LAB 11: Functional Genomics
The primary objectives are:
- Learn how to find and use functional genomic information.
- Explore protein interaction data and visualization tools.
1. IntAct is a database of protein interactions. The IntAct data is pairwise protein interactions and protein complexes and contains collected data from almost 12,000 experiments. From the main page of IntAct search for 'PKB AND human'. PKB is the human serine/threonine-protein kinase. Several database interaction and protein matches will be found. Click on the 'Lists' tab and find the protein entry for human PKB. It has the name "akt1_human" in the IntAct database--AKT1 is the approved name for this gene.
1a. How many different proteins interact with human PKB according to the IntAct database?
1b. Note that your answer in 1a is different from the 38 interactions in the "Number of interactions" column for akt1_human. Why is this?
1c. Examine the interaction record for PKB-GSK3B. What type of experiment demonstrated the interaction?
1d. Back up to the "akt1_human" interaction page. The 'Links' column is where IntAct links to the UniProt KnowledgeBase (UniProtKB) to provide annotation for its proteins. One type of annotation is Gene Ontology (GO). Gene Ontology is a heirarchical controlled vocabulary used to annotate genes. Terms are organized in a heirarchy from general to specific. The same annoation terms are used (to the extent possible) for different organisms. There are three types of GO annotations: Molecular Function, Cellular Component, and Biological Process. GO information for PKB is linked midway down the page. Where is PKB found in the cell?
2. Follow this link to the original IntAct network viewer (the one found at the 'Graph' tab isn't complete yet). This opens the interaction viewer. Search using the IntAct ID for PKB/AKT1, "akt1_human", and it will show interactions centered on PKB/AKT1.
2a. How many proteins are shown to interact directly with AKT1?
2b. The panel at the right show GO terms annotated to proteins that are shown in the interaction graph. This can be a useful way of visualizing common properties of this group of proteins. Click on the Show triangle for ATP binding GO terms. Which of the proteins interacting with AKT1 are also ATP binding proteins (of any type)?
2c. Now pull up the interaction graph for one of AKT1's interaction partners, human GSK3B. You can do this by clicking on GSK3B in the AKT1 graph. If the new graph comes up blank try right-clicking it and selecting 'This frame'->'Reload'. With what other AKT family member has GSK3B been shown to interact?
2d. Clicking on 'Expand' at the left of the interaction graph shows an additional generation of interactions, those for each of the proteins in the current graph. Clicking can also be set to expand one leaf of the current graph. The interaction graph gets busy very quick, but this can be a useful way to look at the interaction network and find indirect links. What proteins are both AKT1 and AKT2 know to interact with?
3. Go back to your original search for PKB and now select mouse Akt1, "akt1_mouse". You will notice that mouse Akt1 has some Interactors that have not been reported for human AKT1. There are two common ways to combine results from two species. If you were studying AKT1 it would be useful to examine known interactions for all of the AKT1 orthologs. A wide search would include all reported interactions across species. The other way to combine the information is to use it to find additional conformation. An interaction between a pair of proteins with multiple citations and interactions identified in multiple species would provide confidence that an interaction is real and conserved. Which of the human AKT1 interactions have also been reported in mouse? Provide a list of the gene names. Example: FOXO1,GAPDH,NAD3.
4. Give the citation for IntAct.
BIO520