Aberrantly elevated steroid receptor coactivator‐1 (SRC‐1) expression and activity are strongly correlated with cancer progression and metastasis. Here we report, for the first time, the development of a proteolysis targeting chimera (PROTAC) that is composed of a selective SRC‐1 binder linked to a specific ligand for UBR box, a unique class of E3 ligases recognizing N‐degrons. We showed that the bifunctional molecule efficiently and selectively induced the degradation of SRC‐1 in cells through the N‐degron pathway. Importantly, given the ubiquitous expression of the UBR protein in most cells, PROTACs targeting the UBR box could degrade a protein of interest regardless of cell types. We also showed that the SRC‐1 degrader significantly suppressed cancer cell invasion and migration in vitro and in vivo. Together, these results demonstrate that the SRC‐1 degrader can be an invaluable chemical tool in the studies of SRC‐1 functions. Moreover, our findings suggest PROTACs based on the N‐degron pathway as a widely useful strategy to degrade disease‐relevant proteins.

The N-degron pathway, formerly the N-end rule pathway, is a protein degradation process that determines the half-life of proteins based on their N-terminal residues. In contrast to the well-established in vivo studies over decades, in vitro studies of this pathway, including biochemical characterization and high-resolution structures, are relatively limited. In this study, we have developed a unique fusion technique using microtubule-associated protein 1A/1B light chain 3B, a key marker protein of autophagy, to tag the N terminus of the proteins involved in the N-degron pathway, which enables high yield of homogeneous target proteins with variable N-terminal residues for diverse biochemical studies including enzymatic and binding assays and substrate identification. Intriguingly, crystallization showed a markedly enhanced probability, even for the N-degron complexes. To validate our results, we determined the structures of select proteins in the N-degron pathway and compared them with the Protein Data Bank–deposited proteins. Furthermore, several biochemical applications of this technique were introduced. Therefore, this technique can be used as a general tool for the in vitro study of the N-degron pathway.

The N‐degron pathway determines the half‐life of proteins in both prokaryotes and eukaryotes by precisely recognizing the N‐terminal residue (N‐degron) of substrates. ClpS proteins from bacteria bind to substrates containing hydrophobic N‐degrons (Leu, Phe, Tyr, and Trp) and deliver them to the caseinolytic protease system ClpAP. This mechanism is preserved in organelles such as mitochondria and chloroplasts. Bacterial ClpS adaptors bind preferentially to Leu and Phe N‐degrons; however, ClpS1 from Arabidopsis thaliana (AtClpS1) shows a difference in that it binds strongly to Phe and Trp N‐degrons and only weakly to Leu. This difference in behavior cannot be explained without structural information due to the high sequence homology between bacterial and plant ClpS proteins. Here, we report the structure of AtClpS1 at 2.0 Å resolution in the presence of a bound N‐degron. The key determinants for α‐amino group recognition are conserved among all ClpS proteins, but the α3‐helix of eukaryotic AtClpS1 is significantly shortened, and consequently, a loop forming a pocket for the N‐degron is moved slightly outward to enlarge the pocket. In addition, amino acid replacement from Val to Ala causes a reduction in hydrophobic interactions with Leu N‐degron. A combination of the fine‐tuned hydrophobic residues in the pocket and the basic gatekeeper at the entrance of the pocket controls the N‐degron selectivity of the plant ClpS protein.

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Aberrantly elevated steroid receptor coactivator‐1 (SRC‐1) expression and activity are strongly correlated with cancer progression and metastasis. Here we report, for the first time, the development of a proteolysis targeting chimera (PROTAC) that is composed of a selective SRC‐1 binder linked to a specific ligand for UBR box, a unique class of E3 ligases recognizing N‐degrons. We showed that the bifunctional molecule efficiently and selectively induced the degradation of SRC‐1 in cells through the N‐degron pathway. Importantly, given the ubiquitous expression of the UBR protein in most cells, PROTACs targeting the UBR box could degrade a protein of interest regardless of cell types. We also showed that the SRC‐1 degrader significantly suppressed cancer cell invasion and migration in vitro and in vivo. Together, these results demonstrate that the SRC‐1 degrader can be an invaluable chemical tool in the studies of SRC‐1 functions. Moreover, our findings suggest PROTACs based on the N‐degron pathway as a widely useful strategy to degrade disease‐relevant proteins.