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A Human Proteome Array Approach to Identifying Key Host Proteins Targeted by Toxoplasma Kinase ROP18.

ABSTRACT: Toxoplasma kinase ROP18 is a key molecule responsible for the virulence of Toxoplasma gondii; however, the mechanisms by which ROP18 exerts parasite virulence via interaction with host proteins remain limited to a small number of identified substrates. To identify a broader array of ROP18 substrates, we successfully purified bioactive mature ROP18 and used it to probe a human proteome array. Sixty eight new putative host targets were identified. Functional annotation analysis suggested that these proteins have a variety of functions, including metabolic process, kinase activity and phosphorylation, cell growth, apoptosis and cell death, and immunity, indicating a pleiotropic role of ROP18 kinase. Among these proteins, four candidates, p53, p38, UBE2N, and Smad1, were further validated. We demonstrated that ROP18 targets p53, p38, UBE2N, and Smad1 for degradation. Importantly, we demonstrated that ROP18 phosphorylates Smad1 Ser-187 to trigger its proteasome-dependent degradation. Further functional characterization of the substrates of ROP18 may enhance understanding of the pathogenesis of Toxoplasma infection and provide new therapeutic targets. Similar strategies could be used to identify novel host targets for other microbial kinases functioning at the pathogen-host interface.

SUBMITTER: Yang Z

PROVIDER: S-EPMC5341007 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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A Human Proteome Array Approach to Identifying Key Host Proteins Targeted by Toxoplasma Kinase ROP18.

Yang Zhaoshou Z Hou Yongheng Y Hao Taofang T Rho Hee-Sool HS Wan Jun J Luan Yizhao Y Gao Xin X Yao Jianping J Pan Aihua A Xie Zhi Z Qian Jiang J Liao Wanqin W Zhu Heng H Zhou Xingwang X

Molecular & cellular proteomics : MCP 20170113 3

Toxoplasma kinase ROP18 is a key molecule responsible for the virulence of Toxoplasma gondii; however, the mechanisms by which ROP18 exerts parasite virulence via interaction with host proteins remain limited to a small number of identified substrates. To identify a broader array of ROP18 substrates, we successfully purified bioactive mature ROP18 and used it to probe a human proteome array. Sixty eight new putative host targets were identified. Functional annotation analysis sugge ...[more]

PMID: 28087594

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Project description:BackgroundApoptosis plays a critical role in the embryonic development, homeostasis of immune system and host defense against intracellular microbial pathogens. Infection by the obligate intracellular pathogen Toxoplasma gondii can both inhibit and induce host cell apoptosis; however, the parasitic factors involved remain unclear. The T. gondii virulence factor ROP18 (TgROP18) has been reported to regulate host cell apoptosis; nevertheless, results for this regulation have been rarely reported or have provided contradictory findings. Human purinergic receptor 1 (P2X1) is an ATP-gated ion channel that responds to ATP stimulation and functions in cell apoptosis mediation. The precise roles of TgROP18 in T. gondii pathogenesis, and the relationship between TgROP18 and host P2X1 in host cell apoptosis are yet to be revealed.MethodsApoptosis rates were determined by flow cytometry (FCM) and TUNEL assay. The interaction between TgROP18 and the host P2X1 was measured by fluorescence resonance energy transfer (FRET) and co-immunoprecipitation (co-IP) assay. Calcium influx and mitochondrial membrane depolarization were determined by FCM after JC-1 staining. The translocation of cytochrome C (Cyt C), Bax and Bcl2 proteins, expression of the apoptotic proteins PARP and caspase activation were detected by western blotting.ResultsThe apoptosis rates of glial or immune cells (human SF268, mouse RAW264.7 and human THP-1 cells) infected by any T. gondii strain (RH-type I, ME49-type II and VEG-type III) were significantly inhibited compared with their uninfected controls. TgROP18 inhibited ATP-induced apoptosis of SF268 with P2X1 expression, but had no effect on RAW264.7 or THP-1 cells without detectable P2X1 expression. It was further identified that TgROP18 interacted with P2X1, and overexpression of ROP18 in COS7 cells significantly inhibited cell apoptosis mediated by P2X1. Moreover, TgROP18 also inhibited P2X1-mediated Ca2+ influx, translocation of cytochrome C from the mitochondria to the cytosol, and ATP-triggered caspase activation.ConclusionsToxoplasma gondii infection inhibits ATP-induced host cell apoptosis, regardless of strain virulence and host cell lines. TgROP18 targets the purinergic receptor P2X1 of the SF268 human neural cells and inhibits ATP-induced apoptosis through the mitochondrial pathway, suggesting a sensor role for the host proapoptotic protein P2X1 in this process.

Dataset Information

A Human Proteome Array Approach to Identifying Key Host Proteins Targeted by Toxoplasma Kinase ROP18.

Publications

A Human Proteome Array Approach to Identifying Key Host Proteins Targeted by <i>Toxoplasma</i> Kinase ROP18.

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