RESUMEN
Among a series of benzopyridone-based scaffolds investigated as human transient receptor potential vanilloid 1 (TRPV1) ligands, two isomeric benzopyridone scaffolds demonstrated a consistent and distinctive functional profile in which 2-oxo-1,2-dihydroquinolin-5-yl analogues (e.g., 2) displayed high affinity and potent antagonism, whereas 1-oxo-1,2-dihydroisoquinolin-5-yl analogues (e.g., 3) showed full agonism with high potency. Our computational models provide insight into the agonist-antagonist boundary of the analogues suggesting that the Arg557 residue in the S4-S5 linker might be important for sensing the agonist binding and transmitting signals. These results provide structural insights into the TRPV1 and the protein-ligand interactions at a molecular level.
Asunto(s)
Descubrimiento de Drogas , Piridonas/química , Canales Catiónicos TRPV/agonistas , Canales Catiónicos TRPV/antagonistas & inhibidores , Animales , Humanos , Estructura Molecular , Relación Estructura-Actividad , Urea/químicaRESUMEN
A series of 1-indazol-3-(1-phenylpyrazol-5-yl)methyl ureas were investigated as hTRPV1 antagonists. The structure-activity relationship study was conducted systematically for both the indazole A-region and the 3-trifluoromethyl/t-butyl pyrazole C-region to optimize the antagonism toward the activation by capsaicin. Among them, the antagonists 26, 50 and 51 displayed highly potent antagonism with Ki(CAP) = 0.4-0.5 nM. Further, in vivo studies in mice indicated that these derivatives both antagonized capsaicin induced hypothermia, consistent with their in vitro activity, and themselves did not induce hyperthermia. In the formalin model, 51 showed anti-nociceptive activity in a dose-dependent manner.
Asunto(s)
Indazoles/farmacología , Compuestos de Metilurea/farmacología , Pirazoles/farmacología , Canales Catiónicos TRPV/antagonistas & inhibidores , Analgésicos/síntesis química , Analgésicos/farmacología , Animales , Temperatura Corporal/efectos de los fármacos , Células CHO , Capsaicina/farmacología , Cricetulus , Humanos , Indazoles/síntesis química , Compuestos de Metilurea/síntesis química , Ratones , Estructura Molecular , Pirazoles/síntesis química , Relación Estructura-Actividad , Canales Catiónicos TRPV/agonistasRESUMEN
A series of indane-type acetamide and propanamide analogues were investigated as TRPV1 antagonists. The analysis of structure-activity relationship indicated that indane A-region analogues exhibited better antagonism than did the corresponding 2,3-dihydrobenzofuran and 1,3-benzodioxole surrogates. Among them, antagonist 36 exhibited potent and selective antagonism toward capsaicin for hTRPV1 and mTRPV1. Further, in vivo studies indicated that antagonist 36 showed excellent analgesic activity in both phases of the formalin mouse pain model and inhibited the pain behavior completely at a dose of 1 mg/kg in the 2nd phase.
Asunto(s)
Amidas/química , Indanos/química , Canales Catiónicos TRPV/antagonistas & inhibidores , Acetamidas/química , Acetamidas/metabolismo , Acetamidas/uso terapéutico , Amidas/metabolismo , Amidas/uso terapéutico , Analgésicos/química , Analgésicos/uso terapéutico , Animales , Capsaicina/química , Capsaicina/metabolismo , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Humanos , Ratones , Dolor/inducido químicamente , Dolor/tratamiento farmacológico , Piridinas/química , Relación Estructura-Actividad , Canales Catiónicos TRPV/metabolismoRESUMEN
In order to discover a novel type of analgesic, we investigated dual activity ligands with TRPV1 antagonism and mu-opioid receptor affinity with the goal of eliciting synergistic analgesia while avoiding the side effects associated with single targeting. Based on a combination approach, a series of 4-benzyl-4-(dimethylamino)piperidinyl analogues were designed, synthesized and evaluated for their receptor activities. Among them, compound 49 exhibited the most promising dual-acting activity toward TRPV1 and the mu-opioid receptor in vitro. In vivo,49 displayed potent, dose-dependent antinociceptive activity in both the 1st and 2nd phases in the formalin assay. Consistent with its postulated mechanism, we confirmed that in vivo, as in vitro, compound 49 both antagonized TRPV1 and functioned as a mu-opioid agonist. This result indicates that dual-acting TRPV1 antagonist/mu-opioid ligands can be made and represent a new and promising class of analgesic.
Asunto(s)
Analgésicos Opioides/farmacología , Descubrimiento de Drogas , Dolor/tratamiento farmacológico , Receptores Opioides/metabolismo , Canales Catiónicos TRPV/antagonistas & inhibidores , Animales , Células CHO , Células Cultivadas , Cricetulus , Relación Dosis-Respuesta a Droga , Humanos , Ligandos , Masculino , Ratones , Ratones Endogámicos ICR , Estructura Molecular , Dolor/metabolismo , Relación Estructura-Actividad , Canales Catiónicos TRPV/metabolismoRESUMEN
BACKGROUND: Single-port laparoscopic approaches are well established in the field of minimally invasive surgery; however, single-port laparoscopic distal pancreatectomy (SPLDP) has not been evaluated in a large number of distal pancreatic neoplasms. We aimed to compare single-port laparoscopic distal pancreatectomy outcomes with conventional laparoscopic distal pancreatectomy (LDP) outcomes. METHODS: We retrospectively evaluated the medical records of 101 patients who underwent SPLDP (n = 26) or LDP (n = 75). We performed 1:1 propensity score matching between the two groups. Consequently, 26 patients were included in each group. We analyzed the learning curve based on the operation time in SPLDP. RESULTS: Single-port laparoscopic distal pancreatectomy could be performed with fewer trocars (P < 0.001) and assistants (P < 0.001). However, compared to the LDP group, mean operation time was longer (278.9 vs. 178.7 min, P < 0.001) and splenic vessel preservation rates were lower (0% vs. 46.2%, P < 0.001) in the SPLDP group. The mean pain visual analogue scale score was significantly lower at postoperative day 1 (P < 0.001) and day 2 (P < 0.001) in the SPLDP group. The learning curve was determined in the 12th case for SPLDP. CONCLUSIONS: Single-port laparoscopic distal pancreatectomy is comparable in safety to conventional laparoscopic approaches for distal pancreatic neoplasms, with fewer trocars, assistants and less pain; however, operation time was longer.
Asunto(s)
Laparoscopía/educación , Laparoscopía/métodos , Curva de Aprendizaje , Pancreatectomía/educación , Pancreatectomía/métodos , Neoplasias Pancreáticas/cirugía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Tempo Operativo , Dimensión del Dolor , Complicaciones Posoperatorias/sangre , Complicaciones Posoperatorias/diagnóstico por imagen , Puntaje de Propensión , Estudios RetrospectivosRESUMEN
Atopic dermatitis (AD) is a chronic inflammatory skin disease whose prevalence is increasing worldwide. Filaggrin (FLG) is essential for the development of the skin barrier, and its genetic mutations are major predisposing factors for AD. In this study, we developed a convenient and practical method to detect FLG mutations in AD patients using peptide nucleic acid (PNA) probes labelled with fluorescent markers for rapid analysis. Fluorescence melting curve analysis (FMCA) precisely identified FLG mutations based on the distinct difference in the melting temperatures of the wild-type and mutant allele. Moreover, PNA probe-based FMCA easily and accurately verified patient samples with both heterozygote and homozygote FLG mutations, providing a high-throughput method to reliable screen AD patients. Our method provides a convenient, rapid and accurate diagnostic tool to identify potential AD patients allowing for early preventive treatment, leading to lower incidence rates of AD, and reducing total healthcare expenses.
Asunto(s)
Análisis Mutacional de ADN/métodos , Sondas de ADN , Dermatitis Atópica/diagnóstico , Dermatitis Atópica/genética , Proteínas de Filamentos Intermediarios/genética , Alelos , Estudios de Casos y Controles , Proteínas Filagrina , Fluorescencia , Heterocigoto , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Homocigoto , Humanos , Mutación , Ácidos Nucleicos de Péptidos/genética , Temperatura de TransiciónRESUMEN
The conjugation of amino acids to the protein N termini is universally observed in eukaryotes and prokaryotes, yet its functions remain poorly understood. In eukaryotes, the amino acid l-arginine (l-Arg) is conjugated to N-terminal Asp (Nt-Asp), Glu, Gln, Asn, and Cys, directly or associated with posttranslational modifications. Following Nt-arginylation, the Nt-Arg is recognized by UBR boxes of N-recognins such as UBR1, UBR2, UBR4/p600, and UBR5/EDD, leading to substrate ubiquitination and proteasomal degradation via the N-end rule pathway. It has been a mystery, however, why studies for the past five decades identified only a handful of Nt-arginylated substrates in mammals, although five of 20 principal amino acids are eligible for arginylation. Here, we show that the Nt-Arg functions as a bimodal degron that directs substrates to either the ubiquitin (Ub)-proteasome system (UPS) or macroautophagy depending on physiological states. In normal conditions, the arginylated forms of proteolytic cleavage products, D101-CDC6 and D1156-BRCA1, are targeted to UBR box-containing N-recognins and degraded by the proteasome. However, when proteostasis by the UPS is perturbed, their Nt-Arg redirects these otherwise cellular wastes to macroautophagy through its binding to the ZZ domain of the autophagic adaptor p62/STQSM/Sequestosome-1. Upon binding to the Nt-Arg, p62 acts as an autophagic N-recognin that undergoes self-polymerization, facilitating cargo collection and lysosomal degradation of p62-cargo complexes. A chemical mimic of Nt-Arg redirects Ub-conjugated substrates from the UPS to macroautophagy and promotes their lysosomal degradation. Our results suggest that the Nt-Arg proteome of arginylated proteins contributes to reprogramming global proteolytic flux under stresses.
Asunto(s)
Arginina/metabolismo , Autofagia/fisiología , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteolisis , Proteínas de Unión al ARN/metabolismo , Aminoaciltransferasas/genética , Aminoaciltransferasas/metabolismo , Animales , Autofagia/efectos de los fármacos , Proteína BRCA1/metabolismo , Femenino , Células HEK293 , Células HeLa , Humanos , Hidroxicloroquina/farmacología , Ratones , Ratones Endogámicos C57BL , Complejo de la Endopetidasa Proteasomal/metabolismo , Dominios Proteicos , Ubiquitina/metabolismoRESUMEN
Macroautophagy mediates the selective degradation of proteins and non-proteinaceous cellular constituents. Here, we show that the N-end rule pathway modulates macroautophagy. In this mechanism, the autophagic adapter p62/SQSTM1/Sequestosome-1 is an N-recognin that binds type-1 and type-2 N-terminal degrons (N-degrons), including arginine (Nt-Arg). Both types of N-degrons bind its ZZ domain. By employing three-dimensional modeling, we developed synthetic ligands to p62 ZZ domain. The binding of Nt-Arg and synthetic ligands to ZZ domain facilitates disulfide bond-linked aggregation of p62 and p62 interaction with LC3, leading to the delivery of p62 and its cargoes to the autophagosome. Upon binding to its ligand, p62 acts as a modulator of macroautophagy, inducing autophagosome biogenesis. Through these dual functions, cells can activate p62 and induce selective autophagy upon the accumulation of autophagic cargoes. We also propose that p62 mediates the crosstalk between the ubiquitin-proteasome system and autophagy through its binding Nt-Arg and other N-degrons.Soluble misfolded proteins that fail to be degraded by the ubiquitin proteasome system (UPS) are redirected to autophagy via specific adaptors, such as p62. Here the authors show that p62 recognises N-degrons in these proteins, acting as a N-recognin from the proteolytic N-end rule pathway, and targets these cargos to autophagosomal degradation.
Asunto(s)
Autofagosomas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína Sequestosoma-1/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales , Animales , Arginina/metabolismo , Autofagia , Sitios de Unión , Western Blotting , Células Cultivadas , Células HEK293 , Células HeLa , Humanos , Ratones Noqueados , Microscopía Confocal , Modelos Moleculares , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Dominios Proteicos , Proteolisis , Proteína Sequestosoma-1/química , Proteína Sequestosoma-1/genética , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Although proteasome inhibitors (PIs) are used as anticancer drugs to treat various cancers, their relative therapeutic efficacy on stem cells vs. bulk cancers remains unknown. Here, we show that stem cells derived from gliomas, GSCs, are up to 1,000-fold more sensitive to PIs (IC50, 27-70 nM) compared with their differentiated controls (IC50, 47 to ¼100 µM). The stemness of GSCs correlates to increased ubiquitination, whose misregulation readily triggers apoptosis. PI-induced apoptosis of GSCs is independent of NF-κB but involves the phosphorylation of c-Jun N-terminal kinase as well as the transcriptional activation of endoplasmic reticulum (ER) stress-associated proapoptotic mediators. In contrast to the general notion that ER stress-associated apoptosis is signaled by prolonged unfolded protein response (UPR), GSC-selective apoptosis is instead counteracted by the UPR ATF3 is a key mediator in GSC-selective apoptosis. Pharmaceutical uncoupling of the UPR from its downstream apoptosis sensitizes GSCs to PIs in vitro and during tumorigenesis in mice. Thus, a combinational treatment of a PI with an inhibitor of UPR-coupled apoptosis may enhance targeting of stem cells in gliomas.
Asunto(s)
Glioma/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Biomarcadores , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioma/tratamiento farmacológico , Glioma/genética , Glioma/patología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Modelos Biológicos , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas , Ubiquitinación/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The N-terminal amino acid of a protein is an essential determinant of ubiquitination and subsequent proteasomal degradation in the N-end rule pathway. Using para-chloroamphetamine (PCA), a specific inhibitor of the arginylation branch of the pathway (Arg/N-end rule pathway), we identified that blocking the Arg/N-end rule pathway significantly impaired the fusion of autophagosomes with lysosomes. Under ER stress, ATE1-encoded Arg-tRNA-protein transferases carry out the N-terminal arginylation of the ER heat shock protein HSPA5 that initially targets cargo proteins, along with SQSTM1, to the autophagosome. At the late stage of autophagy, however, proteasomal degradation of arginylated HSPA5 might function as a critical checkpoint for the proper progression of autophagic flux in the cells. Consistently, the inhibition of the Arg/N-end rule pathway with PCA significantly elevated levels of MAPT and huntingtin aggregates, accompanied by increased numbers of LC3 and SQSTM1 puncta. Cells treated with the Arg/N-end rule inhibitor became more sensitized to proteotoxic stress-induced cytotoxicity. SILAC-based quantitative proteomics also revealed that PCA significantly alters various biological pathways, including cellular responses to stress, nutrient, and DNA damage, which are also closely involved in modulation of autophagic responses. Thus, our results indicate that the Arg/N-end rule pathway may function to actively protect cells from detrimental effects of cellular stresses, including proteotoxic protein accumulation, by positively regulating autophagic flux.
Asunto(s)
Arginina/metabolismo , Autofagia , Proteínas/toxicidad , Animales , Autofagosomas/efectos de los fármacos , Autofagosomas/metabolismo , Autofagia/efectos de los fármacos , Biomarcadores/metabolismo , Chaperón BiP del Retículo Endoplásmico , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Proteínas de Choque Térmico/metabolismo , Humanos , Proteína Huntingtina/metabolismo , Marcaje Isotópico , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Fusión de Membrana/efectos de los fármacos , Ratones , Modelos Biológicos , Complejo de la Endopetidasa Proteasomal/metabolismo , Agregado de Proteínas/efectos de los fármacos , Proteolisis/efectos de los fármacos , Proteómica , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , p-Cloroanfetamina/farmacología , Proteínas tau/metabolismoRESUMEN
The N-end rule pathway is a proteolytic system, in which single N-terminal residues act as a determinant of a class of degrons, called N-degrons. In the ubiquitin (Ub)-proteasome system, specific recognition components, called N-recognins, recognize N-degrons and accelerate polyubiquitination and proteasomal degradation of the substrates. In this study, we show that the pathway regulates the activity of the macroautophagic receptor SQSTM1/p62 (sequestosome 1) through N-terminal arginylation (Nt-arginylation) of endoplasmic reticulum (ER)-residing molecular chaperones, including HSPA5/GRP78/BiP, CALR (calreticulin), and PDI (protein disulfide isomerase). The arginylation is co-induced with macroautophagy (hereafter autophagy) as part of innate immunity to cytosolic DNA and when misfolded proteins accumulate under proteasomal inhibition. Following cytosolic relocalization and arginylation, Nt-arginylated HSPA5 (R-HSPA5) is targeted to autophagosomes and degraded by lysosomal hydrolases through the interaction of its N-terminal Arg (Nt-Arg) with ZZ domain of SQSTM1. Upon binding to Nt-Arg, SQSTM1 undergoes a conformational change, which promotes SQSTM1 self-polymerization and interaction with LC3, leading to SQSTM1 targeting to autophagosomes. Cargoes of R-HSPA5 include cytosolic misfolded proteins destined to be degraded through autophagy. Here, we discuss the mechanisms by which the N-end rule pathway regulates SQSTM1-dependent selective autophagy.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Arginina/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Animales , Chaperón BiP del Retículo Endoplásmico , Humanos , Modelos Biológicos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ubiquitina/metabolismoRESUMEN
Cancer stem cells (CSCs), or alternatively called tumor initiating cells (TICs), are a subpopulation of tumor cells, which possesses the ability to self-renew and differentiate into bulk tumor mass. An accumulating body of evidence suggests that CSCs contribute to the growth and recurrence of tumors and the resistance to chemo- and radiotherapy. CSCs achieve self-renewal through asymmetric division, in which one daughter cell retains the self-renewal ability, and the other is destined to differentiation. Recent studies revealed the mechanisms of asymmetric division in normal stem cells (NSCs) and, to a limited degree, CSCs as well. Asymmetric division initiates when a set of polarity-determining proteins mark the apical side of mother stem cells, which arranges the unequal alignment of mitotic spindle and centrosomes along the apical-basal polarity axis. This subsequently guides the recruitment of fate-determining proteins to the basal side of mother cells. Following cytokinesis, two daughter cells unequally inherit centrosomes, differentiation-promoting fate determinants, and other proteins involved in the maintenance of stemness. Modulation of asymmetric and symmetric division of CSCs may provide new strategies for dual targeting of CSCs and the bulk tumor mass. In this review, we discuss the current understanding of the mechanisms by which NSCs and CSCs achieve asymmetric division, including the functions of polarity- and fate-determining factors.
RESUMEN
We show that ATE1-encoded Arg-transfer RNA transferase (R-transferase) of the N-end rule pathway mediates N-terminal arginylation of multiple endoplasmic reticulum (ER)-residing chaperones, leading to their cytosolic relocalization and turnover. N-terminal arginylation of BiP (also known as GRP78), protein disulphide isomerase and calreticulin is co-induced with autophagy during innate immune responses to cytosolic foreign DNA or proteasomal inhibition, associated with increased ubiquitylation. Arginylated BiP (R-BiP) is induced by and associated with cytosolic misfolded proteins destined for p62 (also known as sequestosome 1, SQSTM1) bodies. R-BiP binds the autophagic adaptor p62 through the interaction of its N-terminal arginine with the p62 ZZ domain. This allosterically induces self-oligomerization and aggregation of p62 and increases p62 interaction with LC3, leading to p62 targeting to autophagosomes and selective lysosomal co-degradation of R-BiP and p62 together with associated cargoes. In this autophagic mechanism, Nt-arginine functions as a delivery determinant, a degron and an activating ligand. Bioinformatics analysis predicts that many ER residents use arginylation to regulate non-ER processes.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Arginina/metabolismo , Autofagia , Retículo Endoplásmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Aminoaciltransferasas/genética , Aminoaciltransferasas/metabolismo , Animales , Línea Celular Tumoral , Células Cultivadas , Embrión de Mamíferos/citología , Chaperón BiP del Retículo Endoplásmico , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Proteínas de Choque Térmico/genética , Humanos , Immunoblotting , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones Noqueados , Microscopía Confocal , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Interferencia de ARN , Homología de Secuencia de Aminoácido , Proteína Sequestosoma-1RESUMEN
OBJECTIVES: To evaluate changes in cuff pressure and position of tapered-cuff and cylindrical-cuff tracheal tubes (TTs) during laparoscopic cholecystectomy; to compare the frequency of endobronchial intubation and airway complaints with the two types of TT. METHODS: Patients were randomly assigned to intubation with a TT with a cylindrical cuff (group C) or a tapered cuff (group T). Anaesthesia was maintained with sevoflurane, O(2) and N(2)O after intubation. TT cuffs were inflated to 20 cm H(2)O; changes in cuff pressure and volume were measured up to 30 min after the start of N(2)O use. Distance between the TT tip and the carina was measured before and after pneumoperitoneum and position change. Frequency of sore throat, dysphagia and hoarseness in the two groups was recorded. RESULTS: Cuff volume and pressure were significantly lower in group T than in group C (n = 32 per group). Cuff pressure and volume significantly increased 30 min after start of N(2)O use in both groups. Distance from the TT tip to the carina decreased during surgery in both groups. There were no cases of endobronchial intubation, and there were no between-group differences in the frequency of airway complaints. CONCLUSIONS: The tapered cuff was associated with a smaller change in cuff pressure during laparoscopic cholecystectomy than the cylindrical cuff, and therefore may be associated with increased preservation of tracheal mucosal perfusion.
Asunto(s)
Abdomen/cirugía , Intubación Intratraqueal/instrumentación , Laparoscopía , Presión , Adulto , Anestesia , Colecistectomía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Cuidados PosoperatoriosRESUMEN
The N-end rule pathway is a cellular proteolytic system that utilizes specific N-terminal residues as degradation determinants, called N-degrons. N-degrons are recognized and bound by specific recognition components (N-recognins) that mediate polyubiquitination of low-abundance regulators and selective proteolysis through the proteasome. Our earlier work identified UBR4/p600 as one of the N-recognins that promotes N-degron-dependent proteasomal degradation. In this study, we show that UBR4 is associated with cellular cargoes destined to autophagic vacuoles and is degraded by the lysosome. UBR4 loss causes multiple misregulations in autophagic pathways, including an increased formation of LC3 puncta. UBR4-deficient mice die during embryogenesis primarily due to defective vascular development in the yolk sac (YS), wherein UBR4 is associated with a bulk lysosomal degradation system that absorbs maternal proteins from the YS cavity and digests them into amino acids. Our results suggest that UBR4 plays a role not only in selective proteolysis of short-lived regulators through the proteasome, but also bulk degradation through the lysosome. Here, we discuss a possible mechanism of UBR4 as a regulatory component in the delivery of cargoes destined to interact with the autophagic core machinery.
Asunto(s)
Autofagia , Proteolisis , Animales , Proteínas de Unión a Calmodulina/metabolismo , Embrión de Mamíferos/metabolismo , Células HEK293 , Humanos , Ratones , Modelos BiológicosRESUMEN
Cancer stem cells (CSCs) are a subpopulation of tumor cells that can self-renew, metastasize, and promote cancer recurrence. A comprehensive characterization of the CSC proteome has been hampered due to their scarcity and rapid differentiation. Here, we present a systematic analysis of the cell-surface proteome using a CSC-like cell line derived from MDA-MB453 breast cancer cells, which exhibited a CD44(+) /CD24(-) (where CD is cluster of differentiation) phenotype and chemoresistance. We identified differentially expressed proteins in CSC-like cells, including upregulated plasma membrane proteins such as CD44, CD133, epidermal growth factor receptor (EGFR), CD147, cadherin 1, integrins, and catenin (cadherin-associated protein), beta 1 (CTNNB1), using an in-situ biotinylation approach followed by MS analysis. We examined the role of CD147 in the promotion of CSC growth and survival, and demonstrated that inhibition of CD147 with a monoclonal antibody induced significant inhibition of cell growth. siRNA-mediated silencing of CD147 gene expression restored the sensitivity of CSC-like cells to 5-fluorouracil (5-FU), along with decreasing the expression of thymidylate synthase, p-AKT, and ß-catenin, while increasing the expression of p-glycogen synthase kinase (GSK)3ß. Increased CD147 expression in the CSC-like cells, as seen by proteomic analysis, and the functional consequences of CD147 overexpression in CSC-like cells suggest that CD147 may be one of the critical cell-surface proteins involved in promoting chemoresistance and survival in CSCs.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Basigina/metabolismo , Resistencia a Antineoplásicos , Fluorouracilo/farmacología , Células Madre Neoplásicas/metabolismo , Basigina/genética , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Proteínas de la Membrana/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Fenotipo , Proteoma/metabolismo , Proteómica , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal , Regulación hacia ArribaRESUMEN
We characterized the cellular properties of cancer stem-like cells (CSLCs) isolated from immortalized MDA-MB453 human breast cancer cells in culture. We showed that although the expression of Octamer-binding transcription factor-4 (OCT4) correlates to stemness in these CSLCs, OCT4 knockdown does not induce their differentiation. Our results suggest that the differentiation program in MDA-MB453 CSLCs is blocked at a step upstream of the transcription of the OCT4 promoter, allowing CSLCs to maintain their population through asymmetric cell division during many repeated passages. Comparative expression analysis indicates that only a subset of genes and signaling pathways known to be associated with survival and maintenance of CSCs are selectively expressed in CSLCs, as compared with non-CSLCs fractionated from the same parental MDA-MB453 cells. These results suggest that selective expression of a limited number of genes may be sufficient for establishment and maintenance of CSLCs with high tumorigenicity.
Asunto(s)
Neoplasias de la Mama/patología , Células Madre Neoplásicas/metabolismo , Autofagia , Biomarcadores , Línea Celular Tumoral , Estrés del Retículo Endoplásmico , Femenino , Perfilación de la Expresión Génica , Histonas/metabolismo , Humanos , Factor 3 de Transcripción de Unión a Octámeros/análisis , Factor 3 de Transcripción de Unión a Octámeros/genética , Tretinoina/farmacología , UbiquitinaciónRESUMEN
The N-end rule pathway is a proteolytic system in which destabilizing N-terminal residues of short-lived proteins act as degradation determinants (N-degrons). Substrates carrying N-degrons are recognized by N-recognins that mediate ubiquitylation-dependent selective proteolysis through the proteasome. Our previous studies identified the mammalian N-recognin family consisting of UBR1/E3α, UBR2, UBR4/p600, and UBR5, which recognize destabilizing N-terminal residues through the UBR box. In the current study, we addressed the physiological function of a poorly characterized N-recognin, 570-kDa UBR4, in mammalian development. UBR4-deficient mice die during embryogenesis and exhibit pleiotropic abnormalities, including impaired vascular development in the yolk sac (YS). Vascular development in UBR4-deficient YS normally advances through vasculogenesis but is arrested during angiogenic remodeling of primary capillary plexus associated with accumulation of autophagic vacuoles. In the YS, UBR4 marks endoderm-derived, autophagy-enriched cells that coordinate differentiation of mesoderm-derived vascular cells and supply autophagy-generated amino acids during early embryogenesis. UBR4 of the YS endoderm is associated with a tissue-specific autophagic pathway that mediates bulk lysosomal proteolysis of endocytosed maternal proteins into amino acids. In cultured cells, UBR4 subpopulation is degraded by autophagy through its starvation-induced association with cellular cargoes destined to autophagic double membrane structures. UBR4 loss results in multiple misregulations in autophagic induction and flux, including synthesis and lipidation/activation of the ubiquitin-like protein LC3 and formation of autophagic double membrane structures. Our results suggest that UBR4 plays an important role in mammalian development, such as angiogenesis in the YS, in part through regulation of bulk degradation by lysosomal hydrolases.