Lysine acetylation is widespread on proteins of diverse function and localization in the protozoan parasite Toxoplasma gondii

Abstract

While histone proteins are the founding members of lysine acetylation substrates, it is now clear that hundreds of other proteins can be acetylated in multiple compartments of the cell. Our knowledge of the scope of this modification throughout the kingdom of life is beginning to emerge, as proteome-wide lysine acetylation has been documented in prokaryotes, Arabidopsis thaliana, Drosophila melanogaster, and human cells. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify parasite peptides enriched by immunopurification with acetyl-lysine antibody, we produced the first proteome-wide analysis of acetylation for a protozoan organism, the opportunistic apicomplexan parasite Toxoplasma gondii. The results show that lysine acetylation is abundant in the actively proliferating tachyzoite form of the parasite, which causes acute toxoplasmosis. Our approach successfully identified known acetylation marks on Toxoplasma histones and α-tubulin and detected over 400 novel acetylation sites on a wide variety of additional proteins, including those with roles in transcription, translation, metabolism, and stress responses. Importantly, an extensive set of parasite-specific proteins, including those found in organelles unique to Apicomplexa, is acetylated in the parasite. Our data provide a wealth of new information that improves our understanding of the evolution of this vital regulatory modification while potentially revealing novel therapeutic avenues. We conclude from this study that lysine acetylation was prevalent in the early stages of eukaryotic cell evolution and occurs on proteins involved in a remarkably diverse array of cellular functions, including those that are specific to parasites.

Fig 1

Overview of the Toxoplasma tachyzoite acetylome. (A) A Western blot analysis of tachyzoite lysate (25 μg) using acetyl-lysine antibody demonstrates the presence of many acetylated proteins in addition to histones (∼14 to 17 kDa). (B) Diagram depicting the experimental design to characterize the parasite acetylome. Intracellular parasites (∼30 h postinfection) were purified from host cells, lysed in urea lysis buffer, and sonicated. The resulting lysate was proteolytically treated to generate a peptide suspension to be used in immunoaffinity purification to enrich acetylated peptides (a red spot denotes an acetylated peptide). Identification of acetylated peptides was achieved with mass spectrometry. (C) Lysine acetylation occurs on proteins involved in virtually every cellular process, but the majority of acetylation occurs on proteins involved in metabolism, translation, and chromatin biology. The pie chart on the right shows the functional categorization of the 308 acetylated residues found across the 187 acetylated proteins, which are displayed in the left pie chart. Acetylation of the 103 lysines across 87 hypothetical proteins was omitted in these pie charts.

Fig 2

Acetylation sites on Toxoplasma lysine acetyltransferases. Acetylation sites (red spots) and phosphorylated sites (blue spots) (38) found in the TgGCN5 and TgMYST family KATs. Acetylation was found within the ADA2 interaction domain of each TgGCN5 (the domain between the KAT and the bromodomain [BRM]). TgGCN5-A was also acetylated within the BRM, and TgGCN5-B was found to be acetylated at multiple lysines within its C-terminal tail. The TgMYST KATs contain a chromodomain (ChRM) and a zinc finger (ZnF) and are expressed as two forms (long and short) due to two translational start sites (the second start codons are marked by a dark green line). Both TgMYST-A and TgMYST-B are acetylated within the N-terminal extension of the longer form of the proteins. An acetyl mark on K288 was also found on TgMYST-A, which is conserved in a number of MYST homologues and may regulate KAT activity of the enzyme. Acetylation was also detected on the TAF1/TAF250 homologue at K1881. No acetylated residues were detected on predicted homologues of Elp3 or Hat1.

Fig 3

Properties of acetylated peptides. (A) Sequence motifs of Toxoplasma acetylation sites ±7 amino acids from the targeted lysine residue. Motifs were compiled using all acetylated peptides or only those found in histones, nonhistone proteins, or mitochondrial proteins. (B) Heat map of amino acid compositions of acetylation sites in Toxoplasma, displaying amino acids that are significantly enriched (green) or depleted (red) compared to the general amino acid composition of the Toxoplasma proteome.