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ERC

RUBINET Report Summary

Project ID: 268930
Funded under: FP7-IDEAS-ERC
Country: Switzerland

Final Report Summary - RUBINET (Regulation of cell growth and division by selective degradation mechanisms)

Regulation of cell growth and division by selective degradation mechanisms

Cell growth and division are tightly regulated by phosphorylation and selective degradation of cellular components. Whereas autophagy is mainly responsible to eliminate long-lived proteins and organelles, the ubiquitin-proteasome system (UPS) rapidly degrades and/or regulates proteins when fast adaptation is needed. Defects in these degradation systems account for numerous human diseases, including cancer, muscle dystrophies, viral and bacterial infections and neurodegeneration. While rapid progress has been made in the identification of UPS and autophagy components, little is known about their regulation and the molecular mechanisms that ensure substrate specificity. In this interdisciplinary project, we have successfully used microscopy-based RNAi screening to identify novel substrate adaptors of cullin/RING-based E3 ubiquitin ligases (CRLs), in particular CRL3 and CRL4 adaptors required to maintain genome stability by regulating faithful DNA-replication and/or chromosome segregation. In addition, we have identified and characterized novel CRL3 adaptors involved in cell migration and intracellular trafficking, respectively. In parallel, we developed a quantitative, label-free SWATH-MS-based pipeline that allows to comprehensively identify ubiquitinylated proteins. Using this powerful method, we have not only identified physiological substrates but also linked them to specific CRLs. Moreover, we validated the functional importance and underlying mechanism of their ubiquitinylation in cells. With respect to CRL regulation, we examined the structural and kinetic parameters underlying CRL regulation by the CSN/Cop9-signalosome complex, which cleaves the Nedd8-modification from the cullin subunit, thereby inactivating CRL-activity. Interestingly, these studies revealed that an unexpected product eviction mechanism plays an important role in catalysis.
To complement these cell biological, biochemical and structural analysis, we have developed a microscopy-based platform coupled to computer-controlled microfluidic devices that allows quantifying dynamic cellular responses in single cells. Using this system, we investigated mechanisms that spatially and temporally regulate of MAP kinase pathways activated in response to stress conditions such as osmolarity changes or nutrient deprivation, or in response to yeast pheromones. Importantly, we discovered an unexpected stochastic sensing mechanism that drives oriented cell polarization in a chemical pheromone gradient, which provides molecular understanding for detecting and interpreting spatial signals in a wide range of systems. We also characterized a new ubiquitin-dependent mechanism restricting MAP-kinase signaling in response to yeast pheromones and identified components that may regulate autophagy and cargo selection during their packaging into autophagosomes. In particular, we focused on mechanisms governing the activation and downstream mechanisms of Atg1/ULK1 function.

Contact

Matthias Peter
Tel.: +41 44 633 65 86
E-mail
Record Number: 189309 / Last updated on: 2016-09-16