Final Report Summary - QUASI (Quantifying signal transduction)
The QUASI project aimed to assess the dynamic and quantitative operation of the signalling transduction pathways, hence the need to measure and quantify all steps, if possible, in signalling pathways considering the amplitude and period of the pathway activity, the spatial distribution and subcellular movements of signalling components as well as their assembly into larger complexes that insulate signalling and control cross-talk between pathways. By optimising and applying various tools and methods QUASI has been able to generate such data which constitute the crucial parameters in order to correctly target signalling pathways with current existing drugs or with the design of new ones. In order to achieve the actual time line of signalling events, it was necessary, whenever possible, to monitor the processes in individual living cells rather than in a whole cell population. The project investigated especially the High osmolarity glycerol (HOG) as well as the mating pheromone response signalling pathways.
The project achieved the following objectives:
- monitoring activated protein kinases in the cell by adopting two approaches, namely generating a range of immunoreagents to quantify key phosphorylation events as well as a method based on stable isotope labelling and mass spectroscopy;
- developing suitable fluorescent reporters and bio-imaging tools in order to determine dynamic signalling events in real-time in single living cells;
- enhancing the tools for monitoring transient protein interaction and protein complex formation in the living cell, namely by chemical cross-linking followed by mass spectrometry as well as by microscopy;
- designing novel Adenosine triphosphate (ATP) analogues and protein kinase mutations which are sensitive to specific inhibitory compounds in order to specifically, rapidly and temporarily inhibit signalling components in the living cells;
- identifying direct protein kinase targets and quantify kinase-substrate reactions. The right combination of kinase such as HOG1 mutant variant and ATP analogue would result in the phosphorylation and labelling of only the immediate protein kinase targets;
- establishing mathematical models that represent how the pheromone and HOG signalling pathways as are activated, a model that predicts cross-talk features and provides information regarding the glycerol accumulation and links to cell cycle regulation;
- generating visualisation tools, namely animations of pheromone and HOG pathways.
The project achieved the following objectives:
- monitoring activated protein kinases in the cell by adopting two approaches, namely generating a range of immunoreagents to quantify key phosphorylation events as well as a method based on stable isotope labelling and mass spectroscopy;
- developing suitable fluorescent reporters and bio-imaging tools in order to determine dynamic signalling events in real-time in single living cells;
- enhancing the tools for monitoring transient protein interaction and protein complex formation in the living cell, namely by chemical cross-linking followed by mass spectrometry as well as by microscopy;
- designing novel Adenosine triphosphate (ATP) analogues and protein kinase mutations which are sensitive to specific inhibitory compounds in order to specifically, rapidly and temporarily inhibit signalling components in the living cells;
- identifying direct protein kinase targets and quantify kinase-substrate reactions. The right combination of kinase such as HOG1 mutant variant and ATP analogue would result in the phosphorylation and labelling of only the immediate protein kinase targets;
- establishing mathematical models that represent how the pheromone and HOG signalling pathways as are activated, a model that predicts cross-talk features and provides information regarding the glycerol accumulation and links to cell cycle regulation;
- generating visualisation tools, namely animations of pheromone and HOG pathways.