Pore Forming Toxins with potential applications for Biomedicine and Nanobiotechnology
The study of two pore-forming proteins, sticholysins I and II (StI/II, Sts) produced by the Caribbean Sea anemone Stichodactyla helianthus (Lanio et al, 2001:187) has been the major focus of many researchers during the last years. They comprise a group of highly hemolytic cysteine-less proteins, with molecular weights around 20 kDa, high isoelectric points (>9.5), and a preference for sphingomyelin-containing membranes. One of the most remarkable features of these soluble proteins is that they can readily bind to cells and model membranes such as lipidic monolayers and vesicles, forming pores of constant size (Martínez et al, 2001:1547; Tejuca et al, 2001:125). The formation of these transmembrane pores disrupts ion gradients, leading to osmotic swelling and ultimately to cell death. In fact, they are extremely cytotoxic and lytic to a variety of cells and their vesicular organelles, which determine their potential applications for biomedicine. One of these applications is related with the construction of immunotoxins for selective killing of undesired cells, such as parasites (Tejuca et al, 1999: 489) or tumour cells (Tejuca et al, 2004:731, Tejuca et al, 2009:15). Immunotoxins (ITs) are chimeric molecules in which a cell binding ligand, such as a monoclonal antibody (mAb) or a growth factor are coupled to a killer toxin in order to address its activity towards a specific undesired cell. The most common toxic moieties used in the construction of ITs are those able to inhibit protein synthesis. However, these toxins require internalization and translocation to the cytosol to achieve the cytotoxic effect. Once internalized, most of the IT molecules are routed to lysosomes and further degraded. The use of cytolysins from anemones (actinoporins) in the construction of ITs is an attractive alternative to overcome some of the problems of classical ITs since they achieve their toxic action through a mechanism of membrane pore formation. However, the non-specific toxicity of the actinoporin-based ITs obtained so far, which is associated with the toxic moiety, is the major limitation to attain further applications of these molecules. An approach designed to overcome this non-specific toxicity is the construction of inactive ITs with built-in biological "triggers" that will active the toxin following a biological stimulus. Particularly, engineered cytolysins can be activated by the action of tumour-specific proteinases. In this direction, we work in the design of actinoporin-based ITs that can be specifically activated by tumour proteinases. Another goal of our group is to use actinoporin-based ITs as components of chemotherapeutic cocktails. Considering these basic ideas several approaches are being developed in our laboratory related with the study and use of Sts and synthetic peptides (Casallanovo et al, 2006:169; Cilli et al, 2007:1201) that mimic regions functionally relevant for these actinoporins.
Pore forming toxins show also interesting properties for the design of novel cytosolic delivery systems of macromolecules, which has been previously performed using many bacterial toxins (Dietrich, 2001:23). This is one of the current scientific problems directly related with development of vaccines against cancer and other diseases. The research projects related with construction of ITs and novel cytosolic delivery systems of macromolecules are carried out with the contribution of several foreign and national institutions such as Center of Molecular Immunology (CIM) and the Center for Genetic Engineering and Biotechnology (CIGB).
Altogether, the studies performed during more than 20 years (Alvarez et al, 2009, Tejuca et al, 2009) are an example of how the basic knowledge gained so far in the study of the structure - function relationship of these proteins (Tejuca et al, 1996: 14947; Pazos et al, 2003: 571; Pazos et al, 2006:1083) can contribute to the development of new approaches with potential applications in Biomedicine.
Bionanotechnology has been described as one of the most promising alternative for future development in different fields of life and society. One of their applications is associated to the development of new therapeutic formulations that can be addressed to specific cells; it means the construction of intelligent drugs which would be to an improved therapeutic index. The aim of one of the most important research fields of our center is to obtain and characterize liposomal vesicles for different purposes. Liposomes are artificially prepared nanometric vesicles (although some of them can be bigger) that have a roughly spherical shape with an aqueous core surrounded by one or more lipid bilayers. Due to their ability to stimulate an immune response by addressing antigens to immune system cells, these vesicles are employed in immunizations and in vaccines designs. One of the first results using liposomes as immunoadjuvants in our country was performed in collaboration with CIM. This project allowed the obtainment of antibodies anti-gangliosides through immobilization of glycololipids antigens in liposomes (Vázquez et al, 1995:551). Remarkable results have also been obtained by our group in the study of the immunmodulating properties of liposomal lipids for antigens such as EGF (Luzardo et al, 2002: 147; Lanio et al, 2008:1) and other antigens of the Dermatophagoides siboney mite (Calderón et al, 2006:38), showing the efficacy of these systems.
Likewise, the Center of Protein Studies (CEP) collaborates with other projects related with the characterization and functionalization of magnetics particles developed at the Institute of Sciences and Technology of Materials from the University of Havana (Figueroa-Espí et al, 2009:47). We have recently reviewed the results obtained so far by our group as well as the perspectives in the use of liposomes as nanobiotechnological tools (Lanio et al, 2009:23) and the construction of immunotoxins (Tejuca et al, 2009:15).
General Coordinator of the group:
Pore Forming Toxins with potential applications for Biomedicine and Nanobiotechnology
PhD. María Eliana Lanio Ruiz, Profesor Titular ()
Research Fields
Research Field: Pore forming toxins and their mimetic peptides
PhD. Carlos Alvarez Varcárcel, Senior Professor (): Head of the group: Pore Forming Toxins and their mimetic peptides. The aim of this group is to gain insight into the mechanism of pore formation by sticholysins at the molecular level. This group performs structure-function relationship studies of synthetic peptides that mimic the N-terminal region of these toxins and study the consequences of the interaction of these proteins with the membrane for the cells.
PhD. Uris Ros Quincoces, Assistant Professor (). She makes her post-doctoral research in the study of the influence of the membrane properties in the conformational and functional properties of synthetic peptides that reproduce the N-terminal region of StI and StII.
B.S. Lohans Pedrero Puentes, Instructor Professor (). Her thesis is focused to study the effect of membrane lipid composition on toxins binding and permeabilizing activity.
MSc. Carmen Soto Febles, Auxiliary Professor (). She makes her doctoral thesis in the study of the determinants of the cellular membrane that modulate the binding and permeabilizing activity of Sts and the intracellular mechanisms triggered by the toxin action.
Research Line: Cystein mutants as tools for structural and functional studies of sticholysins
PhD. Isabel Fabiola Pazos Santos, Senior Professor (). Head of the group: cystein mutants as tools for structural and functional studies of Sts. This research line is focused to cloning, expression and characterization of recombinant variants of Sts, as well as in the design of cystein mutants that allow gain insight into the mechanism of pore formation by Sts.
PhD. Aisel Valle Garay, Assistant Professor (). He makes his post-doctoral research using cystein mutants of StI in order to obtain new molecular details of functional mechanism of the actinoporins.
Research line: Liposomes and Sticholysins: their applications in nanobiotechnology
PhD. María Eliana Lanio Ruiz, Senior Professor (). Head of the research group related with the nanobiotech applications of Sts, such as, the design of immunotoxins for selective killing of cancer cells and novel cytosolic delivery systems of macromolecules based on liposomes and Sts.
PhD. María del Carmen Luzardo, Senior Professor (). Leads the researches related with the use of liposomes as adjuvant for the allergens model.
MSc. Yoelys Cruz Leal, Researcher (). She makes her PhD thesis in the study of the possible roles of B1 cells in the immunoadjuvants capacity of liposomes.
MSc. Rady Laborde Quintana, Instructor Professor (). Her doctoral thesis is related with the design of novel cytosolic delivery systems of macromolecules based on liposomes and Sts.
B.S. Yadira de la Patria Hervias, MSc Student (). Her thesis is related with the selective depletion (elimination) of memory B lymphocytes specific for Ovalbumin using the fusion protein Ova-PE40.
PhD. María Eugenia Alonso Biosca, Senior Professor (). She supervises the immunological researches related with Sts.