Probing the Role of Actin Arginylation Through Synthetic Biology

The shape and structure of cells requires the cytoskeleton. Actin is an abundant cytoskeletal protein that is present in all cells. Actin plays important roles in cell shape establishment / maintenance, cell division, cell movement, muscle contraction, and heart beating. In many animals, Actin in non-muscle cells undergoes a particular modification termed arginylation. Through this modification, the amino-acid arginine is attached to the front end of actin protein. Why only certain actins get arginylated and what function is accomplished by actin arginylation are not understood. We will use a combination of synthetic biology, biochemistry, fish and mammalian cell biology to understand the function performed by arginylation of actin. We propose that a subset of actin functions may be impacted by arginylation. Since mis-regulation of actin cytoskeleton function is correlated with several human diseases, understanding actin arginylation and its function can provide insights into actin-linked diseases.

The actin cytoskeleton plays fundamental roles in muscle and non-muscle cell types. Non-muscle cells express two highly related actin isoforms, termed beta and gamma, which differ only by 4 amino-acids. Despite the high sequence identity, beta, but not gamma, actin undergoes an N-terminal modification termed arginylation. In this process, the first and second amino acids of actin (Methionine and Aspartic Acid) are removed and an Arginine residue is ligated to the N-terminus of beta-actin such that Arginine becomes the first amino acid of the modified actin. How arginylation of beta actin affects actin function and whether arginylated beta-actin localizes to distinct cellular locations has not been conclusively established. It is unknown if synthetically arginylated gamma-actin can perform functions performed by arginylated beta-actin. It is also unknown if cells require a balance of arginylated and non-arginylated actin or if arginylated actin will suffice for all cytoskeletal functions.

We have developed a method to purify actin isoforms, including arginylated actin isoforms. We will use this system to purify arginylated and non-arginylated beta and gamma actins. We will investigate their biochemical properties as substrates for Arp2/3 and formin mediated actin filament nucleation as well as for myosin dependent gliding. We will label these actins and inject them into mammalian and zebrafish cells to assess if arginylated and non-arginylated actins show differential localizations. We will identify the proteins that interact differentially with arginylated and non-arginylated actin polymers. To address if arginylated gamma-actin can suppress actin cytoskeletal defects of these cells, we will inject or express arginylated gamma-actin in cells defective in the arginyl transferase (Ate1) . Finally, we will generate cells in which all polymerization competent actin are arginylated to address if cells require a balance of arginylated and non-arginylated actins.

Impact Summary

Our research fits the BBSRC strategic priority areas of "Healthy ageing across the lifecourse" as well as "Synthetic biology", and falls under the overall themes of "Bioscience for health" and "Exploiting New Ways of Working". The work will advance fundamental knowledge to improve human health and train a skilled workforce.

Economic competitiveness and quality of life: The work will contribute to scientific knowledge, and provide novel insights into normal functioning of an important cytoskeletal protein relevant to many aspects of eukaryotic cell biology and also to human disease. Therefore, the work can potentially lead to improved health and enhance the quality of life.

Training: A PDRA and RA will be trained in advanced molecular genetics, protein expression, biochemistry, mammalian and zebrafish cell biology, and advanced imaging, which are applicable to academia and industry settings. The PDRA will develop science communication and public engagement skills, and have the opportunity for a wide range of transferable skills courses that are designed to maximize individual potential and employability. This will contribute to the training of a highly skilled workforce.

Communications and public engagement: The public will be made aware of our work through exhibitions, open days, and school visits. We will make our material accessible and suitable for our intended audience.

Collaborations and Co-Production: Data generated from the proposed work will provide sufficient opportunities for future collaborations. The approaches described and our findings will be of interest to researchers in academic and clinical settings, and to industry. For example, cytosolic actin defects have been reported to cause human diseases and thus the work can lead to collaborations with clinicians. We will be alert to such possibilities and establish new collaborations with relevant groups as opportunities arise.

Data Sharing and Dissemination of information: The findings will be disseminated via seminars and presentations at scientific conferences at conferences where academia and industry are represented. Results will be published in appropriate journals and in line with current BBSRC policy. New resources generated during the project will be deposited with a public repository and made available to researchers and industry. Biochemical and proteomic datasets will be shared through publications, and made available via web-based portals.

Resource Generation: New resources generated of interest to other groups/research projects (new plasmids, synthetic proteins) will be made available after publication to industry and academia through public repositories.

Exploitation and Application: If findings of potential commercial interest are identified, we will work with Warwick Ventures, the Technology Transfer Office of the University of Warwick, in order to protect IPR before dissemination, and to explore routes to exploitation via commercial partnerships and pathways.

Impact activity deliverables: The work described will increase our understanding of cytoskeletal organization and function, with potential implications for a variety of human diseases, thus increasing scientific knowledge of value to the clinic and pharma sectors. A significant impact deliverable is outreach and public engagement.