Dissecting the molecular function of TECPR2

• Applicant: Professor Dr. Christian Behrends, Ph.D.
• Subject Area: Cell Biology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 437166575

Hereditary sensory and autonomic neuropathy 9 (HSAN9) is a rare fatal neurological disease caused by mis- and nonsense mutations in the gene encoding for Tectonin β-propeller repeat containing protein 2 (TECPR2). While we previously found that TECPR2 is required for lysosomal consumption of autophagosomes and ER-to-Golgi transport, it remains elusive how exactly TECPR2 is involved in autophagy and secretion and what downstream sequels arise from defective TECPR2 due to its involvement in these processes. In the past funding period, we addressed these questions by determining which proteins depend on TECPR2 for their trafficking out of the ER and sorting within the cell. Hence, we employed a series of spatial proteomic approaches to systematically examine defects along the secretory pathway in an HSAN9-mimicking cell model. This global analysis unveiled profound alterations at distinct trafficking steps including ER exit site formation, COPII-mediated transport to the Golgi, sorting to the lysosome or plasma membrane and secretion into the extracellular space. Intriguingly, a number of proteins affected at these compartments are indeed potentially relevant for HSAN9 pathogenesis. In addition, we identified a number of ER- and Golgi-associated TECPR2 binding partners whose interactions are lost when TECPR2’s C-terminus is missing due to pathogenic mutations, thus supporting a loss-of-function mechanism for HSAN9. However, so far, mechanistic insights into how TECPR2 coordinates its association with components of the different trafficking and sorting machineries remained unclear. Hence, key elements which might help to understand TECPR2’s molecular function are not fully understood. In the next funding period, we therefore wish to focus on the identification and characterisation of TECPR2 binding sites that mediate critical interactions with partner proteins and cellular membranes and that thereby coordinate the involvement of TECPR2 in trafficking and protein sorting. To do so, we will combine structure prediction guided mutagenesis and biochemical assays with reconstitution experiments in knockout cells, interaction and proximity proteomics as well as confocal microscopy. Moreover, we will clarify if and how TECPR2 is regulated by ATG8 family proteins and the newly identified TECPR2 binding partner casein kinase 2. Lastly, after having examined the relevance of scaffolding and regulatory modules of TECPR2 in model cells with experimental perturbation, we will validate our findings regarding TECPR2’s function in patho-physiologically relevant cells. Thus, the expected results will yield mechanistic insights into the biochemical and cellular role of TECPR2 and deepen our understanding of the molecular consequences of TECPR2 deficiency. Both aspects have the potential to contribute to rationalizing strategies to rescue or bypass TECPR2 malfunction which is urgently needed to develop treatment options for HSAN9 patients.

• DFG Programme: Research Grants