Comprehensive Histone Phosphorylation Analysis and Identification of Pf14-3-3 Protein as a Histone H3 Phosphorylation Reader in Malaria Parasites

Abstract

The important role of histone posttranslational modifications, particularly methylation and acetylation, in Plasmodium falciparum gene regulation has been established. However, the role of histone phosphorylation remains understudied. Here, we investigate histone phosphorylation utilizing liquid chromatography and tandem mass spectrometry to analyze histones extracted from asexual blood stages using two improved protocols to enhance preservation of PTMs. Enrichment for phosphopeptides lead to the detection of 14 histone phospho-modifications in P. falciparum. The majority of phosphorylation sites were observed at the N-terminal regions of various histones and were frequently observed adjacent to acetylated lysines. We also report the identification of one novel member of the P. falciparum histone phosphosite binding protein repertoire, Pf14-3-3I. Recombinant Pf14-3-3I protein bound to purified parasite histones. In silico structural analysis of Pf14-3-3 proteins revealed that residues responsible for binding to histone H3 S10ph and/or S28ph are conserved at the primary and the tertiary structure levels. Using a battery of H3 specific phosphopeptides, we demonstrate that Pf14-3-3I preferentially binds to H3S28ph over H3S10ph, independent of modification of neighbouring residues like H3S10phK14ac and H3S28phS32ph. Our data provide key insight into histone phosphorylation sites. The identification of a second member of the histone modification reading machinery suggests a widespread use of histone phosphorylation in the control of various nuclear processes in malaria parasites.

Nuclear Pores and Perinuclear Expression Sites ofvarand Ribosomal DNA Genes Correspond to Physically Distinct Regions in Plasmodium falciparum

“…Protein extracts were separated on a 4 to 12% SDS-PAGE gel (Criterion, Bio-Rad) and analyzed by Western blotting using the rabbit antiPfNup116 at 1:500. Horseradish peroxidase (HRP)-conjugated anti-P. falciparum aldolase (ab38905; Abcam) at 1:1,000 and rabbit anti-histone 3 (ab1791; Abcam) at 1:2,000 were used to assess purity of cytoplasmic and nuclear fractions as described previously (21). Secondary antibody detection was done with anti-rabbit-HRP (GE health care) at 1:5,000.…”

Apicomplexa and Histone Variants: What’s New?

“…All sequences can be found in EuPathDB (https://eupathdb.org/eupathdb/). most histones, including H3 and H3.3 [52]. These marks are frequently seen in combination with neighboring lysine acetylation (and methylation).…”

A Plasmodium Falciparum Bromodomain Protein Regulates Invasion Gene Expression

“…Many histone modifications have been mapped across the P. falciparum genome (Bá rtfai et al, 2010;Cui et al, 2007;Gupta et al, 2013;Jiang et al, 2013;Lopez-Rubio et al, 2009;Salcedo-Amaya et al, 2009), and many proteins with predicted histone-binding domains have been identified bioinformatically (Bischoff and Vaquero, 2010). However, very few factors which bind to modified histones have been characterized (Dastidar et al, 2013;Flueck et al, 2009;Pé rez-Toledo et al, 2009).…”