CHEK2 gene codes for serin/threonine kinase Chk2 (Checkpoint kinase 2). In response to genomic DNA damage, Chk2 phosphorylates its substrates (proteins Cdc25C, BRCA1 or p53), whose activation leads either to cell cycle arrest, DNA damage repair or induction of apoptosis. Germline mutations in CHEK2 gene increase risk of cancer development. Analysis of high risk breast cancer patients in Czech Republic reveals rare CHEK2 mutations (mainly missense) with yet unknown clinical significance. This work focuses on functional impact of these variants and analysis of kinase activity of variant isoforms of Chk2 kinase. For this purpose, recombinant constructs were expressed in bacterial cells of E. coli. Enzymatic activity of Chk2 kinase isoforms in crude cell lysates was measured by the phosphorylation of Chk2 arteficial substrate spectrophotometrically. Results of in vitro kinase assay were correlated to the results of in silico prediction software. The results show that from 15 analyzed mutations (together with one in frame deletion), kinase activity was abrogated in all variants affecting the kinase domain of Chk2, in concordance with in silico predictions. The same result has been found for a FHA domain variant p.R145Q. No significant changes in kinase activity were observed in case of two FHA domain variants...

ABCB11/BSEP est le transporteur des acides biliaires, localisé au niveau du pôle canaliculaire des hépatocytes. Les mutations de ce gène sont responsables de la cholestase familiale intrahépatique progressive de type 2.Au cours de ma thèse, j’ai évalué la capacité des aminoglycosides et du PTC124 à induire la translecture de codons stop prématurés, l’adressage et la fonction de mutants non-sens et faux sens de Bsep ainsi que l’effet d’une bithérapie (translecture+chaperone).Dans nos modèles cellulaires, la gentamicine était capable d’induire la translecture du codon-stop prématuré du mutant non-sens BsepR1090X dans les lignées NIH3T3, HEK293 et Can 10. La protéine entière générée était partiellement détectée aux membranes plasmiques des cellules HEK293 et canaliculaires des cellules Can 10 et était partiellement fonctionnelle puisqu’elle était responsable d’une augmentation de l’activité de transport de 3H-taurocholate (3H-TC) dans les clones MDCK. Ces effets étaient potentialisés par l’addition de drogues chaperones telles que le 4-phenylbutyrate (4-PB).J’ai également mis en évidence la capacité de nouveaux composés dérivés du 4-PB (MHMPB, OTNC et HMPB) à corriger l’adressage et à augmenter le transport de 3H-TC du mutant faux sens BsepR1128C à des concentrations plus faibles que le 4-PB. Enfin, j’ai pu montrer que d'autres drogues chaperones (GPB, PA, SAHA et C18), pouvaient corriger l’adressage canaliculaire de BsepR1128C et augmenter son activité de transport de 3H-TC dans les clones MDCK. / ABCB11/BSEP is the main bile acids transporter located at the canalicular pole of hepatocytes. Mutations of ABCB11 are responsible for progressive familial intrahepatic cholestasis type 2.During my phD, I evaluated the ability of aminoglycosides and PTC124 to induce readthrough of premature termination codons, targeting and function of nonsense and missense mutants of Bsep and also the effect of combined therapy (readthrough + chaperone).In our expermental models, gentamicin increased readthrough of p.R1090X mutation NIH3T3, HEK293 and Can 10 lines. The resulting full-length protein was detected at the plasma membrane of HEK293 and at the canalicular membrane of Can 10 cells; and was partially functional since it was responsible for increasing the transport activity of 3H-taurocholate (3H-TC) in MDCK clones. These effects were potentiated by the addition of chaperone drugs such as 4-phenylbutyrate (4-PB).I have also demonstrated the ability of new 4-PB derived compounds (MHMPB, OTNC and HMPB) to correct mistrafficking and to increase 3H-TC transport of BsepR1128C missense mutant at lower concentrations than 4-PB. Finally, I showed that other chaperone drugs (GPB, PA, SAHA, and C18) were able to correct mistrafiking of BsepR1128C and to increase its 3H-TC transport activity in MDCK clones.

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