Phosphorylation and Methylation of Proteasomal Proteins of the Haloarcheon Haloferax volcanii

Proteasomes are composed of 20S core particles (CPs) of α- and β-type subunits that associate with regulatory particle AAA ATPases such as the proteasome-activating nucleotidase (PAN) complexes of archaea. In this study, the roles and additional sites of post-translational modification of proteasomes were investigated using the archaeon Haloferax volcanii as a model. Indicative of phosphorylation, phosphatase-sensitive isoforms of α1 and α2 were detected by 2-DE immunoblot. To map these and other potential sites of post-translational modification, proteasomes were purified and analyzed by tandem mass spectrometry (MS/MS). Using this approach, several phosphosites were mapped including α1 Thr147, α2 Thr13/Ser14 and PAN-A Ser340. Multiple methylation sites were also mapped to α1, thus, revealing a new type of proteasomal modification. Probing the biological role of α1 and PAN-A phosphorylation by site-directed mutagenesis revealed dominant negative phenotypes for cell viability and/or pigmentation for α1 variants including Thr147Ala, Thr158Ala and Ser58Ala. An H. volcanii Rio1p Ser/Thr kinase homolog was purified and shown to catalyze autophosphorylation and phosphotransfer to α1. The α1 variants in Thr and Ser residues that displayed dominant negative phenotypes were significantly reduced in their ability to accept phosphoryl groups from Rio1p, thus, providing an important link between cell physiology and proteasomal phosphorylation

Structural Insights into Fibronectin Type III Domain-mediated Signaling

The alternatively spliced type-III extradomain B (EIIIB) of Fibronectin (FN) is only expressed during embryogenesis, wound healing and tumorigenesis. The biological function of this domain remains unclear. We describe here the first crystal structure of the interface between alternatively-spliced domain EIIIB and its adjacent FN type-III domain 8 (FN B-8). The opened CC′ loop of EIIIB and the rotation and tilt of EIIIB domain allows good access to the FG loop of FN-8 which is normally hindered by the CC′ loop of FN-7. In addition, the AGEGIP sequence of the CC′ loop of EIIIB replaces the NGQQGN sequence of the CC′ loop of FN-7. Finally, the CC” loop of EIIIB forms an acidic groove with FN-8. These structural findings warrant future studies directed at identifying potential binding partners for FN B-8 interface, linking EIIIB to skeletal and cartilagenous development, wound healing, and tumorigenesis, respectively

Genetic Analysis of the Functions and Interactions of Components of the LevQRST Signal Transduction Complex of Streptococcus mutans

Transcription of the genes for a fructan hydrolase (fruA) and a fructose/mannose sugar:phosphotransferase permease (levDEFG) in Streptococcus mutans is activated by a four-component regulatory system consisting of a histidine kinase (LevS), a response regulator (LevR) and two carbohydrate-binding proteins (LevQT). The expression of the fruA and levD operons was at baseline in a levQ mutant and substantially decreased in a levT null mutant, with lower expression with the cognate inducers fructose or mannose, but slightly higher expression in glucose or galactose. A strain expressing levQ with two point mutations (E170A/F292S) did not require inducers to activate gene expression and displayed altered levD expression when growing on various carbohydrates, including cellobiose. Linker-scanning (LS) mutagenesis was used to generate three libraries of mutants of levQ, levS and levT that displayed various levels of altered substrate specificity and of fruA/levD gene expression. The data support that LevQ and LevT are intimately involved in the sensing of carbohydrate signals, and that LevQ appears to be required for the integrity of the signal transduction complex, apparently by interacting with the sensor kinase LevS

Composition dependence of lithium diffusion in lithium silicide: a density functional theory study

The lithiation process of silicon was investigated by using ab initio molecular dynamics. Diffusion coefficients of Li in Li–Si alloys were calculated to be in the range between 2.08×10−9 and 3.53×10−7 cm2 s−1 at room temperature. The results showed that the Li mobility is strongly dependent on the composition of the LixSi alloys. The Li diffusivity in a LixSi alloy can be enhanced by two orders of magnitude when x is increased from 1.0 to 3.75, which can be explained by the instability of the Si network, owing to charge transfer from Li to Si

Membrane Composition Changes and Physiological Adaptation by Streptococcus mutans Signal Recognition Particle Pathway Mutants

Previously, we presented evidence that the oral cariogenic species Streptococcus mutans remains viable but physiologically impaired and sensitive to environmental stress when genes encoding the minimal conserved bacterial signal recognition particle (SRP) elements are inactivated. Two-dimensional gel electrophoresis of isolated membrane fractions from strain UA159 and three mutants (Δffh, ΔscRNA, and ΔftsY) grown at pH 7.0 or pH 5.0 allowed us to obtain insight into the adaptation process and the identities of potential SRP substrates. Mutant membrane preparations contained increased amounts of the chaperones DnaK and GroES and ClpP protease but decreased amounts of transcription- and translation-related proteins, the β subunit of ATPase, HPr, and several metabolic and glycolytic enzymes. Therefore, the acid sensitivity of SRP mutants might be caused in part by diminished ATPase activity, as well as the absence of an efficient mechanism for supplying ATP quickly at the site of proton elimination. Decreased amounts of LuxS were also observed in all mutant membranes. To further define physiological changes that occur upon disruption of the SRP pathway, we studied global gene expression in S. mutans UA159 (parent strain) and AH333 (Δffh mutant) using microarray analysis. Transcriptome analysis revealed up-regulation of 81 genes, including genes encoding chaperones, proteases, cell envelope biosynthetic enzymes, and DNA repair and replication enzymes, and down-regulation of 35 genes, including genes concerned with competence, ribosomal proteins, and enzymes involved in amino acid and protein biosynthesis. Quantitative real-time reverse transcription-PCR analysis of eight selected genes confirmed the microarray data. Consistent with a demonstrated defect in competence and the suggested impairment of LuxS-dependent quorum sensing, biofilm formation was significantly decreased in each SRP mutant

Differential recovery of membrane proteins after extraction by aqueous methanol and trifluoroethanol

10.1002/pmic.200600579Proteomics7101654-166

Analysis of the surface, secreted, and intracellular proteome of Propionibacterium acnes

Propionibacterium acnes, plays an important role in acne vulgaris and other diseases. However, understanding of the exact mechanisms of P. acnes pathogenesis is limited. Few studies have investigated its proteome, which is essential for vaccine development. Here, we comprehensively investigate the proteome of P. acnes strain ATCC 6919, including secreted, cell wall, membrane, and cytosolic fractions in three types of growth media. A total of 531 proteins were quantified using an Orbitrap mass spectrometer and bioinformatically categorized for localization and function. Several, including PPA1939, a highly expressed surface and secreted protein, were identified as potential vaccine candidates. Keywords: Acne, Proteomics, Mass spectrometry, Cell wal

First evidence of a membrane-bound, tyramine and beta-phenylethylamine producing, tyrosine decarboxylase in Enterococcus faecalis: a two-dimensional electrophoresis proteomic study

The soluble and membrane proteome of a tyramine producing Enterococcus faecalis, isolated from an Italian goat cheese, was investigated. A detailed analysis revealed that this strain also produces small amounts of beta-phenylethylamine. Kinetics of tyramine and beta-phenylethylamine accumulation, evaluated in tyrosine plus phenylalanine-enriched cultures (stimulated condition), suggest that the same enzyme, the tyrosine decarboxylase (TDC), catalyzes both tyrosine and phenylalanine decarboxylation: tyrosine was recognized as the first substrate and completely converted into tyramine (100% yield) while phenylalanine was decarboxylated to beta-phenylethylamine (10% yield) only when tyrosine was completely depleted. The presence of an aspecific aromatic amino acid decarboxylase is a common feature in eukaryotes, but in bacteria only indirect evidences of a phenylalanine decarboxylating TDC have been presented so far. Comparative proteomic investigations, performed by 2-DE and MALDI-TOF/TOF MS, on bacteria grown in conditions stimulating tyramine and beta-phenylethylamine biosynthesis and in control conditions revealed 49 differentially expressed proteins. Except for aromatic amino acid biosynthetic enzymes, no significant down-regulation of the central metabolic pathways was observed in stimulated conditions, suggesting that tyrosine decarboxylation does not compete with the other energy-supplying routes. The most interesting finding is a membrane-bound TDC highly over-expressed during amine production. This is the first evidence of a true membrane-bound TDC, longly suspected in bacteria on the basis of the gene sequence