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

Mol Cell Proteomics. 2017 Mar;16(3):469-484. doi: 10.1074/mcp.M116.063602. Epub 2017 Jan 13.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Molecular Sequence Annotation
  • Phosphorylation
  • Protein Array Analysis / methods*
  • Protein Interaction Maps
  • Protein-Serine-Threonine Kinases / metabolism*
  • Proteome / metabolism*
  • Proteomics / methods*
  • Smad1 Protein / metabolism
  • Tumor Suppressor Protein p53 / metabolism
  • Ubiquitin-Conjugating Enzymes / metabolism

Substances

  • Proteome
  • SMAD1 protein, human
  • Smad1 Protein
  • TP53 protein, human
  • Tumor Suppressor Protein p53
  • UBE2N protein, human
  • Ubiquitin-Conjugating Enzymes
  • Protein-Serine-Threonine Kinases
  • ROP18 protein, Toxoplasma gondii