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. 2017 Feb 28;8(1):e02189-16.
doi: 10.1128/mBio.02189-16.

Toxoplasma DJ-1 Regulates Organelle Secretion by a Direct Interaction With Calcium-Dependent Protein Kinase 1

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Free PMC article

Toxoplasma DJ-1 Regulates Organelle Secretion by a Direct Interaction With Calcium-Dependent Protein Kinase 1

Matthew A Child et al. mBio. .
Free PMC article

Abstract

Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson's disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-Å resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, and that this interaction potentiates CDPK1 kinase activity. Finally, we show that genetic deletion of TgDJ-1 results in upregulation of CDPK1 expression and that disruption of the CDPK1/TgDJ-1 complex in vivo prevents normal exocytosis of parasite virulence-associated organelles called micronemes. Overall, our data suggest that TgDJ-1 functions as a noncanonical kinase-regulatory scaffold that integrates multiple intracellular signals to tune microneme exocytosis in T. gondiiIMPORTANCE Apicomplexan parasites such as Toxoplasma and Plasmodium are obligate intracellular parasites that require the protective environment of a host cell in order to replicate and survive within a host organism. These parasites secrete effector proteins from specialized apical organelles to select and invade a chosen host cell. The secretion of these organelles is a tightly regulated process coordinated by endogenous small molecules and calcium-dependent protein kinases. We previously identified the Toxoplasma orthologue of the highly conserved protein DJ-1 as a regulator of microneme secretion, but the molecular basis for this was not known. We have now identified the molecular mechanism for how TgDJ-1 regulates microneme secretion. TgDJ-1 interacts with the kinase responsible for the secretion of these organelles (calcium-dependent kinase 1) and synergizes with calcium to potentiate kinase activity. This interaction is direct, phosphodependent, and necessary for the normal secretion of these important organelles.

Figures

FIG 1
FIG 1
The structure of TgDJ-1 reveals a conserved arginine dyad that coordinates a direct phosphospecific interaction with CDPK1. (A) Surface representation of the TgDJ-1 structure, with the two monomers represented in gray and green. The target of WRR-086 modification, Cys127, is highlighted in yellow, and the oxidized Cys104 in the nucleophilic pocket is shown in stick representation. (B and C) The Arg27/Arg47 dyad coordinates a water molecule with the oxidized Cys104 (B), which is replaced by a PO43− in the structure of reduced HsDJ-1 (PDB accession no. 3BWE) (C). The structure of HsDJ-1 was overlaid with TgDJ-1, and a portion is shown, with the residues equivalent to Arg27/Arg47 shown in light blue. For clarity, TgDJ-1 Cys104 is not shown in panel C. (D) Coomassie and Western blot analysis of pulldown of TgDJ-1 by GST-CDPK1 or GST alone. (E) Coomassie and Western blot analysis of pulldown of TgDJ-1 using either GST-CDPK1 (CDPK1) or GST-CDPK1 coexpressed with lambda phosphatase (CDPK1 + L/phos.). Arrowheads indicate size shifts for the two purified GST-tagged CDPK1 species. (F) Coomassie and Western blot analysis of pulldown of wild-type TgDJ-1 (WT), TgDJ-1R47A (R47A), or TgDJ-1R27A/R47A (R27A/R47A) using either GST-CDPK1 (CDPK1) or GST-CDPK1 coexpressed with lambda phosphatase (CDPK1 + L/phos.). IB, immunoblot.
FIG 2
FIG 2
The association and stability of the TgDJ-1/CDPK1 complex are sensitive to secondary messengers. (A) Western blot analysis of the ability of HA-tagged CDPK1 to coprecipitate FLAG-tagged TgDJ-1 under low-calcium (+5 mM EGTA) (−calcium) or high-calcium (+5 mM CaCl2) (+calcium) conditions and reductive (R) or oxidative (Ox) conditions. IP, immunoprecipitation. IB, immunoblot. (B) Western blot analysis of the stability of isolated TgDJ-1/CDPK1 complex in the presence of 5 mM calcium (± calcium), hydrogen peroxide (+H2O2), or both.
FIG 3
FIG 3
TgDJ-1 potentiates CDPK1 kinase activity, with the effect dependent upon, and synergistic with, calcium. (A) Kinase activity assay to optimize CDPK1 concentration. Scatter plot of data points from a kinase assay optimizing the concentration of CDPK1 kinase required to generate robust signal for 10 µM syntide and 10 µM ATP. (B) Kinase activity assay to optimize calcium concentration for CDPK1 activation. Scatter plot of data points from a kinase assay testing the concentration of calcium required to fully activate CDPK1. (C) Kinase activity assay testing the effect of TgDJ-1 upon CDPK1 activity. Scatter plot of data points from a kinase assay where TgDJ-1 was titrated against CDPK1, either in the presence (CDPK1+DJ-1) or absence of the peptide substrate (no syntide). (D) Kinase assay testing the effect of calcium on pre-complexed CDPK1+DJ-1. Scatter plot of kinase assay data points where CDPK1 alone (CDPK1 only), TgDJ-1 alone (DJ-1 only), or TgDJ-1+CDPK1 (DJ-1+CDPK1) was incubated with EDTA, 50 nM, or 500 µM calcium. Two concentrations of TgDJ-1 were used: 0.26 µM (low DJ-1) or 2.6 µM (high DJ-1). Significance was calculated for a two-way ANOVA statistical analysis (with Dunnett’s multiple comparison test) comparing each group of means (EDTA, 50 nM or 500 µM calcium) with the CDPK1 only control within the group. ns, not significant; ****, P ≤ 0.0001. (E) Kinase assay testing the effect of incubating CDPK1 with TgDJ-1 in the presence of a titration of calcium. Histogram of kinase assay data points for CDPK1 only, or TgDJ-1+CDPK1 pre-incubated with 5, 50, 500, or 5,000 µM calcium. Data presented are the means of three replicates, with error bars indicating ± SD. Significance was calculated for an ordinary one-way ANOVA statistical analysis (with Dunnett’s multiple comparison test) comparing each mean with the CDPK1 only control. ns, not significant; ****, P ≤ 0.0001. (F) Kinase assay testing the capacity of different TgDJ-1 mutants to potentiate CDPK1 activity. Histogram of kinase assay data points for CDPK1 alone (CDPK1 only), or CDPK1 incubated with 10 µM wild-type, C104A, R47A, or R27A/R47A mutant TgDJ-1 protein. Data presented are the means of three replicates, with error bars indicating ± SD. Significance was calculated for an ordinary one-way ANOVA statistical analysis (with Dunnett’s multiple comparison test) comparing each mean with wild-type TgDJ-1 (WT). ns, not significant; ****, P ≤ 0.0001. The panel beneath the histogram is a Coomassie stained gel indicating equal input of the different TgDJ-1 mutant proteins in the kinase assay reactions. For panels A, B, C, and D, data presented are the means of three replicates, with error bars indicating ± SD. RLU, relative light units.
FIG 4
FIG 4
Phenotypic characterization of ΔTgdj-1 parasites. (A) Strategy to replace the native dj-1 locus with the drug-selectable HXGPRT cassette and generate the ΔTgdj-1 parasite line. (B) PCR confirmation of loss of the dj-1 locus in two individual clones (C10 and F6) isolated from two independent transfections (Tf1 and Tf2). (C) Western blot confirmation of the loss of TgDJ-1 protein expression in the same ΔTgDJ-1 parasite clones. SAG1, loading control. (D) Plaque assays comparing growth of wild-type (WT) and TgDJ-1 knockout (ΔTgDJ-1) parasites. (E) Gliding motility assays comparing the 2D motility of wild-type (WT) and TgDJ-1 knockout (ΔTgdj-1) parasites. (F) Microneme secretion assays comparing the levels of basal secretion of MIC2 over time for the wild-type (WT) strain and two individual ΔTgDJ-1 clones (C10 and F6) isolated from two independent transfections. GRA7, loading control. (G) Quantification of the microneme secretion assays. Asterisk indicates the level of significance as determined by Student’s t test; data represent means ± SD for results from n = 3 experiments. (H) Attachment/invasion assay comparing wild-type (WT) and TgDJ-1 knockout (ΔTgdj-1) parasites. The histogram presents the quantification of raw parasite numbers for attachment/invasion assays for the wild-type (WT) strain versus ΔTgdj-1 clone F6. Asterisk indicates the level of significance as determined by Student’s t test; data represent means ± SD for results from n = 3 experiments.
FIG 5
FIG 5
TgDJ-1 regulates microneme secretion. (A) Western blots of microneme secretion assays comparing levels of basal secretion of MIC2 over time for wild-type (WT), ΔTgdj-1 clone F6, and ΔTgdj-1 parasites complemented with wild-type Tgdj-1 (ΔTgdj-1WTcomp). GRA7, loading control. (B) Quantification of basal microneme secretion assays; data represent means ± SD for results from n = 3 experiments. Significance was calculated for a two-way statistical ANOVA for comparison of the different strains (WT, ΔTgdj-1, and ΔTgdj-1WTcomp) (****, P ≤ 0.0001) and the different time points (15, 30, and 60 min) (****, P ≤ 0.0001). Post hoc pairwise comparisons of the different strains at each time point were performed using Tukey’s multiple-comparison test and the data presented in Table S1. (C) Western blots of microneme secretion assays comparing ethanol-induced secretion of MIC2 with different concentrations of ethanol for the wild-type strain (WT), the TgDJ-1 knockout strain (ΔTgDJ-1), and the TgDJ-1 knockout strain complemented with wild-type TgDJ-1 (ΔTgDJ-1WTcomp). GRA7, loading control. (D) Quantification of induced microneme secretion assays; data represent means ± SD for results from n = 3 experiments. Significance was calculated for a two-way statistical ANOVA for comparison of the different strains (WT, ΔTgdj-1, and ΔTgdj-1WTcomp) (****, P ≤ 0.0001) and the different proportions of EtOH (0%, 0.1%, and 1%) (****, P ≤ 0.0001). Post hoc pairwise comparisons of the different strains at each ethanol concentration were performed using Tukey’s multiple-comparison test and the data presented in Table S2. (E) Western blot comparing expression levels of CDPK1 in wild-type (WT), ΔTgdj-1, and ΔTgdj-1WTcomp parasites. SAG1, loading control. (F) Quantification of CDPK1 expression levels. Data represent means ± SD for results from n = 3 experiments. Significance was calculated for an ordinary one-way statistical ANOVA to assess the difference between the means (***, P ≤ 0.001). Results of a post hoc Tukey’s multiple-comparison test for pairwise comparisons of wild-type (WT), Tgdj-1, and ΔTgdj-1WTcomp parasites are presented. ns, not significant. **, P ≤ 0.01. ***, P ≤ 0.001.
FIG 6
FIG 6
Engineered drug-insensitive TgDJ-1 alleles confirm the functional contribution of the TgDJ-1/CDPK1 complex to microneme secretion. (A) Schematic of the strategy to engineer and introduce drug-insensitive alleles to generate merodiploid parasite strains. (B) Western blot analysis using both anti-CDPK1 and anti-FLAG antibodies for expression of CDPK1 and FLAG-tagged TgDJ-1 in isogenic parasite strains expressing either TgDJ-1C127A (C127A), TgDJ-1C127A/C104A (C104A), TgDJ-1C127A/R47A (R47A), or TgDJ-1C127A/R27A/R47A (R27A/R47A). This nomenclature for the merodiploid parasites is used throughout. (C) Indirect immunofluorescence assays examining the localization of intracellular TgDJ-1 merodiploid parasites. Gliding-associated protein 45 (GAP45) is used as a marker for the inner-membrane complex/plasma membrane; DAPI, nuclear stain. Scale bar = 5 µM. (D) Plaque assays comparing growth of wild-type and TgDJ-1 merodiploid parasites. (E) Quantification of Western blot analysis of induced microneme secretion assays performed in the presence of a titration of WRR-086. Data represent means ± SD for n = 3 experiments. Significance was calculated for a two-way ANOVA statistical analysis comparing wild-type and all merodiploid strains. Significance for comparison of the different strain group means = **** (P ≤ 0.0001); and the different WRR-086 concentrations (0, 1, 5, 10 µM) = **** (P ≤ 0.0001). Post-hoc pairwise comparison of the different strains for each WRR-086 concentration was performed using Tukey’s multiple comparison test and the data presented in Table S3.
FIG 7
FIG 7
Model molecular pathway for the regulation of microneme secretion by TgDJ-1 and CDPK1. (A) CDPK1 is thought to autophosphorylate as part of its mechanism of activation. The negatively charged phosphate is indicated (-P-). (B) Under reducing conditions, TgDJ-1 associates with the phosphorylated, calcium-depleted conformer of CDPK1 to produce state B, with the negative charge of the phosphate recognized and coordinated by the conserved arginine dyad on TgDJ-1. These positively charged arginine residues are indicated (+). (C) The addition of calcium produces the final biologically active TgDJ-1/CDPK1 complex, as illustrated, with TgDJ-1 synergizing with calcium to potentiate CDPK1 activity. This potentiated complex is required to maintain normal microneme secretion. (D) The combination of a calcium and ROS signal (possibly H2O2) triggers dissociation of the complex, returning CDPK1 to the unbound, lower-activity state, allowing the kinase to recycle to reassociate with a pool of reduced TgDJ-1 protein. (E) The small-molecule inhibitor WRR-086 binds to TgDJ-1 and potentially prevents association with CDPK1. This prevents of the formation of the TgDJ-1/kinase complex shown in panel B, with the net result being that the CDPK1 cannot achieve the TgDJ-1-potentiated activity state, leading to a block in microneme secretion as previously reported (18).

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