Abstract 1392 Identification of CAND1 and CAND2 interacting proteins using BioID Article Swipe

Cullin-associated Nedd8-dissociated 1 and 2 (CAND1 and CAND2) are key regulators of cullin-RING ubiquitin ligases (CRLs). CRLs are composed of a cullin scaffold, a RING protein, a substrate receptor, and adaptor proteins. They catalyze the ubiquitination of proteins, marking the protein for degradation in the proteasome. Dysfunctional CAND1/2 and the resulting dysregulation of protein ubiquitination can lead to cancer and neurodegenerative disorders. The Skp, Cullin, F-box containing complex (SCF) is a CRL that equips S-phase kinase-associated protein 1 (SKP1) as an adaptor protein, and F-box proteins as substrate receptors. CAND1/2 facilitates the disassembly of SCF by binding to its cullin scaffold and allows for the efficient recycling and dynamic assembly of diverse SCF complexes that identify specific targets for degradation. As vital protein exchange factors and activators in SCF and other CRLs, CAND1/2 ultimately enables CRLs to effectively aid in the adaptation of cells to changing cellular demands by regulating protein levels. While certain stable CAND1/2 interactions are known, other less stable or transient interactions are unknown, which may be regulators or novel regulating targets of CAND1/2. We used the BioID proximity labeling system to investigate these unknown CAND1/2 interactions. BioID is a promiscuous biotin ligase that biotinylates nearby proteins and allows us to capture CAND1/2 interactions within the cell. Due to CAND1/2's large molecular weight, we generated N- and C-terminal BioID fusions to encompass the whole protein. We expressed the fusion proteins in CAND1/2 double-knockout HEK293 cells. Upon biotin addition, BioID-fused CAND1/2 biotinylated proteins that came within the labeling radius of the bait proteins. Using streptavidin's strong biotin affinity, we isolated biotinylated proteins and analyzed them using mass spectrometry. Multiple Western blot analyses of our affinity purifications validated our BioID system. We confirmed the presence of known interacting proteins and tested different conditions for optimal affinity purification. This included changing the composition of the reaction buffer and chemically adding protease resistance to the streptavidin beads, minimizing streptavidin peptide noise in the mass spectrometry results. Non-denaturing conditions yielded the most effective affinity purification. With the optimized method for biotinylated protein isolation, we sent our samples for mass spectrometry analysis, using an unfused BioID protein as a reference sample. For the results, we screened proteins based on increased enrichment compared to the reference sample, endogenous biotinylation status, and presence in already published literature. Our results revealed interesting findings, such as the enrichment of proteins involved in the UFMylation pathway and the SUMOylation pathway, suggesting the involvement of CAND1 and CAND2 in these post-translational modification pathways. Further studies will allow us to validate the candidate interacting proteins and elucidate CAND1/2 roles in other pathways. This work was supported by the National Institute of General Medical Sciences [R35GM138016] and the American Heart Association [20CDA35270030].