Opto-MDMi: a dual-lock optogenetic system for robust activation of endogenous p53

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors apply the AsLOV2 domain to control the localisation and the exposure of two peptides (PMI and PMI-M3) that compete with Mdm2/MdmX for binding to p53, thus freeing p53 from these negative regulators and allowing its levels to rise. The authors follow an established strategy in optogenetics, which is to combine two layers of regulation for tighter control: (1) caging the peptide into the Ja helix of AsLOV2; 2) sequestration of the peptide away from its site of action using the LOVTRAP system.

Strengths:

The authors show that a reporter is activated when cells are exposed to light. A strength is in the lower background that was achieved after adding the second layer of regulation.

Weaknesses:

This study claims to be focused on the control of endogenous p53; however, endogenous p53 levels are not quantified. Moreover, endogenous p53 target genes are also not analysed. Only a synthetic reporter is quantified, which has been placed in the genome of HCT116 cells after the creation of a stable cell line. Microscopy images show only one or a maximum of two cells. Finally, the authors claim their strategy is a general one that can be applied to control other peptides, but they do not show this generality in this paper.

Reviewer #2 (Public review):

The authors developed Opto-MDMi, an optogenetic system for light-controlled activation of endogenous p53. The main idea is to target the p53-MDM2/MDMX regulatory interaction using PMI inhibitory peptides. This is a nice strategy because it avoids overexpression of p53, which can have adverse effects that might confound the study of p53 activity. The authors first tested a LOVTRAP-based localization strategy, which showed some efficacy but also showed basal activation. They then developed a LOV2-PMI peptide-caging module to control the activity of the PMI peptide itself, testing for interactions first in vitro and then in vivo. Finally, they combined the two systems into a dual-lock design, where LOVTRAP controls localization and LOV2-PMI controls peptide activity. This combination led to somewhat more potent stimulation of p53 activity.

Another useful aspect of the paper is the detailed description of the development and testing of the LOV2-PMI peptide-caging module, which may aid in the design of other LOV2-based peptide-caging designs.

Strengths

Overall, the paper is novel and rigorous, and the claims are supported by the data. The optoMDMi tool seems ready for implementation, for example, to manipulate and study the role of p53 signaling dynamics. A few points of clarification would strengthen the work.

Weaknesses

The authors develop many tool variants, but there is some lack of clarity over how all of these tools compare to each other, and which ones interested users should use. The work would also be strengthened by showing modulation of endogenous p53 in more than one cell line.