Characterization of Specific N-alpha-Acetyltransferase 50 (Naa50) Inhibitors Identified Using a DNA Encoded Library
ACS MEDICINAL CHEMISTRY LETTERS
Authors: Kung, Pei-Pei; Bingham, Patrick; Burke, Benjamin J.; Chen, Qiuxia; Cheng, Xuemin; Deng, Ya-Li; Dou, Dengfeng; Feng, Junli; Gallego, Gary M.; Gehring, Michael R.; Grant, Stephan K.; Greasley, Samantha; Harris, Anthony R.; Maegley, Karen A.; Meier, Jordan; Meng, Xiaoyun; Montano, Jose L.; Morgan, Barry A.; Naughton, Brigitte S.; Palde, Prakash B.; Paul, Thomas A.; Richardson, Paul; Sakata, Sylvie; Shaginian, Alex; Sonnenburg, William K.; Subramanyam, Chakrapani; Timofeevski, Sergei; Wan, Jinqiao; Yan, Wen; Stewart, Albert E.
Abstract
Two novel compounds were identified as Naa50 binders/inhibitors using DNA-encoded technology screening. Biophysical and biochemical data as well as cocrystal structures were obtained for both compounds (3a and 4a) to understand their mechanism of action. These data were also used to rationalize the binding affinity differences observed between the two compounds and a MLGP peptide-containing substrate. Cellular target engagement experiments further confirm the Naa50 binding of 4a and demonstrate its selectivity toward related enzymes (Naa10 and Naa60). Additional analogs of inhibitor 4a were also evaluated to study the binding mode observed in the cocrystal structures.
An Organellar N alpha-Acetyltransferase, Naa60, Acetylates Cytosolic N Termini of Transmembrane Proteins and Maintains Golgi Integrity
CELL REPORTS
Authors: Aksnes, Henriette; Van Damme, Petra; Goris, Marianne; Starheim, Kristian K.; Marie, Michael; Stove, Svein Isungset; Hoel, Camilla; Kalvik, Thomas Vikestad; Hole, Kristine; Glomnes, Nina; Furnes, Clemens; Ljostveit, Sonja; Ziegler, Mathias; Niere, Marc; Gevaert, Kris; Arnesen, Thomas
Abstract
N-terminal acetylation is a major and vital protein modification catalyzed by N-terminal acetyltransferases (NATs). NatF, or N alpha-acetyltransferase 60 (Naa60), was recently identified as a NAT in multicellular eukaryotes. Here, we find that Naa60 differs from all other known NATs by its Golgi localization. A new membrane topology assay named PROMPT and a selective membrane permeabilization assay established that Naa60 faces the cytosolic side of intracellular membranes. An Nt-acetylome analysis of NAA60-knockdown cells revealed that Naa60, as opposed to other NATs, specifically acetylates transmembrane proteins and has a preference for N termini facing the cytosol. Moreover, NAA60 knockdown causes Golgi fragmentation, indicating an important role in the maintenance of the Golgi's structural integrity. This work identifies a NAT associated with membranous compartments and establishes N-terminal acetylation as a common modification among transmembrane proteins, a thus-far poorly characterized part of the N-terminal acetylome.
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