Optimization of ZEBRA protein as an innovative delivery system for therapeutic molecules

In recent years, the understanding of disease molecular mechanisms has led to the identification of genes and proteins that are altered in disease state and many therapeutic targets have been found located within cells. The protective and hydrophobic nature of plasma membrane prevents therapeutic drugs from entering cells. Cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) have emerged as a group of non-invasive delivery vectors for various hydrophilic macromolecules, and several in vitro and in vivo applications as pharmaceutical carriers have been reported. A novel cell-penetrating peptide deriving from the Epstein-Barr virus ZEBRA transcription factor has been recently characterized in our laboratory. A reductionist study of full-length ZEBRA protein has allowed to identify the amino acid region (named as Minimal Domain, MD) implicated in cellular uptake. This peptide is able to cross the mammalian cell membranes via a direct translocation mechanism even when fused to cargo molecules such as eGFP reporter protein. The direct penetration mechanism represents a great advantage for therapeutic applications as the cargo molecules can be directly delivered into cells cytoplasm in a biological active form. The aim of this thesis is to explore the cell-penetrating properties of the MD peptide and evaluate its applications as therapeutic protein delivery system. This work is structured in three parts.The first part describes the study on the optimization of MD peptide sequence by size-reduction and the evaluation of its amino acid composition role in the translocation process across the cell membrane. This study has led to the identification of a shorter MD sequence (MD11) with unvaried mechanism of translocation. The second section describes a MD11-based therapeutic approach aiming at repair a dysfunction of the protein synthesis identified in most cancers. The regulation of the protein synthesis has a crucial role in governing the eukaryotic cell growth and subtle defects in the translational machinery can alter the cellular physiology and lead to cell malignancy. Among the different factors intervening in the regulation of this process, the eukaryotic initiation factor 3 (eIF3) contributes to oncogenesis and maintenance of the cancer state. This complex is composed of 13 subunits (designated eIF3 a-m). The expression of eIF3 subunits is altered in several cancers, and in particular the f subunit (eIF3f) is significantly down-regulated in pancreas, vulva, breast, melanoma, ovary and small intestine tumors. The eIF3f ectopic expression by transient gene transfection inhibits cellular protein synthesis and induces apoptosis in melanoma and pancreatic cancer cells. Starting from these observations, we developed an innovative therapeutic approach for cancer treatment in which the missing eIF3f protein is produced in vitro in fusion to MD11, and delivered to cells. These results have demonstrated that the MD11- based eIF3f transfer system may represent a powerful strategy to suppress the tumor-cell proliferation. The last part of this thesis explores the cell-penetrating property of MD11 in yeast cells, and in particular in the pathogenic fungus Candida albicans. The presented results demonstrate the versatility of MD11, functioning as vectors in both yeast and mammalian cells and as carrier for proteins with biological activity.The MD11 potential as protein delivery system is evident; however some improvements regarding the fusion protein formulation and in vivo studies should be realized to validate the effectiveness of its therapeutic application.