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P-NADs: PUX-based NAnobody Degraders for Ubiquitin-Independent Degradation of Target Proteins

Wang, J.; Chistov, G.; Zhang, J.; Huntington, B.; Salem, I.; Sandholu, A.; Arold, S. T.

2023-12-20 synthetic biology
10.1101/2023.12.20.572486 bioRxiv
Show abstract

Targeted protein degradation (TPD) allows cells to maintain a functional proteome and to rapidly adapt to changing conditions. Methods that repurpose TPD for the deactivation of specific proteins have demonstrated significant potential in therapeutic and research applications. Most of these methods are based on proteolysis targeting chimaera (PROTAC) which link the protein target to an E3 ubiquitin ligase binding moiety, resulting in the ubiquitin-based degradation of the target protein. In this study, we introduce a method for ubiquitin-independent TPD based on nanobody-conjugated plant ubiquitin regulatory X domain-containing (PUX) adaptor proteins. We show that the PUX-based NAnobody Degraders (P-NADs) can unfold a target protein through the Arabidopsis and human orthologues of the CDC48 unfoldase without the need for ubiquitination or initiating motifs. Despite originating from plants, P-NAD plasmids can be transfected into a human cell line, where produced proteins use the endogenous CDC48 machinery for ubiquitin-independent TPD. Thus, P-NADs pave the road for ubiquitin-independent therapeutic TPD approaches. The P-NAD design combined with established in vitro and cellular assays make this system also a versatile platform for elucidating functional aspects of CDC48-based TPD in plants and animals.

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