p53 Interacts with VDAC1, Modulating Its Expression Level and Oligomeric State to Activate Apoptosis

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

The revised manuscript has addressed most of my technical concerns.

Comments on the Quality of English Language

fine

Author Response

Response : The manuscript was edited by a professional English-language editor

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

The authors have addressed most of the issues, and they added several expriments to provide more results to support the conclusion. I think the manuscript are better qualified now with stronger evidence and improved presentation. So I think it is suitable to be published on Biomolecules now. 

Author Response

 The figures are high quality; however, they may not appear at their full resolution in the website version.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript from Shoshan-Barmatz et al describes the interaction of the p53 tumor suppressor with the mitochondrial pore complex protein VDAC1.  It suggests that p53 drives VDAC1 complex formation, activation, and promotes apoptosis.  The paper is well written, and the data mostly supports the conclusion.  The contribution to the field may be limited as many of these observations have been previously published.  The manuscript might benefit from a more complete consideration of previous work.  It might also be worth including a discussion of the relevance of VDAC2 and 3 in these processes.  

 

Specific issues:

Not all figures Some clearly state the number of replicates and a description of statistical significance.   

It is unclear if the VDAC1 siRNA is specific or also suppresses VDAC2 and 3.

Parts of Figure 2 are not particularly convincing.  The over-expressed GFP-p53 appears to be primarily cytoplasmic/mitochondrial.  However, the endogenous p53 by IHC appears to be almost all nuclear in all the cells (Hela presumably).  An explanation for this apparent discrepancy is important.  The interpretation of the results as suggesting nuclear localization of p53 when VDAC is suppressed could be mistaken as it could also be stabilization or enhanced expression of p53.  A fractionation/Western blot experiment might be decisive.  It is possible that Hela cells may not be the best system to examine endogenous p53 due to the presence of papilloma virus proteins.  

Figure 3- the levels of p53 in the Western blot suppressed for VDAC do not appear to correlate well with the quantification.   The quantification suggests and increase, the western blot gives the appearance of a decrease.  Can this be explained ?

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, Gigi and co-workers reveal the pathway that p53 induces cell apoptosis through modulation the expression and oligomerization of VDAC1. The p53 is a well-known unit in cell fate regulation and tumor genesis, and the VDAC1 is also important in mitochondria-related biological functions. However, I think the data and evidence showed in this paper is not sufficient to support the conclusion. There are a lot of hypothesis in the manuscript based on amphibolous image and data. For example, Co-IP experiment or colocalization immunofluorescent assay is more solid to confirm the interaction between p53 and VDAC1. In figure 2a, the morphology of mitochondria in Hela cells looks weird, it should not aggregate around the nuclei. In Figure 2d, they claim the p53 transfer into nuclear position after KD of VDAC1, but it seems just some fluorescent intensity change, not the localization. In Figure 4a they used AV-PI staining and FACS analysis, same as what they did in Figure 3c, why the parameter in the presented FACS result are the size of the cells but not the fluorescent signal of the AV-PI? In Figure 5, a band of VDAC1 with DTT and without any p53 is necessary to confirm the effect of the reducer. Overall, I believe there are many more evidences should be collected to make the conclusion more reliable, especially for such an important mechanism. 

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript investigates the interaction between p53 and VDAC1, highlighting the role of p53-induced oligomerization of VDAC1 in promoting apoptosis. While the authors present multiple lines of evidence supporting the oligomerization of VDAC1 in the presence of p53, several critical issues remain that must be addressed prior to consideration for publication. Detailed comments are outlined below:

1. Typographical Error:
• Line 93: "vDAC1" should be corrected to "VDAC1."
2. Methodological Concerns with Crosslinking Experiments:
• Line 185: The description of the crosslinking experiments lacks clarity. Specifically, the manuscript does not explain how protein concentrations in cells were measured prior to crosslinking. Accurate quantification is essential for interpreting these experiments and ensuring reproducibility.
3. VDAC1 Extraction Method:
• Lines 216–220: VDAC1 is an integral membrane protein. The authors need to clarify how VDAC1 was extracted from cells without solubilizing the membrane, as the current description is insufficient.
4. Purification Method Detail:
• Line 239: While the authors refer to previous literature for the VDAC1 purification protocol, a brief description should be included in the methods section to ensure reproducibility and clarity for readers unfamiliar with the referenced work.
5. VDAC1 Conductance Interpretation:
• Lines 295–296: The observed reduction in VDAC1 conductance in the presence of p53 is ambiguous. It is unclear whether this decrease results from stabilization of the channel in a low-conductance state or from oligomerization (especially if multiple functional channels were reconstituted into the bilayer). Further clarification is needed.
6. Figure 5 – Controls and Data Representation:
• The binding experiments in Figure 5 lack appropriate positive and negative controls, making it difficult to distinguish between specific and non-specific interactions between p53 and VDAC1. These controls are essential before any firm conclusions about direct interaction can be drawn.
• Panel D: The channel traces should include expanded segments to clearly demonstrate channel opening and closing events. Currently, the traces suggest the channels may be constitutively open, which requires clarification.
7. Figure Legend Clarity:
• Line 306: There appears to be either a typographical error or an unexplained discrepancy in the legend. If the data in panels A and B were fitted, the authors must explain the fitting approach.
8. Oligomerization and Conductance Relationship:
• Line 313: It is unclear how p53-induced oligomerization leads to decreased conductance, especially if only one or two channels are present in the membrane. This mechanism requires further explanation and experimental validation.
9. Figure 2, Panel D – Transfection Variability:
• Line 340: There is a double scale label in panel D, which should be corrected. Additionally, as these are transfection-based experiments, the authors should include staining or quantification of VDAC1 expression to confirm consistent silencing. Transfection efficiency can vary between experiments and must be controlled.
10. Figure 5 – Western Blot Interpretation and Quantification:

• The western blot data raise several concerns:
• Western blotting can be affected by transfer efficiency, antibody quality, and imaging exposure time. To validate the oligomerization results, Coomassie-stained gels should be included as a more reliable method of total protein quantification.
• The molecular weights of the observed bands do not correspond to expected oligomeric forms. Given that monomeric VDAC1 is ~35 kDa, dimers and tetramers should appear at ~70 kDa and ~140 kDa, respectively. However, the presented blots show dimers around ~60 kDa and tetramers below 100 kDa, which is inconsistent and may indicate crosslinking artifacts or gel inconsistencies.
• The total protein amount appears to differ significantly between uncrosslinked and crosslinked samples, even though equal amounts were reportedly used. This discrepancy needs to be resolved, and proper quantification of total protein across conditions should be performed to support the conclusions.