Article

Functional Dissection of the Apicomplexan Glideosome Molecular Architecture

Department of Microbiology and Molecular Medicine, Centre Medical Universitaire, University of Geneva, CH-1211 Geneva 4, Switzerland.
Cell host & microbe (Impact Factor: 12.33). 10/2010; 8(4):343-57. DOI: 10.1016/j.chom.2010.09.002
Source: PubMed

ABSTRACT

The glideosome of apicomplexan parasites is an actin- and myosin-based machine located at the pellicle, between the plasma membrane (PM) and inner membrane complex (IMC), that powers parasite motility, migration, and host cell invasion and egress. It is composed of myosin A, its light chain MLC1, and two gliding-associated proteins, GAP50 and GAP45. We identify GAP40, a polytopic protein of the IMC, as an additional glideosome component and show that GAP45 is anchored to the PM and IMC via its N- and C-terminal extremities, respectively. While the C-terminal region of GAP45 recruits MLC1-MyoA to the IMC, the N-terminal acylation and coiled-coil domain preserve pellicle integrity during invasion. GAP45 is essential for gliding, invasion, and egress. The orthologous Plasmodium falciparum GAP45 can fulfill this dual function, as shown by transgenera complementation, whereas the coccidian GAP45 homolog (designated here as) GAP70 specifically recruits the glideosome to the apical cap of the parasite.

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    • "Some of these proteins have been previously reported to be palmitoylated: in the case of TgCDPK3, studies have shown that palmitoylation not only occurs but it also affects the localization of the protein and thus its function in the parasite's ability to egress the host-cell [35]. Likewise, mutagenesis studies on TgGAP45, have demonstrated that both N-and C-terminal acylations are important for proper anchoring of the protein to the parasite's pellicle and affect vital processes such as gliding, invasion and egress [36]. Considering the variety of functions in which palmitoylation is involved , it was expected that the same diversity was reflected in the localization of the affected proteins. "
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    ABSTRACT: Protein palmitoylation has been shown to be an important post-translational modification in eukaryotic cells. This modification alters the localization and/or the function of the targeted protein. In the recent years protein palmitoylation has risen in importance in apicomplexan parasites as well. In Toxoplasma gondii, some proteins have been reported to be modified by palmitate. With the development of new techniques that allow the isolation of palmitoylated proteins, this significant post-translational modification has begun to be studied in more detail in T. gondii. Here we describe the palmitoylome of the tachyzoite stage of T. gondii using a combination of the acyl-biotin exchange chemistry method and mass spectrometry analysis. We identified 401 proteins found in multiple cellular compartments, with a wide range of functions that vary from metabolic processes, gliding and host-cell invasion to even regulation of transcription and translation. Besides, we found that more rhoptry proteins than the ones already described for Toxoplasma are palmitoylated, suggesting an important role for this modification in the invasion mechanism of the host-cell. This study documents that protein palmitoylation is a common modification in T. gondii that could have an impact on different cellular processes.
    Full-text · Article · Jan 2016 · Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics
    • "To confirm that proteins identified in our proteomic analysis were in fact palmitoylated, we carried out direct validation studies in which specific proteins from our dataset were isolated by immunoprecipitation and the presence of the palmitate analog (17-ODYA) confirmed biochemically. MLC1 was previously suggested to be palmitoylated (Fré nal et al., 2010). We metabolically labeled MLC1-FLAG-expressing parasites (Leung et al., 2014) with 17-ODYA, immunoprecipitated MLC1 with the FLAG epitope, used Click chemistry to attach an azido-rhodamine fluorophore , and analyzed the labeled proteins by SDS-PAGE (Figure 3A). "
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    ABSTRACT: 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 AMA1 palmitoylation enhances the release of AMA1 and other invasion-related proteins from apical secretory organelles, suggesting a previously unrecognized role for AMA1. These findings suggest that palmitoylation is ubiquitous throughout the T. gondii proteome and reveal insights into the biology of this important human pathogen.
    No preview · Article · Oct 2015 · Cell host & microbe
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    • "GAP50 was originally assigned to the parasite inner membrane complex (IMC), an alveolar double-membrane structure underneath the parasite plasmalemma, which mediates both cell motility and stability. Whereas the C-terminal transmembrane helix of GAP50 is anchored within the outer IMC membrane, the N-terminal portion protrudes into the IMC lumen (Baum et al., 2006; Sanders et al., 2007; Frénal et al., 2010; Bosch et al., 2012). Although not motile, the gametocytes of P. falciparum possess an IMC, which here functions to stabilize their crescent shapes (Dearnley et al., 2012; Kono et al., 2012; Simon et al., 2013). "
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    ABSTRACT: The acquisition of regulatory proteins is a means of blood-borne pathogens to avoid destruction by the human complement. We recently showed that the gametes of the human malaria parasite Plasmodium falciparum bind factor H (FH) from the blood meal of the mosquito vector to assure successful sexual reproduction, which takes places in the mosquito midgut. While these findings provided a first glimpse of a complex mechanism used by Plasmodium to control the host immune attack, it is hitherto not known, how the pathogenic blood stages of the malaria parasite evade destruction by the human complement. We now show that the human complement system represents a severe threat for the replicating blood stages, particularly for the reinvading merozoites, with complement factor C3b accumulating on the surfaces of the intraerythrocytic schizonts as well as of free merozoites. C3b accumulation initiates terminal complement complex formation, in consequence resulting in blood stage lysis. To inactivate C3b, the parasites bind FH as well as related proteins FHL-1 and CFHR-1 to their surface, and FH-binding is trypsin-resistant. Schizonts acquire FH via two contact sites, which involve CCP modules 5 and 20. Blockage of FH-mediated protection via anti-FH antibodies results in significantly impaired blood stage replication, pointing to the plasmodial complement evasion machinery as a promising malaria vaccine target. This article is protected by copyright. All rights reserved.
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