PREreview of Biochemical analysis of deacetylase activity of rice sirtuin OsSRT1, a class IV member in plants

This PREreview is the result of a virtual, live-streamed preprint journal club with 21 participants organized and hosted by PREreview and eLife.

The authors of this preprint have made advances in attempting to understand the functions of plant sirtuins, in this case, OsSRT1 from Oryza sativa indica. Working towards this goal, they have performed biochemical experiments to understand the kinetics of plant sirtuins and the roles of variable C-terminal and N-terminal domains in the activity of the enzyme. Moreover, the authors studied and described the role of OsSRT1 in the deacetylation of histones, H3 and H4, and non-histone target OsPARP1, and the changes in this activity in response to stress conditions, as well as the different substrate specificity between plants and human homologous proteins. The authors used recombinant expression in bacteria to produce the proteins, and used protein biochemistry separation techniques to purify the proteins before biochemical analysis. Furthermore, they performed a pull-down assay using Ni-NTA IMAC coupled with Western blotting to determine protein-protein interactions between their target proteins and potential deacetylated substrates. The N-terminal domain of OsSRT1 was reported to bind weakly to ligands and showed no activity compared to the C-terminal containing constructs. These results suggest the need for the C-terminus in catalysis.

Several participants of the live-streamed preprint review found the possible role of plant sirtuins in the DNA damage repair pathways particularly interesting. During the discussion, a few concerns and questions have emerged, and we would like to share them here in the hope that they can help the authors.

Major issues

• Several of the claims made in the text are not supported by the presented data. The authors should refrain from using the phrase ‘data is unavailable’ and should include supporting data in the Supplementary Information section. For example in Results #2, the authors say “as this enzyme exists as a monomer in solution based on size exclusion data” but the data is not included. The lines “C-terminal domain, which is required for catalysis.“; though the delta-c-terminal shows some activity…” and “as OSTRT1 is localized in the nucleus?” requires data or reference.
• Overall, many of the assays performed by the authors are inadequately described, both in terms of the methodology and the rationale. Please provide a brief summary of the assay before describing the results to help the readers understand what is written or has been done.
• The authors say “Further we have tried to correlate these deacetylation events with different stress conditions” (page 2). Yet the stress experiments are not described in the Methods section. There are no concentrations of chemicals, and time points. The authors need to clearly state in the Methods how experiments presented in Fig. 5 were performed.
• Quantification from a dot blot is not acceptable and requires the authors to state a qualitative relation, rather than a quantitative one.
• Western blots do not have loading controls in some figures (1b, 2a). The authors must add loading controls and show the corresponding uncropped Ponceau-stained gel images. Did the authors quantify the band intensity in figures 1c and 2b? This is not clear. Explanation of the method in more detail would be beneficial.
• Protein purification is only referenced and details are not mentioned. Please discuss the expression and purification protocol in more detail. When a previous publication is referred to for the methodology, it is helpful to still briefly summarize the method in a sentence or two qualitatively.
• Overall this manuscript appears to have multiple tones rather than one universal tone. Editing is suggested to improve on this delivery.
• To do the manuscript justice, the authors should aim to re-write the abstract to include the following in no more than 250 words, (1) a lay introduction to the background and the question they’ve addressed in this manuscript, (2) a non-technical summary of the key experiments and findings from this work, and (3) importance and implications of their findings.

Minor issues

• Some figures are not annotated properly (Fig1A) and format can be improved (Figs 1C, 2A). Please properly indicate the experimental or loading controls. For example in 2B: is it just no enzyme or a different protein with the same enzyme?
• The experiments carried out ‘in vitro’ and ‘in vivo’ could be more clearly delineated throughout the whole manuscript.
• Figures 1 and 2 could be combined. Please place proper labels in all figures and use simplified legends.
• Figures 4A and 4B are not referred to in the main text. The figure titles should be described in the manuscript better (full names of conditions). Lanes in Fig. 1B and 2A should be labeled directly in the figure and not by lane number. Second line of the western blot in Fig. 1A is not labeled.
• ‘uM’ in Y-axes is edited manually post hoc. Please insert proper labels when first generating the graph.
• The ‘H134Y mutant’ is used in different figure panels but the purpose of using it is unclear as it is only mentioned in passing in the Methods section. Please elaborate on why it is being used.
• Please add the reference(s) for this claim in the Introduction section: “RNAi studies show that sirtuins are involved in stress response in rice plant….”
• Please provide data for the quality and stability of the truncated constructs. Additionally, protein gel filtration profiles or SDS-PAGE profiles for purified proteins are missing. Please include them as supplemental data.
• Data for NAD+ requirement for activity could be added in the manuscript.
• Sequence comparison with regards to human homologs could be added.
• Please include the name of the program/software used for homology modeling and the template used for homology modeling.
• “We have performed Ni pull down experiments to study the ability of these OsSRT1 constructs to bind the ligands.” Please clarify what you mean by “ligands” in this specific context.
• Please include the primers and sequence of the DNA constructs in the Methods.
• Including the overall structure of a representative sirtuin with different domains indicated, in both the substrate-free and substrate-bound forms, might help in better appreciating the detailed overview of different sirtuins provided in the introduction.
• The conclusions introduce many new concepts that are not mentioned at the beginning of the manuscript. The authors could introduce these topics in the introductions section to enable the reader to consider them while reading the manuscripts results and findings.
• The last paragraph of the results seems more fitting to be included under conclusions.
• The authors could make the title more specific to the manuscript’s work and findings. The title “Biochemical analysis of deacetylase activity of rice sirtuin OsSRT1, a class IV member in plants” is quite general and does not do full justice to the manuscript.

We thank the authors for sharing their work as a preprint and are very interested in the research findings. We hope our feedback above will be helpful as they consider any revisions to the manuscript or future lines of work.