Abstract
SummaryThe affinity-directed protein missile (AdPROM) system utilizes specific polypeptide binders of intracellular proteins of interest (POIs) conjugated to an E3 ubiquitin ligase moiety to enable targeted proteolysis of the POI. However, a chemically tuneable AdPROM system is more desirable. Here, we use Halo-tag/VHL-recruiting proteolysis-targeting chimera (HaloPROTAC) technology to develop a ligand-inducible AdPROM (L-AdPROM) system. When we express an L-AdPROM construct consisting of an anti-GFP nanobody conjugated to the Halo-tag, we achieve robust degradation of GFP-tagged POIs only upon treatment of cells with the HaloPROTAC. For GFP-tagged POIs, ULK1, FAM83D, and SGK3 were knocked in with a GFP-tag using CRISPR/Cas9. By substituting the anti-GFP nanobody for a monobody that binds H- and K-RAS, we achieve robust degradation of unmodified endogenous RAS proteins only in the presence of the HaloPROTAC. Through substitution of the polypeptide binder, the highly versatile L-AdPROM system is useful for the inducible degradation of potentially any intracellular POI.
Highlights
Developments in RNA interference (RNAi) and CRISPR/Cas9 technologies have enabled the manipulation of specific proteins of interest (POIs) to study and understand their biological functions (Elbashir et al, 2001; Cong et al, 2013; Doudna and Charpentier, 2014; Sander and Joung, 2014)
GFP-ULK1 and FAM83D-GFP Are Degraded with HaloPROTAC-E in Cells Expressing FLAG-aGFP6M-Halo First, we developed a ligand-inducible affinity-directed protein missile (AdPROM) (L-AdPROM) construct, consisting of anti-GFP nanobody (aGFP) conjugated to the Halo-tag and tagged with a FLAG reporter, for the degradation of GFP-tagged POIs only in the presence of HaloPROTAC-E (Figure 1A)
In the presence of POI-GFP, FLAG-aGFP6M-Halo binds POI-GFP with high affinity. Treating these cells with HaloPROTAC-E recruits FLAG-aGFP6MHalo bound to POI-GFP to von HippelLindau (VHL)
Summary
Developments in RNA interference (RNAi) and CRISPR/Cas technologies have enabled the manipulation of specific proteins of interest (POIs) to study and understand their biological functions (Elbashir et al, 2001; Cong et al, 2013; Doudna and Charpentier, 2014; Sander and Joung, 2014). The ubiquitin-proteasome system (UPS) plays a fundamental role in the degradation of proteins to maintain cellular homeostasis (Roos-Mattjus and Sistonen, 2004; Pines and Lindon, 2005). Through sequential actions of the E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases, target proteins are covalently labeled with ubiquitin chains, marking them for recognition and degradation by the proteasome (Scheffner et al, 1995). The Cullin (CUL) really interesting new gene (RING) E3 ligase (CRL) family plays a fundamental role in regulating protein turnover in cells through the UPS (Wenzel et al, 2011; Zhao et al, 2012). CRLs are activated through NEDDylation, where the small ubiquitin-like modifier NEDD8 (neural precursor cell expressed developmentally downregulated protein 8) is covalently attached to a lysine residue of the CUL (Soucy et al, 2009). VHL binds to hydroxy-proline-modified hypoxia-inducible factor 1a (HIF1a) and brings HIF1a in close proximity to RBX1 for its
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