The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6.

The glycoprotein sclerostin has been identified as a negative regulator of bone growth. It exerts its function by interacting with the Wnt co-receptor LRP5/6, blocks the binding of Wnt factors and thereby inhibits Wnt signalling. Neutralizing anti-sclerostin antibodies are able to restore Wnt activity and enhance bone growth thereby presenting a new osteoanabolic therapy approach for diseases such as osteoporosis. We have generated various Fab antibodies against human and murine sclerostin using a phage display set-up. Biochemical analyses have identified one Fab developed against murine sclerostin, AbD09097 that efficiently neutralizes sclerostin's Wnt inhibitory activity. In vitro interaction analysis using sclerostin variants revealed that this neutralizing Fab binds to sclerostin's flexible second loop, which has been shown to harbour the LRP5/6 binding motif. Affinity maturation was then applied to AbD09097, providing a set of improved neutralizing Fab antibodies which particularly bind human sclerostin with enhanced affinity. Determining the crystal structure of AbD09097 provides first insights into how this antibody might recognize and neutralize sclerostin. Together with the structure-function relationship derived from affinity maturation these new data will foster the rational design of new and highly efficient anti-sclerostin antibodies for the therapy of bone loss diseases such as osteoporosis.

Gating of the two-pore cation channel AtTPC1 in the plant vacuole is based on a single voltage-sensing domain.

The two-pore cation channel TPC1 operates as a dimeric channel in animal and plant endomembranes. Each subunit consists of two homologous Shaker-like halves, with 12 transmembrane domains in total (S1-S6, S7-S12). In plants, TPC1 channels reside in the vacuolar membrane, and upon voltage stimulation, give rise to the well-known slow-activating SV currents. Here, we combined bioinformatics, structure modelling, site-directed mutagenesis, and in planta patch clamp studies to elucidate the molecular mechanisms of voltage-dependent channel gating in TPC1 in its native plant background. Structure-function analysis of the Arabidopsis TPC1 channel in planta confirmed that helix S10 operates as the major voltage-sensing site, with Glu450 and Glu478 identified as possible ion-pair partners for voltage-sensing Arg537. The contribution of helix S4 to voltage sensing was found to be negligible. Several conserved negative residues on the luminal site contribute to calcium binding, stabilizing the closed channel. During evolution of plant TPC1s from two separate Shaker-like domains, the voltage-sensing function in the N-terminal Shaker-unit (S1-S4) vanished.

Venus Flytrap HKT1-Type Channel Provides for Prey Sodium Uptake into Carnivorous Plant Without Conflicting with Electrical Excitability.

The animal diet of the carnivorous Venus flytrap, Dionaea muscipula, contains a sodium load that enters the capture organ via an HKT1-type sodium channel, expressed in special epithelia cells on the inner trap lobe surface. DmHKT1 expression and sodium uptake activity is induced upon prey contact. Here, we analyzed the HKT1 properties required for prey sodium osmolyte management of carnivorous Dionaea. Analyses were based on homology modeling, generation of model-derived point mutants, and their functional testing in Xenopus oocytes. We showed that the wild-type HKT1 and its Na(+)- and K(+)-permeable mutants function as ion channels rather than K(+) transporters driven by proton or sodium gradients. These structural and biophysical features of a high-capacity, Na(+)-selective ion channel enable Dionaea glands to manage prey-derived sodium loads without confounding the action potential-based information management of the flytrap.

Hyperactivation of constitutively dimerized oncogenic EGF receptors by autocrine loops.

The epidermal growth factor (EGF) receptor (EGFR) has a key role in normal embryonic development, adult tissue homeostasis and many pathological processes, in particular tumour formation. Aberrant EGFR activation occurs in many cancer types, and inhibition of this receptor is a promising anti-tumour strategy. Besides overexpression of the wild-type receptor, mutated oncogenic EGFR variants are often associated with malignant transformation. In human non-small-cell lung cancers, kinase mutants of the EGFR are rather common. Human glioblastoma often express the truncated EGFRvIII version as well as other dimerized and permanently activated mutants of the receptor, which are considered as tumour drivers. Similarly, the mutated and dimerized EGFR variant Xiphophorus melanoma receptor kinase (Xmrk) is causative for the development of malignant pigment cell tumours in medaka and Xiphophorus melanoma models. It is generally believed that oncogenic receptors that are active due to dimerizing mutations are ligand independent. Here, we show that different EGFR variants from fish and human efficiently induce autocrine loops by inducing EGFR ligands such as amphiregulin and HB-EGF. Importantly, the pre-dimerized oncogenic EGFR versions Xmrk from Xiphophorus and human EGFR(C600F), though already active in absence of ligands, respond to ligand stimulation with enhanced oncogenic signalling. In summary, our data show that autocrine or paracrine loops are still acting on pre-dimerized oncogenic EGFRs and contribute to their pro-tumorigenic signalling.

Selecting highly structure-specific antibodies using structured synthetic mimics of the cystine knot protein sclerostin.

Antibodies directed against specific regions of a protein have traditionally been raised against full proteins, protein domains or simple unstructured peptides, containing contiguous stretches of primary sequence. We have used a new approach of selecting antibodies against restrained peptides mimicking defined epitopes of the bone modulator protein sclerostin, which has been identified as a negative regulator of the Wnt pathway. For a fast exploration of activity defining epitopes, we produced a set of synthetic peptide constructs mimicking native sclerostin, in which intervening loops from the cystine-knot protein sclerostin were truncated and whose sequences were optimized for fast and productive refolding. We found that the second loop within the cystine knot could be replaced by unnatural sequences, both speeding up folding, and increasing yield. Subsequently, we used these constructs to pan the HuCAL phage display library for antibodies capable of binding the native protein, thereby restricting recognition to the desired epitope regions. It is shown that the antibodies that were obtained recognize a complex epitope in the protein that cannot be mimicked with linear peptides. Antibodies selected against peptides show similar recognition specificity and potency as compared with antibodies obtained from full-length recombinant protein.

Evidence for an influence of TCF7L2 polymorphism rs7903146 on insulin resistance and sensitivity indices in overweight children and adolescents during a lifestyle intervention.

OBJECTIVE: The T allele of the single-nucleotide polymorphism rs7903146 in TCF7L2 (transcription factor 7 like 2 gene) is associated with type 2 diabetes mellitus. The aim of this study was to analyze whether there is an allele-dosage effect on changes of insulin resistance and sensitivity indices in overweight children participating in a lifestyle intervention. METHODS: We genotyped rs7903146 in 236 overweight children (mean age 10.7 years, mean body mass index (BMI) 28.1 kg/m2) completing a 1-year lifestyle intervention. Degree of overweight as BMI-SDS, homeostasis model assessment for insulin resistance (HOMA-IR) and the insulin sensitivity index QUICKI were measured before and after intervention. RESULTS: Lifestyle intervention resulted in an overweight reduction of -0.29+/-0.02 BMI-SDS. HOMA-IR (-0.63+/-0.22) and QUICKI (+0.008+/-0.003) indices improved significantly (P<0.05) in the course of the intervention in the 155 children with a decrease of BMI-SDS. There was an additive negative effect of T allele on changes of HOMA-IR (P=0.041) and QUICKI (P=0.001) in linear regression analyses adjusted to changes of weight status, age, gender and pubertal stage. CONCLUSION: Overweight children showed a negative dosage-allele effect per T allele at single-nucleotide polymorphism rs7903146 in TCF7L2 concerning an improvement of insulin resistance and sensitivity after overweight reduction in a lifestyle intervention. This finding further suggests that this polymorphism might be involved in glucose metabolism.

Biochemical and structural analysis of the interaction between the UBA(2) domain of the DNA repair protein HHR23A and HIV-1 Vpr.

The DNA repair protein HHR23A is a highly conserved protein that functions in nucleotide excision repair. HHR23A contains two ubiquitin associated domains (UBA) that are conserved in a number of proteins with diverse functions involved in ubiquitination, UV excision repair, and signaling pathways via protein kinases. The cellular binding partners of UBA domains remain unclear; however, we previously found that the HHR23A UBA(2) domain interacts specifically with the HIV-1 Vpr protein. Analysis of the low resolution solution structure of HHR23A UBA(2) revealed a hydrophobic loop region of the UBA(2) domain that we predicted was the interface for protein/protein interactions. Here we present results of in vitro binding studies that demonstrate the requirement of this hydrophobic loop region for interaction with human immunodeficiency virus (HIV-1) Vpr. A single point mutation of the Pro at residue 333 to a Glu totally abolishes the binding of HIV-1 Vpr to UBA(2). High resolution NMR structures of the binding deficient UBA(2) mutant P333E as well as of the wild-type UBA(2) domain were determined to compare the effect of this mutation on the structure. Small but significant differences are observed only locally at the site of the mutation. The biochemical and structural analysis confirms the function of the HHR23A UBA(2) GFP-loop as the protein/protein interacting domain.

Non-steady-state diffusion in a multilayered tissue initiated by manipulation of chemical activity at the boundaries.

Diffusion of ionic and nonionic species in multilayered tissues plays an important role in the metabolic processes that take place in these tissues. To create a mathematical model of these diffusion processes, we have chosen as an example hydrogen-bicarbonate ion pair diffusion within the mammalian cornea. This choice was based on the availability of experimental data on this system. The diffusion coefficient of the hydrogen-bicarbonate ion pair in corneal stroma and epithelium is calculated from the observed change in pH in the stroma when conditions at the corneal anterior epithelial surface are changed while the posterior surface is continually bathed with a Ringer's solution in equilibrium with a CO2-gas air mixture. Matching experimental results to a mathematical model of the cornea as a two-layer diffusion system yields, at 37 degrees C, a diffusion coefficient of the hydrogen-bicarbonate ion pair of 2.5 x 10(-6) cm2/s in the stroma and 0.4 x 10(-6) cm2/s in the epithelium. Application of the Nernst-Einstein equation to these data gives the following diffusion coefficients in the two layers: 1) stroma, D(H+) = 11.8 x 10(-6) cm2/s; D(HCO3-) = 1.5 x 10(-6) cm2/s; and 2) epithelium, D(H+) = 1.9 x 10(-6) cm2/s; D(HCO3-) = 0.22 x 10(-6) cm2/s.