## 4 Discussion  
p53 protein levels are mainly regulated by the E3 ubiquitin ligase Mdm2, which triggers p53 ubiquitination and consequent proteasomal degradation. Mdm2 additionally inhibits p53 by binding to its N-terminal transactivation domain (TAD), which is thus not free for contacting the transcription machinery. This second mechanism of p53 inhibition is shared by other proteins, such as MdmX, which do not lead to p53 degradation. As some tumors are characterized by wild type p53 but aberrant overexpression/activity of one or more of its negative regulators, a promising strategy to combat them is to restore normal p53 signaling by stopping the aberrant inhibition the protein is subject to. Peptides that bind to Mdm2/MdmX on the same surface used to bind to p53 have been previously developed and shown to be promising. Here we engineered optogenetic control of the PMI peptide using LEXY, a light-inducible nuclear protein export system previously developed by us. LEXY enables dictating with blue light the  
Figure 3: **LEXY allows controlling with light endogenous p53 and p21 levels. ** (a, b) Western blot showing the levels of p53 (**a**) and p21 (**b**) in HCT166 cells. Mock indicates cells transfected with empty pcDNA3.1(+). \(\beta\)-actin was used as loading control. (**c**). Bar plot showing the quantification of three independent western blots with samples as in (a) and (b). Levels are shown normalized to the levels of the mock, dark control. NS p \(\geq\) 0.05, *** p \(<\) 0.001, _t_-test. localization of any peptide or protein fused to it, in this case PMI and mCherry. This strategy could be applied because we found that PMI needs to be nuclear to function (Figure 1b,c). It was interesting to see that, in HCT166 cells, nuclearly localized PMI led to higher p21 levels than the control p53-mCherry (with or without additional NLS) (Figure 1b,c). We speculate that this may be due to some interference of the fluorescent protein with p53 transcriptional activity. It was surprising to see that the MIP peptide, which was reported to elevate p53 levels in HCT116 cells [37], did not have any effect under our experimental conditions (Supplementary Figure S1). As we did not find in the literature other reports confirming the efficacy of this peptide in living cells beyond the work by [PERSON] and colleagues, we reckon further experiments are needed to clarify this issue. The approach based on LEXY developed here can be used to answer biological questions regarding p53 biology, such as what is the relationship between cellular outcome and p53 levels. The advantage of using light to elevate endogenous p53 levels is that cells with precisely tuned p53 levels can be generated within a single population, making the interpretation of the results easier than when comparing cells cultured separately. Moreover, optogenetics is the best approach to answer questions regarding the effect of the position of cells within a tissue on the cellular outcome, as individual cells can be illuminated. It would be, therefore, possible to study if having higher p53 levels causes different outcomes depending on whether the cells lie in the middle or at the periphery of a tissue. PMI-LEXY represents an alternative to the existing opto-p53 which we previously established [35]. opto-p53 is the fusion of p53 to LEXY (mCherry was included for visualization purposes). This construct is transcriptionally active and allows controlling p53 target genes by controlling directly the localization of p53, not its levels. In this case, p53-LEXY, that is, an exogenous p53 protein, is the main actuator of the response. In the case of PMI-LEXY, instead, endogenous p53 is the main actuator of the response. The approach based on PMI-LEXY mimics the natural situation, while that based on p53-LEXY suffers from the possibility that alternative pathways are triggered by the high cytoplasmic p53 levels which arise during the illumination phase.