Thesis presented March 24, 2023

Abstract:
Prokineticin-1 (PROK1) protein, also known as EG-VEGF (Endocrine gland derived-vascular endothelial growth factor) belongs to the prokineticin (PROK) family, whose members are directly involved in the control of key processes such as inflammation and angiogenesis. PROK1 acts via two GPCR receptors, PROKR1 and PROKR2 and regulates several physiological and pathological processes, including cancer and cystic fibrosis (CF). My research team recently demonstrated that the expression of PROK1 and PROKR2 were increased in the female reproductive cancer, gestational choriocarcinoma (GC) and in CF. The antagonization of PROKR2 in an animal model of GC decreased tumor growth and its progression, suggesting that inhibition of the PROK1/PROKR2 system may constitute a promising therapeutic target for all PROK1-dependent cancers and pro-inflammatory diseases. However, anti-PROKR2 antagonists are not specific for this receptor, exhibit long-term toxicity and may be associated with the development of side effects as they block a shared receptor with other members of the PROKs family.
The objectives of my thesis project aimed at i) developing a new strategy that targets the inhibition of the ligand, PROK1, and ii) characterizing the molecular and cellular features that differentiate PROKR1 from PROKR2 in order to propose new therapeutic strategies that will specifically target one or the other receptor.
The development of the PROK1 blocking strategy required the cloning of the human Prok1 sequence in different plasmids, the implementation of several eukaryotic or prokaryotic expression and production systems, and the development of numerous purification techniques in order to resolve its three-dimensional structure (STD). In silico analyses were also performed to accelerate the STD resolution of PROK1, to characterize the sites of its interaction with PROKR2, and to perform molecular docking in order to identify chemical molecules able to inhibit the PROK1-PROKR2 interaction. A functional assay was also developed to test in vitro the most promising molecules. The functional test required the development of two stable cell lines expressing PROKR1 or PROKR2.
The cellular and molecular characterization of PROKR1 and PROKR2 revealed post-translational differences regarding the receptor’s half-life, its stability, trafficking and folding levels. Based on these observations, we initiate the investigation of the mechanisms behind these differences, firstly by targeting the roles of N- and C-terminus in these processes, particularly in the dimerizations of PROKR1and PROKR2.
Altogether my thesis project allowed the production of a recombinant PROK1 protein, the resolution of its structure and the identification of the amino acids that are involved in its interaction with PROKR2. The development of cell lines expressing PROKR1 or PROKR2 allowed, i) the set-up of a functional test of the activity of prokineticins, ii) the identification of three inhibiting molecules of the PROK1 ligand and iii) the demonstration that, in spite the existence of a huge identity between PROKR1 and PROKR2, PROKR1 would be more stable at the plasma membrane; undergo less degradation at the lysosome level and its C-terminal part may confer the dimerization between PROKRs receptors.
In conclusion, my thesis project established new molecular tools for the study of the prokineticins system; identified new inhibiting molecules of the PROK1-PROKR2 interaction, and compared for the first time PROKRs cellular and biochemical features. All these data will allow better targeting of PROKRs and their ligands in order to propose more specific pharmacotherapies for PROK-dependent cancers and inflammatory diseases, such as preeclampsia and cystic fibrosis.

Keywords:
Placenta, EG-VEGF, PROKR2, PROKR1, PROK1, choriocarcinoma

On-line thesis. ​