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Sticholysins from the sea anemone Stichodactyla Helianthus: interaction with nucleic acids for biotechnology application

Grant number: 16/01379-1
Support type:Research Grants - Visiting Researcher Grant - International
Duration: June 07, 2016 - August 06, 2016
Field of knowledge:Biological Sciences - Biophysics
Principal Investigator:Rosangela Itri
Grantee:Rosangela Itri
Visiting researcher: María Eliana Lanio Ruiz
Visiting researcher institution: Universidad de La Habana (UH), Cuba
Home Institution: Instituto de Física (IF). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Sticholysins I and II (Sts/StI and StII) are pore-forming proteins (PFP), belonging to the protein family actinoporins, produced by the sea anemone Stichodactyla helianthus. Actinoporins are secreted in soluble form and incorporated into membranes where they form the oligomeric transmembrane pores with a radius of 1 nm, leading to cell lysis. They are characterized by having a high Ip (> 9.0), a molecular mass of about 20 kDa and contain no cysteine residues. Sts exhibit high similarity and identity in their amino acids sequences, however St II is more active than St I. The tridimensional structure of Sts is characterized by a compact ² sandwich of 10 ² strands flanked on the opposite sides by two alfa-helices, one of them located at the N-terminus.The N-terminal sequence seems to be the best candidate for pore formation and appears to play a determining role in the difference of activity among them. Due to the high Ip of Sts they exhibit an expanded positively charged surface at neutral pH. Considering this characteristic and their permabilizing activity, Sts could behave as nucleic acids-condensing polycationic agents able to mediate their endosomal escape in order to express or repress target-gene expression in cells. In fact, results obtained by the group of profa. Lanio (University of Havana, Cuba) suggest this possibility: St II is able to interact with DNA forming DNA-StII complexes at different N/P ratios exhibiting difference with those formed by a classical polycation polyethylenimine (PEI). DNA-St II complexes retained the hemolytic activity of the protein (data not published). These results supported the idea that Sts could work as a DNA delivery system into the cells. The structural and thermodynamic characterizations underlying the formation of the complexes DNA-St II are very important and necessary. In this regard, the scientific visit of profa. Lanio at USP to work in close collaboration with profa. Itri from IF-USP and prof. Ciancaglini from Chemistry Department of FFCLRP-USP has the main aim to investigate the main structural properties of the complexes formed by DNA-St II by Small Angle X-Ray Scattering (SAXS) at IFUSP coupled to the binding parameters between DNA and St II evaluated by Isotermic Titulation Calorimetry (ITC) at FFCLRP-USP. Further, once the structural features of the complexes are determined, we also intend to evaluate the permeabilizing activity of St II free and associated to DNA using giant unilamellar vesicles (GUVs) as model membranes observed by optical microscopy (IFUSP). The interaction between St II and its DNA complex with membranes is part of the PhD project from Raffaela de Rosa (FAPESP fellow, under supervision of profa. Itri). (AU)