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1.
Biochem Biophys Res Commun ; 586: 129-136, 2022 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-34839191

RESUMEN

Taxol is commonly used chemotherapy regimen for esophageal squamous cell carcinoma (ESCC). Study of the underlying mechanisms of Taxol chemoresistance provides better understanding of esophageal cancer treatment and may provide a rational molecular target for diagnosis and intervention. Here we showed FBXO31, which was reported to be highly expressed in ESCC and significantly associated with poor prognosis, could regulate ESCC chemosensitivity to Taxol. Silencing of FBXO31 in ESCC cells sensitized cells to Taxol treatment, evidenced by FACS analysis and TUNEL assay, showing as an increased apoptotic population in FBXO31-knockdown cells compared to the control cells. The mass spectrometry data and coimmunoprecipitation results showed FBXO31 could bind with cofilin-1. Cofilin-1 knockdown in FBXO31-overexpression cells reversed FBXO31-induced suppression of cell apoptosis, suggesting FBXO31-mediated Taxol chemoresistance is associated with cofilin-1. Furthermore, in vivo experiments confirmed that knockdown of FBXO31 sensitized ESCC to Taxol treatment. This finding substantiated a pivotal role of FBOX31 in ESCC chemoresistance, indicating that FBXO31 may be a potential indicator or target for drug resistance in ESCC.


Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Cofilina 1/genética , Resistencia a Antineoplásicos/genética , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas de Esófago/genética , Proteínas F-Box/genética , Paclitaxel/farmacología , Proteínas Supresoras de Tumor/genética , Animales , Apoptosis/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cofilina 1/metabolismo , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/metabolismo , Carcinoma de Células Escamosas de Esófago/patología , Proteínas F-Box/antagonistas & inhibidores , Proteínas F-Box/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Desnudos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
2.
Nat Chem Biol ; 17(6): 684-692, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33846619

RESUMEN

Heparan sulfate (HS) proteoglycans bind extracellular proteins that participate in cell signaling, attachment and endocytosis. These interactions depend on the arrangement of sulfated sugars in the HS chains generated by well-characterized biosynthetic enzymes; however, the regulation of these enzymes is largely unknown. We conducted genome-wide CRISPR-Cas9 screens with a small-molecule ligand that binds to HS. Screening of A375 melanoma cells uncovered additional genes and pathways impacting HS formation. The top hit was the epigenetic factor KDM2B, a histone demethylase. KDM2B inactivation suppressed multiple HS sulfotransferases and upregulated the sulfatase SULF1. These changes differentially affected the interaction of HS-binding proteins. KDM2B-deficient cells displayed decreased growth rates, which was rescued by SULF1 inactivation. In addition, KDM2B deficiency altered the expression of many extracellular matrix genes. Thus, KDM2B controls proliferation of A375 cells through the regulation of HS structure and serves as a master regulator of the extracellular matrix.


Asunto(s)
Proteínas F-Box/antagonistas & inhibidores , Estudio de Asociación del Genoma Completo , Heparitina Sulfato/metabolismo , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Algoritmos , Sistemas CRISPR-Cas , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Descubrimiento de Drogas , Matriz Extracelular/genética , Ensayos Analíticos de Alto Rendimiento , Humanos , Unión Proteica/genética , RNA-Seq , Sulfotransferasas/antagonistas & inhibidores
3.
Neurotherapeutics ; 18(2): 1295-1315, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33415686

RESUMEN

Many epigenetic regulators are involved in pain-associated spinal plasticity. Coactivator-associated arginine methyltransferase 1 (CARM1), an epigenetic regulator of histone arginine methylation, is a highly interesting target in neuroplasticity. However, its potential contribution to spinal plasticity-associated neuropathic pain development remains poorly explored. Here, we report that nerve injury decreased the expression of spinal CARM1 and induced allodynia. Moreover, decreasing spinal CARM1 expression by Fbxo3-mediated CARM1 ubiquitination promoted H3R17me2 decrement at the K+ channel promoter, thereby causing K+ channel epigenetic silencing and the development of neuropathic pain. Remarkably, in naïve rats, decreasing spinal CARM1 using CARM1 siRNA or a CARM1 inhibitor resulted in similar epigenetic signaling and allodynia. Furthermore, intrathecal administration of BC-1215 (a novel Fbxo3 inhibitor) prevented CARM1 ubiquitination to block K+ channel gene silencing and ameliorate allodynia after nerve injury. Collectively, the results reveal that this newly identified spinal Fbxo3-CARM1-K+ channel gene functional axis promotes neuropathic pain. These findings provide essential insights that will aid in the development of more efficient and specific therapies against neuropathic pain.


Asunto(s)
Epigénesis Genética/fisiología , Proteínas F-Box/antagonistas & inhibidores , Neuralgia/terapia , Manejo del Dolor/métodos , Canales de Potasio , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Animales , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Femenino , Masculino , Neuralgia/genética , Neuralgia/metabolismo , Canales de Potasio/genética , Canales de Potasio/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , ARN Interferente Pequeño/administración & dosificación , Ratas , Ratas Sprague-Dawley , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Asta Dorsal de la Médula Espinal/efectos de los fármacos , Asta Dorsal de la Médula Espinal/metabolismo
4.
Artículo en Inglés | MEDLINE | ID: mdl-33431581

RESUMEN

Molecular genetic and structural studies have revealed the mechanisms of fundamental components of key auxin regulatory pathways consisting of auxin biosynthesis, transport, and signaling. Chemical biology methods applied in auxin research have been greatly expanded through the understanding of auxin regulatory pathways. Many small-molecule modulators of auxin metabolism, transport, and signaling have been generated on the basis of the outcomes of genetic and structural studies on auxin regulatory pathways. These chemical modulators are now widely used as essential tools for dissecting auxin biology in diverse plants. This review covers the structures, primary targets, modes of action, and applications of chemical tools in auxin biosynthesis, transport, and signaling.


Asunto(s)
Bioquímica/métodos , Ácidos Indolacéticos/química , Proteínas de Arabidopsis/agonistas , Proteínas de Arabidopsis/antagonistas & inhibidores , Proteínas F-Box/agonistas , Proteínas F-Box/antagonistas & inhibidores , Profármacos , Receptores de Superficie Celular/agonistas , Receptores de Superficie Celular/antagonistas & inhibidores , Transducción de Señal
5.
Biochem Biophys Res Commun ; 558: 224-230, 2021 06 18.
Artículo en Inglés | MEDLINE | ID: mdl-32933748

RESUMEN

The NF-κB transcription factor is involved in inflammation and cell proliferation, survival, and transformation. It is a heterodimer made of p50 or p52 and a member of the Rel family of proteins. p50 and p52 are derived from limited ubiquitin- and proteasome-mediated proteolytic processing of the larger precursors p105 and p100, respectively. Both precursors can be either processed or completely degraded by the ubiquitin-proteasome system. Previous work in our laboratory identified KPC1 as a ubiquitin ligase that mediates processing of p105 to the p50 subunit. Overexpression of the ligase leads to increased level of p50 with a resultant marked tumor-suppressive effect. In the present study, we identify FBXO7, a known ubiquitin ligase that binds to p105 and ubiquitinates it, but surprisingly, leads to its accumulation and to that of p65 - the Rel partner of p50 - and to increased cell proliferation. Importantly, a ΔF-Box mutant of FBXO7 which is inactive has similar effects on accumulation of p105 and cell proliferation, strongly suggesting that p105 is a pseudo substrate of FBXO7.


Asunto(s)
Proteínas F-Box/metabolismo , Subunidad p50 de NF-kappa B/metabolismo , Secuencia de Aminoácidos , Línea Celular , Proliferación Celular/fisiología , Estabilidad de Enzimas , Proteínas F-Box/antagonistas & inhibidores , Proteínas F-Box/genética , Células HEK293 , Células HeLa , Humanos , Células K562 , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Subunidad p50 de NF-kappa B/antagonistas & inhibidores , Subunidad p50 de NF-kappa B/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteolisis , ARN Interferente Pequeño/genética , Especificidad por Sustrato , Factor de Transcripción ReIA/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitinación
6.
JCI Insight ; 5(11)2020 06 04.
Artículo en Inglés | MEDLINE | ID: mdl-32493843

RESUMEN

Mitochondrial quality control is mediated by the PTEN-induced kinase 1 (PINK1), a cytoprotective protein that is dysregulated in inflammatory lung injury and neurodegenerative diseases. Here, we show that a ubiquitin E3 ligase receptor component, FBXO7, targets PINK1 for its cellular disposal. FBXO7, by mediating PINK1 ubiquitylation and degradation, was sufficient to induce mitochondrial injury and inflammation in experimental pneumonia. A computational simulation-based screen led to the identification of a small molecule, BC1464, which abrogated FBXO7 and PINK1 association, leading to increased cellular PINK1 concentrations and activities, and limiting mitochondrial damage. BC1464 exerted antiinflammatory activity in human tissue explants and murine lung inflammation models. Furthermore, BC1464 conferred neuroprotection in primary cortical neurons, human neuroblastoma cells, and patient-derived cells in several culture models of Parkinson's disease. The data highlight a unique opportunity to use small molecule antagonists that disrupt PINK1 interaction with the ubiquitin apparatus to enhance mitochondrial quality, limit inflammatory injury, and maintain neuronal viability.


Asunto(s)
Proteínas F-Box/antagonistas & inhibidores , Proteínas Mitocondriales/metabolismo , Fármacos Neuroprotectores/farmacología , Proteínas Quinasas/metabolismo , Proteolisis/efectos de los fármacos , Ubiquitinación/efectos de los fármacos , Animales , Línea Celular Tumoral , Estabilidad de Enzimas , Proteínas F-Box/metabolismo , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/patología , Ratones , Fármacos Neuroprotectores/química , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Neumonía/tratamiento farmacológico , Neumonía/metabolismo , Neumonía/patología
7.
Development ; 147(12)2020 06 17.
Artículo en Inglés | MEDLINE | ID: mdl-32467239

RESUMEN

Molecular chaperones often work collaboratively with the ubiquitylation-proteasome system (UPS) to facilitate the degradation of misfolded proteins, which typically safeguards cellular differentiation and protects cells from stress. In this study, however, we report that the Hsp70/Hsp90 chaperone machinery and an F-box protein, MEC-15, have opposing effects on neuronal differentiation, and that the chaperones negatively regulate neuronal morphogenesis and functions. Using the touch receptor neurons (TRNs) of Caenorhabditis elegans, we find that mec-15(-) mutants display defects in microtubule formation, neurite growth, synaptic development and neuronal functions, and that these defects can be rescued by the loss of Hsp70/Hsp90 chaperones and co-chaperones. MEC-15 probably functions in a Skp-, Cullin- and F-box- containing complex to degrade DLK-1, which is an Hsp90 client protein stabilized by the chaperones. The abundance of DLK-1, and likely other Hsp90 substrates, is fine-tuned by the antagonism between MEC-15 and the chaperones; this antagonism regulates TRN development, as well as synaptic functions of GABAergic motor neurons. Therefore, a balance between the UPS and the chaperones tightly controls neuronal differentiation.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas F-Box/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Microtúbulos/metabolismo , Neuritas/fisiología , Animales , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Proteínas de Caenorhabditis elegans/genética , Proteínas F-Box/antagonistas & inhibidores , Proteínas F-Box/genética , Neuronas GABAérgicas/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Chaperonas Moleculares/antagonistas & inhibidores , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutagénesis , Neuronas Aferentes/metabolismo , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Interferencia de ARN , ARN Bicatenario , Ubiquitina/metabolismo , Ubiquitinación
8.
Theranostics ; 10(9): 4150-4167, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32226545

RESUMEN

E3 ubiquitin ligases play a critical role in cellular mechanisms and cancer progression. F-box protein is the core component of the SKP1-cullin 1-F-box (SCF)-type E3 ubiquitin ligase and directly binds to substrates by various specific domains. According to the specific domains, F-box proteins are further classified into three sub-families: 1) F-box with leucine rich amino acid repeats (FBXL); 2) F-box with WD 40 amino acid repeats (FBXW); 3) F-box only with uncharacterized domains (FBXO). Here, we summarize the substrates of F-box proteins, discuss the important molecular mechanism and emerging role of F-box proteins especially from the perspective of cancer development and progression. These findings will shed new light on malignant tumor progression mechanisms, and suggest the potential role of F-box proteins as cancer biomarkers and therapeutic targets for future cancer treatment.


Asunto(s)
Proteínas F-Box , Neoplasias/metabolismo , Animales , Línea Celular Tumoral , Proteínas F-Box/antagonistas & inhibidores , Proteínas F-Box/metabolismo , Humanos , Ligandos , Ratones , Transducción de Señal , Ubiquitinación
9.
J Invest Dermatol ; 140(6): 1154-1165.e5, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-31705877

RESUMEN

We performed a small interfering RNA screen to identify targets for cutaneous squamous cell carcinoma (cSCC) therapy in the ubiquitin/ubiquitin-like system. We provide evidence for selective anti-cSCC activity of knockdown of the E3 ubiquitin ligase MARCH4, the ATPase p97/VCP, the deubiquitinating enzyme USP8, the cullin-RING ligase (CRL) 4 substrate receptor CDT2/DTL, and components of the anaphase-promoting complex/cyclosome (APC/C). Specifically attenuating CRL4CDT2 by CDT2 knockdown can be more potent in killing cSCC cells than targeting CRLs or CRL4s in general by RBX1 or DDB1 depletion. Suppression of the APC/C or forced APC/C activation by targeting its repressor EMI1 are both potential therapeutic approaches. We observed that cSCC cells can be selectively killed by small-molecule inhibitors of USP8 (DUBs-IN-3/compound 22c) and the NEDD8 E1 activating enzyme/CRLs (MLN4924/pevonedistat). A substantial proportion of cSCC cell lines are very highly MLN4924-sensitive. Pathways that respond to defects in proteostasis are involved in the anti-cSCC activity of p97 suppression. Targeting USP8 can reduce the expression of growth factor receptors that participate in cSCC development. EMI1 and CDT2 depletion can selectively cause DNA re-replication and DNA damage in cSCC cells.


Asunto(s)
Carcinoma de Células Escamosas/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Carcinoma de Células Escamosas/patología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Ciclopentanos/farmacología , Ciclopentanos/uso terapéutico , Ensayos de Selección de Medicamentos Antitumorales , Endopeptidasas/genética , Endopeptidasas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas F-Box/antagonistas & inhibidores , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Terapia Molecular Dirigida/métodos , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , ARN Interferente Pequeño/metabolismo , Neoplasias Cutáneas/patología , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Enzimas Activadoras de Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/metabolismo
10.
Invest New Drugs ; 38(1): 20-28, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-30887251

RESUMEN

F-box proteins, a type of substrate-recognition complexes consisting of SKP1-cullin 1-F-box protein (SCF) E3 ligase, can critically affect many cellular processes because of the ubiquitylation and subsequent degradation of target proteins. This study investigated the effect of FBXO22 on melanoma angiogenesis, migration, and invasion. Results showed that FBXO22 staining intensity was increased in malignant melanoma (MM) compared with that in skin tissue (P˂0.001). The percentage of high FBXO22 expression in MM (74.3%) was markedly higher than that in paracancerous and skin tissues (0%) (P˂0.001). FBXO22 was also overexpressed in MM tissues compared with that in normal skin tissues. FBXO22 knockdown in vitro inhibited MM cell migration, invasion, and angiogenesis (P < 0.001). In vivo studies confirmed that using nude mice with knocked down FBXO22 reduced the formation of blood vessels and decreased the positive rate of CD31 (P < 0.05). HIF-1α expression varied with FBXO22, indicating that FBXO22 regulated the expression of HIF-1α and VEGFA and that FBXO22 was a regulator of HIF-1α and VEGF for the control of tumor angiogenesis. In conclusion, FBXO22 promoted the migration and invasion of tumor cells and melanoma angiogenesis via HIF-1α upregulation. This study demonstrated that FBXO22 knockdown suppressed tumor progression and metastasis, suggesting that FBXO22 might be developed as a novel target for treating patients with MM.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Proteínas F-Box/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Melanoma/patología , Neovascularización Patológica/patología , Receptores Citoplasmáticos y Nucleares/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Apoptosis , Biomarcadores de Tumor/genética , Ciclo Celular , Movimiento Celular , Proliferación Celular , Proteínas F-Box/antagonistas & inhibidores , Proteínas F-Box/genética , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Melanoma/genética , Melanoma/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Pronóstico , ARN Interferente Pequeño/genética , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/genética , Transducción de Señal , Células Tumorales Cultivadas , Ubiquitinación , Factor A de Crecimiento Endotelial Vascular/genética , Ensayos Antitumor por Modelo de Xenoinjerto
11.
J Biochem ; 166(6): 517-527, 2019 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-31778188

RESUMEN

Dysregulation of microRNAs (miRNAs) plays a key role during the pathogenesis of chemoresistance in lung cancer (LCa). Previous study suggests that miR-324-5p may serve as a unique miRNA signature for LCa, but its role and the corresponding molecular basis remain largely explored. Herein, we report that miR-324-5p expression was significantly increased in cisplatin (CDDP)-resistant LCa tissues and cells, and this upregulation predicted a poor post-chemotherapy prognosis in LCa patients. miR-324-5p was further shown to impact CDDP response: Ectopic miR-324-5p expression in drug-naïve LCa cells was sufficient to attenuate sensitivity to CDDP and to confer more robust tumour growth in CDDP-challenged nude mice. Conversely, ablation of miR-324-5p expression in resistant cells effectively potentiated CDDP-suppressed cell growth in vitro and in vivo. Using multiple approaches, we further identified the tumour suppressor FBXO11 as the direct down-stream target of miR-324-5p. Stable expression of FBXO11 could abrogate the pro-survival effects of miR-324-5p in CDDP-challenged LCa cells. Together, these findings suggest that miR-324-5p upregulation mediates, at least partially, the CDDP resistance by directly targeting FBXO11 signalling in LCa cells. In-depth elucidation of the molecular basis underpinning miR-324-5p action bears potential implications for mechanism-based strategies to improve CDDP responses in LCa.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Cisplatino/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas F-Box/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , MicroARNs/farmacología , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Regulación hacia Arriba/efectos de los fármacos , Células A549 , Animales , Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad
12.
PLoS Pathog ; 15(7): e1007946, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31348812

RESUMEN

By binding to the adaptor protein SKP1 and serving as substrate receptors for the SKP1 Cullin, F-box E3 ubiquitin ligase complex, F-box proteins regulate critical cellular processes including cell cycle progression and membrane trafficking. While F-box proteins are conserved throughout eukaryotes and are well studied in yeast, plants, and animals, studies in parasitic protozoa are lagging. We have identified eighteen putative F-box proteins in the Toxoplasma genome of which four have predicted homologs in Plasmodium. Two of the conserved F-box proteins were demonstrated to be important for Toxoplasma fitness and here we focus on an F-box protein, named TgFBXO1, because it is the most highly expressed by replicative tachyzoites and was also identified in an interactome screen as a Toxoplasma SKP1 binding protein. TgFBXO1 interacts with Toxoplasma SKP1 confirming it as a bona fide F-box protein. In interphase parasites, TgFBXO1 is a component of the Inner Membrane Complex (IMC), which is an organelle that underlies the plasma membrane. Early during replication, TgFBXO1 localizes to the developing daughter cell scaffold, which is the site where the daughter cell IMC and microtubules form and extend from. TgFBXO1 localization to the daughter cell scaffold required centrosome duplication but before kinetochore separation was completed. Daughter cell scaffold localization required TgFBXO1 N-myristoylation and was dependent on the small molecular weight GTPase, TgRab11b. Finally, we demonstrate that TgFBXO1 is required for parasite growth due to its function as a daughter cell scaffold effector. TgFBXO1 is the first F-box protein to be studied in apicomplexan parasites and represents the first protein demonstrated to be important for daughter cell scaffold function.


Asunto(s)
Proteínas F-Box/fisiología , Proteínas Protozoarias/fisiología , Toxoplasma/crecimiento & desarrollo , Toxoplasma/patogenicidad , Animales , Proteínas F-Box/antagonistas & inhibidores , Proteínas F-Box/genética , Técnicas de Silenciamiento del Gen , Genes Protozoarios , Humanos , Dominios y Motivos de Interacción de Proteínas , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/genética , Proteínas Quinasas Asociadas a Fase-S/fisiología , Toxoplasma/genética
13.
Cell ; 178(2): 316-329.e18, 2019 07 11.
Artículo en Inglés | MEDLINE | ID: mdl-31257023

RESUMEN

Approximately 30% of human lung cancers acquire mutations in either Keap1 or Nfe2l2, resulting in the stabilization of Nrf2, the Nfe2l2 gene product, which controls oxidative homeostasis. Here, we show that heme triggers the degradation of Bach1, a pro-metastatic transcription factor, by promoting its interaction with the ubiquitin ligase Fbxo22. Nrf2 accumulation in lung cancers causes the stabilization of Bach1 by inducing Ho1, the enzyme catabolizing heme. In mouse models of lung cancers, loss of Keap1 or Fbxo22 induces metastasis in a Bach1-dependent manner. Pharmacological inhibition of Ho1 suppresses metastasis in a Fbxo22-dependent manner. Human metastatic lung cancer display high levels of Ho1 and Bach1. Bach1 transcriptional signature is associated with poor survival and metastasis in lung cancer patients. We propose that Nrf2 activates a metastatic program by inhibiting the heme- and Fbxo22-mediated degradation of Bach1, and that Ho1 inhibitors represent an effective therapeutic strategy to prevent lung cancer metastasis.


Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Neoplasias Pulmonares/patología , Factor 2 Relacionado con NF-E2/metabolismo , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/antagonistas & inhibidores , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Línea Celular Tumoral , Movimiento Celular , Proteínas F-Box/antagonistas & inhibidores , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Femenino , Hemo-Oxigenasa 1/antagonistas & inhibidores , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Humanos , Estimación de Kaplan-Meier , Proteína 1 Asociada A ECH Tipo Kelch/antagonistas & inhibidores , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 2 Relacionado con NF-E2/genética , Metástasis de la Neoplasia , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Activación Transcripcional
14.
Biochem Biophys Res Commun ; 514(2): 558-564, 2019 06 25.
Artículo en Inglés | MEDLINE | ID: mdl-31060780

RESUMEN

Traumatic spinal cord injury (SCI) is a major cause of death and lifelong disability in the world. However, the pathological process of SCI has not been fully understood. F-box/WD repeat-containing protein 5 (FBXW5), a subunit of the SCF-type E3 ubiquitin ligase complex, plays an essential role in regulating various pathologies. However, little is known about the effects of FBXW5 on the progression of SCI. In this study, using a rodent model with SCI, we found that FBXW5 expression was markedly down-regulated in spinal dorsal horn of rats after SCI surgery. Rats with FBXW5 knockdown showed the improved paw withdrawal latency responding to thermal stimuli on the ipsilateral side while showed no significant influence on the basal threshold on the contralateral side. In addition, SCI-induced increase of pro-inflammatory cytokines, including tumor necrosis factor α (TNF-α), interleukin (IL)-1ß and IL-6, was obviously decreased by FBXW5 knockdown, along with microglia inactivation as evidenced by the reduced expression of Iba-1. Moreover, immunofluorescent staining suggested that FBXW5 was co-localized with Iba-1 in spinal cord tissues of SCI rats. Furthermore, p38, Jun kinase (JNK) and extracellular signal-regulated kinase (ERK)-1/2 activation was significantly increased by SCI in spinal dosal horn of rats. Notably, FBXW5 knockdown markedly reduced the expression of phosphorylated p38 and JNK without affecting ERK1/2 activity in SCI rats. What's more, suppressing p38 and JNK activation significantly alleviated SCI-induced abnormal behavior in rats, along with reduced expression of pro-inflammatory cytokines. Taken together, these results provided evidence that down-regulation of FBXW5 was involved in the prevention of SCI.


Asunto(s)
Proteínas F-Box/genética , Hiperalgesia/genética , MAP Quinasa Quinasa 4/genética , Microglía/metabolismo , Traumatismos de la Médula Espinal/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Progresión de la Enfermedad , Proteínas F-Box/antagonistas & inhibidores , Proteínas F-Box/metabolismo , Regulación de la Expresión Génica , Terapia Genética/métodos , Hiperalgesia/metabolismo , Hiperalgesia/patología , Hiperalgesia/prevención & control , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Masculino , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Microglía/patología , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Asta Dorsal de la Médula Espinal/lesiones , Asta Dorsal de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/terapia , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
15.
Cell Death Dis ; 10(5): 351, 2019 04 25.
Artículo en Inglés | MEDLINE | ID: mdl-31024008

RESUMEN

F-box only protein 8 (FBX8), as a critical component of the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases, has been associated with several malignancies through interacting with a member of proteins. However, the substrates of FBX8 for destruction in the progression of colorectal carcinoma (CRC) need to be explored. Here, we show that loss of FBX8 accelerates chemical-induced colon tumorigenesis. FBX8 directly targets GSTP1 for ubiquitin-mediated proteasome degradation in CRC. GSTP1 promotes the proliferation, invasion, and metastasis of CRC cells. Furthermore, GSTP1 is upregulated in CRC tissue samples and predicts poor prognosis of CRC patients. The inactivation of FBX8 negatively correlated with increased levels and stability of GSTP1 in clinical CRC tissues and FBX8 knockout transgenic mice. These findings identify a novel ubiquitination pathway as FBX8-GSTP1 axis that regulates the progression of CRC, which might be a potential prognostic biomarker for CRC patients.


Asunto(s)
Neoplasias Colorrectales/patología , Proteínas F-Box/metabolismo , Gutatión-S-Transferasa pi/metabolismo , Animales , Biomarcadores/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Neoplasias Colorrectales/inducido químicamente , Neoplasias Colorrectales/mortalidad , Regulación hacia Abajo , Proteínas F-Box/antagonistas & inhibidores , Proteínas F-Box/genética , Gutatión-S-Transferasa pi/antagonistas & inhibidores , Gutatión-S-Transferasa pi/genética , Humanos , Estimación de Kaplan-Meier , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Pronóstico , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ubiquitinación
16.
Cell Mol Life Sci ; 76(11): 2217-2229, 2019 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-30980108

RESUMEN

As the female gamete, meiotic oocytes provide not only half of the genome but also almost all stores for fertilization and early embryonic development. Because de novo mRNA transcription is absent in oocyte meiosis, protein-level regulations, especially the ubiquitin proteasome system, are more crucial. As the largest family of ubiquitin E3 ligases, Skp1-Cullin-F-box complexes recognize their substrates via F-box proteins with substrate-selected specificity. However, the variety of F-box proteins and their unknown substrates hinder our understanding of their functions. In this report, we find that Fbxo30, a new member of F-box proteins, is enriched in mouse oocytes, and its expression level declines substantially after the metaphase of the first meiosis (MI). Notably, depletion of Fbxo30 causes significant chromosome compaction accompanied by chromosome segregation failure and arrest at the MI stage, and this arrest is not caused by over-activation of spindle assembly checkpoint. Using immunoprecipitation and mass spectrometric analysis, we identify stem-loop-binding protein (SLBP) as a novel substrate of Fbxo30. SLBP overexpression caused by Fbxo30 depletion results in a remarkable overload of histone H3 on chromosomes that excessively condenses chromosomes and inhibits chromosome segregation. Our finding uncovers an unidentified pathway-controlling chromosome segregation and cell progress.


Asunto(s)
Segregación Cromosómica , Cromosomas de los Mamíferos/metabolismo , Proteínas F-Box/genética , Histonas/genética , Meiosis , Proteínas Nucleares/genética , Oocitos/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Animales , Cromosomas de los Mamíferos/ultraestructura , Proteínas F-Box/antagonistas & inhibidores , Proteínas F-Box/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Histonas/metabolismo , Ratones , Ratones Endogámicos ICR , Proteínas Nucleares/metabolismo , Oocitos/ultraestructura , Cultivo Primario de Células , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN , Transducción de Señal , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo
17.
Mol Med Rep ; 19(2): 1049-1055, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30483744

RESUMEN

MicroRNAs (miRNAs) are acknowledged as essential regulators in human cancer types, including glioblastoma (GBM). However, the functions of microRNA­3666 (miR­3666) in GBM remain unclear. In the present study, it was identified that the expression of miR­3666 was significantly downregulated in GBM tissues compared with adjacent normal tissues by reverse transcription­quantitative polymerase chain reaction. Additionally, miR­3666 was downregulated in GBM cell lines. Furthermore, it was observed that the miR­3666 expression level in patients with GBM was associated with prognosis. With functional experiments, it was identified that overexpression of miR­3666 significantly inhibited the proliferation, migration and invasion of GBM cells in vitro by Cell Counting kit­8 and Transwell assays. Ectopic expression of miR­3666 significantly arrested GBM cells in the G0 phase by fluorescence activated cell sorting. In terms of the underlying mechanism, it was identified that lysine­specific demethylase 2A (KDM2A) is a direct target of miR­3666 in GBM cells. Overexpression of miR­3666 significantly decreased the expression of KDM2A in GBM cells. Furthermore, it was observed that knockdown of KDM2A significantly suppressed the proliferation, migration and invasion of GBM cells. Collectively, the present results demonstrated that the miR­3666/KDM2A axis serves an important role in the progression of GBM, which provides novel insight into the development of therapeutic strategies for GBM treatment.


Asunto(s)
Neoplasias Encefálicas/genética , Proteínas F-Box/genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Histona Demetilasas con Dominio de Jumonji/genética , MicroARNs/genética , Adulto , Secuencia de Bases , Sitios de Unión , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Ciclo Celular/genética , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Proteínas F-Box/antagonistas & inhibidores , Proteínas F-Box/metabolismo , Femenino , Glioblastoma/metabolismo , Glioblastoma/mortalidad , Glioblastoma/patología , Humanos , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Histona Demetilasas con Dominio de Jumonji/metabolismo , Masculino , MicroARNs/metabolismo , Persona de Mediana Edad , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Análisis de Supervivencia
18.
Cell Death Dis ; 9(11): 1123, 2018 11 08.
Artículo en Inglés | MEDLINE | ID: mdl-30409964

RESUMEN

Epithelial ovarian carcinoma (EOC) is the most lethal gynecologic malignancy. However, the molecular mechanisms remain unclear. In this study, we found that miR-146b was downregulated in EOC and its expression level was negatively correlated with the pathological staging. Follow-up functional experiments illustrated that overexpression of miR-146b significantly inhibited cell migration and invasion, and increased cell proliferation, but it also improved the response to chemotherapeutic agents. Mechanistically, we demonstrated that miR-146b exerted its function mainly through inhibiting F-box and leucine-rich repeat protein 10 (FBXL10), and upregulated the Cyclin D1, vimentin (VIM), and zona-occludens-1 (ZO-1) expression in EOC. These findings indicate that miR-146b-FBXL10 axis is an important epigenetic regulation pathway in EOC. Low miR-146b may contribute to cancer progression from primary stage to advanced stage, and may be the promising therapeutic target of EOC.


Asunto(s)
Carcinoma Epitelial de Ovario/genética , Resistencia a Antineoplásicos/genética , Proteínas F-Box/genética , Regulación Neoplásica de la Expresión Génica , Histona Demetilasas con Dominio de Jumonji/genética , MicroARNs/genética , Neoplasias Ováricas/genética , Adulto , Animales , Antineoplásicos/farmacología , Carcinoma Epitelial de Ovario/tratamiento farmacológico , Carcinoma Epitelial de Ovario/metabolismo , Carcinoma Epitelial de Ovario/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Ciclina D1/genética , Ciclina D1/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Epigénesis Genética , Proteínas F-Box/antagonistas & inhibidores , Proteínas F-Box/metabolismo , Femenino , Humanos , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Histona Demetilasas con Dominio de Jumonji/metabolismo , Ratones , Ratones Desnudos , MicroARNs/metabolismo , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Paclitaxel/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Carga Tumoral/efectos de los fármacos , Vimentina/genética , Vimentina/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-1/metabolismo
19.
JCI Insight ; 3(19)2018 10 04.
Artículo en Inglés | MEDLINE | ID: mdl-30282819

RESUMEN

The acute respiratory distress syndrome (ARDS) causes an estimated 70,000 US deaths annually. Multiple pharmacologic interventions for ARDS have been tested and failed. An unmet need is a suitable laboratory human model to predictively assess emerging therapeutics on organ function in ARDS. We previously demonstrated that the small molecule BC1215 blocks actions of a proinflammatory E3 ligase-associated protein, FBXO3, to suppress NF-κB signaling in animal models of lung injury. Ex vivo lung perfusion (EVLP) is a clinical technique that maintains lung function for possible transplant after organ donation. We used human lungs unacceptable for transplant to model endotoxemic injury with EVLP for 6 hours. LPS infusion induced inflammatory injury with impaired oxygenation of pulmonary venous circulation. BC1215 treatment after LPS rescued oxygenation and decreased inflammatory cytokines in bronchoalveolar lavage. RNA sequencing transcriptomics from biopsies taken during EVLP revealed robust inflammatory gene induction by LPS with a strong signal for NF-κB-associated transcripts. BC1215 treatment reduced the LPS induction of genes associated with inflammatory and host defense gene responses by Gene Ontology (GOterm) and pathways analysis. BC1215 also significantly antagonized LPS-mediated NF-κB activity. EVLP may provide a unique human platform for preclinical study of chemical entities such as FBXO3 inhibitors on tissue physiology.


Asunto(s)
Bencilaminas/farmacología , Proteínas F-Box/antagonistas & inhibidores , Pulmón/efectos de los fármacos , Perfusión/métodos , Piridinas/farmacología , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Adolescente , Adulto , Bencilaminas/uso terapéutico , Evaluación Preclínica de Medicamentos/métodos , Proteínas F-Box/metabolismo , Femenino , Humanos , Lipopolisacáridos/toxicidad , Pulmón/patología , Masculino , Persona de Mediana Edad , Piridinas/uso terapéutico , Síndrome de Dificultad Respiratoria/inducido químicamente , Síndrome de Dificultad Respiratoria/patología , Transducción de Señal/efectos de los fármacos
20.
Cell Death Dis ; 9(8): 811, 2018 07 24.
Artículo en Inglés | MEDLINE | ID: mdl-30042425

RESUMEN

Presynaptic active zone proteins play a crucial role in regulating synaptic plasticity. Although the ubiquitin-proteasome system underlying the degradation of the presynaptic active zone protein is well established, the contribution of this machinery to regulating spinal plasticity during neuropathic pain development remains unclear. Here, using male Sprague Dawley rats, we demonstrated along with behavioral allodynia, neuropathic injury induced a marked elevation in the expression levels of an active zone protein Munc13-1 in the homogenate and synaptic plasma membrane of the ipsilateral dorsal horn. Moreover, nerve injury-increased Munc13-1 expression was associated with an increase in the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) in ipsilateral dorsal horn neurons. This neuropathic injury-induced accumulation of Munc13-1 colocalized with synaptophysin but not homer1 in the dorsal horn. Focal knockdown of spinal Munc13-1 expression attenuated behavioral allodynia and the increased frequency, not the amplitude, of mEPSCs in neuropathic rats. Remarkably, neuropathic injury decreased spinal Fbxo45 expression, Fbxo45-Munc13-1 co-precipitation, and Munc13-1 ubiquitination in the ipsilateral dorsal horn. Conversely, focal knockdown of spinal Fbxo45 expression in naive animals resulted in behavioral allodynia in association with similar protein expression and ubiquitination in the dorsal horn as observed with neuropathic injury rats. Furthermore, both neuropathic insults and intrathecal injection of tumor necrosis factor-α (TNF-α) impeded spinal Fbxo45-dependent Munc13-1 ubiquitination, which was reversed by intrathecal TNF-α-neutralizing antibody. Our data revealed that spinal TNF-α impedes Fbxo45-dependent Munc13-1 ubiquitination that accumulates Munc13-1 in the presynaptic area and hence facilitates the synaptic excitability of nociceptive neurotransmission underlying neuropathic pain.


Asunto(s)
Proteínas F-Box/metabolismo , Hiperalgesia/patología , Proteínas del Tejido Nervioso/metabolismo , Asta Dorsal de la Médula Espinal/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Ubiquitinación/efectos de los fármacos , Animales , Anticuerpos Neutralizantes/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Proteínas F-Box/antagonistas & inhibidores , Proteínas F-Box/genética , Ácido Glutámico/metabolismo , Hiperalgesia/etiología , Hiperalgesia/metabolismo , Masculino , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Técnicas de Placa-Clamp , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Asta Dorsal de la Médula Espinal/patología , Asta Dorsal de la Médula Espinal/fisiología , Nervios Espinales/lesiones , Factor de Necrosis Tumoral alfa/inmunología
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