Project number 28

High-resolution biochemical and microscopic analysis of the necrosome

Necrosis was originally considered as an accidental type of cell death but recent discoveries revealed that there are multiple types of necrotic cell death (Vanden Berghe et al. NRMCB 2014). Many of them have underlying and targetable pathways, such as pyroptosis (capase-11, gasdermin D) and necroptosis (see below). Both cell death modalities are associated with cytokine production and release Damage Associated Molecular Patterns (DAMPs), which modulate the immune response.  Receptor interacting protein kinase 1 and 3 (RIPK1, RIPK3) and Mixed Lineage Kinase Like (MLKL) are key players in necrosome assembly, an essential protein complex in the onset of necroptosis. However, the exact interplay of RIPK1 and RIPK3 activities in necrosome assembly remain controversial. Together with the complex nature of RIPK function, this suggests different necrosome “flavors”, basically calling for a deeper understanding of the process. The identification of additional regulatory and effector molecules remains therefore an essential objective.
We propose to investigate the precise composition of the necrosome and to study functions attributed to both RIPK1, RIPK3 and MLKL in space and time in TNF-induced necroptosis using our recent cellular biosensor models.
This project will benefit from advanced microscopy techniques (e.g. FLiM-FRET,and CLEM) to gain high spatial-temporal resolution imaging of the necrosome at the single cell level. We will further use new proteomics approaches (e.g. APEX2 and Virotrap) to characterize necrosome composition with unrivaled precision.  Functional analysis of novel components will be pursued for the endogenous necrosome by genome editing and the advanced microscopy modalities mentioned above.
All the biochemical studies including proteomics, as well as genome editing experiments will be performed under the supervision of Sven Eyckerman. The microscopy aspects in the project will be guided by Franck Riquet in the group of Peter Vandenabeele.

Keywords
necroptosis, protein complex, microscopy, proteomics

Supervisors
Peter Vandenabeele, VIB Inflammation Research Center, UGent, Gent
Sven Eyckerman, VIB Medical Biotechnology Center, UGent, Gent