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Referee comments: Referee 1 (Lan Ma)
Posted by PLoS_ONE_Group on 01 Feb 2008 at 17:40 GMT
Referee 1's review (Lan Ma):
07-PONE-RA-02620 RECONSTITUTION OF MDM2-DEPENDENT POST-TRANSLATIONAL MODIFICATIONS OF P53 IN YEAST
The authors studied Mdm2-dependent post-translational regulation of p53 by expressing human p53 and Mdm2 proteins in yeast cell S. cerevisiae. They showed that some basic regulations of p53 by Mdm2 such as binding, ubiquitination and sumoylation were preserved in yeast, even though the p53 and Mdm2 genes are foreign to the yeast genome. These results indicated that the endogenous ubiquitination pathways in yeast could interact with p53 and Mdm2 to implement ubiquitination. In addition, their in vivo microscopy data showed that p53 and Mdm2 co-localized to PML-like nuclear bodies in live yeast cells and that sumoylation of p53 was necessary for this co-localization. In its essence, the paper attempts to reconstitute a reduced system of p53 and Mdm2 in yeast and lay a basis for further reconstructing the p53-Mdm2 regulatory network.
The idea of dissecting the complex p53 network into minimal regulatory modules is appealing --- while some computational work has shown that the overall function of a complex network can be achieved by summing up the functions of its independently operating subsystems, the experimental counterpart like this one is highly desired. It is encouraging to see that some basic Mdm2-dependent regulations of p53 such as ubiquitination and sumoylation can be replicated in a foreign host. Therefore, this work illustrates important potentials for utilizing this system to discover new features of p53 system. My main concern is that the model system appears to be a simplified system that can only be used to study the post-translational regulation of p53 by Mdm2, thus the real significance and potential impact of developing such a system is not yet clear.
This manuscript could be improved by addressing the following points:
(1) Comparing to the natural p53-Mdm2 network, in this system the interaction between p53 and Mdm2 is just one-directional. That is, only Mdm2 can regulate p53 but not vice versa. As a consequence, this system cannot complete the negative feedback loop which has been shown to be vital for the proper functioning of p53. This seems to be a big hurdle to overcome in order to in the future study the dynamics of p53 such as stress-induced oscillations. As stated in the last paragraph "Importantly, we have now set the basis for further reconstruct the p53-Mdm2 network", building this simple system is just a starting point for including new interactions and/or components in the p53 network so that more features can be recapitulated. Yet, it is not clear to this reviewer how the reconstructing of p53-Mdm2 network can be achieved. The authors should give some concrete suggestions on what further experimental steps could be taken along this line to implement the negative feedback loop.
(2) Kwek et al Oncogene (20), 2587 (2001) showed that p53 mutated at lysine 386 presented a similar pattern of intranuclear localization as WT p53 in 293, MCF7 and HT1080 cells. However, data in this paper showed that the nuclear localization of lysine 386 mutated non-sumoylatable p53 is abolished. Does it mean that the nuclear localization mechanism of p53 behaves in a different way in the reconstituted system here than in other natural p53 host cells? The authors should discuss this discrepancy.
(3) Figure 5D shows that non-sumoylatable p53 was distributed across the whole cell when co-expressed with Mdm2. Can the authors speculate the possible reason why non-sumoylatable p53 is expelled out of nucleus? We know that WT p53 alone is localized to nucleus (Figure 1 C & D), how about the distribution of non-sumoylatable p53?
(4) Comparing Figure 3B and Figure5D, it appears that the distribution of p53 and Mdm2 has almost the same pattern. In Figure 3B, the binding of p53 is impaired while in Figure 5D p53 is non-sumoylatable. The authors should point out that this similarity between Figure3B and 5D is actually due to the correlation between the steps of binding and sumoylation in the regulatory network of p53. That is, proper binding between p53 and Mdm2 is necessary for sumoylation of p53 as shown in Figure 5B and other literature such as Chen & Chen, Oncogene (22) 5348 (2003).
(5) In Figure 3, it would be helpful to show an immunoelectron microscopy figure of cells expressing only WT p53, which could demonstrate whether the distribution of WT p53 alone in the nucleus is doughnut-like shape or not. Addition of this information could help readers better understand how the particular structure of p53-Mdm2 complexes is formed when p53 and Mdm2 are co-expressed.
(6) Figure 4B shows that when WT p53 is co-expressed with PML, instead of largely localized to nucleus as WT p53 alone does (Fig.3A), it seems to diffuse across the whole cell. Does this data indicate that PML inhibit the localization of p53 to nucleus? Combining the data that p53-Mdm2 localizes to the nucleus when p53, Mdm2 and PML are co-expressed (Fig. 4D), can we infer that Mdm2 can counter-act the negative effect of PML on p53? Some clarification would be helpful.
(7) Figure 1 shows that WT p53 is degraded when co-expressed with WT Mdm2. Was p53-ECFP also degraded by Mdm2-EYFP? If so, how did this degradation of p53 affect the nuclear co-localization of p53-Mdm2? Was the co-localization of p53-Mdm2 stable over time or not?
N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication the manuscript has been revised in light of these comments and to address other editorial requirements.