Thiol redox proteomics seen with fluorescent eyes: the detection of cysteine oxidative modifications by fluorescence derivatization and 2-DE

There is increasing evidence that several reversible oxidative post-translational modifications of protein cysteines participate in cell signalling. Specific proteomic techniques are required to identify these modifications and to study their regulation in different cell processes, that are collectively known as thiol redox proteomics. Recently, fluorescence derivatization methods have been developed that enable these post-translational modifications to be studied using proteomic workflows based on two-dimensional electrophoresis, which is a relatively accessible and affordable technique. As well as enabling a large number of samples to be processed, two-dimensional electrophoresis has the advantage that it does not rely on the intensive use of mass spectrometers. This methodology allows to "visualise" redox changes in a broad context and, although identification of the modified residues is not so straightforward, complementary derivatization can overcome this drawback. Here we review the different derivatization strategies that have been employed in these studies, comparing their advantages and potential limitations. We also review the applications and results obtained, with particular emphasis on those involving (patho)physiological stimuli, thereby showing the potential of these techniques to study the thiol redox proteome.     

Brucella abortus inhibits IFN-γ-induced FcγRI expression and FcγRI-restricted phagocytosis via toll-like receptor 2 on human monocytes/macrophages

The strategies that allow Brucella abortus to persist for years inside macrophages subverting host immune responses are not completely understood. Immunity against this bacterium relies on the capacity of IFN-γ to activate macrophages, endowing them with the ability to destroy intracellular bacteria. We report here that infection with B. abortus down-modulates the expression of the type I receptor for the Fc portion of IgG (FcγRI, CD64) and FcγRI-restricted phagocytosis regulated by IFN-γ in human monocytes/macrophages. Both phenomena were not dependent on bacterial viability, since they were also induced by heat-killed B. abortus (HKBA), suggesting that they were elicited by a structural bacterial component. Accordingly, a prototypical B. abortus lipoprotein (L-Omp19), but not its unlipidated form, inhibited both CD64 expression and FcγRI-restricted phagocytosis regulated by IFN-γ. Moreover, a synthetic lipohexapeptide that mimics the structure of the protein lipid moiety also inhibited CD64 expression, indicating that any Brucella lipoprotein could down-modulate CD64 expression and FcγRI-restricted phagocytosis. Pre-incubation of monocytes/macrophages with anti-TLR2 mAb blocked the inhibition of the CD64 expression mediated by HKBA and L-Omp19. These results, together with our previous observations establish that B. abortus utilizes its lipoproteins to inhibit the monocytes/macrophages activation mediated by IFN-γ and to subvert host immunonological responses.     

Ligand‐induced formation of transient dimers of mammalian 12/15‐lipoxygenase: A key to allosteric behavior of this class of enzymes?

Mammalian lipoxygenases (LOXs) have been implicated in cellular defense response and are important for physiological homeostasis. Since their discovery, LOXs have been believed to function as monomeric enzymes that exhibit allosteric properties. In aqueous solutions, the rabbit 12/15-LOX is mainly present as hydrated monomer but changes in the local physiochemical environment suggested a monomer-dimer equilibrium. Because the allosteric character of the enzyme can hardly be explained using a single ligand binding-site model, we proposed that the binding of allosteric effectors may shift the monomer-dimer equilibrium toward dimer formation. To test this hypothesis, we explored the impact of an allosteric effector [13(S)-hydroxyoctadeca-9(Z),11(E)-dienoic acid] on the structural properties of rabbit 12/15-LOX by small-angle X-ray scattering. Our data indicate that the enzyme undergoes ligand-induced dimerization in aqueous solution, and molecular dynamics simulations suggested that LOX dimers may be stable in the presence of substrate fatty acids. These data provide direct structural evidence for the existence of LOX dimers, where two noncovalently linked enzyme molecules might work in unison and, therefore, such mode of association might be related to the allosteric character of 12/15-LOX. Introduction of negatively charged residues (W181E + H585E and L183E + L192E) at the intermonomer interface disturbs the hydrophobic dimer interaction of the wild-type LOX, and this structural alteration may lead to functional distortion of mutant enzymes.     

Comparison of soil microbial communities from two distinct karst areas in Hungary

Karst areas belong to the most exposed terrestrial ecosystems, therefore their study have a priority task in Hungary, as well. The aim of this study was to compare the structure, activity and diversity of soil microbial communities from two distinct Hungarian karst areas (Aggtelek NP and Tapolca-basin). Soil samples were taken three times from 6 distinct sites, from different depths. Soil microbial biomass C (MBC), microbial biomass N (MBN), basal respiration (BRESP) and substrate induced respiration (SIR) were measured. The phylogenetic diversity of bacterial communities was compared by Denaturing Gradient Gel Electrophoresis (DGGE). The highest MBC, MBN, BRESP and SIR values were measured in the rendzina soil from Aggtelek. On the basis of biomass and respiration measurements, microbial communities differentiated mainly according to soil depths whereas DGGE profiles of bacterial communities resulted in groups mainly according to sampling sites.     

Novel Clostridium thermocellum Type I Cohesin-Dockerin Complexes Reveal a Single Binding Mode

Protein-protein interactions play a pivotal role in a large number of biological processes exemplified by the assembly of the cellulosome. Integration of cellulosomal components occurs through the binding of type I cohesin modules located in a non-catalytic molecular scaffold to type I dockerin modules located at the C terminus of cellulosomal enzymes. The majority of type I dockerins display internal symmetry reflected by the presence of two essentially identical cohesin-binding surfaces. Here we report the crystal structures of two novel Clostridium thermocellum type I cohesin-dockerin complexes (CohOlpC-Doc124A and CohOlpA-Doc918). The data revealed that the two dockerins, Doc918 and Doc124A, are unusual because they lack the structural symmetry required to support a dual binding mode. Thus, in both cases, cohesin recognition is dominated by residues located at positions 11, 12, and 19 of one of the dockerin binding surfaces. The alternative binding mode is not possible (Doc918) or highly limited (Doc124A) because residues that assume the critical interacting positions, when dockerins are reoriented by 180°, make steric clashes with the cohesin. In common with a third dockerin (Doc258) that also presents a single binding mode, Doc124A directs the appended cellulase, Cel124A, to the surface of C. thermocellum and not to cellulosomes because it binds preferentially to type I cohesins located at the cell envelope. Although there are a few exceptions, such as Doc918 described here, these data suggest that there is considerable selective pressure for the evolution of a dual binding mode in type I dockerins that direct enzymes into cellulosomes.     

Proteomic analysis of phytopathogenic fungus <Emphasis Type="Italic">Botrytis cinerea</Emphasis> as a potential tool for identifying pathogenicity factors,therapeutic targets and for basic research

Botrytis cinerea is a phytopathogenic fungus causing disease in a substantial number of economically important crops. In an attempt to identify putative fungal virulence factors, the two-dimensional gel electrophoresis (2-DE) protein profile from two B. cinerea strains differing in virulence and toxin production were compared. Protein extracts from fungal mycelium obtained by tissue homogenization were analyzed. The mycelial 2-DE protein profile revealed the existence of qualitative and quantitative differences between the analyzed strains. The lack of genomic data from B. cinerea required the use of peptide fragmentation data from MALDI-TOF/TOF and ESI ion trap for protein identification, resulting in the identification of 27 protein spots. A significant number of spots were identified as malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The different expression patterns revealed by some of the identified proteins could be ascribed to differences in virulence between strains. Our results indicate that proteomic analysis are becoming an important tool to be used as a starting point for identifying new pathogenicity factors, therapeutic targets and for basic research on this plant pathogen in the postgenomic era.     

Novel Alternative Splicing Predicts a Truncated Isoform of the NMDA Receptor Subunit 1 (NR1) in Embryonic Rat Brain

The expression of mesencephalic brain derived neurotrophic factor (BDNF) has been shown to be regulated by dopaminergic neuronal functioning and glutamate receptors (GluRs). In turn, BDNF participates in the regulation of mesencephalic GluRs’ expression. In the present study we analyzed, using semi-quantitative RT-PCR, the effect of BDNF as well as of the GluRs agonists NMDA and trans-(±)-1-Amino-(1S,3R)-cyclopentane dicarboxylic acid (t-ACPD), on the expression levels of the NMDA GluR subunit 1 (NR1) mRNA, using rat cultured mesencephalic neurons. In the course of this study, a novel rat mRNA splice variant of NR1 was identified. This new NR1 mRNA isoform is characterized by the insertion of an 82 base pair intron containing an inframe stop codon, thus predicting the expression of a putative truncated protein of 465 amino acids. The RT-PCR and in situ hybridization reveals that the novel NR1 mRNA is expressed in various brain regions of the rat embryo, whereas no expression was detected in the adult rat brain. The modulation of the novel NR1 mRNA isoform by both BDNF and the metabotropic GluR agonist t-ACPD, suggests that the resulting putative NR1 truncated protein may be relevant in the regulatory network of glutamatergic neurotransmission in the developing central nervous system.