Integrative omics identifies conserved and pathogen-specific responses of sepsis-causing bacteria.

Even in the setting of optimal resuscitation in high-income countries severe sepsis and septic shock have a mortality of 20-40%, with antibiotic resistance dramatically increasing this mortality risk. To develop a reference dataset enabling the identification of common bacterial targets for therapeutic intervention, we applied a standardized genomic, transcriptomic, proteomic and metabolomic technological framework to multiple clinical isolates of four sepsis-causing pathogens: Escherichia coli, Klebsiella pneumoniae species complex, Staphylococcus aureus and Streptococcus pyogenes. Exposure to human serum generated a sepsis molecular signature containing global increases in fatty acid and lipid biosynthesis and metabolism, consistent with cell envelope remodelling and nutrient adaptation for osmoprotection.

Exploring modular reengineering strategies to redesign the teicoplanin non-ribosomal peptide synthetase.

Non-ribosomal peptide synthesis is an important biosynthesis pathway in secondary metabolism. In this study we have investigated modularisation and redesign strategies for the glycopeptide antibiotic teicoplanin. Using the relocation or exchange of domains within the NRPS modules, we have identified how to initiate peptide biosynthesis and explored the requirements for the functional reengineering of both the condensation/adenylation domain and epimerisation/condensation domain interfaces.

Structures of a non-ribosomal peptide synthetase condensation domain suggest the basis of substrate selectivity.

Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate.

Redesign of Substrate Selection in Glycopeptide Antibiotic Biosynthesis Enables Effective Formation of Alternate Peptide Backbones.

Nonribosomal peptide synthesis is capable of utilizing a wide range of amino acid residues due to the selectivity of adenylation (A)-domains. Changing the selectivity of A-domains could lead to new bioactive nonribosomal peptides, although remodeling efforts of A-domains are often unsuccessful. Here, we explored and successfully reengineered the specificity of the module 3 A-domain from glycopeptide antibiotic biosynthesis to change the incorporation of 3,5-dihydroxyphenylglycine into 4-hydroxyphenylglycine.

Understanding the early stages of peptide formation during the biosynthesis of teicoplanin and related glycopeptide antibiotics.

The biosynthesis of the glycopeptide antibiotics (GPAs) demonstrates the exceptional ability of nonribosomal peptide (NRP) synthesis to generate diverse and complex structures from an expanded array of amino acid precursors. Whilst the heptapeptide cores of GPAs share a conserved C terminus, including the aromatic residues involved cross-linking and that are essential for the antibiotic activity of GPAs, most structural diversity is found within the N terminus of the peptide. Furthermore, the origin of the (D)-stereochemistry of residue 1 of all GPAs is currently unclear, despite its importance for antibiotic activity.

A proof-reading mechanism for non-proteinogenic amino acid incorporation into glycopeptide antibiotics.

Non-ribosomal peptide biosynthesis produces highly diverse natural products through a complex cascade of enzymatic reactions that together function with high selectivity to produce bioactive peptides. The modification of non-ribosomal peptide synthetase (NRPS)-bound amino acids can introduce significant structural diversity into these peptides and has exciting potential for biosynthetic redesign. However, the control mechanisms ensuring selective modification of specific residues during NRPS biosynthesis have previously been unclear.

Exploring the Tetracyclization of Teicoplanin Precursor Peptides through Chemoenzymatic Synthesis.

The glycopeptide antibiotics (GPAs) serve as an important example of the interplay of two powerful enzymatic classes in secondary metabolism: the coupling of nonribosomal peptide synthesis with oxidative aromatic cross-linking performed by cytochrome P450 enzymes. This interplay is responsible for the generation of the highly cross-linked peptide aglycone at the core of this compound class that is required for antibiotic activity and, as such, serves as an important point for the exploration of chemoenzymatic routes to understand the selectivity and mechanism of this complex cascade. Here, we demonstrate the effective reconstitution of enzymatic tetracyclization of synthetic teicoplanin-derived heptapeptides and furthermore discern the importance of the OxyE enzyme in maintaining effective cyclization of such peptides bearing 3,5-dihydroxyphenylglycine residues at position 3 in their structures.

The Diiron Monooxygenase CmlA from Chloramphenicol Biosynthesis Allows Reconstitution of β-Hydroxylation during Glycopeptide Antibiotic Biosynthesis.

β-Hydroxylation plays an important role in the nonribosomal peptide biosynthesis of many important natural products, including bleomycin, chloramphenicol, and the glycopeptide antibiotics (GPAs). Various oxidative enzymes have been implicated in such a process, with the mechanism of incorporation varying from installation of hydroxyl groups in amino acid precursors prior to adenylation to direct amino acid oxidation during peptide assembly. In this work, we demonstrate the utility and scope of the unusual nonheme diiron monooxygenase CmlA from chloramphenicol biosynthesis for the β-hydroxylation of a diverse range of carrier protein bound substrates by adapting this enzyme as a non-native -acting enzyme within NRPS-mediated GPA biosynthesis.

LFQ-Analyst: An Easy-To-Use Interactive Web Platform To Analyze and Visualize Label-Free Proteomics Data Preprocessed with MaxQuant.

Relative label-free quantification (LFQ) of shotgun proteomics data using precursor (MS1) signal intensities is one of the most commonly used applications to comprehensively and globally quantify proteins across biological samples and conditions. Due to the popularity of this technique, several software packages, such as the popular software suite MaxQuant, have been developed to extract, analyze, and compare spectral features and to report quantitative information of peptides, proteins, and even post-translationally modified sites. However, there is still a lack of accessible tools for the interpretation and downstream statistical analysis of these complex data sets, in particular for researchers and biologists with no or only limited experience in proteomics, bioinformatics, and statistics.

Enzymatic Cascade To Evaluate the Tricyclization of Glycopeptide Antibiotic Precursor Peptides as a Prequel to Biosynthetic Redesign.

Natural products are the greatest source of antimicrobial agents, although their structural complexity often renders synthetic production and diversification of key classes impractical. One pertinent example is the glycopeptide antibiotics (GPAs), which are highly challenging to synthesize due to their heavily cross-linked structures. Here, we report an optimized method that generates >75% tricyclic peptides from synthetic precursors in order to explore the acceptance of novel GPA precursor peptides by these key existent biosynthetic enzymes.

Kistamicin biosynthesis reveals the biosynthetic requirements for production of highly crosslinked glycopeptide antibiotics.

Kistamicin is a divergent member of the glycopeptide antibiotics, a structurally complex class of important, clinically relevant antibiotics often used as the last resort against resistant bacteria. The extensively crosslinked structure of these antibiotics that is essential for their activity makes their chemical synthesis highly challenging and limits their production to bacterial fermentation. Kistamicin contains three crosslinks, including an unusual 15-membered A-O-B ring, despite the presence of only two Cytochrome P450 Oxy enzymes thought to catalyse formation of such crosslinks within the biosynthetic gene cluster.

Proteotranscriptomic Measurements of E6-Associated Protein (E6AP) Targets in DU145 Prostate Cancer Cells.

Prostate cancer is a common cause of cancer-related death in men. E6AP (E6-Associated Protein), an E3 ubiquitin ligase and a transcription cofactor, is elevated in a subset of prostate cancer patients. Genetic manipulations of E6AP in prostate cancer cells expose a role of E6AP in promoting growth and survival of prostate cancer cells and However, the effect of E6AP on prostate cancer cells is broad and it cannot be explained fully by previously identified tumor suppressor targets of E6AP, promyelocytic leukemia protein and p27.

Structural basis for substrate selection by the translocation and assembly module of the β-barrel assembly machinery.

The assembly of proteins into bacterial outer membranes is a key cellular process that we are only beginning to understand, mediated by the β-barrel assembly machinery (BAM). Two crucial elements of that machinery are the core BAM complex and the translocation and assembly module (TAM), with each containing a member of the Omp85 superfamily of proteins: BamA in the BAM complex, TamA in the TAM. Here, we used the substrate protein FimD as a model to assess the selectivity of substrate interactions for the TAM relative to those of the BAM complex.

Neurotoxicity in Sri Lankan Russell's Viper (Daboia russelii) Envenoming is Primarily due to U1-viperitoxin-Dr1a, a Pre-Synaptic Neurotoxin.

Russell's vipers are snakes of major medical importance in Asia. Russell's viper (Daboia russelii) envenoming in Sri Lanka and South India leads to a unique, mild neuromuscular paralysis, not seen in other parts of the world where the snake is found. This study aimed to identify and pharmacologically characterise the major neurotoxic components of Sri Lankan Russell's viper venom.

Structure of the poly-C9 component of the complement membrane attack complex.

The membrane attack complex (MAC)/perforin-like protein complement component 9 (C9) is the major component of the MAC, a multi-protein complex that forms pores in the membrane of target pathogens. In contrast to homologous proteins such as perforin and the cholesterol-dependent cytolysins (CDCs), all of which require the membrane for oligomerisation, C9 assembles directly onto the nascent MAC from solution. However, the molecular mechanism of MAC assembly remains to be understood.

Fetuin B Is a Secreted Hepatocyte Factor Linking Steatosis to Impaired Glucose Metabolism.

Liver steatosis is associated with the development of insulin resistance and the pathogenesis of type 2 diabetes. We tested the hypothesis that protein signals originating from steatotic hepatocytes communicate with other cells to modulate metabolic phenotypes. We show that the secreted factors from steatotic hepatocytes induce pro-inflammatory signaling and insulin resistance in cultured cells.

Changes in protein abundance are observed in bacterial isolates from a natural host.

Bacterial proteomic studies frequently use strains cultured in synthetic liquid media over many generations. It is uncertain whether bacterial proteins expressed under these conditions will be the same as the repertoire found in natural environments, or when bacteria are infecting a host organism. Thus, genomic and proteomic characterization of bacteria derived from the host environment in comparison to reference strains grown in the lab, should aid understanding of pathogenesis.

Particles on the move: intracellular trafficking and asymmetric mitotic partitioning of nanoporous polymer particles.

Nanoporous polymer particles (NPPs) prepared by mesoporous silica templating show promise as a new class of versatile drug/gene delivery vehicles owning to their high payload capacity, functionality, and responsiveness. Understanding the cellular dynamics of such particles, including uptake, intracellular trafficking, and distribution, is an important requirement for their development as therapeutic carriers. Herein, we examine the spatiotemporal map of the cellular processing of submicrometer-sized disulfide-bonded poly(methacrylic acid) (PMASH) NPPs in HeLa cells using both flow cytometry and fluorescence microscopy.

The proteome browser web portal.

In 2010, the Human Proteome Organization launched the Human Proteome Project (HPP), aimed at identifying and characterizing the proteome of the human body. To support complete coverage, one arm of the project will take a gene- or chromosomal-centric strategy (C-HPP) aimed at identifying at least one protein product from each protein-coding gene. Despite multiple large international biological databases housing genomic and protein data, there is currently no single system that integrates updated pertinent information from each of these data repositories and assembles the information into a searchable format suitable for the type of global proteomics effort proposed by the C-HPP.

Solubilisation of the armadillo-repeat protein β-catenin using a zwitterionic detergent allows resolution of phosphorylated forms by 2DE.

β-catenin is a member of the armadillo repeat family of proteins and has important functions in cell-cell adhesion and Wnt signalling. Different protein species of β-catenin have been shown to exist in the cell and the relative proportions of these species are altered upon stimulation of cells with Wnt-3a (Gottardi and Gumbiner, 2004). In order to determine whether posttranslational modifications (PTMs) of β-catenin underlie these different protein species, we have used 2DE separation and immunoblotting with an antibody specific for β-catenin.

Proteomic profiling of secretome and adherent plasma membranes from distinct mammary epithelial cell subpopulations.

The stem cell niche comprises stem cells (SCs), stromal cells, soluble factors, extracellular matrix constituents and vascular networks. The ability to identify signals that regulate SC self-renewal and differentiation is confounded by the difficulty in isolating pure SC niche components in sufficient quantities to enable their biochemical characterisation. Here, we report the extracellular (secretome) and adherent plasma membrane proteomes of three distinct epithelial cell subpopulations isolated and immortalized from the mouse mammary gland--basal and mammary stem cell (basal/MaSC), luminal progenitor (LP) and mature luminal (ML) cell lines.

Tandem application of cationic colloidal silica and Triton X-114 for plasma membrane protein isolation and purification: towards developing an MDCK protein database.

Plasma membrane (PM) proteins are attractive therapeutic targets because of their accessibility to drugs. Although genes encoding PM proteins represent 20-30% of eukaryotic genomes, a detailed characterisation of their encoded proteins is underrepresented, due, to their low copy number and the inherent difficulties in their isolation and purification as a consequence of their high hydrophobicity. We describe here a strategy that combines two orthogonal methods to isolate and purify PM proteins from Madin Darby canine kidney (MDCK) cells.

Purification of basolateral integral membrane proteins by cationic colloidal silica-based apical membrane subtraction.

Epithelial cell polarity mediates many essential biological functions and perturbation of the apical/basolateral divide is a hallmark of epithelial to mesenchymal transition in carcinoma. Therefore, correct targeting of proteins to the apical and basolateral surfaces is essential to proper epithelial cell function. However, proteomic characterisation of apical/basolateral sorting has been largely ignored, due to ineffectual separation techniques and contamination of plasma-membrane preparations with housekeeping proteins.

Stem cell markers: insights from membrane proteomics?

Stem cells (SCs) are defined by their combined abilities to both self-renew indefinitely in vitro and differentiate into adult cell types. One of the major driving forces of SC research is that SCs may provide a potentially unlimited source for cell-replacement therapies in regenerative medicine. However, the identification of SCs and their progenies at different stages, and the success of cell-replacement therapies, which form the basis of SC engineering, will depend on the ability to characterize and ultimately isolate homogeneous primary stem or progenitor cell populations to a large degree.