HDP

PF3D7_1446800 (HDP) is an essential Plasmodium protein whose function has been the subject of two divergent mechanistic models built on different experimental foundations (PMID:18437218, PMID:42567866). In vitro and heterologous/non-endogenous-tagging studies characterized HDP as a highly potent heme detoxification protein that converts free heme into hemozoin: recombinant protein possesses two heme-binding sites with a heme-protein spectrum (PMID:24025682), and histidine residues His122, His172, His175, and His197 act as molecular tethers that position two hemes for dimer formation seeding hemozoin crystal growth (PMID:25138161), with heme-to-hemozoin conversion proceeding through a transient cyclic heme-dimer intermediate and depending on the N-terminal region (PMID:35934300, PMID:40759288). In this model HDP reaches the food vacuole via an 'Outbound-Inbound' trafficking route through the infected-erythrocyte cytosol (PMID:18437218), associates with a food-vacuole protease complex containing falcipain 2/2', plasmepsins II and IV, and histo-aspartic protease to couple hemoglobin degradation to hemozoin formation (PMID:23471987), and contributes to hemoglobin uptake through interactions with hemoglobin and PTEX translocon components including PfEXP-2 (PMID:36238365). A subsequent study using endogenous tagging and genetic knockout instead localizes HDP to the mitochondrion and places it within the mitoribosomal large subunit, where its loss abolishes mitochondrial electron flow through complexes III and IV and causes developmental arrest rescued by bypassing respiratory-chain-dependent pyrimidine biosynthesis, while having no detectable effect on hemozoin formation (PMID:42567866). The prior food-vacuole/heme-detoxification assignments therefore require re-evaluation against this endogenous-tagging and knockout evidence (PMID:42567866).

1. 2008 High

   Establishing whether a dedicated parasite factor drives heme-to-hemozoin conversion, this work identified HDP as a potent heme-detoxifying protein trafficked to the food vacuole and showed the locus is essential.

   Evidence In vitro hemozoin formation assay, cellular fractionation/trafficking, and failed gene-disruption in P. falciparum

   PMID:18437218

   Open questions at the time

   • Essentiality inferred from failed disruption, not conditional depletion
   • Trafficking route inferred rather than directly imaged for endogenous protein
   • In vitro activity does not establish the physiological site of action
2. 2012 Medium

   To connect HDP to antimalarial action, reduced HDP expression was linked to loss of hemozoin formation and artemisinin resistance, implying hemozoin production is part of the artemisinin mechanism.

   Evidence In vivo drug-pressure selection in P. yoelii with hemozoin, HDP expression, and glutathione-pathway readouts

   PMID:22403683

   Open questions at the time

   • Correlative expression change rather than defined genetic lesion
   • Does not exclude other adaptive changes in resistant strains
   • Rodent-parasite model of unclear translation to human parasites
3. 2013 High

   To define the biochemical context of heme processing, HDP was shown to form part of a food-vacuole protease complex and to cooperate with falcipain 2 in converting hemoglobin to hemozoin, and separately to possess two intrinsic heme-binding sites.

   Evidence Co-IP/MS, gel filtration, glycerol gradient cosedimentation and in vitro reconstitution [#1]; recombinant protein UV-visible spectroscopy [#2] in P. falciparum

   PMID:23471987 PMID:24025682

   Open questions at the time

   • Complex composition from co-fractionation does not establish direct binary contacts for all members
   • His-tag altered heme-binding, complicating interpretation of recombinant spectra
   • Physiological necessity of the complex for hemozoin formation not tested by depletion
4. 2014 High

   To resolve the catalytic chemistry, specific histidines were shown to tether and position two hemes for dimer formation that seeds hemozoin crystallization.

   Evidence Site-directed mutagenesis of His122/His172/His175/His197 with hemozoin assay and UV-visible/resonance Raman spectroscopy in P. falciparum

   PMID:25138161

   Open questions at the time

   • Each mutation only halved activity, leaving residual mechanism unexplained
   • No crystal structure of the heme-bound protein
   • Assays performed in vitro with recombinant protein
5. 2015 Medium

   Cross-species conservation of the heme-detoxification model was tested by characterizing the P. vinckei ortholog's high-affinity heme binding and histidine-dependent hemozoin formation.

   Evidence Recombinant PvHDP heme-binding and hemozoin assays, DEPC histidine modification, subcellular fractionation and stage western blots in P. vinckei

   PMID:25891072

   Open questions at the time

   • Cytosolic localization from fractionation not corroborated by imaging
   • Ortholog behavior may not fully mirror P. falciparum protein
   • No genetic test of function
6. 2017 Medium

   To link HDP function to hemoglobin processing in cells, a hemoglobin-binding domain was mapped and a peptide against it phenocopied cysteine-protease-inhibitor food-vacuole defects.

   Evidence In silico modeling, biochemical binding assays, and peptide competition in live P. falciparum 3D7 with morphological readout

   PMID:28949547

   Open questions at the time

   • Peptide phenotype does not prove the endogenous domain acts in the food vacuole
   • Binding-site assignment partly in silico
   • No direct depletion of HDP
7. 2022 High

   Conditional depletion and reciprocal interaction studies extended HDP's proposed role to hemoglobin uptake via PTEX-associated partners, and kinetic/truncation work refined the heme-to-hemozoin reaction as multi-step and N-terminus-dependent.

   Evidence GFP-trap Co-IP, GlmS knockdown with hemoglobin-uptake and morphology readouts [#7]; pyridine hemochrome kinetics and N-terminal truncation constructs [#8] in P. falciparum

   PMID:35934300 PMID:36238365 PMID:40759288

   Open questions at the time

   • Localization by antibody/IP overlaps compartments not resolved to a single site
   • Interaction with PTEX components not shown to be direct
   • Kinetic intermediate identity inferred, not structurally captured
8. 2026 High

   Endogenous tagging and knockout overturned the food-vacuole/heme-detoxification model, placing HDP in the mitochondrion as a mitoribosomal large-subunit component essential for mitochondrial protein synthesis and respiration.

   Evidence Endogenous tagging with live imaging, genetic knockout, bioenergetic assays, drug-sensitivity, quantitative proteomics and mitoribosome structural modeling in P. falciparum

   PMID:42567866

   Open questions at the time

   • Direct structural placement in the mitoribosome rests on modeling plus proteomics
   • Reconciliation with prior in vitro heme-binding and complex data not experimentally addressed here
   • Whether any residual food-vacuole pool exists is not resolved

• It remains unresolved how the earlier heme-binding, histidine-catalysis, and food-vacuole-complex observations relate to the mitochondrial/mitoribosomal function established by endogenous tagging and knockout.
• No study directly reconciles the in vitro hemozoin activity with the knockout phenotype showing no effect on hemozoin
• No structure of endogenous HDP in either proposed context
• Species divergence (P. vinckei, P. yoelii) not re-examined with endogenous methods