Protein level knockdown of transmembrane proteins (360G-Wellcome-211595_Z_18_Z)

TrimAway is a newly described protein-level depletion method for degrading specific endogenous proteins. The technique relies on the experimental introduction of antibodies to the cell, which elicits rapid degradation of target antigens via the cytoplasmic Fc receptor and E3 ubiquitin ligase TRIM21. This enables acute depletion of proteins, enabling the functional characterisation of previously intractable proteins. TrimAway has been shown to act against diverse cytosolic substrates including membrane-anchored GFP. However, it is currently unknown whether TrimAway is capable of targeting transmembrane proteins. To address this, we will attempt to degrade three representative transmembrane proteins using TrimAway. With one, seven and twelve transmembrane passes respectively, the impact of topology on degradation rates will be determined. Targets, and control GFP, will be expressed as C-terminal myc-tagged constructs and expressed in human cells competent for TrimAway. Anti-myc antibody will be electroporated into the cells according to established protocols and the fate of target proteins will be monitored by western blot. Our targets have been selected as well-studied, disease-relevant proteins whose function may be illuminated by acute depletion. The results will help define the limits of the TrimAway technique and shed light on the cell's ability to degrade membrane proteins via the ubiquitin-proteasome system.