Folding and assembly pathway of the auto-inhibited dimeric state of TAp63α

• Applicants: Professor Dr. Volker Dötsch; Dr. Günter Kramer
• Subject Area: Biochemistry ; Biophysics
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 564271668

Surveillance of genetic quality in oocytes is the task of p63, a member of the p53 family. The longest p63 isoform, TAp63α, is highly expressed in oocytes during the dictyate arrest phase which in humans lasts up to 50 years. We have shown that during this dictyate arrest phase TAp63α is kept in a closed, inactive and only dimeric conformation. Detection of DNA double-strand breaks results in the activation of a kinase cascade involving the two kinases CHK2 and CK1 which results in the opening of the closed state and the formation of the open and tetrameric conformation. The tetrameric form is capable of high affinity binding to DNA which results in the upregulation of a pro-apoptotic transcriptional program and death of the damaged oocyte. As DNA damage is also triggered by chemotherapeutics this mechanism leads to premature ovarian insufficiency (POI) in female cancer patients. We have further shown that once the tetrameric state is formed, dephosphorylation does not convert the active tetrameric state back into the inactive dimeric state, as the tetrameric state is the thermodynamically more stable one. The inactive dimeric state thus constitutes a high energy, kinetically trapped conformation and the activation process is irreversible. These data are supported by our results showing that infertility in female patients can be caused by mutations that render TAp63a constitutively tetrameric. All these data predict that the inactive dimeric state most likely forms co-translationally to prevent that active tetramer would accumulate and kill the oocyte. We have started to investigate this proposed co-translational folding mechanism and have shown by disome-selective profiling that di-ribosomes are formed when TAp63a gets expresseed both in vivo (H1299 celles) as well as in vitro (rabbit reticulocyte lysate). Disome formation starts after the first 90 residues have been synthesized which correlates well with the predicted appearance of a b-strand of the proposed inhibitory b-sheet that keeps TAp63a in the dimeric inactive conformation. Here we propose to investigate the initial steps of this co-translational folding mechanism further. We will further characterize nascent chain segments that are required to form disomes by selective disome profiling using different p63 mutants. We will also identify mRNA and nascent protein features that result in slower translation to allow the already translated domains to fold and for the nascent chains to interact with other nascent chains and investigate the involvement of chaperones in the folding process. Finally, we will use peptide interaction studies to directly investigate the interaction between peptide segments identified in the selective disome profiling as crucial for the co-translational folding process.

• DFG Programme: Research Grants