ABSTRACT
Targeted protein degradation is a groundbreaking modality in drug discovery; however, the regulatory mechanisms are still not fully understood. Here, we identify cellular signaling pathways that modulate the targeted degradation of the anticancer target BRD4 and related neosubstrates BRD2/3 and CDK9 induced by CRL2VHL- or CRL4CRBN -based PROTACs. The chemicals identified as degradation enhancers include inhibitors of cellular signaling pathways such as poly-ADP ribosylation (PARG inhibitor PDD00017273), unfolded protein response (PERK inhibitor GSK2606414), and protein stabilization (HSP90 inhibitor luminespib). Mechanistically, PARG inhibition promotes TRIP12-mediated K29/K48-linked branched ubiquitylation of BRD4 by facilitating chromatin dissociation of BRD4 and formation of the BRD4-PROTAC-CRL2VHL ternary complex; by contrast, HSP90 inhibition promotes BRD4 degradation after the ubiquitylation step. Consequently, these signal inhibitors sensitize cells to the PROTAC-induced apoptosis. These results suggest that various cell-intrinsic signaling pathways spontaneously counteract chemically induced target degradation at multiple steps, which could be liberated by specific inhibitors.
Subject(s)
Cell Cycle Proteins , Proteolysis , Signal Transduction , Transcription Factors , Ubiquitination , Humans , Signal Transduction/drug effects , Proteolysis/drug effects , Transcription Factors/metabolism , Transcription Factors/genetics , Cell Cycle Proteins/metabolism , HSP90 Heat-Shock Proteins/metabolism , Cell Line, Tumor , Apoptosis/drug effects , Cyclin-Dependent Kinase 9/metabolism , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Bromodomain Containing ProteinsABSTRACT
Ubiquitin regulates various cellular functions by posttranslationally modifying substrates with diverse ubiquitin codes. Recent discoveries of new ubiquitin chain topologies, types of bonds, and non-protein substrates have substantially expanded the complexity of the ubiquitin code. Here, we describe the ubiquitin system covering the basic principles and recent discoveries related to mechanisms, technologies, and biological importance.
Subject(s)
Protein Processing, Post-Translational , Proteostasis , Ubiquitination , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Humans , AnimalsABSTRACT
Targeted protein degradation through chemical hijacking of E3 ubiquitin ligases is an emerging concept in precision medicine. The ubiquitin code is a critical determinant of the fate of substrates. Although two E3s, CRL2VHL and CRL4CRBN, frequently assemble with proteolysis-targeting chimeras (PROTACs) to attach lysine-48 (K48)-linked ubiquitin chains, the diversity of the ubiquitin code used for chemically induced degradation is largely unknown. Here we show that the efficacy of cIAP1-targeting degraders depends on the K63-specific E2 enzyme UBE2N. UBE2N promotes degradation of cIAP1 induced by cIAP1 ligands and subsequent cancer cell apoptosis. Mechanistically, UBE2N-catalyzed K63-linked ubiquitin chains facilitate assembly of highly complex K48/K63 and K11/K48 branched ubiquitin chains, thereby recruiting p97/VCP, UCH37 and the proteasome. Degradation of neo-substrates directed by cIAP1-recruiting PROTACs also depends on UBE2N. These results reveal an unexpected role for K63-linked ubiquitin chains and UBE2N in degrader-induced proteasomal degradation and demonstrate the diversity of the ubiquitin code used for chemical hijacking.
Subject(s)
Ubiquitin-Protein Ligases , Ubiquitin , Ubiquitin/metabolism , Ubiquitination , Ubiquitin-Protein Ligases/metabolism , Proteasome Endopeptidase Complex/metabolism , ProteolysisABSTRACT
Targeted protein degradation is an emerging therapeutic paradigm. Small-molecule degraders such as proteolysis-targeting chimeras (PROTACs) induce the degradation of neo-substrates by hijacking E3 ubiquitin ligases. Although ubiquitylation of endogenous substrates has been extensively studied, the mechanism underlying forced degradation of neo-substrates is less well understood. We found that the ubiquitin ligase TRIP12 promotes PROTAC-induced and CRL2VHL-mediated degradation of BRD4 but is dispensable for the degradation of the endogenous CRL2VHL substrate HIF-1α. TRIP12 associates with BRD4 via CRL2VHL and specifically assembles K29-linked ubiquitin chains, facilitating the formation of K29/K48-branched ubiquitin chains and accelerating the assembly of K48 linkage by CRL2VHL. Consequently, TRIP12 promotes the PROTAC-induced apoptotic response. TRIP12 also supports the efficiency of other degraders that target CRABP2 or TRIM24 or recruit CRBN. These observations define TRIP12 and K29/K48-branched ubiquitin chains as accelerators of PROTAC-directed targeted protein degradation, revealing a cooperative mechanism of branched ubiquitin chain assembly unique to the degradation of neo-substrates.
