Receptor deorphanization in an echinoderm reveals kisspeptin evolution and relationship with SALMFamide neuropeptides.

Background: Kisspeptins are neuropeptides that regulate reproductive maturation in mammals via G-protein-coupled receptor-mediated stimulation of gonadotropin-releasing hormone secretion from the hypothalamus. Phylogenetic analysis of kisspeptin-type receptors indicates that this neuropeptide signaling system originated in a common ancestor of the Bilateria, but little is known about kisspeptin signaling in invertebrates.

Results: Contrasting with the occurrence of a single kisspeptin receptor in mammalian species, here, we report the discovery of an expanded family of eleven kisspeptin-type receptors in a deuterostome invertebrate - the starfish Asterias rubens (phylum Echinodermata).

Substrate and Stereochemical Control of Peptidoglycan Cross-Linking by Transpeptidation by PBP1B.

Penicillin binding proteins (PBPs) catalyzing transpeptidation reactions that stabilize the peptidoglycan component of the bacterial cell wall are the targets of β-lactams, the most clinically successful antibiotics to date. However, PBP-transpeptidation enzymology has evaded detailed analysis, because of the historical unavailability of kinetically competent assays with physiologically relevant substrates and the previously unappreciated contribution of protein cofactors to PBP activity. By re-engineering peptidoglycan synthesis, we have constructed a continuous spectrophotometric assay for transpeptidation of native or near native peptidoglycan precursors and fragments by PBP1B, allowing us to (a) identify recognition elements of transpeptidase substrates, (b) reveal a novel mechanism of stereochemical editing within peptidoglycan transpeptidation, (c) assess the impact of peptidoglycan substrates on β-lactam targeting of transpeptidation, and (d) demonstrate that both substrates have to be bound before transpeptidation occurs.

Ancient role of vasopressin/oxytocin-type neuropeptides as regulators of feeding revealed in an echinoderm.

Background: Vasopressin/oxytocin (VP/OT)-type neuropeptides are well known for their roles as regulators of diuresis, reproductive physiology and social behaviour. However, our knowledge of their functions is largely based on findings from studies on vertebrates and selected protostomian invertebrates. Little is known about the roles of VP/OT-type neuropeptides in deuterostomian invertebrates, which are more closely related to vertebrates than protostomes.

Discovery and functional characterisation of a luqin-type neuropeptide signalling system in a deuterostome.

Neuropeptides are diverse and evolutionarily ancient regulators of physiological/behavioural processes in animals. Here we have investigated the evolution and comparative physiology of luqin-type neuropeptide signalling, which has been characterised previously in protostomian invertebrates. Phylogenetic analysis indicates that luqin-type receptors and tachykinin-type receptors are paralogous and probably originated in a common ancestor of the Bilateria.

Urbilaterian origin of paralogous GnRH and corazonin neuropeptide signalling pathways.

Gonadotropin-releasing hormone (GnRH) is a key regulator of reproductive maturation in humans and other vertebrates. Homologs of GnRH and its cognate receptor have been identified in invertebrates-for example, the adipokinetic hormone (AKH) and corazonin (CRZ) neuropeptide pathways in arthropods. However, the precise evolutionary relationships and origins of these signalling systems remain unknown.

Site Specific Genetic Incorporation of Azidophenylalanine in Schizosaccharomyces pombe.

The diversity of protein functions is impacted in significant part by the chemical properties of the twenty amino acids, which are used as building blocks for nearly all proteins. The ability to incorporate unnatural amino acids (UAA) into proteins in a site specific manner can vastly expand the repertoire of protein functions and also allows detailed analysis of protein function. In recent years UAAs have been incorporated in a site-specific manner into proteins in a number of organisms.

Reticulomics: Protein-Protein Interaction Studies with Two Plasmodesmata-Localized Reticulon Family Proteins Identify Binding Partners Enriched at Plasmodesmata, Endoplasmic Reticulum, and the Plasma Membrane.

The endoplasmic reticulum (ER) is a ubiquitous organelle that plays roles in secretory protein production, folding, quality control, and lipid biosynthesis. The cortical ER in plants is pleomorphic and structured as a tubular network capable of morphing into flat cisternae, mainly at three-way junctions, and back to tubules. Plant reticulon family proteins (RTNLB) tubulate the ER by dimerization and oligomerization, creating localized ER membrane tensions that result in membrane curvature.

Composition of distinct sub-proteomes in Myxococcus xanthus: metabolic cost and amino acid availability.

Subsets of proteins involved in distinct functional processes are subject to different selective pressures. We investigated whether there is an amino acid composition bias (AACB) inherent in discrete subsets of proteins, and whether we could identify changing patterns of AACB during the life cycle of the social bacterium Myxococcus xanthus. We quantitatively characterised the cellular, soluble secreted, and outer membrane vesicle (OMV) sub-proteomes of M.

Exploring the functional soil-microbe interface and exoenzymes through soil metaexoproteomics.

Functionally important proteins at the interface of cell and soil are of potentially low abundance when compared with commonly recovered intracellular proteins. A novel approach was developed and used to extract the metaexoproteome, the subset of proteins found outside the cell, in the context of a soil enriched with the nitrogen-containing recalcitrant polymer chitin. The majority of proteins recovered was of bacterial origin and localized to the outer membrane or extracellular milieu.

Discovery of a novel neurophysin-associated neuropeptide that triggers cardiac stomach contraction and retraction in starfish.

Feeding in starfish is a remarkable process in which the cardiac stomach is everted over prey and then retracted when prey tissue has been resorbed. Previous studies have revealed that SALMFamide-type neuropeptides trigger cardiac stomach relaxation and eversion in the starfish Asterias rubens. We hypothesized, therefore, that a counteracting neuropeptide system controls cardiac stomach contraction and retraction.

Comparison of one- and two-dimensional liquid chromatography approaches in the label-free quantitative analysis of Methylocella silvestris.

The proteome of the bacterium Methylocella silvestris has been characterized using reversed phase ultra high pressure liquid chromatography (UPLC) and two-dimensional reversed phase (high pH)-reversed phase (low pH) UPLC prior to mass spectrometric analysis. Variations in protein expression levels were identified with the aid of label-free quantification in a study of soluble protein extracts from the organism grown using methane, succinate, or propane as a substrate. The number of first dimensional fractionation steps has been varied for 2D analyses, and the impact on data throughput and quality has been demonstrated.

A Role for Sigma Factor σ(E) in Corynebacterium pseudotuberculosis Resistance to Nitric Oxide/Peroxide Stress.

Pathogenic intracellular bacteria can respond to antimicrobial mechanisms of the host cell through transient activation of stress-responsive genes by alternative sigma (σ) factors of the RNA polymerase. We evaluated the contribution of the extracytoplasmic function sigma factor σ(E) for Corynebacterium pseudotuberculosis resistance to stress conditions resembling those found intracellularly during infection. A sigE-null mutant strain (ΔsigE) of this bacterium was more susceptible in vitro to acidic pH, cell surface stressors, and biologically relevant concentrations of nitric oxide (NO).

Assimilatory potential of Helicoverpa armigera reared on host (Chickpea) and nonhost (Cassia tora) diets.

Adaptation to plant allelochemicals is a crucial aspect of herbivore chemical ecology. To understand an insect ecology, we studied an effect of nonhost Cassia tora seed-based diet (Ct) on growth, development, and molecular responses in Helicoverpa armigera. We employed a comparative approach to investigate the proteomic differences in gut, hemolymph, and frass of H.

A combined approach for comparative exoproteome analysis of Corynebacterium pseudotuberculosis.

Background: Bacterial exported proteins represent key components of the host-pathogen interplay. Hence, we sought to implement a combined approach for characterizing the entire exoproteome of the pathogenic bacterium Corynebacterium pseudotuberculosis, the etiological agent of caseous lymphadenitis (CLA) in sheep and goats.

Results: An optimized protocol of three-phase partitioning (TPP) was used to obtain the C.

Conformational stability of Syrian hamster prion protein PrP(90-231).

Many transmissible spongiform encephalopathies (TSEs) are believed to be caused by a misfolded form of the normal cellular prion protein (PrP(C)) known as PrP(Sc). While PrP(Sc) is known to be exceptionally stable and resistant to protease degradation, PrP(C) has not shown these same unusual characteristics. However, using ion mobility spectrometry mass spectrometry (IMS-MS), we found evidence for at least one very stable conformation of a truncated form of recombinant PrP(C) consisting of residues 90-231, which resists unfolding in the absence of solvent at high injection energies and at temperatures in excess of 600 K.

Structural analysis of prion proteins by means of drift cell and traveling wave ion mobility mass spectrometry.

The prion protein (PrP) is implicitly involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). The conversion of normal cellular PrP (PrP(C)), a protein that is predominantly alpha-helical, to a beta-sheet-rich isoform (PrP(Sc)), which has a propensity to aggregate, is the key molecular event in prion diseases. During its short life span, PrP can experience two different pH environments; a mildly acidic environment, whilst cycling within the cell, and a neutral pH when it is glycosyl phosphatidylinositol (GPI)-anchored to the cell membrane.

A comparison of labeling and label-free mass spectrometry-based proteomics approaches.

The proteome of the recently discovered bacterium Methylocella silvestris has been characterized using three profiling and comparative proteomics approaches. The organism has been grown on two different substrates enabling variations in protein expression to be identified. The results obtained using the experimental approaches have been compared with respect to number of proteins identified, confidence in identification, sequence coverage and agreement of regulated proteins.

Characterization of phosphorylated peptides using traveling wave-based and drift cell ion mobility mass spectrometry.

Phosphorylation is one the most studied and important post translational modifications. Nano electrospray mass spectrometry coupled with traveling wave (T-Wave)-based ion mobility has been used to filter for phosphorylated peptides in tryptic protein digests. T-Wave parameters have been optimized to maximize the separation between phosphorylated and non-phosphorylated peptides.

End-group characterisation of poly(propylene glycol)s by means of electrospray ionisation-tandem mass spectrometry (ESI-MS/MS).

The end-group functionalisation of a series of poly(propylene glycol)s has been characterised by means of electrospray ionisation-tandem mass spectrometry (ESI-MS/MS). A series of peaks with mass-to-charge ratios that are close to that of the precursor ion were used to generate information on the end-group functionalities of the poly(propylene glycol)s. Fragment ions resulting from losses of both of the end groups were noted from some of the samples.

A proteomic approach to the identification of the major virion structural proteins of the marine cyanomyovirus S-PM2.

In this study, an MS-based proteomics approach to characterizing the virion structural proteins of the novel marine 'photosynthetic' phage S-PM2 is presented. The virus infects ecologically important cyanobacteria of the genus Synechococcus that make a substantial contribution to primary production in the oceans. The S-PM2 genome encodes 236 ORFs, some of which exhibit similarity to known phage virion structural proteins, but the majority (54%) show no detectable homology to known proteins from other organisms.

Novel software for the assignment of peaks from tandem mass spectrometry spectra of synthetic polymers.

Novel software has been developed to aid the interpretation of tandem mass spectrometry (MS/MS) data from synthetic polymers. The software is particularly focused toward aiding the end-group determination of these materials by significantly speeding up the interpretation process. This allows information on the initiator and/or chain transfer agents, used to generate the polymer, and the mechanism of termination to be inferred from the data much more rapidly.

Proteomics in Cancer Research and Diagnosis; An Update.

The availability of reference genomes for, amongst others, man and mouse, coupled with the ability to manipulate the expression of single genes/proteins in model organisms, has fuelled a revolution in biomedical research. Non-invasive diagnosis and treatment, disease prediction and personalised medicine are all expected developments to result from the genomics revolution. Building on this resource, we are now able to employ high-throughput techniques for profiling the expression of thousands of genes/proteins in tissues, body fluids and even individual cells and have evolved a whole new science-systems biology that seeks to reveal the biological information in such data-rich studies.

Noncovalent Shiga-like toxin assemblies: characterization by means of mass spectrometry and tandem mass spectrometry.

Shiga-like toxin 1 (SLTx), produced by enterohemorrhagic strains of Escherichia coli (EHEC), belongs to a family of structurally and functionally related AB(5) protein toxins that are associated with human disease. EHEC infection often gives rise to hemolytic colitis, while toxin-induced kidney damage is one of the major causes of hemolytic uremic syndrome (HUS) and acute renal failure in children. As such, an understanding and analysis of the noncovalent interactions that maintain the quaternary structure of this toxin are fundamentally important since such interactions have significant biochemical and medical implications.

Improved system for protein engineering of the hydroxylase component of soluble methane monooxygenase.

Soluble methane monooxygenase (sMMO) of Methylosinus trichosporium OB3b is a three-component oxygenase that catalyses the O(2)- and NAD(P)H-dependent oxygenation of methane and numerous other substrates. Despite substantial interest in the use of genetic techniques to study the mechanism of sMMO and manipulate its substrate specificity, directed mutagenesis of active-site residues was previously impossible because no suitable heterologous expression system had been found for expression in a highly active form of the hydroxylase component, which is an (alphabetagamma)(2) complex containing the binuclear iron active site. A homologous expression system that enabled the expression of recombinant wild-type sMMO in a derivative of M.