Mass spectrometric mapping of linker histone H1 variants reveals multiple acetylations, methylations, and phosphorylation as well as differences between cell culture and tissue

Posttranslational modifications of histones are involved in regulation of chromatin structure and gene activity. Whereas the modifications of the core histones H2A, H2B, H3, and H4 have been extensively studied, our knowledge of H1 modifications remained mainly limited to its phosphorylation. Here we analyzed the composition of histone H1 variants and their modifications in two human cell lines and nine mouse tissues. Use of a hybrid linear ion trap-orbitrap mass spectrometer facilitated assignment of modifications by high resolution and low ppm mass accuracy for both the precursor and product mass spectra. Across different tissues we identified a range of phosphorylation, acetylation, and methylation sites. We also mapped sites of ubiquitination and report identification of formylated lysine residues. Interestingly many of the mapped modifications are located within the globular domain of the histones at sites that are thought to be involved in binding to nucleosomal DNA. Investigation of mouse tissue in addition to cell lines uncovered a number of interesting differences. For example, whereas methylation sites are frequent in tissues, this type of modification was much less abundant in cultured cells and escaped detection. Our study significantly extends the known spectrum of linker histone variability.     

Candida albicans Sun41p, a putative glycosidase, is involved in morphogenesis, cell wall biogenesis, and biofilm formation

The SUN gene family has been defined in Saccharomyces cerevisiae and comprises a fungus-specific family of proteins which show high similarity in their C-terminal domains. Genes of this family are involved in different cellular processes, like DNA replication, aging, mitochondrial biogenesis, and cytokinesis. In Candida albicans the SUN family comprises two genes, SUN41 and SIM1. We demonstrate that C. albicans mutants lacking SUN41 show similar defects as found for S. cerevisiae, including defects in cytokinesis. In addition, the SUN41 mutant showed a higher sensitivity towards the cell wall-disturbing agent Congo red, whereas no difference was observed in the presence of calcofluor white. Compared to the wild type, SUN41 deletion strains exhibited a defect in biofilm formation, a reduced adherence on a Caco-2 cell monolayer, and were unable to form hyphae on solid medium under the conditions tested. Interestingly, Sun41p was found to be secreted in the medium of cells growing as blastospores as well as those forming hyphae. Our results support a function of SUN41p as a glycosidase involved in cytokinesis, cell wall biogenesis, adhesion to host tissue, and biofilm formation, indicating an important role in the host-pathogen interaction.     

Retinoic acid–induced survival effects in SH-SY5Y neuroblastoma cells

Neuroblastoma is a malignant childhood cancer arising from the embryonic sympathoadrenal lineage of the neural crest. Retinoic acid (RA) is included in the multimodal therapy of patients with high-risk neuroblastoma to eliminate minimal residual disease. However, the formation of RA-resistant cells substantially lowers 5-year overall survival rates. To examine mechanisms that lead to treatment failure, we chose human SH-SY5Y cells, which are known to tolerate incubation with RA by activating the survival kinases Akt and extracellular signal-regulated kinase 1/2. Characterization of downstream pathways showed that both kinases increased the phosphorylation of the ubiquitin ligase mouse double minute homolog 2 (Mdm2) and thereby enhanced p53 degradation. When p53 signaling was sustained by blocking complex formation with Mdm2 or enhancing c-Jun N-terminal kinase (JNK) activation, cell viability was significantly reduced. In addition, Akt-mediated phosphorylation of the cell-cycle regulator p21 stimulated complex formation with caspase-3, which also contributed to cell protection. Thus, treatment with RA augmented survival signaling and attenuated basal apoptotic pathways in SH-SY5Y cells, which increased cell viability.     

The peroxynitrite pathway in development: phenytoin and benzo[a]pyrene embryopathies in inducible nitric oxide synthase knockout mice

Nitric oxide generated by nitric oxide synthases (NOSs) can react with reactive oxygen species (ROS), forming peroxynitrite, which may contribute to the ROS-initiated macromolecular damage implicated in the embryopathic effects of both endogenous and drug-enhanced oxidative stress. Inducible NOS (iNOS) is nonconstitutive in most tissues, and its embryonic expression and developmental importance are unknown. Herein, during organogenesis (Gestational Days 9 and 10), wild-type B6129PF2 embryos in culture were highly susceptible to the ROS-initiating teratogens phenytoin and benzo[a]pyrene, whereas iNOS knockout embryos were substantially but not completely protected (p < .05), implicating iNOS in the embryopathic mechanism. However, in contrast to prostaglandin H synthase-catalyzed teratogen bioactivation and ROS formation, which occurs within the embryo, in vivo iNOS expression was limited to placental tissue. These results suggest that the diffusion of nitric oxide from placental progenitor tissue (ectoplacental cone) to embryonic target tissues contributes to the embryopathic effects of ROS-initiating teratogens in embryo culture, which may constitute a mechanism by which embryonic determinants of ROS-mediated teratogenesis can be modulated by maternal extra-embryonic processes.     

Conformational analysis of octa- and tetrabromo tetraphenylporphyrins and their Ni(II) and Tb(III) complexes

Molecular mechanics (MM) calculations were used to analyze the puckering of metalloporphyrins as a function of metal ion size and the position of substituents on the porphyrin periphery, on a three series of octa- and tetrabromo tetraphenylporphyrins: without metal, and with Ni(II), and Tb(III) as representative small and large metal ions, respectively. Molecular energy optimization calculations were carried out using the Consistent Force Field (CFF) program, with the parameters developed previously and new parameters for bromine atom. Normal-coordinate structural decomposition (NSD) analysis was performed on the equilibrium structures obtained by MM calculations. The conformers are also stereochemically characterized, compared with available X-ray structures and with the conformers obtained in our previous MM study using chloro instead of bromo beta-pyrrole substituents.     

High expression of green fluorescent protein in Pichia pastoris leads to formation of fluorescent particles

Wild type gene for green fluorescent protein (GFP) was stably integrated into the Pichia pastoris genome and yielded an expression level of over 40% of total cellular protein. The high cytoplasmic concentration of fluorescent (properly folded and processed) GFP caused the formation of fluorescent spherical structures, which could be observed by fluorescence or confocal microscopy after controlled permeabilization of the yeast cells with 0.2% N-lauroyl sarcosine (NLS). Fluorescent GFP particles were also isolated after removal of the cell wall and found to be quite resistant to 0.2% N-lauroyl sarcosine. SDS-PAGE analysis of the isolated fluorescent particles revealed the presence of an 80 kDa protein (alcohol oxidase) and GFP (30%). We conclude that GFP is able to enter spontaneously into the peroxisomes and is inserted into densely packed layers of alcohol oxidase. Consequently, the formation of similar fluorescent particles can also be expected in other organisms when using high-level expression systems. As GFP is widely used in fusion with other proteins as a reporter for protein localization and for many other applications in biotechnology, care must be taken to avoid false interpretations of targeting or trafficking mechanisms inside the cells. In addition, when whole cells or cytoplasmic fractions are used for the quantitative determination of GFP levels, incorrect and misleading values of GFP could be obtained due to the formation of fluorescent particles containing material inside which is not available for fluorescence measurements.     

Carbon isotope variability and sedimentology of the Upper Permian carbonate rocks and changes across the Permian-Triassic boundary in the Masore section (Western Slovenia)

Carbonate and total organic carbon stable isotope analyses of the Upper Permian and Lower Triassic succession in the Masore section in western Slovenia indicate a high storage of organic matter during the Upper Permian, as well as the well known worldwide light carbon isotope event across the P/Tr boundary. The perturbations in the global carbon cycle observed in the investigated section span an approximately 50 cm thick interval (from –11 cm below to +41 cm above the lithostratigraphically determined P/Tr boundary), and coincide more or less with changes in lithology, as well as with an abrupt disappearance of Upper Permian marine fauna. In this section changes in the sedimentary environment are most probably related to Upper Permian—Lower Triassic sea level changes. The carbonate and organic carbon negative peak anomaly could be explained by accelerated changes in the end Permian carbon cycle, due to some co-occurring events, such as pronounced erosion and oxidation of organic carbon, a possible release of methane from stored hydrates, and volcanic activity, as well as by a sudden reduction in primary productivity triggered by not yet completely satisfactorily explained mechanisms.     

p27 binds cyclin-CDK complexes through a sequential mechanism involving binding-induced protein folding

p27 controls cell proliferation by binding and regulating nuclear cyclin-dependent kinases (CDKs). In addition, p27 interacts with other nuclear and cytoplasmic targets and has diverse biological functions. We seek to understand how the structural and dynamic properties of p27 mediate its several functions. We show that, despite showing disorder before binding its targets, p27 has nascent secondary structure that may have a function in molecular recognition. Binding to Cdk2-cyclin A is accompanied by p27 folding, and kinetic data suggest a sequential mechanism that is initiated by binding to cyclin A. p27 regulates CDK-cyclin complexes involved directly in cell cycle control and does not interact with other closely related CDKs. We show that p27-cyclin interactions are an important determinant of this specificity and propose that the homologous cell cycle regulators p21 and p57 function by a similar sequential, folding-on-binding mechanism.     

Exclusion of repetitive DNA elements from gnathostome Hox clusters

Despite their homology and analogous function, the Hox gene clusters of vertebrates and invertebrates are subject to different constraints on their structural organization. This is demonstrated by a drastically different distribution of repetitive DNA elements in the Hox cluster regions. While gnathostomes have a strong tendency to exclude repetitive DNA elements from the inside of their Hox clusters, no such trend can be detected in the Hox gene clusters of protostomes. Repeats "invade" the gnathostome Hox clusters from the 5' and 3' ends while the core of the clusters remains virtually free of repetitive DNA. This invasion appears to be correlated with relaxed constraints associated with gene loss after cluster duplications.