Quantitative Proteomic Analysis Reveals JMJD6 and DNAJB11 as Endogenous Substrates of E3 Ligase RFFL

The ubiquitin-proteasome system contributes to protein quality control, involving E3 ligases that ubiquitinate proteins and leading to their degradation. The dysregulation of protein degradation results in the abnormal accumulation of proteins and is implicated in the pathology of diverse diseases, making targeted protein degradation a promising therapeutic strategy. Here, we focus on RFFL, an endosome-associated RING E3 ligase involved in mitochondrial homeostasis and the clearance of misfolded cystic fibrosis transmembrane conductance regulator proteins. Using label-free quantitative mass spectrometry based proteomics for interactome and differential expression analyses, we systematically investigated and identified putative substrates of RFFL. For more confident identification, we performed these analyses on three cell lines that we generated: an RFFL knockout cell line generated using CRISPR/Cas9, another cell line rescuing RFFL expression when complemented with KO cells with stably expressing RFFL cDNA, and wild-type cells. We validated JMJD6 and DNAJB11 as substrates of endogenous RFFL, providing orthogonal validation and confidence in our screening approach. We demonstrated that RFFL ubiquitinates and degrades JMJD6 and DNAJB11 via the proteasomal pathway using in vivo assays. Interestingly, we also discovered a hitherto unknown role of RFFL in lipid metabolism. Collectively, this study provides the first comprehensive and unbiased analysis of RFFL substrates employing multiple complementary approaches.