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

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

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

cAMP-Dependent Signaling Pathways as Potential Targets for Inhibition of Plasmodium falciparum Blood Stages

We review the role of signaling pathways in regulation of the key processes of merozoite egress and red blood cell invasion by Plasmodium falciparum and, in particular, the importance of the second messengers, cAMP and Ca2+, and cyclic nucleotide dependent kinases. cAMP-dependent protein kinase (PKA) is comprised of cAMP-binding regulatory, and catalytic subunits. The less well conserved cAMP-binding pockets should make cAMP analogs attractive drug leads, but this approach is compromised by the poor membrane permeability of cyclic nucleotides. We discuss how the conserved nature of ATP-binding pockets makes ATP analogs inherently prone to off-target effects and how ATP analogs and genetic manipulation can be useful research tools to examine this. We suggest that targeting PKA interaction partners as well as substrates, or developing inhibitors based on PKA interaction sites or phosphorylation sites in PKA substrates, may provide viable alternative approaches for the development of anti-malarial drugs. Proximity of PKA to a substrate is necessary for substrate phosphorylation, but the P. falciparum genome encodes few recognizable A-kinase anchor proteins (AKAPs), suggesting the importance of PKA-regulatory subunit myristylation and membrane association in determining substrate preference. We also discuss how Pf14-3-3 assembles a phosphorylation-dependent signaling complex that includes PKA and calcium dependent protein kinase 1 (CDPK1) and how this complex may be critical for merozoite invasion, and a target to block parasite growth. We compare altered phosphorylation levels in intracellular and egressed merozoites to identify potential PKA substrates. Finally, as host PKA may have a critical role in supporting intracellular parasite development, we discuss its role at other stages of the life cycle, as well as in other apicomplexan infections. Throughout our review we propose possible new directions for the therapeutic exploitation of cAMP-PKA-signaling in malaria and other diseases caused by apicomplexan parasites

A PM10 chemically characterised nation-wide dataset for Italy. Geographical influence on urban air pollution and source apportionment

: Urban textures of the Italian cities are peculiarly shaped by the local geography generating similarities among cities placed in different regions but comparable topographical districts. This suggested the following scientific question: can such different topographies generate significant differences on the PM10 chemical composition at Italian urban sites that share similar geography despite being in different regions? To investigate whether such communalities can be found and are applicable at Country-scale, we propose here a novel methodological approach. A dataset comprising season-averages of PM10 mass concentration and chemical composition data was built, covering the decade 2005-2016 and referring to urban sites only (21 cities). Statistical analyses, estimation of missing data, identification of latent clusters and source apportionment modelling by Positive Matrix Factorization (PMF) were performed on this unique dataset. The first original result is the demonstration that a dataset with atypical time resolution can be successfully exploited as an input matrix for PMF obtaining Country-scale representative chemical profiles, whose physical consistency has been assessed by different tests of modelling performance. Secondly, this dataset can be considered a reference repository of season averages of chemical species over the Italian territory and the chemical profiles obtained by PMF for urban Italian agglomerations could contribute to emission repositories. These findings indicate that our approach is powerful, and it could be further employed with datasets typically available in the air pollution monitoring networks

The Pseudomonas aeruginosa Chemotaxis Methyltransferase CheR1 Impacts on Bacterial Surface Sampling

The characterization of factors contributing to the formation and development of surface-associated bacterial communities known as biofilms has become an area of intense interest since biofilms have a major impact on human health, the environment and industry. Various studies have demonstrated that motility, including swimming, swarming and twitching, seems to play an important role in the surface colonization and establishment of structured biofilms. Thereby, the impact of chemotaxis on biofilm formation has been less intensively studied. Pseudomonas aeruginosa has a very complex chemosensory system with two Che systems implicated in flagella-mediated motility. In this study, we demonstrate that the chemotaxis protein CheR1 is a methyltransferase that binds S-adenosylmethionine and transfers a methyl group from this methyl donor to the chemoreceptor PctA, an activity which can be stimulated by the attractant serine but not by glutamine. We furthermore demonstrate that CheR1 does not only play a role in flagella-mediated chemotaxis but that its activity is essential for the formation and maintenance of bacterial biofilm structures. We propose a model in which motility and chemotaxis impact on initial attachment processes, dispersion and reattachment and increase the efficiency and frequency of surface sampling in P. aeruginosa

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Molekulare Mechanismen der cAMP-vermittelten Signaltransduktion

Deutsche Forschungsgemeinschaft (DFG): Projekt He 1818/4Die vorliegende Dissertation basiert zwar auf meiner eingereichten kumulativen Dissertation, allerdings ohne die Original Artikel, die bei dem jeweiligen Verlag/Journal (ggf. kostenpflichtig) eingesehen werden können. Die bis zum Zeitpunkt meiner Dissertation noch nicht akzeptierten Manuskripte Publikation V und VI sind jetzt bei BMC veröffentlicht und finden sich unter: -----> Publikation VI - Moll, D., Prinz, A., Brendel, C. M., Berrera, M., Guske, K., Zaccolo, M., Genieser, H. G. and Herberg, F. W., "Biochemical characterization and cellular imaging of a novel, membrane permeable fluorescent cAMP analog", BMC Biochem 9, 18 (2008). -----> Publikation V - Bertinetti, D., Schweinsberg, S., Hanke, S. E., Schwede, F., Bertinetti, O., Drewianka, S., Genieser, H. G. and Herberg, F. W., "Chemical tools selectively target components of the PKA system", BMC Chem Biol 9 (1), 3 (2009)

Crystal structures of the carboxyl cGMP binding domain of the Plasmodium falciparum cGMP-dependent protein kinase reveal a novel capping triad crucial for merozoite egress.

The Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is a key regulator across the malaria parasite life cycle. Little is known about PfPKG's activation mechanism. Here we report that the carboxyl cyclic nucleotide binding domain functions as a "gatekeeper" for activation by providing the highest cGMP affinity and selectivity. To understand the mechanism, we have solved its crystal structures with and without cGMP at 2.0 and 1.9 Å, respectively. These structures revealed a PfPKG-specific capping triad that forms upon cGMP binding, and disrupting the triad reduces kinase activity by 90%. Furthermore, mutating these residues in the parasite prevents blood stage merozoite egress, confirming the essential nature of the triad in the parasite. We propose a mechanism of activation where cGMP binding allosterically triggers the conformational change at the αC-helix, which bridges the regulatory and catalytic domains, causing the capping triad to form and stabilize the active conformation

Enzymkinetik in der Schule

Daniela Bertinetti wurde finanziell durch das DFG Projekt He1818-4 unterstütztDie hier vorliegende Fassung dieses Artikels entspricht der eingereichten Manuskriptversion mit den gewünschten Korrekturen des Verlages und kleinen Modifikationen zur besseren Lesbarkeit des Artikels. Es handelt sich NICHT um die gesetzte und vom Verlag bearbeitet Version