Not just fat : investigating the proteome of cetacean blubber tissue

Mammalian adipose tissue is increasingly being recognized as an endocrine organ involved in the regulation of a number of metabolic processes and pathways. It responds to signals from different hormone systems and the central nervous system, and expresses a variety of protein factors with important paracrine and endocrine functions. This study presents a first step towards the systematic analysis of the protein content of cetacean adipose tissue, the blubber, in order to investigate the kinds of proteins present and their relative abundance. Full depth blubber subsamples were collected from dead-stranded harbour porpoises (Phocoena phocoena) (n = 21). Three total protein extraction methods were trialled, and the highest total protein yields with the lowest extraction variability were achieved using a RIPA cell lysis and extraction buffer based protocol. Extracted proteins were separated using 1D Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE), and identified using nanoflow Liquid Chromatography Electrospray Ionization in tandem with Mass Spectrometry (nLC-ESI–MS/MS). A range of proteins were identified (n = 295) and classed into eight functional groups, the most abundant of which were involved in cell function and metabolism (45%), immune response and inflammation (15%) and lipid metabolism (11%). These proteins likely originate both from the various cell types within the blubber tissue itself, and from the circulation. They therefore have the potential to capture information on the cellular and physiological stresses experienced by individuals at the time of sampling. The importance of this proteomic approach is two-fold: Firstly, it could help to assign novel functions to marine mammal blubber in keeping with current understanding of the multi-functional role of adipose tissue in other mammals. Secondly, it could lead to the development of a suite of biomarkers to better monitor the physiological state and health of live individuals though remote blubber biopsy sampling.Publisher PDFPeer reviewe

On the interaction between human IQGAP1 and actin

DM thanks the School of Biological Sciences, Queen’s University, Belfast for a summer studentship and EH thanks the Department of Employment and Learning, Northern Ireland for a postgraduate studentship. The work was funded in part by grants from the BBSRC (BB/D000394/1 To DJT) and by the Wellcome Trust [grant number GR06281AIA] which funded the purchase of the QStar XL mass spectrometer at the BBSRC Mass Spectrometry and Proteomics Facility, University of St Andrews and funded SLS.IQGAPs are eukaryotic proteins which integrate signals from various sources and pass these on the cytoskeleton. Understanding how they do this requires information on the interfaces between the proteins. Here, it is shown that the calponin homology domain of human IQGAP1 (CHD1) can be crosslinked with α-actin. The stoichiometry of the interaction was 1:1. A molecular model was built of the complex and associated bioinformatics analyses predicted that the interaction is likely to involve an electrostatic interaction between Lys-240 of α-actin and Glu-30 of CHD1. These residues are predicted to be accessible and are not involved in many intra-protein interactions; they are thus available for interaction with binding partners. They are both located in regions of the proteins which are predicted to be flexible and disordered; interactions between signalling molecules often involve flexible, disordered regions. The predicted binding region in CHD1 is well conserved in many eukaryotic IQGAP-like proteins. In some cases (e.g Dictyostelium discoideum and Saccharomyces cerevisiae) protein sequence conservation is weak, but molecular modelling reveals that a region of charged, polar residues in a flexible N-terminus is structurally well conserved. Therefore we conclude that the calponin homology domains of IQGAP1-like proteins interact initially through the electrostatic interaction identified here and that there may be subsequent conformational changes to form the final complex.PostprintPeer reviewe

Bunyamwera orthobunyavirus glycoprotein precursor is processed by cellular signal peptidase and signal peptide peptidase

This study was supported by Wellcome Trust Grant 099220/B/12/Z (to R.M.E.) and Grant 094476/Z/10/Z that funded the purchase of the TripleTOF 5600 mass spectrometer at the Biomedical Sciences Research Complex (BSRC) of University of St. Andrews.Bunyamwera virus (BUNV) is the prototype of the Orthobunyavirus genus and Bunyaviridae family that contains important human and animal pathogens. The cleavage mechanism of orthobunyavirus glycoprotein precursor (GPC) and the host proteases involved have not been clarified. Here we found that NSm and Gc contain their own internal signal peptides, which mediate the GPC cleavage by host signal peptidase and signal peptide peptidase (SPP). Furthermore, the NSm domain-I plays an important postcleavage role in cell fusion. Our data clarified the implication of host proteases in the processing of the orthobunyavirus GPC. This work identifies SPP as a potential intervention target, and the knowledge we gained will benefit preventive strategies against other orthobunyavirus infections.PostprintPeer reviewe

Rapid evolution and gene expression : a rapidly-evolving Mendelian trait that silences field crickets has widespread effects on mRNA and protein expression

A major advance in modern evolutionary biology is the ability to start linking phenotypic evolution in the wild with genomic changes that underlie that evolution. We capitalised on a rapidly-evolving Hawaiian population of crickets (Teleogryllus oceanicus) to test hypotheses about the genomic consequences of a recent Mendelian mutation of large effect which disrupts the development of sound-producing structures on male forewings. The resulting silent phenotype, flatwing, persists because of natural selection imposed by an acoustically-orienting parasitoid, but it interferes with mate attraction. We examined gene expression differences in developing wing buds of wild-type and flatwing male crickets using RNA-seq and quantitative proteomics. Most differentially expressed (DE) transcripts were down-regulated in flatwing males (625 up vs. 1716 down), whereas up and down-regulated proteins were equally represented (30 up and 34 down). Differences between morphs were clearly not restricted to a single pathway, and we recovered annotations associated with a broad array of functions that would not be predicted a priori. Using a candidate gene detection test based on homology we identified 30% of putative Drosophila wing development genes in the cricket transcriptome, but only 10% were DE. In addition to wing related annotations, endocrine pathways and several biological processes such as reproduction, immunity and locomotion were DE in the mutant crickets at both biological levels. Our results illuminate the breadth of genetic pathways that are potentially affected in the early stages of adaptation.PostprintPeer reviewe

Pironetin reacts covalently with cysteine-316 of α-tubulin to destabilize microtubule

This work was funded by grants to C.L. from the National Natural Science Foundation of China (81373283 and U1402222). J.H.N. is supported as an award holder of Chinese National Thousand Talents Program, Wellcome Trust Senior Investigator Award (WT100209MA) and Royal Society Wolfson Merit Award. X.Z. is supported by Sichuan Province Thousand Talents Scheme in China and the State Key Program of National Natural Science of China (21534008).Molecules which alter the normal dynamics of microtubule assembly and disassembly include many anticancer drugs in clinical use. So far all such therapeutics target β-tubulin and structural biology has explained the basis of their action and permitted design of new drugs. However by shifting the profile of β-tubulin isoforms, cancer cells become resistant to treatment. Compounds that bind to α-tubulin are less well characterized and unexploited. The natural product pironetin is known to bind to α-tubulin and is a potent inhibitor of microtubule polymerization. Previous reports had identified that pironetin reacts with lysine-352 residue however analogues designed on this model had much lower potency which was difficult to explain, hindering further development. We report crystallographic and mass spectrometric data that reveal that pironetin forms a covalent bond to cysteine-316 in α-tubulin via a Michael addition reaction. These data provide a basis for the rational design of α-tubulin targeting chemotherapeutics.Publisher PDFPeer reviewe

Quantitative proteomic changes in LPS-activated monocyte-derived dendritic cells : a SWATH-MS study

We would like to thank Fiona Cooke for her help with collection of blood samples. We wish to thank the Wellcome Trust for funding the purchase of the TripleTOF 5600+ mass spectrometer (grant number 094476/Z/10/Z) and their Institutional Strategic Support Fund (grant number 097831/Z/11/Z) for funding a PhD studentship (to D.W.-M.). This work was also supported by Arthritis Research UK (grant number 21261).Dendritic cells are key immune cells that respond to pathogens and co-ordinate many innate and adaptive immune responses. Quantitative mass spectrometry using Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) was performed here to determine the global alterations in monocyte-derived dendritic cells (moDCs) in response to stimulation with lipopolysaccharide (LPS). A moDC library of 4,666 proteins was generated and proteins were quantified at 0, 6 and 24 h post-LPS stimulation using SWATH-MS. At 6 h and 24 h post-LPS exposure, the relative abundance of 227 and 282 proteins was statistically significantly altered (p-value≤0.05), respectively. Functional annotation of proteins exhibiting significant changes in expression between the various time points led to the identification of clusters of proteins implicated in distinct cellular processes including interferon and interleukin signalling, endocytosis, the ER-phagosome pathway and antigen-presentation. Major histocompatibility complex (MHC) class I proteins were highly upregulated at 24 h, in SWATH-MS, whilst MHC class II proteins exhibited comparatively less change over this period. This study provides new detailed insight into the global proteomic changes that occur in moDCs during antigen processing and presentation and further demonstrates the potential of SWATH-MS for the quantitative study of proteins involved in cellular processes.Publisher PDFPeer reviewe

Synthesis of Next Generation Maleimide Radical Labels

This work was funded by EPSRC, Grant Number EP/LO22044/1, a Royal Society University Research Fellowship to JEL , Royal Society Research Grant RG120645 and support from Active Spectrum Inc.The synthesis and characterization of four new nitroxide-radical-containing next-generation maleimides are presented. Each new label has a single leaving group which is either a phenoxyl or bromide. The linker between the maleimide and the nitroxide-containing framework is either a racemic mixture of a short chain or an achiral longer chain. These molecules have been designed to site-specifically label vicinal cysteines in proteins for magnetic resonance studies. The characterization of the final products includes crystallography and the labeling of sperm whale myoglobin protein.PostprintPeer reviewe

Life-long epigenetic programming of cortical architecture by maternal ‘Western’ diet during pregnancy

Funding: European Research Council (SECRET-CELLS, ERC-2015-AdG-695136; T.H.); Wellcome Trust grant number 094476/Z/10/Z, which funded the purchase of the TripleTOF 5600 mass spectrometer at the BSRC Mass Spectrometry and Proteomics Facility, University of St. Andrews.The evolution of human diets led to preferences toward polyunsaturated fatty acid (PUFA) content with ‘Western’ diets enriched in ω-6 PUFAs. Mounting evidence points to ω-6 PUFA excess limiting metabolic and cognitive processes that define longevity in humans. When chosen during pregnancy, ω-6 PUFA-enriched ‘Western’ diets can reprogram maternal bodily metabolism with maternal nutrient supply precipitating the body-wide imprinting of molecular and cellular adaptations at the level of long-range intercellular signaling networks in the unborn fetus. Even though unfavorable neurological outcomes are amongst the most common complications of intrauterine ω-6 PUFA excess, cellular underpinnings of life-long modifications to brain architecture remain unknown. Here, we show that nutritional ω-6 PUFA-derived endocannabinoids desensitize CB1 cannabinoid receptors, thus inducing epigenetic repression of transcriptional regulatory networks controlling neuronal differentiation. We found that cortical neurons lose their positional identity and axonal selectivity when mouse fetuses are exposed to excess ω-6 PUFAs in utero. Conversion of ω-6 PUFAs into endocannabinoids disrupted the temporal precision of signaling at neuronal CB1 cannabinoid receptors, chiefly deregulating Stat3-dependent transcriptional cascades otherwise required to execute neuronal differentiation programs. Global proteomics identified the immunoglobulin family of cell adhesion molecules (IgCAMs) as direct substrates, with DNA methylation and chromatin accessibility profiling uncovering epigenetic reprogramming at >1400 sites in neurons after prolonged cannabinoid exposure. We found anxiety and depression-like behavioral traits to manifest in adult offspring, which is consistent with genetic models of reduced IgCAM expression, to suggest causality for cortical wiring defects. Overall, our data uncover a regulatory mechanism whose disruption by maternal food choices could limit an offspring’s brain function for life.PostprintPeer reviewe

Toward the effective surveillance of hypospadias.

Concern about apparent increases in the prevalence of hypospadias--a congenital male reproductive-tract abnormality--in the 1960s to 1980s and the possible connection to increasing exposures to endocrine-disrupting chemicals have underlined the importance of effective surveillance of hypospadias prevalence in the population. We report here the prevalence of hypospadias from 1980 to 1999 in 20 regions of Europe with EUROCAT (European Surveillance of Congenital Anomalies) population-based congenital anomaly registers, 14 of which implemented a guideline to exclude glanular hypospadias. We also report data from the England and Wales National Congenital Anomaly System (NCAS). Our results do not suggest a continuation of rising trends of hypospadias prevalence in Europe. However, a survey of the registers and a special validation study conducted for the years 1994-1996 in nine EUROCAT registers as well as NCAS identified a clear need for a change in the guidelines for registration of hypospadias. We recommend that all hypospadias be included in surveillance, but that information from surgeons be obtained to verify location of the meatus, and whether surgery was performed, in order to interpret trends. Investing resources in repeated special surveys may be more cost-effective than continuous population surveillance. We conclude that it is doubtful whether we have had the systems in place worldwide for the effective surveillance of hypospadias in relation to exposure to potential endocrine-disrupting chemicals

Improved detection of higher molecular weight proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry on polytetrafluoroethylene surfaces

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) of proteins was performed on a range of polytetrafluoroethylene (PTFE) surfaces. Sinapinic acid and alpha-cyano-4-hydroxycinnamic acid matrices were compared and the order of application varied to identify the best combination for each surface. It is demonstrated that the use of a PTFE surface improves the intensity of signals obtained for higher molecular weight proteins. Copyright (C) 2003 John Wiley Sons, Ltd.</p