Extensive lysine acetylation occurs in evolutionarily conserved metabolic pathways and parasite-specific functions during Plasmodium falciparum intraerythrocytic development

Abstract

Lysine acetylation has emerged as a major post-translational modification involved in diverse cellular functions. Using a combination of immunoisolation and liquid chromatography coupled to accurate mass spectrometry, we determined the first acetylome of the human malaria parasite Plasmodium falciparum during its active proliferation in erythrocytes with 421 acetylation sites identified in 230 proteins. Lysine-acetylated proteins are distributed in the nucleus, cytoplasm, mitochondrion and apicoplast. Whereas occurrence of lysine acetylation in a similarly wide range of cellular functions suggests conservation of lysine acetylation through evolution, the Plasmodium acetylome also revealed significant divergence from those of other eukaryotes and even the closely related parasite Toxoplasma. This divergence is reflected in the acetylation of a large number of Plasmodium-specific proteins and different acetylation sites in evolutionarily conserved acetylated proteins. A prominent example is the abundant acetylation of proteins in the glycolysis pathway but relatively deficient acetylation of enzymes in the citrate cycle. Using specific transgenic lines and inhibitors, we determined that the acetyltransferase PfMYST and lysine deacetylases play important roles in regulating the dynamics of cytoplasmic protein acetylation. The Plasmodium acetylome provides an exciting start point for further exploration of functions of acetylation in the biology of malaria parasites.

Fig. 1. Analysis of acetylation in P. falciparum

(A) Overall acetylation of P. falciparum in the intraerythrocytic development stages. Proteins were isolated from ring (R), early trophozoite (ET), late trophozoite (LT) and schizont (S). The right panel shows the reaction of anti-acetyllysine antibodies to LT proteins blocked by acetylated BSA. (B) Acetylation of cytoplasmic proteins in trophozoites of GFP-control and parasite lines with overexpression of a truncated inactive KAT PfMYST (F1C3-GFP) and overexpression of the full-length active PfMYST (F1-GFP). (C) Effect of HDAC inhibitors (TSA and nicotinamide) on cytoplasmic protein acetylation. Parasites were treated from ring stage with 1 to 4 times of IC₅₀ of TSA or 5 to 15 mM of nicotinamide for 15 h. Equal amounts of protein lysates were separated by SDS-PAGE, and the acetylated proteins were detected with anti-acetyllysine antibodies. Equal protein loading was evidenced by Western blotting with anti-HSP70 antibodies. The graphs under the respective Western blots show the relative signal intensities determined by densitometry (mean + standard deviation) from three replicates.

Fig. 2. Identification of protein acetylation in P. falciparum trophozoites

(A) Two representative MS results showing the modification of H3 and H3.3 at K28 and K37. Each peptide was fragmented by MS/MS and the fragments observed were consistent with the sequence of the peptide as shown on top of each MS/MS spectrum. Note that b ions are counting from N-terminus and y ions from C-terminus. Overall the Ms/Ms data unambiguously confirmed that both peptides were diacetylated and they differ by two amino acids. (B) Localization of acetylated proteins in different cellular compartments. (C) Verification of acetylation of five proteins by immuno-precipitation and Western-blot with anti-acetyllysine antibodies.

Fig. 3. Acetylation occurs in proteins involving diverse functions in P. falciparum

(A) Functional classification of lysine-acetylated proteins from all or cytosolic and nuclear fractions. (B) Enrichment analysis. The red bars indicate the proteins at those functional groups are significantly overrepresented, whereas the green bars indicate underrepresented functional groups. P-values were calculated using simulations and were then transformed using the negative natural log for visualization.

Fig. 4. Acetylation of enzymes in the purine, pyrimidine and polyamine metabolic pathways in P. falciparum. Purine pathway

AMP, adenosine 5′-monophosphate; IMP, inosine 5′-monophosphate; XMP, xanthosine 5′-monophosphate; GMP, guanosine 5′-monophosphate; MTA, methylthioadenosine; MTI, methylthioinosine; AdS, adenoylsuccinate; PfADA, P. falciparum adenosine deaminase; PfPNP, P. falciparum purine nucleoside phosphorylase; PfHGXPRT, P. falciparum hypoxanthine-guanine-xanthine phosphoribosyl transferase; PfAMPDA, P. falciparum adenosine 5′-monophosphate deaminase; PfIMPDH, P. falciparum inosine 5′-monophosphate dehydrogenase; PfGMPs, P. falciparum guanosine 5′-monophosphate synthase; PfAdSS, adenylosuccinate synthetase; PfAdSL, adenylosuccinate lyase. Pyrimidine pathway: OMP, orotidine 5′-monophosphate; UMP, uridine 5′-monophosphate; UTP, uridine 5′-triphosphate; CTP, cytidine 5′-triphosphate; PfCPSII, P. falciparum carbamoyl phosphate synthetase II; PfATCase, P. falciparum aspartate carbamoyltransferase; PfDHOase, P. falciparum dihydroorotase; PfDHODH, P. falciparum dihydroorotate dehydrogenase; PfOPRT, P. falciparum orotate phosphoribosyltransferase; PfODC, P. falciparum orotidine 5′-monophosphate decarboxylase; PfCTPs, P. falciparum cytidine 5′-triphosphate synthase. Polyamine Pathway: AdoMet, S-adenosylmethionine; AdoHC, S-adenosylhomocysteine; HC, homocysteine; Met, methionine; dcAdoMet, decarboxylated S-adenosylmethionine; PfSpdSyn, P. falciparum spermidine synthase; PfODCAdoMetDC, P. falciparum ornithine decarboxylase/S-adenosylmethionine decarboxylase; PfMetTfase, P. falciparum methyltransferase(s); PfAHC, P. falciparum S-adenosyl homocysteinase; PfMetSyn, P. falciparum methionine synthase; SAMS, P. falciparum S-adenosylmethionine synthase. The arrows indicate the direction of net flux. The metabolically un-favored direction is depicted with light arrows on reversible steps. Multiple arrows indicate pathways not shown entirely. The enzymes are boxed and acetylated enzymes are shown in green blocks.

Fig. 5. Acetylation sites in P. falciparum KATs and their associated proteins

The structures of two key KATs, PfGCN5 and PfMYST, and their associated proteins, PfADA2, Pf10_0079, Pf14_0315 are depicted. The acetylated sites are shown with short bars with numbers indicating the positions of lysine residues. KAT: lysine acetyltransferase activity domain, ADA2: ADA2 binding domain, BRM: bromodomain, ADA2LD: ADA2 like-domain, ChRM: chromodomain, Znf: zinc finger. PHD: PHD domain.

Fig. 6. Effect of lysine acetylation on HAT activities of PfGCN5 and PfMYST

GFP-tagged PfGCN5 and PfMYST were immunoprecipitated from P. falciparum trophozoites cultured without or with the inhibitor mixture (TSA and nicotinamide). Blots with anti-GFP antibodies indicate approximately equal amounts of purified PfGCN5 or PfMYST used in the HAT assays. The overall acetylation levels of the purified proteins were estimated by Western blots with anti-acetyllysine antibodies. HAT activities of purified PfGCN5 and PfMYST were determined using core histones and nucleosomal histones.

Fig. 7. Acetylation motifs and conservation of acetylation sites

(A) Relative abundance of each amino acid residues surrounding sites of acetylated lysine in all acetylated proteins, cytosolic proteins, nuclear proteins and histones. (B) Lysine conservation of P. falciparum acetylated lysines in other pathogen species. Acetylated lysines are significantly more conserved than non-acetylated lysines.