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TORCH Report Summary

Project ID: 614552
Funded under: FP7-IDEAS-ERC
Country: Switzerland

Mid-Term Report Summary - TORCH (TOR and Cellular Homeostasis)

The Target Of Rapamycin (TOR) proteins are ser/thr kinases conserved in Eukarya. We discovered that TOR proteins nucleate two distinct protein complexes, TORC1 and TORC2, and, subsequently, that these complexes are broadly conserved from yeast to man. Work from many groups has since demonstrated that TORC1 and TORC2 regulate many, widely varying, aspects of cell and organism physiology. Consequently, TOR inhibitors, such as rapamycin and derivatives, have found clinically use in the treatment of cancer, cardio-vasculature disease and to prevent organ rejection. However, these therapeutics are not the hoped for magic bullets necessitating further research into TOR signaling and the identification of novel nodes in these pathways that can be potentially targeted for therapeutic gain.
We continue to exploit the model eukaryote Saccharomyces cerevisiae (bakers’ yeast) to understand the structure and function of the signal transduction pathways in which the TOR complexes operate. Important to this effort is our recent appreciation that these kinases function in feedback loops (Eltschinger and Loewith, 2016). The implication of this is that downstream effectors of these kinases will in many cases also be upstream regulators as well. The identification of kinase effectors is relatively straight forward provided one possesses good tool compounds with which to acutely and specifically inhibit your kinase of interest, and ample access to state-of-the-art mass spectrometry facilities. We now have both. TORC1 is inhibited by rapamycin, however, this macrolide does not inhibit TORC2, for reasons that were mysterious. Using hybrid structure biology approaches (cryo-electron microscopy and cross-linking mass spectrometry) we reconstructed the structure of TORC2 to 26Å resolution (Gaubitz et al, 2015). From this structure we identified the TORC2-specific protein responsible for the resistance to rapamycin. An unbiased mutational analysis of the gene encoding this protein eventually provided us with an allele that retained function in TORC2 but simultaneously rendered the complex sensitive to rapamycin (Gaubitz et al 2015). Using this and other genetic tricks we can now independently inhibit either TORC1 or TORC2 with rapamycin. Sponsored by this ERC Consolidator grant an Oribtrap Fusion Mass Spectrometer was purchased. Importantly, this purchase is now supported by three skilled collaborators (an analytical chemist Post Doc, a mass-spec-experienced molecular biology post doc and a bioinformatics PhD student) that were subsequently attracted to the group. With this infrastructure, we can now follow, time-dependent changes in the yeast phosphoproteome following acute TORC1-, TORC2-, or TORC1- and TORC2- inhibition, and how this is affected by metabolic status of the cells. Mining of these data and subsequent biochemical follow-up experiments will follow.
In parallel to these efforts we have pursued structural investigation of TORC1. From these efforts we have discovered the mechanism by which carbon-derived signals affect activity of this complex. This exciting work will be submitted for review in a high impact journal in the coming days.

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