RESUMEN

Ventilator-induced lung injury is a serious complication in mechanically ventilated patients. Neddylation, the post-translational modification of neural precursor cell-expressed developmentally down-regulated 8 (NEDD8) conjugation, regulates numerous biological functions. However, its involvement and therapeutic significance in ventilator-induced lung injury remains unknown. Therefore, this study aimed to examine the kinetics and contribution of activated neddylation and the impact of neddylation inhibition in mice subjected to high tidal volume (HTV) ventilation in vivo and human pulmonary alveolar epithelial cells stimulated through cyclic stretching (CS) in vitro. The neddylation and expression of ubiquitin conjugating enzyme 3 (UBA3), a NEDD8-activating enzyme (NAE) catalytic subunit, were time-dependently upregulated in HTV-ventilated mice. Additionally, the NAE inhibitor MLN4924 considerably attenuated acute lung injury induced by HTV ventilation, manifesting as reduced inflammation and oxidative stress. Furthermore, MLN4924 effectively reduced the secretion of inflammatory cytokines from Ly6Chigh monocytes and neutrophils, subsequently decreasing endothelial permeability. Moreover, our study revealed an upregulation of the neddylation pathway, oxidative stress, and apoptosis during CS of alveolar epithelial cells. However, blockade of neddylation via MLN4924 or through UBA3 knockdown suppressed this upregulation. Overall, the inhibition of neddylation may alleviate HTV-induced acute lung injury by preventing CS-induced damage to alveolar epithelial cells. This indicates that the neddylation pathway plays a critical role in the progression of ventilator-induced lung injury. These findings may provide a new therapeutic target for treating ventilator-induced lung injury.

RESUMEN

The objective of our study was to develop a genetically encoded biosensor for quantification of Nedd8, a post-translational modifier that regulates cellular signals through conjugation to other proteins. Perturbations in the balance of free (i.e., unconjugated) and conjugated Nedd8 caused by defects in Nedd8 enzymes or cellular stress are implicated in various diseases. Despite the biological and biomedical importance of Nedd8 dynamics, no method exists for direct quantification of free Nedd8, hindering the study of Nedd8 and activities of its associated enzymes. Genetically encoded biosensors are established as tools to study other dynamic systems, but limitations of current biosensor design methods make them poorly suited for free Nedd8 quantification. We have developed a modular method to design genetically encoded biosensors that employs a target binding domain and two reporter domains positioned on opposite sides of the target binding site. Target quantification is based on competition between target binding and the interaction of the reporter domains. We applied our design strategy to free Nedd8 quantification by developing a selective binder for free Nedd8 and combining it with fluorescent or split nanoluciferase reporters. Our sensors produced quantifiable and specific signals for free Nedd8 and enabled real-time monitoring of deneddylation by DEN1 with a physiological substrate. Our sensor design will be useful for high-throughput screening for deneddylation inhibitors, which have potential in treatment of cancers such as acute lymphoblastic leukemia. The modular design strategy can be extended to develop genetically encoded quantitative biosensors for other proteins of interest.

RESUMEN

BACKGROUND: Gliomas are the most common primary malignant tumours of the central nervous system, and neddylation may be a potential target for the treatment of gliomas. Our study analysed neddylation's potential role in gliomas of different pathological types and its correlation with immunotherapy. METHODS: Genes required for model construction were sourced from existing literature, and their expression data were extracted from the TCGA and CGGA databases. LASSO regression was employed to identify genes associated with the prognosis of glioma patients in TCGA and to establish a clinical prognostic model. Biological changes in glioma cell lines following intervention with hub genes were evaluated using the CCK-8 assay and transwell assay. The genes implicated in the model construction were validated across various cell lines using Western blot. We conducted analyses to examine correlations between model scores and clinical data, tumor microenvironments, and immune checkpoints. Furthermore, we investigated potential differences in molecular functions and mechanisms among different groups. RESULTS: We identified 249 genes from the Reactome database and analysed their expression profiles in the TCGA and CGGA databases. After using LASSO-Cox, four genes (BRCA1, BIRC5, FBXL16 and KLHL25, p < 0.05) with significant correlations were identified. We selected FBXL16 for validation in in vitro experiments. Following FBXL16 overexpression, the proliferation, migration, and invasion abilities of glioma cell lines all showed a decrease. Then, we constructed the NEDD Index for gliomas. The nomogram indicated that this model could serve as an independent prognostic marker. Analysis of the tumour microenvironment and immune checkpoints revealed that the NEDD index was also correlated with immune cell infiltration and the expression levels of various immune checkpoints. CONCLUSION: The NEDD index can serve as a practical tool for predicting the prognosis of glioma patients, and it is correlated with immune cell infiltration and the expression levels of immune checkpoints.

Asunto(s)

Biomarcadores de Tumor , Neoplasias Encefálicas , Regulación Neoplásica de la Expresión Génica , Glioma , Humanos , Glioma/genética , Glioma/inmunología , Glioma/patología , Pronóstico , Línea Celular Tumoral , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/patología , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Proteína NEDD8/genética , Proteína NEDD8/metabolismo , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología , Proteínas de Punto de Control Inmunitario/genética , Proteínas de Punto de Control Inmunitario/metabolismo , Proliferación Celular/genética , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Femenino , Perfilación de la Expresión Génica/métodos , Bases de Datos Genéticas , Movimiento Celular/genética , Masculino

RESUMEN

Drug-induced liver injury (DILI) is a significant cause of acute liver failure (ALF) and liver transplantation in the Western world. Acetaminophen (APAP) overdose is a main contributor of DILI, leading to hepatocyte cell death through necrosis. Here, we identified that neddylation, an essential post-translational modification involved in the mitochondria function, was upregulated in liver biopsies from patients with APAP-induced liver injury (AILI) and in mice treated with an APAP overdose. MLN4924, an inhibitor of the neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8)-activating enzyme (NAE-1), ameliorated necrosis and boosted liver regeneration in AILI. To understand how neddylation interferes in AILI, whole-body biotinylated NEDD8 (bioNEDD8) and ubiquitin (bioUB) transgenic mice were investigated under APAP overdose with and without MLN4924. The cytidine diphosphate diacylglycerol (CDP-DAG) synthase TAM41, responsible for producing cardiolipin essential for mitochondrial activity, was found modulated under AILI and restored its levels by inhibiting neddylation. Understanding this ubiquitin-like crosstalk in AILI is essential for developing promising targeted inhibitors for DILI treatment.

Asunto(s)

Acetaminofén , Cardiolipinas , Enfermedad Hepática Inducida por Sustancias y Drogas , Ciclopentanos , Proteína NEDD8 , Pirimidinas , Acetaminofén/efectos adversos , Animales , Proteína NEDD8/metabolismo , Proteína NEDD8/genética , Humanos , Pirimidinas/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Cardiolipinas/metabolismo , Ratones , Ciclopentanos/farmacología , Masculino , Hígado/metabolismo , Hígado/patología , Hígado/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Transgénicos , Hepatocitos/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Transducción de Señal/efectos de los fármacos , Enzimas Activadoras de Ubiquitina/metabolismo , Enzimas Activadoras de Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores

RESUMEN

The Hippo-YAP signaling pathway plays a central role in many biological processes such as regulating cell fate, organ size, and tissue growth, and its key components are spatiotemporally expressed and posttranslationally modified during these processes. Neddylation is a posttranslational modification that involves the covalent attachment of NEDD8 to target proteins by NEDD8-specific E1-E2-E3 enzymes. Whether neddylation is involved in Hippo-YAP signaling remains poorly understood. Here, we provide evidence supporting the critical role of NEDD8 in facilitating the Hippo-YAP signaling pathway by mediating neddylation of the transcriptional coactivator yes-associated protein 1 (YAP1). Overexpression of NEDD8 induces YAP1 neddylation and enhances YAP1 transactivity, but inhibition of neddylation suppresses YAP1 transactivity and attenuates YAP1 nuclear accumulation. Furthermore, inhibition of YAP1 signaling promotes MLN4924-induced ovarian granulosa cells apoptosis and disruption of nedd8 in zebrafish results in downregulation of yap1-activated genes and upregulation of yap1-repressed genes. Further assays show that the xiap ligase promotes nedd8 conjugates to yap1 and that yap1 neddylation. In addition, we identify lysine 159 as a major neddylation site on YAP1. These findings reveal a novel mechanism for neddylation in the regulation of Hippo-YAP signaling.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Ciclopentanos , Proteína NEDD8 , Proteínas Serina-Treonina Quinasas , Transducción de Señal , Factores de Transcripción , Proteínas Señalizadoras YAP , Pez Cebra , Proteína NEDD8/metabolismo , Proteína NEDD8/genética , Humanos , Animales , Proteínas Señalizadoras YAP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Pez Cebra/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Ciclopentanos/metabolismo , Vía de Señalización Hippo , Apoptosis , Pirimidinas/farmacología , Células HEK293 , Femenino , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Procesamiento Proteico-Postraduccional

RESUMEN

Similar to ubiquitin, the ubiquitin-like protein NEDD8 is not only conjugated to other proteins but is itself subject to posttranslational modifications including lysine acetylation. Yet, compared to ubiquitin, only little is known about the biochemical and structural consequences of site-specific NEDD8 acetylation. Here, we generated site-specifically mono-acetylated NEDD8 variants for each known acetylation site by genetic code expansion. We show that, in particular, acetylation of K11 has a negative impact on the usage of NEDD8 by the NEDD8-conjugating enzymes UBE2M and UBE2F and that this is likely due to electrostatic and steric effects resulting in conformational changes of NEDD8. Finally, we provide evidence that p300 acts as a position-specific NEDD8 acetyltransferase.

RESUMEN

Plasmodium parasites, the causative agents of malaria, rely on sophisticated cellular mechanisms to survive and proliferate within their hosts. Plasmodium complex life cycle requires posttranslational modifications (PTMs) to control cellular activities. Neddylation is a type of PTM in which NEDD8 is covalently attached to target proteins and plays an important role in cell cycle control and metabolism. Covalent attachment to its substrates requires the Nedd8-activating enzyme, E1; the NEDD8-conjugating enzyme, E2; and the ligase, E3. In Plasmodium, protein neddylation is essential for parasite development during the stage I-II transition from zygote to ookinete differentiation and malaria transmission. Here, we discuss the current understanding of protein neddylation in Plasmodium, which is involved in malaria transmission.

Asunto(s)

Malaria , Proteína NEDD8 , Plasmodium , Procesamiento Proteico-Postraduccional , Humanos , Proteína NEDD8/metabolismo , Proteína NEDD8/genética , Plasmodium/metabolismo , Plasmodium/fisiología , Animales , Malaria/parasitología , Malaria/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Estadios del Ciclo de Vida

RESUMEN

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.

Asunto(s)

Retinopatía Diabética , Vesículas Extracelulares , Células Madre Mesenquimatosas , MicroARNs , Proteína NEDD8 , Factor 2 Relacionado con NF-E2 , Especies Reactivas de Oxígeno , Animales , Humanos , Ratones , Proteínas Cullin/metabolismo , Proteínas Cullin/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/genética , Retinopatía Diabética/patología , Retinopatía Diabética/genética , Retinopatía Diabética/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Productos Finales de Glicación Avanzada/metabolismo , Células Madre Mesenquimatosas/metabolismo , Ratones Endogámicos C57BL , MicroARNs/genética , MicroARNs/metabolismo , Proteína NEDD8/metabolismo , Proteína NEDD8/genética , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal

RESUMEN

Doxorubicin (DOX) is a widely utilized chemotherapeutic agent in clinical oncology for treating various cancers. However, its clinical use is constrained by its significant side effects. Among these, the development of cardiomyopathy, characterized by cardiac remodeling and eventual heart failure, stands as a major concern following DOX chemotherapy. In our current investigation, we have showcased the efficacy of MLN4924 in mitigating doxorubicin-induced cardiotoxicity through direct inhibition of the NEDD8-activating enzyme, NAE. MLN4924 demonstrated the ability to stabilize mitochondrial function post-doxorubicin treatment, diminish cardiomyocyte apoptosis, alleviate oxidative stress-induced damage in the myocardium, enhance cardiac contractile function, mitigate cardiac fibrosis, and impede cardiac remodeling associated with heart failure. At the mechanistic level, MLN4924 intervened in the neddylation process by inhibiting the NEDD8 activating enzyme, NAE, within the murine cardiac tissue subsequent to doxorubicin treatment. This intervention resulted in the suppression of NEDD8 protein expression, reduction in neddylation activity, and consequential manifestation of cardioprotective effects. Collectively, our findings posit MLN4924 as a potential therapeutic avenue for mitigating doxorubicin-induced cardiotoxicity by attenuating heightened neddylation activity through NAE inhibition, thereby offering a viable and promising treatment modality for afflicted patients.

Asunto(s)

Cardiotoxicidad , Ciclopentanos , Doxorrubicina , Miocitos Cardíacos , Proteína NEDD8 , Pirimidinas , Animales , Ratones , Apoptosis/efectos de los fármacos , Cardiotoxicidad/tratamiento farmacológico , Cardiotoxicidad/patología , Cardiotoxicidad/prevención & control , Cardiotoxicidad/etiología , Cardiotoxicidad/metabolismo , Ciclopentanos/farmacología , Ciclopentanos/uso terapéutico , Doxorrubicina/efectos adversos , Ratones Endogámicos C57BL , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Proteína NEDD8/metabolismo , Proteína NEDD8/antagonistas & inhibidores , Estrés Oxidativo/efectos de los fármacos , Pirimidinas/farmacología , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Enzimas Activadoras de Ubiquitina/metabolismo , Enzimas Activadoras de Ubiquitina/genética

RESUMEN

Adult T-cell leukemia (ATL), caused by HTLV-1, is the most lethal hematological malignancy. NEDD8-activating enzyme (NAE) is a component of the NEDD8 conjunction pathway that regulates cullin-RING ubiquitin ligase (CRL) activity. HTLV-1-infected T cells expressed higher levels of NAE catalytic subunit UBA3 than normal peripheral blood mononuclear cells. NAE1 knockdown inhibited proliferation of HTLV-1-infected T cells. The NAE1 inhibitor MLN4924 suppressed neddylation of cullin and inhibited the CRL-mediated turnover of tumor suppressor proteins. MLN4924 inhibited proliferation of HTLV-1-infected T cells by inducing DNA damage, leading to S phase arrest and caspase-dependent apoptosis. S phase arrest was associated with CDK2 and cyclin A downregulation. MLN4924-induced apoptosis was mediated by the upregulation of pro-apoptotic and downregulation of anti-apoptotic proteins. Furthermore, MLN4924 inhibited NF-κB, AP-1, and Akt signaling pathways and activated JNK. Therefore, neddylation inhibition is an attractive strategy for ATL therapy. Our findings support the use of MLN4924 in ATL clinical trials.

Asunto(s)

Apoptosis , Proliferación Celular , Ciclopentanos , Virus Linfotrópico T Tipo 1 Humano , Proteína NEDD8 , FN-kappa B , Proteínas Proto-Oncogénicas c-akt , Pirimidinas , Transducción de Señal , Factor de Transcripción AP-1 , Enzimas Activadoras de Ubiquitina , Humanos , Pirimidinas/farmacología , FN-kappa B/metabolismo , Factor de Transcripción AP-1/metabolismo , Ciclopentanos/farmacología , Transducción de Señal/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Apoptosis/efectos de los fármacos , Proteína NEDD8/metabolismo , Proliferación Celular/efectos de los fármacos , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Enzimas Activadoras de Ubiquitina/metabolismo , Enzimas Activadoras de Ubiquitina/genética , Linfocitos T/metabolismo , Linfocitos T/efectos de los fármacos , Ubiquitinas/metabolismo , Proteínas Cullin/metabolismo

RESUMEN

Neddylation, akin to ubiquitination, represents a post­translational modification of proteins wherein neural precursor cell­expressed developmentally downregulated protein 8 (NEDD8) is modified on the substrate protein through a series of reactions. Neddylation plays a pivotal role in the growth and proliferation of animal cells. In colorectal cancer (CRC), it predominantly contributes to the proliferation, metastasis and survival of tumor cells, decreasing overall patient survival. The strategic manipulation of the NEDD8­mediated neddylation pathway holds immense therapeutic promise in terms of the potential to modulate the growth of tumors by regulating diverse biological responses within cancer cells, such as DNA damage response and apoptosis, among others. MLN4924 is an inhibitor of NEDD8, and its combined use with platinum drugs and irinotecan, as well as cycle inhibitors and NEDD activating enzyme inhibitors screened by drug repurposing, has been found to exert promising antitumor effects. The present review summarizes the recent progress made in the understanding of the role of NEDD8 in the advancement of CRC, suggesting that NEDD8 is a promising anti­CRC target.

Asunto(s)

Neoplasias Colorrectales , Ubiquitinas , Animales , Humanos , Apoptosis , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Proteína NEDD8/genética , Proteína NEDD8/metabolismo , Procesamiento Proteico-Postraduccional , Ubiquitinas/genética

RESUMEN

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.

Asunto(s)

Plasmodium berghei , Ubiquitinas , Animales , Ubiquitinas/genética , Ubiquitinas/metabolismo , Plasmodium berghei/genética , Plasmodium berghei/metabolismo , Proteínas Cullin/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo

RESUMEN

The overexpression of neddylation modification is frequently observed in human tumor cells. Targeting the neddylation pathway has been recognized as a promising anticancer therapeutic strategy, thus discovering potent and selective neddylation inhibitors is of great importance. In this study, we designed and synthesized a series of novel neddylation inhibitors bearing benzothiazole scaffolds by molecular hybridization strategy and all compounds were evaluated for antiproliferative activity against MGC-803, MCF-7, A549 and KYSE-30 cell lines. In vitro anti-tumor studies showed that the most promising compound X-10, effectively suppressed MGC-803 cells growth and migration, induced apoptosis and arrested cell cycle at G2/M phase. Importantly, by directly interacting with NAE1, X-10 blocked NAE1 activity, specifically preventing neddylation of Cullin 3 and Cullin 1, and produced a dose-response decline in the level of UBC12-NEDD8 complex. Overall, our data indicate that X-10 inhibits the process of neddylation, making it a potentially agent for both cancer prevention and therapy purposes.

Asunto(s)

Neoplasias Gástricas , Humanos , Neoplasias Gástricas/tratamiento farmacológico , Ciclo Celular , Benzotiazoles/farmacología , Ciclopentanos/farmacología , Línea Celular Tumoral , Apoptosis

RESUMEN

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.

Asunto(s)

Ligandos , Ubiquitina-Proteína Ligasas , Ubiquitinación , Animales , Humanos , Ratones , beta Catenina/metabolismo , Proteínas con Repetición de beta-Transducina/metabolismo , Proteínas Cullin/metabolismo , Suramina/farmacología , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/efectos de los fármacos , Proteína NEDD8/metabolismo

RESUMEN

Aging in humans is associated with abdominal distribution and remodeling of body fat and a parallel gradual increase in the prevalence of metabolic diseases such as obesity, type 2 diabetes mellitus and fatty liver disease, as well as the risk of developing metabolic complications. Current treatments might be improved by understanding the detailed mechanisms underlying the onset of age-related metabolic disorders. Neddylation, a post-translational modification that adds the ubiquitin-like protein NEDD8 to substrate proteins, has recently been linked to age-related metabolic diseases, opening new avenues of investigation and raising a potential target for treatment of these diseases. In this review, we will focus on the potential role of NEDD8-mediated neddylation in age-related metabolic dysregulation, insulin resistance, obesity, type 2 diabetes mellitus and fatty liver. We propose that alterations in NEDD8-mediated neddylation contribute to triggering insulin resistance and the development of age-related metabolic dysregulation, thus highlighting NEDD8 as a promising therapeutic target for preventing age-related metabolic diseases.

Asunto(s)

Diabetes Mellitus Tipo 2 , Resistencia a la Insulina , Humanos , Proteína NEDD8 , Ubiquitinas/metabolismo , Obesidad

RESUMEN

Neddylation is a post-translational modification process, similar to ubiquitination, that controls several biological processes. Notably, it is often aberrantly activated in neoplasms and plays a critical role in the intricate dynamics of the tumor microenvironment (TME). This regulatory influence of neddylation permeates extensively and profoundly within the TME, affecting the behavior of tumor cells, immune cells, angiogenesis, and the extracellular matrix. Usually, neddylation promotes tumor progression towards increased malignancy. In this review, we highlight the latest understanding of the intricate molecular mechanisms that target neddylation to modulate the TME by affecting various signaling pathways. There is emerging evidence that the targeted disruption of the neddylation modification process, specifically the inhibition of cullin-RING ligases (CRLs) functionality, presents a promising avenue for targeted therapy. MLN4924, a small-molecule inhibitor of the neddylation pathway, precisely targets the neural precursor cell-expressed developmentally downregulated protein 8 activating enzyme (NAE). In recent years, significant advancements have been made in the field of neddylation modification therapy, particularly the integration of MLN4924 with chemotherapy or targeted therapy. This combined approach has demonstrated notable success in the treatment of a variety of hematological and solid tumors. Here, we investigated the inhibitory effects of MLN4924 on neddylation and summarized the current therapeutic outcomes of MLN4924 against various tumors. In conclusion, this review provides a comprehensive, up-to-date, and thorough overview of neddylation modifications, and offers insight into the critical importance of this cellular process in tumorigenesis.

RESUMEN

Immunosuppressive cells play important roles in generating an immunosuppressive tumor microenvironment and facilitating tumor immune escape. However, the molecular mechanisms underlying their immunosuppressive effects remain unclear. UBA3, the sole catalytic subunit of the neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8)-activating enzyme E1, is highly expressed in various human malignancies, along with an activated neddylation pathway. In this study, we investigated the relationships between the UBA3-dependent neddylation pathway and the infiltration of several immunosuppressive cell populations in lung adenocarcinoma (LUAD). We explored the regulatory mechanisms of UBA3 in LUAD cells by using mRNA sequencing and functional enrichment analyses. Correlations between neddylation and immune infiltrates were assessed by Western blotting, real-time PCR, and analyses of public databases. We found elevated levels of UBA3 expression in LUAD tissues compared to adjacent normal tissues. Blocking UBA3 and the neddylation pathway promoted the accumulation of the phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (p-IκBα), inhibiting the gene expression of tumor cell-derived cytokines such as C-C motif chemokine ligand (CCL) 2, C-X-C motif ligand (CXCL)1, CXCL2, colony-stimulating factor (CSF) 1, CSF2 interleukin (IL)-6, and IL-1B. Moreover, the overexpression of UBA3 in LUAD cells was associated with the secretion of these cytokines, and the recruitment and infiltration of immunosuppressive cells including tumor-associated macrophages (TAMs), plasmacytoid dendritic cells (pDCs), Th2 cells and T-regulatory cells (Tregs). This could facilitate the tumor immune escape and malignant progression of LUAD. Our findings provide new insights into the role of UBA3 in establishing an immunosuppressive tumor microenvironment by modulating nuclear factor kappa B (NF-кB) signaling and the neddylation pathway.

Asunto(s)

Adenocarcinoma del Pulmón , Neoplasias Pulmonares , Enzimas Activadoras de Ubiquitina , Humanos , Adenocarcinoma del Pulmón/metabolismo , Citocinas , Ligandos , Neoplasias Pulmonares/metabolismo , Proteína NEDD8 , FN-kappa B , Microambiente Tumoral , Enzimas Activadoras de Ubiquitina/metabolismo

RESUMEN

PURPOSE: Overactivated neddylation is considered to be a common event in cancer. Long non-coding RNAs (lncRNAs) can regulate cancer development by mediating post-translational modifications. However, the role of lncRNA in neddylation modification remains unclear. METHODS: LncRNA cytochrome P450 family 1 subfamily B member 1 antisense RNA 1 (CYP1B1-AS1) expression in breast cancer tissues was evaluated by RT-PCR and TCGA BRCA data. Gain and loss of function experiments were performed to explore the role of CYP1B1-AS1 in breast cancer cell proliferation and apoptosis in vitro and in vivo. Luciferase assay, CHIP-qPCR assay, transcriptome sequencing, RNA-pulldown assay, mass spectrometry, RIP-PCR and Western blot were used to investigate the regulatory factors of CYP1B1-AS1 expression and the molecular mechanism of CYP1B1-AS1 involved in neddylation modification. RESULTS: We found that CYP1B1-AS1 was down-regulated in breast cancer tissues and correlated with prognosis. In vivo and in vitro functional experiments confirmed that CYP1B1-AS1 inhibited cell proliferation and induced apoptosis. Mechanistically, CYP1B1-AS1 was regulated by the transcription factor, forkhead box O1 (FOXO1), and could be upregulated by inhibiting the PI3K/FOXO1 pathway. Moreover, CYP1B1-AS1 bound directly to NEDD8 activating enzyme E1 subunit 1 (NAE1) to regulate protein neddylation. CONCLUSION: This study reports for the first time that CYP1B1-AS1 inhibits protein neddylation to affect breast cancer cell proliferation, which provides a new strategy for the treatment of breast cancer by lncRNA targeting neddylation modification.

Asunto(s)

Neoplasias de la Mama , ARN Largo no Codificante , Humanos , Femenino , ARN sin Sentido , ARN Largo no Codificante/genética , Neoplasias de la Mama/genética , Apoptosis/genética , Proliferación Celular/genética , Proteína Forkhead Box O1/genética , Citocromo P-450 CYP1B1

RESUMEN

To explore effects of aging-related genes (ARGs) on the prognosis of Acute Myeloid Leukemia (AML), a seven-ARGs signature was developed and validated in AML patients. The numbers of seven-ARG sequences were selected to construct the survival prognostic signature in TCGA-LAML cohort, and two GEO datasets were used independently to verify the prognostic values of signature. According to seven-ARGs signature, patients were categorized into two subgroups. Patients with high-risk prognostic score were defined as HRPS-group/high-risk group, while others were set as LRPS-group/low-risk group. HRPS-group presented adverse overall survival (OS) than LRPS-group in TCGA-AML cohort (HR=3.39, P<0.001). In validation, the results emphasized a satisfactory discrimination in different time points, and confirmed the poor OS of HRPS-group both in GSE37642 (HR=1.96, P=0.001) and GSE106291 (HR=1.88, P<0.001). Many signal pathways, including immune- and tumor-related processes, especially NF-κB signaling, were highly enriched in HRPS-group. Coupled with high immune-inflamed infiltration, the HRPS-group was highly associated with the driver gene and oncogenic signaling pathway of TP53. Prediction of blockade therapy targeting immune checkpoint indicated varied benefits base on the different ARGs signature score, and the results of predicted drug response suggested that Pevonedistat, an inhibitor of NEDD8-activating enzyme, targeting NF-κB signaling, may have potential therapeutic value for HRPS-group. Compared with clinical factors alone, the signature had an independent value and more predictive power of AML prognosis. The 7-ARGs signature may help to guide clinical-decision making to predict drug response, and survival in AML patients.

Asunto(s)

Leucemia Mieloide Aguda , FN-kappa B , Humanos , Pronóstico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Envejecimiento , Toma de Decisiones Clínicas

RESUMEN

As a protein that resembles ubiquitin, neural precursor cell expressed developmentally downregulated 8 (NEDD8) takes part in neddylation, which modifies substrates in a manner similar to ubiquitination and alters the activity of target proteins. Neddylation may affect the activity of multiple signaling pathways, have a regulatory role in tumor formation, progression and metastasis, and influence the prognosis of cancer treatment. The present review summarizes the regulatory roles of NEDD8 in the MDM2­p53, NF­κB, PI3K/AKT/mTOR, hypoxia­inducible factor, Hippo and receptor tyrosine kinase signaling pathways, as well as in the development and progression of lung cancer.

Asunto(s)

Neoplasias Pulmonares , Ubiquitinas , Humanos , Proteína NEDD8/genética , Proteína NEDD8/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ubiquitinación