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 Background Our Plan Our Plan Results Results Future work Future work

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

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

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

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

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

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

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

10. Our Plan Detect changes of FRET (Förster Resonance Energy Transfer) signals Detect changes of FRET (Förster Resonance Energy Transfer) signals http://www.rowland.harvard.edu/labs/bacteria/images/fret2.jpg PIAS1 STAT1 PIAS1STAT1 440 nm480 nm440 nm 527 nm

11. Hypothetical Inhibitor(s) Inhibitor “X” separates the two proteins Inhibitor “X” separates the two proteins Resulting in loss of energy transfer Resulting in loss of energy transfer We want to find what “X” is We want to find what “X” is X PIAS1 STAT1PIAS1STAT1 440 nm480 nm 440 nm 527 nm http://www.rowland.harvard.edu/labs/bacteria/images/fret2.jpg

12. What We Accomplished YFPSTAT1 pcDNA 3.1 PIAS1CFP pcDNA 3.1

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

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

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

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

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