Bacillus Thuringiensis Parasporin-5 Expression In Eukaryotic Cells And Its Antitumor Molecular Mechanism Research

B.thuringiensis(Bt) is a ubiquitous Gram-positive bacterium,forming parasporal crystal in the late stage of spore formation. The spore crystal proteins have insecticidal or antitumor activity, so-called insecticidal crystal proteins(ICPs) or anticancer crystal protein(Parasporin or PS). To date, Bt has been a research subject of more than a century, and its insecticidal crystal protein has been widely used in various biological controls. However, PS proteins were only reported in the last decade, due to its unique chemical structure, broad spectrum of antitumor activity and low level of cytotoxicity, PS proteins have been attracted lots of attention.The molecular mechanism underlying the antitumor activity of PS proteins are not clear at present.In this study, we employed mammalian cell expression system to probe the antitumor activity of PS5, by passing the cell entry step. We synthesized codon-optimized PS5 gene and constructed expression plasmid for its expression in tumor cell lines. When cells were transfected with plasmids carrying PS5, we found that PS5 expression potently induced vesicle formation, promoted apoptosis and significantly inhibited cell growth. Biochemical analysis showed that PS5 expression is sufficient to induce hallmark events of apoptosis,including DNA fragmentation and PARP1 cleavage. Fluorescence microscopy indicated that PS5 primarily resided in the nucleus, with a minor portion localizing in the cytosol.To define the molecular action by which PS5 induces cell death,we performed Immuno precipitation and analyzed PS5-interacting proteins by tandem mass spectrometry. This analysis identified multiple cellular proteins that are implicated in vesicle formation and trafficking, including ESYT1, KIF1 A, DNTC1, AFAD and RHG05.While the roles of these cellular proteins call further investigation, my work suggests that PS5 targets multiple cellular components of the vesicle system to induce cell death. In conclusion, this study provides a new direction and experimental system to investigate the molecular mechanism of PS protein in killing tumor cells that may be useful for targeted antitumor therapy.