Protein-Protein Interactions

1. Protein-Protein Interactions From Transgene to Protein, Station IVb http://teachline.ls.huji.ac.il/~92984/ T. Bruck, Dr. E.R. Bennett, E. Podoly

2.  Introduction • Interactome •Driving Forces •Energy  Methods •Experimental •Computational  Proteins

3. Genomics > Proteomics > Interactomics The Post-Genomic Era Proteins rarely function in isolation. Protein-Protein interactions (PPIs) are an integral part of cellular processes and functions. Understanding and characterization of data coming from proteomic experiments is one of the major challenges of the post-genomic era: This is the time of the Interactome.

4. Interactome – The Protein’s Social Utility Interactomics is a study of interaction network, aimed at mapping the molecular interactions in cells using computational and experimental methods. proteins

5. The Driving Forces of Interactions I. Covalent Interactions · Disulfide bonds II. Non-Covalent Interactions · Hydrophobic · Hydrogen bonds · Electrostatic

6. The Driving Forces of Interactions Covalent bondsresult from sharing pairs of electrons between atoms. Disulfide bonds are formed between the thiol groups of cysteine residues. Non-covalent bonds involve dispersed variations of electromagnetic interactions: Hydrophobic interactionsare formed between non-polar atoms. Electrostatic interactions are formed between charged atoms. Hydrogen bonds are formed between an electronegative atom and a hydrogen atom bonded to another electronegative atom.

7. Energy of Interactions Noncovalent interactions are weak as compared with a covalent bond, but: 1. They work jointly to hold two interacting surfaces together. 2. A surface topography enables substantial areas of two interacting surfaces to approach each other closely.

8. Methods to Study PPIs Tracking PPIs and delineating their detailed information are both interest for researchers in many fields. Multitude of methods detect PPIs, each has its own strengths and weaknesses. Specificity: most of the interactions detected occur in reality. Sensitivity: many interactions that occur in reality are detected.

9. Two-Hybrid System (Y2H) A genetic method based on transcription factor structure in yeast. A functional transcription factor consists of two separable domains:  DNA binding domain (BD)  Activation domain (AD) In Y2H system, these two domains are separated and each is fused to the protein of interest (X and Y, respectively). Physical interaction between BD-X and AD-Y reconstitutes the transcription factor that activates the transcription of reporter gene.

10. Fluorescence Resonance Energy Transfer* Monitoring interactions of two proteins, one of them is labeled with a donor and the other with an acceptor, by following the energy transfer between them. Protein A is labeled with CFP while protein B is labeled with YFP. If the A and B are adjacent to each other, an energy transfer happens. * Termed also: "Förster Resonance Energy Transfer“, after the German scientist Theodor Förster

11. Co - ImmunoPrecipitation An antibody specific to protein X is added to a cell lysate. The antibody-protein complex is pelleted with protein-G sepharose. Unbound protein are washed. Identification of proteins in the pellet can be determined by western blot/sequencing a purified protein band/antibody against protein Y.

12. Size Exclusion Chromatography* Porous beads separate different sizes of molecules. Small proteins enter the pores and have a longer path and longer transist time than bigger proteins. * Termed also: Gel Filtration Chromatography

13. Size Exclusion Chromatography Protein Complexes will have even shorter path and transist time compared to the its protein components.

14. Affinity Chromatography Beads coated with antibodies that bind their antigen. A tagged protein of interest binds to beads coated with antibodies against its tag. A cell lysate is passed through the affinity column.

15. Affinity Chromatography Binding partners are not eluted. Tag can be broken by an enzyme, following by proteins’ identification as before.

16. Surface Plasmon Resonance* SPR reflectivity measures the changes in the local index of refraction upon adsorption of the target molecule to the metal surface. * Termed also: Dual Polarisation Interferometry

17. Comparison of Methods Specificity C/E/P Tag/Ig Weaknesses Strengths Sensitivity Screening In vivo Y2H High Low +/- False pos. +/-/- Time Consuming Kinetics Equilibrium FRET +/+/+ High +/- High Co-IP -/-/- -/+ High High Simplicity Time Consuming Size Ch. Med. High +/+/+ -/- Preparative Time Consuming +/+/+ Affinity Ch. Screening High +/+ Med. Immobilization dependent T.C Apparatus Kinetics Equilibrium SPR +/+/+ Med. High Kinetics Equilibrium Fluorescence +/+/+ Med. High Apparatus Crosslinker dependent Crosslinking

19. Bioinformatic Approaches http://dip.doe-mbi.ucla.edu/

20. Bioinformatic Approaches http://160.80.34.4/mint/Welcome.do

21. Bioinformatic Approaches http://mips.gsf.de/genre/proj/mpact/yeast/query/interaction

22. Bioinformatic Approaches http://www.thebiogrid.org/

23. Bioinformatic Approaches http://www.himap.org/main/main.jsp

24. Bioinformatic Approaches http://www.ihop-net.org/UniPub/iHOP/

25. 1º 2º 3º 4º Quanternary Organization: A Case Study of PPIs The workshop focuses not in the proteins themselves but in their homo-oligomerization state as a tool to study the field of PPI and a new methodology to approach this field.

26. 15-Lipoxygenase A 94-kDa protein with iron binding site. Three alternatively spliced transcript variants encoding two distinct isoforms have been described. Activity: Arachidonate + O2→ 15S-hydroperoxyeicosatetraenoic acid Function: Arachidonic acid metabolism & linoleic acid metabolism. Interactions: Unknown. Oligomerization: Monomer.

27. Beta-Enolase A 47-kDa protein with magnesium binding site (required for catalysis and dimerization). Three isozyme subunits (alpha, beta, gamma) are known. Activity: 2-phospho-D-glycerate → 2-phosphoenolpyruvate + H2O Function: Glycolysis, Muscle development and regeneration. Interactions:AChE. Oligomerization: Homodimers or heterodimers.

28. Pyruvate Kinase Muscle Isozyme A 57-kDa protein with four metal binding sites (magnesium and potassium). Three alternatively spliced transcript variants encoding two distinct isoforms have been reported and four mammalian isozymes: L (liver), R (red cells), M1 (muscle, heart and brain) and M2 (early fetal tissues). Activity:ADP + 2-phosphoenolpyruvate→ ATP + pyruvate Function: Glycolytic pathway. Interactions: Thyroid hormone & Opa protein. Oligomerization: Homotetramer; On subunit dissociation, a dimeric intermediate is formed.

29. PEP ADP Ser/ fructose 1,6-P2 ATP pyruvate PK Oligomerization The tetramer:dimer ratio of PK is not constant, and determines whether glucose carbons are converted to energy (tetrameric) or into synthetic processes (dimeric). Oxygen starvation or highly accumulated glycolytic intermediates, such as fructose 1,6-bisphosphate or the amino acid serine, induce the reassociation of the dimeric form to the tetrameric form.