Abstract
Peroxiredoxins are thiol peroxidases which reduce hydrogen peroxide (H2O2) to water in our cells. They play an important role in cell signalling, protect macromolecules from oxidative damage, and are implicated in aging and multiple diseases. The catalytic mechanism of peroxiredoxins is well understood; however, their active oligomeric state within cells remains uncertain. Peroxiredoxins have been proposed to dynamically switch between a dimeric state and a decameric/dodecameric ring composed of five/six dimers. Factors influencing this potential switch could be a combination of the protein’s oxidation state, concentration, environmental pH or post-translational modifications.
Our aim is to develop tools to track the oligomeric state dynamics of human peroxiredoxin 2 (Prx2) in the cytoplasm of living cells. A synthetic yeast surface display library was screened with decameric Prx2 or a mutant, dimeric, Prx2 to discover nanobodies that specifically recognise either oligomeric state. Three nanobodies that specifically bind to dimeric but not decameric Prx2 were identified via co-elution size exclusion chromatography (SEC). Immunoprecipitation followed by western blotting found that two of these nanobodies can selectively isolate Prx2 from HEK293 cell lysates.
An alternative approach was used to develop binders for the Prx2 decamer, based on the BindCraft pipeline which leverages AlphaFold2, MPNN and PyRosetta to hallucinate novel protein binders to a specified target. A pool of potential binders has been identified and will be transformed into yeast and screened for binding to the Prx2 decamer. Successful binder sequences identified from the pool will be expressed in E. coli and their binding assessed using SEC.
These oligomeric state-specific binding molecules will enable us to study the dynamics of Prx2 in living cells, and identify the influence oligomeric state has on the facilitation of H2O2 signalling, or their oxidative protection role. This information will advance our knowledge of the molecular mechanisms behind the roles of Prx2 in disease and ageing contexts.