Petr Halada

Hepcidin, the hormone of iron metabolism, is bound specifically to α-2-macroglobulin in blood

Hepcidin is a major regulator of iron metabolism. Hepcidin-based therapeutics/diagnostics could play roles in hematology in the future, and thus, hepcidin transport is crucial to understand. In this study, we identify alpha2-macroglobulin (alpha2-M) as the specific hepcidin-binding molecule in blood. Interaction of 125I-hepcidin with alpha2-M was identified using fractionation of plasma proteins followed by native gradient polyacrylamide gel electrophoresis and mass spectrometry. Hepcidin binding to nonactivated alpha2-M displays high affinity (Kd 177 +/- 27 nM), whereas hepcidin binding to albumin was nonspecific and displayed nonsaturable kinetics. Surprisingly, the interaction of hepcidin with activated alpha2-M exhibited a classical sigmoidal binding curve demonstrating cooperative binding of 4 high-affinity (Kd 0.3 microM) hepcidin-binding sites. This property probably enables efficient sequestration of hepcidin and its subsequent release or inactivation that may be important for its effector functions. Because alpha2-M rapidly targets ligands to cells via receptor-mediated endocytosis, the binding of hepcidin to alpha2-M may influence its functions. In fact, the alpha2-M-hepcidin complex decreased ferroportin expression in J774 cells more effectively than hepcidin alone. The demonstration that alpha2-M is the hepcidin transporter could lead to better understanding of hepcidin physiology, methods for its sensitive measurement and the development of novel drugs for the treatment of iron-related diseases.

Characteristics of Gloeophyllum trabeum Alcohol Oxidase, an Extracellular Source of H2O2 in Brown Rot Decay of Wood

ABSTRACT A novel alcohol oxidase (AOX) has been purified from mycelial pellets of the wood-degrading basidiomycete Gloeophyllum trabeum and characterized as a homooctameric nonglycosylated protein with native and subunit molecular masses of 628 and 72.4 kDa, containing noncovalently bonded flavin adenine dinucleotide. The isolated AOX cDNA contained an open reading frame of 1,953 bp translating into a polypeptide of 651 amino acids displaying 51 to 53% identity with other published fungal AOX amino acid sequences. The enzyme catalyzed the oxidation of short-chain primary aliphatic alcohols with a preference for methanol (Km = 2.3 mM, kcat = 15.6 s−1). Using polyclonal antibodies and immunofluorescence staining, AOX was localized on liquid culture hyphae and extracellular slime in sections from degraded wood and on cotton fibers. Transmission electron microscopy immunogold labeling localized the enzyme in the hyphal periplasmic space and wall and on extracellular tripartite membranes and slime, while there was no labeling of hyphal peroxisomes. AOX was further shown to be associated with membranous or slime structures secreted by hyphae in wood fiber lumina and within the secondary cell walls of degraded wood fibers. The differences in AOX targeting compared to the known yeast peroxisomal localization were traced to a unique C-terminal sequence of the G. trabeum oxidase, which is apparently responsible for the proteins different translocation. The extracellular distribution and the enzymes abundance and preference for methanol, potentially available from the demethylation of lignin, all point to a possible role for AOX as a major source of H2O2, a component of Fentons reagent implicated in the generally accepted mechanisms for brown rot through the production of highly destructive hydroxyl radicals.

Protein Patterns of Pig Oocytes During In Vitro Maturation

Abstract In vitro maturation (IVM) of fully grown mammalian oocytes is characterized by initial germinal vesicle (GV) breakdown and rearrangement of microtubule network during the first meiosis (MI), followed by extrusion of the first polar body and block of the oocytes in metaphase of the second meiosis (MII). Only fully matured oocytes are capable of undergoing fertilization and the initiation of zygotic development. These observations are mostly based on morphological evaluation; however, the molecular events responsible for these processes are not known. In this study, we have launched the analysis of pig oocytes during in vitro maturation using a proteomics approach. First, oocyte proteins have been separated by two-dimensional gel electrophoresis and identified by mass spectrometry. Remarkably, several proteins, including peroxiredoxins, ubiquitin carboxyl-terminal hydrolase isozyme L1, and spermine synthase, are even more abundant than actin, usually the most abundant protein in somatic cells. Furthermore, we have initiated comparative analysis of the oocytes at different stages of maturation to characterize candidate proteins, which are differentially expressed during in vitro maturation. To date, we have identified antiquitin (D7A1), the member of aldehyde dehydrogenase family7 that has been significantly increased in MI and MII stages compared with GV oocytes. To our knowledge, this is the first pig oocyte proteome available so far that may be used as a reference map. The proteins that are differentially regulated during IVM may present potential biomarkers of oocyte maturation and quality. It is a useful inventory toward a deeper understanding of the mechanisms underlying reproduction and development.

Proteome Mining of Human Follicular Fluid Reveals a Crucial Role of Complement Cascade and Key Biological Pathways in Women Undergoing in Vitro Fertilization

In vitro fertilization (IVF) is fraught with problems and currently proteomics approaches are being tried out to examine the microenvironment of the follicle in order to assess biological and immunological parameters that may affect its development. Additionally, better understanding of reproductive process may help increase IVF birth rate per embryo transfer and at the same time avoid spontaneous miscarriages or life threatening conditions such as ovarian hyperstimulation syndrome. The primary aim of this study was to search for specific differences in protein composition of human follicular fluid (HFF) and plasma in order to identify proteins that accumulate or are absent in HFF. Depletion of abundant proteins combined with multidimensional protein fractionation allowed the study of middle- and lower-abundance proteins. Paired comparison study examining HFF with plasma/serum from women undergoing successful IVF revealed important differences in the protein composition which may improve our knowledge of the follicular microenvironment and its biological role. This study showed involvement of innate immune function of complement cascade in HFF. Complement inhibition and the presence of C-terminal fragment of perlecan suggested possible links to angiogenesis which is a vital process in folliculogenesis and placental development. Differences in proteins associated with blood coagulation were also found in the follicular milieu. Several specific proteins were observed, many of which have not yet been associated with follicle/oocyte maturation. These proteins together with their regulatory pathways may play a vital role in the reproductive process.

Identification of the covalent flavin adenine dinucleotide-binding region in pyranose 2-oxidase from Trametes multicolor.

We present the first report on characterization of the covalent flavinylation site in flavoprotein pyranose 2-oxidase. Pyranose 2-oxidase from the basidiomycete fungus Trametes multicolor, catalyzing C-2/C-3 oxidation of several monosaccharides, shows typical absorption maxima of flavoproteins at 456, 345, and 275 nm. No release of flavin was observed after protein denaturation, indicating covalent attachment of the cofactor. The flavopeptide fragment resulting from tryptic/chymotryptic digestion of the purified enzyme was isolated by anion-exchange and reversed-phase high-performance liquid chromatography. The flavin type, attachment site, and mode of its linkage were determined by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy of the intact flavopeptide, without its prior enzymatic degradation to the central aminoacyl moiety. Mass spectrometry identified the attached flavin as flavin adenine dinucleotide (FAD). Post-source decay analysis revealed that the flavin is covalently bound to histidine residue in the peptide STHW, consistent with the results of N-terminal amino acid sequencing by Edman degradation. The type of the aminoacyl flavin covalent link was determined by NMR spectroscopy, resulting in the structure 8alpha-(N(3)-histidyl)-FAD.

Proteomics approach in classifying the biochemical basis of the anticancer activity of the new olomoucine-derived synthetic cyclin-dependent kinase inhibitor, bohemine.

The aim of this study was to use two‐dimensional electrophoresis (2‐DE) coupled with multivariate principal component analysis (PCA) to characterize the quantitative changes in the protein composition of the CEM T‐lymphoblastic leukemia cell line after treatment with bohemine (BOH), a synthetic olomoucin‐derived cyclin‐dependent kinase inhibitor (CDKI). Cell classification, reflecting protein patterns, clearly distinguished two main groups: one group consists of 9, 12 and 24 h treated BOH cells while the second is represented by the 0 and 24 h control untreated cells and the 6 h BOH‐exposed CEM lymphoblasts. Discriminant protein spots differentially expressed in the BOH‐treated CEM cells were selected for identification by matrix assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS) or electrospray ionization‐tandem MS (ESI‐MS/MS). Five of the selected protein spots were unequivocally identified as α‐enolase, triosephosphate isomerase, eukaryotic initiation factor 5A, and α‐ and β‐subunits of Rho GDP‐dissociation inhibitor 1. These proteins, all significantly downregulated in CEM T‐lymphoblast leukemia in the course of BOH treatment, are known to play an important role in cellular functions such as glycolysis, protein biosynthesis, and cytoskeleton rearrangement. These results indicate that the cellular effects of olomoucine‐derived CDKIs are not dependent on their ability to inhibit CDKs and could be mediated by several factors such as a decrease in protein synthesis and/or glycolysis which in turn diminishes the ability of cancer cells to function.

The Conserved Lysine 860 in the Additional Fatty-acylation Site of Bordetella pertussis Adenylate Cyclase Is Crucial for Toxin Function Independently of Its Acylation Status

The Bordetella pertussis RTX (repeat in toxin family protein) adenylate cyclase toxin-hemolysin (ACT) acquires biological activity upon a single amide-linked palmitoylation of the ε-amino group of lysine 983 (Lys983) by the accessory fatty-acyltransferase CyaC. However, an additional conserved RTX acylation site can be identified in ACT at lysine 860 (Lys860), and this residue becomes palmitoylated when recombinant ACT (r-Ec-ACT) is produced together with CyaC in Escherichia coli K12. We have eliminated this additional acylation site by replacing Lys860 of ACT with arginine, leucine, and cysteine residues. Two-dimensional gel electrophoresis and microcapillary high performance liquid chromatography/tandem mass spectrometric analyses of mutant proteins confirmed that the two sites are acylated independently in vivo and that mutations of Lys860 did not affect the quantitative acylation of Lys983 by palmitoyl (C16:0) and palmitoleil (cis Δ9 C16:1) fatty-acyl groups. Nevertheless, even the most conservative substitution of lysine 860 by an arginine residue caused a 10-fold decrease of toxin activity. This resulted from a 5-fold reduction of cell association capacity and a further 2-fold reduction in cell penetration efficiency of the membrane-bound K860R toxin. These results suggest that lysine 860 plays by itself a crucial structural role in membrane insertion and translocation of the toxin, independently of its acylation status.

Properties of pyranose dehydrogenase purified from the litter‐degrading fungus Agaricus xanthoderma

We purified an extracellular pyranose dehydrogenase (PDH) from the basidiomycete fungus Agaricus xanthoderma using ammonium sulfate fractionation and ion‐exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric glycoprotein (5% carbohydrate) containing a covalently bound FAD as its prosthetic group. The PDH polypeptide consists of 575 amino acids and has a molecular mass of 65 400 Da as determined by MALDI MS. On the basis of the primary structure of the mature protein, PDH is a member of the glucose–methanol–choline oxidoreductase family. We constructed a homology model of PDH using the 3D structure of glucose oxidase from Aspergillus niger as a template. This model suggests a novel type of bi‐covalent flavinylation in PDH, 9‐S‐cysteinyl, 8‐α‐N3‐histidyl FAD. The enzyme exhibits a broad sugar substrate tolerance, oxidizing structurally different aldopyranoses including monosaccharides and oligosaccharides as well as glycosides. Its preferred electron donor substrates are d‐glucose, d‐galactose, l‐arabinose, and d‐xylose. As shown by in situ NMR analysis, d‐glucose and d‐galactose are both oxidized at positions C2 and C3, yielding the corresponding didehydroaldoses (diketoaldoses) as the final reaction products. PDH shows no detectable activity with oxygen, and its reactivity towards electron acceptors is rather limited, reducing various substituted benzoquinones and complexed metal ions. The azino‐bis‐(3‐ethylbenzthiazolin‐6‐sulfonic acid) cation radical and the ferricenium ion are the best electron acceptors, as judged by the catalytic efficiencies (kcat/Km). The enzyme may play a role in lignocellulose degradation.

Aspartic protease nepenthesin-1 as a tool for digestion in hydrogen/deuterium exchange mass spectrometry.

Hydrogen/deuterium exchange coupled to mass spectrometry (HXMS) utilizes enzymatic digestion of proteins to localize the information about altered exchange patterns in protein structure. The ability of the protease to produce small peptides and overlapping fragments and provide sufficient coverage of the protein sequence is essential for localizing regions of interest. Recently, it was shown that there is an interesting group of proteolytic enzymes from carnivorous pitcher plants of the genus Nepenthes. In this report, we describe successful immobilization and the use of one of these enzymes, nepenthesin-1, in HXMS workflow. In contrast to pepsin, it has different cleavage specificities, and despite its high inherent susceptibility to reducing and denaturing agents, it is very stable upon immobilization and withstands even high concentration of guanidine hydrochloride and reducing agents. We show that denaturing agents can alter digestion by reducing protease activity and/or substrate solubility, and additionally, they influence the trapping of proteolytic peptides onto the reversed phase resin.

Construction of a Francisella tularensis two-dimensional electrophoresis protein database.

We have started the construction of a two‐dimensional database of the proteome of Francisella tularensis, a bacterium that is responsible for the highly pathogenic disease tularemia. The genome of this intracellular pathogen is not completely sequenced yet and, currently, information about only 66 proteins is available from NCBI database. We have analyzed the F. tularensis live vaccine strain by two‐dimensional gel electrophoresis with immobilized pH 3–10 gradient in the first dimension and 9–16% gradient or tricine SDS‐PAGE in the second dimension. In both cases about 2000 spots were detected. Furthermore, we compared the protein pattern of the nonvirulent F. tularensis live vaccine strain with protein profiles of two wild type clinical isolates and more than 50 differentially expressed proteins were counted. The separated proteins are going to be identified by peptide mass fingerprinting. However, due to the lack of complete genome sequence data only eight proteins were unambiguously identified. Among them, acid phosphatase and the most basic isoform of a hypothetical 23 kDa protein are characteristic only for virulent strains.