Rapid and reversible knockdown of endogenously tagged endosomal proteins via an optimized HaloPROTAC degrader

Hannah Tovell, Andrea Testa, Chiara Maniaci, Houjiang Zhou, Alan Prescott, Thomas Macartney, Alessio Ciulli (Lead / Corresponding author), Dario Alessi (Lead / Corresponding author)

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Abstract

Inducing post-translational protein knockdown is an important approach to probe biology and validate drug targets. An efficient strategy to achieve this involves expression of a protein of interest fused to an exogenous tag, allowing tag-directed chemical degraders to mediate protein ubiquitylation and proteasomal degradation. Here, we combine improved HaloPROTAC degrader probes with CRISPR/Cas9 genome editing technology to trigger rapid degradation of endogenous target proteins. Our optimized probe, HaloPROTAC-E, a chloroalkane conjugate of high-affinity VHL binder VH298, induced reversible degradation of two endosomally localized proteins, SGK3 and VPS34, with a DC 50 of 3-10 nM. HaloPROTAC-E induced rapid (∼50% degradation after 30 min) and complete (D max of ∼95% at 48 h) depletion of Halo-tagged SGK3, blocking downstream phosphorylation of the SGK3 substrate NDRG1. HaloPROTAC-E more potently induced greater steady state degradation of Halo tagged endogenous VPS34 than the previously reported HaloPROTAC3 compound. Quantitative global proteomics revealed that HaloPROTAC-E is remarkably selective inducing only degradation of the Halo tagged endogenous VPS34 complex (VPS34, VPS15, Beclin1, and ATG14) and no other proteins were significantly degraded. This study exemplifies the combination of HaloPROTACs with CRISPR/Cas9 endogenous protein tagging as a useful method to induce rapid and reversible degradation of endogenous proteins to interrogate their function.

Original languageEnglish
Pages (from-to)882-892
Number of pages11
JournalACS Chemical Biology
Volume14
Issue number5
Early online date12 Apr 2019
DOIs
Publication statusPublished - 17 May 2019

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Degradation
Clustered Regularly Interspaced Short Palindromic Repeats
Proteins
Ubiquitination
Phosphorylation
Proteomics
Proteolysis
Binders
Technology
Genes
Pharmaceutical Preparations
Substrates

Cite this

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title = "Rapid and reversible knockdown of endogenously tagged endosomal proteins via an optimized HaloPROTAC degrader",
abstract = "Inducing post-translational protein knockdown is an important approach to probe biology and validate drug targets. An efficient strategy to achieve this involves expression of a protein of interest fused to an exogenous tag, allowing tag-directed chemical degraders to mediate protein ubiquitylation and proteasomal degradation. Here, we combine improved HaloPROTAC degrader probes with CRISPR/Cas9 genome editing technology to trigger rapid degradation of endogenous target proteins. Our optimized probe, HaloPROTAC-E, a chloroalkane conjugate of high-affinity VHL binder VH298, induced reversible degradation of two endosomally localized proteins, SGK3 and VPS34, with a DC 50 of 3-10 nM. HaloPROTAC-E induced rapid (∼50{\%} degradation after 30 min) and complete (D max of ∼95{\%} at 48 h) depletion of Halo-tagged SGK3, blocking downstream phosphorylation of the SGK3 substrate NDRG1. HaloPROTAC-E more potently induced greater steady state degradation of Halo tagged endogenous VPS34 than the previously reported HaloPROTAC3 compound. Quantitative global proteomics revealed that HaloPROTAC-E is remarkably selective inducing only degradation of the Halo tagged endogenous VPS34 complex (VPS34, VPS15, Beclin1, and ATG14) and no other proteins were significantly degraded. This study exemplifies the combination of HaloPROTACs with CRISPR/Cas9 endogenous protein tagging as a useful method to induce rapid and reversible degradation of endogenous proteins to interrogate their function.",
author = "Hannah Tovell and Andrea Testa and Chiara Maniaci and Houjiang Zhou and Alan Prescott and Thomas Macartney and Alessio Ciulli and Dario Alessi",
note = "This work was supported by the Medical Research Council [grant number MC_UU_12016/2 (to D.R.A.)]; the European Research Council (ERC) under the European Union’s Seventh Framework Programme (FP7/2007–2013) as a Starting Grant to A.C. [grant agreement No. ERC–2012–StG–311460 DrugE3CRLs]; the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (Boehringer Ingelheim, GlaxoSmithKline, and Merck KGaA, to D.R.A.). H.T. is supported by an AstraZeneca BBSRC Studentship.",
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T1 - Rapid and reversible knockdown of endogenously tagged endosomal proteins via an optimized HaloPROTAC degrader

AU - Tovell, Hannah

AU - Testa, Andrea

AU - Maniaci, Chiara

AU - Zhou, Houjiang

AU - Prescott, Alan

AU - Macartney, Thomas

AU - Ciulli, Alessio

AU - Alessi, Dario

N1 - This work was supported by the Medical Research Council [grant number MC_UU_12016/2 (to D.R.A.)]; the European Research Council (ERC) under the European Union’s Seventh Framework Programme (FP7/2007–2013) as a Starting Grant to A.C. [grant agreement No. ERC–2012–StG–311460 DrugE3CRLs]; the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (Boehringer Ingelheim, GlaxoSmithKline, and Merck KGaA, to D.R.A.). H.T. is supported by an AstraZeneca BBSRC Studentship.

PY - 2019/5/17

Y1 - 2019/5/17

N2 - Inducing post-translational protein knockdown is an important approach to probe biology and validate drug targets. An efficient strategy to achieve this involves expression of a protein of interest fused to an exogenous tag, allowing tag-directed chemical degraders to mediate protein ubiquitylation and proteasomal degradation. Here, we combine improved HaloPROTAC degrader probes with CRISPR/Cas9 genome editing technology to trigger rapid degradation of endogenous target proteins. Our optimized probe, HaloPROTAC-E, a chloroalkane conjugate of high-affinity VHL binder VH298, induced reversible degradation of two endosomally localized proteins, SGK3 and VPS34, with a DC 50 of 3-10 nM. HaloPROTAC-E induced rapid (∼50% degradation after 30 min) and complete (D max of ∼95% at 48 h) depletion of Halo-tagged SGK3, blocking downstream phosphorylation of the SGK3 substrate NDRG1. HaloPROTAC-E more potently induced greater steady state degradation of Halo tagged endogenous VPS34 than the previously reported HaloPROTAC3 compound. Quantitative global proteomics revealed that HaloPROTAC-E is remarkably selective inducing only degradation of the Halo tagged endogenous VPS34 complex (VPS34, VPS15, Beclin1, and ATG14) and no other proteins were significantly degraded. This study exemplifies the combination of HaloPROTACs with CRISPR/Cas9 endogenous protein tagging as a useful method to induce rapid and reversible degradation of endogenous proteins to interrogate their function.

AB - Inducing post-translational protein knockdown is an important approach to probe biology and validate drug targets. An efficient strategy to achieve this involves expression of a protein of interest fused to an exogenous tag, allowing tag-directed chemical degraders to mediate protein ubiquitylation and proteasomal degradation. Here, we combine improved HaloPROTAC degrader probes with CRISPR/Cas9 genome editing technology to trigger rapid degradation of endogenous target proteins. Our optimized probe, HaloPROTAC-E, a chloroalkane conjugate of high-affinity VHL binder VH298, induced reversible degradation of two endosomally localized proteins, SGK3 and VPS34, with a DC 50 of 3-10 nM. HaloPROTAC-E induced rapid (∼50% degradation after 30 min) and complete (D max of ∼95% at 48 h) depletion of Halo-tagged SGK3, blocking downstream phosphorylation of the SGK3 substrate NDRG1. HaloPROTAC-E more potently induced greater steady state degradation of Halo tagged endogenous VPS34 than the previously reported HaloPROTAC3 compound. Quantitative global proteomics revealed that HaloPROTAC-E is remarkably selective inducing only degradation of the Halo tagged endogenous VPS34 complex (VPS34, VPS15, Beclin1, and ATG14) and no other proteins were significantly degraded. This study exemplifies the combination of HaloPROTACs with CRISPR/Cas9 endogenous protein tagging as a useful method to induce rapid and reversible degradation of endogenous proteins to interrogate their function.

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U2 - 10.1021/acschembio.8b01016

DO - 10.1021/acschembio.8b01016

M3 - Article

VL - 14

SP - 882

EP - 892

JO - ACS Chemical Biology

JF - ACS Chemical Biology

SN - 1554-8929

IS - 5

ER -