M18AAP

M18AAP (PF3D7_0932300) is a metal-dependent aspartyl aminoexopeptidase of intra-erythrocytic P. falciparum that is required for parasite proliferation and is implicated in the terminal degradation of peptides in the parasite cytosol (PMID:17720817). The enzyme displays strict exopeptidase specificity, cleaving only N-terminal acidic residues (glutamate or aspartate), operates optimally at neutral pH, and depends on metal ions for both activity and stability (PMID:17720817, PMID:22709581). Biochemical sizing indicated a ~560-kDa oligomer, and X-ray crystallography resolved a dodecameric tetrahedral assembly that encloses 12 active sites within a central catalytic cavity accessed through four entry points guarded by flexible loops, with active-site features distinct from mammalian homologs (PMID:17720817, PMID:22709581). M18AAP localizes predominantly to the parasite cytosol but is also detected in the parasitophorous vacuole, indicating trafficking out of the parasite, and antisense knockdown is lethal (PMID:17720817). Recombinant M18AAP binds several human erythrocyte membrane and cytoskeletal proteins including spectrin in vitro, and its spectrin-binding region is absent in human, yeast, and other Plasmodium homologs (PMID:18721457). The enzyme is inhibitable by small molecules with associated antiplasmodial activity, supporting it as a candidate antimalarial target (PMID:24619116).

1. 2007 Medium

   Establishing where the enzyme acts and whether it matters was the first question; localization plus knockdown defined M18AAP as an essential cytosolic (and partly PV-trafficked) enzyme of blood-stage parasites.

   Evidence Immunocytochemistry and immunoblot fractionation of transgenic parasites with antisense RNA knockdown in P. falciparum

   PMID:17720817

   Open questions at the time

   • Mechanism of trafficking to the parasitophorous vacuole unresolved
   • Precise cause of lethality (which pathway fails) not defined
   • Endogenous physiological substrates not identified
2. 2007 High

   Defining the catalytic identity of the enzyme was needed; biochemical reconstitution showed strict N-terminal acidic-residue specificity, neutral pH optimum, metal dependence, and an oligomeric assembly.

   Evidence Recombinant expression, native PAGE/size-exclusion sizing, defined-substrate activity and metal-dependence assays for P. falciparum M18AAP

   PMID:17720817

   Open questions at the time

   • Identity of the required metal ion not resolved
   • Relationship of the biochemical oligomer to a cellular assembly unclear at this stage
3. 2007 Medium

   Placing M18AAP within compartmentalized amino-acid generation showed it acts in the cytosol distinct from food-vacuole aminopeptidases while contributing to efficient proliferation.

   Evidence Localization of epitope-tagged aminopeptidases, food-vacuole fractionation, bestatin growth inhibition and amino-acid supplementation in P. falciparum

   PMID:17895246

   Open questions at the time

   • Direct demonstration that M18AAP acts on hemoglobin-derived peptides in vivo is lacking
   • Functional interplay with the food-vacuole enzymes not resolved
4. 2008 Medium

   Testing a possible host-interaction role revealed the enzyme binds erythrocyte membrane/cytoskeletal proteins, with a spectrin-binding region unique among homologs.

   Evidence Blot overlay of recombinant P. falciparum M18AAP against erythrocyte membrane proteins plus coupled activity assays

   PMID:18721457

   Open questions at the time

   • Binding shown only in vitro by a single method (blot overlay)
   • Functional consequence of erythrocyte-protein binding in vivo unknown
   • No reciprocal validation of the interactions
5. 2012 High

   The architectural basis of catalysis was unresolved until crystallography revealed a dodecameric tetrahedral cage enclosing 12 active sites accessed through gated entry points, with distinct active-site features versus mammalian homologs.

   Evidence X-ray crystallography and positional-scanning fluorogenic substrate profiling of P. falciparum M18AAP

   PMID:22709581

   Open questions at the time

   • How the flexible gating loops select substrates mechanistically not established
   • Structural basis of erythrocyte-protein binding not resolved
   • No structure with a physiological substrate or inhibitor bound
6. 2014 Medium

   Establishing druggability, small-molecule inhibitors of recombinant enzyme with antiplasmodial activity supported M18AAP as an antimalarial target and defined a noncompetitive inhibition mode.

   Evidence High-throughput fluorescence enzymatic assay with Ki determination, cathepsin L1 counter-screen, and parasite growth inhibition in P. falciparum

   PMID:24619116

   Open questions at the time

   • On-target mechanism of killing in parasites not directly demonstrated
   • Micromolar potency leaves selectivity/potency unoptimized
   • Binding site of the noncompetitive inhibitors not mapped
7. 2011 Low

   A natural-product screen identified a plant-derived triazinone oxime as an in vitro inhibitor of the enzyme.

   Evidence GC-MS identification, coupled enzyme assay, and depletion experiment with recombinant P. falciparum M18AAP (crude extract context)

   PMID:21687598

   Open questions at the time

   • Inhibition measured in crude extract with indirect depletion, not with purified compound kinetics
   • No cellular antiplasmodial confirmation for the isolated compound
   • Not independently replicated

• The endogenous in vivo substrates and the precise metabolic role of M18AAP in hemoglobin-derived peptide catabolism remain to be directly demonstrated.
• No in vivo substrate identified
• Contribution to cytosolic peptide degradation inferred but not directly measured
• Physiological relevance of erythrocyte-protein binding unknown