title: A Human Proteome Array Approach to Identifying Key Host Proteins Targeted byToxoplasmaKinase ROP18.
abstract: Toxoplasmakinase ROP18 is a key molecule responsible for the virulence ofToxoplasma 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 ofToxoplasmainfection 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.
authors.affiliation: From the ‡Department of Biochemistry and Molecular Biology, Sun Yat-Sen University Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, China.
authors.forename: Zhaoshou
authors.initials: Z
authors.lastname: Yang
authors.affiliation: From the ‡Department of Biochemistry and Molecular Biology, Sun Yat-Sen University Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, China.
authors.forename: Yongheng
authors.initials: Y
authors.lastname: Hou
authors.affiliation: From the ‡Department of Biochemistry and Molecular Biology, Sun Yat-Sen University Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, China.
authors.forename: Taofang
authors.initials: T
authors.lastname: Hao
authors.affiliation: the §Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Hee-Sool
authors.initials: HS
authors.lastname: Rho
authors.affiliation: the ¶Department of Ophthalmology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287.
authors.forename: Jun
authors.initials: J
authors.lastname: Wan
authors.affiliation: the ‖State Key Lab of Ophthalmology, Guangdong Provincial Key Lab of Ophthalmology and Visual Science, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060, China.|the **School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China.
authors.forename: Yizhao
authors.initials: Y
authors.lastname: Luan
authors.affiliation: ‡‡The Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510630, China; and.
authors.forename: Xin
authors.initials: X
authors.lastname: Gao
authors.affiliation: §§The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, China.
authors.forename: Jianping
authors.initials: J
authors.lastname: Yao
authors.affiliation: From the ‡Department of Biochemistry and Molecular Biology, Sun Yat-Sen University Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, China.
authors.forename: Aihua
authors.initials: A
authors.lastname: Pan
authors.affiliation: the ‖State Key Lab of Ophthalmology, Guangdong Provincial Key Lab of Ophthalmology and Visual Science, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060, China.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Xie
authors.affiliation: the ¶Department of Ophthalmology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287.
authors.forename: Jiang
authors.initials: J
authors.lastname: Qian
authors.affiliation: From the ‡Department of Biochemistry and Molecular Biology, Sun Yat-Sen University Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, China; zhouxw2@mail.sysu.edu.cn hzhu4@jhmi.edu liaowq5@mail.sysu.edu.cn.
authors.forename: Wanqin
authors.initials: W
authors.lastname: Liao
authors.affiliation: the §Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; zhouxw2@mail.sysu.edu.cn hzhu4@jhmi.edu liaowq5@mail.sysu.edu.cn.
authors.forename: Heng
authors.initials: H
authors.lastname: Zhu
authors.affiliation: From the ‡Department of Biochemistry and Molecular Biology, Sun Yat-Sen University Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, China; zhouxw2@mail.sysu.edu.cn hzhu4@jhmi.edu liaowq5@mail.sysu.edu.cn.
authors.forename: Xingwang
authors.initials: X
authors.lastname: Zhou
mesh_terms: D002460:Cell Line; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D058977:Molecular Sequence Annotation; D010766:Phosphorylation; D040081:Protein Array Analysis / Q000379:methods*; D060066:Protein Interaction Maps; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015800:Protozoan Proteins; D051898:Smad1 Protein / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism
chemical_list: D020543:Proteome; D015800:Protozoan Proteins; C497399:SMAD1 protein, human; D051898:Smad1 Protein; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C471607:UBE2N protein, human; D044763:Ubiquitin-Conjugating Enzymes; D017346:Protein Serine-Threonine Kinases; C556793:ROP18 protein, Toxoplasma gondii