An Affinity-directed PROtein Missile (AdPROM) system for targeted proteolysis

Luke Fulcher, Thomas Macartney, Polyxeni Bozatzi, Annika Hornberger, Alejandro Rojas-Fernandez, Gopal Sapkota (Lead / Corresponding author)

Research output: Contribution to journalArticle

21 Citations (Scopus)
146 Downloads (Pure)

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 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.
Original languageEnglish
Article number160255
Pages (from-to)1-9
Number of pages9
JournalOpen Biology
Volume6
Issue number10
DOIs
Publication statusPublished - 26 Oct 2016

Keywords

  • Ubiquitination
  • Proteolysis
  • nanobody
  • VPS34
  • VHL

Fingerprint Dive into the research topics of 'An Affinity-directed PROtein Missile (AdPROM) system for targeted proteolysis'. Together they form a unique fingerprint.

  • Student Theses

    File

    Profiles

    No photo of Gopal Sapkota

    Sapkota, Gopal

    • MRC PPU - Scientific Programme Leader (MRC)

    Person: Research

    Cite this

    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. [160255]. https://doi.org/10.1098/rsob.160255