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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

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

introduction
Introduction
  • 1918 flu pandemic
  • JAK/STAT pathway
  • SUMO regulation
  • Goals and Strategies
  • Methods
  • Results
  • Conclusion
spanish flu pandemic of 1918
Spanish Flu Pandemic of 1918

http://en.wikipedia.org/wiki/Image:W_curve.png

jak stat pathway
JAK/STAT Pathway
  • Cytokines like interferon send messages throughout the body

http://en.wikipedia.org/wiki/Image:Jakstat_pathway.svg

jak stat pathway5
JAK/STAT Pathway
  • JAK phosphorylates the receptor and STAT
  • STAT dimer stimulates transcription

http://en.wikipedia.org/wiki/Image:Jakstat_pathway.svg

jak stat pathway6
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

sumo regulation

SENP

E3 (PIAS)

ATP

S

E2

E1

S

STAT

STAT

SUMO

SUMO

SUMO

SUMO

SUMO

SUMO regulation
  • SUMO protein cascade
  • PIAS and SENP function
slide8
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
strategy
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
methods
Methods
  • Mutations at/around these amino acids
wild type protein synthesis
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

incorporating pbpa in vivo
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
trna requirements
tRNA requirements
  • Codon mutated to TAG (stop codon)
  • Unrecognized by endogenous synthetases
aminoacyl trna synthetase requirements
Aminoacyl-tRNA Synthetase Requirements
  • Only aminoacylates the engineered tRNA
  • Only accepts unnatural amino acid into active site
methods mutation
Methods: Mutation
  • Mutations through PCR primers/hybridization
  • Restriction digestion of DNA
  • Ligation to mammalian expression vector
methods expression
Methods: Expression
  • Transfection of STAT1/alanine SUMO1/SENP2
  • Lyse cells and extract protein

http://www.amaxa.com/uploads/pics/StableTransfection_W.gif

methods expression18
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

methods analysis
Methods: Analysis
  • Analysis using Western Blot

Insert protein here

big

STAT/SUMO

SUMO/SENP

SUMO

small

results and future work
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
conclusion
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
acknowledgements
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
ackowledgements
Ackowledgements
  • Dr. Jiayu Liao
references
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