NGFN-PLUS
Protein interaction networks
| Coordinator: | Prof. Dr. Erich Wanker | |
| Institution: | Max-Delbrück-Centrum für Molekulare Medizin (MDC) Berlin-Buch | |
| Homepage: | www.mdc-berlin.de |
Protein tyrosine kinases (PTKs) are important regulators of intracellular signal transduction pathways whose normal activity is subject to strict control and regulation. Alterations of PTK signaling through mutations or other genetic variations often leads to deregulated kinase activity and to malignant transformations of the cell.
In subproject 6 a comprehensive protein interaction network for the 32 human cytoplasmic PTKs and 27 selected Mutanom target proteins and their respective mutant equivalents was to be created. Among these were proteins such as PIK3CA, PTEN and SMAD4.
For the interaction screening a well-established high-throughput (HT) yeast 2-hybrid (Y2H) method was used.
First, the known cDNA clones of all human cytoplasmic PTKs were cloned into Gateway-compatible vectors. After sequence validation, cancer-relevant mutations were inserted. PTK clones and the other Mutanom target proteins, along with their mutant versions, were then tested against the 17,000 human proteins of our clone library using the Y2H technology. Thereby, for the PTK clones and mutants 7252, for the Mutanom target proteins and mutants 7997 protein-protein interactions (PPIs) could be identified.
A representative portion of these two PPI sets could be successfully validated using a semi-automated LUMIER assay. Another part of the Mutanom target protein PPIs could be confirmed also by HT FRET methods (Fig. 1).
The generated PPI data were analyzed and evaluated using bioinformatic methods and used to systematically identify potential modulators of cancer-related signaling pathways in cell-based reporter experiments, such as RNAi knock-down assays and overexpression studies.
In the subproject, a valuable scientific resource has been created to identify new tumor suppressors, oncogenes, and interaction partners of known cancer target proteins. In addition, the research results contribute to the elucidation of new oncogenic kinase signaling pathways and to the development of new approaches for cancer drug development.
Fig. 1: FRET-validiertes PPI-Netzwerk für das krebsrelevante Protein PIK3CA.
Additional relevant Internet link:
The Mutanom project
Further Coordinators:
In subproject 6 a comprehensive protein interaction network for the 32 human cytoplasmic PTKs and 27 selected Mutanom target proteins and their respective mutant equivalents was to be created. Among these were proteins such as PIK3CA, PTEN and SMAD4.
For the interaction screening a well-established high-throughput (HT) yeast 2-hybrid (Y2H) method was used.
First, the known cDNA clones of all human cytoplasmic PTKs were cloned into Gateway-compatible vectors. After sequence validation, cancer-relevant mutations were inserted. PTK clones and the other Mutanom target proteins, along with their mutant versions, were then tested against the 17,000 human proteins of our clone library using the Y2H technology. Thereby, for the PTK clones and mutants 7252, for the Mutanom target proteins and mutants 7997 protein-protein interactions (PPIs) could be identified.
A representative portion of these two PPI sets could be successfully validated using a semi-automated LUMIER assay. Another part of the Mutanom target protein PPIs could be confirmed also by HT FRET methods (Fig. 1).
The generated PPI data were analyzed and evaluated using bioinformatic methods and used to systematically identify potential modulators of cancer-related signaling pathways in cell-based reporter experiments, such as RNAi knock-down assays and overexpression studies.
In the subproject, a valuable scientific resource has been created to identify new tumor suppressors, oncogenes, and interaction partners of known cancer target proteins. In addition, the research results contribute to the elucidation of new oncogenic kinase signaling pathways and to the development of new approaches for cancer drug development.
Fig. 1: FRET-validiertes PPI-Netzwerk für das krebsrelevante Protein PIK3CA.
Additional relevant Internet link:
The Mutanom project
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