DC 4: New approaches to screen for novel direct mTORC1 targets

Project part of Work Package 1

Objectives: Several mTORC1 substrates with functions in anabolic and catabolic processes have been uncovered but their contribution and interplay in mediating mTORC1’s pathophysiological outputs is poorly understood, and we likely still miss a plethora of targets involved in these processes. State of the art shotgun phosphoproteomic approaches do not provide the full picture as they do not allow to distinguish whether phosphorylations are mediated by the kinase-of-interest itself or by an unknown downstream effector kinase. We have developed an on-column in vitro kinase assay (OBIKA) that allows the unbiased screen of complex, proteome-scale samples for direct kinase targets. The PhD candidate will combine OBIKA with in vivo phosphoproteomics to comprehensively identify mTORC1 substrates. Kinases directly interact with their substrates to perform phosphorylation reactions. Proximity labelling based on the miniTurbo-approach will allow to investigate stimulus-dependent protein neighbourhoods of mTORC1. To study the relevance of newly identified proteins and phosphosites in metabolic regulation, the doctoral candidate will CRISPR or overexpress up to 10 candidates and analyse their effects on mTORC1-driven cell growth, proliferation and metabolism. Inactive Ala and phospho-mimicking Asp/Glu variants will allow to study the relevance of single phosphosites. The potential of new molecular targets to serve as drug targets will be evaluated jointly with medical chemistry labs in the MENTOR.

Expected Results: The PhD candidate will identify new direct mTORC1 target sites that control cell growth and proliferation and evaluate their suitability for pharmacological intervention.

Prospects of career development: Over the past 15 years, alumni from the Dengjel lab became group leaders in academia, project leaders in pharma companies and technological companies, and clinical research associates. Due to detailed knowledge in mass spectrometry-based proteomics the job opportunities for the doctoral candidates in companies and academia is large. It will be further expanded by the multidisciplinary nature of the Research and Educational program, leading to potential recruitments in new fields (biomarkers analysis, cellular models, drug discovery and development) in both academia and industries.

Planned secondments: 3 months at UKESSEN for metabolome analysis of cells with phosphosite variants of novel mTORC1 targets. 3 months at Topadur for analysis of mTOR inhibitor.