Enhanced ApcMin/+ adenoma formation after epithelial CUL4B deletion by recruitment of myeloid-derived suppressor cells.

Colorectal cancer (CRC) stands as a prevalent malignancy globally. A pivotal event in CRC pathogenesis involves the loss-of-function mutation in the APC gene, leading to the formation of benign polyps. Despite the well-established role of APC, the contribution of CUL4B to CRC initiation in the pre-tumorous stage remains poorly understood. In this investigation, we generated a murine model by crossing ApcMin/+ mice with Cul4bΔIEC mice to achieve specific deletion of Cul4b in the gut epithelium against an ApcMin/+ background. By employing histological methods, RNA-sequencing (RNA-seq), and flow cytometry, we assessed alterations and characterized the immune microenvironment. Our results unveiled that CUL4B deficiency in gut epithelium expedited ApcMin/+ adenoma formation. Notably, CUL4B in adenomas restrained the accumulation of tumor-infiltrating myeloid-derived suppressor cells (MDSCs). In vivo inhibition of MDSCs significantly delayed the growth of CUL4B deleted ApcMin/+ adenomas. Furthermore, the addition of MDSCs to in vitro cultured ApcMin/+; Cul4bΔIEC adenoma organoids mitigated their alterations. Mechanistically, CUL4B directly interacted with the promoter of Csf3, the gene encoding granulocyte-colony stimulating factor (G-CSF) by coordinating with PRC2. Inhibiting CUL4B epigenetically activated the expression of G-CSF, promoting the recruitment of MDSCs. These findings offer novel insights into the tumor suppressor-like roles of CUL4B in regulating ApcMin/+ adenomas, suggesting a potential therapeutic strategy for CRC initiation and progression in the context of activated Wnt signaling.

[The effect of ubiquitin ligase Cullin3 on the proliferation, migration and invasion of triple-negative breast cancer cells and exploration of its mechanism].

Objective: To investigate the effects of ubiquitin ligase Cullin3 (CUL3) on the proliferation, migration and invasion ability of triple-negative breast cancer (TNBC) cells and its mechanism of action. Methods: Bioinformatics-based methods were used to obtain CUL3 gene and protein expression data in TNBC tissues, and to assess the expression of CUL3 in tumour tissues of TNBC patients (n=160) and in normal breast tissues (n=572), and its relationship with clinical prognosis. The effects of overexpression of CUL3 on the proliferation, migration and invasion ability of TNBC cells in vitro were detected by CCK8 cell proliferation assay, scratch assay and transwell assay; proteins that might interact with CUL3 were screened by immunoprecipitation combined with mass spectrometry analysis, and the substrate protein regulated by CUL3 was identified as Glutathione S-Transferase Pi 1 (GSTP1); the effects of overexpression of GSTP1 on the migration and invasion ability of TNBC cells were detected by scratch assay and Transwell assay, and it was explored whether overexpression of CUL3 could reverse the effects of GSTP1 on the migration and invasion ability of cells; and the effects of overexpression of GSTP1 on the migration and invasion ability of cells were detected by Western blot and IP (Immunoprecipitation) to detect the effect of CUL3 on the ubiquitination modification of GSTP1 protein, and to verify the molecular mechanism by which CUL3 regulates the expression of GSTP1 to affect TNBC migration and invasion. Results: CUL3 expression was significantly higher in TNBC (P<0.000 1), and high CUL3 expression was closely associated with poor prognosis of TNBC patients (OS, P=0.018; RFS, P=0.008); overexpression of CUL3 significantly increased the proliferation of TNBC cells (F=11.97, P=0.002 for the 231-cell group, F=51.92, P<0.001 for the 468-cell group), migration [74.7±4.0 and 128.0±6.1 perforating cells in the overexpression groups of 231 and 468 cell lines, compared with 21.0±2.7 and 70.0±6.6 in the blank control (NC) group, and the t-values of 231 and 468 cell groups were-19.24 and-11.23, with P-values<0.001] and invasive ability (48 h cell proliferation rates were 56.6%±4.4% and 51.6%±3.7% in the 231 and 468 cell line overexpression groups, compared with 40.5%±2.9% and 32.9%±4.8% in the NC group, respectively, t=-5.26, P=0.006 3 in the 231 cell group; t=-5.38 in the 468 cell group, P=0.005 8); GSTP1 expression was reduced in TNBC, and up-regulation of GSTP1 inhibited TNBC cell migration (the number of membrane-penetrating cells in the overexpression groups of 231 and 468 cell lines were 16.3±6.5 and 33.0±6.2, respectively, compared with 34.3±2.5 and 77.3±5.0 in the NC group, and t=5.44 in the 231 cell group, P=0.006; 468 cell group t=7.20, P=0.002) and invasion (48 h cell proliferation rates of 49.6%±1.7% and 36.2%±1.4% in the 231 and 468 cell line overexpression groups, compared to 59.4%±4.7% and 53.0%±1.7% in the NC group, t=3.42, P=0.027 in the 231 cell group; 468 cell group t=13.18, P<0.001), whereas up-regulation of CUL3 reversed the effects of GSTP1 on cell migration (37.0±1.0 and 67.0±5.3 membrane-penetrating cells in the overexpression groups of 231 and 468 cell lines, respectively, 231 cell group t=-3.97, P=0.017; 468 cell group t=-6.12, P=0.004), and invasion (48 h cell proliferation rates of 71.9%±3.6% and 59.4%±2.1% in the 231 and 468 cell line overexpression groups, respectively, with t-values of -9.61 and -16.01 in the 231 and 468 cell groups, respectively, P-values<0.001) inhibitory effects; and CUL3, by increasing GSTP1 ubiquitylation modification, promotes ubiquitin-proteasome system to degrade GSTP1 protein, thereby reducing the stability of GSTP1 protein. Conclusion: Overexpression of CUL3 promotes TNBC development by promoting GSTP1 ubiquitination degradation inducing cell migration and invasion.

Protein neddylation and its role in health and diseases.

NEDD8 (Neural precursor cell expressed developmentally downregulated protein 8) is an ubiquitin-like protein that is covalently attached to a lysine residue of a protein substrate through a process known as neddylation, catalyzed by the enzyme cascade, namely NEDD8 activating enzyme (E1), NEDD8 conjugating enzyme (E2), and NEDD8 ligase (E3). The substrates of neddylation are categorized into cullins and non-cullin proteins. Neddylation of cullins activates CRLs (cullin RING ligases), the largest family of E3 ligases, whereas neddylation of non-cullin substrates alters their stability and activity, as well as subcellular localization. Significantly, the neddylation pathway and/or many neddylation substrates are abnormally activated or over-expressed in various human diseases, such as metabolic disorders, liver dysfunction, neurodegenerative disorders, and cancers, among others. Thus, targeting neddylation becomes an attractive strategy for the treatment of these diseases. In this review, we first provide a general introduction on the neddylation cascade, its biochemical process and regulation, and the crystal structures of neddylation enzymes in complex with cullin substrates; then discuss how neddylation governs various key biological processes via the modification of cullins and non-cullin substrates. We further review the literature data on dysregulated neddylation in several human diseases, particularly cancer, followed by an outline of current efforts in the discovery of small molecule inhibitors of neddylation as a promising therapeutic approach. Finally, few perspectives were proposed for extensive future investigations.

MSC-derived small extracellular vesicles mitigate diabetic retinopathy by stabilizing Nrf2 through miR-143-3p-mediated inhibition of neddylation.

Diabetic retinopathy (DR) is a highly hazardous and widespread complication of diabetes mellitus (DM). The accumulated reactive oxygen species (ROS) play a central role in DR development. The aim of this research was to examine the impact and mechanisms of mesenchymal stem cell (MSC)-derived small extracellular vesicles (sEV) on regulating ROS and retinal damage in DR. Intravitreal injection of sEV inhibited Cullin3 neddylation, stabilized Nrf2, decreased ROS, reduced retinal inflammation, suppressed Müller gliosis, and mitigated DR. Based on MSC-sEV miRNA sequencing, bioinformatics software, and dual-luciferase reporter assay, miR-143-3p was identified to be the key effector for MSC-sEV's role in regulating neural precursor cell expressed developmentally down-regulated 8 (NEDD8)-mediated neddylation. sEV were able to be internalized by Müller cells. Compared to advanced glycation end-products (AGEs)-induced Müller cells, sEV coculture decreased Cullin3 neddylation, activated Nrf2 signal pathway to combat ROS-induced inflammation. The barrier function of endothelial cells was impaired when endothelial cells were treated with the supernatant of AGEs-induced Müller cells, but was restored when treated with supernatant of AGEs-induced Müller cells cocultured with sEV. The protective effect of sEV was, however, compromised when miR-143-3p was inhibited in sEV. Moreover, the protective efficacy of sEV was diminished when NEDD8 was overexpressed in Müller cells. These findings showed MSC-sEV delivered miR-143-3p to inhibit Cullin3 neddylation, stabilizing Nrf2 to counteract ROS-induced inflammation and reducing vascular leakage. Our findings suggest that MSC-sEV may be a potential nanotherapeutic agent for DR, and that Cullin3 neddylation could be a new target for DR therapy.

Dynamic role of CUL4B in radiation-induced intestinal injury-regeneration.

CUL4B, a crucial scaffolding protein in the largest E3 ubiquitin ligase complex CRL4B, is involved in a broad range of physiological and pathological processes. While previous research has shown that CUL4B participates in maintaining intestinal homeostasis and function, its involvement in facilitating intestinal recovery following ionizing radiation (IR) damage has not been fully elucidated. Here, we utilized in vivo and in vitro models to decipher the role of CUL4B in intestinal repair after IR-injury. Our findings demonstrated that prior to radiation exposure, CUL4B inhibited the ubiquitination modification of PSME3, which led to the accumulation of PSME3 and subsequent negative regulation of p53-mediated apoptosis. In contrast, after radiation, CUL4B dissociated from PSME3 and translocated into the nucleus at phosphorylated histones H2A (γH2AX) foci, thereby impeding DNA damage repair and augmenting p53-mediated apoptosis through inhibition of BRCA1 phosphorylation and RAD51. Our study elucidated the dynamic role of CUL4B in the repair of radiation-induced intestinal damage and uncovered novel molecular mechanisms underlying the repair process, suggesting a potential therapeutic strategy of intestinal damage after radiation therapy for cancers.

Hyperthermia and cisplatin combination therapy promotes caspase-8 accumulation and activation to enhance apoptosis and pyroptosis in cancer cells.

BACKGROUND: Hyperthermia can play a synergistic role with chemotherapy in combination therapy. Although the association between caspase activation, apoptosis, and pyroptosis have been published for both cisplatin (CDDP) and hyperthermia therapies independently, the interactions between these molecular pathways in combination therapy are unknown. The present study aimed to investigate the possible interactions between caspase 8 activation, apoptosis, and pyroptosis in combination therapy. METHODS: Cells were treated with CDDP (15 µg/ml), followed by hyperthermia at optimized temperature (42.5 °C) in water-bath. After combination therapy, cell viability was analyzed by CCK-8, and cell death was analyzed by Annexin-V-FITC/PI and caspases activation. Immuno-staining and co-immuno-precipitation were used to examine the interaction between p62 and caspase-8. Pyroptosis was investigated by western blotting and transmission electron microscopy. E3 ligase Cullin 3 was knockdown by siRNA. In addition, caspase-8 activation was modulated by CRISPR-Cas9 gene-editing or pharmacological inhibition. RESULTS: Combination therapy promoted K63-linked polyubiquitination of caspase-8 and cellular accumulation of caspase-8. In turn, polyubiquitinated caspase-8 interacted with p62 and led to the activation of caspase-3. Knockdown of the E3 ligase Cullin 3 by siRNA reduced caspase-8 polyubiquitination and activation. In addition, combination therapy induced release of the pore-forming N-terminus from gasdermins and promoted pyroptosis along with caspase-8 accumulation and activation. Knockdown of caspase-8 by CRISPR/Cas9 based gene editing reduced the sensitivity of tumor cells to apoptosis and pyroptosis. CONCLUSIONS: Our study presented a novel mechanism in which hyperthermia synergized with chemotherapy in promoting apoptosis and pyroptosis in a caspase-8 dependent manner.

Buffalo colostrum peptide mitigates Parkinson's disease pathophysiology through Cullin-3 inhibition.

Colostrum/Milk is a chief repertoire of antioxidant peptides. Nuclear factor-erythroid 2 related factor 2 (Nrf2) is a viable target for Parkinson's Disease (PD), as this pathway deduced to be impaired in PD. Cullin-3 is one of the crucial E3 ligase responsible for its regulation. The present study screened peptide libraries of buffalo colostrum & milk peptides for Cullin-3 inhibition, thus ensuing activation of Nrf2 to alleviate the molecular etiopathology in PD using the C. elegans as a model. The structure was modelled, binding sites analyzed and peptide-interactions analyzed by docking. Among the 55 sequences (≤1 kDa), the peptide SFVSEVPEL having the highest dock score (-16.919) was synthesized and evaluated for its effects on oxidative stress markers, antioxidant enzymes, neurochemical marker and Nrf2/Skn-1 levels. The lead peptide alleviated the oxidative pathophysiology and behavioural deficits associated with PD in C. elegans.

Icariin ameliorates osteoporosis in ovariectomized rats by targeting Cullin 3/Nrf2/OH pathway for osteoclast inhibition.

Osteoporosis, characterized by low bone mass and bone microarchitecture breakdown, has become a growing public health problem. The increase in oxidative stress could lead to an imbalance between osteoblasts-mediated osteogenesis and osteoclast-mediated bone resorption, which gives rise to osteoporosis. Nrf2 is a master transcription factor that regulates oxidative stress and has recently been reported to take part in the development of osteoporosis. Icariin, a leading active flavonoid in herbal Epimedium pubescens, has significant antioxidant activity in and is widely applied for treating bone diseases. In this study, we aimed to explore the effect of icariin on osteoclastogenesis and its potential mechanism from the perspective of oxidative stress inhibition, using ovariectomized (OVX) rats and RANKL-induced RAW264.7 cells. Our results demonstrated that icariin-treated OVX rats exhibited higher bone density, fewer tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, and lower ROS levels in bone tissues than vehicle-treated OVX rats. Also, icariin suppressed osteoclast differentiation and inhibited the expression of osteoclastogenesis-related genes, such as NFATc1, Ctsk, Trap, and c-Fos, in RANKL-induced RAW264.7 cells. Icariin also reduced intracellular ROS levels by increasing the expression of nuclear Nrf2 and HO-1. Further mechanistic studies showed icariin inhibited Cullin 3 expression and could delay Nrf2 degradation by reducing the ubiquitination of endogenous Nrf2 in RANKL-stimulated RAW264.7 cells, and these effects were markedly reversed by cullin three overexpression. These findings suggest icariin alleviated osteoporosis by suppressing osteoclastogenesis via targeting the Cullin 3/Nrf2/OH signaling pathway. Our study implied that icariin may be a potential candidate to treat osteoporosis.

Inhibition of glycogen synthase kinase-3 enhances NRF2 protein stability, nuclear localisation and target gene transcription in pancreatic beta cells.

Accumulation of reactive oxygen species (i.e., oxidative stress) is a leading cause of beta cell dysfunction and apoptosis in diabetes. NRF2 (NF-E2 p45-related factor-2) regulates the adaptation to oxidative stress, and its activity is negatively regulated by the redox-sensitive CUL3 (cullin-3) ubiquitin ligase substrate adaptor KEAP1 (Kelch-like ECH-associated protein-1). Additionally, NRF2 is repressed by the insulin-regulated Glycogen Synthase Kinase-3 (GSK3). We have demonstrated that phosphorylation of NRF2 by GSK3 enhances ß-TrCP (beta-transducin repeat-containing protein) binding and ubiquitylation by CUL1 (cullin-1), resulting in increased proteasomal degradation of NRF2. Thus, we hypothesise that inhibition of GSK3 activity or ß-TrCP binding upregulates NRF2 and so protects beta cells against oxidative stress. We have found that treating the pancreatic beta cell line INS-1 832/13 with the KEAP1 inhibitor TBE31 significantly enhanced NRF2 protein levels. The presence of the GSK3 inhibitor CT99021 or the ß-TrCP-NRF2 protein-protein interaction inhibitor PHAR, along with TBE31, resulted in prolonged NRF2 stability and enhanced nuclear localisation (P < 0.05). TBE31-mediated induction of NRF2-target genes encoding NAD(P)H quinone oxidoreductase 1 (Nqo1), glutamate-cysteine ligase modifier (Gclm) subunit and heme oxygenase (Hmox1) was significantly enhanced by the presence of CT99021 or PHAR (P < 0.05) in both INS-1 832/13 and in isolated mouse islets. Identical results were obtained using structurally distinct GSK3 inhibitors and inhibition of KEAP1 with sulforaphane. In summary, we demonstrate that GSK3 and ß-TrCP/CUL1 regulate the proteasomal degradation of NRF2, enhancing the impact of KEAP1 regulation, and so contributes to the redox status of pancreatic beta cells. Inhibition of GSK3, or ß-TrCP/CUL1 binding to NRF2 may represent a strategy to protect beta cells from oxidative stress.

Oncogenic mutations of KRAS modulate its turnover by the CUL3/LZTR1 E3 ligase complex.

KRAS is a proto-oncogene encoding a small GTPase. Mutations contribute to ∼30% of human solid tumours, including lung adenocarcinoma, pancreatic, and colorectal carcinomas. Most KRAS activating mutations interfere with GTP hydrolysis, essential for its role as a molecular switch, leading to alterations in their molecular environment and oncogenic signalling. However, the precise signalling cascades these mutations affect are poorly understood. Here, APEX2 proximity labelling was used to profile the molecular environment of WT, G12D, G13D, and Q61H-activating KRAS mutants under starvation and stimulation conditions. Through quantitative proteomics, we demonstrate the presence of known KRAS interactors, including ARAF and LZTR1, which are differentially captured by WT and KRAS mutants. Notably, the KRAS mutations G12D, G13D, and Q61H abrogate their association with LZTR1, thereby affecting turnover. Elucidating the implications of LZTR1-mediated regulation of KRAS protein levels in cancer may offer insights into therapeutic strategies targeting KRAS-driven malignancies.

Structural insights into the ubiquitylation strategy of the oligomeric CRL2FEM1B E3 ubiquitin ligase.

Cullin-RING E3 ubiquitin ligase (CRL) family members play critical roles in numerous biological processes and diseases including cancer and Alzheimer's disease. Oligomerization of CRLs has been reported to be crucial for the regulation of their activities. However, the structural basis for its regulation and mechanism of its oligomerization are not fully known. Here, we present cryo-EM structures of oligomeric CRL2FEM1B in its unneddylated state, neddylated state in complex with BEX2 as well as neddylated state in complex with FNIP1/FLCN. These structures reveal that asymmetric dimerization of N8-CRL2FEM1B is critical for the ubiquitylation of BEX2 while FNIP1/FLCN is ubiquitylated by monomeric CRL2FEM1B. Our data present an example of the asymmetric homo-dimerization of CRL. Taken together, this study sheds light on the ubiquitylation strategy of oligomeric CRL2FEM1B according to substrates with different scales.

Neddylation is essential for malaria transmission in Plasmodium berghei.

Neddylation is a type of posttranslational modification known to regulate a wide range of cellular processes by covalently conjugating the ubiquitin-like protein Nedd8 to target proteins at lysine residues. However, the role of neddylation in malaria parasites has not been determined. Here, for the first time, we showed that neddylation plays an essential role in malaria transmission in Plasmodium berghei. We found that disruption of Nedd8 did not affect blood-stage propagation, gametocyte development, gamete formation, or zygote formation while abolishing the formation of ookinetes and further transmission of the parasites in mosquitoes. These phenotypic defects in Nedd8 knockout parasites were complemented by reintroducing the gene that restored mosquito transmission to wild-type levels. Our data establish the role of P. berghei Nedd8 in malaria parasite transmission.IMPORTANCENeddylation is a process by which Nedd8 is covalently attached to target proteins through three-step enzymatic cascades. The attachment of Nedd8 residues results in a range of diverse functions, such as cell cycle regulation, metabolism, immunity, and tumorigenesis. The potential neddylation substrates are cullin (CUL) family members, which are implicated in controlling the cell cycle. Cullin neddylation leads to the activation of cullin-RING ubiquitin ligases, which regulate a myriad of biological processes through target-specific ubiquitylation. Neddylation possibly regulates meiosis in zygotes, which subsequently develop into ookinetes. Our findings point to an essential function of this neddylation pathway and highlight its possible importance in designing novel intervention strategies.

Exploiting the Cullin E3 Ligase Adaptor Protein SKP1 for Targeted Protein Degradation.

Targeted protein degradation with proteolysis targeting chimeras (PROTACs) is a powerful therapeutic modality for eliminating disease-causing proteins through targeted ubiquitination and proteasome-mediated degradation. Most PROTACs have exploited substrate receptors of Cullin-RING E3 ubiquitin ligases such as cereblon and VHL. Whether core, shared, and essential components of the Cullin-RING E3 ubiquitin ligase complex can be used for PROTAC applications remains less explored. Here, we discovered a cysteine-reactive covalent recruiter EN884 against the SKP1 adapter protein of the SKP1-CUL1-F-box containing the SCF complex. We further showed that this recruiter can be used in PROTAC applications to degrade neo-substrate proteins such as BRD4 and the androgen receptor in a SKP1- and proteasome-dependent manner. Our studies demonstrate that core and essential adapter proteins within the Cullin-RING E3 ubiquitin ligase complex can be exploited for targeted protein degradation applications and that covalent chemoproteomic strategies can enable recruiter discovery against these targets.

JunD-miR494-CUL3 axis promotes radioresistance and metastasis by facilitating EMT and restraining PD-L1 degradation in esophageal squamous cell carcinoma.

Therapy resistance and metastatic progression jointly determine the fatal outcome of cancer, therefore, elucidating their crosstalk may provide new opportunities to improve therapeutic efficacy and prevent recurrence and metastasis in esophageal squamous cell carcinoma (ESCC). Here, we have established radioresistant ESCC cells with the remarkable metastatic capacity, and identified miR-494-3p (miR494) as a radioresistant activator. Mechanistically, we demonstrated that cullin 3 (CUL3) is a direct target of miR494, which is transcriptionally regulated by JunD, and highlighted that JunD-miR494-CUL3 axis promotes radioresistance and metastasis by facilitating epithelial-mesenchymal transition (EMT) and restraining programmed cell death 1 ligand 1 (PD-L1) degradation. In clinical specimens, miR494 is significantly up-regulated and positively associated with T stage and lymph node metastasis in ESCC tissues and serum. Notably, patients with higher serum miR494 expression have poor prognosis, and patients with higher CUL3 expression have more conventional dendritic cells (cDCs) and plasmacytoid DCs (pDCs), less cancer-associated fibroblasts (CAF2/4), and tumor endothelial cells (TEC2/3) infiltration than patients with lower CUL3 expression, suggesting that CUL3 may be involved in tumor microenvironment (TME). Overall, miR494 may serve as a potential prognostic predictor and therapeutic target, providing a promising strategy for ESCC treatment.

Pharmacological inhibition of neddylation impairs long interspersed element 1 retrotransposition.

Aberrant long interspersed element 1 (LINE-1 or L1) activity can cause insertional mutagenesis and chromosomal rearrangements and has been detected in several types of cancers. Here, we show that neddylation, a post-translational modification process, is essential for L1 transposition. The antineoplastic drug MLN4924 is an L1 inhibitor that suppresses NEDD8-activating enzyme activity. Neddylation inhibition by MLN4924 selectively impairs ORF2p-mediated L1 reverse transcription and blocks the generation of L1 cDNA. Consistent with these results, MLN4924 treatment suppresses the retrotransposition activity of the non-autonomous retrotransposons short interspersed nuclear element R/variable number of tandem repeat/Alu and Alu, which rely on the reverse transcription activity of L1 ORF2p. The E2 enzyme UBE2M in the neddylation pathway, rather than UBE2F, is required for L1 ORF2p and retrotransposition. Interference with the functions of certain neddylation-dependent Cullin-really interesting new gene E3 ligases disrupts L1 reverse transcription and transposition activity. Our findings provide insights into the regulation of L1 retrotransposition and the identification of therapeutic targets for L1 dysfunctions.

SPOP point mutations regulate substrate preference and affect its function.

The adaptor SPOP recruits substrates to CUL3 E3 ligase for ubiquitination and degradation. Structurally, SPOP harbors a MATH domain for substrate recognition, and a BTB domain responsible for binding CUL3. Reported point mutations always occur in SPOP's MATH domain and are through to disrupt affinities of SPOP to substrates, thereby leading to tumorigenesis. In this study, we identify the tumor suppressor IRF2BP2 as a novel substrate of SPOP. SPOP enables to attenuate IRF2BP2-inhibited cell proliferation and metastasis in HCC cells. However, overexpression of wild-type SPOP alone suppresses HCC cell proliferation and metastasis. In addition, a HCC-derived mutant, SPOP-M35L, shows an increased affinity to IRF2BP2 in comparison with wild-type SPOP. SPOP-M35L promotes HCC cell proliferation and metastasis, suggesting that M35L mutation possibly reprograms SPOP from a tumor suppressor to an oncoprotein. Taken together, this study uncovers mutations in SPOP's MATH lead to distinct functional consequences in context-dependent manners, rather than simply disrupting its interactions with substrates, raising a noteworthy concern that we should be prudent to select SPOP as therapeutic target for cancers.

CUL4A Ubiquitin Ligase Is an Independent Predictor of Overall Survival in Pancreatic Adenocarcinoma.

BACKGROUND/AIM: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with dismal prognosis. Genomic instability due to defects in cell-cycle regulation/mitosis or deficient DNA-damage repair is a major driver of PDAC progression with clinical relevance. Deregulation of licensing of DNA replication leads to DNA damage and genomic instability, predisposing cells to malignant transformation. While overexpression of DNA replication-licensing factors has been reported in several human cancer types, their role in PDAC remains largely unknown. We aimed here to examine the expression and prognostic significance of the DNA replication-licensing factors chromatin licensing and DNA replication factor 1 (CDT1), cell-division cycle 6 (CDC6), minichromosome maintenance complex component 7 (MCM7) and also of the ubiquitin ligase regulator of CDT1, cullin 4A (CUL4A), in PDAC. MATERIALS AND METHODS: Expression levels of CUL4, CDT1, CDC6 and MCM7 were evaluated by immunohistochemistry in 76 formalin-fixed paraffin-embedded specimens of PDAC patients in relation to DNA-damage response marker H2AX, clinicopathological parameters and survival. We also conducted bioinformatics analysis of data from online available databases to corroborate our findings. RESULTS: CUL4A and DNA replication-licensing factors were overexpressed in patients with PDAC and expression of CDT1 positively correlated with H2AX. Expression of CUL4A and CDT1 positively correlated with lymph node metastasis. Importantly, elevated CUL4A expression was associated with reduced overall survival and was an independent indicator of poor prognosis on multivariate analysis. CONCLUSION: Our findings implicate CUL4A, CDT1, CDC6 and MCM7 in PDAC progression and identify CUL4A as an independent prognostic factor for this disease.

CUL4B mutations impair human cortical neurogenesis through PP2A-dependent inhibition of AKT and ERK.

Mutation in CUL4B gene is one of the most common causes for X-linked intellectual disability (XLID). CUL4B is the scaffold protein in CUL4B-RING ubiquitin ligase (CRL4B) complex. While the roles of CUL4B in cancer progression and some developmental processes like adipogenesis, osteogenesis, and spermatogenesis have been studied, the mechanisms underlying the neurological disorders in patients with CUL4B mutations are poorly understood. Here, using 2D neuronal culture and cerebral organoids generated from the patient-derived induced pluripotent stem cells and their isogenic controls, we demonstrate that CUL4B is required to prevent premature cell cycle exit and precocious neuronal differentiation of neural progenitor cells. Moreover, loss-of-function mutations of CUL4B lead to increased synapse formation and enhanced neuronal excitability. Mechanistically, CRL4B complex represses transcription of PPP2R2B and PPP2R2C genes, which encode two isoforms of the regulatory subunit of protein phosphatase 2 A (PP2A) complex, through catalyzing monoubiquitination of H2AK119 in their promoter regions. CUL4B mutations result in upregulated PP2A activity, which causes inhibition of AKT and ERK, leading to premature cell cycle exit. Activation of AKT and ERK or inhibition of PP2A activity in CUL4B mutant organoids rescues the neurogenesis defect. Our work unveils an essential role of CUL4B in human cortical development.

Dynamic molecular architecture and substrate recruitment of cullin3-RING E3 ligase CRL3KBTBD2.

Phosphatidylinositol 3-kinase α, a heterodimer of catalytic p110α and one of five regulatory subunits, mediates insulin- and insulin like growth factor-signaling and, frequently, oncogenesis. Cellular levels of the regulatory p85α subunit are tightly controlled by regulated proteasomal degradation. In adipose tissue and growth plates, failure of K48-linked p85α ubiquitination causes diabetes, lipodystrophy and dwarfism in mice, as in humans with SHORT syndrome. Here we elucidated the structures of the key ubiquitin ligase complexes regulating p85α availability. Specificity is provided by the substrate receptor KBTBD2, which recruits p85α to the cullin3-RING E3 ubiquitin ligase (CRL3). CRL3KBTBD2 forms multimers, which disassemble into dimers upon substrate binding (CRL3KBTBD2-p85α) and/or neddylation by the activator NEDD8 (CRL3KBTBD2~N8), leading to p85α ubiquitination and degradation. Deactivation involves dissociation of NEDD8 mediated by the COP9 signalosome and displacement of KBTBD2 by the inhibitor CAND1. The hereby identified structural basis of p85α regulation opens the way to better understanding disturbances of glucose regulation, growth and cancer.

Proapoptotic effect of WS-299 induced by NOXA accumulation and NRF2-counterbalanced oxidative stress damage through targeting RBX1-UBE2M interaction in gastric cancers.

The abnormal activation of Cullin RING E3 Ligases (CRLs) is closely associated with the occurrence and development of various cancers. Targeting the neddylation pathway represents an effective approach for cancer treatment. In this work, we reported that WS-299, structurally featuring a coumarin moiety attached to the triazolopyrimidine, exhibited excellent anti-proliferative activity in MGC-803 and HGC-27 cells. WS-299 exerted potent anticancer effects by inhibiting clone formation, EdU incorporation and inducing cell cycle arrest. WS-299 inhibited CUL3/5 neddylation and caused an obvious accumulation of Nrf2 and NOXA, substrates of CRL3 and CRL5, respectively. Biochemical studies showed that WS-299 inhibited CUL3 neddylation by inhibiting RBX1-UBE2M interaction. The anti-proliferative effect of WS-299 was mainly induced by NOXA-mediated apoptosis. Of note, Nrf2 attenuated WS-299-induced reactive oxygen species (ROS) levels. Furthermore, Nrf2 accumulation also had an antagonistic effect on NOXA-induced apoptosis. Therefore, WS-299 and siNrf2 synergistically increased ROS levels, apoptotic cells and suppressed tumor growth in vivo. Taken together, our research clarified the anti-cancer mechanisms of WS-299 through targeting the RBX1-UBE2M protein-protein interaction and inhibiting the neddylation modification of CUL3 and CUL5. More importantly, our studies also demonstrated that combination of WS-299 with shNrf2 could be an effective strategy for treating gastric cancers.