RESUMEN
Inhibition of the cyclic-AMP degrading enzyme phosphodiesterase type 4 (PDE4) in the brains of animal models is protective in Alzheimer's disease (AD). We show for the first time that enzymes from the subfamily PDE4D not only colocalize with beta-amyloid (Aß) plaques in a mouse model of AD but that Aß directly associates with the catalytic machinery of the enzyme. Peptide mapping suggests that PDE4D is the preferential PDE4 subfamily for Aß as it possesses a unique binding site. Intriguingly, exogenous addition of Aß to cells overexpressing the PDE4D5 longform caused PDE4 activation and a decrease in cAMP. We suggest a novel mechanism where PDE4 longforms can be activated by Aß, resulting in the attenuation of cAMP signalling to promote loss of cognitive function in AD.
Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , AMP Cíclico , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Neuronas , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Péptidos beta-Amiloides/metabolismo , AMP Cíclico/metabolismo , Ratones , Neuronas/metabolismo , Humanos , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/genética , Unión Proteica , Activación Enzimática , Ratones Transgénicos , Placa Amiloide/metabolismo , Placa Amiloide/patologíaRESUMEN
Cyclic AMP is a ubiquitous second messenger used to transduce intracellular signals from a variety of Gs-coupled receptors. Compartmentalisation of protein intermediates within the cAMP signaling pathway underpins receptor-specific responses. The cAMP effector proteins protein-kinase A and EPAC are found in complexes that also contain phosphodiesterases whose presence ensures a coordinated cellular response to receptor activation events. Popeye domain containing (POPDC) proteins are the most recent class of cAMP effectors to be identified and have crucial roles in cardiac pacemaking and conduction. We report the first observation that POPDC proteins exist in complexes with members of the PDE4 family in cardiac myocytes. We show that POPDC1 preferentially binds the PDE4A sub-family via a specificity motif in the PDE4 UCR1 region and that PDE4s bind to the Popeye domain of POPDC1 in a region known to be susceptible to a mutation that causes human disease. Using a cell-permeable disruptor peptide that displaces the POPDC1-PDE4 complex we show that PDE4 activity localized to POPDC1 modulates cycle length of spontaneous Ca2+ transients firing in intact mouse sinoatrial nodes.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico , AMP Cíclico , Animales , Proteínas Portadoras/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Ratones , Hidrolasas Diéster Fosfóricas/metabolismo , Sistemas de Mensajero Secundario , Transducción de SeñalRESUMEN
zDHHC S-acyltransferases are enzymes catalyzing protein S-acylation, a common post-translational modification on proteins frequently affecting their membrane targeting and trafficking. The ankyrin repeat (AR) domain of zDHHC17 (HIP14) and zDHHC13 (HIP14L) S-acyltransferases, which is involved in both substrate recruitment and S-acylation-independent functions, was recently shown to bind at least six proteins, by specific recognition of a consensus sequence in them. To further refine the rules governing binding to the AR of zDHHC17, we employed peptide arrays based on zDHHC AR-binding motif (zDABM) sequences of synaptosomal-associated protein 25 (SNAP25) and cysteine string protein α (CSPα). Quantitative comparisons of the binding preferences of 400 peptides allowed us to construct a position-specific scoring matrix (PSSM) for zDHHC17 AR binding, with which we predicted and subsequently validated many putative zDHHC17 interactors. We identified 95 human zDABM sequences with unexpected versatility in amino acid usage; these sequences were distributed among 90 proteins, of which 62 have not been previously implicated in zDHHC17/13 binding. These zDABM-containing proteins included all family members of the SNAP25, sprouty, cornifelin, ankyrin, and SLAIN-motif containing families; seven endogenous Gag polyproteins sharing the same binding sequence; and several proteins involved in cytoskeletal organization, cell communication, and regulation of signaling. A dozen of the zDABM-containing proteins had more than one zDABM sequence, whereas isoform-specific binding to the AR of zDHHC17 was identified for the Ena/VASP-like protein. The large number of zDABM sequences within the human proteome suggests that zDHHC17 may be an interaction hub regulating many cellular processes.
Asunto(s)
Aciltransferasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteoma/metabolismo , Aciltransferasas/química , Proteínas Adaptadoras Transductoras de Señales/química , Repetición de Anquirina , Línea Celular , Proteínas del Choque Térmico HSP40/química , Proteínas del Choque Térmico HSP40/metabolismo , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/química , Péptidos/química , Péptidos/metabolismo , Análisis por Matrices de Proteínas/métodos , Unión Proteica , Proteoma/química , Proteína 25 Asociada a Sinaptosomas/química , Proteína 25 Asociada a Sinaptosomas/metabolismoRESUMEN
Aberrant WNT signaling drives colorectal cancer (CRC). Here, we identify TIAM1 as a critical antagonist of CRC progression through inhibiting TAZ and YAP, effectors of WNT signaling. We demonstrate that TIAM1 shuttles between the cytoplasm and nucleus antagonizing TAZ/YAP by distinct mechanisms in the two compartments. In the cytoplasm, TIAM1 localizes to the destruction complex and promotes TAZ degradation by enhancing its interaction with ßTrCP. Nuclear TIAM1 suppresses TAZ/YAP interaction with TEADs, inhibiting expression of TAZ/YAP target genes implicated in epithelial-mesenchymal transition, cell migration, and invasion, and consequently suppresses CRC cell migration and invasion. Importantly, high nuclear TIAM1 in clinical specimens associates with increased CRC patient survival. Together, our findings suggest that in CRC TIAM1 suppresses tumor progression by regulating YAP/TAZ activity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Movimiento Celular , Neoplasias Colorrectales/metabolismo , Células Epiteliales/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Mucosa Intestinal/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfoproteínas/metabolismo , Transporte Activo de Núcleo Celular , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Células CACO-2 , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Citoplasma/metabolismo , Células Epiteliales/patología , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Factores de Intercambio de Guanina Nucleótido/deficiencia , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Mucosa Intestinal/patología , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Invasividad Neoplásica , Fenotipo , Fosfoproteínas/genética , Proteolisis , Interferencia de ARN , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Transactivadores , Factores de Transcripción , Transcripción Genética , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Transfección , Vía de Señalización Wnt , Proteínas Señalizadoras YAP , Pez Cebra/embriología , Proteínas con Repetición de beta-Transducina/genética , Proteínas con Repetición de beta-Transducina/metabolismoRESUMEN
The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is a sequence-specific DNA-binding protein that plays an essential role in viral episome replication and segregation, by recruiting the cellular complex of DNA replication onto the origin (oriP) and by tethering the viral DNA onto the mitotic chromosomes. Whereas the mechanisms of viral DNA replication are well documented, those involved in tethering EBNA1 to the cellular chromatin are far from being understood. Here, we have identified regulator of chromosome condensation 1 (RCC1) as a novel cellular partner for EBNA1. RCC1 is the major nuclear guanine nucleotide exchange factor for the small GTPase Ran enzyme. RCC1, associated with chromatin, is involved in the formation of RanGTP gradients critical for nucleo-cytoplasmic transport, mitotic spindle formation and nuclear envelope reassembly following mitosis. Using several approaches, we have demonstrated a direct interaction between these two proteins and found that the EBNA1 domains responsible for EBNA1 tethering to the mitotic chromosomes are also involved in the interaction with RCC1. The use of an EBNA1 peptide array confirmed the interaction of RCC1 with these regions and also the importance of the N-terminal region of RCC1 in this interaction. Finally, using confocal microscopy and Förster resonance energy transfer analysis to follow the dynamics of interaction between the two proteins throughout the cell cycle, we have demonstrated that EBNA1 and RCC1 closely associate on the chromosomes during metaphase, suggesting an essential role for the interaction during this phase, perhaps in tethering EBNA1 to mitotic chromosomes.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Mitosis , Proteínas Nucleares/metabolismo , Dominios y Motivos de Interacción de Proteínas , Secuencias de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Cromosomas Humanos/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/química , Antígenos Nucleares del Virus de Epstein-Barr/genética , Transferencia Resonante de Energía de Fluorescencia , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Células HEK293 , Células HeLa , Humanos , Metafase , Microscopía Confocal , Proteínas Nucleares/química , Proteínas Nucleares/genética , Análisis por Matrices de Proteínas , Mapeo de Interacción de Proteínas , Huso Acromático/metabolismoRESUMEN
Cyclic AMP (cAMP)-specific phosphodiesterase-4 (PDE4) enzymes underpin compartmentalised cAMP signalling by localising to distinct signalling complexes. PDE4 long isoforms can be phosphorylated by mitogen-activated protein kinase-activated protein kinase 2 (MK2), which attenuates activation of such enzymes through their phosphorylation by protein kinase A. Here we show that MK2 interacts directly with PDE4 long isoforms and define the sites of interaction. One is a unique site that locates within the regulatory upstream conserved region 1 (UCR1) domain and contains a core Phe141, Leu142 and Tyr143 (FLY) cluster (PDE4A5 numbering). Located with the second site is a critical core Phe693, Glu694, Phe695 (FQF) motif that is also employed in the sequestering of PDE4 long forms by an array of other signalling proteins, including the signalling scaffold ß-arrestin, the tyrosyl kinase Lyn, the SUMOylation E2 ligase UBC9, the dynein regulator Lis1 (PAFAH1B1) and the protein kinase Erk. We propose that the FQF motif lies at the heart of a multifunctional docking (MFD) site located within the PDE4 catalytic unit. It is clear from our data that, as well as aiding fidelity of interaction, the MFD site confers exclusivity of binding between PDE4 and a single specific partner protein from the cohort of signalling proteins whose interaction with PDE4 involves the FQF motif.
Asunto(s)
Dominio Catalítico , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/química , Péptidos y Proteínas de Señalización Intracelular/química , Simulación del Acoplamiento Molecular , Proteínas Serina-Treonina Quinasas/química , 1-Alquil-2-acetilglicerofosfocolina Esterasa/química , 1-Alquil-2-acetilglicerofosfocolina Esterasa/genética , 1-Alquil-2-acetilglicerofosfocolina Esterasa/metabolismo , Secuencias de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Expresión Génica , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/química , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/química , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , beta-Arrestinas/química , beta-Arrestinas/genética , beta-Arrestinas/metabolismo , Familia-src Quinasas/química , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
OBJECTIVES: Although many traumatic incidents result in changes to appearance, little research has examined the experience of individuals distressed by such changes in connection with psychological processes involved in posttraumatic stress disorder (PTSD). This study aimed to examine how PTSD and appearance concern associated with burn injury are experienced when both difficulties co-occur. METHOD: The qualitative method of interpretative phenomenological analysis (IPA) was used to provide a framework for building nuanced accounts of individual experience. In-depth analysis was conducted with interview data obtained from 8 women, who were purposively selected on the basis of being distressed in relation to burn scarring, and having symptoms of PTSD. RESULTS: Participants described how changes in appearance were experienced as maintaining a sense of threat through social stigma, and acting as a trigger for re-experiencing the traumatic incident that had caused the burn injury. As such, appearance concern and PTSD symptomatology appeared intertwined within the participants' accounts of their postburn injury recovery. CONCLUSIONS: This is the first study to consider some of the processes through which PTSD and appearance concern might be mutually maintained. The results suggest that psychosocial interventions need to be tailored to simultaneously address processes related to concerns about change in appearance and also with traumatic re-experiencing. (PsycINFO Database Record
Asunto(s)
Trastorno Dismórfico Corporal/psicología , Quemaduras/psicología , Trastornos por Estrés Postraumático/psicología , Adolescente , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Investigación Cualitativa , Estigma SocialRESUMEN
Alzheimer's disease (AD) is the most common form of dementia in the elderly and its prevalence is set to increase rapidly in coming decades. However, there are as yet no available drugs that can halt or even stabilize disease progression. One of the main pathological features of AD is the presence in the brain of senile plaques mainly composed of aggregated ß amyloid (Aß), a derivative of the longer amyloid precursor protein (APP). The amyloid hypothesis proposes that the accumulation of Aß within neural tissue is the initial event that triggers the disease. Here we review research efforts that have attempted to inhibit the generation of the Aß peptide through modulation of the activity of the proteolytic secretases that act on APP and discuss whether this is a viable therapeutic strategy for treating AD.
RESUMEN
Up-regulation of Hsp20 protein levels in response to amyloid fibril formation is considered a key protective response against the onset of Alzheimer's disease (AD). Indeed, the physical interaction between Hsp20 and Aß is known to prevent Aß oligomerisation and protects neuronal cells from Aß mediated toxicity, however, details of the molecular mechanism and regulatory cell signalling events behind this process have remained elusive. Using both conventional MTT end-point assays and novel real time measurement of cell impedance, we show that Hsp20 protects human neuroblastoma SH-SY5Y cells from the neurotoxic effects of Aß. In an attempt to provide a mechanism for the neuroprotection afforded by Hsp20, we used peptide array, co-immunoprecipitation analysis and NMR techniques to map the interaction between Hsp20 and Aß and report a binding mode where Hsp20 binds adjacent to the oligomerisation domain of Aß, preventing aggregation. The Hsp20/Aß interaction is enhanced by Hsp20 phosphorylation, which serves to increase association with low molecular weight Aß species and decrease the effective concentration of Hsp20 required to disrupt the formation of amyloid oligomers. Finally, using a novel fluorescent assay for the real time evaluation of morphology-specific Aß aggregation, we show that phospho-dependency of this effect is more pronounced for fibrils than for globular Aß forms and that 25mers corresponding to the Hsp20 N-terminal can be used as Aß aggregate inhibitors. Our report is the first to provide a molecular model for the Hsp20/Aß complex and the first to suggest that modulation of the cAMP/cGMP pathways could be a novel route to enhance Hsp20-mediated attenuation of Aß fibril neurotoxicity.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/farmacología , Proteínas del Choque Térmico HSP20/metabolismo , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Arginina/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Inmunoprecipitación , Espectroscopía de Resonancia Magnética , Mutación/genética , Neuroblastoma/patología , Mapeo Peptídico , Fosforilación/efectos de los fármacos , Factores de TiempoRESUMEN
Beta arrestins are molecular scaffolds that can bring together three-component mitogen-activated protein kinase signalling modules to promote signal compartmentalisation. We use peptide array technology to define novel interfaces between components within the c-Jun N-terminal kinase (JNK)/beta arrestin signalling complex. We show that beta arrestin 1 and beta arrestin 2 associate with JNK3 via the kinase N-terminal domain in a region that, surprisingly, does not harbour a known 'common docking' motif. In the N-domain and C-terminus of beta arrestin 1 and beta arrestin 2 we identify two novel apoptosis signal-regulating kinase 1 binding sites and in the N-domain of the beta arrestin 1 and beta arrestin 2 we identify a novel MKK4 docking site.
Asunto(s)
Arrestinas/química , Arrestinas/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 10 Activada por Mitógenos/química , Proteína Quinasa 10 Activada por Mitógenos/metabolismo , Péptidos/metabolismo , Análisis por Matrices de Proteínas/métodos , Secuencia de Aminoácidos , Animales , Arrestinas/genética , MAP Quinasa Quinasa 4/química , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Proteína Quinasa 10 Activada por Mitógenos/genética , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/genética , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , beta-ArrestinasRESUMEN
We identify a compartmentalized signaling system that identifies a functional role for the GTP exchange factor, exchange protein activated by cAMP (EPAC) coupled to Rap2 in the nucleus. In this system, cAMP regulates the nuclear/cytoplasmic trafficking of DNA-dependent protein kinase (DNA-PK), a critical kinase that acts to repair double-stranded breaks (DSBs) in damaged DNA and to phosphorylate the cell survival kinase, PKB/Akt. Intersecting regulatory inputs for cAMP employ EPAC to transduce positive effects, namely the Rap2-dependent nuclear exit and activation of DNA-PK, whereas protein kinase A (PKA) provides the negative input by antagonizing these actions. We identify this as a compartmentalized regulatory system where modulation of cAMP input into the stimulatory, EPAC and inhibitory, PKA intersecting arms is provided by spatially discrete, cAMP degradation systems. The distribution of DNA-PK between nuclear and cytoplasmic compartments can thus potentially be influenced by relative inputs of cAMP signaling through the EPAC and PKA pathways. Through this signaling system EPAC activation can thereby impact on the Ser-473 phosphorylation status of PKB/Akt and the repair of etoposide-induced DSBs.
Asunto(s)
Núcleo Celular/enzimología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Secuencia de Aminoácidos , Roturas del ADN de Doble Cadena , Activación Enzimática , Células HeLa , Humanos , Espacio Intracelular/metabolismo , Datos de Secuencia Molecular , Péptidos/química , Hidrolasas Diéster Fosfóricas/metabolismo , Fosforilación , Fosfoserina/metabolismo , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rap/metabolismoRESUMEN
Cells of early mammalian embryos have the potential to develop into any adult cell type, and are thus said to be pluripotent. Pluripotency is lost during embryogenesis as cells commit to specific developmental pathways. Although restriction of developmental potential is often associated with repression of inappropriate genetic programmes, the role of epigenetic silencing during early lineage commitment remains undefined. Here, we used mouse embryonic stem cells to study the function of epigenetic silencing in pluripotent cells. Embryonic stem cells lacking Mbd3 - a component of the nucleosome remodelling and histone deacetylation (NuRD) complex - were viable but failed to completely silence genes that are expressed before implantation of the embryo. Mbd3-deficient embryonic stem cells could be maintained in the absence of leukaemia inhibitory factor (LIF) and could initiate differentiation in embryoid bodies or chimeric embryos, but failed to commit to developmental lineages. Our findings define a role for epigenetic silencing in the cell-fate commitment of pluripotent cells.