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