Protein-Protein Interaction (PPI)A review

Introduction

• Protein–protein interactions occur when two or more proteins bind together.

• Proteins control and mediate many of the biological activities of cells by these interactions.

• Information about PPIs improves our understanding of diseases and can provide the basis for new therapeutic approaches.

• Practical applications include: 1. Analysis of metabolic and signal transduction;

mainly to find out disease pattern.2. Pharmacogenetics research; the study of drug

transporters, drug receptors, and drug targets.

The types of protein interactions 1

1. Binary PPI

2. Scaffolding proteins:eg: GAB 2, a Scaffolding

Protein in cancer

The types of protein interactions 2

1. Metabolic and signaling pathways

2. Morphogenic pathways; in which groups of proteins participate in the same cellular function during a developmental process.

3. Structural complexes ; molecular machines in which numerous macromolecules are brought together.

Signaling pathway

Morphogenic pathway

Structural complexes

PPIs Identification Methods

• Yeast two-hybrid system• split ubiquitin system

• split lactamase / split galactosidase system

• split yellow fluorescent protein (YFP) system

Experimental

((In vivo

• Co-immunoprecipitation• Tagged Fusion Proteins

• X-ray Diffraction

• Biacore

• Phage display

Experimental

((In vitro

• BIND

• DIP

• MINT

• IntAct

Computational

(In silico)

o Yeast two-hybrid (H2Y) system

• The most common screening approach.

• Testing for physical interactions between two proteins .

• Is based on the properties of the yeast GAL4 protein, which consists of separable domains responsible for DNA-binding and transcriptional activation.

• Plasmids encoding two hybrid proteins, one consisting of the GAL4 DNA-binding domain fused to protein X and the other consisting of the GAL4 activation domain fused to protein Y, are constructed and introduced into yeast.

• Interaction between proteins X and Y leads to the transcriptional activation of a reporter gene containing a binding site for GAL4.

Experimental—in vivo

Y2H for binary PPI identification

• X= prey

• Y= bait

• AD: activation Domain

• DBD: DNA Binding

Domain

• Reporter gene:LacZ reporter - Blue/White

Screening

Y2H assay against library

• (a) A model of the yeast two-hybrid system.

• (G) general transcription machinery.

• (b) A library yeast two-hybrid screen. A collection of preys are screened with a bait of interest.

• (c) A matrix yeast two-hybrid screen used to generate a protein-protein interaction network (in 96-well microtiter).

o Co-immunopercipitation (Co-IP)

• A popular technique to identify PPIs by using target protein-specific antibodies to indirectly capture proteins that are bound to a specific target protein.

• These protein complexes can then be analyzed to identify new binding partners, binding affinities, the kinetics of binding and the function of the target protein.

• Unlike Y2H, is used also for the analysis of protein complexes.

Experimental—in vitro

Over all of experimental methods

• ‘atomic observation’ assyas : in which the protein interaction is detected

using, for example, X-ray crystallography. These experiments can yield

specific information on the atoms or residues involved in the interaction.

• ‘direct interaction observation’ : where protein interaction between two

partners can be detected as in a two-hybrid experiment.

• ‘multi-protein complexes observation’: using methods such as immuno-

precipitation. This type of experiment does not unveil the chemical detail

of the interactions or even reveal which proteins are in direct contact but

gives information as to which proteins are found in a complex at a given

time.

• http://bind.ca

• A free, open-source database for archiving and exchanging molecular assembly information.

• The database contains - Interactions- Molecular complexes- Pathways

oBIND(Biomolecular Interaction Network Database)

Computational—in silico

• BIND Interaction Viewer Java showing how molecules can be connected in the database from molecular complex to small molecule.

• Yellow: protein• Purple: small molecule; • white: molecular complex;

• This session was seeded by the interaction between human LAT and Grb2 proteins involved in cell signaling in the T-cell.

Other databases with web add.

• DIP (Database of Interacting Proteins)http://dip.doe-mbi.ucla.edu

• MINThttp://mint.bio.uniroma2.it/mint

• DLRP (Database of Ligand-Receptor Partners)http://dip.doe-mbi.ucla.edu/dip/DLRP.cgi

• STRINGhttp://string-db.org

• Human Protein Interaction Databasehttp://www.hpid.org

Visualization of networks

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