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

  1. Nilabhra Mitra
  2. Sanghamitra Dey

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Abstract

The role of plant sirtuins is slowly unwinding. There are only reports of H3K9Ac deacetylation by OsSRT1. This belongs to class IV sirtuin family with a longer C-terminus. Here C-terminus is required for ligand binding and catalysis. OsSRT1 can deacetylate the lys residues at the N terminal tail of both H3 and H4. It can also target the non-histone target, OsPARP1 playing a role in DNA damage repair pathway. Changes in the extent of different histone deacetylation by OsSRT1 is also related with different abiotic stress conditions. NAM and ADP-ribose has negative effect on OsSRT1 deacetylation.

Highlights

  • OsSRT1 is capable of deacetylating various lysine residues of histone H3 and H4 as well as OsPARP1.

  • The extra long C-terminus of OsSRT1 is required for its substrate binding and thus, its catalysis.

  • On plant’s exposure to H 2 O 2 and Arsenic toxicity, there is a relationship between increased expression of OsSRT1 and increased deacetylation of H3 and H4.

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    1. PREreview

      This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/6780154.

      Mitra, N. and Dey, S. have biochemically characterized the deacetylase activity of rice sirtuin, OsSRT1, and provided a glimpse of its role in the context of abiotic stresses in plants. They have asked fundamental questions about the role of different domains of this protein in its enzymatic function and addressed those using mutagenesis and biochemical assays. However, I have a few concerns about some results. I have described these below:

      General

      1. Towards the end of the Introduction, it is said that "OsSRT1 is capable of influencing the DNA repair pathway as the constituent proteins involved in this pathway can interact with OsSRT1 and eventually get deacetylated". However, this study does not unambiguously show the effect of OsSRT1 on DNA damage repair pathways. Thus, I suggest conducting additional experiments (such as single cell RNA-sequencing to show OsSRT1 mediated upregulation of DNA damage response genes when subjected to abiotic stress or fluorescence-based experiments to show OsSRT1 mediated increase in recruitment of DNA damage repair enzymes to the damage site) to show the role of OsSRT1 on DNA damage repair pathways. Alternatively, the authors should consider removing this aspect from the Introduction but elaborating more on this in the Conclusions / Discussions section.
      2. In Figure 1A, the labels for sample and control are missing. Also, the figure legend lacks key details about the experiment. For example, it does not explain what H134Y is, whether it is a mutation on OsSRT1 or on H3, and what it contributes to the results. In Figure 1B, the deacetylation of several acetylated H3 lysine residues is shown. This figure also provides the histone deacetylation by HsSIRT6 as a control. Here, adding a lane showing the H3 deacetylation by HsSIRT7 will provide a more complete picture. Similarly, adding HsSIRT6 and HsSIRT7 mediated H4 deacetylation as controls in Figure 2A will provide a better picture.
      3. The text points out the specificity of OsSRT1 towards NAD+ for its catalytic function. This is a rather interesting finding and I believe, much like myself, many readers would like to look at the results of this experiment. I would strongly recommend authors to add this result as a figure. Also, addressing the structural basis for this specificity might be an aspect that can be explored further.
      4. In this study, the effects of various drugs, such as resveratrol on the activity of OsSRT1 have been studied. However, the motivation for testing these drugs and an interpretation of the findings is missing.
      5. Figure 4B requires a more detailed legend. Again, the reason for using H134Y is not mentioned. Also, the text does not talk about the activity of these constructs on PARP1. I believe a reader would like to see a comparison between the activities of these constructs, from OsSRT1 and OsSRT2. What additional function does the longer C-terminus of OsSRT1 carry out? Do both OsSRT1 and OsSRT2 exhibit similar deacetylation substrate specificities? Which of the domains confer the deacetylation substrate specificity to these proteins?

       

      Minor comments

      1. The abstract lacks a clear distinction between what was previously known and what this study contributes to existing knowledge. I would suggest to add a few sentences to the end of the abstract emphasizing the downstream implications of these findings.
      2. In the second line of Introduction, it is said that: "Among all the eukaryotes, human sirtuins are the most studied proteins" which is misleading. I would recommend rephrasing it to something like "Sirtuins from humans are among the most studied isoforms of this protein family".
      3. In the second paragraph of the Introduction, there is a section that says "Most of the plants, monocots and dicots, have these two members (SRT1 and SRT2), except maize having only one sirtuins, SRT1. This class III member is also present in few other plants, including rice". Here, the second sentence is hanging as this sentence leaves the reader confused if rice and the most other plants mentioned here have only SRT1? Following this, it is said that "OsSRT1 (483aa) has homology with both HsSIRT6 (355aa) and HsSIRT7 (410aa), thus, belonging to class IV sirtuin family" which further leaves a confusion whether SRT1 is a class III or a class IV sirtuin. Also, I would further suggest adding a few sentences to the introduction, summarizing the differences between the different classes of sirtuins.
      4. The residue numbers for CC, ΔN1 and ΔN2 does not match between the text and Figure 4A. Figure 4C is cited before Figure 4B.
      5. In various places in the manuscript, the phrase "OsSRT1 deacetylation" is used. This phrase is misleading as it seems to point towards the deacetylation of OsSRT1, which is not the case here. I would suggest to change it to something like "OsSRT1 mediated deacetylation".
      6. In the Conclusions section, it is difficult to follow what previous knowledge does this study build on and what are the key insights from this study. However, here, the implications of the study are well highlighted.
    2. PREreview

      This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/6323771.

      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.

    3. Version published to 10.1101/2021.11.05.467508v1 on bioRxiv