Reverse Interactomics

演讲者：王神宇

By Dehua Pei and Anne-Sophie Wavreille

1. Introduction of interactomics

2. What is reverse interactomics

3. Advantages and limitations of the reverse

interactomics

4. Conclusions

1. Introduction of interactomics

Interactomics : The systematic mapping of the protein–protein interaction network in a living cell.

The most extensively used experimental approach:

Yeast two-hybrid system

Co-immunoprecipitation

Protein pulldown

Yeast two-hybrid system:

Co-immunoprecipitation:

Protein pulldown:

Post-translational modifications, such as tyrosine phosphorylation, which does not normally occur in yeast.

Yeast two-hybrid system:

Co-immunoprecipitation & Protein pulldown:

Usually, limited to the identification of the most abundant and high-affinity binding proteins. For less abundant proteins, a single affinity purification step (immunoprecipitation or pull-down) usually does not produce samples of sufficient purity and/or quantity to allow for positive proteinidentification.

2. What is reverse interactomics:

A significant fraction of protein–protein interactions are mediated by small modular domains (e.g., SH2, PTB, SH3, PDZ, WW and FHA domains), which recognize short linear peptide motifs in their target proteins.

Reverse Interactomics Approach: To decode protein–protein interactions mediated by these modular domains, it is a chemical/bioinformatics method.

3. Advantages and limitations of the reverse interactomics:

Provides not only the identity of an interacting partner, but also the molecular basis of the interaction;

Advantages:

Detect low abundance protein partners, because a candidate protein is identified by in silico searches (which are not affected by low abundance) and its validation is achieved through Western blot analysis, which is highly sensitive and specific.

Limitations:

The initial database searches often produce many candidate proteins, which must be prioritized prior to cellular assays.

False negatives are also possible, because a physiological interaction may not utilize the most optimal sequence motif.

Conclusions:

The reverse interactomics method provides a powerful, complementary approach for identifying the protein targets of a binding domain or enzyme and the peptide motif(s) responsible for the interaction.

It also provides an exciting example of how chemical tools can be employed to help elucidate complex biological systems.

Questions are appreciated!