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. Molecular &

Genome-Wide Bimolecular Fluorescence Complementation-Based Proteomic Analysis of Toxoplasma gondii ROP18’s Human Interactome Shows Its Key Role in Regulation of Cell Immunity and Apoptosis

“…Among the ROP18 I -interacting proteins, CNBP, DCTD, NUP160, and PRAC, which had been previously confirmed as ROP18 I -interacting proteins by a previous human proteome array ( 22 ), were also identified in our HT-BiFC assay, indicating the reliability and quality of our results. In addition to these known interactions, 488 interactions of ROP18 I with human proteins were newly defined in our study.…”

“…However, YTH generates a high occurrence of false positives and requires that the interacting proteins accumulate in the yeast nucleus ( 21 ). More recently, by using a protein array approach, Yang et al have identified 68 substrates of the Tg ROP18 kinase, and four of them have been validated as the host targets ( 22 ). Although protein array is useful for comprehensive screens of protein functions, it requires pure functional proteins that are difficult to obtain because of the difficulties in expressing the proteins in a soluble form with correct folding ( 23 ).…”

An Integrated Systems Biology Approach Identifies the Proteasome as a Critical Host Machinery for ZIKV and DENV Replication

“…In-depth in vivo studies confirmed that these phosphorylation events played an important role in promoting viral DNA replication in all four viruses. In another study, we observed that a secreted protein kinase ROP18, encoded by Toxoplasma gondii, could phosphorylate multiple components in the MAPK pathway [29]. A third example is the observation that KSHV-encoded LANA protein could bind to all three components of the NER damage recognition/verification complex XPA-RPA (i.e., XPA, RPA1 and RPA2) [47].…”

“…The Human Proteome Microarray v3.0 (HuProt TM array), comprised of 20,240 immobilized human proteins from >15,000 full-length genes, was used to identify human-viral PPI networks [29]. Each viral protein was fluorescently labeled and individually probed to the HuProt array.…”

Toxoplasma gondiiROP18 Inhibits Human Glioblastoma Cell Apoptosis through Mitochondrial Pathway by Targeting Host Cell P2X1

“…It has been reported that overexpression of Tg ROP18 significantly suppresses human embryo kidney epithelial cell 293T apoptosis induced by Actinomycin D at 48hrs post transfection [15], and Tg ROP18 targets P53 for degradation to inhibit host cell apoptosis [30]. On the contrary, Tg ROP18 is also reported to induce the apoptosis of murine neuroblastoma N2a cells through the endoplasmic reticulum stress-mediated apoptosis pathway at 24hrs post infection [14].…”