HaloPROTAC3 does not trigger the degradation of the halotagged parasitophorous vacuole membrane protein UIS4 during Plasmodium liver stage development
Targeted protein degradation (TPD) is a novel strategy for developing therapeutics against pathogens. Prior to causing malaria, Plasmodium parasites replicate within hepatocytes as liver stages, surrounded by a parasitophorous vacuole membrane (PVM). We hypothesized that TPD can be employed to trigger host-driven degradation of essential liver stage PVM proteins and lead to parasite death. To explore this, we took advantage of the proteolysis-targeting-chimera HaloPROTAC3, a molecule that recruits the host von Hippel-Lindau (VHL) E3 ligase to the HaloTag (HT). Parasites expressing HT fused to the host cytosol-exposed domain of the PVM protein UIS4 (UIS4-HT) were generated in Plasmodium berghei and Plasmodium cynomolgi, but only P. berghei UIS4-HT enabled productive liver stage infection experiments in vitro. Although HaloPROTAC3 triggered the degradation of HT proteins in host cells, it had no impact on the survival of P. berghei UIS4-HT liver stages. Furthermore, HaloPROTAC3 bound to P. berghei UIS4-HT but did not recruit VHL or trigger ubiquitination of the PVM. Overall, although this study did not establish whether host-driven TPD can degrade Plasmodium PVM proteins, it highlights the challenges of developing TPD approaches against novel targets and offers insights for advancing this therapeutic strategy against pathogens.
Melanie Lam, Alexandra Probst, Laura Torres, Ashley A Lantigua, Matthew E Fishbaugher, Jyothsna R Kumar, Manuel Saldivia, Allison Torres, Shreeya Hegde, Maya Aleshnick, Charlie Jennison, Sarah G H Roberson, Chester J Joyner, Ashley M Vaughan, Brandon K Wilder, Carole Manneville, Erika L Flannery, David Marcellin, Beat Nyfeler, Zacharias Thiel, Sebastian A Mikolajczak, Anke Harupa, Gabriel Mitchell. Sci Rep. 2025 May 26;15(1):18323. doi: 10.1038/s41598-025-98257-9.