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1.
Nat Commun ; 12(1): 2005, 2021 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-33790271

RESUMEN

Förster resonant energy transfer (FRET) is a powerful mechanism to probe associations in situ. Simultaneously performing more than one FRET measurement can be challenging due to the spectral bandwidth required for the donor and acceptor fluorophores. We present an approach to distinguish overlapping FRET pairs based on the photochromism of the donor fluorophores, even if the involved fluorophores display essentially identical absorption and emission spectra. We develop the theory underlying this method and validate our approach using numerical simulations. To apply our system, we develop rsAKARev, a photochromic biosensor for cAMP-dependent protein kinase (PKA), and combine it with the spectrally-identical biosensor EKARev, a reporter for extracellular signal-regulated kinase (ERK) activity, to deliver simultaneous readout of both activities in the same cell. We further perform multiplexed PKA, ERK, and calcium measurements by including a third, spectrally-shifted biosensor. Our work demonstrates that exploiting donor photochromism in FRET can be a powerful approach to simultaneously read out multiple associations within living cells.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/química , Proteínas Luminiscentes/química , Algoritmos , Animales , Técnicas Biosensibles/métodos , Células COS , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente/métodos , Imagen de Lapso de Tiempo/métodos
2.
Nat Commun ; 12(1): 2176, 2021 04 12.
Artículo en Inglés | MEDLINE | ID: mdl-33846315

RESUMEN

The hexosamine pathway (HP) is a key anabolic pathway whose product uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor for glycosylation processes in mammals. It modulates the ER stress response and HP activation extends lifespan in Caenorhabditis elegans. The highly conserved glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the rate-limiting HP enzyme. GFAT-1 activity is modulated by UDP-GlcNAc feedback inhibition and via phosphorylation by protein kinase A (PKA). Molecular consequences of GFAT-1 phosphorylation, however, remain poorly understood. Here, we identify the GFAT-1 R203H substitution that elevates UDP-GlcNAc levels in C. elegans. In human GFAT-1, the R203H substitution interferes with UDP-GlcNAc inhibition and with PKA-mediated Ser205 phosphorylation. Our data indicate that phosphorylation affects the interactions of the two GFAT-1 domains to control catalytic activity. Notably, Ser205 phosphorylation has two discernible effects: it lowers baseline GFAT-1 activity and abolishes UDP-GlcNAc feedback inhibition. PKA controls the HP by uncoupling the metabolic feedback loop of GFAT-1.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Retroalimentación Fisiológica , Hexosaminas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Estrés del Retículo Endoplásmico , Mutación con Ganancia de Función , Cinética , Fosforilación , Unión Proteica , Dominios Proteicos , Serina/genética , Uridina Difosfato N-Acetilglucosamina/metabolismo
3.
Nat Commun ; 12(1): 1837, 2021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33758202

RESUMEN

Oocytes are held in meiotic prophase for prolonged periods until hormonal signals trigger meiotic divisions. Key players of M-phase entry are the opposing Cdk1 kinase and PP2A-B55δ phosphatase. In Xenopus, the protein Arpp19, phosphorylated at serine 67 by Greatwall, plays an essential role in inhibiting PP2A-B55δ, promoting Cdk1 activation. Furthermore, Arpp19 has an earlier role in maintaining the prophase arrest through a second serine (S109) phosphorylated by PKA. Prophase release, induced by progesterone, relies on Arpp19 dephosphorylation at S109, owing to an unknown phosphatase. Here, we identified this phosphatase as PP2A-B55δ. In prophase, PKA and PP2A-B55δ are simultaneously active, suggesting the presence of other important targets for both enzymes. The drop in PKA activity induced by progesterone enables PP2A-B55δ to dephosphorylate S109, unlocking the prophase block. Hence, PP2A-B55δ acts critically on Arpp19 on two distinct sites, opposing PKA and Greatwall to orchestrate the prophase release and M-phase entry.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Meiosis , Oocitos/metabolismo , Fosfoproteínas/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Proteína Quinasa CDC2/metabolismo , Cromatografía Liquida , Femenino , Meiosis/efectos de los fármacos , Meiosis/genética , Meiosis/fisiología , Proteínas Nucleares/metabolismo , Ácido Ocadaico/toxicidad , Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas/genética , Fosforilación , Progesterona/farmacología , Proteína Fosfatasa 2/antagonistas & inhibidores , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/aislamiento & purificación , Proteínas Recombinantes , Espectrometría de Masas en Tándem , Proteínas de Xenopus/antagonistas & inhibidores , Proteínas de Xenopus/genética , Proteínas de Xenopus/aislamiento & purificación , Xenopus laevis
4.
Nat Cell Biol ; 23(3): 268-277, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33664495

RESUMEN

The sympathetic nervous system-catecholamine-uncoupling protein 1 (UCP1) axis plays an essential role in non-shivering adaptive thermogenesis. However, whether there exists a direct effector that physically connects catecholamine signalling to UCP1 in response to acute cold is unknown. Here we report that outer mitochondrial membrane-located AIDA is phosphorylated at S161 by the catecholamine-activated protein kinase A (PKA). Phosphorylated AIDA translocates to the intermembrane space, where it binds to and activates the uncoupling activity of UCP1 by promoting cysteine oxidation of UCP1. Adipocyte-specific depletion of AIDA abrogates UCP1-dependent thermogenesis, resulting in hypothermia during acute cold exposure. Re-expression of S161A-AIDA, unlike wild-type AIDA, fails to restore the acute cold response in Aida-knockout mice. The PKA-AIDA-UCP1 axis is highly conserved in mammals, including hibernators. Denervation of the sympathetic postganglionic fibres abolishes cold-induced AIDA-dependent thermogenesis. These findings uncover a direct mechanistic link between sympathetic input and UCP1-mediated adaptive thermogenesis.


Asunto(s)
Adipocitos Marrones/metabolismo , Tejido Adiposo Pardo/inervación , Proteínas de Transferencia de Fosfolípidos/metabolismo , Sistema Nervioso Simpático/fisiología , Termogénesis , Proteína Desacopladora 1/metabolismo , Adiponectina/genética , Adiponectina/metabolismo , Animales , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Metabolismo Energético , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidación-Reducción , Proteínas de Transferencia de Fosfolípidos/deficiencia , Proteínas de Transferencia de Fosfolípidos/genética , Fosforilación , Transducción de Señal , Proteína Desacopladora 1/deficiencia , Proteína Desacopladora 1/genética
5.
Eur J Med Chem ; 216: 113318, 2021 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-33730624

RESUMEN

Identifying a pharmacological agent that targets only one of more than 500 kinases present in humans is an important challenge. One potential solution to this problem is the development of bivalent kinase inhibitors, which consist of two connected fragments, each bind to a dissimilar binding site of the bisubstrate enzyme. The main advantage of bivalent (type V) kinase inhibitors is generating more interactions with target enzymes that can enhance the molecules' selectivity and affinity compared to single-site inhibitors. Earlier type V inhibitors were not suitable for the cellular environment and were mostly used in in vitro studies. However, recently developed bivalent compounds have high kinase affinity, high biological and chemical stability in vivo. This review summarized the hetero-bivalent kinase inhibitors described in the literature from 2014 to the present. We attempted to classify the molecules by serine/threonine and tyrosine kinase inhibitors, and then each target kinase and its hetero-bivalent inhibitor was assessed in depth. In addition, we discussed the analysis of advantages, limitations, and perspectives of bivalent kinase inhibitors compared with the monovalent kinase inhibitors.


Asunto(s)
Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor EphA1/antagonistas & inhibidores , Receptor EphA1/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/metabolismo
6.
Am J Physiol Heart Circ Physiol ; 320(4): H1646-H1656, 2021 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-33635165

RESUMEN

Apelin receptor (APJ) activation by apelin-13 (APLN-13) engages both Gαi proteins and ß-arrestins, stimulating distinct intracellular pathways and triggering physiological responses like enhanced cardiac contractility. Substituting the C-terminal phenylalanine of APLN-13 with α-methyl-l-phenylalanine [(l-α-Me)Phe] or p-benzoyl-l-phenylalanine (Bpa) generates biased analogs inducing APJ functional selectivity toward Gαi proteins. Using these original analogs, we proposed to investigate how the canonical Gαi signaling of APJ regulates the cardiac function and to assess their therapeutic impact in a rat model of isoproterenol-induced myocardial dysfunction. In vivo and ex vivo infusions of either Bpa or (l-α-Me)Phe analogs failed to enhance rats' left ventricular (LV) contractility compared with APLN-13. Inhibition of Gαi with pertussis toxin injection optimized the cardiotropic effect of APLN-13 and revealed the inotropic impact of Bpa. Moreover, both APLN-13 and Bpa efficiently limited the forskolin-induced and PKA-dependent phosphorylation of phospholamban at the Ser16 in neonatal rat ventricular myocytes. However, only Bpa significantly reduced the inotropic effect of forskolin infusion in isolated-perfused heart, highlighting its efficient bias toward Gαi. Compared with APLN-13, Bpa also markedly improved isoproterenol-induced myocardial systolic and diastolic dysfunctions. Bpa prevented cardiac weight increase, normalized both ANP and BNP mRNA expressions, and decreased LV fibrosis in isoproterenol-treated rats. Our results show that APJ-driven Gαi/adenylyl cyclase signaling is functional in cardiomyocytes and acts as negative feedback of the APLN-APJ-dependent inotropic response. Biased APJ signaling toward Gαi over the ß-arrestin pathway offers a promising strategy in the treatment of cardiovascular diseases related to myocardial hypertrophy and high catecholamine levels.NEW & NOTEWORTHY By using more potent Gαi-biased APJ agonists that strongly inhibit cAMP production, these data point to the negative inotropic effect of APJ-mediated Gαi signaling in the heart and highlight the potential protective impact of APJ-dependent Gαi signaling in cardiovascular diseases associated with left ventricular hypertrophy.


Asunto(s)
Receptores de Apelina/agonistas , Apelina/farmacología , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Miocitos Cardíacos/efectos de los fármacos , Disfunción Ventricular Izquierda/prevención & control , Función Ventricular Izquierda/efectos de los fármacos , Adenilil Ciclasas/metabolismo , Animales , Apelina/análogos & derivados , Receptores de Apelina/metabolismo , Proteínas de Unión al Calcio/metabolismo , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Preparación de Corazón Aislado , Isoproterenol , Ligandos , Masculino , Miocitos Cardíacos/metabolismo , Fosforilación , Ratas Sprague-Dawley , Transducción de Señal , Disfunción Ventricular Izquierda/inducido químicamente , Disfunción Ventricular Izquierda/metabolismo , Disfunción Ventricular Izquierda/fisiopatología
7.
J Mol Biol ; 433(8): 166875, 2021 04 16.
Artículo en Inglés | MEDLINE | ID: mdl-33556408

RESUMEN

The coronavirus nucleocapsid protein (N) controls viral genome packaging and contains numerous phosphorylation sites located within unstructured regions. Binding of phosphorylated SARS-CoV N to the host 14-3-3 protein in the cytoplasm was reported to regulate nucleocytoplasmic N shuttling. All seven isoforms of the human 14-3-3 are abundantly present in tissues vulnerable to SARS-CoV-2, where N can constitute up to ~1% of expressed proteins during infection. Although the association between 14-3-3 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, its molecular mechanism and the specific critical phosphosites are unknown. Here, we show that phosphorylated SARS-CoV-2 N protein (pN) dimers, reconstituted via bacterial co-expression with protein kinase A, directly associate, in a phosphorylation-dependent manner, with the dimeric 14-3-3 protein, but not with its monomeric mutant. We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various efficiencies and deduce the apparent KD to selected isoforms, showing that these are in a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to Ser197, which is conserved among related zoonotic coronaviruses and located within the functionally important, SR-rich region of N. The relatively tight 14-3-3/pN association could regulate nucleocytoplasmic shuttling and other functions of N via occlusion of the SR-rich region, and could also hijack cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention.


Asunto(s)
Proteínas 14-3-3/química , Proteínas 14-3-3/metabolismo , /metabolismo , Secuencia de Aminoácidos , Sitios de Unión/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Escherichia coli , Humanos , Mutación , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Fosfoserina/metabolismo , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , ARN Viral/metabolismo , Especificidad por Sustrato
8.
Nat Immunol ; 22(3): 336-346, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33574616

RESUMEN

The anatomic location and immunologic characteristics of brain tumors result in strong lymphocyte suppression. Consequently, conventional immunotherapies targeting CD8 T cells are ineffective against brain tumors. Tumor cells escape immunosurveillance by various mechanisms and tumor cell metabolism can affect the metabolic states and functions of tumor-infiltrating lymphocytes. Here, we discovered that brain tumor cells had a particularly high demand for oxygen, which affected γδ T cell-mediated antitumor immune responses but not those of conventional T cells. Specifically, tumor hypoxia activated the γδ T cell protein kinase A pathway at a transcriptional level, resulting in repression of the activatory receptor NKG2D. Alleviating tumor hypoxia reinvigorated NKG2D expression and the antitumor function of γδ T cells. These results reveal a hypoxia-mediated mechanism through which brain tumors and γδ T cells interact and emphasize the importance of γδ T cells for antitumor immunity against brain tumors.


Asunto(s)
Neoplasias Encefálicas/inmunología , Citotoxicidad Inmunológica , Glioblastoma/inmunología , Linfocitos Intraepiteliales/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Escape del Tumor , Microambiente Tumoral , Animales , Apoptosis , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Antígenos CD8/genética , Antígenos CD8/metabolismo , Línea Celular Tumoral , Técnicas de Cocultivo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación Neoplásica de la Expresión Génica , Genes Codificadores de la Cadena delta de los Receptores de Linfocito T , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Linfocitos Intraepiteliales/metabolismo , Linfocitos Intraepiteliales/patología , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones Desnudos , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Fenotipo , Transducción de Señal , Hipoxia Tumoral
9.
Mol Cell ; 81(4): 675-690.e8, 2021 02 18.
Artículo en Inglés | MEDLINE | ID: mdl-33453167

RESUMEN

Neural network computations are usually assumed to emerge from patterns of fast electrical activity. Challenging this view, we show that a male fly's decision to persist in mating hinges on a biochemical computation that enables processing over minutes to hours. Each neuron in a recurrent network contains slightly different internal molecular estimates of mating progress. Protein kinase A (PKA) activity contrasts this internal measurement with input from the other neurons to represent accumulated evidence that the goal of the network has been achieved. When consensus is reached, PKA pushes the network toward a large-scale and synchronized burst of calcium influx that we call an eruption. Eruptions transform continuous deliberation within the network into an all-or-nothing output, after which the male will no longer sacrifice his life to continue mating. Here, biochemical activity, invisible to most large-scale recording techniques, is the key computational currency directing behavior and motivational state.


Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Drosophila/metabolismo , Red Nerviosa/metabolismo , Neuronas/metabolismo , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas de Drosophila/genética , Drosophila melanogaster
10.
Int J Mol Sci ; 22(3)2021 Jan 25.
Artículo en Inglés | MEDLINE | ID: mdl-33503999

RESUMEN

Inherited retinal degenerative diseases (IRDs), which ultimately lead to photoreceptor cell death, are characterized by high genetic heterogeneity. Many IRD-associated genetic defects affect 3',5'-cyclic guanosine monophosphate (cGMP) levels. cGMP-dependent protein kinases (PKGI and PKGII) have emerged as novel targets, and their inhibition has shown functional protection in IRDs. The development of such novel neuroprotective compounds warrants a better understanding of the pathways downstream of PKGs that lead to photoreceptor degeneration. Here, we used human recombinant PKGs in combination with PKG activity modulators (cGMP, 3',5'-cyclic adenosine monophosphate (cAMP), PKG activator, and PKG inhibitors) on a multiplex peptide microarray to identify substrates for PKGI and PKGII. In addition, we applied this technology in combination with PKG modulators to monitor kinase activity in a complex cell system, i.e. the retinal cell line 661W, which is used as a model system for IRDs. The high-throughput method allowed quick identification of bona fide substrates for PKGI and PKGII. The response to PKG modulators helped us to identify, in addition to ten known substrates, about 50 novel substrates for PKGI and/or PKGII which are either specific for one enzyme or common to both. Interestingly, both PKGs are able to phosphorylate the regulatory subunit of PKA, whereas only PKGII can phosphorylate the catalytic subunit of PKA. In 661W cells, the results suggest that PKG activators cause minor activation of PKG, but a prominent increase in the activity of cAMP-dependent protein kinase (PKA). However, the literature suggests an important role for PKG in IRDs. This conflicting information could be reconciled by cross-talk between PKG and PKA in the retinal cells. This must be explored further to elucidate the role of PKGs in IRDs.


Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Susceptibilidad a Enfermedades , Degeneración Retiniana/etiología , Degeneración Retiniana/metabolismo , Secuencia de Aminoácidos , Biomarcadores , Proteínas Portadoras/química , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Activación Enzimática , Predisposición Genética a la Enfermedad , Humanos , Cinética , Unión Proteica , Degeneración Retiniana/patología , Especificidad por Sustrato
11.
Nat Commun ; 12(1): 413, 2021 01 18.
Artículo en Inglés | MEDLINE | ID: mdl-33462202

RESUMEN

Long-term potentiation (LTP) at hippocampal CA1 synapses can be expressed by an increase either in the number (N) of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors or in their single channel conductance (γ). Here, we have established how these distinct synaptic processes contribute to the expression of LTP in hippocampal slices obtained from young adult rodents. LTP induced by compressed theta burst stimulation (TBS), with a 10 s inter-episode interval, involves purely an increase in N (LTPN). In contrast, either a spaced TBS, with a 10 min inter-episode interval, or a single TBS, delivered when PKA is activated, results in LTP that is associated with a transient increase in γ (LTPγ), caused by the insertion of calcium-permeable (CP)-AMPA receptors. Activation of CaMKII is necessary and sufficient for LTPN whilst PKA is additionally required for LTPγ. Thus, two mechanistically distinct forms of LTP co-exist at these synapses.


Asunto(s)
Región CA1 Hipocampal/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Potenciales Postsinápticos Excitadores/fisiología , Potenciación a Largo Plazo/fisiología , Receptores AMPA/metabolismo , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Masculino , Memoria a Largo Plazo/fisiología , Técnicas de Placa-Clamp , Ratas , Ritmo Teta/fisiología
12.
Ecotoxicol Environ Saf ; 207: 111480, 2021 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-33254385

RESUMEN

Environmental or occupational exposure of Cadmium (Cd) is concerned to be a threat to human health. The kidney is main target of Cd accumulation, which increases the risk of renal cell carcinoma (RCC). In addition, low content of Cd had been determined in kidney cancer, however, the roles of presence of Cd in renal tumors progression are still unclear. The present study is proposed to determine the effect of low-dose Cd exposure on the renal cancer cells and aimed to clarify the underlying mechanisms. The cell viability, cytotoxicity, and the migratory effect of low-dose Cd on the renal cancer cells were detected. Moreover, the roles of reactive oxygen species (ROS), Ca2+, and cyclic AMP (cAMP)/protein kinase A (PKA)-cyclooxygenase2 (COX2) signaling, as well as COX2 catalytic product prostaglandin E2 (PGE2) on cell migration and invasion were identified. Our results suggested that low dose Cd exposure promoted migration of renal cancer Caki-1 cells, which was not dependent on Cd-induced ROS and intracellular Ca2+ levels. Cd exposure induced cAMP/PKA-COX2, which mediated cell migration and invasion, and decreased expressions of epithelial-mesenchymal transition (EMT) marker, E-cadherin, but increased expressions of N-cadherin and Vimentin. Moreover, Cd-induced secretion of PGE2 feedback on activation of cAMP/PKA-COX2 signaling, also promoted EMT, migration and invasion of renal cancer Caki-1 cells. This study might contribute to understanding of the mechanism of Cd-induce progression of renal cancer and future studies on the prevention and therapy of renal cell carcinomas.


Asunto(s)
Cadmio/toxicidad , Contaminantes Ambientales/toxicidad , Transición Epitelial-Mesenquimal/fisiología , Antígenos CD , Cadherinas/metabolismo , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Humanos , Neoplasias Renales , Transducción de Señal/efectos de los fármacos , Vimentina/metabolismo
13.
Biochim Biophys Acta Mol Cell Res ; 1868(1): 118884, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33039554

RESUMEN

Low complexity regions are involved in the assembly and disassembly of P-bodies (PBs). Saccharomyces cerevisiae contains three genes encoding the protein kinase A (PKA) catalytic subunit: TPK1, TPK2 and TPK3. Tpk2 and Tpk3 isoforms localize to PBs upon glucose starvation showing different mechanisms and kinetics of accumulation. In contrast to the other two isoforms, Tpk2 harbors a glutamine-rich prion-like domain (PrLD) at the N-terminus. Here we show that the appearance of Tpk2 foci in response to glucose starvation, heat stress or stationary phase was dependent on its PrLD. Moreover, the PrLD of Tpk2 was necessary for efficient PB and stress granule aggregation during stress conditions and in quiescent cells. Deletion of PrLD does not affect the in vitro and in vivo kinase activity of Tpk2 or its interaction with the regulatory subunit Bcy1. We present evidence that the PrLD of Tpk2 serves as a scaffold domain for PB assembly in a manner that is independent of Pat1 phosphorylation by PKA. In addition, a mutant strain where Tpk2 lacks PrLD showed a decrease of turnover of mRNA during glucose starvation. This work therefore provides new insight into the mechanism of stress-induced cytoplasmic mRNP assembly, and the role of isoform specific domains in the regulation of PKA catalytic subunit specificity and dynamic localization to cytoplasmic RNPs granules.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas de Unión al ARN/genética , Proteínas de Saccharomyces cerevisiae/genética , Estrés Fisiológico/genética , Dominio Catalítico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/química , Citoplasma/genética , Citoplasma/metabolismo , Gránulos Citoplasmáticos/genética , Regulación Fúngica de la Expresión Génica/genética , Fosforilación/genética , Priones/genética , Saccharomyces cerevisiae/genética
14.
Nature ; 590(7846): 451-456, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33361810

RESUMEN

Reinforcement learning models postulate that neurons that release dopamine encode information about action and action outcome, and provide a teaching signal to striatal spiny projection neurons in the form of dopamine release1. Dopamine is thought to guide learning via dynamic and differential modulation of protein kinase A (PKA) in each class of spiny projection neuron2. However, the real-time relationship between dopamine and PKA in spiny projection neurons remains untested in behaving animals. Here we monitor the activity of dopamine-releasing neurons, extracellular levels of dopamine and net PKA activity in spiny projection neurons in the nucleus accumbens of mice during learning. We find positive and negative modulation of dopamine that evolves across training and is both necessary and sufficient to explain concurrent fluctuations in the PKA activity of spiny projection neurons. Modulations of PKA in spiny projection neurons that express type-1 and type-2 dopamine receptors are dichotomous, such that these neurons are selectively sensitive to increases and decreases, respectively, in dopamine that occur at different phases of learning. Thus, PKA-dependent pathways in each class of spiny projection neuron are asynchronously engaged by positive or negative dopamine signals during learning.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dopamina/metabolismo , Aprendizaje , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/enzimología , Neuronas Dopaminérgicas/metabolismo , Femenino , Fluorescencia , Neuronas GABAérgicas/efectos de los fármacos , Neuronas GABAérgicas/enzimología , Neuronas GABAérgicas/metabolismo , Aprendizaje/efectos de los fármacos , Masculino , Ratones , Plasticidad Neuronal/efectos de los fármacos , Núcleo Accumbens/citología , Fotometría , Receptores Dopaminérgicos/clasificación , Receptores Dopaminérgicos/metabolismo
15.
J Agric Food Chem ; 69(1): 212-222, 2021 Jan 13.
Artículo en Inglés | MEDLINE | ID: mdl-33353303

RESUMEN

ι-Carrageenan performs diversified biological activities but has low bioavailability. ι-Carrageenan tetrasaccharide (ιCTs), a novel marine oligosaccharide prepared by the marine enzyme Cgi82A, was investigated for its effects on insulin resistance in high-fat and high-sucrose diet mice. Oral administration of ιCTs (ιCTs-L 30.0 mg/kg·bw, ιCTs-H 90.0 mg/kg·bw) decreased fasting blood glucose by 35.1% ± 1.41 (P < 0.01) and 27.4% ± 0.420 (P < 0.05), and enhanced glucose tolerance. Besides, ιCTs-L ameliorated islet vacuolization, decreased the ß cell apoptosis by 21.8% ± 0.200 (P < 0.05), and promoted insulin secretion by 5.41% ± 0.0173 (P < 0.01) through pancreatic hematoxylin and eosin (H&E) staining, TUNEL staining, and insulin-glucagon immunostaining analysis. Interestingly, ιCTs-L and ιCTs-H treatment increased the incretin GLP-1 content in serum by 22.1% ± 0.402 (P < 0.01) and 10.7% ± 0.0935 (P < 0.05) respectively, through regulating the bile acid levels, which contributed to the inhibition of ß cell apoptosis. Mechanically, ιCTs upregulated the expression of the GLP-1 receptor (GLP-1R) and protein kinase A (PKA) in the GLP-1/cAMP/PKA signaling pathway, and further inhibited the expression of cytochrome C and caspase 3 in the mitochondrial apoptotic pathway. In conclusion, this study suggested that ιCTs alleviated insulin resistance by GLP-1-mediated inhibition of ß cell apoptosis and proposed a new strategy for developing potential functional foods that prevent insulin resistance.


Asunto(s)
Apoptosis/efectos de los fármacos , Carragenina/administración & dosificación , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Péptido 1 Similar al Glucagón/metabolismo , Células Secretoras de Insulina/citología , Mitocondrias/efectos de los fármacos , Oligosacáridos/administración & dosificación , Extractos Vegetales/administración & dosificación , Animales , Glucemia/metabolismo , Carragenina/química , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatología , Péptido 1 Similar al Glucagón/genética , Receptor del Péptido 1 Similar al Glucagón/genética , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Humanos , Resistencia a la Insulina , Secreción de Insulina/efectos de los fármacos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Oligosacáridos/análisis , Extractos Vegetales/análisis , Transducción de Señal/efectos de los fármacos
16.
Arch Biochem Biophys ; 698: 108731, 2021 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-33359563

RESUMEN

Microbial pathogens, such as Trypanosoma brucei, have an enormous impact on global health and economic systems. Protein kinase A of T. brucei is an attractive drug target as it is an essential enzyme which differs significantly from its human homolog. The hinge region of this protein's regulatory domain is vital for enzymatic function, but its conformation is unknown. Here, the secondary structure of this region has been characterized using NMR and CD spectroscopies. More specifically, three overlapping peptides corresponding to residues T187-I211, G198-Y223 and V220-S245 called peptide 1, peptide 2 and peptide 3, respectively, were studied. The peptide 1 and peptide 2 are chiefly unfolded; only low populations (<10%) of α-helix were detected under the conditions studied. In contrast, the peptide 3 contains a long α-helix whose population is significantly higher; namely, 36% under the conditions studied. Utilizing the dihedral φ and ψ angles calculated on the basis of the NMR data, the conformation of the peptide 3 was calculated and revealed an α-helix spanning residues E230-N241. This α-helix showed amphiphilicity and reversible unfolding and refolding upon heating and cooling. Most fascinating, however, is its capacity to inhibit the activity of the catalytic domain of Trypanosoma equiperdum protein kinase A even though it is quite distinct from the canonical inhibitor motif. Based on this property, we advance that peptoids based on the peptide 3 α-helix could be novel leads for developing anti-trypanosomal therapeutics.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/química , Fragmentos de Péptidos/química , Inhibidores de Proteínas Quinasas/química , Trypanosoma brucei brucei/enzimología , Secuencia de Aminoácidos , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Pruebas de Enzimas , Conformación Proteica en Hélice alfa , Replegamiento Proteico , Desplegamiento Proteico , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Alineación de Secuencia , Porcinos
17.
Mol Med Rep ; 23(2)2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33300086

RESUMEN

Intermedin (IMD) is a calcitonin/calcitonin­related peptide that elicits cardioprotective effects in a variety of heart diseases, such as cardiac hypertrophy and heart failure. However, the molecular mechanism of IMD remains unclear. The present study investigated the effects of IMD on neonatal rat ventricular myocytes treated with thapsigargin. The results of the present study demonstrated that thapsigargin induced apoptosis in cardiomyocytes in a dose­ and time­dependent manner. Thapsigargin induced endoplasmic reticulum stress, as determined by increased expression levels of 78­kDa glucose­regulated protein, C/EBP­homologous protein and caspase­12, which were dose­dependently attenuated by pretreatment with IMD. In addition, IMD treatment counteracted the thapsigargin­induced suppression of sarco/endoplasmic reticulum Ca2+­ATPase (SERCA) activity and protein expression levels, and cytoplasmic Ca2+ overload. IMD treatment also augmented the phosphorylation of phospholamban, which is a crucial regulator of SERCA. Additionally, treatment with the protein kinase A antagonist H­89 inhibited the IMD­mediated cardioprotective effects, including SERCA activity restoration, anti­Ca2+ overload, endoplasmic reticulum stress inhibition and antiapoptosis effects. In conclusion, the results of the present study suggested that IMD may protect cardiomyocytes against thapsigargin­induced endoplasmic reticulum stress and the associated apoptosis at least partly by activating the protein kinase A/SERCA pathway.


Asunto(s)
Adrenomedulina/metabolismo , Señalización del Calcio/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Neuropéptidos/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Tapsigargina/farmacología , Adrenomedulina/genética , Animales , Señalización del Calcio/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Estrés del Retículo Endoplásmico/genética , Femenino , Masculino , Neuropéptidos/genética , Ratas , Ratas Sprague-Dawley , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética
18.
Clin Sci (Lond) ; 134(24): 3259-3282, 2020 12 23.
Artículo en Inglés | MEDLINE | ID: mdl-33346357

RESUMEN

The 3'-5'-cyclic adenosine monophosphate (cAMP)/PKA pathway represents a major target for pharmacological intervention in multiple disease conditions. Although the last decade saw the concept of highly compartmentalized cAMP/PKA signaling consolidating, current means for the manipulation of this pathway still do not allow to specifically intervene on discrete cAMP/PKA microdomains. Since compartmentalization is crucial for action specificity, identifying new tools that allow local modulation of cAMP/PKA responses is an urgent need. Among key players of cAMP/PKA signaling compartmentalization, a major role is played by A-kinase anchoring proteins (AKAPs) that, by definition, anchor PKA, its substrates and its regulators within multiprotein complexes in well-confined subcellular compartments. Different tools have been conceived to interfere with AKAP-based protein-protein interactions (PPIs), and these primarily include peptides and peptidomimetics that disrupt AKAP-directed multiprotein complexes. While these molecules have been extensively used to understand the molecular mechanisms behind AKAP function in pathophysiological processes, less attention has been devoted to their potential application for therapy. In this review, we will discuss how AKAP-based PPIs can be pharmacologically targeted by synthetic peptides and peptidomimetics.


Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Péptidos/uso terapéutico , Animales , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Terapia Molecular Dirigida , Transducción de Señal
19.
Int J Mol Sci ; 21(24)2020 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-33339131

RESUMEN

The cytoskeleton has a primary role in cardiomyocyte function, including the response to mechanical stimuli and injury. The small heat shock protein 20 (Hsp20) conveys protective effects in cardiac muscle that are linked to serine-16 (Ser16) Hsp20 phosphorylation by stress-induced PKA, but the link between Hsp20 and the cytoskeleton remains poorly understood. Herein, we demonstrate a physical and functional interaction of Hsp20 with the cytoskeletal protein 14-3-3. We show that, upon phosphorylation at Ser16, Hsp20 translocates from the cytosol to the cytoskeleton where it binds to 14-3-3. This leads to dissociation of 14-3-3 from the F-actin depolymerization regulator cofilin-2 (CFL2) and enhanced F-actin depolymerization. Importantly, we demonstrate that the P20L Hsp20 mutation associated with dilated cardiomyopathy exhibits reduced physical interaction with 14-3-3 due to diminished Ser16 phosphorylation, with subsequent failure to translocate to the cytoskeleton and inability to disassemble the 14-3-3/CFL2 complex. The topological sequestration of Hsp20 P20L ultimately results in impaired regulation of F-actin dynamics, an effect implicated in loss of cytoskeletal integrity and amelioration of the cardioprotective functions of Hsp20. These findings underscore the significance of Hsp20 phosphorylation in the regulation of actin cytoskeleton dynamics, with important implications in cardiac muscle physiology and pathophysiology.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citoesqueleto/metabolismo , Proteínas del Choque Térmico HSP20/metabolismo , Miocardio/metabolismo , Proteínas 14-3-3/metabolismo , Actinas/metabolismo , Animales , Cofilina 2/metabolismo , Células HEK293 , Proteínas del Choque Térmico HSP20/genética , Humanos , Ratones , Mutación , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional
20.
PLoS One ; 15(12): e0244253, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33347508

RESUMEN

In order to elucidate involvement of cyclic AMP and intracellular Ca2+,[Ca2+]i, in the modulation of aqueous humour formation (AHF), we studied the effects of terbutaline, forskolin and 8-Br-cAMP in the isolated bovine eye. We also studied the interaction of cAMP on calcium signaling in cultured ciliary epithelial (CE) cells. Drug effects on AHF were measured by fluorescein dilution. Drug effects on [Ca2+]i were studied by the fura-2 fluorescence ratio technique. Terbutaline (100 nmol-100 M), forskolin (30 nM-100 M) or 8-Br-cAMP (100 nM- 10 µM), administered in the arterial perfusate produced significant reductions in AHF. The AH reducing effect of terbutaline was blocked by a selective inhibitor of protein kinase A (KT-5720). ATP (100 M) caused a rapid, transient (peak) increase in [Ca2+]i followed by a sustained plateau phase lasting more than 5 minutes. Preincubation of the cells (6 min) with terbutaline, forskolin or 8-Br-cAMP significantly reduced the peak calcium response to ATP. The sustained plateau phase of the response, on the other hand, was augmented by each of the agents. KT-5720 partially reversed the inhibitory effect of terbutaline on the peak and totally inhibited its effect on the plateau phase. These data indicate: (a) that AHF in the bovine eye can be manipulated through cyclic AMP, operating via protein kinase A, (b) that protein kinase A can affect [Ca2+]i homeostasis, (c) that calcium release from the intracellular store, not the entry, affects AHF, and (d) that interaction of [Ca2+]i with cAMP plays a role in modulating AH secretion.


Asunto(s)
Humor Acuoso/metabolismo , Secreciones Corporales/efectos de los fármacos , Calcio/metabolismo , Colforsina/farmacología , AMP Cíclico/farmacología , Terbutalina/farmacología , Animales , Humor Acuoso/efectos de los fármacos , Broncodilatadores/farmacología , Bovinos , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo
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