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1.
Cell Death Dis ; 13(4): 350, 2022 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-35428778

RESUMEN

Patients with late-stage and human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) continue to have a very poor prognosis. The development of more effective novel therapies that improve overall survival and overcome drug resistance is an urgent priority. Here we report that HNSCC tumors significantly overexpress NEDD8 and exhibit high sensitivity to the first-in-class NEDD8-activating enzyme (NAE) inhibitor pevonedistat. Additional studies established that disruption of NEDD8-mediated protein turnover with pevonedistat dramatically augmented cisplatin-induced DNA damage and apoptosis in HNSCC models. Further analysis revealed that the specific pevonedistat target CUL4A played an essential role in driving the synergy of the pevonedistat and cisplatin combination. Targeted inhibition of CUL4A resulted in significant downregulation in Damage Specific DNA binding protein 2 (DDB2), a DNA-damage recognition protein that promotes nucleotide excision repair and resistance to cisplatin. Silencing of CUL4A or DDB2 enhanced cisplatin-induced DNA damage and apoptosis in a manner similar to that of pevonedistat demonstrating that targeted inhibition of CUL4A may be a novel approach to augment cisplatin therapy. Administration of pevonedistat to mice bearing HNSCC tumors significantly decreased DDB2 expression in tumor cells, increased DNA damage and potently enhanced the activity of cisplatin to yield tumor regression and long-term survival of all animals. Our findings provide strong rationale for clinical investigation of CUL4A inhibition with pevonedistat as a novel strategy to augment the efficacy of cisplatin therapy for patients with HNSCC and identify loss of DDB2 as a key pharmacodynamic mediator controlling sensitivity to this regimen.


Asunto(s)
Cisplatino , Proteínas Cullin , Proteínas de Unión al ADN , Neoplasias de Cabeza y Cuello , Carcinoma de Células Escamosas de Cabeza y Cuello , Animales , Línea Celular Tumoral , Cisplatino/farmacología , Proteínas Cullin/antagonistas & inhibidores , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Sinergismo Farmacológico , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Ratones , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patología
2.
Nat Commun ; 12(1): 6662, 2021 11 18.
Artículo en Inglés | MEDLINE | ID: mdl-34795264

RESUMEN

SPOP, an E3 ubiquitin ligase, acts as a prostate-specific tumor suppressor with several key substrates mediating oncogenic function. However, the mechanisms underlying SPOP regulation are largely unknown. Here, we have identified G3BP1 as an interactor of SPOP and functions as a competitive inhibitor of Cul3SPOP, suggesting a distinctive mode of Cul3SPOP inactivation in prostate cancer (PCa). Transcriptomic analysis and functional studies reveal a G3BP1-SPOP ubiquitin signaling axis that promotes PCa progression through activating AR signaling. Moreover, AR directly upregulates G3BP1 transcription to further amplify G3BP1-SPOP signaling in a feed-forward manner. Our study supports a fundamental role of G3BP1 in disabling the tumor suppressive Cul3SPOP, thus defining a PCa cohort independent of SPOP mutation. Therefore, there are significantly more PCa that are defective for SPOP ubiquitin ligase than previously appreciated, and these G3BP1high PCa are more susceptible to AR-targeted therapy.


Asunto(s)
Proteínas Cullin/antagonistas & inhibidores , ADN Helicasas/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Neoplasias de la Próstata/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Receptores Androgénicos/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Antagonistas de Receptores Androgénicos/farmacología , Animales , Carcinogénesis , Línea Celular Tumoral , Movimiento Celular , Supervivencia Celular/efectos de los fármacos , Proteínas Cullin/metabolismo , ADN Helicasas/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , ARN Helicasas/genética , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal/efectos de los fármacos , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo
3.
Eur J Med Chem ; 226: 113896, 2021 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-34624825

RESUMEN

Targeting neddylation pathway has been recognized as an attractive anticancer therapeutic strategy, thus discovering potent and selective neddylation inhibitors is highly desirable. Our work reported the discovery of novel cinnamyl piperidine compounds and their antitumor activity in vitro and in vivo. Among these compounds, compound 4g was identified as a novel neddylation inhibitor and decreased the neddylation levels of cullin 1, cullin 3 and cullin 5. Mechanistic studies demonstrated that compound 4g could inhibit the migration ability of gastric cancer cells and induce apoptosis partly mediated by the Nrf2-Keap1 pathway. Furthermore, in vivo anti-tumor studies showed that 4g effectively inhibited tumor growth without obvious toxicity. Collectively, the cinnamyl piperidine derivatives could serve as new lead compounds for developing highly effective neddylation inhibitors for gastric cancer therapy.


Asunto(s)
Antineoplásicos/farmacología , Cinamatos/farmacología , Proteínas Cullin/antagonistas & inhibidores , Descubrimiento de Drogas , Piperidinas/farmacología , Neoplasias Gástricas/tratamiento farmacológico , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cinamatos/síntesis química , Cinamatos/química , Proteínas Cullin/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Piperidinas/síntesis química , Piperidinas/química , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Relación Estructura-Actividad , Células Tumorales Cultivadas
4.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artículo en Inglés | MEDLINE | ID: mdl-33946721

RESUMEN

Microalgae-based biodiesel production has many advantages over crude oil extraction and refinement, thus attracting more and more concern. Protein ubiquitination is a crucial mechanism in eukaryotes to regulate physiological responses and cell development, which is highly related to algal biodiesel production. Cullins as the molecular base of cullin-RING E3 ubiquitin ligases (CRLs), which are the largest known class of ubiquitin ligases, control the life activities of eukaryotic cells. Here, three cullins (CrCULs) in the green microalgae Chlamydomonas reinhardtii were identified and characterized. To investigate the roles of CrCULs in lipid metabolism, the gene expression profiles of CrCULs under nutrition starvation were examined. Except for down-regulation under nitrogen starvation, the CrCUL3 gene was induced by sulfur and iron starvation. CrCUL2 seemed insensitive to nitrogen and sulfur starvation because it only had changes after treatment for eight days. CrCUL4 exhibited an expression peak after nitrogen starvation for two days but this declined with time. All CrCULs expressions significantly increased under iron deficiency at two and four days but decreased thereafter. The silencing of CrCUL2 and CrCUL4 expression using RNAi (RNA interference) resulted in biomass decline and lipids increase but an increase of 20% and 28% in lipid content after growth for 10 days, respectively. In CrCUL2 and CrCUL4 RNAi lines, the content of fatty acids, especially C16:0 and C18:0, notably increased as well. However, the lipid content and fatty acids of the CrCUL3 RNAi strain slightly changed. Moreover, the subcellular localization of CrCUL4 showed a nuclear distribution pattern. These results suggest CrCUL2 and CrCUL4 are regulators for lipid accumulation in C. reinhardtii. This study may offer an important complement of lipid biosynthesis in microalgae.


Asunto(s)
Proteínas Algáceas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Proteínas Cullin/metabolismo , Lípidos/biosíntesis , Proteínas Algáceas/antagonistas & inhibidores , Proteínas Algáceas/genética , Secuencia de Aminoácidos , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/crecimiento & desarrollo , Proteínas Cullin/antagonistas & inhibidores , Proteínas Cullin/genética , Ácidos Grasos/metabolismo , Metabolismo de los Lípidos/genética , Modelos Moleculares , Filogenia , Interferencia de ARN , Transcriptoma
5.
Drug Des Devel Ther ; 15: 1-8, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33442232

RESUMEN

Cullin-RING E3 ligases (CRLs) are the largest family of E3 ubiquitin ligases, responsible for about 20% of the protein degradation by the ubiquitin-proteasome system (UPS). Given their vital roles in multiple cellular processes, and over-activation in many human cancers, CRLs are validated as promising targets for anti-cancer therapies. Activation of CRLs requires cullin neddylation, a process catalysed by three neddylation enzymes. Recently, our group established an AlphaScreen-based in vitro cullin neddylation assay and employed it for high-throughput screening to search for small-molecule inhibitors targeting cullin neddylation. During our pilot screen, gossypol, a natural product extracted from cottonseeds, was identified as one of the most potent neddylation inhibitors of cullin-1 and cullin-5. We further demonstrated that gossypol blocks cullin neddylation by binding to cullin-1/-5 to inactivate CRL1/5 ligase activity, leading to accumulation of MCL-1 and NOXA, the substrates of CRL1 and CRL5, respectively. The combination of gossypol and an MCL-1 inhibitor synergistically enhanced the anti-proliferative effect in multiple human cancer cell lines. Our study unveiled a rational combination of two previously known inhibitors of the Bcl-2 family for enhanced anti-cancer efficacy and identified a novel activity of gossypol as an inhibitor of CRL1 and CRL5 E3s, thus providing a new possibility in the development of novel CRL inhibitors for anti-cancer therapy.


Asunto(s)
Proteínas Cullin/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Gosipol/farmacología , Proteínas Cullin/metabolismo , Humanos , Ubiquitinación/efectos de los fármacos
6.
Acta Pharmacol Sin ; 42(4): 508-517, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-32724175

RESUMEN

Hypertension is the most prevalent health condition worldwide, affecting ~1 billion people. Gordon's syndrome is a form of secondary hypertension that can arise due to a number of possible mutations in key genes that encode proteins in a pathway containing the With No Lysine [K] (WNK) and its downstream target kinases, SPS/Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress responsive kinase 1 (OSR1). This pathway regulates the activity of the thiazide-sensitive sodium chloride cotransporter (NCC), which is responsible for NaCl reabsorption in the distal nephron. Therefore, mutations in genes encoding proteins that regulate the NCC proteins disrupt ion homeostasis and cause hypertension by increasing NaCl reabsorption. Thiazide diuretics are currently the main treatment option for Gordon's syndrome. However, they have a number of side effects, and chronic usage can lead to compensatory adaptations in the nephron that counteract their action. Therefore, recent research has focused on developing novel inhibitory molecules that inhibit components of the WNK-SPAK/OSR1-NCC pathway, thereby reducing NaCl reabsorption and restoring normal blood pressure. In this review we provide an overview of the currently reported molecular inhibitors of the WNK-SPAK/OSR1-NCC pathway and discuss their potential as treatment options for Gordon's syndrome.


Asunto(s)
Inhibidores de Proteínas Quinasas/uso terapéutico , Seudohipoaldosteronismo/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Animales , Presión Sanguínea/efectos de los fármacos , Proteínas de Unión al Calcio/antagonistas & inhibidores , Proteínas Cullin/antagonistas & inhibidores , Diuréticos/uso terapéutico , Humanos , Proteínas de Microfilamentos/antagonistas & inhibidores , Unión Proteica/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Seudohipoaldosteronismo/metabolismo , Transducción de Señal/fisiología , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Proteína Quinasa Deficiente en Lisina WNK 1/antagonistas & inhibidores , Proteína Quinasa Deficiente en Lisina WNK 1/metabolismo
7.
Behav Brain Res ; 399: 113051, 2021 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-33279641

RESUMEN

Heterologous sensitization of adenylyl cyclase (AC) is defined by an enhanced cAMP response following persistent activation of Gαi/o-coupled receptors. This phenomenon was first observed in cellular models, and later reported in animal models of inflammatory pain or following chronic exposure to drugs of abuse including opioids and cocaine. Recently, we used genome-wide siRNA screening to identify Cullin3 signaling as a mediator of AC sensitization in cellular models. We also showed that pharmacological inhibition of Cullin3 with the neddylation inhibitor, MLN4924, abolished heterologous sensitization of several AC isoforms, including AC1, AC2, AC5, and AC6. Because ACs, especially AC1, have been implicated in alcohol-induced locomotor sensitization and inflammatory pain, we assessed the potential activity of MLN4924 in both murine models. We found that MLN4924 (30 mg/kg, i.p.) accumulated in the brain and reduced both locomotor sensitization induced by repeated alcohol administration and allodynia in an inflammatory pain model. Based on our previous findings that MLN4924 potently blocks AC sensitization in cellular models, we propose that the activity of MLN4924 in both animal models potentially occurs through blocking AC sensitization. Our findings provide the basis for understanding the molecular mechanism and yield a new pathway for drug development for pathological disorders associated with AC sensitization.


Asunto(s)
Alcoholismo/tratamiento farmacológico , Depresores del Sistema Nervioso Central/farmacología , Sensibilización del Sistema Nervioso Central/efectos de los fármacos , Proteínas Cullin/antagonistas & inhibidores , Ciclopentanos/farmacología , Inhibidores Enzimáticos/farmacología , Etanol/farmacología , Hiperalgesia/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Locomoción/efectos de los fármacos , Proteína NEDD8 , Pirimidinas/farmacología , Alcoholismo/complicaciones , Animales , Depresores del Sistema Nervioso Central/administración & dosificación , Ciclopentanos/administración & dosificación , Ciclopentanos/farmacocinética , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacocinética , Etanol/administración & dosificación , Hiperalgesia/inducido químicamente , Inflamación/inducido químicamente , Masculino , Ratones , Ratones Endogámicos BALB C , Pirimidinas/administración & dosificación , Pirimidinas/farmacocinética
8.
PLoS One ; 15(6): e0234708, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32555680

RESUMEN

Fibroblast growth factor receptors (FGFRs) are frequently altered in a variety of human cancer cells and are overexpressed in hepatocellular carcinoma (HCC). Several literatures have proven that they are efficacious for HCC therapy, however, the underlying mechanism remains unclear. Here, we found FGFR4 was overexpressed in HCC cell lines HepG2 and Hep3B and we used PD173074, an FGFR4 inhibitor, to explore the role of FGFR4 and its underlying mechanism in these cell lines. The results showed that PD173074 significantly arrested HepG2 and Hep3B cells in G1 phase and inhibited cell proliferation. Furthermore, Western blot analysis revealed that PD173074 decreased the levels of P-FRS2α, P-ERK, CDK2, cyclin E and NF-κB (p65) in the nucleus while it increased the levels of ubiquitin and CUL3, an E3 ubiquitin ligase which involves in cyclin E degradation. Meanwhile, the data from RT-qPCR showed that PD173074 also decreased miR-141 level. In conclusion, these results suggest that FGFR4 is involved in HCC by ERK/CUL3/cyclin E signaling pathway, and the finding may provide a potential theoretical basis for treatment by targeting FGFR4 in HCC.


Asunto(s)
Proteínas Cullin/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Pirimidinas/farmacología , Ubiquitina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteínas Cullin/antagonistas & inhibidores , Proteínas Cullin/genética , Ciclina E/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Proteínas de la Membrana/metabolismo , MicroARNs/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Transcripción ReIA/antagonistas & inhibidores , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo
9.
Comb Chem High Throughput Screen ; 23(8): 814-826, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32407263

RESUMEN

BACKGROUND: Parkinson's disease ranks second, after Alzheimer's as the major neurodegenerative disorder, for which no cure or disease-modifying therapies exist. Ample evidence indicate that PD manifests as a result of impaired anti-oxidative machinery leading to neuronal death wherein Cullin-3 has ascended as a potential therapeutic target for diseases involving damaged anti-oxidative machinery. OBJECTIVE: The design of target specific inhibitors for the Cullin-3 protein might be a promising strategy to increase the Nrf2 levels and to decrease the possibility of "off-target" toxic properties. METHODS: In the present study, an integrated computational and wet lab approach was adopted to identify small molecule inhibitors for Cullin-3. The rational drug designing process comprised homology modeling and derivation of the pharmacophore for Cullin-3, virtual screening of Zinc natural compound database, molecular docking and Molecular dynamics based screening of ligand molecules. In vivo validations of an identified lead compound were conducted in the PD model of C. elegans. RESULTS AND DISCUSSION: Our strategy yielded a potential inhibitor; (Glide score = -12.31), which was evaluated for its neuroprotective efficacy in the PD model of C. elegans. The inhibitor was able to efficiently defend against neuronal death in PD model of C. elegans and the neuroprotective effects were attributed to its anti-oxidant activities, supported by the increase in superoxide dismutase, catalase and the diminution of acetylcholinesterase and reactive oxygen species levels. In addition, the Cullin-3 inhibitor significantly restored the behavioral deficits in the transgenic C. elegans. CONCLUSION: Taken together, these findings highlight the potential utility of Cullin-3 inhibition to block the persistent neuronal death in PD. Further studies focusing on Cullin-3 and its mechanism of action would be interesting.


Asunto(s)
Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Proteínas Cullin/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Fármacos Neuroprotectores/química , Estrés Oxidativo/efectos de los fármacos , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/prevención & control , Acetilcolinesterasa/metabolismo , Animales , Antioxidantes/farmacología , Caenorhabditis elegans , Bases de Datos Farmacéuticas , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/farmacología , Humanos , Modelos Animales , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Factor 2 Relacionado con NF-E2/metabolismo , Fármacos Neuroprotectores/farmacología , Especies Reactivas de Oxígeno/metabolismo
10.
Adv Exp Med Biol ; 1217: 317-347, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31898236

RESUMEN

Cullin-RING ligases (CRLs), the largest family of E3 ubiquitin ligases, have become an attractive target for drug discovery, primarily due to their ability to regulate the degradation of numerous functionally and structurally diverse proteins, thereby controlling a myriad of biological processes. As the abnormal expressions of CRLs and their substrate proteins are associated with human diseases, elucidating their roles in these physiological and pathological processes will facilitate CRL-targeting drug development for the treatment of these diseases. Notably, these studies are also providing new concepts for the design of potential small-molecule therapeutics targeting CRLs and for the use of CRLs to degrade "undruggable" proteins. In this chapter, we systematically review the development of small molecules that target CRLs and especially emphasize the applications of CRLs in a chemical chimera for protein degradation, termed proteolysis-targeting chimeras (PROTACs).


Asunto(s)
Proteínas Cullin/antagonistas & inhibidores , Proteínas Cullin/metabolismo , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Proteolisis/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Enfermedad , Humanos
11.
Am J Physiol Cell Physiol ; 317(3): C457-C465, 2019 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-31216194

RESUMEN

Cell motility and migration play critical roles in various physiological processes and disease states. Here, we show that the BBBsome, a macromolecule composed of eight Bardet-Biedl syndrome (BBS) proteins including BBS1, is a critical determinant of cell migration and wound healing. Fibroblast cells derived from mice or humans harboring a homozygous missense mutation (BBS1M390R/M390R) that disrupt the BBSome exhibit defects in migration and wound healing. Furthermore, we demonstrate that BBS1M390R/M390R mice have significantly delayed wound closure. In line with this, we provide data suggesting that BBS1M390R/M390R fibroblasts have impaired platelet-derived growth factor-AA (PDGF) receptor-α signaling, a key regulator of directional cell migration acting as a chemoattractant during postnatal migration responses such as wound healing. In addition, we show that BBS1M390R/M390R fibroblasts have upregulated RhoA expression and activity. The relevance of RhoA upregulation is demonstrated by the ability of RhoA-kinase inhibitor Y27632 to partially rescue the migration defect of BBS1M390R/M390R fibroblasts cells. We also show that accumulation of RhoA protein in BBS1M390R/M390R fibroblasts cells is associated with reduction and inactivation of the ubiquitin ligase Cullin-3. Consistent with this, Cullin-3 inhibition with MLN4924 is sufficient to reduce migration of normal fibroblasts. These data implicate the BBSome in cell motility and tissue repair through a mechanism that involves PDGF receptor signaling and Cullin-3-mediated control of RhoA.


Asunto(s)
Síndrome de Bardet-Biedl , Movimiento Celular/fisiología , Proteínas Cullin/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Transducción de Señal/fisiología , Proteína de Unión al GTP rhoA/fisiología , Animales , Síndrome de Bardet-Biedl/genética , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Proteínas Cullin/antagonistas & inhibidores , Ciclopentanos/farmacología , Inhibidores Enzimáticos/farmacología , Femenino , Técnicas de Sustitución del Gen/métodos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Pirimidinas/farmacología , Proteína de Unión al GTP rhoA/antagonistas & inhibidores
13.
Mol Med Rep ; 18(3): 2531-2540, 2018 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-30015884

RESUMEN

Cullin 4A (CUL4A) is the major component of cullin­RING­based E3 ubiquitin­protein ligase complexes, which regulate the ubiquitination of target proteins. The overexpression of CUL4A has been associated with the development and progression of various cancer types. However, a detailed understanding of the role of CUL4A in human liver cancer has not been determined by previous studies. In the present study, the association between human liver cancer and CUL4A expression was investigated. The expression of CUL4A in liver cancer tissues and paracancerous tissues of patients was investigated by reverse transcription­quantitative polymerase chain reaction, western blotting and immunohistochemical staining. Overexpression and knockdown of CUL4A were induced with an overexpression vector and small interfering RNA transfection, respectively, in human liver cancer cell lines, and the effects on cell proliferation were analyzed by a Cell Counting Kit­8 assay to investigate the role of CUL4A in human liver cancer. Cell migration, invasion, apoptosis and the cell cycle were also analyzed following transfection. The results of the present study revealed that the mRNA and protein expression of CUL4A was increased in the liver cancer tissues compared with the paracancerous tissues of 3 patients. Additionally, the results demonstrated that downregulation of CUL4A expression inhibited cell proliferation, migration and invasion, and increased the percentage of cell apoptosis, in HEPG2 and MHCC97­H cells, while CUL4A overexpression led to the opposite effects. Therefore, the results of the current study indicated that CUL4A may serve an important role in the development and progression of human liver cancer, and highlights the potential of CUL4A as a novel target in the diagnosis and treatment of human liver cancer and potentially other cancer types.


Asunto(s)
Proteínas Cullin/metabolismo , Neoplasias Hepáticas/patología , Apoptosis , Cadherinas/metabolismo , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Movimiento Celular , Claudina-3/metabolismo , Proteínas Cullin/antagonistas & inhibidores , Proteínas Cullin/genética , Células Hep G2 , Humanos , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/metabolismo , Ocludina/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo
14.
J Med Chem ; 61(7): 2680-2693, 2018 04 12.
Artículo en Inglés | MEDLINE | ID: mdl-29547696

RESUMEN

We previously discovered and validated a class of piperidinyl ureas that regulate defective in cullin neddylation 1 (DCN1)-dependent neddylation of cullins. Here, we report preliminary structure-activity relationship studies aimed at advancing our high-throughput screen hit into a tractable tool compound for dissecting the effects of acute DCN1-UBE2M inhibition on the NEDD8/cullin pathway. Structure-enabled optimization led to a 100-fold increase in biochemical potency and modestly increased solubility and permeability as compared to our initial hit. The optimized compounds inhibit the DCN1-UBE2M protein-protein interaction in our TR-FRET binding assay and inhibit cullin neddylation in our pulse-chase NEDD8 transfer assay. The optimized compounds bind to DCN1 and selectively reduce steady-state levels of neddylated CUL1 and CUL3 in a squamous cell carcinoma cell line. Ultimately, we anticipate that these studies will identify early lead compounds for clinical development for the treatment of lung squamous cell carcinomas and other cancers.


Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Proteínas Cullin/antagonistas & inhibidores , Proteína NEDD8/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Carcinoma de Células Escamosas/tratamiento farmacológico , Línea Celular Tumoral , Cristalografía por Rayos X , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Ensayos Analíticos de Alto Rendimiento , Humanos , Péptidos y Proteínas de Señalización Intracelular , Neoplasias Pulmonares/tratamiento farmacológico , Modelos Moleculares , Conformación Molecular , Proteína NEDD8/metabolismo , Unión Proteica , Proteínas , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Enzimas Ubiquitina-Conjugadoras/antagonistas & inhibidores
15.
Biochem Biophys Res Commun ; 499(1): 17-23, 2018 04 30.
Artículo en Inglés | MEDLINE | ID: mdl-29550478

RESUMEN

A disintegrin and metalloproteinase (ADAM) family are crucial enzymes for ectodomain shedding of multiple substrates and are involved in diverse biologic and pathologic processes. However, the molecular mechanism underlying substrate selectivity of ADAMs is poorly understood. In this study, we observed that disruption of actin polymerization by pharmacological inhibitors, latrunculin A (LatA) and cytochalasin D (CyD), induced ectodomain shedding of epidermal growth factor (EGF) family ligands. Induced shedding activity by LatA or CyD was suppressed by a metalloprotease inhibitor KB-R7785, indicating that ADAMs-mediated shedding is tightly controlled by actin cytoskeleton. We also investigated roles of cullin family, a component of cullin-RING based E3 ubiquitin ligases, in ectodomain shedding, since cullin family is implicated in the regulation of cytoskeletal dynamics. Knockdown of cullin 3 (Cul3) by a specific siRNA inhibited ectodomain shedding of amphiregulin (AREG), a member of EGF family, and responses were associated with activation of RhoA GTPase and induction of stress fiber formation. On the other hand, the RhoA inhibitor C3 transferase rescued AREG shedding reduced by Cul3 knockdown. These results describe a novel molecular mechanism of Cul3 to regulate AREG shedding by modulating cytoskeletal dynamics in a RhoA dependent manner.


Asunto(s)
Proteína ADAM17/genética , Citoesqueleto de Actina/metabolismo , Anfirregulina/genética , Proteínas Cullin/genética , Fibroblastos/metabolismo , Proteína ADAM17/antagonistas & inhibidores , Proteína ADAM17/metabolismo , ADP Ribosa Transferasas/farmacología , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/ultraestructura , Anfirregulina/metabolismo , Animales , Toxinas Botulínicas/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/antagonistas & inhibidores , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Proteínas Cullin/antagonistas & inhibidores , Proteínas Cullin/metabolismo , Citocalasina D/antagonistas & inhibidores , Citocalasina D/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Regulación de la Expresión Génica , Glicina/análogos & derivados , Glicina/farmacología , Humanos , Ácidos Hidroxámicos/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacología , Tiazolidinas/antagonistas & inhibidores , Tiazolidinas/farmacología , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismo
16.
Fly (Austin) ; 12(2): 118-126, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29355077

RESUMEN

The COP9 signalosome inhibits the activity of Cullin-RING E3 ubiquitin ligases by removing Nedd8 modifications from their Cullin subunits. Neddylation renders these complexes catalytically active, but deneddylation is also necessary for them to exchange adaptor subunits and avoid auto-ubiquitination. Although deneddylation is thought to be the primary function of the COP9 signalosome, additional activities have been ascribed to some of its subunits. We recently showed that COP9 subunits protect the transcriptional repressor and tumor suppressor Capicua from two distinct modes of degradation. Deneddylation by the COP9 signalosome inactivates a Cullin 1 complex that ubiquitinates Capicua following its phosphorylation by MAP kinase in response to Epidermal Growth Factor Receptor signaling. The CSN1b subunit also stabilizes unphosphorylated Capicua to control its basal level, independently of the deneddylase function of the complex. Here we further examine the importance of deneddylation for COP9 functions in vivo. We use an uncleavable form of Nedd8 to show that preventing deneddylation does not reproduce the effects of loss of COP9. In contrast, in the presence of COP9, conjugation to uncleavable Nedd8 renders Cullins unable to promote the degradation of their substrates. Our results suggest that irreversible neddylation prolongs COP9 binding to and inhibition of Cullin-based ubiquitin ligases.


Asunto(s)
Complejo del Señalosoma COP9/metabolismo , Proteínas Cullin/antagonistas & inhibidores , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteína NEDD8/metabolismo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Animales , Complejo del Señalosoma COP9/genética , Células Cultivadas , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteína NEDD8/genética , Proteolisis , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación
17.
Int J Biol Macromol ; 106: 532-543, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-28802844

RESUMEN

Cullin (CUL)-RING E3 ubiquitin ligases (CRLs) are attractive therapeutic targets as they regulate diverse biological processes important for cancer cell survival by conferring substrate selectivity for ubiquitination and degradation. Given the complexity of CRL complexes, steps toward the structure-based design of small-molecule inhibitors to modulate their activity have remained elusive. In this study, we explored the structural assembly and interaction details of closely related CUL scaffolds (CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5 and CUL7) with RBX1 to screen potent small molecules against CRLs. The RING-Box (RBX1 and RBX2) proteins heterodimerize with CULs and dynamically facilitate the ubiquitination process. The docked complexes of conserved CUL C-terminal domains exhibited a common RBX1 binding pattern through the incorporation of intermolecular ß-sheet and α/ß core, stabilized by hydrophobic contacts. The comparative binding pattern analysis of CUL-RBX1 interfaces revealed a unique structural motif (VLYRLWLN) that directs the binding of RBX1 N-terminal ß-strand. Through reinvigorating the subtle structural dynamics of bound complexes and application of structure-based drug design approaches, we proposed a set of inhibitors which could be further optimized to target CRL activity. One reference compound (C64) was extensively characterized for selective binding at the RBX1-binding grooves/VLYRLWLN of CUL1-7. We speculate that mechanistic information of the individual residual contributions through structure-guided approaches could be pivotal for the rational design of more promising and active drug candidates against CRLs.


Asunto(s)
Antineoplásicos/química , Proteínas Portadoras/química , Proteínas Cullin/química , Diseño de Fármacos , Bibliotecas de Moléculas Pequeñas/química , Ubiquitina-Proteína Ligasas/química , Secuencia de Aminoácidos , Antineoplásicos/metabolismo , Sitios de Unión , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Proteínas Cullin/antagonistas & inhibidores , Proteínas Cullin/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Bibliotecas de Moléculas Pequeñas/metabolismo , Especificidad por Sustrato , Termodinámica , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación
18.
Curr Biol ; 27(22): 3544-3553.e5, 2017 Nov 20.
Artículo en Inglés | MEDLINE | ID: mdl-29103937

RESUMEN

Maintenance of protein homeostasis, or proteostasis, is crucial for organismal health. Disruption of proteostasis can lead to the accumulation of protein aggregates, which are associated with aging and many human diseases such as Alzheimer's disease [1-3]. Through analysis of the C. elegans host response to intracellular infection, we describe here a novel response pathway that enhances proteostasis capacity and appears to act in parallel to well-studied proteostasis pathways. These findings are based on analysis of the transcriptional response to infection by the intracellular pathogen Nematocida parisii [4]. The response to N. parisii is strikingly similar to the response to infection by the Orsay virus, another natural intracellular pathogen of C. elegans, and is distinct from responses to extracellular pathogen infection [4-6]. We have therefore named this common transcriptional response the intracellular pathogen response (IPR), and it includes upregulation of several predicted ubiquitin ligase complex components such as the cullin cul-6. Through a forward genetic screen we found pals-22, a gene of previously unknown function, to be a repressor of the cul-6/cullin gene and other IPR gene expression. Interestingly, pals-22 mutants have increased thermotolerance and reduced levels of stress-induced polyglutamine aggregates, likely due to upregulated IPR gene expression. We found the enhanced stress resistance of pals-22 mutants to be dependent on cul-6, suggesting that pals-22 mutants have increased activity of a CUL-6/cullin-containing ubiquitin ligase complex. pals-22 mutant phenotypes appear independent of the well-studied heat shock and insulin signaling pathways, indicating that the IPR is a distinct pathway that protects animals from proteotoxic stress.


Asunto(s)
Proteostasis/genética , Proteostasis/fisiología , Animales , Antinematodos , Caenorhabditis elegans/genética , Caenorhabditis elegans/microbiología , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Proteínas de Caenorhabditis elegans/biosíntesis , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Cullin/antagonistas & inhibidores , Proteínas Cullin/biosíntesis , Citoplasma/metabolismo , Represión Enzimática , Interacciones Huésped-Patógeno/fisiología , Microsporidios , Ubiquitina/metabolismo
19.
Cell Physiol Biochem ; 43(3): 1152-1167, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28977802

RESUMEN

BACKGROUND/AIMS: ALT1 is a novel long non-coding RNA derived from the alternatively spliced transcript of the deleted in lymphocytic leukemia 2 (DLEU2). To date, ALT1 biological roles in human vascular endothelial cells have not been reported. METHODS: ALT1 was knocked down by siRNAs. Cell proliferation was analyzed by cck-8. The existence and sequence of human ALT1 were identified by 3' rapid amplification of cDNA ends. The interaction between lncRNA and proteins was analyzed by RNA-Protein pull down assay, RNA immunoprecipitation, and mass spectrometry analysis. RESULTS: ALT1 was expressed in human umbilical vein endothelial cells (HUVECs). The expression of ALT1 was significantly downregulated in contact-inhibited HUVECs and in hypoxia-induced, growth-arrested HUVECs. Knocking down of ALT1 inhibited the proliferation of HUVECs by G0/G1 cell cycle arrest. We observed that angiotensin converting enzyme Ⅱ(ACE2) was a direct target gene of ALT1. Knocking-down of ALT1 or its target gene ACE2 could efficiently decrease the expression of cyclin D1 via the enhanced ubiquitination and degradation, in which HIF-1α and protein von Hippel-Lindau (pVHL) might be involved. CONCLUSION: The results suggested the human long non-coding RNA ALT1 is a novel regulator for cell cycle of HUVECs via ACE2 and cyclin D1 pathway.


Asunto(s)
Peptidil-Dipeptidasa A/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Enzima Convertidora de Angiotensina 2 , Apoptosis , Proteínas Portadoras/metabolismo , Hipoxia de la Célula , Proliferación Celular , Proteínas Cullin/antagonistas & inhibidores , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Proteínas del Citoesqueleto , Regulación hacia Abajo , Puntos de Control de la Fase G1 del Ciclo Celular , Células Endoteliales de la Vena Umbilical Humana , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inmunoprecipitación , MicroARNs/metabolismo , Chaperonas Moleculares , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/genética , Interferencia de ARN , ARN Largo no Codificante , ARN Interferente Pequeño/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Transferasas , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Ubiquitinación
20.
Br J Cancer ; 116(4): 489-500, 2017 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-28095394

RESUMEN

BACKGROUND: Colorectal carcinoma (CRC) is the third most common cancer worldwide. Platinum-based anticancer compounds still constitute one mainstay of systemic CRC treatment despite limitations due to adverse effects and resistance development. Trabectedin has shown promising antitumor effects in CRC, however, again resistance development may occur. In this study, we aimed to develop strategies to circumvent or even exploit acquired trabectedin resistance in novel CRC treatment regimens. METHODS: Human HCT116 CRC cells were selected for acquired trabectedin resistance in vitro and characterised by cell biological as well as bioinformatic approaches. In vivo xenograft experiments were conducted. RESULTS: Selection of HCT116 cells for trabectedin resistance resulted in p53-independent hypersensitivity of the selected subline against cisplatin. Bioinformatic analyses of mRNA microarray data suggested deregulation of nucleotide excision repair and particularly loss of the ubiquitin ligase CUL4A in trabectedin-selected cells. Indeed, transient knockdown of CUL4A sensitised parental HCT116 cells towards cisplatin. Trabectedin selected but not parental HCT116 xenografts were significantly responsive towards cisplatin treatment. CONCLUSIONS: Trabectedin selection-mediated CUL4A loss generates an Achilles heel in CRC cancer cells enabling effective cisplatin treatment. Hence, inclusion of trabectedin in cisplatin-containing cancer treatment regimens might cause profound synergism based on reciprocal resistance prevention.


Asunto(s)
Cisplatino/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Proteínas Cullin/genética , Dioxoles/uso terapéutico , Resistencia a Antineoplásicos/genética , Tetrahidroisoquinolinas/uso terapéutico , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Proteínas Cullin/antagonistas & inhibidores , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Genes p53 , Células HCT116 , Humanos , ARN Interferente Pequeño/farmacología , Trabectedina
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