Presentation on theme: " The purpose of this presentation is to explore a somewhat virgin field, that of pore-forming toxins- with an added emphasis on the research potential."— Presentation transcript:
The purpose of this presentation is to explore a somewhat virgin field, that of pore-forming toxins- with an added emphasis on the research potential of cholesterol dependent cytolysins. The novelty of this field is obvious, yet the potential in cancer research seems to be unlimited. Given the ever expanding demand for cancer treatment and research, this presentation should briefly highlight an expanding field that could become the epicenter of cancer research.
What are Pore Forming Toxins? ◦ These are toxins, typically produced by bacteria, that induce unregulated pore formation in biological membranes. As a result the structural integrity of the biological membrane is compromised, leading to unregulated cytoplasmic exchange, a disruption in the established ion gradient and most likely apoptosis.
Categorization: There are four general groups that pore-forming toxins fall into, depending on several distinct characteristics: ◦ Beta pore-forming ◦ AB or Binary ◦ Small pore-forming ◦ Cholesterol dependent cytolysins (large pore- forming)
Dimorphic proteins that undergo a complex conformational change when interacting with a biological membrane. Beta pore-forming toxins are soluble monomer proteins that become multimeric. Seen above is an example of a multimeric protein: composed of two or more monomers. They can be either homogeneous or heterogeneous
Mechanism of action: After the Beta pore-forming toxin binds to the biological membrane, the Beta portion of the proteins ‘flips’ into the membrane. Shown left A is a view of the protein inserting into the lipid bilayer and B is an overview of the membrane; you can see the resulting pore, central, has already formed.
Above: a simplified example of a Beta forming toxin mechanism. The grey represents the lipid membrane. The top picture displays the pore forming abilities of the toxins after the Beta undecapeptide enters the lipid bilayer. Notice that domain four is in direct contact with the lipid memnrane while domain 3 is in contact with the formed pore. The bottom example displays the insertion of the Beta sheet into the lipid membrane. Each color is representative of a separate protein domain-Beta pore-forming proteins typically encompass domains 1-4.
Examples: ◦ Clostridium perfringens Gas gangrene Food poisoning ◦ Alpha-hemolysin Secreted by some bacteria in an attempt to lyse commonly to gain nutrients ◦ PVL- Panton-Valentine leukocidin Increased virulence of Staphyloccus aureus Commonly associated with MERSA (Methicillin resistant Staphylococcus aureus)
Proteins that utilize an ‘AB’ mechanism to gain entry into host cells. These toxins are composed of two distinct binding proteins acting in suite. The protein subunit A works enzymatically while the B subunit binds to the lipid membrane. These toxins are typically produced from spore forming bacteria through synergistic mechanisms.
Examples: ◦ Bacillus anthracis Bioterrorism agent Cutaneous, gastrointestinal, pulmonary ◦ Clostridium botulinum Causes paralysis Used in Botox, typically to paralyzes the face (notice at left the subjects ‘face’ looks similar to a mask)
Typically an inducible defense mechanism of certain strains of bacteria. ◦ Can act as a defense mechanism, specifically a defense against non-beneficial phagocytosis ◦ Can provoke cell responses that may be in some way beneficial for the bacterium, such as obtaining nutrients ◦ Bacteria can devote upwards of 20% of their energy demands towards producing these toxins
Proteins in which cholesterol is absolutely necessary for lipid membrane binding to occur, though these proteins may not directly bind to the cholesterol molecules directly. Lipid rafts are thought to increase the likely hood of potential binding, possibly as a result of increased cholesterol concentrations. Given the nature of the human lipid membrane, these CDCs are of particular interest.
Cholesterol and the human lipid membrane: a brief review Cholesterol allows the lipid membrane to establish and maintain a constant permeability while coherently keeping the membrane fluid. In the display at left, the numerous red structures in the membrane represent cholesterol.
The two hallmark characteristics of all CDCs: ◦ A. An absolute dependence on the presence of membrane cholesterol ◦ B. Resulting in the formation of extraordinarily large membrane pores, up to approximately 300A 1 Anstrom = 1 X 10 -10 meters
How do we know cholesterol-dependence? ◦ A series of test under differing environmental conditions were in which the findings were: Hemolysis by CDCs is inhibited by the addition of free cholesterol to the toxin prior to incubating it with erythrocyte membranes. Some CDCs have been shown to directly bind to cholesterol No binding took place to synthetic membranes lacking cholesterol
Through these experimental results you can adequately deduce that cholesterol is necessary for these cytolysins to bind to a biological membrane. The specific contribution of membrane bound cholesterol to the denaturation mechanism of CDCs appears to be convoluted however- it is not entirely clear if cholesterol functions as the receptor in the classical sense in which there is a 1:1stoichmetric relationship. Additional research is still needed.
From experimental results it has been show that the binding affinity of CDCs to biological membranes is almost 50 mol % of the total membrane lipids. If was found that at 40 mol% there was little to no binding, yet at 55 mol% maximal binding occurred. This observation suggests that the interaction between the CDCs and the membrane bound cholesterol is not simply the result of one toxin molecule binding to one cholesterol molecule, since cholesterol was not limiting under any of the conditions studied. These results thus support the theory that a complex, unknown reaction mechanism occurs between CDCs and membrane cholesterol.
Examples ◦ Sea anemone Simple water dwelling cnidarian organism Is of particular interest to cancer research, specifically with respect to its cytolysin membrane binding abilities
What is cancer? ◦ This is a broad term that describes uncontrolled cell growth, that establishes cell division beyond normal limits. Often times the cancer will metastasis to other portions of the human body. Above: Right lung infected with cancer, compared to a normal left lung. Take home message: don’t smoke !
So what do CDCs have to do with it? ◦ Immunotoxins are molecules, typically of an antibody bound to a killer toxin. These immunotoxins, ITs, attempt to utilize a ligand bound to a toxin to target and attack specific undesirable cells, for the purposes of this presentation, tumour cells. Acting to enzymatically inhibit protein synthesis, clinical phase II trials utilizing ITs have recorded positive results; in one instance a complete remission in a high proportion of hairy-cell leukemia patients.
So what do CDCs have to do with it? ◦ The problem: solid tumours, as compared to hairy-cell leukemia, are much more tightly packed- making it almost impossible for classical ITs to penetrate deep into the tumour cells and be effective. In response research has been conducted on membrane-active immunotoxins, specifically through the utilization of CDCs.
So what do CDCs have to do with it? ◦ The CDCs produced by the Sea anemone, bound to site-specific antibodies, have been found through experimentation to be the most viable option for cancer research. The CDCs produced by the sea anemone bind to the tumour and induce pore formation, which coherently allows for the ITs to penetrate deep into the tumour, thereby reducing its effective size.
The early results ◦ Through initial animal trials and in vitro studies, it was found that these new membrane active immunotoxins, through the use of sea anemone CDCs, were able to successfully penetrate solid tumour cells by inducing pore formation. Although a great deal of additional research is requried, the field of membrane- active immunotoxins appears to have a bright future.
So where’s the pot of Gold? ◦ If you’re thinking that these reported results sound too good to be true, its probably because there has been a minor caveat excluded. The main problem with the ITs constructed in this fashion is the lack of specificity associated with the toxin moiety. In other words, the CDCs may not only bind to your tumour, but also any healthy cell it encounters along the way also.
A question left unanswered ◦ Possibly the biggest question left unresolved, requiring additional research, is the specificity of the immunotoxins produced. If the toxin is not capable of binding in a case specific fashion on a cellular level, the therapeutic index of cytolysin would be slim, as many healthy cells would be destroyed consequently in the process. One is left to wonder if the benefits of cytolysin would outweigh the costs; would the toxins of the sea anemone kill the patient before it reduces the malignant tumour to a reasonable size?
Above: A display of several clinical drugs, how far along they are in testing (how soon they will be available to the general public) and the specific type of cancer they target.
Summation: Even though some questions may remain, the potential impact of PFTs on medicine, and more importantly everyday life, could be prolific. A promising medical breakthrough of this magnitude attributed to a toxin produced by an organism as simple as a sea anemone should display the irony of human history. As a society we are only now realizing the potential benefits nature has to offer. Discoveries such as this will hopefully influence mass opinions on natural benefits, especially when reflecting on the lack of serious funding and research throughout history.
Abrami, L., M. Fivaz, E. Decroly, N. G. Seidah, F. Jean, G. Thomas, S. H. Leppla, J. T. Buckley, and F. G. van der Goot. 1998. The pore-forming toxin proaerolysin is activated by furin. J. Biol. Chem. 273:32656-32661. Bhakdi, S., U. Weller, I. Walev, E. Martin, D. Jonas, and M. Palmer. 1993. A guide to the use of pore-forming toxins for controlled permeabilization of cell membranes. Med. Microbiol. Immunol. (Berlin) 182:167-175. Cowell, J. L., and A. W. Bernheimer. 1978. Role of cholesterol in the action of cereolysin on membranes. Arch. Biochem. Biophys. 190:603- 610. Czajkowsky, D. M., E. M. Hotze, Z. Shao, and R. K. Tweten. 2004. Vertical collapse of a cytolysin prepore moves its transmembrane β- hairpins to the membrane. EMBO J. 23:3206-3215. Gaillard, J. L., P. Berche, J. Mounier, S. Richard, and P. Sansonetti. 1987. In vitro model of penetration and intracellular growth of Listeria monocytogenes in the human enterocyte-like cell line Caco-2. Infect. Immun. 55:2822-2829. Giddings, K. S., A. E. Johnson, and R. K. Tweten. 2003. Redefining cholesterol's role in the mechanism of the cholesterol-dependent cytolysins. Proc. Natl. Acad. Sci. USA 100:11315-11320. Giddings, K. S., J. Zhao, P. J. Sims, and R. K. Tweten. 2004. Hum. CD59 is a receptor for the cholesterol-dependent cytolysin intermedilysin. Nat. Struct. Mol. Biol. 12:1173-1178. Heuck, A. P., R. K. Tweten, and A. E. Johnson. 2003. Assembly and topography of the prepore complex in cholesterol-dependent cytolysins. J. Biol. Chem. 278:31218-31225. Heuck, A. P., R. K. Tweten, and A. E. Johnson. 2001. Beta-barrel pore-forming toxins: intriguing dimorphic proteins. Biochemistry 40:9065- 9073. Hotze, E., and R. K. Tweten. 2002. The cholesterol dependent cytolysins: current perspectives on membrane assembly and insertion, p. 23- 37. In A. Ménez (ed.), Perspectives in molecular toxinology. John Wiley & Sons, Ltd., West Sussex, England. Mitchell, T. J., P. W. Andrew, F. K. Saunders, A. N. Smith, and G. J. Boulnois. 1991. Complement activation and antibody binding by pneumolysin via a region of the toxin homologous to a human acute-phase protein. Mol. Microbiol. 5:1883-1888. Nakamura, M., N. Sekino, M. Iwamoto, and Y. Ohno-Iwashita. 1995. Interaction of theta-toxin (perfringolysin O), a cholesterol-binding cytolysin, with liposomal membranes: change in the aromatic side chains upon binding and insertion. Biochemistry 34:6513-6520. Palmer, M., R. Harris, C. Freytag, M. Kehoe, J. Tranum-Jensen, and S. Bhakdi. 1998. Assembly mechanism of the oligomeric streptolysin O pore: the early membrane lesion is lined by a free edge of the lipid membrane and is extended gradually during oligomerization. EMBO J. 17:1598-1605. Shatursky, O., A. P. Heuck, L. A. Shepard, J. Rossjohn, M. W. Parker, A. E. Johnson, and R. K. Tweten. 1999. The mechanism of membrane insertion for a cholesterol dependent cytolysin: a novel paradigm for pore-forming toxins. Cell 99:293-299. Waheed, A. A., Y. Shimada, H. F. Heijnen, M. Nakamura, M. Inomata, M. Hayashi, S. Iwashita, J. W. Slot, and Y. Ohno-Iwashita. 2001. Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts). Proc. Natl. Acad. Sci. USA 98:4926- 4931.