Affinage · Toxoplasma gondii

ASP3

aspartyl protease ASP3 · UniProt A0A125YQD9

Annotated
2026-09-08
6 papers in source corpus 3 papers cited in narrative 3 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

TgASP3 is an aspartyl protease of the endosomal-like/post-Golgi compartment of Toxoplasma gondii that functions as an essential maturase for microneme and rhoptry proteins, with its proteolytic activity required for rhoptry discharge during host cell invasion and for plasma membrane lysis during egress (PMID:28898199). N-terminomic profiling of ASP3-depleted parasites established a defined set of secretory effector substrates whose maturation depends on this enzyme both in vivo and in vitro (PMID:28898199). TgASP3 operates within a sequential maturation pathway, performing early cleavages of micronemal effectors upstream of the final trimming step carried out by TgCPC1, whose loss blocks downstream processing of ASP3-primed substrates without affecting ASP3 itself (PMID:37326431). A conserved flap-region phenylalanine (F344) gates access to the catalytic cavity and determines inhibitor/substrate recognition: mutation of F344 raises the IC50 of the antimalarial inhibitor 49c, and drug-resistance selection reproducibly converts F344 to cysteine (PMID:29519896).

Mechanistic history

Synthesis pass · year-by-year structured walk · 3 steps
  1. 2017 High

    Established that TgASP3 is the maturase responsible for processing microneme and rhoptry proteins, answering what enzymatic step licenses these secretory effectors for organellar storage and discharge.

    Evidence Conditional depletion with TAILS N-terminomics, in vitro protease assays, and subcellular localization in T. gondii

    PMID:28898199

    Open questions at the time
    • Structural basis of substrate cleavage not resolved
    • Mechanism coupling ASP3 maturation to rhoptry discharge and membrane lysis not defined
    • Full substrate repertoire beyond identified microneme/rhoptry proteins not delineated
  2. 2018 High

    Identified the flap-region residue F344 as the determinant of inhibitor and substrate recognition, explaining how the catalytic cavity is gated and how resistance arises.

    Evidence Homology modeling, docking, F344 site-directed mutagenesis, and drug-resistance selection with IC50 measurements in T. gondii

    PMID:29519896

    Open questions at the time
    • No experimental crystal/cryo-EM structure to confirm the docking model
    • Effect of F344 variation on physiological substrate cleavage versus inhibitor binding not disentangled
  3. 2023 Medium

    Placed TgASP3 within an ordered proteolytic maturation cascade, showing it performs early cleavages upstream of the final TgCPC1 trimming step.

    Evidence Genetic deletion of TgCPC1 with substrate maturation-state readouts and invasion/egress assays in T. gondii

    PMID:37326431

    Open questions at the time
    • Direct substrate handoff between ASP3, TgCPL, and TgCPC1 not biochemically reconstituted
    • Whether ASP3 and TgCPL act redundantly or on distinct sites not resolved

Open questions

Synthesis pass · forward-looking unresolved questions
  • The three-dimensional structure of TgASP3 and the molecular mechanism linking effector maturation to the physical events of rhoptry discharge and membrane lysis remain undefined.
  • No experimentally determined enzyme structure
  • No reconstitution of the full ASP3-TgCPL-TgCPC1 cascade
  • Regulation of ASP3 activity and trafficking within the endosomal-like compartment unknown

Mechanism profile

Synthesis pass · controlled-vocabulary classification
Partners
TGCPC1 (DOWNSTREAM MATURASE IN THE SAME SECRETORY PROCESSING CASCADE)TGCPL (ENDOLYSOSOMAL PROTEASE ACTING IN THE SAME EFFECTOR MATURATION PATHWAY)

Evidence

Reading pass · 3 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2017 TgASP3 resides in the endosomal-like compartment (post-Golgi) of T. gondii and acts as a maturase for microneme and rhoptry proteins; depletion of ASP3 blocks rhoptry discharge during invasion and host cell plasma membrane lysis during egress. Substrates were identified by N-terminome profiling (TAILS) comparing wild-type and ASP3-depleted parasites, and maturation was confirmed both in vivo and in vitro. Conditional depletion (knockdown), terminal amine isotopic labelling of substrates (TAILS/N-terminomics), in vitro protease assays, subcellular fractionation/localization imaging eLife High 28898199
2018 TgASP3 phylogenetically clusters with Plasmodium falciparum Plasmepsins IX and X and shares a conserved phenylalanine residue (F344) in the flap region that is critical for binding the antimalarial inhibitor 49c; mutation of F344 increases IC50 of 49c 2–15-fold, and selection of resistant T. gondii strains consistently converts F344 to cysteine, establishing this flap residue as a key determinant of substrate/inhibitor recognition. Homology modeling, molecular docking, site-directed mutagenesis (F344 variants), drug-resistance selection in T. gondii, IC50 measurements The EMBO journal High 29519896
2023 TgASP3 acts upstream of TgCPC1 in the secretory protein maturation pathway; TgASP3 (post-Golgi) and TgCPL (endolysosomal system) perform earlier proteolytic cleavages on micronemal effectors, while TgCPC1 mediates the final trimming step. Loss of TgCPC1 does not affect TgASP3 itself but blocks downstream maturation of effectors that TgASP3 had partially processed. Genetic deletion of TgCPC1, comparison of maturation states of secretory proteins relative to TgASP3/TgCPL-dependent cleavages, invasion/egress phenotypic assays mBio Medium 37326431

Source papers

Stage 0 corpus · 6 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2017 A druggable secretory protein maturase of Toxoplasma essential for invasion and egress. eLife 80 28898199
2004 Host cell invasion by the apicomplexans: the significance of microneme protein proteolysis. Current opinion in microbiology 72 15358257
2017 Immunization with Toxoplasma gondii aspartic protease 3 increases survival time of infected mice. Acta tropica 17 28238685
2018 Modeling and resistant alleles explain the selectivity of antimalarial compound 49c towards apicomplexan aspartyl proteases. The EMBO journal 14 29519896
2023 A cathepsin C-like protease mediates the post-translation modification of Toxoplasma gondii secretory proteins for optimal invasion and egress. mBio 4 37326431
2023 A cathepsin C-like protease post-translationally modifies Toxoplasma gondii secretory proteins for optimal invasion and egress. bioRxiv : the preprint server for biology 0 36712013