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Transient Protein-Protein
Interactions (TPPI)
Presented By:
Muhammad
Shoaib Amjad
11 arid 3758
PhD Botany
1st Semester
Contents
 Protein-protein interactions
 Transient protein interaction
 Transient protein and drug interaction
 Experimental techniques used for detecting TPPIs
 List of databases
 Summary
 References
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Protein-Protein Interaction (PPI)
 Interaction of two proteins
 Play an essential role in the proper functioning of
living cells
 The forces responsible for these interactions include
 Electrostatic forces
 Hydrogen bonds
 Van der waals forces
 Hydrophobic effects
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Types of Potien-Protien interactions
PPIs can be classified on the bases of
Composition
 Homo and hetero-oligomeric complexes
Affinity
 Non-obligate and obligate complexes
Stability/Life time
 Transient and permanent
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Transient and Permanent
 Permanent
 Stable
 Irreversible
 Strong
 Long life
 Electrostatic force
 Example: α/β tubulin dimer and many enzymeinhibitor complexes
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Transiet protein-protein interactions
(TPPI)
TPPIs are involved in many biological processes:

Signal transduction

Protein complexes or molecular machinery

Protein carrier

Protein modifications (phosphorylation)

Hormone receptor binding

Allostery of enzymes

Inhibition of proteases

Correction of misfold protein by chaperones
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Transient protein and drug
interaction
 Transient interactions might be important in
 drug mechanisms in two ways: the drugs that
1. act on TPPIs
2. act transiently on their multiple targets
 A cancer-related example for the former type
of drugs is nutlins
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Transient protein and drug
interaction
Nutlins
MDM2
MDM2
p53
p53
Tumor
suppressed
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Experimental Techniques
 Yeast two-hybrid screens
 Mass spectrometry
 Intracellular localization of proteins with
fluorescence markers
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Yeast Two-hybrid
 Researchers insert a gene in yeast for a "bait" protein alongside
DNA for half of an "activator" protein.
 The other half of the activator DNA is then inserted alongside
DNA for random "prey" proteins.
 The yeast cells are then grown up and the proteins are allowed to
interact.
 If bait and prey proteins bind, the two halves of the activator
protein be close enough to work together to turn on another yeast
gene that turns the cell blue, signaling a match.
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How does it work?
 Uses yeast as a model for eukaryotic protein
interactions
 A library is screened or a protein is characterized using
a bait construct
 Interactions are identified by the transcription of
reporter genes
 Positives are selected using differential media
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Transcriptio
n
Activating
Region
Bait Protein
Prey Protein
Reporter Gene
DNA-Binding
Domain
DNA-Binding Site
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What is the yeast two-hybrid system
used for?
 Identifies novel protein-protein interactions
 Can identify protein cascades
 Identifies mutations that affect protein-protein
binding
 Can identify interfering proteins in known
interactions
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Steps to Screen a Library
 Create the Bait Plasmid Construct from the gene
of interest and the DNA binding domain of Gal4
or LexA or other suitable domain
 Transform with the bait construct a yeast strain
lacking the promoter for the reporter genes and
select for transformed yeast
 Transform the yeast again with the library
plasmids
 Select for interaction
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Sequence analysis
 Isolate plasmid from yeast and transform E. coli
 Purify plasmid from E. coli and sequence
 Blast sequence against database for known proteins
or construct a possible protein sequence from the
DNA sequence and compare to other proteins
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Sample Plasmid
From Golemis Lab Homepage
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Reporter Genes
 LacZ reporter - Blue/White Screening
 HIS3 reporter - Screen on His+ media
 LEU2 reporter - Screen on Leu+ media
 ADE2 reporter - Screen on Ade+ media
 URA3 reporter - Screen on Ura+ media
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False Positives
 False positives are the largest problem with the
yeast two-hybrid system
 Can be caused by:
 Non-specific binding of the prey
 Ability to induce transcription without interaction with
the bait (Majority of false positives)
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Elimination of False Positives
 Sequence Analysis
 Plasmid Loss Assays
 Retransformation of both strain with bait plasmid and
strain without bait plasmid
 Test for interaction with an unrelated protein as bait
 Two (or more) step selections
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Advantages
 Immediate availability of the cloned gene of the




interacting protein
Only a single plasmid construction is required
Interactions are detected in vivo
Weak, transient interactions can be detected
Can accumulate a weak signal over time
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Examples of Uses of the Yeast
Two-Hybrid System
 Identification of caspase substrates
 Interaction of Calmodulin and L-Isoaspartyl
Methyltransferase
 Genetic characterization of mutations in E2F1
 Peptide hormone-receptor interactions
 Pha-4 interactions in C. elegans
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The Study of Protein-protein
Interaction by Mass Spectrometry
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Summary
 The components of transient complexes associate
and dissociate rapidly while transiently interacting
with each other to function dynamically in crucial
regulatory and signaling pathways.
 The identification and analysis of these complexes
have become more manageable with the emerging
sensitive
and
high-resolution
experimental
techniques accompanied by the high-throughput
computational methods.
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 As the coverage of these techniques increase,
they can provide a good template to understand
and design new transient complexes.
 An example for such advanced techniques is,
PRISM,
which
uses
available
transient
interactions as a template set and searches
structural and evolutionary similarities between
the template set and the target proteins to be
predicted.
References
 James R. Perkins, I. Diboun, B.H. Dessailly, J. G. Lees and C. Orengo.
2010. Transient Protein- Protein Interactions: Structural Functional And
Network Properties. Cell:1233-1244.
 Saliha E. A. O., H. B. Engin, A. Gursoy and O. Keskin. 2011. Transient
Protein- Protein Interactions. Protein Interaction Designed And
selection: 1-14
 Costel C. D., K. Deinhardt, G. Zhang, H. S. Cardasis and M. V. C.A.
Neubert. 2011. Identifying transient protein–protein interactions in
EphB2 signaling by blue native PAGE and mass spectrometry
Proteomics J:11, 4514–4528
 Lakshmipuram S. S., R. M. Bhaskara, J. Sharma and N. Srinivasan.
2012. Roles of residues in the interface of transient protein-protein
complexes before complexation. Scientific reports: 334.
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