1. Determining the Efficacy of the KillerRed/IL-13.E11Y Fusion Protein: A Cytotoxic, Photo-activated Protein Designed to Target Glioblastomas Fusion E. ColiKR E. Coli Count CFU/mL wt E. Coli Expose to 545nm at 6mW/cm2 for duration of 1 h Count CFU/mL Determine Binding affinity Alpha Screen Binding Affinity Assay Isolated Fusion Protein Erik Knight Department of Biological Sciences, York College of Pennsylvania, York, PA Introduction Human glioblastoma cells overexpress human interleukin-13α2 receptors on their cell surfaces. These receptors have been shown to have an elevated binding affinity to a mutant human interleukin-13.E11Y. Killerred is a synthetic photosensitizer: a protein that produces reactive oxygen species (ROS) when excited with 540-580nm wavelength of light. ROS are naturally occurring molecules that are cytotoxic in excess amounts. Oxidative stress from an abundance of ROS include: interference of cell signaling, breakdown of cellular membrane, and ultimately cell death. Previous work has yielded a mutant strain of E.coli that inducibly produce a fusion protein of hIL-13.E11Y and Killerred. The amount of ROS produced by the fusion protein and its ability to bind to the IL-13 α2 receptor and cause cell death have not yet been investigated. Proposed Results Fusion E. Coli Expose to 545nm at 6mW/cm2 for duration of 1 h Conduct ROS Glo Assay Measure Luminescence (RLU) KR E. Coli wt E. Coli Experimental Design Isolated IL-13.E11Y Literature Review Williams et. al. have conducted a study using Killerred to alter neural function in C. elegans. They have found that the Killerred was cytotoxic in the presence of superoxide dismutase (SOD) and even more toxic in the absence. Furthermore, through use of a strain of C. elegans with a knockout for the apoptosis inducing gene, the cause of cell death was determined to be cell lysis. The same article also tested the localization of ROS damage by targeting specific neurons in worms. Oxidative damage was found to be generalized to only targeted cells. Mintz et al. found a strong correlation between overexpression of Il-13α2 receptors on the surface of astrocytomas, especially gliomas. Thompson et al. studied the relationship between several human interleukin 13 mutants and their ability to bind. Mutant IL-13.E11Y was found to have a high affinity to bind to the Il-13α2 receptor compared to the wild type. Proposed Results Continued Both groups should experience significant amount of cell death should occur after 1 hour of treatment. Complete culture death should take place after 24 hours. Luminescence given off by the E. Coli producing just Killerred should be comparable to the amount given off by the fusion protein producing strain. Wild type E. Coli should emit significantly less. The binding affinity assay will show the preferential binding between Il-13α2 and the mutant Il-13.E11Y as compared to wt hil-13 Furthermore, there should be no significant difference between the binding of the fusion protein and the binding of the mutant il-13 to the il-13α2 receptor. http://www.rosetreestrust.co.uk/wp-content/uploads/2014/05/Glioma-3.jpg Literature Cited https://www.promega.com/resources/pubhub/a-luminescent-assay-for-detection-of-reactive- oxygen-species/ http://www.perkinelmer.com/catalog/category/id/alphascreen%20assays%20and%20reagents Carpentier, P., Violet, S., Blanchoin, L. Structural basis for the phototoxicity of the fluorescent protein KillerRed. 2009. FEBS Letters. 583: 2839-2842. Mintz, A., Gibo, D.M., et. Al. IL-13R2 is a Glioma-Restricted Receptor for Interleukin-13. 2002. Neoplasia. 4 (5): 388-399. Thompson J. and Debinski W. 1999. Mutants of interleukin 13 with altered reactivity toward interleukin 13 receptors. Journal of Biological Chemistry. (274):29944–29950. Williams, D. C., Ramirez, P. M. Rapid and Permanent Neuronal Inactivation In Vivo via SubcellularGeneration of Reactive Oxygen with the Use of KillerRed. 2013. Cell Reports. 5: 553-563. Wassenaar, T.M., Beimfohr, C., Geske, T., Zimmermann, K. Voluntarily exposure to a single, high dose of probiotic Escherichia coli results in prolonged colonization. 2014. Beneficial Microbes. Objectives To quantify the amount of Reactive Oxygen Species produced To assess the binding ability of the fusion protein to the IL-13 α2 receptor To determine the fusion proteins efficacy in causing cell death Acknowledgments I would like to thank Dr. Thompson for sharing his expertise on the subject that helped me outline this project. Also I would like to thank Jacquelyn Bedsaul for laying the ground work I built my study upon.