A Phytochemical Screen for Modulators of Protein Arginylation

Protein arginylation is emerging as an important regulator of many physiological processes. Catalyzed by arginyl-transferase 1 (ATE1), the conjugation of arginine onto proteins bearing acidic N-terminal amino acids facilitates their degradation via the ubiquitin proteasome system. Studies have shown that ATE1 prevents the accumulation of damaged proteins associated neurodegeneration and plays a role in obesity. Thus, the modulation of ATE1 holds promise for treating these increasingly common diseases. We recently generated a fluorescent reporter to measure ATE1 activity in cells. This reporter produces two fluorescent proteins from a single transcript: Ndeg-GFP, which is degraded by ATE1, and mCherry-Ub, a stable reference. This enables ratiometric fluorescence to normalize off-target effects on transcription, translation, or cell fitness. Activators of ATE1 decrease the GFP/mCherry ratio, whereas inhibitors increase the ratio. Here, we have begun initial screening with this reporter by testing a number of phytochemicals for their effects on ATE1 and protein degradation.