An affinity-directed protein missile system for targeted proteolysis
Open accessAbstract
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.
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Targeted degradation of GFP-VPS34 also led to the degradation of endogenous UVRAG, a protein which forms a stable complex with VPS34.
“Excitingly, targeted degradation of GFP-VPS34 also led to the degradation of endogenous UVRAG, a protein which forms a stable complex with VPS34 [18].”
From Advances in targeted degradation of endogenous proteins (2019) · cites this paper as [18]
AdPROM combines the endogenous CUL2 CRL machinery with high-affinity polypeptide binders for target protein recognition.
“Recent data from our own laboratory described a novel and efficient proteolytic system that combines the endogenous cullin 2 (CUL2) CRL machinery, with high-affinity polypeptide binders for target protein recognition [18, 19].”
From Advances in targeted degradation of endogenous proteins (2019) · cites this paper as [18]
AdPROM can induce degradation of endogenous GFP-tagged PAWS1 and VPS34 using nanobodies recognizing GFP fused with VHL.
“Alternatively, for POIs for which the binding nanobodies/monobodies do not yet exist, we generated GFP-tagged knockin POIs through CRISPR/Cas9 gene editing technology, and utilized nanobodies recognizing GFP fused with VHL to induce degradation of endogenous GFP-tagged PAWS1 and VPS34 [18].”
From Advances in targeted degradation of endogenous proteins (2019) · cites this paper as [18]
Degradation of VPS34 by AdPROM resulted in reduction in levels of endosomal phosphatidylinositol (3,4,5)-trisphosphate.
“As expected, the degradation of VPS34 by AdPROM resulted in reduction in levels of endosomal phosphatidylinositol (3,4,5)-trisphosphate [18].”
From Advances in targeted degradation of endogenous proteins (2019) · cites this paper as [18]
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References 39 · Cited by 91
- Authors
- Fulcher, L. J., Macartney, T., Bozatzi, P., Hornberger, A., Rojas-Fernandez, A., & Sapkota, G. P.
- Affiliations
- University of Dundee (GB)
- Center for Interdisciplinary Studies on the Nervous System and Institute of Medicine, Universidad Austral de Chile, Valdivia, Chile
- Corresponding author
- Sapkota, Gopal P.
- Venue
- Open Biology
- Publisher
- The Royal Society
- Published
- 2016-10
- Volume
- 6
- Issue
- 10
- Pages
- 160255
- ISSN
- 2046-2441 (ISSN-L)
- PMC
- PMC5090066
- OpenAlex
- W2544450870
- Cited by
- 91
- References
- 39
- Citations by year
- 2026: 3 · 2025: 13 · 2024: 12 · 2023: 10 · 2022: 5 · 2021: 10 · 2020: 16 · 2019: 7 · 2018: 5 · 2017: 10
- FWCI
- 3.81
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Open Biology (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Discovery Research Portal (University of Dundee) (publishedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Medical Research Council
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Degradation and Inhibitors
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Keywords
- targeted proteolysis, von Hippel-Lindau protein, protein degradation, anti-GFP nanobody, CRISPR/Cas9 knock-in, Vps34, UVRAG, phosphatidylinositol 3-phosphate, proteasome, tetracycline-inducible expression
- MeSH terms
- Humans, Recombinant Fusion Proteins, Protein Engineering, Tumor Suppressor Proteins, Cell Line, Tumor, Von Hippel-Lindau Tumor Suppressor Protein, Smad1 Protein, Gene Knock-In Techniques, HEK293 Cells, Class III Phosphatidylinositol 3-Kinases, Proteolysis
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