Dataset Information

Antibody RING-Mediated Destruction (ARMeD) of endogenous proteins

ABSTRACT: To help understand the cellular function of a gene the encoding DNA or mRNA transcript can be manipulated and the consequences observed. However, these approaches do not have a direct effect on the protein product of the gene, which is either permanently abrogated or depleted at a rate defined by the half-life of the protein. Therefore, complete gene deletions and RNA interference of proteins with a very long half-life risk obscuring the direct consequences via compensatory or secondary cellular responses. We therefore sought to develop a single-component system that could induce the rapid degradation of the specific endogenous protein itself. A genetically introduced inducible construct of the RING domain of the ubiquitin E3 ligase RNF4 with a protein-specific camelid nanobody mediates destruction of the target by the ubiquitin proteasome system, a process we describe as Antibody RING-Mediated Destruction (ARMeD). The technique is acutely specific as we observed no off-target protein destruction. Furthermore, bacterially produced nanobody-RING fusion proteins electroporated into cells induce degradation of target within minutes. With the increasing availability of protein-specific nanobodies this method will allow the rapid and specific degradation of a wide range of endogenous proteins.

ORGANISM(S): Homo Sapiens

SUBMITTER: David Dickerson

PROVIDER: PXD016193 | panorama | Thu May 28 00:00:00 BST 2020

REPOSITORIES: PanoramaPublic

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Publications

Antibody RING-Mediated Destruction of Endogenous Proteins.

Ibrahim Adel F M AFM Shen Linnan L Tatham Michael H MH Dickerson David D Prescott Alan R AR Abidi Naima N Xirodimas Dimitris P DP Hay Ronald T RT

Molecular cell 20200525 1

To understand gene function, the encoding DNA or mRNA transcript can be manipulated and the consequences observed. However, these approaches do not have a direct effect on the protein product of the gene, which is either permanently abrogated or depleted at a rate defined by the half-life of the protein. We therefore developed a single-component system that could induce the rapid degradation of the specific endogenous protein itself. A construct combining the RING domain of ubiquitin E3 ligase R ...[more]

PMID: 32454028

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Project description:Targeted proTargeted protein degradation has recently emerged as a novel option in drug discovery. Natural protein half-life is expected to affect the efficacy of degrading agents, but it has not been systematically explored to what extent it influences target protein degradation. Using mathematical modelling of protein degradation, we demonstrate that the natural half-life of a target protein has a dramatic effect on the level of protein degradation induced by a PROTAC which can pose significant hurdles to screening efforts. Moreover, we show that upon screening for degraders of short-lived proteins, agents that stall protein synthesis, such as GSPT1 degraders and generally cytotoxic compounds, deceptively appear as protein degrading agents. This is exemplified by the disappearance of short-lived proteins such as MCL1 and MDM2 upon GSPT1 degradation and upon treatment with cytotoxic agents such as doxorubicin. These findings have implications for target selection as well as for the type of control experiments required before concluding that a novel agent works as a bone-fide targeted protein degrader. tein degradation has recently emerged as a novel option in drug discovery. Natural protein half-life is expected to affect the efficacy of degrading agents, but it has not been systematically explored to what extent it influences target protein degradation. Using mathematical modelling of protein degradation, we demonstrate that the natural half-life of a target protein has a dramatic effect on the level of protein degradation induced by a PROTAC which can pose significant hurdles to screening efforts. Moreover, we show that upon screening for degraders of short-lived proteins, agents that stall protein synthesis, such as GSPT1 degraders and generally cytotoxic compounds, deceptively appear as protein degrading agents. This is exemplified by the disappearance of short-lived proteins such as MCL1 and MDM2 upon GSPT1 degradation and upon treatment with cytotoxic agents such as doxorubicin. These findings have implications for target selection as well as for the type of control experiments required before concluding that a novel agent works as a bone-fide targeted protein degrader.

Dataset Information

Antibody RING-Mediated Destruction (ARMeD) of endogenous proteins

Publications

Antibody RING-Mediated Destruction of Endogenous Proteins.

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