Peroxiredoxins: Unveiling the potent antioxidants leading our fight against disease

There is a lot of evidence which shows that oxidative stress and damage is a precursor for, or directly causative to a range of common diseases such as cardiovascular disease, cancer, diabetes, metabolic disorders, atherosclerosis, IBD and more. While ROS (reactive oxygen species) and oxidation mechanisms are solidified in literature, research in regard to our defences against them have been limited by technology. Peroxiredoxins are a family of thiol peroxidases which are highly expressed in almost all living creatures, responsible for ~90% of the H₂O₂ reduction in humans. Peroxiredoxins are already well established as antioxidants, with strong links already supported between peroxiredoxin activity and oxidative stress diseases / ageing. However, the relationship between oligomeric state and in vivo activity is poorly understood. In recent literature, human peroxiredoxins have also been linked to cellular signalling pathways. Until recently, proteins involved in the reduction of ROS, specifically H₂O₂ reduction were only observed in vitro, this due to a variety of confounding factors, with quantitative and qualitative research largely including western blotting, PAGE and cellular lysis, requiring the destruction of the live cells. This created vast limitations in this area of research, as the complicated dynamics of ROS reductases, binding partners, interactions and oligomeric states were not only difficult to study in vitro, but knowledge about them in vivo did not exist.

There are 6 isoforms of peroxiredoxin in humans, with ‘1’ and ‘2’ residing in the cytosol. The subclass ‘2-cys’ peroxiredoxins use the bridging of two cysteine residues on their surface via the formation of disulphide bonds to reduce H₂O₂ (ROS) to water, this disulphide bond formation is associated with a change in oligomeric structure, with wild-type proteins being found in monomeric, dimeric and decameric complexes.

There were three aims within this research to study Prx1 and Prx2. Firstly, fluorescently tagged peroxiredoxins 1 and 2 were produced in human cells using endogenous CRISPR methods, creating genetically modified cell lines that continuously express mNeonGreen fused peroxiredoxins. The second objective involved exogenous methods, over expressing mCerulean fused peroxiredoxin constructs from transfected plasmids. Lastly, western blotting and fluorescence techniques were employed with these fluorescently tagged peroxiredoxins to understand their oligomeric state at basal conditions and in response to H₂O₂ exposure.

Fluorescence microscopy confirmed the successful introduction of a fluorescent probe to endogenous peroxiredoxin 1 and 2 within Hap-1 cells, and western blotting confirmed that the plasmids transfected into HT-1080 cells successfully produced mCerulean fused peroxiredoxin 1 and 2. This helped to establish baseline knowledge about the intracellular interactions between tagged and wild type peroxiredoxins, as well as show differences in behaviour for peroxiredoxins 1 and 2 when exposed to H₂O₂ (oxidising agent). The successful implementation of a fluorescent protein to the N-terminus of both endogenously and exogenously produced peroxiredoxins 1 and 2 will lead to the use of fluorescence methods such as homo-FRET and fluorescence correlation spectroscopy to be used to monitor the proteins, in an attempt to learn more about their interactions and roles in different oligomeric states.