content has not been described. Here we identify a molecular mechanism through which bacteria-produced uracil facilitates pathogen-clearing defecation. Ingestion of uracil increases defecation frequency, requiring the Duox pathway and TrpA1. The TrpA1(A) transcript spliced with exon10b (TrpA1(A)10b) that is present in a subset of midgut enteroendocrine cells (EECs) is critical for uracil-dependent defecation. TRPA1(A)10b heterologously expressed in Xenopus oocytes is an excellent HOCl receptor characterized with elevated sensitivity and fast activation kinetics of macroscopic HOCl-evoked currents compared to those of the alternative TRPA1(A)10a isoform. Consistent with TrpA1's role in defecation, uracil-excreting Erwinia carotovora showed higher persistence in TrpA1-deficient guts. Taken together, our results propose that the uracil/Duox pathway promotes bacteria expulsion from the gut through the HOCl-sensitive receptor, TRPA1(A)10b, thereby minimizing the chances that bacteria adapt to survive host defense systems.


Asunto(s)
Proteínas de Drosophila/biosíntesis , Enfermedades Transmitidas por los Alimentos/genética , Interacciones Huésped-Patógeno/genética , NADPH Oxidasas/biosíntesis , Canales Catiónicos TRPC/biosíntesis , Animales , Bacterias/metabolismo , Bacterias/patogenicidad , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/microbiología , Enfermedades Transmitidas por los Alimentos/microbiología , Regulación de la Expresión Génica , Humanos , Ácido Hipocloroso/metabolismo , Canales Iónicos , NADPH Oxidasas/genética , Oocitos/microbiología , Especies Reactivas de Oxígeno/metabolismo , Canal Catiónico TRPA1 , Canales Catiónicos TRPC/genética , Xenopus
8.
J Biol Chem ; 286(28): 25301-8, 2011 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-21622564

RESUMEN

The dopamine D2 receptor (D2R) plays a crucial role in the regulation of diverse key physiological functions, including motor control, reward, learning, and memory. This receptor is present in vivo in two isoforms, D2L and D2S, generated from the same gene by alternative pre-mRNA splicing. Each isoform has a specific role in vivo, underlining the importance of a strict control of its synthesis, yet the molecular mechanism modulating alternative D2R pre-mRNA splicing has not been completely elucidated. Here, we identify heterogeneous nuclear ribonucleoprotein M (hnRNP M) as a key molecule controlling D2R splicing. We show that binding of hnRNP M to exon 6 inhibited the inclusion of this exon in the mRNA. Importantly, the splicing factor Nova-1 counteracted hnRNP M effects on D2R pre-mRNA splicing. Indeed, mutations of the putative Nova-1-binding site on exon 6 disrupted Nova-1 RNA assembly and diminished the inhibitory effect of Nova-1 on hnRNP M-dependent exon 6 exclusion. These results identify Nova-1 and hnRNP M as D2R pre-mRNA-binding proteins and show their antagonistic role in the alternative splicing of D2R pre-mRNA.


Asunto(s)
Empalme Alternativo/fisiología , Antígenos de Neoplasias/metabolismo , Exones/fisiología , Ribonucleoproteína Heterogénea-Nuclear Grupo M/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptores de Dopamina D2/biosíntesis , Animales , Antígenos de Neoplasias/genética , Células COS , Bovinos , Chlorocebus aethiops , Perros , Ribonucleoproteína Heterogénea-Nuclear Grupo M/genética , Humanos , Ratones , Células 3T3 NIH , Proteínas del Tejido Nervioso/genética , Antígeno Ventral Neuro-Oncológico , Pan troglodytes , Unión Proteica , Precursores del ARN/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Ratas , Receptores de Dopamina D2/genética
9.
J Cell Sci ; 123(Pt 20): 3547-57, 2010 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-20930143

RESUMEN

The transcription factor CLOCK-BMAL1 is a core component of the molecular clock machinery that drives circadian gene expression and physiology in mammals. Recently, we reported that this heterodimeric transcription factor functions as a signaling molecule in response to the resetting stimuli via the Ca²+-dependent protein kinase C pathway. Here, we demonstrate that the CREB-binding protein (CBP) plays a key role in rapid activation of the CLOCK-BMAL1 heterodimer that leads to phase resetting of the circadian clock. Under physiological conditions, a bimolecular fluorescence complementation (BiFC) assay revealed that CLOCK and BMAL1 dimerize in the cytoplasm and subsequently translocate into the nucleus in response to serum stimuli (mean time duration was 29.2 minutes and mean velocity 0.7 µm/minute). Concomitantly, BMAL1 rapidly recruited CBP on Per1 promoter E-box, but not p300 (a functional analog of CBP), in the discrete nuclear foci. However, recruitment of CBP by cAMP/Ca²+ response element-binding (CREB) protein on CRE was not markedly increased upon delivery of the resetting stimuli. Furthermore, overexpression of CBP greatly potentiated the CLOCK-BMAL1-mediated Per1 transcription, and this effect was completely abolished by site-directed mutation of E-box elements, but not by the mutation of CRE in the Per1 promoter. Furthermore, molecular knockdown of CBP severely dampened circadian oscillation of clock gene expression triggered by the resetting stimuli. These findings suggest that CBP recruitment by BMAL1 mediates acute transactivation of CLOCK-BMAL1, thereby inducing immediate-early Per1 transcription and phase resetting of the circadian clock.


Asunto(s)
Factores de Transcripción ARNTL/metabolismo , Proteínas CLOCK/metabolismo , Proteína de Unión a CREB/metabolismo , Relojes Circadianos/fisiología , Factores de Transcripción ARNTL/genética , Animales , Proteínas CLOCK/genética , Células COS , Proteína de Unión a CREB/genética , Núcleo Celular/metabolismo , Chlorocebus aethiops , Inmunoprecipitación de Cromatina , Relojes Circadianos/genética , Citoplasma/metabolismo , Immunoblotting , Inmunoprecipitación , Ratones , Células 3T3 NIH , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Unión Proteica/genética , Unión Proteica/fisiología , Proteína Quinasa C/metabolismo , Multimerización de Proteína , ARN Interferente Pequeño
10.
Exp Mol Med ; 54(9): 1412-1422, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-36175485

RESUMEN

In this review, we discuss the ways in which recent studies of low-complexity (LC) domains have challenged our understanding of the mechanisms underlying cellular organization. LC sequences, long believed to function in the absence of a molecular structure, are abundant in the proteomes of all eukaryotic organisms. Over the past decade, the phase separation of LC domains has emerged as a fundamental mechanism driving dynamic multivalent interactions of many cellular processes. We review the key evidence showing the role of phase separation of individual proteins in organizing cellular assemblies and facilitating biological function while implicating the dynamics of phase separation as a key to biological validity and functional utility. We also highlight the evidence showing that pathogenic LC proteins alter various phase separation-dependent interactions to elicit debilitating human diseases, including cancer and neurodegenerative diseases. Progress in understanding the biology of phase separation may offer useful hints toward possible therapeutic interventions to combat the toxicity of pathogenic proteins.


Asunto(s)
Enfermedades Neurodegenerativas , Proteoma , Humanos , Enfermedades Neurodegenerativas/metabolismo
11.
Cell Death Dis ; 11(3): 203, 2020 Mar 23.
Artículo en Inglés | MEDLINE | ID: mdl-32251281

RESUMEN

A correction to this paper has been published and can be accessed via a link at the top of the paper.

12.
Exp Mol Med ; 52(4): 604-614, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32269286

RESUMEN

HCN channels regulate excitability and rhythmicity in the hippocampal CA1 pyramidal cells. Perturbation in the HCN channel current (Ih) is associated with neuropsychiatric disorders, such as autism spectrum disorders. Recently, protein arginine methyltransferase 7 (PRMT7) was shown to be highly expressed in the hippocampus, including the CA1 region. However, the physiological function of PRMT7 in the CA1 neurons and the relationship to psychiatric disorders are unclear. Here we showed that PRMT7 knockout (KO) mice exhibit hyperactivity and deficits in social interaction. The firing frequency of the CA1 neurons in the PRMT7 KO mice was significantly higher than that in the wild-type (WT) mice. Compared with the WT CA1 neurons, the PRMT7 KO CA1 neurons showed a more hyperpolarized resting potential and a higher input resistance, which were occluded by the Ih-current inhibitor ZD7288; these findings were consistent with the decreased Ih and suggested the contribution of Ih-channel dysfunction to the PRMT7 KO phenotypes. The HCN1 protein level was decreased in the CA1 region of the PRMT7 KO mice in conjunction with a decrease in the expression of Shank3, which encodes a core scaffolding protein for HCN channel proteins. A brief application of the PRMT7 inhibitor DS437 did not reproduce the phenotype of the PRMT7 KO neurons, further indicating that PRMT7 regulates Ih by controlling the channel number rather than the open probability. Moreover, shRNA-mediated PRMT7 suppression reduced both the mRNA and protein levels of SHANK3, implying that PRMT7 deficiency might be responsible for the decrease in the HCN protein levels by altering Shank3 expression. These findings reveal a key role for PRMT7 in the regulation of HCN channel density in the CA1 pyramidal cells that may be amenable to pharmacological intervention for neuropsychiatric disorders.


Asunto(s)
Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/fisiopatología , Regulación de la Expresión Génica , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Proteína-Arginina N-Metiltransferasas/deficiencia , Conducta Social , Potenciales de Acción , Animales , Conducta Animal , Biomarcadores , Línea Celular , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Ratones , Ratones Noqueados , Técnicas de Placa-Clamp , Células Piramidales/metabolismo
13.
Mol Cell Biol ; 26(19): 7318-30, 2006 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16980631

RESUMEN

CLOCK and BMAL1 are bHLH-PAS-containing transcription factors that bind to E-box elements and are indispensable for expression of core circadian clock components such as the Per and Cry genes. A key step in expression is the heterodimerization of CLOCK and BMAL1 and their accumulation in the nucleus with an approximately 24-h periodicity. We show here that nucleocytoplasmic shuttling of BMAL1 is essential for transactivation and for degradation of the CLOCK/BMAL1 heterodimer. Using serial deletions and point mutants, we identified a functional nuclear localization signal and Crm1-dependent nuclear export signals in BMAL1. Transient-transfection experiments revealed that heterodimerization of CLOCK and BMAL1 accelerates their turnover, as well as E-box-dependent clock gene transcription. Moreover, in embryonic mouse fibroblasts, robust transcription of Per2 is tightly associated with massive degradation of the CLOCK/BMAL1 heterodimer. CRY proteins suppressed this process during the transcription-negative phase and led to nuclear accumulation of the CLOCK/BMAL1 heterodimer. Thus, these findings suggest that the decrease of BMAL1 abundance during the circadian cycle reflects robust transcriptional activation of clock genes rather than inhibition of BMAL1 synthesis.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Procesamiento Proteico-Postraduccional , Transactivadores/metabolismo , Activación Transcripcional , Factores de Transcripción ARNTL , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas CLOCK , Núcleo Celular/metabolismo , Células Cultivadas , Ritmo Circadiano/fisiología , Citoplasma/metabolismo , Dimerización , Células HeLa , Humanos , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Células 3T3 NIH , Señales de Exportación Nuclear , Señales de Localización Nuclear/química , Estructura Terciaria de Proteína , Transporte de Proteínas , Transcripción Genética/genética , Ubiquitina/metabolismo
14.
Cell Death Dis ; 10(12): 903, 2019 12 02.
Artículo en Inglés | MEDLINE | ID: mdl-31787756

RESUMEN

Endoplasmic reticulum (ER) stress signaling plays a critical role in the control of cell survival or death. Persistent ER stress activates proapoptotic pathway involving the ATF4/CHOP axis. Although accumulating evidences support its important contribution to cardiovascular diseases, but its mechanism is not well characterized. Here, we demonstrate a critical role for PRMT1 in the control of ER stress in cardiomyocytes. The inhibition of PRMT1 augments tunicamycin (TN)-triggered ER stress response in cardiomyocytes while PRMT1 overexpression attenuates it. Consistently, PRMT1 null hearts show exacerbated ER stress and cell death in response to TN treatment. Interestingly, ATF4 depletion attenuates the ER stress response induced by PRMT1 inhibition. The methylation-deficient mutant of ATF4 with the switch of arginine 239 to lysine exacerbates ER stress accompanied by enhanced levels of proapoptotic cleaved Caspase3 and phosphorylated-γH2AX in response to TN. The mechanistic study shows that PRMT1 modulates the protein stability of ATF4 through methylation. Taken together, our data suggest that ATF4 methylation on arginine 239 by PRMT1 is a novel regulatory mechanism for protection of cardiomyocytes from ER stress-induced cell death.


Asunto(s)
Factor de Transcripción Activador 4/metabolismo , Estrés del Retículo Endoplásmico , Miocitos Cardíacos/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Factor de Transcripción Activador 4/química , Factor de Transcripción Activador 4/genética , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Línea Celular , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Metilación/efectos de los fármacos , Mutación/genética , Miocitos Cardíacos/efectos de los fármacos , Especificidad de Órganos , Unión Proteica/efectos de los fármacos , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Ratas , Factor de Transcripción CHOP/metabolismo , Tunicamicina/farmacología , Regulación hacia Arriba/efectos de los fármacos
15.
Exp Mol Med ; 51(10): 1-14, 2019 10 10.
Artículo en Inglés | MEDLINE | ID: mdl-31601786

RESUMEN

The sodium leak channel NALCN is a key player in establishing the resting membrane potential (RMP) in neurons and transduces changes in extracellular Ca2+ concentration ([Ca2+]e) into increased neuronal excitability as the downstream effector of calcium-sensing receptor (CaSR). Gain-of-function mutations in the human NALCN gene cause encephalopathy and severe intellectual disability. Thus, understanding the regulatory mechanisms of NALCN is important for both basic and translational research. This study reveals a novel mechanism for NALCN regulation by arginine methylation. Hippocampal dentate granule cells in protein arginine methyltransferase 7 (PRMT7)-deficient mice display a depolarization of the RMP, decreased threshold currents, and increased excitability compared to wild-type neurons. Electrophysiological studies combined with molecular analysis indicate that enhanced NALCN activities contribute to hyperexcitability in PRMT7-/- neurons. PRMT7 depletion in HEK293T cells increases NALCN activity by shifting the dose-response curve of NALCN inhibition by [Ca2+]e without affecting NALCN protein levels. In vitro methylation studies show that PRMT7 methylates a highly conserved Arg1653 of the NALCN gene located in the carboxy-terminal region that is implicated in CaSR-mediated regulation. A kinase-specific phosphorylation site prediction program shows that the adjacent Ser1652 is a potential phosphorylation site. Consistently, our data from site-specific mutants and PKC inhibitors suggest that Arg1653 methylation might modulate Ser1652 phosphorylation mediated by CaSR/PKC-delta, leading to [Ca2+]e-mediated NALCN suppression. Collectively, these data suggest that PRMT7 deficiency decreases NALCN methylation at Arg1653, which, in turn, decreases CaSR/PKC-mediated Ser1652 phosphorylation, lifting NALCN inhibition, thereby enhancing neuronal excitability. Thus, PRMT7-mediated NALCN inhibition provides a potential target for the development of therapeutic tools for neurological diseases.


Asunto(s)
Canales Iónicos/genética , Proteínas de la Membrana/genética , Neuronas/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Animales , Arginina/genética , Arginina/metabolismo , Encefalopatías/genética , Encefalopatías/patología , Señalización del Calcio/genética , Células HEK293 , Hipocampo/metabolismo , Humanos , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Potenciales de la Membrana/genética , Potenciales de la Membrana/fisiología , Metilación , Ratones , Fosforilación , Procesamiento Proteico-Postraduccional , Receptores Sensibles al Calcio/genética
16.
Exp Mol Med ; 50(12): 1-10, 2018 12 06.
Artículo en Inglés | MEDLINE | ID: mdl-30523262

RESUMEN

Bmal1 is one of the key molecules that controls the mammalian molecular clock. In humans, two isoforms of Bmal1 are generated by alternative RNA splicing. Unlike the extensively studied hBmal1b, the canonical form of Bmal1 in most species, the expression and/or function of another human-specific isoform, hBmal1a, are poorly understood. Due to the lack of the N-terminal nuclear localization signal (NLS), hBMAL1a does not enter the nucleus as hBMAL1b does. However, despite the lack of the NLS, hBMAL1a still dimerizes with either hCLOCK or hBMAL1b and thereby promotes cytoplasmic retention or protein degradation, respectively. Consequently, hBMAL1a interferes with hCLOCK:hBMAL1b-induced transcriptional activation and the circadian oscillation of Period2. Moreover, when the expression of endogenous hBmal1a is aborted by CRISPR/Cas9-mediated knockout, the rhythmic expression of hPer2 and hBmal1b is restored in cultured HeLa cells. Together, these results suggest a role for hBMAL1a as a negative regulator of the mammalian molecular clock.


Asunto(s)
Factores de Transcripción ARNTL/metabolismo , Relojes Circadianos/fisiología , Isoformas de ARN/metabolismo , Factores de Transcripción ARNTL/genética , Animales , Proteínas CLOCK/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Dimerización , Retroalimentación Fisiológica , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Señales de Localización Nuclear/genética , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Proteolisis , Isoformas de ARN/genética , Empalme del ARN
17.
Exp Mol Med ; 50(12): 1-9, 2018 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-30559345

RESUMEN

Estrogen has diverse effects on cardiovascular function, including regulation of the contractile response to vasoactive substances such as serotonin. The serotonin system recently emerged as an important player in the regulation of vascular tone in humans. However, hyperreactivity to serotonin appears to be a critical factor for the pathophysiology of hypertension. In this study, we examined the modulatory mechanisms of estrogen in serotonin-induced vasoconstriction by using a combinatory approach of isometric tension measurements, molecular biology, and patch-clamp techniques. 17ß-Estradiol (E2) elicited a significant and concentration-dependent relaxation of serotonin-induced contraction in deendothelialized aortic strips isolated from male rats. E2 triggered a relaxation of serotonin-induced contraction even in the presence of tamoxifen, an estrogen receptor antagonist, suggesting that E2-induced changes are not mediated by estrogen receptor. Patch-clamp studies in rat arterial myocytes showed that E2 prevented Kv channel inhibition induced by serotonin. Serotonin increased Src activation in arterial smooth muscle required for contraction, which was significantly inhibited by E2. The estrogen receptor-independent inhibition of Src by E2 was confirmed in HEK293T cells that do not express estrogen receptor. Taken together, these results suggest that estrogen exerts vasodilatory effects on serotonin-precontracted arteries via Src, implying a critical role for estrogen in the prevention of vascular hyperreactivity to serotonin.


Asunto(s)
Aorta/patología , Estrógenos/metabolismo , Arterias Mesentéricas/patología , Vasoconstricción/fisiología , Animales , Regulación hacia Abajo , Células HEK293 , Humanos , Masculino , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Canales de Potasio con Entrada de Voltaje/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Estrógenos/metabolismo , Serotonina/metabolismo , Familia-src Quinasas/metabolismo
18.
Mol Cells ; 38(10): 911-7, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26447139

RESUMEN

Citronellal, a well-known plant-derived mosquito repellent, was previously reported to repel Drosophila melanogaster via olfactory pathways involving but not directly activating Transient Receptor Potential Ankyrin 1 (TRPA1). Here, we show that citronellal is a direct agonist for Drosophila and human TRPA1s (dTRPA1 and hTRPA1) as well as Anopheles gambiae TRPA1 (agTRPA1). Citronellal-induced activity is isoform-dependent for Drosophila and Anopheles gambiae TRPA1s. The recently identified dTRPA1(A) and ag-TRPA1(A) isoforms showed citronellal-provoked currents with EC50s of 1.0 B1 0.2 and 0.1 B1 0.03 mM, respectively, in Xenopus oocytes, while the sensitivities of TRPA1(B)s were much inferior to those of TRPA1(A)s. Citronellal dramatically enhanced the feeding-inhibitory effect of the TRPA1 agonist N-methylmaleimide (NMM) in Drosophila at an NMM concentration that barely repels flies. Thus, citronellal can promote feeding deterrence of fruit flies through direct action on gustatory dTRPA1, revealing the first isoform-specific function for TRPA1(A).


Asunto(s)
Aldehídos/farmacología , Proteínas de Drosophila/agonistas , Drosophila melanogaster/efectos de los fármacos , Conducta Alimentaria/efectos de los fármacos , Repelentes de Insectos/farmacología , Monoterpenos/farmacología , Proteínas del Tejido Nervioso/agonistas , Canales Catiónicos TRPC/agonistas , Canales de Potencial de Receptor Transitorio/agonistas , Potenciales de Acción/efectos de los fármacos , Monoterpenos Acíclicos , Animales , Anopheles/efectos de los fármacos , Canales de Calcio , Drosophila melanogaster/metabolismo , Drosophila melanogaster/fisiología , Humanos , Canales Iónicos , Maleimidas/farmacología , Oocitos , Isoformas de Proteínas/agonistas , Canal Catiónico TRPA1 , Xenopus laevis
19.
Science ; 345(6201): 1139-45, 2014 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-25081482

RESUMEN

Many RNA regulatory proteins controlling pre-messenger RNA splicing contain serine:arginine (SR) repeats. Here, we found that these SR domains bound hydrogel droplets composed of fibrous polymers of the low-complexity domain of heterogeneous ribonucleoprotein A2 (hnRNPA2). Hydrogel binding was reversed upon phosphorylation of the SR domain by CDC2-like kinases 1 and 2 (CLK1/2). Mutated variants of the SR domains changing serine to glycine (SR-to-GR variants) also bound to hnRNPA2 hydrogels but were not affected by CLK1/2. When expressed in mammalian cells, these variants bound nucleoli. The translation products of the sense and antisense transcripts of the expansion repeats associated with the C9orf72 gene altered in neurodegenerative disease encode GRn and PRn repeat polypeptides. Both peptides bound to hnRNPA2 hydrogels independent of CLK1/2 activity. When applied to cultured cells, both peptides entered cells, migrated to the nucleus, bound nucleoli, and poisoned RNA biogenesis, which caused cell death.


Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Astrocitos/metabolismo , Nucléolo Celular/metabolismo , Dipéptidos/metabolismo , Demencia Frontotemporal/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Proteínas/genética , Empalme Alternativo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Astrocitos/patología , Proteína C9orf72 , Muerte Celular , Células Cultivadas , Dipéptidos/genética , Dipéptidos/farmacología , Transportador 2 de Aminoácidos Excitadores , Demencia Frontotemporal/genética , Demencia Frontotemporal/patología , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato , Fosforilación , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/metabolismo , ARN sin Sentido/antagonistas & inhibidores , ARN sin Sentido/biosíntesis , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesis , ARN Ribosómico/antagonistas & inhibidores , ARN Ribosómico/biosíntesis , Secuencias Repetitivas de Aminoácido , Transcripción Genética
20.
Exp Neurobiol ; 20(1): 18-28, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22110358

RESUMEN

As a consequence of the Earth's rotation, almost all organisms experience day and night cycles within a 24-hr period. To adapt and synchronize biological rhythms to external daily cycles, organisms have evolved an internal time-keeping system. In mammals, the master circadian pacemaker residing in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus generates circadian rhythmicity and orchestrates numerous subsidiary local clocks in other regions of the brain and peripheral tissues. Regardless of their locations, these circadian clocks are cell-autonomous and self-sustainable, implicating rhythmic oscillations in a variety of biochemical and metabolic processes. A group of core clock genes provides interlocking molecular feedback loops that drive the circadian rhythm even at the single-cell level. In addition to the core transcription/translation feedback loops, post-translational modifications also contribute to the fine regulation of molecular circadian clocks. In this article, we briefly review the molecular mechanisms and post-translational modifications of mammalian circadian clock regulation. We also discuss the organization of and communication between central and peripheral circadian oscillators of the mammalian circadian clock.

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