KBTBD4 Cancer Hotspot Mutations Drive Neomorphic Degradation of HDAC1/2 Corepressor Complexes.

Cancer mutations can create neomorphic protein-protein interactions to drive aberrant function 1 . As a substrate receptor of the CULLIN3-RBX1 E3 ubiquitin ligase complex, KBTBD4 is recurrently mutated in medulloblastoma (MB) 2 , the most common embryonal brain tumor in children, and pineoblastoma 3 . These mutations impart gain-of-function to KBTBD4 to induce aberrant degradation of the transcriptional corepressor CoREST 4 . However, their mechanism of action remains unresolved. Here, we elucidate the mechanistic basis by which KBTBD4 mutations promote CoREST degradation through engaging HDAC1/2, the direct neomorphic target of the substrate receptor. Using deep mutational scanning, we systematically map the mutational landscape of the KBTBD4 cancer hotspot, revealing distinct preferences by which insertions and substitutions can promote gain-of-function and the critical residues involved in the hotspot interaction. Cryo-electron microscopy (cryo-EM) analysis of two distinct KBTBD4 cancer mutants bound to LSD1-HDAC1-CoREST reveals that a KBTBD4 homodimer asymmetrically engages HDAC1 with two KELCH-repeat propeller domains. The interface between HDAC1 and one of the KBTBD4 propellers is stabilized by the MB mutations, which directly insert a bulky side chain into the active site pocket of HDAC1. Our structural and mutational analyses inform how this hotspot E3-neo-substrate interface can be chemically modulated. First, our results unveil a converging shape complementarity-based mechanism between gain-of-function E3 mutations and a molecular glue degrader, UM171. Second, we demonstrate that HDAC1/2 inhibitors can block the mutant KBTBD4-HDAC1 interface, the aberrant degradation of CoREST, and the growth of KBTBD4-mutant MB models. Altogether, our work reveals the structural and mechanistic basis of cancer mutation-driven neomorphic protein-protein interactions and pharmacological strategies to modulate their action for therapeutic applications.

Asymmetric Engagement of Dimeric CRL3 KBTBD4 by the Molecular Glue UM171 Licenses Degradation of HDAC1/2 Complexes.

UM171 is a potent small molecule agonist of ex vivo human hematopoietic stem cell (HSC) self-renewal, a process that is tightly controlled by epigenetic regulation. By co-opting KBTBD4, a substrate receptor of the CULLIN3-RING E3 ubiquitin ligase complex, UM171 promotes the degradation of members of the CoREST transcriptional corepressor complex, thereby limiting HSC attrition. However, the direct target and mechanism of action of UM171 remain unclear. Here, we reveal that UM171 acts as a molecular glue to induce high-affinity interactions between KBTBD4 and HDAC1 to promote the degradation of select HDAC1/2 corepressor complexes. Through proteomics and chemical inhibitor studies, we discover that the principal target of UM171 is HDAC1/2. Cryo-electron microscopy (cryo-EM) analysis of dimeric KBTBD4 bound to UM171 and the LSD1-HDAC1-CoREST complex unveils an unexpected asymmetric assembly, in which a single UM171 molecule enables a pair of KBTBD4 KELCH-repeat propeller domains to recruit HDAC1 by clamping on its catalytic domain. One of the KBTBD4 propellers partially masks the rim of the HDAC1 active site pocket, which is exploited by UM171 to extend the E3-neo-substrate interface. The other propeller cooperatively strengthens HDAC1 binding via a separate and distinct interface. The overall neomorphic interaction is further buttressed by an endogenous cofactor of HDAC1-CoREST, inositol hexakisphosphate, which makes direct contacts with KBTBD4 and acts as a second molecular glue. The functional relevance of the quaternary complex interaction surfaces defined by cryo-EM is demonstrated by in situ base editor scanning of KBTBD4 and HDAC1. By delineating the direct target of UM171 and its mechanism of action, our results reveal how the cooperativity offered by a large dimeric CRL E3 family can be leveraged by a small molecule degrader and establish for the first time a dual molecular glue paradigm.

Total chemical synthesis of sumoylated histone H4 reveals negative biochemical crosstalk with histone ubiquitylation.

An efficient total chemical synthesis of site-specifically sumoylated histone H4 was undertaken to generate homogenously modified mononucleosomes. These were tested as substrates in biochemical assays with the histone H2B-specific ubiquitin ligases Rad6 and Bre1, which revealed the strong inhibition of H2B ubiquitylation by SUMO. This novel negative biochemical crosstalk between SUMO and ubiquitin was also confirmed to exist in human cells.

INOSITOL (1,3,4) TRIPHOSPHATE 5/6 KINASE1-dependent inositol polyphosphates regulate auxin responses in Arabidopsis.

The combinatorial phosphorylation of myo-inositol results in the generation of different inositol phosphates (InsPs), of which phytic acid (InsP6) is the most abundant species in eukaryotes. InsP6 is also an important precursor of the higher phosphorylated inositol pyrophosphates (PP-InsPs), such as InsP7 and InsP8, which are characterized by a diphosphate moiety and are also ubiquitously found in eukaryotic cells. While PP-InsPs regulate various cellular processes in animals and yeast, their biosynthesis and functions in plants has remained largely elusive because plant genomes do not encode canonical InsP6 kinases. Recent work has shown that Arabidopsis (Arabidopsis thaliana) INOSITOL (1,3,4) TRIPHOSPHATE 5/6 KINASE1 (ITPK1) and ITPK2 display in vitro InsP6 kinase activity and that, in planta, ITPK1 stimulates 5-InsP7 and InsP8 synthesis and regulates phosphate starvation responses. Here we report a critical role of ITPK1 in auxin-related processes that is independent of the ITPK1-controlled regulation of phosphate starvation responses. Those processes include primary root elongation, root hair development, leaf venation, thermomorphogenic and gravitropic responses, and sensitivity to exogenously applied auxin. We found that the recombinant auxin receptor complex, consisting of the F-Box protein TRANSPORT INHIBITOR RESPONSE1 (TIR1), ARABIDOPSIS SKP1 HOMOLOG 1 (ASK1), and the transcriptional repressor INDOLE-3-ACETIC ACID INDUCIBLE 7 (IAA7), binds to anionic inositol polyphosphates with high affinity. We further identified a physical interaction between ITPK1 and TIR1, suggesting a localized production of 5-InsP7, or another ITPK1-dependent InsP/PP-InsP isomer, to activate the auxin receptor complex. Finally, we demonstrate that ITPK1 and ITPK2 function redundantly to control auxin responses, as deduced from the auxin-insensitive phenotypes of itpk1 itpk2 double mutant plants. Our findings expand the mechanistic understanding of auxin perception and suggest that distinct inositol polyphosphates generated near auxin receptors help to fine-tune auxin sensitivity in plants.

A novel interleukin-1 receptor-associated kinase 4 from blunt snout bream (Megalobrama amblycephala) is involved in inflammatory response via MyD88-mediated NF-κB signal pathway.

Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a crucial role in the Toll-like receptor/IL-1R signal pathway, which mediates the downstream signal transduction involved in innate and adaptive immunity. In the present study, an IRAK4 homologue (named as MaIRAK4) from blunt snout bream (Megalobrama amblycephala) was cloned and characterized. The open reading frame (ORF) of MaIRAK4 contains 1422 nucleotides, encoding a putative protein of 473 amino acids. Protein structural analysis revealed that MaIRAK4 has an N-terminal death domain (DD) and a central kinase domain (S_TKc), similar to those of mammals and other fishes. Multiple sequence alignment demonstrated that MaIRAK4 is highly homologous with that of grass carp (97.67%). The qRT-PCR analysis showed that MaIRAK4 expressed widely in all examined tissues, including heart, liver, spleen, kidney, head-kidney, gill, intestine and muscle, with the highest expression in the liver and spleen. After stimulation with LPS, MaIRAK4 expression upregulated significantly and reached a peak at 6 h and 12 h post LPS stimulation in the spleen and head-kidney, respectively. After challenge with Aeromonas hydrophila, MaIRAK4 expression peaked at 48 h and 72 h in spleen/head-kidney and liver, respectively. These results implied that MaIRAK4 is involved in the host defense against bacterial infection. Subcellular localization analysis indicated that MaIRAK4 distributed in the cytoplasm. Co-immunoprecipitation and subcellular co-localization assay revealed that MaIRAK4 can combine with MaMyD88 through DD domain. MaIRAK4 overexpression can induce slightly the NF-κB promoter activity in HEK 293 cells. However, the activity of NF-κB promoter was dramatically enhanced after co-transfection with MaIRAK4 and MaMyD88 plasmids. The results showed that MaIRAK4 was involved in NF-κB signal pathway mediated by maMyD88. The expression level of pro-inflammatory cytokines (IL-1ß, IL-6, IL-8 and TNF-α) decreased significantly after the siRNA-mediated knockdown of MaIRAK4. Together, these results suggest that MaIRAK4 plays an important function in the innate immunity of M. amblycephala by inducing cytokines expression.

Defining molecular glues with a dual-nanobody cannabidiol sensor.

"Molecular glue" (MG) is a term coined to describe the mechanism of action of the plant hormone auxin and subsequently used to characterize synthetic small molecule protein degraders exemplified by immune-modulatory imide drugs (IMiDs). Prospective development of MGs, however, has been hampered by its elusive definition and thermodynamic characteristics. Here, we report the crystal structure of a dual-nanobody cannabidiol-sensing system, in which the ligand promotes protein-protein interaction in a manner analogous to auxin. Through quantitative analyses, we draw close parallels among the dual-nanobody cannabidiol sensor, the auxin perception complex, and the IMiDs-bound CRL4CRBN E3, which can bind and ubiquitinate "neo-substrates". All three systems, including the recruitment of IKZF1 and CK1α to CRBN, are characterized by the lack of ligand binding activity in at least one protein partner and an under-appreciated preexisting low micromolar affinity between the two proteinaceous subunits that is enhanced by the ligand to reach the nanomolar range. These two unifying features define MGs as a special class of proximity inducers distinct from bifunctional compounds and can be used as criteria to guide target selection for future rational discovery of MGs.

Basis for metabolite-dependent Cullin-RING ligase deneddylation by the COP9 signalosome.

The Cullin-RING ligases (CRLs) are the largest family of ubiquitin E3s activated by neddylation and regulated by the deneddylase COP9 signalosome (CSN). The inositol polyphosphate metabolites promote the formation of CRL-CSN complexes, but with unclear mechanism of action. Here, we provide structural and genetic evidence supporting inositol hexakisphosphate (IP6) as a general CSN cofactor recruiting CRLs. We determined the crystal structure of IP6 in complex with CSN subunit 2 (CSN2), based on which we identified the IP6-corresponding electron density in the cryoelectron microscopy map of a CRL4A-CSN complex. IP6 binds to a cognate pocket formed by conserved lysine residues from CSN2 and Rbx1/Roc1, thereby strengthening CRL-CSN interactions to dislodge the E2 CDC34/UBE2R from CRL and to promote CRL deneddylation. IP6 binding-deficient Csn2K70E/K70E knockin mice are embryonic lethal. The same mutation disabled Schizosaccharomyces pombe Csn2 from rescuing UV-hypersensitivity of csn2-null yeast. These data suggest that CRL transition from the E2-bound active state to the CSN-bound sequestered state is critically assisted by an interfacial IP6 small molecule, whose metabolism may be coupled to CRL-CSN complex dynamics.

Structural dynamics of the human COP9 signalosome revealed by cross-linking mass spectrometry and integrative modeling.

The COP9 signalosome (CSN) is an evolutionarily conserved eight-subunit (CSN1-8) protein complex that controls protein ubiquitination by deneddylating Cullin-RING E3 ligases (CRLs). The activation and function of CSN hinges on its structural dynamics, which has been challenging to decipher by conventional tools. Here, we have developed a multichemistry cross-linking mass spectrometry approach enabled by three mass spectometry-cleavable cross-linkers to generate highly reliable cross-link data. We applied this approach with integrative structure modeling to determine the interaction and structural dynamics of CSN with the recently discovered ninth subunit, CSN9, in solution. Our results determined the localization of CSN9 binding sites and revealed CSN9-dependent structural changes of CSN. Together with biochemical analysis, we propose a structural model in which CSN9 binding triggers CSN to adopt a configuration that facilitates CSN-CRL interactions, thereby augmenting CSN deneddylase activity. Our integrative structure analysis workflow can be generalized to define in-solution architectures of dynamic protein complexes that remain inaccessible to other approaches.

Structural plasticity of D3-D14 ubiquitin ligase in strigolactone signalling.

The strigolactones, a class of plant hormones, regulate many aspects of plant physiology. In the inhibition of shoot branching, the α/ß hydrolase D14-which metabolizes strigolactone-interacts with the F-box protein D3 to ubiquitinate and degrade the transcription repressor D53. Despite the fact that multiple modes of interaction between D14 and strigolactone have recently been determined, how the hydrolase functions with D3 to mediate hormone-dependent D53 ubiquitination remains unknown. Here we show that D3 has a C-terminal α-helix that can switch between two conformational states. The engaged form of this α-helix facilitates the binding of D3 and D14 with a hydrolysed strigolactone intermediate, whereas the dislodged form can recognize unmodified D14 in an open conformation and inhibits its enzymatic activity. The D3 C-terminal α-helix enables D14 to recruit D53 in a strigolactone-dependent manner, which in turn activates the hydrolase. By revealing the structural plasticity of the SCFD3-D14 ubiquitin ligase, our results suggest a mechanism by which the E3 coordinates strigolactone signalling and metabolism.

Allosteric Activation of Ubiquitin-Specific Proteases by ß-Propeller Proteins UAF1 and WDR20.

Ubiquitin-specific proteases (USPs) constitute the largest family of deubiquitinating enzymes, whose catalytic competency is often modulated by their binding partners through unknown mechanisms. Here we report on a series of crystallographic and biochemical analyses of an evolutionarily conserved deubiquitinase, USP12, which is activated by two ß-propeller proteins, UAF1 and WDR20. Our structures reveal that UAF1 and WDR20 interact with USP12 at two distinct sites far from its catalytic center. Without increasing the substrate affinity of USP12, the two ß-propeller proteins potentiate the enzyme through different allosteric mechanisms. UAF1 docks at the distal end of the USP12 Fingers domain and induces a cascade of structural changes that reach a critical ubiquitin-contacting loop adjacent to the catalytic cleft. By contrast, WDR20 anchors at the base of this loop and remotely modulates the catalytic center of the enzyme. Our results provide a mechanistic example for allosteric activation of USPs by their regulatory partners.

Inositol hexakisphosphate (IP6) generated by IP5K mediates cullin-COP9 signalosome interactions and CRL function.

The family of cullin-RING E3 Ligases (CRLs) and the constitutive photomorphogenesis 9 (COP9) signalosome (CSN) form dynamic complexes that mediate ubiquitylation of 20% of the proteome, yet regulation of their assembly/disassembly remains poorly understood. Inositol polyphosphates are highly conserved signaling molecules implicated in diverse cellular processes. We now report that inositol hexakisphosphate (IP6) is a major physiologic determinant of the CRL-CSN interface, which includes a hitherto unidentified electrostatic interaction between the N-terminal acidic tail of CSN subunit 2 (CSN2) and a conserved basic canyon on cullins. IP6, with an EC50 of 20 nM, acts as an intermolecular "glue," increasing cullin-CSN2 binding affinity by 30-fold, thereby promoting assembly of the inactive CRL-CSN complexes. The IP6 synthase, Ins(1,3,4,5,6)P5 2-kinase (IPPK/IP5K) binds to cullins. Depleting IP5K increases the percentage of neddylated, active Cul1 and Cul4A, and decreases levels of the Cul1/4A substrates p27 and p21. Besides dysregulating CRL-mediated cell proliferation and UV-induced apoptosis, IP5K depletion potentiates by 28-fold the cytotoxic effect of the neddylation inhibitor MLN4924. Thus, IP5K and IP6 are evolutionarily conserved components of the CRL-CSN system and are potential targets for cancer therapy in conjunction with MLN4924.

Gln40 deamidation blocks structural reconfiguration and activation of SCF ubiquitin ligase complex by Nedd8.

The full enzymatic activity of the cullin-RING ubiquitin ligases (CRLs) requires a ubiquitin-like protein (that is, Nedd8) modification. By deamidating Gln40 of Nedd8 to glutamate (Q40E), the bacterial cycle-inhibiting factor (Cif) family is able to inhibit CRL E3 activities, thereby interfering with cellular functions. Despite extensive structural studies on CRLs, the molecular mechanism by which Nedd8 Gln40 deamidation affects CRL functions remains unclear. We apply a new quantitative cross-linking mass spectrometry approach to characterize three different types of full-length human Cul1-Rbx1 complexes and uncover major Nedd8-induced structural rearrangements of the CRL1 catalytic core. More importantly, we find that those changes are not induced by Nedd8(Q40E) conjugation, indicating that the subtle change of a single Nedd8 amino acid is sufficient to revert the structure of the CRL catalytic core back to its unmodified form. Our results provide new insights into how neddylation regulates the conformation and activity of CRLs.

Rate Motifs Tune Auxin/Indole-3-Acetic Acid Degradation Dynamics.

Ubiquitin-mediated protein degradation is a common feature in diverse plant cell signaling pathways; however, the factors that control the dynamics of regulated protein turnover are largely unknown. One of the best-characterized families of E3 ubiquitin ligases facilitates ubiquitination of auxin (aux)/indole-3-acetic acid (IAA) repressor proteins in the presence of auxin. Rates of auxin-induced degradation vary widely within the Aux/IAA family, and sequences outside of the characterized degron (the minimum region required for auxin-induced degradation) can accelerate or decelerate degradation. We have used synthetic auxin degradation assays in yeast (Saccharomyces cerevisiae) and in plants to characterize motifs flanking the degron that contribute to tuning the dynamics of Aux/IAA degradation. The presence of these rate motifs is conserved in phylogenetically distant members of the Arabidopsis (Arabidopsis thaliana) Aux/IAA family, as well as in their putative Brassica rapa orthologs. We found that rate motifs can act by enhancing interaction between repressors and the E3, but that this is not the only mechanism of action. Phenotypes of transgenic plants expressing a deletion in a rate motif in IAA28 resembled plants expressing degron mutations, underscoring the functional relevance of Aux/IAA degradation dynamics in regulating auxin responses.

VIH2 Regulates the Synthesis of Inositol Pyrophosphate InsP8 and Jasmonate-Dependent Defenses in Arabidopsis.

Diphosphorylated inositol polyphosphates, also referred to as inositol pyrophosphates, are important signaling molecules that regulate critical cellular activities in many eukaryotic organisms, such as membrane trafficking, telomere maintenance, ribosome biogenesis, and apoptosis. In mammals and fungi, two distinct classes of inositol phosphate kinases mediate biosynthesis of inositol pyrophosphates: Kcs1/IP6K- and Vip1/PPIP5K-like proteins. Here, we report that PPIP5K homologs are widely distributed in plants and that Arabidopsis thaliana VIH1 and VIH2 are functional PPIP5K enzymes. We show a specific induction of inositol pyrophosphate InsP8 by jasmonate and demonstrate that steady state and jasmonate-induced pools of InsP8 in Arabidopsis seedlings depend on VIH2. We identify a role of VIH2 in regulating jasmonate perception and plant defenses against herbivorous insects and necrotrophic fungi. In silico docking experiments and radioligand binding-based reconstitution assays show high-affinity binding of inositol pyrophosphates to the F-box protein COI1-JAZ jasmonate coreceptor complex and suggest that coincidence detection of jasmonate and InsP8 by COI1-JAZ is a critical component in jasmonate-regulated defenses.

Lewis base mediated halogenation/semipinacol rearrangement of diazo compounds: new access to α-halo-quaternary ketones.

A novel halogenation/semipinacol rearrangement of α-diazo alcohol catalyzed by Lewis base has been developed through a carbene-free mechanism. This semipinacol transposition, initiated by an electrophilic halogenation (X = Cl(+), Br(+), and I(+)) of diazo carbon event, furnished a convenient synthetic route for the efficient synthesis of α-halo-quaternary ketones under mild conditions.

Highly enantioselective synthesis of bisoxindoles with two vicinal quaternary stereocenters via Lewis base mediated addition of oxindoles to isatin-derived ketimines.

A highly efficient asymmetric organocatalytic addition of 3-substituted oxindole to isatin-derived ketimine is reported with excellent stereocontrol (>99 : 1 dr, >99% ee) under mild conditions. This method provides access to the bisoxindole structure moiety with two vicinal quaternary stereogenic centers.

Visible-light-induced direct C(sp3)-H difluromethylation of tetrahydroisoquinolines with the in situ generated difluoroenolates.

An effective approach to C1-difluoromethylated tetrahydroisoquinoline derivatives has been developed through C-H functionalization of tertiary amines by visible-light photoredox catalysis. This method uses stable, easily obtained α,α-difluorinated gem-diol as the CF2 source. The corresponding products were obtained in moderate to high yields at ambient temperature.

Organocatalytic one-pot synthesis of highly substituted pyridazines from Morita-Baylis-Hillman carbonates and diazo compounds.

A biologically inspired organocatalytic one-pot synthesis of highly functionalized pyridazines, which are ubiquitous structural units in a number of biologically active compounds, has been developed by starting from readily available diazo compounds and Morita-Baylis-Hillman (MBH) carbonates. Under mild reaction conditions, this synthetic route tolerated significant substrate variation to deliver a broad range of substituted products, including CF3 -substituted pyridazines derivatives. Moreover, the introduction of trifluoromethyl groups into the ring of pyridazine could be completed conveniently from 2,2,2-trifluorodiazoethane.