Proteome-wide profiling of protein lysine acetylation in Aspergillus flavus

Abstract

Protein lysine acetylation is a prevalent post-translational modification that plays pivotal roles in various biological processes in both prokaryotes and eukaryotes. Aspergillus flavus, as an aflatoxin-producing fungus, has attracted tremendous attention due to its health impact on agricultural commodities. Here, we performed the first lysine-acetylome mapping in this filamentous fungus using immune-affinity-based purification integrated with high-resolution mass spectrometry. Overall, we identified 1383 lysine-acetylation sites in 652 acetylated proteins, which account for 5.18% of the total proteins in A. flavus. According to bioinformatics analysis, the acetylated proteins are involved in various cellular processes involving the ribosome, carbon metabolism, antibiotic biosynthesis, secondary metabolites, and the citrate cycle and are distributed in diverse subcellular locations. Additionally, we demonstrated for the first time the acetylation of fatty acid synthase α and β encoded by aflA and aflB involved in the aflatoxin-biosynthesis pathway (cluster 54), as well as backbone enzymes from secondary metabolite clusters 20 and 21 encoded by AFLA_062860 and AFLA_064240, suggesting important roles for acetylation associated with these processes. Our findings illustrating abundant lysine acetylation in A. flavus expand our understanding of the fungal acetylome and provided insight into the regulatory roles of acetylation in secondary metabolism.

Fig 1. Lysine acetylation status is analyzed by using SDS-PAGE and western blotting.

(A) Confirmation of acetylated proteins present in A. flavus. 15μg protein samples were loaded for SDS-PAGE analysis. Acetylated Lys antibody (PTM Biolabs) was used in a 1:1000 dilution. (B) Distribution of lysine-acetylated peptides based on the number of acetylation sites.

Fig 2. Motif analysis of Kac peptides.

(A) Acetylation motifs and conservation of acetylation sites. The height of each letter corresponds to the frequency with which that amino acid residue is found at that position. (B) Heat map representing the amino acid composition of the Kac sites, showing the frequency of the different types of amino acids surrounding Kac sites. (C) Cellular distribution of acetylated proteins and sites. (D) Sequence logo plots of normalized amino acid frequencies ±10 amino acids from the lysine acetylation site in cellular compartments. (E) Comparison analysis of acetylation motifs between Aspergillus flavus, Phytophthora sojae, Botrytis cinerea, and Bacillus amyloliquefaciens.

Fig 3

(A) GO, (B) subcellular-localization analysis, and (C) KEGG-pathway enrichment of the identified Kac proteins. (D) Comparison analysis of enriched pathways between Aspergillus flavus, Phytophthora sojae, Botrytis cinerea, and Bacillus amyloliquefaciens.

Fig 4. Acetylation of metabolic enzymes identified as involved in glycolysis/gluconeogenesis and the citric acid cycle.

The identified numbers of lysine-acetylated enzymes and proteins are shown in red. The identified Kac proteins found in mammalian cells are marked with●, those in Escherichia coli with ★, and those in Saccharopolyspora erythraea with▲.