Identification of TgAtg8-TgAtg3 interaction in Toxoplasma gondii

Acta Trop. 2015 Sep 22. pii: S0001-706X(15)30110-8. doi: 10.1016/j.actatropica.2015.09.013. [Epub ahead of print]

 

Chen D¹, Lin J², Liu Y², Li X³, Chen G⁴, Hua Q⁵, Nie Q⁶, Hu X⁷, Tan F⁸.

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Abstract

Autophagy is a catabolic process in eukaryotic cells involved in the targeted degradation of cellular organelles and the cytoplasm. Recent works in Toxoplasma gondii suggest that the autophagy processes may serve as an important pathway in modulating parasite survival or death. As an important modulator of Atg8 lipidation and autophagy, Atg8-Atg3 interaction has been attracting increasing attention. However, there is no direct evidence that TgAtg8-TgAtg3 interaction occurs in the parasite. In this study, we firstly found TgAtg8 partially colocalized with TgAtg3 in GFP-TgAtg8 transgenic strains using IFA. Then, lysates from GFP-TgAtg8 tachyzoites were directly subject to large-scale tandem affinity purification with anti-GFP antibody. Western blot and tandem mass spectrometry (MS/MS) analysis determined the interaction between TgAtg8 and TgAtg3. Additionally, we performed real-time interaction analysis with a surface plasmon resonance biosensor using BIAcore system. As expected, the result demonstrated a concentration-dependent increases in resonance signals and indicated the TgAtg8 could bind directly TgAtg3 in vitro. Noteworthily, A KD of 34.9nM obtained from TgAtg8-TgAtg3 interaction indicate a high-affinity between Atg8-Atg3 in Toxoplasma. Furthermore, homology modeling and sequence alignment showed that TgAtg8 has greatest sequence and structural conservation. Within TgAtg3, this protein possesses the core E2 enzymatic activity structure and a truncated handle region which may contain AIM sequence. Taken together, our findings would help elucidate the formation mechanism of autophagosome in Toxoplasma and provide a possibility for looking into parasitic drug targets.

Copyright © 2015. Published by Elsevier B.V.

KEYWORDS:

Atg3; Atg8; Protein-Protein interaction; Surface plasmon resonance; Toxoplasma gondii; autophagy

PMID:

 

26407821

 

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