Insight into the proteome of the hyperthermophilic Crenarchaeon Ignicoccus hospitalis: the major cytosolic and membrane proteins

Ignicoccus hospitalis, a hyperthermophilic, chemolithoautotrophic Crenarchaeon, is the host of Nanoarchaeum equitans. Together, they form an intimate association, the first among Archaea. Membranes are of fundamental importance for the interaction of I. hospitalis and N. equitans, as they harbour the proteins necessary for the transport of macromolecules like lipids, amino acids, and cofactors between these organisms. Here, we investigated the protein inventory of I. hospitalis cells, and were able to identify 20 proteins in total. Experimental evidence and predictions let us conclude that 11 are soluble cytosolic proteins, eight membrane or membrane-associated proteins, and a single one extracellular. The quantitatively dominating proteins in the cytoplasm (peroxiredoxin; thermosome) antagonize oxidative and temperature stress which I. hospitalis cells are exposed to at optimal growth conditions. Three abundant membrane protein complexes are found: the major protein of the outer membrane, which might protect the cell against the hostile environment, forms oligomeric complexes with pores of unknown selectivity; two other complexes of the cytoplasmic membrane, the hydrogenase and the ATP synthase, play a key role in energy production and conversion

Absorption and Emission Spectroscopic Characterization of 10-Phenyl-Isoalloxazine Derivatives

The flavoquinone dyes 10-phenyl-isoalloxazine-3-acetic acid ethyl ester (1) and 10-(4-bromo-phenyl)-3-methyl-isoalloxazine (2) in dichloromethane, acetonitrile, and methanol are characterized by absorption and emission spectroscopy. Absorption cross-section spectra, stimulated emission cross-section spectra, fluorescence quantum distributions, quantum yields, lifetimes, and degrees of fluorescence polarization are determined. The blue-light photo-degradation of the dyes is studied. Mass spectroscopic measurements reveal the formation of phenyl-benzo-pteridine (isoalloxazine) derivatives, tetraaza-benzo-aceanthrylene derivatives, dihydro-quinooxaline derivatives, and pyrazino-carbazole derivatives. An enhancement of photo-degradation is observed by the formed photo-fragments

Shotgun Proteomics of Human Dentin with Different Prefractionation Methods

Abstract Human dentin is not only a composite material of a collagenous matrix and mineral to provide strength and elasticity to teeth, but also a precious reservoir full of bioactive proteins. They are released after demineralization caused by bacterial acids in carious lesions, by decalcifying irrigants or dental materials and they modulate tissue responses in the underlying dental pulp. This work describes a first-time analysis of the proteome of human dentin using a shotgun proteomic approach that combines three different protein fractionation methods. Dentin matrix proteins were extracted by EDTA and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), OFFGEL isoelectric focusing (IEF) or strong cation exchange chromatography (SCX). Liquid chromatography tandem mass spectrometry (LC-MS/MS) identified 813 human proteins with high confidence, however, isoelectric focusing turned out to be the most beneficial prefractionation method. All Proteins were categorized based on the PANTHER system and representation analysis revealed 31 classes and subclasses to be overrepresented. The acquired knowledge provides a comprehensive insight into the number of proteins in human dentin as well as their physiological and pathological functions. Thus, the data presented paves the way to the analysis of specific functions of dentin matrix proteins in vivo and their potential in tissue engineering approaches to regenerate dental pulp

Interrelationships between Yeast Ribosomal Protein Assembly Events and Transient Ribosome Biogenesis Factors Interactions in Early Pre-Ribosomes

Early steps of eukaryotic ribosome biogenesis require a large set of ribosome biogenesis factors which transiently interact with nascent rRNA precursors (pre-rRNA). Most likely, concomitant with that initial contacts between ribosomal proteins (r-proteins) and ribosome precursors (pre-ribosomes) are established which are converted into robust interactions between pre-rRNA and r-proteins during the course of ribosome maturation. Here we analysed the interrelationship between r-protein assembly events and the transient interactions of ribosome biogenesis factors with early pre-ribosomal intermediates termed 90S pre-ribosomes or small ribosomal subunit (SSU) processome in yeast cells. We observed that components of the SSU processome UTP-A and UTP-B sub-modules were recruited to early pre-ribosomes independently of all tested r-proteins. On the other hand, groups of SSU processome components were identified whose association with early pre-ribosomes was affected by specific r-protein assembly events in the head-platform interface of the SSU. One of these components, Noc4p, appeared to be itself required for robust incorporation of r-proteins into the SSU head domain. Altogether, the data reveal an emerging network of specific interrelationships between local r-protein assembly events and the functional interactions of SSU processome components with early pre-ribosomes. They point towards some of these components being transient primary pre-rRNA in vivo binders and towards a role for others in coordinating the assembly of major SSU domains

Analysis of (pre-) rRNAs co-purifying with Flag tagged r-proteins of the SSU in the yeast <i>noc4–8</i> mutant strain.

<p>The temperature sensitive <i>noc4–8</i> yeast mutant strain (TY40) was transformed with vectors supporting the constitutive expression of Flag tagged SSU r-proteins (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032552#pone.0032552.s004" target="_blank">Fig. S4</a>). Overnight cultures of transformants were grown for one generation time in full medium at 24°C to an OD of 0.4 and then cultivated for three hours in full medium at either permissive (24°C) or restrictive (37°C) temperature. The respective Flag-tagged r-protein was affinity purified from cellular extracts using anti-Flag M2 beads and co-purifying (pre-) rRNA species were analysed by Northern blotting using oligo 1819, which hybridizes in ribosomal precursor rRNAs between 18S and 5.8S rRNA sequences and detects 35S, 32S, 23S, and 20S pre-rRNAs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032552#pone.0032552.s001" target="_blank">Fig. S1</a>). Oligo 205, which hybridizes within the 18S region, was used to detect 18S rRNA. Equal signal intensities of input (In) and affinity purified (IP) fractions correspond to 3% co-purification of the respective rRNA. The numbers in the lower panels indicate the efficiencies of 20S pre-rRNA purification divided by the efficiencies of 18S rRNA purification to normalize for possible over-all variations in the individual immuno-purification experiments. However, we note that the changes in 18S rRNA co-purification efficiencies between experiments performed with one transformant grown in permissive versus non-permissive conditions were in general below twenty percent. The numbers shown are the average of the results of two to four independent experiments and the values obtained with cells grown in permissive conditions were set to one.</p

Analysis pre-rRNAs co-purifying with Noc4p-TAP after <i>in vivo</i> depletion of r-proteins of the SSU.

<p>The yeast strains carrying galactose inducible alleles of the indicated SSU r-protein genes in combination with TAP-tag fusion alleles of Noc4p were either cultivated in medium containing galactose (Gal) as carbon source or were transferred to glucose containing medium (Glu) and cultivated for additional four hours. Noc4p-TAP was affinity purified via its Protein A moiety using IgG sepharose beads. The amount of purified Noc4p-TAP was monitored by Western blotting (lower panels) and co-purified pre-rRNA species were analysed by Northern blotting (upper panels) using oligo 1819, which hybridizes in ribosomal precursor rRNAs between 18S and 5.8S rRNA sequences and detects 35S, 32S, 23S, and 20S pre-rRNAs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032552#pone.0032552.s001" target="_blank">Fig. S1</a>). Equal signal intensities of input (In) and beads (IP) fractions in Northern blots correspond to 1% co-precipitation of the respective rRNA. Efficiencies of 35S pre-rRNA purification normalized to the values obtained for cells grown in permissive conditions are indicated in the lower panel. For the Western blot analyses equal signal intensities of input (In) and beads (IP) correspond to 20% precipitation of the TAP-tagged bait protein. The strains are ordered in regard to the binding of the respective r-proteins to the three main secondary structure domains of the 18S rRNA. Prokaryotic homologues of rpS11, rpS9, rpS22, rpS13, and rpS5 are primary rRNA <i>in vitro</i> binders. Homologues of rpS15 and rpS14 are secondary/tertiary <i>in vitro</i> binders in the assembly trees initiated by binding of the homologues of rpS13 and rpS5, respectively (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032552#pone-0032552-g001" target="_blank">Fig. 1</a>).</p

Analysis of changes in ribosome biogenesis factor composition of early 40S pre-ribosomes purified from cells after <i>in vivo</i> depletion of SSU r-proteins rpS5, rpS13, or rpS14.

<p>The yeast strain TY1907 (wildtype) expressing chromosome encoded TAP tagged Utp4p, and conditional mutant yeast strains expressing chromosome encoded TAP tagged Utp4p and carrying in addition galactose inducible alleles of RPS5 (TY1524), RPS13 (TY1893), or RPS14 (TY2104) were cultivated in medium containing galactose as carbon source and were subsequently transferred to glucose containing medium and cultivated for additional four hours. Utp4p-TAP was affinity purified from corresponding cellular extracts using IgG coupled magnetic beads. Affinity purified proteins were digested by trypsin and the resulting peptides from each sample were labelled with specific iTRAQ reagents. Labelled peptides of wildtype samples were combined with labelled peptides of samples derived from the conditional mutants of either RPS5, RPS13, or RPS14 and were then further analyzed by LC-MS/MS as described in material and methods. Datasets of in total eight (mutant:wildtype) comparisons were generated. In experiments 1–4 Utp4p-TAP fractions purified from the wildtype strain (TY1907) were compared with Utp4p-TAP fractions purified from the conditional RPS5 mutant (TY1524). In experiments 5–7 Utp4p-TAP fractions purified from the wildtype strain (TY1907) were compared with the ones purified from the conditional RPS13 mutant strain (TY1893). In experiment 8 Utp4p-TAP fractions from the wildtype strain were compared with the one purified from the conditional RPS14 mutant strain (TY2104). Experiments 3 and 6 are duplicates of the LC-MS/MS analysis of experiments 2 and 5, respectively. iTRAQ ratios of SSU processome components identified in 5 or more of the 8 experiments were combined to one dataset and statistical clustering algorithms were applied as described in material and methods. (<b>A</b>) shows a comparison of the similarity of the eight individual experimental datasets in regard to each other and (<b>B</b>) shows a clustering analysis of the identified SSU processome components in regard to their iTRAQ ratios in the eight experiments. In (B) boxes in red colours represent relative enrichment and boxes in green colours relative deprivation of a protein in Utp4p-TAP fractions purified from mutant versus wildtype cells. Boxes in gray colour indicate that no peptide of the respective protein could be identified in the corresponding experiment. Standard names of the identified components are indicated in (B) on the left. On the right the major protein groups described in the text are designated and it is indicated if a component belongs to the UTP-A, UTP-B, or UTP-C SSU processome sub-module. We note that despite the overall highly similar composition of Utp4-TAP purifications from cells <i>in vivo</i> depleted of rps13 (experiments 5–7) or rpS14 (experiment 8) the dataset gave first indications for specific differences between these pre-ribosomal populations, as in the content of Krr1p.</p

Analysis of pre-rRNAs co-purifying with UTP-A or UTP-B components after <i>in vivo</i> depletion of r-proteins of the SSU.

<p>The indicated yeast strains carrying galactose inducible alleles of the indicated SSU r-protein genes in combination with TAP-tag fusion alleles of UTP-A component Utp4p (<b>A</b>), or UTP-B component Pwp2p (<b>B</b>), were either cultivated in medium containing galactose (Gal) as carbon source or were transferred to glucose containing medium (Glu) and cultivated for additional four hours to turn off the expression of the respective r-proteins. TAP-tagged bait proteins were affinity purified via their Protein A moiety using IgG sepharose beads. The amount of purified bait protein was monitored by Western blotting (lower panels) and co-purified pre-rRNA species were analysed by Northern blotting (upper panels) using oligo 1819, which hybridizes in ribosomal precursor rRNAs between 18S and 5.8S rRNA sequences and detects 35S, 32S, 23S, and 20S pre-rRNAs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032552#pone.0032552.s001" target="_blank">Fig. S1</a>). Equal signal intensities of input (In) and beads (IP) fractions in Northern blots correspond to 1% co-precipitation of the respective rRNA. Efficiencies of 35S pre-rRNA purification normalized to the values obtained for cells grown in permissive conditions are indicated in the lower panels. For the Western blot analyses equal signal intensities of input (In) and beads (IP) correspond to 20% precipitation of the TAP-tagged bait protein. The strains are ordered in regard to the binding of the respective r-proteins to the three major secondary structure domains of the 18S rRNA. Prokaryotic homologues of rpS11, rpS9, rpS22, rpS13, and rpS5 are primary rRNA <i>in vitro</i> binders. Prokaryotic homologues of rpS15 and rpS14 are secondary/tertiary <i>in vitro</i> binders of the assembly trees initiated by binding of the homologues of rpS13 and rpS5, respectively (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032552#pone-0032552-g001" target="_blank">Fig. 1</a>).</p

30S <i>in vitro</i> assembly map ordered in accordance to the domain organisation of the 16S rRNA and represented in a 2D projection of the 30S ribosomal subunit (adapted from [<b>4</b>]).

<p>(<b>A</b>) The six different r-protein assembly trees (initiated by primary binding r-proteins) of the <i>E. coli</i> 30S subunit are ordered according to their physical location on the 16S rRNA (in 5′ to 3′ direction) and attributed to 16S rRNA domain organisation (5′, central, and 3′ domain). The r-proteins are classified by their binding hierarchy. Primary binding proteins (1°) are capable of initiating pioneering interactions with rRNA independent of other proteins. The secondary binders (2°) require one or more primary binding proteins for their association with rRNA, while tertiary binding (3°) proteins require both primary and secondary binders for their incorporation into ribosomal subunits. If existing, homologous r-proteins in <i>S. cerevisiae</i> (rpS nomenclature) are shown next to their prokaryotic counterparts. (<b>B</b>) A schematic presentation of the tertiary structure of the 16S rRNA is depicted. Each of the three major secondary structure domains of the 16S rRNA forms distinct morphological features of the 30S subunit. The 16S rRNA 5′ domain forms the shoulder and foot (red), the central domain forms the platform (green) and the 3′ major domain forms the head (blue). The assembly map of (A) is superimposed in this schematic structure visualisation paying attention to the localisation of the respective r-protein. The colour of the circle gives information about the assembly hierarchy of the respective r-protein (see also A). S11/rpS14 is classified in a species-dependent manner as a tertiary binder (<i>E. coli</i>) or a primary binder (<i>Aquifex aeolicus</i>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032552#pone.0032552-Recht1" target="_blank">[60]</a>. Only r-proteins with sequence homologous in <i>S. cerevisiae</i> (rpS nomenclature) are shown. The figure is reproduced and adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032552#pone.0032552-Sykes1" target="_blank">[4]</a> (adaptation from the original assembly map of Nomura and colleagues <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032552#pone.0032552-Held1" target="_blank">[1]</a>).</p