1. Interactions of SUMO within the JAK/STAT transcription pathway A new method to analyze the regulation of the JAK/STAT immune response pathway using protein engineering Adam Cheng Xiulin Shen Prof. Jiayu Liao Department of Bioengineering

2. Introduction ► 1918 flu pandemic ► JAK/STAT pathway ► SUMO regulation ► Goals and Strategies ► Methods ► Results ► Conclusion

3. Spanish Flu Pandemic of 1918 http://en.wikipedia.org/wiki/Image:W_curve.png

4. JAK/STAT Pathway ► Cytokines like interferon send messages throughout the body http://en.wikipedia.org/wiki/Image:Jakstat_pathway.svg

5. JAK/STAT Pathway ► JAK phosphorylates the receptor and STAT ► STAT dimer stimulates transcription http://en.wikipedia.org/wiki/Image:Jakstat_pathway.svg

6. JAK/STAT Pathway ► Outcomes from the JAK/STAT1 Pathway:  Immunoglobulin G  Protein Kinase R (PKR)  TH1 Differentiation (T-Cells) http://en.wikipedia.org/wiki/Image:Jakstat_pathway.svg

7. SUMO regulation ► SUMO protein cascade ► PIAS and SENP function ATP E1 SE2 S E3 (PIAS) STAT SENP SUMO STAT

8. Goal ► Investigate signaling specificity by analyzing SUMO’s interactions using a new strategy of incorporating unnatural amino acids ► Understand the regulation mechanisms of cytokine signaling pathways

9. Strategy ► Mutate essential interactive amino acids between SUMO and SENP to alanine so SENP cannot recognize SUMO ► Mutate neighboring amino acids to p- benzoyl-L-phenylalanine to permanently link and fish out interaction partners

10. Methods ► Mutations at/around these amino acids

11. Crosslinking unnatural amino acid: pBpa – p-benzoyl-L-phenylalanine

12. Wild Type Protein Synthesis ► Essential parts in protein synthesis: http://cropandsoil.oregonstate.edu/classes/c ss430/lecture%209-07/figure-08-01.JPG http://www.bio.dav idson.edu/courses/ genomics/2005/Dry sdale/molecular%2 0function.jpg

13. Incorporating pBpa in vivo ► What is needed for unnatural amino acid incorporation?  1) Unnatural amino acid (pBpa)  2) Engineered tRNA  3) Engineered Aminoacyl-tRNA synthetase

14. tRNA requirements ► Codon mutated to TAG (stop codon) ► Unrecognized by endogenous synthetases

15. Aminoacyl-tRNA Synthetase Requirements ► Only aminoacylates the engineered tRNA ► Only accepts unnatural amino acid into active site

16. Methods: Mutation ► Mutations through PCR primers/hybridization ► Restriction digestion of DNA ► Ligation to mammalian expression vector

17. Methods: Expression ► Transfection of STAT1/alanine SUMO1/SENP2 ► Lyse cells and extract protein http://www.amaxa.com/uploads/pics/StableTransfection_W.gif

18. Methods: Expression ► Transfection of STAT1/pBpa SUMO1/SENP2/TAG tRNA/Aminoacyl-tRNA synthetase ► Add p-benzoyl-L-phenylalanine (1mM) 6h after transfection SUMO/SENP complex

19. Methods: Analysis ► Analysis using Western Blot SUMO STAT/SUMO SUMO/SENP big small Insert protein here

20. Results and Future Work ► All mutations are complete ► Analyze using Western Blot ► Affirm results using another method ► Move techniques to related pathways  Anti-tumor p53 pathway

21. Conclusion ► A new amino acid incorporation strategy was applied ► Two types of SUMO mutations were generated to further the study of the JAK/STAT pathway ► Results will benefit human health

22. Acknowledgements ► Yang Song ► Vicente Nuñez ► Vipul Madahar ► Clarence Pasion ► Xiulin Shen ► Department of Bioengineering ► Maria Franco-Aguilar and UCLEADS ► Dr. Victor Rodgers, Jill Brady, BRITE

23. Ackowledgements ► Dr. Jiayu Liao

24. References ► D. Schmidt and S. Miller. (2003) PIAS/SUMO: Partners in transcriptional regulation. Cell. Mol. Life. Sci. 60:2561-2574 ► Johnson, Erica S. (2004) Protein Modification by SUMO. Annu. Rev. Biochem. 73: 355-382