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.     

Transduction of human embryonic stem cells by ecotropic retroviral vectors

The steadily increasing availability of human embryonic stem (hES) cell lines has created strong interest in applying available tools for gene transfer in murine cells to human systems. Here we present a method for the transduction of hES cells with ecotropic retroviral vectors. hES cells were transiently transfected with a construct carrying the murine retrovirus receptor mCAT1. Subsequently, the cells were exposed to replication-deficient Moloney murine leukemia virus (MoMuLV) derivatives or pseudotyped lentiviral vectors. With oncoretroviral vectors, this procedure yields overall transduction efficiencies of up to 20% and permits selection of permanently transduced clones with high frequency. Selected clones maintained expression of pluripotency-associated markers and exhibited multi-germ layer differentiation both in vitro and in vivo. HES cell-derived somatic cells including neural progeny maintained high levels of transgene expression. Lentiviral vectors pseudotyped with the MoMuLV envelope could be introduced in the same manner with efficiencies of up to 33%. Transgene expression of lentivirally transduced hES cells remained permanent after differentiation even without selection pressure. Bypassing the regulatory issues associated with the use of amphotropic retroviral systems and exploiting the large pool of existing murine vectors, this method provides a safe and versatile tool for gene transfer and lineage analysis in hES cells and their progeny.     

Setting up standards and a reference map for the alkaline proteome of the Gram-positive bacterium Lactococcus lactis

Despite the fact that almost 39% of the theoretical expressed proteins of Lactococcus lactis have a predicted isoelectric point above 7, these proteins have not been studied in previous proteome analyses. In the present study, we set up a reference map of alkaline lactococcal proteins by using immobilized pH gradients (IPG) spanning pH 6 to 12 and 9 to 12, and protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Different electrophoresis systems for isoelectric focusing were evaluated to optimize the first dimension. Best results were obtained by sample application using cup-loading at the anodic side and increasing the final voltage up to 8000 V for IPGs, using N,N-dimethylacrylamide as monomer. After two-dimensional gel electrophoresis of extracts obtained from exponentially growing cells, about 200 protein spots were selected for identification by peptide mass fingerprinting. With MALDI-TOF MS, 153 proteins were identified that were the products of 85 different genes. Their predicted isoelectric points range from as high as 11.31 to as low as 6.34. Ribosomal proteins, hypothetical proteins and proteins with unknown function represent the largest groups of identified proteins. For further classification, the codon adaptation index (CAI) and grand average of hydropathicity (GRAVY) for each lactococcal protein were calculated. The protein with the lowest CAI identified in this study is the manganese ABC transporter ATP-binding protein. Less than 10% of the alkaline lactococcal proteins have a smaller CAI. The highest GRAVY for an identified protein is 0.26. The complete in silico data of Lactococcus lactis as well as clickable reference maps are available at www.wzw.tum.de/proteomik/lactis.     

Novel role for Netrins in regulating epithelial behavior during lung branching morphogenesis

The development of many organs, including the lung, depends upon a process known as branching morphogenesis, in which a simple epithelial bud gives rise to a complex tree-like system of tubes specialized for the transport of gas or fluids. Previous studies on lung development have highlighted a role for fibroblast growth factors (FGFs), made by the mesodermal cells, in promoting the proliferation, budding, and chemotaxis of the epithelial endoderm. Here, by using a three-dimensional culture system, we provide evidence for a novel role for Netrins, best known as axonal guidance molecules, in modulating the morphogenetic response of lung endoderm to exogenous FGFs. This effect involves inhibition of localized changes in cell shape and phosphorylation of the intracellular mitogen-activated protein kinase(s) (ERK1/2, for extracellular signal-regulated kinase-1 and -2), elicited by exogenous FGFs. The temporal and spatial expression of netrin 1, netrin 4, and Unc5b genes and the localization of Netrin-4 protein in vivo suggest a model in which Netrins in the basal lamina locally modulate and fine-tune the outgrowth and shape of emergent epithelial buds.     

Influence of Heparin Mimetics on Assembly of the FGF·FGFR4 Signaling Complex

Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF signaling as they promote FGF·FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM₆), octasaccharide (HM₈), and decasaccharide (HM₁₀), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM₈ and HM₁₀ are significantly more potent than HM₆ in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1·FGFR4 signaling. To understand the molecular basis for these differential activities in FGF1/2·FGFR4 signaling, we used NMR spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence of both a secondary FGF1·FGFR4 interaction site and a direct FGFR4·FGFR4 interaction site thus supporting the formation of the symmetric mode of FGF·FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM₈ relative to HM₆ in stimulating FGF2·FGFR4 signaling correlates with the higher affinity of HM₈ to bind and dimerize FGF2. Notably FGF2·HM₈ exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF·FGFR dimerization model, which incorporates the differential ability of HM to dimerize FGFs.     

Activation of estrogen receptor-beta by a special extract of Rheum rhaponticum (ERr 731), its aglycones and structurally related compounds

The special extract ERr 731^® from the roots of Rheum rhaponticum is the major constituent of Phytoestrol^® N which is used for the treatment of climacteric symptoms in menopausal women. However, the molecular mode of action of ERr 731^® was unknown. For the first time, ERr 731^® and its aglycones trans-rhapontigenin and desoxyrhapontigenin were investigated with regard to the activation of the estrogen receptor- or estrogen receptor-β (ER, ERβ). The related hydroxystilbenes cis-rhapontigenin, resveratrol and piceatannol were studied as comparators. As controls, 17β-estradiol or the selective ER-(propylpyrazoltriol) or ERβ-agonists (diarylpropionitril) were used. Neither in ER-expressing yeast cells, in the ER-responsive Ishikawa cells, nor in human endometrial HEC-1B cells transiently transfected with the ER an activation of ER by ERr 731^® or the other single compounds was detected. Furthermore, an antiestrogenic effect was not observed. In contrast in human endometrial HEC-1B cells transiently transfected with the ERβ, 100 ng/ml ERr 731^® and the single compounds significantly induced the ERβ-coupled luciferase activity in a range comparable to 10⁻⁸ M 17β-estradiol. All effects were abolished with the pure ER antagonist ICI 182780, indicating an ER-specific effect. The ERβ agonistic activity by ERr 731^® could be of importance for its clinical use, as central functions relevant to climacteric complaints are proposed to be mediated via ERβ activation.     

Determination of absolute configuration in 4‐aryl‐3,4‐dihydro‐2(1H)‐pyrimidones by high performance liquid chromatography and CD spectroscopy

The absolute configuration of three 4‐aryl‐3,4‐dihydro‐2(1H)‐pyrimidones (Biginelli compounds, DHPMs) was established by comparison of the typical circular dichroism (CD) spectra of individual enantiomers with reference samples of known absolute configuration. The enantiomers were obtained by semipreparative separation of racemic mixtures on a Chiralcel OD‐H chiral stationary phase. The method was used to establish the enantiopreference of various lipases in biocatalytic kinetic resolution experiments employing activated DHPM esters. Chirality 11:659–662, 1999. © 1999 Wiley‐Liss, Inc.     

Methionine sulfoxide reductases are essential for virulence of Salmonella typhimurium

Production of reactive oxygen species represents a fundamental innate defense against microbes in a diversity of host organisms. Oxidative stress, amongst others, converts peptidyl and free methionine to a mixture of methionine-S- (Met-S-SO) and methionine-R-sulfoxides (Met-R-SO). To cope with such oxidative damage, methionine sulfoxide reductases MsrA and MsrB are known to reduce MetSOs, the former being specific for the S-form and the latter being specific for the R-form. However, at present the role of methionine sulfoxide reductases in the pathogenesis of intracellular bacterial pathogens has not been fully detailed. Here we show that deletion of msrA in the facultative intracellular pathogen Salmonella (S.) enterica serovar Typhimurium increased susceptibility to exogenous H(2)O(2), and reduced bacterial replication inside activated macrophages, and in mice. In contrast, a ΔmsrB mutant showed the wild type phenotype. Recombinant MsrA was active against free and peptidyl Met-S-SO, whereas recombinant MsrB was only weakly active and specific for peptidyl Met-R-SO. This raised the question of whether an additional Met-R-SO reductase could play a role in the oxidative stress response of S. Typhimurium. MsrC is a methionine sulfoxide reductase previously shown to be specific for free Met-R-SO in Escherichia (E.) coli. We tested a ΔmsrC single mutant and a ΔmsrBΔmsrC double mutant under various stress conditions, and found that MsrC is essential for survival of S. Typhimurium following exposure to H(2)O(2,) as well as for growth in macrophages, and in mice. Hence, this study demonstrates that all three methionine sulfoxide reductases, MsrA, MsrB and MsrC, facilitate growth of a canonical intracellular pathogen during infection. Interestingly MsrC is specific for the repair of free methionine sulfoxide, pointing to an important role of this pathway in the oxidative stress response of Salmonella Typhimurium.     

Development of antibiotic resistance and up-regulation of the antimutator gene pfpI in mutator Pseudomonas aeruginosa due to inactivation of two DNA oxidative repair genes (mutY, mutM)

Prevention and correction of oxidative DNA lesions in Pseudomonas aeruginosa is ensured by the DNA oxidative repair system (GO). Single inactivation of mutT, mutY and mutM involved in GO led to elevated mutation rates (MRs) that correlated to increased development of resistance to antibiotics. In this study, we constructed a double mutant in mutY and mutM (PAOMY-Mgm) and characterized the phenotype and the gene expression profile using microarray and RT-PCR. PAOMY-Mgm presented 28-fold increases in MR compared with wild-type reference strain PAO1. In comparison, the PAOMYgm (mutY) single mutant showed only a fivefold increase, whereas the single mutant PAOMMgm (mutM) showed a nonsignificant increase in MR compared with PAO1 and the single mutants. Mutations in the regulator nfxB leading to hyperexpression of MexCD-OprJ efflux pump were found as the mechanism of resistance to ciprofloxacin in the double mutant. A better fitness of the mutator compared with PAO1 was found in growth competition experiments in the presence of ciprofloxacin at concentrations just below minimal inhibitory concentration. Up-regulation of the antimutator gene pfpI, that has been shown to provide protection to oxidative stress, was found in PAOMY-Mgm compared with PAO1. In conclusion, we showed that MutY and MutM are cooperating in the GO of P. aeruginosa, and that oxidative DNA lesions might represent an oxidative stress for the bacteria.     

Crystal structure and functional analysis of MiD49, a receptor for the mitochondrial fission protein Drp1

Mitochondrial fission requires recruitment of dynamin‐related protein 1 (Drp1) to the mitochondrial surface, where assembly leads to activation of its GTP‐dependent scission function. MiD49 and MiD51 are two receptors on the mitochondrial outer membrane that can recruit Drp1 to facilitate mitochondrial fission. Structural studies indicated that MiD51 has a variant nucleotidyl transferase fold that binds an ADP co‐factor essential for activation of Drp1 function. MiD49 shares sequence homology with MiD51 and regulates Drp1 function. However, it is unknown if MiD49 binds an analogous co‐factor. Because MiD49 does not readily crystallize, we used structural predictions and biochemical screening to identify a surface entropy reduction mutant that facilitated crystallization. Using molecular replacement, we determined the atomic structure of MiD49 to 2.4 Å. Like MiD51, MiD49 contains a nucleotidyl transferase domain; however, the electron density provides no evidence for a small‐molecule ligand. Structural changes in the putative nucleotide‐binding pocket make MiD49 incompatible with an extended ligand like ADP, and critical nucleotide‐binding residues found in MiD51 are not conserved. MiD49 contains a surface loop that physically interacts with Drp1 and is necessary for Drp1 recruitment to the mitochondrial surface. Our results suggest a structural basis for the differential regulation of MiD51‐ versus MiD49‐mediated fission.