Abstract
Targeted proteolysis of endogenous proteins is desirable as a research toolkit and in therapeutics. CRISPR/Cas9-mediated gene knockouts are irreversible and often not feasible for many genes. Similarly, RNA interference approaches necessitate prolonged treatments, can lead to incomplete knockdowns and are often associated with off-target effects. Targeted proteolysis can overcome these limitations. In this report, we describe an affinity-directed protein missile (AdPROM) system that harbours the von Hippel–Lindau (VHL) protein, the substrate receptor of the Cullin2 (CUL2) E3 ligase complex, tethered to polypeptide binders that selectively bind and recruit endogenous target proteins to the CUL2-E3 ligase complex for ubiquitination and proteasomal degradation. By using synthetic monobodies that selectively bind the protein tyrosine phosphatase SHP2 and a camelid-derived VHH nanobody that selectively binds the human ASC protein, we demonstrate highly efficient AdPROM-mediated degradation of endogenous SHP2 and ASC in human cell lines. We show that AdPROM-mediated loss of SHP2 in cells impacts SHP2 biology. This study demonstrates for the first time that small polypeptide binders that selectively recognize endogenous target proteins can be exploited for AdPROM-mediated destruction of the target proteins.
Highlights
Achieving targeted proteolysis of endogenous cellular proteins is desirable for research into their function, but is increasingly sought after as an approach to disrupt target protein function in therapeutics
By combining the CUL2 machinery and anti-GFP nanobodies, we recently reported an efficient affinity-directed protein missile (AdPROM) system for proteolysis of endogenous target proteins that were marked by knocking in a GFP-tag using CRISPR/Cas9
In an attempt to test whether the proteolytic AdPROM consisting of von Hippel–Lindau (VHL) tethered to monobodies recognizing human SHP2 could degrade endogenous SHP2, we employed two distinct monobodies that selectively bind the N-Src-homology 2 (SH2) and C-SH2 domains of SHP2, termed aNSa1 (KD 1⁄4 14 nM) and aCS3 (KD 1⁄4 4 nM), respectively [17]
Summary
Achieving targeted proteolysis of endogenous cellular proteins is desirable for research into their function, but is increasingly sought after as an approach to disrupt target protein function in therapeutics. The ubiquitin–proteasome system (UPS) is a major player for the proteolysis of many cellular proteins for maintenance of homeostasis [1,2,3]. The Cullin RING (really interesting new gene) E3 ligase (CRL) family represents one of the key contributors to the UPS for protein turnover in cells [6,7]. Each CRL complex comprises one Cullin (CUL1-7) member, one or more adaptors that bind to a substrate receptor and a RING E3 ligase (RBX1/2) [6,7]. The CUL2-CRL is in a complex with Elongin B and C as adaptors, the von Hippel–Lindau (VHL) protein as the substrate receptor, and RING-box protein 1 (RBX1) as the E3 ligase [8,9]. All CRLs are activated through the covalent attachment of the small ubiquitin-like modifier NEDD8 to lysine residues of the Cullin (a process termed NEDDylation [10]), and the pan-Cullin NEDDylation inhibitor MLN4924 inhibits CRL activation [11]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
