Differences in backbone dynamics of two homologous bacterial albumin-binding modules: implications for binding specificity and bacterial adaptation

Proteins G and PAB are bacterial albumin-binding proteins expressed at the surface of group C and G streptococci and Peptostreptococcus magnus, respectively. Repeated albumin-binding domains, known as GA modules, are found in both proteins. The third GA module of protein G from the group G streptococcal strain G148 (G148-GA3) and the second GA module of protein PAB from P.magnus strain ALB8 (ALB8-GA) exhibit 59% sequence identity and both fold to form three-helix bundle structures that are very stable against thermal denaturation. ALB8-GA binds human serum albumin with higher affinity than G148-GA3, but G148-GA3 shows substantially broader albumin-binding specificity than ALB8-GA. The (15)N nuclear magnetic resonance spin relaxation measurements reported here, show that the two GA modules exhibit mobility on the picosecond-nanosecond time scale in directly corresponding regions (loops and termini). Most residues in G148-GA3 were seen to be involved in conformational exchange processes on the microsecond-millisecond time scale, whereas for ALB8-GA such motions were only identified for the beginning of helix 2 and its preceding loop. Furthermore, and more importantly, hydrogen-deuterium exchange and saturation transfer experiments reveal large differences between the two GA modules with respect to motions on the second-hour time scale. The high degree of similarity between the two GA modules with respect to sequence, structure and stability, and the observed differences in dynamics, binding affinity and binding specificity to different albumins, suggest a distinct correlation between dynamics, binding affinity and binding specificity. Finally, it is noteworthy in this context that the module G148-GA3, which has broad albumin-binding specificity, is expressed by group C and G streptococci known to infect all mammalian species, whereas P.magnus with the ALB8-GA module has been isolated only from humans.     

Evolution of protein complexity: The blue copper-containing oxidases and related proteins

Summary The blue copper proteins and their relatives have been compared by sequence alignments, by comparison of three-dimensional structures, and by construction of phylogenetic trees. The group contains proteins varying in size from 100 residues to over 2,300 residues in a single chain, containing from zero to nine copper atoms, and with a broad variation in function ranging from electron carrier proteins and oxidases to the blood coagulation factors V and VIII. Difference matrices show the sequence difference to be over 90% for many pairs in the group, yet alignment scores and other evidence suggest that they all evolved from a common ancestor. We have attempted to delineate how this evolution took place and in particular to define the mechanisms by which these proteins acquired an ever-increasing complexity in structure and function. We find evidence for six such mechanisms in this group of proteins: domain enlargement, in which a single domain increases in size from about 100 residues up to 210; domain duplication, which allows for a size increase from about 170 to about 1,000 residues; segment elongation, in which a small segment undergoes multiple successive duplications that can increase the chain size 50-fold; domain recruitment, in which a domain coded elsewhere in the genome is added on to the peptide chain; subunit formation, to form multisubunit proteins; and glycosylation, which in some cases doubles the size of the protein molecule. Size increase allows for the evolution of new catalytic properties, in particular the oxidase function, and for the formation of coagulation factors with multiple interaction sites and regulatory properties. The blood coagulation system is examined as an example in which a system of interacting proteins evolved by successive duplications of larger parts of the genome. The evolution of size, functionality, and diversity is compared with the general question of increase in size and complexity in biology.     

甜菜夜蛾不同世代对氯氟氰菊酯抗性减退及多功能氧化酶系活性变化

在室内用人工饲料连续饲养和未用药剂筛选条件下,测定了采自田间对氯氟氰菊酯产生高水平抗性甜菜夜蛾Spodoptera exigua (Hübner)种群的抗药性及其多功能氧化酶系活性的变化情况。用点滴法测定不同世代3龄幼虫抗性结果为：室内F₁代LD₅₀值为 0.9672 μg/头,抗性倍数为4 836.0倍,以后各世代逐渐降低,至F₄₃代LD₅₀值为0.0325μg/头,抗性倍数为162.5倍,抗性水平下降了29.8倍。用浸叶法测定不同世代3龄幼虫抗性结果为：室内F₁代LC₅₀值为185.6 mg/L,抗性倍数为964.7倍,以后各世代也逐渐降低,至F₄₃代LC₅₀值为9.2 mg/L,抗性倍数为47.8倍,抗性水平下降了20.2倍。与敏感品系相比,该田间种群室内饲养至F₄₃代仍处于较高的抗性水平,抗性减退缓慢,很难恢复到敏感水平。测定甜菜夜蛾田间种群室内F₂、F₂₀和F₄₁代及敏感品系5龄幼虫中肠微粒体甲氧试卤灵-O-脱甲基酶、乙氧试卤灵-O-脱乙基酶、芳香基羟基化酶及艾氏剂环氧化酶活性,结果表明：与敏感品系相比,田间种群甲氧试卤灵-O-脱甲基酶和艾氏剂环氧化酶的活性仅F₂代显著较高,F₂₀和F₄₁代差异不显著,乙氧试卤灵-O-脱乙基酶和芳香基羟基化酶的活性F₂、F₂₀和F₄₁代均显著较高。结果提示甜菜夜蛾抗性水平可能与其体内微粒体多功能氧化酶系活性有密切关系。     

Kinetics of haem binding to human albumin and haemopexin: nonspecific interactions of haem with proteins

The kinetics of haem binding to human serum albumin and haemopexin were studied by means of the stopped flow technique. The reaction could be divided into three kinetically clearly distinguished steps: (1) extremely fast reaction of haem with nonspecific binding sites on the surface of the apoprotein molecule; this type of haem binding site seems to exist in proteins in general; (2) by meaas of equilibrium with its monomer, haem is transferred to the specific binding site; this second order reaction takes about 1–2 s, the reaction rate constant amounts to ≈10⁶ l mol^?1 s^?1 both for albumin and haemopexin: (3) conformational changes of haemoprotein molecule, accompanied by changes of absorption spectra in the Soret region; this series of slow monomolecular reactions takes about 20 min. These results are discussed in connection with the mechanism of haem transport from blood to liver cells.     

Simulation of the cavity-binding site of three bacterial multicopper oxidases upon complex stabilization: interactional profile and electron transference pathways

Previous studies have shown that multicopper oxidases (MCOs) oxidize organic and inorganic compounds through oxidation–reduction reactions in which three structurally and functionally arranged copper centers coordinate the uptake of an electron from a reduced substrate. Structural comparisons among three bacterial MCOs, with high structural homology and available three-dimensional information, reveal that the primary structural differences between these MCOs are located near the mononuclear copper center (T1Cu), where substrate oxidation occurs, as opposed to where the reduction of oxygen to water occurs at the trinuclear center. Nevertheless, this substrate oxidation is achieved through an outer-sphere electron transfer mechanism that does not generate a stable substrate–enzyme complex. In this study, MCOs from Thermus thermophilus (Tth-MCO), Bacillus subtilis (CotA), and Escherichia coli (CueO), which have been previously determined through X-ray crystallography, were used as models to analyze the binding modes of these MCOs to three organic molecules, with specific interest in the substrate-binding site. The binding mode of the electron-donor molecule to the electron transfer binding site was primarily attributed to hydrophobic contacts, which likely play an important role in the determination of substrate specificity. Some complexes generated in this study showed an electron donor molecule conformation in which an electron could be directly transferred to the histidines coordinating T1Cu, while for others additional electron transference pathways were also possible through the participation of charged residues during electron transfer.     

Symmetrical aryl linked bis-iminothiazolidinones as new chemical entities for the inhibition of monoamine oxidases: Synthesis,in vitro biological evaluation and molecular modelling analysis

The multifactorial nature of Parkinson’s disease necessitates the development of new chemical entities with inherent ability to address key pathogenic processes. To this end, two series of new symmetrical 1,2- and 1,4-bis(2-aroyl/alkoylimino-5-(2-methoxy-2-oxoethylidene)-4-oxo-thiazolidin-3-yl)benzene derivatives (3a–g and 5a–e) were synthesized in good yields by the cyclization of 1,2- and 1,4-bis(N′-substituted thioureido)benzene intermediates with dimethyl acetylenedicarboxylate (DMAD) in methanol at ambient temperature. The bis-iminothiazolidinone compounds were investigated in vitro for their inhibition of monoamine oxidase (MAO-A & MAO-B) enzymes with the aim to identify new and distinct pharmacophores for the treatment of neurodegenerative disorders like Parkinson’s disease. Most of the designed compounds exhibited good inhibitory efficacy against monoamine oxidases. Compound 5a was identified as the most potent inhibitor of MAO-A depicting an IC₅₀ value of 0.001 μM, a 4-fold stronger inhibitory strength compared to standard inhibitor (clorgyline: IC₅₀ = 0.0045 μM). Molecular docking studies provided insights into enzyme-inhibitor interactions and a rationale for the observed inhibition towards monoamine oxidases.     

Reciprocal regulation of NADPH oxidases and the cyclooxygenase-2 pathway

The objective of this work was to analyze the possible association between cyclooxygenase-2 (COX-2) and NADPH oxidases (NOX) in liver cells, in response to various proinflammatory and toxic insults. First, we observed that treatment of Chang liver (CHL) cells with various COX-2 inducers increased reactive oxygen species (ROS) production concomitant with GSH depletion, phorbol 12-myristate 13-acetate (PMA) being the most effective treatment. Moreover, early changes in the oxidative status induced by PMA were inhibited by glutathione ethyl ester, which also impeded COX-2 induction. In fact, CHL cells expressed NOX1 and NOX4, although only NOX4 expression was up-regulated in the presence of PMA. Knock-down experiments suggested that PMA initiated a pathway in which NOX1 activation controlled COX-2 expression and activity, which, in turn, induced NOX4 expression by activation of the prostaglandin receptor EP4. In addition, CHL cells overexpressing COX-2 showed higher NOX4 expression and ROS content, which were decreased in the presence of the COX-2 inhibitor DFU. Interestingly, we found that addition of prostaglandin E(2) (PGE(2)) also induced NOX4 expression and ROS production, which might promote cell adhesion. Finally, we determined that NOX4 induction by PGE(2) was dependent on ERK1/2 signaling. Taken together, these results indicate that NOX proteins and COX-2 are reciprocally regulated in liver cells.     

Penicillin binding proteins: key players in bacterial cell cycle and drug resistance processes

Bacterial cell division and daughter cell formation are complex mechanisms whose details are orchestrated by at least a dozen different proteins. Penicillin-binding proteins (PBPs), membrane-associated macromolecules which play key roles in the cell wall synthesis process, have been exploited for over 70 years as the targets of the highly successful beta-lactam antibiotics. The increasing incidence of beta-lactam resistant microorganisms, coupled to progress made in genomics, genetics and immunofluorescence microscopy techniques, have encouraged the intensive study of PBPs from a variety of bacterial species. In addition, the recent publication of high-resolution structures of PBPs from pathogenic organisms have shed light on the complex intertwining of drug resistance and cell division processes. In this review, we discuss structural, functional and biological features of such enzymes which, albeit having initially been identified several decades ago, are now being aggressively pursued as highly attractive targets for the development of novel antibiotherapies.     

The tetrazolium reduction method for assessing the viability of individual bacterial cells in aquatic environments: improvements,performance and applications

The electron transport system of respiring organisms reduces 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan. Active bacterial cells may be recognized under the microscope by epifluorescence and by the simultaneous presence, seen under bright light field of optically dense intracellular deposits of INT-formazan. An improved procedure that leads to a sharp definition of cells and formazan deposits is presented here. Cells are concentrated on cellulose membrane filters of 0.1 μm porosity which are rendered further transparent prior to immersion of the cells in a layer of 4′, 6-diaminidino-2-phenylindole (DAPI) s′ fluorochrome. This process leads to two significant improvements: (1) the fluorochrome is not trapped inside the membrane, which decreases the background fluorescence and leads to a better detection of the small cells; (2) the cells are immersed in an aqueous solution, which prevents rapid dissolution of the formazan crystals which would be expected if they were in contact with oily clearing agents. Tests on formazan labelling and on storage of INT-processed samples suggest other precautions for reliable use. Improved in this way, the method is simple, rapid and has numerous applications in environmental studies, ecophysiology and ecotoxicology. Some examples are given, with 2 to 98% of INT reducing cells observed, depending on different environmental conditions.     

The bacterial nucleoid: a highly organized and dynamic structure

Recent advances in bacterial cell biology have revealed unanticipated structural and functional complexity, reminiscent of eukaryotic cells. Particular progress has been made in understanding the structure, replication, and segregation of the bacterial chromosome. It emerged that multiple mechanisms cooperate to establish a dynamic assembly of supercoiled domains, which are stacked in consecutive order to adopt a defined higher-level organization. The position of genetic loci on the chromosome is thereby linearly correlated with their position in the cell. SMC complexes and histone-like proteins continuously remodel the nucleoid to reconcile chromatin compaction with DNA replication and gene regulation. Moreover, active transport processes ensure the efficient segregation of sister chromosomes and the faithful restoration of nucleoid organization while DNA replication and condensation are in progress.     

Antioxidants, Oxidative Damage and Oxygen Deprivation Stress: a Review

Oxidative stress is induced by a wide range of environmentalfactors including UV stress, pathogen invasion (hypersensitivereaction), herbicide action and oxygen shortage. Oxygen deprivationstress in plant cells is distinguished by three physiologicallydifferent states: transient hypoxia, anoxia and reoxygenation.Generation of reactive oxygen species (ROS) is characteristicfor hypoxia and especially for reoxygenation. Of the ROS, hydrogenperoxide (H₂O₂) and superoxide (O₂^·–) are bothproduced in a number of cellular reactions, including the iron-catalysedFenton reaction, and by various enzymes such as lipoxygenases,peroxidases, NADPH oxidase and xanthine oxidase. The main cellularcomponents susceptible to damage by free radicals are lipids(peroxidation of unsaturated fatty acids in membranes), proteins(denaturation), carbohydrates and nucleic acids. Consequencesof hypoxia-induced oxidative stress depend on tissue and/orspecies (i.e. their tolerance to anoxia), on membrane properties,on endogenous antioxidant content and on the ability to inducethe response in the antioxidant system. Effective utilizationof energy resources (starch, sugars) and the switch to anaerobicmetabolism and the preservation of the redox status of the cellare vital for survival. The formation of ROS is prevented byan antioxidant system: low molecular mass antioxidants (ascorbicacid, glutathione, tocopherols), enzymes regenerating the reducedforms of antioxidants, and ROS-interacting enzymes such as SOD,peroxidases and catalases. In plant tissues many phenolic compounds(in addition to tocopherols) are potential antioxidants: flavonoids,tannins and lignin precursors may work as ROS-scavenging compounds.Antioxidants act as a cooperative network, employing a seriesof redox reactions. Interactions between ascorbic acid and glutathione,and ascorbic acid and phenolic compounds are well known. Underoxygen deprivation stress some contradictory results on theantioxidant status have been obtained. Experiments on overexpressionof antioxidant production do not always result in the enhancementof the antioxidative defence, and hence increased antioxidativecapacity does not always correlate positively with the degreeof protection. Here we present a consideration of factors whichpossibly affect the effectiveness of antioxidant protectionunder oxygen deprivation as well as under other environmentalstresses. Such aspects as compartmentalization of ROS formationand antioxidant localization, synthesis and transport of antioxidants,the ability to induce the antioxidant defense and cooperation(and/or compensation) between different antioxidant systemsare the determinants of the competence of the antioxidant system.     

Resolution of electron and proton transfer events in the electrochromism associated with quinone reduction in bacterial reaction centers

We have measured the electrochromic response of the bacteriopheophytin, BPh, and bacteriochlorophyll, BChl, cofactors during the Q_A ^–Q_B Q_AQ_B ^– electron transfer in chromatophores of Rhodobacter (Rb.) capsulatus and Rb. sphaeroides. The electrochromic response rises faster in chromatophores and is more clearly biexponential than it is in isolated reaction centers. The chromatophore spectra can be interpreted in terms of a clear kinetic separation between fast electron transfer and slower non-electron transfer events such as proton transfer or protein relaxation. The electrochromic response to electron transfer exhibits rise times of about 4 µs (70%) and 40 µs (30%) in Rb. capsulatus and 4 µs (60%) and 80 µs (40%) in Rb. sphaeroides. The BPh absorption band is shifted to nearly equivalent positions in the Q_A ^– and nascent Q_B ^– states, indicating that the electrochromic perturbation of BPh absorption from the newly formed Q_B ^– state is comparable to that of Q_A ^– . Subsequently, partial attenuation of the Q_B ^– electrochromism occurs with a time constant on the order of 200 µs. This can be attributed to partial charge compensation by H⁺ (or other counter ion) movement into the Q_B pocket. Electron transfer events were found to be slower in detergent isolated RCs than in chromatophores, more nearly monoexponential, and overlap H⁺ transfer, suggesting that a change in rate-limiting step has occurred upon detergent solubilization.     

Quorum-sensing regulation of gene expression: Fundamental and applied aspects and the role in bacterial communication

Quorum sensing (QS) is a specific type of regulation of gene expression in bacteria; it is dependent on the population density. QS systems include two obligate components: a low-molecular-weight regulator (autoinducer), readily diffusible through the cytoplasmic membrane, and a regulatory receptor protein, which interacts with the regulator. As the bacterial population reaches a critical level of density, autoinducers accumulate to a necessary threshold value and abrupt activation (induction) of certain genes and operons occurs. By means of low-molecular-weight regulators, bacteria accomplish communication between cells belonging to the same or different species, genera, and even families. QS systems have been shown to play a key role in the regulation of various metabolic processes in bacteria and to function as global regulators of the expression of bacterial genes. Data are presented on different types of QS systems present in bacteria of various taxonomic groups, on the species specificity of these systems, and on communication of bacteria by means of QS systems. The possibility is considered of using QS regulation systems as targets while combating bacterial infections; other applied aspects of QS investigation are discussed.     

Shotgun proteomics of bacterial pathogens: advances,challenges and clinical implications

Mass spectrometry-based proteomics is increasingly used in analysis of bacterial pathogens. Simple experimental set-ups based on high accuracy mass spectrometry and powerful biochemical and bioinformatics tools are capable of reliably quantifying levels of several thousand bacterial proteins in a single experiment, reaching the analytical capacity to completely map whole proteomes. Here the authors present the state-of-the-art in bacterial pathogen proteomics and discuss challenges that the field is facing, especially in analysis of low abundant, modified proteins from organisms that are difficult to culture. Constant improvements in speed and sensitivity of mass spectrometers, as well as in bioinformatic and biochemical workflows will soon allow for comprehensive analysis of regulatory mechanisms of pathogenicity and enable routine application of proteomics in the clinical setting.     

Metal-catalyzed oxidation reactions and mass spectrometry: the roles of ascorbate and different oxidizing agents in determining Cu-protein-binding sites

Further study has been made of metal-catalyzed oxidation (MCO) reactions and mass spectrometry as a method to determine the binding site of copper in metalloproteins. The role of ascorbate and a variety of oxidizing agents, including O2, H2O2, and S2O8(2-), have been investigated using Cu/Zn superoxide dismutase (SOD) as a model system. Ascorbate is found to play two competing roles in the MCO reactions. It reduces Cu(II), which initiates and maintains the generation of reactive oxygen species, and it scavenges radicals, which helps to localize oxidation products to amino acids near the metal center. An ascorbate concentration of 100 mM is found to be optimal with regard to localizing oxidation products to only the Cu-binding residues (His44, His46, His61, and His118) of Cu/Zn SOD. This concentration of ascorbate is very similar to the optimum concentration found in our previous studies of different Cu-binding proteins. Another notable result from this study is the observation that S2O8(2-) is more effective as an oxidant than O2 or H2O2 in the MCO reactions. Because S2O8(2-) is more stable in solution than H2O2, using it as an oxidizing agent results in much less nonspecific oxidation to the protein. The overall results of this study suggest that general MCO reaction conditions may exist for determining the metal-binding site of a wide range of Cu-binding proteins.     

Changes in glycosylation of acute-phase proteins in health and disease: Occurrence,regulation and function

The pathophysiological variations in different glycoforms of acute-phase glycoproteins in serum most likely result from changes in the glycosylation process during their biosynthesis in the parenchymal cells of the liver. Biosynthesis in other cells or tissues may contribute, but in general appears to play a minor role. Inflammatory cytokines appear to regulate the process, but glycosylation changes are independent of protein synthesis. In addition, other humoral factors such as corticosteroids and growth factors are involved. The interplay of these factors is determined by the stage of the disease (e.g rheumatoid arthritis), the physiological situation (e.g. pregnancy), or directly or indirectly by extraneous factors such as drugs (e.g. ethanol). Information about the functional implications of the changes is limited, but some reports suggest that for ₁-acid glycoprotein the changes might affect the operation of the immune system.