Acetylation of the Malaria Parasite

Lysine acetylation is a ubiquitous post-translational modification found in many organisms, including the pernicious malaria parasite, Plasmodium falciparum. Recently, Cobbold et al. (2016) used liquid chromatography–mass spectrometry (LC-MS)-based stable isotope labeling by amino acids in cell culture (SILAC) to increase acetylation coverage across the malaria parasite proteome.¹ In doing so, the team expanded the P. falciparum acetylome seven-fold by characterizing 2,876 acetylation sites on 1,146 proteins. These numbers contributed 29 of the 34 previously identified histone acetylation sites and 24 novel histone acetylation sites, totaling 58 sites across all eight histones.

For their experiments, the team cultured and synchronized parasites to the trophozoite stage. They then extracted proteins and quantified them using a BCA assay (Thermo Scientific). Next, they separated peptides into ca. 12 fractions via strong cation exchange (Supelco SCX SPE, Sigma) and used a small portion for mass spectrometry. Taking the remaining portion of the peptide fraction, the team enriched the samples for acetylated lysine via anti-acetyl lysine immunoprecipitation. For the SILAC experiments, the researchers pooled treated and control cell pellets prior to the protein extraction.

The team used both enriched and unenriched peptide fractions for a high-resolution reversed-phase nano-UPLC-MS and MS/MS performed on an Easy-nLC Ultra 1000 nanoflow capillary UPLC system (Thermo Scientific) coupled to a Velos Pro Orbitrap Elite hybrid mass spectrometer (Thermo Scientific). They used Proteome Discoverer software (v. 1.4/2.0, Thermo Scientific) to process MS data and, using the Mascot search engine, searched the resulting information against a concatenated database consisting of the proteomes of P. falciparum (PlasmoDB v. 9.2) and human erythrocytes.

Turning to acetyl-CoA profiling, the team hypothesized that changes in this metabolite would influence acetylation. The investigators analyzed metabolites by extracting and analyzing samples on a Q Exactive mass spectrometer. These experiments, along with gene ontology studies, led to the discovery that lysine acetylation targets a diverse range of protein complexes. Specifically, they found it is enriched within the Apicomplexan AP2 (ApiAP2) DNA-binding protein family.

Next, the team considered acetyl-lysine targets of lysine deacetylase, which is important in histone acetylation transcription and a potential antimalarial target. While the team identified specific acetyl-lysine targets, they maintain that the P. falciparum acetylome requires further exploration for researchers to better understand its regulation.

 

Reference

Cobbold, S.A., et al. (2016) “Proteome-wide analysis reveals widespread lysine acetylation of major protein complexes in the malaria parasite,” Scientific Reports, 6(19722). doi: 10.1038/srep19722.