Koordination der Zellteilung und Differenzierung durch Ubiquitylierung - Funktion und Regulation von E3 Ubiquitin Ligasen ausserhalb des Zellzyklus

• Antragsteller: Dr. Jörg Mansfeld
• Fachliche Zuordnung: Zellbiologie
• Förderung: Förderung von 2012 bis 2018
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 225822023

The faithful coordination of proliferation and differentiation is fundamental for multicellular life and development. The decision whether cells keep dividing or transitionally or permanently exit from the cell cycle is tightly controlled by the ubiquitin E3 ligases of the cycle machinery. Ubiquitin E3 ligases covalently attach ubiquitin molecules to their substrate, which can target them for degradation by the 26S proteasome. A key ubiquitin E3 ligase involved in cell cycle control is the anaphase-promoting complex/ cyclosome (APC/C). Even though the APC/C has been intensively studied for its role in mitosis, emerging evidence indicates that the APC/C and further ubiquitin E3 ligases fulfil additional roles in quiescent cells and during differentiation including neurogenesis and myogenesis. These surprising functions of key cell cycle proteins have only been recently been discovered and we only have a limited understanding how E3 ligase activity and specificity is regulated beyond the cell cycle.I have previously established a quantitative mass spectrometry approach that allows comparing the APC/C and its interactions at specific stages of the cell cycle but can also be applied to cells at different stages of differentiation. Using this approach I will investigate how APC/C activity and specificity is regulated at the transition into quiescence and in differentiated cells using retinal pigmental epithelial cells (RPE1) and myogenesis as models. To functionally characterise identified interactors of the APC/C and to measure APC/C activity in living cells we have developed a method to rapidly ‘knock in’ fluorescent probes into the endogenous loci of genes, thereby avoiding potential artefacts caused by overexpression. Subsequent in vitro ubiquitylation assays relying on purified components will enable us to elucidate the underlying molecular mechanisms that regulate the activity and specificity of the APC/C.To understand how E3 ligases control cellular differentiation we have first to identify their crucial sub-strates at specific times during the differentiation program. Focusing on myogenesis as a differentia-tion model I will identify substrates of the APC/C and further Cullin-RING ligases such as SCF(MAFbx) on a global level to understand how the transition from the proliferating myoblast to differentiated myotubes is executed. To this end I propose a novel technique that I have developed to identify substrates including the ubiquitylated residue of specific E3 ligases in a single experiment. This proposal aims to gain a detailed knowledge of the molecular mechanisms that define the activity and specificity of E3 ligases during cell cycle exit and differentiation to facilitate the development of therapeutic strategies for ubiquitylation-related diseases including cancer and muscle atrophy.

• DFG-Verfahren: Emmy Noether-Nachwuchsgruppen