Transcript SUMOylation of STAT

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
► SUMO regulation
► Goals and Strategies
► Methods
► Results
► Conclusion
Spanish Flu Pandemic of 1918
http://en.wikipedia.org/wiki/Image:W_curve.png
JAK/STAT Pathway
http://en.wikipedia.org/wiki/Image:Jakstat_pathway.svg
► Cytokines
like interferon send messages
throughout the body
JAK/STAT Pathway
http://en.wikipedia.org/wiki/Image:Jakstat_pathway.svg
► JAK
phosphorylates the receptor and STAT
► STAT dimer stimulates transcription
JAK/STAT Pathway
http://en.wikipedia.org/wiki/Image:Jakstat_pathway.svg
► Outcomes
from the JAK/STAT1 Pathway:
 Immunoglobulin G
 Protein Kinase R (PKR)
 TH1 Differentiation (T-Cells)
SUMO regulation
SUMO
E3 (PIAS)
ATP
SUMO
E1
S
SUMO
E2
S
SUMO
SUMO
SENP
STAT
STAT
► SUMO
protein cascade
► PIAS and SENP function
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
► Mutate
essential interactive amino acids
between SUMO and SENP to alanine so
SENP cannot recognize SUMO
► Mutate neighboring amino acids to pbenzoyl-L-phenylalanine to permanently link
and fish out interaction partners
Methods
► Mutations
at/around these amino acids
Crosslinking unnatural amino acid:
pBpa – p-benzoyl-L-phenylalanine
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
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
► Codon
mutated to TAG
(stop codon)
► Unrecognized by
endogenous
synthetases
Aminoacyl-tRNA Synthetase Requirements
► Only
aminoacylates the engineered tRNA
► Only accepts unnatural amino acid into
active site
Methods: Mutation
► Mutations
through PCR
primers/hybridization
► Restriction digestion of DNA
► Ligation to mammalian expression vector
Methods: Expression
► Transfection
of STAT1/alanine SUMO1/SENP2
► Lyse cells and extract protein
http://www.amaxa.com/uploads/pics/StableTransfection_W.gif
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
► Analysis
using Western Blot
Insert protein here
big
STAT/SUMO
SUMO
small
SUMO/SENP
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
►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
► 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
► Dr.
Jiayu Liao
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