Opto-MDMi: a dual-lock optogenetic system for robust activation of endogenous p53
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eLife Assessment
This manuscript describes an important development of several variants of optogenetic tools to control endogenous p53 activity. They are based on peptides competing with Mdm2/MdmX for binding to p53, thus releasing p53 from its negative regulators and stabilizing its cellular levels. In principle, the data are convincing but should be complemented by investigations of p53 target genes at endogenous levels (instead of only reporter constructs). The study therefore remains incomplete but will be of interest to scientists working on optogenetics as well as the p53 field.
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Abstract
Optogenetics has emerged as a powerful technology for manipulating biological functions with high spatiotemporal resolution, yet the precise control of endogenous molecules remains a significant challenge. In this study, we developed Opto-MDMi, a dual-lock optogenetic platform designed to control the activity of endogenous p53, a master regulator of cell cycle and apoptosis. The p53 pathway is strictly governed by its negative regulators, MDM2 and MDMX, which inhibit p53 through direct binding and ubiquitination. Our system integrates two distinct light-responsive modules: Opto-MDMi (LOVTRAP), which regulates the nuclear translocation of p53-activating peptides, and Opto-MDMi (LOV2-PMI), which controls the binding activity of these peptides by photocaging them within the AsLOV2 domain. Through extensive in vitro screening and live-cell assays, we discovered that truncating the Jα helix of LOV2 effectively restricts the movement of fused inhibitory peptides, thereby masking their interaction with MDM2/MDMX under dark conditions. By combining these two regulatory layers into a dual-lock system, we achieved robust light-dependent activation of endogenous p53 while significantly suppressing basal activity in the dark. Our findings not only provide a potent tool for p53 research but also establish a general design principle for optogenetically regulating functional peptides with the LOV2 domain, offering a versatile framework for the future development of optogenetic actuators.
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eLife Assessment
This manuscript describes an important development of several variants of optogenetic tools to control endogenous p53 activity. They are based on peptides competing with Mdm2/MdmX for binding to p53, thus releasing p53 from its negative regulators and stabilizing its cellular levels. In principle, the data are convincing but should be complemented by investigations of p53 target genes at endogenous levels (instead of only reporter constructs). The study therefore remains incomplete but will be of interest to scientists working on optogenetics as well as the p53 field.
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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, …
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.
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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 …
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.
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