scholarly journals The E3 Ubiquitin Ligase NARF Promotes Colony Formation in vitro and Exhibits Enhanced Expression Levels in Glioblastoma Multiforme in vivo

2010 ◽  
Vol 9 (2 and 3) ◽  
Author(s):  
Tucker Anderson ◽  
Christopher Wright ◽  
William Brooks
Keyword(s):  
Glioblastoma Multiforme ◽  
Ubiquitin Ligase ◽  
Colony Formation ◽  
Ring Finger ◽  
Negative Regulator ◽  
Low Grade ◽  
Dependent Manner ◽  
Enhanced Expression

The ubiquitin ligase NARF is a newly identified protein belonging to a small family of structurally similar E3 proteins. NARF is a negative regulator of the canonical Wnt-β-catenin pathway, targeting TCF/LEF family members for proteolytic degradation through poly-ubiquitination. We examined the role that NARF plays in cell division and found that overexpression of NARF in a colony forming assay increases colony formation in a RING finger-dependent manner. Furthermore, we demonstrate that NARF transcripts are expressed at a higher level in the grade IV brain tumor glioblastoma multiforme as compared with low grade astrocytomas. Our data thus indicate that NARF is a positive regulator of cell growth and may be involved in the tumorigenic process.

scholarly journals The p73 Tumor Suppressor Is Targeted by Pirh2 RING Finger E3 Ubiquitin Ligase for the Proteasome-dependent Degradation

2011 ◽  
Vol 286 (41) ◽  
pp. 35388-35395 ◽  
Author(s):  
Yong-Sam Jung ◽  
Yingjuan Qian ◽  
Xinbin Chen
Keyword(s):  
Tumor Suppressor ◽  
Ubiquitin Ligase ◽  
Human Cancer ◽  
Ectopic Expression ◽  
E3 Ubiquitin Ligase ◽  
Ring Finger ◽  
Growth Suppression ◽  
Dependent Manner

The p73 gene, a homologue of the p53 tumor suppressor, is expressed as TA and ΔN isoforms. TAp73 has similar activity as p53 and functions as a tumor suppressor whereas ΔNp73 has both pro- and anti-survival functions. While p73 is rarely mutated in spontaneous tumors, the expression status of p73 is linked to the sensitivity of tumor cells to chemotherapy and prognosis for many types of human cancer. Thus, uncovering its regulators in tumors is of great interest. Here, we found that Pirh2, a RING finger E3 ubiquitin ligase, promotes the proteasome-dependent degradation of p73. Specifically, we showed that knockdown of Pirh2 up-regulates, whereas ectopic expression of Pirh2 down-regulates, expression of endogenous and exogenous p73. In addition, Pirh2 physically associates with and promotes TAp73 polyubiquitination both in vivo and in vitro. Moreover, we found that p73 can be degraded by both 20 S and 26 S proteasomes. Finally, we showed that Pirh2 knockdown leads to growth suppression in a TAp73-dependent manner. Taken together, our findings indicate that Pirh2 promotes the proteasomal turnover of TAp73, and thus targeting Pirh2 to restore TAp73-mediated growth suppression in p53-deficient tumors may be developed as a novel anti-cancer strategy.

scholarly journals ORMDL1 is upregulated and associated with favorable outcome in colorectal cancer

2020 ◽  
Author(s):  
Qian Wang ◽  
Wanjun Liu ◽  
Si Chen ◽  
Qianxin Luo ◽  
Yichen Li ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Colony Formation ◽  
Transmembrane Protein ◽  
Cohort Analysis ◽  
Negative Regulator ◽  
Morphologic Change ◽  
Mesenchymal Transition

AbstractBackgroundORMDL1 gene encodes a transmembrane protein for endoplasmic reticulum and is known as crucial negative regulator for sphingolipid biogenesis. However, it has been rarely studied in tumor-related context. Therefore, its prognostic value and functional significance in colorectal cancer (CRC) remain to be explored.MethodsTCGA CRC cohort analysis, qRT-PCR, and immunohistochemistry (IHC) were used to examine the ORMDL1 expression level. The association between ORMDL1 expression and various clinical characteristics were analyzed by Chi-square tests. CRC patients’ overall survival (OS) was analyzed by Kaplan-Meier analysis. In vitro and in vivo cell-based assays were performed to explore the role of ORMDL1 in cell proliferation, invasion and migration. Transcriptional changes of cells either with ORMDL1 knockdowned or overexpressed were compared and analyzed.ResultsORMDL1 was upregulated in CRC tissues either in TCGA cohort or in our cohort. Interestingly, its expression was significantly lower in patients with metastasis compared to patients without metastasis, and high expression group had longer OS than low expression group. Knockdown of ORMDL1 expression can promote proliferation, colony formation and invasion, while attenuate migration in CRC cell lines. In opposite, forced overexpression of ORMDL1 reduced cell proliferation, colony formation and invasion, while enhanced cell migration. Epithelial-to-mesenchymal transition (EMT) related genes were enriched among differentially expressed genes when ORMDL1 was knockdowned in cells, which was consistent with morphologic change by microscopy observation. Finally, stable knockdown of ORMDL1 can promote cancer cell proliferation in vivo to some extent.ConclusionORMDL1 is upregulated and may serve as biomarker to predict favourable outcome in colorectal cancer.

scholarly journals BTB Protein Keap1 Targets Antioxidant Transcription Factor Nrf2 for Ubiquitination by the Cullin 3-Roc1 Ligase

2005 ◽  
Vol 25 (1) ◽  
pp. 162-171 ◽  
Author(s):  
Manabu Furukawa ◽  
Yue Xiong
Keyword(s):  
Transcription Factor ◽  
Ring Finger ◽  
Negative Regulator ◽  
Protein Accumulation ◽  
Protein Motif ◽  
Terminal Domain ◽  
Catalytic Function ◽  
Cullin 3

ABSTRACT The concentrations and functions of many eukaryotic proteins are regulated by the ubiquitin pathway, which consists of ubiquitin activation (E1), conjugation (E2), and ligation (E3). Cullins are a family of evolutionarily conserved proteins that assemble by far the largest family of E3 ligase complexes. Cullins, via a conserved C-terminal domain, bind with the RING finger protein Roc1 to recruit the catalytic function of E2. Via a distinct N-terminal domain, individual cullins bind to a protein motif present in multiple proteins to recruit specific substrates. Cullin 3 (Cul3), but not other cullins, binds directly with BTB domains to constitute a potentially large number of BTB-CUL3-ROC1 E3 ubiquitin ligases. Here we report that the human BTB-Kelch protein Keap1, a negative regulator of the antioxidative transcription factor Nrf2, binds to CUL3 and Nrf2 via its BTB and Kelch domains, respectively. The KEAP1-CUL3-ROC1 complex promoted NRF2 ubiquitination in vitro and knocking down Keap1 or CUL3 by short interfering RNA resulted in NRF2 protein accumulation in vivo. We suggest that Keap1 negatively regulates Nrf2 function in part by targeting Nrf2 for ubiquitination by the CUL3-ROC1 ligase and subsequent degradation by the proteasome. Blocking NRF2 degradation in cells expressing both KEAP1 and NRF2 by either inhibiting the proteasome activity or knocking down Cul3, resulted in NRF2 accumulation in the cytoplasm. These results may reconcile previously observed cytoplasmic sequestration of NRF2 by KEAP1 and suggest a possible regulatory step between KEAP1-NRF2 binding and NRF2 degradation.

Interaction and Regulation of HSC with Osteopontin Is Mediated through α4β1 and α9β1Integrins, Which Are Differentially Expressed in the HSC Pool.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1330-1330
Author(s):  
David N. Haylock ◽  
Genevieve A. Whitty ◽  
Brenda Williams ◽  
Melonie J. Storan ◽  
Susie K. Nilsson
Keyword(s):  
Cord Blood ◽  
Critical Role ◽  
Negative Regulator ◽  
Hemopoietic Stem Cell ◽  
Null Mouse ◽  
Dependent Manner ◽  
Independent Manner ◽  
Hsc Niche

Abstract Osteoblasts are a key cellular component of the hemopoietic stem cell (HSC) niche and directly regulate the HSC pool. Molecules synthesised by osteoblasts both promote or inhibit HSC proliferation. Osteopontin (Opn) is an osteoblast produced, RGD containing protein with roles in cell adhesion and migration. Until recently, the role of Opn in hemopoiesis was seen as restricted to the regulation of bone turnover. However, from analysis of hemopoiesis in the Opn null mouse, we have demonstrated that Opn plays a critical role in regulating the HSC pool. Furthermore Opn is critical in trans-marrow migration and lodgement of HSC within the BM after transplantation. When added to in vitro HSC cultures, exogenous thrombin-cleaved Opn also inhibits cell proliferation and potently suppresses HSC differentiation. We have now demonstrated that this interaction occurs in an RGD-independent manner via the cryptic SVVYGLR epitope revealed on the N-terminal fragment of Opn following thrombin cleavage. This epitope has previously been shown to bind to α4β1 and α9β1. HSC are known to express α4β1, but we have now shown that within the HSC pool this occurs in a differential manner, mimicking that of CD38, with more committed CD34+CD38+ cord blood progenitors having the highest levels of expression. In addition, we have shown the previously unrecognised characteristic of human marrow and cord blood HSC, the expression of α9β1, which also occurs in a differential manner, but mimicking CD34. Expression of α9β1 is highest on cord blood CD34+CD38− cells, a population highly enriched for HSC. Using the synthetic SVVYGLR peptide in culture, we re-capitulated the thrombin-cleaved Opn induced suppression of HSC differentiation in a dose dependent manner. Antibody blocking experiments demonstrated that binding to this peptide was occurring through both α4β1 and α9β1. In contrast, suppression of HSC proliferation and differentiation did not occur through the upstream alternate α4β1 binding site. Furthermore, we have now demonstrated endogenous binding of Opn to α4β1 and α9β1 to cord blood HSC in vivo. Together, these data provide strong evidence that Opn is an important component of the HSC niche which acts as a physiological negative regulator. Furthermore, our studies identify the previously unrecognised characteristic of HSC, the expression of α9β1, which together with α4β1 provides two receptors on HSC with differing expression signatures and potentially a mechanism for fine tunning the physiological effects of Opn.

scholarly journals β2M Signals Monocytes Through Non-Canonical TGFβ Receptor Signal Transduction

2021 ◽  
Author(s):  
Zachary T Hilt ◽  
Preeti Maurya ◽  
Laura Tesoro ◽  
Daphne N Pariser ◽  
Sara K Ture ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Flow Cytometry ◽  
Nuclear Localization ◽  
Ubiquitin Ligase ◽  
Dependent Manner ◽  
Elevated Plasma ◽  
Imaging Flow Cytometry ◽  
Tgfβ Receptor

Rationale: Circulating monocytes can have pro-inflammatory or pro-reparative phenotypes. The endogenous signaling molecules and pathways that regulate monocyte polarization in vivo are poorly understood. We have shown that platelet derived beta-2 microglobulin (β2M) and transforming growth factor beta (TGFβ) have opposing effects on monocytes by inducing inflammatory and reparative phenotypes respectively, but each bind and signal through the same receptor. We now define the signaling pathways involved. Objective: To determine the molecular mechanisms and signal transduction pathways by which β2M and TGFβ regulate monocyte responses both in vitro and in vivo. Methods and Results: Wild-type (WT) and platelet specific β2M knockout (Plt-β2M -/- ) mice were treated intravenously with either β2M or TGFβ to increase plasma concentrations to those in cardiovascular diseases. Elevated plasma β2M increased pro-inflammatory monocytes, while increased plasma TGFβ increased pro-reparative monocytes. TGFβ receptor (TGFβR) inhibition blunted monocyte responses to both β2M and TGFβ in vivo. Using imaging flow cytometry, we found that β2M decreased monocyte SMAD2/3 nuclear localization, while TGFβ promoted SMAD nuclear translocation, but decreased non-canonical/inflammatory (JNK and NFκB nuclear localization). This was confirmed in vitro using both imaging flow cytometry and immunoblots. β2M, but not TGFβ, promoted ubiquitination of SMAD3 and SMAD4, that inhibited their nuclear trafficking. Inhibition of ubiquitin ligase activity blocked non-canonical SMAD-independent monocyte signaling and skewed monocytes towards a pro-reparative monocyte response. Conclusions: Our findings indicate that elevated plasma β2M and TGFβ dichotomously polarize monocytes. Furthermore, these immune molecules share a common receptor, but induce SMAD-dependent canonical signaling (TGFβ) versus non-canonical SMAD-independent signaling (β2M) in a ubiquitin ligase dependent manner. This work has broad implications as β2M is increased in several inflammatory conditions, while TGFβ is increased in fibrotic diseases.

scholarly journals Epigenetic Inactivation of Acetyl-Co A acetyltransferase 1 promotes the proliferation and metastasis of nasopharyngeal carcinoma via decreasing ketogenesis

2020 ◽  
Author(s):  
Yunliang Lu ◽  
Xiaohui Zhou ◽  
Weilin Zhao ◽  
Zhipeng Liao ◽  
Bo Li ◽  
...  
Keyword(s):  
Cell Lines ◽  
Colony Formation ◽  
Ketone Body ◽  
Epithelial Mesenchymal Transition ◽  
Cpg Island ◽  
Dependent Manner ◽  
Mesenchymal Transition ◽  
Ketone Body Metabolism

Abstract Background Acy1 Coenzyme A Acyltransferases1 (ACAT1) is a key enzyme in the metabolism of ketone bodies, but its expression and biological function in the pathogenesis of NPC remains underexplored. Methods The mRNA and protein expression levels of ACAT1 in NPC and normal control tissues were analyzed by qPCR and immunohistochemistry staining, respectively. GEO database was applied for meta-analysis of ACAT1 mRNA expression and DNA promoter methylation. The role of ACAT1 in NPC proliferation was examined by CCK8 and colony formation assays in vitro and tumorigenicity in vivo. The wound healing and transwell assays were used for analyzing the migratory and invasive ability. cDNA microarray analysis was performed to identify the genes involved in epithelial-mesenchymal transition and dysregulated by ACAT1. These changes were further confirmed by western blot. Results We found that ACAT1 is inactivated in NPC cell lines and primary tissues. DNA microarray data showed higher methylation in the CpG island region of ACAT1 in NPC than normal tissues. The demethylating reagent 5-aza-dC significantly restored the transcription of ACAT1 in NPC cell lines, suggesting that ACAT1 was inactivated by DNA promoter hypermethylation. Ectopic overexpression of ACAT1 remarkably suppressed the proliferation and colony formation of NPC cells in vitro. As well, the tumorigenesis of NPC cells overexpressing ACAT1 was decreased in vivo. In addition, the migratory and invasive capacities of NPC cells was inhibited by ACAT1 overexpression. Importantly, the higher level of ACAT1 was accompanied by an increased expression of CDH1, EPCAM, and a decreased expression of vimentin and SPARC. This strongly indicates that ACAT1 is able to affect the epithelial-mesenchymal transition in NPC, thereby controlling cellular motility. In addition, we found that ACAT1 expression increases the intracellular level of β-HB. Moreover, exogenous β-HB remarkably inhibits the growth of NPC cells in a dose-dependent manner. Conclusions We have discovered that the ketone body metabolism enzyme ACAT1 is epigenetically downregulated in NPC and acts as a potential tumor suppressor in NPC. Our findings highlight the possibility of using the modulation of ketone body metabolism as effective adjuvant therapy for NPC.

scholarly journals Inhibition of murine erythroid colony formation in vitro by interferon gamma and correction by interferon receptor immunoadhesin

Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 911-915 ◽  
Author(s):  
RT Jr Means ◽  
SB Krantz ◽  
J Luna ◽  
SA Marsters ◽  
A Ashkenazi
Keyword(s):  
Animal Model ◽  
Interferon Gamma ◽  
Colony Formation ◽  
Interleukin 1 ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Ifn Gamma ◽  
Dose Dependent

It has been previously reported that inhibition of human erythroid colony-forming units (CFU-E) in vitro by interleukin-1 (IL-1) is an indirect effect, occurring through the production of interferon gamma (IFN gamma). IFN gamma, in turn, inhibits CFU-E colony formation directly, and its inhibitory effect can be overcome by exposure to high concentrations of erythropoietin (EPO). To develop an in vitro animal model for investigating inhibition of erythropoiesis by IFN gamma, the effects of recombinant murine (rm) IFN gamma on highly purified CFU-E from the spleens of mice infected with the anemia strain of the Friend virus (FVA) were studied. rmIFN gamma inhibited CFU-E colony formation in a dose-dependent manner. This inhibition occurred with large (> or = 8 cell) colonies only; smaller colonies were not affected. The inhibitory effect was corrected to 72% of control by high EPO concentrations of 64 U/mL. Murine CFU-E were then cultured with rmIFN gamma in the presence of a soluble murine IFN gamma receptor fused to the hinge and Fc domains of the human IgG1 heavy chain (mIFN gamma R-IgG). Inhibition of CFU-E colony formation by rmIFN gamma (100 U/mL) was corrected by mIFN gamma R-IgG in a dose-dependent manner, with an approximate IC50 of 0.05 nmol/L, and complete or near complete correction at 0.5 nmol/L. Similarly, a human IFN gamma R-IgG greatly reduced the inhibitory effect of recombinant human IFN gamma on human CFU-E. These experiments provide an in vitro animal model for studying the inhibitory effects of IFN gamma on erythropoiesis and indicate that IFN gamma R-IgG may be a useful agent for reducing the toxicity of IFN gamma in vivo.

scholarly journals The CUL1 C-Terminal Sequence and ROC1 Are Required for Efficient Nuclear Accumulation, NEDD8 Modification, and Ubiquitin Ligase Activity of CUL1

2000 ◽  
Vol 20 (21) ◽  
pp. 8185-8197 ◽  
Author(s):  
Manabu Furukawa ◽  
Yanping Zhang ◽  
Joseph McCarville ◽  
Tomohiko Ohta ◽  
Yue Xiong
Keyword(s):  
Nuclear Localization ◽  
Ubiquitin Ligase ◽  
Nuclear Import ◽  
Ring Finger ◽  
Nuclear Accumulation ◽  
Protein Families ◽  
Terminal Sequence ◽  
Ubiquitin Ligase Activity

ABSTRACT Members of the cullin and RING finger ROC protein families form heterodimeric complexes to constitute a potentially large number of distinct E3 ubiquitin ligases. We report here that the highly conserved C-terminal sequence in CUL1 is dually required, both for nuclear localization and for modification by NEDD8. Disruption of ROC1 binding impaired nuclear accumulation of CUL1 and decreased NEDD8 modification in vivo but had no effect on NEDD8 modification of CUL1 in vitro, suggesting that ROC1 promotes CUL1 nuclear accumulation to facilitate its NEDD8 modification. Disruption of NEDD8 binding had no effect on ROC1 binding, nor did it affect nuclear localization of CUL1, suggesting that nuclear localization and NEDD8 modification of CUL1 are two separable steps, with nuclear import preceding and required for NEDD8 modification. Disrupting NEDD8 modification diminishes the IκBα ubiquitin ligase activity of CUL1. These results identify a pathway for regulation of CUL1 activity—ROC1 and the CUL1 C-terminal sequence collaboratively mediate nuclear accumulation and NEDD8 modification, facilitating assembly of active CUL1 ubiquitin ligase. This pathway may be commonly utilized for the assembly of other cullin ligases.

scholarly journals TEB4 is a C4HC3 RING finger-containing ubiquitin ligase of the endoplasmic reticulum

2005 ◽  
Vol 388 (2) ◽  
pp. 647-655 ◽  
Author(s):  
Gerco HASSINK ◽  
Marjolein KIKKERT ◽  
Sjaak van VOORDEN ◽  
Shiow-Ju LEE ◽  
Robbert SPAAPEN ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Degradation ◽  
Ubiquitin Ligase ◽  
Ring Finger ◽  
Transmembrane Domains ◽  
Dependent Manner ◽  
N Terminus ◽  
Ubiquitin Conjugating Enzyme ◽  
New Gene

In the present study, the human TEB4 is identified as a novel ER (endoplasmic reticulum)-resident ubiquitin ligase. TEB4 has homologues in many species and has a number of remarkable properties. TEB4 contains a conserved RING (really interesting new gene) finger and 13 predicted transmembrane domains. The RING finger of TEB4 and its homologues is situated at the N-terminus and has the unconventional C4HC3 configuration. The N-terminus of TEB4 is located in the cytosol. We show that the isolated TEB4 RING domain catalyses ubiquitin ligation in vitro in a reaction that is ubiquitin Lys48-specific and involves UBC7 (ubiquitin-conjugating enzyme 7). These properties are reminiscent of E3 enzymes, which are involved in ER-associated protein degradation. TEB4 is an ER degradation substrate itself, promoting its own degradation in a RING finger- and proteasome-dependent manner.

scholarly journals The Yeast GID Complex, a Novel Ubiquitin Ligase (E3) Involved in the Regulation of Carbohydrate Metabolism

2008 ◽  
Vol 19 (8) ◽  
pp. 3323-3333 ◽  
Author(s):  
Olivier Santt ◽  
Thorsten Pfirrmann ◽  
Bernhard Braun ◽  
Jeannette Juretschke ◽  
Philipp Kimmig ◽  
...  
Keyword(s):  
Carbohydrate Metabolism ◽  
Ubiquitin Ligase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Ring Finger ◽  
Vitro Assay ◽  
Fructose 1,6 Bisphosphatase ◽  
Key Enzyme ◽  
Ubiquitin Ligase E3

Glucose-dependent regulation of carbon metabolism is a subject of intensive studies. We have previously shown that the switch from gluconeogenesis to glycolysis is associated with ubiquitin-proteasome linked elimination of the key enzyme fructose-1,6-bisphosphatase. Seven glucose induced degradation deficient (Gid)-proteins found previously in a genomic screen were shown to form a complex that binds FBPase. One of the subunits, Gid2/Rmd5, contains a degenerated RING finger domain. In an in vitro assay, heterologous expression of GST-Gid2 leads to polyubiquitination of proteins. In addition, we show that a mutation in the degenerated RING domain of Gid2/Rmd5 abolishes fructose-1,6-bisphosphatase polyubiquitination and elimination in vivo. Six Gid proteins are present in gluconeogenic cells. A seventh protein, Gid4/Vid24, occurs upon glucose addition to gluconeogenic cells and is afterwards eliminated. Forcing abnormal expression of Gid4/Vid24 in gluconeogenic cells leads to fructose-1,6-bisphosphatase degradation. This suggests that Gid4/Vid24 initiates fructose-1,6-bisphosphatase polyubiquitination by the Gid complex and its subsequent elimination by the proteasome. We also show that an additional gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, is subject to Gid complex-dependent degradation. Our study uncovers a new type of ubiquitin ligase complex composed of novel subunits involved in carbohydrate metabolism and identifies Gid4/Vid24 as a major regulator of this E3.

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