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Von Hahn T.,Klinik fur Gastroenterologie | Von Hahn T.,Institute For Molekularbiologie | Schiene-Fischer C.,Max Planck Forschungsstelle fur Enzymologie der Proteinfaltung | Van N.D.,Klinik fur Gastroenterologie | And 17 more authors.
Gastroenterology | Year: 2012

Background & Aims: Hepatitis C virus (HCV) uses several host factors to infect and replicate in human hepatocytes. Cyclophilin A (CypA) is required for viral replication, and CypA inhibitors are in development. We investigated the effects of nonsynonymous single nucleotide polymorphisms (SNPs) in the region of peptidyl-prolyl isomerase A (PPIA) that encodes CypA on HCV infection and replication of human hepatocytes. Methods: We used a combination of virologic, biochemical, and genetic approaches to investigate the effects of PPIA variants on HCV replication in cultured Huh-7.5 cells. We reduced levels of CypA in these cells using small hairpin RNAs (shRNAs). Results: Using shRNAs, we showed that CypA was required for replication of HCV in Huh-7.5 cells and identified 3 SNPs in PPIA that protected cells from HCV entry or replication. Levels of HCV RNA were reduced 3-4 log in cells homozygous for the variant alleles; release of new particles was also reduced, but viral entry was not affected. The effects of the variant alleles were recessive and stronger for preventing replication of full-length HCV genomes than subgenomes. CypA inhibitors prevented replication of residual HCV in hepatocytes. The variants appeared to destabilize the CypA protein; the single amino acid changes led to rapid degradation of the protein. Conclusions: We identified variants in PPIA that destabilize its product, CypA, and prevent HCV infection and replication. These findings indicate mechanisms by which some cells might be resistant to HCV infection and that CypA is a good therapeutic target. © 2012 AGA Institute.


Kunz C.,University of Leipzig | Jahreis G.,Max Planck Forschungsstelle fur Enzymologie der Proteinfaltung | Gunther R.,University of Leipzig | Berger S.,University of Leipzig | And 2 more authors.
Journal of Peptide Science | Year: 2012

The influence of lithium cations on the cis/trans isomerization of prolyl peptide bonds was investigated in a quantitative manner in trifluoroethanol (TFE) and acetonitrile, employing NMR techniques. The focus was on various environmental and structural aspects, such as lithium cation and water concentrations, the type of the partner amino acid in the prolyl peptide bond, and the peptide sequence length. Comparison of the thermodynamic parameters of the isomerization in LiCl/TFE and TFE shows a lithium cation concentration dependence of the cis/trans ratio, which saturates at cation concentrations >200mM. A pronounced increase in the cis isomer content in the presence of lithium cations occurs with the exception of peptides with Gly-Pro and Asp-Pro moieties. The cation effect appears already at the dipeptide level. The salt concentration can considerably be reduced in solvents with a lower number of nucleophilic centers like acetonitrile. The lithium cation effect decreases with small amounts of water and disappears at a water concentration of about 5%. The isomerization kinetics under the influence of lithium cations suggests a weak cation interaction with the carbonyl oxygen of the peptide bond. © 2012 European Peptide Society and John Wiley & Sons, Ltd.


Hediger T.,Heinrich Heine University Dusseldorf | Frank W.,Heinrich Heine University Dusseldorf | Schumann M.,Max Planck Forschungsstelle fur Enzymologie der Proteinfaltung | Fischer G.,Max Planck Forschungsstelle fur Enzymologie der Proteinfaltung | Braun M.,Heinrich Heine University Dusseldorf
Chemistry and Biodiversity | Year: 2012

A series of 18 differently substituted new aryl hetaryl ketones and thioketones were synthesized in four to six steps from commercial starting materials. The new ketones were evaluated as inhibitors of the peptidyl-prolyl cis-trans isomerase hPin1 with Ki values ranging in the one-digit micromolar to sub-micromolar numbers. A crystal structure revealed the non-planar arrangement of the aryl residues at the carbonyl compound and supports the hypothesis that the new compounds might mimic the transition state of the enzymatic conversion. © 2012 Verlag Helvetica Chimica Acta AG, Zürich.


Garvey M.,Max Planck Forschungsstelle fur Enzymologie der Proteinfaltung | Tepper K.,Max Planck Forschungsstelle fur Enzymologie der Proteinfaltung | Haupt C.,Martin Luther University of Halle Wittenberg | Knupfer U.,Leibniz Institute for Natural Product Research and Infection Biology | And 6 more authors.
Biochemical and Biophysical Research Communications | Year: 2011

The oligomerization of Aβ peptide into amyloid fibrils is a hallmark of Alzheimer's disease. Due to its biological relevance, phosphate is the most commonly used buffer system for studying the formation of Aβ and other amyloid fibrils. Investigation into the characteristics and formation of amyloid fibrils frequently relies upon material formed in vitro, predominantly in phosphate buffers. Herein, we examine the effects on the fibrillation and oligomerization mechanism of Aβ peptide that occur due solely to the influence of phosphate buffer. We reveal that significant differences in amyloid fibrillation are observed due to fibrillation being initiated in phosphate or HEPES buffer (at physiological pH and temperature). Except for the differing buffer ions, all experimental parameters were kept constant. Fibril formation was assessed using fluorescently monitored kinetic studies, microscopy, X-ray fiber diffraction and infrared and nuclear magnetic resonance spectroscopies. Based on this set up, we herein reveal profound effects on the mechanism and speed of Aβ fibrillation. The three histidine residues at positions 6, 13 and 14 of Aβ(1-40) are instrumental in these mechanistic changes. We conclude that buffer plays a more significant role in fibril formation than has been generally acknowledged. © 2011 Elsevier Inc.


Malesevic M.,Max Planck Forschungsstelle fur Enzymologie der Proteinfaltung | Kuhling J.,Max Planck Forschungsstelle fur Enzymologie der Proteinfaltung | Erdmann F.,Max Planck Forschungsstelle fur Enzymologie der Proteinfaltung | Balsley M.A.,George Washington University | And 3 more authors.
Angewandte Chemie - International Edition | Year: 2010

Trlmeslc acid amide serves as a scaffold for a lipophilic cyclophilin inhibitor, a fluorescent rhodamine dye (TAMRA), and a (D-Glu)6 oligopeptide residue. Although the affinity of 1 for intracellular cyclophilin A (CypA) is very high, fluorescence measurements indicate complete exclusion from the cell. CypA-induced Chemotaxis of lymphocytes is inhibited by 1 since extracellular cyclophilins are responsible for the physiological signal Chemilcal Equation Presentation. © 2010 Wiley-VCH Verlag GmbH & Co. KCaA, Weinheim.

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