TY - JOUR A1 - Boschert, Verena A1 - van Dinther, Maarten A1 - Weidauer, Stella A1 - van Pee, Katharina A1 - Muth, Eva-Maria A1 - ten Dijke, Peter A1 - Mueller, Thomas D. T1 - Mutational Analysis of Sclerostin Shows Importance of the Flexible Loop and the Cystine-Knot for Wnt-Signaling Inhibition JF - PLoS ONE N2 - The cystine-knot containing protein Sclerostin is an important negative regulator of bone growth and therefore represents a promising therapeutic target. It exerts its biological task by inhibiting the Wnt (wingless and int1) signaling pathway, which participates in bone formation by promoting the differentiation of mesenchymal stem cells to osteoblasts. The core structure of Sclerostin consists of three loops with the first and third loop (Finger 1 and Finger 2) forming a structured \(\beta\)-sheet and the second loop being unstructured and highly flexible. Biochemical data showed that the flexible loop is important for binding of Sclerostin to Wnt co-receptors of the low-density lipoprotein related-protein family (LRP), by interacting with the Wnt co-receptors LRP5 or -6 it inhibits Wnt signaling. To further examine the structural requirements for Wnt inhibition, we performed an extensive mutational study within all three loops of the Sclerostin core domain involving single and multiple mutations as well as truncation of important regions. By this approach we could confirm the importance of the second loop and especially of amino acids Asn92 and Ile94 for binding to LRP6. Based on a Sclerostin variant found in a Turkish family suffering from Sclerosteosis we generated a Sclerostin mutant with cysteines 84 and 142 exchanged thereby removing the third disulfide bond of the cystine-knot. This mutant binds to LRP6 with reduced binding affinity and also exhibits a strongly reduced inhibitory activity against Wnt1 thereby showing that also elements outside the flexible loop are important for inhibition of Wnt by Sclerostin. Additionally, we examined the effect of the mutations on the inhibition of two different Wnt proteins, Wnt3a and Wnt1. We could detect clear differences in the inhibition of these proteins, suggesting that the mechanism by which Sclerostin antagonizes Wnt1 and Wnt3a is fundamentally different. KW - Wnt signaling cascade KW - binding analysis KW - osteoblasts KW - sequence motif analysis KW - biological locomotion KW - signal inhibition KW - cell binding assay KW - luciferase Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-129862 VL - 8 IS - 11 ER - TY - JOUR A1 - Doppler, Kathrin A1 - Appeltshauser, Luise A1 - Krämer, Heidrun H. A1 - King Man Ng, Judy A1 - Meinl, Edgar A1 - Villmann, Carmen A1 - Brophy, Peter A1 - Dib-Hajj, Sulayman D. A1 - Waxman, Stephen G. A1 - Weishaupt, Andreas A1 - Sommer, Claudia T1 - Contactin-1 and Neurofascin-155/-186 Are Not Targets of Auto-Antibodies in Multifocal Motor Neuropathy JF - PLoS One N2 - Multifocal motor neuropathy is an immune mediated disease presenting with multifocal muscle weakness and conduction block. IgM auto-antibodies against the ganglioside GM1 are detectable in about 50% of the patients. Auto-antibodies against the paranodal proteins contactin-1 and neurofascin-155 and the nodal protein neurofascin-186 have been detected in subgroups of patients with chronic inflammatory demyelinating polyneuropathy. Recently, auto-antibodies against neurofascin-186 and gliomedin were described in more than 60% of patients with multifocal motor neuropathy. In the current study, we aimed to validate this finding, using a combination of different assays for auto-antibody detection. In addition we intended to detect further auto-antibodies against paranodal proteins, specifically contactin-1 and neurofascin-155 in multifocal motor neuropathy patients’ sera. We analyzed sera of 33 patients with well-characterized multifocal motor neuropathy for IgM or IgG anti-contactin-1, anti-neurofascin-155 or -186 antibodies using enzyme-linked immunosorbent assay, binding assays with transfected human embryonic kidney 293 cells and murine teased fibers. We did not detect any IgM or IgG auto-antibodies against contactin-1, neurofascin-155 or -186 in any of our multifocal motor neuropathy patients. We conclude that auto-antibodies against contactin-1, neurofascin-155 and -186 do not play a relevant role in the pathogenesis in this cohort with multifocal motor neuropathy. KW - motor proteins KW - enzyme-linked immunoassays KW - binding analysis KW - neuropathy KW - nerve fibers KW - cell binding assay KW - antibodies KW - enzyme assays Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126156 VL - 10 IS - 7 ER - TY - JOUR A1 - Merget, Benjamin A1 - Sotriffer, Christoph A. T1 - Slow-Onset Inhibition of Mycobacterium tuberculosis InhA: Revealing Molecular Determinants of Residence Time by MD Simulations JF - PLoS One N2 - An important kinetic parameter for drug efficacy is the residence time of a compound at a drug target, which is related to the dissociation rate constant koff. For the essential antimycobacterial target InhA, this parameter is most likely governed by the ordering of the flexible substrate binding loop (SBL). Whereas the diphenyl ether inhibitors 6PP and triclosan (TCL) do not show loop ordering and thus, no slow-binding inhibition and high koff values, the slightly modified PT70 leads to an ordered loop and a residence time of 24 minutes. To assess the structural differences of the complexes from a dynamic point of view, molecular dynamics (MD) simulations with a total sampling time of 3.0 µs were performed for three ligand-bound and two ligand-free (perturbed) InhA systems. The individual simulations show comparable conformational features with respect to both the binding pocket and the SBL, allowing to define five recurring conformational families. Based on their different occurrence frequencies in the simulated systems, the conformational preferences could be linked to structural differences of the respective ligands to reveal important determinants of residence time. The most abundant conformation besides the stable EI* state is characterized by a shift of Ile202 and Val203 toward the hydrophobic pocket of InhA. The analyses revealed potential directions for avoiding this conformational change and, thus, hindering rapid dissociation: (1) an anchor group in 2'-position of the B-ring for scaffold stabilization, (2) proper occupation of the hydrophobic pocket, and (3) the introduction of a barricade substituent in 5'-position of the diphenyl ether B-ring. KW - crystal structure KW - ethers KW - oxygen KW - cofactors (biochemistry) KW - binding analysis KW - biochemical simulations KW - hydrogen bonding mycobacterium tuberculosis Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125607 VL - 10 IS - 5 ER - TY - JOUR A1 - Paletta, Daniel A1 - Fichtner, Alina Suzann A1 - Starick, Lisa A1 - Porcelli, Steven A. A1 - Savage, Paul B. A1 - Herrmann, Thomas T1 - Species Specific Differences of CD1d Oligomer Loading In Vitro JF - PLoS One N2 - CD1d molecules are MHC class I-like molecules that present glycolipids to iNKT cells. The highly conserved interaction between CD1d:α-Galactosylceramide (αGC) complexes and the iNKT TCR not only defines this population of αβ T cells but can also be used for its direct identification. Therefore, CD1d oligomers are a widely used tool for iNKT cell related investigations. To this end, the lipid chains of the antigen have to be inserted into the hydrophobic pockets of the CD1d binding cleft, often with help of surfactants. In this study, we investigated the influence of different surfactants (Triton X-100, Tween 20, Tyloxapol) on in vitro loading of CD1d molecules derived from four different species (human, mouse, rat and cotton rat) with αGC and derivatives carrying modifications of the acyl-chain (DB01-1, PBS44) and a 6-acetamido-6-deoxy-addition at the galactosyl head group (PBS57). We also compared rat CD1d dimers with tetramers and staining of an iNKT TCR transductant was used as readout for loading efficacy. The results underlined the importance of CD1d loading efficacy for proper analysis of iNKT TCR binding and demonstrated the necessity to adjust loading conditions for each oligomer/glycolipid combination. The efficient usage of surfactants as a tool for CD1d loading was revealed to be species-specific and depending on the origin of the CD1d producing cells. Additional variation of surfactant-dependent loading efficacy between tested glycolipids was influenced by the acyl-chain length and the modification of the galactosyl head group with PBS57 showing the least dependence on surfactants and the lowest degree of species-dependent differences. KW - cell staining KW - major histocompatibility complex KW - binding analysis KW - oligomers KW - glycolipids KW - lipids KW - surfactants KW - T cells Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-124879 VL - 10 IS - 11 ER -