Search Results (39)

Ubiquitination is a dynamic and tightly regulated post-translational modification essential for modulating protein stability, trafficking, and function to preserve cellular homeostasis. This process is orchestrated through a hierarchical enzymatic cascade involving three key enzymes: the E1 ubiquitin-activating enzyme, the E2 ubiquitin-conjugating enzyme, and the E3 ubiquitin ligase. The final step of ubiquitination is catalyzed by the E3 ubiquitin ligase, which facilitates the transfer of ubiquitin from the E2 enzyme to the substrate, thereby dictating which proteins undergo ubiquitination. Emerging evidence underscores the critical roles of ubiquitin ligases in neurodevelopment, regulating fundamental processes such as neuronal polarization, axonal outgrowth, synaptogenesis, and synaptic function. Mutations in genes encoding ubiquitin ligases and the consequent dysregulation of these pathways have been increasingly implicated in a spectrum of neurodevelopmental disorders, including autism spectrum disorder, intellectual disability, and attention-deficit/hyperactivity disorder. This review synthesizes current knowledge on the molecular mechanisms underlying neurodevelopment regulated by Cullin-RING ubiquitin ligases—the largest subclass of ubiquitin ligases—and their involvement in the pathophysiology of neurodevelopmental disorders. A deeper understanding of these mechanisms holds significant promise for informing novel therapeutic strategies, ultimately advancing clinical outcomes for individuals affected by neurodevelopmental disorders. Full article

Cullin 2 (Cul2), a core component of the Cullin-RING E3 ubiquitin ligase complex, is integral to regulating distinct biological processes. However, its role in innate immune defenses remains poorly understood. In this study, we investigated the functional significance of Cul2 in the immune deficiency (IMD) signaling-mediated antimicrobial immune reactions in Drosophila melanogaster (fruit fly). We demonstrated that loss-of-function of Cul2 led to a marked reduction in antimicrobial peptide induction following bacterial infection, which was associated with increased fly mortality and bacterial load. The proteomic analysis further revealed that loss-of-function of Cul2 reduced the expression of Effete (Eff), a key E2 ubiquitin-conjugating enzyme during IMD signaling. Intriguingly, ectopic expression of eff effectively rescued the immune defects caused by loss of Cul2. Taken together, the results of our study underscore the critical role of Cul2 in ensuring robust IMD signaling activation, highlighting its importance in the innate immune defense against microbial infection in Drosophila. Full article

Recent discoveries revealed mechanistic insights into the control of adipogenesis by the Constitutive Photomorphogenesis 9 Signalosome (CSN) and its variants, CSN^CSN7A and CSN^CSN7B, which differ in the paralog subunits, CSN7A and CSN7B. CSN^CSN7A and CSN^CSN7B variants form permanent complexes with cullin-RING-ubiquitin ligases 3 and 4A (CRL3 and CRL4A), respectively. These complexes can be found in most eukaryotic cells and represent a critical reservoir for cellular functions. In an early stage of adipogenesis, mitotic clonal expansion (MCE), CSN-CRL1, and CSN^CSN7B-CRL4A are blocked to ubiquitinate the cell cycle inhibitor p27^KIP, leading to cell cycle arrest. In addition, in MCE CSN-CRL complexes rearrange the cytoskeleton for adipogenic differentiation and CRL3^KEAP1 ubiquitylates the inhibitor of adipogenesis C/EBP homologous protein (CHOP) for degradation by the 26S proteasome, an adipogenesis-specific proteolysis. During terminal adipocyte differentiation, the CSN^CSN7A-CRL3 complex is recruited to a lipid droplet (LD) membrane by RAB18. Currently, the configuration of the substrate receptors of CSN^CSN7A-CRL3 on LDs is unclear. CSN^CSN7A-CRL3 is activated by neddylation on the LD membrane, an essential adipogenic step. Damage to CSN/CUL3/CUL4A genes is associated with diverse diseases, including obesity. Due to the tremendous impact of CSN-CRLs on adipogenesis, we need strategies for adequate treatment in the event of malfunctions. Full article

Ubiquitylation is a post-translational modification that modulates protein function and stability. It is orchestrated by the concerted action of three types of enzymes, with substrate specificity governed by ubiquitin ligases (E3s), which may exist as single proteins or as part of multi-protein complexes. Although Cullin (CUL) proteins lack intrinsic enzymatic activity, they participate in the formation of active ubiquitin ligase complexes, known as Cullin-Ring ubiquitin Ligases (CRLs), through their association with ROC1 or ROC2, along with substrate adaptor and receptor proteins. Mammalian genomes encode several CUL proteins (CUL1–9), each contributing to distinct CRLs. Among these CUL proteins, CUL1, CUL3, and CUL4 are believed to be the most ancient and evolutionarily conserved from yeast to mammals, with CUL4 uniquely duplicated in vertebrates. Genetic evidence strongly implicates CUL4-based ubiquitin ligases (CRL4s) in chromatin regulation across various species and suggests that, in vertebrates, CRL4s have also acquired a cytosolic role, which is facilitated by a cytosol-localizing paralog of CUL4. Substrates identified through biochemical studies have elucidated the molecular mechanisms by which CRL4s regulate chromatin and cytosolic processes. The substantial body of knowledge on CUL4 biology amassed over the past two decades provides a unique opportunity to explore the functional evolution of CRL4. In this review, we synthesize the available structural, genetic, and biochemical data on CRL4 from various model organisms and discuss the conserved and novel functions of CRL4s. Full article

Ubiquitination is one of the most important post-translational modifications in eukaryotes. The ubiquitination cascade includes ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). The E3 ligases, responsible for substrate recognition, are the most abundant and varied proteins in the cascade and the most studied. SKP1-CUL1-F-Box (SCF)-type E3 ubiquitin ligases are multi-subunit RING (Really Interesting New Gene) E3 ubiquitin ligases, composed of CUL1 (Cullin 1), RBX1 (RING BOX 1), SKP1 (S-phase Kinase-associated Protein 1), and F-box proteins. In vitro ubiquitination assays, used for studying the specific recognition of substrate proteins by E3 ubiquitin ligases, require the purification of all components involved in the cascade, and for assays with SCF-type E3 ligases, additional proteins (several SCF complex subunits). Here, the Duet expression system was used to co-express E1, E2, ubiquitin, ubiquitylation target (substrate), and the four subunits of a SCF-type E3 ligase in E. coli. When these proteins co-exist in bacterial cells, ubiquitination occurs and can be detected by Western Blot. The effectiveness of this bacterial system for detecting ubiquitination cascade activity was demonstrated by replicating both AtSCF^TIR1-mediated and human SCF^FBXO28-mediated ubiquitylation in bacteria. This system provides a basic but adaptable platform for the study of SCF-type E3 ubiquitin ligases. Full article

Mezigomide is an oral cereblon E3 ligase modulator (CELMoD) that is under clinical investigation in patients with relapsed/refractory (RR) multiple myeloma (MM). Like other CELMoD compounds, mezigdomide acts by altering the conformation of cereblon within the cullin 4A ring ligase–cereblon (CRL4CRBN) E3 ubiquitin ligase complex, thereby recruiting novel protein substrates for selective proteasomal degradation. These include two critical lymphoid transcription factors, Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3), also known as Ikaros and Aiolos, which have important roles in the development and differentiation of hematopoietic cells, in MM pathobiology, and in suppressing the expression of interferon-stimulating genes and T-cell stimulation. Among the CELMoDs, mezigdomide has the greatest cereblon-binding potency, plus the greatest potency for the degradation of Ikaros and Aiolos and subsequent downstream antimyeloma effects. Preclinical studies of mezigdomide have demonstrated its anti-proliferative and apoptotic effects in MM, along with its immune-stimulatory effects and its synergistic activity with other antimyeloma agents, including in lenalidomide-/pomalidomide-resistant MM cell lines and mouse xenograft models. Early-phase clinical trial data indicate notable activity in heavily pretreated patients with RRMM, including those with triple-class-refractory disease, together with a tolerable and manageable safety profile. This review summarizes current preclinical and clinical findings with mezigdomide and its potential future roles in the treatment of MM. Full article

E3 ubiquitin ligases play a pivotal role in ubiquitination, a crucial post-translational modification process. Anaphase-promoting complex (APC), a large cullin-RING E3 ubiquitin ligase, regulates the unidirectional progression of the cell cycle by ubiquitinating specific target proteins and triggering plant immune responses. Several E3 ubiquitin ligases have been identified owing to advancements in sequencing and annotation of the wheat genome. However, the types and functions of APC E3 ubiquitin ligases in wheat have not been reported. This study identified 14 members of the APC gene family in the wheat genome and divided them into three subgroups (CCS52B, CCS52A, and CDC20) to better understand their functions. Promoter sequence analysis revealed the presence of several cis-acting elements related to hormone and stress responses in the APC E3 ubiquitin ligases in wheat. All identified APC E3 ubiquitin ligase family members were highly expressed in the leaves, and the expression of most genes was induced by the application of methyl jasmonate (MeJA). In addition, the APC gene family in wheat may play a role in plant defense mechanisms. This study comprehensively analyzes APC genes in wheat, laying the groundwork for future research on the function of APC genes in response to viral infections and expanding our understanding of wheat immunity mechanisms. Full article

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. Full article

FBXW7 is a critical regulator of cell cycle, cell signaling, and development. A highly conserved F-box protein and component of the SKP1–Cullin–F-box (SCF) complex, FBXW7 functions as a recognition subunit within a Cullin–RING E3 ubiquitin ligase responsible for ubiquitinating substrate proteins and targeting them for proteasome-mediated degradation. In human cells, FBXW7 promotes degradation of a large number of substrate proteins, including many that impact disease, such as NOTCH1, Cyclin E, MYC, and BRAF. A central focus for investigation has been to understand the molecular mechanisms that allow the exquisite substrate specificity exhibited by FBXW7. Recent work has produced a clearer understanding of how FBXW7 physically interacts with both high-affinity and low-affinity substrates. We review new findings that provide insights into the consequences of “hotspot” missense mutations of FBXW7 that are found in human cancers. Finally, we discuss how the FBXW7–substrate interaction, and the kinases responsible for substrate phosphorylation, contribute to patterned protein degradation in C. elegans development. Full article

Autophagy is a tightly regulated catabolic process involved in the degradation and recycling of proteins and organelles. Ubiquitination plays an important role in the regulation of autophagy. Vacuole Membrane Protein 1 (VMP1) is an essential autophagy protein. The expression of VMP1 in pancreatic cancer stem cells carrying the activated Kirsten rat sarcoma viral oncogene homolog (KRAS) triggers autophagy and enables therapy resistance. Using biochemical and cellular approaches, we identified ubiquitination as a post-translational modification of VMP1 from the initial steps in autophagosome biogenesis. VMP1 remains ubiquitinated as part of the autophagosome membrane throughout autophagic flux until autolysosome formation. However, VMP1 is not degraded by autophagy, nor by the ubiquitin–proteasomal system. Mass spectrometry and immunoprecipitation showed that the cell division cycle protein cdt2 (Cdt2), the substrate recognition subunit of the E3 ligase complex associated with cancer, cullin–RING ubiquitin ligase complex 4 (CRL4), is a novel interactor of VMP1 and is involved in VMP1 ubiquitination. VMP1 ubiquitination decreases under the CRL inhibitor MLN4924 and increases with Cdt2 overexpression. Moreover, VMP1 recruitment and autophagosome formation is significantly affected by CRL inhibition. Our results indicate that ubiquitination is a novel post-translational modification of VMP1 during autophagy in human tumor cells. VMP1 ubiquitination may be of clinical relevance in tumor-cell-therapy resistance. Full article

The ubiquitin proteasome system (UPS) is critically important for cellular homeostasis and affects virtually all key functions in normal and neoplastic cells. Currently, a comprehensive review of the role of the UPS in ependymoma (EPN) brain tumors is lacking but may provide valuable new information on cellular networks specific to different EPN subtypes and reveal future therapeutic targets. We have reviewed publicly available EPN gene transcription datasets encoding components of the UPS pathway. Reactome analysis of these data revealed genes and pathways that were able to distinguish different EPN subtypes with high significance. We identified differential transcription of several genes encoding ubiquitin E2 conjugases associated with EPN subtypes. The expression of the E2 conjugase genes UBE2C, UBE2S, and UBE2I was elevated in the ST_EPN_RELA subtype. The UBE2C and UBE2S enzymes are associated with the ubiquitin ligase anaphase promoting complex (APC/c), which regulates the degradation of substrates associated with cell cycle progression, whereas UBE2I is a Sumo-conjugating enzyme. Additionally, elevated in ST_EPN_RELA were genes for the E3 ligase and histone deacetylase HDAC4 and the F-box cullin ring ligase adaptor FBX031. Cluster analysis demonstrated several genes encoding E3 ligases and their substrate adaptors as EPN subtype specific genetic markers. The most significant Reactome Pathways associated with differentially expressed genes for E3 ligases and their adaptors included antigen presentation, neddylation, sumoylation, and the APC/c complex. Our analysis provides several UPS associated factors that may be attractive markers and future therapeutic targets for the subtype-specific treatment of EPN patients. Full article

Cullin 3 (CUL3) is the scaffold of Cullin3 Ring E3-ligases (CRL3s), which use various BTB-adaptor proteins to ubiquitinate numerous substrates targeting their proteasomal degradation. CUL3 mutations, responsible for a severe form of familial hyperkalemia and hypertension (FHHt), all result in a deletion of exon 9 (amino-acids 403-459) (CUL3-∆9). Surprisingly, while CUL3-∆9 is hyperneddylated, a post-translational modification that typically activates CRL complexes, it is unable to ubiquitinate its substrates. In order to understand the mechanisms behind this loss-of function, we performed comparative label-free quantitative analyses of CUL3 and CUL3-∆9 interactome by mass spectrometry. It was observed that CUL3-∆9 interactions with COP9 and CAND1, both involved in CRL3 complexes’ dynamic assembly, were disrupted. These defects result in a reduction in the dynamic cycling of the CRL3 complexes, making the CRL3-∆9 complex an inactive BTB-adaptor trap, as demonstrated by SILAC experiments. Collectively, the data indicated that the hyperneddylated CUL3-∆9 protein is inactive as a consequence of several structural changes disrupting its dynamic interactions with key regulatory partners. Full article

Parkinson’s disease (PD) is a debilitating neurodegenerative disease that causes a great clinical burden. However, its exact molecular pathologies are not fully understood. Whilst there are a number of avenues for research into slowing, halting, or reversing PD, one central idea is to enhance the clearance of the proposed aetiological protein, oligomeric α-synuclein. Oligomeric α-synuclein is the main constituent protein in Lewy bodies and neurites and is considered neurotoxic. Multiple E3 ubiquitin-protein ligases, including the NEDD4 (neural precursor cell expressed developmentally downregulated protein 4) family, parkin, SIAH (mammalian homologues of Drosophila seven in absentia), CHIP (carboxy-terminus of Hsc70 interacting protein), and SCF^FXBL5 SCF ubiquitin ligase assembled by the S-phase kinase-associated protein (SKP1), cullin-1 (Cul1), a zinc-binding RING finger protein, and the F-box domain/Leucine-rich repeat protein 5-containing protein FBXL5), have been shown to be able to ubiquitinate α-synuclein, influencing its subsequent degradation via the proteasome or lysosome. Here, we explore the link between NEDD4 ligases and PD, which is not only via α-synuclein but further strengthened by several additional substrates and interaction partners. Some members of the NEDD4 family of ligases are thought to crosstalk even with PD-related genes and proteins found to be mutated in familial forms of PD. Mutations in NEDD4 family genes have not been observed in PD patients, most likely because of their essential survival function during development. Following further in vivo studies, it has been thought that NEDD4 ligases may be viable therapeutic targets in PD. NEDD4 family members could clear toxic proteins, enhancing cell survival and slowing disease progression, or might diminish beneficial proteins, reducing cell survival and accelerating disease progression. Here, we review studies to date on the expression and function of NEDD4 ubiquitin ligases in the brain and their possible impact on PD pathology. Full article

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. Full article

The ubiquitin–proteasome pathway (UPP) is an important protein degradation pathway that can participate in the regulation of the physiological process of organisms by specifically removing abnormal peptides and degrading cell regulators. UPP mainly involves three enzymes, among which the E3 ubiquitin ligase function is central to UPP. E3 ubiquitin ligases can recruit substrate protein for ubiquitination, and they have various forms. Among them, the Skp1–Cul1–F-box (SCF) complex is the most representative member of the cullin RING ubiquitin ligases type in RING-domain E3 ligases, being mainly composed of Cullin 1, Skp1, Rbx1, and F-box proteins. The F-box protein is the key component for SCF to perform specific functions. The F-box protein is one of the largest protein families in plants, and its family members are involved in the regulation of many key physiological processes, such as growth and development of plants and the response to external stimuli. Herein, we briefly review the structure, classification, function, and hormone signaling pathways of F-box proteins. Full article