PREreview of Heterooligomerization drives structural plasticity of eukaryotic peroxiredoxins

This review resulted from the graduate-level course "How to Read and Evaluate Scientific Papers and Preprints" from the University of São Paulo, which aimed to provide students with the opportunity to review scientific articles, develop critical and constructive discussions on the endless frontiers of knowledge, and understand the peer review process.

Peroxiredoxins are essential thiol peroxidases, with functions that include detoxification, redox signaling and chaperone activity. Present throughout the eukaryotic kingdom, these proteins are known to form dimers and homooligomers. In this work, the authors show the formation of heterooligomers between different Prx isoforms. Furthermore, their stability and enzymatic activity was characterized. This phenomenon was confirmed in yeast and human cells indicating it may be widespread amongst eukaryotes. This is, in sum, a very interesting work with a very important topic that can have many consequences for the understanding of the role of peroxiredoxins in normal and stress conditions to the cell. The heterooligomers could be a way to modulate the Prx activity and function in redox signaling and as chaperones. We appreciate how the authors took care to approach the topic from different perspectives, i.e in yeast and human cells and also with kinetics experiments. This increases the robustness of the conclusions presented in the paper. Notwithstanding, we have some minor suggestions to offer to the authors’ consideration. For the sake of clarity we have divided the observations between experimental suggestions and minor textual and graphical suggestions.

Experimental suggestions

• For the experiment with HEK293 WT: Is it possible to analyze the heterooligomer fractions with proteomic tools to identify and distinguish the heterooligomers with different stoichiometric ratios?;

• In the topic “Native human PRDX1 and PRDX2 form heterooligomers in HEK293 cell” experiments were done to assess the heterooligomer enzymatic activity in cells exposed to H2O2 using roGFP2 assay. We were wondering if it is possible to study this condition using proteomic analysis to see the abundance of heterooligomers and compare to a non exposed condition and maybe also identify the stoichiometric ratios in both conditions;

• In Western blot experiments, it should be specified whether equivalent amounts of protein were loaded in each lane to ensure comparability of the results. We understand that this is presented in the supplementary material figure 2, however given the relevance of this information, we believe it should be moved to the method section;

• The co-expression of Tsa1 and Tsa2 tagged with different tags (His6 and Strep) was considered, and sequential purifications were performed to detect potential interactions. However, we would like to suggest that a negative control be added to Figure 2. This can be something like the purification of an unrelated protein tagged with Strep to verify the specificity of the observed interaction. The absence of such controls makes it difficult to interpret the results, as it does not allow for the exclusion of nonspecific interactions or purification artifacts;

• In the section "Heterooligomerization enables remarkable structural plasticity" the authors explore the structural diversity of heterodecamers formed by Tsa1 and Tsa2. To further this analysis, it would be valuable to conduct additional experiments, such as X-ray crystallography, to identify the specific structures of the Tsa1-Tsa2 heterodecamers that actually form in cells. Moreover, investigating whether the different heterodecamer conformations exhibit distinct enzymatic activities could provide a deeper understanding of the biological and functional relevance of the heterooligomerization between Tsa1 and Tsa2;

• If possible, it would be nice to see the deconvoluted CD spectra of the different heterodimers to better evaluate the structural differences.

Minor suggestions

• The different heterooligomers in figure 3 C are hard to distinguish as they are represented in similar shades of green. If possible shapes could be added to the lines to aid differentiation;

• We would like to kindly suggest, if it is something the authors are interested in, some reading on colour accessibility:

I)https://www.ascb.org/science-news/how-to-make-scientific-figures-accessible-to-readers-with-color-blindness/

II)https://davidmathlogic.com/colorblind/#%23D81B60-%231E88E5-%23FFC107-%23004D40-%236A1A1A

• In Figure 5 D it is again really hard to distinguish the lines;

• The legend of Figure 3 D has really light blue lettering in the legend that is hard to read as it is difficult to make out from the white background. One possibility to resolve this is using black lettering as in the legend of figure 5, for it stands out better (Our point about the distinguishability of the lines themselves still stands). Another possibility would be to keep the coloured lettering and add a black outline to the text so that it pops out better from the white background;

• Figures 2 and 4 have legends that do not match the panels. 2 has an explanation for panel C although there are only panels A and B. Figure 4 is a little more difficult to parse, perhaps the B is misplaced? There are also no panels C to F in the image. The yeast genomic representations may benefit from a more detailed legend.

  We wish the authors the best, and good luck throughout the publishing process.

  Domenico Riolfi Barzotto, Júlia Oliveira Castro and Felipe Alves Garcia 

Competing interests

The authors declare that they have no competing interests.