p53 isoforms have a high aggregation propensity, interact with chaperones and lack binding to p53 interaction partners

Reviewer #1 (Public review):

Summary:

Brdar, Osterburg, Munick, et al. present an interesting cellular and biochemical investigation of different p53 isoforms. The authors investigate the impact of different isoforms on the in-vivo transcriptional activity, protein stability, induction of the stress response, and hetero-oligomerization with WT p53. The results are logically presented and clearly explained. Indeed, the large volume of data on different p53 isoforms will provide a rich resource for researchers in the field to begin to understand the biochemical effects of different truncations or sequence alterations.

Strengths:

The authors achieved their aims to better understand the impact/activity of different p53 is-forms, and their data will support their statements. Indeed, the major strengths of the paper lie in its comprehensive characterization of different p53 isoforms and the different assays that are measured. Notably, this includes p53 transcriptional activity, protein degradation, induction of the chaperone machinery, and hetero-oligomerization with wtp53. This will provide a valuable dataset where p53 researchers can evaluate the biological impact of different isoforms in different cell lines. The authors went to great lengths to control and test for the effect of (1) p53 expression level, (2) promotor type, and (3) cell type. I applaud their careful experiments in this regard.

Weaknesses:

One thing that I would have liked to see more of is the quantification of the various pull-down/gel assays - to better quantify the effect of, e.g., hetero-oligomerization among the various isoforms. In addition, a discussion about the role of isoforms that contain truncations in the IDRs is not available. It is well known that these regions function in an auto-inhibitory manner (e.g. work by Wright/Dyson) and also mediate many PPIs, which likely have functional roles in vivo (e.g. recruiting p53 to various complexes). The discussion could be strengthened by focusing on some of these aspects of p53 as well.

Reviewer #2 (Public review):

Summary:

In this manuscript entitled "p53 isoforms have a high aggregation propensity, interact with chaperones and lack 1 binding to p53 interaction partners", the authors suggest that the p53 isoforms have high aggregation propensity and that they can co-aggregate with canonical p53 (FLp53), p63 and p73 thus exerting a dominant-negative effect.

Strengths:

Overall, the paper is interesting as it provides some characterization of most p53 isoforms DNA binding (when expressed alone), folding structure, and interaction with chaperones. The data presented support their conclusion and bring interesting mechanistic insight into how p53 isoforms may exert some of their activity or how they may be regulated when they are expressed in excess.

Weaknesses:

The main limitation of this manuscript is that the isoforms are highly over-expressed throughout the manuscript, although the authors acknowledge that the level of expression is a major factor in the aggregation phenomenon and "that aggregation will only become a problem if the expression level surpasses a certain threshold level" (lines 273-274 and results shown in Figures S3D, 6E). The p53 isoforms are physiologically expressed in most normal human cell types at relatively low levels which makes me wonder about the physiological relevance of this phenomenon.

Furthermore, it was previously reported that some isoforms clearly induce transcription of target genes which are not observed here. For example, p53β induces p21 expression (Fujita K. et al. p53 isoforms Delta133p53 and p53beta are endogenous regulators of replicative cellular senescence. Nat Cell Biol. 2009 Sep;11(9):1135-42), and Δ133p53α induces RAD51, RAD52, LIG4, SENS1 and SOD1 expression (Gong, L. et al. p53 isoform D113p53/D133p53 promotes DNA double-strand break repair to protect cell from death and senescence in response to DNA damage. Cell Res. 2015, 25, 351-369. / Gong, L. et al. p53 isoform D133p53 promotes the efficiency of induced pluripotent stem cells and ensures genomic integrity during reprogramming. Sci. Rep. 2016, 6, 37281. / Horikawa, I. et al. D133p53 represses p53-inducible senescence genes and enhances the generation of human induced pluripotent stem cells. Cell Death Differ. 2017, 24, 1017-1028. / Gong, L. p53 coordinates with D133p53 isoform to promote cell survival under low-level oxidative stress. J. Mol. Cell Biol. 2016, 8, 88-90. / Joruiz et al. Distinct functions of wild-type and R273H mutant Δ133p53α differentially regulate glioblastoma aggressiveness and therapy-induced senescence. Cell Death Dis. 2024 Jun 27;15(6):454.) which demonstrates that some isoforms can induce target genes transcription and have defined normal functions (e.g. Cellular senescence or DNA repair).

However, in this manuscript, the authors conclude that isoforms are "largely unfolded and not capable of fulfilling a normal cellular function" (line 438), that they do not have "well defined physiological roles" (line 456), and that they only "have the potential to inactivate members of the p53 protein family by forming inactive hetero complexes with wtp53" (line 457-458).

Therefore, I think it is essential that the authors better discuss this major discrepancy between their study and previously published research.