Dipeptidyl peptidases and E3 ligases of N-degron pathways cooperate to regulate protein stability.

N-degrons are short sequences located at protein N-terminus that mediate the interaction of E3 ligases (E3s) with substrates to promote their proteolysis. It is well established that N-degrons can be exposed following protease cleavage to allow recognition by E3s. However, our knowledge regarding how proteases and E3s cooperate in protein quality control mechanisms remains minimal. Using a systematic approach to monitor the protein stability of an N-terminome library, we found that proline residue at the third N-terminal position (hereafter "P+3") promotes instability. Genetic perturbations identified the dipeptidyl peptidases DPP8 and DPP9 and the primary E3s of N-degron pathways, UBR proteins, as regulators of P+3 bearing substrate turnover. Interestingly, P+3 UBR substrates are significantly enriched for secretory proteins. We found that secretory proteins relying on a signal peptide (SP) for their targeting contain a "built-in" N-degron within their SP. This degron becomes exposed by DPP8/9 upon translocation failure to the designated compartments, thus enabling clearance of mislocalized proteins by UBRs to maintain proteostasis.

Ubiquitin-independent proteasomal degradation driven by C-degron pathways.

Although most eukaryotic proteins are targeted for proteasomal degradation by ubiquitination, a subset have been demonstrated to undergo ubiquitin-independent proteasomal degradation (UbInPD). However, little is known about the molecular mechanisms driving UbInPD and the degrons involved. Utilizing the GPS-peptidome approach, a systematic method for degron discovery, we found thousands of sequences that promote UbInPD; thus, UbInPD is more prevalent than currently appreciated. Furthermore, mutagenesis experiments revealed specific C-terminal degrons required for UbInPD. Stability profiling of a genome-wide collection of human open reading frames identified 69 full-length proteins subject to UbInPD. These included REC8 and CDCA4, proteins which control proliferation and survival, as well as mislocalized secretory proteins, suggesting that UbInPD performs both regulatory and protein quality control functions. In the context of full-length proteins, C termini also play a role in promoting UbInPD. Finally, we found that Ubiquilin family proteins mediate the proteasomal targeting of a subset of UbInPD substrates.

Molecular basis for arginine C-terminal degron recognition by Cul2FEM1 E3 ligase.

Degrons are elements within protein substrates that mediate the interaction with specific degradation machineries to control proteolysis. Recently, a few classes of C-terminal degrons (C-degrons) that are recognized by dedicated cullin-RING ligases (CRLs) have been identified. Specifically, CRL2 using the related substrate adapters FEM1A/B/C was found to recognize C degrons ending with arginine (Arg/C-degron). Here, we uncover the molecular mechanism of Arg/C-degron recognition by solving a subset of structures of FEM1 proteins in complex with Arg/C-degron-bearing substrates. Our structural research, complemented by binding assays and global protein stability (GPS) analyses, demonstrates that FEM1A/C and FEM1B selectively target distinct classes of Arg/C-degrons. Overall, our study not only sheds light on the molecular mechanism underlying Arg/C-degron recognition for precise control of substrate turnover, but also provides valuable information for development of chemical probes for selectively regulating proteostasis.