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Suzanne VromanSuzanne Vroman
Katze LabKatze Lab
University of Washington University of Washington
Protein-Protein Protein-Protein Interactions of P58Interactions of P58IPKIPK
Aim 3
Use functional genomics approaches to examine the role of P58 in the presence and absence of virus infection– Identification of P58 interactive partners
Protein Expression – Mammalian, E. coli
Yeast two-hybrid screens
Hsp40
P52rIP
K
Characterization of P58 pathway
P58IPK
P58IPK
PKR ?PERK
Inactive
active
eIF2α eIF2α eIF2α – independent?
Virus Infection ER stress
Enhanced viral replication
Return to ER homeostasis
Regulation of apoptosis and other functions
Post-transcriptional activation
Transcriptional activation
pTriEx expression vector
mammalian promoter
E. coli promoter
HSV/HIS -tags
Bacterial Protein ExpressionBacterial Protein ExpressionTransformation of mP58 and hP58 pTriEx vectors into BL21 E. coli cells
Bacterial Protein ExpressionBacterial Protein ExpressionTransformation of mP58 and hP58 pTriEx vectors into BL21 E. coli cells
Inoculate LB-Amp-Cam Media for expression
Bacterial Protein ExpressionBacterial Protein ExpressionTransformation of mP58 and hP58 pTriEx vectors into BL21 E. coli cells
Inoculate LB-Amp-Cam Media for expression
Check OD throughout the day (approx. every hour)
Bacterial Protein ExpressionBacterial Protein ExpressionTransformation of mP58 and hP58 pTriEx vectors into BL21 E. coli cells
Inoculate LB-Amp-Cam Media for expression
Check OD throughout the day (approx. every hour)
- At OD of .6 add IPTG inducer and grow for 3-5 hrs.
Bacterial Protein ExpressionBacterial Protein ExpressionTransformation of mP58 and hP58 pTriEx vectors into BL21 E. coli cells
Inoculate LB-Amp-Cam Media for expression
Check OD throughout the day (approx. every hour)
- At OD of .6 add IPTG inducer and grow for 3-5 hrs.
Freeze and harvest pellet
Transient Transfection:Transient Transfection:Mammalian Protein Mammalian Protein
ExpressionExpressionPlasmid vector DNA
hP58, mP58pTriEx
Transfection(lipofection)Reagent
Transient Transfection:Transient Transfection:Mammalian Protein Mammalian Protein
ExpressionExpressionPlasmid vector DNA
hP58, mP58pTriEx
Transfection(lipofection)Reagent
DNA lipid liposome
Transient Transfection:Transient Transfection:Mammalian Protein Mammalian Protein
ExpressionExpressionPlasmid vector DNA
hP58, mP58pTriEx
Transfection(lipofection)Reagent
DNA lipid liposome
Endocytosis
Cell293/293T
Transient Transfection:Transient Transfection:Mammalian Protein Mammalian Protein
ExpressionExpressionPlasmid vector DNA
hP58, mP58pTriEx
Transfection(lipofection)Reagent
DNA lipid liposome
EndocytosisDNA released
Cell293/293T
Transient Transfection:Transient Transfection:Mammalian Protein Mammalian Protein
ExpressionExpressionPlasmid vector DNA
hP58, mP58pTriEx
Transfection(lipofection)Reagent
DNA lipid liposome
EndocytosisDNA released
Cell293/293T
-24 hours
-48 hours
Lysis/cell harvest
Transient Transfection:Transient Transfection:Mammalian Protein Mammalian Protein
ExpressionExpressionPlasmid vector DNA
hP58, mP58pTriEx
Transfection(lipofection)Reagent
DNA lipid liposome
EndocytosisDNA released
Cell293/293T
-24 hours
-48 hours
Lysis/cell harvest
Anti P58 western blotting
Targeted proteomics to Targeted proteomics to identify interacting partnersidentify interacting partners
Affinity chromatography Affinity chromatography and mass spectrometryand mass spectrometry– Discovery of new proteins Discovery of new proteins
that interact with the given that interact with the given “bait” protein“bait” protein
– Complex protein Complex protein environment native to the environment native to the environment of the bait environment of the bait protein protein
Immobilized protein (P58)
“bait”
Protein mixture
“prey”
Dual-Reporter 2-Hybrid Dual-Reporter 2-Hybrid AssayAssay
HIS3
Gal4BD Gal4A
D
baitbaitpreyprey
-“Bait” protein fused to a DNA binding domain of a transcription factor and placed in a yeast expression vector
-A library of proteins (the “prey) are fused to an activation domain of another transcription factor, and placed in separate yeast expression vector
-When the bait and prey are transformed in yeast, the binding domain binds to a DNA-binding site on a promoter sequence that drives a reporter protein (HIS3)
-An interaction between bait and prey brings the activation domain in the vicinity of the DNA binding domain
Possible outcomes of protein interaction data
Scenario Possible interpretations
Present in all Non-specific
Present in mockInvolve in P58 regulation
Novel P58IPK regulatory pathway
Present during influenza virus infectionInvolved in P58 activation?
Novel antiviral pathway
Present during ER stressInvolved in PERK inhibition
Novel UPR pathway?Present during ER stress and influenza
infection Common P58 IPK regulator or effector
Present in all
Vector alone
Possible outcomes of protein interaction data
Scenario Possible interpretations
Present in all Non-specific
Present in mockInvolve in P58 regulation
Novel P58IPK regulatory pathway
Present during influenza virus infectionInvolved in P58 activation?
Novel antiviral pathway
Present during ER stressInvolved in PERK inhibition
Novel UPR pathway?Present during ER stress and influenza
infection Common P58 IPK regulator or effector
Present in all
Mock infection/treatment
Present in mock
Possible outcomes of protein interaction data
Scenario Possible interpretations
Present in all Non-specific
Present in mockInvolve in P58 regulation
Novel P58IPK regulatory pathway
Present during influenza virus infectionInvolved in P58 activation?
Novel antiviral pathway
Present during ER stressInvolved in PERK inhibition
Novel UPR pathway?Present during ER stress and influenza
infection Common P58 IPK regulator or effector
Present in all
Influenza infection
Present in mock
Present in infection
Present during ER stress and infection
Possible outcomes of protein interaction data
Scenario Possible interpretations
Present in all Non-specific
Present in mockInvolve in P58 regulation
Novel P58IPK regulatory pathway
Present during influenza virus infectionInvolved in P58 activation?
Novel antiviral pathway
Present during ER stressInvolved in PERK inhibition
Novel UPR pathway?Present during ER stress and influenza
infection Common P58 IPK regulator or effector
Present in all
ER stress
Present in mock
Present in infection
Present during ER stress and infection
Present in ER stress