BCH 4101 Lecture Notes - Lecture 13: Dihydrofolate Reductase, Interactome, Human Genome Project
28 Apr 2018
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November 22, 2017
Proteome and Interaction Networks
After the Human Genome Project
Sequencing projects reveal much fewer genes than originally predicted
-Number of genes can’t explain complexity
How do we explain complexity?
-Alternative splicing: way of creating different proteins from the same gene
-Post-translational modifications: changes protein function depending on cellular location
-Genetic factors influencing gene expression
-Interactome
•Protein interactions: once you start combining proteins, the number of possible effects of all the possible
complexes increases
Interactome
Complete set of molecular interactions in a cell (assembled into networks)
Within/between biochemical families (proteins, nucleic acids, metabolites, etc.)
-Within: protein-protein, DNA-DNA, etc.
-Between: protein-DNA, etc.
Protein-protein interactions (PPI or PIN) and protein-DNA interactions (gene regulatory networks)
Interactomics
Three main methods to map a protein-protein interactome (PPI):
-Measure binary (pairwise) interactions: looks at the interaction between two proteins (ex. A with B, B with C, etc.)
•2 methods
-Protein complementation assay (PCA)
-Yeast two-hybrid analysis
-Measure protein complexes: looks at the interaction between at least three proteins (ex. Preinitiation complex)
•1 method: mass spectrometry based co-purification analysis
Protein Complementation Assay (PCA)
PCA based on the Dihydrofolate reductase (DHFR) enzyme
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November 22, 2017
-DHFR reduces dihydrofolic acid into the essential vitamin folate
•Necessary for cell functioning
•Cell can’t replicate without it unless supplemented with nucleic acids
-Methotrexate (MTX) is a competitive inhibitor of DHFR
•Methotrexate has a very similar structure fo dihydrofolate (DHF)
•Will bind to the enzyme and prevent its catalytic function
-PCA uses a DHFR mutant that does to bind methotrexate
Based on the premise that you can separate the DHFR enzyme into two separate parts, rendering it nonfunctional
-When the two parts are brought into close proximity, they will refold into a functional enzyme
Will fuse protein X to one half of the enzyme, fuse protein Y to the other half of the enzyme
-If the two proteins interact, the enzyme will be able to re-fuse
•Only when the two proteins interact, will the enzyme be brought back together
Perform a selection assay:
-Add the two halves of the enzyme into a host cell
-In the two proteins interact, the reconstituted enzyme will work, nucleotides would be produced, and the cells will
be able to grow
-Add MTX: will kill cells that do not have the altered enzyme (cells that did not uptake the assay), but will not kills
cells that have a functioning PCA DHFR
•Recall: PCA uses modified DHFR that is unaffected by MTX
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Can plate multiple interactions and see which cells survive in order to determine which combinations of proteins
interact
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November 22, 2017
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Yeast 2-Hybrid Assay
Binds proteins two transcription factors
TFs have two domains:
-DNA-binding domain: binds DNA, can’t activate transciption
-Trans-activation domain: activates transcription, can’t bind DNA
Fuse one protein to each domain of the transcription factors; the TF will only become functional if the two proteins
interact
-Transcription will only occur if the two proteins interact
Use TFs that influence a reporter gene (ex. lacZ gene)
-Proteins interact —> transcription of lacZ gene —> formation of blue colonies
-Proteins do not interact —> no transcription of the lacZ gene —> formation of white colonies
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lacZ
DNA binding domain
blue
No transcription (white)
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Document Summary
Sequencing projects reveal much fewer genes than originally predicted. Alternative splicing: way of creating different proteins from the same gene. Post-translational modi cations: changes protein function depending on cellular location. Interactome: protein interactions: once you start combining proteins, the number of possible effects of all the possible complexes increases. Complete set of molecular interactions in a cell (assembled into networks) Within/between biochemical families (proteins, nucleic acids, metabolites, etc. ) Protein-protein interactions (ppi or pin) and protein-dna interactions (gene regulatory networks) Three main methods to map a protein-protein interactome (ppi): Measure binary (pairwise) interactions: looks at the interaction between two proteins (ex. A with b, b with c, etc. : 2 methods. Measure protein complexes: looks at the interaction between at least three proteins (ex. Preinitiation complex: 1 method: mass spectrometry based co-puri cation analysis. Pca based on the dihydrofolate reductase (dhfr) enzyme.
