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1.
Biomolecules ; 11(6)2021 05 22.
Artículo en Inglés | MEDLINE | ID: mdl-34067263

RESUMEN

Although ubiquitination is widely assumed to be the only regulated step in the ubiquitin-proteasome pathway, recent studies have demonstrated several important mechanisms that regulate the activities of the 26S proteasome. Most proteasomes in cells are inactive but, upon binding a ubiquitinated substrate, become activated by a two-step mechanism requiring an association of the ubiquitin chain with Usp14 and then a loosely folded protein domain with the ATPases. The initial activation step is signaled by Usp14's UBL domain, and many UBL-domain-containing proteins (e.g., Rad23, Parkin) also activate the proteasome. ZFAND5 is a distinct type of activator that binds ubiquitin conjugates and the proteasome and stimulates proteolysis during muscle atrophy. The proteasome's activities are also regulated through subunit phosphorylation. Agents that raise cAMP and activate PKA stimulate within minutes Rpn6 phosphorylation and enhance the selective degradation of short-lived proteins. Likewise, hormones, fasting, and exercise, which raise cAMP, activate proteasomes and proteolysis in target tissues. Agents that raise cGMP and activate PKG also stimulate 26S activities but modify different subunit(s) and stimulate also the degradation of long-lived cell proteins. Both kinases enhance the selective degradation of aggregation-prone proteins that cause neurodegenerative diseases. These new mechanisms regulating proteolysis thus have clear physiological importance and therapeutic potential.


Asunto(s)
Atrofia Muscular/enzimología , Enfermedades Neurodegenerativas/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Humanos , Proteínas/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo
2.
Cell ; 183(6): 1682-1698.e24, 2020 12 10.
Artículo en Inglés | MEDLINE | ID: mdl-33232692

RESUMEN

In order to analyze how a signal transduction network converts cellular inputs into cellular outputs, ideally one would measure the dynamics of many signals within the network simultaneously. We found that, by fusing a fluorescent reporter to a pair of self-assembling peptides, it could be stably clustered within cells at random points, distant enough to be resolved by a microscope but close enough to spatially sample the relevant biology. Because such clusters, which we call signaling reporter islands (SiRIs), can be modularly designed, they permit a set of fluorescent reporters to be efficiently adapted for simultaneous measurement of multiple nodes of a signal transduction network within single cells. We created SiRIs for indicators of second messengers and kinases and used them, in hippocampal neurons in culture and intact brain slices, to discover relationships between the speed of calcium signaling, and the amplitude of PKA signaling, upon receiving a cAMP-driving stimulus.


Asunto(s)
Colorantes Fluorescentes/metabolismo , Genes Reporteros , Imagen Óptica , Transducción de Señal , Animales , Calcio/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Hipocampo/metabolismo , Humanos , Ratones , Neuronas/metabolismo , Péptidos/metabolismo , Proteínas/metabolismo , Células Piramidales/metabolismo
3.
Sci Rep ; 10(1): 1551, 2020 01 31.
Artículo en Inglés | MEDLINE | ID: mdl-32005857

RESUMEN

We have previously reported that transient knock-down of ATPase inhibitory factor 1 (IF1) by siRNA upregulates ATP levels and subsequently augments insulin secretion in model pancreatic ß-cells INS-1E. Here we investigated how long-term IF1-overexpression impacts pancreatic ß-cell bioenergetics and insulin secretion. We generated INS-1E cell line stably overexpressing native IF1. We revealed that IF1 overexpression leads to a substantial decrease in ATP levels and reduced glucose-stimulated insulin secretion. A decrease in total cellular ATP content was also reflected in decreased free ATP cytosolic and mitochondrial levels, as monitored with ATeam biosensor. Consistently, cellular respiration of IF1-overexpressing cells was decreased. 3D structured illumination microscopy (SIM) revealed a higher amount of insulin granules with higher volume in IF1-overexpressing cells. Similar effects occurred when cells were incubated at low glucose concentrations. Noteworthy, activation of PKA by dibutyryl cAMP entirely abolished the inhibitory effect of IF1 overexpression on ATP production and insulin secretion. Mitochondrial network morphology and cristae ultrastructure in INS-1E overexpressing IF1 remained mostly unchanged. Finally, we show that INS-1E cells decrease their IF1 protein levels relative to ATP synthase α-subunit in response to increased glucose. In conclusion, IF1 actively downregulates INS-1E cellular metabolism and reduces their ability to secrete insulin.


Asunto(s)
Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Proteínas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , CMP Cíclico/análogos & derivados , CMP Cíclico/metabolismo , Regulación hacia Abajo , Glucosa/metabolismo , Proteínas/genética , ARN Interferente Pequeño/genética , Ratas , Transducción de Señal , Regulación hacia Arriba
4.
J Cell Biochem ; 120(10): 18193-18208, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31172579

RESUMEN

DDX3 is a host viral factor that can inhibit the hepatitis B virus-induced innate immune responses. In this study, the 20 bioactive compounds have screened the effects on DDX3 and we found that 5-HT upregulated DDX3 promoter activity via the 5-HT7 receptor on liver hepatocellular cells (HepG2 cells) by using a luciferase assay, reverse transcription-polymerase chain reaction analysis, and Western blot analysis. Furthermore, we are trying to elucidate the pathways involved in the stimulating effect of 5-HT on DDX3 expression to induce innate immune responses against hepatitis B virus infection. A knockdown of the 5-HT7 receptor by transfection si-5-HT7 receptors or si-control into HepG2 cells treated by 5-HT (or 5-HT plus agonist) confirmed the role of the 5-HT7 receptor in DDX3 expression. The IFN-ß-Luc expression and level of hepatitis B virus surface Antigen (HBsAg) showed that DDX3 mediated by the 5-HT7 agonist (AS-19) increased IFN-ß expression and inhibited HBV replication. Luciferase assays showed the involvement of 5-HT7 receptors in DDX3 expression via cAMP/AC/PKA pathways by using protein kinase A (PKA) and adenylyl cyclase inhibitor (MDL 12330A). AS-19 mediated DDX3 promoter activated PKA extracellular signal-regulated kinase ERK signaling the p53 phosphorylation (-1080/-1070) resulted in upregulation of DDX3 promoter transactivation via the 5-HT7 receptors agonist. Overall, 5-HT7 was found to be a new potential target to inhibit hepatitis B infection by activating AC/PKA/ERK pathways by phosphorylating p53 via the 5-HT7 agonist response by mediating DDX3 expression.


Asunto(s)
ARN Helicasas DEAD-box/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas/metabolismo , Receptores de Serotonina/genética , Serotonina/farmacología , Adenilil Ciclasas/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/virología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , ARN Helicasas DEAD-box/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células Hep G2 , Virus de la Hepatitis B/fisiología , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/virología , Fosforilación/efectos de los fármacos , Interferencia de ARN , Receptores de Serotonina/metabolismo , Agonistas de Receptores de Serotonina/farmacología , Transducción de Señal/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Replicación Viral/efectos de los fármacos
5.
Biol Psychiatry ; 86(2): 131-142, 2019 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-31076080

RESUMEN

BACKGROUND: The basolateral amygdala (BLA) has been widely implicated in the pathophysiology of major depressive disorder. A-kinase anchoring protein 150 (AKAP150) directs kinases and phosphatases to synaptic glutamate receptors, controlling synaptic transmission and plasticity. However, the role of the AKAP150 in the BLA in major depressive disorder remains poorly understood. METHODS: Depressive-like behaviors in C57BL/6J mice were developed by chronic restraint stress (CRS). Mice received either intra-BLA injection of lentivirus-expressing Akap5 short hairpin RNA or Ht-31, a peptide to disrupt the interaction of AKAP150 and protein kinase A (PKA), followed by depressive-like behavioral tests. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid glutamate receptor (AMPAR)-mediated miniature excitatory postsynaptic currents were recorded by whole-cell patch-clamp techniques. RESULTS: Chronic stress exposure induced depressive-like behaviors, which were accompanied by an increase in total and synaptic AKAP150 expression in the BLA. Accordingly, CRS facilitated the association of AKAP150 with PKA, but not of calcineurin in the BLA. Intra-BLA infusion of lentivirus-expressing Akap5 short hairpin RNA or Ht-31 prevented depressive-like behaviors and normalized phosphorylation of serine 845 and surface expression of AMPAR subunit 1 (GluA1) in the BLA of CRS mice. Finally, blockage of AKAP150-PKA complex signaling rescued the changes in AMPAR-mediated miniature excitatory postsynaptic currents in depressive-like mice. CONCLUSIONS: These results suggest that AKAP150-PKA directly modulates BLA neuronal synaptic strength, and that AKAP150-PKA-GluA1 streamline signaling complex is responsible for CRS-induced disruption of synaptic AMPAR-mediated transmission and depressive-like behaviors in mice.


Asunto(s)
Proteínas de Anclaje a la Quinasa A/genética , Complejo Nuclear Basolateral/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Depresión/genética , Depresión/psicología , Estrés Psicológico/genética , Estrés Psicológico/psicología , Proteínas de Anclaje a la Quinasa A/efectos de los fármacos , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/efectos de los fármacos , Depresión/etiología , Suspensión Trasera/psicología , Ratones , Ratones Endogámicos C57BL , Proteínas/farmacología , Receptores AMPA/biosíntesis , Receptores AMPA/genética , Restricción Física , Estrés Psicológico/complicaciones , Natación/psicología , Transmisión Sináptica
6.
PLoS One ; 14(4): e0214606, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30990811

RESUMEN

PURPOSE: The aim of this study was to investigate the effect of phosphotyrosine interaction domain containing 1 (PID1) on the insulin-induced activation of the AKT (protein kinase B)/protein kinase A (PKA)/hormone-sensitive lipase (HSL) pathway and lipolysis. METHODS: Sprague-Dawley rats were fed either chow or a high-fat diet (HFD). The levels of insulin, glycerol, free fatty acids (FFAs) and PID1 mRNA expression were measured in the 2 groups. Furthermore, we examined the role of PID1 in the regulation of the AKT/PKA/HSL cascade and lipolysis in the 3T3-L1 cell line. RESULTS: Adipose tissue from HFD rats exhibited elevated PID1 expression, which showed a positive correlation with insulin levels and lipolysis. In 3T3-L1 adipocytes, we found that the antilipolytic effect of insulin is mediated by AKT and that phosphorylated AKT results in the promotion of PDE3B expression, the dephosphorylation of PKA and HSL and the suppression of glycerol release. However, overexpression of PID1 and treatment with 1 µM isoproterenol and 100 nM insulin for 24 h resulted in an increased release of glycerol and a noticeable inhibition of AKT phosphorylation, PDE3B expression and the phosphorylation of PKA/HSL in 3T3-L1 cells. In contrast, knockdown of PID1 and treatment with the above reagents inhibited lipolysis and activated the phosphorylation of AKT, which resulted in the dephosphorylation of PKA and HSL. CONCLUSIONS: Our findings indicate that PID1 in adipose tissue increases lipolysis by altering the antilipolytic action of insulin. This suggests that PID1 may represent a new therapeutic target to ameliorate adipocyte lipolysis and hence improve insulin sensitivity.


Asunto(s)
Tejido Adiposo/metabolismo , Insulina/farmacología , Lipólisis/efectos de los fármacos , Proteínas/metabolismo , Transducción de Señal/efectos de los fármacos , Células 3T3-L1 , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Dieta Alta en Grasa , Insulina/metabolismo , Isoproterenol/farmacología , Masculino , Ratones , Fosforilación/efectos de los fármacos , Proteínas/antagonistas & inhibidores , Proteínas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Regulación hacia Arriba/efectos de los fármacos
7.
Methods Enzymol ; 615: 501-526, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30638540

RESUMEN

Experimental screening for protein-ligand interactions is a central task in drug discovery. Nuclear magnetic resonance (NMR) spectroscopy enables the determination of binding affinities, as well as the measurement of structural and dynamic parameters governing the interaction. With traditional liquid-state NMR relying on a nuclear spin polarization on the order of 10-5, hyperpolarization methods such as dissolution dynamic nuclear polarization (D-DNP) can increase signals by several orders of magnitude. The resulting increase in sensitivity has the potential to reduce requirements for the concentration of protein and ligands, improve the accuracy of the detection of interaction by allowing the use of near-stoichiometric conditions, and increase throughput. This chapter introduces a selection of basic techniques for the application of D-DNP to screening. Procedures for hyperpolarization are briefly reviewed, followed by the description of NMR methods for detection of binding through changes in chemical shift and relaxation parameters. Experiments employing competitive binding with a known ligand are shown, which can be used to determine binding affinity or yield structural information on the pharmacophore. The specific challenges of working with nonrenewable hyperpolarization are reviewed, and solutions including the use of multiplexed NMR detection are described. Altogether, the methods summarized in this chapter are intended to allow for the efficient detection of binding affinity, structure, and dynamics facilitated through substantial signal enhancements provided by hyperpolarization.


Asunto(s)
Ligandos , Espectroscopía de Resonancia Magnética/métodos , Proteínas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Unión Proteica , Conformación Proteica , Proteínas/química , Solubilidad
8.
Arch Oral Biol ; 90: 91-99, 2018 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-29597062

RESUMEN

OBJECTIVES: Phosphodiesterases comprise a superfamily of enzymes that hydrolyze and inactivate cyclic AMP (cAMP) and/or cyclic GMP (cGMP), thereby regulating cellular signaling mechanisms. We herein investigated the production of phosphodiesterase 2A (PDE2A) in the mouse submandibular gland. DESIGN: The expression and localization of the mRNA and protein of PDE2A were examined in the submandibular gland of male and female mice using the reverse transcription-polymerase chain reaction, in situ hybridization, Western blotting, and immunohistochemistry. RESULTS: Among the different species of phosphodiesterases examined in the mouse submandibular gland, PDE2A, which hydrolyzes cAMP and cGMP, exhibited a marked sexual difference; it was more abundantly expressed in females. The mRNA and protein signals for PDE2A were intense in all acinar and duct portions, including the striated duct, in females, whereas in males, these signals were markedly weaker in the granular convoluted duct, the counterpart of the female striated duct, than in acini and other duct portions. Furthermore, the signals for protein kinases A and G1, which are intracellular effectors of cAMP and cGMP, respectively, were markedly weaker in the male granular convoluted duct. CONCLUSIONS: These results suggest that cyclic nucleotide-dependent signaling mechanisms function poorly in granular convoluted duct cells in the mouse submandibular gland.


Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/biosíntesis , Glándula Submandibular/enzimología , Glándula Submandibular/metabolismo , Animales , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/genética , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Hidrolasas Diéster Fosfóricas/clasificación , Hidrolasas Diéster Fosfóricas/metabolismo , Proteínas/metabolismo , ARN Mensajero/metabolismo , Caracteres Sexuales , Factores Sexuales , Transducción de Señal , Glándula Submandibular/citología
9.
Sci Rep ; 8(1): 50, 2018 01 08.
Artículo en Inglés | MEDLINE | ID: mdl-29311589

RESUMEN

Inhibitor-1 is converted into a potent inhibitor of native protein phosphatase-1 (PP1) when Thr35 is phosphorylated by cAMP-dependent protein kinase (PKA). However, PKA-phosphorylated form of inhibitor-1 displayed a weak activity in inhibition of recombinant PP1. The mechanism for the impaired activity of PKA-phosphorylated inhibitor-1 toward inhibition of recombinant PP1 remained elusive. By using NMR spectroscopy in combination with site-directed mutagenesis and inhibitory assay, we found that the interaction between recombinant PP1 and the consensus PP1-binding motif of PKA-thiophosphorylated form of inhibitor-1 was unexpectedly weak. Unlike binding to native PP1, the subdomains 1 (residues around and including the phosphorylated Thr35) and 2 (the consensus PP1-binding motif) of PKA-thiophosphorylated form of inhibitor-1 do not exhibit a synergistic effect in inhibition of recombinant PP1. This finding implied that a slight structural discrepancy exists between native and recombinant PP1, resulting in PKA-thiophosphorylated form of inhibitor-1 displaying a different affinity to native and recombinant enzyme.


Asunto(s)
Espectroscopía de Resonancia Magnética , Proteína Fosfatasa 1/química , Proteínas/química , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Modelos Moleculares , Fosforilación , Unión Proteica , Conformación Proteica , Proteína Fosfatasa 1/metabolismo , Proteínas/metabolismo , Relación Estructura-Actividad
10.
Biochem J ; 474(19): 3355-3371, 2017 09 24.
Artículo en Inglés | MEDLINE | ID: mdl-28947610

RESUMEN

The ubiquitin proteasome system degrades the great majority of proteins in mammalian cells. Countless studies have described how ubiquitination promotes the selective degradation of different cell proteins. However, there is a small but the growing literature that protein half-lives can also be regulated by post-translational modifications of the 26S proteasome. The present study reviews the ability of several kinases to alter proteasome function through subunit phosphorylation. For example, PKA (protein kinase A) and DYRK2 (dual-specificity tyrosine-regulated kinase 2) stimulate the proteasome's ability to degrade ubiquitinated proteins, peptides, and adenosine triphosphate, while one kinase, ASK1 (apoptosis signal-regulating kinase 1), inhibits proteasome function during apoptosis. Proteasome phosphorylation is likely to be important in regulating protein degradation because it occurs downstream from many hormones and neurotransmitters, in conditions that raise cyclic adenosine monophosphate or cyclic guanosine monophosphate levels, after calcium influx following synaptic depolarization, and during phases of the cell cycle. Beyond its physiological importance, pharmacological manipulation of proteasome phosphorylation has the potential to combat various diseases. Inhibitors of phosphodiesterases by activating PKA or PKG (protein kinase G) can stimulate proteasomal degradation of misfolded proteins that cause neurodegenerative or myocardial diseases and even reduce the associated pathology in mouse models. These observations are promising since in many proteotoxic diseases, aggregation-prone proteins impair proteasome function, and disrupt protein homeostasis. Conversely, preventing subunit phosphorylation by DYRK2 slows cell cycle progression and tumor growth. However, further research is essential to determine how phosphorylation of different subunits by these (or other) kinases alters the properties of this complex molecular machine and thus influence protein degradation rates.


Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas/metabolismo , Animales , Apoptosis , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Humanos , Neuronas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Proteolisis
11.
Pain ; 158(10): 2012-2024, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-28767511

RESUMEN

Several studies implicated cyclic adenosine monophosphate (cAMP) as an important second messenger for regulating nociceptor sensitization, but downstream targets of this signaling pathway which contribute to neuronal plasticity are not well understood. We used a Cre/loxP-based strategy to disable the function of either HCN2 or PKA selectively in a subset of peripheral nociceptive neurons and analyzed the nociceptive responses in both transgenic lines. A near-complete lack of sensitization was observed in both mutant strains when peripheral inflammation was induced by an intradermal injection of 8br-cAMP. The lack of HCN2 as well as the inhibition of PKA eliminated the cAMP-mediated increase of calcium transients in dorsal root ganglion neurons. Facilitation of Ih via cAMP, a hallmark of the Ih current, was abolished in neurons without PKA activity. Collectively, these results show a significant contribution of both genes to inflammatory pain and suggest that PKA-dependent activation of HCN2 underlies cAMP-triggered neuronal sensitization.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Canales de Potasio/metabolismo , Células Receptoras Sensoriales/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Bradiquinina/farmacología , Calcio/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Ganglios Espinales/citología , Hiperalgesia/fisiopatología , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Inflamación/inducido químicamente , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Canal de Sodio Activado por Voltaje NAV1.8/genética , Canal de Sodio Activado por Voltaje NAV1.8/metabolismo , Umbral del Dolor , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Canales de Potasio/genética , Proteínas/genética , Proteínas/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Transducción de Señal
12.
SLAS Discov ; 22(1): 77-85, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-27628689

RESUMEN

Connexin 43 (Cx43), the predominant gap junction (GJ) protein, directly interacts with the A-kinase-anchoring protein (AKAP) Ezrin in human cytotrophoblasts and a rat liver epithelial cells (IAR20). The Cx43-Ezrin-protein kinase (PKA) complex facilitates Cx43 phosphorylation by PKA, which triggers GJ opening in cytotrophoblasts and IAR20 cells and may be a general mechanism regulating GJ intercellular communication (GJIC). Considering the importance of Cx43 GJs in health and disease, they are considered potential pharmaceutical targets. The Cx43-Ezrin interaction is a protein-protein interaction that opens possibilities for targeting with peptides and small molecules. For this reason, we developed a high-throughput cell-based assay in which GJIC can be assessed and new compounds characterized. We used two pools of IAR20 cells, calcein loaded and unloaded, that were mixed and allowed to attach. Next, GJIC was monitored over time using automated imaging via the IncuCyte imager. The assay was validated using known GJ inhibitors and anchoring peptide disruptors, and we further tested new peptides that interfered with the Cx43-Ezrin binding region and reduced GJIC. Although an AlphaScreen assay can be used to screen for Cx43-Ezrin interaction inhibitors, the cell-based assay described is an ideal secondary screen for promising small-molecule hits to help identify the most potent compounds.


Asunto(s)
Comunicación Celular , Conexina 43/metabolismo , Proteínas del Citoesqueleto/metabolismo , Uniones Comunicantes/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Animales , Comunicación Celular/efectos de los fármacos , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática/efectos de los fármacos , Fluoresceínas/metabolismo , Uniones Comunicantes/efectos de los fármacos , Imagenología Tridimensional , Unión Proteica/efectos de los fármacos , Proteínas/farmacología , Ratas
13.
Proc Biol Sci ; 283(1830)2016 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-27147099

RESUMEN

Although mammalian spermatozoa only acquire functional maturity as they are conveyed through the male (epididymal maturation) and female (capacitation) reproductive tracts, the degree of post-testicular development necessary to achieve fertilization in other vertebrate species remains far less clear. Indeed, despite reports that the epididymis of birds and reptiles is capable of secreting proteins that bind and modify the sperm surface characteristics, it remains unclear whether capacitation is a pre-requisite for fertilization in these species. Using the ancient reptilian Australian saltwater crocodile as a model, this study was undertaken to explore whether reptile sperm do undergo capacitation-like changes following ejaculation. Our studies revealed that crocodile spermatozoa experienced a rapid and sustained, cyclic-AMP mediated increase in progressive motility following incubation under conditions optimized for the induction of capacitation in mammalian species such as the mouse and human. This response was coupled with elevated levels of phosphorylation associated with both protein kinase A and tyrosine kinase substrates, the latter of which were predominantly localized within the sperm flagellum. In findings that also accord with mammalian spermatozoa, we confirmed a homologue of outer dense fibre 2 as one of the principal substrates for tyrosine phosphorylation. Overall, our findings support the concept that crocodile spermatozoa do undergo a process that is homologous to capacitation in preparation for fertilization of an ovum.


Asunto(s)
Caimanes y Cocodrilos , Semen/citología , Capacitación Espermática , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Masculino , Fosfotirosina/metabolismo , Proteínas/metabolismo , Motilidad Espermática , Espermatozoides/metabolismo
14.
Cell Rep ; 12(12): 2143-55, 2015 Sep 29.
Artículo en Inglés | MEDLINE | ID: mdl-26387949

RESUMEN

The mitochondrial H(+)-ATP synthase synthesizes most of cellular ATP requirements by oxidative phosphorylation (OXPHOS). The ATPase Inhibitory Factor 1 (IF1) is known to inhibit the hydrolase activity of the H(+)-ATP synthase in situations that compromise OXPHOS. Herein, we demonstrate that phosphorylation of S39 in IF1 by mitochondrial protein kinase A abolishes its capacity to bind the H(+)-ATP synthase. Only dephosphorylated IF1 binds and inhibits both the hydrolase and synthase activities of the enzyme. The phosphorylation status of IF1 regulates the flux of aerobic glycolysis and ATP production through OXPHOS in hypoxia and during the cell cycle. Dephosphorylated IF1 is present in human carcinomas. Remarkably, mouse heart contains a large fraction of dephosphorylated IF1 that becomes phosphorylated and inactivated upon in vivo ß-adrenergic stimulation. Overall, we demonstrate the essential function of the phosphorylation of IF1 in regulating energy metabolism and speculate that dephosho-IF1 might play a role in signaling mitohormesis.


Asunto(s)
Adenosina Trifosfato/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Mitocondrias Cardíacas/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Proteínas/metabolismo , Animales , Sitios de Unión , Bucladesina/farmacología , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Pruebas de Enzimas , Regulación de la Expresión Génica , Glucólisis/efectos de los fármacos , Glucólisis/genética , Células HCT116 , Humanos , Isoquinolinas/farmacología , Cinética , Ratones , Mitocondrias Cardíacas/efectos de los fármacos , ATPasas de Translocación de Protón Mitocondriales/química , ATPasas de Translocación de Protón Mitocondriales/genética , Modelos Moleculares , Miocardio/citología , Miocardio/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , Fosforilación , Unión Proteica , Proteínas/química , Proteínas/genética , Transducción de Señal , Sulfonamidas/farmacología
15.
Cell Metab ; 22(5): 851-60, 2015 Nov 03.
Artículo en Inglés | MEDLINE | ID: mdl-26411340

RESUMEN

Fat and muscle lipolysis involves functional interactions of adipose triglyceride lipase (ATGL), α-ß hydrolase domain-containing protein 5 (ABHD5), and tissue-specific perilipins 1 and 5 (PLIN1 and PLIN5). ABHD5 potently activates ATGL, but this lipase-promoting activity is suppressed when ABHD5 is bound to PLIN proteins on lipid droplets. In adipocytes, protein kinase A (PKA) phosphorylation of PLIN1 rapidly releases ABHD5 to activate ATGL, but mechanisms for rapid regulation of PLIN5-ABHD5 interaction in muscle are unknown. Here, we identify synthetic ligands that release ABHD5 from PLIN1 or PLIN5 without PKA activation and rapidly activate adipocyte and muscle lipolysis. Molecular imaging and affinity probe labeling demonstrated that ABHD5 is directly targeted by these synthetic ligands and additionally revealed that ABHD5-PLIN interactions are regulated by endogenous ligands, including long-chain acyl-CoA. Our results reveal a new locus of lipolysis control and suggest ABHD5 ligands might be developed into novel therapeutics that directly promote fat catabolism.


Asunto(s)
1-Acilglicerol-3-Fosfato O-Aciltransferasa/metabolismo , Proteínas Portadoras/metabolismo , Lipólisis/genética , Fosfoproteínas/metabolismo , Proteínas/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferasa/genética , Células 3T3-L1 , Acilcoenzima A/metabolismo , Adipocitos/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Humanos , Ligandos , Ratones , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Perilipina-1 , Perilipina-5 , Fosfoproteínas/genética , Proteínas/genética
16.
Reprod Domest Anim ; 50(5): 705-11, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26294224

RESUMEN

Capacitation is a biological phenomenon occurring prior to fertilization and is a multiple event process. Many physiological and biochemical changes takes place during the process; these changes are related to lipid composition of membrane, intracellular modulation of ion concentration, protein phosphorylation, sperm movement and membrane permeability. These events occur when the sperm is exposed to the new environment of ion concentration in the female reproductive tract. Ions such as bicarbonate and calcium facilitate capacitation by activating adenylyl cyclase, thus initiating protein kinase A (PKA) signalling cascade. Extracellular-regulated kinase pathway is activated by ligand binding to the membrane receptors and intracellular activation by reactive oxygen species (ROS). Activation of these pathways leads to the phosphorylation of different proteins, which is associated with events such as capacitation, hyperactivation and acrosome reaction that are essential for successful fertilization. Extensive studies were carried out on protein phosphorylation in relation to capacitation, but its role still remains ambiguous.


Asunto(s)
Transducción de Señal/fisiología , Capacitación Espermática/fisiología , Reacción Acrosómica , Adenilil Ciclasas/metabolismo , Animales , Bicarbonatos , Calcio , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática , Femenino , Fertilización/fisiología , Masculino , Mamíferos , Fosforilación , Proteínas/metabolismo , Especies Reactivas de Oxígeno , Espermatozoides/fisiología
17.
Angew Chem Int Ed Engl ; 54(22): 6511-5, 2015 May 26.
Artículo en Inglés | MEDLINE | ID: mdl-25877959

RESUMEN

Structure-based drug design (SBDD) is a powerful and widely used approach to optimize affinity of drug candidates. With the recently introduced INPHARMA method, the binding mode of small molecules to their protein target can be characterized even if no spectroscopic information about the protein is known. Here, we show that the combination of the spin-diffusion-based NMR methods INPHARMA, trNOE, and STD results in an accurate scoring function for docking modes and therefore determination of protein-ligand complex structures. Applications are shown on the model system protein kinase A and the drug targets glycogen phosphorylase and soluble epoxide hydrolase (sEH). Multiplexing of several ligands improves the reliability of the scoring function further. The new score allows in the case of sEH detecting two binding modes of the ligand in its binding site, which was corroborated by X-ray analysis.


Asunto(s)
Diseño de Fármacos , Ligandos , Proteínas/química , Sitios de Unión , Cristalografía por Rayos X , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Difusión , Epóxido Hidrolasas/antagonistas & inhibidores , Epóxido Hidrolasas/metabolismo , Glucógeno Fosforilasa/antagonistas & inhibidores , Glucógeno Fosforilasa/metabolismo , Espectroscopía de Resonancia Magnética , Simulación del Acoplamiento Molecular , Unión Proteica , Proteínas/metabolismo
18.
Birth Defects Res A Clin Mol Teratol ; 103(1): 12-9, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24917297

RESUMEN

The primary cilium is critical in sonic hedgehog (Shh)-dependent ventral patterning of the vertebrate neural tube. Most mutants that cause disruption of the cilium result in decreased Shh signaling in the neural tube. In contrast, mutations in the intraflagellar complex A (IFT-A) and the tubby family protein, Tulp3, result in increased Shh signaling in the neural tube. Proteomic analysis of Tulp3-binding proteins first pointed to the role of the IFT-A complex in trafficking Tulp3 into the cilia. Tulp3 directs trafficking of rhodopsin family G-protein-coupled receptors (GPCRs) to the cilia, suggesting the role of a GPCR in mediating the paradoxical effects of the Tulp3/IFT-A complex in causing increased Shh signaling. Gpr161 has recently been identified as a Tulp3/IFT-A-regulated GPCR that localizes to the primary cilium. A null knock-out mouse model of Gpr161 phenocopies Tulp3 and IFT-A mutants, and causes increased Shh signaling throughout the neural tube. In the absence of Shh, the bifunctional Gli transcription factors are proteolytically processed into repressor forms in a protein kinase A (PKA) -dependent and cilium-dependent manner. Gpr161 activity results in increased cAMP levels in a Gαs -coupled manner, and determines processing of Gli3. Shh signaling also results in removal of Gpr161 from the cilia, suggesting that Gpr161 functions in a positive feedback loop in the Shh pathway. As PKA-null and Gαs mutant embryos also exhibit increased Shh signaling in the neural tube, Gpr161 is a strong candidate for a GPCR that regulates ciliary cAMP levels, and activates PKA in close proximity to the cilia.


Asunto(s)
Tipificación del Cuerpo/fisiología , Cilios/metabolismo , Flagelos/metabolismo , Tubo Neural/fisiología , Proteínas/fisiología , Receptores Acoplados a Proteínas G/fisiología , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular , Péptidos y Proteínas de Señalización Intracelular , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Organogénesis , Transducción de Señal , Proteína Gli3 con Dedos de Zinc
19.
Neurobiol Learn Mem ; 114: 101-112, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24882624

RESUMEN

Protein kinase A (PKA) and other signaling molecules are spatially restricted within neurons by A-kinase anchoring proteins (AKAPs). Although studies on compartmentalized PKA signaling have focused on postsynaptic mechanisms, presynaptically anchored PKA may contribute to synaptic plasticity and memory because PKA also regulates presynaptic transmitter release. Here, we examine this issue using genetic and pharmacological application of Ht31, a PKA anchoring disrupting peptide. At the hippocampal Schaffer collateral CA3-CA1 synapse, Ht31 treatment elicits a rapid decay of synaptic responses to repetitive stimuli, indicating a fast depletion of the readily releasable pool of synaptic vesicles. The interaction between PKA and proteins involved in producing this pool of synaptic vesicles is supported by biochemical assays showing that synaptic vesicle protein 2 (SV2), Rim1, and SNAP25 are components of a complex that interacts with cAMP. Moreover, acute treatment with Ht31 reduces the levels of SV2. Finally, experiments with transgenic mouse lines, which express Ht31 in excitatory neurons at the Schaffer collateral CA3-CA1 synapse, highlight a requirement for presynaptically anchored PKA in pathway-specific synaptic tagging and long-term contextual fear memory. These results suggest that a presynaptically compartmentalized PKA is critical for synaptic plasticity and memory by regulating the readily releasable pool of synaptic vesicles.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Hipocampo/metabolismo , Memoria/fisiología , Plasticidad Neuronal/fisiología , Terminales Presinápticos/metabolismo , Sinapsis/metabolismo , Proteínas de Anclaje a la Quinasa A/metabolismo , Animales , Miedo/fisiología , Proteínas de Unión al GTP/metabolismo , Hipocampo/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Memoria/efectos de los fármacos , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Plasticidad Neuronal/efectos de los fármacos , Terminales Presinápticos/efectos de los fármacos , Proteínas/farmacología , Sinapsis/efectos de los fármacos , Proteína 25 Asociada a Sinaptosomas/metabolismo
20.
Cell ; 152(1-2): 210-23, 2013 Jan 17.
Artículo en Inglés | MEDLINE | ID: mdl-23332756

RESUMEN

The primary cilium is required for Sonic hedgehog (Shh) signaling in vertebrates. In contrast to mutants affecting ciliary assembly, mutations in the intraflagellar transport complex A (IFT-A) paradoxically cause increased Shh signaling. We previously showed that the IFT-A complex, in addition to its canonical role in retrograde IFT, binds to the tubby-like protein, Tulp3, and recruits it to cilia. Here, we describe a conserved vertebrate G-protein-coupled receptor, Gpr161, which localizes to primary cilia in a Tulp3/IFT-A-dependent manner. Complete loss of Gpr161 in mouse causes midgestation lethality and increased Shh signaling in the neural tube, phenocopying Tulp3/IFT-A mutants. Constitutive Gpr161 activity increases cAMP levels and represses Shh signaling by determining the processing of Gli3 to its repressor form. Conversely, Shh signaling directs Gpr161 to be internalized from cilia, preventing its activity. Thus, Gpr161 defines a morphogenetic pathway coupling protein kinase A activation to Shh signaling during neural tube development.


Asunto(s)
Cilios/metabolismo , Embrión de Mamíferos/metabolismo , Tubo Neural/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular , Péptidos y Proteínas de Señalización Intracelular , Ratones , Datos de Secuencia Molecular , Filogenia , Proteínas/metabolismo , Receptores Acoplados a Proteínas G/química , Alineación de Secuencia
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