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We used linker histone-depleted normal human fibroblast nuclei as templates to study how phosphorylation affects histone H5 binding to chromatin in situ. Permeabilized cells were treated with 0.7 M NaCl to extract the native linker histones. Histone H5 was purified from chicken erythrocytes and phosphorylated in vitro by recombinant cdk5/p35 kinase. High performance capillary electrophoresis (HPCE) showed that the phosphorylated protein contained a mixture of multiply phosphorylated forms. Control experiments, using mass spectrometry, revealed that up to five SPXK motifs in the C terminus were phosphorylated, but also that about 10% of the protein contained one phosphoserine in the N-terminus. Reconstitution of H1-depleted fibroblast nuclei with nonphosphorylated or phosphorylated H5 was performed at physiological ionic strength. The bound H5 was then extracted using NaCl concentrations in the range of 0.15 to 0.7 M. The release of the H5 molecules was monitored by DAPI staining and image cytofluorometry. Our results show that H5 phosphorylation substantially reduced its affinity for chromatin in situ, which support previous observations indicating that C-terminal phosphorylation may be essential for the biological functions of linker histones. © 2012 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

Protein C inhibitor (PCI) is a serpin with broad protease reactivity. It binds glycosaminoglycans and certain phospholipids that can modulate its inhibitory activity. PCI can penetrate through cellular membranes via binding to phosphatidylethanolamine. The exact mechanism of PCI internalization and the intracellular role of the serpin are not well understood. Here we showed that testisin, a glycosylphosphatidylinositol-anchored serine protease, cleaved human PCI and mouse PCI (mPCI) at their reactive sites as well as at sites close to their N terminus. This cleavage was observed not only with testisin in solution but also with cell membrane-anchored testisin on U937 cells. The cleavage close to the N terminus released peptides rich in basic amino acids. Synthetic peptides corresponding to the released peptides of human PCI (His¹-Arg11) and mPCI (Arg¹-Ala18) functioned as cell-penetrating peptides. Because intact mPCI but not testisin-cleaved mPCI was internalized by Jurkat T cells, a truncated mPCI mimicking testisin-cleaved mPCI was created. The truncated mPCI lacking 18 amino acids at the N terminus was not taken up by Jurkat T cells. Therefore our model suggests that testisin or other proteases could regulate the internalization of PCI by removing its N terminus. This may represent one of the mechanisms regulating the intracellular functions of PCI. [ABSTRACT FROM AUTHOR]

Abstract: The Transient Receptor Potential Vanilloid 1 (TRPV1, vanilloid receptor 1) ion channel plays a key role in the perception of thermal and inflammatory pain, however, its molecular environment in dorsal root ganglia (DRG) is largely unexplored. Utilizing a panel of sequence-directed antibodies against TRPV1 protein and mouse DRG membranes, the channel complex from mouse DRG was detergent-solubilized, isolated by immunoprecipitation and subsequently analyzed by mass spectrometry. A number of potential TRPV1 interaction partners were identified, among them cytoskeletal proteins, signal transduction molecules, and established ion channel subunits. Based on stringent specificity criteria, the voltage-gated K+ channel beta 2 subunit (Kvβ2), an accessory subunit of voltage-gated K+ channels, was identified of being associated with native TRPV1 channels. Reverse co-immunoprecipitation and antibody co-staining experiments confirmed TRPV1/Kvβ2 association. Biotinylation assays in the presence of Kvβ2 demonstrated increased cell surface expression levels of TRPV1, while patch-clamp experiments resulted in a significant increase of TRPV1 sensitivity to capsaicin. Our work shows, for the first time, the association of a Kvβ subunit with TRPV1 channels, and suggests that such interaction may play a role in TRPV1 channel trafficking to the plasma membrane. [Copyright &y& Elsevier]

ICln is a multifunctional protein involved in regulatory mechanisms as different as membrane ion transport and RNA splicing. The protein is water-soluble, and during regulatory volume decrease after cell swelling, it is able to migrate from the cytosol to the cell membrane. Purified, water-soluble ICln is able to insert into lipid bilayers to form ion channels. Here, we show that ICln159, a truncated ICln mutant, which is also able to form ion channels in lipid bilayers, belongs to the pleckstrin homology (PH) domain superfold family of proteins. The ICln PH domain shows unusual properties as it lacks the electrostatic surface polarization seen in classical PH domains. However, similar to many classical PH domain-containing proteins, ICln interacts with protein kinase C, and in addition, interacts with cAMP-dependent protein kinase and cGMP-dependent protein kinase type II but not cGMP-dependent protein kinase type Iβ. A major phosphorylation site for all three kinases is Ser-45 within the ICln PH domain. Furthermore, ICln159 interacts with LSm4, a protein involved in splicing and mRNA degradation, suggesting that the ICln159 PH domain may serve as a protein-protein interaction platform. [ABSTRACT FROM AUTHOR]

Aspergillus fumigatus is the predominant mould pathogen for humans. Adaption to host-imposed iron limitation has previously been demonstrated to be essential for its virulence. [2Fe-2S] clusters are crucial as cofactors of several metabolic pathways and mediate cytosolic/nuclear iron sensing in fungi including A. fumigatus . [2Fe-2S] cluster trafficking has been shown to involve BolA family proteins in both mitochondria and the cytosol/nucleus. Interestingly, both A. fumigatus homologues, termed Bol1 and Bol3, possess mitochondrial targeting sequences, suggesting the lack of cytosolic/nuclear versions. Here, we show by the combination of mutational, proteomic and fluorescence microscopic analyses that expression of the Bol3 encoding gene leads to dual localization of gene products to mitochondria and the cytosol/nucleus via alternative translation initiation downstream of the mitochondrial targeting sequence, which appears to be highly conserved in various Aspergillus species. Lack of either mitochondrial Bol1 or Bol3 was phenotypically inconspicuous while lack of cytosolic/nuclear Bol3 impaired growth during iron limitation but not iron sensing which indicates a particular importance of [2Fe-2S] cluster trafficking during iron limitation. Remarkably, cytosolic/nuclear Bol3 differs from the mitochondrial version only by N-terminal acetylation, a finding that was only possible by mutational hypothesis testing.

N-Chlorotaurine sodium (NCT) is a promising microbicidal agent for topical treatment of infections. Its targets of attack in Escherichia coli have been investigated by proteomics. Incubation in 1% NCT for 10 and 30 min revealed a change of the charge and a separation of numerous proteins into a series of spots with a different pI. Charge differences could be related to oxidation of cysteine residues to their corresponding sulfonic acids. Heat shock protein 60 appeared, while ribosome-releasing factor, d-ribose periplasmic binding protein, and malonyl-CoA transacylase spots decreased. These results indicate penetration of oxidation capacity into the bacteria and destruction of essential proteins by NCT.

Ovarian cancer remains still associated with poor prognosis because it is diagnosed predominantly at advanced stages. Ovarian-specific tumor markers do not yet exist for early detection of the disease. At the search of diagnostic markers for ovarian cancer, proteomic-based approaches have focused on novel investigations of neoplastic processes in tumor patients. Cystic fluids of malignant and benign ovarian tumors and serum from the corresponding patients were collected and processed for two-dimensional gel electrophoresis. Proteins were visualized on the gels by silver staining. At the low molecular mass level between 10 and 20 kDa, selected protein spots were additionally processed for nanospray mass spectrometry and partial amino acid sequencing. For protein identification, the sequencing results were compared with computer information from a protein data bank. Protein patterns from cystic fluids of ovarian carcinomas differed significantly from those of benign cysts and revealed additional polypeptides at low molecular mass level between 10 and 20 kDa. Protein patterns from serum of patients with malignant ovarian tumors also contained additional polypeptides between 10 and 20 kDa that were not detected in serum from patients with benign cysts. The additional proteins in serum were present in similar electrophoretic positions compared with those found in the cystic fluid of the corresponding ovarian carcinomas. Protein spots in the range of 10-20 kDa were selected for partial amino acid sequencing. Two protein spots were identified as calgranulin A and three spots as calgranulin B. Either both proteins or only calgranulin A or B were present in cystic fluid from ovarian carcinomas and serum of the corresponding patients. These two proteins were absent or not detectable in fluid from benign ovarian cysts and in serum from those patients. Our investigations concerning protein patterns in cystic fluid of malignant and benign ovarian tumors provide new information about alterations in protein synthesis linked to neoplastic events of the ovary. With the proteomic strategy, new tumor markers are characterized and may serve for diagnostic purposes of patients with ovarian cancer.

A maize histone deacetylase gene was identified as a homolog of yeast Hda1. The predicted protein corresponds to a previously purified maize deacetylase that is active as a protein monomer with a molecular weight of 48,000 and is expressed in all tissues of germinating embryos. Hda1 is synthesized as an enzymatically inactive protein with an apparent molecular weight of 84,000 that is processed to the active 48-kD form by proteolytic removal of the C-terminal part, presumably via a 65-kD intermediate. The enzymatically inactive 84-kD protein also is part of a 300-kD protein complex of unknown function. The proteolytic cleavage of ZmHda1 is regulated during maize embryo germination in vivo. Expression of the recombinant full-length protein and the 48-kD form confirmed that only the smaller enzyme form is active as a histone deacetylase. In line with this finding, we show that the 48-kD protein is able to repress transcription efficiently in a reporter gene assay, whereas the full-length protein, including the C-terminal part, lacks full repression activity. This report on the processing of Hda1-p84 to enzymatically active Hda1-p48 demonstrates that proteolytic cleavage is a mechanism to regulate the function of Rpd3/Hda1-type histone deacetylases.

Histones are the most intensively studied group of basic nuclear proteins and are of great importance with regard to the organization of chromatin structure and control of gene activity. The complexity of this protein family places very high demands on the analytical methods applied. The advent of high-performance capillary electrophoresis provided a promising new tool for their separation, which is an essential prerequisite for studying the biological role of this protein family. Problems inherent to histone analysis due to their unique physical and chemical properties are reviewed, and the pros and cons of distinct strategies developed for CE separation of these proteins are discussed.

We have recently reported that anti-SLA seropositive autoimmune hepatitis (AIH) patients develop autoantibodies against glutathione S-transferase (GST). GSTs are multifunctional enzymes mediating hepatic detoxification of cytotoxic and genotoxic compounds and are also involved in biliary secretion. We have observed varying reactivity of individual AIH sera towards several GST isoenzymes. Since the GST subunits have very similar molar masses and therefore are not satisfactorily resolved by one-dimensional gel electrophoresis, we have performed their fractionation by reverse-phase high performance liquid chromatography (HPLC) to better separate the individual GST isoenzymes. 4 individual GST subunits were isolated as judged by electrophoretic analysis of the 4 distinct peaks. The identity of isolated proteins was unequivocally determined by protein sequencing. Isolated subtypes were loaded on 15% SDS gels and blotted. Immunoblotting was performed with eleven anti-SLA positive sera that displayed differential reactivity with total GSTs. Fractionation of the GSTs by HPLC did not impair their ability to react with specific autoantibodies. Interestingly, the majority of GST-positive AIH sera reacted with one or two GST subtypes, only two sera recognized 3 subunits. Ya was most prevalent autoantigen. Autoantibodies against Yb2 were detected solely in one serum. This pattern of reactivity indicates that individual patients' sera discriminate between GST subunits despite their sequence homology. It is well known that the GST variants differ within their amino-terminal part while the residual moiety is highly conserved. It would suggest that autoantibodies recognize distinct epitopes located within amino-terminus of individual GST variants.