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Hydrogen peroxide (H2O2) has been widely used to study the oxidative stress response. However, H2O2 is unstable and easily decomposes into H2O and O2. Consequently, a wide range of exposure times and treatment concentrations has been described in the literature. In the present study, we established a ferrous oxidation-xylenol orange (FOX) assay, which was originally described for food and body liquids, as a method for the precise quantification of H2O2 concentrations in cell culture media. We observed that the presence of FCS and high cell densities significantly accelerate the decomposition of H2O2, therefore acting as a protection against cell death by accidental necrosis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The parvulin-type peptidyl-prolyl cis/trans isomerases (PPIases) have been shown to be involved in tumor progression and the pathogenesis of Alzheimer's disease and were therefore a subject of intense research. Here, we describe a role for parvulin 17 in microtubule assembly. Co-precipitation experiments and sedimentation assays demonstrated that parvulin 17 interacts with tubulin in a GTP-dependent manner and thereby promotes the formation of microtubules, as shown by transmission electron microscopy and a microtubule polymerization assay. The microtubule-assembly-promoting properties of parvulin 17 seem to depend on its PPIase activity. Thus, catalytic deficient variants of parvulin 17 were not able to promote microtubule formation. Accordingly, inhibitors of parvulin 17 activity also prevent parvulin-catalyzed tubulin polymerization. The analysis of tubulin interaction sites on parvulin using peptide microarrays revealed that tubulin interacts with the substrate binding pocket of parvulin. Additionally, β-tubulin peptide scan on microarrays demonstrates interaction of parvulin 17 with an Arg-Pro-Asp motif corresponding to proline residue 87 of β-tubulin. Confocal laser scanning microscopy points to a function of parvulin 17 in microtubule dynamics as well. Parvulin 17 is predominantly found in the cytosol and colocalizes with microtubules., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Proline-directed protein phosphorylation was shown to depend on the capacity of the targeted Ser(Thr)-Pro bond to exhibit conformational polymorphism. The cis/trans isomer specificity underlying ERK2-catalyzed phosphate transfer leads to a complete discrimination of the cis Ser(Thr)-Pro conformer of oligopeptide substrates. We investigated in vitro the ERK2-catalyzed phosphorylation of Aspergillus oryzae RNase T1 containing two Ser-Pro bonds both of which share high stabilization energy in their respective native state conformation, the cis Ser54-Pro and the trans Ser72-Pro moiety. Despite trans isomer specificity of ERK2, a doubly phosphorylated RNase T1 was found as the final reaction product. Similarly, the RNase T1 S54G/P55N and RNase T1 P73V variants, which retain the prolyl bond conformations of the RNase T1-wt, were both monophosphorylated with a catalytic efficiency kcat/KM of 425 M(-1) s(-1) and 1228 M(-1) s(-1), respectively. However, initial phosphorylation rates did not depend linearly on the ERK2 concentration. The phosphorylation rate of the resulting plateau region at high ERK2 concentrations can be increased up to threefold for the RNase T1 P73V variant in the presence of the peptidyl-prolyl cis/trans isomerase Cyclophilin 18, indicating a conformational interconversion as the rate limiting step in the catalyzed phosphate group transfer. Using peptidyl-prolyl cis/trans isomerases with different substrate specificity, we identified a native state conformational equilibrium of the Ser54-Pro bond with the minor trans Ser54-Pro bond as the phosphorylation-sensitive moiety. This technique can therefore be used for a determination of the ratio and the interconversion rates of prolyl bond isomers in the native state of proteins.

Human parvulin 14 (hPar14) is a folding helper enzyme belonging to the parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases). This enzyme is thought to play a role in cell-cycle and chromatin remodeling. Although hPar14 was nuclearly localized and bound to double-stranded DNA, the molecular basis of the subcellular localization and the functional regulation remained unknown. Here we show that subcellular localization and DNA-binding ability of hPar14 is regulated by posttranslational modification of its N-terminal domain. As proved by MALDI-TOF mass spectrometry and MS/MS fragmentation, hPar14 is phosphorylated at Ser19 in vitro and in vivo. In human HeLa cells the protein is most likely modified by casein kinase 2 (CK2). Phosphorylation of hPar14 is inhibited both in vitro and in vivo by 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), a specific inhibitor of CK2 activity. Mutation of Ser19 to Ala abolishes phosphorylation and alters the subcellular localization of hPar14 from predominantly nuclear to significantly cytoplasmic. Immunostaining shows that a Glu19 mutant of hPar14, which mimics the phosphorylated state of Ser19, is localized around the nuclear envelope, but does not penetrate into the nucleoplasm. In contrast to wild-type hPar14, the in vitro DNA-binding affinity of the Glu19 mutant is strongly reduced, suggesting that only the dephosphorylated protein is the active DNA-binding form of hPar14 in the nucleus.