DOCSLIB.ORG

(ZPBP2) and Several Proteins Containing BX7B Motifs in Human Sperm May Have Hyaluronic Acid Binding Or Recognition Properties

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
(ZPBP2) and Several Proteins Containing BX7B Motifs in Human Sperm May Have Hyaluronic Acid Binding Or Recognition Properties This is a repository copy of Zona pellucida-binding protein 2 (ZPBP2) and several proteins containing BX7B motifs in human sperm may have hyaluronic acid binding or recognition properties. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/146678/ Version: Accepted Version Article: Torabi, F, Bogle, OA, Estanyol, JM et al. (2 more authors) (2017) Zona pellucida-binding protein 2 (ZPBP2) and several proteins containing BX7B motifs in human sperm may have hyaluronic acid binding or recognition properties. Molecular Human Reproduction, 23 (12). pp. 803-816. ISSN 1360-9947 https://doi.org/10.1093/molehr/gax053 (c) 2017, The Author. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. This is an author produced version of a paper published in Molecular Human Reproduction. Uploaded in accordance with the publisher's self-archiving policy. Reuse Items deposited in White Rose Research Online are protected by copyright, with all rights reserved unless indicated otherwise. They may be downloaded and/or printed for private study, or other acts as permitted by national copyright laws. The publisher or other rights holders may allow further reproduction and re-use of the full text version. This is indicated by the licence information on the White Rose Research Online record for the item. Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ Draft Manuscript Submitted to MHR for Peer Review Draft Manuscript For Review. Reviewers should submit their review at http://mc.manuscriptcentral.com/molehr Could zona pellucida binding protein 2 (ZPBP2) or proteins containing BX7B motifs in human sperm have hyaluronic acid binding or recognition properties For Review Only Journal: Molecular Human Reproduction Manuscript ID MHR- 7-0 62.R2 Manuscript Type: Original Research Date Submitted by the Author: n/a Complete ,ist of Authors: Torabi, Forough. /niversity of ,eeds, ,eeds Institute of Cardiovascular and Metabolic Medicine 0ogle, Orleigh. Faculty of Medicine, Hospital Clinic and /niversity of 0arcelona, Human 1enetics Research 1roup 2stanyol /,,AT2, Josep M. /niversity of 0arcelona, 3roteomic /nit, CCiT, /niversitat de 0arcelona Oliva, Rafael. Faculty of Medicine, Hospital Clinic and /niversity of 0arcelona, Human 1enetics Research 1roup Miller, David. /niversity of ,eeds, ,eeds Institute of Cardiovascular and Metabolic Medicine Hyaluronic acid 6HA7, Hyaluronic Acid 0inding 3rotein 6HA037, ,in8 module, 4ey 5ords: ,C-MS/MS, 0970 motif http://molehr.oxfordjournals.org/ Page 1 of 176 Draft Manuscript Submitted to MHR for Peer Review 1 Could zona pellucida binding protein 2 (ZPBP2) or several proteins containing BX7B 2 motifs in human sperm have hyaluronic acid binding or recognition properties 3 4 Running title: Identification and characterisation of putative sperm Hyaladherins using mass 5 spectrometry 6 7 F. Torabi, O. A. Bogle, J. ,. Estanyol, R. Oliva, .. ,iller 8 Abstract 9 Bac/ground: Hyaluronic acid binding proteins (hyaladherins) can bind hyaluronic acid (HA) 10 surrounding the cumulus-oophorusFor Review complex, are disti nctOnly from hyases such as PH 20 ( PAM1) and 11 are expressed by mature spermatozoa. Although HABP1 and CD44 are reasonably well 12 characterised hyaladherins and the former has been implicated in sperm-oocyte interactions, the 13 overall significance of sperm hyaladherins for male fertility is still poorly understood. 14 ,ethods: As information on sperm hyaladherins is limited, a protein affinity +panning, procedure was 15 used to facilitate their enrichment by partitioning of whole sperm protein homogenates (N.4) into 16 binding and non-binding fractions and their subse/uent characterisation by li/uid chromatography 17 tandem mass spectrometry (0C-M 1M ). e/uences of proteins from both fractions were submitted 18 to PDBsum to loo2 for orthologous entries (PDB codes) and all returned codes were /ueried against 19 the matching protein using A ( e/uences Annotated by tructure) loo2ing for structural 20 similarities between them. A systematic search for other common features of hyaladherins was also 21 underta2en. 22 Results: The presence of B47B se/uence motifs found in several well-described hyaladherins 23 including RHAMM was used to assess efficacy of potential hyaladherin partitioning by the HA 24 substrate. The data showed that 708 (14128) and 34.78 (28181) of proteins in the bound and 25 unbound fractions, respectively, contained these motifs (one-tailed ; score . 1.47; p . 0.074), 26 indicating wea2 discrimination by the substrate. Querying PDBsum with se/uences for all bound 27 proteins returned several PDB codes matching ;PBP2 with the HA-binding 0in2 domain of the 28 hyaladherin, CD44. Western blot analysis confirmed the affinity partitioning of proteins indicated by 1 http://molehr.oxfordjournals.org/ Draft Manuscript Submitted to MHR for Peer Review Page 2 of 176 29 the 0C-M 1M results with ADAM32 (containing two B47B motifs) and ;PBP2 (containing a 0in2- 30 li2e HA-binding domain) present only in the binding fraction. 31 32 .iscussion and Conclusions 33 The protein extraction methods or the HA substrate used to pan them in this study were probably 34 not ideal, as hyaladherins expected to be present in sperm homogenates (such as CD44 and 35 RHAMM) were not detected. The results, however, provide evidence that ;PBP2, found only in the 36 bound fraction mayFor have hyaladherin-li2e Review properties, whichOnly may reflect the evolutionary bac2ground 37 context of contemporary sperm–egg interaction mechanisms. 38 0ey1ords: Hyaluronic acid (HA), Hyaladherins, CD44, 0in2 module, B47B motif, ;PBP2, ADAM32. 39 40 41 Introduction 42 In natural reproductive cycles, eAaculated spermatozoa encounter physiological selection pressures 43 during their Aourney across the female reproductive tract, where sperm encounter several biological 44 +chec2points,, ensuring that only the +fitter, cells reach the oocyte (I2awa et al. , 2010, uarez and 45 Pacey, 2006). In assisted reproductive technologies (ART), the success of embryo development and 46 pregnancy outcome also depends on sperm /uality ( a22as et al. , 2000) and the selection of viable 47 sperm, including sperm with high DNA integrity and chromatin maturity is crucial (He2matdoost et al. 48 , 200C). To a large extent, sperm selection for ART particularly with IC I depends on the 49 embryologists, experience of pic2ing the best sperm and is based on microscopic parameters of 50 sperm motility, viability and morphology. perm abnormalities, however, particularly those occurring 51 at the molecular level cannot always be detected by microscopic observation alone (Palermo et al. , 52 1CC2). Celi2-Ozenci et al. (2004), for example, showed sperm with normal motility and morphology 53 may have chromosomal abnormalities and IC I provides no barrier preventing sperm with chromatin 54 and other defects from participating in the fertilisation process. Negative effects on IC I outcomes of 55 the use of poor /uality sperm may include miscarriage, an increased ris2 of congenital abnormalities 56 and childhood cancer (Celi2-Ozenci et al. , 2004, Eopal2rishnan et al. , 2000, Halliday, 2012, Faleel 2 http://molehr.oxfordjournals.org/ Page 3 of 176 Draft Manuscript Submitted to MHR for Peer Review 57 and Ghan, 2013, 0arsen et al. , 2013). These issues have led to the development of alternative 58 methods of sperm selection for IC I based on functional properties, potentially mimic2ing the natural 59 processes occurring in the reproductive tract of healthy individuals and sperm binding to hyaluronic 60 acid (HA) is one such method. 61 62 HA is a non-sulphated glycosaminoglycan with numerous biological functions in the extracellular 63 matrix and on the cell surface (Amemiya et al. , 2007). HA is abundant in the female reproductive 64 tract and may be involvedFor in sperm-eggReview interactions (EhoshOnly et al. , 2007). The cumulus oophorus 65 complex (COC) is also HA-rich (;huo and Gimata, 2001) and HA permeates the zona pellucida and 66 the perivitelline space of mammalian oocytes (Handevoort et al. , 1CC7). During in vivo fertilisation, 67 mature spermatozoa bind hyaluronic acid in the extracellular matrix of the COC via hyaladherins 68 and subse/uently release unrelated hyases (for example, PH201 PAM1) facilitating HA digestion 69 and penetration of the cumulus mass. Immature spermatozoa, however, do not bind to HA or may 70 bind it more wea2ly (Huszar et al. , 2003, Nasr-Esfahani et al. , 2008). 71 72 Hyaladherins including CD44 (BaAorath et al. , 1CC8, Underhill, 1CC2) and RHAMM (Hardwic2 et al. , 73 1CC2, Yang et al. , 1CC4), both present in eAaculate spermatozoa (Amaral et al. , 2014) are 74 categorised by whether they contain a 0in2 domain or module (a se/uence of L100 amino acids 75 composed of two alpha-helices, two triple-stranded anti-parallel beta-sheets and two disulphide 76 bonds (Barta et al. , 1CC3) or a B47B motif (where the MBNs are arginine (R) or lysine (G) residues 77 and the M4N is a se/uence of seven non-acidic and at least one basic amino acids), a covalent bond 78 or combinations thereof (Amemiya et al., 2007, Day and Prestwich, 2002, Yang et al. , 1CC4). CD44, 79 for example, contains both a 0in2 module and a B47B motif. 80 81 Investigating sperm hyaladherins is Austified in relation to understanding the fertilisation potential of 82 sperm and the causes of male infertility. A previous study in our laboratory provided evidence for the 83 complexity of hyaladherin expression in eAaculate human spermatozoa and showed that sperm 84 binding to HA was enhanced by capacitation (Torabi et al. , 2016) and may involve un2nown 3 http://molehr.oxfordjournals.org/ Draft Manuscript Submitted to MHR for Peer Review Page 4 of 176 85 hyaladherins. To improve our understanding, proteins extracted from washed and homogenised 86 eAaculate human spermatozoa were subAected to affinity panning on a HA-coated surface (Amemiya 87 et al., 2007).
Load more

Recommended publications
  • Proteomic Analyses Reveal a Role of Cytoplasmic Droplets As an Energy Source During Sperm Epididymal Maturation
    Proteomic analyses reveal a role of cytoplasmic droplets as an energy source during sperm epididymal maturation Shuiqiao Yuana,b, Huili Zhenga, Zhihong Zhengb, Wei Yana,1 aDepartment of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, 89557; and bDepartment of Laboratory Animal Medicine, China Medical University, Shenyang, 110001, China Corresponding author. Email: [email protected] Supplemental Information contains one Figure (Figure S1), three Tables (Tables S1-S3) and two Videos (Videos S1 and S2) files. Figure S1. Scanning electron microscopic images of purified murine cytoplasmic droplets. Arrows point to indentations resembling the resealed defects at the detaching points when CDs come off the sperm flagella. Scale bar = 1µm Table S1 Mass spectrometry-based identifiaction of proteins highly enriched in murine cytoplasmic droplets. # MS/MS View:Identified Proteins (105) Accession Number Molecular Weight Protein Grouping Ambiguity Dot_1_1 Dot_2_1 Dot_3_1 Dot_4_1Dot_5_1 Dot_1_2 Dot_2_2 Dot_3_2 Dot_4_2 Dot_5_2 1 IPI:IPI00467457.3 Tax_Id=10090 Gene_Symbol=Ldhc L-lactate dehydrogenase C chain IPI00467457 36 kDa TRUE 91% 100% 100% 100% 100% 100% 100% 100% 100% 2 IPI:IPI00473320.2 Tax_Id=10090 Gene_Symbol=Actb Putative uncharacterized protein IPI00473320 42 kDa TRUE 75% 100% 100% 100% 100% 89% 76% 100% 100% 100% 3 IPI:IPI00224181.7 Tax_Id=10090 Gene_Symbol=Akr1b7 Aldose reductase-related protein 1 IPI00224181 36 kDa TRUE 100% 100% 76% 100% 100% 4 IPI:IPI00228633.7 Tax_Id=10090 Gene_Symbol=Gpi1 Glucose-6-phosphate
    [Show full text]
  • Cellular and Molecular Signatures in the Disease Tissue of Early
    Cellular and Molecular Signatures in the Disease Tissue of Early Rheumatoid Arthritis Stratify Clinical Response to csDMARD-Therapy and Predict Radiographic Progression Frances Humby1,* Myles Lewis1,* Nandhini Ramamoorthi2, Jason Hackney3, Michael Barnes1, Michele Bombardieri1, Francesca Setiadi2, Stephen Kelly1, Fabiola Bene1, Maria di Cicco1, Sudeh Riahi1, Vidalba Rocher-Ros1, Nora Ng1, Ilias Lazorou1, Rebecca E. Hands1, Desiree van der Heijde4, Robert Landewé5, Annette van der Helm-van Mil4, Alberto Cauli6, Iain B. McInnes7, Christopher D. Buckley8, Ernest Choy9, Peter Taylor10, Michael J. Townsend2 & Costantino Pitzalis1 1Centre for Experimental Medicine and Rheumatology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK. Departments of 2Biomarker Discovery OMNI, 3Bioinformatics and Computational Biology, Genentech Research and Early Development, South San Francisco, California 94080 USA 4Department of Rheumatology, Leiden University Medical Center, The Netherlands 5Department of Clinical Immunology & Rheumatology, Amsterdam Rheumatology & Immunology Center, Amsterdam, The Netherlands 6Rheumatology Unit, Department of Medical Sciences, Policlinico of the University of Cagliari, Cagliari, Italy 7Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow G12 8TA, UK 8Rheumatology Research Group, Institute of Inflammation and Ageing (IIA), University of Birmingham, Birmingham B15 2WB, UK 9Institute of
    [Show full text]
  • CSE642 Final Version
    Eindhoven University of Technology MASTER Dimensionality reduction of gene expression data Arts, S. Award date: 2018 Link to publication Disclaimer This document contains a student thesis (bachelor's or master's), as authored by a student at Eindhoven University of Technology. Student theses are made available in the TU/e repository upon obtaining the required degree. The grade received is not published on the document as presented in the repository. The required complexity or quality of research of student theses may vary by program, and the required minimum study period may vary in duration. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain Eindhoven University of Technology MASTER THESIS Dimensionality Reduction of Gene Expression Data Author: S. (Sako) Arts Daily Supervisor: dr. V. (Vlado) Menkovski Graduation Committee: dr. V. (Vlado) Menkovski dr. D.C. (Decebal) Mocanu dr. N. (Nikolay) Yakovets May 16, 2018 v1.0 Abstract The focus of this thesis is dimensionality reduction of gene expression data. I propose and test a framework that deploys linear prediction algorithms resulting in a reduced set of selected genes relevant to a specified case. Abstract In cancer research there is a large need to automate parts of the process of diagnosis, this is mainly to reduce cost, make it faster and more accurate.
    [Show full text]
  • Genome Sequencing Unveils a Regulatory Landscape of Platelet Reactivity
    ARTICLE https://doi.org/10.1038/s41467-021-23470-9 OPEN Genome sequencing unveils a regulatory landscape of platelet reactivity Ali R. Keramati 1,2,124, Ming-Huei Chen3,4,124, Benjamin A. T. Rodriguez3,4,5,124, Lisa R. Yanek 2,6, Arunoday Bhan7, Brady J. Gaynor 8,9, Kathleen Ryan8,9, Jennifer A. Brody 10, Xue Zhong11, Qiang Wei12, NHLBI Trans-Omics for Precision (TOPMed) Consortium*, Kai Kammers13, Kanika Kanchan14, Kruthika Iyer14, Madeline H. Kowalski15, Achilleas N. Pitsillides4,16, L. Adrienne Cupples 4,16, Bingshan Li 12, Thorsten M. Schlaeger7, Alan R. Shuldiner9, Jeffrey R. O’Connell8,9, Ingo Ruczinski17, Braxton D. Mitchell 8,9, ✉ Nauder Faraday2,18, Margaret A. Taub17, Lewis C. Becker1,2, Joshua P. Lewis 8,9,125 , 2,14,125✉ 3,4,125✉ 1234567890():,; Rasika A. Mathias & Andrew D. Johnson Platelet aggregation at the site of atherosclerotic vascular injury is the underlying patho- physiology of myocardial infarction and stroke. To build upon prior GWAS, here we report on 16 loci identified through a whole genome sequencing (WGS) approach in 3,855 NHLBI Trans-Omics for Precision Medicine (TOPMed) participants deeply phenotyped for platelet aggregation. We identify the RGS18 locus, which encodes a myeloerythroid lineage-specific regulator of G-protein signaling that co-localizes with expression quantitative trait loci (eQTL) signatures for RGS18 expression in platelets. Gene-based approaches implicate the SVEP1 gene, a known contributor of coronary artery disease risk. Sentinel variants at RGS18 and PEAR1 are associated with thrombosis risk and increased gastrointestinal bleeding risk, respectively. Our WGS findings add to previously identified GWAS loci, provide insights regarding the mechanism(s) by which genetics may influence cardiovascular disease risk, and underscore the importance of rare variant and regulatory approaches to identifying loci contributing to complex phenotypes.
    [Show full text]
  • Experimental Eye Research 129 (2014) 93E106
    Experimental Eye Research 129 (2014) 93e106 Contents lists available at ScienceDirect Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer Transcriptomic analysis across nasal, temporal, and macular regions of human neural retina and RPE/choroid by RNA-Seq S. Scott Whitmore a, b, Alex H. Wagner a, c, Adam P. DeLuca a, b, Arlene V. Drack a, b, Edwin M. Stone a, b, Budd A. Tucker a, b, Shemin Zeng a, b, Terry A. Braun a, b, c, * Robert F. Mullins a, b, Todd E. Scheetz a, b, c, a Stephen A. Wynn Institute for Vision Research, The University of Iowa, Iowa City, IA, USA b Department of Ophthalmology and Visual Sciences, Carver College of Medicine, The University of Iowa, Iowa City, IA, USA c Department of Biomedical Engineering, College of Engineering, The University of Iowa, Iowa City, IA, USA article info abstract Article history: Proper spatial differentiation of retinal cell types is necessary for normal human vision. Many retinal Received 14 September 2014 diseases, such as Best disease and male germ cell associated kinase (MAK)-associated retinitis pigmen- Received in revised form tosa, preferentially affect distinct topographic regions of the retina. While much is known about the 31 October 2014 distribution of cell types in the retina, the distribution of molecular components across the posterior pole Accepted in revised form 4 November 2014 of the eye has not been well-studied. To investigate regional difference in molecular composition of Available online 5 November 2014 ocular tissues, we assessed differential gene expression across the temporal, macular, and nasal retina and retinal pigment epithelium (RPE)/choroid of human eyes using RNA-Seq.
    [Show full text]
  • Supplementary Data Genbank Or OSE Vs RO NIA Accession Gene Name Symbol FC B-Value H3073C09 11.38 5.62 H3126B09 9.64 6.44 H3073B0
    Supplementary Data GenBank or OSE vs RO NIA accession Gene name Symbol FC B-value H3073C09 11.38 5.62 H3126B09 9.64 6.44 H3073B08 9.62 5.59 AU022767 Exportin 4 Xpo4 9.62 6.64 H3073B09 9.59 6.48 BG063925 Metallothionein 2 Mt2 9.23 18.89 H3064B07 9.21 6.10 H3073D08 8.28 6.10 AU021923 Jagged 1 Jag1 7.89 5.93 H3070D08 7.54 4.58 BG085110 Cysteine-rich protein 1 (intestinal) Crip1 6.23 16.40 BG063004 Lectin, galactose binding, soluble 1 Lgals1 5.95 10.36 BG069712 5.92 2.34 BG076976 Transcribed locus, strongly similar to NP_032521.1 lectin, galactose binding, soluble 1 5.64 8.36 BG062930 DNA segment, Chr 11, Wayne State University 99, expressed D11Wsu99e 5.63 8.76 BG086474 Insulin-like growth factor binding protein 5 Igfbp5 5.50 15.95 H3002d11 5.13 20.77 BG064706 Keratin complex 1, acidic, gene 19 Krt1-19 5.06 9.07 H3007A09 5.05 2.46 H3065F02 4.84 5.43 BG081752 4.81 1.25 H3010E09 4.71 11.90 H3064c11 4.43 1.00 BG069711 Transmembrane 4 superfamily member 9 Tm4sf9 4.29 1.23 BG077072 Actin, beta, cytoplasmic Actb 4.29 3.01 BG079788 Hemoglobin alpha, adult chain 1 Hba-a1 4.26 6.63 BG076798 4.23 0.80 BG074344 Mesothelin Msln 4.22 6.97 C78835 Actin, beta, cytoplasmic Actb 4.16 3.02 BG067531 4.15 1.61 BG073468 Hemoglobin alpha, adult chain 1 Hba-a1 4.10 6.23 H3154H07 4.08 5.38 AW550167 3.95 5.66 H3121B01 3.94 5.94 H3124f12 3.94 5.64 BG073608 Hemoglobin alpha, adult chain 1 Hba-a1 3.84 5.32 BG073617 Hemoglobin alpha, adult chain 1 Hba-a1 3.84 5.75 BG072574 Hemoglobin alpha, adult chain 1 Hba-a1 3.82 5.93 BG072211 Tumor necrosis factor receptor superfamily,
    [Show full text]
  • Whole-Exome SNP Array Identifies 15 New Susceptibility Loci for Psoriasis
    ARTICLE Received 27 Sep 2014 | Accepted 28 Feb 2015 | Published 9 Apr 2015 | Updated 13 Mar 2018 DOI: 10.1038/ncomms7793 OPEN Whole-exome SNP array identifies 15 new susceptibility loci for psoriasis Xianbo Zuo1,2,3,4,5,6,7,*, Liangdan Sun1,2,3,4,5,6,7,*, Xianyong Yin1,2,3,4,5,6,7,*, Jinping Gao1,4,5,6,7,*, Yujun Sheng1,4,5,6,7,*, Jinhua Xu2,4, Jianzhong Zhang8, Chundi He9, Ying Qiu10, Guangdong Wen8, Hongqing Tian11, Xiaodong Zheng1,4,5,6,7, Shengxiu Liu1,4,5,6,7,WenjunWang1,4,5,6,7,WeiranLi1,4,5,6,7,YuyanCheng1,4,5,6,7, Longdan Liu1,4,5,6,7, Yan Chang1,4,5,6,7,ZaixingWang1,4,5,6,7,ZenggangLi1,4,5,6,7,LongnianLi1,4,5,6,7, Jianping Wu1,4,5,6,7,LingFang1,4,5,6,7, Changbing Shen1,4,5,6,7, Fusheng Zhou1,4,5,6,7,BoLiang1,4,5,6,7,Gang Chen1,4,5,6,7,HuiLi1,4,5,6,7, Yong Cui4,5,6,7,AieXu12,XueqinYang13,FeiHao14, Limin Xu15,XingFan1,4,5,6,7, Yuzhen Li16,RinaWu17, Xiuli Wang18, Xiaoming Liu19,MinZheng20, Shunpeng Song21,BihuaJi22, Hong Fang23, Jianbin Yu24,YongxinSun25,YanHui26,FurenZhang11,RongyaYang27,SenYang1,4,5,6,7 & Xuejun Zhang1,2,3,4,5,6,7 Genome-wide association studies (GWASs) have reproducibly associated B40 susceptibility loci with psoriasis. However, the missing heritability is evident and the contributions of coding variants have not yet been systematically evaluated. Here, we present a large-scale whole- exome array analysis for psoriasis consisting of 42,760 individuals.
    [Show full text]
  • Investigation of Host Factors Involved in Legionella Pneumophila Virulence
    Investigation of host factors involved in Legionella pneumophila virulence Sze Ying Ong Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy October 2017 Department of Microbiology and Immunology The University of Melbourne at the Peter Doherty Institute of Infection and Immunity Produced on archival quality paper ABSTRACT Legionella pneumophila is an environmental organism that can become accidental bacterial pathogen when inhaled into the lungs of humans. On entering the lungs, L. pneumophila is phagocytosed by alveolar macrophages. However, instead of being removed accordingly by the host immune system, the bacteria rapidly establish a Legionella-containing vacuole (LCV) and replicate intracellularly. The ability to establish the LCV relies on a type IV secretion system, also known as the Dot/Icm system. The Dot/Icm system is essential for virulence and delivers a large repertoire (> 300) of effector proteins into infected host cells. These effector proteins modulate a wide range of host processes such as vesicle trafficking, host protein translation, regulation of GTPases and apoptosis. Despite the phenomenal number (one of the highest among known bacterial pathogens) of effector proteins translocated by L. pneumophila into host cells, effector secretion is not detected until the bacterium contacts a host cell. This is in contrast to other bacterial pathogens such as enteropathogenic Escherichia coli or Salmonella sp., which can be induced to secrete effector proteins via a type III secretion system into liquid bacteriological cultures. The interactions that occur between the host cells and L. pneumophila in order to activate the Dot/Icm system are poorly understood. In this study, we used an RNAi approach to screen for host factors that contribute to Dot/Icm effector protein translocation.
    [Show full text]
  • 24651 Story.Indd
    Aetiology of Depression: Insights from epidemiological and genetic research Olivera Story-Jovanova Acknowledgments: Financial support for the publication of this thesis by the Department of Epidemiology of the Erasmus MC, is gratefully acknowledged. ISBN: 978-94-6233-909-5 Cover: Concept by Olivera Story-Jovanova, design by Chris van Wolferen-Ketel, photography by Michal Macku. Layout: Gildeprint, Enschede. Printing: Gildeprint, Enschede. © Olivera Story-Jovanova, 2018 For all articles published, the copyright has been transferred to the respective publisher. No part of this thesis may be stored in a retrieval system, or transmitted in any form or by any means, without written permission from the author or, when appropriate, from the publisher. Aetiology of Depression: Insights from epidemiological and genetic research Etiologie van depressie: Inzichten vanuit de epidemiologisch en de genetisch onderzoek Proefschrift ter verkrijging van de graad van doctor aan de Erasmus Universiteit Rotterdam op gezag van de rector magnificus Prof. dr. H.A.P. Pols en volgens besluit van het College voor Promoties. De openbare verdediging zal plaatsvinden op 4 April 2018 om 15:30 uur door Olivera Story-Jovanova geboren te Skopje, Macedonia PROMOTIECOMMISSIE Promotor Prof. dr. H. Tiemeier Overige leden Prof. D. Boomsma Prof. K. Berger Prof. C. van Duijn Copromotor Dr. N. Amin Paranimfen Ayesha Sajjad Dina Atlas For my husband, children, sister, parents, grandparents, my parents in law and all you who believed in me. You are a gift of unconditional love, acceptance, joy and wisdom. I am thankful for that! ACKNOWLEDGEMENTS The research described in this thesis was performed within the frame work of the Rotterdam Study.
    [Show full text]
  • Characterizing Genomic Duplication in Autism Spectrum Disorder by Edward James Higginbotham a Thesis Submitted in Conformity
    Characterizing Genomic Duplication in Autism Spectrum Disorder by Edward James Higginbotham A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Molecular Genetics University of Toronto © Copyright by Edward James Higginbotham 2020 i Abstract Characterizing Genomic Duplication in Autism Spectrum Disorder Edward James Higginbotham Master of Science Graduate Department of Molecular Genetics University of Toronto 2020 Duplication, the gain of additional copies of genomic material relative to its ancestral diploid state is yet to achieve full appreciation for its role in human traits and disease. Challenges include accurately genotyping, annotating, and characterizing the properties of duplications, and resolving duplication mechanisms. Whole genome sequencing, in principle, should enable accurate detection of duplications in a single experiment. This thesis makes use of the technology to catalogue disease relevant duplications in the genomes of 2,739 individuals with Autism Spectrum Disorder (ASD) who enrolled in the Autism Speaks MSSNG Project. Fine-mapping the breakpoint junctions of 259 ASD-relevant duplications identified 34 (13.1%) variants with complex genomic structures as well as tandem (193/259, 74.5%) and NAHR- mediated (6/259, 2.3%) duplications. As whole genome sequencing-based studies expand in scale and reach, a continued focus on generating high-quality, standardized duplication data will be prerequisite to addressing their associated biological mechanisms. ii Acknowledgements I thank Dr. Stephen Scherer for his leadership par excellence, his generosity, and for giving me a chance. I am grateful for his investment and the opportunities afforded me, from which I have learned and benefited. I would next thank Drs.
    [Show full text]
  • Genome-Wide Screening Identifies Genes and Biological Processes
    Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 10-12-2018 Genome-Wide Screening Identifies Genes and Biological Processes Implicated in Chemoresistance and Oncogene-Induced Apoptosis Tengyu Ko Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Cancer Biology Commons, Cell Biology Commons, and the Genomics Commons Recommended Citation Ko, Tengyu, "Genome-Wide Screening Identifies Genes and Biological Processes Implicated in Chemoresistance and Oncogene- Induced Apoptosis" (2018). LSU Doctoral Dissertations. 4715. https://digitalcommons.lsu.edu/gradschool_dissertations/4715 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. GENOME-WIDE SCREENING IDENTIFIES GENES AND BIOLOGICAL PROCESSES IMPLICATED IN CHEMORESISTANCE AND ONCOGENE- INDUCED APOPTOSIS A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical and Veterinary Medical Sciences through the Department of Comparative Biomedical Sciences by Tengyu Ko B.S., University of California, Santa Barbara 2010 December 2018 ACKNOWLEDGEMENTS I would like to express my sincerest gratitude to my major supervisor Dr. Shisheng Li for giving me the opportunity to join his team and the freedom to pursue projects. I appreciate all of his thoughts and efforts. Truly, none of these findings would be possible without his supervisions, supports, insightful discussions, and patience.
    [Show full text]
  • Supporting Information
    Supporting Information Foucault et al. 10.1073/pnas.1006855107 SI Materials and Methods KpnI plus BamHI and cloned under the control of the Ptet pro- Bacterial Strains, Plasmids, and Growth Conditions. The origin and moter of pAT890(pAT29ΩPtet) digested by the same enzymes to properties of bacterial strains and plasmids are summarized in generate plasmid pAT891(pAT29ΩPtet lucR)(Table S1). Escherichia coli Table S1. Top10 (Invitrogen) was used as a host pAT895(pGhost9Ωint′P lucR int″). To integrate the luciferase gene for recombinant plasmids, and kanamycin (50 μg/mL) was the tet in the chromosome of JH2-2::I, an int′Ptet lucR int″ fragment was selective agent for cloning PCR products in the pCR-Blunt E. coli constructed in the thermosensitive vector pGhost9 (Fig. S1). vector (Invitrogen). TG1 RepA (1) was used as a host for The int′ fragment amplified with the intB1 and intB2 primers constructions in the pGhost9 vector (2). Ampicillin (75 μg/mL), μ μ (Table S2) from total DNA of JH2-2::I was inserted in the XhoI spectinomycin (60 g/mL), and erythromycin (150 g/mL) were and PstI sites of pCR-Blunt to generate plasmid pAT892(pCR- added to culture media to prevent loss of plasmids derived from BluntΩint′). pGEX-6P-2ΩlucR, pAT29, and pGhost9, respectively. Strains The Ptet lucR insert of pAT891(pAT29ΩPtetlucR) amplified were grown in brain heart infusion broth or agar (Difco) at 37 °C. with primers Ptet2 and LucR-rev was cloned downstream from int′ Plasmid Constructions. The plasmids were constructed as follows. between the PstI and BamHI restriction sites of plasmid Ωint′ Ωint′ Construction of pAT886 and pAT889.
    [Show full text]