TRIP12 promotes small-molecule-induced degradation through K29/K48-branched ubiquitin chains

•TRIP12 facilitates PROTAC-induced and CRL2VHL-mediated degradation of BRD4•TRIP12 specifically assembles K29-linked Ub chains•TRIP12 CRL2VHL cooperatively assemble K29/K48-branched chains on BRD4•Targeted uses unique mechanisms rather than a simple “hijack” Targeted protein is an emerging therapeutic paradigm. Small-molecule degraders such as proteolysis-targeting chimeras (PROTACs) induce the neo-substrates by hijacking E3 ubiquitin ligases. Although ubiquitylation endogenous substrates has been extensively studied, mechanism underlying forced less well understood. We found that ligase TRIP12 promotes BRD4 but dispensable for substrate HIF-1?. associates with via chains, facilitating formation accelerating assembly K48 linkage CRL2VHL. Consequently, apoptotic response. also supports efficiency other target CRABP2 or TRIM24 recruit CRBN. These observations define accelerators PROTAC-directed targeted degradation, revealing cooperative branched chain to neo-substrates. 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Ebert B.L. screen identifies machinery lenalidomide-dependent CRL4CRBN activity.Blood. 132: 1293-1303Crossref multiple steps after activation (i.e., what type how it encoded decoded accomplish targeting) characterized. contrast genuine substrates, evolutionarily optimized, need otherwise stable proteins, we hypothesized mediating driven may require regulators, hence mechanism. study, sought elucidate focusing BRD4. BRD4, member family, regulator transcription inhibition induces apoptosis cancer cells Using VHL-based BD-containing 4 (BRD4)-targeting MZ1 ARV771 (Raina HECT-type thyroid hormone receptor-interacting 12 (TRIP12) turned out generating cooperation results identified operating chemically induced degradation. identify protein(s) modulate PROTACs, purified BRD4-interacting HT1080 expressing FLAG-BRD4 VHL treated vehicle (Figure S1A). Label-free quantification liquid chromatography-mass spectrometry (LC-MS) (Table S1) revealed CRL2VHL, CUL2, Elongin B, C, NEDD8 co-purified MZ1-dependent manner, (Figures 1A 1B ). chromatin regulators cyclin T1, JMJD6, Mediator constitutively interacted our surprise, was MZ1-treated 1A; Table S1). relative abundance immunoprecipitants increased 4.9-fold presence 1B). several ligases, interactions were largely unchanged absence confirm proteomic screening, examined association intact cells. did interact steady state 1C, lane 5), co-immunoprecipitate behavior similar lanes 6–7). encompasses shorter isoform lacking N-terminal sequences (isoform 4: 192 kDa) longer isoforms identical 1: 220 kDa; 223 3: 225 kDa); preferentially 1C). protein. ubiquitylates RNF168 (Gudjonsson 2012Gudjonsson Altmeyer Savic V. Toledo Dinant Grøfte Bartkova Poulsen Oka Bekker-Jensen al.TRIP12 UBR5 suppress spreading damaged chromosomes.Cell. 150: 697-709Abstract (217) ASXL1 (Kweon 2019Kweon S.M. 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Then, ARV771, another BRD4-targeting cis-MZ1. ARV771-induced BRD4/BRD2 2D, 3–6), no cis-MZ1 7–10). noticed although HIF-1?, those BRD2, HIF-1? implies differentially verify possibility, siRNA 2E, 2 6), deferoxamine (DFO) inactivates proline hydroxylases inhibit VHL-HIF-1? interaction (Jaakkola 2001Jaakkola Mole D.R. Tian Y.M. Gielbert Gaskell S.J. Kriegsheim Hebestreit H.F. Mukherji al.Targeting HIF-alpha O2-regulated prolyl hydroxylation.Science. 292: 468-472Crossref (4232) 3 7), inhibitor MG132 8). experiment inhibited DFO MG132, knockdown. release treatment. accumulated washout DFO-containing media 2F). selectively (BRD4/2), 2G), implying PROTAC-specific next unlike dependent associated Hence, vitro. Purified full-length elongated free vitro manner HECT domain E1/E2 3A S2A). performed absolute eight parallel monitoring (PRM), MS2 fragment ion-based heavy-labeled signature peptides internal standards almost exclusively 3B S2B). Ubiquitylation assays lysine-to-arginine mutants single-lysine 3C). Previous studies reported amino acid (aa) determinants (Maspero 2013Maspero Valentini Mari Cecatiello Soffientini Pasqualato Polo Structure ubiquitin-loaded reveals catalytic priming.Nat. Struct. Mol. 20: 696-701Crossref (105) serine residue K63-type 3D, left). Strikingly, valine converted right). data reveal mammalian K29 reasoned could elongate BRD4-conjugated Ubs. test possibility system, searched sites From mass spectrometric analysis S2), (K445 K456) BD2 MZ1-depe