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

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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

Fingerprint

Proteolysis
Missiles
proteolysis
Proteins
proteins
Class III Phosphatidylinositol 3-Kinases
protein transport
Proteasome Endopeptidase Complex
proteasome endopeptidase complex
Degradation
Clustered Regularly Interspaced Short Palindromic Repeats
Single-Domain Antibodies
Fluorescence
degradation
Ubiquitin-Protein Ligases
fluorescence
Ubiquitination
Protein Transport
Osteosarcoma
ubiquitin-protein ligase

Keywords

  • Ubiquitination
  • Proteolysis
  • nanobody
  • VPS34
  • VHL

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
Fulcher, Luke ; Macartney, Thomas ; Bozatzi, Polyxeni ; Hornberger, Annika ; Rojas-Fernandez, Alejandro ; Sapkota, Gopal. / An Affinity-directed PROtein Missile (AdPROM) system for targeted proteolysis. In: Open Biology. 2016 ; Vol. 6, No. 10. pp. 1-9.
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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, vol. 6, no. 10, 160255, pp. 1-9. https://doi.org/10.1098/rsob.160255

An Affinity-directed PROtein Missile (AdPROM) system for targeted proteolysis. / Fulcher, Luke; Macartney, Thomas; Bozatzi, Polyxeni; Hornberger, Annika; Rojas-Fernandez, Alejandro; Sapkota, Gopal (Lead / Corresponding author).

In: Open Biology, Vol. 6, No. 10, 160255, 26.10.2016, p. 1-9.

Research output: Contribution to journalArticle

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T1 - An Affinity-directed PROtein Missile (AdPROM) system for targeted proteolysis

AU - Fulcher, Luke

AU - Macartney, Thomas

AU - Bozatzi, Polyxeni

AU - Hornberger, Annika

AU - Rojas-Fernandez, Alejandro

AU - Sapkota, Gopal

N1 - Funding: MRC (MC_UU_12016/3)

PY - 2016/10/26

Y1 - 2016/10/26

N2 - 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.

AB - 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.

KW - Ubiquitination

KW - Proteolysis

KW - nanobody

KW - VPS34

KW - VHL

U2 - 10.1098/rsob.160255

DO - 10.1098/rsob.160255

M3 - Article

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EP - 9

JO - Open Biology

JF - Open Biology

SN - 2046-2441

IS - 10

M1 - 160255

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