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Project titleNovel Fe2+-dependent bioluminescent system of marine bristle worms Chaetopterus variopedatus: structure, function and applications.

AffiliationShemyakin - Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences,

KeywordsBioluminescence, luciferin, luciferase, bioimaging, organic peroxides, biosensor, Chaetopterus

Annotation
The fundamental scientific task, within the framework of which the proposed project is carried out, is the study of the mechanisms of bioluminescence, i.e. the physicochemical basis of the process of energy conversion of chemical energy into light in living organisms. The aim of the present project is a thorough investigation of the luciferin-luciferase bioluminescence system of marine polychaetes Chaetopterus. It is well established that in a living cell in the process of ferroptosis, a special type of programmed cell death, ferrous ions and hydroperoxides of unsaturated fatty acids are simultaneously present. Also, the accumulation of ferrous ions and the formation of lipid peroxides, leading to cell damage, can be observed in a number of neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Friedreich's ataxia and other pathological processes, including amyotrophic lateral sclerosis, periventricular leukomalacia, ischemic stroke, traumatic brain injury and acute kidney injury. The course of the above diseases is presumably associated with the triggering of ferroptosis cascade. At the same time, since cancer cells contain an increased amount of ferrous ions, the induction of ferroptosis is a promising strategy for the therapy of tumors resistant to apoptosis. The study of the mechanisms of ferroptosis and the search for new inducers and suppressors of ferroptosis are essential for the creation of new drug treatments for neurodegenerative and oncological diseases. However, for large-scale screening of new inducers (suppressors) of ferroptosis, it is necessary to detect this process in living cells or in laboratory animals. In the previous study of the Chaetopterus bioluminescence system, it was found that the substrate of Chaetopterus bioluminescence reaction is an organic peroxide (the chemical nature of the substrate is lipid peroxide). Since the process of ferroptosis is characterised by both the accumulation of ferrous ions and the formation of organic peroxides, then ferroptosis will trigger the development of luminescent signal in cells expressing Fe2+-dependent Chaetopterus luciferase. Thus, the Chaetopterus bioluminescence system is a promising tool for monitoring ferroptosis in living organisms. During the implementation of the 2018 project, significant strides were made in identifying the components of Fe2+-dependent Chaetopterus variopedatus bioluminescence system: the structures of luciferins were established, a method for their synthesis was developed, and luciferase genes were cloned. The objectives of the current project are the adaptation of the bioluminescent system of Chaetopterus variopedatus for expression in heterologous organisms, the elucidation of the chemical basis of luciferin oxidation, and the deciphering of luciferase spatial structure. To achieve the set goals, it is necessary to accomplish a number of tasks: to carry out the structural studies of the products of enzymatic oxidation of luciferin, to establish the chemical mechanism of bioluminescence; to elucidate the presence and structure of the luciferase cofactor - a possible emitter of light in the bioluminescent reaction of C. variopedatus; to obtain an active recombinant C. variopedatus luciferase; to decipher the spatial structure of C. variopedatus luciferase; based on the the obtained data, to evaluate the potential for applications of Chaetopterus variopedatus new bioluminescent system for bioimaging. The pioneering quality of the project and its novelty provide full set of possibilities for intellectual property protection of its results, concerned with practical use of the novel bioluminescent system (new luciferins, luciferases, bioluminescent reaction cofactors and methods of their application) for the development of various new test systems and sensors based on bioluminescence. The unique properties of the bioluminescence system under investigation (the only known system regulated by iron) and the obtained data (a fundamentally new chemical structure of luciferin and luciferase, not homologous to any known proteins) support the undoubted scientific novelty of the project.

Expected results
As a result of the project, a complete structural and functional organization of a new type of Fe2+-regulated bioluminescence system of marine polychaetes Chaetopterus variopedatus will be described. The cofactors and post-translational modifications required for the functioning of Chaetopterus variopedatus luciferase will be established. An active recombinant luciferase from Chaetopterus variopedatus will be obtained and its possible applications in bioimaging will be demonstrated. The mechanism of bioluminescence reaction of a new unique natural luciferin will be deciphered, the structures of the products of its enzymatic oxidation will be established. In addition to answering the fundamental questions, the structure elucidation of all components of polychaetes Chaetopterus bioluminescence system and the deciphering mechanism of action of the new natural luciferin will further allow the development of new unique systems for visualization of biochemical processes in living cells, and will become integral for the development of new luminescence-based analytical technologies that could be employed to measure the enzymes activities and qualitative and quantitative analysis of various proteins and low molecular weight analytes. The results of the work will be published in leading scientific journals such as the journals Nature Publishing Group, Journal of American Chemical Society, Angewandte Chemie, Photochemical and Photobiological Sciences, Biochemistry, PNAS, etc. The pioneering nature of the project and its novelty provide the possibility of patent protection for all practically significant results associated with the use of a new bioluminescent system (new luciferin, luciferase, bioluminescence cofactor and methods of their application) for the development of various bioimaging systems.

Annotation of the results obtained in 2022
In the second year of an ongoing project, we showed that Chaetopterus variopedatus luciferase undergoes significant post-translational modifications. Firstly, the active protein does not contain an N-terminal signal sequence. Secondly, it also lacks 24 C-terminal amino acid residues. Thirdly, we found that Chaetopterus luciferase is glycosylated, and carbohydrate residues can be removed by PNGase F. This suggests that they are attached to the amino groups of asparagine residues in the luciferase. We have shown that the removal of carbohydrate residues results in the loss of luciferase activity. Consequently, the heterologous expression of this protein in its active form is handicapped. We showed that neither Chaetopterus luciferin, nor luciferase, nor bioluminescent reaction products contain a classical light emitter (a system of conjugated double bonds capable of fluorescence) and proposed a fundamentally new mechanism of C. variopedatus bioluminescence. We have shown that Chaetopterus luciferase substrates can be products of ferroptosis, a recently discovered pathway for programmed cell death, and Chaetopterus luciferase is a promising tool for detecting ferroptosis in living organisms.

Publications

1. Zagitova RI, Purtov KV, Shcheglov AS, Mineev KS, Dubinnyi MA, Myasnyanko IN, Belozerova OA, Pakhomova VG, Petushkov VN, Rodionova NS, Lushpa VA, Guglya EB, Kovalchuk S, Kozhemyako VB, Mirza JD, Oliveira AG, Yampolsky IV, Kaskova ZM, Tsarkova AS. Conjugated Dienoic Acid Peroxides as Substrates in Chaetopterus Bioluminescence System. International Journal of Molecular Science, 2023, 24, 9466 (year - 2023) https://doi.org/10.3390/ijms24119466

2. Lyakhovich M., Burakova L., Mineev K., Petushkov V., Zagitova R., Tsarkova A. , Kovalchuk S., Yampolsky I., Vysotski E., Kaskova Z. Mechanism of coelenterazine photoinactivation of Beroe abyssicola photoprotein FEBS Open Bio, 12, Suppl. S1, 244 (year - 2022) https://doi.org/10.1002/2211-5463.13440

3. Purtov KV, Petushkov VN , Rodionova NS, Chepurnykh TV , Shcheglov AS , Ziganshin RH, Tsarkova AS Habitat-dependent variations of Chaetopterus variopedatus polychaete luciferases. The EuroBiotech Journal, Volume 7, Issue 1, page 32. (year - 2023) https://doi.org/10.2478/ebtj-2023-0004

4. Zagitova R., Purtov K., Shcheglov A., Belozerova O., Tsarkova A. Chaetopterus variopedatus bioluminescence system low-molecular-weight components FEBS Open Bio, 12, Suppl. S1, 311 (year - 2022) https://doi.org/10.1002/2211-5463.13440

Annotation of the results obtained in 2021
In the first year of the ongoing project, a synthesis method was developed and active luciferin analogues with functional groups were obtained that made it possible to trace their transformation into enzymatic reaction products using mass spectrometric detection. Our assumptions about the factors affecting the activity of the structures were confirmed; as a result, we managed to achieve an increase in activity by a factor of 3–20, depending on the modifications introduced. The resulting highly active analogs were used to search for bioluminescent reaction products by analyzing reaction mixtures by HPLC with UV and mass detection. No specific reaction products were found, either because they are unstable or because they remain bound to the protein under in vitro conditions. The effect of various antioxidants on the bioluminescent reaction and its resulting products has been studied. We have found that antioxidants, especially “radical scavengers” such as sodium ascorbate, ferrostatin-1 (3-Amino-4-cyclohexylaminobenzoic acid ethyl ester), TEMPO (2,2,6,6-Tetramethylpiperidine 1-oxyl) effectively suppress the bioluminescent response of Chaetopterus. In the course of work on optimizing the refolding of natural C. variopedatus luciferase, we tested various methods for restoring the native structure of proteins. However, none of the methods used by us led to the restoration of the specific activity of the enzyme denatured during the study. Perhaps this is due to the loss of some binding low molecular weight component involved in the formation of the quaternary structure of this luciferase.

Publications

1. Tsarkova AS Luciferins Under Construction: A Review of Known Biosynthetic Pathways Frontiers in Ecology and Evolution, 2021, Volume 9, Article 667829 (year - 2021) https://doi.org/10.3389/fevo.2021.667829

2. Zagitova RI, Purtov KV, Shcheglov AS, Tsarkova AS Low-molecular-weight components of polychaete worm Chaetopterus va variopedatus bioluminescence system The EuroBiotech Journal, 2021, Volume 5, Issue s2, page 102 (year - 2021) https://doi.org/10.2478/ebtj-2021-0031