Identification of TgAtg8–TgAtg3 interaction in Toxoplasma gondii

Chen, Di; Lin, Jiaxin; Liu, Yangyang; Li, Xiangzhi; Chen, Gaozhi; Hua, Qianqian; Nie, Qinqin; Hu, Xin; Tan, Feng

doi:10.1016/j.actatropica.2015.09.013

Cited by 7 publications

(8 citation statements)

References 39 publications

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“…Recombinant protein His 6 -TgAtg8 was expressed and purified according to protocols reported previously (24). To generate both WT and mutant versions of GST-TgAtg8, coding sequences of TgAtg8 R27E , TgAtg8 D44A/K45S/K46A , and TgAtg8 ⌬68 -76 were commercially synthetized (Genewiz, Suzhou, China) and directionally cloned into the pGEX-6p-1 expression vector, using BamH I and NotI to generate the corresponding plasmids pGST-TgAtg8 R27E , pGST-TgAtg8 D44A/K45S/K46A , and pGST-TgAtg8 ⌬68 -76 .…”

Section: Cloning Expression and Purification Of Tgatg8mentioning

confidence: 99%

“…To express WT of TgAtg3, the plasmid pHis 6 -TgAtg3 constructed previously (24) was transformed into BL21 for expression and purification following the protocol published previously. The mutant versions of TgAtg3 and the mutant versions of TgAtg3, pHis 6 -TgAtg3 F239A , pHis 6 -TgAtg3 I242A , pHis 6 -TgAtg3 F239A/I242A and pHis 6 -TgAtg3 W168A/P171A , were synthesized using overlap PCR.…”

Section: Cloning Expression and Purification Of Tgatg3mentioning

confidence: 99%

“…Western blot assays were performed as described previously (24). For the identification of TgAtg8 lipidation, parasite lysates were separated by 6 M urea SDS-PAGE followed by Western blotting.…”

Section: Western Blot Analysismentioning

confidence: 99%

“…GST-TgAtg8 mixed with various peptides was injected over the chip at 30 l/min. The association time was 80 s, and the dissociation time was 300 s. The data were collected and analyzed with ProteOn manager software as described previously (24).…”

Section: Biacore Analysismentioning

confidence: 99%

“…A crystal structural study of Saccharomyces cerevisiae Atg3 (ScAtg3) and its interaction with Atg8 showed that Atg3 specifically binds to Atg8 through an Atg8 family-interacting motif (AIM) sequence, WXXL, which is a short linear sequence that has been found in a growing number of proteins interacting with Atg8 homologs (22,23). We showed previously that TgAtg8 is able to interact with TgAtg3 in vivo and in vitro, but the detailed interaction mechanism is unknown (24). To better understand the TgAtg8 -TgAtg3 interaction in T. gondii, we set out to discover the potential AIM sequence in TgAtg3 that interacts with TgAtg8 by combining an overlapping peptide array with AlphaScreen assay technology.…”

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confidence: 99%

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

Liu

Zhang

Wang

et al. 2018

Journal of Biological Chemistry

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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.

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Section: Cloning Expression and Purification Of Tgatg8mentioning

confidence: 99%

Section: Cloning Expression and Purification Of Tgatg3mentioning

confidence: 99%

Section: Western Blot Analysismentioning

confidence: 99%

Section: Biacore Analysismentioning

confidence: 99%

mentioning

confidence: 99%

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

Liu

Zhang

Wang

et al. 2018

Journal of Biological Chemistry

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Structure-based drug design, synthesis and biological assays of P. falciparum Atg3–Atg8 protein–protein interaction inhibitors

Villa

Legnani

Colombo

et al. 2018

J Comput Aided Mol Des

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The proteins involved in the autophagy (Atg) pathway have recently been considered promising targets for the development of new antimalarial drugs. In particular, inhibitors of the protein-protein interaction (PPI) between Atg3 and Atg8 of Plasmodium falciparum retarded the blood- and liver-stages of parasite growth. In this paper, we used computational techniques to design a new class of peptidomimetics mimicking the Atg3 interaction motif, which were then synthesized by click-chemistry. Surface plasmon resonance has been employed to measure the ability of these compounds to inhibit the Atg3-Atg8 reciprocal protein-protein interaction. Moreover, P. falciparum growth inhibition in red blood cell cultures was evaluated as well as the cyto-toxicity of the compounds.

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Toxoplasma gondii autophagy-related protein ATG7 maintains apicoplast inheritance by stabilizing and lipidating ATG8

Wu,

Ying,

Lin

et al. 2024

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Diseas

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Identification of TgAtg8–TgAtg3 interaction in Toxoplasma gondii

Cited by 7 publications

References 39 publications

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

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

Structure-based drug design, synthesis and biological assays of P. falciparum Atg3–Atg8 protein–protein interaction inhibitors

Toxoplasma gondii autophagy-related protein ATG7 maintains apicoplast inheritance by stabilizing and lipidating ATG8

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