Interactions of SUMO within Interactions of SUMO within the JAK/STAT transcription the JAK/STAT transcription

pathwaypathwayA new method to analyze the regulation of the JAK/STAT A new method to analyze the regulation of the JAK/STAT immune response pathway using protein engineeringimmune response pathway using protein engineering

Adam ChengAdam Cheng

Xiulin ShenXiulin Shen

Prof. Jiayu LiaoProf. Jiayu Liao

Department of BioengineeringDepartment of Bioengineering

IntroductionIntroduction

►1918 flu pandemic1918 flu pandemic► JAK/STAT pathwayJAK/STAT pathway►SUMO regulationSUMO regulation►Goals and StrategiesGoals and Strategies►MethodsMethods►ResultsResults►ConclusionConclusion

Spanish Flu Pandemic of Spanish Flu Pandemic of 19181918

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

JAK/STAT PathwayJAK/STAT Pathway

► Cytokines like interferon send Cytokines like interferon send messages throughout the bodymessages throughout the body

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

JAK/STAT PathwayJAK/STAT Pathway

► JAK phosphorylates the receptor and STATJAK phosphorylates the receptor and STAT► STAT dimer stimulates transcriptionSTAT dimer stimulates transcription

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

JAK/STAT PathwayJAK/STAT Pathway

►Outcomes from the JAK/STAT1 Pathway:Outcomes from the JAK/STAT1 Pathway: Immunoglobulin GImmunoglobulin G Protein Kinase R (PKR)Protein Kinase R (PKR) TH1 Differentiation (T-Cells)TH1 Differentiation (T-Cells)

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

SUMO regulationSUMO regulation

►SUMO protein cascadeSUMO protein cascade►PIAS and SENP functionPIAS and SENP function

ATP

E1 S E2 S

E3 (PIAS)

STAT

SENP

SUMO SUMO

SUMOSUMO

SUMO

STAT

GoalGoal

► Investigate signaling specificity by Investigate signaling specificity by analyzing SUMO’s interactions using a analyzing SUMO’s interactions using a new strategy of incorporating new strategy of incorporating unnatural amino acidsunnatural amino acids

►Understand the regulation Understand the regulation mechanisms of cytokine signaling mechanisms of cytokine signaling pathwayspathways

StrategyStrategy

►Mutate essential interactive amino Mutate essential interactive amino acids between SUMO and SENP to acids between SUMO and SENP to alanine so SENP cannot recognize alanine so SENP cannot recognize SUMOSUMO

►Mutate neighboring amino acids to p-Mutate neighboring amino acids to p-benzoyl-L-phenylalanine to benzoyl-L-phenylalanine to permanently link and fish out permanently link and fish out interaction partnersinteraction partners

MethodsMethods

►Mutations at/around these amino acidsMutations at/around these amino acids

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

Wild Type Protein SynthesisWild Type Protein Synthesis

► Essential parts in protein synthesis:Essential parts in protein synthesis:

http://cropandsoil.oregonstate.edu/classes/css430/lecture%209-07/figure-08-01.JPG

http://www.bio.davidson.edu/courses/genomics/2005/Drysdale/molecular%20function.jpg

Incorporating pBpa Incorporating pBpa in vivoin vivo

►What is needed for unnatural amino What is needed for unnatural amino acid incorporation?acid incorporation? 1) Unnatural amino acid (pBpa)1) Unnatural amino acid (pBpa) 2) Engineered tRNA2) Engineered tRNA 3) Engineered Aminoacyl-tRNA synthetase3) Engineered Aminoacyl-tRNA synthetase

tRNA requirementstRNA requirements

► Codon mutated to Codon mutated to TAG (stop codon)TAG (stop codon)

► Unrecognized by Unrecognized by endogenous endogenous synthetasessynthetases

Aminoacyl-tRNA Synthetase Aminoacyl-tRNA Synthetase RequirementsRequirements

►Only aminoacylates the engineered Only aminoacylates the engineered tRNAtRNA

►Only accepts unnatural amino acid into Only accepts unnatural amino acid into active siteactive site

Methods: MutationMethods: Mutation

►Mutations through PCR Mutations through PCR primers/hybridizationprimers/hybridization

►Restriction digestion of DNARestriction digestion of DNA►Ligation to mammalian expression Ligation to mammalian expression

vectorvector

Methods: ExpressionMethods: Expression► Transfection of STAT1/alanine SUMO1/SENP2Transfection of STAT1/alanine SUMO1/SENP2► Lyse cells and extract proteinLyse cells and extract protein

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

Methods: ExpressionMethods: Expression► Transfection of STAT1/pBpa SUMO1/SENP2/TAG Transfection of STAT1/pBpa SUMO1/SENP2/TAG

tRNA/Aminoacyl-tRNA synthetasetRNA/Aminoacyl-tRNA synthetase► Add p-benzoyl-L-phenylalanine (1mM) 6h after Add p-benzoyl-L-phenylalanine (1mM) 6h after

transfectiontransfection

SUMO/SUMO/SENP SENP

complexcomplex

Methods: AnalysisMethods: Analysis► Analysis using Western BlotAnalysis using Western Blot

SUMO

STAT/SUMOSUMO/SENP

big

small

Insert protein hereInsert protein here

Results and Future WorkResults and Future Work

►All mutations are completeAll mutations are complete►Analyze using Western BlotAnalyze using Western Blot►Affirm results using another methodAffirm results using another method►Move techniques to related pathwaysMove techniques to related pathways

Anti-tumor p53 pathwayAnti-tumor p53 pathway

ConclusionConclusion

►A new amino acid incorporation A new amino acid incorporation strategy was applied strategy was applied

►Two types of SUMO mutations were Two types of SUMO mutations were generated to further the study of the generated to further the study of the JAK/STAT pathwayJAK/STAT pathway

►Results will benefit human healthResults will benefit human health

AcknowledgementsAcknowledgements

►Yang SongYang Song►Vicente NuVicente Nuññezez►Vipul MadaharVipul Madahar►Clarence PasionClarence Pasion►Xiulin ShenXiulin Shen►Department of BioengineeringDepartment of Bioengineering►Maria Franco-Aguilar and UCLEADSMaria Franco-Aguilar and UCLEADS►Dr. Victor Rodgers, Jill Brady, BRITEDr. Victor Rodgers, Jill Brady, BRITE

AckowledgementsAckowledgements

►Dr. Jiayu LiaoDr. Jiayu Liao

ReferencesReferences

► D. Schmidt and S. Miller. (2003) PIAS/SUMO: Partners in D. Schmidt and S. Miller. (2003) PIAS/SUMO: Partners in transcriptional regulation. Cell. Mol. Life. Sci. transcriptional regulation. Cell. Mol. Life. Sci. 6060:2561-2574:2561-2574

► Johnson, Erica S. (2004) Protein Modification by SUMO. Johnson, Erica S. (2004) Protein Modification by SUMO. Annu. Rev. Biochem. Annu. Rev. Biochem. 73:73: 355-382 355-382