RESUMO
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.
Assuntos
Cardiomegalia/prevenção & controle , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Miócitos Cardíacos/enzimologia , Sistemas do Segundo Mensageiro , Adenoviridae/genética , Animais , Animais Recém-Nascidos , Cardiomegalia/enzimologia , Cardiomegalia/genética , Cardiomegalia/patologia , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/antagonistas & inibidores , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/genética , Modelos Animais de Doenças , Vetores Genéticos , Masculino , Microdomínios da Membrana/enzimologia , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Fosforilação , Interferência de RNA , Ratos Sprague-Dawley , Ratos Wistar , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Fatores de Tempo , Transdução Genética , TransfecçãoRESUMO
Leucine-rich repeat kinase 2 (LRRK2) is a multidomain protein implicated in Parkinson disease (PD); however, the molecular mechanism and mode of action of this protein remain elusive. cAMP-dependent protein kinase (PKA), along with other kinases, has been suggested to be an upstream kinase regulating LRRK2 function. Using MS, we detected several sites phosphorylated by PKA, including phosphorylation sites within the Ras of complex proteins (ROC) GTPase domain as well as some previously described sites (S910 and S935). We systematically mapped those sites within LRRK2 and investigated their functional consequences. S1444 in the ROC domain was confirmed as a target for PKA phosphorylation using ROC single-domain constructs and through site-directed mutagenesis. Phosphorylation at S1444 is strikingly reduced in the major PD-related LRRK2 mutations R1441C/G/H, which are part of a consensus PKA recognition site ((1441)RASpS(1444)). Furthermore, our work establishes S1444 as a PKA-regulated 14-3-3 docking site. Experiments of direct binding to the three 14-3-3 isotypes gamma, theta, and zeta with phosphopeptides encompassing pS910, pS935, or pS1444 demonstrated the highest affinities to phospho-S1444. Strikingly, 14-3-3 binding to phospho-S1444 decreased LRRK2 kinase activity in vitro. Moreover, substitution of S1444 by alanine or by introducing the mutations R1441C/G/H, abrogating PKA phosphorylation and 14-3-3 binding, resulted in increased LRRK2 kinase activity. In conclusion, these data clearly demonstrate that LRRK2 kinase activity is modulated by PKA-mediated binding of 14-3-3 to S1444 and suggest that 14-3-3 interaction with LRRK2 is hampered in R1441C/G/H-mediated PD pathogenesis.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Mutação , Doença de Parkinson/metabolismo , Proteínas Serina-Treonina Quinases/genética , Alanina/química , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Espectrometria de Massas , Mutagênese Sítio-Dirigida , Fosforilação , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
We exploit the mechanical action of surface acoustic waves (SAW) to differentially lyse human cancer cells in a chemical-free manner. The extent to which cells were disrupted is reported for a range of SAW parameters, and we show that the presence of 10 µm polystyrene beads is required to fully rupture cells and their nuclei. We show that SAW is capable of subcellular fractionation through the chemical-free isolation of nuclei from whole cells. The concentration of protein was assessed in lysates with a sensitive microfluidic antibody capture (MAC) chip. An antibody-based sandwich assay in a microfluidic microarray format was used to detect unlabeled human tumor suppressor protein p53 in crude lysates, without any purification step, with single-molecule resolution. The results are digital, enabling sensitive quantification of proteins with a dynamic range >4 orders of magnitude. For the conditions used, the efficiency of SAW-induced mechanical lysis was determined to be 12.9% ± 0.7% of that for conventional detergent-based lysis in yielding detectable protein. A range of possible loss mechanisms that could lead to the drop in protein yield are discussed. Our results show that the methods described here are amenable to an integrated point-of-care device for the assessment of tumor protein expression in fine needle aspirate biopsies.
Assuntos
Fracionamento Celular/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Som , Proteína Supressora de Tumor p53/análise , Linhagem Celular Tumoral , Desenho de Equipamento , HumanosRESUMO
RATIONALE: cAMP and cGMP are intracellular second messengers involved in heart pathophysiology. cGMP can potentially affect cAMP signals via cGMP-regulated phosphodiesterases (PDEs). OBJECTIVE: To study the effect of cGMP signals on the local cAMP response to catecholamines in specific subcellular compartments. METHODS AND RESULTS: We used real-time FRET imaging of living rat ventriculocytes expressing targeted cAMP and cGMP biosensors to detect cyclic nucleotides levels in specific locales. We found that the compartmentalized, but not the global, cAMP response to isoproterenol is profoundly affected by cGMP signals. The effect of cGMP is to increase cAMP levels in the compartment where the protein kinase (PK)A-RI isoforms reside but to decrease cAMP in the compartment where the PKA-RII isoforms reside. These opposing effects are determined by the cGMP-regulated PDEs, namely PDE2 and PDE3, with the local activity of these PDEs being critically important. The cGMP-mediated modulation of cAMP also affects the phosphorylation of PKA targets and myocyte contractility. CONCLUSIONS: cGMP signals exert opposing effects on local cAMP levels via different PDEs the activity of which is exerted in spatially distinct subcellular domains. Inhibition of PDE2 selectively abolishes the negative effects of cGMP on cAMP and may have therapeutic potential.
Assuntos
Catecolaminas/fisiologia , AMP Cíclico/fisiologia , GMP Cíclico/fisiologia , Miócitos Cardíacos/metabolismo , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/biossíntese , Miócitos Cardíacos/citologia , Miócitos Cardíacos/enzimologia , RatosRESUMO
An expression-uncoupled tandem affinity purification assay is introduced which differs from the standard TAP assay by uncoupling the expression of the TAP-bait protein from the target cells. Here, the TAP-tagged bait protein is expressed in Escherichia coli and purified. The two concatenated purification steps of the classical TAP are performed after addition of the purified bait to brain tissue homogenates, cell and nuclear extracts. Without prior genetic manipulation of the target, upscaling, free choice of cell compartments and avoidance of expression triggered heat shock responses could be achieved in one go. By the strategy of separating bait expression from the prey protein environment numerous established, mostly tissue-specific binding partners of the protein kinase A catalytic subunit Cbeta1 were identified, including interactions in binary, ternary and quaternary complexes. In addition, the previously unknown small molecule inhibitor-dependent interaction of Cbeta1 with the cell cycle and apoptosis regulatory protein-1 was verified. The uncoupled tandem affinity purification procedure presented here expands the application range of the in vivo TAP assay and may serve as a simple strategy for identifying cell- and tissue-specific protein complexes.
Assuntos
Proteínas de Transporte/metabolismo , Clonagem Molecular/métodos , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteômica/métodos , Marcadores de Afinidade , Animais , Proteínas Reguladoras de Apoptose , Química Encefálica , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Linhagem Celular , Núcleo Celular/química , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Humanos , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos TestesRESUMO
Programmed degradation of mitochondria by mitophagy, an essential process to maintain mitochondrial homeostasis, is not completely understood. Here we uncover a regulatory process that controls mitophagy and involves the cAMP-degrading enzyme phosphodiesterase 2A2 (PDE2A2). We find that PDE2A2 is part of a mitochondrial signalosome at the mitochondrial inner membrane where it interacts with the mitochondrial contact site and organizing system (MICOS). As part of this compartmentalised signalling system PDE2A2 regulates PKA-mediated phosphorylation of the MICOS component MIC60, resulting in modulation of Parkin recruitment to the mitochondria and mitophagy. Inhibition of PDE2A2 is sufficient to regulate mitophagy in the absence of other triggers, highlighting the physiological relevance of PDE2A2 in this process. Pharmacological inhibition of PDE2 promotes a 'fat-burning' phenotype to retain thermogenic beige adipocytes, indicating that PDE2A2 may serve as a novel target with potential for developing therapies for metabolic disorders.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Mitocôndrias/metabolismo , Mitofagia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/genética , Imunofluorescência , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Mitocôndrias/genética , Proteínas Mitocondriais/metabolismo , Mitofagia/genética , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genéticaRESUMO
The primary goal of bioprocess cell line development is to obtain high product yields from robustly growing and well-defined clonal cell lines in timelines measured in weeks rather than months. Likewise, high-throughput screening of B cells and hybridomas is required for most cell line engineering workflows. A substantial bottleneck in these processes is detecting and isolating rare clonal cells with the required characteristics. Traditionally, this was achieved by the resource-intensive method of limiting dilution cloning, and more recently aided by semiautomated technologies such as cell sorting (e.g., fluorescence-activated cell sorting) and colony picking. In this paper we report on our novel Cyto-Mine Single Cell Analysis and Monoclonality Assurance System, which overcomes the limitations of current technologies by screening hundreds of thousands of individual cells for secreted target proteins, and then isolating and dispensing the highest producers into microtiter plate wells (MTP). The Cyto-Mine system performs this workflow using a fully integrated, microfluidic Cyto-Cartridge. Critically, all reagents and Cyto-Cartridges used are animal component-free (ACF) and sterile, thus allowing fast, robust, and safe isolation of desired cells.
Assuntos
Células Clonais/citologia , Ensaios de Triagem em Larga Escala/métodos , Análise de Célula Única/métodos , Software , Animais , Antígenos/metabolismo , Células CHO , Células Imobilizadas/citologia , Cricetulus , Citometria de Fluxo , Transferência Ressonante de Energia de Fluorescência , Humanos , Processamento de Imagem Assistida por Computador , Imunoglobulina G/metabolismo , CamundongosRESUMO
An Enterococcus faecalis mutant strain with a reduced ability for biofilm formation and primary attachment when compared to the high biofilm-forming wild-type strain was characterized by molecular biological and proteomic approaches. A point mutation in the srt-1 gene, which encodes a sortase-type enzyme and is part of the recently described bee (biofilm enhancer in Enterococcus) gene cluster, could be identified in the mutant strain. The Srt-1 deficiency resulted in a loss of the Bee-2 protein within a high molecular weight complex in cell surface protein extracts, as determined by mass spectrometry. These findings strongly suggest a specific linkage of Bee-2 to Bee-1 and Bee-3 within a complex by Srt-1. Furthermore, the identification of specific pilin motifs conserved in surface proteins of gram-positive bacteria indicated a possible involvement of the bee genes in the formation of pili structures, and may thus play a role in enhancing biofilm formation in Enterococcus faecalis.
Assuntos
Biofilmes/crescimento & desenvolvimento , Enterococcus faecalis/genética , Fímbrias Bacterianas/genética , Família Multigênica , Adulto , Aderência Bacteriana , Proteínas de Bactérias/análise , Enterococcus faecalis/química , Enterococcus faecalis/isolamento & purificação , Enterococcus faecalis/fisiologia , Feminino , Humanos , Mutação Puntual , Proteoma/análise , Vagina/microbiologiaRESUMO
With the completion of the major genome projects, one focus in biomedical research has shifted from the analysis of the rather static genome to the highly dynamic proteome. The sequencing of whole genomes did not lead to much anticipated insights into disease mechanisms; however, it paved the way for proteomics by providing the databases for protein identification by peptide mass fingerprints. The relative protein distribution within a cell or tissue is subject to change upon external and internal stimuli. Signal transduction events extend beyond a simple change in protein levels; rather they are governed by posttranslational modifications (PTMs), which provide a quick and efficient way to modulate cellular signals. Because most PTMs change the mass of a protein, they are amenable to analysis by mass spectrometry. Their investigation adds a level of functionality to proteomics, which can be expected to greatly aid in the understanding of the complex cellular machinery involved in signal transduction, metabolism, differentiation or in disease. This review provides an overview on posttranslational modifications exemplified on the model system cAMP-dependent protein kinase. Strategies for detection of selected PTMs are described and discussed in the context of protein kinase function.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Acilação , Sequência de Aminoácidos , Asparagina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/química , Modelos Moleculares , Dados de Sequência Molecular , Ácido Mirístico/metabolismo , Fosforilação , Espectrometria de Massas em TandemRESUMO
Subcellular localization of PKA (cAMP-dependent protein kinase or protein kinase A) is determined by protein-protein interactions between its R (regulatory) subunits and AKAPs (A-kinase-anchoring proteins). In the present paper, we report the development of the Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen) as a means to characterize AKAP-based peptide competitors of PKA anchoring. In this assay, the prototypic anchoring disruptor Ht31 efficiently competed in RIIalpha isoform binding with RII-specific and dual-specificity AKAPs (IC50 values of 1.4+/-0.2 nM and 6+/-1 nM respectively). In contrast, RIalpha isoform binding to a dual-specific AKAP was less efficiently competed (IC50 of 156+/-10 nM). Characterization of two RI-selective anchoring disruptors, RIAD (RI-anchoring disruptor) and PV-38 revealed that RIAD (IC50 of 13+/-1 nM) was 20-fold more potent than PV-38 (IC50 of 304+/-17 nM) and did not compete in the RIIalpha-AKAP interaction. We also observed that the kinetics of RII displacement from pre-formed PKA-AKAP complexes and competition of RII-AKAP complex formation by Ht31 differed by an order of magnitude when the component parts were mixed in vitro. No such difference in potency was seen for RIalpha-AKAP complexes. Thus the AlphaScreen assay may prove to be a valuable tool for detailed characterization of a variety of PKA-AKAP complexes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Biologia Molecular/métodos , Proteínas de Ancoragem à Quinase A , Proteínas Quinases Dependentes de AMP Cíclico/química , Estrutura Terciária de Proteína , Subunidades Proteicas , Proteínas/metabolismoRESUMO
cAMP-dependent protein kinase (PKA) plays a key role in intracellular signalling. cAMP antagonists, acting as suppressors of PKA activity by preventing PKA-holoenzyme dissociation, have received increasing attention because of their potential use in diagnostics as well as for therapeutic purposes. A large number of cAMP analogs have been described over the last three decades and methodology has been established to monitor cAMP agonists action by either following enzymatic activity or holoenzyme dissociation. This is not the case for cAMP antagonists, where only a few substances have been demonstrated to exhibit effects in the low micromolar range, for example, Rp-8-Br-cAMPS. A main drawback in the development of new compounds is the lack of technologies to assess antagonist action in an in vitro situation as well as in living cells. Here we quantify the effect of several cAMP analogs applying three different biochemical/biophysical assay setups and one in-cell assay. This includes two methods monitoring subunit dissociation in a test tube, namely AlphaScreen, a bead-based proximity assay, and surface plasmon resonance, determining the association and dissociation patterns of the two PKA subunits in real time in response to antagonists. BRET(2), performed in living cells in a 96-well format, allows testing for the efficacy of membrane-permeable cAMP analogs based on a genetically engineered cAMP sensor. Using novel and established experimental strategies side by side, the action of cAMP and cAMP analogs was tested on type Ialpha PKA holoenzyme, thus generating methodology to screen drug libraries for potential cAMP antagonists with high accuracy, reproducibility as well as potential for automation.
Assuntos
AMP Cíclico/antagonistas & inibidores , Animais , Células COS , Chlorocebus aethiops , AMP Cíclico/agonistas , Avaliação Pré-Clínica de Medicamentos/métodos , Ativação Enzimática/efeitos dos fármacos , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Camundongos , Espectrofotometria , Ressonância de Plasmônio de SuperfícieRESUMO
Direct optical detection provides an excellent means to investigate interactions of molecules in biological systems. The dynamic equilibria inherent to these systems can be described in greater detail by recording the kinetics of a biomolecular interaction. Optical biosensors allow direct detection of interaction patterns without the need for labeling. An overview covering several commercially available biosensors is given, with a focus on instruments based on surface plasmon resonance (SPR) and reflectometric interference spectroscopy (RIFS). Potential assay formats and experimental design, appropriate controls, and calibration procedures, especially when handling low molecular weight substances, are discussed. The single steps of an interaction analysis combined with practical tips for evaluation, data processing, and interpretation of kinetic data are described in detail. In a practical example, a step-by-step procedure for the analysis of a low molecular weight compound interaction with serum protein, determined on a commercial SPR sensor, is presented.
Assuntos
Técnicas Biossensoriais/métodos , Ligação Proteica , Ligação Competitiva , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/estatística & dados numéricos , Interpretação Estatística de Dados , Humanos , Técnicas In Vitro , Cinética , Ligantes , Peso Molecular , Óptica e Fotônica/instrumentação , Albumina Sérica/metabolismo , Análise Espectral/instrumentação , Análise Espectral/métodos , Ressonância de Plasmônio de Superfície/instrumentação , Ressonância de Plasmônio de Superfície/métodos , Varfarina/metabolismoRESUMO
Soft ionization methods for the introduction of labile biomolecules into a mass spectrometer are of fundamental importance to biomolecular analysis. Previously, electrospray ionization (ESI) and matrix assisted laser desorption-ionization (MALDI) have been the main ionization methods used. Surface acoustic wave nebulization (SAWN) is a new technique that has been demonstrated to deposit less energy into ions upon ion formation and transfer for detection than other methods for sample introduction into a mass spectrometer (MS). Here we report the optimization and use of SAWN as a nebulization technique for the introduction of samples from a low flow of liquid, and the interfacing of SAWN with liquid chromatographic separation (LC) for the analysis of a protein digest. This demonstrates that SAWN can be a viable, low-energy alternative to ESI for the LC-MS analysis of proteomic samples.
Assuntos
Produtos Biológicos/química , Cromatografia Líquida , Espectrometria de Massas , Proteômica , Humanos , Peptídeos/química , Proteínas/química , Proteômica/métodosRESUMO
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.