Lipidome analysis of rotavirus-infected cells confirms the close interaction of lipid droplets with viroplasms

Rotaviruses (RVs) cause acute gastroenteritis in infants and young children, and are globally distributed. Within the infected host cell, RVs establish replication complexes in viroplasms (‘viral factories’) to which lipid droplet organelles are recruited. To further understand this recently discovered phenomenon, the lipidomes of RV-infected and uninfected MA104 cells were investigated. Cell lysates were subjected to equilibrium ultracentrifugation through iodixanol gradients. Fourteen different classes of lipids were differentiated by mass spectrometry. The concentrations of virtually all lipids were elevated in RV-infected cells. Fractions of low density (1.11–1.15 g ml−1), in which peaks of the RV dsRNA genome and lipid droplet- and viroplasm-associated proteins were observed, contained increased amounts of lipids typically found concentrated in the cellular organelle lipid droplets, confirming the close interaction of lipid droplets with viroplasms. A decrease in the ratio of the amounts of surface to internal components of lipid droplets upon RV infection suggested that the lipid droplet–viroplasm complexes became enlarged.

Structural and Functional Variation within the Alanine-Rich Repetitive Domain of Streptococcal Antigen I/II

ABSTRACT Members of the antigen I/II family of cell surface proteins are highly conserved, multifunctional adhesins that mediate interactions of oral streptococci with other oral bacteria, with cell matrix proteins (e.g., type I collagen), and with salivary glycoproteins, e.g., gp340. The interaction of gp340 (formerly designated salivary agglutinin) with Streptococcus mutans requires an alanine-rich repetitive domain (A region) of antigen I/II that is highly conserved in all members of this family of proteins. In this report, we show that the A regions from the two Streptococcus gordonii M5 antigen I/II proteins (SspA and SspB) interact differently with the salivary gp340 glycoprotein and appear to be structurally distinct. Recombinant polypeptides encompassing the A region of SspA or from a highly related S. mutans antigen I/II protein (SpaP) competitively inhibited the interaction of gp340 with intact S. gordonii and S. mutans cells, respectively. In contrast, an A region polypeptide from SspB was inactive, and furthermore, it did not bind to purified gp340 in vitro. Circular dichroism spectra suggested that all three polypeptides were highly α-helical and may form coiled-coil structures. However, the A region of SspB underwent a conformational change and exhibited reduced α-helical structure at pH 8.5, whereas the A region polypeptides from SspA and SpaP were relatively stable under these conditions. Melt curves also indicated that at physiological pH, the A region of SspB lost α-helical structure more rapidly than that of SspA or SpaP when the temperature was increased from 10 to 40°C. Furthermore, the SspB A region polypeptide denatured completely at a temperature that was 7 to 9°C lower than that required for the A region polypeptide of SspA or SpaP. The full-length SspB protein and the three A region peptides migrated in native gel electrophoresis and column chromatography with apparent molecular masses that were approximately 2- to 2.5-fold greater than their predicted molecular masses. However, sedimentation equilibrium ultracentrifugation data showed that the A region peptides sedimented as monomers, suggesting that the peptides may form nonglobular intramolecular coiled-coil structures under the experimental conditions used. Taken together, our results suggest that the A region of SspB is less stable than the corresponding A regions of SspA and SpaP and that this structural difference may explain, at least in part, the functional variation observed in their interactions with salivary gp340.

Purification and Characterization of Pyranose Oxidase from the White Rot Fungus Trametes multicolor

ABSTRACT We purified an intracellular pyranose oxidase from mycelial extracts of the white rot fungus Trametes multicolor by using ammonium sulfate fractionation, hydrophobic interaction, ion-exchange chromatography, and gel filtration. The native enzyme has a molecular mass of 270 kDa as determined by equilibrium ultracentrifugation and is composed of four identical 68-kDa subunits as determined by matrix-assisted laser desorption ionization mass spectrometry. Each subunit contains one covalently bound flavin adenine dinucleotide as its prosthetic group. The enzyme oxidizes several aldopyranoses specifically at position C-2, and its preferred electron donor substrates are d-glucose,d-xylose, and l-sorbose. During this oxidation reaction electrons are transferred to oxygen, yielding hydrogen peroxide. In addition, the enzyme catalyzes the two-electron reduction of 1,4-benzoquinone, several substituted benzoquinones, and 2,6-dichloroindophenol, as well as the one-electron reduction of the ABTS [2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid)] cation radical. As judged by the catalytic efficiencies (k cat/Km ), some of these quinone electron acceptors are much better substrates for pyranose oxidase than oxygen. The optimum pH of the pyranose oxidase-catalyzed reaction depends strongly on the electron acceptor employed and varies from 4 to 8. It has been proposed that the main metabolic function of pyranose oxidase is as a constituent of the ligninolytic system of white rot fungi that provides peroxidases with H2O2. An additional function could be reduction of quinones, key intermediates that are formed during mineralization of lignin.

Complex formation between deoxyhypusine synthase and its protein substrate, the eukaryotic translation initiation factor 5A (eIF5A) precursor

Deoxyhypusine synthase catalyses the first step in the post-translational synthesis of hypusine [Nϵ-(4-amino-2-hydroxybutyl) lysine] in a single cellular protein, the precursor of eukaryotic initiation factor 5A (eIF5A). Deoxyhypusine synthase exists as a tetramer with four potential active sites. The formation of a stable complex between human deoxyhypusine synthase and its protein substrate, human recombinant eIF5A precursor (ec-eIF5A), was examined by affinity chromatography using polyhistidine-tagged (His·Tag) ec-eIF5A, by a gel mobility-shift method, and by analytical ultracentrifugation. Deoxyhypusine synthase was selectively retained by His·Tag-ec-eIF5A immobilized on a resin. The complex of deoxyhypusine synthase and ec-eIF5A was separated from the free enzyme and protein substrate by electrophoresis under non-denaturing conditions. The stoichiometry of the two components in the complex was estimated to be 1 deoxyhypusine synthase tetramer to 1 ec-eIF5A monomer by N-terminal amino acid sequencing of the complex. Equilibrium ultracentrifugation data further supported this 1:1 ratio and indicated a very strong interaction of the enzyme with ec-eIF5A (Kd≤ 0.5 nM). Formation of the complex was not dependent on NAD+ or spermidine and occurred at pH 7.0-9.2. An enzyme-product complex, as well as the deoxyhypusine-containing product (modified ec-eIF5A), was also detected at pH 7.0-9.2 in a complete reaction mixture containing 1 mM spermidine.

Homodimerization and hetero-oligomerization of the single-domain trefoil protein pNR-2/pS2 through cysteine 58

The single-domain human trefoil proteins [pNR-2/pS2 and human intestinal trefoil factor (hITF)] have seven cysteine residues, of which six are involved in maintaining the structure of the trefoil domain. The seventh does not form part of the trefoil domain and is located three residues from the C-terminus. The ability of the pNR-2/pS2 single trefoil domain protein to dimerize was examined by using recombinant protein with either a cysteine or a serine residue at this position by equilibrium ultracentrifugation, laser-assisted desorption MS, gel filtration and PAGE. pNR-2/pS2 Cys58 formed dimers, whereas pNR-2/pS2 Ser58 did not. Experiments in which the dimer was treated with thiol agents demonstrated that the dimer was linked via a disulphide bond and that the intermolecular disulphide bond was more susceptible to reduction than the intramolecular disulphide bonds. To examine whether dimeric pNR-2/pS2 was secreted by oestrogen-responsive breast cancer cells, which are known to express pNR-2/pS2 mRNA, conditioned medium was separated on non-denaturing polyacrylamide gels, transferred to PVDF membrane and reacted with antiserum against pNR-2/pS2. Monomeric and dimeric pNR-2/pS2 were detected but the majority of the protein reactivity was associated with a larger protein. Treatment of this protein with thiol agents suggested that it is an oligomer containing pNR-2/pS2 linked to another protein by a disulphide bond. These studies suggest that the biological action of pNR-2/pS2 single-domain trefoil protein might involve the formation of homodimers or oligomers with other proteins.