Transcript Acknowledgements - Bourns College of Engineering

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
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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?
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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?
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Protein Inhibitor of Activated STAT
Negative regulator of STAT1
Why Inhibit PIAS1-STAT1
Interactions?
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UCLA Mice Study
Control of Immune Response
Potential therapy development for immune system deficiencies
Our Plan
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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
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Transfect fusion plasmid into mammalian cells
Transfect the smaller plasmids:
(YFP-STAT1 & PIAS1-CFP)
OR
Our Plan
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Why Two Alternatives?
ONE COMPLEX:
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Hypothesis
JAK1KD will phosphorylate STAT1 and initiate PIAS1STAT1 interaction
 No need to introduce IFNγ to activate STAT1
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TWO SEPARATE PLASMIDS:
More straight forward approach to experiment
 Need IFNγ to activate STAT1
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Our Plan
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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)
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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
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Transfected the YFP-STAT1
Transfected PIAS1-CFP
What We Accomplished
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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
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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
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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
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Acknowledgements
Dr. Jiayu Liao
 Yang Song
 Vipul Madahar
 Xiulin Shen
 Adam Cheng
 Dr. V. G. J. Rodgers
 BRITE Program
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