GBP2

PF3D7_1006800 (GBP2) is a G-quadruplex-binding protein of Plasmodium that couples recognition of G-rich nucleic acid structures to virulence gene regulation and RNA metabolism (PMID:35273922, PMID:33340385). In vitro it binds specifically to G-rich telomere repeats folded as G-quadruplexes and to a stable G4-forming sequence (AP2_G4), and it also binds G-rich RNA (PMID:35273922, PMID:33340385). Consistent with this dual DNA/RNA activity, GBP2 partially co-localises with the telomeric protein HP1 while also occupying the cytoplasm, and its interactome is enriched for RNA-associated proteins (PMID:35273922). Functionally, knockout of GBP2 alters var gene expression and renders parasites unable to survive exposure to the G4-stabilising ligand pyridostatin, linking its G4-binding activity to regulation of variant surface antigen genes (PMID:33340385). In P. berghei, GBP2 localises to both nucleus and cytoplasm and interacts with translational repression complex components ALBA4, DOZI, and CITH and with a PHAX-domain exportin-1 adaptor, implicating it in mRNA export and translational control (PMID:34604117). Deletion of gbp2 in P. berghei delays asexual growth and sharply reduces male and female gametocyte production, and GBP2 abundance is governed by purine availability, being reduced under purine starvation and associated with altered levels of purine-salvage enzymes (PMID:31958569, PMID:34604117). Beyond these findings, the structural basis of G4 recognition and the direct mRNA targets of GBP2 have not been characterized in the available corpus.

1. 2020 Medium

   Establishing whether GBP2 has a biological role required a loss-of-function test; deletion showed it is needed for both asexual and sexual development and tied its abundance to purine metabolism.

   Evidence gbp2 gene deletion with phenotypic scoring, comparative proteomics, and genetic epistasis with a purine transporter knockout in P. berghei

   PMID:31958569

   Open questions at the time

   • How purine availability mechanistically controls GBP2 levels is unresolved
   • Whether the purine-enzyme changes are direct GBP2 targets or downstream effects is not established
   • Molecular activity underlying the developmental phenotype not defined in this study
2. 2021 Medium

   To connect GBP2 to a molecular substrate, DNA pull-down and knockout linked its G4-binding activity to virulence gene regulation, showing G4 recognition has functional consequences for var expression.

   Evidence AP2_G4 DNA pull-down with LC-MS/MS, in vitro/in vivo G4-binding validation, knockout var-gene analysis, and pyridostatin sensitivity in P. falciparum

   PMID:33340385

   Open questions at the time

   • Whether GBP2 directly binds var-locus G4s in vivo versus acting indirectly is unclear
   • No structural model of the G4-GBP2 interaction
   • Genome-wide G4 occupancy not mapped
3. 2021 Medium

   Defining GBP2's protein partners clarified its cytoplasmic role, placing it with translational repression machinery and an mRNA-export adaptor and distinguishing its developmental requirement from related family members.

   Evidence IP-MS, live-cell imaging, and comparative gene-deletion phenotyping in P. berghei

   PMID:34604117

   Open questions at the time

   • Direct mRNA cargo of GBP2 not identified
   • Whether GBP2 acts within a stable complex or transiently with ALBA4/DOZI/CITH is unresolved
   • Functional contribution of the PHAX-domain adaptor interaction to export not tested directly
4. 2022 Medium

   Resolving the nature of GBP2's nucleic acid targets, chromatin proteomics and binding assays showed it recognises telomeric G-quadruplexes and G-rich RNA and partly co-localises with the telomere protein HP1.

   Evidence PICh, in vitro DNA/RNA G4-binding assays, HP1 co-localisation immunofluorescence, and interactome MS in P. falciparum

   PMID:35273922

   Open questions at the time

   • Functional role of GBP2 at telomeres versus var loci not disentangled
   • Relative in vivo occupancy of DNA versus RNA targets unquantified
   • Whether HP1 co-localisation reflects direct interaction is not shown

• The structural basis of G4 discrimination and the direct in vivo DNA and mRNA targets that mediate GBP2's effects on var expression and gametocytogenesis remain unknown.
• No structure of GBP2 or its G4-bound complex
• Direct mRNA cargo and genome-wide binding sites uncharacterized
• Mechanism linking purine availability to GBP2 abundance undefined