POT, an optogenetics-based endogenous protein degradation system.

Precise regulation of protein abundance is critical for cellular homeostasis, whose dysfunction may directly lead to human diseases. Optogenetics allows rapid and reversible control of precisely defined cellular processes, which has the potential to be utilized for regulation of protein dynamics at various scales. Here, we developed a novel optogenetics-based protein degradation system, namely Peptide-mediated OptoTrim-Away (POT) which employs expressed small peptides to effectively target endogenous and unmodified proteins. By engineering the light-induced oligomerization of the E3 ligase TRIM21, POT can rapidly trigger protein degradation via the proteasomal pathway. Our results showed that the developed POT-PI3K and POT-GPX4 modules, which used the iSH2 and FUNDC1 domains to specifically target phosphoinositide 3-kinase (PI3K) and glutathione peroxidase 4 (GPX4) respectively, were able to potently induce the degradation of these endogenous proteins by light. Both live-cell imaging and biochemical experiments validated the potency of these tools in downregulating cancer cell migration, proliferation, and even promotion of cell apoptosis. Therefore, we believe the POT offers an alternative and practical solution for rapid manipulation of endogenous protein levels, and it could potentially be employed to dissect complex signaling pathways in cell and for targeted cellular therapies.