• Why barcode? High-throughput multiplex sequencing of mitochondrial genomes for molecular systematics

      Timmermans, Martijn J.T.N.; Dodsworth, Steven; Culverwell, C. Lorna; Bocak, Ladislav; Ahrens, Dirk; Littlewood, D.T.J.; Pons, J.; Vogler, Alfried P. (Oxford University Press (OUP), 2010-09-28)
      Mitochondrial genome sequences are important markers for phylogenetics but taxon sampling remains sporadic because of the great effort and cost required to acquire full-length sequences. Here, we demonstrate a simple, cost-effective way to sequence the full complement of protein coding mitochondrial genes from pooled samples using the 454/Roche platform. Multiplexing was achieved without the need for expensive indexing tags (‘barcodes’). The method was trialled with a set of long-range polymerase chain reaction (PCR) fragments from 30 species of Coleoptera (beetles) sequenced in a 1/16th sector of a sequencing plate. Long contigs were produced from the pooled sequences with sequencing depths ranging from 10 to 100 per contig. Species identity of individual contigs was established via three ‘bait’ sequences matching disparate parts of the mitochondrial genome obtained by conventional PCR and Sanger sequencing. This proved that assembly of contigs from the sequencing pool was correct.Our study produced sequences for 21 nearly complete and seven partial sets of protein coding mitochondrial genes. Combined with existing sequences for 25 taxa, an improved estimate of basal relationships in Coleoptera was obtained. The procedure could be employed routinely for mitochondrial genome sequencing at the species level, to provide improved species ‘barcodes’ that currently use the cox1 gene only.
    • WWOX sensitises ovarian cancer cells to paclitaxel via modulation of the ER stress response

      Janczar, Szymon; Nautiyal, Jaya; Xiao, Yi; Curry, Edward; Sun, Mingjun; Zanini, Elisa; Paige, Adam J.W.; Gabra, Hani; Imperial College London; Medical University of Lodz; et al. (Springer Nature, 2017-07-27)
      There are clear gaps in our understanding of genes and pathways through which cancer cells facilitate survival strategies as they become chemoresistant. Paclitaxel is used in the treatment of many cancers, but development of drug resistance is common. Along with being an antimitotic agent paclitaxel also activates endoplasmic reticulum (ER) stress. Here, we examine the role of WWOX (WW domain containing oxidoreductase), a gene frequently lost in several cancers, in mediating paclitaxel response. We examine the ER stress-mediated apoptotic response to paclitaxel in WWOX-transfected epithelial ovarian cancer (EOC) cells and following siRNA knockdown of WWOX. We show that WWOX-induced apoptosis following exposure of EOC cells to paclitaxel is related to ER stress and independent of the antimitotic action of taxanes. The apoptotic response to ER stress induced by WWOX re-expression could be reversed by WWOX siRNA in EOC cells. We report that paclitaxel treatment activates both the IRE-1 and PERK kinases and that the increase in paclitaxel-mediated cell death through WWOX is dependent on active ER stress pathway. Log-rank analysis of overall survival (OS) and progression-free survival (PFS) in two prominent EOC microarray data sets (Tothill and The Cancer Genome Atlas), encompassing ~800 patients in total, confirmed clinical relevance to our findings. High WWOX mRNA expression predicted longer OS and PFS in patients treated with paclitaxel, but not in patients who were treated with only cisplatin. The association of WWOX and survival was dependent on the expression level of glucose-related protein 78 (GRP78), a key ER stress marker in paclitaxel-treated patients. We conclude that WWOX sensitises EOC to paclitaxel via ER stress-induced apoptosis, and predicts clinical outcome in patients. Thus, ER stress response mechanisms could be targeted to overcome chemoresistance in cancer.
    • ZAG and a potential role in systemic lipid homeostastis: examining the evidence from in vitro human studies and patients with chronic illness

      McDermott, Lindsay C.; Jadoon, Ayesha; Cunningham, Phil (Future Medicine, 2012-08-31)
      ZAG, a 42 kDa ubiquitously expressed soluble and secreted protein, consists of an MHC-like fold and binds fatty acids with an affinity similar to that of albumin. In human adipocytes, cytoplasmic and secreted ZAG appear inversely related to fat mass. Immunoblotting and biochemical measurements of human cachectic adipocytes and plasma suggest that ZAG’s lipolytic function may be mediated by βIAR. Plasma ZAG correlates with cholesterol in human populations, as does its single nucleotide polymorphism rs4215, which also associates with obesity. Biochemical and human genetic data, in vitro experiments and theoretical data imply that adipocyte ZAG’s expression is regulated by PPAR-γ and glucocorticoids. Cell biological experiments and data from human tissue indicate that ZAG may induce fatty-acid oxidation in skeletal muscle. Overall, these findings suggest ZAG’s participation in systemic lipid homeostasis. Understanding the molecular mechanisms behind ZAG’s in vivo behavior potentially allows for rational drug design to control body fat mass.
    • Zinc-induced oligomerisation of zinc α2 glycoprotein reveals multiple fatty acid binding sites

      Zahid, Henna; Miah, Layeque; Lau, Andy M.; Brochard, Lea; Hati, Debolina; Bui, Tam T. T.; Drake, Alex F.; Gor, Jayesh; Perkins, Stephen J.; McDermott, Lindsay C.; et al. (Portland Press, 2015-12-09)
      Zinc α2 glycoprotein (ZAG) is an adipokine with a class I major histocompatibility complex protein fold and is associated with obesity and diabetes. Although its intrinsic ligand remains unknown, ZAG binds the dansylated C11 fatty acid, DAUDA, in the groove between the α1 and α2 domains. The surface of ZAG has about 15 weak zinc binding sites deemed responsible for precipitation from human plasma. Here the functional significance of these metal sites was investigated. Analytical ultracentrifugation and circular dichroism showed that zinc, but not other divalent metals, cause ZAG to oligomerise in solution. Thus ZAG dimers and trimers were observed in the presence of 1 mM and 2 mM zinc. Molecular modelling of X-ray scattering curves and sedimentation coefficients indicated a progressive stacking of ZAG monomers, suggesting the ZAG groove may be occluded in these. Using fluorescence-detected sedimentation velocity, these ZAG-zinc oligomers were again observed in the presence of the fluorescent boron dipyrromethene fatty acid C16-BODIPY. Fluorescence spectroscopy confirmed that ZAG binds C16-BODIPY. ZAG binding to C16-BODIPY, but not to DAUDA, was reduced by increased zinc concentrations. We conclude that the lipid binding groove in ZAG contains at least two distinct fatty acid binding sites for DAUDA and C16-BODIPY, similar to the multiple lipid binding seen in the structurally-related immune protein Cd1c. In addition, because high concentrations of zinc occur in the pancreas, the perturbation of these multiple lipid binding sites by zinc may be significant in Type 2 diabetes where dysregulation of ZAG and zinc homeostasis occurs.
    • Zn-alpha2-glycoprotein, an MHC class I-related glycoprotein regulator of adipose tissues: modification or abrogation of ligand binding by site-directed mutagenesis

      McDermott, Lindsay C.; Freel, June A.; West, Anthony P.; Bjorkman, Pamela J.; Kennedy, Malcolm W.; University of Glasgow; California Institute of Technology (American Chemical Society, 2006-01-31)
      Zn-alpha(2)-glycoprotein (ZAG) is a soluble lipid-mobilizing factor associated with cancer cachexia and is a novel adipokine. Its X-ray crystal structure reveals a poly(ethylene glycol) molecule, presumably substituting for a higher affinity natural ligand, occupying an apolar groove between its alpha(1) and alpha(2) domain helices that corresponds to the peptide binding groove in class I MHC proteins. We previously provided evidence that the groove is a binding site for hydrophobic ligands that may relate to the protein's signaling function and that the natural ligands are probably (polyunsaturated) fatty acid-like. Using fluorescence-based binding assays and site-directed mutagenesis, we now demonstrate formally that the groove is indeed the binding site for hydrophobic ligands. We also identify amino acid positions that are involved in ligand binding and those that control the shape and exposure to solvent of the binding site itself. Some of the mutants showed minimal effects on their binding potential, one showed enhanced binding, and several were completely nonbinding. Particularly notable is Arg-73, which projects into one end of the binding groove and is the sole charged amino acid adjacent to the ligand. Replacing this amino acid with alanine abolished ligand binding and closed the groove to solvent. Arg-73 may therefore have an unexpected dual role in binding site access and anchor for an amphiphilic ligand. These data add weight to the distinctiveness of ZAG among MHC class I-like proteins in addition to providing defined binding-altered mutants for cellular signaling studies and potential medical applications.