RESUMEN

Pluripotency is established in E4.5 preimplantation epiblast. Embryonic stem cells (ESCs) represent the immortalization of pluripotency, however, their gene expression signature only partially resembles that of developmental ground-state. Induced PRAMEL7 expression, a protein highly expressed in the ICM but lowly expressed in ESCs, reprograms developmentally advanced ESC+serum into ground-state pluripotency by inducing a gene expression signature close to developmental ground-state. However, how PRAMEL7 reprograms gene expression remains elusive. Here we show that PRAMEL7 associates with Cullin2 (CUL2) and this interaction is required to establish ground-state gene expression. PRAMEL7 recruits CUL2 to chromatin and targets regulators of repressive chromatin, including the NuRD complex, for proteasomal degradation. PRAMEL7 antagonizes NuRD-mediated repression of genes implicated in pluripotency by decreasing NuRD stability and promoter association in a CUL2-dependent manner. Our data link proteasome degradation pathways to ground-state gene expression, offering insights to generate in vitro models to reproduce the in vivo ground-state pluripotency.

Asunto(s)

Células Madre Pluripotentes , Células Madre Pluripotentes/metabolismo , Células Madre Embrionarias/metabolismo , Transcriptoma , Cromatina/metabolismo , Diferenciación Celular/genética

RESUMEN

Human immunodeficiency virus type 2 (HIV-2) is known to be less pathogenic than HIV-1. However, the mechanism(s) underlying the decreased HIV-2 pathogenicity is not fully understood. Herein, we report that ß-chemokine CCL2 expression was increased in HIV-1-infected human monocyte-derived macrophages (MDM) but decreased in HIV-2-infected MDM when compared to uninfected MDM. Inhibition of CCL2 expression following HIV-2 infection occurred at both protein and mRNA levels. By microarray analysis, quantitative PCR, and Western blotting, we identified that Signal Transducer and Activator of Transcription 1 (STAT1), a critical transcription factor for inducing CCL2 gene expression, was also reduced in HIV-2-infected MDM. Blockade of STAT1 in HIV-infected MDM using a STAT1 inhibitor significantly reduced the production of CCL2. In contrast, transduction of STAT1-expressing pseudo-retrovirus restored CCL2 production in HIV-2-infected MDM. These findings support the concept that CCL2 inhibition in HIV-2-infected MDM is meditated by reduction of STAT1. Furthermore, we showed that STAT1 reduction in HIV-2-infected MDM was regulated by the CUL2/RBX1 ubiquitin E3 ligase complex-dependent proteasome pathway. Knockdown of CUL2 or RBX1 restored the expression of STAT1 and CCL2 in HIV-2-infected MDM. Taken together, our findings suggest that differential regulation of the STAT1-CCL2 axis may be one of the mechanisms underlying the different pathogenicity observed for HIV-1 and HIV-2.

Asunto(s)

Quimiocina CCL2 , Infecciones por VIH , VIH-1 , VIH-2 , Humanos , Células Cultivadas , Regulación de la Expresión Génica , Seropositividad para VIH , VIH-1/genética , VIH-2/genética , Macrófagos , Virulencia , Replicación Viral , Quimiocina CCL2/metabolismo , Infecciones por VIH/metabolismo , Infecciones por VIH/virología

RESUMEN

Background: MicroRNA-154-5p (miR-154-5p) plays a role in tumorigenesis in diverse human malignancies. Nevertheless, little is known about the mechanism by which miR-154-5p alters the growth and metastasis of cervical cancer. This research aimed to analyze the role of miR-154-5p in the pathology of cervical cancer in vitro and in vivo. Methods: The level of miR-154-5p in human papillomavirus 16 positive cervical cancer cells was examined by real-time quantitative polymerase chain reaction. Bioinformatics predicted the downstream targets and potential functions of miR-154-5p. Furthermore, lentiviral technology was used to construct SiHa cell lines with stable up- and down-expression levels of miR-154-5p. Its differential expression effects on the progress and metastasis of cervical cancer were analyzed using cell culture and animal models. Results: MiR-154-5p showed low expression in cervical cancer cells. Overexpression of miR-154-5p could markedly inhibit the proliferation, migration, and colony formation ability of SiHa cells, concomitantly leading to G1 arrest of the cell cycle, while silencing miR-154-5p triggered the opposite results. Meanwhile, overexpression of miR-154-5p restrained the growth and metastasis of cervical cancer by silencing CUL2 in vivo. Additionally, miR-154-5p reduced CUL2 level, and overexpression of CUL2 influenced the effect of miR-154-5p in cervical cancer. In conclusion, miR-154-5p restrained the growth and metastasis of cervical cancer by directly silencing CUL2.

Asunto(s)

Proteínas Cullin , MicroARNs , Neoplasias del Cuello Uterino , Animales , Femenino , Humanos , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , MicroARNs/genética , Neoplasias del Cuello Uterino/genética , Proteínas Cullin/genética , Silenciador del Gen , Metástasis de la Neoplasia

RESUMEN

Drosophila ovary has been one of the most mature and excellent systems for studying the in vivo regulatory mechanisms of stem cell fate determination. It has been well-known that the bone morphogenetic protein (BMP) signaling released by the niche cells promotes the maintenance of germline stem cells (GSCs) through inhibiting the transcription of the bag-of-marbles (bam) gene, which encodes a key factor for GSC differentiation. However, whether Bam is regulated at the post-translational level remains largely unknown. Here we show that the E3 ligase Cullin-2 (Cul2) is involved in modulating Bam ubiquitination, which occurs probably at multiple lysine residues of Bam's C-terminal region. Genetic evidence further supports the notion that Cul2-mediated Bam ubiquitination and turnover are essential for GSC maintenance and proper germline development. Collectively, our data not only uncovers a novel regulatory mechanism by which Bam is controlled at the post-translational level, but also provides new insights into how Cullin family protein determines the differentiation fate of early germ cells.

Asunto(s)

Drosophila , Ubiquitina-Proteína Ligasas , Femenino , Animales , Proteínas Cullin/genética , Células Germinativas , Diferenciación Celular/genética

RESUMEN

The dysregulation of transcription factors is widely associated with tumorigenesis. As the most well-defined transcription factor in multiple types of cancer, c-Myc can transform cells by transactivating various downstream genes. Given that there is no effective way to directly inhibit c-Myc, c-Myc targeting strategies hold great potential for cancer therapy. In this study, we found that WSB1, which has a highly positive correlation with c-Myc in 10 cancer cell lines and clinical samples, is a direct target gene of c-Myc, and can positively regulate c-Myc expression, which forms a feedforward circuit promoting cancer development. RNA sequencing results from Bel-7402 cells confirmed that WSB1 promoted c-Myc expression through the ß-catenin pathway. Mechanistically, WSB1 affected ß-catenin destruction complex-PPP2CA assembly and E3 ubiquitin ligase adaptor ß-TRCP recruitment, which inhibited the ubiquitination of ß-catenin and transactivated c-Myc. Of interest, the effect of WSB1 on c-Myc was independent of its E3 ligase activity. Moreover, overexpressing WSB1 in the Bel-7402 xenograft model could further strengthen the tumor-driven effect of c-Myc overexpression. Thus, our findings revealed a novel mechanism involved in tumorigenesis in which the WSB1/c-Myc feedforward circuit played an essential role, highlighting a potential c-Myc intervention strategy in cancer treatment.

RESUMEN

BACKGROUND: Pretreatment assessments for glioblastoma (GBM) patients, especially elderly or frail patients, are critical for treatment planning. However, genetic profiling with intracranial biopsy carries a significant risk of permanent morbidity. We previously demonstrated that the CUL2 gene, encoding the scaffold cullin2 protein in the cullin2-RING E3 ligase (CRL2), can predict GBM radiosensitivity and prognosis. CUL2 expression levels are closely regulated with its copy number variations (CNVs). This study aims to develop artificial neural networks (ANNs) for pretreatment evaluation of GBM patients with inputs obtainable without intracranial surgical biopsies. METHODS: Public datasets including Ivy-GAP, The Cancer Genome Atlas Glioblastoma (TCGA-GBM), and the Chinese Glioma Genome Atlas (CGGA) were used for training and testing of the ANNs. T1 images from corresponding cases were studied using automated segmentation for features of heterogeneity and tumor edge contouring. A ratio comparing the surface area of tumor borders versus the total volume (SvV) was derived from the DICOM-SEG conversions of segmented tumors. The edges of these borders were detected using the canny edge detector. Packages including Keras, Pytorch, and TensorFlow were tested to build the ANNs. A 4-layered ANN (8-8-8-2) with a binary output was built with optimal performance after extensive testing. RESULTS: The 4-layered deep learning ANN can identify a GBM patient's overall survival (OS) cohort with 80%-85% accuracy. The ANN requires 4 inputs, including CUL2 copy number, patients' age at GBM diagnosis, Karnofsky Performance Scale (KPS), and SvV ratio. CONCLUSION: Quantifiable image features can significantly improve the ability of ANNs to identify a GBM patients' survival cohort. Features such as clinical measures, genetic data, and image data, can be integrated into a single ANN for GBM pretreatment evaluation.

RESUMEN

BACKGROUND: Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide. MicroRNAs (miRNAs) have been widely validated as potential biomarkers for cancer treatment and diagnosis. AIMS: This paper intends to study the effect and specific mechanism of miR-574-3p/CUL2 axis in GC. METHODS: The miR-574-3p expression in GC tissues and cell lines was analyzed by reverse transcription polymerase chain reaction (RT-PCR). GC cell (N87) proliferation, migration and invasion were determined by the Brdu assay and Transwell assay, respectively. The tumor xenotransplantation model was established in vivo to test the effect of miR-574-3p or Cullin 2 (CUL2) on tumor growth. The relationship between miR-574-3p and CUL2 was predicated by bioinformatic analysis and verified by dual-luciferase reporter assay and RIP experiment. The expression of CUL2, hypoxia-induced transcription factor-1α (HIF-1α) as well as E-cadherin, Snail and Vimentin was monitored by western blot and immunohistochemistry. RESULTS: miR-574-3p was overexpressed in GC tissues and cells. Forced upregulation of miR-574-3p enhanced proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of GC cells (N87), while downregulation of miR-574-3p resulted in reverse effects. Additionally, miR-574-3p promoted N87 cells growth and EMT in vivo. CUL2 was negatively regulated by miR-574-3p in N87 cells, and upregulation of CUL2 repressed the malignant behaviors of N87 cells. Moreover, CUL2 directly interacted with HIF-1α and suppressed HIF-1α expression both in vitro and in vivo. CONCLUSIONS: miR-574-3p targeted CUL2 to upregulate HIF-1α, thus facilitating the progression of GC.

Asunto(s)

Proteínas Cullin , Transición Epitelial-Mesenquimal , Subunidad alfa del Factor 1 Inducible por Hipoxia , MicroARNs , Neoplasias Gástricas , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Proteínas Cullin/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , MicroARNs/genética , Isoformas de Proteínas/genética , Neoplasias Gástricas/patología

RESUMEN

BACKGROUND AND PURPOSE: Positive epidermal growth factor receptor (EGFR) immunoreactivity in glioblastoma multiforme (GBM) often predicts poor radiation response. Meanwhile, all attempts to target EGFR pharmaceutically have been unsuccessful, mainly due to molecular heterogeneity of EGFR expression in GBM. A molecular biology-based and efficient way to access cellular protein levels of EGFR is urgently needed. EGFR, together with HIF-1α and Cyclin B1, is degraded via cullin2-RING E3 ligase (CRL2). It is worthwhile to investigate the possible involvement of CRL2 on GBM survival and radiosensitivity. MATERIALS AND METHODS: Clinicopathological features, radiographic images, survival data, RNA-Seq, copy number variations (CNVs), and other genetic changes were studied on over 3800 glioma and GBM cases, which are derived from 5 independent cohorts. These datasets include the Chinese Glioma Genome Atlas, The Cancer Genome Atlas, Ivy Glioblastoma Atlas Project, Clinical Proteomic Tumor Analysis Consortium, and the 2008 Parson's GBM dataset. RESULTS: Expression of CUL2, which encodes the scaffold protein cullin2 in the CRL2 E3 ligase, can predict GBM progression and survival rate. Cullin2 protein levels are inversely related to those of HIF-1α, VEGF-A, Cyclin B1, and EGFR. Elevated CUL2 expression predicts increased radiosensitivity and dampened signal intensities in perfusion imaging. CUL2 expression are regulated via CNVs, which are inheritable structural DNA variations. CONCLUSION: CUL2 expression levels and CNVs can be integrated as potential biomarkers in facilitating GBM and prognosis and radiosensitivity profiling.

Asunto(s)

Neoplasias Encefálicas , Proteínas Cullin/genética , Glioblastoma , Biomarcadores , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/radioterapia , Línea Celular Tumoral , Variaciones en el Número de Copia de ADN , Glioblastoma/genética , Glioblastoma/radioterapia , Humanos , Pronóstico , Proteómica , Tolerancia a Radiación/genética , Ubiquitina-Proteína Ligasas/genética

RESUMEN

DNA replication is a complex process that needs to be executed accurately before cell division in order to maintain genome integrity. DNA replication is divided into three main stages: initiation, elongation and termination. One of the key events during initiation is the assembly of the replicative helicase at origins of replication, and this mechanism has been very well described over the last decades. In the last six years however, researchers have also focused on deciphering the molecular mechanisms underlying the disassembly of the replicative helicase during termination. Similar to replisome assembly, the mechanism of replisome disassembly is strictly regulated and well conserved throughout evolution, although its complexity increases in higher eukaryotes. While budding yeast rely on just one pathway for replisome disassembly in S phase, higher eukaryotes evolved an additional mitotic pathway over and above the default S phase specific pathway. Moreover, replisome disassembly has been recently found to be a key event prior to the repair of certain DNA lesions, such as under-replicated DNA in mitosis and inter-strand cross-links (ICLs) in S phase. Although replisome disassembly in human cells has not been characterised yet, they possess all of the factors involved in these pathways in model organisms, and de-regulation of many of them are known to contribute to tumorigenesis and other pathological conditions.

Asunto(s)

Replicación del ADN , Neoplasias/metabolismo , Fase S , Saccharomycetales , Animales , Caenorhabditis elegans , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Supervivencia Celular , ADN/metabolismo , Genoma , Humanos , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Mitosis , Mapeo de Interacción de Proteínas , Origen de Réplica , Saccharomyces cerevisiae , Saccharomycetales/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Xenopus laevis

RESUMEN

Posttranslational protein modifications play an important role in regulating protein stability and cellular function. There are at least eight Cullin family members. Among them, Cullin-2 forms a functional E3 ligase complex with elongin B, elongin C, RING-box protein 1 (RBX1, also called ROC1), as well as the substrate recognition subunit (SRS) to promote the substrate ubiquitination and degradation. In this book chapter, we will review Cullin-2 E3 ligase complexes that include various SRS proteins, including von Hippel Lindau (pVHL), leucine-rich repeat protein-1 (LRR-1), preferentially expressed antigen of melanoma (PRAME), sex-determining protein FEM-1 and early embryogenesis protein ZYG-11. We will focus on the VHL signaling pathway in clear cell renal cell carcinoma (ccRCC), which may reveal various therapeutic avenues in treating this lethal cancer.

Asunto(s)

Carcinoma de Células Renales/enzimología , Proteínas Cullin/metabolismo , Neoplasias Renales/enzimología , Animales , Carcinoma de Células Renales/tratamiento farmacológico , Humanos , Neoplasias Renales/tratamiento farmacológico , Unión Proteica , Factores de Transcripción/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo

RESUMEN

BACKGROUND/AIM: The cullin (CUL) family of proteins is involved in the ubiquitin/mediated degradation of proteins, regulating cell proliferation, cell-cycle control, migration, invasion and metastasis in the process of tumor progression. The aim of the present study was to examine if there is any correlation between the immunohistochemical (IHC) expression of Cullin-1 and -2 proteins in colorectal cancer tissue specimens with several clinicopathological variables. MATERIALS AND METHODS: Between January 2012 and December 2014, 96 consecutive adenocarcinoma patients were submitted to oncological colectomy, as the first therapeutic option, with a curative intent. CUL-1 and -2 protein expression was examined with IHC on paraffin-embedded tissue sections. CUL-1 and -2 protein positivity, was correlated with patients' age, gender, stage, histological grade, proliferative capacity (Ki-67 labeling index) and mutant p53 protein expression. The positivity for CUL-1, CUL-2, mutant p53 protein and Ki-67 index, was determined by the percentage of their IHC expression in the total number of cancer cells. RESULTS: Choosing as a cut-off point for CUL-1 positivity the 10%, a statistically significant relationship of the expression of the mutant p53 protein (p=0.04) and the co-expression of CUL-2 (p=0.003) were noticed. By setting the cut-off limit for CUL-2 expression to 10%, no statistically significant differences were observed between its expression and the examined clinicopathological variables. However, by increasing the cut-off limit for CUL-2 expression to 30%, a statistically significant correlation of its expression to the mutated p53 protein was noticed (p=0.047). Co-expression of CUL-1 and -2 in more than 10%, significantly correlated to the coexistence of adenomatous polyps along the large bowel (p=0.0329). Multivariate analysis of CUL-1 and -2 co-expression in more than 10% disclosed their expression as an independent factor for adenomatous polyps development in the large bowel (p=0.035, RR=2.1). CONCLUSION: CUL-1 overexpression may happen early in the process of carcinogenesis mainly affecting the vulnerable p53(+) large bowel cells, arresting them in the G1 phase of cell-cycle, while it may also induce the expression of CUL-2. Co-expression of CUL-1 and CUL-2, in the arrested (in G1 phase) large bowel cells, promotes carcinogenesis up to adenomatous polyp formation. Since no relationship between cullins expression and development of cancer on adenoma was found, the results of the present study may be useful explaining the initiation but not the progression of carcinogenesis in colorectal cancer. Further molecular and clinical studies are needed in order to delineate the clinical importance of these proteins in the management of colorectal cancer patients.

Asunto(s)

Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Proteínas Cullin/metabolismo , Anciano , Biomarcadores de Tumor , Neoplasias Colorrectales/mortalidad , Proteínas Cullin/genética , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias

RESUMEN

Cullin RING E3 ubiquitin ligases (CRLs) function in the ubiquitin proteasome system to catalyze the transfer of ubiquitin from E2 conjugating enzymes to specific substrate proteins. CRLs are large dynamic complexes and attractive drug targets for the development of small-molecule inhibitors and chemical inducers of protein degradation. The atomic details of whole CRL assembly and interactions that dictate subunit specificity remain elusive. Here we present the crystal structure of a pentameric CRL2VHL complex, composed of Cul2, Rbx1, Elongin B, Elongin C, and pVHL. The structure traps a closed state of full-length Cul2 and a new pose of Rbx1 in a trajectory from closed to open conformation. We characterize hotspots and binding thermodynamics at the interface between Cul2 and pVHL-EloBC and identify mutations that contribute toward a selectivity switch for Cul2 versus Cul5 recognition. Our findings provide structural and biophysical insights into the whole Cul2 complex that could aid future drug targeting.

Asunto(s)

Proteínas Portadoras/química , Proteínas Cullin/química , Elonguina/química , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/química , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Proteínas Cullin/metabolismo , Elonguina/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Complejos Multiproteicos/química , Mutación , Conformación Proteica , Termodinámica , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo

Asunto(s)

Proteínas de Ciclo Celular , Ubiquitinación , Ciclo Celular , Proteínas Nucleares/genética

RESUMEN

We recently reported an Affinity-directed PROtein Missile (AdPROM) system for the targeted proteolysis of endogenous proteins of interest (POI) ( Fulcher et al., 2016 and 2017). AdPROM consists of the Von Hippel Lindau (VHL) protein, a Cullin 2 E3 ligase substrate receptor (Bosu and Kipreos, 2008), conjugated to a high affinity polypeptide binder (such as a camelid nanobody) that recognises the target protein in cells. When introduced in cells, the target protein is recruited to the CUL2 E3 ubiquitin ligase complex for ubiquitin-mediated proteasomal degradation. For target protein recruitment, we have utilised both camelid-derived VHH domain nanobodies as well as synthetic polypeptide monobodies based on the human type III fibronectin domain ( Sha et al., 2013 ; Fridy et al., 2014 ; Schmidt et al., 2016 ). In this protocol, we describe detailed methodology involved in generating AdPROM constructs and their application in human cell lines for target protein destruction. AdPROM allows functional characterisation of the POI and its efficiency of target protein destruction overcomes many limitations of RNA-interference approaches, which necessitate long treatments and are associated with off-target effects, and CRISPR/Cas9 gene editing, which is not always feasible.

RESUMEN

BACKGROUND: Cullin-RING E3 ubiquitin ligase complexes play a central role in targeting cellular proteins for ubiquitination-dependent protein turnover through 26S proteasome. Cullin-2 is a member of the Cullin family, and it serves as a scaffold protein for Elongin B and C, Rbx1 and various substrate recognition receptors to form E3 ubiquitin ligases. MAIN BODY OF THE ABSTRACT: First, the composition, structure and the regulation of Cullin-2 based E3 ubiquitin ligases were introduced. Then the targets, the biological functions of complexes that use VHL, Lrr-1, Fem1b, Prame, Zyg-11, BAF250, Rack1 as substrate targeting subunits were described, and their involvement in diseases was discussed. A small molecule inhibitor of Cullins as a potential anti-cancer drug was introduced. Furthermore, proteins with VHL box that might bind to Cullin-2 were described. Finally, how different viral proteins form E3 ubiquitin ligase complexes with Cullin-2 to counter host viral defense were explained. CONCLUSIONS: Cullin-2 based E3 ubiquitin ligases, using many different substrate recognition receptors, recognize a number of substrates and regulate their protein stability. These complexes play critical roles in biological processes and diseases such as cancer, germline differentiation and viral defense. Through the better understanding of their biology, we can devise and develop new therapeutic strategies to treat cancers, inherited diseases and viral infections.

RESUMEN

The Cullin2-type ubiquitin ligases belong to the Cullin-Ring Ligase (CRL) family, which is a crucial determinant of proteasome-based degradation processes in eukaryotes. Because of the finding of von Hippel-Lindau tumor suppressor (VHL), the Cullin2-type ubiquitin ligases gain focusing in the research of many diseases, especially in tumors. These multisubunit enzymes are composed of the Ring finger protein, the Cullin2 scaffold protein, the Elongin B&C linker protein and the variant substrate recognition subunits (SRSs), among which the Cullin2 scaffold protein is the determining factor of the enzyme mechanism. Substrate recognition of Cullin2-type ubiquitin ligases depends on SRSs and results in the degradation of diseases associated substrates by intracellular signaling events. This review focuses on the diversity and the multifunctionality of SRSs in the Cullin2-type ubiquitin ligases, including VHL, LRR-1, FEM1b, PRAME and ZYG11. Recently, as more SRSs are being discovered and more aspects of substrate recognition have been illuminated, insight into the relationship between Cul2-dependent SRSs and substrates provides a new area for cancer research.

Asunto(s)

Proteínas Cullin/fisiología , Antígenos de Neoplasias/fisiología , Proteínas Portadoras/fisiología , Proteínas de Ciclo Celular/fisiología , Proteínas Cullin/química , Humanos , Subunidades de Proteína , Proteínas Represoras/fisiología , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/fisiología

RESUMEN

The Suppressor of Cytokine Signaling (SOCS) proteins are critical, highly conserved feedback inhibitors of signal transduction cascades. The family of SOCS proteins is divided into two groups: ancestral and vertebrate-specific SOCS proteins. Vertebrate-specific SOCS proteins have been heavily studied as a result of their strong mutant phenotypes. However, the ancestral clade remains less studied, a potential result of genetic redundancies in mammals. Use of the genetically tractable organism Drosophila melanogaster enables in vivo assessment of signaling components and mechanisms with less concern about the functional redundancy observed in mammals. In this study, we investigated how the SOCS family member Suppressor of Cytokine Signaling at 36E (Socs36E) attenuates Janus Kinase/Signal Transducer and Activator of Transcription (Jak/STAT) activation during specification of motile border cells in Drosophila oogenesis. We found that Socs36E genetically interacts with the Cullin2 (Cul2) scaffolding protein. Like Socs36E, Cul2 is required to limit the number of motile cells in egg chambers. We demonstrated that loss of Cul2 in the follicle cells significantly increased nuclear STAT protein levels, which resulted in additional cells acquiring invasive properties. Further, reduction of Cul2 suppressed border cell migration defects that occur in a Stat92E-sensitized genetic background. Our data incorporated Cul2 into a previously described Jak/STAT-directed genetic regulatory network that is required to generate a discrete boundary between cell fates. We also found that Socs36E is able to attenuate STAT activity in the egg chamber when it does not have a functional SOCS box. Collectively, this work contributes mechanistic insight to a Jak/STAT regulatory genetic circuit, and suggests that Socs36E regulates Jak/STAT signaling via a Cul2-dependent mechanism, as well as by a Cullin-independent manner, in vivo.

Asunto(s)

Proteínas Cullin/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Ovario/crecimiento & desarrollo , Ovario/metabolismo , Factores de Transcripción STAT/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Animales , Animales Modificados Genéticamente , Movimiento Celular/genética , Movimiento Celular/fisiología , Proteínas Cullin/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Genes de Insecto , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Quinasas Janus/genética , Quinasas Janus/metabolismo , Mutación , Oogénesis/genética , Oogénesis/fisiología , Ovario/citología , Factores de Transcripción STAT/genética , Transducción de Señal , Proteínas Supresoras de la Señalización de Citocinas/genética

RESUMEN

Signaling from a niche consisting of somatic cells is essential for maintenance of germline stem cells (GSCs) in the ovary of Drosophila. Decapentaplegic (Dpp), a type of bone morphogenetic protein (BMP) signal, emanating from the niche, is the most important signal for this process. Cullin proteins constitute the core of a multiprotein E3-ligase important for their functions viz. degradation or modification of proteins necessary for different cellular processes. We have found that a Cullin protein called Cullin-2 (Cul-2) expresses in both somatic and germline cells of the Drosophila ovary. Reduction of Cul-2 in somatic cells causes upregulation of Dpp signal and produces accumulation of extra GSC-like cells inside germarium, the anteriormost structure of the ovary. Our results suggest that Cullin-2 protein present in the somatic cells is involved in a non cell-autonomous regulation of the extent of Dpp signaling and thus controls the differentiation of GSCs to cystoblasts (CBs).

Asunto(s)

Proteínas Cullin/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/crecimiento & desarrollo , Ovario/fisiología , Células Madre/citología , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular , Cruzamientos Genéticos , Regulación hacia Abajo , Receptores ErbB/metabolismo , Femenino , Genotipo , Microscopía Fluorescente , Fenotipo , Interferencia de ARN , Transducción de Señal

RESUMEN

Hypoxia, or low oxygen tension, is a unique environmental stress that induces global changes in a complex regulatory network of transcription factors and signaling proteins to coordinate cellular adaptations in metabolism, proliferation, DNA repair, and apoptosis. Several lines of evidence now establish microRNAs (miRNAs), which are short noncoding RNAs that regulate gene expression through posttranscriptional mechanisms, as key elements in this response to hypoxia. Oxygen deprivation induces a distinct shift in the expression of a specific group of miRNAs, termed hypoxamirs, and emerging evidence indicates that hypoxia regulates several facets of hypoxamir transcription, maturation, and function. Transcription factors such as hypoxia-inducible factor are upregulated under conditions of low oxygen availability and directly activate the transcription of a subset of hypoxamirs. Conversely, hypoxia selectively represses other hypoxamirs through less well characterized mechanisms. In addition, oxygen deprivation has been directly implicated in epigenetic modifications such as DNA demethylation that control specific miRNA transcription. Finally, hypoxia also modulates the activity of key proteins that control posttranscriptional events in the maturation and activity of miRNAs. Collectively, these findings establish hypoxia as an important proximal regulator of miRNA biogenesis and function. It will be important for future studies to address the relative contributions of transcriptional and posttranscriptional events in the regulation of specific hypoxamirs and how such miRNAs are coordinated in order to integrate into the complex hierarchical regulatory network induced by hypoxia.

Asunto(s)

Regulación de la Expresión Génica , Hipoxia/genética , MicroARNs/genética , Factores de Transcripción/genética , Adaptación Fisiológica , Animales , Apoptosis , Epigénesis Genética , Humanos , Hipoxia/metabolismo , Hipoxia/fisiopatología , MicroARNs/agonistas , MicroARNs/antagonistas & inhibidores , MicroARNs/metabolismo , Oxígeno/farmacología , Transducción de Señal , Factores de Transcripción/metabolismo , Transcripción Genética