AAP

The 'AAP' designation in Toxoplasma gondii spans two mechanistically distinct proteins. TgAAP is a cytoplasmic, metal-dependent aspartyl aminopeptidase that cleaves N-terminal acidic residues, preferring glutamate and aspartate (PMID:27678060); CRISPR/Cas9 knockout reduces parasite attachment/invasion, replication, and substrate-specific aminopeptidase activity without abolishing growth, marking it a contributory but non-essential enzyme (PMID:27678060). AAP7 is a separate apical annuli protein that traffics LMBD3 to the plasma membrane, connecting plasma-membrane-embedded LMBD3 to IMC suture-embedded AAP proteins; its depletion causes LMBD3 to mislocalize among secretory organelles (PMID:41256620). AAP7 depletion also leads to accumulation of polyglutamylated subpellicular microtubules (SPMTs) at the basal end of the parasite, indicating AAP7 influences SPMT turnover (PMID:41256620).

1. 2016 High

   Establishing the enzymatic identity of TgAAP answered whether the protein is a functional aminopeptidase and defined its substrate preference, positioning it as a metal-dependent aspartyl aminopeptidase acting on N-terminal acidic residues.

   Evidence In vitro enzymatic assay of recombinant protein expressed in E. coli

   PMID:27678060

   Open questions at the time

   • Physiological substrates in the parasite not identified
   • Metal-ion identity and catalytic mechanism not resolved
2. 2016 Medium

   Localizing TgAAP to the tachyzoite cytoplasm placed its aminopeptidase activity in the cytosolic compartment rather than a secretory or membrane site.

   Evidence Indirect immunofluorescence of wild-type parasites

   PMID:27678060

   Open questions at the time

   • Cytoplasmic interactors and downstream targets not defined
3. 2016 Medium

   Genetic knockout tested whether TgAAP activity is functionally required, showing it contributes to attachment/invasion and replication but is dispensable for viability.

   Evidence CRISPR/Cas9 knockout with invasion, replication, and aminopeptidase activity assays

   PMID:27678060

   Open questions at the time

   • Molecular link between aminopeptidase activity and invasion/replication phenotype unresolved
   • Compensating peptidases not excluded
4. 2025 Medium

   Characterizing AAP7 defined a distinct apical annuli protein whose role is trafficking LMBD3 to the plasma membrane, linking the annuli architecture to plasma-membrane cargo delivery.

   Evidence Auxin-inducible degron depletion of AAP7 with fluorescence localization of LMBD3 (preprint)

   PMID:41256620

   Open questions at the time

   • Direct physical interaction between AAP7 and LMBD3 not demonstrated
   • Molecular basis of the novel trafficking route unknown
5. 2025 Medium

   Connecting AAP7 depletion to a secretion defect identified impaired GRA17 dense granule secretion as the critical functional consequence, causally validated by GRA17 overexpression rescue of the bubble vacuole phenotype.

   Evidence Conditional depletion, dense granule secretion assays, and GRA17 overexpression rescue (preprint)

   PMID:41256620

   Open questions at the time

   • Mechanism by which AAP7 promotes GRA17 secretion not defined
   • Whether the effect is direct or through LMBD3 trafficking unresolved
6. 2025 Low

   Observing polyglutamylated microtubule accumulation upon AAP7 depletion raised a possible role in SPMT turnover.

   Evidence Conditional depletion with immunofluorescence of polyglutamylated tubulin (preprint)

   PMID:41256620

   Open questions at the time

   • Single indirect marker readout with no direct mechanistic link to AAP7
   • Not independently confirmed
   • Causality versus secondary consequence of secretion defect unresolved