Identification of novel bacterial plasminogen-binding proteins in the human pathogen Mycobacterium tuberculosis

Binding and activation of human plasminogen (Plg) to generate the proteolytic enzyme plasmin (Plm) have been associated with the invasive potential of certain bacteria. In this work, proteomic analysis together with ligand blotting assays identified several major Plg-binding spots in Mycobacterium tuberculosis soluble extracts (SEs) and culture filtrate proteins. The identity of 15 different proteins was deduced by N-terminal and/or MS and corresponded to DnaK, GroES, GlnA1, Ag85 complex, Mpt51, Mpt64, PrcB, MetK, SahH, Lpd, Icl, Fba, and EF-Tu. Binding of Plg to recombinant M. tuberculosis DnaK, GlnA1, and Ag85B was further confirmed by ELISA and ligand blotting assays. The binding was inhibited by epsilon-aminocaproic acid, indicating that the interaction involved lysine residues. Plg bound to recombinant mycobacterial proteins was activated to Plm by tissue-type Plg activator. In contrast with recombinant proteins, M. tuberculosis SE enhanced several times the Plg activation mediated by the activator. Interestingly, GlnA1 was able to bind the extracellular matrix (ECM) protein fibronectin. Together these results show that M. tuberculosis posses several Plg receptors suggesting that bound Plg to bacteria surface, can be activated to Plm, endowing bacteria with the ability to break down ECM and basal membranes proteins contributing to tissue injury in tuberculosis.     

Supramolecular structure of self-assembling alamethicin analog studied by ESR and PELDOR

Three analogs of alamethicin F50/5, labelled with the TOAC (='2,2,6,6-tetramethylpiperidin-1-oxyl-4-amino-4-carboxylic acid') spin label at positions 1 (Alm1), 8 (Alm8), and 16 (Alm16), resp., were studied by Electron-Spin-Resonance (ESR) and Pulsed Electron-Electron Double-Resonance (PELDOR) techniques in solvents of different polarity to investigate the self-assembly of amphipathic helical peptides in membrane-mimicking environments. In polar solvents, alamethicin forms homogeneous solutions. In the weakly polar chloroform/toluene 1 : 1 mixture, however, this peptide forms aggregates that are detectable at 293 K by ESR in liquid solution, as well as by PELDOR in frozen, glassy solution at 77 K. In liquid solution, free alamethicin molecules and their aggregates show rotational-mobility correlation times tau(r) of 0.87 and 5.9 ns, resp. Based on these values and analysis of dipole-dipole interactions of the TOAC labels in the aggregates, as determined by PELDOR, the average number N of alamethicin molecules in the aggregates is estimated to be less than nine. A distance-distribution function between spin labels in the supramolecular aggregate was obtained. This function exhibits two maxima: a broad one at a distance of 3.0 nm, and a wide one at a distance of ca. 7 nm. A molecular-dynamics (MD)-based model of the aggregate, consisting of two parallel tetramers, each composed of four molecules arranged in a 'head-to-tail' fashion, is proposed, accounting for the observed distances and their distribution.     

Extracting protein regulatory networks with graphical models

During the last decade the development of high-throughput biotechnologies has resulted in the production of exponentially expanding quantities of biological data, such as genomic and proteomic expression data. One fundamental problem in systems biology is to learn the architecture of biochemical pathways and regulatory networks in an inferential way from such postgenomic data. Along with the increasing amount of available data, a lot of novel statistical methods have been developed and proposed in the literature. This article gives a non-mathematical overview of three widely used reverse engineering methods, namely relevance networks, graphical Gaussian models, and Bayesian networks, whereby the focus is on their relative merits and shortcomings. In addition the reverse engineering results of these graphical methods on cytometric protein data from the RAF-signalling network are cross-compared via AUROC scatter plots.     

Proteome analysis of whole saliva: a new tool for rheumatic diseases--the example of Sjögren's syndrome

Sj?gren's syndrome (pSS) is a systemic disease that affects salivary glands directly, and is therefore expected to influence the composition of human whole saliva (WS) fluid. The aim of this study was to characterize the WS proteins of pSS patients using a proteomic approach to assess a valid procedure to examine the global changes of the salivary protein profiles in connective tissue disorders. The WS proteins expressed in patients affected by pSS and healthy volunteers were analyzed using the 2-DE technique. The WS protein pattern was altered in pSS patients compared to controls, with a decrease in some of the typical salivary proteins. Particularly, a remarkable alteration of carbonic anhydrase VI was observed. Moreover, a comparison of WS protein profile of pSS patients with the one obtained from controls revealed a set of differentially expressed proteins. These proteins were related to acute and chronic inflammation while some others were involved in oxidative stress injury. These findings are in line with the systemic immuno-inflammatory aspects of pSS and open the possibility for a systematic search of diagnostic biomarkers and targets for therapeutic intervention in pSS.     

Integrated analytical approach in veal calves administered the anabolic androgenic steroids boldenone and boldione: urine and plasma kinetic profile and changes in plasma protein expression

Surveillance of illegal use of steroids hormones in cattle breeding is a key issue to preserve human health. To this purpose, an integrated approach has been developed for the analysis of plasma and urine from calves treated orally with a single dose of a combination of the androgenic steroids boldenone and boldione. A quantitative estimation of steroid hormones was obtained by LC-APCI-Q-MS/MS analysis of plasma and urine samples obtained at various times up to 36 and 24 h after treatment, respectively. These experiments demonstrated that boldione was never found, while boldenone alpha- and beta-epimers were detected in plasma and urine only within 2 and 24 h after drug administration, respectively. Parallel proteomic analysis of plasma samples was obtained by combined 2-DE, MALDI-TOF-MS and muLC-ESI-IT-MS/MS procedures. A specific protein, poorly represented in normal plasma samples collected before treatment, was found upregulated even 36 h after hormone treatment. Extensive mass mapping experiments proved this component as an N-terminal truncated form of apolipoprotein A1 (ApoA1), a protein involved in cholesterol transport. The expression profile of ApoA1 analysed by Western blot analysis confirmed a significant and time dependent increase of this ApoA1 fragment. Then, provided that further experiments performed with a growth-promoting schedule will confirm these preliminary findings, truncated ApoA1 may be proposed as a candidate biomarker for steroid boldenone and possibly other anabolic androgens misuse in cattle veal calves, when no traces of hormones are detectable in plasma or urine.     

Mechanical response and conformational changes of alpha-actinin domains during unfolding: a molecular dynamics study

Alpha-actinin is a cytoskeleton-binding protein involved in the assembly and regulation of the actin filaments. In this work molecular dynamics method was applied to investigate the mechanical behaviour of the human skeletal muscle α-actinin. Five configurations were unfolded at an elongation speed of 0.1 nm/ps in order to investigate the conformational changes occurring during the extension process. Moreover, a sensitivity analysis at different velocities was performed for one of the R2–R3 spectrin-like repeat configuration extracted in order to evaluate the effect of the pulling speed on the mechanical behaviour of the molecule. Two different behaviours were recognized with respect to the pulling speed. In particular, at speed higher than 0.025 nm/ps a continuous rearrangement without evident force peaks was obtained, on the contrary at lower speed evident peaks in the range 500–750 pN were detected. R3 repeat resulted more stable than R2 during mechanical unfolding, due to the lower hydrophobic surface available to the solvent. The characterization of the R2–R3 units can be useful for the development of cytoskeleton network models based on stiffness values obtained by analyses performed at the molecular level.     

The functions of Sco proteins from genome-based analysis

Sco proteins are widespread proteins found in eukaryotic as well as in many prokaryotic organisms. The 3D structure of representatives from human, yeast, and Bacillus subtilis has been determined, showing a thioredoxin-like fold. Sco proteins have been implicated mainly as copper transporters involved in the assembly of the CuA cofactor in cytochrome c oxidase. Some mutations have been identified in humans that lead to defective cytochrome c oxidase formation and thus to fatal illnesses. However, it appears that the physiological function of Sco proteins goes beyond assembly of the CuA cofactor. Extensive analysis of completely sequenced prokaryotic genomes reveals that 18% of them contain either Sco proteins but not CuA-containing proteins or vice versa. In addition, in several cases, multiple Sco-encoding genes occur even if only a single potential Sco target is encoded in the genome. Genomic context analysis indeed points to a more general role for Sco proteins in copper transport, also to copper enzymes lacking a CuA cofactor. To obtain further insight into the possible role of Sco in the assembly of other cofactors, a search for Cox11 proteins, which are important for CuB biosynthesis, was also performed. A general framework for the action of Sco proteins is proposed, based on the hypothesis that they can couple metal transport and thiol/disulfide-based oxidoreductase activity, as well as select between either of these two cellular functions. This model reconciles the variety of experimental observations made on these proteins over the years, and can constitute a basis for further studies.