SPIN4 Is a Principal Endogenous Substrate of the E3 Ubiquitin Ligase DCAF16

Area of Science:

• Cellular biology
• Proteomics
• Ubiquitination pathways

Background:

• DCAF16 is a key component of Cullin-RING E3 ubiquitin ligases.
• Understanding DCAF16 substrates is crucial for its role in targeted protein degradation.
• Electrophilic PROTACs can target DCAF16 for nuclear-restricted protein degradation.

Purpose of the Study:

• To identify endogenous protein substrates of DCAF16.
• To elucidate the specific relationship between DCAF16 and its substrates.
• To characterize the functional implications of the DCAF16-substrate interaction.

Main Methods:

• Comparative proteomics using untargeted mass spectrometry on DCAF16-wild type and DCAF16-knockout cells.
• Co-immunoprecipitation assays to confirm protein interactions.
• Ubiquitination assays and site-directed mutagenesis to identify ubiquitination sites and binding residues.

Main Results:

• Spindlin-4 (SPIN4), a Tudor domain-containing protein, was identified as significantly upregulated in DCAF16-knockout cells.
• DCAF16 directly interacts with and ubiquitinates SPIN4, but not other SPIN proteins.
• A unique lysine residue in SPIN4 was identified as critical for DCAF16 binding.
• SPIN4 preferentially binds trimethylated histone H3K4.

Conclusions:

• DCAF16 and SPIN4 form a dedicated E3 ligase-substrate complex.
• This complex regulates the turnover and presumed functions of SPIN4 in human cells.
• The findings provide insights into the specificity of DCAF16-mediated ubiquitination.