As mentioned in our previous report, Manizheh Izadi (the PhD student who is developing this project) went to the Rockefeller University during 2015 and spent 5 months in Prof. Robert Roeder’s laboratory. There, she was able to express and purify different versions of PGC-1alpha4 (GST- and Flag-fusions) using bacteria and insect cell systems. The purified fusion proteins were used as baits in protein complex purification experiments using nuclear extracts from HeLa cells. After protein complex purification, the isolated proteins were submitted for identification by mass spectrometry to our collaborators at Harvard Medical School. This approach was extremely successful in identifying a variety of nuclear proteins that interact with PGC-1alpha4 in HeLa cells. Among which are transcription factors, other coactivators, and splicing factors. We are in the process of validating these interactions through co-immunoprecipitation experiments followed by immunoblotting using specific antibodies for the different interactors. We will follow up on select proteins to understand how they regulate PGC-1alpha4 function. Part of these results have been submitted for publication to the Journal of Biological Chemistry (paper currently under revision).
We have used the same purified PGC-1alpha4 proteins to identify interactors in mouse skeletal muscle extracts. However, there were significant difficulties in obtaining high quality nuclear extracts from mouse muscle tissue. We did perform the protein complex purification, followed by identification by mass spectrometry. Unfortunately, these experiments were less successful than the described above. Mainly due to high abundance of muscle structural proteins in the extracts. We are currently trying to optimize the extract preparation.
Additional news from the lab:
Last November Manizheh Izadi defended her PhD halftime control with great success and was complimented on the significant amount of work developed so far.
I am happy to report that I have completed my “Docentur” and am now Associate Professor of Molecular Physiology.