Protein homeostasis is critical for cellular function and is maintained through a balance of protein synthesis and degradation. Targeted protein degradation (TPD) enables the selective reduction of protein target levels. However, as most established TPD methods rely on the ubiquitin proteasomal system (UPS), their effectiveness could be limited by the expression of the E3 ligase in different settings. In this study, we developed a TPD tool that utilizes ubiquitin-independent pathway, allowing for the degradation of target proteins without reliance on the ubiquitin-proteasome system (UPS). To achieve this, we first optimized the degradation efficiency of the Dnd1 degron. Leveraging this stronger degron, we established a versatile platform for targeted protein degradation by fusing the degron to the C-terminus of nanobodies. As a proof of concept, we used the LaG16-degron fusion to regulate eGFP levels in HEK cells and Xenopus oocytes, observing that LaG16-degron binding, but not LaG16 alone, significantly reduced eGFP levels. To provide an additional layer of control over degradation, we incorporated an optogenetic transcription system for light-induced expression of our degradation tool, thereby achieving conditional suppression of the EGFP-fusion proteins upon exposure to blue light. Finally, we demonstrated the ability to modulate endogenous signaling by targeting β-catenin with a BC2–degron fusion, resulting in a measurable perturbation of Wnt pathway activity. Together, this work demonstrated a generalized, ubiquitin-independent TPD strategy. We envision that this system can provide unique advantages in controlling endogenous protein levels in live cells.
Huang et al. (Sun,) studied this question.