RESUMEN
BACKGROUND: KRAS mutation is one of the dominant gene mutations in colorectal cancer (CRC). Up to present, targeting KRAS for CRC treatment remains a clinical challenge. WNT974 (LGK974) is a porcupine inhibitor that interferes Wnt signaling pathway. Artesunate (ART) is a water-soluble semi-synthetic derivative of artemisinin. METHODS: The synergistic effect of ART and WNT974 combination in reducing CRC cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RT-PCR was utilized for the mRNA levels of KRAS, CUL7, ANAPC2, UBE2M, RNF123, SYVN1, or ß-TrCP. Western blot assay was utilized for the protein levels of NRAS, HRAS, KRAS, ANAPC2, ß-TrCP, GSK-3ß, p-Akt (Ser473), t-Akt, p-PI3K (Tyr458), t-PI3K, p-mTOR (Ser2448), t-mTOR. Xenograft mouse model assay was performed for the anti-CRC effect of combination of ART and WNT974 in vivo. IHC assay was utilized for the levels of KRAS, ß-TrCP, GSK-3ß or ANAPC2 in tumor tissues. RESULTS: Our study shows that the combination of WNT974 and ART exhibits synergistic effect in reducing CRC growth. The combination treatment significantly reduces KRAS protein level and activity in CRC cells. Interestingly, the combination treatment increases E3 ligases ANAPC2 expression. Our data show that overexpression of ANAPC2 significantly reduces KRAS protein levels, which is reversed by MG132. Knockdown of ANAPC2 in CRC abolishes the combination treatment-reduce KRAS expression. Besides, the treatment also increases the expressions of GSK-3ß and E3 ligase ß-TrCP that is known to degrade GSK-3ß-phosphorylated KRAS protein. Knockdown of ß-TrCP- and inhibition of GSK-3ß abolish the combination treatment-induce KRAS ubiquitination and reduction in expression. Last but not least, combination treatment suppresses PI3K/Akt/m-TOR signaling pathway. CONCLUSIONS: Our data clearly show that the combination treatment significantly enhances KRAS protein degradation via the ubiquitination ubiquitin-proteasome pathway, which is also demonstrated in xenograft mouse model. The study provides strong scientific evidence for the development of the combination of WNT974 and ART as KRAS-targeting therapeutics for CRC treatment. Video Abstract.
Asunto(s)
Ubiquitina-Proteína Ligasas , Proteínas con Repetición de beta-Transducina , Animales , Subunidad Apc2 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Artesunato/farmacología , Línea Celular Tumoral , Proteínas Cullin , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Pirazinas , Piridinas , Serina-Treonina Quinasas TOR/metabolismo , Enzimas Ubiquitina-Conjugadoras , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinas/metabolismo , Vía de Señalización Wnt , Proteínas con Repetición de beta-Transducina/genética , Proteínas con Repetición de beta-Transducina/metabolismoRESUMEN
Neuromuscular junctions (NMJs) are peripheral synapses between motoneurons and skeletal muscle fibers that are critical for the control of muscle contraction. Dysfunction of these synapses has been implicated in congenital myasthenic syndrome (CMS). In vertebrates, agrin-LRP4-MuSK signaling plays a critical role in acetylcholine receptor (AChR) clustering and NMJ formation. The adaptor protein DOK7 is the downstream substrate of MuSK and also a cytoplasmic activator of MuSK. The role of DOK7 in the promotion of AChR clustering and the mechanisms involved have been well studied; however, the negative regulation of DOK7 after MuSK activation remains unknown. Anaphase-promoting complex 2 (APC2), the core subunit of APC/C E3 ligase complex, was originally believed to regulate cell-cycle transitions. Here, we show that APC2 is enriched at post-synapse of NMJs in postmitotic myotubes. In response to agrin stimulation, APC2 negatively regulates AChR clustering by promoting the ubiquitination of DOK7 at lysine 243 for its proteolytic degradation, which relies on MuSK kinase activity and the phosphorylation of tyrosine 106 in DOK7. Thus, this study provides a mechanism whereby agrin signaling is negatively regulated as part of vertebrate NMJ homeostasis.
Asunto(s)
Agrina/metabolismo , Subunidad Apc2 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Proteolisis , Transducción de Señal , Ubiquitinación , Agrina/genética , Animales , Subunidad Apc2 del Ciclosoma-Complejo Promotor de la Anafase/genética , Ciclo Celular , Línea Celular , Ratones , Fibras Musculares Esqueléticas/citología , Proteínas Musculares/genéticaRESUMEN
Mycobacterium tuberculosis is an intracellular pathogen that uses several strategies to interfere with the signalling functions of host immune molecules. Many other bacterial pathogens exploit the host ubiquitination system to promote pathogenesis1,2, but whether this same system modulates the ubiquitination of M. tuberculosis proteins is unknown. Here we report that the host E3 ubiquitin ligase ANAPC2-a core subunit of the anaphase-promoting complex/cyclosome-interacts with the mycobacterial protein Rv0222 and promotes the attachment of lysine-11-linked ubiquitin chains to lysine 76 of Rv0222 in order to suppress the expression of proinflammatory cytokines. Inhibition of ANAPC2 by specific short hairpin RNA abolishes the inhibitory effect of Rv0222 on proinflammatory responses. Moreover, mutation of the ubiquitination site on Rv0222 impairs the inhibition of proinflammatory cytokines by Rv0222 and reduces virulence during infection in mice. Mechanistically, lysine-11-linked ubiquitination of Rv0222 by ANAPC2 facilitates the recruitment of the protein tyrosine phosphatase SHP1 to the adaptor protein TRAF6, preventing the lysine-63-linked ubiquitination and activation of TRAF6. Our findings identify a previously unrecognized mechanism that M. tuberculosis uses to suppress host immunity, and provide insights relevant to the development of effective immunomodulators that target M. tuberculosis.
Asunto(s)
Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Interacciones Huésped-Patógeno/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis/inmunología , Ubiquitinación , Ciclosoma-Complejo Promotor de la Anafase/química , Animales , Subunidad Apc2 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Células Cultivadas , Citocinas/antagonistas & inhibidores , Citocinas/inmunología , Citocinas/metabolismo , Femenino , Mediadores de Inflamación/antagonistas & inhibidores , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Lisina/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Transducción de Señal , Factor 6 Asociado a Receptor de TNF/antagonistas & inhibidores , Factor 6 Asociado a Receptor de TNF/metabolismo , Factor de Transcripción AP-1/metabolismo , Tuberculosis/microbiología , Virulencia/inmunologíaRESUMEN
The importance of the Wnt-signaling pathway on the regulation and maintenance of the intestinal stem cell (ISC) population is well recognized. However, our current knowledge base is founded on models using systems of gross deregulation of the Wnt-signaling pathway. Given the importance of this signaling pathway on intestinal homeostasis, there is a need to explore the role of more subtle alterations in Wnt-signaling levels within this tissue. Herein, we have used a model of Apc2 loss to meet this aim. Apc2 is a homolog of Apc which can also form a destruction complex capable of binding ß-catenin, albeit less efficiently than Apc. We show that systemic loss of Apc2 results in an increase in the number of cells displaying nuclear ß-catenin at the base of the intestinal crypt. This subsequently impacts the expression levels of several ISC markers and the fitness of ISCs as assessed by organoid formation efficiency. This work provides the first evidence that the function and fitness of ISCs can be altered by even minor misregulation of the Wnt-signaling pathway. Our data highlights the importance of correct maintenance of this crucial signaling pathway in the maintenance and function of the ISC population. Stem Cells 2018;36:114-122.
Asunto(s)
Subunidad Apc2 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/citología , Células Madre/citología , Células Madre/metabolismo , Vía de Señalización Wnt , Animales , Subunidad Apc2 del Ciclosoma-Complejo Promotor de la Anafase/deficiencia , Subunidad Apc2 del Ciclosoma-Complejo Promotor de la Anafase/genética , Apoptosis/fisiología , Intestino Delgado/citología , Intestino Delgado/metabolismo , Ratones , Ratones Noqueados , Modelos AnimalesRESUMEN
In mammalian females, germ cells remain arrested as primordial follicles. Resumption of meiosis is heralded by germinal vesicle breakdown, condensation of chromosomes, and their eventual alignment on metaphase plates. At the first meiotic division, anaphase-promoting complex/cyclosome associated with Cdc20 (APC/CCdc20) activates separase and thereby destroys cohesion along chromosome arms. Because cohesion around centromeres is protected by shugoshin-2, sister chromatids remain attached through centromeric/pericentromeric cohesin. We show here that, by promoting proteolysis of cyclins and Cdc25B at the germinal vesicle (GV) stage, APC/C associated with the Cdh1 protein (APC/CCdh1) delays the increase in Cdk1 activity, leading to germinal vesicle breakdown (GVBD). More surprisingly, by moderating the rate at which Cdk1 is activated following GVBD, APC/CCdh1 creates conditions necessary for the removal of shugoshin-2 from chromosome arms by the Aurora B/C kinase, an event crucial for the efficient resolution of chiasmata.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromosomas , Meiosis , Animales , Subunidad Apc2 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Aurora Quinasa B/metabolismo , Aurora Quinasa C/metabolismo , Proteína Quinasa CDC2/metabolismo , Proteínas Cdc20/fisiología , Proteínas Cdh1/metabolismo , Centrómero , Proteínas Cromosómicas no Histona/metabolismo , Femenino , Centro Germinal , Masculino , Ratones , Ratones Noqueados , Modelos Teóricos , Separasa/metabolismo , Fosfatasas cdc25/fisiología , CohesinasRESUMEN
BACKGROUND: Assisted reproductive technologies are associated with altered methylation in term placenta. However, it is unclear whether methylation patterns are the result of fertility treatments or intrauterine environment. Thus, we set out to determine whether there are differences in the first-trimester placenta that may be altered by the underlying fertility treatments. Genome-wide DNA methylation analyses from chorionic villus sampling (CVS) from matched singleton pregnancies conceived using in vitro fertilization (IVF), non-IVF fertility treatment (NIFT), or those conceived spontaneously were performed using Illumina Infinium HumanMethylation450 BeadChip from 15 matched CVS samples. Nanofluidic quantitative polymerase chain reaction (qPCR) of differently methylated genes was performed in a confirmatory cohort of 23 IVF conceptions and 24 NIFT conceptions. RESULTS: Global methylation was similar among the IVF, NIFT, and spontaneous conceptions. However, differential methylation from IVF and NIFT pregnancies was present at 34 CpG sites, which was significantly different. Of those, 14 corresponded to known genes, with methylation changes detected at multiple loci in 3 genes, anaphase-promoting complex subunit 2 ( ANAPC2), C-X-C motif chemokine ligand 14 ( CXCL14), and regulating synaptic membrane exocytosis 1 ( RIMS1). Nanofluidic qPCR of differentially methylated genes identified pre T-cell antigen receptor alpha ( PTCRA) to be significantly downregulated in IVF versus NIFT conceptions. CONCLUSION: Although global methylation patterns are similar, there are differences in methylation of specific genes in IVF compared to NIFT conceptions, leading to altered gene expression. PTCRA was differentially methylated and downregulated in IVF conceptions, warranting further investigation. It remains to be determined whether these changes affect placentation and whether it is due to the more profound underlying infertility requiring IVF, yet these data provide unique insight into the first-trimester placental epigenome.
Asunto(s)
Vellosidades Coriónicas/metabolismo , Metilación de ADN , Fertilización In Vitro , Infertilidad/metabolismo , Primer Trimestre del Embarazo/metabolismo , Adulto , Subunidad Apc2 del Ciclosoma-Complejo Promotor de la Anafase/genética , Subunidad Apc2 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Femenino , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Perfilación de la Expresión Génica , Sitios Genéticos , Humanos , Infertilidad/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Embarazo , Primer Trimestre del Embarazo/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismoRESUMEN
PURPOSE: Anaphase promoting complex (APC/C) is an E3 ligase enzyme, which ubiquinates various proteins involved in the cell cycle. This protein complex may have a pivotal role in the cell cycle control affecting pathological conditions such as cancer. APC7 and APC2 subunits of the APC/C complex are involved in the substrate recognition and the catalytic reaction, respectively. MATERIALS AND METHODS: In this study, quantitative Real-time PCR was used to analyse APC2 and APC7 expression in different cancer cell lines as well as AML patient's blood cells. RESULTS: The results showed that APC2 and APC7 subunits were both over expressed in cancer cell lines (p=0.008). The mean expression ratio of APC2 and APC7 in different cancer cells were 2.60±0.22 and 4.83±0.11, respectively. An increase in expression of APC2 and APC7 was seen among 12 out of 14 AML patients (85%). There was a significant positive correlation between APC2 upregulation and the detection of splenomegaly in the patients (r=0.808, p=0.001). CONCLUSION: This was the first study suggesting that APC/C upregulation may contribute to the pathogenesis of cancer and can be used as a molecular biomarker to predict the progression and the prognosis of AML.
Asunto(s)
Subunidad Apc2 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Subunidad Apc7 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Subunidad Apc2 del Ciclosoma-Complejo Promotor de la Anafase/genética , Subunidad Apc7 del Ciclosoma-Complejo Promotor de la Anafase/genética , Línea Celular Tumoral , Femenino , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoAsunto(s)
Subunidad Apc11 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Subunidad Apc2 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas Cdc20/metabolismo , Biosíntesis de Péptidos Independientes de Ácidos Nucleicos/genética , Poliubiquitina/biosíntesis , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina/genética , Ubiquitinación/fisiología , HumanosRESUMEN
Protein modification with ubiquitin chains is an essential signaling event catalyzed by E3 ubiquitin ligases. Most human E3s contain a signature RING domain that recruits a ubiquitin-charged E2 and a separate domain for substrate recognition. How RING-E3s can build polymeric ubiquitin chains while binding substrates and E2s at defined interfaces remains poorly understood. Here, we show that the RING-E3 APC/C catalyzes chain elongation by strongly increasing the affinity of its E2 for the distal acceptor ubiquitin in a growing conjugate. This function of the APC/C requires its coactivator as well as conserved residues of the E2 and ubiquitin. APC/C's ability to track the tip of an emerging conjugate is required for APC/C-substrate degradation and accurate cell division. Our results suggest that RING-E3s tether the distal ubiquitin of a growing chain in proximity to the active site of their E2s, allowing them to assemble polymeric conjugates without altering their binding to substrate or E2.
Asunto(s)
Subunidad Apc11 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Subunidad Apc2 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas Cdc20/metabolismo , Biosíntesis de Péptidos Independientes de Ácidos Nucleicos/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina/genética , Dominio Catalítico , Proteínas Cdc20/genética , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Activación Enzimática , Células HeLa , Humanos , Unión Proteica , Estructura Terciaria de Proteína , Interferencia de ARN , ARN Interferente Pequeño , Ubiquitina/biosíntesis , UbiquitinaciónRESUMEN
Polyubiquitination by E2 and E3 enzymes is a predominant mechanism regulating protein function. Some RING E3s, including anaphase-promoting complex/cyclosome (APC), catalyze polyubiquitination by sequential reactions with two different E2s. An initiating E2 ligates ubiquitin to an E3-bound substrate. Another E2 grows a polyubiquitin chain on the ubiquitin-primed substrate through poorly defined mechanisms. Here we show that human APC's RING domain is repurposed for dual functions in polyubiquitination. The canonical RING surface activates an initiating E2-ubiquitin intermediate for substrate modification. However, APC engages and activates its specialized ubiquitin chain-elongating E2 UBE2S in ways that differ from current paradigms. During chain assembly, a distinct APC11 RING surface helps deliver a substrate-linked ubiquitin to accept another ubiquitin from UBE2S. Our data define mechanisms of APC/UBE2S-mediated polyubiquitination, reveal diverse functions of RING E3s and E2s, and provide a framework for understanding distinctive RING E3 features specifying ubiquitin chain elongation.
Asunto(s)
Subunidad Apc11 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Subunidad Apc2 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Biosíntesis de Péptidos Independientes de Ácidos Nucleicos , Poliubiquitina/biosíntesis , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación/fisiología , Secuencia de Aminoácidos , Subunidad Apc4 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Puntos de Control del Ciclo Celular , Células HeLa , Humanos , Datos de Secuencia Molecular , Poliubiquitina/genética , Estructura Terciaria de ProteínaRESUMEN
The Mdm2 proto-oncoprotein is the primary negative regulator for p53. While it is believed that Mdm2 degradation is regulated via its own E3 ubiquitin ligase activity, recent development of knock-in mouse models demonstrates that Mdm2 E3 ligase function is dispensable for self-degradation in vivo. Here, we show that the anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase for Mdm2 degradation. We demonstrate that APC2, a scaffold subunit of APC/C, binds to Mdm2 and is required for Mdm2 polyubiquitination and proteasomal degradation. Downregulation of APC2 by RNAi results in transcription-independent accumulation of Mdm2 and attenuation of stress-induced p53 stabilization, leading to decreased senescence and increased cell survival. Furthermore, APC2 expression is frequently downregulated in human cancers; in tumor cell lines, APC2 downregulation correlates with Mdm2 overexpression. Our study shows the regulation of Mdm2 by the E3 ubiquitin ligase APC/C and has important therapeutic implications for tumors with Mdm2 overexpression.
Asunto(s)
Subunidad Apc2 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Animales , Subunidad Apc2 del Ciclosoma-Complejo Promotor de la Anafase/genética , Carcinogénesis/metabolismo , Línea Celular Tumoral , Humanos , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteína p53 Supresora de Tumor/metabolismo , UbiquitinaciónRESUMEN
Three types of mitosis, which are open, closed or semi-open mitosis, function in eukaryotic cells, respectively. The open mitosis involves breakage of the nuclear envelope before nuclear division, whereas the closed mitosis proceeds with an intact nuclear envelope. To understand the mechanism and significance of three types of mitotic division in eukaryotes, we investigated the process of semi-open mitosis, in which the nuclear envelope is only partially broken, in the fission yeast Schizosaccharomyces japonicus. In anaphase-promoting complex/cyclosome (APC/C) mutants of Sz. japonicus, the nuclear envelope remained relatively intact during anaphase, resulting in impaired semi-open mitosis. As a suppressor of apc2 mutant, a mutation of Oar2, which was a 3-oxoacyl-[acyl carrier protein] reductase, was obtained. The level of the Oar2, which had two destruction-box motifs recognized by APC/C, was increased in APC/C mutants. Furthermore, the defective semi-open mitosis observed in an apc2 mutant was restored by mutated oar2+. Based on these findings, we propose that APC/C regulates the dynamics of the nuclear envelope through degradation of Oar2 dependent on APC/C during the metaphase-to-anaphase transition of semi-open mitosis in Sz. japonicus.