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A Targeted Protein Degradation Cell-Based Screening for Nanobodies Selective toward the Cellular RHOB GTP-Bound Conformation

Abstract : The selective downregulation of activated intracellular proteins is a key challenge in cell biology. RHO small GTPases switch between a guanosine diphosphate (GDP)-bound and a guanosine triphosphate (GTP)-bound state that drives downstream signaling. At present, no tool is available to study endogenous RHO-GTPinduced conformational changes in live cells. Here, we established a cell-based screen to selectively degrade RHOB-GTP using F-box-intracellular single-domain antibody fusion. We identified one intracellular antibody (intrabody) that shows selective targeting of endogenous RHOB-GTP mediated by interactions between the CDR3 loop of the domain antibody and the GTP-binding pocket of RHOB. Our results suggest that, while RHOB is highly regulated at the expression level, only the GTPbound pool, but not its global expression, mediates RHOB functions in genomic instability and in cell invasion. The F-box/intrabody-targeted protein degradation represents a unique approach to knock down the active form of small GTPases or other proteins with multiple cellular activities.
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https://hal.archives-ouvertes.fr/hal-02378315
Contributor : Jean-Denis Pedelacq <>
Submitted on : Tuesday, November 24, 2020 - 3:06:16 PM
Last modification on : Monday, November 30, 2020 - 4:12:36 PM
Long-term archiving on: : Thursday, February 25, 2021 - 8:09:22 PM

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Nicolas Béry, Laura Keller, Marjorie Soulié, Rémi Gence, Anne-Laure Iscache, et al.. A Targeted Protein Degradation Cell-Based Screening for Nanobodies Selective toward the Cellular RHOB GTP-Bound Conformation. Cell Chemical Biology, Cell Press, 2019, 26 (11), pp.1544-1558.e6. ⟨10.1016/j.chembiol.2019.08.009⟩. ⟨hal-02378315⟩

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