CYP19B

PF3D7_1115600 (CYP19B) is an abundant P. falciparum cyclophilin-family protein that participates in protein-folding quality control and stress signalling, and contributes to antimalarial drug resistance (PMID:7588714, PMID:36696458). It was originally characterised as one of the two major cyclosporin A (CsA)-binding proteins of the parasite, comprising up to 0.5% of total parasite protein and peaking in schizont-stage parasites; despite carrying an apparent N-terminal ER-directed signal sequence, CYP19B localises primarily to the parasite cytosol (PMID:12906882). Recombinant CYP19B was first shown to be an active peptidyl-prolyl cis-trans isomerase (PPIase) inhibited by CsA (IC50 ~10 nM) whose signal peptide can be cleaved and glycosylated in a microsomal translocation system (PMID:7588714); a later comparative study of the parasite cyclophilin family instead detected molecular chaperone activity (prevention of thermal aggregation) rather than isomerase activity for recombinant CYP19B (PMID:22546550). CYP19B interacts with Hsp70, and CsA-family ligands disrupt trafficking of the rhoptry protein RAP1, linking CYP19B-containing complexes to protein transport (PMID:26156578). Functionally, CYP19B acts as a negative regulator of the integrated stress response: its overexpression modulates phosphorylated eIF2α levels and confers limited but significant resistance to artemisinin and piperaquine, with a promoter short-tandem-repeat polymorphism linked to its upregulation in drug-resistant field isolates (PMID:36696458). Mutations in CYP19B recovered from CsA-resistant clones place it in the pathway by which a CsA–cyclophilin complex inhibits parasite calcineurin, although direct calcineurin-inhibition rescue in that study was demonstrated only for the sibling cyclophilin, not CYP19B itself (PMID:15811524).

1. 1995 High

   Establishing whether CYP19B is an enzyme addressed its basic biochemical identity: recombinant protein was shown to be a CsA-inhibitable PPIase, and its signal peptide was shown to be a functional ER-translocation/glycosylation signal.

   Evidence Recombinant expression in E. coli, quantified in vitro PPIase assay, in vitro translation/translocation across microsomes

   PMID:7588714

   Open questions at the time

   • In vitro translocation/glycosylation does not establish the in vivo compartment of the native protein
   • No physiological PPIase substrate identified
2. 2003 High

   Whether CYP19B is a major CsA target in the parasite and where it resides was resolved by showing it is one of two dominant cyclosporin-binding proteins, highly abundant, schizont-enriched, and cytosolic despite its signal sequence.

   Evidence Cyclosporin-Affigel affinity chromatography, peptide mass fingerprinting, subcellular fractionation, recombinant PPIase assay

   PMID:12906882

   Open questions at the time

   • Cytosolic localisation is inferred from fractionation, not imaging
   • Reason the ER signal sequence does not retain the protein in the secretory pathway is unexplained
3. 2005 Medium

   To connect CYP19B to CsA action, mutations in functionally significant residues were found in CsA-resistant clones, implicating CYP19B in the CsA–cyclophilin/calcineurin inhibition pathway.

   Evidence Sequencing of CsA-resistant clones plus in vitro calcineurin phosphatase inhibition assay

   PMID:15811524

   Open questions at the time

   • Functional calcineurin-inhibition rescue was shown only for the sibling CYP19A, not CYP19B itself
   • Causal link between the CYP19B mutations and the resistance phenotype not demonstrated by allelic replacement
4. 2012 Medium

   A comparative family-wide biochemical survey re-examined CYP19B activity, detecting molecular chaperone (anti-aggregation) activity but no isomerase activity under the common assay conditions used.

   Evidence Recombinant expression in a common E. coli system, PPIase assay, thermal aggregation chaperone assay

   PMID:22546550

   Open questions at the time

   • Negative PPIase result conflicts with the earlier positive assay and may reflect construct/assay differences
   • Chaperone substrates are model proteins, not physiological clients
5. 2015 Medium

   Interaction studies began to define CYP19B's protein-network context, showing direct binding to Hsp70 and linking CYP-ligand treatment to disruption of RAP1 trafficking.

   Evidence Co-immunoprecipitation and yeast two-hybrid for Hsp70; drug-treatment phenotypic assay for RAP1 transport

   PMID:26156578

   Open questions at the time

   • RAP1 trafficking defect used ligands affecting both CYP19A and CYP19B, so the effect is not uniquely attributable to CYP19B
   • Functional consequence of the Hsp70 interaction not established
6. 2015 Low

   An interactome screen placed CYP19B as a putative partner of the PP1c/Inhibitor-2 complex in both asexual and sexual stages, hinting at regulation by dephosphorylation.

   Evidence mCherry-tagged PP1c/I2 affinity pulldown with mass spectrometry in P. berghei schizonts and gametocytes

   PMID:35162991

   Open questions at the time

   • Single pulldown/MS without reciprocal validation for CYP19B
   • No functional consequence of the PP1c/I2 association tested
   • Shown in P. berghei, not P. falciparum
7. 2023 High

   The physiological role in drug resistance was defined by showing CYP19B is a negative regulator of the integrated stress response whose overexpression modulates eIF2α phosphorylation and confers resistance to artemisinin and piperaquine.

   Evidence Stable CYP19B overexpression in P. falciparum, in vitro drug susceptibility assays, eIF2α-P immunoblotting, promoter STR polymorphism analysis

   PMID:36696458

   Open questions at the time

   • Direct enzymatic/biochemical mechanism by which CYP19B lowers eIF2α-P is not defined
   • Which kinase/phosphatase CYP19B acts upon is unidentified
   • Resistance conferred is limited in magnitude

• How CYP19B's biochemical activity (isomerase versus chaperone), its Hsp70/PP1c-I2 associations, and its dampening of eIF2α phosphorylation are mechanistically integrated into a single molecular pathway remains unresolved.
• No physiological PPIase or chaperone substrate identified
• The molecular step linking CYP19B to eIF2α dephosphorylation is unknown
• Reconciliation of positive and negative in vitro PPIase results is lacking