Cullin-RING E3 Ligases Research Articles

Identification of a PGXPP degron motif in dishevelled and structural basis for its binding to the E3 ligase KLHL12.

Wnt signalling is dependent on dishevelled proteins (DVL1-3), which assemble an intracellular Wnt signalosome at the plasma membrane. The levels of DVL1-3 are regulated by multiple Cullin-RING E3 ligases that mediate their ubiquitination and degradation. The BTB-Kelch protein KLHL12 was the first E3 ubiquitin ligase to be identified for DVL1-3, but the molecular mechanisms determining its substrate interactions have remained unknown. Here, we mapped the interaction of DVL1-3 to a ‘PGXPP' motif that is conserved in other known partners and substrates of KLHL12, including PLEKHA4, PEF1, SEC31 and DRD4. To determine the binding mechanism, we solved a 2.4 Å crystal structure of the Kelch domain of KLHL12 in complex with a DVL1 peptide that bound with low micromolar affinity. The DVL1 substrate adopted a U-shaped turn conformation that enabled hydrophobic interactions with all six blades of the Kelch domain β-propeller. In cells, the mutation or deletion of this motif reduced the binding and ubiquitination of DVL1 and increased its stability confirming this sequence as a degron motif for KLHL12 recruitment. These results define the molecular mechanisms determining DVL regulation by KLHL12 and establish the KLHL12 Kelch domain as a new protein interaction module for a novel proline-rich motif.

Targeting Cullin-RING E3 ligases for anti-cancer therapy: efforts on drug discovery

Cullin-RING E3 ligases (CRLs) are the major components of ubiquitin-proteasome system, responsible for ubiquitylation and subsequent degradation of thousands of cellular proteins. CRLs play vital roles in the regulation of multiple cellular processes, including cell cycle, cell apoptosis, DNA replication, signalling transduction among the others, and are frequently dysregulated in many human cancers. The discovery of specific neddylation inhibitors, represented by MLN4924, has validated CRLs as promising targets for anti-cancer therapies with a growing market. Recent studies have focused on the discovery of the CRLs inhibitors by a variety of approaches, including high through-put screen, virtual screen or structure-based drug design. The field is, however, still facing the major challenging, since CRLs are a large multi-unit protein family without typical active pockets to facilitate the drug design, and enzymatic activity is mainly dependent on undruggable protein-protein interactions and dynamic conformation changes. Up to now, most reported CRLs inhibitors are aiming at targeting the F-box family proteins (e.g., SKP2, β-TrCP and FBXW7), the substrate recognition subunit of SCF E3 ligases. Other studies reported few small molecule inhibitors targeting the UBE2M-DCN1 interaction, which specifically inhibits CRL3/CRL1 by blocking the cullin neddylation. On the other hand, several CRL activators have been reported, such as plant auxin and immunomodulatory imide drugs, thalidomide. Finally, proteolysis-targeting chimeras (PROTACs) has emerged as a new technology in the field of drug discovery, specifically targeting the undruggable protein-protein interaction. The technique connects the small molecule that selectively binds to a target protein to a CRL E3 via a chemical linker to trigger the degradation of target protein. The PROTAC has become a hotspot in the field of E3-ligase-based anti-cancer drug discovery.

Molecular Mechanisms of the Teratogenic Effects of Thalidomide.

Thalidomide was sold worldwide as a sedative over 60 years ago, but it was quickly withdrawn from the market due to its teratogenic effects. Thalidomide was later found to have therapeutic effects in several diseases, although the molecular mechanisms remained unclear. The discovery of cereblon (CRBN), the direct target of thalidomide, a decade ago greatly improved our understanding of its mechanism of action. Accumulating evidence has shown that CRBN functions as a substrate of Cullin RING E3 ligase (CRL4CRBN), whose specificity is controlled by ligands such as thalidomide. For example, lenalidomide and pomalidomide, well-known thalidomide derivatives, degrade the neosubstrates Ikaros and Aiolos, resulting in anti-proliferative effects in multiple myeloma. Recently, novel CRBN-binding drugs have been developed. However, for the safe handling of thalidomide and its derivatives, a greater understanding of the mechanisms of its adverse effects is required. The teratogenic effects of thalidomide occur in multiple tissues in the developing fetus and vary in phenotype, making it difficult to clarify this issue. Recently, several CRBN neosubstrates (e.g., SALL4 (Spalt Like Transcription Factor 4) and p63 (Tumor Protein P63)) have been identified as candidate mediators of thalidomide teratogenicity. In this review, we describe the current understanding of molecular mechanisms of thalidomide, particularly in the context of its teratogenicity.

Gossypol inhibits cullin neddylation by targeting SAG-CUL5 and RBX1-CUL1 complexes

Cullin-RING E3 ligase (CRL) is the largest family of E3 ubiquitin ligase, responsible for ubiquitylation of ∼20% of cellular proteins. CRL plays an important role in many biological processes, particularly in cancers due to abnormal activation. CRL activation requires neddylation, an enzymatic cascade transferring small ubiquitin-like protein NEDD8 to a conserved lysine residue on cullin proteins. Recent studies have validated that neddylation is an attractive anticancer target. In this study, we report the establishment of an Alpha-Screen-based high throughput screen (HTS) assay for in vitro CUL5 neddylation, and screened a library of 17,000 compounds including FDA approved drugs, natural products and synthetic drug-like small-molecule compounds. Gossypol, a natural compound derived from cotton seed, was identified as an inhibitor of cullin neddylation. Biochemical studies showed that gossypol blocked neddylation of both CUL5 and CUL1 through direct binding to SAG-CUL5 or RBX1-CUL1 complex, and CUL5-H572 plays a key role for gossypol binding. On cellular level, gossypol inhibited cullin neddylation in a variety of cancer cell lines and selectively caused accumulation of NOXA and MCL1, the substrates of CUL5 and CUL1, respectively, in multiple cancer cell lines. Combination of gossypol with specific MCL1 inhibitor synergistically suppress growth of human cancer cells. Our study revealed a previously unknown anti-cancer mechanism of gossypol with potential to develop a new class of neddylation inhibitors.

The Negative Cross-Talk between SAG/RBX2/ROC2 and APC/C E3 Ligases in Regulation of Cell Cycle Progression and Drug Resistance

APC/C is a well characterized E3 ligase that couples with UBE2C and UBE2S E2s for ubiquitylation of the targeted substrates via the K11 linkage. Our recent data showed that SAG/RBX2/ROC2, a RING component of Cullin-RING E3 ligase (CRL), also complexes with these two E2s (UBE2C/2S) for K11-linked substrate polyubiquitylation. Whether these two E3s cross-talk with each other and the associated biological consequence are previously unknown. Here we report that these two E3s inhibit each other during cell cycle progression, which could lead to drug resistance. Specifically, SAG competes with APC2 for UBE2C/UBE2S binding to act as an endogenous inhibitor of APC/C, thereby regulating the G2/M progression. SAG inactivation triggers premature activation of APC/C, leading to mitotic slippage, as well as the resistance of cancer cells to anti-microtubule drugs both in vitro and in vivo models. On the other hand, SAG itself is a substrate of APC/CCDH1 for targeted degradation at the G1 phase. The degradation-resistant mutant of SAG-R82A/L85A accelerates the G1 to S progression, thereby promoting proliferation/survival of lung cancer cells. Our study reveals that the negative crosstalk between SAG and APC/C is likely a mechanism to ensure the fidelity of cell cycle progression, and manipulations to abrogate this fine balance would lead to enhanced proliferation and drug resistance.

NEDD8nucleates a multivalent cullin-RING-UBE2D ubiquitin ligation assembly.

Virtually all eukaryotic processes are regulated by cullin-RING E3 ligase (CRL)-catalyzed protein ubiquitylation1, which is exquisitely controlled by cullin modification with the ubiquitin (UB)-like protein NEDD82–6. However, how CRLs catalyze ubiquitylation, and the basis for NEDD8 activation, remain unknown. We report the cryo EM structure of a chemically-trapped complex representing the ubiquitylation intermediate whereby neddylated CRL1β-TRCP promotes UB transfer from the E2 UBE2D to its recruited substrate phosphorylated IκBα. The structure shows that NEDD8 acts as a nexus binding disparate cullin elements and the RING-activated UB-linked UBE2D. Concomitant local structural remodeling and large-scale CRL domain movements converge to juxtapose the substrate and ubiquitylation active site. The results explain how a distinctive UB-like protein alters the functions of its targets, and show how numerous NEDD8-dependent interprotein interactions and conformational changes synergistically configure a catalytic CRL architecture that is both robust for rapid substrate ubiquitylation and fragile to enable ensuing cullin-RING functions.

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.

SlCAND1, encoding cullin-associated Nedd8-dissociated protein 1, regulates plant height, flowering time, seed germination, and root architecture in tomato

Silencing of SlCAND1 expression resulted in dwarfish, loss of apical dominance, early flowering, suppression of seed germination, and abnormal root architecture in tomato Cullin-RING E3 ligases (CRLs)-dependent ubiquitin proteasome system mediates degradation of numerous proteins that controls a wide range of developmental and physiological processes in eukaryotes. Cullin-associated Nedd8-dissociated protein 1 (CAND1) acts as an exchange factor allowing substrate recognition part exchange and plays a vital role in reactivating CRLs. The present study reports on the identification of SlCAND1, the only one CAND gene in tomato. SlCAND1 expression is ubiquitous and positively regulated by multiple plant hormones. Silencing of SlCAND1 expression using RNAi strategy resulted in a pleiotropic and gibberellin/auxin-associated phenotypes, including dwarf plant with reduced internode length, loss of apical dominance, early flowering, low seed germination percentage, delayed seed germination speed, short primary root, and increased lateral root proliferation and elongation. Moreover, application of exogenous GA3 or IAA could partly rescue some SlCAND1-silenced phenotypes, and the expression levels of gibberellin/auxin-related genes were altered in SlCAND1-RNAi lines. These facts revealed that SlCAND1 is required for gibberellin/auxin-associated regulatory network in tomato. Although SlCAND1 is crucial for multiple developmental processes during vegetative growth stage, SlCAND1-RNAi lines didn't exhibit visible effect on fruit development and ripening. Meanwhile, we discussed that multiple physiological functions of SlCAND1 in tomato are different to previous report of its ortholog in Arabidopsis. Our study adds a new perspective on the functional roles of CAND1 in plants, and strongly supports the hypothesis that CAND1 and its regulated ubiquitin proteasome system are pivotal for plant vegetative growth but possibly have different roles in diverse plant species.

Cullin-RING Ligase Regulation by the COP9 Signalosome: Structural Mechanisms and New Physiologic Players.

The Cullin-RING E3 ligases (CRLs) are major ubiquitylation machineries regulated by reversible cycles of neddylation and deneddylation. The deneddylase COP9 Signalosome (CSN) terminates CRL catalytic cycle. CSN also provides a docking platform for several kinases and deubiquitinases that might play a role in regulating CRL. Recently, remarkable progress has been made in elucidating the biochemical principles and physiological implications of such exquisite regulation. The cryo-EM structures of CRL-CSN complexes provide the biochemical basis of their cognate interactions and reveal potential regulatory mechanisms during complex disassembly. Moreover, novel players beyond the canonical eight subunits of CSN were identified. This includes CSNAP, a potential 9th CSN subunit with regulatory functions, and the metabolite inositol hexakisphosphate (IP6), which enhances CRL-CSN complex formation, with IP6-metabolizing enzymes possibly instilling dynamics to the CRL-CSN system. Here, we review and summarize these new mechanistic insights along with progress in understanding CSN biology based on model organisms with genetically edited CSN subunits.

Small molecule NSC1892 targets the CUL4A/4B-DDB1 interactions and causes impairment of CRL4DCAF4 E3 ligases to inhibit colorectal cancer cell growth.

Cullin 4A and 4B (CUL4A and 4B) function as oncogenes in colorectal cancer (CRC) cells. Both of them conservatively associate with DNA damage-binding protein 1 (DDB1) and DDB1-CUL4-associated factor 4 (DCAF4) to form Cullin-RING E3 ligases known as CRL4DCAF4, which specifically ubiquitinate and degrade tumor suppressor ST7 (suppression of tumorigenicity 7). Knockdown either CUL4A/4B or DDB1 significantly inhibits tumor cell growth in vitro and in vivo. Thus, targeting these CRL4DCAF4 components and their interactions may be an effective strategy for the therapy of CRC. In this study, we developed an in vitro AlphaScreen assay to identify small molecules targeting the CUL4A-DDB1 interaction. We obtained a compound NSC1892, which strongly disrupted the CUL4A-DDB1 interaction (IC50 = 1.8 μM). Oncogenic phenotype analyses indicated that NSC1892 showed significant cytotoxicity to decrease cell proliferation, colony formation and invasion in CRC cells. Biochemical analyses demonstrated that NSC1892 treatment did not change CUL4A and CUL4B protein levels, but caused the degradation of DDB1, thereby leading to the impaired assembly of CRL4DCAF4 E3 ligases and resulting in the accumulation of ST7. The administration of NSC1892 in mice also significantly inhibited tumor growth through degrading DDB1 and accumulating ST7. Interestingly, NSC1892 also showed promising cytotoxicity to decrease the growth of other CUL4A- or CUL4B-overexpressing tumor cells such as SKOV3 ovarian cells and Saos2 osteosarcoma cells. Our results provide a new avenue for the development of a therapeutic compound targeting tumors through disrupting the CUL4-DDB1 interaction.

Activity of TAS4464, a novel NEDD8 activating enzyme E1 inhibitor, against multiple myeloma via inactivation of nuclear factor κB pathways.

The ubiquitin proteasome pathway is essential for the proliferation and survival of multiple myeloma (MM) cells. TAS4464, a novel highly potent inhibitor of NEDD8 activating enzyme, selectively inactivates cullin‐RING ubiquitin E3 ligases, resulting in accumulation of their substrates. Here, we examined 14 MM cell lines treated with TAS4464. TAS4464 induced growth arrest and cell death in the MM cell lines even in the presence of bone marrow stromal cells. It also induced the accumulation of phospho‐inhibitor of κBα and phospho‐p100, impaired the activities of nuclear factor κB (NF‐κB) transcription factors p65 and RelB, and decreased the expression of NF‐κB target genes, suggesting that TAS4464 inhibits both the canonical and non–canonical NF‐κB pathways. TAS4464 had similar effects in an in vivo human‐MM xenograft mouse model in which it was also observed to have strong antitumor effects. TAS4464 synergistically enhanced the antitumor activities of the standard MM chemotherapies bortezomib, lenalidomide/dexamethasone, daratumumab and elotuzumab. Together, these results suggest that the anti–MM activity of TAS4464 occurs via inhibition of the NF‐κB pathways, and that treatment with TAS4464 is a potential approach for treating MM by single and combination therapies.

Dynamic ubiquitination determines transcriptional activity of the plant immune coactivator NPR1.

Activation of systemic acquired resistance in plants is associated with transcriptome reprogramming induced by the unstable coactivator NPR1. Immune-induced ubiquitination and proteasomal degradation of NPR1 are thought to facilitate continuous delivery of active NPR1 to target promoters, thereby maximising gene expression. Because of this potentially costly sacrificial process, we investigated if ubiquitination of NPR1 plays transcriptional roles prior to its proteasomal turnover. Here we show ubiquitination of NPR1 is a progressive event in which initial modification by a Cullin-RING E3 ligase promotes its chromatin association and expression of target genes. Only when polyubiquitination of NPR1 is enhanced by the E4 ligase, UBE4, it is targeted for proteasomal degradation. Conversely, ubiquitin ligase activities are opposed by UBP6/7, two proteasome-associated deubiquitinases that enhance NPR1 longevity. Thus, immune-induced transcriptome reprogramming requires sequential actions of E3 and E4 ligases balanced by opposing deubiquitinases that fine-tune activity of NPR1 without strict requirement for its sacrificial turnover.

The Proteasome Lid Triggers COP9 Signalosome Activity during the Transition of Saccharomyces cerevisiae Cells into Quiescence.

The class of Cullin–RING E3 ligases (CRLs) selectively ubiquitinate a large portion of proteins targeted for proteolysis by the 26S proteasome. Before degradation, ubiquitin molecules are removed from their conjugated proteins by deubiquitinating enzymes, a handful of which are associated with the proteasome. The CRL activity is triggered by modification of the Cullin subunit with the ubiquitin-like protein, NEDD8 (also known as Rub1 in Saccharomyces cerevisiae). Cullin modification is then reversed by hydrolytic action of the COP9 signalosome (CSN). As the NEDD8–Rub1 catalytic cycle is not essential for the viability of S. cerevisiae, this organism is a useful model system to study the alteration of Rub1–CRL conjugation patterns. In this study, we describe two distinct mutants of Rpn11, a proteasome-associated deubiquitinating enzyme, both of which exhibit a biochemical phenotype characterized by high accumulation of Rub1-modified Cdc53–Cullin1 (yCul1) upon entry into quiescence in S. cerevisiae. Further characterization revealed proteasome 19S-lid-associated deubiquitination activity that authorizes the hydrolysis of Rub1 from yCul1 by the CSN complex. Thus, our results suggest a negative feedback mechanism via proteasome capacity on upstream ubiquitinating enzymes.

Structural basis of Cullin 2 RING E3 ligase regulation by the COP9 signalosome

Cullin-Ring E3 Ligases (CRLs) regulate a multitude of cellular pathways through specific substrate receptors. The COP9 signalosome (CSN) deactivates CRLs by removing NEDD8 from activated Cullins. Here we present structures of the neddylated and deneddylated CSN-CRL2 complexes by combining single-particle cryo-electron microscopy (cryo-EM) with chemical cross-linking mass spectrometry (XL-MS). These structures suggest a conserved mechanism of CSN activation, consisting of conformational clamping of the CRL2 substrate by CSN2/CSN4, release of the catalytic CSN5/CSN6 heterodimer and finally activation of the CSN5 deneddylation machinery. Using hydrogen-deuterium exchange (HDX)-MS we show that CRL2 activates CSN5/CSN6 in a neddylation-independent manner. The presence of NEDD8 is required to activate the CSN5 active site. Overall, by synergising cryo-EM with MS, we identify sensory regions of the CSN that mediate its stepwise activation and provide a framework for understanding the regulatory mechanism of other Cullin family members.

CSNAP, the smallest CSN subunit, modulates proteostasis through cullin-RING ubiquitin ligases.

The cullin-RING ubiquitin E3 ligase (CRL) family consists of ~250 complexes that catalyze ubiquitylation of proteins to achieve cellular regulation. All CRLs are inhibited by the COP9 signalosome complex (CSN) through both enzymatic (deneddylation) and nonenzymatic (steric) mechanisms. The relative contribution of these two mechanisms is unclear. Here, we decouple the mechanisms using CSNAP, the recently discovered ninth subunit of the CSN. We find that CSNAP reduces the affinity of CSN toward CRL complexes. Removing CSNAP does not affect deneddylation, but leads to global effects on the CRL, causing altered reproductive capacity, suppressed DNA damage response, and delayed cell cycle progression. Thus, although CSNAP is only 2% of the CSN mass, it plays a critical role in the steric regulation of CRLs by the CSN.

Structural Basis for Recruitment of DAPK1 to the KLHL20 E3 Ligase

SummaryBTB-Kelch proteins form the largest subfamily of Cullin-RING E3 ligases, yet their substrate complexes are mapped and structurally characterized only for KEAP1 and KLHL3. KLHL20 is a related CUL3-dependent ubiquitin ligase linked to autophagy, cancer, and Alzheimer's disease that promotes the ubiquitination and degradation of substrates including DAPK1, PML, and ULK1. We identified an “LPDLV”-containing motif in the DAPK1 death domain that determines its recruitment and degradation by KLHL20. A 1.1-Å crystal structure of a KLHL20 Kelch domain-DAPK1 peptide complex reveals DAPK1 binding as a loose helical turn that inserts deeply into the central pocket of the Kelch domain to contact all six blades of the β propeller. Here, KLHL20 forms salt-bridge and hydrophobic interactions including tryptophan and cysteine residues ideally positioned for covalent inhibitor development. The structure highlights the diverse binding modes of β-propeller domains versus linear grooves and suggests a new target for structure-based drug design.

Abstract 5083: BSCHL-2046, a lead chalcone derivative from a newly synthesized library, is a potent neddylation inhibitor against the growth of prostate cancer cell lines

Abstract Members of the chalcone family of molecules and their derivatives have been shown to inhibit various forms of cancer cells. In particular, Flavokawain A has been shown to inhibit prostate cancer cell lines. In order to capitalize on the bioactive nature of chalcone derivatives, we created a library of chalcone derivatives based on the scaffold of Flavokawain A but with an additional anti-cancer moiety. Here we have demonstrated that BSCHL-2046, the most potent member of the library, inhibits cell growth of prostate cancer cell lines including LNCaP, C4-2B, DU145, 22Rv1, and PC3 with IC50’s ranging from 0.44µM-7.5µM. BSCHL-2046 inhibited Ubc12 neddylation in an in vitro assay at an IC50 of 1.5µM and demonstrated complete inhibition at a concentration of 6µM. BSCHL-2046 also reduced Nedd-8 conjugations to both Cullin-1 and Ubc12 in vivo in DU145 cells. The inhibition of neddylation by BSCHL-2046 resulted in an increased expression of p27 and CDT-1. Using molecular modeling, BSCHL-2046 was predicted to bind directly to the Cullin-RING E3 ligase complex. The Cellular Thermal Shift Assay (CETSA, an in vivo assay) demonstrated that BSCHL-2046 significantly decreased heat-induced protein degradation of E3 subunits, supporting BSCHL-2046 directly binding to the complex. Furthermore, BSCHL-2046 at a concentration of 500nM and 1.25µM displayed a 75% to complete inhibition of colony formation of C4-2B cells in soft agar. Taken together, these results demonstrate BSCHL-2046 and its structural analogs are potent neddylation inhibitors that deserve further investigation for the prevention and treatment of prostate cancer. Note: This abstract was not presented at the meeting. Citation Format: Matthew A. Tippin, Dong Jun Fu, Liankun Song, Victor Pham, Xaolin Zi. BSCHL-2046, a lead chalcone derivative from a newly synthesized library, is a potent neddylation inhibitor against the growth of prostate cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5083.

ARIH2 Is a Vif-Dependent Regulator of CUL5-Mediated APOBEC3G Degradation in HIV Infection

The Cullin-RING E3 ligase (CRL) family is commonly hijacked by pathogens to redirect the host ubiquitin proteasome machinery to specific targets. During HIV infection, CRL5 is hijacked by HIV Vif to target viral restriction factors of the APOBEC3 family for ubiquitination and degradation. Here, using a quantitative proteomics approach, we identify the E3 ligase ARIH2 as a regulator of CRL5-mediated APOBEC3 degradation. The CUL5Vif/CBFß complex recruits ARIH2 where it acts to transfer ubiquitin directly to the APOBEC3 targets. ARIH2 is essential for CRL5-dependent HIV infectivity in primary CD4+ Tcells. Furthermore, we show that ARIH2 cooperates with CRL5 to prime other cellular substrates for polyubiquitination, suggesting this may represent a general mechanism beyond HIV infection and APOBEC3 degradation. Taken together, these data identify ARIH2 as a co-factor in the Vif-hijacked CRL5 complex that contributes to HIV infectivity and demonstrate the operation of the E1-E2-E3/E3-substrate ubiquitination mechanism in a viral infection context.

Role of the COP9 Signalosome (CSN) in Cardiovascular Diseases.

The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) is an evolutionarily conserved multi-protein complex, consisting of eight subunits termed CSN1-CSN8. The main biochemical function of the CSN is the control of protein degradation via the ubiquitin-proteasome-system through regulation of cullin-RING E3-ligase (CRL) activity by deNEDDylation of cullins, but the CSN also serves as a docking platform for signaling proteins. The catalytic deNEDDylase (isopeptidase) activity of the complex is executed by CSN5, but only efficiently occurs in the three-dimensional architectural context of the complex. Due to its positioning in a central cellular pathway connected to cell responses such as cell-cycle, proliferation, and signaling, the CSN has been implicated in several human diseases, with most evidence available for a role in cancer. However, emerging evidence also suggests that the CSN is involved in inflammation and cardiovascular diseases. This is both due to its role in controlling CRLs, regulating components of key inflammatory pathways such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and complex-independent interactions of subunits such as CSN5 with inflammatory proteins. In this case, we summarize and discuss studies suggesting that the CSN may have a key role in cardiovascular diseases such as atherosclerosis and heart failure. We discuss the implicated molecular mechanisms ranging from inflammatory NF-κB signaling to proteotoxicity and necrosis, covering disease-relevant cell types such as myeloid and endothelial cells or cardiomyocytes. While the CSN is considered to be disease-exacerbating in most cancer entities, the cardiovascular studies suggest potent protective activities in the vasculature and heart. The underlying mechanisms and potential therapeutic avenues will be critically discussed.

Potent 5-Cyano-6-phenyl-pyrimidin-Based Derivatives Targeting DCN1-UBE2M Interaction.

Neddylation of the Cullin-RING E3 ligases (CRLs) regulates the homeostasis of approximately 20% of cellular proteins. Defective in cullin neddylation 1 (DCN1), as a co-E3 ligase, interacts with UBE2M to enhance the activation of CRLs, and this interaction is emerging as a therapeutic target for human diseases. Here, we present a series of pyrimidin-based small molecular inhibitors targeting DCN1-UBE2M interaction. After finding a novel inhibitor DC-1 with IC50 = 1.2 μM, we performed a series of chemical optimizations, which finally led to the discovery of a potent thiazole containing 5-cyano-6-phenylpyrimidin-based inhibitor DC-2 (IC50 = 15 nM). Next, using protein and cellular thermal shift assays, coimmunoprecipitation, molecular docking, and site-specific mutation experiments, we further proved that DC-2 specifically inhibited the interaction of UBE2M and DCN1 at molecule and cellular levels, resulting in the decrease of cullin3 neddylation and accumulation of its substrate, NRF2. Our findings indicate that DC-2 may serve as a novel lead compound for specific derivatives targeting DCN1-UBE2M interaction.