BIOLOG Life Science Institute

Bremen, Germany

BIOLOG Life Science Institute

Bremen, Germany

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Kusch J.,Friedrich - Schiller University of Jena | Thon S.,Friedrich - Schiller University of Jena | Schulz E.,Schmalkalden University of Applied Sciences | Biskup C.,Friedrich - Schiller University of Jena | And 4 more authors.
Nature Chemical Biology | Year: 2012

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetrameric membrane proteins that generate electrical rhythmicity in specialized neurons and cardiomyocytes. The channels are primarily activated by voltage but are receptors as well, binding the intracellular ligand cyclic AMP. The molecular mechanism of channel activation is still unknown. Here we analyze the complex activation mechanism of homotetrameric HCN2 channels by confocal patch-clamp fluorometry and kinetically quantify all ligand binding steps and closed-open isomerizations of the intermediate states. For the binding affinity of the second, third and fourth ligand, our results suggest pronounced cooperativity in the sequence positive, negative and positive, respectively. This complex interaction of the subunits leads to a preferential stabilization of states with zero, two or four ligands and suggests a dimeric organization of the activation process: within the dimers the cooperativity is positive, whereas it is negative between the dimers. © 2012 Nature America, Inc. All rights reserved.

Nache V.,Friedrich - Schiller University of Jena | Zimmer T.,Friedrich - Schiller University of Jena | Wongsamitkul N.,Friedrich - Schiller University of Jena | Schmauder R.,Friedrich - Schiller University of Jena | And 5 more authors.
Science Signaling | Year: 2012

Olfactory cyclic nucleotide-gated (CNG) ion channels are essential contributors to signal transduction of olfactory sensory neurons. The activity of the channels is controlled by the cyclic nucleotides guanosine 3′,5′-monophosphate (cGMP) and adenosine 3′,5′- monophosphate (cAMP). The olfactory CNG channels are composed of two CNGA2 subunits, one CNGA4 and one CNGB1b subunit, each containing a cyclic nucleotide-binding domain. Using patch-clamp fluorometry, we measured ligand binding and channel activation simultaneously and showed that cGMP activated olfactory CNG channels not only by binding to the two CNGA2 subunits but also by binding to the CNGA4 subunit. In a channel in which the CNGA2 subunits were compromised for ligand binding, cGMP binding to CNGA4 was sufficient to partly activate the channel. In contrast, in heterotetrameric channels, the CNGB1b subunit did not bind cGMP, but channels with this subunit showed activation by cAMP. Thus, the modulatory subunits participate actively in translating ligand binding to activation of heterotetrameric olfactory CNG channels and enable the channels to differentiate between cyclic nucleotides.

Wolter S.,Hannover Medical School | Dove S.,University of Regensburg | Golombek M.,Hannover Medical School | Schwede F.,Biolog Life Science Institute | Seifert R.,Hannover Medical School
Naunyn-Schmiedeberg's Archives of Pharmacology | Year: 2014

There is increasing evidence for a role of cytidine 3′,5′-cyclic monophosphate (cCMP) as second messenger. In a recent study, we showed that cCMP activates both purified guanosine 3′,5′-cyclic monophosphate (cGMP)-dependent protein kinase Iα (PKG Iα) and adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) isoenzymes with the regulatory subunits RIα and RIIα. Moreover, the membrane-permeant cCMP analog dibutyryl (DB)-cCMP induces effective vasodilation and inhibition of platelet aggregation via PKG Iα, but not via PKA. These data prompted us to conduct a systematic analysis of the effects of cyclic nucleotide (cNMP) analogs on purified PKG Iα and PKA RIα and RIIα We also studied the effect of DB-cCMP on PKA-dependent phosphorylation of the transcription factor cAMP response-binding protein (CREB) in S49 wild-type lymphoma cells and S49 kin- cells, devoid of the catalytic subunit of PKA. The major cellular metabolite of the prodrug DB-cCMP, N4-monobutyryl (4-MB)-cCMP, was a partial and low-potency activator of purified PKG Iα and a full and moderate-potency activator of PKA RIα and RIIα. Sp-cCMPS and Sp-cAMPS activated PKA RIα and RIIα with much higher potency and efficacy than PKG Iα. Molecular modeling suggested that the cytidine ring interacts with PKG Iα mainly via hydrophobic interactions, while the butyryl group projects away from the kinase. In contrast to DB-cAMP, DB-cCMP did not induce PKA-dependent phosphorylation in intact cells. Taken together, our data show that N4-monobutyryl-cCMP (4-MB-cCMP) activates PKA RIα and PKA RIIα more potently and with higher efficacy than PKG Iα in vitro but not in vivo. cNMP phosphorothioates constitute a starting point for the development of PKA activators with high selectivity relative to PKG. © 2014 Springer-Verlag Berlin Heidelberg.

Brunskole Hummel I.,Hannover Medical School | Brunskole Hummel I.,University of Regensburg | Reinartz M.T.,Hannover Medical School | Kalble S.,Hannover Medical School | And 4 more authors.
PLoS ONE | Year: 2013

In neutrophils, activation of the β2-adrenergic receptor (β2AR), a Gs-coupled receptor, inhibits inflammatory responses, which could be therapeutically exploited. The aim of this study was to evaluate the effects of various β2AR ligands on adenosine-3′,5′-cyclic monophosphate (cAMP) accumulation and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced superoxide anion (O2 •-) production in human neutrophils and to probe the concept of ligand-specific receptor conformations (also referred to as functional selectivity or biased signaling) in a native cell system. This is an important question because so far, evidence for functional selectivity has been predominantly obtained with recombinant systems, due to the inherent difficulties to genetically manipulate human native cells. cAMP concentration was determined by HPLC/tandem mass spectrometry, and O2 •- formation was assessed by superoxide dismutase-inhibitable reduction of ferricytochrome c. β2AR agonists were generally more potent in inhibiting fMLP-induced O2 •- production than in stimulating cAMP accumulation. (-)-Ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the cAMP assay, but partially inhibited fMLP-induced O2 •- production. Moreover, (-)-adrenaline was equi-efficacious in both assays whereas the efficacy of salbutamol was more than two-fold higher in the O2 •- assay. Functional selectivity was visualized by deviations of ligand potencies and efficacies from linear correlations for various parameters. We obtained no evidence for involvement of protein kinase A in the inhibition of fMLP-induced O2 •- production after β2AR-stimulation although cAMP-increasing substances inhibited O2 •- production. Taken together, our data corroborate the concept of ligand-specific receptor conformations with unique signaling capabilities in native human cells and suggest that the β2AR inhibits O2 •- production in a cAMP-independent manner. © 2013 Brunskole Hummel et al.

Hanke S.E.,University of Kassel | Bertinetti D.,University of Kassel | Badel A.,University of Kassel | Schweinsberg S.,University of Kassel | And 2 more authors.
New Biotechnology | Year: 2011

cAMP (Adenosine-3',5'-cyclic monophosphate) is a general second messenger controlling distinct targets in eukaryotic cells. In a (sub)proteomic approach, two classes of phosphorothioate cAMP affinity tools were used to isolate and to identify signalling complexes of the main cAMP target, cAMP dependent protein kinase (PKA). Agonist analogues (here: Sp-cAMPS) bind to the regulatory subunits of PKA (PKA-R), together with their interaction partners, and cause dissociation of a holoenzyme complex comprising PKA-R and catalytic subunits of PKA (PKA-C). Antagonist analogues (here: Rp-cAMPS) bind to the holoenzyme without dissociating the complex and were developed to identify interaction partners that bind to the entire complex or to PKA-C. More than 80 different proteins were isolated from tissue extracts including several PKA isoforms and known as well as potentially new interaction partners. Nevertheless, unspecific binding of general nucleotide binding proteins limited the outcome of this chemical proteomics approach. Surface plasmon resonance (SPR) was employed to optimise the entire workflow of pull down proteomics and to quantify the effects of different nucleotides (ATP, ADP, GTP and NADH) on PKA-R binding to affinity material. We could demonstrate that the addition of NADH to lysates improved specificity in pull down experiments. Using a combination of SPR studies and pull down experiments it was shown unambiguously that it is possible to specifically elute protein complexes with cAMP or cGMP from cAMPS analogue matrices. The side-by-side analysis of the PKA-R interactome and the holoenzyme complexed with interacting proteins will contribute to a further dissection of the multifaceted PKA signalling network. © 2010 Elsevier B.V.

Borner S.,University of Würzburg | Schwede F.,BIOLOG Life Science Institute | Schlipp A.,University of Würzburg | Berisha F.,University of Würzburg | And 4 more authors.
Nature Protocols | Year: 2011

Real-time measurements of second messengers in living cells, such as cAMP, are usually performed by ratiometric fluorescence resonance energy transfer (FRET) imaging. However, correct calibration of FRET ratios, accurate calculations of absolute cAMP levels and actual permeabilities of different cAMP analogs have been challenging. Here we present a protocol that allows precise measurements of cAMP concentrations and kinetics by expressing FRET-based cAMP sensors in cells and modulating them with an inhibitor of adenylyl cyclase activity and a cell-permeable cAMP analog that fully inhibits and activates the sensors, respectively. Using this protocol, we observed different basal cAMP levels in primary mouse cardiomyocytes, thyroid cells and in 293A cells. The protocol can be generally applied for calibration of second messenger or metabolite concentrations measured by FRET, and for studying kinetics and pharmacological properties of their membrane-permeable analogs. The complete procedure, including cell preparation and FRET measurements, takes 3-6 d. © 2011 Nature America, Inc. All rights reserved.

Stokman G.,Leiden University | Qin Y.,Leiden University | Genieser H.-G.,Biolog Life Science Institute | Schwede F.,Biolog Life Science Institute | And 5 more authors.
Journal of the American Society of Nephrology | Year: 2011

Renal ischemia-reperfusion injury is associated with the loss of tubular epithelial cell-cell and cell-matrix interactions which contribute to renal failure. The Epac-Rap signaling pathway is a potent regulator of cell-cell and cell-matrix adhesion. The cyclic AMP analogue 8-pCPT-2′-O-Me-cAMP has been shown to selectively activate Epac, whereas the addition of an acetoxymethyl (AM) ester to 8-pCPT-2′-O-MecAMP enhanced in vitro cellular uptake. Here we demonstrate that pharmacological activation of Epac-Rap signaling using acetoxymethyl-8-pCPT-2′-O-Me-cAMP preserves cell adhesions during hypoxia in vitro, maintaining the barrier function of the epithelial monolayer. Intrarenal administration in vivo of 8-pCPT-2′-O-Me-cAMP also reduced renal failure in a mouse model for ischemia-reperfusion injury. This was accompanied by decreased expression of the tubular cell stress marker clusterin-α, and lateral expression of β-catenin after ischemia indicative of sustained tubular barrier function. Our study emphasizes the undervalued importance of maintaining tubular epithelial cell adhesion in renal ischemia and demonstrates the potential of pharmacological modulation of cell adhesion as a new therapeutic strategy to reduce the extent of injury in kidney disease and transplantation. Copyright © 2011 by the American Society of Nephrology.

Leech C.A.,New York University | Dzhura I.,New York University | Chepurny O.G.,New York University | Kang G.,New York University | And 3 more authors.
Progress in Biophysics and Molecular Biology | Year: 2011

Insulin secretion from pancreatic β cells is stimulated by glucagon-like peptide-1 (GLP-1), a blood glucose-lowering hormone that is released from enteroendocrine L cells of the distal intestine after the ingestion of a meal. GLP-1 mimetics (e.g., Byetta) and GLP-1 analogs (e.g., Victoza) activate the β cell GLP-1 receptor (GLP-1R), and these compounds stimulate insulin secretion while also lowering levels of blood glucose in patients diagnosed with type 2 diabetes mellitus (T2DM). An additional option for the treatment of T2DM involves the administration of dipeptidyl peptidase-IV (DPP-IV) inhibitors (e.g., Januvia, Galvus). These compounds slow metabolic degradation of intestinally released GLP-1, thereby raising post-prandial levels of circulating GLP-1 substantially. Investigational compounds that stimulate GLP-1 secretion also exist, and in this regard a noteworthy advance is the demonstration that small molecule GPR119 agonists (e.g., AR231453) stimulate L cell GLP-1 secretion while also directly stimulating β cell insulin release. In this review, we summarize what is currently known concerning the signal transduction properties of the β cell GLP-1R as they relate to insulin secretion. Emphasized are the cyclic AMP, protein kinase A, and Epac2-mediated actions of GLP-1 to regulate ATP-sensitive K + channels, voltage-dependent K + channels, TRPM2 cation channels, intracellular Ca 2+ release channels, and Ca 2+-dependent exocytosis. We also discuss new evidence that provides a conceptual framework with which to understand why GLP-1R agonists are less likely to induce hypoglycemia when they are administered for the treatment of T2DM. © 2011 Elsevier Ltd.

Chepurny O.G.,New York University | Kelley G.G.,New York University | Dzhura I.,New York University | Leech C.A.,New York University | And 6 more authors.
American Journal of Physiology - Endocrinology and Metabolism | Year: 2010

Potential insulin secretagogue properties of an acetoxymethyl ester of a cAMP analog (8-pCPT-2′-O-Me-cAMP-AM) that activates the guanine nucleotide exchange factors Epac1 and Epac2 were assessed using isolated human islets of Langerhans. RT-QPCR demonstrated that the predominant variant of Epac expressed in human islets was Epac2, although Epac1 was detectable. Under conditions of islet perifusion, 8-pCPT-2′-O-Me-cAMP-AM (10 μM) potentiated first-and second-phase 10 mM glucose-stimulated insulin secretion (GSIS) while failing to influence insulin secretion measured in the presence of 3 mM glucose. The insulin secretagogue action of 8-pCPT-2′-O-MecAMP-AM was associated with depolarization and an increase of [Ca2+]i that reflected both Ca2+ influx and intracellular Ca2+ mobilization in islet β-cells. As expected for an Epac-selective cAMP analog, 8-pCPT-2′-O-Me-cAMP-AM (10 μM) failed to stimulate phosphorylation of PKA substrates CREB and Kemptide in human islets. Furthermore, 8-pCPT-2′-O-Me-cAMP-AM (10 μM) had no significant ability to activate AKAR3, a PKA-regulated biosensor expressed in human islet cells by viral transduction. Unexpectedly, treatment of human islets with an inhibitor of PKA activity (H-89) or treatment with a cAMP antagonist that blocks PKA activation (Rp-8-CPT-cAMPS) nearly abolished the action of 8-pCPT-2′-O-MecAMP-AM to potentiate GSIS. It is concluded that there exists a permissive role for PKA activity in support of human islet insulin secretion that is both glucose dependent and Epac regulated. This permissive action of PKA may be operative at the insulin secretory granule recruitment, priming, and/or postpriming steps of Ca2+-dependent exocytosis. Copyright © 2010 American Physiological Society.

Reinecke D.,Hannover Medical School | Schwede F.,Biolog Life Science Institute | Genieser H.-G.,Biolog Life Science Institute | Seifert R.,Hannover Medical School
PLoS ONE | Year: 2013

As second messengers, the cyclic purine nucleotides adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP) play an essential role in intracellular signaling. Recent data suggest that the cyclic pyrimidine nucleotides cytidine 3′,5′-cyclic monophosphate (cCMP) and uridine 3′,5′-cyclic monophosphate (cUMP) also act as second messengers. Hydrolysis by phosphodiesterases (PDEs) is the most important degradation mechanism for cAMP and cGMP. Elimination of cUMP and cCMP is not completely understood, though. We have shown that human PDEs hydrolyze not only cAMP and cGMP but also cyclic pyrimidine nucleotides, indicating that these enzymes may be important for termination of cCMP- and cUMP effects as well. However, these findings were acquired using a rather expensive HPLC/mass spectrometry assay, the technical requirements of which are available only to few laboratories. N'-Methylanthraniloyl-(MANT-)labeled nucleotides are endogenously fluorescent and suitable tools to study diverse protein/nucleotide interactions. In the present study, we report the synthesis of new MANT-substituted cyclic purine- and pyrimidine nucleotides that are appropriate to analyze substrate specificity and kinetics of PDEs with more moderate technical requirements. MANT-labeled nucleoside 3′,5′-cyclic monophosphates (MANT-cNMPs) are shown to be substrates of various human PDEs and to undergo a significant change in fluorescence upon cleavage, thus allowing direct, quantitative and continuous determination of hydrolysis via fluorescence detection. As substrates of several PDEs, MANT-cNMPs show similar kinetics to native nucleotides, with some exceptions. Finally, they are shown to be also appropriate tools for PDE inhibitor studies. © 2013 Reinecke et al.

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