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.