Chemical tools selectively target components of the PKA system

<p>Abstract</p> <p>Background</p> <p>In the eukaryotic cell the cAMP-dependent protein kinase (PKA) is a key enzyme in signal transduction and represents the main target of the second messenger cAMP. Here we describe the design, synthesis and characterisation of specifically tailored cAMP analogs which can be utilised as a tool for affinity enrichment and purification as well as for proteomics based analyses of cAMP binding proteins.</p> <p>Results</p> <p>Two sets of chemical binders were developed based on the phosphorothioate derivatives of cAMP, Sp-cAMPS and Rp-cAMPS acting as cAMP-agonists and -antagonists, respectively. These compounds were tested via direct surface plasmon resonance (SPR) analyses for their binding properties to PKA R-subunits and holoenzyme. Furthermore, these analogs were used in an affinity purification approach to analyse their binding and elution properties for the enrichment and improvement of cAMP binding proteins exemplified by the PKA R-subunits. As determined by SPR, all tested Sp-analogs provide valuable tools for affinity chromatography. However, Sp-8-AEA-cAMPS displayed (i) superior enrichment properties while maintaining low unspecific binding to other proteins in crude cell lysates, (ii) allowing mild elution conditions and (iii) providing the capability to efficiently purify all four isoforms of active PKA R-subunit in milligram quantities within 8 h. In a chemical proteomics approach both sets of binders, Rp- and Sp-cAMPS derivatives, can be employed. Whereas Sp-8-AEA-cAMPS preferentially binds free R-subunit, Rp-AHDAA-cAMPS, displaying antagonist properties, not only binds to the free PKA R-subunits but also to the intact PKA holoenzyme both from recombinant and endogenous sources.</p> <p>Conclusion</p> <p>In summary, all tested cAMP analogs were useful for their respective application as an affinity reagent which can enhance purification of cAMP binding proteins. Sp-8-AEA-cAMPS was considered the most efficient analog since Sp-8-AHA-cAMPS and Sp-2-AHA-cAMPS, demonstrated incomplete elution from the matrix, as well as retaining notable amounts of bound protein contaminants. Furthermore it could be demonstrated that an affinity resin based on Rp-8-AHDAA-cAMPS provides a valuable tool for chemical proteomics approaches.</p

DNA catabolites in triathletes: effects of supplementation with an aronia–citrus juice (polyphenols-rich juice)

In this study we analyzed whether our aronia–citrus juice (ACJ, the composition is based on a mixture of 95% citrus juice with 5% of Aronia melanocarpa juice), rich in polyphenols, and physical exercise had an effect on seven catabolites of DNA identified in plasma and on a urine isoprostane (8-iso-PGF2α). Sixteen elite triathletes on a controlled diet for triathlon training (45 days) were used in this clinical trial. Our results show a decrease in the 8-hydroxy-2′-deoxyguanosine concentration due to chronic physical exercise. The ACJ intake and physical exercise maintained the guanosine-3′,5′-cyclic monophosphate plasmatic concentrations and decreased the concentration of 8-hydroxyguanine as well as urinary values of 8-iso-PGF2α. Finally, we observed a significant increase in the 8-nitroguanosine levels in triathletes after ACJ intake, compared to the placebo stage. It is concluded that the combination of the intake of ACJ, rich in polyphenolic compounds, with adequate training was able to influence the plasmatic and urinary values of oxidative stress biomarkers. This suggests a positive effect on the oxidative damage and potential associations with DNA repair mechanisms.LAGF was awarded a pre-doctoral FPI fellowship (BES2012-060185) by the Spanish government. This study was supported by the project AGL2011-23690 (CICYT) (Spanish Ministry of Economy and Competitiveness). This work has been partially funded by the “Fundación Séneca de la Región de Murcia” Grupo de Excelencia 19900/GERM/15

Biochemical characterization and cellular imaging of a novel, membrane permeable fluorescent cAMP analog

&lt;p&gt;&lt;b&gt;Background&lt;/b&gt;&lt;/p&gt; &lt;p&gt;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-Iα, PKA-IIα, PKA-IIβ) 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.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Results&lt;/b&gt;&lt;/p&gt; &lt;p&gt;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 RIIα and RIIβ subunits increases the amplitude of excitation and absorption maxima significantly; no major change was observed with RIα. In vitro binding of the compound to RIα subunit and activation of the PKA-Iα 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 μM of the fluorescent cAMP analog is available to the indicator after one hour of incubation and that about 600 μM of the compound had to be added to intact cells to half-maximally dissociate a PKA type IIα sensor.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Conclusion&lt;/b&gt;&lt;/p&gt; &lt;p&gt;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.&lt;/p&gt

Cell-permeable Non-hydrolyzable cAMP Derivatives as Tools for Analysis of Signaling Pathways Controlling Gene Regulation in Dictyostelium

A novel class of cAMP derivatives were tested for binding to surface cAMP receptors (CAR), protein kinase A (PKA), and cAMP-phosphodiesterase (PDE) and for induction of three classes of cAMP regulated genes in Dictyostelium discoideum. These derivatives carry sulfur substitutions for either the axial (Sp) or equatorial (Rp) exocyclic oxygen atoms, while further modifications were introduced to provide specificity for binding to either CAR or PKA, and/or to increase lipophilicity and render the derivatives membrane-permeable. All derivatives bind weakly to PDE and are almost not degraded during incubation with Dictyostelium cells. One cAMP derivative, 6-thioethylpurineriboside 3',5‘-monophosphorothioate, Sp-isomer (Sp-6SEtcPuMPS), fulfills the criteria for selective activation of PKA in vivo. The compound enters Dictyostelium cells and reaches an intracellular concentration of 1 µM, sufficient to activate PKA, at an extracellular concentration of 30 µM, which is insufficient to activate CAR. Expression of cAMP-regulated prespore and prestalk genes and the aggregative PDE gene are effectively induced by CAR agonists and very poorly by PKA agonists. Even Sp-6SEtcPuMPS is ineffective to induce gene expression. These data not only indicate that surface cAMP receptors are the first targets for cAMP-induced gene expression, but argue against direct induction of expression of these genes by cAMP-induced PKA activation

Щодо утворення сімейств атомарних радіальних базисних функцій

Наведено схему побудови сімейств атомарних радіальних базисних функцій, які є нескінченно диференційовними фінітними розв'язками функціонально-диференціальних рівнянь, породжених операторами Лапласа та Гельмгольца.The scheme of building a family of atomic radial basis functions which are infinitely differentiable finite solutions of the functional-differential equations containing the Laplace and Helmholtz operators is introduced

Nucleoside analogue activators of cyclic AMP-independent protein kinase A of Trypanosoma

Protein kinase A (PKA), the main effector of cAMP in eukaryotes, is a paradigm for the mechanisms of ligand-dependent and allosteric regulation in signalling. Here we report the orthologous but cAMP-independent PKA of the protozoan Trypanosoma and identify 7-deazanucleosides as potent activators (EC50 >= 6.5 nM) and high affinity ligands (K-D >= 8 nM). A co-crystal structure of trypanosome PKA with 7-cyano-7-deazainosine and molecular docking show how substitution of key amino acids in both CNB domains of the regulatory subunit and its unique C-terminal alpha D helix account for this ligand swap between trypanosome PKA and canonical cAMP-dependent PKAs. We propose nucleoside-related endogenous activators of Trypanosoma brucei PKA (TbPKA). The existence of eukaryotic CNB domains not associated with binding of cyclic nucleotides suggests that orphan CNB domains in other eukaryotes may bind undiscovered signalling molecules. Phosphoproteome analysis validates 7-cyano-7-deazainosine as powerful cell-permeable inducer to explore cAMP-independent PKA signalling in medically important neglected pathogens

Quantification of cAMP and cGMP analogs in intact cells: pitfalls in enzyme immunoassays for cyclic nucleotides

Immunoassays are routinely used as research tools to measure intracellular cAMP and cGMP concentrations. Ideally, this application requires antibodies with high sensitivity and specificity. The present work evaluates the cross-reactivity of commercially available cyclic nucleotide analogs with two non-radioactive and one radioactive cAMP and cGMP immunoassay. Most of the tested cyclic nucleotide analogs showed low degree competition with the antibodies; however, with Rp-cAMPS, 8-Br-cGMP and 8-pCPT-cGMP, a strong cross-reactivity with the corresponding cAMP and cGMP, respectively, immunoassays was observed. The determined EIA-binding constants enabled the measurement of the intracellular cyclic nucleotide concentrations and revealed a time- and lipophilicity-dependent cell membrane permeability of the compounds in the range of 10–30% of the extracellular applied concentration, thus allowing a more accurate prediction of the intracellular analog levels in a given experiment