INFORMATION ABOUT PROJECT,
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COMMON PART

Project Number19-14-11008

Project titleDetermination of the bioluminescence mechanism of higher fungi and development of its applications towards new noninfringing group of analytical methods for medicine and biotechnology

Project LeadGitelson Josef

AffiliationInstitute of Biophysics of Siberian Branch of Russian Academy of Sciences,

Research area 04 - BIOLOGY AND LIFE SCIENCES, 04-209 - Biotechnology (including biological nanotechnology)

Keywordsbioluminescence, fungi, biotechnology, test systems, luciferase, luminous fungis, Neonothopanus nambi, luciferin, luciferase, luminescent system

PROJECT CONTENT

Annotation
Bioluminescent research methods are very diverse and are used in vitro and in vivo for the analysis of metabolites, immunological studies, for investigation of gene expression and for environmental monitoring. Bioluminescence has been widely used in medicine to study diseases such as diabetes, cancer, infectious and neurodegenerative diseases [Magalhães C.M., Esteves da Silva J.C., Pinto da Silva L. (2016) Chemiluminescence and Bioluminescence as an Excitation Source in the Photodynamic Therapy of Cancer: A Critical Review Chemphyschem. 7(15):2286-94], as well as for screening of drugs [Kelkar M., De A. (2012) Bioluminescence based in vivo screening technologies. Curr Opin Pharmacol. 12(5):592-600], for bioimaging of living systems in real time (the resulting bioluminescent transgenic animals are later used in preclinical studies) [(Coleman S., and McGregor A. (2015) A bright future for bioluminescent imaging in viral research. Future Virol. ; 10(2): 169–183]; bioimaging is also widely used to study protein-protein interactions. Since various classes of luciferases fundamentally differ from one another by the protein structure and substrate specificity, their greater diversity will increase the methodological range of applications for fundamental and applied purposes. The luciferase of fungi Neonothapanus nambi discovered in our laboratory as task of the RSF project belongs to a fundamentally new family of proteins that do not have a homology with any previously described proteins. We have shown the bioluminescent activity of this luciferase upon heterologous expression of the gene in Pichia pastoris cells and mammalian cells. As the aim of this project, we are planning several tasks of fundamental importance as well as of practical application. We would obtain mammalian cell lines stably expressing the luciferase of the fungi Neonothapanus nambi. Such cell lines have been successfully used in preclinical studies of antitumor therapy [De A., Loening A.M., S.S. (2007) An Improved Bioluminescence Resonance Energy Transfer Strategy for Imaging Intracellular Events in Single Cells and Living Subjects. Cancer Res. Aug 1; 67(15): 7175–7183)]. Since fungal luciferase is a membrane protein, various media that mimic the membrane environment where the protein has its native structure and activity, and which are excellent for structural and biophysical investigations, will be studied in the course of the project. To increase the brightness of luciferase in such studies the mutagenesis of the luciferase gene, including a modification of the N-terminal region will be carried out. To evaluate the luminescent activity of the mutants obtained, as well as for the detection of luciferase in a denatured and inactive state, we propose to obtain antibodies to this protein, which will greatly expand the scope of application of this luciferase and the possibility of setting different controls. In addition, it is planned to conduct a large-scale selection of conditions for the crystallization of the luciferase. Crystallization of membrane proteins is associated with considerable difficulties [Parker J.L., Newstead S. (2016) Membrane Protein Crystallisation: Current Trends and Future Perspectives. Adv Exp Med Biol.; 922: 61–72], since it is necessary to solubilize them while maintaining the native structure and stability. At the moment, the structures of only about 600 membrane proteins from more than 50,000 known proteins associated with membranes are resolved. The information obtained through the crystallization of each new membrane protein can improve the precision of algorithms for prediction of such structures and similar protein domains.

Expected results
During the extension of the project, we plan to modify the luciferase gene to increase its luminescent activity and stability in aqueous media. In particular, we will pay close attention to the modifications of N-terminus of this protein, since, according to bioinformatic predictions, this is the site where the amino acid residues that participate in the interaction of luciferase with the membrane is located. Presumably, modifications to the N-terminal region may affect the solubility of this protein, as well as its brightness and stability of its structure. In the course of the project, we plan to apply mutagenesis techniques to create modified variants of the fungal luciferase gene, as well as to develop a technique for high-throughput screening of mutants with the desired properties. Currently, the luminescence level of fungi luciferase in the heterologous system of mammalian cell culture is comparable to the luminescence level of one the firefly that is often used in bioimaging. [Proc Natl Acad Sci U S A. 2018 115(50):12728-12732. Genetically encodable bioluminescent system from fungi. Kotlobay AA, Sarkisyan KS, Mokrushina YA, Marcet-Houben M, Serebrovskaya EO, Markina NM, Gonzalez Somermeyer L, Gorokhovatsky AY, Vvedensky A, Purtov KV, Petushkov VN, Rodionova NS, Chepurnyh TV, Fakhranurova LI, Guglya EB, Ziganshin R, Tsarkova AS, Kaskova ZM, Shender V, Abakumov M, Abakumova TO, Povolotskaya IS, Eroshkin FM, Zaraisky AG, Mishin AS, Dolgov SV, Mitiouchkina TY, Kopantzev EP, Waldenmaier HE, Oliveira AG, Oba Y, Barsova E, Bogdanova EA, Gabaldón T, Stevani CV, Lukyanov S, Smirnov IV, Gitelson JI, Kondrashov FA, Yampolsky IV]. In the case of creation of a brighter version of fungal luciferase, the number of possible methods of its application will rise up, and would include combination with other known luciferases. Currently, methods based on the luciferin-luciferase reaction are extremely popular among biological and medical research around the world.[ J Immunol Methods. 2017 447:1-13. A bioluminescent caspase-1 activity assay rapidly monitors inflammasome activation in cells. O'Brien M, Moehring D, Muñoz-Planillo R, Núñez G, Callaway J, Ting J, Scurria M, Ugo T, Bernad L, Cali J, Lazar D.; Amino Acids. 2016 48(5):1151-60. Novel bioluminescent binding assays for interaction studies of protein/peptide hormones with their receptors. Liu YL, Guo ZY.; Biochemistry. 2017 56(39):5178-5184. Bioluminescent Probes for Imaging Biology beyond the Culture Dish. Rathbun CM, Prescher JA.] Therefore, the presence of a bright variant of the fungi luciferase, which specifically binds only to its substrate, will expand the range of bioimaging techniques and, in some cases, increase the sensitivity and scope of the applications of these methods. During extension of the project, we will produce a library of vectors for creation of lentiviral particles for transduction of mammalian cell cultures; a collection of relevant viral particles, and a collection of mammalian cell cultures which are stably expressing wild-type luciferase gene or its modified variants (for example, ones fused with fluorescent protein to improve visualization and microscopic examination capabilities or the gene encoding N-terminus modifications to increase the brightness of the product). To solve practical problems associated with the detection of luciferase and determine its specific activity, we will obtain antibodies to luciferase. The resulting antibodies will be used to conduct western blot hybridization and histochemical reactions of mammalian cell cultures transformed with the luciferase gene. Using mutants with improved characteristics, we will continue screening of the conditions for the crystallization of luciferase, which would further allow us to establish the active center and mechanism of the enzymatic oxidation reaction of the fungal luciferin [Kaskova Z.M. et al (2017) Mechanism and color modulation of fungal bioluminescence.Science Advances, Vol. 3, no. 4, e1602847]. Thus, the tasks set in the project - the estimation of the structure of the new luciferase of higher fungi and the development of methods for its biomedical application correspond to the topics of advanced world research in the field. Successful implementation of the tasks will significantly improve the ergonomics of the methods of diagnosis currently available in medicine and would make easier the monitoring of common dangerous diseases.

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