• T-cell reconstitution after thymus xenotransplantation induces hair depigmentation and loss

      Furmanski, Anna L.; O'Shaughnessy, Ryan F.L.; Saldana, Jose Ignacio; Blundell, Michael P.; Thrasher, Adrian J.; Sebire, Neil; Davies, E Graham; Crompton, Tessa (Elsevier, 2013-01-10)
      Here we present a mouse model for T-cell targeting of hair follicles, linking the pathogenesis of alopecia to that of depigmentation disorders. Clinically, thymus transplantation has been successfully used to treat T-cell immunodeficiency in congenital athymia, but is associated with autoimmunity. We established a mouse model of thymus transplantation by subcutaneously implanting human thymus tissueinto athymic C57BL/6 nude mice. These xenografts supported mouse T-cell development. Surprisingly, we did not detect multiorgan autoimmune disease. However, in all transplanted mice, we noted a striking depigmentation and loss of hair follicles. Transfer of T cells from transplanted nudes to syngeneic black-coated RAG−/- recipients caused progressive, persistent coat-hair whitening, which preceded patchy hair loss in depigmented areas. Further transfer experiments revealed that these phenomena could be induced by CD4+ T cells alone. Immunofluorescent analysis suggested that Trp2+ melanocyte-lineage cells were decreased in depigmented hair follicles, and pathogenic T cells upregulated activation markers when exposed to C57BL/6 melanocytes in vitro, suggesting that these T cells are not tolerant to self-melanocyte antigens. Our data raise interesting questions about the mechanisms underlying tissue-specific tolerance to skin antigens.
    • Tandem oligomeric expression of metallothionein enhance heavy metal tolerance and bioaccumulation in Escherichia coli.

      Ma, Wenli; Li, Xuefen; Wang, Qi; Ren, Zhumei; Crabbe, M. James C.; Wang, Lan; Shanxi University; University of Oxford; University of Bedfordshire (Elsevier, 2019-06-13)
      Metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, metal-binding proteins, which play important roles in metal homeostasis and heavy metal detoxification. In our previous study, a novel full length MT cDNA was successfully cloned from the freshwater crab (Sinopotamon henanense). In the present study, tandem repeats of two and three copies of the crab MT gene were integrated by overlap extension PCR (SOEPCR) and expressed in Escherichia coli. The SUMO fusion expression system was adopted to increase the stability and solubility of the recombinant MT proteins. The recombinant proteins were purified and their metal-binding abilities were further analyzed by the ultraviolet absorption spectral scan. Furthermore, the metal tolerance and bioaccumulation of E. coli cells expressing oligomeric MTs were determined. Results showed that the recombinant plasmids pET28a-SUMO-2MT and pET28a-SUMO-3MT were successfully constructed. SDS-PAGE analysis showed that the SUMO-2MT and SUMO-3MT were expressed mainly in the soluble forms. Oligomeric MTs expression significantly enhanced Cu, Cd or Zn tolerance and accumulation in E. coli in the order: SUMO-3MT˃SUMO-2MT˃SUMO-MT˃control. Cells harboring pET28a-SUMO -3MT exhibited the highest Cu, Cd or Zn bioaccumulation at 5.8-fold, 3.1-fold or 6.7-fold higher than that of the control cells. Our research could lay a foundation for large-scale preparation of MTs and provide a scientific basis for bioremediation of heavy metal pollution by oligomeric MTs.
    • Targeted editing of SlMAPK6 using CRISPR/Cas9 technology to promote the development of axillary buds in tomato plants

      Li, Yunzhou; Yue, Ningbo; Basit, Abdul; Li, Yulong; Zhang, Dalong; Qin, Lei; Crabbe, M. James C.; Xu, Wen; Wang, Yong; Yan, Jianmin; et al. (Canadian Center of Science and Education, 2021-01-15)
      The mitogen-activated protein kinase (MAPK) cascade signaling system has been relatively conserved throughout the evolution of eukaryotes and is involved in the regulation of growth and development and metabolism. In this study, dwarf tomato plants were used as the research material. First, the tissue-specific expression of SlMAPK6 was measured in wild-type plants by quantitative RT-PCR. The results showed that SlMAPK6 was highly expressed in the tissues of the stems, leaves and flowers but was expressed at low levels in the tissues of the roots, sepals and fruits. Second, SlMAPK6-knockout lines CRISPR-3 and CRISPR-7 were obtained by CRISPR-Cas9 technology and Agrobacterium-mediated transformation. Compared with wild-type, the mutant lines CRISPR-3 and CRISPR-7 showed significant phenotypic characteristics, such as increased numbers of axillary buds and true leaves, thickened stems, and longer leaflets. In addition, to explore the molecular mechanism by which MAPK regulates axillary bud growth, we also showed that SlMAPK6 positively regulates the strigolactone synthesis genes SlCCD7 and SlCCD8 and the gibberellin (GA) synthesis genes GA20ox3 and GA3ox1 and negatively regulates the axillary bud development-related genes Ls, BL and BRC1b/TCP8 and the GA synthesis inhibitory gene GAI. Therefore, SlMAPK6 appears to regulate the synthesis of strigolactone and GA to induce the growth and development of tomato axillary buds.
    • TCR-alpha CDR3 loop audition regulates positive selection

      Ferreira, Cristina; Furmanski, Anna L.; Millrain, Maggie M.; Bartok, Istvan; Guillaume, Philippe; Lees, Rosemary; Simpson, Elizabeth; MacDonald, H. Robson; Dyson, Julian (American Association of Immunologists, 2006-08-03)
      How positive selection molds the T cell repertoire has been difficult to examine. In this study, we use TCR-β-transgenic mice in which MHC shapes TCR-α use. Differential AV segment use is directly related to the constraints placed on the composition of the CDR3 loops. Where these constraints are low, efficient selection of αβ pairs follows. This mode of selection preferentially uses favored AV-AJ rearrangements and promotes diversity. Increased constraint on the α CDR3 loops leads to inefficient selection associated with uncommon recombination events and limited diversity. Further, the two modes of selection favor alternate sets of AJ segments. We discuss the relevance of these findings to the imprint of self-MHC restriction and peripheral T cell activation.
    • Thymus transplantation for complete DiGeorge syndrome: European experience

      Davies, E Graham; Cheung, Melissa; Gilmour, Kimberly; Maimaris, Jesmeen; Curry, Joe; Furmanski, Anna L.; Sebire, Neil; Halliday, Neil; Mengrelis, Konstantinos; Adams, Stuart; et al. (Elsevier, 2017-04-08)
      Background: Thymus transplantation is a promising strategy for the treatment of athymic complete DiGeorge syndrome (cDGS). Methods: Twelve patients with cDGS were transplanted with allogeneic cultured thymus. Objective: To confirm and extend the results previously obtained in a single centre. Results: Two patients died of pre-existing viral infections without developing thymopoeisis and one late death occurred from autoimmune thrombocytopaenia. One infant suffered septic shock shortly after transplant resulting in graft loss and the need for a second transplant. Evidence of thymopoeisis developed from 5-6 months after transplantation in ten patients. The median (range) of circulating naïve CD4 counts (x10663 /L) were 44(11-440) and 200(5-310) at twelve and twenty-four months post-transplant and T-cell receptor excision circles were 2238 (320-8807) and 4184 (1582 -24596) per106 65 T-cells. Counts did not usually reach normal levels for age but patients were able to clear pre-existing and later acquired infections. At a median of 49 months (22-80), eight have ceased prophylactic antimicrobials and five immunoglobulin replacement. Histological confirmation of thymopoeisis was seen in seven of eleven patients undergoing biopsy of transplanted tissue including five showing full maturation through to the terminal stage of Hassall body formation. Autoimmune regulator (AIRE) expression was also demonstrated. Autoimmune complications were seen in 7/12 patients. In two, early transient autoimmune haemolysis settled after treatment and did not recur. The other five suffered ongoing autoimmune problems including: thyroiditis (3); haemolysis (1), thrombocytopaenia (4) and neutropenia (1). Conclusions: This study confirms the previous reports that thymus transplantation can reconstitute T cells in cDGS but with frequent autoimmune complications in survivors.
    • Time-calibrated phylogenetic trees establish a lag between polyploidisation and diversification in Nicotiana (Solanaceae)

      Clarkson, James J.; Dodsworth, Steven; Chase, Mark W.; Royal Botanic Gardens, Kew; University of Western Australia (Springer-Verlag Wien, 2017-05-08)
      We investigate the timing of diversification in allopolyploids of Nicotiana (Solanaceae) utilising sequence data of maternal and paternal origin to look for evidence of a lag phase during which diploidisation took place. Bayesian relaxed clock phylogenetic methods show recent allopolyploids are a result of several unique polyploidisation events, and older allopolyploid sections have undergone subsequent speciation at the polyploid level (i.e. a number of these polyploid species share a singular origin). The independently formed recent polyploid species in the genus all have mean age estimates below 1 million years ago (Ma). Nicotiana  section Polydicliae (two species) evolved 1.5 Ma, N. section Repandae (four species) formed 4 Ma, and N. section Suaveolentes (*35 species) is about 6 million years old. A general trend of higher speciation rates in older polyploids is evident, but diversification dramatically increases at approximately 6 Ma (in section Suaveolentes). Nicotiana sect. Suaveolentes has spectacularly radiated to form 35 species in Australia and some Pacific islands following a lag phase of almost 6 million years. Species have filled new ecological niches and undergone extensive diploidisation (e.g. chromosome fusions bringing the ancestral allotetraploid number, n = 24, down to n = 15 and ribosomal loci numbers back to diploid condition). Considering the progenitors of Suaveolentes inhabit South America, this represents the colonisation of Australia by polyploids that have subsequently undergone a recent radiation into new environments. To our knowledge, this study is the first report of a substantial lag phase being investigated below the family level.
    • Transcriptome profiling of the UV-B stress response in the desert shrub Lycium ruthenicum

      Chen, Haikui; Feng, Yang; Wang, Lina; Yonezawa, Takahiro; Crabbe, M. James C.; Zhang, Xiu; Zhong, Yang; Beifang University of Nationalities, Yinchuan; Fudan University, Shanghai; University of Oxford; et al. (Springer, 2015-02-15)
      Ultraviolet-B (UV-B) is a natural component of the solar radiation. Due to its high energy, low dosages of UV-B can bring huge potential damage effect to organisms. Despite much research that has analyzed the gene expression changes of plants that under UV-B radiation, the transcriptome response of Lycium ruthenicum under the UV-B induction is still unavailable. The aim of our study was to identify UV-B responsive genes and gain an insight into the underlying genetic basis of the pathobiology of UV-B related damage. We collected leaf samples from L. ruthenicum with and without UV-B exposure, and then performed a transcriptome profiling to comprehensively investigate their expression signatures. By employing the high throughput RNA-sequencing analysis of samples with and without UV-B exposure, we identified 1,913 up-regulated and 536 down-regulated genes at least by twofold changes. The activity of antioxidant enzyme related genes, including the superoxide dismutase, was decreased, genes related to the synthesis of secondary metabolites and defense responses, such as cinnamyl alcohol dehydrogenase, chalcone-flavanone isomerase and dihydroflavonol reductase were also downregulated. The expression patterns of 14 randomly selected genes resulted from quantitative real-time PCR were basically consistent with their transcript abundance changes identified by RNA-sequencing. We found that several biological pathways related to biotic and abiotic stresses, including cell defense, photosynthesis processes, energy metabolism, were involved in the process of UV-B stress response. A genome-wide screening of gene deregulation under UV-B induction would provide an insight into the understanding of the molecular bases and pathogenesis of UV-B responses.
    • The transfer of iron between ceruloplasmin and transferrins

      White, Kenneth N.; Conesa, Celia; Sánchez, Lourdes; Amini, Maryam; Farnaud, Sébastien; Lorvoralak, Chanakan; Evans, Robert W.; London Metropolitan University; Universidad de Zaragoza; Brunel University; et al. (Elsevier, 2012-03-28)
      It is over 60years since the discovery and isolation of the serum ferroxidase ceruloplasmin. In that time much basic information about the protein has been elucidated including its catalytic and kinetic properties as an enzyme, expression, sequence and structure. The importance of its biological role is indicated in genetic diseases such as aceruloplasminemia where its function is lost through mutation. Despite this wealth of data, fundamental questions about its action remain unanswered and in this article we address the question of how ferric iron produced by the ferroxidase activity of ceruloplasmin could be taken up by transferrins or lactoferrins. Overlapping peptide libraries for human ceruloplasmin have been probed with a number of different lactoferrins to identify putative lactoferrin-binding regions on human ceruloplasmin. Docking software, 3D-Garden, has been used to model the binding of human lactoferrin to human ceruloplasmin. Upon probing the human ceruloplasmin library with human lactoferrin, three predominantly acidic lactoferrin-binding peptides, located in domains 2, 5 and 6 of human ceruloplasmin, were identified. The docking software identified a complex such that the N-lobe of human apo-lactoferrin interacts with the catalytic ferroxidase centre on human ceruloplasmin. In vitro binding studies and molecular modelling indicate that lactoferrin can bind to ceruloplasmin such that a direct transfer of ferric iron between the two proteins is possible. A direct transfer of ferric iron from ceruloplasmin to lactoferrin would prevent both the formation of potentially toxic hydroxyl radicals and the utilization of iron by pathogenic bacteria. BACKGROUND METHODS RESULTS GENERAL SIGNIFICANCE
    • Tripartite efflux pumps: energy is required for dissociation, but not assembly or opening of the outer membrane channel of the pump

      Janganan, Thamarai K.; Bavro, Vassiliy N; Zhang, Li; Borges-Walmsley, Maria Ines; Walmsley, Adrian R.; University of Durham; University of Birmingham (Wiley, 2013-05-30)
      The MtrCDE multidrug pump, from Neisseria gonorrhoeae, is assembled from the inner and outer membrane proteins MtrD and MtrE, which are connected by the periplasmic membrane fusion protein MtrC. Although it is clear that MtrD delivers drugs to the channel of MtrE, it remains unclear how drug delivery and channel opening are connected. We used a vancomycin sensitivity assay to test for opening of the MtrE channel. Cells expressing MtrE or MtrE-E434K were insensitive to vancomycin; but became moderately and highly sensitive to vancomycin respectively, when coexpressed with MtrC, suggesting that the MtrE channel opening requires MtrC binding and is energy-independent. Cells expressing wild-type MtrD, in an MtrCE background, were vancomycin-insensitive, but moderately sensitive in an MtrCE-E434K background. The mutation of residues involved in proton translocation inactivated MtrD and abolished drug efflux, rendered both MtrE and MtrE-E434K vancomycin-insensitive; imply that the pump-component interactions are preserved, and that the complex is stable in the absence of proton flux, thus sealing the open end of MtrE. Following the energy-dependent dissociation of the tripartite complex, the MtrE channel is able to reseal, while MtrE-E434K is unable to do so, resulting in the vancomycin-sensitive phenotype. Thus, our findings suggest that opening of the OMP via interaction with the MFP is energy-independent, while both drug export and complex dissociation require active proton flux.
    • Tropomyosin isoforms show unexpected differential effects on actin polymerization

      Maytum, Robin; Dudekula, Khadar B.; University of Bedfordshire; University of Edinburgh (American Chemical Society, 2017-02-03)
      Tropomyosin is a rod-like coiled-coil protein that forms a continuous filament that is weakly associated, but firmly-attached to the surface of the actin filaments in all eukaryotic cells. Simple eukaryotes such as yeasts have only one or two different tropomyosin isoforms which are known to be essential and perform roles in regulating the actin cytoskeleton. However higher eukaryotes have larger numbers of tropomyosins, the number of which appear linked to organismal complexity. Mammals have 4 genes producing over 40 different isoforms by alternative splicing. In higher organisms tropomyosin is best known and characterized in the regulation of striated muscle contraction. The role of tropomyosin outside of muscle is less well understood. It is generally thought to have a regulatory role in controlling interactions of actin-binding proteins and in providing additional stability to actin-filaments. In the latter case has been considered that tropomyosin binds to actin-filaments some time after their formation, both making them mechanically stiffer and protecting them from breakdown. We have produced a range of recombinant tropomyosins from all four mammalian genes and characterized their actin-binding affinities in a cosedimentation assay. We have then used them to systematically study the effects of different isoforms of tropomyosin on actin polymerization for the first time. We have monitored actin polymerization by the well-characterised change in fluorescence of a pyrene-label attached to actin. Actin polymerisation is monitored by measuring the significant fluorescence enhancement on polymerization. Our results characterize the actin-affinities of some of the TPM3 and TPM4 isoforms for the first time, These are in the same general range as mammalian isoforms previously characterized by our group and others. We demonstrate differential effects of the different isoforms on actin-polymerisation for the first time. The data unexpectedly show the most significant effects of the different isoforms appears to be in the early initiation / elongation stages of polymerizations. This is unexpected as tropomyosin is only considered to have significant affinity for actin filaments through itself forming a polymer along the surface of an actin filament. Different isoforms appear capable of both enhancing and inhibiting the early stages of polymerization, with examples of the shorter 6-actin spanning TPM1 gene isoforms showing a significant reduction in the lag-phase of early polymerization. These differential effects on different isoforms provides a new role for tropomyosin in not only stabilizing filaments, but also in helping catalyze their formation.
    • Tropomyosin isoforms show unexpected differential effects on actin polymerization

      Maytum, Robin; Dudekula, Khadar B. (Cell Press, 2017-02-03)
      Tropomyosin is a rod-like coiled-coil protein that forms a continuous filament that is weakly associated, but firmly-attached to the surface of the actin filaments in all eukaryotic cells. Simple eukaryotes such as yeasts have only one or two different tropomyosin isoforms which are known to be essential and perform roles in regulating the actin cytoskeleton. However higher eukaryotes have larger numbers of tropomyosins, the number of which appear linked to organismal complexity. Mammals have 4 genes producing over 40 different isoforms by alternative splicing.
    • A universal probe set for targeted sequencing of 353 nuclear genes from any flowering plant designed using k-medoids clustering

      Johnson, Matthew G.; Pokorny, Lisa; Dodsworth, Steven; Botigue, Laura R.; Cowan, Robyn S.; Devault, Alison; Eiserhardt, Wolf L.; Epitawalage, Niroshini; Forest, Felix; Kim, Jan T.; et al. (Oxford University Press (OUP), 2018-12-10)
      Sequencing of target-enriched libraries is an efficient and cost-effective method for obtaining DNA sequence data from hundreds of nuclear loci for phylogeny reconstruction. Much of the cost of developing targeted sequencing approaches is associated with the generation of preliminary data needed for the identification of orthologous loci for probe design. In plants, identifying orthologous loci has proven difficult due to a large number of whole-genome duplication events, especially in the angiosperms (flowering plants).We used multiple sequence alignments from over 600 angiosperms for 353 putatively single-copy protein-coding genes identified by the One Thousand Plant Transcriptomes Initiative to design a set of targeted sequencing probes for phylogenetic studies of any angiosperm group. To maximize the phylogenetic potential of the probes, while minimizing the cost of production, we introduce a k-medoids clustering approach to identify the minimum number of sequences necessary to represent each coding sequence in the final probe set. Using this method, 5–15 representative sequences were selected per orthologous locus, representing the sequence diversity of angiosperms more efficiently than if probes were designed using available sequenced genomes alone. To test our approximately 80,000 probes, we hybridized libraries from 42 species spanning all higher-order groups of angiosperms, with a focus on taxa not present in the sequence alignments used to design the probes. Out of a possible 353 coding sequences, we recovered an average of 283 per species and at least 100 in all species. Differences among taxa in sequence recovery could not be explained by relatedness to the representative taxa selected for probe design, suggesting that there is no phylogenetic bias in the probe set. Our probe set, which targeted 260 kbp of coding sequence, achieved a median recovery of 137 kbp per taxon in coding regions, a maximum recovery of 250 kbp, and an additional median of 212 kbp per taxon in flanking non-coding regions across all species. These results suggest that the Angiosperms353 probe set described here is effective for any group of flowering plants and would be useful for phylogenetic studies from the species level to higher-order groups, including the entire angiosperm clade itself.
    • Using genomic repeats for phylogenomics: A case study in wild tomatoes (Solanum section Lycopersicon: Solanaceae)

      Dodsworth, Steven; Chase, Mark W.; Särkinen, Tiina; Knapp, Sandra; Leitch, Andrew R. (Blackwell Publishing Ltd, 2015-07-17)
      High-throughput sequencing data have transformed molecular phylogenetics and a plethora of phylogenomic approaches are now readily available. Shotgun sequencing at low genome coverage is a common approach for isolating high-copy DNA, such as the plastid or mitochondrial genomes, and ribosomal DNA. These sequence data, however, are also rich in repetitive elements that are often discarded. Such data include a variety of repeats present throughout the nuclear genome in high copy number. It has recently been shown that the abundance of repetitive elements has phylogenetic signal and can be used as a continuous character to infer tree topologies. In the present study, we evaluate repetitive DNA data in tomatoes (Solanum section Lycopersicon) to explore how they perform at the inter- and intraspecific levels, utilizing the available data from the 100 Tomato Genome Sequencing Consortium. The results add to previous examples from angiosperms where genomic repeats have been used to resolve phylogenetic relationships at varying taxonomic levels. Future prospects now include the use of genomic repeats for population-level analyses and phylogeography, as well as potentially for DNA barcoding.
    • 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.