The von Hippel-Lindau (VHL) protein serves to recruit the hypoxia inducible factor alpha (HIF1α) protein under normoxia to the CUL2 E3 ubiquitin ligase for its ubiquitylation and degradation through the proteasome. In this report, we modify VHL to engineer an Affinity directed PROtein Missile (AdPROM) system to direct specific endogenous target proteins for proteolysis in mammalian cells. The proteolytic AdPROM construct harbours a cameloid anti-green fluorescence protein (aGFP) nanobody that is fused to VHL for either constitutive or tetracycline-inducible expression. For target proteins, we exploit CRISPR/Cas9 to rapidly generate human kidney HEK293 and U2OS osteosarcoma homozygous knockin cells harbouring GFP tags at the VPS34 (vacuolar protein sorting 34) and PAWS1 (protein associated with SMAD1, aka FAM83G) loci respectively. Using these cells, we demonstrate that the expression of the VHL-aGFP AdPROM system results in near-complete degradation of the endogenous GFP-VPS34 and PAWS1-GFP proteins through the proteasome. Additionally, we show that Tet- inducible destruction of GFP-VPS34 results in the degradation of its associated partner, UVRAG, and reduction in levels of cellular phosphatidylinositol 3-phosphate.
Identification and characterisation of FAM83 proteins as key regulators of CK1 protein kinases in cells: FAM83D recruits CK1α to the mitotic spindle for proper spindle positioningAuthor: Fulcher, L. J., 2019
Student thesis: Doctoral Thesis › Doctor of Philosophy
Fulcher, L., Macartney, T., Bozatzi, P., Hornberger, A., Rojas-Fernandez, A., & Sapkota, G. (2016). An Affinity-directed PROtein Missile (AdPROM) system for targeted proteolysis. Open Biology, 6(10), 1-9. . https://doi.org/10.1098/rsob.160255