Target identification of the novel Toxoplasma gondii rhoptry kinase RON13

ABSTRACT: The Toxoplasma gondii rhoptry neck protein RON13 was first identified as a substrate of aspartyl protease 3 by Terminal Amine Isotopic Labelling of Substrates (TAILS8, Dogga et al. 2017). It harbours a kinase domain the activity of which is important for host cell invasion. To identify the targets of RON13, we engineered a transgenic T. gondii line in the RH ΔKu80 background exploiting the tetracycline repressor system (Meissner et al. 2001). Here, the change of promoter resulted in the overall downregulation of RON13 expression leading to parasites that display significantly diminished infection capability. This defect can be rescued by complementation with an active version of RON13 kinase. We have performed phosphopeptide enrichment and shotgun proteomics using the parental line and three mutant strains: RON13 knock-down (RON13-KD), RON13 knock-down complemented with wildtype RON13 as well as RON13 knock-down complemented with the catalytically dead kinase in which an essential aspartate was replaced by alanine. Comparison of the results between the parental line and RON13-KD allowed us to identify phosphosites that were differentially phosphorylated. Validation of those phosphosites was performed using the comparison between the two complemented lines with the active or the inactive versions of RON13.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Toxoplasma Gondii Rh

TISSUE(S): Foreskin Fibroblast Cell Line

SUBMITTER: Chandra Ramakrishnan  

LAB HEAD: Prof. Adrian B. Hehl

PROVIDER: PXD018056 | Pride | 2021-05-13

REPOSITORIES: Pride