Fig. 3: Mutated NAA60 proteins fail to associate with the Golgi, are highly unstable and lack intrinsic enzymatic activity. | Nature Communications

Fig. 3: Mutated NAA60 proteins fail to associate with the Golgi, are highly unstable and lack intrinsic enzymatic activity.

From: Biallelic NAA60 variants with impaired N-terminal acetylation capacity cause autosomal recessive primary familial brain calcifications

Fig. 3

a Predicted proteins resulting from the PFBC variants in NAA60 described here. WT NAA60 contains 242 amino acid (aa) residues and is part of the N-terminal acetyltransferase (NAT) family. These proteins share a distinct secondary structural GNAT ___domain (aa 13–182 of NAA60, indicated in yellow), comprising a critical binding site for the acetyl-CoA donor (aa 108–113 of NAA60, indicated in orange). In addition, NAA60 has a C-terminal extension unlike other NATs in which two Golgi membrane-binding amphipathic helices are found (aa 190–224, indicated in light blue). b FLAG-NAA60 wild-type and FLAG-NAA60-PFBC patient variants were expressed in RPE-1 cells, immunostained, and imaged with a Leica SP8 confocal microscope using the Lightning module for optimized resolution. The scale bar is 20 µm for all except for the magnified frames. Images are representative >100 transfected cells. c FLAG-NAA60 wild-type and FLAG-NAA60-PFBC variants were expressed in HEK293FT cells, and six hours prior to harvesting, cells were treated with 5 µM proteasomal inhibitor MG132. Result shown is representative of at least three independent experiments. d The indicated cells were lysed and endogenous NAA60 levels were assessed by western blot. HAP1 Ctrl and NAA60 KO cells were used as control for the antibody. Lym. = primary lymphoblasts. Fibroblasts = primary dermal fibroblasts. Representative of three experiments. e NAA60 variant proteins lack intrinsic enzymatic activity. WT and variant constructs were expressed in HEK293T cells and immunoprecipitated (IP) to produce the proteins for an enzyme activity test as illustrated. NAA60 IP products were tested for their ability to Nt-acetylate the NAA60-type substrate peptide MAPL in a classical in vitro [14 C] Nt-acetylation assay. For each condition, at least three independent replica experiments were included (n = 3 for F1 and F2/3, n = 9 for F4 and F5), pretests were performed to be able to apply equal enzyme inputs, and a final normalization to enzyme input was performed. **** indicates p < 0.0001 by impaired two-tailed t-test with Welch’s correction testing each mutant against the WT. Source data are provided in the Source Data file. Parts of the figure were drawn by using elements modified with text, markings, and annotations from Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 (https://creativecommons.org/licenses/by/3.0/).

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