Broad Range Amino Acid Specificity of RNA-dependent Lipid Remodeling by Multiple Peptide Resistance Factors

Aminoacylphosphatidylglycerol synthases (aaPGSs) are multiple peptide resistance factors that transfer amino acids from aminoacyl-tRNAs to phosphatidylglycerol (PG) in the cytoplasmic membrane. Aminoacylation of PG is used by bacteria to decrease the net negative charge of the cell envelope, diminishing affinity for charged molecules and allowing for adaptation to environmental changes. Lys-PGS, which transfers lysine to PG, is essential for the virulence of certain pathogens, providing resistance to both host cationic antimicrobial peptides and therapeutic antibiotics. Ala-PGS was also recently described, but little is known about the possible activities of other members of the highly diverse aaPGS family of proteins. Systematic deletion of the predicted membrane-inserted domains of several aaPGSs revealed that the carboxyl-terminal hydrophilic domain alone is sufficient for aminoacylphosphatidylglycerol transferase catalytic activity. In contrast to previously characterized aaPGSs, the Enterococcus faecium enzyme used an expanded repertoire of amino acids to modify PG with Ala, Arg, or Lys. Reexamination of previously characterized aaPGSs also revealed broader than anticipated substrate specificity, for example Bacillus subtilis Lys-PGS was shown to also catalyze Ala-PG synthesis. The relaxed substrate specificities of these aaPGSs allows for more elaborate remodeling of membrane lipids than previously thought, potentially providing bacteria that harbor these enzymes resistance to a broad spectrum of antibiotics and environmental stresses.To adapt to changing environmental conditions, such as those encountered during host infection, bacteria must alter the properties of their cellular envelope by adjusting the composition and abundance of their membrane lipids. Bacteria choose from a variety of lipid components that vary in fatty acid chain length and saturation levels and that bear a repertoire of different polar head groups. Aminoacylphosphatidylglycerol synthases (aaPGSs)² are enzymes embedded within the bacterial membrane that are responsible for the transfer of amino acids from aminoacyl-tRNAs (aa-tRNAs) to the polar head groups of phosphatidylglycerol (PG). Addition of amino acids to membrane PG is a critical mechanism evolved by bacteria to decrease the net negative charge of the cell membrane (1). This alteration of charge diminishes the membrane affinity for cationic antimicrobial peptides used by the host immune system (i.e. defensins), and for other bactericidal agents (2). During infection, aaPGSs have been shown to be essential for the virulence of several pathogenic microorganisms by facilitating the evasion of antibiotic activity. Their role in virulence and their broad distribution in bacterial species make aaPGSs attractive targets for new therapeutic strategies to combat pathogenic microbes.Lys-PG in the membrane allows for the evasion of neutrophils and enhances the virulence of Staphylococcus aureus both in mice (3) and in endovascular infection of rabbits (4). Similar observations were made for Listeria monocytogenes, in which Lys-PG enhances infectivity of epithelial cells and macrophages in mice (5, 6). Moreover, Lys-PG provides S. aureus with resistance to other classes of cationic bactericidal agents such as vancomycin (glycopeptide) and daptomycin (lipopeptide) (7, 8), which are often used as a last resort to treat infections. A new aaPGS with altered specificity was recently discovered in Clostridium perfringens and Pseudomonas aeruginosa (9, 10). This enzyme, Ala-PGS, is responsible for the formation of Ala-PG in the membrane. Despite the net neutral charge of Ala-PG, this modification has been shown to enhance bacterial resistance to certain cationic antimicrobial peptides (10). Both Lys-PG and Ala-PG have also been shown to enhance the resistance of S. aureus and P. aeruginosa to several negatively charged β-lactams (e.g. oxacillin, methicillin, cefsulodin) (7, 10, 11). These latter findings suggest that aaPGs confer antibiotic resistance both by diminishing the net negative charge of the membrane and by modulating more general biophysical properties such as membrane fluidity and permeability (12). In addition, aaPGs not only decrease membrane permeability to cationic antimicrobial peptides, but also to protons and osmolytes (e.g. lactate), providing bacteria with resistance to challenging osmotic or acidic growth conditions such as those encountered during fermentative growth (10, 13, 14).Despite recent progress, many questions remain unanswered concerning PG aminoacylation in bacteria. Since the identification of Lys-PGS and Ala-PGS and the discovery of resistances associated with these enzymes the amino acid specificities of only seven aaPGSs have been directly characterized (2, 5, 9, 10, 13, 15–18). Predicted aaPGS sequences from different organisms display a remarkable level of secondary structural diversity. To date, 348 putative aaPGS genes in 213 distinct species have been identified in 96 different genera of microorganisms, covering almost all known groups of bacteria. Also, 49 of the available genome sequences encode two or more aaPGS paralogs. This dual occurrence is most often encountered in Gram-positive bacteria, particularly in members of the Actinobacteria class (e.g. Mycobacterium, Streptomyces, etc.). Here, we describe structure/function-based assignment of aaPGS catalytic domains that subsequently allowed us to identify new amino acid specificities of PG aminoacylation. The coexistence of several such activities in the same organism expands the set of unique aaPGSs that are available for tuning of membrane properties and may provide these bacteria with resistance to a far broader spectrum of antimicrobial agents and environmental conditions than previously appreciated. In addition, our work shows that although some aaPGSs display strict substrate specificity (e.g. Lys-PGS from C. perfringens), some aaPGSs display relaxed substrate specificity in vitro. For example, Enterococcus faecium aaPGS exhibits a triple specificity for Ala-, Arg-, and Lys-tRNA, and Bacillus subtilis displays a dual specificity for Lys- and, Ala-tRNA.     

Structure-Guided Combinatorial Engineering Facilitates Affinity and Specificity Optimization of Anti-CD81 Antibodies

Hepatitis C viral infection is the major cause of chronic hepatitis that affects as many as 71 million people worldwide. Rather than target the rapidly shifting viruses and their numerous serotypes, four independent antibodies were made to target the host antigen CD81 and were shown to block hepatitis C viral entry. The single-chain variable fragment of each antibody was crystallized in complex with the CD81 large extracellular loop in order to guide affinity maturation of two distinct antibodies by phage display. Affinity maturation of antibodies using phage display has proven to be critical to therapeutic antibody development and typically involves modification of the paratope for increased affinity, improved specificity, enhanced stability or a combination of these traits. One antibody was engineered for increased affinity for human CD81 large extracellular loop that equated to increased efficacy, while the second antibody was engineered for cross-reactivity with cynomolgus CD81 to facilitate animal model testing. The use of structures to guide affinity maturation library design demonstrates the utility of combining structural analysis with phage display technologies.     

Three Recombinant Engineered Antibodies against Recombinant Tags with High Affinity and Specificity

We describe three recombinant engineered antibodies against three recombinant epitope tags, constructed with divalent binding arms to recognize divalent epitopes and so achieve high affinity and specificity. In two versions, an epitope is inserted in tandem into a protein of interest, and a homodimeric antibody is constructed by fusing a high-affinity epitope-binding domain to a human or mouse Fc domain. In a third, a heterodimeric antibody is constructed by fusing two different epitope-binding domains which target two different binding sites in GFP, to polarized Fc fragments. These antibody/epitope pairs have affinities in the low picomolar range and are useful tools for many antibody-based applications.     

Binding Mechanism of the Peptidoglycan Hydrolase Acm2: Low Affinity,Broad Specificity

Peptidoglycan hydrolases are bacterial secreted enzymes that cleave covalent bonds in the cell-wall peptidoglycan, thereby fulfilling major physiological functions during cell growth and division. Although the molecular structure and functional roles of these enzymes have been widely studied, the molecular details underlying their interaction with peptidoglycans remain largely unknown, mainly owing to the paucity of appropriate probing techniques. Here, we use atomic force microscopy to explore the binding mechanism of the major autolysin Acm2 from the probiotic bacterium Lactobacillus plantarum. Atomic force microscopy imaging shows that incubation of bacterial cells with Acm2 leads to major alterations of the cell-surface nanostructure, leading eventually to cell lysis. Single-molecule force spectroscopy demonstrates that the enzyme binds with low affinity to structurally different peptidoglycans and to chitin, and that glucosamine in the glycan chains is the minimal binding motif. We also find that Acm2 recognizes mucin, the main extracellular component of the intestinal mucosal layer, thereby suggesting that this enzyme may also function as a cell adhesion molecule. The binding mechanism (low affinity and broad specificity) of Acm2 may represent a generic mechanism among cell-wall hydrolases for guiding cell division and cell adhesion.     

Binding Mechanism of the Peptidoglycan Hydrolase Acm2: Low Affinity,Broad Specificity

Peptidoglycan hydrolases are bacterial secreted enzymes that cleave covalent bonds in the cell-wall peptidoglycan, thereby fulfilling major physiological functions during cell growth and division. Although the molecular structure and functional roles of these enzymes have been widely studied, the molecular details underlying their interaction with peptidoglycans remain largely unknown, mainly owing to the paucity of appropriate probing techniques. Here, we use atomic force microscopy to explore the binding mechanism of the major autolysin Acm2 from the probiotic bacterium Lactobacillus plantarum. Atomic force microscopy imaging shows that incubation of bacterial cells with Acm2 leads to major alterations of the cell-surface nanostructure, leading eventually to cell lysis. Single-molecule force spectroscopy demonstrates that the enzyme binds with low affinity to structurally different peptidoglycans and to chitin, and that glucosamine in the glycan chains is the minimal binding motif. We also find that Acm2 recognizes mucin, the main extracellular component of the intestinal mucosal layer, thereby suggesting that this enzyme may also function as a cell adhesion molecule. The binding mechanism (low affinity and broad specificity) of Acm2 may represent a generic mechanism among cell-wall hydrolases for guiding cell division and cell adhesion.     

Virus-Binding Proteins Recovered from Bacterial Culture Derived from Activated Sludge by Affinity Chromatography Assay Using a Viral Capsid Peptide

The contamination of water environments by pathogenic viruses has raised concerns about outbreaks of viral infectious diseases in our society. Because conventional water and wastewater treatment systems are not effective enough to inactivate or remove pathogenic viruses, a new technology for virus removal needs to be developed. In this study, the virus-binding proteins (VBPs) in a bacterial culture derived from activated sludge were successfully recovered. The recovery of VBPs was achieved by applying extracted crude proteins from a bacterial culture to an affinity column in which a custom-made peptide of capsid protein from the poliovirus type 1 (PV1) Mahoney strain (H₂N-DNPASTTNKDKL-COOH) was immobilized as a ligand. VBPs exhibited the ability to adsorb infectious particles of PV1 Sabin 1 as determined by enzyme-linked immunosorbent assay. The evaluation of surface charges of VBPs with ion-exchange chromatography found that a majority of VBP molecules had a net negative charge under the conditions of affinity chromatography. On the other hand, a calculated isoelectric point implied that the viral peptide in the affinity column was also charged negatively. As a result, the adsorption of the VBPs to the viral peptide in the affinity column occurred with a strong attractive force that was able to overcome the electrostatic repulsive force. Two-dimensional electrophoresis revealed that the isolated VBPs include a number of proteins, and their molecular masses were widely distributed but smaller than 100 kDa. Amino acid sequences of N termini of five VBPs were determined. Homology searches for the N termini against all protein sequences in the National Center for Biotechnology Information (NCBI) database showed that the isolated VBPs in this study were newly discovered proteins. These VBPs that originated with bacteria in activated sludge might be stable, because they are existing in the environment of wastewater treatments. Therefore, a virus removal technology utilizing VBPs as viral adsorbents can be developed, since it is possible to replicate VBPs by protein cloning techniques.     

Ligand Specificity of a High Affinity Cytokinin-binding Protein

A soluble cytokinin-binding protein from wheat germ that has a high affinity for a range of purine cytokinins also interacts with a variety of nonpurine compounds that can affect cytokinin-modified processes in animal or plant cells or which bind to proteins known to interact with certain cytokinins. A variety of structurally disparate compounds which inhibit chloroplast photosystem II activity (including phenylurea, carbanilate, and alkylamino-2-chloro-sym-triazine compounds) displace kinetin from the protein in an apparently competitive fashion. However, various energy transfer inhibitors (including organotin compounds and N,N′-dicy-clohexylcarbodiimide) also inhibit kinetin binding to the protein. N⁶,2-0′-Dibutyryl-3′,5′-cyclic AMP and 1-methyl-3-isobutylxanthine (the effects of which on fibroblast morphology and motility can be mimicked by cytokinins) are inhibitors of kinetin binding to the protein. A variety of compounds that can have antimitotic effects (including 1-methyl-3-isobutylxanthine and certain alkylated cyclic nucleotide, carbanilate, and tryptamine compounds) displace kinetin from the protein. However, a variety of indole derivatives also displace kinetin from the cytokinin-binding protein, which in a qualitative sense has a broad ligand specificity.     

Specificity of Milk Peptide Utilization by Lactococcus lactis

To study the substrate specificity of the oligopeptide transport system of Lactococcus lactis for its natural substrates, the growth of L. lactis MG1363 was studied in a chemically defined medium containing milk peptides or a tryptic digest of α_s2-casein as the source of amino acids. Peptides were separated into acidic, neutral, and basic pools by solid-phase extraction or by cation-exchange liquid chromatography. Their ability to sustain growth and the time course of their utilization demonstrated the preferential use of hydrophobic basic peptides with molecular masses ranging between 600 and 1,100 Da by L. lactis MG1363 and the inability to use large, acidic peptides. These peptide utilization preferences reflect the substrate specificity of the oligopeptide transport system of the strain, since no significant cell lysis was inferred. Considering the free amino acid content of milk and these findings on peptide utilization, it was demonstrated that the cessation of growth of L. lactis MG1363 in milk was due to deprivation of leucine and methionine.     

The Specificity of Peptide Chain Extension by N-Carboxyanhydrides

We have used amino acids activated by carbonyldiimidazole to study the enantiospecificity of peptide elongation in aqueoussolution. Peptide `primers' Glu₁₀ and Ala₃Glu₁₀were elongated with the enantiomers of arginine, glutamic acid,asparagine, phenylalanine, serine and valine. The homochiral addition was always the more efficient reaction; the enantiospecificity was large in some cases but very small in others. In every case Ala₃Glu₁₀ was elongated more efficiently than Glu₁₀.     

Development and Evaluation of a Pseudovirus-Luciferase Assay for Rapid and Quantitative Detection of Neutralizing Antibodies against Enterovirus 71

The level of neutralizing antibodies (NtAb) induced by vaccine inoculation is an important endpoint to evaluate the efficacy of EV71 vaccine. In order to evaluate the efficacy of EV71 vaccine, here, we reported the development of a novel pseudovirus system expression firefly luciferase (PVLA) for the quantitative measurement of NtAb. We first evaluated and validated the sensitivity and specificity of the PVLA method. A total of 326 serum samples from an epidemiological survey and 144 serum specimens from 3 clinical trials of EV71 vaccines were used, and the level of each specimen''s neutralizing antibodies (NtAb) was measured in parallel using both the conventional CPE-based and PVLA-based assay. Against the standard neutralization assay based on the inhibition of the cytopathic effect (CPE), the sensitivity and specificity of the PVLA method are 98% and 96%, respectively. Then, we tested the potential interference of NtAb against hepatitis A virus, Polio-I, Polio-II, and Polio-III standard antisera (WHO) and goat anti-G10/CA16 serum, the PVLA based assay showed no cross-reactivity with NtAb against other specific sera. Importantly, unlike CPE based method, no live replication-competent EV71 is used during the measurement. Taken together, PVLA is a rapid and specific assay with higher sensitivity and accuracy. It could serve as a valuable tool in assessing the efficacy of EV71 vaccines in clinical trials and disease surveillance in epidemiology studies.     

Distinct Cellular Assembly Stoichiometry of Polycomb Complexes on Chromatin Revealed by Single-molecule Chromatin Immunoprecipitation Imaging

Epigenetic complexes play an essential role in regulating chromatin structure, but information about their assembly stoichiometry on chromatin within cells is poorly understood. The cellular assembly stoichiometry is critical for appreciating the initiation, propagation, and maintenance of epigenetic inheritance during normal development and in cancer. By combining genetic engineering, chromatin biochemistry, and single-molecule fluorescence imaging, we developed a novel and sensitive approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) to enable investigation of the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was validated by using chromatin complexes with known stoichiometry. The stoichiometry of subunits within a polycomb complex and the assembly stoichiometry of polycomb complexes on chromatin have been extensively studied but reached divergent views. Moreover, the cellular assembly stoichiometry of polycomb complexes on chromatin remains unexplored. Using Sm-ChIPi, we demonstrated that within mouse embryonic stem cells, one polycomb repressive complex (PRC) 1 associates with multiple nucleosomes, whereas two PRC2s can bind to a single nucleosome. Furthermore, we obtained direct physical evidence that the nucleoplasmic PRC1 is monomeric, whereas PRC2 can dimerize in the nucleoplasm. We showed that ES cell differentiation induces selective alteration of the assembly stoichiometry of Cbx2 on chromatin but not other PRC1 components. We additionally showed that the PRC2-mediated trimethylation of H3K27 is not required for the assembly stoichiometry of PRC1 on chromatin. Thus, these findings uncover that PRC1 and PRC2 employ distinct mechanisms to assemble on chromatin, and the novel Sm-ChIPi technique could provide single-molecule insight into other epigenetic complexes.     

运用染色质免疫沉淀技术筛选AP-2α的靶基因

在后基因组时代,DNA-蛋白质的相互作用是研究基因表达调控的一个重要领域.染色质免疫沉淀技术(chromatin immunoprecipitation assay,简称CHIP)是目前唯一研究体内DNA与蛋白质相互作用的方法.对与ChIP有关的实验条件进行了优化,获得了较优的实验条件,并运用ChIP实验筛选出了转录因子activator protein-2 alpha (AP-2a)的未知靶基因,对于进一步研究AP-2a的功能和调控网络打下了基础.     

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design^1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols^1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.     

Immunoprecipitation Assay of Alpha-fetoprotein Synthesis by Cultured Mouse Hepatoma Cells Treated with Estrogens and Glucocorticords

This investigation was to study the biosynthesis of ³H-labeled alpha-fetoprotein (AFP) by cultured mouse hepatoma (HEPA-2) cells. Both the function and regulation of this oncodevelopmental gene are unknown. However, evidence indicates that mechanisms controlling the expression of AFP involve aspects of both normal embryonic development and neoplastic transformation. The secretion of AFP was analyzed during different phases of the growth cycle to provide information on AFP production using standard culture conditions. The highest rate of secretion occurred during the stationary phase, followed by the late logarithmic and early logarithmic phases of growth, respectively. The production of AFP was then determined following the addition of glucocorticords and estrogens in an attempt to understand hormonal factors that may be involved. Studies utilizing estradiol-17β indicated that the secretion of AFP did not appear to be sensitive to this steroid even though sucrose density gradient analysis of HEPA-2 cytosol, for estrogenic receptors, revealed competitive binding moieties in the 8S and 4S regions of the gradient. In contrast, the secretion of the total complement of proteins, including AFP, was significantly stimulated by the glucocorticords, dexamethasone and corticosterone. Analysis of HEPA-2 cytosol for glucocorticord receptors revealed binding components in the 7S and 3–4S regions of the gradient. The ³H-dexamethasone binding appeared to be stereospecific since nonlabeled dexamethasone, but not nonlabeled estradiol-17β, effectively displaced the bound radioactivity. The glucocorticoid-binding component in HEPA-2 therefore displayed characteristics reported for glucocorticord receptors in normal liver and other hepatomas.