The full idea behind our project and its impact.
We propose utilizing this fusion protein in sunscreen, so it can be easily accessed and applied. Current forms of protection from UV radiation in sunscreen are through UV filters, such as benzophenone [1]. Although sunscreens are still effective in protecting against UV radiation, they only mitigate UV-induced damage and don’t typically reverse already existing damage.
For our project, we decided to focus on maximizing the ability of photolyase, an enzyme found in various bacteria and plant species, to repair common types of DNA damage, particularly cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts, within human skin cells. We plan to combine a cell penetrating peptide (specifically the TAT peptide from the HIV virus), a nuclear localization signal (from the SV40 Large T-antigen), and either a 6-4 photolyase protein (from Arabidopsis thaliana) or a CPD photolyase protein (from zebrafish) in one fusion protein, with linker sequences between each component to allow the protein to fold properly. The cell penetrating peptide will allow the fusion protein to enter the cell through ionic interactions with the cell membrane [2]. The TAT peptide in particular has been found to have the ability to penetrate human skin cells [3] Then, the NLS will guide the protein to the nucleus, which will be the main location of the DNA damage. [4]. Finally, the photolyase protein, depending on whether it is CPD photolyase or 6-4 photolyase, will repair a certain kind of DNA damage caused by UV radiation [5]. We will be using the common linker amino acid sequence GGGGS in between the three parts [6].
By combining these three components, we can create a system where the protein can be produced by bacteria and enter the cell on its own. It will then be able to repair the DNA damage caused by UV radiation itself rather than merely preventing it, as opposed to the pre existing approaches to this issue. We plan to incorporate this fusion protein into sunscreen so that, in combination with preventative measures, it will protect both astronauts and people on Earth from the growing problem of UV radiation. Although sunscreens with photolyase already exist, there is not much benefit that the photolyase adds as of yet, though their existence shows that photolyase, despite not naturally occurring in humans, does not negatively affect human skin [7].
Other proteins in human skin, such as fibronectin, have been found to be acting as natural sunscreens [8]. However, in the case of the fusion protein, we may need to employ another protein delivery system in order to ensure that it is able to make it inside the skin cells. We may be able to experiment with using polymer matrices, including collagen hydrogels, to carry the proteins and protect them from proteolysis. Collagen is already very biocompatible, although it does degrade quickly. Collagen I monomers can form cross-linking structures [9], and the fusion proteins can be protected from within these cross-linking structures and diffuse into the skin [10]. Other hydrogels have been successfully integrated into sunscreens as well, particularly hyaluronic acid and tannic acid hydrogels [11], so this could be applied to collagen hydrogels as well.
If we wanted to implement fusion proteins in sunscreen, we would likely need to test its efficacy on bacteria, and then mice. We would need to thoroughly test it to ensure that there are no harmful side effects. We would also need to test to ensure that the collagen hydrogel can effectively protect the proteins from degradation, and that the proteins would actually enter the cell. If we created a sunscreen product, it would typically take about 6 months to a year in order for it to get approved [12].
[1] Amar, S. K., Srivastav, A. K., Dubey, D., Chopra, D., Singh, J., & Mujtaba, S. F. (2019). Sunscreen-induced expression and identification of photosensitive marker proteins in human keratinocytes under UV radiation. Toxicology and Industrial Health, 35(7), 457-465. https://doi.org/10.1177/0748233719862128
[2] Vives, E., Richard, J., Rispal, C., & Lebleu, B. (2003). TAT peptide internalization: Seeking the mechanism of entry. Current Protein & Peptide Science, 4(2), 125-132. https://doi.org/10.2174/1389203033487306
[3] Johnson, J. L., Lowell, B. C., Ryabinina, O. P., Stephen Lloyd, R., & McCullough, A. K. (2011). TAT-Mediated delivery of a DNA repair enzyme to skin cells rapidly initiates repair of uv-induced DNA damage. Journal of Investigative Dermatology, 131(3), 753-761. https://doi.org/10.1038/jid.2010.300
[4] Kalderon, D., Roberts, B. L., Richardson, W. D., & Smith, A. E. (1984). A short amino acid sequence able to specify nuclear location. Cell, 39(3), 499-509. https://doi.org/10.1016/0092-8674(84)90457-4
[5] Liu, Z., Wang, L., & Zhong, D. (2015). Dynamics and mechanisms of DNA repair by photolyase. Physical Chemistry Chemical Physics, 17(18), 11933-11949. https://doi.org/10.1039/c4cp05286b
[6] Trinh, R., Gurbaxani, B., Morrison, S. L., & Seyfzadeh, M. (2004). Optimization of codon pair use within the (GGGGS)3 linker sequence results in enhanced protein expression. Molecular Immunology, 40(10), 717-722. https://doi.org/10.1016/j.molimm.2003.08.006
[7] Miot, H. A., Miot, L. D. B., Silva, M. G., & Marques, M. E. A. (2009). Physiopathology of ultraviolet radiation. Anais Brasileiros de Dermatologia, 84(4), 335-345. https://www.scielo.br/j/abd/a/C3kY4LS3BdqYbdjcK5hrbVf/
[8] D’Orazio, J., Jarrett, S., Amaro-Ortiz, A., & Scott, T. (2013). UV radiation and the skin. International Journal of Molecular Sciences, 14(6), 12222-12248. https://doi.org/10.1016/j.redox.2015.04.003
[9] Du, L., Fan, M., & Mei, H. (2021). Application of nanotechnology in biofilm engineering: Methods and prospects. Microbial Biotechnology, 13(5), 100098. https://doi.org/10.1016/j.mtbio.2021.100098
[10] Deming, T. J. (2007). Polypeptide materials: New synthetic methods and applications in biomaterials. Progress in Polymer Science, 32(8-9), 858-875. https://doi.org/10.1016/j.prsogpolymsci.2007.04.001
[11] Xu, L., Zhang, C., Zhang, Y., Yan, X., & Wang, Y. (2021). The application of biopolymers for skin repair and protection: Recent advances in biopolymer technology. International Journal of Biological Macromolecules, 182, 243-259. https://doi.org/10.1016/j.ijbiomac.2021.09.169
[12] Prime Matter Labs. (n.d.). What to expect when bringing a sunscreen product to market. Prime Matter Labs. https://www.primematterlabs.com/resources/what-to-expect-when-bringing-a-sunscreen-product-to-market