Identifying the modulators of protein arginylation



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Protein arginylation is emerging as an important regulator of developmental and physiological processes. Arginylation, catalyzed by Arginyltransferase 1 (ATE1), is the post-translational conjugation of arginine to proteins bearing acidic N-terminal amino acids such as aspartate or glutamate [1]. This facilitates their degradation via the N-degron pathway of the ubiquitin proteasome system (UPS). Recent studies in our lab and others have shown that ATE1 is required for the removal of specific fragments associated neurodegeneration [2, 3] and that the loss of ATE1 is associated with disruption of fat metabolism and resistance to diet-induced obesity [4]. Thus, the modulation of ATE1 holds promise for treating these increasingly common human diseases. The N-degron pathway of the UPS recognizes proteins bearing either N-terminal hydrophobic or N-terminal basic amino acids such as arginine. These N-terminal amino acids function as degradation signals called N-degrons. Previously, we discovered that ATE1 is required for the degradation of TDP43247, a specific fragment of the human Transactive response DNA Binding Protein 43 (TDP43) associated with amyotrophic lateral sclerosis and other forms of dementia [2, 3]. In our efforts to identify modulators of ATE1, we generated a dual-fluorescent reporter sensitive to its activity. For this, the green fluorescent protein (GFP) was expressed bearing an N-degron derived from TDP43247 (247-DLIIKGISVHISNAEPK-263)[2, 3]. This reporter was named “Ndeg-GFP” for N-terminal degradation signal-GFP. N-deg-GFP is rapidly and efficiently arginylated by ATE1, causing its destabilization through the UPS. Ndeg-GFP is expressed in a linear fusion with mCherry-Ub using the ubiquitin reference technique which allows co-translational cleavage by intracellular de-ubiquitylases (DUBs) [5]. This generates both a stable mCherry-Ub and Ndeg-GFP, whose stability is inversely related to ATE1 activity. Ratiometric GFP/mCherry fluorescence allows for a quantitative measurement of in vivo ATE1 activity and controls for off-target effects that cause changes in transcription, translation, or overall cell fitness. Activators of ATE1 decrease the GFP/mCherry ratio, whereas inhibitors increase this ratio. We have utilized this dual-fluorescent reporter system across various settings to investigate modulators of ATE1 activity. Our endeavors include screening phytochemicals and small molecule compounds to discern their impact on ATE1-mediated protein degradation. Additionally, we have explored diverse number of environmental conditions to ascertain the response of ATE1 to stress. Our data suggest that ATE1 plays a critical role in cell fate determination during pathological conditions. Moreover, we have also outlined several strategies that employ our reporter system that can help uncover novel insights into the spatial, temporal, and contextual regulation of ATE1. These initiatives aim to uncover modulators of ATE1, potentially offering therapeutic targets for diseases associated with ATE1, such as obesity or neurodegeneration [6].



Biology, Molecular