Structural and mechanistic insights into the CAND1-mediated SCF substrate receptor exchange.

Modular SCF (SKP1-CUL1-Fbox) ubiquitin E3 ligases orchestrate multiple cellular pathways in eukaryotes. Their variable SKP1-Fbox substrate receptor (SR) modules enable regulated substrate recruitment and subsequent proteasomal degradation. CAND proteins are essential for the efficient and timely exchange of SRs. To gain structural understanding of the underlying molecular mechanism, we reconstituted a human CAND1-driven exchange reaction of substrate-bound SCF alongside its co-E3 ligase DCNL1 and visualized it by cryo-EM. We describe high-resolution structural intermediates, including a ternary CAND1-SCF complex, as well as conformational and compositional intermediates representing SR- or CAND1-dissociation. We describe in molecular detail how CAND1-induced conformational changes in CUL1/RBX1 provide an optimized DCNL1-binding site and reveal an unexpected dual role for DCNL1 in CAND1-SCF dynamics. Moreover, a partially dissociated CAND1-SCF conformation accommodates cullin neddylation, leading to CAND1 displacement. Our structural findings, together with functional biochemical assays, help formulate a detailed model for CAND-SCF regulation.

OsDWD1 E3 ligase-mediated OsNPR1 degradation suppresses basal defense in rice.

Many ubiquitin E3 ligases function in plant immunity. Here, we show that Oryza sativa (rice) DDB1 binding WD (OsDWD1) suppresses immune responses by targeting O. sativa non-expresser of pathogenesis-related gene 1 (OsNPR1) for degradation. Knock-down and overexpression experiments in rice plants showed that OsDWD1 is a negative regulator of the immune response and that OsNPR1 is a substrate of OsDWD1 and a substrate receptor of OsCRL4. After constructing the loss-of-function mutant OsDWD1R239A , we showed that the downregulation of OsNPR1 seen in rice lines overexpressing wild-type (WT) OsDWD1 (OsDWD1WT -ox) was compromised in OsDWD1R239A -ox lines, and that OsNPR1 upregulation enhanced resistance to pathogen infection, confirming that OsCRL4OsDWD1 regulates OsNPR1 protein levels. The enhanced disease resistance seen in OsDWD1 knock-down (OsDWD1-kd) lines contrasted with the reduced disease resistance in double knock-down (OsDWD1/OsNPR1-kd) lines, indicating that the enhanced disease resistance of OsDWD1-kd resulted from the accumulation of OsNPR1. Moreover, an in vivo heterologous protein degradation assay in Arabidopsis thaliana ddb1 mutants confirmed that the CUL4-based E3 ligase system can also influence OsNPR1 protein levels in Arabidopsis. Although OsNPR1 was degraded by the OsCRL4OsDWD1 -mediated ubiquitination system, the phosphodegron-motif-mutated NPR1 was partially degraded in the DWD1-ox protoplasts. This suggests that there might be another degradation process for OsNPR1. Taken together, these results indicate that OsDWD1 regulates OsNPR1 protein levels in rice to suppress the untimely activation of immune responses.

CUL3BPM E3 ubiquitin ligases regulate MYC2, MYC3, and MYC4 stability and JA responses.

The jasmonate (JA)-pathway regulators MYC2, MYC3, and MYC4 are central nodes in plant signaling networks integrating environmental and developmental signals to fine-tune JA defenses and plant growth. Continuous activation of MYC activity is potentially lethal. Hence, MYCs need to be tightly regulated in order to optimize plant fitness. Among the increasing number of mechanisms regulating MYC activity, protein stability is arising as a major player. However, how the levels of MYC proteins are modulated is still poorly understood. Here, we report that MYC2, MYC3, and MYC4 are targets of BPM (BTB/POZ-MATH) proteins, which act as substrate adaptors of CUL3-based E3 ubiquitin ligases. Reduction of function of CUL3BPM in amiR-bpm lines, bpm235 triple mutants, and cul3ab double mutants enhances MYC2 and MYC3 stability and accumulation and potentiates plant responses to JA such as root-growth inhibition and MYC-regulated gene expression. Moreover, MYC3 polyubiquitination levels are reduced in amiR-bpm lines. BPM3 protein is stabilized by JA, suggesting a negative feedback regulatory mechanism to control MYC activity, avoiding harmful runaway responses. Our results uncover a layer for JA-pathway regulation by CUL3BPM-mediated degradation of MYC transcription factors.

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.

Recent insight into the role of RING-finger E3 ligases in glioma.

The ubiquitin proteasome system (UPS) serves as the major posttranslational modification system for the maintenance of protein homeostasis. The ubiquitin ligases (E3s) are responsible for the recognition and recruitment of specific substrate proteins for polyubiquitination. Really interesting new gene (RING) finger E3s account for the majority of E3s. The human genome encodes more than 600 RING E3s, which are divided into three subclasses: single polypeptide E3s, cullin-RING ligases (CRLs) and other multisubunit E3s. The abnormal regulation of RING E3s has been reported to disrupt normal biological processes and induce the occurrence of many human malignancies. Glioma is the most common type of malignant primary brain tumor. In the last few decades, patient prognosis has improved as novel targeted therapeutic agents have developed. In this review, we will summarize the current knowledge about the dysregulation of RING E3s and the altered stability of their substrates in glioma. We will further introduce and discuss the current status and future perspectives of the application of small inhibitors and proteolysis-targeting chimeric molecules (PROTACs) interfering with RING E3s as potential anticancer agents for glioma.

Cullin-RING Ligases as Promising Targets for Gastric Carcinoma Treatment.

Gastric carcinoma has serious morbidity and mortality, which seriously threats human health. The studies on gastrointestinal cell biology have shown that the ubiquitination modification that occurs after protein translation plays an essential role in the pathogenesis of gastric carcinoma. Protein ubiquitination is catalyzed by E3 ubiquitin ligase and can regulate various substrate proteins in different cellular pathways. Cullin-RING E3 ligase (CRLs) is a representative of the E3 ubiquitin ligase family, which requires cullin (CUL) neddylation modification for activation to regulate homeostasis of ~20% of cellular proteins. The substrate molecules regulated by CRLs are often involved in many cell progressions such as cell cycle progression, cell apoptosis, DNA damage and repair. Given that CRLs play an important role in modulation of biological activities, so targeting a certain CULs member neddylation may be an attractive strategy for selectively controlling the cellular proteins levels to achieve the goal of cancer treatment. In this review, we will discuss the roles of CULs and Ring protein in gastric carcinoma and summarize the current neddylation modulators for gastric carcinoma treatment.

Effective targeting of the ubiquitin-like modifier NEDD8 for lung adenocarcinoma treatment.

Protein neddylation, a process of conjugating neural precursor cell expressed, developmentally downregulated 8 (NEDD8) to substrates, plays a tumor-promoting role in lung carcinogenesis. Our previous study showed MLN4924, an inhibitor of NEDD8 activating enzyme (E1), significantly inhibits the growth of multiple cancer cells. However, resistance can develop to MLN4924 by mutation. Therefore, it is important to further understand how NEDD8 acts in lung cancer. In the present study, we demonstrated NEDD8 is overactivated in lung cancers and confers a worse patient overall survival. Furthermore, we report that in lung adenocarcinoma cells, NEDD8 depletion significantly suppressed lung cancer cell growth and progression both in vitro and in vivo. Mechanistic studies revealed that NEDD8 depletion induced the accumulation of a panel of tumor-suppressive cullin-RING ubiquitin ligase substrates (e.g., p21, p27, and Wee1) via blocking their degradation, triggering cell cycle arrest at G2 phase, thus inducing apoptosis or senescence in a cell-line-dependent manner. The present study demonstrates the role of NEDD8 in regulating the malignant phenotypes of lung cancer cells and further validates NEDD8 as a potential therapeutic target in lung cancer.

Characterization of the mammalian family of DCN-type NEDD8 E3 ligases.

Cullin-RING ligases (CRL) are ubiquitin E3 enzymes that bind substrates through variable substrate receptor proteins and are activated by attachment of the ubiquitin-like protein NEDD8 to the cullin subunit. DCNs are NEDD8 E3 ligases that promote neddylation. Mammalian cells express five DCN-like (DCNL) proteins but little is known about their specific functions or interaction partners. We found that DCNLs form stable stoichiometric complexes with CAND1 and cullins that can only be neddylated in the presence of a substrate adaptor. These CAND-cullin-DCNL complexes might represent 'reserve' CRLs that can be rapidly activated when needed. We further found that all DCNLs interact with most cullin subtypes, but that they are probably responsible for the neddylation of different subpopulations of any given cullin. This is consistent with the fact that the subcellular localization of DCNLs in tissue culture cells differs and that they show unique tissue-specific expression patterns in mice. Thus, the specificity between DCNL-type NEDD8 E3 enzymes and their cullin substrates is only apparent in well-defined physiological contexts and related to their subcellular distribution and restricted expression.

Enzyme-substrate relationships in the ubiquitin system: approaches for identifying substrates of ubiquitin ligases.

Protein ubiquitylation is an important post-translational modification, regulating aspects of virtually every biochemical pathway in eukaryotic cells. Hundreds of enzymes participate in the conjugation and deconjugation of ubiquitin, as well as the recognition, signaling functions, and degradation of ubiquitylated proteins. Regulation of ubiquitylation is most commonly at the level of recognition of substrates by E3 ubiquitin ligases. Characterization of the network of E3-substrate relationships is a major goal and challenge in the field, as this expected to yield fundamental biological insights and opportunities for drug development. There has been remarkable success in identifying substrates for some E3 ligases, in many instances using the standard protein-protein interaction techniques (e.g., two-hybrid screens and co-immunoprecipitations paired with mass spectrometry). However, some E3s have remained refractory to characterization, while others have simply not yet been studied due to the sheer number and diversity of E3s. This review will discuss the range of tools and techniques that can be used for substrate profiling of E3 ligases.

Stabilization of GTSE1 by cyclin D1-CDK4/6 promotes cell proliferation: relevance in cancer prognosis.

Cyclin D1 is the activating subunit of the cell cycle kinases CDK4 and CDK6, and its dysregulation is a well-known oncogenic driver in many human cancers. The biological function of cyclin D1 has been primarily studied by focusing on the phosphorylation of the retinoblastoma (RB) gene product. Here, using an integrative approach combining bioinformatic analyses and biochemical experiments, we show that GTSE1 (G2 and S phases expressed protein 1), a protein positively regulating cell cycle progression, is a previously unknown substrate of cyclin D1-CDK4/6. The phosphorylation of GTSE1 mediated by cyclin D1-CDK4/6 inhibits GTSE1 degradation, leading to high levels of GTSE1 also during the G1 phase of the cell cycle. Functionally, the phosphorylation of GTSE1 promotes cellular proliferation and is associated with poor prognosis within a pan-cancer cohort. Our findings provide insights into cyclin D1's role in cell cycle control and oncogenesis beyond RB phosphorylation.

Advances in the potential roles of Cullin-RING ligases in regulating autoimmune diseases.

Cullin-RING ligases (CRLs) are the largest class of E3 ubiquitin ligases regulating the stability and subsequent activity of a large number of important proteins responsible for the development and progression of various diseases, including autoimmune diseases (AIDs). However, the detailed mechanisms of the pathogenesis of AIDs are complicated and involve multiple signaling pathways. An in-depth understanding of the underlying regulatory mechanisms of the initiation and progression of AIDs will aid in the development of effective therapeutic strategies. CRLs play critical roles in regulating AIDs, partially by affecting the key inflammation-associated pathways such as NF-κB, JAK/STAT, and TGF-ß. In this review, we summarize and discuss the potential roles of CRLs in the inflammatory signaling pathways and pathogenesis of AIDs. Furthermore, advances in the development of novel therapeutic strategies for AIDs through targeting CRLs are also highlighted.

Determining the Effects of Neddylation on Cullin-RING Ligase-Dependent Protein Ubiquitination.

As the largest family of ubiquitin (Ub) E3 ligases, cullin-RING ligases (CRLs) play crucial roles in various cellular processes, and their activities are tightly regulated by orchestrated mechanisms. Neddylation, the conjugation of a Ub-like protein NEDD8 to a target protein such as the cullin, represents a key regulatory mechanism for CRLs. Biochemical and structural studies of a few CRLs have revealed that cullin neddylation alters the CRL conformation and activates CRL-dependent protein ubiquitination. Here, using CUL2-RING ligase (CRL2) as an example, we describe our protocols for the preparation of recombinant CUL2 with or without NEDD8 conjugation, which is further used to quantitatively determine the effect of neddylation on CRL2-dependent protein ubiquitination in vitro. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and purification of CUL2•RBX1 from Escherichia coli Support Protocol: Further purification of CUL2•RBX1 with additional chromatography on an FPLC system Basic Protocol 2: Reconstitution of cullin neddylation for quantitative ubiquitination assay in vitro.

Targeting the COP9 signalosome for cancer therapy.

The COP9 signalosome (CSN) is a highly conserved protein complex composed of 8 subunits (CSN1 to CSN8). The individual subunits of the CSN play essential roles in cell proliferation, tumorigenesis, cell cycle regulation, DNA damage repair, angiogenesis, and microenvironmental homeostasis. The CSN complex has an intrinsic metalloprotease that removes the ubiquitin-like activator NEDD8 from cullin-RING ligases (CRLs). Binding of neddylated CRLs to CSN is sensed by CSN4 and communicated to CSN5 with the assistance of CSN6, thus leading to the activation of deneddylase. Therefore, CSN is a crucial regulator at the intersection between neddylation and ubiquitination in cancer progression. Here, we summarize current understanding of the roles of individual CSN subunits in cancer progression. Furthermore, we explain how the CSN affects tumorigenesis through regulating transcription factors and the cell cycle. Finally, we discuss individual CSN subunits as potential therapeutic targets to provide new directions and strategies for cancer therapy.

Roles of Cullin-RING Ubiquitin Ligases in Cardiovascular Diseases.

Maintenance of protein homeostasis is crucial for virtually every aspect of eukaryotic biology. The ubiquitin-proteasome system (UPS) represents a highly regulated quality control machinery that protects cells from a variety of stress conditions as well as toxic proteins. A large body of evidence has shown that UPS dysfunction contributes to the pathogenesis of cardiovascular diseases. This review highlights the latest findings regarding the physiological and pathological roles of cullin-RING ubiquitin ligases (CRLs), an essential player in the UPS, in the cardiovascular system. To inspire potential therapeutic invention, factors regulating CRL activities are also discussed.

Neddylation regulation of mitochondrial structure and functions.

Mitochondria are the powerhouse of a cell. The structure and function of mitochondria are precisely regulated by multiple signaling pathways. Neddylation, a post-translational modification, plays a crucial role in various cellular processes including cellular metabolism via modulating the activity, function and subcellular localization of its substrates. Recently, accumulated data demonstrated that neddylation is involved in regulation of morphology, trafficking and function of mitochondria. Mechanistic elucidation of how mitochondria is modulated by neddylation would further our understanding of mitochondrial regulation to a new level. In this review, we first briefly introduce mitochondria, then neddylation cascade, and known protein substrates subjected to neddylation modification. Next, we summarize current available data of how neddylation enzymes, its substrates (including cullins/Cullin-RING E3 ligases and non-cullins) and its inhibitor MLN4924 regulate the structure and function of mitochondria. Finally, we propose the future perspectives on this emerging and exciting field of mitochondrial research.

Phosphorylation regulates cullin-based ubiquitination in tumorigenesis.

Cullin-RING ligases (CRLs) recognize and interact with substrates for ubiquitination and degradation, and can be targeted for disease treatment when the abnormal expression of substrates involves pathologic processes. Phosphorylation, either of substrates or receptors of CRLs, can alter their interaction. Phosphorylation-dependent ubiquitination and proteasome degradation influence various cellular processes and can contribute to the occurrence of various diseases, most often tumorigenesis. These processes have the potential to be used for tumor intervention through the regulation of the activities of related kinases, along with the regulation of the stability of specific oncoproteins and tumor suppressors. This review describes the mechanisms and biological functions of crosstalk between phosphorylation and ubiquitination, and most importantly its influence on tumorigenesis, to provide new directions and strategies for tumor therapy.

Ubiquitylation of the ER-Shaping Protein Lunapark via the CRL3KLHL12 Ubiquitin Ligase Complex.

Cullin-RING ligases (CRLs) control key cellular processes by promoting ubiquitylation of a multitude of soluble cytosolic and nuclear proteins. Subsets of CRL complexes are recruited and activated locally at cellular membranes; however, few CRL functions and substrates at these distinct cellular compartments are known. Here, we use a proteomic screen to identify proteins that are ubiquitylated at cellular membranes and found that Lunapark, an endoplasmic reticulum (ER)-shaping protein localized to ER three-way junctions, is ubiquitylated by the CRL3KLHL12 ubiquitin ligase. We demonstrate that Lunapark interacts with mechanistic target of rapamycin complex-1 (mTORC1), a central cellular regulator that coordinates growth and metabolism with environmental conditions. We show that mTORC1 binds Lunapark specifically at three-way junctions, and lysosomes, where mTORC1 is activated, make contact with three-way junctions where Lunapark resides. Inhibition of Lunapark ubiquitylation results in neurodevelopmental defects indicating that KLHL12-dependent ubiquitylation of Lunapark is required for normal growth and development.

Cullin Ring Ubiquitin Ligases (CRLs) in Cancer: Responses to Ionizing Radiation (IR) Treatment.

Treatment with ionizing radiation (IR) remains the cornerstone of therapy for multiple cancer types, including disseminated and aggressive diseases in the palliative setting. Radiotherapy efficacy could be improved in combination with drugs that regulate the ubiquitin-proteasome system (UPS), many of which are currently being tested in clinical trials. The UPS operates through the covalent attachment of ATP-activated ubiquitin molecules onto substrates following the transfer of ubiquitin from an E1, to an E2, and then to the substrate via an E3 enzyme. The specificity of ubiquitin ligation is dictated by E3 ligases, which select substrates to be ubiquitylated. Among the E3s, cullin ring ubiquitin ligases (CRLs) represent prototypical multi-subunit E3s, which use the cullin subunit as a central assembling scaffold. CRLs have crucial roles in controlling the cell cycle, hypoxia signaling, reactive oxygen species clearance and DNA repair; pivotal factors regulating the cancer and normal tissue response to IR. Here, we summarize the findings on the involvement of CRLs in the response of cancer cells to IR, and we discuss the therapeutic approaches to target the CRLs which could be exploited in the clinic.

Murine Myocardial Transcriptome Analysis Reveals a Critical Role of COPS8 in the Gene Expression of Cullin-RING Ligase Substrate Receptors and Redox and Vesicle Trafficking Pathways.

Background: The COP9 signalosome (CSN) consisting of 8 unique protein subunits (COPS1 through COPS8) serves as the cullin deneddylase, regulating the catalytic dynamics of cullin RING ligases (CRLs), the largest family of ubiquitin ligases Background: The COP9 signalosome (CSN) consisting of 8 unique protein subunits (COPS1 through COPS8) serves as the cullin deneddylase, regulating the catalytic dynamics of cullin RING ligases (CRLs), the largest family of ubiquitin ligases. Supported primarily by the decrease of substrate receptor (SR) proteins of CRLs in cells deficient of a CSN subunit, CSN-mediated cullin deneddylation is believed to prevent autoubiquitination and self-destruction of the SR in active CRLs. However, it is unclear whether the decrease in SRs is solely due to protein destabilization. Moreover, our prior studies have demonstrated that cardiac specific knockout of Cops8 (Cops8-CKO) impairs autophagosome maturation and causes massive necrosis in cardiomyocytes but the underlying mechanism remains poorly understood. Given that Cops8 is nucleus-enriched and a prior report showed its binding to the promoter of several genes and association of its ablation with decreased mRNA levels of these genes, we sought to determine the dynamic changes of myocardial transcriptome in mice with perinatal Cops8-CKO and to explore their functional implications. Methods and Results: Myocardial transcriptomes of Cops8flox/flox , Cops8flox/+::Myh6-Cre, and Cops8flox/flox::Myh6-Cre littermate mice at postnatal 2 and 3 weeks were analyzed. The data were imported into an in-house analysis pipeline using Bioconductor for quantile normalization and statistical analysis. Differentially expressed genes (DEGs) between groups at each time point or between time points within the group were revealed by t-test. Genes with p < 0.05 after Benjamini and Hochberg false discovery rate correction for multiple hypothesis testing were considered as significant DEGs. We found that (1) the Ingenuity Pathway Analysis (IPA) revealed significant enrichment of DEGs in multiple pathways, especially those responding to oxidative stress, in homozygous Cops8-CKO hearts at both 2 and 3 weeks, corroborating the occurrence of massive cardiomyocyte necrosis at 3 weeks; (2) the decreases in multiple CRL SR proteins were associated with decreased transcript levels; and (3) enrichment of DEGs in the chromatin remodeling pathway and the microtubule motility and vesicle trafficking pathways. Conclusions: Our data are consistent with the notion that Cops8/CSN plays a role in the transcriptional regulation of CRL SRs and in the redox and vesicle trafficking pathways.

Radiosensitization by the investigational NEDD8-activating enzyme inhibitor MLN4924 (pevonedistat) in hormone-resistant prostate cancer cells.

Salvage radiotherapy (SRT) is the first-line treatment for prostate cancer patients with biochemical recurrence following radical prostatectomy, and new specific radiosensitizers are in urgent need to enhance SRT effect. MLN4924 (also known as Pevonedistat), a specific inhibitor of NEDD8-activating enzyme, has recently entered phase I/II clinical trials in several malignancies. By inhibiting cullin neddylation, MLN4924 inactivates Cullin-RING ligases (CRL), which have been validated as an attractive radiosensitizing target. In our study, we demonstrate that MLN4924 can be used as a potent radiosensitizer in hormone-resistant prostate cancer cells. We found that MLN4924 inhibited cullin neddylation and sensitized prostate cancer cells to irradiation (IR). Mechanistically, MLN4924 enhanced IR-induced G2 cell-cycle arrest, by inducing accumulation of WEE1/p21/p27, three well-known CRL substrates. Importantly, siRNA knockdown of WEE1/p21/p27 partially abrogated MLN4924-induced G2 cell-cycle arrest, indicating a causal role of WEE1/p21/p27 in MLN4924-induced radiosensitization. Further mechanistic studies revealed that induction of DNA damage and apoptosis also contributed to MLN4924 radiosensitization in hormone-resistant prostate cancer cells. Our findings lay the foundation for future application of MLN4924 as a potential radiosensitizer in hormone refractory prostate cancer (HRPC).