A protein silencing switch by ligand-induced proteasome-targeting intrabodies |
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Authors: | Melchionna Teresa Cattaneo Antonino |
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Institution: | 1 International School for Advanced Studies (SISSA), Basovizza s.s.14, km 163, 34012, Trieste, Italy 2 Lay Line Genomics SpA, Basovizza s.s.14, km 163, 34012, Trieste, Italy 3 European Brain Research Institute (EBRI), Via del Fosso di Fiorano 64/65, 00143, Rome, Italy |
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Abstract: | The selective knock-down of cellular proteins has proven useful for in vivo studies of protein function and RNAi methods are readily available for this purpose. However, interfering directly at the protein level may have distinct advantages, with the intracellular targeting of antibodies (intrabodies) representing an attractive option, although not a general one. We demonstrate a novel, general strategy named suicide (or silencing) intrabody technology (SIT), based on the inducible degradation of intrabodies, which are equipped with proteasome-targeting sequences and thus converted into suicide intrabodies. We show that suicide intrabodies are able to redirect the target cellular proteins upon stimulus administration to the proteolytic machinery, thus resulting in selective protein knock-down. Remarkably, suicide intrabody acts in a catalytic fashion. SIT is a ligand-inducible strategy, potentially applicable to any protein of interest and does not require the engineering of cellular proteolytic enzymes. SIT represents a general approach to confer “neutralizing” properties to any intrabody, a valuable feature, given the present impossibility to select a priori intrinsically neutralizing antibodies. This knock-down strategy, together with available methods to isolate functional intrabodies, should allow the large-scale investigation of intracellular protein networks. |
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Keywords: | FACS fluorescence-activated cell sorting GFP green fluorescent protein oNPG ortho-nitrophenyl-β d-galactoside" target="_blank">d-galactoside SIT suicide (or silencing) intrabody technology TNFα tumor necrosis factor alpha UPP ubiquitin/proteasome pathway |
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