Global profiling of lysine 2-hydroxyisobutyrylome in Toxoplasma gondii using affinity purification mass spectrometry.

Lysine 2-hydroxyisobutyrylation (Khib) is a recently discovered and evolutionarily conserved form of protein post-translational modification (PTM) found in mammalian and yeast cells. Previous studies have shown that Khib plays roles in the activity of gene transcription and Khib-containing proteins are closely related to the cellular metabolism. In this study, a global Khib-containing analysis using the latest databases (ToxoDB 46, 8322 sequences, downloaded on April 16, 2020) and sensitive immune-affinity enrichment coupled with liquid chromatography-tandem mass spectrometry was performed. A total of 1078 Khib modification sites across 400 Khib-containing proteins were identified in tachyzoites of Toxoplasma gondii RH strain. Bioinformatics and functional enrichment analysis showed that Khib-modified proteins were associated with various biological processes, such as ribosome, glycolysis/gluconeogenesis, and central carbon metabolism. Interestingly, many proteins of the secretory organelles (e.g., microneme, rhoptry, and dense granule) that play roles in the infection cycle of T. gondii were found to be Khib-modified, suggesting the involvement of Khib in key biological process during T. gondii infection. We also found that histone proteins, key enzymes related to cellular metabolism, and several glideosome components had Khib sites. These results expanded our understanding of the roles of Khib in T. gondii and should promote further investigations of how Khib regulates gene expression and key biological functions in T. gondii.