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Licht A.,Humboldt University of Berlin | Bulut H.,Free University of Berlin | Scheffel F.,Humboldt University of Berlin | Daumke O.,Max Delbruck Zentrum fur Molekulare Medizin | And 4 more authors.
Journal of Molecular Biology | Year: 2011

Solute receptors (binding proteins) are indispensable components of canonical ATP-binding cassette importers in prokaryotes. Here, we report on the characterization and crystal structures in the closed and open conformations of AcbH, the solute receptor of the putative carbohydrate transporter AcbFG which is encoded in the acarbose (acarviosyl-1,4-maltose) biosynthetic gene cluster from Actinoplanes sp. SE50/110. Binding assays identified AcbH as a high-affinity monosaccharide-binding protein with a dissociation constant (Kd) for β-d-galactopyranose of 9.8 ± 1.0a nM. Neither galactose-containing di- and trisaccharides, such as lactose and raffinose, nor monosaccharides including d-galacturonic acid, l-arabinose, d-xylose and l-rhamnose competed with [14C]galactose for binding to AcbH. Moreover, AcbH does not bind d-glucose, which is a common property of all but one d-galactose-binding proteins characterized to date. Strikingly, determination of the X-ray structure revealed that AcbH is structurally homologous to maltose-binding proteins rather than to glucose-binding proteins. Two helices are inserted in the substrate-binding pocket, which reduces the cavity size and allows the exclusive binding of monosaccharides, specifically β-d-galactopyranose, in the 4C1 conformation. Site-directed mutagenesis of three residues from the binding pocket (Arg82, Asp361 and Arg362) that interact with the axially oriented O4-H hydroxyl of the bound galactopyranose and subsequent functional analysis indicated that these residues are crucial for galactose binding. To our knowledge, this is the first report of the tertiary structure of a solute receptor with exclusive affinity for β-d-galactopyranose. The putative role of a galactose import system in the context of acarbose metabolism in Actinoplanes sp. is discussed. © 2010 Elsevier Ltd. All rights reserved.

Amm I.,University of Stuttgart | Sommer T.,Max Delbruck Zentrum fur Molekulare Medizin | Wolf D.H.,University of Stuttgart
Biochimica et Biophysica Acta - Molecular Cell Research | Year: 2014

Mistakes are part of our world and constantly occurring. Due to transcriptional and translational failures, genomic mutations or diverse stress conditions like oxidation or heat misfolded proteins are permanently produced in every compartment of the cell. As misfolded proteins in general lose their native function and tend to aggregate several cellular mechanisms have been evolved dealing with such potentially toxic protein species. Misfolded proteins are mostly recognized by chaperones on the basis of their exposed hydrophobic patches and, if unable to refold them to their native state, are targeted to proteolytic pathways. Most prominent are the ubiquitin-proteasome system and the autophagic vacuolar (lysosomal) system, eliminating misfolded proteins from the cellular environment. A major task of this quality control system is the specific recognition and separation of the misfolded from the correctly folded protein species and the folding intermediates, respectively, which are on the way to the correct folded state but exhibit properties of misfolded proteins. In this review we focus on the recognition process and subsequent degradation of misfolded proteins via the ubiquitin-proteasome system in the different cell compartments of eukaryotic cells. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf. © 2013 Elsevier B.V.

Isachenko V.,Universitatsfrauenklinik Ulm | Isachenko E.,Universitatsfrauenklinik Ulm | Nawroth F.,Fertility Center Hamburg | Wiegratz I.,Universitatsfrauenklinik Frankfurt Am Main | And 4 more authors.
Geburtshilfe und Frauenheilkunde | Year: 2010

Purpose: Cryopreservation, the most important stage of the cryobanking of ovarian tissue, can be carried out using one of two methods: conventional ("slow") freezing, and vitrification (direct and immediate immersion into liquid nitrogen). For human oocytes and embryos vitrification is more effective compared to conventional freezing. However, these comparative data are limited for human ovarian tissue. The aim of this study was to compare conventional freezing of ovarian tissue with vitrification. Material and Methods: Ovarian tissue from 5 patients was transported to the laboratory within 20 min at 32 to 34°C, divided into smaller pieces (1×1 to 1,5×0,7 to 1mm) and randomly distributed into three groups: Group 1: control (fresh tissue), group 2: pieces after vitrification/warming, Group 3: pieces after conventional freezing/thawing. All pieces were cultured in vitro for 12 days. The viability of the tissue was evaluated by the development of the follicles and GAPDH gene expression after in vitro culture. Results: 93, 74 and 78% of the follicles of groups 1, 2 or 3 were morphologically normal. Molecular analysis showed that the intensity of GAPDH gene expression in the tissue after conventional freezing was greatly increased compared to after vitrification. Conclusion: It was concluded that for the cryopreservation of human ovarian tissue conventional freezing is more suitable than vitrification. © 2010 Georg Thieme Verlag KG Stuttgart, New York.

Mansour W.,Technion - Israel Institute of Technology | Nakasone M.A.,Technion - Israel Institute of Technology | Nakasone M.A.,University of Maryland University College | Von Delbruck M.,Max Delbruck Zentrum fur Molekulare Medizin | And 7 more authors.
Journal of Biological Chemistry | Year: 2015

Background: Deconjugation of polyubiquitin is an essential step in preparing substrates for proteolysis by the 26S proteasome. Results: Proteasome-associated DUBs, Rpn11 and Ubp6, process long Lys11- or Lys63-linked polyUb more efficiently than Lys48 linkages. Conclusion: 26S proteasomes can completely disassemble a mixed/branched polyUb conjugate. Significance: These observations call into question what constitutes an efficient signal for proteasome targeting versus proteolysis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Hinrichs S.,Justus Liebig University | Heger J.,Justus Liebig University | Schreckenberg R.,Justus Liebig University | Wenzel S.,Justus Liebig University | And 6 more authors.
Cardiovascular Research | Year: 2011

AimsRenin and peroxisome proliferator-activated receptor (PPAR-γ) interact directly with cardiomyocytes and influence protein synthesis. We investigated their effects and interaction on the size of cardiomyocytes. Methods and resultsEffects of renin and PPAR-γ activation were studied in cultured adult rat ventricular cardiomyocytes, transgenic mice with a cardiomyocyte-restricted knockout of PPAR-γ, and transgenic rats overexpressing renin, TGR(mRen2)27. The length and width of cardiomyocytes were analysed 24 h after administration of factors. Renin caused an unexpected effect on the length of cardiomyocytes that was inhibited by mannose-6-phosphate and monensin, but not by administration of glucose-6-phosphate. Endothelin-1 used as a classical pro-hypertrophic agonist increased cell width but not cell length. Renin caused an activation of p38 and p42/44 mitogen-activated protein (MAP) kinases. The latter activation was impaired by mannose-6-phosphate. Inhibition of p42/44 but not of p38 MAP kinase activation attenuated the effect of renin on cell length. In contrast, activation of PPAR-γ reduced cell length. Feeding wild-type mice with pioglitazone, a PPAR-γ agonist, reduced cell length. Cardiomyocytes isolated from PPAR-γ knockout mice were longer, and their length was not affected by pioglitazone. Cardiomyocytes isolated from TGR(mRen2)27 rats were longer than those of non-transgenic littermates. Cell length was reduced by feeding these mice with pioglitazone. Pioglitazone affected cell length independent of blood pressure. ConclusionThe length of cardiomyocytes is controlled by the activation of cardiac-specific mannose-6-phosphate/insulin-like growth factor II receptors and activation of PPAR-γ. This type of cell size modification differs from that of any other known pro-hypertrophic agonists. © 2010 The Author.

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