Targeted Protein Degradation by Nanobody-UBX Degraders (NUDs)

Abstract

Targeted protein degradation refers to a technique that selectively eliminates specific proteins within cells. This can be achieved by recruiting E3 ligases to specific proteins, which label the target with ubiquitin and lead to their eventual degradation by the proteasome. Cdc48/p97, a highly conserved AAA+ ATPase, is an essential player in the ubiquitin-proteasome system. It works as an unfoldase and segregase, unfolding ubiquitinated proteins and driving their removal from protein complexes, chromatin, or membranes to be sent for proteasomal degradation. Cdc48/p97 cooperates with many UBX domain-containing adaptor proteins, which use their UBX domain to bind to Cdc48/p97 and enable it to perform various functions. To expand on previous targeted protein degradation techniques, we designed nanobody-UBX degraders (NUDs) that recruit Cdc48/p97 to unfold target proteins without requiring ubiquitination. By establishing in vitro assays, we have uncovered insights into the mechanisms of substrate unfolding by Cdc48 in conjunction with NUDs and its endogenous adaptors Ufd1-Npl4 and PUX proteins. Our findings suggest the potential for a novel targeted protein degradation strategy that leverages the unfolding mechanism of Cdc48/p97 and its interactions with adaptors.