Characterization of the molecular mechanism of the autophagy-related Atg8–Atg3 protein interaction in Toxoplasma gondii

Abstract

Toxoplasmosis is caused by an obligate intracellular parasite, the protozoan Discovery of novel drugs against infection could circumvent the toxicity of existing drugs and resistance to current treatments. The autophagy-related protein 8 (Atg8)-Atg3 interaction in is a promising drug target because of its importance for regulating Atg8 lipidation. We reported previously that TgAtg8 and TgAtg3 interact directly. Here we validated that substitutions of conserved residues of TgAtg8 interacting with the Atg8 family-interacting motif (AIM) in Atg3 disrupt the TgAtg8-TgAtg3 interaction and reduce TgAtg8 lipidation and autophagosome formation. These findings were consistent with results reported previously for Atg8, suggesting functional conservation of Atg8 in and Moreover, using peptide and AlphaScreen assays, we identified the AIM sequence in TgAtg3 that binds TgAtg8. We determined that the core TgAtg3 AIM contains a Phe-Ala-Asp-Ile (FADI) signature distinct from the WLLP signature in the AIM of Atg3. Furthermore, an alanine-scanning assay revealed that the TgAtg8-TgAtg3 interaction in also depends strongly on several residues surrounding the core TgAtg3 AIM, such as Asn, Asp, and Cys These results indicate that distinct AIMs in Atg3 contribute to differences between and Atg8-Atg3 interactions. By elucidating critical residues involved in the TgAtg8-TgAtg3 interaction, our work paves the way for the discovery of potential anti-toxoplasmosis drugs. The quantitative and straightforward AlphaScreen assay developed here may enable high-throughput screening for small molecules disrupting the TgAtg8-TgAtg3 interaction.

Toxoplasma TgAtg8-TgAtg3 Interaction Primarily Contributes to Apicoplast Inheritance and Parasite Growth in Tachyzoite

“…Our previous work focused on identifying the core AIM in TgAtg3 through biochemical analyses in vitro and determined that the 239 FADI 242 of TgAtg3 is responsible for mediating TgAtg8-TgAtg3 interaction ( 28 ). To evaluate whether the TgAtg8-TgAtg3 interaction is involved in tachyzoite growth and apicoplast inheritance, we first generated a conditionally inducible knockdown cell line of TgAtg3 (iTgAtg3) using a transactivator (TATi)-based, tetracycline (Tet)-regulated system ( 29 , 30 ).…”

“…The iTgAtg3 line was then complemented with either N-terminal, triple-hemagglutinin (3HA)-tagged, wild-type (WT) gene (3HA-TgAtg3 WT c) or a mutant allele (3HA-TgAtg3 Mut c) which harbored F293A/I242A double-point mutations identified to disrupt TgAtg8-TgAtg3 interaction ( 28 ). These constructs were integrated into the uracil phosphoribosyltransferase (UPRT) locus by double-homologous recombination using CRISPR/Cas9, and incorporation into the genome was confirmed by PCR detection and sequencing (Fig.…”

“…Our previous study showed that the mutation of TgAtg3 AIM significantly reduces the ability of TgAtg3 to bind to TgAtg8 in vitro ( 28 ). In the present study, we evaluated further the impact of TgAtg3 AIM on TgAtg8-TgAtg3 interaction using both 3HA-TgAtg3 WT c and 3HA-TgAtg3 Mut c strains via in vivo immunoprecipitation.…”

“…Several studies in Plasmodium falciparum have suggested that the Atg8-Atg3 interaction may be an attractive new drug target in apicomplexan parasites ( 25 – 27 ). Using a series of biochemistry assays in vitro , we have previously verified the importance of the TgAtg8-TgAtg3 interaction in TgAtg8 lipidation and demonstrated that the core AIM of TgAtg3, 239 FADI 242 , which is distinct from Plasmodium , plays an important role in mediating this interaction ( 28 ); however, the function of the TgAtg8-TgAtg3 interaction in the Toxoplasma life cycle remains poorly understood. Based on our previous findings, we separately generated TgAtg3 F293A/I242A and TgAtg8 R27E mutants in this study to provide the first direct evidence that the TgAtg8-TgAtg3 interaction is essential to in vivo TgAtg8 lipidation and apicoplast inheritance, and that disruption of this interaction leads to the delayed death phenotype.…”

Binding Features and Functions of ATG3

“…Atg3 A IM affects the Cvt pathway and the formation of Atg8–PE but does not affect the formation of the Atg8–Atg3 thioester intermediate ( Yamaguchi et al, 2010 ). Interestingly, Atg3 A IM in Toxoplasma gondii contains an FADI (Phe–Ala–Asp–Ile) sequence and a WLLP (Trp–Leu–Leu–Pro) sequence, which is different from that of yeast Atg3 A IM ( Chen et al, 2016 ; Liu et al, 2018 ; Varberg et al, 2018 ). Such findings indicate that Atg3 A IM may be species specific; thus, antitoxoplasmosis drugs and anti- Plasmodium drugs that target Atg8–Atg3 could be developed.…”