MYRIAD NIAID Biodefense Proteomics Research Centers 2nd ...
Upcoming SlideShare
Loading in...5
×
 

MYRIAD NIAID Biodefense Proteomics Research Centers 2nd ...

on

  • 573 views

 

Statistics

Views

Total Views
573
Views on SlideShare
572
Embed Views
1

Actions

Likes
0
Downloads
1
Comments
0

1 Embed 1

https://blackboard.jhu.edu 1

Accessibility

Categories

Upload Details

Uploaded via as Microsoft PowerPoint

Usage Rights

© All Rights Reserved

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Processing…
Post Comment
Edit your comment
  • Genomic DNA was sheared using two methods: sonication and enzymatic (CviJ1 under star conditions). CviJ I cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends.CviJ I** restricts PyGCPy and PuGCPu, in addition to PuGCPy.
  • 23 kD protein from F. tularnesis is thought to suppress proinflammatory activation of the host cell.
  • Genomic DNA was sheared using two methods: sonication and enzymatic (CviJ1 under star conditions). CviJ I cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends.CviJ I** restricts PyGCPy and PuGCPu, in addition to PuGCPy.
  • Average insert size 200 AA = 600 nt

MYRIAD NIAID Biodefense Proteomics Research Centers 2nd ... MYRIAD NIAID Biodefense Proteomics Research Centers 2nd ... Presentation Transcript

  • NIAID Biodefense Proteomics Research Centers 2nd Programmatic Meeting Jerry Lanchbury, Ph.D. Myriad Genetics, Inc.
  • Myriad Genetics, Inc. 650 Employees Location: Salt Lake City, UT Biotherapeutics & Predictive Medicine
  • Academic Collaborators F. tularensis Dr. Martha B. Furie Dept. of Pathology SUNY at Stony Brook
    • Y. pestis
    • Dr. James B. Bliska
    • Dept. of Molecular Genetics and Microbiology Center for Infectious Diseases
    • SUNY at Stony Brook
    • B. anthracis
    • Dr. Kenneth A. Bradley
    • Dept. of Microbiology Immunology and Molecular Genetics
    • UCLA
    Vaccinia and Variola major Dr. Grant McFadden The John P. Robart Research Institue University of Western Ontario
  • Discovery of viral pathogen-host interactions : Example of HIV-1 Viral Budding Pathway
    • Yeast two-hybrid screen of pathogen proteins vs. human libraries
    • Validation of interactions using cell-based and biochemical assays (collaboration with Sundquist lab at University of Utah)
    • Expansion of viral budding pathway by identification of interactions between key host proteins
    • Identification of novel therapeutic strategies such as disruption of protein interactions and chemical modulation of newly identified protein regulators
  • Tsg101 and the VPS Pathway are Essential for HIV-1 Budding J.E. Garrus, U.K. von Schwedler, O.W. Pornillos, S.G. Morham, K.H. Zavitz, H.E. Wang, D.A. Wettstein, K.M. Stray, M. Côté, R.L. Rich, D.G. Myszka, and W.I. Sundquist. Cell 107 : 55 (2001) HIV-1 Gag p6 Tsg101 The p6 fragment of the HIV-1 Gag polypeptide was used in two-hybrid screens of human libraries. Six protein interaction partners were identified, including the Tsg101 protein that was later shown to be required for viral budding.
  • Pathogen-host proteomics goals
    • Discover pathogen-human protein interactions for selected Category A Select Agents
    • Validate selected interactions for target utility (small molecules, biologics, diagnostics)
    • To conduct a selective survey of protein-protein interactions occurring between host and selected proteins from B. anthracis , Y. pestis , F. tularensis , Variola major, and Vaccinia proteins using a directed yeast two-hybrid approach.
    • To comprehensively survey protein interactions that occur between category A pathogens using a random yeast two-hybrid approach.
  • Year 1 Progress
    • Completed initial survey of protein-protein interactions of selected pathogenic proteins from Y. pestis , B. anthracis , F. tularenesis .
    • Directed yeast two-hybrid screen of 266 vaccinia ORFs.
    • Initiated random yeast two-hybrid screen for B. anthracis
    • Begun validation of pathogen-host interactions
        • B. anthracis (PA 4 and LF 5)
        • Y. pestis (YPKA 3)
  • Directed vs. Random Search Directed Search Random Search Randomly Fragment DNA PCR amplify gene of interest Bacterial Genomic DNA Liquid Mating with Host Library Recombine into plasmid in frame with GAL4 DNA binding domain Recombine into ORF selective plasmid creating a library of GAL4 DNA binding fusion proteins Bacterial Genomic DNA Liquid Mating with Host Library
  • Directed Yeast Two-Hybrid Process Flowchart Sequence verify DBD constructs Test constructs For Sefl-activation Add human AD library Solid mating Of DBD and AD yeast Double genetic Selection on Solid plates Pick Positive colonies Sequence DBD And AD from yeast Analyze DBD And AD sequences Select Representative clones Purify DBD And AD constructs Transform Constructs into Naïve yeast Sequence DBD And AD constructs Reconfirm Y2H interaction (luminescence) Compare Sequences to originals Analyze Confirmation data Release positives Create DBD constructs Manual process Liquid-handling robot Luminometer Automated sequencers Informatics process
  • Pathogen Directed Search Summary
  • Smallpox
  • Vaccinia Directed Search
    • 266 ORFs searched
    • 12 ORFs returned novel host interactions
    • 113 novel prey
    • 25 additional ORFs return prey and are in final analysis
    • 14% of Vaccinia ORFs show novel host protein-protein interactions from our studies
  • B. anthracis biology J. Clin. Invest. 112 :656-658 (2003) Protective Antigen 10 baits created 7 host interactors Lethal Factor 10 baits created 8 host interactors
  • B. anthracis Directed Search Results
    • Protective Antigen
      • Prey subcellular localization
        • 2 membrane associated proteins
        • 2 nuclear proteins
        • 2 cytoplasmic proteins
        • 1 extracellular protein
    • Lethal Factor
      • Prey subcellular localization
        • 5 membrane associated proteins
        • 3 cytoplasmic proteins
  • Yersinia pestis - Model of type III secretion From Cornelis, G.R., 2002, J.C.B. LcrV LcrV LcrV LcrV
  • Yersinia pestis biology 8 baits created 2 host interactors 10 baits created 4 host interactors LCRV 6 baits created 18 host interactors Annu. Rev. Microbiol. 2005. 59:69–89
  • Y. pestis Directed Search Results
    • YPKA
      • Prey subcellular localization
        • 3 cytoplasmic proteins
        • 1 other
    • YopM
      • Prey subcellular localization
        • 2 other
    • LCRV
      • Prey subcellular localization
        • 2 membrane associated proteins
        • 9 cytoplasmic proteins
        • 1 nuclear protein
        • 1 extracellular protein
        • 5 others
  • Directed vs. Random Search Directed Search Random Search Randomly Fragment DNA PCR amplify gene of interest Bacterial Genomic DNA Liquid Mating with Host Library Recombine into plasmid in frame with GAL4 DNA binding domain Recombine into ORF selective plasmid creating a library of GAL4 DNA binding fusion proteins Bacterial Genomic DNA Liquid Mating with Host Library
  • Random Two-Hybrid Statistics
    • Survey of category A pathogens using a random two-hybrid approach
      • Myriad will survey each bacterial genome such that between 5 and 10-fold coverage will be obtained
      • A minimum of 121,000 random two-hybrid searches will be completed
  • Random Two-Hybrid Statistics
  • Random Yeast Two-Hybrid Process Plate DBD library Pick DBD colonies into 96-well plate Add human spleen AD library Liquid mating of DBD and AD yeast Double genetic Selection on Solid plates Pick Positive colonies Sequence DBD And AD from yeast Analyze DBD and AD sequences Select proteins for directed analysis Generate DBD library In yeast Manual process Liquid-handling robot Luminometer Automated sequencers Informatics process
  • Current Status of Random Yeast Two-Hybrid
    • 6840 searches have been started in the random B. anthracis search (12% of the searches have been initiated)
    • For each search an average of 1 million diploids were generated
    • About 6.0 % of the searches returned positives
      • 1.2 % appear to be self-activating.
  • Next Steps
    • Complete B. anthracis random searches
    • Initiate Y. pestis and F. tularensis random searches
    • Acquire 15 identified Variola proteins not represented in Vaccinia and search those using a directed approach
    • Nominate additional pathogen proteins to search using a directed approach
    • selected B. anthracis host protein-protein interactions
  • Next steps II
    • To conduct initial validation experiments that establish the relative importance of the highlighted pathogen-host interactions with a view to selecting target interactions for the development of preventative or therapeutic vaccines and small molecule drugs
      • After analysis of the two-hybrid results, individual expert investigators for each organism will select proteins of interest for analysis
      • Ken Bradley has begun experiments to validate
      • James Bliska will begin experiments to validate selected Y. pestis host protein-protein interactions
  • Acknowledgements
    • Pronet Group - Jerry Lanchbury, Donna Shattuck, Sasha Gutin, Mark McKeller, Ching Yu Hsueh, Brian Morris, Max Dufford, David Larson, Rachelle Robertson, Linda Wong, Yuan Wan, Georges Frech, Jorja DeGrado, Jian Chen, Chris Neff, Yang Chen, Christina Davenport, Deane Woodland, Jennifer Hannan, Andrew Morris, Wei Xiong, Ryan Doering, Todd Peterson, Melinda Jones, Sheila Towne, Jonathan Nelson, Kwang Tae Jimmy Park, Nafei Xu,