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.     

Cutting edge: culture with high doses of viral peptide induces previously unprimed CD8(+) T cells to produce cytokine

Culturing naive T cells with 50 microM selected HIV-1 envelope peptides for 6 days in the presence of IL-2 drives the emergence of a substantial CD8(+) population that secretes IFN-gamma following short-term stimulation with 1 microM peptide. This response is H-2K(b) restricted, epitope specific, and requires the continuing presence of peptide. The same effect was found for known H-2D(b)-restricted peptides from two influenza virus proteins. The great majority of these influenza-specific CD8(+)IFN-gamma(+) T cells neither stained with the cognate tetramer nor expressed the TCR Vbeta bias that is characteristic of the CD8(+) set expanded in vivo during an infection. Thus, multipoint binding of low affinity TCRs on naive CD8(+) T cells can drive peptide-specific cytokine production. However, at least for two influenza-derived epitopes, the avidity of the TCR-MHC peptide interaction appears to be insufficient to stabilize a tetrameric complex of MHC class I glycoprotein plus peptide on the lymphocyte surface.     

Identification of MHC class II-associated peptides that promote the presentation of toxic shock syndrome toxin-1 to T cells

Previous studies have shown that the DM-deficient cell line, T2-I-A(b), is very inefficient at presenting toxic shock syndrome toxin 1 (TSST-1) to T cells, suggesting that I-A(b)-associated peptides play an essential role in the presentation of this superantigen. Consistent with this, the loading of an I-A(b)-binding peptide, staphylococcal enterotoxin B 121-136, onto T2-I-A(b) cells enhanced TSST-1 presentation >1000-fold. However, despite extensive screening, no other peptides have been identified that significantly promote TSST-1 presentation. In addition, the peptide effect on TSST-1 presentation has been demonstrated only in the context of the tumor cell line T2-I-A(b). Here we show that peptides that do not promote TSST-1 presentation can be converted into "promoting" peptides by the progressive truncation of C-terminal residues. These studies result in the identification of two peptides derived from IgGV heavy chain and I-Ealpha proteins that are extremely strong promoters of TSST-1 presentation (47,500- and 12,000-fold, respectively). We have also developed a system to examine the role of MHC class II-associated peptides in superantigen presentation using splenic APC taken directly ex vivo. The data confirmed that the length of the MHC class II-bound peptide plays a critical role in the presentation of TSST-1 by splenic APC and showed that different subpopulations of APC are equally peptide dependent in TSST-1 presentation. Finally, we demonstrated that the presentation of staphylococcal enterotoxin A, like TSST-1, is peptide dependent, whereas staphylococcal enterotoxin B presentation is peptide independent.     

A chimeric human-bovine parainfluenza virus type 3 expressing measles virus hemagglutinin is attenuated for replication but is still immunogenic in rhesus monkeys

The chimeric recombinant virus rHPIV3-N(B), a version of human parainfluenza virus type 3 (HPIV3) that is attenuated due to the presence of the bovine PIV3 nucleocapsid (N) protein open reading frame (ORF) in place of the HPIV3 ORF, was modified to encode the measles virus hemagglutinin (HA) inserted as an additional, supernumerary gene between the HPIV3 P and M genes. This recombinant, designated rHPIV3-N(B)HA, replicated like its attenuated rHPIV3-N(B) parent virus in vitro and in the upper and lower respiratory tracts of rhesus monkeys, indicating that the insertion of the measles virus HA did not further attenuate rHPIV3-N(B) in vitro or in vivo. Monkeys immunized with rHPIV3-N(B)HA developed a vigorous immune response to both measles virus and HPIV3, with serum antibody titers to both measles virus (neutralizing antibody) and HPIV3 (hemagglutination inhibiting antibody) of over 1:500. An attenuated HPIV3 expressing a major protective antigen of measles virus provides a method for immunization against measles by the intranasal route, a route that has been shown with HPIV3 and respiratory syncytial virus vaccines to be relatively refractory to the neutralizing and immunosuppressive effects of maternally derived virus-specific serum antibodies. It should now be possible to induce a protective immune response against measles virus in 6-month-old infants, an age group that in developing areas of the world is not responsive to the current measles virus vaccine.     

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.