BIS183 Lecture 15 - Protein-Protein Interactions I

Why are we interested in protein-protein interactions?

• Protein complexes (multiple working together)

  • transcription factors

  • Enzyme complexes

• Sense of how the proteome is organized

• Receptor-ligand pairs

Yeast Two Hybrid Methodology

• Two hybrid proteins - takes advantage of TF modularity

  • DNA Binding Domain

  • Activating Domain

Bait

• Protein of interest + GAL4 DBD

Prey

• Protein Y + GAL4 AD

Introduce Bait and prey

Look for expression of reporters

• HIS3 - Helps grow on media lacking HIS3

• LACZ - turns blue in presence of LAC Z substrate

What does autoactivation look like in Y2H assay?

• BAIT alone (Protein of interest +DBD) is sufficient to drive trxn of a reporter

• due to Yeast protein + AD complex interactions

• BAD → if bait strain results in autoactivation, cannot be used

What are the 4 different results possible for Y2H Assay

1) Reporter construct alone

• HIS3 - no growth

• LACZ - white

2) Bait alone

• HIS3 - no growth

• LACZ - white

3) Bait + Prey - Interact

• HIS3 - no growth

• LACZ - white

4) Bait + Prey - DON’T interact

• no growth

• white

Describe the Permissive and Binary aspects of Y2H

Permissive

• Grows yeast on media containing HIS (media regularly DOESN’T contain HIS) → lets yeast survive and Bait + prey successfully introduced for yeast

Binary

• Only test interaction between one protein and another protein

• Can’t test more than two at once

How can “Guilt by Association” be used to infer protein function in Y2H Assay?

• Look at the trxn of associated proteins

Guilt by Association

• proteins interact with each other

• in clustering methods → look at similar transcripts

• Proteins that interact with each other may have similar function in the body

Quesitons regarding the validation of networks and Overalp between two studies

How much overlap do you think there was between these studies?

• Look at the overlap between bait and prey in studies

• 17% overlap

How do you determine what a “true” or real interaction is?

• Must determine if interactions occur in vivo or in cell

What do you think the best way is to biologically validate a protein-protein interaction in a study? 6 things

1) Literature (previous reports)

2) MIPS - Munich Infor center for Genes and Protein

3) Mine available transcription datasets is protein transcripts found in same time + place

4) If proteins are found in the same cells (AD or Tag proteins)

5) Determine if proteins physically interact → Affinity Purification

6) Function → genetics

What are the advantages and disadvantages of the Y2H Assay

Advantages

• Sensitivity

  • overproduction of proteins in yeast

  • Detects weaks interactions

• Flexibility

• Large number of variable inserts can be examined at once

Disadvantages

• Labor and material intensive

• Array based Y2H - need efficient elaborate pooling and deconvolution schemes

• False positive (in assay, but not in vivo), false negative (not in assay, but in vivo)

Exmaples of False negatives in Y2H

• Real interactions are missed

  • Failures in nuclear localization

  • Membrane proteins and secretory proteins not amenable to system

  • Autoactivation

  • Improper folding

  • Missing cofactors

  • Missing post-translational modifications

  • Differences in vector systems

Examples fo False Positives in Y2H

• Non-relevant interaction - overexpression

• Autoactivation

Describe the steps for Tandem Affinity Purification

Purpose: to extract on the Protein of interest, IP-based purification tech to study P=P interactions

1) Bait protein

• Creates complex containing:

  • Protein

  • CaBP - Cal modulin binding protein (binds CA²+)

  • TEV Cleavage Site Protease (Enable cleavage by TEV protease)

  • Protein A (Able to be recognized by Igv beads)

2) Introduce construct into cells

• In vitro - translate protein _incubate protein with a protein cocktail from lysed cells

3) Bait protein forms complex with other cellular protein

• Identify other protein so your bait interacts with (advantage - proteins are found within one complex)

4) Cell extract, pass over column with IgG beads

5) Cleave complex with TEV protease

• Very pure (two tandem purifications)

• reduced chance of pulling down non-specific proteins

6) Trypsin digestion of protein complex - break protein to small pieces

7) Mass spectrometry analysis - ID the proteins, list of proteins with which your bait has complexed

COMPLEX; in vivo interactions

Help us better understand the proteome

Describe the Identification of Novel RNA Pol II Subunit

Top row - Tagged subunit protein with TAP

Column (y-axis) - proteins that interact with IWR protein

Rpb → RNA polymerase binding