The information is prepared on the basis of data from the information-analytical system RSF, informative part is represented in the author's edition. All rights belong to the authors, the use or reprinting of materials is permitted only with the prior consent of the authors.

Project titleDry preservation of biological materials using protective proteins from an anhydrobiotic insect

KeywordsAnhydrobiosis, dry preservation of enzymes, cells and antibodies, CRISPR-Cas9 genome editing, trehalose, the sleeping chironomid, desiccation, LEA proteins, Sf9 cells, polymerases, chromosome conformation capture

Annotation
In order to move toward new technologies of dry preservation for biological materials used in biomedical applications and agriculture, we will utilize protective proteins (anhydroprotectants) from an anhydrobiotic insect Polypedilum vanderplanki to improve the resistance of model insect cells, antibodies and enzymes to freezing and dehydration. The whole project will be will be implemented by close collaboration among Russian and Japanese research teams and will include two main steps: 1. The optimal set of protective proteins to form molecular shield will be identified by combination of omics analysis and gene editing experiments using updated genome data of the anhydrobiotic chironomid . We will adapt CRISPR/Cas9 gene editing system to the Pv11 cell line derived from the chironomid, and using original reporter assay (recombinant luciferase stability under desiccation in the cells), we will analyze the impact on desiccation tolerance of several candidate protective proteins, which are specific to the anhydrobiotic chironomid : such as LEA proteins, antioxidants and isoaspartyl-methyltransferases. Using previously identified highly effective promoter, we will analyze the subcellular localization of protective recombinant LEA proteins both in Pv11 cells and in the Spodoptera cell line Sf9. Finally, using Hi-C technology, we identify the co-localization of candidate protective genes in Topologically Associated Domains (TAD) in the genome and their regulation upon desiccation. These steps, combined with existing detailed gene expression data will allow us to select the most promising candidates for in vivo and in vitro experiments. 2. The selected pool of protective proteins will be used for in vivo (recombinant expression of proteins in the model cell line Sf9, combined with trehalose treatment) and in vitro (using several model DNA polymerases and antibodies) experiments. The effect of Pv-derived proteins in combination with trehalose will be analyzed on the stability of the model cells and enzymes to desiccation and freezing in order to develop the approach to enhance the preservability of important biomaterials for agriculture, medicine and industry.

Expected results
Russia and Japan’s research groups will work in complementary mode, to achieve several key goals during the project. The first stage of the project will be focused on the basic mechanisms of anhydrobiosis in the sleeping chironomid and on the development of applied methods for functional analysis of related protective biomolecules. Here we state several key objectives: 1. Improvement of the assembly and annotation of the sleeping chironomid genome, to obtain a detailed physical map of the genome. Analysis of the repository genes using new data sets of small RNAs and non-polyA transcripts. 2. Analysis the spatial organization of chromatin in Pv11 cells under normal conditions and in the desiccated state using chromosome conformation capture approach. Identifying topologically associating domains structure in the chironomid genome for a better elucidation of the protective genes in Pv acting together. 3. Development the protocols of CRISPR/Cas9-mediated genome editing for Pv11 cells and targeted analysis of the effect of the knockout of keys anhydrobiosis-associated genes on desiccation tolerance and on the ability of Pv11 cells to provide exogenous protection to desiccation-sensitive recombinant proteins. 4. Analysis of the subcellular localization of LEA proteins in Pv11 cells, and in the model insect cell line Sf9 to identify the most promising members of LEA family for in vitro experiments. In the second stage of the project, accumulated results will be used for the development of methods for cells and proteins preservation, fitting practical needs: 5. We anticipate the improvement of the resistance to mild freezing and desiccation of the model insect cell line Sf9, commonly used for recombinant protein production using baculovirus, by simultaneous introduction of recombinant protective proteins from Pv11 together with trehalose. 6. Enhanced methods for the dry preservation of desiccation-sensitive recombinant DNA polymerases and antibodies, using both in vitro and in cell approach.

Annotation of the results obtained in 2019
At this stage of the project, we analyzed the intracellular distribution of LEA protective proteins both in chironomid cells and during their exogenous expression in desiccation-sensitive model Sf9 insect cells. We found that intracellular localization of proteins is not conservative in several cases, which must be considered during genetic engineering work using chironomid protective proteins. At the same time, a significant amount of LEA proteins retained intracellular localization regardless of cell type, which opens the possibility of planning specific protection of individual cell organelles from dehydration. We also characterized a new unusual group of proteins-coding genes in the midge, the origin of which is associated with classical LEA proteins, but structurally different from them. These are membrane proteins (Lea island located, Lil), characterized by increased expression during desiccation, indicating their role in P. vanderplanki anhydrobiosis. These genes are orphan and no orthologs have been found in other living species, including dipteran insects, which include chironomids. Thus, it can be argued that we found a new group of proteins that specifically interacting with cell membranes and likely protecting the membranes during desiccation. At the same time, the expression of separately recombinant LEA and Lil proteins did not increase the resistance of Sf9 model cells to desiccation, which suggests that the formation of a protective molecular shield in the cell requires the synergistic activity of various groups of chironomid proteins. We also analyzed non-coding long and small RNA in the chironomid, and determined those that are associated in their transcriptional activity with genes actively involved in anhydrobiosis. Remarkably, the large part of non-coding RNA associated with anhydrobiosis was located in a certain part of the genome - on the fourth chromosome, in the same place as other unique chironomid genes associated with tolerance to dehydration.

Publications

1. Alina Ryabova, Richard Cornette, Alexander Cherkasov, Masahiko Watanabe, Takashi Okuda, Elena Shagimardanova, Takahiro Kikawada, Oleg Gusev Combined metabolome and transcriptome analysis reveals key components of complete desiccation tolerance in an anhydrobiotic insect Proceedings of the National Academy of Sciences of the United States of America (PNAS), 117, 32, 19209-19220 (year - 2020) https://doi.org/10.1073/pnas.2003650117

2. Miyata Y., Tokumoto S., Sogame Y., Deviatiiarov R., Okada J. Cornette R., Gusev O. Shagimardanova E., Sakurai M., Kikawada T. Identification of a novel strong promoter from the anhydrobiotic midge, Polypedilum vanderplanki, with conserved function in various insect cell lines SCIENTIFIC REPORTS, Volume 9, Номер статьи 7004 (year - 2019) https://doi.org/10.1038/s41598-019-43441-x

3. Sabina A. Kondratyeva, Alexander A. Nesmelov, Alexander V. Cherkasov, Yugo Miyata, Shoko Tokumoto, Takahiro Kikawada, Oleg A. Gusev, Elena I. Shagimardanova Intracellular localization and gene expression analysis provide new insights on LEA proteins’ diversity in anhydrobiotic midge PLOS ONE, - (year - 2019) https://doi.org/10.1101/825133

4. Taisiya A. Voronina, Alexander A. Nesmelov, Sabina A. Kondratyeva , Ruslan M. Deviatiiarov , Yugo Miyata , Shoko Tokumoto , Richard Cornette , Oleg A. Gusev, Takahiro Kikawada, Elena I. Shagimardanova New group of transmembrane proteins associated with desiccation tolerance in the anhydrobiotic midge Polypedilum vanderplanki Scientific Reports, 10, 11633 (year - 2020) https://doi.org/10.1038/s41598-020-68330-6

5. Takahiro G Yamada Yusuke Hiki Noriko F Hiroi Elena Shagimardanova Oleg Gusev Richard Cornette Takahiro Kikawada Akira Funahashi Identification of a master transcription factor and a regulatory mechanism for desiccation tolerance in the anhydrobiotic cell line Pv11 PLOS ONE, - (year - 2020)

Annotation of the results obtained in 2017
The project is carried out jointly with colleagues from the National Institute of Agrobiological Sciences (part of NARO), Tsukuba, Japan and is aimed to employ the opportunity of dry storage of biological material using protective proteins of an anhydrobiotic midge P. vanderplanki. The midge is the most complex organism capable to anhydrobiosis: tolerance to long-term complete water loss. Previously we characterized a set of proteins and genes contributing to formation of molecular shied. The implementation of this project is an attempt to use the obtained basic knowledge about the mechanisms of sustainability to create a new biotechnological approach. At the same time, the tasks of basic science are also addressed. In 2017, assembly of the chironomid genome was significantly improved by integrating sequencing data from the genomic DNA of the culture of Pv11 cells obtained using Illumina technology and long reads from PacBio platform, as well as the involvement of RNA sequencing data at different stages of dehydration. This, first, allowed us to correct many errors in characterizing the genes involved in response to desiccation stress (for example, heat shock proteins). Secondly, we improved the annotation of genes, which is important, for a detailed characterization of the genome and, as a consequence, to clarify the mechanisms of adaptation to complete loss of water. It will also allow applying Hi-C technology to evaluate the spatial organization of chromatin under normal conditions and in modeling dehydration. Hi-C protocol has been developed and optimized at this stage for Pv11 cells, which will allow to carry chromatin interaction analysis in response to desiccation in the next stages of the project implementation. Another objective of the study was to obtain sequencing data for the analysis of the involvement of small and non-coding RNA in anhydrobiosis in P. vanderplanki. For this purpose, using a recognized model of anhydrobiosis, the culture of an anhydrobiotic insect Pv11 cells was used to isolate small RNAs, and libraries were obtained under normal conditions and after treatment with trehalose (initiation of anhydrobiosis-associated changes). Previously, it was shown that the patterns of gene expression after the addition of trehalose to the culture of Pv11 cells are similar to response of the larvae to desiccation. The received data will also be analyzed at the next stages of the project implementation. From the point of view of applied science, genetic constructs bearing the sequence of all known Lea-protein genes were obtained. Transfection protocols for these constructs were developed for the culture of Pv11 and Sf9 cells. This is a very important step for the subsequent evaluation of the improvement in cell survival during overexpression of protective proteins in cells. In addition, this will allow an assessment of the intracellular localization of Lea-proteins and closer to a refinement of their functions.

Publications

1. Nesmelov A.A., Shagimardanova E.I., Kikawada T., Gusev O.A. Involvement of Heat Shock Proteins in Invertebrate Anhydrobiosis Heat Shock Proteins in Stress, Springer, Dordrecht, Netherlands, - (year - 2018)

2. - Таланты насекомых. У иных козявок людям стоит поучиться живучести газета Поиск, Поиск. Наука, №41 (2017) (year - )

Annotation of the results obtained in 2018
An African non-biing midge (chironomid) Polypedilum is a unique insect able to survive complete desiccation. A combination of omics technologies allowed us to key some molecular mechanisms that ensure the induction of anhydrobiosis, where metabolic processes are completely stopped. The use of Hi-C method allowed to significantly improve the genome assembly of P. vanderplanki reaching the level of chromosomes-grade super-scaffolds. This version of the genomic assembly is so far the most complete for P.vanderplanki and is optimal for use in the analysis of transcriptome data. At the same time we developed new Midgebase 2.0 database on the Zenbu platform (http://fantom.gsc.riken.jp/zenbu/) which contains all existing versions of genome assembly, and a set of available data (RNA-seq at different stages of anhydrobiosis, life cycle, Cage data etc). In future other kinds of omics data will be added to the platform. The platform is convenient for data analysis and visualization in the interactive mode and allows the study of gene expression at different stages of the chironomid life cycle or in the dehydration cycle. A detailed analysis of long non-coding RNA revealed a number of candidates which might play an important role in the survival during anhydrobiosis. Cell culture experiments made it possible to divide all Lea proteins, which are known to be a key participant in the anhydrobiosis process into groups, according to their intra-cellular localization. This allows to get closer to understanding the function of each individual representative of the Lea family of proteins in anhydrobiotic chironomid cells.

Publications

1. Mazin P.V., Shagimardanova E.I., Kozlova O.S., Cherkasov A.A., Sutormin R., Stepanova V.V., Stupnikov A., Logacheva M.D., Penin A.A., Sogame Y., Cornette R., Tokumoto S., Miyata Y., Kikawada T., Gelfand M.S., Gusev O.A. Cooption of heat shock regulatory system for anhydrobiosis in the sleeping chironomid Polypedilum vanderplanki Proceedings of the National Academy of Sciences of the United States of America, №10, V.115, P: E2477–E2486 (year - 2018) https://doi.org/10.1073/pnas.1719493115

2. Cherkasov A.V., Ryabova A.V., Battulin N., Ananeva A.V., Gusev O.A., Shagimardanova E.I. Three-dimensional organization of genome of sleeping chironomid Polypedilum vanderplanki Febs Open Bio, Том: 8 Стр.: 136-136 Приложение: 1 Аннотация к встрече: P.02-018-W (year - 2018)

3. Nesmelov A.A., Voronina T., Kondratyeva S.A., Shagimardanova E.I. Localization of LEA proteins in the cells of anhydrobiotic insect Febs Open Bio, Том: 8 Стр.: 292-292 Приложение: 1 Аннотация к встрече: P.09-265-M (year - 2018)

4. Nesmelov A.A., Shagimardanova E.I., Kikawada T., Gusev O.A. Involvement of Heat Shock Proteins in Invertebrate Anhydrobiosis Heat Shock Proteins and Stress. Heat Shock Proteins. Springer, Cham, In: Asea A., Kaur P. (eds) Heat Shock Proteins and Stress. Heat Shock Proteins, vol 15.P.179-192 (year - 2018) https://doi.org/10.1007/978-3-319-90725-3_10