ABSTRACT
AIMS: The parasympathetic nervous system is thought to play a key role in atrial fibrillation (AF). Since parasympathetic signalling is primarily mediated by the heterotrimeric G-protein, Galpha(i)betagamma, we hypothesized that targeted inhibition of Galpha(i) interactions in the posterior left atrium (PLA) would modify the substrate for vagal AF. METHODS AND RESULTS: Cell-penetrating(cp)-Galpha(i)1/2 and cp-Galpha(i)3 C-terminal peptides were assessed for their ability to attenuate cholinergic-parasympathetic signalling in isolated feline atrial myocytes and in canine left atrium (LA). Confocal fluorescence microscopy indicated that cp-Galpha(i)1/2 and/or cp-Galpha(i)3 peptides moderated carbachol attenuation of cellular Ca(2+) transients in isolated atrial myocytes. High-density epicardial mapping of dog PLA, left atrial pulmonary veins (PVs), and left atrial appendage (LAA) indicated that the delivery of cp-Galpha(i)1/2 peptide or cp-Galpha(i)3 peptide into the PLA prolonged effective refractory periods at baseline and during vagal stimulation in the PLA and to varying extents also in the LAA and PV regions. After delivery of cp-Galpha(i) peptides into the PLA, AF inducibility during vagal stimulation was significantly diminished. CONCLUSION: These results demonstrate the feasibility of using specific G(i)-protein inhibition to achieve selective parasympathetic denervation in the PLA, with a resulting change in vagal responsiveness across the entire LA.
Subject(s)
Atrial Fibrillation/drug therapy , Cardiovascular Agents/pharmacology , GTP-Binding Protein alpha Subunits, Gi-Go/antagonists & inhibitors , Myocytes, Cardiac/drug effects , Parasympathectomy/methods , Peptides/pharmacology , Vagus Nerve/drug effects , Action Potentials , Animals , Atrial Fibrillation/metabolism , Atrial Fibrillation/physiopathology , Calcium Signaling/drug effects , Carbachol/pharmacology , Cats , Cholinergic Agonists/pharmacology , Cyclic AMP/metabolism , Dogs , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , GTP-Binding Protein beta Subunits/metabolism , GTP-Binding Protein gamma Subunits/metabolism , Heart Atria/drug effects , Heart Atria/innervation , Heart Atria/metabolism , Microscopy, Confocal , Myocytes, Cardiac/metabolism , Potassium/metabolism , Receptor, Muscarinic M2/metabolism , Refractory Period, Electrophysiological , Time Factors , Vagus Nerve/metabolism , Vagus Nerve/physiopathologyABSTRACT
Rad51 is the central catalyst of homologous recombination in eukaryotes and is thus critical for maintaining genomic integrity. Recent crystal structures of filaments formed by Rad51 and the closely related archeal RadA and eubacterial RecA proteins place the ATPase site at the protomeric interface. To test the relevance of this feature, we mutated conserved residues at this interface and examined their effects on key activities of Rad51: ssDNA-stimulated ATP hydrolysis, DNA binding, polymerization on DNA substrates and catalysis of strand-exchange reactions. Our results show that the interface seen in the crystal structures is very important for nucleoprotein filament formation. H352 and R357 of yeast Rad51 are essential for assembling the catalytically competent form of the enzyme on DNA substrates and coordinating its activities. However, contrary to some previous suggestions, neither of these residues is critical for ATP hydrolysis.
Subject(s)
Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Substitution , DNA, Single-Stranded/metabolism , Microscopy, Atomic Force , Mutagenesis, Site-Directed , Nucleotides/metabolism , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Rad51 Recombinase/chemistry , Saccharomyces cerevisiae Proteins/chemistryABSTRACT
Rad51, the major eukaryotic homologous recombinase, is important for the repair of DNA damage and the maintenance of genomic diversity and stability. The active form of this DNA-dependent ATPase is a helical filament within which the search for homology and strand exchange occurs. Here we present the crystal structure of a Saccharomyces cerevisiae Rad51 filament formed by a gain-of-function mutant. This filament has a longer pitch than that seen in crystals of Rad51's prokaryotic homolog RecA, and places the ATPase site directly at a new interface between protomers. Although the filament exhibits approximate six-fold symmetry, alternate protein-protein interfaces are slightly different, implying that the functional unit of Rad51 within the filament may be a dimer. Additionally, we show that mutation of His352, which lies at this new interface, markedly disrupts DNA binding.
Subject(s)
DNA-Binding Proteins/chemistry , Rec A Recombinases/chemistry , Adenosine Triphosphatases/chemistry , Adenosine Triphosphate/chemistry , Crystallography, X-Ray , DNA/chemistry , DNA/metabolism , DNA Damage , Histidine/chemistry , Hydrogen-Ion Concentration , Hydrolysis , Models, Molecular , Mutation , Phosphorylation , Protein Binding , Protein Conformation , Rad51 Recombinase , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins , Time Factors , Tyrosine/chemistryABSTRACT
Integration host factor (IHF) is a DNA-bending protein that recognizes its cognate sites through indirect readout. Previous studies have shown that binding of wild-type (WT)-IHF is disrupted by a T to A mutation at the center position of a conserved TTR motif in its binding site, and that substitution of betaGlu44 with Ala prevented IHF from discriminating between A and T at this position. We have determined the crystal structures and relative binding affinities for all combinations of WT-IHF and IHF-betaGlu44Ala bound to the WT and mutant DNAs. Comparison of these structures reveals that DNA twist plays a major role in DNA recognition by IHF, and that this geometric parameter is dependent on the dinucleotide step and not on the bound IHF variant.
Subject(s)
DNA, Bacterial/chemistry , DNA, Bacterial/metabolism , Integration Host Factors/chemistry , Integration Host Factors/metabolism , Amino Acid Substitution , Base Pairing/physiology , Binding Sites , Crystallography, X-Ray , Integration Host Factors/genetics , Models, Molecular , Mutagenesis , Protein ConformationABSTRACT
The fundamental processes by which proteins recognize and bind to nucleic acids are critical to understanding cellular function. To explore the factors involved in protein-DNA recognition, we used hydrostatic pressure to perturb the binding of the BamHI endonuclease to cognate DNA, both in experiment and in molecular dynamic simulations. A new technique of high-pressure gel mobility shift analysis was used to test the effects of elevated hydrostatic pressure on the binding of BamHI to its cognate recognition sequence. Upon application of a pressure of 500 bar, the equilibrium dissociation constant of BamHI binding to the cognate site was found to increase nearly 10-fold. A challenge has been to link this type of pure thermodynamic measurement to functional events occurring at the molecular level. Thus, we used molecular dynamic simulations at both ambient and elevated pressures to reveal details of the direct and water-mediated interactions between BamHI and cognate DNA, which allow explanation of the effects of pressure on site-specific protein-DNA binding and complex stability.
