E1B-55-Kilodalton Protein Is Not Required To Block p53-Induced Transcription during Adenovirus Infection

ABSTRACT The adenovirus E1B-55-kDa protein binds and inactivates the tumor suppressor protein p53. However, the role of this interaction during infection is still poorly understood and was therefore examined here. Infection with a virus carrying the E1B-55-kDa mutation R239A, preventing the interaction with p53, led to the accumulation of p53. However, p53 target genes were not activated in the infected cells, although p53 phosphorylation did occur and the p53 antagonists Mdm2 and ΔNp73 did not accumulate. Deletion of E4orf6, alone or in combination with E1B-55-kDa, did not allow the induction of p53-responsive genes either. In transient reporter assays, the viral E1A-13S protein antagonized p53 activity; mutational analysis suggested that this depends partially on p300 binding, but it depends even more strongly on the interaction of E1A with the p400/TRRAP protein complex. However, viruses expressing E1A mutants lacking these binding activities, in combination with E1B-55-kDa R239A, still abolished p53 activity. In contrast, when the mutation of E1B-55-kDa at R239A was combined with a deletion of the apoptosis inhibitor E1B-19-kDa, infected cells showed more extensive apoptosis than after infection with single mutants, suggesting that accumulated p53, albeit transcriptionally inactive, might nonetheless enhance apoptosis. Despite extensive apoptosis of the infected cells, the deletion of E1B-19-kDa, in combination with the E1B-55-kDa mutation or in the presence of the constitutively active p53 mutant p53mt24-28, reduced virus replication less than fivefold. In conclusion, adenovirus does not need direct binding of E1B-55-kDa to inactivate p53, and forced p53 activity with consecutive apoptosis does not severely impair virus replication.

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381: Promoter Hypermethylation Profile of Kidney Cancer with New Pro-Apoptotic P53 Target Genes and Possible Clinical Implications

The Journal of Urology ◽

10.1016/s0022-5347(18)32637-5 ◽

2006 ◽

Vol 175 (4S) ◽

pp. 125-125

Author(s):

Frank Christoph ◽

Steffen Weikert ◽

Carsten Kempkensteffen ◽

Martin Schostak ◽

Hans Krause ◽

...

Keyword(s):

Kidney Cancer ◽

Target Genes ◽

Promoter Hypermethylation ◽

Clinical Implications ◽

P53 Target Genes

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Differential Activation of p53 Target Genes in Breast Cancer

10.21236/ada405348 ◽

2002 ◽

Author(s):

Edward Thornborrow ◽

James Manfredi

Keyword(s):

Breast Cancer ◽

Target Genes ◽

Differential Activation ◽

P53 Target Genes

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PPARα Signaling is Activated by Cocoa in Mouse Liver

Natural Product Communications ◽

10.1177/1934578x1300800507 ◽

2013 ◽

Vol 8 (5) ◽

pp. 1934578X1300800

Author(s):

Marco Fidaleo ◽

Claudia Sartori

Keyword(s):

Mouse Liver ◽

Target Genes ◽

Signal Modulation ◽

Sod1 Gene ◽

Serum Triglycerides ◽

Oxidative Balance ◽

Gene Level ◽

Direct Binding ◽

Somatic Index ◽

Oxidation System

In this study we evaluated in mouse liver the effects of cocoa on PPARα signaling. To this aim, mouse diet was supplemented with 10%, w/w, cocoa for one and two weeks. We quantified the expression of PPARα target genes and PPARα gene level and some parameters related to PPARα activation (hepato-somatic index, peroxisomal β-oxidation system and catalase activity). Moreover, we evaluated antioxidant capacity of cocoa by detecting the expression of CAT and SOD1 genes (known to be involved in oxidative balance) and hypolipidemic properties on serum triglycerides. We made a parallel treatment with 0.025%, w/w, ciprofibrate, a well-known PPARα activator, to quantify signal modulation by cocoa. It is known that PPARα activation by ciprofibrate is mediated by direct binding to the receptor and strongly induces expression of target genes. Our results show that cocoa weakly up-regulates PPARα target genes as a consequence of the modulation of the PPARα gene level and does not improve the triglyceride profile in blood. Finally, cocoa increased SOD1 gene expression suggesting an antioxidant effect.

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Nuclear Pore Component Nup98 Is a Potential Tumor Suppressor and Regulates Posttranscriptional Expression of Select p53 Target Genes

Molecular Cell ◽

10.1016/j.molcel.2012.09.020 ◽

2012 ◽

Vol 48 (5) ◽

pp. 799-810 ◽

Cited By ~ 45

Author(s):

Stephan Singer ◽

Ruiying Zhao ◽

Anthony M. Barsotti ◽

Anette Ouwehand ◽

Mina Fazollahi ◽

...

Keyword(s):

Tumor Suppressor ◽

Target Genes ◽

Nuclear Pore ◽

P53 Target Genes ◽

Potential Tumor Suppressor

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Editorial: [Exciting Discoveries About New p53 Target Genes, Cancer Drugs and Diagnostic Tools, and Mechanisms of Various Human Diseases]

Current Molecular Medicine ◽

10.2174/156652412802480934 ◽

2012 ◽

Vol 12 (8) ◽

pp. 899-900

Author(s):

David Wan-Cheng

Keyword(s):

Target Genes ◽

Human Diseases ◽

Diagnostic Tools ◽

Cancer Drugs ◽

P53 Target Genes

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A Combined Genetic-Proteomic Approach Identifies Residues within Dengue Virus NS4B Critical for Interaction with NS3 and Viral Replication

Journal of Virology ◽

10.1128/jvi.00867-15 ◽

2015 ◽

Vol 89 (14) ◽

pp. 7170-7186 ◽

Cited By ~ 26

Author(s):

Laurent Chatel-Chaix ◽

Wolfgang Fischl ◽

Pietro Scaturro ◽

Mirko Cortese ◽

Stephanie Kallis ◽

...

Keyword(s):

Amino Acid ◽

Dengue Virus ◽

Virus Replication ◽

Strong Evidence ◽

Drug Target ◽

Nonstructural Protein ◽

Rna Replication ◽

Replication Cycle ◽

Infected Cells ◽

Ns3 Protease

ABSTRACTDengue virus (DENV) infection causes the most prevalent arthropod-borne viral disease worldwide. Approved vaccines are not available, and targets suitable for the development of antiviral drugs are lacking. One possible drug target is nonstructural protein 4B (NS4B), because it is absolutely required for virus replication; however, its exact role in the DENV replication cycle is largely unknown. With the aim of mapping NS4B determinants critical for DENV replication, we performed a reverse genetic screening of 33 NS4B mutants in the context of an infectious DENV genome. While the majority of these mutations were lethal, for several of them, we were able to select for second-site pseudoreversions, most often residing in NS4B and restoring replication competence. To identify all viral NS4B interaction partners, we engineered a fully viable DENV genome encoding an affinity-tagged NS4B. Mass spectrometry-based analysis of the NS4B complex isolated from infected cells identified the NS3 protease/helicase as a major interaction partner of NS4B. By combining the genetic complementation map of NS4B with a replication-independent expression system, we identified the NS4B cytosolic loop—more precisely, amino acid residue Q134—as a critical determinant for NS4B-NS3 interaction. An alanine substitution at this site completely abrogated the interaction and DENV RNA replication, and both were restored by pseudoreversions A69S and A137V. This strict correlation between the degree of NS4B-NS3 interaction and DENV replication provides strong evidence that this viral protein complex plays a pivotal role during the DENV replication cycle, hence representing a promising target for novel antiviral strategies.IMPORTANCEWith no approved therapy or vaccine against dengue virus infection, the viral nonstructural protein 4B (NS4B) represents a possible drug target, because it is indispensable for virus replication. However, little is known about its precise structure and function. Here, we established the first comprehensive genetic interaction map of NS4B, identifying amino acid residues that are essential for virus replication, as well as second-site mutations compensating for their defects. Additionally, we determined the NS4B viral interactome in infected cells and identified the NS3 protease/helicase as a major interaction partner of NS4B. We mapped residues in the cytosolic loop of NS4B as critical determinants for interaction with NS3, as well as RNA replication. The strong correlation between NS3-NS4B interaction and RNA replication provides strong evidence that this complex plays a pivotal role in the viral replication cycle, hence representing a promising antiviral drug target.

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Abstract 1839: MicroRNA-499 Promotes the Differentiation of Cardiac Progenitor Cells into Myocytes

Circulation ◽

10.1161/circ.118.suppl_18.s_395-a ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Toru Hosoda ◽

Konrad Urbanek ◽

Adriana Bastos Carvalho ◽

Claudia Bearzi ◽

Silvana Bardelli ◽

...

Keyword(s):

Gap Junctions ◽

Progenitor Cells ◽

Target Genes ◽

Brdu Incorporation ◽

Cardiac Progenitor Cells ◽

Partial Recovery ◽

Rt Pcr ◽

Protein Levels ◽

Cell Proliferation And Differentiation ◽

Reporter Assays

Myocardial regeneration mediated by cardiac progenitor cells (CPCs) results in the partial recovery of the infarcted heart but the newly formed myocytes within the necrotic tissue have fetal-neonatal characteristics. In contrast, CPC activation in the remote viable myocardium results in the formation of mature myocytes, suggesting that CPC differentiation is conditioned by the surrounding cells. Thus, the hypothesis is raised that microRNAs (miRs) that are highly expressed in myocytes and are absent in CPCs, may translocate through gap junctions to adjacent CPCs promoting their differentiation. By employing miR array and Q-RT-PCR, miR-499 was found to be ~500-fold more expressed in myocytes than CPCs. Additionally, we demonstrated that miR-499 translocates from neighboring cells to CPCs through the formation of gap junctions. The translocated miR-499 was functional and repressed the expression of target genes. Among 200 putative targets of miR-499, we have elected to study Sox6 and Rod1. The validation of these putative miR-499-targets was obtained by reporter assays; cells transfected with miR-499 together with plasmids carrying luciferase and the 3′-UTR region of Sox6 or Rod1 show the expected decrease in luciferase activity. Transcripts of Sox6 and Rod1 were measured by Q-RT-PCR in myocytes and CPCs; Sox6 mRNA was 2-fold higher and Rod1 mRNA was 98% lower in myocytes than CPCs. However, the protein levels of Sox6 and Rod1 were significantly lower in myocytes than CPCs suggesting that miR-499 promotes degradation and/or inhibition of translation of these target genes. To document miR-499 function, CPCs were transfected with a miR-499-expression vector and cell proliferation and differentiation were evaluated 3 days later. BrdU incorporation decreased 60% and the cells displayed a marked upregulation of the myocyte-specific transcription factors Nkx2.5 and MEF2C. Similar results were obtained when Sox6 and Rod1 were selectively blocked with siRNA. In both cases, the number of Nkx2.5- and MEF2C-positive cells increased 2–3-fold. Thus, our data indicate that miR-499 translocates via gap junction from myocytes to CPCs where miR-499 is a crucial modulator of the differentiation of CPCs into cardiomyocytes through the repression of Sox6 and Rod1.

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CK1α ablation in keratinocytes induces p53-dependent, sunburn-protective skin hyperpigmentation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1702763114 ◽

2017 ◽

Vol 114 (38) ◽

pp. E8035-E8044 ◽

Cited By ~ 12

Author(s):

Chung-Hsing Chang ◽

Che-Jung Kuo ◽

Takamichi Ito ◽

Yu-Ya Su ◽

Si-Tse Jiang ◽

...

Keyword(s):

Target Genes ◽

Neutralizing Antibody ◽

Double Knockout ◽

Melanocyte Stimulating Hormone ◽

Kit Ligand ◽

Skin Physiology ◽

P53 Activation ◽

P53 Target Genes ◽

Single Knockout

Casein kinase 1α (CK1α), a component of the β-catenin destruction complex, is a critical regulator of Wnt signaling; its ablation induces both Wnt and p53 activation. To characterize the role of CK1α (encoded byCsnk1a1) in skin physiology, we crossed mice harboring floxedCsnk1a1with mice expressing K14–Cre–ERT2to generate mice in which tamoxifen induces the deletion ofCsnk1a1exclusively in keratinocytes [single-knockout (SKO) mice]. As expected, CK1α loss was accompanied by β-catenin and p53 stabilization, with the preferential induction of p53 target genes, but phenotypically most striking was hyperpigmentation of the skin, importantly without tumorigenesis, for at least 9 mo afterCsnk1a1ablation. The number of epidermal melanocytes and eumelanin levels were dramatically increased in SKO mice. To clarify the putative role of p53 in epidermal hyperpigmentation, we established K14–Cre–ERT2CK1α/p53 double-knockout (DKO) mice and found that coablation failed to induce epidermal hyperpigmentation, demonstrating that it was p53-dependent. Transcriptome analysis of the epidermis revealed p53-dependent up-regulation of Kit ligand (KitL). SKO mice treated with ACK2 (a Kit-neutralizing antibody) or imatinib (a Kit inhibitor) abrogated the CK1α ablation-induced hyperpigmentation, demonstrating that it requires the KitL/Kit pathway. Pro-opiomelanocortin (POMC), a precursor of α-melanocyte–stimulating hormone (α-MSH), was not activated in the CK1α ablation-induced hyperpigmentation, which is in contrast to the mechanism of p53-dependent UV tanning. Nevertheless, acute sunburn effects were successfully prevented in the hyperpigmented skin of SKO mice. CK1α inhibition induces skin-protective eumelanin but no carcinogenic pheomelanin and may therefore constitute an effective strategy for safely increasing eumelanin via UV-independent pathways, protecting against acute sunburn.

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Screening candidate microR-15a- IRAK2 regulatory pairs for predicting the response to Staphylococcus aureus-induced mastitis in dairy cows

Journal of Dairy Research ◽

10.1017/s0022029919000785 ◽

2019 ◽

Vol 86 (4) ◽

pp. 425-431 ◽

Cited By ~ 3

Author(s):

Zhi Chen ◽

Jingpeng Zhou ◽

Xiaolong Wang ◽

Yang Zhang ◽

Xubin Lu ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Differential Expression ◽

High Throughput Sequencing ◽

Target Genes ◽

Interleukin 1 ◽

Differentially Expressed ◽

Luciferase Reporter ◽

Expression Of Genes ◽

Chinese Holstein Cows ◽

Reporter Assays

AbstractWe established a mastitis model using exogenous infection of the mammary gland of Chinese Holstein cows with Staphylococcus aureus and extracted total RNA from S. aureus-infected and healthy mammary quarters. Differential expression of genes due to mastitis was evaluated using Affymetrix technology and results revealed a total of 1230 differentially expressed mRNAs. A subset of affected genes was verified via Q-PCR and pathway analysis. In addition, Solexa high-throughput sequencing technology was used to analyze profiles of miRNA in infected and healthy quarters. These analyses revealed a total of 52 differentially expressed miRNAs. A subset of those results was verified via Q-PCR. Bioinformatics techniques were used to predict and analyze the correlations among differentially expressed miRNA and mRNA. Results revealed a total of 329 pairs of negatively associated miRNA/mRNA, with 31 upregulated pairs of mRNA and 298 downregulated pairs of mRNA. Differential expression of miR-15a and interleukin-1 receptor-associated kinase-like 2 (IRAK2), were evaluated by western blot and luciferase reporter assays. We conclude that miR-15a and miR-15a target genes (IRAK2) constitute potential miRNA–mRNA regulatory pairs for use as biomarkers to predict a mastitis response.

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