Proteomic translation and degradation screen of HECT E3 ligases reveals functional redundancies and exclusivity

Homologous to E6AP C-terminus (HECT) E3 ubiquitin ligases are crucial for maintaining cellular proteostasis by regulating ubiquitination and subsequent degradation of substrates. These enzymes are integral to various biological processes, including protein trafficking, subcellular localization, and immune responses. Defects in HECT E3 ligase functionality have been implicated in various cancers, neurodegenerative diseases, and viral infections.

With the advent of PROteolysis TArgeting Chimaera (PROTAC) technology, HECT E3 ligases have emerged as promising targets for therapeutic intervention. Despite the wide range of pharmacological possibilities, only a subset of the 28 annotated human HECT E3 ligases have been studied in depth. Consequently, much remains unknown about their substrates and regulatory mechanisms.

To address this, we performed a high-throughput translation/degradation proteomic screen coupled with siRNA-induced knockdown of 27/28 HECT E3 ligases. Expanding on our previously established multiplexed enhanced protein dynamic (mePROD) approach, we combined an siRNA screen with pulsed stable isotope labelling in cells (pSILAC) and isobaric tagging (TMT) to temporally quantify protein degradation and synthesis for each HECT E3 ligase upon knockdown. We were able to quantify >8,000 proteins/siRNA in both translation and degradation proteoform states from a single well of a 96-well cell culture plate. Moreover, knockdown was confirmed for the majority of siRNA targets in proteomics data, further increasing the confidence in the efficacy of our optimized high-throughput experimental approach.

Seventeen out of 27 siRNAs showed no drastic difference in overall protein translation and degradation or cell viability, suggesting a narrow range of downstream modules and functional synergism, with a few unique set of differentially regulated proteins corresponding to each HECT E3 ligase. Ten out of 27 siRNAs severely impacted the overall proteome turnover, with a broad effect on protein translation and degradation, suggesting exclusive dependence on these specific HECT E3 ligases and their crucial role in the regulation of cellular proteostasis.

UBR5 has previously been reported to regulate a large number of transcription factors and nuclear proteins. Our data corroborates this, with evidence of reduced protein degradation and cell viability. Similarly, our data confirm known modalities in HECTD2-4, NEDD4, HUWE1 and provides evidence for novel functional substrates with potentially wide therapeutic relevance ranging from cancer, cardiac hypertrophy, inflammation to antidepressants.

In conclusion, we provide here a high-throughput approach to study protein synthesis and degradation that can be combined with siRNA, CRISPR and compound screens. Our findings provide a valuable resource for understanding HECT-mediated protein degradation and paves the way for drug design in various therapeutic contexts.