Global Analysis of Palmitoylated Proteins in Toxoplasma gondii

Abstract

Summary Post-translational modifications (PTMs) such as palmitoylation are critical for the lytic cycle of the protozoan parasite Toxoplasma gondii. While palmitoylation is involved in invasion, motility, and cell morphology, the proteins that utilize this PTM remain largely unknown. Using a chemical proteomic approach, we report a comprehensive analysis of palmitoylated proteins in T. gondii, identifying a total of 282 proteins, including cytosolic, membrane-associated and transmembrane proteins. From this large set of palmitoylated targets, we validate palmitoylation of proteins involved in motility (myosin light chain 1, myosin A), cell morphology (PhIL1), and host-cell invasion (apical membrane antigen 1, AMA1). Further studies reveal that blocking palmitoylation enhances the release of AMA1 and other invasion-related proteins from apical secretory organelles, suggesting that AMA1 controls this secretion process. These findings suggest that palmitoylation is ubiquitous throughout the T. gondii proteome and reveal insights into the biology of this important human pathogen.

Selective Enrichment and Direct Analysis of Protein S-Palmitoylation Sites

“…CuAAC and NH 2 OH thioester hydrolysis conditions have been reported in the litterature, 12,18 but required further investigation to avoid non-specific protein degradation. Quenching the CuAAC reaction with EDTA appeared essential to prevent protein degradation (SI Figure S3A-B), 30 and conditions of the NH 2 OH treatment were further optimized to ensure known S-fatty-acylated proteins, CANX, 11 Fyn, 31 IFITM3 10 labeled with alk-16 were sensitive to the NH 2 OH treatment.…”

“…17 To discriminate S-fatty-acylated proteins from N/O-fatty-acylated proteins, several studies have employed hydroxylamine (NH 2 OH) to selectively hydrolyze thioesters bonds of alk-16 labeled proteins (Figure 1A). 12,18 …”

Protein Palmitoylation Plays an Important Role in Trichomonas vaginalis Adherence

“…Although several Palmitoylproteome analyses have been described in the last years, only few have been using protozoan parasites (12,(53)(54)(55). Interestingly, in all parasites where the role of palmitoylation has been described, it appears to be involved in vital processes such as invasion and motility (12,(53)(54)(55). In this context, here we performed the to speculate that zinc-binding domain would not be required for general palmitoylation activity.…”

“…Specifically, T. vaginalis shared 30 from the 124 palmitoylated proteins found in Trypanosoma brucei, 57 from the 565 of Toxoplasma gondii and 58 from the 495 present in Plasmodium falciparum (12,(53)(54)(55) ( Fig. 3B and Table 1.S).…”

“…Despite these serious consequences, the underlying biochemical processes that lead to T. vaginalis pathogenesis are poorly understood. In this sense, recent works have unraveled the role of protein S-palmitoylation as a significant post-translational modification (PTM) that regulates invasion and motility in different protozoan parasites such as Plasmodium falciparum, Trypanosoma cruzi, Giardia lamblia and Toxoplasma gondii (11,12) as well as adherence to host cell in the fungus C. neoformans (13). S-palmitoylation refers to the covalent attachment of a 16-carbon chain palmitic acid to cysteine residues of a protein through a thioester bond.…”