scholarly journals The Keap1-BTB Protein Is an Adaptor That Bridges Nrf2 to a Cul3-Based E3 Ligase: Oxidative Stress Sensing by a Cul3-Keap1 Ligase

2004 ◽  
Vol 24 (19) ◽  
pp. 8477-8486 ◽  
Author(s):  
Sara B. Cullinan ◽  
John D. Gordan ◽  
Jianping Jin ◽  
J. Wade Harper ◽  
J. Alan Diehl
Keyword(s):  
Oxidative Stress ◽  
Nuclear Import ◽  
Target Genes ◽  
E3 Ligase ◽  
Nuclear Accumulation ◽  
Nrf2 Transcription Factor ◽  
Domain Protein ◽  
Stress Sensing

ABSTRACT The Nrf2 transcription factor promotes survival following cellular insults that trigger oxidative damage. Nrf2 activity is opposed by the BTB/POZ domain protein Keap1. Keap1 is proposed to regulate Nrf2 activity strictly through its capacity to inhibit Nrf2 nuclear import. Recent work suggests that inhibition of Nrf2 may also depend upon ubiquitin-mediated proteolysis. To address the contribution of Keap1-dependent sequestration versus Nrf2 proteolysis, we identified the E3 ligase that regulates Nrf2 ubiquitination. We demonstrate that Keap1 is not solely a cytosolic anchor; rather, Keap1 is an adaptor that bridges Nrf2 to Cul3. We demonstrate that Cul3-Keap1 complexes regulate Nrf2 polyubiquitination both in vitro and in vivo. Inhibition of either Keap1 or Cul3 increases Nrf2 nuclear accumulation, leading to promiscuous activation of Nrf2-dependent gene expression. Our data demonstrate that Keap1 restrains Nrf2 activity via its capacity to target Nrf2 to a cytoplasmic Cul3-based E3 ligase and suggest a model in which Keap1 coordinately regulates both Nrf2 accumulation and access to target genes.

scholarly journals Modulating PKCα Activity to Target Wnt/β-Catenin Signaling in Colon Cancer

Cancers ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 693 ◽  
Author(s):  
Sébastien Dupasquier ◽  
Philippe Blache ◽  
Laurence Picque Lasorsa ◽  
Han Zhao ◽  
Jean-Daniel Abraham ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Target Genes ◽  
Tumor Development ◽  
Orphan Receptor ◽  
Nuclear Accumulation ◽  
Kinase C ◽  
Extracellular Signal

Inactivating mutations of the tumor suppressor Adenomatosis Polyposis Coli (APC), which are found in familial adenomatosis polyposis and in 80% of sporadic colorectal cancers (CRC), result in constitutive activation of the Wnt/β-catenin pathway and tumor development in the intestine. These mutations disconnect the Wnt/β-catenin pathway from its Wnt extracellular signal by inactivating the APC/GSK3-β/axin destruction complex of β-catenin. This results in sustained nuclear accumulation of β-catenin, followed by β-catenin-dependent co-transcriptional activation of Wnt/β-catenin target genes. Thus, mechanisms acting downstream of APC, such as those controlling β-catenin stability and/or co-transcriptional activity, are attractive targets for CRC treatment. Protein Kinase C-α (PKCα) phosphorylates the orphan receptor RORα that then inhibits β-catenin co-transcriptional activity. PKCα also phosphorylates β-catenin, leading to its degradation by the proteasome. Here, using both in vitro (DLD-1 cells) and in vivo (C57BL/6J mice) PKCα knock-in models, we investigated whether enhancing PKCα function could be beneficial in CRC treatment. We found that PKCα is infrequently mutated in CRC samples, and that inducing PKCα function is not deleterious for the normal intestinal epithelium. Conversely, di-terpene ester-induced PKCα activity triggers CRC cell death. Together, these data indicate that PKCα is a relevant drug target for CRC treatment.

scholarly journals Control of Thioredoxin Reductase Gene (trxB) Transcription by SarA in Staphylococcus aureus

2009 ◽  
Vol 192 (1) ◽  
pp. 336-345 ◽  
Author(s):  
Anand Ballal ◽  
Adhar C. Manna
Keyword(s):  
Oxidative Stress ◽  
Staphylococcus Aureus ◽  
Dna Binding ◽  
Target Genes ◽  
Thioredoxin Reductase ◽  
Negative Regulator ◽  
Binding Studies ◽  
Oxidizing Agents

ABSTRACT Thioredoxin reductase (encoded by trxB) protects Staphylococcus aureus against oxygen or disulfide stress and is indispensable for growth. Among the different sarA family mutants analyzed, transcription of trxB was markedly elevated in the sarA mutant under conditions of aerobic as well as microaerophilic growth, indicating that SarA acts as a negative regulator of trxB expression. Gel shift analysis showed that purified SarA protein binds directly to the trxB promoter region DNA in vitro. DNA binding of SarA was essential for repression of trxB transcription in vivo in S. aureus. Northern blot analysis and DNA binding studies of the purified wild-type SarA and the mutant SarAC9G with oxidizing agents indicated that oxidation of Cys-9 reduced the binding of SarA to the trxB promoter DNA. Oxidizing agents, in particular diamide, could further enhance transcription of the trxB gene in the sarA mutant, suggesting the presence of a SarA-independent mode of trxB induction. Analysis of two oxidative stress-responsive sarA regulatory target genes, trxB and sodM, with various mutant sarA constructs showed a differential ability of the SarA to regulate expression of the two above-mentioned genes in vivo. The overall data demonstrate the important role played by SarA in modulating expression of genes involved in oxidative stress resistance in S. aureus.

scholarly journals Oncogenic hijacking of a developmental transcription factor evokes vulnerability toward oxidative stress in Ewing sarcoma

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Aruna Marchetto ◽  
Shunya Ohmura ◽  
Martin F. Orth ◽  
Maximilian M. L. Knott ◽  
Maria V. Colombo ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Transcription Factor ◽  
Ewing Sarcoma ◽  
Target Genes ◽  
Transcriptome Profiling ◽  
In Vivo Experiments ◽  
Ets Family ◽  
Pdx Models

AbstractEwing sarcoma (EwS) is an aggressive childhood cancer likely originating from mesenchymal stem cells or osteo-chondrogenic progenitors. It is characterized by fusion oncoproteins involving EWSR1 and variable members of the ETS-family of transcription factors (in 85% FLI1). EWSR1-FLI1 can induce target genes by using GGAA-microsatellites as enhancers.Here, we show that EWSR1-FLI1 hijacks the developmental transcription factor SOX6 – a physiological driver of proliferation of osteo-chondrogenic progenitors – by binding to an intronic GGAA-microsatellite, which promotes EwS growth in vitro and in vivo. Through integration of transcriptome-profiling, published drug-screening data, and functional in vitro and in vivo experiments including 3D and PDX models, we discover that constitutively high SOX6 expression promotes elevated levels of oxidative stress that create a therapeutic vulnerability toward the oxidative stress-inducing drug Elesclomol.Collectively, our results exemplify how aberrant activation of a developmental transcription factor by a dominant oncogene can promote malignancy, but provide opportunities for targeted therapy.

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 Natural Nrf2 Activators from Juices, Wines, Coffee, and Cocoa

Beverages ◽  
2020 ◽  
Vol 6 (4) ◽  
pp. 68
Author(s):  
Mallique Qader ◽  
Jian Xu ◽  
Yuejun Yang ◽  
Yuancai Liu ◽  
Shugeng Cao
Keyword(s):  
Oxidative Stress ◽  
Defense Mechanisms ◽  
Target Genes ◽  
Antioxidant Activities ◽  
Reactive Oxygen ◽  
Polyphenolic Compounds ◽  
Cellular Systems ◽  
In Vivo Studies

Juices, wine, coffee, and cocoa are rich sources of natural polyphenolic compounds that have potent antioxidant activities proven by in vitro and in vivo studies. These polyphenolic compounds quench reactive oxygen and nitrogen species (RONS) or reactive free radicals and act as natural antioxidants which are also able to protect against reactive oxygen species (ROS)-mediated oxidative damage, which elevates cellular antioxidant capacity to induce antioxidant defense mechanisms by modulating transcription factors. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a transcription factor encoded in humans. It is activated as a result of oxidative stress and induces the expression of its target genes. This is one of the most important cellular defense mechanisms against oxidative stress. However, the oxidative stress alone is not enough to activate Nrf2. Hence phytochemicals, especially polyphenolics, act as natural Nrf2 activators. Herein, this review discusses the natural products identified in juices, coffee, cocoa and wines that modulate Nrf2 activity in cellular systems.

scholarly journals Identification and Verification of the Effect of miR-143 on the Cardioprotective Role of Phosphocreatine in Doxorubicin- Induced Cardiotoxicity by Integrated Bioinformatics Analysis

2021 ◽  
Author(s):  
Chi Zhou ◽  
Zi-Mo Zhou ◽  
Ling Hu ◽  
Ya-Yuan Yang ◽  
Xiang-Wen Meng ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Target Genes ◽  
Bioinformatics Analysis ◽  
Mrna Translation ◽  
Rat Cardiomyocytes ◽  
Adult Rat

Abstract Purpose MicroRNAs (miRNAs) have been reported to play pivotal role in drugs-induced cardiotoxicity act as biomarkes, diagnostic tools and endogenous repressors of gene expression by lowering mRNA stability and interfering with mRNA translation. However, the effect of miRNAs on doxorubicin-induced cardiotoxicity still not clear. In the present study, we identified several key candidate miRNAs involving doxorubicin (DOX)-induced cardiotoxicity in rat myocardial tissues and adult rat cardiomyocytes from the Gene Expression Omnibus (GEO) database via integrated bioinformatics analysis, and the possible effect of miR-143 in the protection of DOX-induced cardiotoxicity by phosphocreatine was subsequently investigated in vivo and in vitro. Methods GSE36239 miRNA expression profiles of DOX-induced cardiotoxicity in rat myocardial tissues and adult rat cardiomyocytes (ARC) were extracted fromGEO datasets. |log2FC| > 1 and P < 0.05 were set as screening criteria, miRNAs expressed in myocardial tissues or ARC were selected as different expression miRNA (DEMs), and subsequently the key miRNAs were obtained from candidate DEMs between myocardial tissues and ARC with Venny 2.1 software. Target genes of miR-143 were predicted with Targetscan and miRBase in the species of homo sapiens, and candidate genes were obtained with Venny 2.1. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses were carried out. Final, the expression and potential role of miR-143 were verified in DOX-induced cardiotoxicity of rat and cardiomyocytes H9c2. Results A total 24 DEMs were captured , including 15 up-regulated and 9 down-regulated genes in rat myocardial tissues and 42 DEMs were discovered, including 13 up-regulated and 29 down-regulated in ARC. Ultimately, 6 DEMs were determined in rat myocardial tissues and ARC by venny 2.1 software. 46 target genes of miR-143, one of the 6 DEMs, were captured from the predict results of Targetscan and miRBase with venny 2.1. The target genes were notably enriched in biological processes (BP) such as cell proliferation and migration. KEGG pathway analysis showed the target genes were enriched in HIF-1 and PI3K-Akt signaling pathway, which closely related to the oxidative stress and cardiomyocytes apoptosis. Further, western blot and RT-PCR results showed DOX-induced oxidative stress down-regulated the expression of miR-143 and Nrf2, SOD and BCL2, and up-regulated Bax and Cleaved caspase 3, while they could been reversed by the intervention of phosphocreatine (PCr) or N-acetyl-L-cystine (NAC) in DOX-induced cardiotoxicity in vivo and in vitro.Conclusion Our data showed that DOX-induced oxidative stress could decrease the expression of miR-143, promote apoptosis of cardiomyocytes, while PCr or NAC mediated antioxidation could reverse the expression down-regulation of miR-143, alleviated apoptosis in DOX-induced cardiotoxicity. Our findings elucidated the regulatory network involving miR-143 in DOX-induced cardiotoxicity, and might unveiled a potential biomarker and molecular mechanisms, which could be helpful to the diagnosis and treatment of DOX-induced cardiotoxicity.

scholarly journals The Redox State of SECIS Binding Protein 2 Controls Its Localization and Selenocysteine Incorporation Function

2006 ◽  
Vol 26 (13) ◽  
pp. 4895-4910 ◽  
Author(s):  
Laura V. Papp ◽  
Jun Lu ◽  
Frank Striebel ◽  
Derek Kennedy ◽  
Arne Holmgren ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Nuclear Export ◽  
Stop Codon ◽  
Binding Protein ◽  
Redox Homeostasis ◽  
Nuclear Export Signal ◽  
Nuclear Accumulation ◽  
Antioxidant Systems

ABSTRACT Selenoproteins are central controllers of cellular redox homeostasis. Incorporation of selenocysteine (Sec) into selenoproteins employs a unique mechanism to decode the UGA stop codon. The process requires the Sec insertion sequence (SECIS) element, tRNASec, and protein factors including the SECIS binding protein 2 (SBP2). Here, we report the characterization of motifs within SBP2 that regulate its subcellular localization and function. We show that SBP2 shuttles between the nucleus and the cytoplasm via intrinsic, functional nuclear localization signal and nuclear export signal motifs and that its nuclear export is dependent on the CRM1 pathway. Oxidative stress induces nuclear accumulation of SBP2 via oxidation of cysteine residues within a redox-sensitive cysteine-rich domain. These modifications are efficiently reversed in vitro by human thioredoxin and glutaredoxin, suggesting that these antioxidant systems might regulate redox status of SBP2 in vivo. Depletion of SBP2 in cell lines using small interfering RNA results in a decrease in Sec incorporation, providing direct evidence for its requirement for selenoprotein synthesis. Furthermore, Sec incorporation is reduced substantially after treatment of cells with agents that cause oxidative stress, suggesting that nuclear sequestration of SBP2 under such conditions may represent a mechanism to regulate the expression of selenoproteins.

scholarly journals Ribosomal Protein L12 Uses a Distinct Nuclear Import Pathway Mediated by Importin 11

2002 ◽  
Vol 22 (4) ◽  
pp. 1266-1275 ◽  
Author(s):  
Scott M. Plafker ◽  
Ian G. Macara
Keyword(s):  
Ribosomal Protein ◽  
Nuclear Import ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Protein Import ◽  
Nuclear Translocation ◽  
Nuclear Accumulation ◽  
Transient Transfection Assay

ABSTRACT Ribosome biogenesis requires the nuclear translocation of ribosomal proteins from their site of synthesis in the cytoplasm to the nucleus. Analyses of the import mechanisms have revealed that most ribosomal proteins can be delivered to the nucleus by multiple transport receptors (karyopherins or importins). We now provide evidence that ribosomal protein L12 (rpL12) is distinguished from the bulk of ribosomal proteins because it accesses the importin 11 pathway as a major route into the nucleus. rpL12 specifically and directly interacted with importin 11 in vitro and in vivo. Both rpL12 binding to and import by importin 11 were inhibited by another importin 11 substrate, UbcM2, indicating that these two cargoes may bind overlapping sites on the transport receptor. In contrast, the import of rpL23a, a ribosomal protein that uses the general ribosomal protein import system, was not competed by UbcM2, and in an in vitro binding assay, importin 11 did not bind to the nuclear localization signal of rpL23a. Furthermore, in a transient transfection assay, the nuclear accumulation of rpL12 was increased by coexpressed importin 11, but not by other importins. These data are consistent with importin 11 being a mediator of rpL12 nuclear import. Taken together, these results indicate that rpL12 uses a distinct nuclear import pathway that may contribute to a mechanism for regulating ribosome synthesis and/or maturation.

scholarly journals The Alternative Sigma Factor RpoE2 Is Involved in the Stress Response to Hypochlorite and in vivo Survival of Haemophilus influenzae

2021 ◽  
Vol 12 ◽  
Author(s):  
Marufa Nasreen ◽  
Aidan Fletcher ◽  
Jennifer Hosmer ◽  
Qifeng Zhong ◽  
Ama-Tawiah Essilfie ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Sigma Factor ◽  
Target Genes ◽  
Cell Envelope ◽  
Sigma Factors ◽  
Methionine Sulfoxide Reductase ◽  
Respiratory Pathogen ◽  
Human Pathogens

Extracytoplasmic function (ECF) sigma factors underpin the ability of bacteria to adapt to changing environmental conditions, a process that is particularly relevant in human pathogens that inhabit niches where human immune cells contribute to high levels of extracellular stress. Here, we have characterized the previously unstudied RpoE2 ECF sigma factor from the human respiratory pathogen H. influenzae (Hi) and its role in hypochlorite-induced stress. Exposure of H. influenzae to oxidative stress (HOCl, H2O2) increased rpoE2 gene expression, and the activity of RpoE2 was controlled by a cytoplasmic 67-aa anti-sigma factor, HrsE. RpoE2 regulated the expression of the periplasmic MsrAB peptide methionine sulfoxide reductase that, in H. influenzae, is required for HOCl resistance, thus linking RpoE2 to HOCl stress. Interestingly, a HiΔrpoE2 strain had wild-type levels of resistance to oxidative stress in vitro, but HiΔrpoE2 survival was reduced 26-fold in a mouse model of lung infection, demonstrating the relevance of this sigma factor for H. influenzae pathogenesis. The HiRpoE2 system has some similarity to the ECF sigma factors described in Streptomyces and Neisseria sp. that also control the expression of msr genes. However, HiRpoE2 regulation extended to genes encoding other periplasmic damage repair proteins, an operon containing a DoxX-like protein, and also included selected OxyR-controlled genes. Based on our results, we propose that the highly conserved HiRpoE2 sigma factor is a key regulator of H. influenzae responses to oxidative damage in the cell envelope region that controls a variety of target genes required for survival in the host.

scholarly journals Arabidopsis SUMO E3 Ligase SIZ1 Interacts with HDA6 and Negatively Regulates HDA6 Function during Flowering

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3001
Author(s):  
Sujuan Gao ◽  
Xueqin Zeng ◽  
Jianhao Wang ◽  
Yingchao Xu ◽  
Chunwei Yu ◽  
...  
Keyword(s):  
Target Genes ◽  
Environmental Changes ◽  
Histone H3 ◽  
E3 Ligase ◽  
Histone Deacetylase 6 ◽  
Histone H3 Acetylation ◽  
Sumo E3 Ligase ◽  
H3 Acetylation

The changes in histone acetylation mediated by histone deacetylases (HDAC) play a crucial role in plant development and response to environmental changes. Mammalian HDACs are regulated by post-translational modifications (PTM), such as phosphorylation, acetylation, ubiquitination and small ubiquitin-like modifier (SUMO) modification (SUMOylation), which affect enzymatic activity and transcriptional repression. Whether PTMs of plant HDACs alter their functions are largely unknown. In this study, we demonstrated that the Arabidopsis SUMO E3 ligase SAP AND MIZ1 DOMAIN-CONTAINING LIGASE1 (SIZ1) interacts with HISTONE DEACETYLASE 6 (HDA6) both in vitro and in vivo. Biochemical analyses indicated that HDA6 is not modified by SUMO1. Overexpression of HDA6 in siz1-3 background results in a decreased level of histone H3 acetylation, indicating that the activity of HDA6 is increased in siz1-3 plants. Chromatin immunoprecipitation (ChIP) assays showed that SIZ1 represses HDA6 binding to its target genes FLOWERING LOCUS C (FLC) and MADS AFFECTING FLOWERING 4 (MAF4), resulting in the upregulation of FLC and MAF4 by increasing the level of histone H3 acetylation. Together, these findings indicate that the Arabidopsis SUMO E3 ligase SIZ1 interacts with HDA6 and negatively regulates HDA6 function.

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