Abstract
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 knock-in cells harbouring GFP tags at the VPS34 (vacuolar protein sorting 34) and protein associated with SMAD1 (PAWS1, 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.
Highlights
The degradation of many proteins by the ubiquitin-proteasome system (UPS) plays a fundamental role in protein turnover and the maintenance of cellular homeostasis [1,2]
By combining the efficiency of CRIPSR/Cas9 genome editing to rapidly tag endogenous proteins with GFP, and using a single vector in which anti-GFP nanobody is tethered to von Hippel–Lindau (VHL) to target the GFP-tagged proteins for CRL-mediated proteasomal degradation, we provide proof-of-principle evidence that the affinity-directed protein missile (AdPROM) system can target selective proteins and efficiently degrade them in cells, either constitutively or in a Tet-inducible manner
AGFP-VHL did not lead to degradation of these proteins, suggesting that this orientation is incapable of recruiting the target proteins close to RBX1 in the CUL2-complex for ubiquitylation
Summary
The degradation of many proteins by the ubiquitin-proteasome system (UPS) plays a fundamental role in protein turnover and the maintenance of cellular homeostasis [1,2]. The system uses the sequential action of E1-ubiquitinactivating enzyme, E2 ubiquitin-conjugating enzymes and E3-ubiquitin ligases to attach ubiquitin chains onto target proteins and mark them for degradation by the proteasome [3,4]. The CULs are bound in a complex by selective adaptor and substrate-receptor subunits as well as one RING E3 ligase protein, RBX1/2 [8,11,12]. All CRLs are activated by the covalent attachment of a ubiquitin-like modifier termed NEDD8 through Neddylation, which requires its cognate E1, E2 and E3 enzymes analogous to the ubiquitylation process [13]. The nature and number of substrate receptor subunits for each CRL complex define the range of targets they ubiquitylate for proteasomal degradation in cells.
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