Interdisciplinary investigation of the human dipeptidyl peptidase III mutants R620C, R623W and R623L

Abstract

Ljudska dipeptidil–peptidaza III (hDPP III) uz katalitičku aktivnost cijepanja dipeptida s N–kraja oligopeptida ima ulogu regulacije Keap1–Nrf2 signalnog puta. Keap1 protein veže Nrf2, transkripcijski faktor ključan za antioksidativni odgovor stanice te ga usmjerava prema ubikvitinaciji. Interakcija između domene Kelch proteina Keap1 i faktora Nrf2 ostvaruje se preko ETGE motiva na Nrf2, a isti motiv sadrži hDPP III. Dokazano je da vezanje hDPP III za Keap1 sprječava deaktivaciju Nrf2 što rezultira pojačanom ekspresijom antioksidativnih gena. Nemogućnost deaktivacije faktora Nrf2 povećava otpornost prema antitumorskim terapijama. Na internetskom portalu za genomiku raka cBioPortal nalaze se genomske sekvence mutanata hDPP III pronađene u stanicama raka. U ovom radu, istraživan je utjecaj tri supstitucije iz navedene baze, R620C, R623L i R623W na peptidaznu aktivnost i interakciju hDPP III s proteinom Keap1. U istraživanjima su korištene biokemijske i biofizičke metode, te molekulsko dinamičke simulacije divljeg tipa proteina i mutanta R623W.Besides the catalytic activity of cleaving dipeptides from the N-terminus of oligopeptides, human dipeptidyl peptidase III (hDPP III) has a role in regulation of the Keap1-Nrf2 signaling pathway which is a part of the cell's response to oxidative stress. Human DPP III competes with Nrf2 for binding to Keap1 through its ETGE motif. Keap1 directs Nrf2 to ubiqutination but binding of hDPP III to Keap1 hinder Nrf2 deactivation resulting in higher expression of antioxidative genes. A consequence of Nrf2 activation in tumor cells is the increased resistance to antitumor therapies. On the portal for cancer genomics (cBio Portal) genome sequences of the following hDPPP III mutants were found and chosen for further investigation: R620C, R623L and R623W. The influence of these mutations on both the peptidase activity and the interaction with Keap1 was investigated by biochemical and biophysical methods, as well as molecular dynamics simulations