Quantitative insights into protein turnover and ubiquitination with HiBiT and NanoBRET

Protein turnover and ubiquitination are fundamental processes regulating cellular homeostasis, proteostasis, and targeted protein degradation. Here, we present an optimized methodology for studying protein stability, ubiquitination dynamics, and degron function using HiBiT (11-amino-acid peptide tag) and NanoBRET (bioluminescence resonance energy transfer BRET) assays, specifically tailored for investigating Cullin-RING E3 ligase receptors, including von Hippel-Lindau (VHL) and FBXL15. HiBiT enables real-time quantification of protein abundance and degradation kinetics, making it ideal for CHX chase experiments and degron validation, while NanoBRET allows live-cell monitoring of ubiquitination events, facilitating the assessment of substrate ubiquitination efficiency and E3 ligase interactions. Given the relevance of protein termini in degradation, we leveraged HiBiT tagging to mask terminal degrons, enabling controlled analysis of N- and C-terminal degron function in protein stability. Using FBXL15 as a model, we demonstrate that degron accessibility significantly impacts turnover rates. Additionally, we applied NanoBRET to explore non-canonical ubiquitination mechanisms, using VHL as a case study to assess ubiquitination dynamics in a live-cell-compatible system. This study establishes HiBiT and NanoBRET as versatile tools for investigating Cullin-RING receptor stability and terminal degron function, providing a valuable methodological resource for dissecting protein turnover, ubiquitination pathways, and post-translational modifications in a quantitative and physiologically relevant manner.