1 / 17

Development of High-throughput Screening Assay for Small Molecule Inhibitor(s) of PIAS1

Development of High-throughput Screening Assay for Small Molecule Inhibitor(s) of PIAS1. Bourns College of Engineering Department of Bioengineering Vicente Nunez Dr. Jiayu Liao. Outline. Background Our Plan Results Future work. JAK-STAT Pathway. Focus on STAT1-PIAS1 Interactions.

jenn
Télécharger la présentation

Development of High-throughput Screening Assay for Small Molecule Inhibitor(s) of PIAS1

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Development of High-throughput Screening Assay for Small Molecule Inhibitor(s) of PIAS1 Bourns College of Engineering Department of Bioengineering Vicente Nunez Dr. Jiayu Liao

  2. Outline • Background • Our Plan • Results • Future work

  3. JAK-STAT Pathway Focus on STAT1-PIAS1 Interactions D. Levy and J. Darnell, Nat Rev Mol Cell Biol (2002) 3: 651-662

  4. What is STAT1? • Signal Transducers and Activators of Transcription • A member of a family of related proteins • Several roles in immune cell regulations and transcription of anti-viral genes

  5. What is PIAS1? • Protein Inhibitor of Activated STAT • Negative regulator of STAT1

  6. Why Inhibit PIAS1-STAT1 Interactions? • UCLA Mice Study • Control of Immune Response • Potential therapy development for immune system deficiencies

  7. Our Plan • Have STAT1 & PIAS1 proteins expressed in mammalian cells • Tag these proteins with other proteins that show fluorescence • Determine what molecules inhibit the binding of PIAS1 to STAT1

  8. Our Plan • Transfect fusion plasmid into mammalian cells • Transfect the smaller plasmids: (YFP-STAT1 & PIAS1-CFP) OR

  9. Our Plan • Why Two Alternatives? • ONE COMPLEX: • Hypothesis • JAK1KD will phosphorylate STAT1 and initiate PIAS1-STAT1 interaction • No need to introduce IFNγ to activate STAT1 • TWO SEPARATE PLASMIDS: • More straight forward approach to experiment • Need IFNγ to activate STAT1

  10. Our Plan • Detect changes of FRET (Förster Resonance Energy Transfer) signals 440 nm 480 nm 440 nm 527 nm PIAS1 STAT1 PIAS1 STAT1 http://www.rowland.harvard.edu/labs/bacteria/images/fret2.jpg

  11. Hypothetical Inhibitor(s) • Inhibitor “X” separates the two proteins • Resulting in loss of energy transfer • We want to find what “X” is 440 nm 480 nm 440 nm 527 nm STAT1 PIAS1 STAT1 PIAS1 X http://www.rowland.harvard.edu/labs/bacteria/images/fret2.jpg

  12. What We Accomplished PIAS1 CFP YFP STAT1 pcDNA 3.1 pcDNA 3.1 pcDNA 3.1

  13. What We Accomplished • Transfected the YFP-STAT1 • Transfected PIAS1-CFP

  14. What We Accomplished • Observed fluorescence from cells expressing the YFP-STAT1 protein

  15. Future Work • Finish characterizing the PIAS1-CFP plasmid • Finish building the 5 fragment construct • Transfect it into mammalian cells • Undertake FRET Assay on both strategies • Develop into high throughput screening assay

  16. In Conclusion • Finding PIAS1 inhibitor(s) will potentially benefit human health • Potential development of treatments for immune system deficiencies • Further our understanding of PIAS1’s role in the JAK-STAT pathway of immune regulation

  17. Acknowledgements Dr. Jiayu Liao Yang Song Vipul Madahar Xiulin Shen Adam Cheng Dr. V. G. J. Rodgers BRITE Program

More Related