Protein protein interactions-ppt

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1. 1
Identification of Protein-Protein Interactions by
Yeast 2-Hybrid System & Immunoprecipitation

2. Protein-Protein interactionsProtein-Protein interactions
Important field in cell biology Important field in cell biology && biochemistry biochemistry
Localization and traffickingLocalization and trafficking
Posttranslational modifications Posttranslational modifications
Signaling networksSignaling networks
Important field in viral replication very difficult to predictImportant field in viral replication very difficult to predict

two main patterns:two main patterns:

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domain-domain interactionsdomain-domain interactions

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domain-peptide interactionsdomain-peptide interactions
  
2

3. 3

4. Co-immunoprecipitation protocolCo-immunoprecipitation protocol
Protein A binds to antibodiesProtein A binds to antibodies
Sepharose beads coated with protein ASepharose beads coated with protein A
Specific antibody binds to the protein of interest Specific antibody binds to the protein of interest
The complex is precipitated by binding to the beads via The complex is precipitated by binding to the beads via
protein Aprotein A
Proteins are released from beads by boiling Proteins are released from beads by boiling
Analysis by SDS-PAGE and/or other methodsAnalysis by SDS-PAGE and/or other methods
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5. 5

6. Detection of protein interactions by microarraysDetection of protein interactions by microarrays
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7. Principle of protein microarray technologyPrinciple of protein microarray technology
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8. Reporter Gene
Bait
Protein
Binding
Domain
Prey
Protein
Activation
Domain
The yeast two-hybrid systemThe yeast two-hybrid system……
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Two hybrid proteins are generated with transcription Two hybrid proteins are generated with transcription
factor domainsfactor domains
Both fusions are expressed in a yeast cell that carries a Both fusions are expressed in a yeast cell that carries a
reporter gene whose expression is under the control of reporter gene whose expression is under the control of
binding sites for the DNA-binding domainbinding sites for the DNA-binding domain

9. Reporter Gene
Bait
Protein
Binding
Domain
Prey
Protein
Activation
Domain
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Interaction of bait and prey proteins localizes the activation Interaction of bait and prey proteins localizes the activation
domain to the reporter gene, thus activating transcription. domain to the reporter gene, thus activating transcription.
Since the reporter gene typically codes for a survival Since the reporter gene typically codes for a survival
factor, yeast colonies will grow only when an interaction factor, yeast colonies will grow only when an interaction
occurs. occurs.

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21. Bait SelectionBait Selection
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Proteins known to be expressed in B cells and/or cardiac Proteins known to be expressed in B cells and/or cardiac
myocytes.myocytes.
Proteins known or suspected to be involved in signaling Proteins known or suspected to be involved in signaling
pathways in B cells and/or cardiac myocytes.pathways in B cells and/or cardiac myocytes.
Proteins believed important in GPCR or BCR signaling Proteins believed important in GPCR or BCR signaling
leading to PIP3 generation. leading to PIP3 generation.

22. Bait DesignBait Design
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1-100 (P sites)
100-200 (Pro regions)
1-240 (N-term)
150-350 (linker)
348-457 (SH2)
210-457 (C-term)
110116134146 350 436
PPP SH2Pro Pro
Phosphorylation
sites
Proline-rich
SH3 binding Src homology 2 domain
BLNK

23. B Cell Receptor PathwayB Cell Receptor Pathway
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24. B Cell Receptor PathwayB Cell Receptor Pathway
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25. BB Cell Receptor PathwayCell Receptor Pathway
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Pak
Nck
Pix/Cool
Git2
14-3-3z
Endophilin2
BAN-P
PDE4B3
Ruk1
-
PP2C
gamma

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29. 29

30. Iden CD19
Actin
CytoskeletonydYe
PDK1y2HY g oSmee&u
PI3Kg (p110)pf-
Btk 2
WISHIty
CAPdb
DblI Yo22
CamK II !-p
NdkBi!i"#
cdc42Id##
CD22$ a
Fyn%#
SOS2
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31. Iden CD19pf-
Btk
Actin
Cytoskeleton%#
SOS2 2
WISHIty
CAPdb
DblI Yo22
CamK II !-p
NdkBi!i"#
cdc42ydYe
PDK1Id##
CD22$ a
Fyny2HY g oSmee&u
PI3Kg (p110)en&&&en&
19070197'&((Hnn
6755399''&e(H)
6671538"'HH(e"
4633514)('&H#(
8567325eH("#'&&
13542677#&)n""H&
20894430#'H#'n')
26326968H&'"#'#
3064262
Sam68
Protein 4.1G
AIP
Cbl-b
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33. ReferencesReferences
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Bonifacino JS, Dell’Angelica EC, Springer TA. 2001. Immunoprecipitation. Bonifacino JS, Dell’Angelica EC, Springer TA. 2001. Immunoprecipitation.
Curr. Protoc. Mol. Biol. Chapter 10:Unit 10.16Curr. Protoc. Mol. Biol. Chapter 10:Unit 10.16..
Protein Microarray techniques. S. Funke Dipl.Biol. Universitäts-Protein Microarray techniques. S. Funke Dipl.Biol. Universitäts-
Augenklinik Mainz. Leitung: PD Dr. Dr. F. Grus. Langenbeckstr. 1, 55131 Augenklinik Mainz. Leitung: PD Dr. Dr. F. Grus. Langenbeckstr. 1, 55131
MainzMainz
Yeast Two-Hybrid, a Powerful Tool for Systems Biology. Anna Brückner, Yeast Two-Hybrid, a Powerful Tool for Systems Biology. Anna Brückner,
Cécile Polge, Nicolas Lentze, et al. Int. J. Mol. Sci. 2009, 10, 2763-2788; Cécile Polge, Nicolas Lentze, et al. Int. J. Mol. Sci. 2009, 10, 2763-2788;
doi:10.3390/ijms10062763 doi:10.3390/ijms10062763