Authors:

Chloe Chui

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About Paper:

Cullin-associated Nedd8-dissociated 1 and 2 (CAND1 and CAND2) are important regulators of cullin-RING ubiquitin ligases, which play key roles in protein ubiquitination through directly interacting with the cullin enzymatic cores. Misregulation of the ubiquitination pathway can lead to growth defects and diseases, including cancer. Although some interactions with CAND1 and CAND2 have been identified, there are many transient interactions that are unknown. In this study, we aimed to identify proteins that interact with CAND1 and CAND 2 via BioID proximity dependent labeling. To identify candidate interacting proteins, we fused the BioID with our bait proteins (CAND1 and CAND2) and expressed the fusion protein in HEK293 cells where CAND1 and CAND2 genes were knocked out. When the cells were supplied with biotin, BioID-fused CAND1/2 was able to promiscuously biotinylate proteins that typically interact with the bait proteins. By performing an affinity purification, we then isolated the biotinylated proteins and analyzed them via mass spectrometry. Upon receiving the mass spectrometry results, we will confirm the success of the BioID labeling by confirming that known interactions are present and identify new proteins that potentially interact with CAND1/2. Follow up experiments will be conducted to confirm interactions between CAND1/2 and the candidate proteins, and the confirmed interacting proteins will be further investigated for their role in regulating cullin-RING ubiquitin ligases and protein ubiquitination.

Source:

Purdue University / 2023

Topics:

No topics listed

Co-authors:

Chloe Chui