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Thalidomide analogues exert their therapeutic effects by binding to the CRL4CRBN E3 ubiquitin ligase, promoting ubiquitination and subsequent proteasomal degradation of specific protein substrates. Drug-induced degradation of IKZF1 and IKZF3 in B-cell malignancies demonstrates the clinical utility of targeting disease-relevant transcription factors for degradation. Here, we found that avadomide (CC-122) induces CRBN-dependent ubiquitination and proteasomal degradation of ZMYM2 (ZNF198), a transcription factor involved in balanced chromosomal rearrangements with FGFR1 and FLT3 in aggressive forms of hematologic malignancies. The minimal drug-responsive element of ZMYM2 is a zinc-chelating MYM domain and is contained in the N-terminal portion of ZMYM2 that is universally included in the derived fusion proteins. We demonstrate that avadomide has the ability to induce proteasomal degradation of ZMYM2–FGFR1 and ZMYM2–FLT3 chimeric oncoproteins, both in vitro and in vivo. Our findings suggest that patients with hematologic malignancies harboring these ZMYM2 fusion proteins may benefit from avadomide treatment.Significance:We extend the potential clinical scope of thalidomide analogues by the identification of a novel avadomide-dependent CRL4CRBN substrate, ZMYM2. Avadomide induces ubiquitination and degradation of ZMYM2–FGFR1 and ZMYM2–FLT3, two chimeric oncoproteins involved in hematologic malignancies, providing a proof of concept for drug-induced degradation of transcription factor fusion proteins by thalidomide analogues.

Supplementary Table 1. Proteomic data

Cereblon (CRBN) is an ubiquitin ligase (E3) adaptor protein co-opted by CRBN E3 Ligase Modulatory Drugs (CELMoD) agents that target therapeutically-relevant proteins for degradation. Prior crystallographic studies defined the drug-binding site within CRBN’s Thalidomide Binding Domain (TBD), but the allostery of drug-induced neosubstrate binding remains unclear. We therefore performed cryo-EM analyses of the DDB1∼CRBN apo-complex, and compared these structures with DDB1∼CRBN in the presence of CELMoD compounds alone and complexed with neosubstrates. Association of CELMoD compounds to the TBD is necessary and sufficient for triggering CRBN allosteric rearrangement from an “open” conformation to the canonical “closed” conformation. Importantly, the neosubstrate Ikaros only stably associates with the closed CRBN conformation, illustrating the importance of allostery for CELMoD compound efficacy, and informing structure-guided design strategies to improve therapeutic efficacy.

A number of clinically validated drugs have been developed by repurposing the CUL4-DDB1-CRBN-RBX1 (CRL4CRBN) E3 ubiquitin ligase complex with molecular glue degraders to eliminate disease-driving proteins. Here, we present the identification of a first-in-class GSPT1-selective cereblon E3 ligase modulator, CC-90009. Biochemical, structural, and molecular characterization demonstrates that CC-90009 coopts the CRL4CRBN to selectively target GSPT1 for ubiquitination and proteasomal degradation. Depletion of GSPT1 by CC-90009 rapidly induces acute myeloid leukemia (AML) apoptosis, reducing leukemia engraftment and leukemia stem cells (LSCs) in large-scale primary patient xenografting of 35 independent AML samples, including those with adverse risk features. Using a genome-wide CRISPR-Cas9 screen for effectors of CC-90009 response, we uncovered the ILF2 and ILF3 heterodimeric complex as a novel regulator of cereblon expression. Knockout of ILF2/ILF3 decreases the production of full-length cereblon protein via modulating CRBN messenger RNA alternative splicing, leading to diminished response to CC-90009. The screen also revealed that the mTOR signaling and the integrated stress response specifically regulate the response to CC-90009 in contrast to other cereblon modulators. Hyperactivation of the mTOR pathway by inactivation of TSC1 and TSC2 protected against the growth inhibitory effect of CC-90009 by reducing CC-90009-induced binding of GSPT1 to cereblon and subsequent GSPT1 degradation. On the other hand, GSPT1 degradation promoted the activation of the GCN1/GCN2/ATF4 pathway and subsequent apoptosis in AML cells. Collectively, CC-90009 activity is mediated by multiple layers of signaling networks and pathways within AML blasts and LSCs, whose elucidation gives insight into further assessment of CC-90009s clinical utility. These trials were registered at www.clinicaltrials.gov as #NCT02848001 and #NCT04336982).

Thalidomide-dependent degradation of the embryonic transcription factor SALL4 by the CRL4CRBN E3 ubiquitin ligase is a plausible major driver of thalidomide teratogenicity. The structure of the second zinc finger of SALL4 in complex with pomalidomide, cereblon and DDB1 reveals the molecular details of recruitment. Sequence differences and a shifted binding position relative to Ikaros offer a path to the rational design of cereblon-binding drugs with reduced teratogenic risk.

Targeted protein degradation is garnering increased attention as a therapeutic modality due in part to its promise of modulating targets previously considered undruggable. Cereblon E3 Ligase Modulating Drugs (CELMoDs) are one of the most well-characterized therapeutics employing this modality. CELMoDs hijack Cereblon E3 ligase activity causing neosubstrates to be ubiquitinated and degraded in the proteasome. Here, we describe a suite of assays-cellular substrate degradation, confirmation of CELMoD mechanism of action, in vitro ubiquitination, and Cereblon binding-that can be used to characterize CELMoD-mediated degradation of Cereblon neosubstrates. While the assays presented herein can be run independently, when combined they provide a strong platform to support the discovery and optimization of CELMoDs and fuel validation of targets degraded by this drug modality.

Thalidomide analogues exert their therapeutic effects by binding to the CRL4CRBN E3 ubiquitin ligase, promoting ubiquitination and subsequent proteasomal degradation of specific protein substrates. Drug-induced degradation of IKZF1 and IKZF3 in B-cell malignancies demonstrates the clinical utility of targeting disease-relevant transcription factors for degradation. Here, we found that avadomide (CC-122) induces CRBN-dependent ubiquitination and proteasomal degradation of ZMYM2 (ZNF198), a transcription factor involved in balanced chromosomal rearrangements with FGFR1 and FLT3 in aggressive forms of hematologic malignancies. The minimal drug-responsive element of ZMYM2 is a zinc-chelating MYM domain and is contained in the N-terminal portion of ZMYM2 that is universally included in the derived fusion proteins. We demonstrate that avadomide has the ability to induce proteasomal degradation of ZMYM2–FGFR1 and ZMYM2–FLT3 chimeric oncoproteins, both in vitro and in vivo. Our findings suggest that patients with hematologic malignancies harboring these ZMYM2 fusion proteins may benefit from avadomide treatment. Significance: We extend the potential clinical scope of thalidomide analogues by the identification of a novel avadomide-dependent CRL4CRBN substrate, ZMYM2. Avadomide induces ubiquitination and degradation of ZMYM2–FGFR1 and ZMYM2–FLT3, two chimeric oncoproteins involved in hematologic malignancies, providing a proof of concept for drug-induced degradation of transcription factor fusion proteins by thalidomide analogues.

Targeted protein degradation is garnering increased attention as a therapeutic modality due in part to its promise of modulating targets previously considered undruggable. Cereblon E3 Ligase Modulating Drugs (CELMoDs) are one of the most well-characterized therapeutics employing this modality. CELMoDs hijack Cereblon E3 ligase activity causing neosubstrates to be ubiquitinated and degraded in the proteasome. Here, we describe a suite of assays-cellular substrate degradation, confirmation of CELMoD mechanism of action, in vitro ubiquitination, and Cereblon binding-that can be used to characterize CELMoD-mediated degradation of Cereblon neosubstrates. While the assays presented herein can be run independently, when combined they provide a strong platform to support the discovery and optimization of CELMoDs and fuel validation of targets degraded by this drug modality.

There is a growing interest in using targeted protein degradation as a therapeutic modality in view of its potential to expand the druggable proteome. One avenue to using this modality is via molecular glue based Cereblon E3 Ligase Modulating Drug compounds. Here, we report the identification of the transcription factor ZBTB16 as a Cereblon neosubstrate. We also report two new Cereblon modulators, CC-3060 and CC-647, that promote ZBTB16 degradation. Unexpectedly, CC-3060 and CC-647 target ZBTB16 for degradation by primarily engaging distinct structural degrons on different zinc finger domains. The reciprocal fusion proteins, ZBTB16-RARα and RARα-ZBTB16, which cause a rare acute promyelocytic leukemia, contain these same structural degrons and can be targeted for proteasomal degradation with Cereblon modulator treatment. Thus, a targeted protein degradation approach via Cereblon modulators may represent a novel therapeutic strategy in acute promyelocytic leukemia where ZBTB16/RARA rearrangements are critical disease drivers.

CELMoDs (cereblon E3 ligase-modulating drugs) are low-molecular-weight small molecules that induce the recruitment of specific protein targets to the cereblon-CRL4 ubiquitin ligase for ubiquitination and subsequent cellular degradation. CELMoDs act to scaffold direct protein–protein interactions between cereblon and substrate, working through a so-called “molecular glue” mechanism. We discuss recent discoveries in CELMoD mechanism of action, including approaches taken to expand the scope of cereblon neosubstrates, and discuss the future and breadth of this emerging class of molecules.

Targeted protein degradation via small-molecule modulation of cereblon offers vast potential for the development of new therapeutics. Cereblon-binding therapeutics carry the safety risks of thalidomide, which caused an epidemic of severe birth defects characterized by forelimb shortening or phocomelia. Here we show that thalidomide is not teratogenic in transgenic mice expressing human cereblon, indicating that binding to cereblon is not sufficient to cause birth defects. Instead, we identify SALL4 as a thalidomide-dependent cereblon neosubstrate. Human mutations in SALL4 cause Duane-radial ray, IVIC, and acro-renal-ocular syndromes with overlapping clinical presentations to thalidomide embryopathy, including phocomelia. SALL4 is degraded in rabbits but not in resistant organisms such as mice because of SALL4 sequence variations. This work expands the scope of cereblon neosubstrate activity within the formerly 'undruggable' C2H2 zinc finger family and offers a path toward safer therapeutics through an improved understanding of the molecular basis of thalidomide-induced teratogenicity.

The drugs lenalidomide and pomalidomide bind to the protein cereblon, directing the CRL4-CRBN E3 ligase toward the transcription factors Ikaros and Aiolos to cause their ubiquitination and degradation. Here we describe CC-220 (compound 6), a cereblon modulator in clinical development for systemic lupus erythematosis and relapsed/refractory multiple myeloma. Compound 6 binds cereblon with a higher affinity than lenalidomide or pomalidomide. Consistent with this, the cellular degradation of Ikaros and Aiolos is more potent and the extent of substrate depletion is greater. The crystal structure of cereblon in complex with DDB1 and compound 6 reveals that the increase in potency correlates with increased contacts between compound 6 and cereblon away from the modeled binding site for Ikaros/Aiolos. These results describe a new cereblon modulator which achieves greater substrate degradation via tighter binding to the cereblon E3 ligase and provides an example of the effect of E3 ligase binding affinity with relevance to other drug discovery efforts in targeted protein degradation.

The 26S proteasome is essential for proteostasis and the regulation of vital processes through ATP-dependent degradation of ubiquitinated substrates. To accomplish the multi-step degradation process, the proteasome’s regulatory particle, consisting of lid and base subcomplexes, undergoes major conformational changes whose origin is unknown. Investigating the Saccharomyces cerevisiae proteasome, we found that peripheral interactions between the lid subunit Rpn5 and the base AAA+ ATPase ring are important for stabilizing the substrate-engagement-competent state and coordinating the conformational switch to processing states upon substrate engagement. Disrupting these interactions perturbs the conformational equilibrium and interferes with degradation initiation, while later processing steps remain unaffected. Similar defects in early degradation steps are observed when eliminating hydrolysis in the ATPase subunit Rpt6, whose nucleotide state seems to control proteasome conformational transitions. These results provide important insight into interaction networks that coordinate conformational changes with various stages of degradation, and how modulators of conformational equilibria may influence substrate turnover.

Thalidomide-dependent degradation of the embryonic transcription factor SALL4 by the CRL4

The 26S proteasome is essential for protein homeostasis and the regulation of vital cellular processes through ATP-dependent degradation of ubiquitinated substrates. To accomplish the multi-step reaction of protein degradation, the proteasome’s regulatory particle, consisting of the lid and base subcomplexes, undergoes major conformational changes whose origin and control are largely unknown. Investigating the Saccharomyces cerevisiae 26S proteasome, we found that peripheral interactions between the lid subunit Rpn5 and the base AAA+ ATPase ring play critical roles in stabilizing the substrate-engagement-competent state and coordinating the conformational switch to processing states after a substrate has been engaged. Disrupting these interactions perturbs the conformational equilibrium and interferes with degradation initiation, while later steps of substrate processing remain unaffected. Similar defects in the early degradation steps are also observed when eliminating hydrolysis in the ATPase subunit Rpt6, whose nucleotide state seems to control conformational transitions of the proteasome. These results provide important insight into the network of interactions that coordinate conformational changes with various stages of proteasomal degradation, and how modulators of conformational equilibria may influence substrate turnover.

Immunomodulatory drugs bind to cereblon (CRBN) to confer differentiated substrate specificity on the CRL4(CRBN) E3 ubiquitin ligase. Here we report the identification of a new cereblon modulator, CC-885, with potent anti-tumour activity. The anti-tumour activity of CC-885 is mediated through the cereblon-dependent ubiquitination and degradation of the translation termination factor GSPT1. Patient-derived acute myeloid leukaemia tumour cells exhibit high sensitivity to CC-885, indicating the clinical potential of this mechanism. Crystallographic studies of the CRBN-DDB1-CC-885-GSPT1 complex reveal that GSPT1 binds to cereblon through a surface turn containing a glycine residue at a key position, interacting with both CC-885 and a 'hotspot' on the cereblon surface. Although GSPT1 possesses no obvious structural, sequence or functional homology to previously known cereblon substrates, mutational analysis and modelling indicate that the cereblon substrate Ikaros uses a similar structural feature to bind cereblon, suggesting a common motif for substrate recruitment. These findings define a structural degron underlying cereblon 'neosubstrate' selectivity, and identify an anti-tumour target rendered druggable by cereblon modulation.

The cereblon modulating agents (CMs) including lenalidomide, pomalidomide and CC-220 repurpose the Cul4-RBX1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase complex to induce the degradation of specific neomorphic substrates via polyubiquitination in conjunction with E2 ubiquitin-conjugating enzymes, which have until now remained elusive. Here we show that the ubiquitin-conjugating enzymes UBE2G1 and UBE2D3 cooperatively promote the K48-linked polyubiquitination of CRL4CRBN neomorphic substrates via a sequential ubiquitination mechanism. Blockade of UBE2G1 diminishes the ubiquitination and degradation of neomorphic substrates, and consequent antitumor activities elicited by all tested CMs. For example, UBE2G1 inactivation significantly attenuated the degradation of myeloma survival factors IKZF1 and IKZF3 induced by lenalidomide and pomalidomide, hence conferring drug resistance. UBE2G1-deficient myeloma cells, however, remained sensitive to a more potent IKZF1/3 degrader CC-220. Collectively, it will be of fundamental interest to explore if loss of UBE2G1 activity is linked to clinical resistance to drugs that hijack the CRL4CRBN to eliminate disease-driving proteins., eLife digest Cells routinely breakdown damaged or unwanted proteins to recycle their building blocks. In humans, most of these unwanted proteins are first tagged with a chain of smaller proteins called ubiquitin, in a process known as ubiquitination. Three kinds of enzymes – named E1, E2 and E3 – act one after the other to recruit and transfer ubiquitin onto the protein. Any problem with this protein-disposal system may cause diseases including cancers. Several drugs such as thalidomide are known to hijack the ubiquitination process by binding to the E3 enzyme. Instead of targeting unwanted proteins, the E3 enzyme-drug complex targets proteins that are driving a disease. These drugs are particularly useful for treating blood cancers. The problem is patients often become resistant to these drugs, and not always because the activity of the E3 enzyme is impaired. An alternative suspect would be an E2 enzyme, but the role of these enzymes remains unclear. Lu et al. have now asked whether a faulty E2 enzyme can lead to drug resistance in a form of blood cancer called multiple myeloma. The experiments tested how proteins relevant for the growth of cancerous myeloma cells were degraded in the presence of different drugs. Genes for the E2 enzymes were inactivated one at a time using a gene editing approach to see which ones would affect the degradation of the proteins and result in drug resistance. Two E2 enzymes, UBE2G1 and UBE2D3, were found to be critical. UBE2D3 first links the disease-driving proteins with one ubiquitin before UBE2G1 can subsequently assemble a chain of ubiquitin proteins. If either of these E2 enzymes was missing from myeloma cells treated with drugs, the disease-driving proteins could not be properly tagged with ubiquitin. This interfered with the degradation of the proteins and allowed the myeloma cells to continue to grow. Yet, myeloma cells that did not have UBE2G1 and were resistant to certain drugs could still respond to other more potent drugs. This suggests that the success of the drugs depends on UBE2G1. Therefore, a better understanding of the activity of this E2 enzyme may be useful for the development of future anticancer drugs.

SUMMARY Substrates are targeted for proteasomal degradation through the attachment of ubiquitin chains that need to be removed by proteasomal deubiquitinases prior to substrate processing. In budding yeast, the deubiquitinase Ubp6 trims ubiquitin chains and affects substrate processing by the proteasome, but the underlying mechanisms and its location within the holoenzyme remained elusive. Here we show that Ubp6 activity strongly responds to interactions with the base ATPase and the conformational state of the proteasome. Electron-microscopy analyses reveal that ubiquitin-bound Ubp6 contacts the N-ring and AAA+ ring of the ATPase hexamer, in close proximity to the deubiquitinase Rpn11. Ubiquitin-bound Ubp6 inhibits substrate deubiquitination by Rpn11, stabilizes the substrate-engaged conformation of the proteasome, and allosterically interferes with the engagement of a subsequent substrate. Ubp6 may thus act as an ubiquitin-dependent timer to coordinate individual processing steps at the proteasome and modulate substrate degradation.

Lenalidomide- and pomalidomide-based therapies are effective drugs in the treatment of patients with multiple myeloma (MM), however most patients with MM eventually relapse or become resistant. CC-92480, a novel cereblon (CRBN) E3 ligase modulator (CELMoD) with multiple activities including potent immunomodulation and single-agent antiproliferative effects, is being investigated in a phase 1 clinical trial (CC-92480-MM-001; NCT03374085) for patients with relapsed/refractory MM (RRMM). The present study investigates the preclinical data and mechanism of action of CC-92480 in MM models. CELMoD agents bound to CRBN confer differentiated substrate-degradation specificity on the CRL4CRBN E3 ubiquitin ligase. CRBN-modulator agents mediate destruction of Ikaros and Aiolos, transcription factors that contribute to myeloma cell survival. CC-92480 was found to produce rapid, deep, and sustained degradation of Ikaros and Aiolos, with superior antimyeloma activity. Accordingly, in a CRBN protein competitive binding assay, CC-92480 displaced a Cy-5-labeled CELMoD analog from CRBN with a 50% inhibitory concentration (IC50) value of 0.03 μM, whereas lenalidomide competed with an IC50 value of 1.27 μM in the same assay, demonstrating a higher binding affinity of CC-92480 for CRBN. Additionally, CC-92480 promoted the recruitment of Ikaros to the CRBN E3 ligase complex more effectively than pomalidomide in 2 orthogonal CRBN/Ikaros binding assays; it also triggered a more extensive cellular ubiquitination of Ikaros, and a faster, more efficient depletion of cellular Ikaros and Aiolos than pomalidomide. In various MM cell lines, including those with acquired resistance to lenalidomide or pomalidomide and low levels of CRBN, CC-92480 produced robust degradation of Ikaros and Aiolos followed by strong reduction of 2 additional and highly critical transcription factors, c-Myc and interferon regulatory factor 4, which are linked to the induction of apoptosis as measured by cleaved caspase-3. The tumoricidal activity of CC-92480 was shown to be CRBN dependent, since the effect was prevented by complete loss of CRBN or by the stabilization of Ikaros and Aiolos. CC-92480 displayed broad and potent antiproliferative activity across a panel of 20 MM cell lines that are either sensitive, have acquired resistance, or are refractory to lenalidomide or pomalidomide; the cell lines also contained diverse chromosomal translocations and oncogenic drivers typically found in MM patients. Approximately half of the MM cell lines evaluated were highly sensitive to CC-92480, with IC50 values for antiproliferative activity ranging from 0.04 to 5 nM; only 2 cell lines had IC50 values > 100 nM. CC-92480 inhibits cell proliferation and induces apoptosis in MM cell lines that are not sensitive to lenalidomide or pomalidomide. This panel of cell lines includes both refractory cell lines and resistant cell lines generated through continuous exposure to lenalidomide and pomalidomide that acquired low levels of CRBN protein or mutations in the CRBN gene. CC-92480 also induced deep destruction of Ikaros and Aiolos in cultures of peripheral blood mononuclear cells (PBMCs), which led to the activation of T cells and increased production of the cytokines interleukin-2 and interferon gamma. These responses occurred at the range of CC-92480 concentrations that show potent tumoricidal effect against MM cells. The T cell activation and enhanced cytokine production by CC-92480 led to the potent and effective immune-mediated killing of MM cells in co-cultures with PBMCs. CC-92480 is a potent antiproliferative and proapoptotic novel CELMoD with enhanced autonomous cell-killing activity in MM cells that are either sensitive, resistant, or have acquired resistance to lenalidomide and pomalidomide. CC-92480 has a unique and rapid degradation profile stemming from the enhanced efficiency to drive the formation of a protein-protein interaction between Ikaros and Aiolos and CRBN, inducing cytotoxic effects in a CRL4CRBN-dependent fashion that leads ultimately to the induction of apoptosis, even in the context of low or mutated CRBN protein. Additionally, similar to lenalidomide, CC-92480 conserves immunomodulatory activity against MM cells. These data support the clinical investigation of CC-92480 in patients with RRMM. Disclosures Lopez-Girona: Celgene Corporation: Employment. Havens:Pfizer: Employment, Equity Ownership; Celgene: Equity Ownership. Lu:Celgene Corporation: Employment, Equity Ownership. Rychak:Celgene Corporation: Employment, Equity Ownership. Mendy:Celgene Corporation: Employment. Gaffney:Celgene: Employment. Surka:Celgene: Employment, Equity Ownership. Lu:Celgene Corporation: Employment, Equity Ownership. Matyskiela:Celgene corporation: Employment. Khambatta:Celgene: Employment. Wong:Celgene Corporation: Employment, Equity Ownership. Hansen:Celgene Corporation: Employment. Pierce:Celgene Corporation: Employment, Equity Ownership. Cathers:Global Blood Therapeutics (GBT): Employment; Celgene Corporation: Equity Ownership. Carmichael:Celgene plc: Employment, Equity Ownership.

The 26S proteasome is the major eukaryotic ATP-dependent protease, responsible for regulating the proteome through degradation of ubiquitin-tagged substrates. Its regulatory particle, containing the heterohexameric AAA+ ATPase motor and the essential deubiquitinase Rpn11, recognizes substrates, removes their ubiquitin chains, and translocates them into the associated peptidase after unfolding, but detailed mechanisms remain unknown. Here we present the first structure of the 26S proteasome from S. cerevisiae during substrate degradation, showing that the regulatory particle switches from a pre-engaged to a translocation-competent conformation. This conformation is characterized by a rearranged ATPase ring with uniform subunit interfaces, a widened central channel coaxially aligned with the peptidase, and a spiral orientation of pore loops that suggests a rapid progression of ATP-hydrolysis events around the ring. Importantly, Rpn11 moves from an occluded position to directly above the central pore, facilitating substrate deubiquitination concomitant with translocation.

The proteasome is the major ATP-dependent protease in eukaryotic cells, but limited structural information restricts a mechanistic understanding of its activities. The proteasome regulatory particle, consisting of the lid and base subcomplexes, recognizes and processes polyubiquitinated substrates. Here we used electron microscopy and a new heterologous expression system for the lid to delineate the complete subunit architecture of the regulatory particle from yeast. Our studies reveal the spatial arrangement of ubiquitin receptors, deubiquitinating enzymes and the protein unfolding machinery at subnanometre resolution, outlining the substrate’s path to degradation. Unexpectedly, the ATPase subunits within the base unfoldase are arranged in a spiral staircase, providing insight into potential mechanisms for substrate translocation through the central pore. Large conformational rearrangements of the lid upon holoenzyme formation suggest allosteric regulation of deubiquitination. We provide a structural basis for the ability of the proteasome to degrade a diverse set of substrates and thus regulate vital cellular processes. Determination of the proteasome regulatory particle structure by electron microscopy. In eukaryotes, the ubiquitin–proteasome system is the major pathway for selective protein degradation. The 19S regulatory particle of the proteasome consists of lid and base subcomplexes, and functions in recognition, deubiquitination, unfolding and translocation of substrates into the 20S core particle for proteolysis. The complete subunit architecture of the regulatory particle has now been determined by electron microscopy. The structure provides insights into the potential mechanisms for substrate unfolding and translocation by ATP-dependent proteases into the proteolytic core.

Cik1, in association with the kinesin Kar3, controls both the mitotic spindle and nuclear fusion during mating. Here, we show that there are two Cik1 isoforms, and that the mitotic form includes an N-terminal domain required for ubiquitination by the Anaphase-Promoting Complex/Cyclosome (APC/C). During vegetative growth, Cik1 is expressed during mitosis and regulates the mitotic spindle, allowing for accurate chromosome segregation. After mitosis, APC/C(Cdh1) targets Cik1 for ubiquitin-mediated proteolysis. Upon exposure to the mating pheromone alpha factor, a smaller APC/C-resistant Cik1 isoform is expressed from an alternate transcriptional start site. This shorter Cik1 isoform is stable and cannot be ubiquitinated by APC/C(Cdh1). Moreover, the two Cik1 isoforms are functionally distinct. Cells that express only the long isoform have defects in nuclear fusion, whereas cells expressing only the short isoform have an increased rate of chromosome loss. These results demonstrate a coupling of transcriptional regulation and APC/C-mediated proteolysis.

The protein cereblon is part of the CRL4-CRBN E3 ubiquitin ligase complex, and has been shown to be the molecular target for the drugs lenalidomide and pomalidomide. These drugs bind to the surface of cereblon, triggering the recruitment of substrate proteins to the ligase complex where they can be ubiquitinated and subsequently degraded by the proteosome. By this mechanism, lenalidomide and pomalidomide cause the degradation of the zinc finger transcription factors Ikaros and Aiolos, which mediate the antimyeloma activity of these compounds. Here we describe the discovery of CC-885, a novel cereblon modulator with potent and broad-spectrum antiproliferative activity against a panel of tumor cell lines. Acute myeloid leukemia (AML) cell lines and patient-derived AML cells show particular sensitivity to CC-885 treatment. CC-885 achieves this activity by causing the degradation of G1 to S phase transition 1 (GSPT1), a translation termination factor required for the release of nascent peptides from the ribosome. A crystal structure of cereblon in complex with the ligase adapter protein DDB1, as well as CC-885 and GSPT1, reveals that GSPT1 interacts with both CC-885 and the surface of cereblon. The principal molecular feature on GSPT1 that binds to cereblon is a beta-hairpin incorporating a glycine residue that docks against CC-885. Surprisingly, we found evidence that a similar molecular feature mediates Ikaros recruitment, even though there is no common structural fold or sequence homology other than the key glycine residue. We thereby define the common molecular feature, or degron, shared by the known cereblon neomorphic substrates. We further describe a novel therapeutic target, GSPT1, with promise in cancer such as AML. These results further show that cereblon-mediated protein degradation can be directed against new proteins and that this mechanism enables the targeting of multiple protein classes that may be considered undruggable with conventional approaches. Citation Format: Mary Matyskiela, Gang Lu, Takumi Ito, Barbra Pagarigan, Chin-Chu Lu, Karen Miller, Wei Fang, Nai-Yu Wang, Derek Nguyen, Jack Houston, Gilles Carmel, Tam Tran, Mariko Riley, Lyn'Al Nosaka, Gabriel Lander, Svetlana Gaidarova, Shuichan Xu, Alexander Ruchelman, Hiroshi Handa, James Carmichale, Thomas O. Daniel, Brian E. Cathers, Antonia Lopez-Girona, Philip Chamberlain. Ligand-directed degradation of GSPT1 by a novel cereblon modulator drives potent antitumor effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr SY37-02. doi:10.1158/1538-7445.AM2017-SY37-02

Energy-dependent proteases of the AAA+ family catalyze the highly specific protein degradation involved in cellular protein quality control and the regulation of numerous vital processes, yet the detailed mechanisms coupling ATP hydrolysis with mechanical substrate unfolding and translocation remain poorly understood.Our cryo-EM structural studies of the eukaryotic 26S proteasome show that its heterohexameric AAA+ ATPase ring adopts a conformation with pronounced spiral-staircase arrangement of subunits in the absence of substrate, but transitions into a translocation-competent conformation upon substrate engagement. This substrate-engaged ring conformation is characterized by uniform interfaces between the six ATPase subunit, a widened central channel coaxially aligned with the peptidase, and a rearranged, more planar spiral orientation of ATPase subunits that suggests a highly coordinated rapid progression of ATP-hydrolysis events around the ring.This coordinated ATP hydrolysis mechanism is further supported by our optical tweezers single-molecule studies of the related bacterial protease ClpXP and may be a general feature of AAA+ translocases. ClpXP translocates substrate polypeptides in steps with constant frequency but variable length, depending on the number of ATP-hydrolyzing subunits.

The 26S proteasome is a 2.5 MDa, 32-subunit ATP-dependent protease that is responsible for the degradation of ubiquitinated protein targets in all eukaryotic cells. This proteolytic machine consists of a barrel-shaped peptidase capped by a large regulatory particle, which contains a heterohexameric AAA+ unfoldase as well as several structural modules of previously unknown function. Recent electron microscopy studies have allowed major breakthroughs in understanding the architecture of the regulatory particle, revealing that the additional modules provide a structural framework to position critical, ubiquitin-interacting subunits and thus allow the 26S proteasome to function as a universal degradation machine for a wide variety of protein substrates. The electron microscopy studies have also uncovered surprising asymmetries in the spatial arrangement of proteasome subunits, yet the functional significance of these architectural features remains unclear. This review will summarize the recent findings on 26S proteasome structure and discuss the mechanistic implications for substrate binding, deubiquitination, unfolding, and degradation.

The anaphase-promoting complex (APC) is a ubiquitin-protein ligase required for the completion of mitosis in all eukaryotes. Recent mechanistic studies reveal how this remarkable enzyme combines specificity in substrate binding with flexibility in ubiquitin transfer, thereby allowing the modification of multiple lysines on the substrate as well as specific lysines on ubiquitin itself.

The anaphase-promoting complex or cyclosome (APC/C) is a ubiquitin ligase essential for the completion of mitosis in all eukaryotic cells. Substrates are recruited to the APC/C by activator proteins (Cdc20 or Cdh1), but it is not known where substrates are bound during catalysis. We explored this problem by analyzing mutations in the tetratricopeptide repeat (TPR)-containing APC/C subunits. We identified residues in Cdc23 and Cdc27 that are required for APC/C binding to Cdc20 and Cdh1 and for APC/C function in vivo. Mutation of these sites increased the rate of activator dissociation from the APC/C but did not affect reaction processivity, suggesting that the mutations have little effect on substrate dissociation from the active site. Further studies revealed that activator dissociation from the APC/C is inhibited by substrate, and that substrates are not bound solely to activator during catalysis but interact bivalently with an additional binding site on the APC/C core.

The antiangiogenic agent fumagillin (Fg) and its analog TNP-470 bind to intracellular metalloprotease methionine aminopeptidase-2 (MetAP-2) and inhibit endothelial cell growth in a p53-dependent manner. To confirm the role of MetAP-2 in endothelial cell proliferation and to validate it as a physiological target for the Fg class of antiangiogenic agents, we have generated a conditional MetAP-2 knockout mouse. Ubiquitous deletion of the MetAP-2 gene ( MAP2 ) resulted in an early gastrulation defect, which is bypassed in double MetAP-2/p53 knockout embryos. Targeted deletion of MAP2 specifically in the hemangioblast lineage resulted in abnormal vascular development, and these embryos die at the midsomite stage. In addition, knockdown of MetAP-2 using small interfering RNA or homologous recombination specifically suppresses the proliferation of cultured endothelial cells. Together, these results demonstrate an essential role for MetAP-2 in angiogenesis and indicate that MetAP-2 is responsible for the endothelial cell growth arrest induced by Fg and its derivatives.

The anaphase-promoting complex or cyclosome (APC) is an unusually complicated ubiquitin ligase, composed of 13 core subunits and either of two loosely associated regulatory subunits, Cdc20 and Cdh1. We analyzed the architecture of the APC using a recently constructed budding yeast strain that is viable in the absence of normally essential APC subunits. We found that the largest subunit, Apc1, serves as a scaffold that associates independently with two separable subcomplexes, one that contains Apc2 (Cullin), Apc11 (RING), and Doc1/Apc10, and another that contains the three TPR subunits (Cdc27, Cdc16, and Cdc23). We found that the three TPR subunits display a sequential binding dependency, with Cdc27 the most peripheral, Cdc23 the most internal, and Cdc16 between. Apc4, Apc5, Cdc23, and Apc1 associate interdependently, such that loss of any one subunit greatly reduces binding between the remaining three. Intriguingly, the cullin and TPR subunits both contribute to the binding of Cdh1 to the APC. Enzymatic assays performed with APC purified from strains lacking each of the essential subunits revealed that only cdc27Δ complexes retain detectable activity in the presence of Cdh1. This residual activity depends on the C-box domain of Cdh1, but not on the C-terminal IR domain, suggesting that the C-box mediates a productive interaction with an APC subunit other than Cdc27. We have also found that the IR domain of Cdc20 is dispensable for viability, suggesting that Cdc20 can activate the APC through another domain. We have provided an updated model for the subunit architecture of the APC.

In eukaryotic cells, the proteasome degrades unwanted proteins by recognizing specific polyubiquitin tags covalently attached to these proteins. The precise manner in which these ubiquitin chains are recognized and removed from the targeted proteins prior to proteolysis is poorly understood. This is partly due to a lack of structural information on the ubiquitin-recognizing components of the proteasome 19S regulatory particle. Using a recombinant expression system and electron microscopy, we were able to localize all subunits of the yeast 19S particle, and elucidate the spatial arrangement of ubiquitin receptors, deubiquitinating enzymes, and the protein unfolding machinery. Our studies also revealed large conformational rearrangements in the lid subcomplex upon holoenzyme formation, suggesting an allosteric mechanism for activation of its deubiquitination activity. From these studies, we have a much better understanding of the manner in which the 26S recognizes and deubiquitinates proteins marked for proteolysis.