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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 D. Levy and J. Darnell, Nat Rev Mol Cell Biol (2002) 3: 651-662 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 What is PIAS1? Protein Inhibitor of Activated STAT Negative regulator of STAT1 Why Inhibit PIAS1-STAT1 Interactions? UCLA Mice Study Control of Immune Response Potential therapy development for immune system deficiencies 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 Our Plan Transfect fusion plasmid into mammalian cells Transfect the smaller plasmids: (YFP-STAT1 & PIAS1-CFP) OR Our Plan Why Two Alternatives? ONE COMPLEX: Hypothesis JAK1KD will phosphorylate STAT1 and initiate PIAS1STAT1 interaction No need to introduce IFNγ to activate STAT1 TWO SEPARATE PLASMIDS: More straight forward approach to experiment Need IFNγ to activate STAT1 Our Plan Detect changes of FRET (Förster Resonance Energy Transfer) signals 440 nm 480 nm PIAS1 STAT1 440 nm 527 nm PIAS1 STAT1 http://www.rowland.harvard.edu/labs/bacteria/images/fret2.jpg 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 PIAS1 X STAT1 440 nm 527 nm PIAS1 STAT1 http://www.rowland.harvard.edu/labs/bacteria/images/fret2.jpg What We Accomplished YFP PIAS1 STAT1 pcDNA 3.1 CFP pcDNA 3.1 pcDNA 3.1 What We Accomplished Transfected the YFP-STAT1 Transfected PIAS1-CFP What We Accomplished Observed fluorescence from cells expressing the YFP-STAT1 protein 150000 100000 50000 0 490 500 510 520 530 540 550 560 Em Wavelength in nm (Ex: 476nm) Well Lambda at Maximum M3 570 580 590 600 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 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 Acknowledgements Dr. Jiayu Liao Yang Song Vipul Madahar Xiulin Shen Adam Cheng Dr. V. G. J. Rodgers BRITE Program