-  molecular genetic tool to study protein-protein interactions

-  if two proteins interact, then a reporter gene is transcriptionally activated

-  Gal4 protein, transcriptional activator composed of two domains with distinct functions

1. DNA binding domain, interacts with promotor (bait)

2. Activating domain, interacts with polymerase (prey)

-  the yeast Gal4 gene is split in two parts, each of which fused to one of the interacting partners to be tested

-  if proteins interact: reporter gene is transcribed

reporter gene promoter polymerase 1.

2.

reporter gene promoter polymerase 1.

2. -  HIS3, URA3, lacZ reporters

-  human PPIs: generic organisational principles, understanding disease mechanisms and signalling cascades

-  proteome-wide studies for model organisms, but not for human proteome

-  human proteome: only more specific interaction maps, e.g. TGF- β

-  PPIs (protein-protein interactions) are crucial for all biological processes

-  other approaches: bioinformatic analyses, e.g. hypothesis-driven or based on conserved orthologous interactions

-  here: PPI map describing a representative part of human interactome

-  > 5500 proteins analysed by high-throughput yeast-two hybrid sceens

-  network connecting 1705 proteins via 3186 interactions

-  validated biochemically (pull-down and co-immunoprecipitation) and by bioinformatics analyses

1. Creation of human protein Matrix in yeast

2. High-throughput Y2H

3. Confirmation with individual reaction partners

3510 bait and 3589 prey 2033 bait and 2051 prey

11,183 clones

> 25 million protein pairs examined!

three different reporter genes

verification

- representative sample of interactions, randomly selected

- 116 protein pairs were tested by membrane coimmunoprecipitation: success rate 62%

- 131 protein pairs were tested by pull-down assay: success rate 66%

properties of the Y2H network

- average interaction partners per protein in network: 1.87

- 804 proteins with one partner, 24 hubs-proteins with > 30 partners

- hubs 3x more likely to be essential for cells

- mean shortest path length between any two proteins: 4.85 links

confidence scoring

1.  PPI activates three reporter genes (HIS3, URA3, lacZ)

2.  PPI is found in human interaction clusters

3.  PPI is found in orthologous D. melanogaster clusters

4.  PPI is found in orthologous C. elegans clusters

5.  PPI is found in orthologous S. cerevisiae clusters

6.  PPI is formed of two proteins sharing GO annotation

--> network of high-confidence interactions (3186 among 1706 proteins) with >3 points

- >3000 PPIs

- 911 supported by ‘confidence scoring”

- 159 verified biochemically

- 195 disease proteins with previously unidentified partners

- interactions of 342 previously uncharacterised proteins

- new roles for known proteins

www.mdc/berlin.de/neuroprot/database.htm

… have fun! the end.