Ubiquitin-conjugating Enzyme Research Articles

VEXAS syndrome: an adult-onset autoinflammatory disorder with underlying somatic mutation.

VEXAS syndrome (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) was first described in 2020, where in a cohort of adults with unexplained fever or inflammation, systematic genetic testing was performed and 25 men with a median age of 64 years and somatic mutations in the UBA1 gene were identified. In the current review, we aim to discuss the relevant literature from January 2023 until July 2024 to give new insights into the pathophysiology, epidemiology, diagnosis and treatment of VEXAS. VEXAS affects 1 : 4269 in men over the age of 50. Janus-Kinase-inhibitors (JAKi) and IL-6-inhibitors are more effective immunosuppressants against hyperinflammation. Ruxolitinib is more effective than other JAKi. Azacitidine induces remission in many patients, but only few MDS-associated patients were treated. Allogeneic stem cell transplantation is feasible for selected cases. Infections are the major cause of death. Prognosis is still poor with a 5-year mortality rate of 18-40%. In the current review, we discuss the novelties for VEXAS, including pathogenic pathways, epidemiological data, diagnostic criteria and algorithms, treatment options and complications. We hope that this review may improve rheumatologists understanding of VEXAS. We strongly recommend enrolling VEXAS patients in registries and clinical trials, to improve prognosis of VEXAS in the future.

Dynamic Expression of Genes Encoding Ubiquitin Conjugating Enzymes (E2s) During Neuronal Differentiation and Maturation: Implications for Neurodevelopmental Disorders and Neurodegenerative Diseases

Background: The ubiquitination process plays a crucial role in neuronal differentiation and function. Numerous studies have focused on the expression and functions of E3 ligases during these different stages, far fewer on E2 conjugating enzymes. In mice, as in humans, these E2s belong to 17 conjugating enzyme families. Objectives: We analyzed by real-time PCR the expression dynamics of all known E2 genes during an in vitro differentiation of mouse hippocampal neuronal cultures, and after, we analyzed their stimulation with N-methyl-D-aspartate (NMDA). Results: We found that 36 of the 38 E2 genes were expressed in hippocampal neurons. Many were up-regulated during neuritogenesis and/or synaptogenesis stages, such as Ube2h, Ube2b, and Aktip. Rapid and delayed responses to NMDA stimulation were associated with the increased expression of several E2 genes, such as Ube2i, the SUMO-conjugating E2 enzyme. We also observed similar expression profiles within the same E2 gene family, consistent with the presence of similar transcription factor binding sites in their respective promoter sequences. Conclusions: Our study indicates that specific expression profiles of E2 genes are correlated with changes in neuronal differentiation and activity. A better understanding of the regulation and function of E2s is needed to better understand the role played by the ubiquitination process in physiological mechanisms and pathophysiological alterations involved in neurodevelopmental or neurodegenerative diseases.

TAF15 inhibits p53 nucleus translocation and promotes HCC cell 5-FU resistance via post-transcriptional regulation of UBE2N.

Chemotherapy resistance is an important factor responsible for the low 5-year survival rate of hepatocellular carcinoma (HCC) patients. Ubiquitin-conjugating enzyme E2N (UBE2N) is a cancer-associated ubiquitin-conjugating enzyme that is expressed in HCC tissues, and its high expression is associated with a poor prognosis. This study explored the role played by UBE2N in development of 5-fluorouracil (5-FU) resistance in HCC cells. Three HCC cell lines (HepG2 [p53 wild type], Huh7 [p53 point mutant type], Hep3B [p53 non-expression type]), and one normal liver cell line (MIHA) were used in our present study. The IC50 value of 5-FU was determined using a cell counting kit-8 (CCK-8) assay. Cell viability was assessed by colony formation assays. TUNEL assays and flow cytometry were used to analyze cell apoptosis. RNA pull-down and RNA immunoprecipitation (RIP) assays were performed to confirm the binding relationship between UBE2N mRNA and TAF15 protein. Our results showed that TAF15 and UBE2N were highly expressed in HCC cells. UBE2N inhibited the translocation of p53 protein into the cell nucleus to increase 5-FU resistance, as reflected by an increased IC50 value, an increase in cell viability, and a reduction in cell apoptosis. Overexpression of p53 reduced 5-FU resistance, but that effect could be reversed by UBE2N overexpression. TAF15 protein bound to and stabilized UBE2N mRNA, thereby inhibiting p53 translocation into the nucleus and promoting 5-FU resistance in HCC cells. Collectively, our present study identified a novel mechanism by which TAF15/UBE2N regulates p53 distribution to increase 5-FU resistance. Our results also suggest potential therapeutic strategies for treating HCC.

UBE2C promotes LUAD progression by ubiquitin-dependent degradation of p53 to inactivate the p53/p21 signaling pathway

Lung adenocarcinoma (LUAD) is one of the greatest causes of cancer death worldwide. As a novel potential tumor biomarker, ubiquitin-conjugating enzyme E2C (UBE2C) is a critical factor during the onset and development of human cancers. However, the mechanisms of UBE2C in LUAD are not well understood. In this study, increased expression level of UBE2C was observed in LUAD tumor tissues. High LUAD level portended a worse prognosis of LUAD patients. Down-regulation of UBE2C attenuated the cell proliferation and cycle, migration, and invasion. Consistently, the tumorigenic capacity of LUAD cells in nude mice was significantly suppressed by the knockdown of UBE2C. Knockdown of UBE2C inhibited the degradation of p53 protein via an ubiquitin-proteasome pathway, thereby increasing p53 and p21 protein expression. Moreover, the inhibition of LUAD cell malignant phenotypes caused by UBE2C knockdown was attenuated on account of the inactivation of p53/p21 signaling pathway. In conclusion, UBE2C facilitates cell malignant behaviour in LUAD by ubiquitin-dependent degradation of p53 to suppress the p53/p21 signaling pathway. UBE2C is potentially developed as a therapeutic target for patients with LUAD.

Novel use of Siltuximab in a patient with VEXAS Syndrome.

VEXAS syndrome (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) is an increasingly recognized disorder that occurs due to somatic mutations of a ubiquitin-activating enzyme encoded by ubiquitin-like modifier activating enzyme 1 gene, UBA1. Clinical findings associated with VEXAS syndrome include recurrent fevers, polychondritis, periorbital edema, pleural effusions, myocarditis and/or pericarditis, hepatosplenomegaly, myelodysplastic syndrome, cytopenias, inflammatory arthritis, neutrophilic dermatosis, and deep venous thrombosis. Novel renal manifestations like interstitial nephritis are infrequent, and to our knowledge, acute renal failure due to C3 glomerulonephritis (C3GN) has not yet been reported. Overwhelming systemic inflammation can result in morbid end-organ damage and death. While there is no formal guideline or established protocol for its management, treatment of VEXAS syndrome with tocilizumab, an interleukin-6 (IL-6)-directed therapy, has been described in the literature. Here, we report a case of a 71-year-old male patient presenting with C3GN as an initial manifestation of VEXAS syndrome and explore the rationale for our approach to treatment with IL-6 blockade. Our patient was initially treated with two inpatient doses of tocilizumab with successful transition to siltuximab in the outpatient setting. He continues to benefit from ongoing siltuximab treatment for more than one year to date without any safety issues or relapse of VEXAS syndrome.

UBC18 E2 conjugating enzyme depends on SINAT1 E3 ligase to destabilize the ESCRT component FREE1 in plant iron deficiency responses.

E2 ubiquitin-conjugating enzymes play a crucial role in the ubiquitination process by catalyzing ubiquitin transfer. Although the function of ubiquitin-protein ligases (E3s) in plants response to diverse abiotic stress by targeting specific substrates has been well studied, the involvement of E2s in environmental responses and their downstream targets are not well understood. In this study, we demonstrated that the E2 ubiquitin-conjugating enzyme 18 (UBC18) influences the stability of FREE1 to modulate iron deficiency stress. UBC18 affects the ubiquitination of FREE1 and promotes its degradation, and overexpression of UBC18 decreases plants' sensitivity to iron deficiency by reducing FREE1 level, whereas the ubc18 mutant exhibits sensitivity due to elevated FREE1 accumulation. This study also identified that lysine residues K227, K295, K315, and K540 are required for FREE1 ubiquitination and stability regulation. Mutating these lysine residues in FREE1 resulted in plants' sensitivity to iron starvation. Taken together, our findings shed light on the mechanism of UBC18 in responding to iron deficiency stress by modulating the abundance of FREE1, and further elucidate the role of ubiquitination sites in FREE1 stability regulation and the plant iron deficiency response.

Features of Vacuoles, E1 enzyme, X-linked, autoinflammatory, and somatic mutation syndrome associated with the c.121A>C, p.(Met41Leu) UBA1 genetic variant: A systematic review

Abstract Introduction/Objective Vacuoles, E1 enzyme, X-linked, autoinflammatory, and somatic mutation (VEXAS) syndrome was first described in 2020. VEXAS is driven by a mutation in the UBA1 gene on hematopoietic progenitor cells that encodes the E1 ubiquitin-activating protein. Loss of this protein leads to various inflammatory clinical presentations ranging from dermatological, pulmonary, rheumatologic and hematologic. Many UBA1 variants have been identified. We did a systematic review focusing on a particular UBA1 variant gene mutation underlying this disease process. Methods/Case Report Databases were searched for articles discussing VEXAS syndrome until April 2024. Only case reports and case series in the English language were included. Data on patient demographics, clinical presentation, laboratory values, bone marrow findings, dermatologic presentation and skin biopsy findings were extracted. Results (if a Case Study enter NA) 332 articles discussing VEXAS syndrome were retrieved, of which 115 were case reports or case series. These were screened for patients carrying a p.Met41Leu UBA1 gene mutation and only the 25 patients with this genetic variant were ultimately included in our study. The average age was 68 years (59-81 years). 24/25 (96%) patients reported dermatologic symptoms ranging from erythema to urticarial eruptions to erythema multiforme. Fever was the second most common presenting complaint (76%) followed by arthritis (68%), chondritis (36%) and thromboembolic disease (28%). Macrocytic anemia was a common laboratory finding (65%). MDS was seen in 52% of the patients. All patients had hematopoietic progenitor cells with vacuoles. Sweet’s syndrome was the most common initial diagnosis (52%) as skin biopsies in most patients showed dermal edema and neutrophilic dermatoses/infiltration. Conclusion The c.121A>C, p.(Met41Leu) UBA1 variant is frequently related to cutaneous manifestations. In patients with clinical features of an inflammatory disorder with dermatological and hematologic abnormalities, testing for the p.Met41Leu UBA1 mutation might lead to an earlier diagnosis and prompt initiation of effective treatment.

Urticarial vasculitis

Purpose of review Urticarial vasculitis is a rare condition manifesting with a variety of clinical presentations ranging from skin limited lesions to life-threatening systemic illnesses. This review aims to highlight the recent findings on the etiology, diagnostic modalities, and therapeutic strategies and course of urticarial vasculitis. Recent findings In addition to well established triggers, urticarial vasculitis (UV) cases associated with severe acute respiratory syndrome coronavirus 2 (SARS-Cov2) disease and COVID-19 vaccines, vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome, and adenosine deaminase (ADA) deficiency have been reported. A clinical-dermoscopic model for differentiating urticarial vasculitis has been developed with purpuric patches and globules favoring UV diagnosis and thus diminishing the need for histopathology. The efficacy of treatment modalities has been reviewed, and antihistamines, systemic corticosteroids, omalizumab, cyclophosphamide, tocilizumab, anti-interleukin (IL)-1 agents, and rituximab were shown to have the highest success rates. Regarding the durability of remission, rituximab, dapsone, and MMF were related to long-lasting treatment free responses. The course of hypocomplementemic urticarial vasculitis was investigated in an epidemiological study, revealing 5- and 10-year survival rates of 92% and 83%, respectively. Chronic obstructive pulmonary disease, septicemia, and end-stage renal disease were identified as causes of mortality. Summary With the aid of dermoscopy, a noninvasive tool, differentiation from chronic spontaneous urticaria can be made, and the need for histopathological examination can be diminished. Although clear definitions and consensus criteria for performing disease severity are lacking, careful screening is needed to tailor the treatment on an individual basis. Emerging infections like SARS-CoV 2, vaccines, and autoinflammatory disorders like VEXAS syndrome and ADA deficiency are new associations. The optimal use of well established agents like systemic corticosteroids and immunomodulators are mainstay treatment modalities, whereas IL-1 inhibitors, omalizumab, rituximab and Janus Kinase inhibitors may represent viable alternatives in selected cases.

Mechanism of nucleosomal H2A K13/15 monoubiquitination and adjacent dual monoubiquitination by RNF168.

The DNA damage repair regulatory protein RNF168, a monomeric RING-type E3 ligase, has a crucial role in regulating cell fate and DNA repair by specific and efficient ubiquitination of the adjacent K13 and K15 (K13/15) sites at the H2A N-terminal tail. However, understanding how RNF168 coordinates with its cognate E2 enzyme UbcH5c to site-specifically ubiquitinate H2A K13/15 has long been hampered by the lack of high-resolution structures of RNF168 and UbcH5c~Ub (ubiquitin) in complex with nucleosomes. Here we developed chemical strategies and determined the cryo-electron microscopy structures of the RNF168-UbcH5c~Ub-nucleosome complex captured in transient H2A K13/15 monoubiquitination and adjacent dual monoubiquitination reactions, providing a 'helix-anchoring' mode for monomeric E3 ligase RNF168 on nucleosome in contrast to the 'compass-binding' mode of dimeric E3 ligases. Our work not only provides structural snapshots of H2A K13/15 site-specific monoubiquitination and adjacent dual monoubiquitination but also offers a near-atomic-resolution structural framework for understanding pathogenic amino acid substitutions and physiological modifications of RNF168.

TRAF7 determines circadian period through ubiquitination and degradation of DBP.

D-site binding protein, DBP, is a clock-controlled transcription factor and drives daily rhythms of physiological processes through the regulation of an array of genes harboring a DNA binding motif, D-box. DBP protein levels show a circadian oscillation with an extremely robust peak/trough ratio, but it is elusive how the temporal pattern is regulated by post-translational regulation. In this study, we show that DBP protein levels are down-regulated by the ubiquitin-proteasome pathway. Analysis using 19 dominant-negative forms of E2 enzymes have revealed that UBE2G1 and UBE2T mediate the degradation of DBP. A proteomic analysis of DBP-interacting proteins and database screening have identified Tumor necrosis factor Receptor-Associated Factor 7 (TRAF7), a RING-type E3 ligase, that forms a complex with UBE2G1 and/or UBE2T. Ubiquitination analysis have revealed that TRAF7 enhances K48-linked polyubiquitination of DBP in cultured cells. Overexpression of TRAF7 down-regulates DBP protein level, while knockdown of TRAF7 up-regulates DBP in cultured cells. Knockout of TRAF7 in NIH3T3 cells have revealed that TRAF7 mediates the time-of-the-day-dependent regulation of DBP levels. Furthermore, TRAF7 has a period-shortening effect on the cellular clock. Together, TRAF7 plays an important role in circadian clock oscillation through destabilization of DBP.

SENP1 mediates zinc-induced ZnT6 deSUMOylation at Lys-409 involved in the regulation of zinc metabolism in Golgi apparatus

Zinc (Zn) transporters contribute to the maintenance of intracellular Zn homeostasis in vertebrate, whose activity and function are modulated by post-translational modification. However, the function of small ubiquitin-like modifier (SUMOylation) in Zn metabolism remains elusive. Here, compared with low Zn group, a high-Zn diet significantly increases hepatic Zn content and upregulates the expression of metal-response element-binding transcription factor-1 (MTF-1), Zn transporter 6 (ZnT6) and deSUMOylation enzymes (SENP1, SENP2, and SENP6), but inhibits the expression of SUMO proteins and the E1, E2, and E3 enzymes. Mechanistically, Zn triggers the activation of the MTF-1/SENP1 pathway, resulting in the reduction of ZnT6 SUMOylation at Lys 409 by small ubiquitin-like modifier 1 (SUMO1), and promoting the deSUMOylation process mediated by SENP1. SUMOylation modification of ZnT6 has no influence on its localization but reduces its protein stability. Importantly, deSUMOylation of ZnT6 is crucial for controlling Zn export from the cytosols into the Golgi apparatus. In conclusion, for the first time, we elucidate a novel mechanism by which SUMO1-catalyzed SUMOylation and SENP1-mediated deSUMOylation of ZnT6 orchestrate the regulation of Zn metabolism within the Golgi apparatus.

Neddylation and Its Target Cullin 3 Are Essential for Adipocyte Differentiation.

The ongoing obesity epidemic has raised awareness of the complex physiology of adipose tissue. Abnormal adipocyte differentiation results in the development of systemic metabolic disorders such as insulin resistance and diabetes. The conjugation of NEDD8 (neural precursor cell expressed, developmentally downregulated 8) to target protein, termed neddylation, has been shown to mediate adipogenesis. However, much remains unknown about its role in adipogenesis. Here, we demonstrated that neddylation and its targets, the cullin (CUL) family members, are differentially regulated during mouse and human adipogenesis. Inhibition of neddylation by MLN4924 significantly reduced adipogenesis of 3T3-L1 and human stromal vascular cells. Deletion of NAE1, a subunit of the only NEDD8 E1 enzyme, suppressed neddylation and impaired adipogenesis. Neddylation deficiency did not affect mitotic cell expansion. Instead, it disrupted CREB/CEBPβ/PPARγ signaling, essential for adipogenesis. Interestingly, among the neddylation-targeted CUL family members, deletion of CUL3, but not CUL1, CUL2, or CUL4A, largely replicated the adipogenic defects observed with neddylation deficiency. A PPARγ agonist minimally rescued the adipogenic defects caused by the deletion of NAE1 and CUL3. In conclusion, our study demonstrates that neddylation and its targeted CUL3 are crucial for adipogenesis. These findings provide potential targets for therapeutic intervention in obesity and metabolic disorders.

Promotion of RNF168-Mediated Nucleosomal H2A Ubiquitylation by Structurally defined K63-Polyubiquitylated Linker Histone H1.

The chemical synthesis of histones with homogeneous modifications is a potent approach for quantitatively deciphering the functional crosstalk between different post-translational modifications (PTMs). Here, we developed an expedient site-specific (poly)ubiquitylation strategy (CAEPL, Cysteine-Aminoethylation coupled with Enzymatic Protein Ligation), which integrates the Cys-aminoethylation reaction with the process of ubiquitin-activating enzyme UBA1-assisted native chemical ligation. Using this strategy, we successfully prepared monoubiquitylated and K63-linked di- and tri-ubiquitylated linker histone H1.0 proteins, which were incorporated into individual chromatosomes. Quantitative biochemical analysis of different RNF168 constructs on ubiquitylated chromatosomes with different ubiquitin chain lengths demonstrated that K63-linked polyubiquitylated H1.0 could directly stimulate RNF168 ubiquitylation activity by enhancing the affinity between RNF168 and chromatosome. Subsequent cryo-EM structural analysis of the RNF168/UbcH5c-Ub/H1.0-K63-Ub3 chromatosome complex revealed the potential recruitment orientation between RNF168 UDM1 domain and K63-linked ubiquitin chain on H1.0. Finally, we explored the impact of H1.0 ubiquitylation on RNF168 activity in the context of asymmetric H1.0-K63-Ub3 di-nucleosome substrate, revealing a comparable stimulation effect of both the inter- and intra-nucleosomal crosstalk. Overall, our study highlights the significance of access to structurally-defined polyubiquitylated H1.0 by CAEPL strategy, enabling in-depth mechanistic investigations of in-trans PTM crosstalk between linker histone H1.0 and core histone H2A ubiquitylation.

Tsg101 UEV Interaction with Nedd4 HECT Relieves E3 Ligase Auto-Inhibition, Promoting HIV-1 Assembly and CA-SP1 Maturation Cleavage

Tsg101, a component of the endosomal sorting complex required for transport (ESCRT), is responsible for recognition of events requiring the machinery, as signaled by cargo tagging with ubiquitin (Ub), and for recruitment of downstream acting subunits to the site. Although much is known about the latter function, little is known about its role in the earlier event. The N-terminal domain of Tsg101 is a structural homologue of Ub conjugases (E2 enzymes) and the protein associates with Ub ligases (E3 enzymes) that regulate several cellular processes including virus budding. A pocket in the domain recognizes a motif, PT/SAP, that permits its recruitment. PT/SAP disruption makes budding dependent on Nedd4L E3 ligases. Using HIV-1 encoding a PT/SAP mutation that makes budding Nedd4L-dependent, we identified as critical for rescue the residues in the catalytic (HECT) domain of the E3 enzyme that lie in proximity to sites in Tsg101 that bind Ub non-covalently. Mutation of these residues impaired rescue by Nedd4L but the same mutations had no apparent effect in the context of a Nedd4 isomer, Nedd4-2s, whose N-terminal (C2) domain is naturally truncated, precluding C2-HECT auto-inhibition. Surprisingly, like small molecules that disrupt Tsg101 Ub-binding, small molecules that interfered with Nedd4 substrate recognition arrested budding at an early stage, supporting the conclusion that Tsg101–Ub–Nedd4 interaction promotes enzyme activation and regulates Nedd4 signaling for viral egress. Tsg101 regulation of E3 ligases may underlie its broad ability to function as an effector in various cellular activities, including viral particle assembly and budding.

The activation of the Notch signaling pathway by UBE2C promotes the proliferation and metastasis of hepatocellular carcinoma

UBE2C, a ubiquitin-conjugating enzyme, functions as an oncogene in different types of human cancers. Nonetheless, the exact influence of UBE2C on the development of HCC via regulation of ubiquitination remains uncertain. Here, we found that UBE2C displayed elevated levels of expression in HCC and was associated with an unfavorable prognosis, as evidenced by the analysis of the TCGA database and the examination of clinical specimens. The role of UBE2C in HCC revealed its ability to promote the growth and metastasis of HCC. Mechanistically, UBE2C activated Notch signaling, as evidenced by the upregulation of N1ICD and Hes1, crucial components of the Notch pathway, and activation of the RBP-JK luciferase reporter by UBE2C. Finally, rescue experiments demonstrated that the oncogenic role of UBE2C was eliminated through treatment with the Notch inhibitor DAPT, while overexpression of N1ICD alleviated the anticarcinogenic impact of knockdown of UBE2C. Altogether, the results of our study indicate that UBE2C plays a role in the activation of Notch signaling and could potentially serve as a viable target for therapeutic interventions in HCC.

A case of VEXAS (vacuoles, E1 enzyme, X‐linked, autoinflammatory, somatic) syndrome presenting as progressive multisystem involvement with parenchymal infiltrates following infection with Epstein Barr virus

AbstractVEXAS (vacuoles, E1 enzyme, X‐linked, autoinflammatory, and somatic) syndrome is a rare multisystem disease affecting predominantly males over 50 and manifesting as widespread progressive inflammatory sequelae and haematological dysfunction. We describe a patient who presented with systemic symptoms of fevers, night sweats and weight loss, and developed progressive inflammatory sequelae including cutaneous lesions, haematological dysfunction, lymphadenopathy, migratory inflammatory arthropathies, with new pulmonary infiltrates, following infection with Epstein Barr Virus. Laboratory investigations, bronchoscopy, bone marrow biopsy and imaging were consistent with an inflammatory aetiology. The constellation of organ system involvement, laboratory, biopsy, and imaging results were suspicious for VEXAS syndrome, and this diagnosis was confirmed by identification of a somatic mutation in the UBA1 gene following extensive exclusion of infectious and autoimmune causes. Interestingly the onset of the VEXAS syndrome coincided with serological confirmation of Epstein Barr Virus raising the importance of further exploration into the underlying aetiology of VEXAS syndrome.

Combination screen in multi-cell type tumor spheroids reveals interaction between aryl hydrocarbon receptor antagonists and E1 ubiquitin-activating enzyme inhibitor

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates genes of drug transporters and metabolic enzymes to detoxify small molecule xenobiotics. It has a complex role in cancer biology, influencing both the progression and suppression of tumors by modulating malignant properties of tumor cells and anti-tumor immunity, depending on the specific tumor type and developmental stage. This has led to the discovery and development of selective AhR modulators, including BAY 2416964 which is currently in clinical trials. To identify small molecule anticancer agents that might be combined with AhR antagonists for cancer therapy, a high-throughput combination screen was performed using multi-cell type tumor spheroids grown from malignant cells, endothelial cells, and mesenchymal stem cells. The AhR selective antagonists BAY 2416964, GNF351, and CH-223191 were tested individually and in combination with twenty-five small molecule anticancer agents. As single agents, BAY 2416964 and CH-223191 showed minimal activity, whereas GNF351 reduced the viability of some spheroid models at concentrations greater than 1 µM. The activity of most combinations aligned well with the single agent activity of the combined agent, without apparent contributions from the AhR antagonist. All three AhR antagonists sensitized tumor spheroids to TAK-243, an E1 ubiquitin-activating enzyme inhibitor. These combinations were active in spheroids containing bladder, breast, ovary, kidney, pancreas, colon, and lung tumor cell lines. The AhR antagonists also potentiated pevonedistat, a selective inhibitor of the NEDD8-activating enzyme E1 regulatory subunit, in several tumor spheroid models. In contrast, the AhR antagonists did not enhance the cytotoxicity of the proteasome inhibitor bortezomib.

CBL-b E3 ligase-mediated neddylation and activation of PARP-1 induce vascular calcification.

Vascular calcification (VC) refers to the accumulation of mineral deposits on the walls of arteries and veins, and it is closely associated with increased mortality in cardiovascular disease patients, particularly among high-risk patients with diabetes and chronic kidney disease (CKD). Neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8) is a ubiquitin-like protein that plays a pivotal role in various cellular functions, primarily through its conjugation to target proteins and subsequent relay of biological signals. However, the role of NEDDylation in VC has not been investigated. In our study, we observed that MLN4924, an inhibitor of the NEDD8-activating E1 enzyme, effectively impedes the progression of VC. LC‒MS/MS analysis revealed that poly(ADP‒ribose) polymerase 1 (PARP-1) is subjected to NEDD8 conjugation, leading to an increase in PARP-1 activity during VC. We subsequently revealed that PARP-1 NEDDylation is mediated by the E3 ligase CBL proto-oncogene B (CBL-b) and is reversed by NEDD8-specific protease 1 (NEDP-1) during VC. Furthermore, the CBL-b C373 peptide effectively mitigated the inactive form of the E3 ligase activity of CBL-b, ultimately preventing VC. These findings provide compelling evidence that the NEDD8-dependent activation of PARP-1 represents a novel mechanism underlying vascular calcification and suggests a promising new therapeutic target for VC.

Efficacy and safety of targeted therapies in VEXAS syndrome: retrospective study from the FRENVEX

ObjectivesVacuoles, E1 enzyme, X-linked, autoinflammatory and somatic (VEXAS) syndrome is an adult-onset autoinflammatory disease associated with somatic ubiquitin-like modifier-activating enzyme 1 (UBA1) mutations. We aimed to evaluate the efficacy and...

UBA1 dysfunction in VEXAS and cancer.

UBA1, an X-linked gene, encodes one of the only two ubiquitin E1 enzymes, playing a pivotal role in initiating one of the most essential post-translational modifications. In late 2020, partial loss-of-function mutations in UBA1 within hematopoietic stem and progenitor cells were found to be responsible for VEXAS Syndrome, a previously unidentified hematoinflammatory disorder predominantly affecting older males. The condition is characterized by severe inflammation, cytopenias, and an association to hematologic malignancies. In this research perspective, we comprehensively review the molecular significance of UBA1 loss of function as well as advancements in VEXAS research over the past four years for each of the VEXAS manifestations - inflammation, cytopenias, clonality, and possible oncogenicity. Special attention is given to contrasting the M41 and non-M41 mutations, aiming to elucidate their differential effects and to identify targetable mechanisms responsible for each of the symptoms. Finally, we explore the therapeutic landscape for VEXAS Syndrome, discussing the efficacy and potential of clone-targeting drugs based on the pathobiology of VEXAS. This includes azacitidine, currently approved for myelodysplastic neoplasms (MDS), novel UBA1 inhibitors being developed for a broad spectrum of cancers, Protein Kinase R-like Endoplasmic Reticulum Kinase (PERK) inhibitors, and auranofin, a long-established drug for rheumatoid arthritis. This perspective bridges basic research to clinical symptoms and therapeutics.