Antibody RING-Mediated Destruction (ARMeD) of endogenous proteins
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ABSTRACT: To 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.
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Permanent Cell Line Cell, Cell Culture
SUBMITTER: Mike Tatham
LAB HEAD: Ronald T. Hay
PROVIDER: PXD018113 | Pride | 2020-05-29
REPOSITORIES: Pride
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