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EPAC1 activation by cAMP stabilizes CFTR at the membrane by promoting its interaction with NHERF1.
Cyclic AMP (cAMP) activates protein kinase A (PKA) but also the guanine nucleotide exchange factor 'exchange protein directly activated by cAMP' (EPAC1; also known as RAPGEF3). Although phosphorylation by PKA is known to regulate CFTR channel gating - the protein defective in cystic fibrosis - the contribution of EPAC1 to CFTR regulation remains largely undefined. Here, we demonstrate that in human airway epithelial cells, cAMP signaling through EPAC1 promotes CFTR stabilization at the plasma membrane by attenuating its endocytosis, independently of PKA activation. EPAC1 and CFTR colocalize and interact through protein adaptor NHERF1 (also known as SLC9A3R1). This interaction is promoted by EPAC1 activation, triggering its translocation to the plasma membrane and binding to NHERF1. Our findings identify a new CFTR-interacting protein and demonstrate that cAMP activates CFTR through two different but complementary pathways - the well-known PKA-dependent channel gating pathway and a new mechanism regulating endocytosis that involves EPAC1. The latter might constitute a novel therapeutic target for treatment of cystic fibrosis.
Microplate selection technique (MPST). A new method for selecting mouse transfectants expressing human gene products.
This paper examines the analytical power of fluorescence activated cell sorting and immunorosetting technique as compared with the newly devised microplate selection technique in identifying transfected murine L cells expressing human surface molecules. The microplate selection technique relies on the mechanical transfer of transfected cells to a terasaki microplate, where an indirect immunofluorescence assay is carried out. It is a simple procedure not requiring costly equipment and with a detection capacity equivalent to that of the fluorescence activated cell sorter. The microplate selection technique proved to be sensitive enough to detect all the transfectants produced during the present study.
Compartmentalization and Regulation of Cyclic Nucleotide Signaling in The CNS
© 2014 by John Wiley & Sons, Inc. All rights reserved. In the central nervous system (CNS), the second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) transduce the biochemical and electrical signals of several hormones and neurotransmitters. By integrating these signals, cAMP and cGMP regulate important neuronal functions, such as neuronal excitability, axon guidance, neurotransmitters release, metabolism, and learning and memory processes, via activation of multiple parallel signaling pathways. How such an intricate pattern of signaling cascades can achieve specificity of response has been questioned for a long time. This chapter describes the mechanisms that control cell signaling via tight spatial compartmentalization of receptors, cyclases, second messengers, and their kinase effectors, as well as of their molecular targets.
cAMP signaling: Methods and protocols
© Springer Science+Business Media New York 2015. All rights reserved. This detailed volume encompasses new technological developments that specifically address questions related to adenosine 3,5-monophosphate (cAMP) compartmentalization, that probe relevant protein-protein interactions, that increase the spatial and temporal resolution of cAMP signal detection, and that can facilitate integration of the mounting complexity of the information that is becoming available on this signaling system. cAMP, the prototypical intracellular second messenger, regulates a large variety of cellular functions and biological processes, including gene transcription, cell metabolism, proliferation, development, as well as more specialized functions depending on the cell type, so the realization of its extremely complex spatial organization and local regulation is providing novel mechanistic insight into cell physiology and is producing a novel framework for the identification of disease mechanisms. Written in the highly successful Methods in Molecular Biology series format, chapters include introduction to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Practical and authoritative, cAMP Signaling: Methods and Protocols serves as a vital resource for researchers working in this expanding, complex field.
Involvement of the multilineage CD38 molecule in a unique pathway of cell activation and proliferation.
We report clear evidence that the interaction of the CD38 molecule with the specific mAb A10 on normal human cells and lines modulates the expression of surface activation markers relevant to T, NK, and plasma cell biology and functions. Moreover A10 mAb binding is followed by proliferation effects on all the target cells analyzed, and the phenomenon is accessory cell and IL-2 dependent. The effects of A10 mAb synergizing both CD2 and CD3 activation pathways indicate that CD38 signal transduction mechanism(s) are apparently different from the aforementioned. Nevertheless the decreased A10-driven proliferation after CD3-Ti modulation suggests a possible functional interdependence between these activation pathways. Taken together, the results indicate that the CD38 molecule might play a physiologic role in T, NK, and plasma cell regulation.
The effect of high-frequency random mutagenesis on in vitro protein evolution: a study on TEM-1 beta-lactamase.
For a number of years a major limitation in genetic analysis of protein function has been the inability to introduce multiple substitutions at distant sites that would enable the selection of clusters of mutations required for improved or novel biological functions. In order to achieve this, we have recently developed a novel mutagenesis procedure in which the triphosphate derivatives of a pyrimidine (6-(2-deoxy-beta-d-ribofuranosyl)-3, 4-dihydro-8H-pyrimido-[4,5-c][1,2]oxazin-7-one; dP) and a purine (8-oxo-2'-deoxyguanosine; 8-oxodG) nucleoside analogue are employed in DNA synthesis reactions in vitro. The procedure allows control of the mutational load and can yield frequencies of amino acid residue substitutions at least one order of magnitude greater than those previously achieved. Here we report the results of an experiment in which we have hypermutated the bacterial enzyme TEM-1 beta-lactamase and selected small pools (<1.5x10(5)) of clones for enzymatic activity against the beta-lactam antibiotic cefotaxime. The experiment resulted in the isolation of a number of TEM-1 mutants with greatly improved activity against cefotaxime. Among these, clone 3D.5 (E104K:M182T:G238S) exhibited a minimum inhibitory concentration for cefotaxime 20,000-fold higher than wild-type TEM-1 and a catalytic efficiency (kcat/Km) 2383 times higher than the wild-type enzyme. Thus, small pools of hypermutated sequences enabled the selection of one of the most active extended beta-lactamases described so far. These results argue against the accepted view that multiple rounds of low-rate mutagenesis and stepwise selection are essential for in vitro protein evolution and extend the scope of directed molecular evolution to proteins for which no genetic selection is available.
Isolation of high affinity human antibodies directly from large synthetic repertoires.
Antibody fragments of moderate affinity (approximately microM) can be isolated from repertoires of approximately 10(8) immunoglobulin genes by phage display and rounds of selection with antigen, and the affinities improved by further rounds of mutation and selection. Here, as an alternative strategy, we attempted to isolate high affinity human antibodies directly from large repertoires. We first created highly diverse repertoires of heavy and light chains entirely in vitro from a bank of human V gene segments and then, by recombination of the repertoires in bacteria, generated a large (close to 6.5 x 10(10)) synthetic repertoire of Fab fragments displayed on filamentous phage. From this repertoire we isolated Fab fragments which bound to a range of different antigens and haptens, and with affinities comparable with those of antibodies from a secondary immune response in mice (up to 4 nM). Although the VH-26 (DP-47) segment was the most commonly used segment in both artificial and natural repertoires, there were also major differences in the pattern of segment usage. Such comparisons may help dissect the contributions of biological mechanisms and structural features governing V gene usage in vivo.
An approach to random mutagenesis of DNA using mixtures of triphosphate derivatives of nucleoside analogues.
We describe a new method for random mutagenesis of DNA based on the use of a mixture of triphosphates of nucleoside analogues. The method relies on DNA amplification in vitro with Taq polymerase and in the presence of the 5'-triphosphates of 6-(2-deoxy-beta-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido-[4,5-C] [1,2]oxazin-7-one(dP) and of 8-oxo-2' deoxyguanosine (8-oxodG). The newly synthesised triphosphate derivative of dP (dPTP) is an excellent substrate for Taq polymerase (Km = 22 microM versus Km = 9.5 microM for TTP); it is incorporated in place of TTP and, with a approximately fourfold lower efficiency, in place of dCTP. After 30 cycles of DNA amplification, equimolar mixtures of the four normal dNTPs and dPTP yield the following frequencies of the four transition mutations: A-->G (4.4 x 10(-2), T-->C (4.3 x 10(-2), G-->A (1.1 x 10(-2) and C-->T (1.0 x 10(-2). The triphosphate derivative of 8-oxodG (8-oxodGTP) is incorporated opposite template adenine and yields two transition mutations (A-->C and T-->G) at frequencies of 0.8 x 10(-2) and 1.2 x 10(-2) respectively. Reaction mixtures containing dPTP and 8-oxodGTP results in both dP and 8-oxodG-induced mutations and an extensive array of codon changes in the absence of insertions and deletions. The method described differs from previous mutagenesis procedures in three respects: (1) it enables very high frequencies of base substitutions (up to 1.9 x 10(-1) (2) it allows control of the mutational load via the number of DNA amplification cycles and (3) it yields both transition and transversion mutations. The procedure may find application in the generation of libraries of DNA and protein mutants from which species with improved or novel activities may be selected.
From cells to genes: how to make antibodies useful in human diagnosis and therapy
Monoclonal antibodies (mAb) have great potential value for in vivo diagnosis and therapy in humans. Their antigenic nature is however responsible for severe side effects and successful applications in the clinic have prove to be more limited than was originally hoped. This review summarize both cell biology and molecular biology approaches developed in order to overcome these limitations. Improving methodologies to immortalize cell lines producing human mAbs are reported with a particular attention to the techniques aimed at rescuing B cells expressing high-affinity human antibodies. A major part of this review is devoted to the protein engineering work. Genetic manipulation of mouse monoclonals to produce humanized antibodies and preparation of bacteriophage libraries displaying Ig repertoires are examined. The possibility to extend these approaches to the production of in vitro repertoires and to obviate the in vivo immunization step is also discussed. © 1993 Springer-Verlag.
Cardiac hypertrophy is inhibited by a local pool of cAMP regulated by phosphodiesterase 2
© 2015 American Heart Association, Inc. Rationale: Chronic elevation of 3'-5'-cyclic adenosine monophosphate (cAMP) levels has been associated with cardiac remodeling and cardiac hypertrophy. However, enhancement of particular aspects of cAMP/protein kinase A signaling seems to be beneficial for the failing heart. cAMP is a pleiotropic second messenger with the ability to generate multiple functional outcomes in response to different extracellular stimuli with strict fidelity, a feature that relies on the spatial segregation of the cAMP pathway components in signaling microdomains. Objective: How individual cAMP microdomains affect cardiac pathophysiology remains largely to be established. The cAMP-degrading enzymes phosphodiesterases (PDEs) play a key role in shaping local changes in cAMP. Here we investigated the effect of specific inhibition of selected PDEs on cardiac myocyte hypertrophic growth. Methods and Results: Using pharmacological and genetic manipulation of PDE activity, we found that the rise in cAMP resulting from inhibition of PDE3 and PDE4 induces hypertrophy, whereas increasing cAMP levels via PDE2 inhibition is antihypertrophic. By real-time imaging of cAMP levels in intact myocytes and selective displacement of protein kinase A isoforms, we demonstrate that the antihypertrophic effect of PDE2 inhibition involves the generation of a local pool of cAMP and activation of a protein kinase A type II subset, leading to phosphorylation of the nuclear factor of activated T cells. Conclusions: Different cAMP pools have opposing effects on cardiac myocyte cell size. PDE2 emerges as a novel key regulator of cardiac hypertrophy in vitro and in vivo, and its inhibition may have therapeutic applications.
Bifunctional Ligands for Inhibition of Tight-Binding Protein-Protein Interactions.
The acknowledged potential of small-molecule therapeutics targeting disease-related protein-protein interactions (PPIs) has promoted active research in this field. The strategy of using small molecule inhibitors (SMIs) to fight strong (tight-binding) PPIs tends to fall short due to the flat and wide interfaces of PPIs. Here we propose a biligand approach for disruption of strong PPIs. The potential of this approach was realized for disruption of the tight-binding (KD = 100 pM) tetrameric holoenzyme of cAMP-dependent protein kinase (PKA). Supported by X-ray analysis of cocrystals, bifunctional inhibitors (ARC-inhibitors) were constructed that simultaneously associated with both the ATP-pocket and the PPI interface area of the catalytic subunit of PKA (PKAc). Bifunctional inhibitor ARC-1411, possessing a KD value of 3 pM toward PKAc, induced the dissociation of the PKA holoenzyme with a low-nanomolar IC50, whereas the ATP-competitive inhibitor H89 bound to the PKA holoenzyme without disruption of the protein tetramer.
Modulation of Compartmentalised Cyclic Nucleotide Signalling via Local Inhibition of Phosphodiesterase Activity.
Cyclic nucleotide phosphodiesterases (PDEs) are the only enzymes that degrade the cyclic nucleotides cAMP and cGMP, and play a key role in modulating the amplitude and duration of the signal delivered by these two key intracellular second messengers. Defects in cyclic nucleotide signalling are known to be involved in several pathologies. As a consequence, PDEs have long been recognized as potential drug targets, and they have been the focus of intense research for the development of therapeutic agents. A number of PDE inhibitors are currently available for the treatment of disease, including obstructive pulmonary disease, erectile dysfunction, and heart failure. However, the performance of these drugs is not always satisfactory, due to a lack of PDE-isoform specificity and their consequent adverse side effects. Recent advances in our understanding of compartmentalised cyclic nucleotide signalling and the role of PDEs in local regulation of cAMP and cGMP signals offers the opportunity for the development of novel strategies for therapeutic intervention that may overcome the current limitation of conventional PDE inhibitors.
Cytoskeleton regulators CAPZA2 and INF2 associate with CFTR to control its plasma membrane levels under EPAC1 activation.
Cystic Fibrosis (CF), the most common lethal autosomic recessive disorder among Caucasians, is caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein, a cAMP-regulated chloride channel expressed at the apical surface of epithelial cells. Cyclic AMP regulates both CFTR channel gating through a protein kinase A (PKA)-dependent process and plasma membane (PM) stability through activation of the exchange protein directly activated by cAMP1 (EPAC1). This cAMP effector, when activated promotes the NHERF1:CFTR interaction leading to an increase in CFTR at the PM by decreasing its endocytosis. Here, we used protein interaction profiling and bioinformatic analysis to identify proteins that interact with CFTR under EPAC1 activation as possible regulators of this CFTR PM anchoring. We identified an enrichment in cytoskeleton related proteins among which we characterized CAPZA2 and INF2 as regulators of CFTR trafficking to the PM. We found that CAPZA2 promotes wt-CFTR trafficking under EPAC1 activation at the PM whereas reduction of INF2 levels leads to a similar trafficking promotion effect. These results suggest that CAPZA2 is a positive regulator and INF2 a negative one for the increase of CFTR at the PM after an increase of cAMP and concomitant EPAC1 activation. Identifying the specific interactions involving CFTR and elicited by EPAC1 activation provides novel insights into late CFTR trafficking, insertion and/or stabilization at the PM and highlighs new potential therapeutic targets to tackle CF disease.
Measuring spatiotemporal dynamics of cyclic AMP signaling in real-time using FRET-based biosensors
Cyclic AMP governs many fundamental signaling events in eukaryotic cells. Although cAMP signaling has been a major research focus for a long time, recent technological developments are revealing novel aspects of this paradigmatic pathway. In this chapter, we give an overview over current fluorescence resonance energy transfer (FRET)-based sensors for detection of cAMP dynamics, and their application in monitoring local, compartmentalized cAMP signals within living cells. A basic step-by-step protocol is given for conducting a FRET experiment in primary cells with a unimolecular cAMP sensor, which can easily be adapted to a user's specific requirements. © Springer Science+Business Media, LLC 2011.
Biochemical characterization and cellular imaging of a novel, membrane permeable fluorescent cAMP analog.
BACKGROUND: A novel fluorescent cAMP analog (8-[Pharos-575]- adenosine-3', 5'-cyclic monophosphate) was characterized with respect to its spectral properties, its ability to bind to and activate three main isoenzymes of the cAMP-dependent protein kinase (PKA-Ialpha, PKA-IIalpha, PKA-IIbeta) in vitro, its stability towards phosphodiesterase and its ability to permeate into cultured eukaryotic cells using resonance energy transfer based indicators, and conventional fluorescence imaging. RESULTS: The Pharos fluorophore is characterized by a Stokes shift of 42 nm with an absorption maximum at 575 nm and the emission peaking at 617 nm. The quantum yield is 30%. Incubation of the compound to RIIalpha and RIIbeta subunits increases the amplitude of excitation and absorption maxima significantly; no major change was observed with RIalpha. In vitro binding of the compound to RIalpha subunit and activation of the PKA-Ialpha holoenzyme was essentially equivalent to cAMP; RII subunits bound the fluorescent analog up to ten times less efficiently, resulting in about two times reduced apparent activation constants of the holoenzymes compared to cAMP. The cellular uptake of the fluorescent analog was investigated by cAMP indicators. It was estimated that about 7 muM of the fluorescent cAMP analog is available to the indicator after one hour of incubation and that about 600 muM of the compound had to be added to intact cells to half-maximally dissociate a PKA type IIalpha sensor. CONCLUSION: The novel analog combines good membrane permeability- comparable to 8-Br-cAMP - with superior spectral properties of a modern, red-shifted fluorophore. GFP-tagged regulatory subunits of PKA and the analog co-localized. Furthermore, it is a potent, PDE-resistant activator of PKA-I and -II, suitable for in vitro applications and spatial distribution evaluations in living cells.
Missense mutations in desmocollin-2 N-terminus, associated with arrhythmogenic right ventricular cardiomyopathy, affect intracellular localization of desmocollin-2 in vitro.
BACKGROUND: Mutations in genes encoding desmosomal proteins have been reported to cause arrhythmogenic right ventricular cardiomyopathy (ARVC), an autosomal dominant disease characterised by progressive myocardial atrophy with fibro-fatty replacement. We screened 54 ARVC probands for mutations in desmocollin-2 (DSC2), the only desmocollin isoform expressed in cardiac tissue. METHODS: Mutation screening was performed by denaturing high-performance liquid chromatography and direct sequencing. To evaluate the pathogenic potentials of the DSC2 mutations detected in patients affected with ARVC, full-length wild-type and mutated cDNAs were cloned in eukaryotic expression vectors to obtain a fusion protein with green fluorescence protein (GFP); constructs were transfected in neonatal rat cardiomyocytes and in HL-1 cells. RESULTS: We identified two heterozygous mutations (c.304G>A (p.E102K) and c.1034T>C (p.I345T)) in two probands and in four family members. The two mutations p.E102K and p.I345T map to the N-terminal region, relevant to adhesive interactions. In vitro functional studies demonstrated that, unlike wild-type DSC2, the two N-terminal mutants are predominantly localised in the cytoplasm. CONCLUSION: The two missense mutations in the N-terminal domain affect the normal localisation of DSC2, thus suggesting the potential pathogenic effect of the reported mutations. Identification of additional DSC2 mutations associated with ARVC may result in increased diagnostic accuracy with implications for genetic counseling.
Imaging the cAMP-dependent signal transduction pathway
In recent years, the development of new technologies based on the green fluorescent protein and FRET (fluorescence resonance energy transfer) has introduced a new perspective in the study of cAMP signalling. Real-time imaging of fluorescent biosensors is making it possible to visualize cAMP dynamics directly as they happen in intact, living cells, providing important and original insights for our understanding of the spatiotemporal organization of the cAMP/PKA (protein kinase A) signalling pathway. ©2005 Biochemical Society.
Restricted diffusion of a freely diffusible second messenger: Mechanisms underlying compartmentalized cAMP signalling
It is becoming increasingly evident that the freely diffusible second messenger cAMP can transduce specific responses by localized signalling. The machinery that underpins compartmentalized cAMP signalling is only now becoming appreciated. Adenylate cyclases, the enzymes that synthesize cAMP, are localized at discrete parts of the plasma membrane, and phosphodiesterases, the enzymes that degrade cAMP, can be targeted to selected subcellular compartments. A-kinase-anchoring proteins then serve to anchor PKA (protein kinase A) close to specific targets, resulting in selective activation. The specific activation of such individual subsets of PKA requires that cAMP is made available in discrete compartments. In this presentation, the molecular and structural mechanisms responsible for compartmentalized PKA signalling and restricted diffusion of cAMP will be discussed. ©2006 Biochemical Society.
β-adrenergic- and muscarinic receptor-induced changes in cAMP activity in adult cardiac myocytes detected with FRET-based biosensor
β-Adrenergic receptor activation regulates cardiac myocyte function through the stimulation of cAMP production and subsequent activation of protein kinase A (PKA). Furthermore, muscarinic receptor activation inhibits as well as facilitates these cAMP-dependent effects. However, it has not always been possible to correlate the muscarinic responses with the direct measurement of changes in cellular cAMP activity. Genetically encoded biosensors have recently been developed, making it possible to monitor real-time changes in cAMP and PKA activity at the single cell level. One such biosensor consists of the regulatory and catalytic subunits of PKA labeled with cyan and yellow fluorescent proteins, respectively. Changes in cAMP activity affecting the association of these labeled PKA subunits can be detected as changes in fluorescence resonance energy transfer. In the present study, an adenovirus-based approach was developed to express this recombinant protein complex in adult cardiac myocytes and use it to monitor changes in cAMP activity produced by β-adrenergic and muscarinic receptor activation. The biosensor expressed with the use of this system is able to detect changes in cAMP activity produced by physiologically relevant levels of β-adrenergic receptor activation without disrupting normal functional responses. It was also possible to directly demonstrate the complex temporal pattern of inhibitory and stimulatory changes in cAMP activity produced by muscarinic receptor activation in these cells. The adenovirus-based approach we have developed should facilitate the use of this biosensor in studying cAMP and PKA-dependent signaling mechanisms in a wide variety of cell types. Copyright © 2005 the American Physiological Society.