Chemical ligation tools to access modified homeodomain proteins[پايان نامه فارسي]

Mahdi Samapour

Cell membrane crossing remains a major challenge for drug delivery. A strategy to deliver bioactive cargoes into cells is to attach them to cell-penetrating peptides (CPPs). A main class of CPPs derives from the third helix of the homeodomain (HD) of homeoproteins (HPs). HPs constitute a transcription factor family able to cross cell membranes (a property that is key to their function) and are involved in crucial processes such as neuronal communication, survival, and differentiation. HD is the 60-residue domain of the homeoprotein responsible for DNA binding and it contains the cell penetration motif. The mechanism of entry of CPPs and HPs is still not fully understood. As Engrailed-2 (En2) HP is currently considered a potential therapeutic protein in Parkinson’s disease, the long-term goal of this project is to incorporate modified residues into CPPs derived from En2 HP and into the En2 HP in order to I) study the impact of this incorporation on cell entry to better understand the mechanism and II) design a new generation of CPPs and HPs with enhanced potency. More precisely, as tryptophan is a key residue for cell entry of HPs and derived CPPs, the strategy will be to replace Trp with non-natural analogs to modulate hydrophobic and ion pair π interactions with membrane partners that are involved in an entry inside cells. After identifying an analog leading to a more efficient CPP, this residue will be introduced into the En-2 HD sequence to observe if enhancement of membrane-translocating properties is conserved at the protein scale. The aim of this master project was to develop all the tools for accessing chemically modified HD or HP proteins by chemical ligation combining expressed and synthetic fragments. For this, we first worked on model peptides and then on an En2 HD derivative.