High-affinity AKAP7delta-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides

PKA (protein kinase A) is tethered to subcellular compartments by direct interaction of its regulatory subunits (RI or RII) with AKAPs (A kinase-anchoring proteins). AKAPs preferentially bind RII subunits via their RII-binding domains. RII-binding domains form structurally conserved amphipathic helices with unrelated sequences. Their binding affinities for RII subunits differ greatly within the AKAP family. Amongst the AKAPs that bind RIIalpha subunits with high affinity is AKAP7delta [AKAP18delta; K(d) (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7delta mutant binds RIIalpha subunits with higher affinity than the full-length protein presumably due to loss of an inhibitory region [Henn, Edemir, Stefan, Wiesner, Lorenz, Theilig, Schmidtt, Vossebein, Tamma, Beyermann et al. (2004) J. Biol. Chem. 279, 26654-26665]. In the present study, we demonstrate that peptides (25 amino acid residues) derived from the RII-binding domain of AKAP7delta bind RIIalpha subunits with higher affinity (K(d)=0.4+/-0.3 nM) than either full-length or N-terminally truncated AKAP7delta, or peptides derived from other RII binding domains. The AKAP7delta-derived peptides and stearate-coupled membrane-permeable mutants effectively disrupt AKAP-RII subunit interactions in vitro and in cell-based assays. Thus they are valuable novel tools for studying anchored PKA signalling. Molecular modelling indicated that the high affinity binding of the amphipathic helix, which forms the RII-binding domain of AKAP7delta, with RII subunits involves both the hydrophobic and the hydrophilic faces of the helix. Alanine scanning (25 amino acid peptides, SPOT technology, combined with RII overlay assays) of the RII binding domain revealed that hydrophobic amino acid residues form the backbone of the interaction and that hydrogen bond- and salt-bridge-forming amino acid residues increase the affinity of the interaction.     

Hypothermic reconditioning by gaseous oxygen persufflation after cold storage of porcine kidneys

BackgroundDelayed graft function still represents a major complication in clinical kidney transplantation. Here we tested the possibility to improve functional outcome of cold stored kidneys a posteriori by hypothermic reconditioning using retrograde oxygen persufflation (ROP) immediately prior to reperfusion.MethodsKidneys from female German Landrace pigs were flushed with Histidine–Tryptophan–Ketoglutarate (HTK) solution and cold-stored for 18 h (control).Some grafts were subsequently subjected to 90 min of retrograde oxygen persufflation (ROP) via the renal vein during cold preservation. Early graft function of all kidneys was assessed thereafter by warm reperfusion in vitro (n = 6, resp.).ResultsRenal function upon reperfusion was significantly enhanced by ROP with an approximately twofold increase in renal clearances of creatinine and urea. ROP also led to higher renal vascular flow rates, enhanced urine output and mitigated histological alterations.ConclusionIt is concluded that initial graft function can be improved by 90 min of hypothermic gaseous oxygenation after arrival of the preserved organ in the transplantation clinic.     

Mapping genetic variants associated with Beta-adrenergic responses in inbred mice

β-blockers and β-agonists are primarily used to treat cardiovascular diseases. Inter-individual variability in response to both drug classes is well recognized, yet the identity and relative contribution of the genetic players involved are poorly understood. This work is the first genome-wide association study (GWAS) addressing the values and susceptibility of cardiovascular-related traits to a selective β(1)-blocker, Atenolol (ate), and a β-agonist, Isoproterenol (iso). The phenotypic dataset consisted of 27 highly heritable traits, each measured across 22 inbred mouse strains and four pharmacological conditions. The genotypic panel comprised 79922 informative SNPs of the mouse HapMap resource. Associations were mapped by Efficient Mixed Model Association (EMMA), a method that corrects for the population structure and genetic relatedness of the various strains. A total of 205 separate genome-wide scans were analyzed. The most significant hits include three candidate loci related to cardiac and body weight, three loci for electrocardiographic (ECG) values, two loci for the susceptibility of atrial weight index to iso, four loci for the susceptibility of systolic blood pressure (SBP) to perturbations of the β-adrenergic system, and one locus for the responsiveness of QTc (p<10(-8)). An additional 60 loci were suggestive for one or the other of the 27 traits, while 46 others were suggestive for one or the other drug effects (p<10(-6)). Most hits tagged unexpected regions, yet at least two loci for the susceptibility of SBP to β-adrenergic drugs pointed at members of the hypothalamic-pituitary-thyroid axis. Loci for cardiac-related traits were preferentially enriched in genes expressed in the heart, while 23% of the testable loci were replicated with datasets of the Mouse Phenome Database (MPD). Altogether these data and validation tests indicate that the mapped loci are relevant to the traits and responses studied.     

Targeting antibody responses to the membrane proximal external region of the envelope glycoprotein of human immunodeficiency virus

Although human immunodeficiency type 1 (HIV-1) infection induces strong antibody responses to the viral envelope glycoprotein (Env) only a few of these antibodies possess the capacity to neutralize a broad range of strains. The induction of such antibodies represents an important goal in the development of a preventive vaccine against the infection. Among the broadly neutralizing monoclonal antibodies discovered so far, three (2F5, Z13 and 4E10) target the short and hidden membrane proximal external region (MPER) of the gp41 transmembrane protein. Antibody responses to MPER are rarely observed in HIV-infected individuals or after immunization with Env immunogens. To initiate antibody responses to MPER in its membrane-embedded native conformation, we generated expression plasmids encoding the membrane-anchored ectodomain of gp41 with N-terminal deletions of various sizes. Following transfection of these plasmids, the MPER domains are displayed on the cell surface and incorporated into HIV virus like particles (VLP). Transfected cells displaying MPER mutants bound as efficiently to both 2F5 and 4E10 as cells transfected with a plasmid encoding full-length Env. Mice immunized with VLPs containing the MPER mutants produced MPER-specific antibodies, the levels of which could be increased by the trimerization of the displayed proteins as well as by a DNA prime-VLP boost immunization strategy. Although 2F5 competed for binding to MPER with antibodies in sera of some of the immunized mice, neutralizing activity could not be detected. Whether this is due to inefficient binding of the induced antibodies to MPER in the context of wild type Env or whether the overall MPER-specific antibody response induced by the MPER display mutants is too low to reveal neutralizing activity, remains to be determined.     

From dynamic expression patterns to boundary formation in the presomitic mesoderm

The segmentation of the vertebrate body is laid down during early embryogenesis. The formation of signaling gradients, the periodic expression of genes of the Notch-, Fgf- and Wnt-pathways and their interplay in the unsegmented presomitic mesoderm (PSM) precedes the rhythmic budding of nascent somites at its anterior end, which later develops into epithelialized structures, the somites. Although many in silico models describing partial aspects of somitogenesis already exist, simulations of a complete causal chain from gene expression in the growth zone via the interaction of multiple cells to segmentation are rare. Here, we present an enhanced gene regulatory network (GRN) for mice in a simulation program that models the growing PSM by many virtual cells and integrates WNT3A and FGF8 gradient formation, periodic gene expression and Delta/Notch signaling. Assuming Hes7 as core of the somitogenesis clock and LFNG as modulator, we postulate a negative feedback of HES7 on Dll1 leading to an oscillating Dll1 expression as seen in vivo. Furthermore, we are able to simulate the experimentally observed wave of activated NOTCH (NICD) as a result of the interactions in the GRN. We esteem our model as robust for a wide range of parameter values with the Hes7 mRNA and protein decays exerting a strong influence on the core oscillator. Moreover, our model predicts interference between Hes1 and HES7 oscillators when their intrinsic frequencies differ. In conclusion, we have built a comprehensive model of somitogenesis with HES7 as core oscillator that is able to reproduce many experimentally observed data in mice.