Abstract
Targeted protein degradation is a powerful tool in determining the function of specific proteins or protein complexes. We fused nanobodies to SPOP, an adaptor protein of the Cullin-RING E3 ubiquitin ligase complex, resulting in rapid ubiquitination and subsequent proteasome-dependent degradation of specific nuclear proteins in mammalian cells and zebrafish embryos. This approach is easily modifiable, as substrate specificity is conferred by an antibody domain that can be adapted to target virtually any protein.
Highlights
In a stable cell line expressing histone H2B (H2B)-GFP (Fig. 1), leading us to design several novel synthetic E3 ligases that could be tested for selective nuclear protein degradation (Supplementary Fig. 1)
Cullin-RING E3 ubiquitin ligase (CRL) complexes were selected as the frameworks for designing synthetic ligases, as they are well characterized and directly transfer ubiquitin from the E2 enzyme to the target protein[11,12]
We replaced the substrate recognition domain of SPOP with a nanobody to create Ab-SPOP, a synthetic E3 ubiquitin ligase that polyubiquitinates target nuclear proteins leading to proteasomal degradation
Summary
Development of a nanobody-targeted E3-ubiquitin ligase that degrades nuclear proteins. Ab-SPOP ligase promoted depletion of GFP or GFP fusion proteins within 3 hours of doxycycline-induced expression, before TagRFP was detectable under epifluorescence (Fig. 3 and Supplementary Figs 3–6) This selective depletion of GFP fusion proteins before the appearance of the RFP signal was investigated further using a stable cell line containing a CMV promoter driving cMyc-GFP expression, and a bi-directional TRE promoter controlling expression of both TagRFP and Ab-SPOP. Wild type AB embryos (injection: 100 pg Ab-SPOP RNA; 10 pg TagRFP RNA per 1-cell stage embryo), did not cause defects (Fig. 8) These experiments indicate that Ab-SPOP is not toxic during zebrafish development and rapidly depletes GFP fusion proteins in vivo. Further modifications of Ab-SPOP and the manipulation of related E3 ligases will lead to the development of new reagents targeting diverse proteins within different intracellular compartments
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