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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. Gothenburg, 13 th – 14 th June, 2005.

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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

Gothenburg, 13th – 14th June, 2005


Wp6 2 genomics and microbiology

WP6.2 Genomics and microbiology

Host pathway: Interferon Signalling

> A family of related proteins

> Bind to specific receptors

Activation of Interferon signalling pathways results in a defined gene expression and:

Increased

antigen presentation

Inhibition of

cellular proliferation

Development of an

inflammatory response

An anti-infective state

Gothenburg, 13th – 14th June, 2005


Wp6 2 genomics and microbiology

WP6.2 Genomics and microbiology

Interferon is a key Inflammatory mediator

with a number of notable therapeutic applications

Gothenburg, 13th – 14th June, 2005


Wp6 2 genomics and microbiology

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

Protein Interaction Network

microRNA

Phenotype DB

Microarray/

Proteomic

Profiling

Gothenburg, 13th – 14th June, 2005


Wp6 2 genomics and microbiology

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

UEDIN/INFORMA

Core Information

Host

263 Interferon gamma response genes identified

Storage, Integration & Dissemination

Baldo Oliva

Protein Interaction

Prediction

Literature Review

Ingenuity

Pathway Assist

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

UEDIN ‘novel’ genes (300 – 400) identified by microarray study

Published ‘novel’ genes identified by microarray study

Gothenburg, 13th – 14th June, 2005


Wp6 2 genomics and microbiology

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

UEDIN/INFORMA

Core Information

Host

263 Interferon gamma response genes identified

Storage, Integration & Dissemination

Baldo Oliva

Protein Interaction

Prediction

Literature Review

Ingenuity

Pathway Assist

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

UEDIN ‘novel’ genes (300 – 400) identified by microarray study

Published ‘novel’ genes identified by microarray study

Gothenburg, 13th – 14th June, 2005


Wp6 2 genomics and microbiology

WP6.2 Genomics and microbiology

WP6.2 – Partner Activity

Mar Alba

Host/ Pathogen

Mapping

UEDIN/INFORMA

Core Information

Host

263 Interferon gamma response genes identified

Storage, Integration & Dissemination

Baldo Oliva

Protein Interaction

Prediction

Literature Review

Ingenuity

Pathway Assist

Validation &

assessment of

utility

Biomarker

Prediction

Pathogen

HCMV, MCMV,HIV

Martin Reczko

miRNA prediction

and mapping

Gothenburg, 13th – 14th June, 2005


Wp6 2 genomics and microbiology

WP6.2 Genomics and microbiology

Host/ Pathogen Mapping: Mar Alba

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

Gothenburg, 13th – 14th June, 2005


Wp6 2 genomics and microbiology

WP6.2 Genomics and microbiology

Process:

Output

Input

Human Protein sequences for core interferon genes (FASTA)

HLA-A, HLA-B, HLA-C

HHV5 UL18

Known interaction:

UL18 is a natural killer cell decoy gene

BLASTP

sequence

similarity

comparison

980 HHV5 protein sequences obtained from VIDA database

TNFRSF1

HHV5 UL144

Known interaction:

Immune evasion viroceptor 1

Gothenburg, 13th – 14th June, 2005


Wp6 2 genomics and microbiology

WP6.2 Genomics and microbiology

Further steps:

  • Herpesviruses are a well studied group of pathogens

    > Sequence comparisons carried out previously: Immune related proteins popular targets for research.

    > Can we identify homologies in less well characterised gene products identifed by microarray.

    > What protein homologies are identified when all virus proteins are compared with all readily available protein sequences.

  • For HCMV/ MCMV

    • > Protein to Protein & miRNA screening may be more novel and revealing

  • Can approach be applied productively to HIV, GB virus C, Hepatitis C?

  • Gothenburg, 13th – 14th June, 2005


    Wp6 2 genomics and microbiology

    WP6.2 Genomics and microbiology

    WP6.2 – Partner Activity

    Mar Alba

    Host/ Pathogen

    Mapping

    UEDIN/INFORMA

    Core Information

    Host

    263 Interferon gamma response genes identified

    Storage, Integration & Dissemination

    Baldo Oliva

    Protein Interaction

    Prediction

    Literature Review

    Ingenuity

    Pathway Assist

    Validation &

    assessment of

    utility

    Biomarker

    Prediction

    Pathogen

    HCMV, MCMV,HIV

    Martin Reczko

    miRNA prediction

    and mapping

    Gothenburg, 13th – 14th June, 2005


    Wp6 2 genomics and microbiology

    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

    Gothenburg, 13th – 14th June, 2005


    Wp6 2 genomics and microbiology

    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

    Gothenburg, 13th – 14th June, 2005


    Wp6 2 genomics and microbiology

    WP6.2 Genomics and microbiology

    Viral Immunomodulatory Interactions (IMIM/ UEDIN)

    CMV Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase)

    > Early, nonstructural protein, 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

    > Does not contain a nuclear localisation signal by ‘SignalP’*

    * Center for Biological Sequence Analysis – Technical University of Denmark - http://www.cbs.dtu.dk/index.shtml

    Gothenburg, 13th – 14th June, 2005


    Wp6 2 genomics and microbiology

    WP6.2 Genomics and microbiology

    Viral Immunomodulatory Interactions (IMIM/ UEDIN)

    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.

    Gothenburg, 13th – 14th June, 2005


    Wp6 2 genomics and microbiology

    WP6.2 Genomics and microbiology

    Viral Immunomodulatory Interactions (IMIM/ UEDIN)

    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.

    Gothenburg, 13th – 14th June, 2005


    Wp6 2 genomics and microbiology

    WP6.2 Genomics and microbiology

    Viral Immunomodulatory Interactions (IMIM/ UEDIN)

    Simplified scheme for nuclear import.

    • Upon synthesis of nuclear proteins in the cytoplasm, e.g. the family of importins or transportins bind to the NLS.

    • 2 importin/NLS-protein (or transportin/protein) complex is then actively transported into the nucleus through nuclear pores involving the Ran GTPase cycle.

    Gothenburg, 13th – 14th June, 2005


    Wp6 2 genomics and microbiology

    WP6.2 Genomics and microbiology

    Sub-cellular Logic Interaction map of interferon signalling pathway submodule

    CMV dUTPase

    Gothenburg, 13th – 14th June, 2005


    Wp6 2 genomics and microbiology

    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

    UEDIN/INFORMA

    Core Information

    Host

    263 Interferon gamma response genes identified

    Storage, Integration & Dissemination

    Baldo Oliva

    Protein Interaction

    Prediction

    Literature Review

    Ingenuity

    Pathway Assist

    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

    UEDIN ‘novel’ genes (300 – 400) identified by microarray study

    Published ‘novel’ genes identified by microarray study

    Gothenburg, 13th – 14th June, 2005


    Wp6 2 genomics and microbiology

    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

    UEDIN/INFORMA

    Core Information

    Host

    263 Interferon gamma response genes identified

    Storage, Integration & Dissemination

    Baldo Oliva

    Protein Interaction

    Prediction

    Literature Review

    Ingenuity

    Pathway Assist

    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

    UEDIN ‘novel’ genes (300 – 400) identified by microarray study

    Published ‘novel’ genes identified by microarray study

    Gothenburg, 13th – 14th June, 2005


    Wp6 2 genomics and microbiology

    WP6.2 Genomics and microbiology

    Storage, integration and dissemination of

    pathway/ biomarker information

    Gothenburg, 13th – 14th June, 2005


    Wp6 2 genomics and microbiology

    WP6.2 Genomics and microbiology

    Storage, integration and dissemination of pathway/ biomarker information:

    Potential solution

    Stores information on

    proteins, genes,

    compounds, interactions

    Cognia Molecular

    www.cognia.com

    Web-based

    curation &

    query tools

    Public or Restricted

    Interface

    API available for development

    Integration purposes

    Visualisation

    Pathway Editor#

    Curation of 263

    core pathway elements complete

    #http://mpe.empproject.com

    Gothenburg, 13th – 14th June, 2005


    Wp6 2 genomics and microbiology

    WP6.2 Genomics and microbiology

    • Previous Year 1 aims for knowledgebase development:

    • 1) Web accessible repository of interferon signalling-related information

    • Further aims for knowledgebase 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

    Gothenburg, 13th – 14th June, 2005


    Wp6 2 genomics and microbiology

    WP6.2 Genomics and microbiology

    Explore methods for development of an

    integrated ‘biomarker’ prediction tool

    Gothenburg, 13th – 14th June, 2005


    Wp6 2 genomics and microbiology

    WP6.2 Genomics and microbiology

    ‘Biomarker’ prediction tool – Motivation

    > WP6-2 partners have/use a variety of valuable resources: currently being harnessed independently

    ARCA

    HIV genotype/ clinical HAART database

    BLASTP

    Protein sequence comparison

    GPX

    Microarray database

    IFN pathway DB

    Interferon knowledgebase

    miRNA prediction tool

    DNA sequence analysis and prediction

    PIANA

    Protein-Protein Interaction prediction

    VIDA

    Database of virus information

    Can we combine these genomic and post-genomic resources to provide the clinical or

    scientific researcher with a single intuitive method for identifying ‘biomarkers’?

    i.e. genes or gene products which play a key role in host-pathogen interaction and

    may be exploited scientifically and/ or clinically

    Gothenburg, 13th – 14th June, 2005


    Wp6 2 genomics and microbiology

    WP6.2 Genomics and microbiology

    ‘Biomarker’ prediction tool – How might this be used?

    Example 1 : From a pathogen to Host

    Researcher

    Pathogen e.g. HCMV

    VIDA

    HCMV Protein Sequences

    HCMV DNA Sequence

    All or subset of human protein sequences

    Predicted P to P interactions

    Predicted miRNA precursors

    Predicted miRNA Binding sites

    ‘Biomarker’

    Experimental Validation: Gene Expression analysis, siRNA

    Knowledgebase Annotation: System specific info, Literature

    Sequence Analysis e.g. NLS, Cellular Location

    Practical Applications

    Gothenburg, 13th – 14th June, 2005


    Wp6 2 genomics and microbiology

    WP6.2 Genomics and microbiology

    ‘Biomarker’ prediction tool – How might this be used?

    Example 2 : From a Host to Pathogen

    Researcher

    Host System of interest e.g. Interferon related genes/ gene products

    Knowledgebase

    Annotational Information incl. Sequence, Clinical Readouts etc for host system

    Pathogen(s) selected (e.g. HIV, Hepatitis, GB virus )

    Protein based prediction

    Sequence based prediction

    Host/ Pathogen Biomarkers

    Gothenburg, 13th – 14th June, 2005


    Wp6 2 genomics and microbiology

    WP6.2 Genomics and microbiology

    Milestones and Objectives

    Year 2

    Year 3

    Year 1

    Gothenburg

    Project definition

    (UEDIN, IMIM, LEIC, FORTH, INFORMA)

    Biomarker definition

    Data acquisition and curation

    Protein to protein interactions defined

    miRNA stem loop identification

    Novel genes experimentally identified

    Genotyping/ infection status

    Data Interpretation

    Pathway Biomarker Validation

    POC study on benefit from integrating clinical and bio- informatics

    WP6-2 Meeting

    Define realistic Specification

    Requirements capture for prediction tool

    Development

    Project Plan

    Prep Publications

    Gothenburg, 13th – 14th June, 2005