Biopharmaceutics

Biography

Biography: Giulio Maria Pasinetti

Abstract

Chromatin is the major regulator of gene expression and genome maintenance. Proteins that bind the nucleosome, the repetitive unit of chromatin, and the histone H4 tail are critical to establishing chromatin architecture and phenotypic outcomes. To modulate the chromatin structure, we designed a Nucleosome Binding Peptide – GMIP1- based on the crystal structure of RCC1 protein bound to a mononucleosome (PDB 3MVD). Molecular dynamic showed that GMIP1 tend to form an alpha helix, which suggests a more stable structure. For binding assays, we reconstituted mononucleosomes in vitro with 601 DNA -167pb- and histone octamer purified from chicken erythrocytes. The binding of fluorescent-labeled GMIP1 to a mononucleosome was observed by EMSA. To verify the GMIP1 effect in vivo, we performed MTT assays with different cell lines, such as Hela, Raw and skin fibroblast (CCD 1059). We observed a significant reduction of cells viability and proliferation with 50 uM of GMIP. In order to analyze the role of GMIP1 on the inflammatory pathway, we performed real-time PCR of TNF-alpha RNA extract from Raw cells. GMIP1 strongly reduced TNF-alpha expression and completely inhibited TNF-alpha stimulation by LPS, reducing fivefold the gene expression compared to non-treated cells. New experiments are on the way to check the peptide binding to nucleosome in cell based assays. Herein we present an exogenous Nucleosome binding molecule, a peptide, with intrinsic properties to modulate chromatin architecture and phenotypic outcome.