DOCSLIB.ORG

Co-Recruitment Analysis of the CBL and CBLB Signalosomes in Primary

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Co-Recruitment Analysis of the CBL and CBLB Signalosomes in Primary Co-recruitment analysis of the CBL and CBLB signalosomes in primary T cells identifies CD5 as a key regulator of TCR-induced ubiquitylation Guillaume Voisinne, Antonio García-Blesa, Karima Chaoui, Frédéric Fiore, Elise Bergot, Laura Girard, Marie Malissen, Odile Burlet-Schiltz, Anne Gonzalez de Peredo, Bernard Malissen, et al. To cite this version: Guillaume Voisinne, Antonio García-Blesa, Karima Chaoui, Frédéric Fiore, Elise Bergot, et al.. Co- recruitment analysis of the CBL and CBLB signalosomes in primary T cells identifies CD5 as a key regulator of TCR-induced ubiquitylation. Molecular Systems Biology, EMBO Press, 2016, 12, pp.876. 10.15252/msb.20166837. inserm-01368500 HAL Id: inserm-01368500 https://www.hal.inserm.fr/inserm-01368500 Submitted on 19 Sep 2016 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Published online: July 29, 2016 Article Co-recruitment analysis of the CBL and CBLB signalosomes in primary T cells identifies CD5 as a key regulator of TCR-induced ubiquitylation Guillaume Voisinne1,†, Antonio García-Blesa1,†, Karima Chaoui2, Frédéric Fiore3, Elise Bergot1, Laura Girard1,3, Marie Malissen1,3, Odile Burlet-Schiltz2, Anne Gonzalez de Peredo2, Bernard Malissen1,3,* & Romain Roncagalli1,** Abstract (interactomes) and of signaling complexes (signalosomes) in their physiological cellular context (Gingras et al, 2007; Hein et al, T-cell receptor (TCR) signaling is essential for the function of T cells 2015). Although such large-scale studies have resulted in compre- and negatively regulated by the E3 ubiquitin–protein ligases CBL hensive views of several signalosomes, they provided limited and CBLB. Here, we combined mouse genetics and affinity purifica- information on their internal organization and dynamics of assem- tion coupled to quantitative mass spectrometry to monitor the bly. Such information is, however, particularly valuable to under- dynamics of the CBL and CBLB signaling complexes that assemble stand how the flow of information resulting from engagement of a in normal T cells over 600 seconds of TCR stimulation. We identify given cell surface receptor propagates over time (Collins et al, most previously known CBL and CBLB interacting partners, as well 2013; Zheng et al, 2013). The T-cell antigen receptors (TCRs) as a majority of proteins that have not yet been implicated in found at the surface of T cells carry out the formidable task of those signaling complexes. We exploit correlations in protein asso- identifying minute number of antigenic peptides. Despite a wealth ciation with CBL and CBLB as a function of time of TCR stimulation of information concerning the dozens of molecules involved in for predicting the occurrence of direct physical association TCR signaling, the general principle of information processing between them. By combining co-recruitment analysis with by the TCR signal transduction network remains incompletely biochemical analysis, we demonstrated that the CD5 transmem- understood (Chakraborty & Weiss, 2014; Malissen & Bongrand, 2015). brane receptor constitutes a key scaffold for CBL- and CBLB- TCR signals propagate through the cytosol and the nucleus via a mediated ubiquitylation following TCR engagement. Our results multilayered signal transduction network involving PPI, phosphory- offer an integrated view of the CBL and CBLB signaling complexes lation, and ubiquitylation. Regulatory mechanisms limit and termi- induced by TCR stimulation and provide a molecular basis for their nate TCR-driven signals allowing the immune system to return to a negative regulatory function in normal T cells. basal state after antigen has been cleared (Acuto et al, 2008). The E3 ubiquitin–protein ligases CBL and CBLB have been recognized as Keywords CBL; CBLB; CD5; ubiquitylation important negative regulators of T-cell activation (Huang & Gu, Subject Categories Immunology; Post-translational Modifications, 2008). Following TCR engagement, the protein tyrosine kinases (PTK) Proteolysis & Proteomics; Signal Transduction LCK and ZAP-70 are activated and phosphorylate several trans- DOI 10.15252/msb.20166837 | Received 26 January 2016 | Revised 14 June membrane and cytosolic proteins, leading in turn to the recruitment of 2016 | Accepted 23 June 2016 CBL and CBLB via their tyrosine kinase binding (TKB) domains or Mol Syst Biol. (2016) 12: 876 indirectly via the GRB2 adaptor. As a result, CBL and CBLB are rapidly phosphorylated on tyrosine residues, causing conformational changes allowing the recruitment of E2 ubiquitin-conjugating enzymes and Introduction their juxtaposition to substrates that include the TCR itself, scaffold proteins (also known as adaptors), cytosolic PTK, and phosphatases Affinity purification followed by mass spectrometry (AP-MS) (Schmidt & Dikic, 2005). Modification of the TCR and of its signaling enables analysis of protein–protein interaction (PPI) networks partners with ubiquitin promotes their sorting to multivesicular 1 Centre d’Immunologie de Marseille-Luminy, Aix Marseille Université, Inserm, CNRS, Marseille, France 2 Institut de Pharmacologie et de Biologie Structurale, Département Biologie Structural Biophysique, Protéomique Génopole Toulouse Midi Pyrénées, CNRS UMR 5089, Toulouse Cedex, France 3 Centre d’Immunophénomique, Aix Marseille Université UM2, Inserm US012, CNRS UMS3367, Marseille, France *Corresponding author. Tel: +33 491269478; Fax: +33 491269430; E-mail: [email protected] **Corresponding author. Tel: +33 491269478; Fax: +33 491269430; E-mail: [email protected] †These authors contributed equally to this work ª 2016 The Authors. Published under the terms of the CC BY 4.0 license Molecular Systems Biology 12: 876 | 2016 1 Published online: July 29, 2016 Molecular Systems Biology Assembly dynamics of the CBL and CBLB signalosomes Guillaume Voisinne et al bodies and lysosomal degradation, thereby ensuring termination of we generated two lines of gene-targeted mice expressing One- TCR signaling (Vardhana et al, 2010). CBL and CBLB are more than STrEP-tag (OST) (Junttila et al, 2005) at the carboxyl-terminus of just E3 ubiquitin–protein ligases and possess numerous interactive endogenous CBL and CBLB proteins (Fig EV1A and B). Analysis of elements that nucleate the formation of complexes thought to mice homozygous for the CblOST allele (also known as B6-Cbltm1Mal; regulate cytoskeletal rearrangement (Lee & Tsygankov, 2013). These called “CBLOST” mice here) and for the CblbOST allele (also known interactive elements comprise a proline-rich region that binds SH3 as B6-Cblbtm1Ciphe; called “CBLBOST” mice here) showed that their T domain-containing proteins, multiple tyrosine phosphorylation sites cells developed properly and that they contained normal numbers enabling interactions with SH2 domain-containing proteins, and a of CD4+ and CD8+ T cells (Fig EV1C–F). CD4+ T cells purified ubiquitin association (UBA) domain. from wild-type, CBLOST, and CBLBOST mice and stimulated with Although the presence of several conserved structural domains anti-CD3 antibodies in the presence or absence of anti-CD28 anti- in CBL and CBLB suggests that they exert redundant functions, the bodies showed that expression of the CBL-OST and CBLB-OST mature T cells of mice deprived of CBL or CBLB showed distinct proteins had no detrimental effect on their proliferation (Fig EV1G phenotypes. Mature T cells from CBL-deficient mice are less respon- and H) or production of interleukin 2 (Fig EV1I and J). Immunoblot sive to antigenic stimulation (Naramura et al, 2002). In contrast, analysis showed that before and after activation with anti-CD3 and CBLB deficiency lowers the threshold that needs to be overcome by anti-CD4 antibodies, CBL-OST and CBLB-OST proteins can be puri- antigenic stimulation to trigger T-cell responses (Bachmaier et al, fied from lysates of CBLOST and CBLBOST CD4+ T cells, respectively, 2000; Chiang et al, 2000). As a result, CBLB-deficient T cells prolif- using Sepharose beads coupled to Strep-Tactin, a streptavidin erate and synthesize cytokines following stimulation with subopti- derivative that binds with high affinity to the OST sequence mal concentration of anti-TCR antibody or in the absence of CD28 (Fig 1A). As expected, no detectable material was recovered from costimulation, and in turn, CBLB-deficient mice are prone to wild-type CD4+ T cells. Therefore, primary CD4+ T cells from autoimmunity (Gronski et al, 2004; Chiang et al, 2007; Teh et al, CBLOST and CBLBOST mice are normal and can be used in AP-MS 2010; Zhou et al, 2014). CBLB-deficient T cells have increased analysis. anti-tumor efficacy, suggesting that CBLB might be exploited as a drug target for the purpose of boosting T-cell responses against tumors (Hinterleitner et al, 2012). A Pull-Down B Immature thymocytes Here, we combined mouse genetics and time-resolved, quantita- WT CBLOST CBLBOST tive proteomics analysis to compare the mode of action of CBL and CD3+CD4: -+ -+-+ WT CBLB following TCR-mediated activation of mature T cells. Consid- kDa CBLBOST ering that the transformed T-cell lines
Load more

Recommended publications
  • Transcriptome Analyses of Rhesus Monkey Pre-Implantation Embryos Reveal A
    Downloaded from genome.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Transcriptome analyses of rhesus monkey pre-implantation embryos reveal a reduced capacity for DNA double strand break (DSB) repair in primate oocytes and early embryos Xinyi Wang 1,3,4,5*, Denghui Liu 2,4*, Dajian He 1,3,4,5, Shengbao Suo 2,4, Xian Xia 2,4, Xiechao He1,3,6, Jing-Dong J. Han2#, Ping Zheng1,3,6# Running title: reduced DNA DSB repair in monkey early embryos Affiliations: 1 State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China 2 Key Laboratory of Computational Biology, CAS Center for Excellence in Molecular Cell Science, Collaborative Innovation Center for Genetics and Developmental Biology, Chinese Academy of Sciences-Max Planck Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China 3 Yunnan Key Laboratory of Animal Reproduction, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China 4 University of Chinese Academy of Sciences, Beijing, China 5 Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, Yunnan 650204, China 6 Primate Research Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, 650223, China * Xinyi Wang and Denghui Liu contributed equally to this work 1 Downloaded from genome.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press # Correspondence: Jing-Dong J. Han, Email: [email protected]; Ping Zheng, Email: [email protected] Key words: rhesus monkey, pre-implantation embryo, DNA damage 2 Downloaded from genome.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press ABSTRACT Pre-implantation embryogenesis encompasses several critical events including genome reprogramming, zygotic genome activation (ZGA) and cell fate commitment.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • CBL Mutations in Myeloproliferative Neoplasms Are Also Found in the Gene’S Proline-Rich Domain and in Patients with the V617FJAK2
    Articles and Brief Reports Chronic Myeloproliferative Disorders CBL mutations in myeloproliferative neoplasms are also found in the gene’s proline-rich domain and in patients with the V617FJAK2 Paula Aranaz,1 Cristina Hurtado,1 Ignacio Erquiaga,1 Itziar Miguéliz,1 Cristina Ormazábal,1 Ion Cristobal,2 Marina García-Delgado,1 Francisco Javier Novo,1 and José Luis Vizmanos1 1Department of Genetics, School of Sciences, University of Navarra, Pamplona; and 2CIMA, Center for Applied Medical Research, University of Navarra, Pamplona, Spain ABSTRACT Funding: this work was funded Background with the help of the Spanish Despite the discovery of the p.V617F in JAK2, the molecular pathogenesis of some chronic myelo- Ministry of Science and Innovation proliferative neoplasms remains unclear. Although very rare, different studies have identified CBL (SAF 2007-62473), the PIUNA (Cas-Br-Murine ecotropic retroviral transforming sequence) mutations in V617FJAK2-negative Program of the University of patients, mainly located in the RING finger domain. In order to determine the frequency of CBL Navarra, the Caja Navarra Foundation through the Program mutations in these diseases, we studied different regions of all CBL family genes (CBL, CBLB and “You choose, you decide” (Project CBLC) in a selected group of patients with myeloproliferative neoplasms. We also included 10.830) and ISCIII-RTICC V617FJAK2-positive patients to check whether mutations in CBL and JAK2 are mutually exclusive (RD06/0020/0078). events. PA received a predoctoral grant from the Government of Navarra. Design and Methods Using denaturing high performance liquid chromatography, we screened for mutations in CBL, Manuscript received on CBLB and CBLC in a group of 172 V617FJAK2-negative and 232 V617FJAK2-positive patients July 26, 2011.
    [Show full text]
  • Supplementary Information Method CLEAR-CLIP. Mouse Keratinocytes
    Supplementary Information Method CLEAR-CLIP. Mouse keratinocytes of the designated genotype were maintained in E-low calcium medium. Inducible cells were treated with 3 ug/ml final concentration doxycycline for 24 hours before performing CLEAR-CLIP. One 15cm dish of confluent cells was used per sample. Cells were washed once with cold PBS. 10mls of cold PBS was then added and cells were irradiated with 300mJ/cm2 UVC (254nM wavelength). Cells were then scraped from the plates in cold PBS and pelleted by centrifugation at 1,000g for 2 minutes. Pellets were frozen at -80oC until needed. Cells were then lysed on ice with occasional vortexing in 1ml of lysis buffer (50mM Tris-HCl pH 7.4, 100mM NaCl, 1mM MgCl2, 0.1 mM CaCl2, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS) containing 1X protease inhibitors (Roche #88665) and RNaseOUT (Invitrogen #10777019) at 4ul/ml final concentration. Next, TurboDNase (Invitrogen #AM2238, 10U), RNase A (0.13ug) and RNase T1 (0.13U) were added and samples were incubated at 37oC for 5 minutes with occasional mixing. Samples were immediately placed on ice and then centrifuged at 16,160g at 4oC for 20 minutes to clear lysate. 25ul of Protein-G Dynabeads (Invitrogen #10004D) were used per IP. Dynabeads were pre-washed with lysis buffer and pre- incubated with 3ul of Wako Anti-Mouse-Ago2 (2D4) antibody. The dynabead/antibody mixture was added to the lysate and rocked for 2 hours at 4oC. All steps after the IP were done on bead until samples were loaded into the polyacrylamide gel.
    [Show full text]
  • Anti-CBLB Antibody (ARG57312)
    Product datasheet [email protected] ARG57312 Package: 100 μl anti-CBLB antibody Store at: -20°C Summary Product Description Rabbit Polyclonal antibody recognizes CBLB Tested Reactivity Hu, Ms Tested Application IHC-P, WB Host Rabbit Clonality Polyclonal Isotype IgG Target Name CBLB Antigen Species Human Immunogen Recombinant Protein of Human CBLB. Conjugation Un-conjugated Alternate Names Nbla00127; EC 6.3.2.-; RNF56; Signal transduction protein CBL-B; Casitas B-lineage lymphoma proto- oncogene b; SH3-binding protein CBL-B; Cbl-b; E3 ubiquitin-protein ligase CBL-B; RING finger protein 56 Application Instructions Application table Application Dilution IHC-P 1:50 - 1:200 WB 1:500 - 1:2000 Application Note * The dilutions indicate recommended starting dilutions and the optimal dilutions or concentrations should be determined by the scientist. Positive Control Jurkat Calculated Mw 109 kDa Properties Form Liquid Purification Affinity purification with immunogen. Buffer PBS (pH 7.3), 0.02% Sodium azide and 50% Glycerol. Preservative 0.02% Sodium azide Stabilizer 50% Glycerol Storage instruction For continuous use, store undiluted antibody at 2-8°C for up to a week. For long-term storage, aliquot and store at -20°C. Storage in frost free freezers is not recommended. Avoid repeated freeze/thaw cycles. Suggest spin the vial prior to opening. The antibody solution should be gently mixed before use. Note For laboratory research only, not for drug, diagnostic or other use. www.arigobio.com 1/2 Bioinformation Gene Symbol CBLB Gene Full Name Cbl proto-oncogene B, E3 ubiquitin protein ligase Function E3 ubiquitin-protein ligase which accepts ubiquitin from specific E2 ubiquitin-conjugating enzymes, and transfers it to substrates, generally promoting their degradation by the proteasome.
    [Show full text]
  • Upregulation of E3 Ubiquitin Ligase CBLC Enhances EGFR
    Published OnlineFirst June 26, 2018; DOI: 10.1158/0008-5472.CAN-17-3858 Cancer Tumor Biology and Immunology Research Upregulation of E3 Ubiquitin Ligase CBLC Enhances EGFR Dysregulation and Signaling in Lung Adenocarcinoma Shiao-Ya Hong1, Yu-Rung Kao1, Te-Chang Lee1, and Cheng-Wen Wu1,2,3,4 Abstract CBLC (CBL proto-oncogene c) belongs to the CBL protein ubiquitinated and positively regulated aEGFR stability family, which has E3 ubiquitin ligase activity toward activated through the conjugation of polyubiquitin by K6 and K11 receptor tyrosine kinases. CBLC is frequently upregulated in linkages. This CBLC-mediated polyubiquitination promoted non–small cell lung cancer (NSCLC), yet very little is known either preferential recycling of aEGFR back to the plasma about the functions of CBLC in tumorigenesis. Here we show membrane or trafficking to the cell nucleus. IHC analyses that CBLC is an epigenetically demethylated target and its revealed a positive correlation between phospho-EGFR and expression can be upregulated in NSCLC after treatment with CBLC in lung adenocarcinoma. In summary, we demonstrate a the DNA methylation inhibitor 50-azacytidine. Depletion novel mechanism by which aEGFR escapes lysosomal degra- of CBLC significantly inhibited cell viability and clonogenicity dation in a CBLC/ubiquitin-dependent manner to sustain its in vitro and reduced tumor growth in a xenograft model. CBLC activation. Our work identifies CBLC as a potential diagnostic silencing further sensitized EGFR-mutated NSCLC cells to biomarker and also points to its utilization as a novel thera- treatment with tyrosine kinase inhibitors. Conversely, ectopic peutic target for NSCLC therapy. expression of CBLC enhanced the activation of EGFR and downstream ERK1/2 signaling after ligand stimulation by Significance: This work demonstrates the role of CBLC expres- competing with CBL for EGFR binding.
    [Show full text]
  • Brief Genetics Report
    Brief Genetics Report The Rat Diabetes Susceptibility Locus Iddm4 and at Least One Additional Gene Are Required for Autoimmune Diabetes Induced by Viral Infection Elizabeth P. Blankenhorn,1 Lucy Rodemich,1 Cristina Martin-Fernandez,1 Jean Leif,2 Dale L. Greiner,2 and John P. Mordes2 BBDR rats develop autoimmune diabetes only after challenge with environmental perturbants. These per- turbants include polyinosinic:polycytidylic acid (poly ype 1 diabetes results from inflammatory infiltra- I:C, a ligand of toll-like receptor 3), agents that deplete tion of pancreatic islets and selective ␤-cell regulatory T-cell (Treg) populations, and a non–␤-cell destruction. It is thought to be caused by envi- cytopathic parvovirus (Kilham rat virus [KRV]). The Tronmental factors operating in a genetically sus- dominant diabetes susceptibility locus Iddm4 is re- ceptible host (1,2). Susceptibility loci include the major quired for diabetes induced by treatment with poly I:C histocompatibility complex (MHC), a promoter polymor- plus Treg depletion. Iddm4 is penetrant in congenic phism of the insulin gene, and an allelic variant of CTLA4 heterozygous rats on the resistant WF background and (3). Among candidate environmental perturbants, viral is 79% sensitive and 80% specific as a predictor of infection is one of the most likely (4). How genes interact induced diabetes. Surprisingly, an analysis of 190 BBDR ؋ WF)F2 rats treated with KRV after brief with the environment to transform diabetes susceptibility) exposure to poly I:C revealed that the BBDR-origin into overt disease is unknown. allele of Iddm4 is necessary but not entirely sufficient BBDR rats model virus-induced autoimmune diabetes for diabetes expression.
    [Show full text]
  • Polymorphic Variation in the CBLB Gene in Human Type 1 Diabetes
    Genes and Immunity (2004) 5, 232–235 & 2004 Nature Publishing Group All rights reserved 1466-4879/04 $25.00 www.nature.com/gene BRIEF COMMUNICATION Polymorphic variation in the CBLB gene in human type 1 diabetes R Kosoy1,2, N Yokoi3, S Seino3,4 and P Concannon1,2 1Molecular Genetics Program, Benaroya Research Institute, Seattle, WA, USA; 2Department of Immunology, University of Washington School of Medicine, Seattle, WA, USA; 3Division of Cellular and Molecular Medicine, Kobe University Graduate School of Medicine, Kobe, Japan; 4Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan CBLB was evaluated as a candidate gene for type 1 diabetes (T1D) susceptibility based on its association with autoimmunity in animal models and its role in T-cell costimulatory signaling. Cblb is one of the two major diabetes predisposing loci in the Komeda diabetes-prone (KDP) rat. Cbl-b, a ubiquitin ligase, couples TCR-mediated stimulation with the requirement for CD28 costimulation, regulating T-cell activation. To identify variants with possible effects on gene function as well as haplotype tagging polymorphisms, the human CBLB coding region was sequenced in 16 individuals with T1D: no variants predicted to change the amino-acid sequence were identified. Seven single-nucleotide polymorphism (SNP) markers spanning the CBLB gene were genotyped in multiplex T1D families and assessed for disease association by transmission disequilibrium testing. No significant evidence of association was obtained for either individual markers or marker haplotypes. Genes and Immunity (2004) 5, 232–235. doi:10.1038/sj.gene.6364057 Published online 12 February 2004 Keywords: diabetes; T cell; SNP; haplotype Introduction studies in human T1D families and studies in the NOD mouse model of T1D.9,10 Type I diabetes (T1D) arises from autoimmune destruc- In the current study, we have evaluated another tion of the insulin-secreting b-cells of the pancreas candidate T1D gene, CBLB, which is implicated based requiring insulin replacement therapy for survival.
    [Show full text]
  • Supplementary Material Contents
    Supplementary Material Contents Immune modulating proteins identified from exosomal samples.....................................................................2 Figure S1: Overlap between exosomal and soluble proteomes.................................................................................... 4 Bacterial strains:..............................................................................................................................................4 Figure S2: Variability between subjects of effects of exosomes on BL21-lux growth.................................................... 5 Figure S3: Early effects of exosomes on growth of BL21 E. coli .................................................................................... 5 Figure S4: Exosomal Lysis............................................................................................................................................ 6 Figure S5: Effect of pH on exosomal action.................................................................................................................. 7 Figure S6: Effect of exosomes on growth of UPEC (pH = 6.5) suspended in exosome-depleted urine supernatant ....... 8 Effective exosomal concentration....................................................................................................................8 Figure S7: Sample constitution for luminometry experiments..................................................................................... 8 Figure S8: Determining effective concentration .........................................................................................................
    [Show full text]
  • Brief Genetics Report
    Brief Genetics Report Haplotype Tag Single Nucleotide Polymorphism Analysis of the Human Orthologues of the Rat Type 1 Diabetes Genes Ian4 (Lyp/Iddm1) and Cblb Felicity Payne, Deborah J. Smyth, Rebecca Pask, Bryan J. Barratt, Jason D. Cooper, Rebecca C.J. Twells, Neil M. Walker, Alex C. Lam, Luc J. Smink, Sarah Nutland, Helen E. Rance, and John A. Todd The diabetes-prone BioBreeding (BB) and Komeda dia- animal model is the severe lymphopenia that is essential betes-prone (KDP) rats are both spontaneous animal for the development of the diabetic phenotype and that is models of human autoimmune, T-cell–associated type 1 inherited as a Mendelian trait (3). Life-long and profound diabetes. Both resemble the human disease, and conse- T-cell lymphopenia is characterized by a reduction in quently, susceptibility genes for diabetes found in these peripheral CD4ϩ T-cells, an even greater reduction of two strains can be considered as potential candidate CD8ϩ T-cells (4), and an almost total absence of RT6ϩ genes in humans. Recently, a frameshift deletion in T-cells (5). The lymphopenia gene is involved in the Ian4, a member of the immune-associated nucleotide regulation of apoptosis in the T-cell lineage and is, there- (Ian)-related gene family, has been shown to map to BB fore, responsible for loss of critical T-cells, resulting in rat Iddm1. In the KDP rat, a nonsense mutation in the T-cell regulatory gene, Cblb, has been described as a autoimmunity (6). Recently, two groups have indepen- major susceptibility locus. Following a strategy of ex- dently shown, by positional cloning of Iddm1/lyp, that amining the human orthologues of susceptibility genes lymphopenia is due to a frameshift deletion in Ian4 (also identified in animal models for association with type 1 called Ian5) of the immune-associated nucleotide (Ian)- diabetes, we identified single nucleotide polymorphisms related gene family (6,7), resulting in a truncated protein (SNPs) from each gene by resequencing PCR product product.
    [Show full text]
  • A Causal Gene Network with Genetic Variations Incorporating Biological Knowledge and Latent Variables
    A CAUSAL GENE NETWORK WITH GENETIC VARIATIONS INCORPORATING BIOLOGICAL KNOWLEDGE AND LATENT VARIABLES By Jee Young Moon A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Statistics) at the UNIVERSITY OF WISCONSIN–MADISON 2013 Date of final oral examination: 12/21/2012 The dissertation is approved by the following members of the Final Oral Committee: Brian S. Yandell. Professor, Statistics, Horticulture Alan D. Attie. Professor, Biochemistry Karl W. Broman. Professor, Biostatistics and Medical Informatics Christina Kendziorski. Associate Professor, Biostatistics and Medical Informatics Sushmita Roy. Assistant Professor, Biostatistics and Medical Informatics, Computer Science, Systems Biology in Wisconsin Institute of Discovery (WID) i To my parents and brother, ii ACKNOWLEDGMENTS I greatly appreciate my adviser, Prof. Brian S. Yandell, who has always encouraged, inspired and supported me. I am grateful to him for introducing me to the exciting research areas of statis- tical genetics and causal gene network analysis. He also allowed me to explore various statistical and biological problems on my own and guided me to see the problems in a bigger picture. Most importantly, he waited patiently as I progressed at my own pace. I would also like to thank Dr. Elias Chaibub Neto and Prof. Xinwei Deng who my adviser arranged for me to work together. These three improved my rigorous writing and thinking a lot when we prepared the second chapter of this dissertation for publication. It was such a nice opportunity for me to join the group of Prof. Alan D. Attie, Dr. Mark P. Keller, Prof. Karl W. Broman and Prof.
    [Show full text]
  • The Human Gene Connectome As a Map of Short Cuts for Morbid Allele Discovery
    The human gene connectome as a map of short cuts for morbid allele discovery Yuval Itana,1, Shen-Ying Zhanga,b, Guillaume Vogta,b, Avinash Abhyankara, Melina Hermana, Patrick Nitschkec, Dror Friedd, Lluis Quintana-Murcie, Laurent Abela,b, and Jean-Laurent Casanovaa,b,f aSt. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, NY 10065; bLaboratory of Human Genetics of Infectious Diseases, Necker Branch, Paris Descartes University, Institut National de la Santé et de la Recherche Médicale U980, Necker Medical School, 75015 Paris, France; cPlateforme Bioinformatique, Université Paris Descartes, 75116 Paris, France; dDepartment of Computer Science, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel; eUnit of Human Evolutionary Genetics, Centre National de la Recherche Scientifique, Unité de Recherche Associée 3012, Institut Pasteur, F-75015 Paris, France; and fPediatric Immunology-Hematology Unit, Necker Hospital for Sick Children, 75015 Paris, France Edited* by Bruce Beutler, University of Texas Southwestern Medical Center, Dallas, TX, and approved February 15, 2013 (received for review October 19, 2012) High-throughput genomic data reveal thousands of gene variants to detect a single mutated gene, with the other polymorphic genes per patient, and it is often difficult to determine which of these being of less interest. This goes some way to explaining why, variants underlies disease in a given individual. However, at the despite the abundance of NGS data, the discovery of disease- population level, there may be some degree of phenotypic homo- causing alleles from such data remains somewhat limited. geneity, with alterations of specific physiological pathways under- We developed the human gene connectome (HGC) to over- come this problem.
    [Show full text]