CRL4DCAF13 E3 ubiquitin ligase targets MeCP2 for degradation to prevent DNA hypermethylation and ensure normal transcription in growing oocytes.

The DNA methylation is gradually acquired during oogenesis, a process sustained by successful follicle development. However, the functional roles of methyl-CpG-binding protein 2 (MeCP2), an epigenetic regulator displaying specifical binding with methylated DNA, remains unknown in oogenesis. In this study, we found MeCP2 protein was highly expressed in primordial and primary follicle, but was almost undetectable in secondary follicles. However, in aged ovary, MeCP2 protein is significantly increased in both oocyte and granulosa cells. Overexpression of MeCP2 in growing oocyte caused transcription dysregulation, DNA hypermethylation, and genome instability, ultimately leading to follicle growth arrest and apoptosis. MeCP2 is targeted by DCAF13, a substrate recognition adaptor of the Cullin 4-RING (CRL4) E3 ligase, and polyubiquitinated for degradation in both cells and oocytes. Dcaf13-null oocyte exhibited an accumulation of MeCP2 protein, and the partial rescue of follicle growth arrest induced by Dcaf13 deletion was observed following MeCP2 knockdown. The RNA-seq results revealed that large amounts of genes were regulated by the DCAF13-MeCP2 axis in growing oocytes. Our study demonstrated that CRL4DCAF13 E3 ubiquitin ligase targets MeCP2 for degradation to ensure normal DNA methylome and transcription in growing oocytes. Moreover, in aged ovarian follicles, deceased DCAF13 and DDB1 protein were observed, indicating a potential novel mechanism that regulates ovary aging.

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.

Cullin-3 proteins be a novel biomarkers and therapeutic targets for hyperchloremia induced by oral poisoning.

Oral poisoning can trigger diverse physiological reactions, determined by the toxic substance involved. One such consequence is hyperchloremia, characterized by an elevated level of chloride in the blood and leads to kidney damage and impairing chloride ion regulation. Here, we conducted a comprehensive genome-wide analysis to investigate genes or proteins linked to hyperchloremia. Our analysis included functional enrichment, protein-protein interactions, gene expression, exploration of molecular pathways, and the identification of potential shared genetic factors contributing to the development of hyperchloremia. Functional enrichment analysis revealed that oral poisoning owing hyperchloremia is associated with 4 proteins e.g. Kelch-like protein 3, Serine/threonine-protein kinase WNK4, Serine/threonine-protein kinase WNK1 and Cullin-3. The protein-protein interaction network revealed Cullin-3 as an exceptional protein, displaying a maximum connection of 18 nodes. Insufficient data from transcriptomic analysis indicates that there are lack of information having direct associations between these proteins and human-related functions to oral poisoning, hyperchloremia, or metabolic acidosis. The metabolic pathway of Cullin-3 protein revealed that the derivative is Sulfonamide which play role in, increasing urine output, and metabolic acidosis resulted in hypertension. Based on molecular docking results analysis it found that Cullin-3 proteins has the lowest binding energies score and being suitable proteins. Moreover, no major variations were observed in unbound Cullin-3 and all three peptide bound complexes shows that all systems remain compact during 50 ns simulations. The results of our study revealed Cullin-3 proteins be a strong foundation for the development of potential drug targets or biomarker for future studies.

Cullin 3 RING E3 ligase inactivation causes NRF2-dependent NADH reductive stress, hepatic lipodystrophy, and systemic insulin resistance.

Cullin RING E3 ligases (CRL) have emerged as key regulators of disease-modifying pathways and therapeutic targets. Cullin3 (Cul3)-containing CRL (CRL3) has been implicated in regulating hepatic insulin and oxidative stress signaling. However, CRL3 function in liver pathophysiology is poorly defined. Here, we report that hepatocyte Cul3 knockout results in rapid resolution of steatosis in obese mice. However, the remarkable resistance of hepatocyte Cul3 knockout mice to developing steatosis does not lead to overall metabolic improvement but causes systemic metabolic disturbances. Liver transcriptomics analysis identifies that CRL3 inactivation causes persistent activation of the nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant defense pathway, which also reprograms the lipid transcriptional network to prevent TG storage. Furthermore, global metabolomics reveals that NRF2 activation induces numerous NAD+-consuming aldehyde dehydrogenases to increase the cellular NADH/NAD+ ratio, a redox imbalance termed NADH reductive stress that inhibits the glycolysis-citrate-lipogenesis axis in Cul3 knockout livers. As a result, this NRF2-induced cellular lipid storage defect promotes hepatic ceramide accumulation, elevates circulating fatty acids, and worsens systemic insulin resistance in a vicious cycle. Hepatic lipid accumulation is restored, and liver injury and hyperglycemia are attenuated when NRF2 activation and NADH reductive stress are abolished in hepatocyte Cul3/Nrf2 double-knockout mice. The resistance to hepatic steatosis, hyperglycemia, and NADH reductive stress are observed in hepatocyte Keap1 knockout mice with NRF2 activation. In summary, our study defines a critical role of CRL3 in hepatic metabolic regulation and demonstrates that the CRL3 downstream NRF2 overactivation causes hepatic metabolic maladaptation to obesity and insulin resistance.

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.

Structure and dynamics of a pentameric KCTD5/CUL3/Gßγ E3 ubiquitin ligase complex.

Heterotrimeric G proteins can be regulated by posttranslational modifications, including ubiquitylation. KCTD5, a pentameric substrate receptor protein consisting of an N-terminal BTB domain and a C-terminal domain, engages CUL3 to form the central scaffold of a cullin-RING E3 ligase complex (CRL3KCTD5) that ubiquitylates Gßγ and reduces Gßγ protein levels in cells. The cryo-EM structure of a 5:5:5 KCTD5/CUL3NTD/Gß1γ2 assembly reveals a highly dynamic complex with rotations of over 60° between the KCTD5BTB/CUL3NTD and KCTD5CTD/Gßγ moieties of the structure. CRL3KCTD5 engages the E3 ligase ARIH1 to ubiquitylate Gßγ in an E3-E3 superassembly, and extension of the structure to include full-length CUL3 with RBX1 and an ARIH1~ubiquitin conjugate reveals that some conformational states position the ARIH1~ubiquitin thioester bond to within 10 Å of lysine-23 of Gß and likely represent priming complexes. Most previously described CRL/substrate structures have consisted of monovalent complexes and have involved flexible peptide substrates. The structure of the KCTD5/CUL3NTD/Gßγ complex shows that the oligomerization of a substrate receptor can generate a polyvalent E3 ligase complex and that the internal dynamics of the substrate receptor can position a structured target for ubiquitylation in a CRL3 complex.

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.

Targeting kelch-like (KLHL) proteins: achievements, challenges and perspectives.

Kelch-like proteins (KLHLs) are a large family of BTB-containing proteins. KLHLs function as the substrate adaptor of Cullin 3-RING ligases (CRL3) to recognize substrates. KLHLs play pivotal roles in regulating various physiological and pathological processes by modulating the ubiquitination of their respective substrates. Mounting evidence indicates that mutations or abnormal expression of KLHLs are associated with various human diseases. Targeting KLHLs is a viable strategy for deciphering the KLHLs-related pathways and devising therapies for associated diseases. Here, we comprehensively review the known KLHLs inhibitors to date and the brilliant ideas underlying their development.

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.

RNA-binding proteins potentially regulate the alternative splicing of apoptotic genes during knee osteoarthritis progression.

BACKGROUND: Alternative splicing (AS) is a principal mode of genetic regulation and one of the most widely used mechanisms to generate structurally and functionally distinct mRNA and protein variants. Dysregulation of AS may result in aberrant transcription and protein products, leading to the emergence of human diseases. Although considered important for regulating gene expression, genome-wide AS dysregulation, underlying mechanisms, and clinical relevance in knee osteoarthritis (OA) remain unelucidated. Therefore, in this study, we elucidated and validated AS events and their regulatory mechanisms during OA progression. RESULTS: In this study, we identified differentially expressed genes between human OA and healthy meniscus samples. Among them, the OA-associated genes were primarily enriched in biological pathways such as extracellular matrix organization and ossification. The predominant OA-associated regulated AS (RAS) events were found to be involved in apoptosis during OA development. The expression of the apoptosis-related gene BCL2L13, XAF1, and NF2 were significantly different between OA and healthy meniscus samples. The construction of a covariation network of RNA-binding proteins (RBPs) and RAS genes revealed that differentially expressed RBP genes LAMA2 and CUL4B may regulate the apoptotic genes XAF1 and BCL2L13 to undergo AS events during OA progression. Finally, RT-qPCR revealed that CUL4B expression was significantly higher in OA meniscus samples than in normal controls and that the AS ratio of XAF1 was significantly different between control and OA samples; these findings were consistent with their expected expression and regulatory relationships. CONCLUSIONS: Differentially expressed RBPs may regulate the AS of apoptotic genes during knee OA progression. XAF1 and its regulator, CUL4B, may serve as novel biomarkers and potential therapeutic targets for this disease.

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.

Plasmodium falciparum contains functional SCF and CRL4 ubiquitin E3 ligases, and CRL4 is critical for cell division and membrane integrity.

Protein ubiquitination is essential for cellular homeostasis and regulation of several processes, including cell division and genome integrity. Ubiquitin E3 ligases determine substrate specificity for ubiquitination, and Cullin-RING E3 ubiquitin ligases (CRLs) make the largest group among the ubiquitin E3 ligases. Although conserved and most studied in model eukaryotes, CRLs remain underappreciated in Plasmodium and related parasites. To investigate the CRLs of human malaria parasite Plasmodium falciparum, we generated parasites expressing tagged P. falciparum cullin-1 (PfCullin-1), cullin-2 (PfCullin-2), Rbx1 (PfRbx1) and Skp1 (PfSkp1). PfCullin-1 and PfCullin-2 were predominantly expressed in erythrocytic trophozoite and schizont stages, with nucleocytoplasmic localization and chromatin association, suggesting their roles in different cellular compartments and DNA-associated processes. Immunoprecipitation, in vitro protein-protein interaction, and ubiquitination assay confirmed the presence of a functional Skp1-Cullin-1-Fbox (PfSCF) complex, comprising of PfCullin-1, PfRbx1, PfSkp1, PfFBXO1, and calcyclin binding protein. Immunoprecipitation, sequence analysis, and ubiquitination assay indicated that PfCullin-2 forms a functional human CRL4-like complex (PfCRL4), consisting of PfRbx1, cleavage and polyadenylation specificity factor subunit_A and WD40 repeat proteins. PfCullin-2 knock-down at the protein level, which would hinder PfCRL4 assembly, significantly decreased asexual and sexual erythrocytic stage development. The protein levels of several pathways, including protein translation and folding, lipid biosynthesis and transport, DNA replication, and protein degradation were significantly altered upon PfCullin-2 depletion, which likely reflects association of PfCRL4 with multiple pathways. PfCullin-2-depleted schizonts had poorly delimited merozoites and internal membraned structures, suggesting a role of PfCRL4 in maintaining membrane integrity. PfCullin-2-depleted parasites had a significantly lower number of nuclei/parasite than the normal parasites, indicating a crucial role of PfCRL4 in cell division. We demonstrate the presence of functional CRLs in P. falciparum, with crucial roles for PfCRL4 in cell division and maintaining membrane integrity.

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.

Modulation of Cullin-RING E3 ubiquitin ligase-dependent ubiquitination by small molecule compounds.

Cullin (CUL)-RING (Really Interesting New Gene) E3 ubiquitin (Ub) ligases (CRLs) are the largest E3 family. The E3 CRL core ligase is a subcomplex formed by the CUL C-terminal domain bound with the ROC1/RBX1 RING finger protein, which acts as a hub that mediates and organizes multiple interactions with E2, Ub, Nedd8, and the ARIH family protein, thereby resulting in Ub transfer to the E3-bound substrate. This report describes the modulation of CRL-dependent ubiquitination by small molecule compounds including KH-4-43, #33, and suramin, which target the CRL core ligases. We show that both KH-4-43 and #33 inhibit the ubiquitination of CK1α by CRL4CRBN. However, either compound's inhibitory effect on this reaction is significantly reduced when a neddylated form of CRL4CRBN is used. On the other hand, both #33 and KH-4-43 inhibit the ubiquitination of ß-catenin by CRL1ß-TrCP and Nedd8-CRL1ß-TrCP almost equally. Thus, neddylation of CRL1ß-TrCP does not negatively impact the sensitivity to inhibition by #33 and KH-4-43. These findings suggest that the effects of neddylation to alter the sensitivity of CRL inhibition by KH-4-43/#33 is dependent upon the specific CRL type. Suramin, a compound that targets CUL's basic canyon, can effectively inhibit CRL1/4-dependent ubiquitination regardless of neddylation status, in contrast to the results observed with KH-4-43/#33. This observed differential drug sensitivity of KH-4-43/#33 appears to echo CUL-specific Nedd8 effects on CRLs as revealed by recent high-resolution structural biology efforts. The highly diversified CRL core ligase structures may provide opportunities for specific targeting by small molecule modulators.

Cullin-RING ligases employ geometrically optimized catalytic partners for substrate targeting.

Cullin-RING ligases (CRLs) ubiquitylate specific substrates selected from other cellular proteins. Substrate discrimination and ubiquitin transferase activity were thought to be strictly separated. Substrates are recognized by substrate receptors, such as Fbox or BCbox proteins. Meanwhile, CRLs employ assorted ubiquitin-carrying enzymes (UCEs, which are a collection of E2 and ARIH-family E3s) specialized for either initial substrate ubiquitylation (priming) or forging poly-ubiquitin chains. We discovered specific human CRL-UCE pairings governing substrate priming. The results reveal pairing of CUL2-based CRLs and UBE2R-family UCEs in cells, essential for efficient PROTAC-induced neo-substrate degradation. Despite UBE2R2's intrinsic programming to catalyze poly-ubiquitylation, CUL2 employs this UCE for geometrically precise PROTAC-dependent ubiquitylation of a neo-substrate and for rapid priming of substrates recruited to diverse receptors. Cryo-EM structures illuminate how CUL2-based CRLs engage UBE2R2 to activate substrate ubiquitylation. Thus, pairing with a specific UCE overcomes E2 catalytic limitations to drive substrate ubiquitylation and targeted protein degradation.

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.

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.

Emerging Roles of Cullin-RING Ubiquitin Ligases in Cardiac Development.

Heart development is a spatiotemporally regulated process that extends from the embryonic phase to postnatal stages. Disruption of this highly orchestrated process can lead to congenital heart disease or predispose the heart to cardiomyopathy or heart failure. Consequently, gaining an in-depth understanding of the molecular mechanisms governing cardiac development holds considerable promise for the development of innovative therapies for various cardiac ailments. While significant progress in uncovering novel transcriptional and epigenetic regulators of heart development has been made, the exploration of post-translational mechanisms that influence this process has lagged. Culling-RING E3 ubiquitin ligases (CRLs), the largest family of ubiquitin ligases, control the ubiquitination and degradation of ~20% of intracellular proteins. Emerging evidence has uncovered the critical roles of CRLs in the regulation of a wide range of cellular, physiological, and pathological processes. In this review, we summarize current findings on the versatile regulation of cardiac morphogenesis and maturation by CRLs and present future perspectives to advance our comprehensive understanding of how CRLs govern cardiac developmental processes.

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.

Mechanism of millisecond Lys48-linked poly-ubiquitin chain formation by cullin-RING ligases.

E3 ubiquitin ligases, in collaboration with E2 ubiquitin-conjugating enzymes, modify proteins with poly-ubiquitin chains. Cullin-RING ligase (CRL) E3s use Cdc34/UBE2R-family E2s to build Lys48-linked poly-ubiquitin chains to control an enormous swath of eukaryotic biology. Yet the molecular mechanisms underlying this exceptional linkage specificity and millisecond kinetics of poly-ubiquitylation remain unclear. Here we obtain cryogenic-electron microscopy (cryo-EM) structures that provide pertinent insight into how such poly-ubiquitin chains are forged. The CRL RING domain not only activates the E2-bound ubiquitin but also shapes the conformation of a distinctive UBE2R2 loop, positioning both the ubiquitin to be transferred and the substrate-linked acceptor ubiquitin within the active site. The structures also reveal how the ubiquitin-like protein NEDD8 uniquely activates CRLs during chain formation. NEDD8 releases the RING domain from the CRL, but unlike previous CRL-E2 structures, does not contact UBE2R2. These findings suggest how poly-ubiquitylation may be accomplished by many E2s and E3s.