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WP6.2 Genomics and microbiology

WP6.2 Genomics and microbiology. Overall Objective: To demonstrate how the integration of pathway biology and host/ pathogen genomics and post-genomics can contribute to clinical diagnosis and treatment of infected patients.

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WP6.2 Genomics and microbiology

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  1. WP6.2 Genomics and microbiology Overall Objective: To demonstrate how the integration of pathway biology and host/ pathogen genomics and post-genomics can contribute to clinical diagnosis and treatment of infected patients 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  2. WP6.2 Genomics and microbiology Work Package Focus: Host Interferon Signalling pathway Family of related proteins bind outside cell to specific receptors Interferon Homodimer binding receptor ‘Signalling’ Activation of the Interferon signalling pathwaysresults in changes in expression of 100 to 1000 genes depending on co-stimuli, cell type, environment etc ‘EFFECT’ 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  3. WP6.2 Genomics and microbiology What is the Effect? Development of an inflammatory response Inhibition of cellular proliferation Increased presentation of non-self to immune system An anti-infective state 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  4. WP6.2 Genomics and microbiology Interferon is a key Inflammatory mediator with a number of notable therapeutic applications Treatment of: Chronic Hepatitis C Viral Infection Melanoma Multiple Sclerosis Idiopathic Pulmonary Fibrosis An understanding of intracellular events following interferon receptor binding will enable rational development of therapeutic intervention in the future 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  5. WP6.2 Genomics and microbiology Medical Informatics Bioinformatics Viral Immuno- modulatory Interactions Clinical Host/ Viral Interactions Outcomes: Secretome 1. Further insight into the virus-host interaction 2. Identification of biomarkers for validation and potential therapeutic exploitation 3. Methods for storage, manipulationand exchange of phenotype data Patient-specific Clinical data Protein Interaction Network Host gene expression data microRNA Viral genotying Information Microarray/ Proteomic Profiling 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  6. WP6.2 Genomics and microbiology Objective 1: Initiation of the Study Identification and distribution of a core set of interferon Gamma-related genes/ proteins around which initial studies of WP6.2 will focus Mar Alba Host/ Pathogen Mapping Core Information Host 238 Human/ 226 mouse Interferon gamma response genes identified Baldo Oliva Protein Interaction Prediction Literature Review Ingenuity Pathway Assist Storage, Integration & Dissemination Validation & assessment of utility Biomarker Prediction Pathogen HCMV, MCMV,HIV Martin Reczko miRNA prediction and mapping Jordi Mestres IFN Pathway Drugability 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  7. WP6.2 Genomics and microbiology Objective 1: Initiation of the Study Identification and distribution of a core set of interferon Gamma-related genes/ proteins around which initial studies of WP6.2 will focus Mar Alba Host/ Pathogen Mapping Core Information Host 238 Human/ 226 mouse Interferon gamma response genes identified Baldo Oliva Protein Interaction Prediction Literature Review Ingenuity Pathway Assist Storage, Integration & Dissemination Validation & assessment of utility Biomarker Prediction Pathogen HCMV, MCMV,HIV Martin Reczko miRNA prediction and mapping Actual number of genes involved in interferon response likely to be >1000 Jordi Mestres IFN pathway Drugability UEDIN ‘novel’ genes (300 – 400) identified by microarray study Published ‘novel’ genes identified by microarray study 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  8. WP6.2 Genomics and microbiology WP6.2 – Partner Activity Mar Alba Host/ Pathogen Mapping Core Information Host 238 Human/ 226 mouse Interferon gamma response genes identified Baldo Oliva Protein Interaction Prediction Literature Review Ingenuity Pathway Assist Storage, Integration & Dissemination Validation & assessment of utility Biomarker Prediction Pathogen HCMV, MCMV,HIV Martin Reczko miRNA prediction and mapping Jordi Mestres IFN pathway Drugability 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  9. WP6.2 Genomics and microbiology Mar Alba – IMIM, Barcelona Host/ Pathogen Mapping: Objective: To investigate and characterise Host and Virus protein homologies Outputs: Provides insight into methods pathogen uses to survive and modulate host responses Provides potential targets for clinical intervention 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  10. WP6.2 Genomics and microbiology UL7 ORF of HCMV may be a Lymphocyte Antigen 9 (LY9/ CD229) homolog • Member of CD150 Ig superfamily • Expressed on T and B lymphocytes • Binds homophilically to itself • Localises to contact area of cells during Ag dependent ‘immune synapse’ formation • Related to SLAM receptor family • regulation of lymphocyte activation and cytokine secretion • CD229 may attenuate T cell signalling 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  11. MASDVSSHLLTVTQSRWTIHHMYNKLLILALFTPVILESIIYVSGPQGGNVTLVSNFTSN ISARWFRWDGNDSHLICFYKRGEGLSTPYVGLSLSCAANQITIFNLTLNDSGRYGAEGFT RSGENETFLWYNLTVKPKPLETTTASNVTTIVTTTPTVIGTKSNVTGNASLAPQLRAVAG FLNQTPRENNTHLALVGVIVFIALIVVCIMGWWKLLCSKPKL Leader peptide; Ig-like domain;Transmembrane WP6.2 Genomics and microbiology UL7, a homolog of the lymphocyte cell surface protein CD229 in HCMV UL7 and CD229 V-like domains (31% identity, 49% homology) UL7: VSGPQGGNVTLVSNFTSNISARWFRWDGNDSHLICFYKRGEGLST---PYVG-LSLSCAA VSGGG+VTL N + + W G + + F + E ++Y+GL ++ + CD229: VSGILGGSVTLPLNISVDTEIENVIWIG-PKNALAFARPKENVTIMVKSYLGRLDITKWS UL7: NQITIFNLTLNDSGRYGAEGFTRSGENETFLWYNLTVKP +INLTLND+GYA+ R+ E T+L V CD229: YSLCISNLTLNDAGSYKAQINQRNFEVTTEEEFTLFVYE Preserved residues between members of the SLAM family 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  12. WP6.2 Genomics and microbiology UL7 ORF of HCMV may be a Lymphocyte Antigen 9 (LY9/ CD229) homolog Highest homology in ligand binding region (almost all CD229 conserved residues present) HCMV UL7 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  13. WP6.2 Genomics and microbiology How might this viral product subvert the host immune response? Viral Homolog CD229 Signalling Human Virus Infected Macrophage MHC TCR T lymphocyte Inhibition of cytotoxicity 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  14. WP6.2 Genomics and microbiology UL7 ORF of HCMV may be a Lymphocyte Antigen 9 (LY9/ CD229) homolog Validation in progress (Ana Angulo/ Pablo Engel - Barcelona) • Fluorescent Viral UL7 • anti-UL7 mAb • HCMV UL7 KO COS cells transfected with UL7-EGFP showing membrane expression of construct Output by March 2006: Preliminary data on role of UL7 in viral infection Output by End 2006: Publication on functional role of UL7 in modulation of host during infection 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  15. WP6.2 Genomics and microbiology WP6.2 – Partner Activity Mar Alba Host/ Pathogen Mapping Core Information Host 238 Human/ 226 mouse Interferon gamma response genes identified Baldo Oliva Protein Interaction Prediction Literature Review Ingenuity Pathway Assist Storage, Integration & Dissemination Validation & assessment of utility Biomarker Prediction Pathogen HCMV, MCMV,HIV Martin Reczko miRNA prediction and mapping Jordi Mestres IFN Pathway Drugability 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  16. Graph PIANA WP6.2 Genomics and microbiology PIANA – B Oliva, J Planas, R Aragues Protein InterAction Network Analysis Uniprot PDB DIP STRING MIPS GO COG P to P interactions Clustering Prediction 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  17. WP6.2 Genomics and microbiology Viral Immunomodulatory Interactions (IMIM/ UEDIN) Objectives: To characterise protein-protein interactions: 1. Within host interferon signalling pathway 2. Between virus and host proteins Objective 1. Application of PIANA to identification of virus/ host protein interaction PIANA Yeast interactions experimentally defined by yeast 2 hybrid approach Gene COG code x 1 2 3 x y z x 7 predicted CMV-Human protein interactions e.g. KPNA4 x x 2. Interacting pairs in yeast defined which have at least 1 COG code in common with CMV proteins 1. COG codes assigned to Human CMV proteins 3. Orthologous human partner identified 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  18. WP6.2 Genomics and microbiology Viral Immunomodulatory Interactions (IMIM/ UEDIN) From Virus/ Host perspective (M. Alba, B. Oliva) HCMV protein Human Proteins Cyclin-dependent kinases regulatory subunit 1 (CKS-1) – Cell Cycle Cyclin-dependent kinases regulatory subunit 2 (CKS-2) – Cell Cycle Nucleoprotein interactor 1; NPI-1 [Homo sapiens] – Cell Cycle Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) (dUTP pyrophosphatase) Importin alpha-2 subunit (KPNA2) (SRP1-alpha) (RAG cohort protein 1) - Nuclear Localisation signal binding Importin alpha-4 subunit (KPNA4) (Qip1 protein) – As above Importin alpha-7 subunit (Karyopherin alpha-6) - As above Sperm associated antigen 6 (SPAG6) [Homo sapiens] – Flagellar movement 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  19. WP6.2 Genomics and microbiology Why is UL72/ dUTPase interesting? • Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) • Hydrolyses dUTP to dUMP and pyrophosphate (PPi) • Maintains a low dUTP:dTTP ratio to minimize the misincorporation of uracil into DNA • Uracil misincorporation into DNA could influence DNA conformation and sequence-specific protein binding leading to and inability to replicate CMV UL72 is not a functional dUTPase Caposio, P., Riera, L., Hahn, G., Landolfo, S. & Gribaudo, G. Evidence that the human cytomegalovirus 46-kDa UL72 protein is not an active dUTPase but a late protein dispensable for replication in fibroblasts. Virology 325, 264-76 (2004). 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  20. WP6.2 Genomics and microbiology Evidence from a closely related family member EBV dUTPase In vitro: Purified dUTPase - Inhibits replication of Human PBMC - Upregulates TNF-, IL-1, IL-6, IL-8 and IL-10 - Enhances NK cell lysis of targets - Inhibits synthesis of Interferon  - Primary cell type affected is macrophage In vivo: Mice injected with purified EBV dUTPase - Inhibits replication of mitogen-stimulated lymphocytes ex vivo. - Inhibits interferon-gamma after re-stimulation ex vivo. - Loss of body mass, elevated body temperature, displayed diminished locomotor activity. 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  21. WP6.2 Genomics and microbiology UL72 may bind a key component of Interferon network KPNA4 (importin alpha 3) Functions in import of IRF3 into nucleus from cytoplasm Kumar KP, McBride KM, Weaver BK, Dingwall C, Reich NC.Regulated nuclear-cytoplasmic localization of interferon regulatory factor 3, a subunit of double-stranded RNA-activated factor 1 Mol Cell Biol 2000 Jun 1;20(11):4159-68. 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  22. WP6.2 Genomics and microbiology Sub-cellular Logic Interaction map of interferon signalling pathway submodule CMV dUTPase 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  23. WP6.2 Genomics and microbiology • Validation in progress • (UEDIN/ Ana Angulo/ Martin Messerle) • UL72 KO virus in production • siRNA to UL72 in production Both approaches will be used to assess infected cell phenotype in presence and absence of viral gene product Output by End 2006: Publication on functional role of UL7 in modulation of host during infection 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  24. WP6.2 Genomics and microbiology Future Plans based around 2 publications 1. ‘Validation of PIANA using interferon protein interaction network’ i. Recreation of network nodes and edges from a seed of components ii. Recreation of network edges using PIANA Output by March/ April 2006:Publication submitted 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  25. WP6.2 Genomics and microbiology Future Plans based around 2 publications 2. ‘Expansion of Interferon protein network using PIANA’ • Existing interferon pathway components used to initiate expansion and identification of • i. New Nodes [Biomarkers] • ii. New Edges [Pathways] • Expanded network then compared with existing microarray data generated after cellular treatment with interferon • Work to be undertaken by Joan Planas during mobility exchange April/ May 2006 Output by End June 2006:Manuscript in preparation Output by End 2006:Manuscript submitted 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  26. WP6.2 Genomics and microbiology Mar Alba Host/ Pathogen Mapping Core Information Host 238 Human/ 226 mouse Interferon gamma response genes identified Baldo Oliva Protein Interaction Prediction Literature Review Ingenuity Pathway Assist Storage, Integration & Dissemination Validation & assessment of utility Biomarker Prediction Pathogen HCMV, MCMV,HIV Martin Reczko miRNA prediction and mapping Jordi Mestres IFN Pathway Drugability 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  27. WP6.2 Genomics and microbiology Understanding microRNA regulation of the Host and Virus: Novel biomarkers for clinical intervention? Abundant class of tiny regulatory RNAs (21nt) Function post-transcriptionally to prevent protein production via: mRNA cleavage (‘High’ complementarity) &/or Translational repression (‘Low’ complementarity) Control and influence many aspects of cellular physiology 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  28. WP6.2 Genomics and microbiology Understanding microRNA regulation of the Host and Virus • Objectives • Prediction of novel precursor miRNAs from genomic (Virus and Host) sequences • Prediction of novel miRNA targets • Experimental verification of miRNA expression/ target gene control 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  29. WP6.2 Genomics and microbiology Understanding microRNA regulation of the Host and Virus Work to-date Focussed on answering question: How might a clinically relevant pathogen be subverting host immune response via miRNA expression? development and application of algorithm for prediction of Human CMV miRNA binding sites in genes from interferon pathway 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  30. WP6.2 Genomics and microbiology Understanding microRNA regulation of the Host and Virus 10 key components of interferon pathway found to contain miRNA target sites miRNA-ID HUGO Ensembl-ID miranda- Score ------------------------------------------------ Single targets: miRUL22A-1_3p HSPA5 ENSG00000044574 119 miRUL22A-1_3p CTSS ENSG00000163131 127 miRUL112-1_3p CBFB ENSG00000067955 113 miRUL112-1_3p DAP ENSG00000112977 132 miRUL148D-1_3p SOCS3 ENSG00000184557 113 miR-US5-1_3p CASP8 ENSG00000064012 124 miR-US5-2_3p PSMD12 ENSG00000197170 111 miRUS33-1_5p PRKCA ENSG00000154229 112 miRUL36-1_5p BCL6 ENSG00000113916 123 Double target: miRUL36-1_5p CAMK2A ENSG00000070808 115 miRUS25-2_3p CAMK2A ENSG00000070808 114 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  31. WP6.2 Genomics and microbiology First: understand the host • superimposition of predicted miRNA target networks for entire genome onto the interferon network • To understand how miRNAs may act to control the expression of interferon-related proteins in vivo. Prediction pipeline All potential miRNAs Map onto interferon genes Novel understanding of system wide control 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  32. WP6.2 Genomics and microbiology Next: understand the pathogen 2. Application of a new algorithm to identification of miRNAs in HCMV, MCMV, KSHV and HIV. Implemented and retrained existing pipeline (called mirnafind) for precursor prediction from (unconserved) sequences only. Reproduce the results from Pfeffer et al on the HCMV Tested the performance on the known mouse miRNAs to redefine thresholds and sensitivity as the system was trained on human data 10 miRNA candidate sequences identified in mCMV Currently designing experiments to verify expression and effect of miRNA expression on Interferon pathway 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  33. WP6.2 Genomics and microbiology Finally: understand the host and pathogen 3. Prediction of host target sites for viral miRNA binding Prediction of viral target sites for host miRNA binding Prediction of viral target sites for viral miRNA binding Prediction of host interferon-related target sites for predicted host miRNA binding This screen will be complete by March 2006 4. Laboratory Validation of above predictions Output by End 2006: 2+ manuscripts in preparation 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  34. WP6.2 Genomics and microbiology Mar Alba Host/ Pathogen Mapping Core Information Host 238 Human/ 226 mouse Interferon gamma response genes identified Baldo Oliva Protein Interaction Prediction Literature Review Ingenuity Pathway Assist Storage, Integration & Dissemination Validation & assessment of utility Biomarker Prediction Pathogen HCMV, MCMV,HIV Martin Reczko miRNA prediction and mapping Jordi Mestres IFN pathway drugability 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  35. WP6.2 Genomics and microbiology Datasets Interferon Gamma: JAK/STAT Apoptosis MHC/ Ag IRF subunit Published Data Williams et al etc TNF TLR P38 Kinase Interferon /  Clinical Data Gene lists Gene lists Gene and Edge lists Gene and Edge lists Output ‘Integration’ V0.1 [June] PIANA Validation Gene lists PIANA Gene network expansion Experimental Data Interferon Treated Macrophage time course Infobiomed Data 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  36. WP6.2 Genomics and microbiology • Aims for pathway database environment • Curate current core information and then incorporate • further signalling pathway information (type 1 interferon, • TLR, TNF-alpha) selected via objective criteria. • 2) Curate data generated in course of Infobiomed project Output by June 2006: Interferon pathway database Output by End 2006: Publicly accessible Interferon pathway database 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  37. WP6.2 Genomics and microbiology Infobiomed project Cross work-package collaborative work WP6.1 AZ – Erasmus - IMIM Drugability analyses - Underway Pathway representation - Proposal available Pathway Ontologies [Can we combine Pathways?] Pathway analysis software etc UEDIN – IMIM – FORTH - Informa WP6.2 Explore susceptibility to periodontitis and IFNG genotype Curation of disease susceptibility and interferon pathway FUA - ACTA WP6.3 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  38. WP6.2 Genomics and microbiology A Clinical Context for Pathway Biology activities 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  39. WP6.2 Genomics and microbiology Collaborative project with Professor Peter Hayes (Wellcome Trust Clinical Research Facility, Edinburgh - Ongoing studies of liver transplantation and cirrhosis > 400 patients: Viral loads (e.g. HIV, HBV) Interferon responders vs Non responders ClinicalDB 5 new patients a week requiring interferon therapy 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  40. WP6.2 Genomics and microbiology Pilot Microarray Experiment IFN responders vs Non responders Experimental design to be decided Project Plan May/ June March to May August September + February Training of Nurses in blood RNA extraction SOP Sample Collection Interpretation and Integration of clinical and pathway data Profiling Analysis Sample selection 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  41. WP6.2 Genomics and microbiology In the context of personalised healthcare… IFN responders vs Non responders Gene signature enabling early classification and alteration of regime etc Individual presents in clinic Time 0 Treatment begins 6 weeks 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  42. WP6.2 Genomics and microbiology GPX Microarray DB Clinical DB Pathway DB Sex: Age: Viral Load: Treatment: History: Etc. Output Integration Deliverables: By end 2006 Pilot output complete with assessment of integration approach Manuscript 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

  43. WP6.2 Genomics and microbiology Milestones and Objectives Year 2 Year 3 Year 1 Gothenburg Aveiro Project definition (UEDIN, IMIM, LEIC, FORTH, INFORMA) Data acquisition and curation Pathway Biomarker Validation Data Interpretation Preparation/ submission of Publications Pathway DB development UEDIN/ FORTH Meet WP6-2/ UPM Meet UEDIN/ IMIM Meet WP6-2 Meet WP6-2 Meeting- Barcelona Project Definition Sample collection and profiling Sample analysis and data integration Project Plan Reporting 6th InfoBiomed Consortium Meeting, Barcelona, 26th – 27th January, 2006

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