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Adam Cheng Xiulin Shen Prof. Jiayu Liao Department of Bioengineering

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. Introduction. 1918 flu pandemic JAK/STAT pathway

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Adam Cheng Xiulin Shen Prof. Jiayu Liao Department of Bioengineering

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  1. Interactions of SUMO within the JAK/STAT transcription pathwayA 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. SENP E3 (PIAS) ATP S E2 E1 S STAT STAT SUMO SUMO SUMO SUMO SUMO SUMO regulation • SUMO protein cascade • PIAS and SENP function

  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://www.bio.davidson.edu/courses/genomics/2005/Drysdale/molecular%20function.jpg http://cropandsoil.oregonstate.edu/classes/css430/lecture%209-07/figure-08-01.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 Insert protein here big STAT/SUMO SUMO/SENP SUMO small

  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

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