Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex.

PubMed

Ganaie, Safder S; Zou, Wei; Xu, Peng; Deng, Xuefeng; Kleiboeker, Steve; Qiu, Jianming

2017-05-01

Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both in vivo and in vitro, and was actively recruited within the viral DNA replication centers. Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases.

Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex

PubMed Central

Ganaie, Safder S.; Zou, Wei; Xu, Peng; Deng, Xuefeng; Kleiboeker, Steve

2017-01-01

Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both in vivo and in vitro, and was actively recruited within the viral DNA replication centers. Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases. PMID:28459842

STAT3 inhibition as a therapeutic strategy for leukemia.

PubMed

Kanna, Rubashruti; Choudhary, Gaurav; Ramachandra, Nandini; Steidl, Ulrich; Verma, Amit; Shastri, Aditi

2017-11-22

Leukemia is characterized by selective overgrowth of malignant hematopoietic stem cells (HSC's) that interfere with HSC differentiation. Cytoreductive chemotherapy can kill rapidly dividing cancerous cells but cannot eradicate the malignant HSC pool leading to relapses. Leukemic stem cells have several dysregulated pathways and the Janus kinases (JAKs) and signal transducer and activator of transcription (STAT) pathway are prominent among them. STAT3 is an important transcription factor that regulates cell growth, proliferation, and inhibits apoptosis. High STAT3 expression in leukemia has been associated with an increased risk for relapse and decreased overall survival. Multiple strategies for interfering with STAT3 activity in leukemic cells include inhibition of STAT3 phosphorylation, interfering with STAT3 interactions, preventing nuclear transfer, inhibiting transcription and causing interference in STAT: DNA binding. A better understanding of key interactions and upstream mediators of STAT3 activity will help facilitate the development of effective cancer therapies and may result in durable remissions.

Cross-talk between KLF4 and STAT3 regulates axon regeneration

NASA Astrophysics Data System (ADS)

Qin, Song; Zou, Yuhua; Zhang, Chun-Li

2013-10-01

Cytokine-induced activation of signal transducer and activator of transcription 3 (STAT3) promotes the regrowth of damaged axons in the adult central nervous system (CNS). Here we show that KLF4 physically interacts with STAT3 upon cytokine-induced phosphorylation of tyrosine 705 (Y705) on STAT3. This interaction suppresses STAT3-dependent gene expression by blocking its DNA-binding activity. The deletion of KLF4 in vivo induces axon regeneration of adult retinal ganglion cells (RGCs) via Janus kinase (JAK)-STAT3 signalling. This regeneration can be greatly enhanced by exogenous cytokine treatment, or removal of an endogenous JAK-STAT3 pathway inhibitor called suppressor of cytokine signalling 3 (SOCS3). These findings reveal an unexpected cross-talk between KLF4 and activated STAT3 in the regulation of axon regeneration that might have therapeutic implications in promoting repair of injured adult CNS.

Role of STAT3 in Cancer Metastasis and Translational Advances

PubMed Central

Patil, Prachi; Gude, Rajiv P.

2013-01-01

Signal transducer and activator of transcription 3 (STAT3) is a latent cytoplasmic transcription factor, originally discovered as a transducer of signal from cell surface receptors to the nucleus. It is activated by tyrosine phosphorylation at position 705 leading to its dimerization, nuclear translocation, DNA binding, and activation of gene transcription. Under normal physiological conditions, STAT3 activation is tightly regulated. However, compelling evidence suggests that STAT3 is constitutively activated in many cancers and plays a pivotal role in tumor growth and metastasis. It regulates cellular proliferation, invasion, migration, and angiogenesis that are critical for cancer metastasis. In this paper, we first describe the mechanism of STAT3 regulation followed by how STAT3 is involved in cancer metastasis, then we summarize the various small molecule inhibitors that inhibit STAT3 signaling. PMID:24199193

Characterization of STAT5B phosphorylation correlating with expression of cytokine-inducible SH2-containing protein (CIS).

PubMed

Cooper, John C; Boustead, Jared N; Yu, Chao-Lan

2006-06-01

Cytokine-inducible SH2-containing protein (CIS) is the first identified member of genes encoding for the suppressor of cytokine signaling (SOCS). CIS is also a well-known target gene of signal transducer and activator of transcription 5 (STAT5) pathways, providing normal negative feedback control of signaling by cytokines and growth factors. Three other SOCS genes, SOCS1, SOCS2, and SOCS3, can be silenced by DNA hypermethylation in human cancers, suggesting a potential mechanism for constitutive STAT activation. However, it is not known whether CIS expression is similarly perturbed in tumor cells. We report here the absence of CIS expression in T lymphoma LSTRA that overexpresses the Lck protein tyrosine kinase and exhibits elevated STAT5 activity. Pervanadate-induced CIS expression and STAT5 binding to the CIS promoter in vivo over a short time course implies that mechanisms other than DNA hypermethylation may contribute to defective CIS expression in LSTRA cells. Comparison with cytokine-dependent BaF3 cells stimulated with interleukin-3 (IL-3) further reveals that CIS induction correlates with specific STAT5b post-translational modifications. It exhibits as the slowest migrating form through SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. This distinctly modified STAT5b is the predominant form that binds to the consensus STAT5 sites in the CIS promoter and accumulates in the nucleus. In vitro phosphatase assays and phosphoamino acid analysis suggest the involvement of phosphorylation on residues other than the highly conserved tyrosine and serine sites in this distinct STAT5b mobility shift. All together, our results provide a novel link between incomplete STAT5b phosphorylation and defective SOCS gene expression in cancer cells.

Arctigenin inhibits STAT3 and exhibits anticancer potential in human triple-negative breast cancer therapy

PubMed Central

Shen, Wanxiang; Zhang, Liang; Gu, Xinsheng; Guo, Yang; Tsai, Hsiang-i; Liu, Xuewen; Li, Jian; Zhang, Jingxuan; Li, Shan; Wu, Fuyun; Liu, Ying

2017-01-01

Triple-negative breast cancers (TNBCs) are the most aggressive and hard-to-treat breast tumors with poor prognosis, and exploration for novel therapeutic drugs is impending. Arctigenin (Atn), a bioactive lignan isolated from seeds of Arctium lappa L, has been reported to inhibit many cancer types; however, the effect of Atn on TNBC remains unclear. In this study, we demonstrated that Atn decreased proliferation, and induced apoptosis in TNBC cells. Furthermore, we explored the underlying mechanism of Atn inhibition on TNBC cells. Computational docking and affinity assay showed that Atn bound to the SH2 domain of STAT3. Atn inhibited STAT3 binding to genomic DNA by disrupting hydrogen bond linking between DNA and STAT3. In addition, Atn augmented Taxotere®-induced TNBC cell cytotoxicity. TNBC xenograft tests also confirmed the antitumor effect of Atn in vivo. These characteristics render Atn as a promising candidate drug for further development and for designing new effective STAT3 inhibitors. PMID:27861147

Arctigenin inhibits STAT3 and exhibits anticancer potential in human triple-negative breast cancer therapy.

PubMed

Feng, Tingting; Cao, Wei; Shen, Wanxiang; Zhang, Liang; Gu, Xinsheng; Guo, Yang; Tsai, Hsiang-I; Liu, Xuewen; Li, Jian; Zhang, Jingxuan; Li, Shan; Wu, Fuyun; Liu, Ying

2017-01-03

Triple-negative breast cancers (TNBCs) are the most aggressive and hard-to-treat breast tumors with poor prognosis, and exploration for novel therapeutic drugs is impending. Arctigenin (Atn), a bioactive lignan isolated from seeds of Arctium lappa L, has been reported to inhibit many cancer types; however, the effect of Atn on TNBC remains unclear. In this study, we demonstrated that Atn decreased proliferation, and induced apoptosis in TNBC cells. Furthermore, we explored the underlying mechanism of Atn inhibition on TNBC cells. Computational docking and affinity assay showed that Atn bound to the SH2 domain of STAT3. Atn inhibited STAT3 binding to genomic DNA by disrupting hydrogen bond linking between DNA and STAT3. In addition, Atn augmented Taxotere®-induced TNBC cell cytotoxicity. TNBC xenograft tests also confirmed the antitumor effect of Atn in vivo. These characteristics render Atn as a promising candidate drug for further development and for designing new effective STAT3 inhibitors.

The Inhibition of Stat5 by a Peptide Aptamer Ligand Specific for the DNA Binding Domain Prevents Target Gene Transactivation and the Growth of Breast and Prostate Tumor Cells

PubMed Central

Weber, Axel; Borghouts, Corina; Brendel, Christian; Moriggl, Richard; Delis, Natalia; Brill, Boris; Vafaizadeh, Vida; Groner, Bernd

2013-01-01

The signal transducer and activator of transcription Stat5 is transiently activated by growth factor and cytokine signals in normal cells, but its persistent activation has been observed in a wide range of human tumors. Aberrant Stat5 activity was initially observed in leukemias, but subsequently also found in carcinomas. We investigated the importance of Stat5 in human tumor cell lines. shRNA mediated downregulation of Stat5 revealed the dependence of prostate and breast cancer cells on the expression of this transcription factor. We extended these inhibition studies and derived a peptide aptamer (PA) ligand, which directly interacts with the DNA-binding domain of Stat5 in a yeast-two-hybrid screen. The Stat5 specific PA sequence is embedded in a thioredoxin (hTRX) scaffold protein. The resulting recombinant protein S5-DBD-PA was expressed in bacteria, purified and introduced into tumor cells by protein transduction. Alternatively, S5-DBD-PA was expressed in the tumor cells after infection with a S5-DBD-PA encoding gene transfer vector. Both strategies impaired the DNA-binding ability of Stat5, suppressed Stat5 dependent transactivation and caused its intracellular degradation. Our experiments describe a peptide based inhibitor of Stat5 protein activity which can serve as a lead for the development of a clinically useful compound for cancer treatment. PMID:24276378

Histone H1 and Chromosomal Protein HMGN2 Regulate Prolactin-induced STAT5 Transcription Factor Recruitment and Function in Breast Cancer Cells.

PubMed

Schauwecker, Suzanne M; Kim, J Julie; Licht, Jonathan D; Clevenger, Charles V

2017-02-10

The hormone prolactin (PRL) contributes to breast cancer pathogenesis through various signaling pathways, one of the most notable being the JAK2/signal transducer and activator of transcription 5 (STAT5) pathway. PRL-induced activation of the transcription factor STAT5 results in the up-regulation of numerous genes implicated in breast cancer pathogenesis. However, the molecular mechanisms that enable STAT5 to access the promoters of these genes are not well understood. Here, we show that PRL signaling induces chromatin decompaction at promoter DNA, corresponding with STAT5 binding. The chromatin-modifying protein high mobility group nucleosomal binding domain 2 (HMGN2) specifically promotes STAT5 accessibility at promoter DNA by facilitating the dissociation of the linker histone H1 in response to PRL. Knockdown of H1 rescues the decrease in PRL-induced transcription following HMGN2 knockdown, and it does so by allowing increased STAT5 recruitment. Moreover, H1 and STAT5 are shown to function antagonistically in regulating PRL-induced transcription as well as breast cancer cell biology. While reduced STAT5 activation results in decreased PRL-induced transcription and cell proliferation, knockdown of H1 rescues both of these effects. Taken together, we elucidate a novel mechanism whereby the linker histone H1 prevents STAT5 binding at promoter DNA, and the PRL-induced dissociation of H1 mediated by HMGN2 is necessary to allow full STAT5 recruitment and promote the biological effects of PRL signaling. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Histone H1 and Chromosomal Protein HMGN2 Regulate Prolactin-induced STAT5 Transcription Factor Recruitment and Function in Breast Cancer Cells*

PubMed Central

Schauwecker, Suzanne M.; Kim, J. Julie; Licht, Jonathan D.; Clevenger, Charles V.

2017-01-01

The hormone prolactin (PRL) contributes to breast cancer pathogenesis through various signaling pathways, one of the most notable being the JAK2/signal transducer and activator of transcription 5 (STAT5) pathway. PRL-induced activation of the transcription factor STAT5 results in the up-regulation of numerous genes implicated in breast cancer pathogenesis. However, the molecular mechanisms that enable STAT5 to access the promoters of these genes are not well understood. Here, we show that PRL signaling induces chromatin decompaction at promoter DNA, corresponding with STAT5 binding. The chromatin-modifying protein high mobility group nucleosomal binding domain 2 (HMGN2) specifically promotes STAT5 accessibility at promoter DNA by facilitating the dissociation of the linker histone H1 in response to PRL. Knockdown of H1 rescues the decrease in PRL-induced transcription following HMGN2 knockdown, and it does so by allowing increased STAT5 recruitment. Moreover, H1 and STAT5 are shown to function antagonistically in regulating PRL-induced transcription as well as breast cancer cell biology. While reduced STAT5 activation results in decreased PRL-induced transcription and cell proliferation, knockdown of H1 rescues both of these effects. Taken together, we elucidate a novel mechanism whereby the linker histone H1 prevents STAT5 binding at promoter DNA, and the PRL-induced dissociation of H1 mediated by HMGN2 is necessary to allow full STAT5 recruitment and promote the biological effects of PRL signaling. PMID:28035005

A conserved motif in the linker domain of STAT1 transcription factor is required for both recognition and release from high-affinity DNA-binding sites.

PubMed

Hüntelmann, Bettina; Staab, Julia; Herrmann-Lingen, Christoph; Meyer, Thomas

2014-01-01

Binding to specific palindromic sequences termed gamma-activated sites (GAS) is a hallmark of gene activation by members of the STAT (signal transducer and activator of transcription) family of cytokine-inducible transcription factors. However, the precise molecular mechanisms involved in the signal-dependent finding of target genes by STAT dimers have not yet been very well studied. In this study, we have characterized a sequence motif in the STAT1 linker domain which is highly conserved among the seven human STAT proteins and includes surface-exposed residues in close proximity to the bound DNA. Using site-directed mutagenesis, we have demonstrated that a lysine residue in position 567 of the full-length molecule is required for GAS recognition. The substitution of alanine for this residue completely abolished both binding to high-affinity GAS elements and transcriptional activation of endogenous target genes in cells stimulated with interferon-γ (IFNγ), while the time course of transient nuclear accumulation and tyrosine phosphorylation were virtually unchanged. In contrast, two glutamic acid residues (E559 and E563) on each monomer are important for the dissociation of dimeric STAT1 from DNA and, when mutated to alanine, result in elevated levels of tyrosine-phosphorylated STAT1 as well as prolonged IFNγ-stimulated nuclear accumulation. In conclusion, our data indicate that the kinetics of signal-dependent GAS binding is determined by an array of glutamic acid residues located at the interior surface of the STAT1 dimer. These negatively charged residues appear to align the long axis of the STAT1 dimer in a position perpendicular to the DNA, thereby facilitating the interaction between lysine 567 and the phosphodiester backbone of a bound GAS element, which is a prerequisite for transient gene induction.

Defects along the T(H)17 differentiation pathway underlie genetically distinct forms of the hyper IgE syndrome.

PubMed

Al Khatib, Shadi; Keles, Sevgi; Garcia-Lloret, Maria; Karakoc-Aydiner, Elif; Reisli, Ismail; Artac, Hasibe; Camcioglu, Yildiz; Cokugras, Haluk; Somer, Ayper; Kutukculer, Necil; Yilmaz, Mustafa; Ikinciogullari, Aydan; Yegin, Olcay; Yüksek, Mutlu; Genel, Ferah; Kucukosmanoglu, Ercan; Baki, Ali; Bahceciler, Nerin N; Rambhatla, Anupama; Nickerson, Derek W; McGhee, Sean; Barlan, Isil B; Chatila, Talal

2009-08-01

The hyper IgE syndrome (HIES) is characterized by abscesses, eczema, recurrent infections, skeletal and connective tissue abnormalities, elevated serum IgE, and diminished inflammatory responses. It exists as autosomal-dominant and autosomal-recessive forms that manifest common and distinguishing clinical features. A majority of those with autosomal-dominant HIES have heterozygous mutations in signal transducer and activator of transcription (STAT)-3 and impaired T(H)17 differentiation. To elucidate mechanisms underlying different forms of HIES. A cohort of 25 Turkish children diagnosed with HIES were examined for STAT3 mutations by DNA sequencing. Activation of STAT3 by IL-6 and IL-21 and STAT1 by IFN-alpha was assessed by intracellular staining with anti-phospho (p)STAT3 and -pSTAT1 antibodies. T(H)17 and T(H)1 cell differentiation was assessed by measuring the production of IL-17 and IFN-gamma, respectively. Six subjects had STAT3 mutations affecting the DNA binding, Src homology 2, and transactivation domains, including 3 novel ones. Mutation-positive but not mutation-negative subjects with HIES exhibited reduced phosphorylation of STAT3 in response to cytokine stimulation, whereas pSTAT1 activation was unaffected. Both patient groups exhibited impaired T(H)17 responses, but whereas STAT3 mutations abrogated early steps in T(H)17 differentiation, the defects in patients with HIES with normal STAT3 affected more distal steps. In this cohort of Turkish children with HIES, a majority had normal STAT3, implicating other targets in disease pathogenesis. Impaired T(H)17 responses were evident irrespective of the STAT3 mutation status, indicating that different genetic forms of HIES share a common functional outcome.

Granulin, a novel STAT3-interacting protein, enhances STAT3 transcriptional function and correlates with poorer prognosis in breast cancer

PubMed Central

Yeh, Jennifer E.; Kreimer, Simion; Walker, Sarah R.; Emori, Megan M.; Krystal, Hannah; Richardson, Andrea; Ivanov, Alexander R.; Frank, David A.

2015-01-01

Since the neoplastic phenotype of a cell is largely driven by aberrant gene expression patterns, increasing attention has been focused on transcription factors that regulate critical mediators of tumorigenesis such as signal transducer and activator of transcription 3 (STAT3). As proteins that interact with STAT3 may be key in addressing how STAT3 contributes to cancer pathogenesis, we took a proteomics approach to identify novel STAT3-interacting proteins. We performed mass spectrometry-based profiling of STAT3-containing complexes from breast cancer cells that have constitutively active STAT3 and are dependent on STAT3 function for survival. We identified granulin (GRN) as a novel STAT3-interacting protein that was necessary for both constitutive and maximal leukemia inhibitory factor (LIF)induced STAT3 transcriptional activity. GRN enhanced STAT3 DNA binding and also increased the time-integrated amount of LIF-induced STAT3 activation in breast cancer cells. Furthermore, silencing GRN neutralized STAT3-mediated tumorigenic phenotypes including viability, clonogenesis, and migratory capacity. In primary breast cancer samples, GRN mRNA levels were positively correlated with STAT3 gene expression signatures and with reduced patient survival. These studies identify GRN as a functionally important STAT3-interacting protein that may serve as an important prognostic biomarker and potential therapeutic target in breast cancer. PMID:26000098

Maternal gestational betaine supplementation-mediated suppression of hepatic cyclin D2 and presenilin1 gene in newborn piglets is associated with epigenetic regulation of the STAT3-dependent pathway.

PubMed

Cai, Demin; Yuan, Mengjie; Jia, Yimin; Liu, Haoyu; Hu, Yun; Zhao, Ruqian

2015-12-01

Betaine, which donates methyl groups through methionine metabolism for DNA and protein methylation, is critical for epigenetic gene regulation, especially during fetal development. Here we fed gestational sows with control or betaine supplemented diets (3 g/kg) throughout the pregnancy to explore the effects of maternal betaine on hepatic cell proliferation in neonatal piglets. Neonatal piglets born to betaine-supplemented sows demonstrated a reduction of cell number and DNA content in the liver, which was associated with significantly down-regulated hepatic expression of cell cycle regulatory genes, cyclin D2 (CCND2) and presenilin1 (PSEN1). Moreover, STAT3 binding to the promoter of CCND2 and PSEN1 was also lower in betaine-exposed piglets, accompanied by strong reduction of STAT3 mRNA and protein expression, along with its phosphorylation at Tyr705 and Ser727 residues. Also, prenatal betaine exposure significantly attenuated upstream kinases of STAT3 signaling pathway (phospho-ERK1/2, phospho-SRC and phospho-JAK2) in the livers of neonates. Furthermore, the repressed STAT3 expression in the liver of betaine-exposed piglets was associated with DNA hypermethylation and more enriched repression histone mark H3K27me3 on its promoter, together with significantly up-regulated expression of H3K27me3 and enhancer of zeste homolog 2 (EZH2) proteins, as well as miR-124a, which targets STAT3. Taken together, our results suggest that maternal dietary betaine supplementation during gestation inhibits hepatic cell proliferation in neonatal piglets, at least partly, through epigenetic regulation of hepatic CCND2 and PSEN1 genes via a STAT3-dependent pathway. These neonatal changes in cell cycle and proliferation regulation may lead to lower liver weight and hepatic DNA content at weaning. Copyright © 2015 Elsevier Inc. All rights reserved.

Novel STAT3 phosphorylation inhibitors exhibit potent growth suppressive activity in pancreatic and breast cancer cells

PubMed Central

Lin, Li; Hutzen, Brian; Zuo, Mingxin; Ball, Sarah; Deangelis, Stephanie; Foust, Elizabeth; Pandit, Bulbul; Ihnat, Michael A.; Shenoy, Satyendra S.; Kulp, Samuel; Li, Pui-Kai; Li, Chenglong; Fuchs, James; Lin, Jiayuh

2010-01-01

The constitutive activation of Signal Transducer and Activator of Transcription 3 (STAT3) is frequently detected in most types of human cancer where it plays important roles in survival, drug-resistance, angiogenesis, and other functions. Targeting constitutive STAT3 signaling is thus an attractive therapeutic approach for these cancers. We have recently developed novel small molecule STAT3 inhibitors known as FLLL31 and FLLL32, which are derived from curcumin (the primary bioactive compound of turmeric). These compounds are designed to bind selectively to Janus Kinase 2 (JAK2) and the STAT3 SH2 domain, which serves crucial roles in STAT3 dimerization and signal transduction. Here we show that FLLL31 and FLLL32 are effective inhibitors of STAT3 phosphorylation, DNA binding activity, and transactivation in vitro, leading to the impediment of multiple oncogenic processes and the induction of apoptosis in pancreatic and breast cancer cell lines. FLLL31 and FLLL32 also inhibit colony formation in soft agar, cell invasion, and exhibit synergy with the anti-cancer drug doxorubicin against breast cancer cells. In addition, we show that FLLL32 can inhibit the induction of STAT3 phosphorylation by Interferon-α (IFNα) and Interleukin-6 (IL-6) in breast cancer cells. We also demonstrate that administration of FLLL32 can inhibit tumor growth and vascularity in chicken embryo xenografts as well as substantially reduce tumor volumes in mouse xenografts. Our findings highlight the potential of these new compounds and their efficacy in targeting pancreatic and breast cancers that exhibit constitutive STAT3 signaling. PMID:20215512

The role of the JAK2-STAT3 pathway in pro-inflammatory responses of EMF-stimulated N9 microglial cells

PubMed Central

2010-01-01

Background In several neuropathological conditions, microglia can become overactivated and cause neurotoxicity by initiating neuronal damage in response to pro-inflammatory stimuli. Our previous studies have shown that exposure to electromagnetic fields (EMF) activates cultured microglia to produce tumor necrosis factor (TNF)-α and nitric oxide (NO) through signal transduction involving the activator of transcription STAT3. Here, we investigated the role of STAT3 signaling in EMF-induced microglial activation and pro-inflammatory responses in more detail than the previous study. Methods N9 microglial cells were treated with EMF exposure or a sham treatment, with or without pretreatment with an inhibitor (Pyridone 6, P6) of the Janus family of tyrosine kinases (JAK). The activation state of microglia was assessed via immunoreaction using the microglial marker CD11b. Levels of inducible nitric oxide synthase (iNOS), TNF-α and NO were measured using real-time reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and the nitrate reductase method. Activation of JAKs and STAT3 proteins was evaluated by western blotting for specific tyrosine phosphorylation. The ability of STAT3 to bind to DNA was detected with an electrophoresis mobility shift assay (EMSA). Results EMF was found to significantly induce phosphorylation of JAK2 and STAT3, and DNA-binding ability of STAT3 in N9 microglia. In addition, EMF dramatically increased the expression of CD11b, TNF-α and iNOS, and the production of NO. P6 strongly suppressed the phosphorylation of JAK2 and STAT3 and diminished STAT3 activity in EMF-stimulated microglia. Interestingly, expression of CD11b as well as gene expression and production of TNF-α and iNOS were suppressed by P6 at 12 h, but not at 3 h, after EMF exposure. Conclusions EMF exposure directly triggers initial activation of microglia and produces a significant pro-inflammatory response. Our findings confirm that the JAK2-STAT3 pathway may not mediate this initial microglial activation but does promote pro-inflammatory responses in EMF-stimulated microglial cells. Thus, the JAK2-STAT3 pathway might be a therapeutic target for reducing pro-inflammatory responses in EMF-activated microglia. PMID:20828402

A plausibly causal functional lupus-associated risk variant in the STAT1-STAT4 locus.

PubMed

Patel, Zubin; Lu, Xiaoming; Miller, Daniel; Forney, Carmy R; Lee, Joshua; Lynch, Arthur; Schroeder, Connor; Parks, Lois; Magnusen, Albert F; Chen, Xiaoting; Pujato, Mario; Maddox, Avery; Zoller, Erin E; Namjou, Bahram; Brunner, Hermine I; Henrickson, Michael; Huggins, Jennifer L; Williams, Adrienne H; Ziegler, Julie T; Comeau, Mary E; Marion, Miranda C; Glenn, Stuart B; Adler, Adam; Shen, Nan; Nath, Swapan K; Stevens, Anne M; Freedman, Barry I; Pons-Estel, Bernardo A; Tsao, Betty P; Jacob, Chaim O; Kamen, Diane L; Brown, Elizabeth E; Gilkeson, Gary S; Alarcón, Graciela S; Martin, Javier; Reveille, John D; Anaya, Juan-Manuel; James, Judith A; Sivils, Kathy L; Criswell, Lindsey A; Vilá, Luis M; Petri, Michelle; Scofield, R Hal; Kimberly, Robert P; Edberg, Jeffrey C; Ramsey-Goldman, Rosalind; Bang, So-Young; Lee, Hye-Soon; Bae, Sang-Cheol; Boackle, Susan A; Cunninghame Graham, Deborah; Vyse, Timothy J; Merrill, Joan T; Niewold, Timothy B; Ainsworth, Hannah C; Silverman, Earl D; Weisman, Michael H; Wallace, Daniel J; Raj, Prithvi; Guthridge, Joel M; Gaffney, Patrick M; Kelly, Jennifer A; Alarcón-Riquelme, Marta E; Langefeld, Carl D; Wakeland, Edward K; Kaufman, Kenneth M; Weirauch, Matthew T; Harley, John B; Kottyan, Leah C

2018-04-18

Systemic Lupus Erythematosus (SLE or lupus) (OMIM: 152700) is a chronic autoimmune disease with debilitating inflammation that affects multiple organ systems. The STAT1-STAT4 locus is one of the first and most highly-replicated genetic loci associated with lupus risk. We performed a fine-mapping study to identify plausible causal variants within the STAT1-STAT4 locus associated with increased lupus disease risk. Using complementary frequentist and Bayesian approaches in trans-ancestral Discovery and Replication cohorts, we found one variant whose association with lupus risk is supported across ancestries in both the Discovery and Replication cohorts: rs11889341. In B cell lines from patients with lupus and healthy controls, the lupus risk allele of rs11889341 was associated with increased STAT1 expression. We demonstrated that the transcription factor HMGA1, a member of the HMG transcription factor family with an AT-hook DNA-binding domain, has enriched binding to the risk allele compared to the non-risk allele of rs11889341. We identified a genotype-dependent repressive element in the DNA within the intron of STAT4 surrounding rs11889341. Consistent with expression quantitative trait locus (eQTL) analysis, the lupus risk allele of rs11889341 decreased the activity of this putative repressor. Altogether, we present a plausible molecular mechanism for increased lupus risk at the STAT1-STAT4 locus in which the risk allele of rs11889341, the most probable causal variant, leads to elevated STAT1 expression in B cells due to decreased repressor activity mediated by increased binding of HMGA1.

Comprehensive meta-analysis of Signal Transducers and Activators of Transcription (STAT) genomic binding patterns discerns cell-specific cis-regulatory modules

PubMed Central

2013-01-01

Background Cytokine-activated transcription factors from the STAT (Signal Transducers and Activators of Transcription) family control common and context-specific genetic programs. It is not clear to what extent cell-specific features determine the binding capacity of seven STAT members and to what degree they share genetic targets. Molecular insight into the biology of STATs was gained from a meta-analysis of 29 available ChIP-seq data sets covering genome-wide occupancy of STATs 1, 3, 4, 5A, 5B and 6 in several cell types. Results We determined that the genomic binding capacity of STATs is primarily defined by the cell type and to a lesser extent by individual family members. For example, the overlap of shared binding sites between STATs 3 and 5 in T cells is greater than that between STAT5 in T cells and non-T cells. Even for the top 1,000 highly enriched STAT binding sites, ~15% of STAT5 binding sites in mouse female liver are shared by other STATs in different cell types while in T cells ~90% of STAT5 binding sites are co-occupied by STAT3, STAT4 and STAT6. In addition, we identified 116 cis-regulatory modules (CRM), which are recognized by all STAT members across cell types defining a common JAK-STAT signature. Lastly, in liver STAT5 binding significantly coincides with binding of the cell-specific transcription factors HNF4A, FOXA1 and FOXA2 and is associated with cell-type specific gene transcription. Conclusions Our results suggest that genomic binding of STATs is primarily determined by the cell type and further specificity is achieved in part by juxtaposed binding of cell-specific transcription factors. PMID:23324445

In silico simulations of STAT1 and STAT3 inhibitors predict SH2 domain cross-binding specificity.

PubMed

Szelag, Malgorzata; Sikorski, Krzysztof; Czerwoniec, Anna; Szatkowska, Katarzyna; Wesoly, Joanna; Bluyssen, Hans A R

2013-11-15

Signal transducers and activators of transcription (STATs) comprise a family of transcription factors that are structurally related and which participate in signaling pathways activated by cytokines, growth factors and pathogens. Activation of STAT proteins is mediated by the highly conserved Src homology 2 (SH2) domain, which interacts with phosphotyrosine motifs for specific contacts between STATs and receptors and for STAT dimerization. By generating new models for human (h)STAT1, hSTAT2 and hSTAT3 we applied comparative in silico docking to determine SH2-binding specificity of the STAT3 inhibitor stattic, and of fludarabine (STAT1 inhibitor). Thus, we provide evidence that by primarily targeting the highly conserved phosphotyrosine (pY+0) SH2 binding pocket stattic is not a specific hSTAT3 inhibitor, but is equally effective towards hSTAT1 and hSTAT2. This was confirmed in Human Micro-vascular Endothelial Cells (HMECs) in vitro, in which stattic inhibited interferon-α-induced phosphorylation of all three STATs. Likewise, fludarabine inhibits both hSTAT1 and hSTAT3 phosphorylation, but not hSTAT2, by competing with the highly conserved pY+0 and pY-X binding sites, which are less well-preserved in hSTAT2. Moreover we observed that in HMECs in vitro fludarabine inhibits cytokine and lipopolysaccharide-induced phosphorylation of hSTAT1 and hSTAT3 but does not affect hSTAT2. Finally, multiple sequence alignment of STAT-SH2 domain sequences confirmed high conservation between hSTAT1 and hSTAT3, but not hSTAT2, with respect to stattic and fludarabine binding sites. Together our data offer a molecular basis that explains STAT cross-binding specificity of stattic and fludarabine, thereby questioning the present selection strategies of SH2 domain-based competitive small inhibitors. © 2013 Elsevier B.V. All rights reserved.

Excess thyroid hormone inhibits embryonic neural stem/progenitor cells proliferation and maintenance through STAT3 signalling pathway.

PubMed

Chen, Chunhai; Zhou, Zhou; Zhong, Min; Li, Maoquan; Yang, Xuesen; Zhang, Yanwen; Wang, Yuan; Wei, Aimin; Qu, Mingyue; Zhang, Lei; Xu, Shangcheng; Chen, Shude; Yu, Zhengping

2011-07-01

Hyperthyroidism is prevalent during pregnancy, but little is known about the effects of excess thyroid hormone on the development of embryonic neural stem/progenitor cells (NSCs), and the mechanisms underlying these effects. Previous studies indicate that STAT3 plays a crucial role in determining NSC fate during neurodevelopment. In this study, we investigated the effects of a supraphysiological dose of 3,5,3'-L-triiodothyronine (T3) on the proliferation and maintenance of NSCs derived from embryonic day 13.5 mouse neocortex, and the involvement of STAT3 in this process. Our results suggest that excess T3 treatment inhibits NSC proliferation and maintenance. T3 decreased tyrosine phosphorylation of JAK1, JAK2 and STAT3, and subsequently inhibited STAT3-DNA binding activity. Furthermore, proliferation and maintenance of NSCs were decreased by inhibitors of JAKs and STAT3, indicating that the STAT3 signalling pathway is involved in the process of NSC proliferation and maintenance. Taken together, these results suggest that the STAT3 signalling pathway is involved in the process of T3-induced inhibition of embryonic NSC proliferation and maintenance. These findings provide data for understanding the effects of hyperthyroidism during pregnancy on fetal brain development, and the mechanisms underlying these effects.

Novel Multiplexed Assay for Identifying SH2 Domain Antagonists of STAT Family Proteins

PubMed Central

Takakuma, Kazuyuki; Ogo, Naohisa; Uehara, Yutaka; Takahashi, Susumu; Miyoshi, Nao; Asai, Akira

2013-01-01

Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z’ values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors. PMID:23977103

Novel multiplexed assay for identifying SH2 domain antagonists of STAT family proteins.

PubMed

Takakuma, Kazuyuki; Ogo, Naohisa; Uehara, Yutaka; Takahashi, Susumu; Miyoshi, Nao; Asai, Akira

2013-01-01

Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z' values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors.

Mitochondrial translocation of signal transducer and activator of transcription 5 (STAT5) in leukemic T cells and cytokine-stimulated cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chueh, Fu-Yu; Leong, King-Fu; Yu, Chao-Lan, E-mail: chaolan.yu@rosalindfranklin.edu

2010-11-26

Research highlights: {yields} STAT5 interacts with a mitochondrial protein PDC-E2 in a leukemic T cell line LSTRA. {yields} Tyrosine-phosphorylated STAT5, but not STAT3, is present in LSTRA mitochondria. {yields} Cytokines induce mitochondrial translocation of STAT5, but not STAT1 or STAT3. {yields} Cytokine-induced mitochondrial translocation of tyrosine-phosphorylated STAT5 is transient. {yields} Mitochondrial STAT5 binds to a putative STAT5 site in the mitochondrial DNA in vitro. -- Abstract: Signal transducers and activators of transcription (STATs) were first identified as key signaling molecules in response to cytokines. Constitutive STAT activation also has been widely implicated in oncogenesis. We analyzed STAT5-associated proteins in amore » leukemic T cell line LSTRA, which exhibits constitutive tyrosine phosphorylation and activation of STAT5. A cellular protein was found to specifically interact with STAT5 in LSTRA cells by co-immunoprecipitation. Sequencing analysis and subsequent immunoblotting confirmed the identity of this STAT5-associated protein as the E2 component of mitochondrial pyruvate dehydrogenase complex (PDC-E2). Consistent with this interaction, both subcellular fractionation and immunofluorescence microscopy revealed mitochondrial localization of STAT5 in LSTRA cells. Mitochondrial localization of tyrosine-phosphorylated STAT5 also occurred in cytokine-stimulated cells. A time course experiment further demonstrated the transient kinetics of STAT5 mitochondrial translocation after cytokine stimulation. In contrast, cytokine-induced STAT1 and STAT3 activation did not result in their translocation into mitochondria. Furthermore, we showed that mitochondrial STAT5 bound to the D-loop regulatory region of mitochondrial DNA in vitro. It suggests a potential role of STAT5 in regulating the mitochondrial genome. Proliferative metabolism toward aerobic glycolysis is well known in cancer cells as the Warburg effect and is also observed in cytokine-stimulated cells. Our novel findings of cytokine-induced STAT5 translocation into mitochondria and its link to oncogenesis provide important insights into the underlying mechanisms of this characteristic metabolic shift.« less

Design of Conditionally Active STATs: Insights into STAT Activation and Gene Regulatory Function

PubMed Central

Milocco, Lawrence H.; Haslam, Jennifer A.; Rosen, Jonathan; Seidel, H. Martin

1999-01-01

The STAT (signal transducer and activator of transcription) signaling pathway is activated by a large number of cytokines and growth factors. We sought to design a conditionally active STAT that could not only provide insight into basic questions about STAT function but also serve as a powerful tool to determine the precise biological role of STATs. To this end, we have developed a conditionally active STAT by fusing STATs with the ligand-binding domain of the estrogen receptor (ER). We have demonstrated that the resulting STAT-ER chimeras are estrogen-inducible transcription factors that retain the functional and biochemical characteristics of the cognate wild-type STATs. In addition, these tools have allowed us to evaluate separately the contribution of tyrosine phosphorylation and dimerization to STAT function. We have for the first time provided experimental data supporting the model that the only apparent role of STAT tyrosine phosphorylation is to drive dimerization, as dimerization alone is sufficient to unmask a latent STAT nuclear localization sequence and induce nuclear translocation, sequence-specific DNA binding, and transcriptional activity. PMID:10082558

Lupus Risk Variant Increases pSTAT1 Binding and Decreases ETS1 Expression

PubMed Central

Lu, Xiaoming; Zoller, Erin E.; Weirauch, Matthew T.; Wu, Zhiguo; Namjou, Bahram; Williams, Adrienne H.; Ziegler, Julie T.; Comeau, Mary E.; Marion, Miranda C.; Glenn, Stuart B.; Adler, Adam; Shen, Nan; Nath, Swapan K.; Stevens, Anne M.; Freedman, Barry I.; Tsao, Betty P.; Jacob, Chaim O.; Kamen, Diane L.; Brown, Elizabeth E.; Gilkeson, Gary S.; Alarcón, Graciela S.; Reveille, John D.; Anaya, Juan-Manuel; James, Judith A.; Sivils, Kathy L.; Criswell, Lindsey A.; Vilá, Luis M.; Alarcón-Riquelme, Marta E.; Petri, Michelle; Scofield, R. Hal; Kimberly, Robert P.; Ramsey-Goldman, Rosalind; Joo, Young Bin; Choi, Jeongim; Bae, Sang-Cheol; Boackle, Susan A.; Graham, Deborah Cunninghame; Vyse, Timothy J.; Guthridge, Joel M.; Gaffney, Patrick M.; Langefeld, Carl D.; Kelly, Jennifer A.; Greis, Kenneth D.; Kaufman, Kenneth M.; Harley, John B.; Kottyan, Leah C.

2015-01-01

Genetic variants at chromosomal region 11q23.3, near the gene ETS1, have been associated with systemic lupus erythematosus (SLE), or lupus, in independent cohorts of Asian ancestry. Several recent studies have implicated ETS1 as a critical driver of immune cell function and differentiation, and mice deficient in ETS1 develop an SLE-like autoimmunity. We performed a fine-mapping study of 14,551 subjects from multi-ancestral cohorts by starting with genotyped variants and imputing to all common variants spanning ETS1. By constructing genetic models via frequentist and Bayesian association methods, we identified 16 variants that are statistically likely to be causal. We functionally assessed each of these variants on the basis of their likelihood of affecting transcription factor binding, miRNA binding, or chromatin state. Of the four variants that we experimentally examined, only rs6590330 differentially binds lysate from B cells. Using mass spectrometry, we found more binding of the transcription factor signal transducer and activator of transcription 1 (STAT1) to DNA near the risk allele of rs6590330 than near the non-risk allele. Immunoblot analysis and chromatin immunoprecipitation of pSTAT1 in B cells heterozygous for rs6590330 confirmed that the risk allele increased binding to the active form of STAT1. Analysis with expression quantitative trait loci indicated that the risk allele of rs6590330 is associated with decreased ETS1 expression in Han Chinese, but not other ancestral cohorts. We propose a model in which the risk allele of rs6590330 is associated with decreased ETS1 expression and increases SLE risk by enhancing the binding of pSTAT1. PMID:25865496

Inhibition of the EGFR/STAT3/CEBPD Axis Reverses Cisplatin Cross-resistance with Paclitaxel in the Urothelial Carcinoma of the Urinary Bladder.

PubMed

Wang, Wei-Jan; Li, Chien-Feng; Chu, Yu-Yi; Wang, Yu-Hui; Hour, Tzyh-Chyuan; Yen, Chia-Jui; Chang, Wen-Chang; Wang, Ju-Ming

2017-01-15

Cisplatin (CDDP) is frequently used in combination chemotherapy with paclitaxel for treating urothelial carcinoma of the urinary bladder (UCUB). CDDP cross-resistance has been suggested to develop with paclitaxel, thus hindering successful UCUB treatment. Therefore, elucidating the mechanisms underlying CDDP-induced anticancer drug resistance is imperative and may provide an insight in developing novel therapeutic strategy. Loss-of-function assays were performed to elucidate the role of the EGFR and STAT3 in CDDP-induced CCAAT/enhancer-binding protein delta (CEBPD) expression in UCUB cells. Reporter and in vivo DNA-binding assays were employed to determine whether CEBPD directly regulates ATP binding cassette subfamily B member 1 (ABCB1) and ATP binding cassette subfamily C member 2 (ABCC2) activation. Finally, a xenograft animal assay was used to examine the abilities of gefitinib and S3I-201 (a STAT3 inhibitor) to reverse CDDP and paclitaxel sensitivity. CEBPD expression was maintained in postoperative chemotherapy patients, and this expression was induced by CDDP even in CDDP-resistant UCUB cells. Upon CDDP treatment, CEBPD activated ABCB1 and ABCC2. Furthermore, the EGFR/STAT3 pathway contributed to CDDP-induced CEBPD expression in UCUB cells. Gefitinib and S3I-201 treatment significantly reduced the expression of CEBPD and enhanced the sensitivity of CDDP-resistant UCUB cells to CDDP and paclitaxel. Our results revealed the risk of CEBPD activation in CDDP-resistant UCUB cells and suggested a therapeutic strategy for patients with UCUB or UCUB resisted to CDDP and paclitaxel by combination with either gefitinib or S3I-201. Clin Cancer Res; 23(2); 503-13. ©2016 AACR. ©2016 American Association for Cancer Research.

Arsenic enhances the apoptosis induced by interferon gamma: key role of IRF-1.

PubMed

El Bougrini, J; Pampin, M; Chelbi-Alix, M K

2006-05-15

Interferons (IFNs) and arsenic trioxide (As2O3) are known inhibitors of cell proliferation and have been used in the treatment of certain forms of malignancy. IFNgamma treatment of cells leads to tyrosine phosphorylation of STAT1 followed by dimerization that accumulates in the nucleus. This is followed by DNA binding, activation of target gene transcription, dephosphorylation, and return to the cytoplasm. We have shown earlier that IFNgamma and As2O3 act synergistically in acute promyelocytic leukemia cells to upregulate IRF-1 expression and to induce apoptosis. Here, we show that in the human fibrosarcoma cell line 2fTGH, As2O3 prolongs IFNgamma-induced STAT1 phosphorylation resulting in persistent binding of STAT1 to GAS motif leading to an increase in IRF-1 expression which correlated with both higher anti-proliferative effect and increased apoptosis. These biological responses induced by IFNgamma alone or in combination with As2O3 were abolished when IRF-1 expression was down-regulated by RNA interference, thus demonstrating the key role of IRF-1.

Epigenetic silencing of the NR4A3 tumor suppressor, by aberrant JAK/STAT signaling, predicts prognosis in gastric cancer

NASA Astrophysics Data System (ADS)

Yeh, Chung-Min; Chang, Liang-Yu; Lin, Shu-Hui; Chou, Jian-Liang; Hsieh, Hsiao-Yen; Zeng, Li-Han; Chuang, Sheng-Yu; Wang, Hsiao-Wen; Dittner, Claudia; Lin, Cheng-Yu; Lin, Jora M. J.; Huang, Yao-Ting; Ng, Enders K. W.; Cheng, Alfred S. L.; Wu, Shu-Fen; Lin, Jiayuh; Yeh, Kun-Tu; Chan, Michael W. Y.

2016-08-01

While aberrant JAK/STAT signaling is crucial to the development of gastric cancer (GC), its effects on epigenetic alterations of its transcriptional targets remains unclear. In this study, by expression microarrays coupled with bioinformatic analyses, we identified a putative STAT3 target gene, NR4A3 that was downregulated in MKN28 GC daughter cells overexpressing a constitutively activated STAT3 mutant (S16), as compared to an empty vector control (C9). Bisulphite pyrosequencing and demethylation treatment showed that NR4A3 was epigenetically silenced by promoter DNA methylation in S16 and other GC cell lines including AGS cells, showing constitutive activation of STAT3. Subsequent experiments revealed that NR4A3 promoter binding by STAT3 might repress its transcription. Long-term depletion of STAT3 derepressed NR4A3 expression, by promoter demethylation, in AGS GC cells. NR4A3 re-expression in GC cell lines sensitized the cells to cisplatin, and inhibited tumor growth in vitro and in vivo, in an animal model. Clinically, GC patients with high NR4A3 methylation, or lower NR4A3 protein expression, had significantly shorter overall survival. Intriguingly, STAT3 activation significantly associated only with NR4A3 methylation in low-stage patient samples. Taken together, aberrant JAK/STAT3 signaling epigenetically silences a potential tumor suppressor, NR4A3, in gastric cancer, plausibly representing a reliable biomarker for gastric cancer prognosis.

IFNγ Induces DNA Methylation-Silenced GPR109A Expression via pSTAT1/p300 and H3K18 Acetylation in Colon Cancer.

PubMed

Bardhan, Kankana; Paschall, Amy V; Yang, Dafeng; Chen, May R; Simon, Priscilla S; Bhutia, Yangzom D; Martin, Pamela M; Thangaraju, Muthusamy; Browning, Darren D; Ganapathy, Vadivel; Heaton, Christopher M; Gu, Keni; Lee, Jeffrey R; Liu, Kebin

2015-07-01

Short-chain fatty acids, metabolites produced by colonic microbiota from fermentation of dietary fiber, act as anti-inflammatory agents in the intestinal tract to suppress proinflammatory diseases. GPR109A is the receptor for short-chain fatty acids. The functions of GPR109A have been the subject of extensive studies; however, the molecular mechanisms underlying GPR109A expression is largely unknown. We show that GPR109A is highly expressed in normal human colon tissues, but is silenced in human colon carcinoma cells. The GPR109A promoter DNA is methylated in human colon carcinoma. Strikingly, we observed that IFNγ, a cytokine secreted by activated T cells, activates GPR109A transcription without altering its promoter DNA methylation. Colon carcinoma grows significantly faster in IFNγ-deficient mice than in wild-type mice in an orthotopic colon cancer mouse model. A positive correlation was observed between GPR109A protein level and tumor-infiltrating T cells in human colon carcinoma specimens, and IFNγ expression level is higher in human colon carcinoma tissues than in normal colon tissues. We further demonstrated that IFNγ rapidly activates pSTAT1 that binds to the promoter of p300 to activate its transcription. p300 then binds to the GPR109A promoter to induce H3K18 hyperacetylation, resulting in chromatin remodeling in the methylated GPR109A promoter. The IFNγ-activated pSTAT1 then directly binds to the methylated but hyperacetylated GPR109 promoter to activate its transcription. Overall, our data indicate that GPR109A acts as a tumor suppressor in colon cancer, and the host immune system might use IFNγ to counteract DNA methylation-mediated GPR109A silencing as a mechanism to suppress tumor development. ©2015 American Association for Cancer Research.

IFNγ induces DNA methylation-silenced GPR109A expression via pSTAT1/p300 and H3K18 acetylation in colon cancer

PubMed Central

Bardhan, Kankana; Paschall, Amy V.; Yang, Dafeng; Chen, May R.; Simon, Priscilla S.; Bhutia, Yangzom; Martin, Pamela M.; Thangaraju, Muthusamy; Browning, Darren D.; Ganapathy, Vadivel; Heaton, Christopher M.; Gu, Keni; Lee, Jeffrey R.; Liu, Kebin

2015-01-01

Short-chain fatty acids, metabolites produced by colonic microbiota from fermentation of dietary fiber, act as anti-inflammatory agents in the intestinal tract to suppress proinflammatory diseases. GPR109A is the receptor for short-chain fatty acids. The functions of GPR109A has been the subject of extensive studies, however, the molecular mechanisms underlying GPR109A expression is largely unknown. We show that GPR109A is highly expressed in normal human colon tissues, but is silenced in human colon carcinoma cells. The GPR109A promoter DNA is methylated in human colon carcinoma. Strikingly, we observed that IFNγ, a cytokine secreted by activated T cells, activates GPR109A transcription without altering its promoter DNA methylation. Colon carcinoma grows significantly faster in IFNγ-deficient mice than in wildtype mice in an orthotopic colon cancer mouse model. A positive correlation was observed between GPR109A protein level and tumor-infiltrating T cells in human colon carcinoma specimens, and IFNγ expression level is higher in human colon carcinoma tissues than in normal colon tissues. We further demonstrated that IFNγ rapidly activates pSTAT1 that binds to the promoter of p300 to activate its transcription. p300 then binds to the GPR109A promoters to induce H3K18 hyperacetylation, resulting in chromatin remodeling in the methylated GPR109A promoter. The IFNγ-activated pSTAT1 then directly binds to the methylated but hyperacetylated GPR109 promoters to activate its transcription. Overall, our data indicate that GPR109A acts as a tumor suppressor in colon cancer and the host immune system might use IFNγ to counteract DNA methylation-mediated GPR109A silencing as a mechanism to suppress tumor development. PMID:25735954

Sequential activation of JAKs, STATs and xanthine dehydrogenase/oxidase by hypoxia in lung microvascular endothelial cells.

PubMed

Wang, Guansong; Qian, Pin; Jackson, Fannie R; Qian, Guisheng; Wu, Guangyu

2008-01-01

Xanthine dehydrogenase/oxidase (XDH/XO) is associated with various pathological conditions related to the endothelial injury. However, the molecular mechanism underlying the activation of XDH/XO by hypoxia remains largely unknown. In this report, we determined whether the Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) signaling pathway is involved in hypoxia-induced activation of XDH/XO in primary cultures of lung microvascular endothelial cells (LMVEC). We found that hypoxia significantly increased interleukin 6 (IL6) production in a time-dependent manner in LMVEC. Hypoxia also markedly augmented phosphorylation/activation of JAKs (JAK1, JAK2 and JAK3) and the JAK downstream effectors STATs (STAT3 and STAT5). Hypoxia-induced activation of STAT3 was blocked by IL6 antibodies, the JAK inhibitor AG490 and the suppressor of cytokine signaling 3 (SOCS3), implying that hypoxia-promoted IL6 secretion activates the JAK/STAT pathway in LMVEC. Phosphorylation and DNA-binding activity of STAT3 were also inhibited by the p38 MAPK inhibitor SB203580 and the phosphatidylinositol 3-kinase inhibitor LY294002, suggesting that multiple signaling pathways involved in STAT activation by hypoxia. Importantly, hypoxia promoted XDH/XO activation in LMVEC, which was markedly reversed by inhibiting the JAK-STAT pathway using IL6 antibodies, AG490 and SOCS3. These data demonstrated that JAKs, STATs and XDH/XO were sequentially activated by hypoxia. These data provide the first evidence indicating that the JAK-STAT pathway is involved in hypoxia-mediated XDH/XO activation in LMVEC.

STATs get their move on.

PubMed

Reich, Nancy C

2013-10-01

Understanding the mechanisms that regulate dynamic localization of a protein within a cell can provide critical insight to its functional molecular interactions. Signal transducers and activators of transcription (STATs) play essential roles in development, proliferation, and immune defense. However the consequences of STAT hyperactivity can predispose to diseases including autoimmunity and cancer. To function as transcription factors STATs must gain access to the nucleus, and knowledge of the mechanisms that regulate STAT nuclear trafficking can provide a means to control STAT action. This review presents a synopsis of some of the studies that address the nuclear dynamics of the STAT proteins. Evidence suggests that not all STATs are the same. Nuclear import of STAT1 and STAT4 appears linked to their tyrosine phosphorylation and the formation of parallel dimers via reciprocal phosphotyrosine and Src homology 2 domain interactions. This dimer arrangement generates a conformational nuclear localization signal. STAT2 is imported continually to the nucleus in an unphosphorylated state due to its association with IRF9, but the dominant nuclear export signal of STAT2 shuttles the complex back to the cytoplasm. Following STAT2 tyrosine phosphorylation, it can form dimers with STAT1 to affect nuclear import as the trimeric complex (ISGF3). Distinctly, STAT3, STAT5, and STAT6 are continually imported to the nucleus independent of tyrosine phosphorylation. Mutational studies indicate the nuclear localization signals in these STATs require the conformational structure of their coiled-coil domains. Increases in STAT nuclear accumulation following cytokine stimulation appear coordinate with their ability to bind DNA.

Inhibition on JAK-STAT3 Signaling Transduction Cascade Is Taken by Bioactive Peptide Alpha-S2 Casein Protein from Goat Ethawah Breed Milk

PubMed Central

Rohmah, Rista Nikmatu; Hardiyanti, Ferlany; Fatchiyah, Fatchiyah

2015-01-01

Background: RA is a systemic inflammatory disease that causes developing comorbidity conditions. This condition can cause by overproduction of pro-inflammatory cytokine. In a previous study, we have found bioactive peptide CSN1S2 from Ethawah goat milk for anti-inflammatory for repair the ileum destruction. However, the signaling transduction cascade of bioactive peptides inhibits inflammation still not clear yet. Therefore, we analyzed the signaling transduction cascade via JAK-STAT3 pathway by in vivo and in silico. Methods: The ileum was isolated DNA and amplification with specific primer. The sequence was analyzed using the Sanger sequencing method. Modeling 3D-structure was predicted by SWISS-MODEL and virtual interaction was analyzed by docking system using Pymol and Discovery Studio 4.0 software. Results: This study showed that STAT3 has target gene 480bp. The normal group and normal treating- CSN1S2 of goat milk have similarity from gene bank. Whereas, RA group had transversion mutation that the purine change into pyrimidine even cause frameshift mutation. Interestingly, after treating with the CSN1S2 protein of goat milk shows reverse to the normal acid sequence group. Based on in silico study, from eight peptides, only three peptides of CSN1S2 protein, which carried by PePT1 to enter the small intestine. The fragments are PepT1-41-NMAIHPR-47; PepT1-182-KISQYYQK-189 and PepT1-214-TNAIPYVR-221. We have found just one bioactive peptide of f182-KISQYYQK-189 is able bind to STAT3. The energy binding of f182-KISQYYQK-189 and RA-STAT3 amino acid, it was Σ = -402.43 kJ/mol and the energy binding of f182-KISQYYQK-189 and RAS-STAT3 amino acid is decreasing into Σ = -407.09 kJ/mol. Conclusion: This study suggested that the fragment 182-KISQYYQK-189 peptides from Ethawah goat milk may act as an anti-inflammatory agent via JAK-STAT3 signal transduction cascade at the cellular level. PMID:26483598

An SH2 domain model of STAT5 in complex with phospho-peptides define ``STAT5 Binding Signatures''

NASA Astrophysics Data System (ADS)

Gianti, Eleonora; Zauhar, Randy J.

2015-05-01

The signal transducer and activator of transcription 5 (STAT5) is a member of the STAT family of proteins, implicated in cell growth and differentiation. STAT activation is regulated by phosphorylation of protein monomers at conserved tyrosine residues, followed by binding to phospho-peptide pockets and subsequent dimerization. STAT5 is implicated in the development of severe pathological conditions, including many cancer forms. However, nowadays a few STAT5 inhibitors are known, and only one crystal structure of the inactive STAT5 dimer is publicly available. With a view to enabling structure-based drug design, we have: (1) analyzed phospho-peptide binding pockets on SH2 domains of STAT5, STAT1 and STAT3; (2) generated a model of STAT5 bound to phospho-peptides; (3) assessed our model by docking against a class of known STAT5 inhibitors (Müller et al. in ChemBioChem 9:723-727, 2008); (4) used molecular dynamics simulations to optimize the molecular determinants responsible for binding and (5) proposed unique "Binding Signatures" of STAT5. Our results put in place the foundations to address STAT5 as a target for rational drug design, from sequence, structural and functional perspectives.

Mapping of transcription factor binding regions in mammalian cells by ChIP: Comparison of array- and sequencing-based technologies

PubMed Central

Euskirchen, Ghia M.; Rozowsky, Joel S.; Wei, Chia-Lin; Lee, Wah Heng; Zhang, Zhengdong D.; Hartman, Stephen; Emanuelsson, Olof; Stolc, Viktor; Weissman, Sherman; Gerstein, Mark B.; Ruan, Yijun; Snyder, Michael

2007-01-01

Recent progress in mapping transcription factor (TF) binding regions can largely be credited to chromatin immunoprecipitation (ChIP) technologies. We compared strategies for mapping TF binding regions in mammalian cells using two different ChIP schemes: ChIP with DNA microarray analysis (ChIP-chip) and ChIP with DNA sequencing (ChIP-PET). We first investigated parameters central to obtaining robust ChIP-chip data sets by analyzing STAT1 targets in the ENCODE regions of the human genome, and then compared ChIP-chip to ChIP-PET. We devised methods for scoring and comparing results among various tiling arrays and examined parameters such as DNA microarray format, oligonucleotide length, hybridization conditions, and the use of competitor Cot-1 DNA. The best performance was achieved with high-density oligonucleotide arrays, oligonucleotides ≥50 bases (b), the presence of competitor Cot-1 DNA and hybridizations conducted in microfluidics stations. When target identification was evaluated as a function of array number, 80%–86% of targets were identified with three or more arrays. Comparison of ChIP-chip with ChIP-PET revealed strong agreement for the highest ranked targets with less overlap for the low ranked targets. With advantages and disadvantages unique to each approach, we found that ChIP-chip and ChIP-PET are frequently complementary in their relative abilities to detect STAT1 targets for the lower ranked targets; each method detected validated targets that were missed by the other method. The most comprehensive list of STAT1 binding regions is obtained by merging results from ChIP-chip and ChIP-sequencing. Overall, this study provides information for robust identification, scoring, and validation of TF targets using ChIP-based technologies. PMID:17568005

Novel signal transducer and activator of transcription 3 (STAT3) mutations, reduced T(H)17 cell numbers, and variably defective STAT3 phosphorylation in hyper-IgE syndrome.

PubMed

Renner, Ellen D; Rylaarsdam, Stacey; Anover-Sombke, Stephanie; Rack, Anita L; Reichenbach, Janine; Carey, John C; Zhu, Qili; Jansson, Annette F; Barboza, Julia; Schimke, Lena F; Leppert, Mark F; Getz, Melissa M; Seger, Reinhard A; Hill, Harry R; Belohradsky, Bernd H; Torgerson, Troy R; Ochs, Hans D

2008-07-01

Hyper-IgE syndrome (HIES) is a rare, autosomal-dominant immunodeficiency characterized by eczema, Staphylococcus aureus skin abscesses, pneumonia with pneumatocele formation, Candida infections, and skeletal/connective tissue abnormalities. Recently it was shown that heterozygous signal transducer and activator of transcription 3 (STAT3) mutations cause autosomal-dominant HIES. To determine the spectrum and functional consequences of heterozygous STAT3 mutations in a cohort of patients with HIES. We sequenced the STAT3 gene in 38 patients with HIES (National Institutes of Health score >40 points) from 35 families, quantified T(H)17 cells in peripheral blood, and evaluated tyrosine phosphorylation of STAT3. Most STAT3 mutations in our cohort were in the DNA-binding domain (DBD; 22/35 families) or Src homology 2 (SH2) domain (10/35) and were missense mutations. We identified 2 intronic mutations resulting in exon skipping and in-frame deletions within the DBD. In addition, we identified 2 mutations located in the transactivation domain downstream of the SH2 domain: a 10-amino acid deletion and an amino acid substitution. In 1 patient, we were unable to identify a STAT3 mutation. T(H)17 cells were absent or low in the peripheral blood of all patients who were evaluated (n = 17). IL-6-induced STAT3-phosphorylation was consistently reduced in patients with SH2 domain mutations but comparable to normal controls in patients with mutations in the DBD. Heterozygous STAT3 mutations were identified in 34 of 35 unrelated HIES families. Patients had impaired T(H)17 cell development, and those with SH2 domain mutations had reduced STAT3 phosphorylation.

Two Naturally Occurring Terpenes, Dehydrocostuslactone and Costunolide, Decrease Intracellular GSH Content and Inhibit STAT3 Activation

PubMed Central

Butturini, Elena; Cavalieri, Elisabetta; Carcereri de Prati, Alessandra; Darra, Elena; Rigo, Antonella; Shoji, Kazuo; Murayama, Norie; Yamazaki, Hiroshi; Watanabe, Yasuo; Suzuki, Hisanori; Mariotto, Sofia

2011-01-01

The main purpose of the present study is to envisage the molecular mechanism of inhibitory action ofdehydrocostuslactone (DCE) andcostunolide (CS), two naturally occurring sesquiterpene lactones, towards the activation of signal transducer and activator of transcription 3 (STAT3). We report that, in human THP-1 cell line, they inhibit IL-6-elicited tyrosine phosphorylation of STAT3 and its DNA binding activity with EC50 of 10 µM with concomitantdown-regulation ofthe phosphorylation of the tyrosine Janus kinases JAK1, JAK2 and Tyk2. Furthermore, these compounds that contain an α-β-unsatured carbonyl moiety and function as potent Michael reaction acceptor, induce a rapid drop in intracellular glutathione (GSH) concentration by direct interaction with it, thereby triggering S-glutathionylation of STAT3. Dehydrocostunolide (HCS), the reduced form of CS lacking only the α-β-unsaturated carbonyl group, fails to exert any inhibitory action. Finally, the glutathione ethylene ester (GEE), the cell permeable GSH form, reverts the inhibitory action of DCE and CS on STAT3 tyrosine phosphorylation. We conclude that these two sesquiterpene lactones are able to induce redox-dependent post-translational modification of cysteine residues of STAT3 protein in order to regulate its function. PMID:21625597

Histone deacetylase 3 regulates the inflammatory gene expression programme of rheumatoid arthritis fibroblast-like synoviocytes

PubMed Central

Angiolilli, Chiara; Kabala, Pawel A; Van Baarsen, Iris M; Ferguson, Bradley S; García, Samuel; Malvar Fernandez, Beatriz; McKinsey, Timothy A; Tak, Paul P; Fossati, Gianluca; Mascagni, Paolo; Baeten, Dominique L; Reedquist, Kris A

2017-01-01

Objectives Non-selective histone deacetylase (HDAC) inhibitors (HDACi) have demonstrated anti-inflammatory properties in both in vitro and in vivo models of rheumatoid arthritis (RA). Here, we investigated the potential contribution of specific class I and class IIb HDACs to inflammatory gene expression in RA fibroblast-like synoviocytes (FLS). Methods RA FLS were incubated with pan-HDACi (ITF2357, givinostat) or selective HDAC1/2i, HDAC3/6i, HDAC6i and HDAC8i. Alternatively, FLS were transfected with HDAC3, HDAC6 or interferon (IFN)-α/β receptor alpha chain (IFNAR1) siRNA. mRNA expression of interleukin (IL)-1β-inducible genes was measured by quantitative PCR (qPCR) array and signalling pathway activation by immunoblotting and DNA-binding assays. Results HDAC3/6i, but not HDAC1/2i and HDAC8i, significantly suppressed the majority of IL-1β-inducible genes targeted by pan-HDACi in RA FLS. Silencing of HDAC3 expression reproduced the effects of HDAC3/6i on gene regulation, contrary to HDAC6-specific inhibition and HDAC6 silencing. Screening of the candidate signal transducers and activators of transcription (STAT)1 transcription factor revealed that HDAC3/6i abrogated STAT1 Tyr701 phosphorylation and DNA binding, but did not affect STAT1 acetylation. HDAC3 activity was required for type I IFN production and subsequent STAT1 activation in FLS. Suppression of type I IFN release by HDAC3/6i resulted in reduced expression of a subset of IFN-dependent genes, including the chemokines CXCL9 and CXCL11. Conclusions Inhibition of HDAC3 in RA FLS largely recapitulates the effects of pan-HDACi in suppressing inflammatory gene expression, including type I IFN production in RA FLS. Our results identify HDAC3 as a potential therapeutic target in the treatment of RA and type I IFN-driven autoimmune diseases. PMID:27457515

Expression of STATs and their inhibitors SOCS and PIAS in brain tumors. In vitro and in vivo study.

PubMed

Ehrmann, J; Strakova, N; Vrzalikova, K; Hezova, R; Kolar, Z

2008-01-01

Proteins of STAT family belongs to the transcription factors. Through their binding to the DNA specific sites and consequent regulation of transcription of various genes, these signaling proteins play an important role in many cell functions. Recent studies demonstrated persistent activation of STATs and loss of their natural inhibitors SOCS and PIAS in various human cancers. There is also evidence that experimental pharmacologic or genetic modulation of their function mignt by a new approach in anticancer treatment. The aim of this study was in vitro assesment and analysis of expression of STATs, SOCS and PIAS in glioblastoma cell lines undergoing treatment by PPARgamma agonists/antagonists because PPARgamma and STATs are tightly regulated by an overlapping set of nuclear regulatory proteins. We further analysed immunohistochemical expression of these proteins in vivo, with its correlation to grading in various brain tumors. The results of in vitro study showed decreased expression of phosphorylated form of STAT3 and increase of its inhibitors SOCS3 and PIAS3 in glioblastoma cell lines after treatment with IC50 of PPARgamma agonist ciglitazone. In vivo study failed to reveal changes in STAT3 and SOCS3 expression in either low and high grade astrocytomas, however we detect lower expression of STAT2 in low grade astrocytomas when comparing with high grade astrocytomas and lower expression of STAT3 in ependymomas when comparing with anaplastic ones. The results showed existing relationship between STAT and PPARgamma signaling in glial tumors and further suppport expected important role of STATs in regulation of growth and differentiation in these tumors.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Unterberger, Claudia; Hanson, Steven; Department of Infection, Immunity and Inflammation, University of Leicester, University Road, Leicester LE1 9HN

Little is known about determinants regulating expression of Mannan-binding lectin associated serine protease-2 (MASP-2), the effector component of the lectin pathway of complement activation. Comparative bioinformatic analysis of the MASP2 promoter regions in human, mouse, and rat, revealed conservation of two putative Stat binding sites, termed StatA and StatB. Site directed mutagenesis specific for these sites was performed. Transcription activity was decreased 5-fold when StatB site was mutated in the wildtype reporter gene construct. Gel retardation and competition assays demonstrated that proteins contained in the nuclear extract prepared from HepG2 specifically bound double-stranded StatB oligonucleotides. Supershift analysis revealed Stat3 tomore » be the major specific binding protein. We conclude that Stat3 binding is important for MASP2 promoter activity.« less

JAK2V617F influences epigenomic changes in myeloproliferative neoplasms.

PubMed

Chen, Chih-Cheng; Chiu, Chia-Chen; Lee, Kuan-Der; Hsu, Chia-Chen; Chen, Hong-Chi; Huang, Tim H-M; Hsiao, Shu-Huei; Leu, Yu-Wei

2017-12-16

Negative valine (V) to phenylalanine (F) switch at the Janus kinase (JAK2) 617 codon (V617F) is the dominant driver mutation in patients with myeloproliferative neoplasms (MPNs). JAK2V617F was proved to be sufficient for cell transformation; however, independent mutations might influence the following epigenomic modifications. To assess the JAK2V617F-induced downstream epigenomic changes without interferences, we profiled the epigenomic changes in ectopically expressed JAK2V617F in Ba/F3 cells. Antibodies against phosphorylated signal transducer and activator of transcription 3 (pSTAT3) and enhancer of zeste homolog 2 (EZH2) were used for chromatin-immunoprecipitation sequencing (ChIP-seq) to detect the downstream epigenomic targets in the JAK2-STAT3 signaling pathway. To confirm the JAK2V617F-induced epigenetic changes in vivo, DNA methylation changes in the target loci in patients with MPNs were detected through methylation-specific polymerase chain reaction and were clustered against the changes within controls. We found that ectopically expressed JAK2V617F in Ba/F3 cells reduced the binding specificity; it was associated with cis-regulatory elements and recognized DNA motifs in both pSTAT3-downstream and EZH2-associated targets. Overlapping target loci between the control and JAK2V617F were <3% and 0.4%, respectively, as identified through pSTAT3 and EZH2 ChIP-seq. Furthermore, the methylation changes in the direct target loci (FOXH1, HOXC9, and SRF) were clustered independently from the control locus (L1TD1) and other mutation genes (HMGA2 and Lin28A) in the analyzed MPN samples. Therefore, JAK2V617F influences target binding in both pSTAT3 and EZH2. Without mutations in epigenetic regulators, JAK2V617F can induce downstream epigenomic modifications. Thus, epigenetic changes in JAK2 downstream targets might be trackable in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

STATs get their move on

PubMed Central

Reich, Nancy C

2013-01-01

Understanding the mechanisms that regulate dynamic localization of a protein within a cell can provide critical insight to its functional molecular interactions. Signal transducers and activators of transcription (STATs) play essential roles in development, proliferation, and immune defense. However the consequences of STAT hyperactivity can predispose to diseases including autoimmunity and cancer. To function as transcription factors STATs must gain access to the nucleus, and knowledge of the mechanisms that regulate STAT nuclear trafficking can provide a means to control STAT action. This review presents a synopsis of some of the studies that address the nuclear dynamics of the STAT proteins. Evidence suggests that not all STATs are the same. Nuclear import of STAT1 and STAT4 appears linked to their tyrosine phosphorylation and the formation of parallel dimers via reciprocal phosphotyrosine and Src homology 2 domain interactions. This dimer arrangement generates a conformational nuclear localization signal. STAT2 is imported continually to the nucleus in an unphosphorylated state due to its association with IRF9, but the dominant nuclear export signal of STAT2 shuttles the complex back to the cytoplasm. Following STAT2 tyrosine phosphorylation, it can form dimers with STAT1 to affect nuclear import as the trimeric complex (ISGF3). Distinctly, STAT3, STAT5, and STAT6 are continually imported to the nucleus independent of tyrosine phosphorylation. Mutational studies indicate the nuclear localization signals in these STATs require the conformational structure of their coiled-coil domains. Increases in STAT nuclear accumulation following cytokine stimulation appear coordinate with their ability to bind DNA. PMID:24470978

Opposite nuclear level and binding activity of STAT5B and STAT3 proteins with rat haptoglobin gene under normal and turpentine induced acute phase conditions.

PubMed

Grigorov, I; Lazić, T; Cvetković, I; Milosavljević, T; Petrović, M

2001-01-01

Transcription of the rat gene encoding haptoglobin (Hp) is highly induced during acute phase (AP) response which has been previously shown to be mediated by inducible STAT3 member of the Signal Transducer and Activators of Transcription (STATs) family proteins. In this study, we observed that under normal but not in the turpentine induced AP conditions, another member of the STAT family proteins, STAT5b is expressed and binds to the hormone regulatory element (HRE) of the rat Hp gene. We found that the nuclear amounts of constitutively active STAT5b in rat liver decreased significantly with time of turpentine treatment as opposed to that of cytosol STAT5b, suggesting possible export of constitutive STAT5b from the nucleus. Nuclear accumulation and binding of inducible STAT3 proteins to the rat Hp gene HRE following turpentine treatment implicated that STAT5b negatively regulates Hp gene expression during normal conditions.

Short Stat5-Interacting Peptide Derived from Phospholipase C-β3 Inhibits Hematopoietic Cell Proliferation and Myeloid Differentiation

PubMed Central

Yasudo, Hiroki; Ando, Tomoaki; Xiao, Wenbin; Kawakami, Yuko; Kawakami, Toshiaki

2011-01-01

Constitutive activation of the transcription factor Stat5 in hematopoietic stem/progenitor cells leads to various hematopoietic malignancies including myeloproliferative neoplasm (MPN). Our recent study found that phospholipase C (PLC)-β3 is a novel tumor suppressor involved in MPN, lymphoma and other tumors. Stat5 activity is negatively regulated by the SH2 domain-containing protein phosphatase SHP-1 in a PLC-β3-dependent manner. PLC-β3 can form the multimolecular SPS complex together with SHP-1 and Stat5. The close physical proximity of SHP-1 and Stat5 brought about by interacting with the C-terminal segment of PLC-β3 (PLC-β3-CT) accelerates SHP-1-mediated dephosphorylation of Stat5. Here we identify the minimal sequences within PLC-β3-CT required for its tumor suppressor function. Two of the three Stat5-binding noncontiguous regions, one of which also binds SHP-1, substantially inhibited in vitro proliferation of Ba/F3 cells. Surprisingly, an 11-residue Stat5-binding peptide (residues 988-998) suppressed Stat5 activity in Ba/F3 cells and in vivo proliferation and myeloid differentiation of hematopoietic stem/progenitor cells. Therefore, this study further defines PLC-β3-CT as the Stat5- and SHP-1-binding domain by identifying minimal functional sequences of PLC-β3 for its tumor suppressor function and implies their potential utility in the control of hematopoietic malignancies. PMID:21949826

TLR9 Ligands Induce S100A8 in Macrophages via a STAT3-Dependent Pathway which Requires IL-10 and PGE2

PubMed Central

Hsu, Kenneth; Chung, Yuen Ming; Endoh, Yasumi; Geczy, Carolyn L.

2014-01-01

S100A8 and S100A9 are highly-expressed calcium-binding proteins in neutrophils and monocytes, and in subsets of macrophages in inflammatory lesions. Unmethylated CpG motifs found in bacterial and viral DNA are potent activators of innate immunity via Toll-like receptor 9 (TLR9). S100A8, but not S100A9, mRNA and protein was directly induced by CpG-DNA in murine and human macrophages. Induction in murine macrophages peaked at 16 h. CpG-DNA-induced S100A8 required de novo protein synthesis; IL-10 and Prostaglandin E2 (PGE2) synergistically enhanced expression and promoted earlier gene induction. Inhibitors of endogenous IL-10, PGE2, and the E prostanoid (EP) 4 receptor strongly suppressed S100A8 expression, particularly when combined. Thus, S100A8 induction by E. coli DNA required both IL-10 and PGE2/EP4 signaling. The MAPKs, PI3K and JAK pathways were essential, whereas ERK1/2 appeared to play a direct role. S100A8 induction by CpG-DNA was controlled at the transcriptional level. The promoter region responsible for activation, either directly, or indirectly via IL-10 and PGE2, was located within a −178 to −34-bp region and required STAT3 binding. Because of the robust links connecting IL-10 and PGE2 with an anti-inflammatory macrophage phenotype, the induction profile of S100A8 strongly indicates a role for this protein in resolution of inflammation. PMID:25098409

TLR9 ligands induce S100A8 in macrophages via a STAT3-dependent pathway which requires IL-10 and PGE2.

PubMed

Hsu, Kenneth; Chung, Yuen Ming; Endoh, Yasumi; Geczy, Carolyn L

2014-01-01

S100A8 and S100A9 are highly-expressed calcium-binding proteins in neutrophils and monocytes, and in subsets of macrophages in inflammatory lesions. Unmethylated CpG motifs found in bacterial and viral DNA are potent activators of innate immunity via Toll-like receptor 9 (TLR9). S100A8, but not S100A9, mRNA and protein was directly induced by CpG-DNA in murine and human macrophages. Induction in murine macrophages peaked at 16 h. CpG-DNA-induced S100A8 required de novo protein synthesis; IL-10 and Prostaglandin E2 (PGE2) synergistically enhanced expression and promoted earlier gene induction. Inhibitors of endogenous IL-10, PGE2, and the E prostanoid (EP) 4 receptor strongly suppressed S100A8 expression, particularly when combined. Thus, S100A8 induction by E. coli DNA required both IL-10 and PGE2/EP4 signaling. The MAPKs, PI3K and JAK pathways were essential, whereas ERK1/2 appeared to play a direct role. S100A8 induction by CpG-DNA was controlled at the transcriptional level. The promoter region responsible for activation, either directly, or indirectly via IL-10 and PGE2, was located within a -178 to -34-bp region and required STAT3 binding. Because of the robust links connecting IL-10 and PGE2 with an anti-inflammatory macrophage phenotype, the induction profile of S100A8 strongly indicates a role for this protein in resolution of inflammation.

Formononetin-induced oxidative stress abrogates the activation of STAT3/5 signaling axis and suppresses the tumor growth in multiple myeloma preclinical model.

PubMed

Kim, Chulwon; Lee, Seok-Geun; Yang, Woong Mo; Arfuso, Frank; Um, Jae-Young; Kumar, Alan Prem; Bian, Jinsong; Sethi, Gautam; Ahn, Kwang Seok

2018-05-29

Aberrant reactions of signal transducer and transcriptional activator (STAT) are frequently detected in multiple myeloma (MM) cancers and can upregulate the expression of multiple genes related to cell proliferation, survival, metastasis, and angiogenesis. Therefore, agents capable of inhibiting STAT activation can form the basis of novel therapies for MM patients. In the present study, we investigated whether the potential anti-cancer effects of Formononetin (FT), a naturally occurring isoflavone derived from Astragalus membranaceus, Trifolium pratense, Glycyrrhiza glabra, and Pueraria lobata, against MM cell lines and human multiple myeloma xenograft tumors in athymic nu/nu mice model are mediated through the negative regulation of STAT3 and STAT5 pathways. Data from the in vitro studies indicated that FT could significantly inhibit cell viability, and induce apoptosis. Interestingly, FT also suppressed constitutive STAT3 (tyrosine residue 705 and serine residue 727) and STAT5 (tyrosine residue 694/699) activation, which correlated with the suppression of the upstream kinases (JAK1, JAK2, and c-Src) in MM cells, and this effect was found to be mediated via an increased production of reactive oxygen species (ROS) due to GSH/GSSG imbalance. Also, FT abrogated STAT3 and STAT5 DNA binding capacity and nuclear translocation. FT induced cell cycle arrest, downregulated the expression of STAT3-regulated anti-apoptotic, angiogenetic, and proliferative gene products; and this correlated with induction of caspase-3 activation and cleavage of PARP. Intraperitoneal administration of FT significantly suppressed the tumor growth in the multiple myeloma xenograft mouse model without exhibiting any significant adverse effects. Overall, our findings indicate that FT exhibits significant anti-cancer effects in MM that may be primarily mediated through the ROS-regulated inhibition of the STAT3 and STAT5 signaling cascade. Copyright © 2018 Elsevier B.V. All rights reserved.

Urokinase receptor is associated with the components of the JAK1/STAT1 signaling pathway and leads to activation of this pathway upon receptor clustering in the human kidney epithelial tumor cell line TCL-598.

PubMed

Koshelnick, Y; Ehart, M; Hufnagl, P; Heinrich, P C; Binder, B R

1997-11-07

The urokinase-type plasminogen activator (uPA) binds to cells via a specific receptor attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Despite the lack of a transmembrane domain, the urokinase receptor (uPAR) is capable of transducing extracellular signals affecting growth, migration, and adhesion. Several Tyr kinases of the src family as well as beta1, beta2, and beta3 integrins were found to be associated with the uPAR. We found that in the human kidney epithelial line TCL-598, also components of the JAK1/STAT1 signal transduction pathway including gp130, are associated with uPAR as revealed by coimmunoprecipitation and are co-localized in caveolae. Upon clustering of uPA.uPAR complex by a monoclonal antibody, JAK1 associates with uPAR, which in turn leads to STAT1 phosphorylation, dimerization, specific binding to DNA, and gene activation. To prove the dependence of STAT1 activation on the uPAR, TCL-598 cells were treated with sense and antisense uPAR oligonucleotides. In antisense-treated cells in which uPAR expression was reduced to less then one third, activation of STAT1 by the clustering antibody was abolished while STAT1 activation by interferon-gamma was unaffected. Therefore, in this cell line, uPA.uPAR also utilizes the JAK1/STAT1 pathway for signaling, and gp130 might be the transmembrane adapter for this signal transduction pathway.

Activation of peroxisome proliferator-activated receptor-β/-δ (PPAR-β/-δ) ameliorates insulin signaling and reduces SOCS3 levels by inhibiting STAT3 in interleukin-6-stimulated adipocytes.

PubMed

Serrano-Marco, Lucía; Rodríguez-Calvo, Ricardo; El Kochairi, Ilhem; Palomer, Xavier; Michalik, Liliane; Wahli, Walter; Vázquez-Carrera, Manuel

2011-07-01

It has been suggested that interleukin (IL)-6 is one of the mediators linking obesity-derived chronic inflammation with insulin resistance through activation of STAT3, with subsequent upregulation of suppressor of cytokine signaling 3 (SOCS3). We evaluated whether peroxisome proliferator-activated receptor (PPAR)-β/-δ prevented activation of the IL-6-STAT3-SOCS3 pathway and insulin resistance in adipocytes. Adipocytes and white adipose tissue from wild-type and PPAR-β/-δ-null mice were used to evaluate the effect of PPAR-β/-δ on the IL-6-STAT3-SOCS3 pathway. First, we observed that the PPAR-β/-δ agonist GW501516 prevented both IL-6-dependent reduction in insulin-stimulated Akt phosphorylation and glucose uptake in adipocytes. In addition, this drug treatment abolished IL-6-induced SOCS3 expression in differentiated 3T3-L1 adipocytes. This effect was associated with the capacity of the drug to prevent IL-6-induced STAT3 phosphorylation on Tyr(705) and Ser(727) residues in vitro and in vivo. Moreover, GW501516 prevented IL-6-dependent induction of extracellular signal-related kinase (ERK)1/2, a serine-threonine-protein kinase involved in serine STAT3 phosphorylation. Furthermore, in white adipose tissue from PPAR-β/-δ-null mice, STAT3 phosphorylation (Tyr(705) and Ser(727)), STAT3 DNA-binding activity, and SOCS3 protein levels were higher than in wild-type mice. Several steps in STAT3 activation require its association with heat shock protein 90 (Hsp90), which was prevented by GW501516 as revealed in immunoprecipitation studies. Consistent with this finding, the STAT3-Hsp90 association was enhanced in white adipose tissue from PPAR-β/-δ-null mice compared with wild-type mice. Collectively, our findings indicate that PPAR-β/-δ activation prevents IL-6-induced STAT3 activation by inhibiting ERK1/2 and preventing the STAT3-Hsp90 association, an effect that may contribute to the prevention of cytokine-induced insulin resistance in adipocytes. © 2011 by the American Diabetes Association.

Increased p50/p50 NF-κB Activation in Human Papillomavirus Type 6- or Type 11-Induced Laryngeal Papilloma Tissue

PubMed Central

Vancurova, Ivana; Wu, Rong; Miskolci, Veronika; Sun, Shishinn

2002-01-01

We have observed elevated NF-κB DNA-binding activity in nuclear extracts from human papillomavirus type 6- and 11-infected laryngeal papilloma tissues. The predominant DNA-binding species is the p50/p50 homodimer. The elevated NF-κB activity could be correlated with a reduced level of cytoplasmic IκBβ and could be associated with the overexpression of p21CIP1/WAF1 in papilloma cells. Increased NF-κB activity and cytoplasmic accumulation of p21CIP1/WAF1 might counteract death-promoting effects elicited by overexpressed PTEN and reduced activation of Akt and STAT3 previously noted in these tissues. PMID:11773428

Genome-wide identification of novel expression signatures reveal distinct patterns and prevalence of binding motifs for p53, nuclear factor-κB and other signal transcription factors in head and neck squamous cell carcinoma

PubMed Central

Yan, Bin; Yang, Xinping; Lee, Tin-Lap; Friedman, Jay; Tang, Jun; Van Waes, Carter; Chen, Zhong

2007-01-01

Background Differentially expressed gene profiles have previously been observed among pathologically defined cancers by microarray technologies, including head and neck squamous cell carcinomas (HNSCCs). However, the molecular expression signatures and transcriptional regulatory controls that underlie the heterogeneity in HNSCCs are not well defined. Results Genome-wide cDNA microarray profiling of ten HNSCC cell lines revealed novel gene expression signatures that distinguished cancer cell subsets associated with p53 status. Three major clusters of over-expressed genes (A to C) were defined through hierarchical clustering, Gene Ontology, and statistical modeling. The promoters of genes in these clusters exhibited different patterns and prevalence of transcription factor binding sites for p53, nuclear factor-κB (NF-κB), activator protein (AP)-1, signal transducer and activator of transcription (STAT)3 and early growth response (EGR)1, as compared with the frequency in vertebrate promoters. Cluster A genes involved in chromatin structure and function exhibited enrichment for p53 and decreased AP-1 binding sites, whereas clusters B and C, containing cytokine and antiapoptotic genes, exhibited a significant increase in prevalence of NF-κB binding sites. An increase in STAT3 and EGR1 binding sites was distributed among the over-expressed clusters. Novel regulatory modules containing p53 or NF-κB concomitant with other transcription factor binding motifs were identified, and experimental data supported the predicted transcriptional regulation and binding activity. Conclusion The transcription factors p53, NF-κB, and AP-1 may be important determinants of the heterogeneous pattern of gene expression, whereas STAT3 and EGR1 may broadly enhance gene expression in HNSCCs. Defining these novel gene signatures and regulatory mechanisms will be important for establishing new molecular classifications and subtyping, which in turn will promote development of targeted therapeutics for HNSCC. PMID:17498291

Cytokine-Induction of Tumor Necrosis Factor Receptor 2 (TNFR2) is Mediated by STAT3 in Colon Cancer Cells

PubMed Central

Hamilton, Kathryn E.; Simmons, James G.; Ding, Shengli; Van Landeghem, Laurianne; Lund, P. Kay

2011-01-01

The IL-6/STAT3 and TNFα/NFκB pathways are emerging as critical mediators of inflammation-associated colon cancer. TNFR2 expression is increased in inflammatory bowel diseases, the azoxymethane/dextran sodium sulfate (AOM/DSS) model of colitis-associated cancer, and by combined IL-6 and TNFα. The molecular mechanisms that regulate TNFR2 remain undefined. This study used colon cancer cell lines to test the hypothesis that IL-6 and TNFα induce TNFR2 via STAT3 and/or NFκB. Basal and IL-6 + TNFα-induced TNFR2 were decreased by pharmacological STAT3 inhibition. NFκB inhibition had little effect on IL-6 + TNFα-induced TNFR2, but did inhibit induction of endogenous IL-6 and TNFR2 in cells treated with TNFα alone. Chromatin immunoprecipitation (ChIP) revealed cooperative effects of IL-6 + TNFα to induce STAT3 binding to a -1578 STAT response element in the TNFR2 promoter, but no effect on NFκB binding to consensus sites. Constitutively active STAT3 was sufficient to induce TNFR2 expression. Over-expression of SOCS3, a cytokine-inducible STAT3 inhibitor, which reduces tumorigenesis in preclinical models of colitis-associated cancer, decreased cytokine-induced TNFR2 expression and STAT3 binding to the -1578 STAT response element. SOCS3 over-expression also decreased proliferation of colon cancer cells and dramatically decreased anchorage-independent growth of colon cancer cells, even cells over-expressing TNFR2. Collectively, these studies demonstrate that IL-6 and TNFα-induced TNFR2 expression in colon cancer cells is mediated primarily by STAT3, and provide evidence that TNFR2 may contribute to the tumor-promoting roles of STAT3. PMID:21994466

Dietary diindolylmethane suppresses inflammation-driven lung squamous cell carcinoma in mice

PubMed Central

Song, Jung Min; Qian, Xuemin; Teferi, Fitsum; Pan, Jing; Wang, Yian; Kassie, Fekadu

2014-01-01

Inflammatory conditions of the lung such as chronic obstructive pulmonary disease (COPD) are known to increase lung cancer risk, particularly lung squamous cell carcinoma (LSCC). In the present study, we developed a mouse model of inflammation-driven LSCC that was induced by N-nitroso-trischloroethylurea (NTCU) and enhanced by lipopolysaccharide (LPS), a potent proinflammatory agent contained in tobacco and tobacco smoke, and determined the chemopreventive effects of BioResponse diindolylmethane (DIM) in the same model. Compared to mice treated with NTCU alone, mice treated with the combination of NTCU and LPS had a 9-fold increase in the number of bronchioles with LSCC. Also, compared to mice treated with LPS alone, mice treated with NTCU plus LPS showed significantly increased expression of the inflammatory cytokines IL-1α, IL-6, and TNFα (all three increased about 7-fold). Parallel to the increased cytokine gene expression, the NTCU plus LPS-treated group exhibited significantly enhanced activation of NF-κB, STAT3, ERK, p-38, and Akt, expression of p53, COX-2, and Mcl-1, and NF-κB- and STAT3-DNA binding in the lung. Dietary administration of DIM (10 µmol/g diet or 2460 ppm) to mice treated with NTCU plus LPS reduced the incidence of LSCC by 2-fold, suppressed activation/expression of proinflammatory and procarcinogenic proteins and NF-κB- and STAT3-DNA binding, but not the expression of cytokines and p53. This study highlights the potential significance of our mouse model to identify promising drugs or dietary agents for the chemoprevention of human LSCC and that DIM is a very good candidate for clinical lung cancer chemoprevention trials. PMID:25403850

Signal transducer and activator of transcription 5B (STAT5B) modulates adipocyte differentiation via MOF.

PubMed

Gao, Peng; Zhang, Yuchao; Liu, Yuantao; Chen, Jicui; Zong, Chen; Yu, Cong; Cui, Shang; Gao, Weina; Qin, Dandan; Sun, Wenchuan; Li, Xia; Wang, Xiangdong

2015-12-01

The role and mechanism of signal transducer and activator of transcription 5B (STAT5B) in adipogenesis remain unclear. In this study, our data showed that Males absent on the first (MOF) protein expression was increased during 3 T3-L1 preadipocytes differentiation accompanied with STAT5B expression increasing. Over-expression STAT5B enhanced MOF promoter trans-activation in HeLa cells. Mutagenesis assay and ChIP analysis exhibited that STAT5B was able to bind MOF promoter. Knocking-down STAT5B in 3 T3-L1 preadipocytes led to decreased expression of MOF, but resulted in increased expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα) and fatty acid-binding protein 4 (Fabp4), which were important factors or enzymes for adipogenesis. We also found that knocking-down MOF in 3 T3-L1 preadipocytes resulted in increased expression of PPARγ, C/EBPα and Fabp4, which was in the same trend as STAT5B knocking-down. Over-expression MOF resulted in reduced promoter trans-activation activity of C/EBPα. These results suggest that STAT5B and MOF work as negative regulators in adipogenesis, and STAT5B modulates preadipocytes differentiation partially by regulating MOF expression. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Inhibition of DNA methyltransferase induces G2 cell cycle arrest and apoptosis in human colorectal cancer cells via inhibition of JAK2/STAT3/STAT5 signalling.

PubMed

Xiong, Hua; Chen, Zhao-Fei; Liang, Qin-Chuan; Du, Wan; Chen, Hui-Min; Su, Wen-Yu; Chen, Guo-Qiang; Han, Ze-Guang; Fang, Jing-Yuan

2009-09-01

DNA methyltransferase inhibitors (MTIs) have recently emerged as promising chemotherapeutic or preventive agents for cancer, despite their poorly characterized mechanisms of action. The present study shows that DNA methylation is integral to the regulation of SH2-containing protein tyrosine phosphatase 1 (SHP1) expression, but not for regulation of suppressors of cytokine signalling (SOCS)1 or SOCS3 in colorectal cancer (CRC) cells. SHP1 expression correlates with down-regulation of Janus kinase/signal transducers and activators of transcription (JAK2/STAT3/STAT5) signalling, which is mediated in part by tyrosine dephosphorylation events and modulation of the proteasome pathway. Up-regulation of SHP1 expression was achieved using a DNA MTI, 5-aza-2'-deoxycytidine (5-aza-dc), which also generated significant down-regulation of JAK2/STAT3/STAT5 signalling. We demonstrate that 5-aza-dc suppresses growth of CRC cells, and induces G2 cell cycle arrest and apoptosis through regulation of downstream targets of JAK2/STAT3/STAT5 signalling including Bcl-2, p16(ink4a), p21(waf1/cip1) and p27(kip1). Although 5-aza-dc did not significantly inhibit cell invasion, 5-aza-dc did down-regulate expression of focal adhesion kinase and vascular endothelial growth factor in CRC cells. Our results demonstrate that 5-aza-dc can induce SHP1 expression and inhibit JAK2/STAT3/STAT5 signalling. This study represents the first evidence towards establishing a mechanistic link between inhibition of JAK2/STAT3/STAT5 signalling and the anticancer action of 5-aza-dc in CRC cells that may lead to the use of MTIs as a therapeutic intervention for human colorectal cancer.

Acanthamoeba castellanii STAT protein.

PubMed

Kicinska, Anna; Leluk, Jacek; Jarmuszkiewicz, Wieslawa

2014-01-01

STAT (signal transducers and activators of transcription) proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyostelium STAT proteins: a coiled coil (characteristic for Dictyostelium STAT coiled coil), a STAT DNA-binding domain and a Src-homology domain. The search for protein sequences homologous to A. castellanii STAT revealed 17 additional sequences from lower eukaryotes. Interestingly, all of these sequences come from Amoebozoa organisms that belong to either Mycetozoa (slime molds) or Centramoebida. We showed that there are four separated clades within the slime mold STAT proteins. The A. castellanii STAT protein branches next to a group of STATc proteins from Mycetozoa. We also demonstrate that Amoebozoa form a distinct monophyletic lineage within the STAT protein world that is well separated from the other groups.

Molecular Mechanism Underlying the Action of Substituted Pro-Gly Dipeptide Noopept.

PubMed

Vakhitova, Y V; Sadovnikov, S V; Borisevich, S S; Ostrovskaya, R U; A Gudasheva, T; Seredenin, S B

2016-01-01

This study was performed in order to reveal the effect of Noopept (ethyl ester of N-phenylacetyl-Lprolylglycine, GVS-111) on the DNA-binding activity of transcriptional factors (TF) in HEK293 cells transiently transfected with luciferase reporter constructs containing sequences for CREB, NFAT, NF-κB, p53, STAT1, GAS, VDR, HSF1, and HIF-1. Noopept (10 μM) was shown to increase the DNA-binding activity of HIF-1 only, while lacking the ability to affect that of CREB, NFAT, NF-κB, p53, STAT1, GAS, VDR, and HSF1. Noopept provoked an additional increase in the DNA-binding activity of HIF-1 when applied in conditions of CoCl2-induced HIF- 1 stabilization. The degree of this HIF-positive effect of Noopept was shown to be concentration-dependent. Piracetam (1 mM) failed to affect significantly any of the TF under study. The results of molecular docking showed that Noopept (L-isomer), as well as its metabolite, L-isomer of N-phenyl-acetylprolyl, unlike its pharmacologically ineffective D-isomer, is able to bind to the active site of prolyl hydroxylase 2. Taking into account the important role of the genes activated by HIF-1 in the formation of an adaptive response to hypoxia, data on the ability of Noopept to provoke a selective increase in the DNA-binding activity of HIF-1 explain the wide spectrum of neurochemical and pharmacological effects of Noopept revealed before. The obtained data allow one to propose the HIF-positive effect as the primary mechanism of the activity of this Pro-Gly-containing dipeptide.

Oncostatin M suppresses metastasis of lung adenocarcinoma by inhibiting SLUG expression through coordination of STATs and PIASs signalings.

PubMed

Pan, Chih-Ming; Wang, Mong-Lien; Chiou, Shih-Hwa; Chen, Hsiao-Yun; Wu, Cheng-Wen

2016-09-13

Oncostatin M (OSM) is linked with multiple biological responses including growth and differentiation. Previous reports showed inhibitory effects of OSM in tumor progression while others showed promoting effects. The dual role of OSM in the development of various cancers is still unclear. We previously described OSM-mediated SLUG suppression, leading to repressed metastasis of lung adenocarcinoma (LAC) cells. However, the underlying mechanism remains elusive. Here, we showed that OSM suppresses SLUG express in LAC cells through a STAT1-dependent transcriptional inhibition. Knockdown of STAT1 reversed the OSM-suppressed SLUG expression and rescued the OSM-mediated inhibition of cell proliferation, migration, and invasion in vitro, as well as pulmonary metastasis in vivo. STAT1 suppressed SLUG transcription through binding to its promoter region in response to OSM. Furthermore, PIAS4, a co-repressor of STAT, and HDAC1 were able to bind to STAT1 on SLUG promoter region, resulting in reduced H3K9 acetylation and suppressed SLUG expression upon OSM treatment. In contrast, PIAS3 bound to activated STAT3, another effector of OSM, in response to OSM and blocked the binding of STAT3 to SLUG promoter region, preventing STAT3-dependent activation of SLUG transcription. Our findings suggested that OSM suppresses SLUG expression and tumor metastasis of LAC through inducing the inhibitory effect of the STAT1-dependent pathway and suppressing the activating effect of STAT3-dependent signaling. These results can serve as a scientific basis for the potential therapeutic intervention of OSM in cancer cells.

Inhibitory Effects of Bangladeshi Medicinal Plant Extracts on Interactions between Transcription Factors and Target DNA Sequences

PubMed Central

Lampronti, Ilaria; Khan, Mahmud T.H.; Borgatti, Monica; Bianchi, Nicoletta

2008-01-01

Several transcription factors (TFs) play crucial roles in governing the expression of different genes involved in the immune response, embryo or cell lineage development, cell apoptosis, cell cycle progression, oncogenesis, repair and fibrosis processes and inflammation. As far as inflammation, TFs playing pivotal roles are nuclear factor kappa B (NF-kB), activator protein (AP-1), signal transducer and activator of transcription (STATs), cAMP response element binding protein (CREB) and GATA-1 factors. All these TFs regulate the expression of pro-inflammatory cytokines and are involved in the pathogenesis of a number of human disorders, particularly those with an inflammatory component. Since several medicinal plants can be employed to produce extracts exhibiting biological effects and because alteration of gene transcription represents a very interesting approach to control the expression of selected genes, this study sought to verify the ability of several extracts derived from Bangladeshi medicinal plants in interfering with molecular interactions between different TFs and specific DNA sequences. We first analyzed the antiproliferative activity of 19 medicinal plants on different human cell lines, including erythroleukemia K562, B lymphoid Raji and T lymphoid Jurkat cell lines. Secondly, we employed the electrophoretic mobility shift assay as a suitable technique for a fast screening of plant extracts altering the binding between NF-kB, AP-1, GATA-1, STAT-3, CREB and the relative target DNA elements. PMID:18830455

Diindolylmethane suppresses ovarian cancer growth and potentiates the effect of cisplatin in tumor mouse model by targeting signal transducer and activator of transcription 3 (STAT3)

PubMed Central

2012-01-01

Background Signal transducer and activator of transcription 3 (STAT3) is activated in majority of ovarian tumors and confers resistance to cisplatin treatment in patients with ovarian cancer. We have reported previously that diindolylmethane (DIM) inhibits the growth of ovarian cancer cells. However, to date the exact mechanism by which DIM induces growth suppressive effects has not been clear. In this report the mode of action of DIM is investigated. Methods Six human ovarian cancer cell lines and an ovarian tumor xenograft animal model were used to study the effect of diindolylmethane alone or in combination with cisplatin. Results Diindolylmethane treatment induced apoptosis in all six ovarian cancer cell lines. Phosphorylation of STAT3 at Tyr-705 and Ser-727 was reduced by DIM in a concentration-dependent manner. In addition, diindolylmethane treatment inhibited nuclear translocation, DNA binding, and transcriptional activity of STAT3. Interleukin (IL)-6-induced phosphorylation of STAT3 at Tyr-705 was significantly blocked by DIM. Overexpression of STAT3 by gene transfection blocked DIM-induced apoptosis. In addition, DIM treatment reduced the levels of IL-6 in ovarian cancer cells and in the tumors. DIM treatment also inhibited cell invasion and angiogenesis by suppressing hypoxia-inducible factor 1α (HIF-1α) and vascular epithelial growth factor (VEGF). Importantly, diindolylmethane treatment potentiated the effects of cisplatin in SKOV-3 cells by targeting STAT3. Oral administration of 3 mg diindolylmethane per day and subsequent administration of cisplatin substantially inhibited in vivo tumor growth. Western blotting analysis of tumor lysates indicated increased apoptosis and reduced STAT3 activation. Conclusions These findings provide a rationale for further clinical investigation of DIM alone or in combination for chemoprevention and/or chemotherapy of ovarian cancer. PMID:22280969

A homolog of teleostean signal transducer and activator of transcription 3 (STAT3) from rock bream, Oplegnathus fasciatus: Structural insights, transcriptional modulation, and subcellular localization.

PubMed

Bathige, S D N K; Thulasitha, William Shanthakumar; Umasuthan, Navaneethaiyer; Jayasinghe, J D H E; Wan, Qiang; Nam, Bo-Hye; Lee, Jehee

2017-04-01

Signal transducer and activator of transcription 3 (STAT3) is one of the crucial transcription factors in the Janus kinase (JAK)/STAT signaling pathway, and it was previously considered as acute phase response factor. A number of interleukins (ILs) such as IL-5, IL-6, IL-9, IL-10, IL-12, and IL-22 are known to be involved in activation of STAT3. In addition, various growth factors and pathogenic or oxidative stresses mediate the activation of a wide range of functions via STAT3. In this study, a STAT3 homolog was identified and functionally characterized from rock bream (RbSTAT3), Oplegnathus fasciatus. In silico characterization revealed that the RbSTAT3 amino acid sequence shares highly conserved common domain architectural features including N-terminal domain, coiled coil domain, DNA binding domain, linker domain, and Src homology 2 (SH2) domains. In addition, a fairly conserved transcriptional activation domain (TAD) was located at the C-terminus. Comparison of RbSTAT3 with other counterparts revealed higher identities (>90%) with fish orthologs. The genomic sequence of RbSTAT3 was obtained from a bacterial artificial chromosome (BAC) library, and was identified as a multi-exonic gene (24 exons), as found in other vertebrates. Genomic structural comparison and phylogenetic studies have showed that the evolutionary routes of teleostean and non-teleostean vertebrates were distinct. Quantitative real time PCR (qPCR) analysis revealed that the spatial distribution of RbSTAT3 mRNA expression was ubiquitous and highly detectable in blood, heart, and liver tissues. Transcriptional modulation of RbSTAT3 was examined in blood and liver tissues after challenges with bacteria (Edwardsiella tarda and Streptococcus iniae), rock bream irido virus (RBIV), and immune stimulants (LPS and poly (I:C)). Significant changes in RbSTAT3 transcription were also observed in response to tissue injury. In addition, the transcriptional up-regulation of RbSTAT3 was detected in rock bream heart cells upon recombinant rock bream IL-10 (rRbIL-10) treatment. Subcellular localization and nuclear translocation of rock bream STAT3 following poly (I:C) treatment were also demonstrated. Taken together, the results of the current study provide important evidence for potential roles of rock bream STAT3 in the immune system and wound healing processes. Copyright © 2017 Elsevier B.V. All rights reserved.

Activation of Peroxisome Proliferator–Activated Receptor-β/-δ (PPAR-β/-δ) Ameliorates Insulin Signaling and Reduces SOCS3 Levels by Inhibiting STAT3 in Interleukin-6–Stimulated Adipocytes

PubMed Central

Serrano-Marco, Lucía; Rodríguez-Calvo, Ricardo; El Kochairi, Ilhem; Palomer, Xavier; Michalik, Liliane; Wahli, Walter; Vázquez-Carrera, Manuel

2011-01-01

OBJECTIVE It has been suggested that interleukin (IL)-6 is one of the mediators linking obesity-derived chronic inflammation with insulin resistance through activation of STAT3, with subsequent upregulation of suppressor of cytokine signaling 3 (SOCS3). We evaluated whether peroxisome proliferator–activated receptor (PPAR)-β/-δ prevented activation of the IL-6-STAT3-SOCS3 pathway and insulin resistance in adipocytes. RESEARCH DESIGN AND METHODS Adipocytes and white adipose tissue from wild-type and PPAR-β/-δ-null mice were used to evaluate the effect of PPAR-β/-δ on the IL-6-STAT3-SOCS3 pathway. RESULTS First, we observed that the PPAR-β/-δ agonist GW501516 prevented both IL-6–dependent reduction in insulin-stimulated Akt phosphorylation and glucose uptake in adipocytes. In addition, this drug treatment abolished IL-6–induced SOCS3 expression in differentiated 3T3-L1 adipocytes. This effect was associated with the capacity of the drug to prevent IL-6–induced STAT3 phosphorylation on Tyr705 and Ser727 residues in vitro and in vivo. Moreover, GW501516 prevented IL-6–dependent induction of extracellular signal–related kinase (ERK)1/2, a serine-threonine-protein kinase involved in serine STAT3 phosphorylation. Furthermore, in white adipose tissue from PPAR-β/-δ–null mice, STAT3 phosphorylation (Tyr705 and Ser727), STAT3 DNA-binding activity, and SOCS3 protein levels were higher than in wild-type mice. Several steps in STAT3 activation require its association with heat shock protein 90 (Hsp90), which was prevented by GW501516 as revealed in immunoprecipitation studies. Consistent with this finding, the STAT3-Hsp90 association was enhanced in white adipose tissue from PPAR-β/-δ–null mice compared with wild-type mice. CONCLUSIONS Collectively, our findings indicate that PPAR-β/-δ activation prevents IL-6–induced STAT3 activation by inhibiting ERK1/2 and preventing the STAT3-Hsp90 association, an effect that may contribute to the prevention of cytokine-induced insulin resistance in adipocytes. PMID:21617181

Proinflammatory Cytokines IL-6 and TNF-α Increased Telomerase Activity through NF-κB/STAT1/STAT3 Activation, and Withaferin A Inhibited the Signaling in Colorectal Cancer Cells.

PubMed

Chung, Seyung S; Wu, Yong; Okobi, Quincy; Adekoya, Debbie; Atefi, Mohammad; Clarke, Orette; Dutta, Pranabananda; Vadgama, Jaydutt V

2017-01-01

There are increasing evidences of proinflammatory cytokine involvement in cancer development. Here, we found that two cytokines, IL-6 and TNF- α , activated colorectal cancer cells to be more invasive and stem-like. Combined treatment of IL-6 and TNF- α phosphorylated transcription factors STAT3 in a synergistic manner. STAT3, STAT1, and NF- κ B physically interacted upon the cytokine stimulation. STAT3 was bound to the promoter region of human telomerase reverse transcriptase (hTERT). IL-6 and TNF- α stimulation further enhanced STAT3 binding affinity. Stem cell marker Oct-4 was upregulated in colorectal cancer cells upon IL-6 and TNF- α stimulation. Withaferin A, an anti-inflammatory steroidal lactone, inhibited the IL-6- and TNF- α -induced cancer cell invasion and decreased colonosphere formation. Notably, withaferin A inhibited STAT3 phosphorylation and abolished the STAT3, STAT1, and NF- κ B interactions. Oct-4 expression was also downregulated by withaferin A inhibition. The binding of STAT3 to the hTERT promoter region and telomerase activity showed reduction with withaferin A treatments. Proinflammatory cytokine-induced cancer cell invasiveness is mediated by a STAT3-regulated mechanism in colorectal cancer cells. Our data suggest that withaferin A could be a promising anticancer agent that effectively inhibits the progression of colorectal cancer.

Triplex DNA-binding proteins are associated with clinical outcomes revealed by proteomic measurements in patients with colorectal cancer

PubMed Central

2012-01-01

Background Tri- and tetra-nucleotide repeats in mammalian genomes can induce formation of alternative non-B DNA structures such as triplexes and guanine (G)-quadruplexes. These structures can induce mutagenesis, chromosomal translocations and genomic instability. We wanted to determine if proteins that bind triplex DNA structures are quantitatively or qualitatively different between colorectal tumor and adjacent normal tissue and if this binding activity correlates with patient clinical characteristics. Methods Extracts from 63 human colorectal tumor and adjacent normal tissues were examined by gel shifts (EMSA) for triplex DNA-binding proteins, which were correlated with clinicopathological tumor characteristics using the Mann-Whitney U, Spearman’s rho, Kaplan-Meier and Mantel-Cox log-rank tests. Biotinylated triplex DNA and streptavidin agarose affinity binding were used to purify triplex-binding proteins in RKO cells. Western blotting and reverse-phase protein array were used to measure protein expression in tissue extracts. Results Increased triplex DNA-binding activity in tumor extracts correlated significantly with lymphatic disease, metastasis, and reduced overall survival. We identified three multifunctional splicing factors with biotinylated triplex DNA affinity: U2AF65 in cytoplasmic extracts, and PSF and p54nrb in nuclear extracts. Super-shift EMSA with anti-U2AF65 antibodies produced a shifted band of the major EMSA H3 complex, identifying U2AF65 as the protein present in the major EMSA band. U2AF65 expression correlated significantly with EMSA H3 values in all extracts and was higher in extracts from Stage III/IV vs. Stage I/II colon tumors (p = 0.024). EMSA H3 values and U2AF65 expression also correlated significantly with GSK3 beta, beta-catenin, and NF- B p65 expression, whereas p54nrb and PSF expression correlated with c-Myc, cyclin D1, and CDK4. EMSA values and expression of all three splicing factors correlated with ErbB1, mTOR, PTEN, and Stat5. Western blots confirmed that full-length and truncated beta-catenin expression correlated with U2AF65 expression in tumor extracts. Conclusions Increased triplex DNA-binding activity in vitro correlates with lymph node disease, metastasis, and reduced overall survival in colorectal cancer, and increased U2AF65 expression is associated with total and truncated beta-catenin expression in high-stage colorectal tumors. PMID:22682314

Molecular Mechanism Underlying the Action of Substituted Pro-Gly Dipeptide Noopept

PubMed Central

Vakhitova, Y. V.; Sadovnikov, S. V.; Borisevich, S. S.; Ostrovskaya, R. U.; A.Gudasheva, T.; Seredenin, S. B.

2016-01-01

This study was performed in order to reveal the effect of Noopept (ethyl ester of N-phenylacetyl-Lprolylglycine, GVS-111) on the DNA-binding activity of transcriptional factors (TF) in HEK293 cells transiently transfected with luciferase reporter constructs containing sequences for CREB, NFAT, NF-κB, p53, STAT1, GAS, VDR, HSF1, and HIF-1. Noopept (10 μM) was shown to increase the DNA-binding activity of HIF-1 only, while lacking the ability to affect that of CREB, NFAT, NF-κB, p53, STAT1, GAS, VDR, and HSF1. Noopept provoked an additional increase in the DNA-binding activity of HIF-1 when applied in conditions of CoCl2-induced HIF- 1 stabilization. The degree of this HIF-positive effect of Noopept was shown to be concentration-dependent. Piracetam (1 mM) failed to affect significantly any of the TF under study. The results of molecular docking showed that Noopept (L-isomer), as well as its metabolite, L-isomer of N-phenyl-acetylprolyl, unlike its pharmacologically ineffective D-isomer, is able to bind to the active site of prolyl hydroxylase 2. Taking into account the important role of the genes activated by HIF-1 in the formation of an adaptive response to hypoxia, data on the ability of Noopept to provoke a selective increase in the DNA-binding activity of HIF-1 explain the wide spectrum of neurochemical and pharmacological effects of Noopept revealed before. The obtained data allow one to propose the HIF-positive effect as the primary mechanism of the activity of this Pro-Gly-containing dipeptide. PMID:27099787

Stat1-Vitamin D Receptor Interactions Antagonize 1,25-Dihydroxyvitamin D Transcriptional Activity and Enhance Stat1-Mediated Transcription

PubMed Central

Vidal, Marcos; Ramana, Chilakamarti V.; Dusso, Adriana S.

2002-01-01

The cytokine gamma interferon (IFN-γ) and the calcitropic steroid hormone 1,25-dihydroxyvitamin D (1,25D) are activators of macrophage immune function. In sarcoidosis, tuberculosis, and several granulomatoses, IFN-γ induces 1,25D synthesis by macrophages and inhibits 1,25D induction of 24-hydroxylase, a key enzyme in 1,25D inactivation, causing high levels of 1,25D in serum and hypercalcemia. This study delineates IFN-γ-1,25D cross talk in human monocytes-macrophages. Nuclear accumulation of Stat1 and vitamin D receptor (VDR) by IFN-γ and 1,25D promotes protein-protein interactions between Stat1 and the DNA binding domain of the VDR. This prevents VDR-retinoid X receptor (RXR) binding to the vitamin D-responsive element, thus diverting the VDR from its normal genomic target on the 24-hydroxylase promoter and antagonizing 1,25D-VDR transactivation of this gene. In contrast, 1,25D enhances IFN-γ action. Stat1-VDR interactions, by preventing Stat1 deactivation by tyrosine dephosphorylation, cooperate with IFN-γ/Stat1-induced transcription. This novel 1,25D-IFN-γ cross talk explains the pathogenesis of abnormal 1,25D homeostasis in granulomatous processes and provides new insights into 1,25D immunomodulatory properties. PMID:11909970

Yes-Associated Protein Promotes Angiogenesis via Signal Transducer and Activator of Transcription 3 in Endothelial Cells.

PubMed

He, Jinlong; Bao, Qiankun; Zhang, Yan; Liu, Mingming; Lv, Huizhen; Liu, Yajin; Yao, Liu; Li, Bochuan; Zhang, Chenghu; He, Shuang; Zhai, Guijin; Zhu, Yan; Liu, Xin; Zhang, Kai; Wang, Xiu-Jie; Zou, Ming-Hui; Zhu, Yi; Ai, Ding

2018-02-16

Angiogenesis is a complex process regulating endothelial cell (EC) functions. Emerging lines of evidence support that YAP (Yes-associated protein) plays an important role in regulating the angiogenic activity of ECs. The objective of this study was to specify the effect of EC YAP on angiogenesis and its underlying mechanisms. In ECs, vascular endothelial growth factor reduced YAP phosphorylation time and dose dependently and increased its nuclear accumulation. Using Tie2Cre-mediated YAP transgenic mice, we found that YAP promoted angiogenesis in the postnatal retina and tumor tissues. Mass spectrometry revealed signal transducer and activator of transcription 3 (STAT3) as a potential binding partner of YAP in ECs. Western blot and immunoprecipitation assays indicated that binding with YAP prolonged interleukin 6-induced STAT3 nuclear accumulation by blocking chromosomal maintenance 1-mediated STAT3 nuclear export without affecting its phosphorylation. Moreover, angiopoietin-2 expression induced by STAT3 was enhanced by YAP overexpression in ECs. Finally, a selective STAT3 inhibitor or angiopoietin-2 blockage partly attenuated retinal angiogenesis in Tie2Cre-mediated YAP transgenic mice. YAP binding sustained STAT3 in the nucleus to enhance the latter's transcriptional activity and promote angiogenesis via regulation of angiopoietin-2. © 2018 American Heart Association, Inc.

Secoisolariciresinol diglucoside prevents the oxidative stress-induced apoptosis of myocardial cells through activation of the JAK2/STAT3 signaling pathway.

PubMed

Huang, Guiqiong; Huang, Xiaofang; Liu, Min; Hua, Yue; Deng, Bo; Jin, Wen; Yan, Wen; Tan, Zhangbin; Wu, Yifen; Liu, Bin; Zhou, Yingchun

2018-06-01

Myocardial cell apoptosis mediated by oxidative stress has previously been identified as a key process in ischemic heart disease. Secoisolariciresinol diglucoside (SDG), a polyphenolic plant lignan primarily found in flaxseed, has been demonstrated to effectively protect myocardial cells from apoptosis. In the present study, the role of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) was investigated in mediating the protective effect of SDG. Findings of the present study revealed that treatment with H2O2 reduced cell viability and induced apoptosis in H9C2 rat cardiomyocytes. However, SDG was able to reduce the effect of H2O2 in a dose‑dependent manner. H2O2 reduced the expression level of phosphorylated STAT3 and inhibited the levels of B‑cell lymphoma‑extra‑large and induced myeloid leukemia cell differentiation protein, which are the STAT3 target genes. Conversely, SDG rescued phosphorylation of STAT3 and increased the levels of STAT3 target genes. Treatment with SDG alone led to a dose‑dependent increased phosphorylation of JAK2 and STAT3, without activating Src. Furthermore, the anti‑apoptotic effects of SDG were partially abolished by a JAK2/STAT3 inhibitor. In addition, molecular docking revealed that SDG may bind to the protein kinase domain of JAK2, at a binding energy of ‑8.258 kcal/mol. Molecular dynamics simulations revealed that JAK2‑SDG binding was stable. In conclusion, activation of the JAK2/STAT3 signaling pathway contributed to the anti‑apoptotic activity of SDG, which may be a potential JAK2 activator.

Proinflammatory Cytokines IL-6 and TNF-α Increased Telomerase Activity through NF-κB/STAT1/STAT3 Activation, and Withaferin A Inhibited the Signaling in Colorectal Cancer Cells

PubMed Central

Okobi, Quincy; Adekoya, Debbie; Atefi, Mohammad; Clarke, Orette; Dutta, Pranabananda; Vadgama, Jaydutt V.

2017-01-01

There are increasing evidences of proinflammatory cytokine involvement in cancer development. Here, we found that two cytokines, IL-6 and TNF-α, activated colorectal cancer cells to be more invasive and stem-like. Combined treatment of IL-6 and TNF-α phosphorylated transcription factors STAT3 in a synergistic manner. STAT3, STAT1, and NF-κB physically interacted upon the cytokine stimulation. STAT3 was bound to the promoter region of human telomerase reverse transcriptase (hTERT). IL-6 and TNF-α stimulation further enhanced STAT3 binding affinity. Stem cell marker Oct-4 was upregulated in colorectal cancer cells upon IL-6 and TNF-α stimulation. Withaferin A, an anti-inflammatory steroidal lactone, inhibited the IL-6- and TNF-α-induced cancer cell invasion and decreased colonosphere formation. Notably, withaferin A inhibited STAT3 phosphorylation and abolished the STAT3, STAT1, and NF-κB interactions. Oct-4 expression was also downregulated by withaferin A inhibition. The binding of STAT3 to the hTERT promoter region and telomerase activity showed reduction with withaferin A treatments. Proinflammatory cytokine-induced cancer cell invasiveness is mediated by a STAT3-regulated mechanism in colorectal cancer cells. Our data suggest that withaferin A could be a promising anticancer agent that effectively inhibits the progression of colorectal cancer. PMID:28676732

Pyruvate dehydrogenase complex (PDC) subunits moonlight as interaction partners of phosphorylated STAT5 in adipocytes and adipose tissue.

PubMed

Richard, Allison J; Hang, Hardy; Stephens, Jacqueline M

2017-12-01

STAT5 proteins play a role in adipocyte development and function, but their specific functions are largely unknown. To this end, we used an unbiased MS-based approach to identify novel STAT5-interacting proteins. We observed that STAT5A bound the E1β and E2 subunits of the pyruvate dehydrogenase complex (PDC). Whereas STAT5A typically localizes to the cytosol or nucleus, PDC normally resides within the mitochondrial matrix where it converts pyruvate to acetyl-CoA. We employed affinity purification and immunoblotting to validate the interaction between STAT5A and PDC subunits in murine and human cultured adipocytes, as well as in adipose tissue. We found that multiple PDC subunits interact with hormone-activated STAT5A in a dose- and time-dependent manner that coincides with tyrosine phosphorylation of STAT5. Using subcellular fractionation and immunofluorescence microscopy, we observed that PDC-E2 is present within the adipocyte nucleus where it associates with STAT5A. Because STAT5A is a transcription factor, we used chromatin immunoprecipitation (ChIP) to assess PDC's ability to interact with STAT5 DNA-binding sites. These analyses revealed that PDC-E2 is bound to a STAT5-binding site in the promoter of the STAT5 target gene c ytokine- i nducible SH 2-containing protein ( cish ). We have demonstrated a compelling interaction between STAT5A and PDC subunits in adipocytes under physiological conditions. There is previous evidence that PDC localizes to cancer cell nuclei where it plays a role in histone acetylation. On the basis of our ChIP data and these previous findings, we hypothesize that PDC may modulate STAT5's ability to regulate gene expression by controlling histone or STAT5 acetylation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Curcumin interacts directly with the Cysteine 259 residue of STAT3 and induces apoptosis in H-Ras transformed human mammary epithelial cells.

PubMed

Hahn, Young-Il; Kim, Su-Jung; Choi, Bu-Young; Cho, Kyung-Cho; Bandu, Raju; Kim, Kwang Pyo; Kim, Do-Hee; Kim, Wonki; Park, Joon Sung; Han, Byung Woo; Lee, Jeewoo; Na, Hye-Kyung; Cha, Young-Nam; Surh, Young-Joon

2018-04-23

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is latent but constitutively activated in many types of cancers. It is well known that STAT3 plays a key role in inflammation-associated tumorigenesis. Curcumin is an anti-inflammatory natural compound isolated from the turmeric (Curcuma longa L., Zingiberaceae) that has been extensively used in a traditional medicine over the centuries. In the present study, we have found that curcumin inhibits STAT3 signaling that is persistently overactivated in H-Ras transformed breast epithelial cells (H-Ras MCF10A). Specific cysteine residues present in STAT3 appear to be critical for the activity as well as conformation of this transcription factor. We identified the cysteine residue 259 of STAT3 as a putative site for curcumin binding. Site-directed mutation of this cysteine residue abolished curcumin-induced inactivation of STAT3 and apoptosis in H-Ras MCF10A cells. The α,β-unsaturated carbonyl moiety of curcumin appears to be essential in its binding to STAT3 in H-Ras MCF10A cells. Tetrahydrocurcumin that lacks such electrophilic moiety failed to interact with STAT3 and to induce apoptosis in the same cell line. Taken together, our findings suggest that curcumin can abrogate aberrant activation of STAT3 through direct interaction, thereby inhibiting STAT3-mediated mammary carcinogenesis.

Molecular cloning, transcriptional profiling, and subcellular localization of signal transducer and activator of transcription 2 (STAT2) ortholog from rock bream, Oplegnathus fasciatus.

PubMed

Bathige, S D N K; Umasuthan, Navaneethaiyer; Priyathilaka, Thanthrige Thiunuwan; Thulasitha, William Shanthakumar; Jayasinghe, J D H E; Wan, Qiang; Nam, Bo-Hye; Lee, Jehee

2017-08-30

Signal transducer and activator of transcription 2 (STAT2) is a key element that transduces signals from the cell membrane to the nucleus via the type I interferon-signaling pathway. Although the structural and functional aspects of STAT proteins are well studied in mammals, information on teleostean STATs is very limited. In this study, a STAT paralog, which is highly homologous to the STAT2 members, was identified from a commercially important fish species called rock bream and designated as RbSTAT2. The RbSTAT2 gene was characterized at complementary DNA (cDNA) and genomic sequence levels, and was found to possess structural features common with its mammalian counterparts. The complete cDNA sequence was distributed into 24 exons in the genomic sequence. The promoter proximal region was analyzed and found to contain potential transcription factor binding sites to regulate the transcription of RbSTAT2. Phylogenetic studies and comparative genomic structure organization revealed the distinguishable evolution for fish and other vertebrate STAT2 orthologs. Transcriptional quantification was performed by SYBR Green quantitative real-time PCR (qPCR) and the ubiquitous expression of RbSTAT2 transcripts was observed in all tissues analyzed from healthy fish, with a remarkably high expression in blood cells. Significantly (P<0.05) altered transcription of RbSTAT2 was detected after immune challenge experiments with viral (rock bream irido virus; RBIV), bacterial (Edwardsiella tarda and Streptococcus iniae), and immune stimulants (poly I:C and LPS). Antiviral potential was further confirmed by WST-1 assay, by measuring the viability of rock bream heart cells treated with RBIV. In addition, results of an in vitro challenge experiment signified the influence of rock bream interleukin-10 (RbIL-10) on transcription of RbSTAT2. Subcellular localization studies by transfection of pEGFP-N1/RbSTAT2 into rock bream heart cells revealed that the RbSTAT2 was usually located in the cytoplasm and translocated near to the nucleus upon poly I:C administration. Altogether, these findings suggest that RbSTAT2 is involved in various biologically crucial mechanisms, and provides immune protection to the rock bream. Copyright © 2017 Elsevier B.V. All rights reserved.

STAT3/NF-κB interactions determine the level of haptoglobin expression in male rats exposed to dietary restriction and/or acute phase stimuli.

PubMed

Uskoković, Aleksandra; Dinić, Svetlana; Mihailović, Mirjana; Grdović, Nevena; Arambašić, Jelena; Vidaković, Melita; Bogojević, Desanka; Ivanović-Matić, Svetlana; Martinović, Vesna; Petrović, Miodrag; Poznanović, Goran; Grigorov, Ilijana

2012-01-01

Haptoglobin is a constitutively expressed protein which is predominantly synthesized in the liver. During the acute-phase (AP) response haptoglobin is upregulated along with other AP proteins. Its upregulation during the AP response is mediated by cis-trans interactions between the hormone-responsive element (HRE) residing in the haptoglobin gene and inducible transcription factors STAT3 and C/EBP β. In male rats that have been subjected to chronic 50% dietary restriction (DR), the basal haptoglobin serum level is decreased. The aim of this study was to characterize the trans-acting factor(s) responsible for the reduction of haptoglobin expression in male rats subjected to 50% DR for 6 weeks. Protein-DNA interactions between C/EBP and STAT families of transcription factors and the HRE region of the haptoglobin gene were examined in livers of male rats subjected to DR, as well as during the AP response that was induced by turpentine administration. In DR rats, we observed associations between the HRE and C/EBPα/β, STAT5b and NF-κB p50, and the absence of interactions between STAT3 and NF-kB p65. Subsequent induction of the AP response in DR rats by turpentine administration elicited a normal, almost 2-fold increase in the serum haptoglobin level that was accompanied by HRE-binding of C/EBPβ, STAT3/5b and NF-kB p65/p50, and the establishment of interaction between STAT3 and NF-κB p65. These results suggest that STAT3 and NF-κB p65 crosstalk plays a central role while C/EBPβ acquires an accessory role in establishing the level of haptoglobin gene expression in male rats exposed to DR and AP stimuli.

The role of STATs in lung carcinogenesis: an emerging target for novel therapeutics.

PubMed

Karamouzis, Michalis V; Konstantinopoulos, Panagiotis A; Papavassiliou, Athanasios G

2007-05-01

The signal transducer and activator of transcription (STAT) proteins are a family of latent cytoplasmic transcription factors, which form dimers when activated by cytokine receptors, tyrosine kinase growth factor receptors as well as non-receptor tyrosine kinases. Dimeric STATs translocate to the nucleus, where they bind to specific DNA-response elements in the promoters of target genes, thereby inducing unique gene expression programs often in association with other transcription regulatory proteins. The functional consequence of different STAT proteins activation varies, as their target genes play diverse roles in normal cellular/tissue functions, including growth, apoptosis, differentiation and angiogenesis. Certain activated STATs have been implicated in human carcinogenesis, albeit only few studies have focused into their role in lung tumours. Converging evidence unravels their molecular interplays and complex multipartite regulation, rendering some of them appealing targets for lung cancer treatment with new developing strategies.

C/EBPβ Promotes STAT3 Expression and Affects Cell Apoptosis and Proliferation in Porcine Ovarian Granulosa Cells.

PubMed

Yuan, Xiaolong; Zhou, Xiaofeng; He, Yingting; Zhong, Yuyi; Zhang, Ailing; Zhang, Zhe; Zhang, Hao; Li, Jiaqi

2018-06-13

Previous studies suggest that signal transducer and activator of transcription 3 (STAT3) and CCAAT/enhancer binding protein beta (C/EBPβ) play an essential role in ovarian granulosa cells (GCs) for mammalian follicular development. Several C/EBPβ putative binding sites were previously predicted on the STAT3 promoter in mammals. However, the molecular regulation of C/EBPβ on STAT3 and their effects on cell proliferation and apoptosis remain virtually unexplored in GCs. Using porcine GCs as a model, the 5′-deletion, luciferase report assay, mutation, chromatin immunoprecipitation, Annexin-V/PI staining and EdU assays were applied to investigate the molecular mechanism for C/EBPβ regulating the expression of STAT3 and their effects on the cell proliferation and apoptosis ability. We found that over and interfering with the expression of C/EBPβ significantly increased and decreased the messenger RNA (mRNA) and protein levels of STAT3 , respectively. The dual luciferase reporter assay showed that C/EBPβ directly bound at −1397/−1387 of STAT3 to positively regulate the mRNA and protein expressions of STAT3 . Both C/EBPβ and STAT3 were observed to inhibit cell apoptosis and promote cell proliferation. Furthermore, C/EBPβ might enhance the antiapoptotic and pro-proliferative effects of STAT3 . These results would be of great insight in further exploring the molecular mechanism of C/EBPβ and STAT3 on the function of GCs and the development of ovarian follicles in mammals.

STAT3 and STAT1 mediate IL-11–dependent and inflammation-associated gastric tumorigenesis in gp130 receptor mutant mice

PubMed Central

Ernst, Matthias; Najdovska, Meri; Grail, Dianne; Lundgren-May, Therese; Buchert, Michael; Tye, Hazel; Matthews, Vance B.; Armes, Jane; Bhathal, Prithi S.; Hughes, Norman R.; Marcusson, Eric G.; Karras, James G.; Na, Songqing; Sedgwick, Jonathon D.; Hertzog, Paul J.; Jenkins, Brendan J.

2008-01-01

Deregulated activation of STAT3 is frequently associated with many human hematological and epithelial malignancies, including gastric cancer. While exaggerated STAT3 signaling facilitates an antiapoptotic, proangiogenic, and proproliferative environment for neoplastic cells, the molecular mechanisms leading to STAT3 hyperactivation remain poorly understood. Using the gp130Y757F/Y757F mouse model of gastric cancer, which carries a mutated gp130 cytokine receptor signaling subunit that cannot bind the negative regulator of cytokine signaling SOCS3 and is characterized by hyperactivation of the signaling molecules STAT1 and STAT3, we have provided genetic evidence that IL-11 promotes chronic gastric inflammation and associated tumorigenesis. Expression of IL-11 was increased in gastric tumors in gp130Y757F/Y757F mice, when compared with unaffected gastric tissue in wild-type mice, while gp130Y757F/Y757F mice lacking the IL-11 ligand–binding receptor subunit (IL-11Rα) showed normal gastric STAT3 activation and IL-11 expression and failed to develop gastric tumors. Furthermore, reducing STAT3 activity in gp130Y757F/Y757F mice, either genetically or by therapeutic administration of STAT3 antisense oligonucleotides, normalized gastric IL-11 expression and alleviated gastric tumor burden. Surprisingly, the genetic reduction of STAT1 expression also reduced gastric tumorigenesis in gp130Y757F/Y757F mice and coincided with reduced gastric inflammation and IL-11 expression. Collectively, our data have identified IL-11 as a crucial cytokine promoting chronic gastric inflammation and associated tumorigenesis mediated by excessive activation of STAT3 and STAT1. PMID:18431520

The Use of Protein-DNA, Chromatin Immunoprecipitation, and Transcriptome Arrays to Describe Transcriptional Circuits in the Dehydrated Male Rat Hypothalamus

PubMed Central

Qiu, Jing; Kleineidam, Anna; Gouraud, Sabine; Yao, Song Tieng; Greenwood, Mingkwan; Hoe, See Ziau; Hindmarch, Charles

2014-01-01

The supraoptic nucleus (SON) of the hypothalamus is responsible for maintaining osmotic stability in mammals through its elaboration of the antidiuretic hormone arginine vasopressin. Upon dehydration, the SON undergoes a function-related plasticity, which includes remodeling of morphology, electrical properties, and biosynthetic activity. This process occurs alongside alterations in steady state transcript levels, which might be mediated by changes in the activity of transcription factors. In order to identify which transcription factors might be involved in changing patterns of gene expression, an Affymetrix protein-DNA array analysis was carried out. Nuclear extracts of SON from dehydrated and control male rats were analyzed for binding to the 345 consensus DNA transcription factor binding sequences of the array. Statistical analysis revealed significant changes in binding to 26 consensus elements, of which EMSA confirmed increased binding to signal transducer and activator of transcription (Stat) 1/Stat3, cellular Myelocytomatosis virus-like cellular proto-oncogene (c-Myc)-Myc-associated factor X (Max), and pre-B cell leukemia transcription factor 1 sequences after dehydration. Focusing on c-Myc and Max, we used quantitative PCR to confirm previous transcriptomic analysis that had suggested an increase in c-Myc, but not Max, mRNA levels in the SON after dehydration, and we demonstrated c-Myc- and Max-like immunoreactivities in SON arginine vasopressin-expressing cells. Finally, by comparing new data obtained from Roche-NimbleGen chromatin immunoprecipitation arrays with previously published transcriptomic data, we have identified putative c-Myc target genes whose expression changes in the SON after dehydration. These include known c-Myc targets, such as the Slc7a5 gene, which encodes the L-type amino acid transporter 1, ribosomal protein L24, histone deactylase 2, and the Rat sarcoma proto-oncogene (Ras)-related nuclear GTPase. PMID:25144923

The fruits of Gleditsia sinensis Lam. inhibits adipogenesis through modulation of mitotic clonal expansion and STAT3 activation in 3T3-L1 cells.

PubMed

Lee, Ji-Hye; Go, Younghoon; Lee, Bonggi; Hwang, Youn-Hwan; Park, Kwang Il; Cho, Won-Kyung; Ma, Jin Yeul

2018-08-10

Gleditsia sinensis Lam. (G. sinensis) has been used in Oriental medicine for tumor, thrombosis, inflammation-related disease, and obesity. The pharmacological inhibitory effects of fruits of G. sinensis (GFE) on hyperlipidemia have been reported, but its inhibitory effects on adipogenesis and underlying mechanisms have not been elucidated. Herein we evaluated the anti-adipogenic effects of GFE and described the underlying mechanisms. The effects of ethanol extracts of GFE on adipocyte differentiation were examined in 3T3-L1 cells using biochemical and molecular analyses. During the differentiation of 3T3-L1 cells, GFE significantly reduced lipid accumulation and downregulated master adipogenic transcription factors, including CCAAT/enhancer-binding protein-α and peroxisome proliferator-activated receptor-γ, at mRNA and protein levels. These changes led to the suppression of several adipogenic-specific genes and proteins, including fatty acid synthase, sterol regulatory element-binding protein 1, stearoyl-CoA desaturase-1, and acetyl CoA carboxylase. However, the inhibitory effects of GFE on lipogenesis were only shown when GFE is treated in the early stage of adipogenesis within the first two days of differentiation. As a potential mechanism, during the early stages of differentiation, GFE inhibited cell proliferation by a decrease in the expression of DNA synthesis-related proteins and increased p27 expression and suppressed signal transducer and activator of transcription 3 (STAT3) activation induced in a differentiation medium. GFE inhibits lipogenesis by negative regulation of adipogenic transcription factors, which is associated with GFE-mediated cell cycle arrest and STAT3 inhibition. Copyright © 2018 Elsevier B.V. All rights reserved.

STAT3-Activated GM-CSFRα Translocates to the Nucleus and Protects CLL Cells from Apoptosis

PubMed Central

Li, Ping; Harris, David; Liu, Zhiming; Rozovski, Uri; Ferrajoli, Alessandra; Wang, Yongtao; Bueso-Ramos, Carlos; Hazan-Halevy, Inbal; Grgurevic, Srdana; Wierda, William; Burger, Jan; O'Brien, Susan; Faderl, Stefan; Keating, Michael; Estrov, Zeev

2014-01-01

Here it was determined that Chronic Lymphocytic Leukemia (CLL) cells express the α-subunit but not the β-subunit of the granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR/CSF3R). GM-CSFRα was detected on the surface, in the cytosol, and the nucleus of CLL cells via confocal microscopy, cell fractionation, and GM-CSFRα antibody epitope mapping. Because STAT3 is frequently activated in CLL and the GM-CSFRα promoter harbors putative STAT3 consensus binding sites, MM1 cells were transfected with truncated forms of the GM-CSFRα promoter, then stimulated with IL-6 to activate STAT3 to identify STAT3 binding sites. Chromatin immunoprecipitation (ChIP) and an electoromobility shift assay (EMSA) confirmed STAT3 occupancy to those promoter regions in both IL-6 stimulated MM1 and CLL cells. Transfection of MM1 cells with STAT3 siRNA or CLL cells with STAT3 shRNA significantly down-regulated GM-CSFRα mRNA and protein levels. RNA transcripts, involved in regulating cell-survival pathways, and the proteins KAP1 (TRIM28) and ISG15 co-immunoprecipitated with GM-CSFRα. GM-CSFRα-bound KAP1 enhanced the transcriptional activity of STAT3, whereas ISG15 inhibited the NF-κB pathway. Nevertheless, overexpression of GM-CSFRα protected MM1 cells from dexamethasone-induced apoptosis, and GM-CSFRα knockdown induced apoptosis in CLL cells, suggesting that GM-CSFRα provides a ligand-independent survival advantage. PMID:24836891

STAT3-activated CD36 facilitates fatty acid uptake in chronic lymphocytic leukemia cells

PubMed Central

Rozovski, Uri; Harris, David M.; Li, Ping; Liu, Zhiming; Jain, Preetesh; Ferrajoli, Alessandra; Burger, Jan; Thompson, Phillip; Jain, Nitin; Wierda, William; Keating, Michael J.; Estrov, Zeev

2018-01-01

Although several studies established that unlike normal B cells chronic lymphocytic leukemia (CLL) cells metabolize fatty acids (FA), how CLL cells internalize FA is poorly understood. Because in various cell types CD36 facilitates FA uptake, we wondered whether a similar mechanism is operative CLL. We found that CD36 levels are higher in CLL cells than in normal B cells, and that small interfering RNA, CD36 neutralizing antibodies or sulfosuccinimidyl oleate (SSO) that inhibits CD36 significantly reduced the oxygen consumption of CLL cells incubated with FA. Because CD36 is oeverexpressed and STAT3 is constitutively activated in CLL cells, we wondered whether STAT3 induces CD36 expression. Sequence analysis identified putative STAT3 binding sites in the CD36 gene promoter. Chromatin immunoprecipitation and an electrophoretic mobility shift assay revealed that STAT3 binds to the CD36 gene promoter. A luciferase assay and STAT3-small hairpin RNA, that significantly decreased the levels of CD36 in CLL cells, established that STAT3 activates the transcription of the CD36 gene. Furthermore, SSO induced a dose-dependent apoptosis of CLL cells. Taken together, our data suggest that STAT3 activates CD36 and that CD36 facilitates FA uptake in CLL cells. Whether CD36 inhibition would provide clinical benefits in CLL remains to be determined. PMID:29765537

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goupille, Olivier; Penglong, Tipparat; Thalassemia Research Center, Mahidol University

The bromodomain and extraterminal (BET) domain family proteins are epigenetic modulators involved in the reading of acetylated lysine residues. The first BET protein inhibitor to be identified, (+)-JQ1, a thienotriazolo-1, 4-diazapine, binds selectively to the acetyl lysine-binding pocket of BET proteins. We evaluated the impact on adipogenesis of this druggable targeting of chromatin epigenetic readers, by investigating the physiological consequences of epigenetic modifications through targeting proteins binding to chromatin. JQ1 significantly inhibited the differentiation of 3T3-L1 preadipocytes into white and brown adipocytes by down-regulating the expression of genes involved in adipogenesis, particularly those encoding the peroxisome proliferator-activated receptor (PPAR-γ), themore » CCAAT/enhancer-binding protein (C/EBPα) and, STAT5A and B. The expression of a constitutively activated STAT5B mutant did not prevent inhibition by JQ1. Thus, the association of BET/STAT5 is required for adipogenesis but STAT5 transcription activity is not the only target of JQ1. Treatment with JQ1 did not lead to the conversion of white adipose tissue into brown adipose tissue (BAT). BET protein inhibition thus interferes with generation of adipose tissue from progenitors, confirming the importance of the connections between epigenetic mechanisms and specific adipogenic transcription factors. - Highlights: • JQ1 prevented the differentiation of 3T3-L1 preadipocytes into white adipocytes. • JQ1 affected clonal cell expansion and abolished lipid accumulation. • JQ1 prevented the differentiation of 3T3-L1 preadipocytes into brown adipocytes. • JQ1 treatment did not lead to the conversion of white adipose tissue into brown adipose tissue. • JQ1 decreased STAT5 expression, but STAT5B{sup ca} expression did not restore adipogenesis.« less

Germline variant FGFR4  p.G388R exposes a membrane-proximal STAT3 binding site.

PubMed

Ulaganathan, Vijay K; Sperl, Bianca; Rapp, Ulf R; Ullrich, Axel

2015-12-24

Variant rs351855-G/A is a commonly occurring single-nucleotide polymorphism of coding regions in exon 9 of the fibroblast growth factor receptor FGFR4 (CD334) gene (c.1162G>A). It results in an amino-acid change at codon 388 from glycine to arginine (p.Gly388Arg) in the transmembrane domain of the receptor. Despite compelling genetic evidence for the association of this common variant with cancers of the bone, breast, colon, prostate, skin, lung, head and neck, as well as soft-tissue sarcomas and non-Hodgkin lymphoma, the underlying biological mechanism has remained elusive. Here we show that substitution of the conserved glycine 388 residue to a charged arginine residue alters the transmembrane spanning segment and exposes a membrane-proximal cytoplasmic signal transducer and activator of transcription 3 (STAT3) binding site Y(390)-(P)XXQ(393). We demonstrate that such membrane-proximal STAT3 binding motifs in the germline of type I membrane receptors enhance STAT3 tyrosine phosphorylation by recruiting STAT3 proteins to the inner cell membrane. Remarkably, such germline variants frequently co-localize with somatic mutations in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. Using Fgfr4 single nucleotide polymorphism knock-in mice and transgenic mouse models for breast and lung cancers, we validate the enhanced STAT3 signalling induced by the FGFR4 Arg388-variant in vivo. Thus, our findings elucidate the molecular mechanism behind the genetic association of rs351855 with accelerated cancer progression and suggest that germline variants of cell-surface molecules that recruit STAT3 to the inner cell membrane are a significant risk for cancer prognosis and disease progression.

Integrity of the LXXLL motif in Stat6 is required for the inhibition of breast cancer cell growth and enhancement of differentiation in the context of progesterone

PubMed Central

2014-01-01

Background Progesterone is essential for the proliferation and differentiation of mammary gland epithelium. Studies of breast cancer cells have demonstrated a biphasic progesterone response consisting of an initial proliferative burst followed by sustained growth arrest. However, the transcriptional factors acting with the progesterone receptor (PR) to mediate the effects of progesterone on mammary cell growth and differentiation remain to be determined. Recently, it was demonstrated that signal transducer and activator of transcription 6 (Stat6) is a cell growth suppressor. Similar to progesterone-bound PR, Stat6 acts by inducing the expression of the G1 cyclin-dependent kinase inhibitors p21 and p27. The possible interaction between Stat6 and progesterone pathways in mammary cells was therefore investigated in the present study. Methods ChIP and luciferase were assayed to determine whether Stat6 induces p21 and p27 expression by recruitment at the proximal Sp1-binding sites of the gene promoters. Immunoprecipitation and Western blotting were performed to investigate the interaction between Stat6 and PR-B. The cellular DNA content and cell cycle distribution in breast cancer cells were analyzed by FACS. Results We found that Stat6 interacts with progesterone-activated PR in T47D cells. Stat6 synergizes with progesterone-bound PR to transactivate the p21 and p27 gene promoters at the proximal Sp1-binding sites. Moreover, Stat6 overexpression and knockdown, respectively, increased or prevented the induction of p21 and p27 gene expression by progesterone. Stat6 knockdown also abolished the inhibitory effects of progesterone on pRB phosphorylation, G1/S cell cycle progression, and cell proliferation. In addition, knockdown of Stat6 expression prevented the induction of breast cell differentiation markers, previously identified as progesterone target genes. Finally, Stat6 gene expression levels increased following progesterone treatment, indicating a positive auto-regulatory loop between PR and Stat6. Conclusions Taken together, these data identify Stat6 as a coactivator of PR mediating the growth-inhibitory and differentiation effects of progesterone on breast cancer cells. PMID:24401087

The transcriptional co-activator p/CIP (NCoA-3) is up-regulated by STAT6 and serves as a positive regulator of transcriptional activation by STAT6.

PubMed

Arimura, Akinori; vn Peer, Maartje; Schröder, Andreas J; Rothman, Paul B

2004-07-23

Transcriptional activation by signal transducer and activator of transcription 6 (STAT6) has been shown to require the direct interaction not only with co-activators such as p300 and cAMP-responsive element-binding protein-binding protein (CBP) but also with nuclear co-activator 1, a member of the p160/steroid receptor co-activator family. Among the p160/steroid receptor co-activators, only p/CIP (nuclear co-activator 3) has been shown to be up-regulated by interleukin (IL)-4 in B cells through a STAT-6-dependent mechanism using Gene-Chip analysis. In this study, we have investigated the function of p/CIP in the transcriptional activation by STAT6. We found that p/CIP indirectly interacted with STAT6 via p300, and overexpression of the CBP-interacting domain of p/CIP (p/CIP(947-1084)) prevented the interaction of p/CIP with STAT6 by blocking the binding of p/CIP to p300. Whereas expression of p/CIP(947-1084) resulted in a marked reduction of STAT6-mediated transactivation, overexpression of wild type p/CIP resulted in significant enhancement of it. In addition, p/CIP(947-1084) markedly reduced CD23 expression on B cells stimulated with IL-4, whereas overexpression of wild type p/CIP enhanced it. Chromatin immunoprecipitations demonstrate that IL-4 increases the interaction of p/CIP with the murine immunoglobulin heavy chain germ line epsilon promoter in B cells. These results suggest that p/CIP positively regulates STAT6 transcriptional activation through formation of a STAT6, p300/CBP, and p/CIP complex.

Carbon monoxide-releasing molecule-3 suppresses Prevotella intermedia lipopolysaccharide-induced production of nitric oxide and interleukin-1β in murine macrophages.

PubMed

Choi, Eun-Young; Choe, So-Hui; Hyeon, Jin-Yi; Choi, Jeom-Il; Choi, In Soon; Kim, Sung-Jo

2015-10-05

This study was performed to analyze the effect of carbon monoxide (CO)-releasing molecule-3 (CORM-3) in alleviating the production of proinflammatory mediators in macrophages treated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen associated with periodontal disease, and its possible mechanisms of action. LPS was isolated using the hot phenol-water method. Culture supernatants were assayed for nitric oxide (NO) and interleukin-1β (IL-1β). Gene expression was quantified by real-time PCR, and protein expression by immunoblotting. DNA-binding activities of NF-κB subunits were determined using an ELISA-based kit. CORM-3 suppressed the production of inducible NO synthase (iNOS)-derived NO and IL-1β at both gene transcription and translation levels in P. intermedia LPS-activated RAW264.7 cells. CORM-3 enhanced heme oxygenase-1 (HO-1) expression in cells stimulated with P. intermedia LPS, and inhibition of HO-1 activity by SnPP notably reversed the suppressive effect of CORM-3 on LPS-induced production of NO. LPS-induced phosphorylation of p38 and JNK was not affected by CORM-3. CORM-3 did not influence P. intermedia LPS-induced degradation of IκB-α. Instead, nuclear translocation of NF-κB p65 and p50 subunits was blocked by CORM-3 in LPS-treated cells. In addition, CORM-3 reduced LPS-induced p65 and p50 binding to DNA. Besides, CORM-3 significantly suppressed P. intermedia LPS-induced phosphorylation of STAT1. Overall, this study indicates that CORM-3 suppresses the production of NO and IL-1β in P. intermedia LPS-activated murine macrophages via HO-1 induction and inhibition of NF-κB and STAT1 pathways. The modulation of host inflammatory response by CORM-3 would be an attractive therapeutic approach to attenuate the progression of periodontal disease. Copyright © 2015 Elsevier B.V. All rights reserved.

Transcriptional activation of the suppressor of cytokine signaling-3 (SOCS-3) gene via STAT3 is increased in F9 REX1 (ZFP-42) knockout teratocarcinoma stem cells relative to wild-type cells.

PubMed

Xu, Juliana; Sylvester, Renia; Tighe, Ann P; Chen, Siming; Gudas, Lorraine J

2008-03-14

Rex1 (Zfp42), first identified as a gene that is transcriptionally repressed by retinoic acid (RA), encodes a zinc finger transcription factor expressed at high levels in F9 teratocarcinoma stem cells, embryonic stem cells, and other stem cells. Loss of both alleles of Rex1 by homologous recombination alters the RA-induced differentiation of F9 cells, a model of pluripotent embryonic stem cells. We identified Suppressor of Cytokine Signaling-3 (SOCS-3) as a gene that exhibits greatly increased transcriptional activation in RA, cAMP, and theophylline (RACT)-treated F9 Rex1(-/-) cells (approximately 25-fold) as compared to wild-type (WT) cells ( approximately 2.5-fold). By promoter deletion, mutation, and transient transfection analyses, we have shown that this transcriptional increase is mediated by the STAT3 DNA-binding elements located between -99 to -60 in the SOCS-3 promoter. Overexpression of STAT3 dominant-negative mutants greatly diminishes this SOCS-3 transcriptional increase in F9 Rex1(-/-) cells. This increase in SOCS-3 transcription is associated with a four- to fivefold higher level of tyrosine-phosphorylated STAT3 in the RACT-treated F9 Rex1(-/-) cells as compared to WT. Dominant-negative Src tyrosine kinase, Jak2, and protein kinase A partially reduce the transcriptional activation of the SOCS 3 gene in RACT-treated F9 Rex1 null cells. In contrast, parathyroid hormone peptide enhances the effect of RA in F9 Rex1(-/-) cells, but not in F9 WT. Thus, Rex1, which is highly expressed in stem cells, inhibits signaling via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, thereby modulating the differentiation of F9 cells.

The ebolavirus VP24 interferon antagonist

PubMed Central

Zhang, Adrianna P.P.; Abelson, Dafna M.; Bornholdt, Zachary A.; Liu, Tong; Woods, Jr, Virgil L.; Saphire, Erica Ollmann

2012-01-01

Suppression during the early phases of the immune system often correlates directly with a fatal outcome for the host. The ebolaviruses, some of the most lethal viruses known, appear to cripple initial stages of the host defense network via multiple distinct paths. Two of the eight viral proteins are critical for immunosuppression. One of these proteins is VP35, which binds double-stranded RNA and antagonizes several antiviral signaling pathways.1,2 The other protein is VP24, which binds transporter molecules to prevent STAT1 translocation.3 A more recent discovery is that VP24 also binds STAT1 directly,4 suggesting that VP24 may operate in at least two separate branches of the interferon pathway. New crystal structures of VP24 derived from pathogenic and nonpathogenic ebolaviruses reveal its novel, pyramidal fold, upon which can be mapped sites required for virulence and for STAT1 binding. These structures of VP24, and new information about its direct binding to STAT1, provide avenues by which we may explore its many roles in the viral life cycle, and reasons for differences in pathogenesis among the ebolaviruses. PMID:23076242

STAT3 or USF2 Contributes to HIF Target Gene Specificity

PubMed Central

Pawlus, Matthew R.; Wang, Liyi; Murakami, Aya; Dai, Guanhai; Hu, Cheng-Jun

2013-01-01

The HIF1- and HIF2-mediated transcriptional responses play critical roles in solid tumor progression. Despite significant similarities, including their binding to promoters of both HIF1 and HIF2 target genes, HIF1 and HIF2 proteins activate unique subsets of target genes under hypoxia. The mechanism for HIF target gene specificity has remained unclear. Using siRNA or inhibitor, we previously reported that STAT3 or USF2 is specifically required for activation of endogenous HIF1 or HIF2 target genes. In this study, using reporter gene assays and chromatin immuno-precipitation, we find that STAT3 or USF2 exhibits specific binding to the promoters of HIF1 or HIF2 target genes respectively even when over-expressed. Functionally, HIF1α interacts with STAT3 to activate HIF1 target gene promoters in a HIF1α HLH/PAS and N-TAD dependent manner while HIF2α interacts with USF2 to activate HIF2 target gene promoters in a HIF2α N-TAD dependent manner. Physically, HIF1α HLH and PAS domains are required for its interaction with STAT3 while both N- and C-TADs of HIF2α are involved in physical interaction with USF2. Importantly, addition of functional USF2 binding sites into a HIF1 target gene promoter increases the basal activity of the promoter as well as its response to HIF2+USF2 activation while replacing HIF binding site with HBS from a HIF2 target gene does not change the specificity of the reporter gene. Importantly, RNA Pol II on HIF1 or HIF2 target genes is primarily associated with HIF1α or HIF2α in a STAT3 or USF2 dependent manner. Thus, we demonstrate here for the first time that HIF target gene specificity is achieved by HIF transcription partners that are required for HIF target gene activation, exhibit specific binding to the promoters of HIF1 or HIF2 target genes and selectively interact with HIF1α or HIF2α protein. PMID:23991099

Expression and Purification of Soluble STAT5b/STAT3 Proteins for SH2 Domain Binding Assay.

PubMed

Asai, Akira; Takakuma, Kazuyuki

2017-01-01

When a large hydrophobic full-length protein is expressed in bacteria, it is often challenging to obtain recombinant proteins in the soluble fraction. One way to overcome this challenge is expression of deletion mutants that have improved solubility while maintaining biological activity. In this chapter, we describe a protocol for expression of truncated forms of STAT5b and STAT3 proteins that are soluble and retain SH2-mediated activity for phospho-Tyr peptide recognition.

Quercetin inhibits the poly(dA:dT)-induced secretion of IL-18 via down-regulation of the expressions of AIM2 and pro-caspase-1 by inhibiting the JAK2/STAT1 pathway in IFN-γ-primed human keratinocytes.

PubMed

Lee, Kyung-Mi; Kang, Jung Hoon; Yun, Mihee; Lee, Seong-Beom

2018-06-05

Quercetin, a polyphenol, belongs to a class of flavonoids that exerts anti-inflammatory effects. Interleukin (IL)-18 is a member of the IL-1 family cytokine that regulates immune responses and is implicated in various inflammatory skin diseases. Absent in melanoma 2 (AIM2) is a cytosolic double-stranded (ds) DNA sensor that recognizes the dsDNA of a microbial or host origin. Binding of dsDNA to AIM2 simulates caspase-1-dependent inflammasome activity, which leads to the production of IL-1β and IL-18. Increased levels of AIM2 have been observed in patients with inflammatory skin diseases. In the current study, we investigated the issue of whether or how Quercetin attenuates poly (dA:dT), a synthetic analog of microbial dsDNA, -induced IL-18 secretion in IFN-γ-primed human keratinocytes. Treatment with 5 and 10 μM of Quercetin inhibited the poly (dA:dT)-induced secretion of IL-18 after IFN-γ priming and before poly (dA:dT)-induced AIM2 activation. In addition, treatment with Quercetin at 10 μM, significantly inhibited the phosphorylation of JAK2 and STAT1, and the nuclear translocation of phosphorylated STAT1 in poly (dA:dT)-treated and IFN-γ-primed keratinocytes. These results suggest that treatment with Quercetin inhibits the poly (dA:dT)-induced secretion of IL-18 via down-regulation of the expressions of AIM2 and pro-caspase-1 by inhibiting the JAK2/STAT1 pathway in IFN-γ-primed keratinocytes. Copyright © 2018 Elsevier Inc. All rights reserved.

IFN-gamma priming up-regulates IFN-stimulated gene factor 3 (ISGF3) components, augmenting responsiveness of IFN-resistant melanoma cells to type I IFNs.

PubMed

Wong, L H; Hatzinisiriou, I; Devenish, R J; Ralph, S J

1998-06-01

IFN-stimulated gene factor 3 (ISGF3) mediates transcriptional activation of IFN-sensitive genes (ISGs). The component subunits of ISGF3, STAT1alphabeta, STAT2, and p48-ISGF3gamma, are tyrosine phosphorylated before their assembly into a complex. Subsequently, the ISGF3 complex is translocated to the nucleus. We have recently established that the responsiveness of human melanoma cell lines to type I IFNs correlates directly with their intracellular levels of ISGF3 components, particularly STAT1. In the present study, we show that pretreating IFN-resistant melanoma cell lines with IFN-gamma (IFN-gamma priming) before stimulation with type I IFN also results in increased levels of ISGF3 components and enhanced DNA-binding activation of ISGF3. In addition, IFN-gamma priming of IFN-resistant melanoma cell lines increased expression of type I IFN-induced ISG products, including ISG54, 2'-5'-oligoadenylate synthase, HLA class I, B7-1, and ICAM-1 Ags. Furthermore, IFN-gamma priming enhanced the antiviral effect of IFN-beta on the IFN-resistant melanoma cell line, MM96. These results support a role for IFN-gamma priming in up-regulating ISGF3, thereby augmenting the responsiveness of IFN-resistant melanoma cell lines to type I IFN and providing a molecular basis and justification for using sequential IFN therapy, as proposed by others, to enhance the use of IFNs in the treatment of melanoma.

Tissue-Specific Autoregulation of the stat3 Gene and Its Role in Interleukin-6-Induced Survival Signals in T Cells

PubMed Central

Narimatsu, Masahiro; Maeda, Hisoka; Itoh, Shousaku; Atsumi, Toru; Ohtani, Takuya; Nishida, Keigo; Itoh, Motoyuki; Kamimura, Daisuke; Park, Sung-Joo; Mizuno, Katsunori; Miyazaki, Jun-ichi; Hibi, Masahiko; Ishihara, Katsuhiko; Nakajima, Koichi; Hirano, Toshio

2001-01-01

Signal transducer and activator of transcription 3 (STAT3) mediates signals of various growth factors and cytokines, including interleukin-6 (IL-6). In certain IL-6-responsive cell lines, the stat3 gene is autoregulated by STAT3 through a composite IL-6 response element in its promoter that contains a STAT3-binding element (SBE) and a cyclic AMP-responsive element. To reveal the nature and roles of the stat3 autoregulation in vivo, we generated mice that harbor a mutation in the SBE (stat3mSBE). The intact SBE was crucial for IL-6-induced stat3 gene activation in the spleen, especially in the red pulp region, the kidney, and both mature and immature T lymphocytes. The SBE was not required, however, for IL-6-induced stat3 gene activation in hepatocytes. T lymphocytes from the stat3mSBE/mSBE mice were more susceptible to apoptosis despite the presence of IL-6 than those from wild-type mice. Consistent with this, IL-6-dependent activation of the Pim-1 and junB genes, direct target genes for STAT3, was attenuated in T lymphocytes of the stat3mSBE/mSBE mice. Thus, the tissue-specific autoregulation of the stat3 gene operates in vivo and plays a role in IL-6-induced antiapoptotic signaling in T cells. PMID:11533249

Withaferin A Inhibits STAT3 and Induces Tumor Cell Death in Neuroblastoma and Multiple Myeloma

PubMed Central

Yco, Lisette P; Mocz, Gabor; Opoku-Ansah, John; Bachmann, André S

2014-01-01

Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor that has been implicated in many human cancers and has emerged as an ideal target for cancer therapy. Withaferin A (WFA) is a natural product with promising antiproliferative properties through its association with a number of molecular targets including STAT3. However, the effect of WFA in pediatric neuroblastoma (NB) and its interaction with STAT3 have not been reported. In this study, we found that WFA effectively induces dose-dependent cell death in high-risk and drug-resistant NB as well as multiple myeloma (MM) tumor cells, prevented interleukin-6 (IL-6)–mediated and persistently activated STAT3 phosphorylation at Y705, and blocked the transcriptional activity of STAT3. We further provide computational models that show that WFA binds STAT3 near the Y705 phospho-tyrosine residue of the STAT3 Src homology 2 (SH2) domain, suggesting that WFA prevents STAT3 dimer formation similar to BP-1-102, a well-established STAT3 inhibitor. Our findings propose that the antitumor activity of WFA is mediated at least in part through inhibition of STAT3 and provide a rationale for further drug development and clinical use in NB and MM. PMID:25452693

Withaferin A Inhibits STAT3 and Induces Tumor Cell Death in Neuroblastoma and Multiple Myeloma.

PubMed

Yco, Lisette P; Mocz, Gabor; Opoku-Ansah, John; Bachmann, André S

2014-01-01

Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor that has been implicated in many human cancers and has emerged as an ideal target for cancer therapy. Withaferin A (WFA) is a natural product with promising antiproliferative properties through its association with a number of molecular targets including STAT3. However, the effect of WFA in pediatric neuroblastoma (NB) and its interaction with STAT3 have not been reported. In this study, we found that WFA effectively induces dose-dependent cell death in high-risk and drug-resistant NB as well as multiple myeloma (MM) tumor cells, prevented interleukin-6 (IL-6)-mediated and persistently activated STAT3 phosphorylation at Y705, and blocked the transcriptional activity of STAT3. We further provide computational models that show that WFA binds STAT3 near the Y705 phospho-tyrosine residue of the STAT3 Src homology 2 (SH2) domain, suggesting that WFA prevents STAT3 dimer formation similar to BP-1-102, a well-established STAT3 inhibitor. Our findings propose that the antitumor activity of WFA is mediated at least in part through inhibition of STAT3 and provide a rationale for further drug development and clinical use in NB and MM.

Combinatorial activation and concentration-dependent repression of the Drosophila even skipped stripe 3+7 enhancer

PubMed Central

Struffi, Paolo; Corado, Maria; Kaplan, Leah; Yu, Danyang; Rushlow, Christine; Small, Stephen

2011-01-01

Despite years of study, the precise mechanisms that control position-specific gene expression during development are not understood. Here, we analyze an enhancer element from the even skipped (eve) gene, which activates and positions two stripes of expression (stripes 3 and 7) in blastoderm stage Drosophila embryos. Previous genetic studies showed that the JAK-STAT pathway is required for full activation of the enhancer, whereas the gap genes hunchback (hb) and knirps (kni) are required for placement of the boundaries of both stripes. We show that the maternal zinc-finger protein Zelda (Zld) is absolutely required for activation, and present evidence that Zld binds to multiple non-canonical sites. We also use a combination of in vitro binding experiments and bioinformatics analysis to redefine the Kni-binding motif, and mutational analysis and in vivo tests to show that Kni and Hb are dedicated repressors that function by direct DNA binding. These experiments significantly extend our understanding of how the eve enhancer integrates positive and negative transcriptional activities to generate sharp boundaries in the early embryo. PMID:21865322

Rhodium(II) proximity-labeling identifies a novel target site on STAT3 for inhibitors with potent anti-leukemia activity

PubMed Central

Minus, Matthew B.; Liu, Wei; Vohidov, Farrukh; Kasembeli, Moses M.; Long, Xin; Krueger, Michael; Stevens, Alexandra; Kolosov, Mikhail I.; Sison, Edward Allen R.; Ball, Zachary T.

2015-01-01

Nearly 40% of children with acute myeloid leukemia (AML) suffer relapse due to chemoresistance, often involving upregulation of the oncoprotein STAT3 (signal transducer and activator of transcription 3). In this paper, rhodium(II)-catalyzed, proximity-driven modification identifies the STAT3 coiled-coil domain (CCD) as a novel ligand-binding site, and we describe a new naphthalene sulfonamide inhibitor that targets the CCD, blocks STAT3 function, and halts its disease-promoting effects in vitro, in tumor growth models, and in a leukemia mouse model, validating this new therapeutic target for resistant AML. PMID:26480340

Nuclear localization of pyruvate dehydrogenase complex-E2 (PDC-E2), a mitochondrial enzyme, and its role in signal transducer and activator of transcription 5 (STAT5)-dependent gene transcription.

PubMed

Chueh, Fu-Yu; Leong, King-Fu; Cronk, Robert J; Venkitachalam, Srividya; Pabich, Samantha; Yu, Chao-Lan

2011-07-01

STAT (signal transducer and activator of transcription) proteins play a critical role in cellular response to a wide variety of cytokines and growth factors by regulating specific nuclear genes. STAT-dependent gene transcription can be finely tuned through the association with co-factors in the nucleus. We showed previously that STAT5 (including 5a and 5b) specifically interacts with a mitochondrial enzyme PDC-E2 (E2 subunit of pyruvate dehydrogenase complex) in both leukemic T cells and cytokine-stimulated cells. However, the functional significance of this novel association remains largely unknown. Here we report that PDC-E2 may function as a co-activator in STAT5-dependent nuclear gene expression. Subcellular fractionation analysis revealed that a substantial amount of PDC-E2 was constitutively present in the nucleus of BaF3, an interleukin-3 (IL-3)-dependent cell line. IL-3-induced tyrosine-phosphorylated STAT5 associated with nuclear PDC-E2 in co-immunoprecipitation analysis. These findings were confirmed by confocal immunofluorescence microscopy showing constant nuclear localization of PDC-E2 and its co-localization with STAT5 after IL-3 stimulation. Similar to mitochondrial PDC-E2, nuclear PDC-E2 was lipoylated and associated with PDC-E1. Overexpression of PDC-E2 in BaF3 cells augmented IL-3-induced STAT5 activity as measured by reporter assay with consensus STAT5-binding sites. Consistent with the reporter data, PDC-E2 overexpression in BaF3 cells led to elevated mRNA levels of endogenous SOCS3 (suppressor of cytokine signaling 3) gene, a known STAT5 target. We further identified two functional STAT5-binding sites in the SOCS3 gene promoter important for its IL-3-inducibility. The observation that both cis-acting elements were essential to detect the stimulatory effect by PDC-E2 strongly supports the role of PDC-E2 in up-regulating the transactivating ability of STAT5. All together, our results reveal a novel function of PDC-E2 in the nucleus. It also raises the possibility of nuclear-mitochondrial crosstalk through the interaction between STAT5 and PDC-E2. Copyright © 2011 Elsevier Inc. All rights reserved.

Nuclear localization of pyruvate dehydrogenase complex-E2 (PDC-E2), a mitochondrial enzyme, and its role in signal transducer and activator of transcription 5 (STAT5)-dependent gene transcription

PubMed Central

Chueh, Fu-Yu; Leong, King-Fu; Cronk, Robert J.; Venkitachalam, Srividya; Pabich, Samantha; Yu, Chao-Lan

2011-01-01

STAT (signal transducer and activator of transcription) proteins play a critical role in cellular response to a wide variety of cytokines and growth factors by regulating specific nuclear genes. STAT-dependent gene transcription can be finely tuned through the association with cofactors in the nucleus. We showed previously that STAT5 (including 5a and 5b) specifically interacts with a mitochondrial enzyme PDC-E2 (E2 subunit of pyruvate dehydrogenase complex) in both leukemic T cells and cytokine-stimulated cells. However, the functional significance of this novel association remains largely unknown. Here we report that PDC-E2 may function as a co-activator in STAT5-dependent nuclear gene expression. Subcellular fractionation analysis revealed that a substantial amount of PDC-E2 was constitutively present in the nucleus of BaF3, an interleukin-3 (IL-3)-dependent cell line. IL-3-induced tyrosine-phosphorylated STAT5 associated with nuclear PDC-E2 in co-immunoprecipitation analysis. These findings were confirmed by confocal immunofluorescence microscopy showing constant nuclear localization of PDC-E2 and its co-localization with STAT5 after IL-3 stimulation. Similar to mitochondrial PDC-E2, nuclear PDC-E2 was lipoylated and associated with PDC-E1. Overexpression of PDC-E2 in BaF3 cells augmented IL-3-induced STAT5 activity as measured by reporter assay with consensus STAT5-binding sites. Consistent with the reporter data, PDC-E2 overexpression in BaF3 cells led to elevated mRNA levels of endogenous SOCS3 (suppressor of cytokine signaling 3) gene, a known STAT5 target. We further identified two functional STAT5-binding sites in the SOCS3 gene promoter important for its IL-3-inducibility. The observation that both cis-acting elements were essential to detect the stimulatory effect by PDC-E2 strongly supports the role of PDC-E2 in up-regulating the transactivating ability of STAT5. All together, our results reveal a novel function of PDC-E2 in the nucleus. It also raises the possibility of nuclear-mitochondrial crosstalk through the interaction between STAT5 and PDC-E2. PMID:21397011

Stat3 phosphorylation is required for embryonic stem cells ground state maintenance in 2i culture media.

PubMed

Wang, Dan; Sang, Hui; Zhang, Kaiyue; Nie, Yan; Zhao, Shuang; Zhang, Yan; He, Ningning; Wang, Yuebing; Xu, Yang; Xie, Xiaoyan; Li, Zongjin; Liu, Na

2017-05-09

Embryonic stem cells (ES cells) can be maintained its undifferentiated state with feeder cells or LIF, which can activate Jak/Stat3 pathway. Recently, it has been reported a new culture condition comprising serum-free medium with ERK and GSK3β inhibitors (2i) could drive ES cells into a state of pluripotency more like inner cell mass (ICM) in mouse blastocysts called ground state. However, although 2i could sustain ES cells self-renewal, LIF is routinely added. The roles of Stat3 activation are still unclear now. Here we investigated whether Jak/Stat3 might also contribute to the induction of ground state pluripotency. We introduced a lentiviral construct with 7-repeat Stat3-binding sequence to drive Renilla luciferase into ES cells, which can be used as a reporter to detect Stat3 activation by noninvasive bioluminescence imaging. Using this ES cells, we investigated the role of Stat3 activation in ground state maintenance. The results showed that Stat3 could be activated by 2i. Stattic, a chemical inhibitor of Stat3 phosphorylation, could effectively inhibit Stat3 activation in ES cells. When Stat3 activation was suppressed, ground state related genes were down regulated, and ES cells could not be maintained the ground state pluripotency even in 2i medium. All of these results indicate Stat3 activation is required in ground state maintenance.

Discovery of novel STAT3 small molecule inhibitors via in silico site-directed fragment-based drug design.

PubMed

Yu, Wenying; Xiao, Hui; Lin, Jiayuh; Li, Chenglong

2013-06-13

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been validated as an attractive therapeutic target for cancer therapy. To stop both STAT3 activation and dimerization, a viable strategy is to design inhibitors blocking its SH2 domain phosphotyrosine binding site that is responsible for both actions. A new fragment-based drug design (FBDD) strategy, in silico site-directed FBDD, was applied in this study. A designed novel compound, 5,8-dioxo-6-(pyridin-3-ylamino)-5,8-dihydronaphthalene-1-sulfonamide (LY5), was confirmed to bind to STAT3 SH2 by fluorescence polarization assay. In addition, four out of the five chosen compounds have IC50 values lower than 5 μM for the U2OS cancer cells. 8 (LY5) has an IC50 range in 0.5-1.4 μM in various cancer cell lines. 8 also suppresses tumor growth in an in vivo mouse model. This study has demonstrated the utility of this approach and could be used to other drug targets in general.

Cyclophilins contribute to Stat3 signaling and survival of multiple myeloma cells.

PubMed

Bauer, K; Kretzschmar, A K; Cvijic, H; Blumert, C; Löffler, D; Brocke-Heidrich, K; Schiene-Fischer, C; Fischer, G; Sinz, A; Clevenger, C V; Horn, F

2009-08-06

Signal transducer and activator of transcription 3 (Stat3) is the major mediator of interleukin-6 (IL-6) family cytokines. In addition, Stat3 is known to be involved in the pathophysiology of many malignancies. Here, we show that the cis-trans peptidyl-prolyl isomerase cyclophilin (Cyp) B specifically interacts with Stat3, whereas the highly related CypA does not. CypB knockdown inhibited the IL-6-induced transactivation potential but not the tyrosine phosphorylation of Stat3. Binding of CypB to Stat3 target promoters and alteration of the intranuclear localization of Stat3 on CypB depletion suggested a nuclear function of Stat3/CypB interaction. By contrast, CypA knockdown inhibited Stat3 IL-6-induced tyrosine phosphorylation and nuclear translocation. The Cyp inhibitor cyclosporine A (CsA) caused similar effects. However, Stat1 activation in response to IL-6 or interferon-gamma was not affected by Cyp silencing or CsA treatment. As a result, Cyp knockdown shifted IL-6 signaling to a Stat1-dominated pathway. Furthermore, Cyp depletion or treatment with CsA induced apoptosis in IL-6-dependent multiple myeloma cells, whereas an IL-6-independent line was not affected. Thus, Cyps support the anti-apoptotic action of Stat3. Taken together, CypA and CypB both play pivotal roles, yet at different signaling levels, for Stat3 activation and function. These data also suggest a novel mechanism of CsA action.

Synthetic Oligodeoxynucleotides (ODN) Containing Suppressive TTAGGG Motifs Inhibit AIM2 Inflammasome Activation

PubMed Central

Kaminski, John J.; Schattgen, Stefan A.; Tzeng, Te-Chen; Bode, Christian; Klinman, Dennis M.; Fitzgerald, Katherine A.

2013-01-01

Synthetic oligodeoxynucleotides comprised of the immunosuppressive motif TTAGGG block TLR9 signaling, prevent STAT1 and STAT4 phosphorylation and attenuate a variety of inflammatory responses in vivo. Here, we demonstrate that such suppressive oligodeoxynucleotides (sup ODN) abrogate activation of cytosolic nucleic acid sensing pathways. Pretreatment of dendritic cells and macrophages with the suppressive ODN-A151 abrogated type I IFN, TNFα and ISG induction in response to cytosolic dsDNA. In addition, A151 abrogated caspase-1-dependent IL-1β and IL-18 maturation in dendritic cells stimulated with dsDNA and murine cytomegalovirus (MCMV). Inhibition was dependent on A151’s phosphorothioate backbone while substitution of the guanosine residues for adenosine negatively affected potency. A151 mediates these effects by binding to AIM2 in a manner that is competitive with immune-stimulatory DNA and as a consequence prevents AIM2 inflammasome complex formation. Collectively, these findings reveal a new route by which suppressive ODNs modulate the immune system and unveil novel applications for suppressive ODNs in the treatment of infectious and autoimmune diseases. PMID:23986531

Feedback activation of STAT3 mediates trastuzumab resistance via upregulation of MUC1 and MUC4 expression

PubMed Central

Li, Wei; Fan, Kexing; Qian, Weizhu; Hou, Sheng; Wang, Hao; Dai, Jianxin; Wei, Huafeng; Guo, Yajun

2014-01-01

Although HER2-targeting antibody trastuzumab confers a substantial benefit for patients with HER2-overexpressing breast and gastric cancer, overcoming trastuzumab resistance remains a large unmet need. In this study, we revealed a STAT3-centered positive feedback loop that mediates the resistance of trastuzumab. Mechanistically, chronic exposure of trastuzumab causes the upregulation of fibronection (FN), EGF and IL-6 in parental trastuzumab-sensitive breast and gastric cells and convergently leads to STAT3 hyperactivation. Activated STAT3 enhances the expression of FN, EGF and IL-6, thus constituting a positive feedback loop which amplifies and maintains the STAT3 signal; furthermore, hyperactivated STAT3 signal promotes the expression of MUC1 and MUC4, consequently mediating trastuzumab resistance via maintenance of persistent HER2 activation and masking of trastuzumab binding to HER2 respectively. Genetic or pharmacological inhibition of STAT3 disrupted STAT3-dependent positive feedback loop and recovered the trastuzumab sensitivity partially due to increased apoptosis induction. Combined trastuzumab with STAT3 inhibition synergistically suppressed the growth of the trastuzumab-resistant tumor xenografts in vivo. Taken together, our results suggest that feedback activation of STAT3 constitutes a key node mediating trastuzumab resistance. Combinatorial targeting on both HER2 and STAT3 may enhance the efficacy of trastuzumab or other HER2-targeting agents in HER2-positive breast and gastric cancer. PMID:25327561

Feedback activation of STAT3 mediates trastuzumab resistance via upregulation of MUC1 and MUC4 expression.

PubMed

Li, Guangchao; Zhao, Likun; Li, Wei; Fan, Kexing; Qian, Weizhu; Hou, Sheng; Wang, Hao; Dai, Jianxin; Wei, Huafeng; Guo, Yajun

2014-09-30

Although HER2-targeting antibody trastuzumab confers a substantial benefit for patients with HER2-overexpressing breast and gastric cancer, overcoming trastuzumab resistance remains a large unmet need. In this study, we revealed a STAT3-centered positive feedback loop that mediates the resistance of trastuzumab. Mechanistically, chronic exposure of trastuzumab causes the upregulation of fibronection (FN), EGF and IL-6 in parental trastuzumab-sensitive breast and gastric cells and convergently leads to STAT3 hyperactivation. Activated STAT3 enhances the expression of FN, EGF and IL-6, thus constituting a positive feedback loop which amplifies and maintains the STAT3 signal; furthermore, hyperactivated STAT3 signal promotes the expression of MUC1 and MUC4, consequently mediating trastuzumab resistance via maintenance of persistent HER2 activation and masking of trastuzumab binding to HER2 respectively. Genetic or pharmacological inhibition of STAT3 disrupted STAT3-dependent positive feedback loop and recovered the trastuzumab sensitivity partially due to increased apoptosis induction. Combined trastuzumab with STAT3 inhibition synergistically suppressed the growth of the trastuzumab-resistant tumor xenografts in vivo. Taken together, our results suggest that feedback activation of STAT3 constitutes a key node mediating trastuzumab resistance. Combinatorial targeting on both HER2 and STAT3 may enhance the efficacy of trastuzumab or other HER2-targeting agents in HER2-positive breast and gastric cancer.

Rhodium(II) Proximity-Labeling Identifies a Novel Target Site on STAT3 for Inhibitors with Potent Anti-Leukemia Activity.

PubMed

Minus, Matthew B; Liu, Wei; Vohidov, Farrukh; Kasembeli, Moses M; Long, Xin; Krueger, Michael J; Stevens, Alexandra; Kolosov, Mikhail I; Tweardy, David J; Sison, Edward Allan R; Redell, Michele S; Ball, Zachary T

2015-10-26

Nearly 40 % of children with acute myeloid leukemia (AML) suffer relapse arising from chemoresistance, often involving upregulation of the oncoprotein STAT3 (signal transducer and activator of transcription 3). Herein, rhodium(II)-catalyzed, proximity-driven modification identifies the STAT3 coiled-coil domain (CCD) as a novel ligand-binding site, and we describe a new naphthalene sulfonamide inhibitor that targets the CCD, blocks STAT3 function, and halts its disease-promoting effects in vitro, in tumor growth models, and in a leukemia mouse model, validating this new therapeutic target for resistant AML. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Subcellular localization and functional characterization of a fish IRF9 from crucian carp Carassius auratus.

PubMed

Shi, Jun; Zhang, Yi-Bing; Liu, Ting-Kai; Sun, Fan; Gui, Jian-Fang

2012-08-01

Mammalian interferon (IFN) regulatory factor 9 (IRF-9) has long been recognized as the DNA sequence recognition subunit of IFN-stimulated gene factor 3 (ISGF3) complex, which is critical for type I IFN to induce the expression of IFN-stimulated genes (ISGs) against viral infection. Recent studies have shown that fish IFN exerts antiviral effects by induction of a number of ISGs and also of itself; however, little is known about the role of fish IRF9 in IFN signaling. Here we identify a fish IRF9 orthologue (CaIRF9) from IFN-producing cell line, crucian carp Carassius auratus blastulae embryonic (CAB) cells. Analysis of subcellular distribution of CaIRF9-green fluorescent protein indicates that CaIRF9 is constitutively present in the nucleus, which is driven by two nuclear localization signals (NLS), one locating within DNA-binding domain (DBD) of CaIRF9 and the other immediately behind DBD, although human IRF9 contains only one NLS analogous to the former of CaIRF9. Overexpression of CaIRF9 together with CaSTAT2 not only activates ISRE-containing promoter but also upregulates the expression of fish ISGs. Strikingly, CaIRF9 together with CaSTAT2 also exhibits an ability to activate crucian carp IFN promoter, and blockade of cellular CaIRF9 attenuates IFN itself-induced activation of crucian carp IFN promoter. Taken together, these data suggest that crucian carp IFN induces the expression of ISGs and also of itself possibly by the JAK-STAT signaling pathway that is conserved from fish to mammals. Copyright © 2012 Elsevier Ltd. All rights reserved.

The differentiation and plasticity of Tc17 cells are regulated by CTLA-4-mediated effects on STATs.

PubMed

Arra, Aditya; Lingel, Holger; Kuropka, Benno; Pick, Jonas; Schnoeder, Tina; Fischer, Thomas; Freund, Christian; Pierau, Mandy; Brunner-Weinzierl, Monika C

2017-01-01

As the blockade of inhibitory surface-molecules such as CTLA-4 on T cells has led to recent advances in antitumor immune therapy, there is great interest in identifying novel mechanisms of action of CD8 + T cells to evoke effective cytotoxic antitumor responses. Using in vitro and in vivo models, we investigated the molecular pathways underlying the CTLA-4-mediated differentiation of IL-17-producing CD8 + T cells (Tc17 cells) that strongly impairs cytotoxicity. Our studies demonstrate that Tc17 cells lacking CTLA-4 signaling have limited production of STAT3-target gene products such as IL-17, IL-21, IL-23R and RORγt. Upon re-stimulation with IL-12, these cells display fast downregulation of Tc17 hallmarks and acquire Tc1 characteristics such as IFNγ and TNF-α co-expression, which is known to correlate with tumor control. Indeed, upon adoptive transfer, these cells were highly efficient in the antigen-specific rejection of established OVA-expressing B16 melanoma in vivo . Mechanistically, in primary and re-stimulated Tc17 cells, STAT3 binding to the IL-17 promoter was strongly augmented by CTLA-4, associated with less binding of STAT5 and reduced relative activation of STAT1 which is known to block STAT3 activity. Inhibiting CTLA-4-induced STAT3 activity reverses enhancement of signature Tc17 gene products, rendering Tc17 cells susceptible to conversion to Tc1-like cells with enhanced cytotoxic potential. Thus, CTLA-4 critically shapes the characteristics of Tc17 cells by regulating relative STAT3 activation, which provides new perspectives to enhance cytotoxicity of antitumor responses.

4-Phenylbutyrate stimulates Hsp70 expression through the Elp2 component of elongator and STAT-3 in cystic fibrosis epithelial cells.

PubMed

Suaud, Laurence; Miller, Katelyn; Panichelli, Ashley E; Randell, Rachel L; Marando, Catherine M; Rubenstein, Ronald C

2011-12-30

Sodium 4-phenylbutyrate (4PBA) corrects trafficking of ΔF508-CFTR in Cystic Fibrosis (CF) epithelia, which is hypothesized to, at least in part, result from increased expression of Hsp70 (stress-induced 70 kDa heat shock protein). To identify other 4PBA-regulated proteins that may promote correction of ΔF508 trafficking, we performed differential display RT-PCR on mRNA from IB3-1 CF bronchiolar epithelial cells treated for 0-24 h with 1 mM 4PBA. In this screen, a STAT-3 (signal transducer and activator of transcription-3)-interacting protein, StIP-1 that regulates STAT-3 activation had transiently increased expression. StIP-1 is identical to Elongator protein 2 (Elp2), a component of the Elongator complex that regulates RNA polymerase II. Previous studies have suggested that Elongator regulates Hsp70 mRNA transcription, and that the Hsp70 promoter contains functional STAT-3-binding sites. We therefore tested the hypothesis that 4PBA increases Hsp70 expression by an Elongator- and STAT-3-dependent mechanism. 4PBA treatment of IB3-1 CF bronchiolar epithelial cells caused transiently increased expression of Hsp70 protein, as well as Elp2 protein and mRNA. Elp2 depletion by transfection of small interfering RNAs, reduced both Elp2 and Hsp70 protein expression. 4PBA also caused transient activation of STAT-3, and increased abundance of nuclear proteins that bind to the STAT-3-responsive element of the Hsp70 promoter. Luciferase reporter assays demonstrated that both Elp2 overexpression and 4PBA increase Hsp70 promoter activity, while Elp2 depletion blocked the ability of 4PBA to stimulate Hsp70 promoter activity. Together, these data suggest that Elp2 and STAT-3 mediate, at least in part, the stimulation of Hsp70 expression by 4PBA.

4-Phenylbutyrate Stimulates Hsp70 Expression through the Elp2 Component of Elongator and STAT-3 in Cystic Fibrosis Epithelial Cells*

PubMed Central

Suaud, Laurence; Miller, Katelyn; Panichelli, Ashley E.; Randell, Rachel L.; Marando, Catherine M.; Rubenstein, Ronald C.

2011-01-01

Sodium 4-phenylbutyrate (4PBA) corrects trafficking of ΔF508-CFTR in Cystic Fibrosis (CF) epithelia, which is hypothesized to, at least in part, result from increased expression of Hsp70 (stress-induced 70 kDa heat shock protein). To identify other 4PBA-regulated proteins that may promote correction of ΔF508 trafficking, we performed differential display RT-PCR on mRNA from IB3-1 CF bronchiolar epithelial cells treated for 0–24 h with 1 mm 4PBA. In this screen, a STAT-3 (signal transducer and activator of transcription-3)-interacting protein, StIP-1 that regulates STAT-3 activation had transiently increased expression. StIP-1 is identical to Elongator protein 2 (Elp2), a component of the Elongator complex that regulates RNA polymerase II. Previous studies have suggested that Elongator regulates Hsp70 mRNA transcription, and that the Hsp70 promoter contains functional STAT-3-binding sites. We therefore tested the hypothesis that 4PBA increases Hsp70 expression by an Elongator- and STAT-3-dependent mechanism. 4PBA treatment of IB3-1 CF bronchiolar epithelial cells caused transiently increased expression of Hsp70 protein, as well as Elp2 protein and mRNA. Elp2 depletion by transfection of small interfering RNAs, reduced both Elp2 and Hsp70 protein expression. 4PBA also caused transient activation of STAT-3, and increased abundance of nuclear proteins that bind to the STAT-3-responsive element of the Hsp70 promoter. Luciferase reporter assays demonstrated that both Elp2 overexpression and 4PBA increase Hsp70 promoter activity, while Elp2 depletion blocked the ability of 4PBA to stimulate Hsp70 promoter activity. Together, these data suggest that Elp2 and STAT-3 mediate, at least in part, the stimulation of Hsp70 expression by 4PBA. PMID:22069317

Methanethiosulfonate derivatives as ligands of the STAT3-SH2 domain.

PubMed

Gabriele, Elena; Ricci, Chiara; Meneghetti, Fiorella; Ferri, Nicola; Asai, Akira; Sparatore, Anna

2017-12-01

With the aim to discover new STAT3 direct inhibitors, potentially useful as anticancer agents, a set of methanethiosulfonate drug hybrids were synthesized. The in vitro tests showed that all the thiosulfonic compounds were able to strongly and selectively bind STAT3-SH2 domain, whereas the parent drugs were completely devoid of this ability. In addition, some of them showed a moderate antiproliferative activity on HCT-116 cancer cell line. These results suggest that methanethiosulfonate moiety can be considered a useful scaffold in the preparation of new direct STAT3 inhibitors. Interestingly, an unusual kind of organo-sulfur derivative, endowed with valuable antiproliferative activity, was occasionally isolated. [Formula: see text].

Retinoic acid induces signal transducer and activator of transcription (STAT) 1, STAT2, and p48 expression in myeloid leukemia cells and enhances their responsiveness to interferons.

PubMed

Matikainen, S; Ronni, T; Lehtonen, A; Sareneva, T; Melén, K; Nordling, S; Levy, D E; Julkunen, I

1997-06-01

IFNs are antiproliferative cytokines that have growth-inhibitory effects on various normal and malignant cells. Therefore, they have been used in the treatment of certain forms of cancer, such as chronic myelogenous leukemia and hairy cell leukemia. However, there is little evidence that IFNs would be effective in the treatment of acute myelogenous leukemia, and molecular mechanisms underlying IFN unresponsiveness have not been clarified. Here we have studied the activation and induction of IFN-specific transcription factors signal transducer and activator of transcription (STAT) 1, STAT2, and p48 in all-trans-retinoic acid (ATRA)-differentiated myeloid leukemia cells using promyelocytic NB4, myeloblastic HL-60, and monoblastic U937 cells as model systems. These cells respond to ATRA by growth inhibition and differentiation. We show that in undifferentiated NB4 cells, 2',5'-oligoadenylate synthetase and MxB gene expression is not activated by IFN-alpha, possibly due to a relative lack of signaling molecules, especially p48 protein. However, during ATRA-induced differentiation, steady-state STAT1, STAT2, and especially p48 mRNA and corresponding protein levels were elevated both in NB4 and U937 cells, apparently correlating to an enhanced responsiveness of these cells to IFNs. ATRA treatment of NB4 cells sensitized them to IFN action as seen by increased IFN-gamma activation site DNA-binding activity or by efficient formation of IFN-alpha-specific ISGF3 complex and subsequent oligoadenylate synthetase and MxB gene expression. Lack of p48 expression could be one of the mechanisms of promyelocytic leukemia cell escape from growth-inhibitory effects of IFN-alpha.

Cellular response to alkylating agent MNNG is impaired in STAT1-deficients cells.

PubMed

Ah-Koon, Laurent; Lesage, Denis; Lemadre, Elodie; Souissi, Inès; Fagard, Remi; Varin-Blank, Nadine; Fabre, Emmanuelle E; Schischmanoff, Olivier

2016-10-01

The SN 1 alkylating agents activate the mismatch repair system leading to delayed G2 /M cell cycle arrest and DNA repair with subsequent survival or cell death. STAT1, an anti-proliferative and pro-apoptotic transcription factor is known to potentiate p53 and to affect DNA-damage cellular response. We studied whether STAT1 may modulate cell fate following activation of the mismatch repair system upon exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Using STAT1-proficient or -deficient cell lines, we found that STAT1 is required for: (i) reduction in the extent of DNA lesions, (ii) rapid phosphorylation of T68-CHK2 and of S15-p53, (iii) progression through the G2 /M checkpoint and (iv) long-term survival following treatment with MNNG. Presence of STAT1 is critical for the formation of a p53-DNA complex comprising: STAT1, c-Abl and MLH1 following exposure to MNNG. Importantly, presence of STAT1 allows recruitment of c-Abl to p53-DNA complex and links c-Abl tyrosine kinase activity to MNNG-toxicity. Thus, our data highlight the important modulatory role of STAT1 in the signalling pathway activated by the mismatch repair system. This ability of STAT1 to favour resistance to MNNG indicates the targeting of STAT1 pathway as a therapeutic option for enhancing the efficacy of SN1 alkylating agent-based chemotherapy. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Nuclear localization of activated STAT6 and STAT3 in epidermis of prurigo nodularis.

PubMed

Fukushi, S; Yamasaki, K; Aiba, S

2011-11-01

Prurigo nodularis (PN) is a chronic dermatitis characterized by discrete, raised, and firm papulonodules with intense pruritus. The pathogenesis still remains to be elucidated. To clarify the role of Th1 and Th2 cytokines in the pathogenesis of PN. We examined the cytokine signatures, such as phosphorylation of STAT1, STAT3 and STAT6, HLA-DR and hyaluronan accumulation, to reveal the Th1 and Th2 cytokine influence on the lesional epidermis of PN. We first optimized antigen retrieval methods to detect these signatures with antibodies for phospho-STAT1 (pSTAT1), phospho-STAT3 (pSTAT3), phospho-STAT6 (pSTAT6), HLA-DR and hyaluronic acid binding protein (HABP) on the formalin-fixed paraffin-embedded sections of psoriasis, lichen planus and atopic dermatitis biopsy samples. Activation of STAT1 and STAT6 in epidermis by Th1 and Th2 cytokines was further confirmed in a cultured skin equivalent model treated with interferon-γ or interleukin (IL)-4/IL-13. With the relevant immunostaining methods, we examined the cytokine signatures in 22 cases of PN. The results revealed that (i) the entire epidermis of 19 cases was stained with anti-pSTAT6 antibody, (ii) 21 cases demonstrated nuclear staining with anti-pSTAT3 antibody, (iii) the entire epidermis of 21 cases was stained with HABP, (iv) the epidermis of eight cases showed scattered staining with anti-pSTAT1 antibody, and (v) six cases were positive for HLA-DR membrane expression. These data indicated that Th2 cytokines related to STAT6 activation together with some unknown stimuli that activate STAT3 play a principal role in the pathogenesis of PN. © 2011 The Authors. BJD © 2011 British Association of Dermatologists.

Effect of azithromycin on Prevotella intermedia lipopolysaccharide-induced production of interleukin-6 in murine macrophages.

PubMed

Choi, Eun-Young; Jin, Ji-Young; Choi, Jeom-Il; Choi, In Soon; Kim, Sung-Jo

2014-04-15

Interleukin-6 (IL-6) is a key proinflammatory cytokine which plays a central role in the pathogenesis of periodontal disease. Host modulatory agents targeting at inhibiting IL-6, therefore, appear to be beneficial in slowing the progression of periodontal disease and potentially reducing destructive aspects of the host response. The present study was designed to investigate the effect of the macrolide antibiotic azithromycin on IL-6 generation in murine macrophages treated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in inflammatory periodontal disease, and its mechanisms of action. Azithromycin significantly suppressed IL-6 production as well as its mRNA expression in P. intermedia LPS-activated RAW264.7 cells. LPS-induced activation of JNK and p38 was not affected by azithromycin treatment. Azithromycin failed to prevent P. intermedia LPS from degrading IκB-α. Instead, azithromycin significantly diminished nuclear translocation and DNA binding activity of NF-κB p50 subunit induced with LPS. Azithromycin inhibited P. intermedia LPS-induced STAT1 and STAT3 phosphorylation. In addition, azithromycin up-regulated the mRNA level of SOCS1 in cells treated with LPS. In conclusion, azithromycin significantly attenuated P. intermedia LPS-induced production of IL-6 in murine macrophages via inhibition of NF-κB, STAT1 and STAT3 activation, which is possibly related to the activation of SOCS1 signaling. Further in vivo studies are required to better evaluate the potential of azithromycin in the treatment of periodontal disease. Copyright © 2014 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kamitani, Shinya; Ohbayashi, Norihiko; Ikeda, Osamu

Signal transducers and activators of transcription (STATs) mediate cell proliferation, differentiation, and survival in immune responses, hematopoiesis, neurogenesis, and other biological processes. Recently, we showed that KAP1 is a novel STAT-binding partner that regulates STAT3-mediated transactivation. KAP1 is a universal co-repressor protein for the KRAB zinc finger protein superfamily of transcriptional repressors. In this study, we found KAP1-dependent repression of interferon (IFN)/STAT1-mediated signaling. We also demonstrated that endogenous KAP1 associates with endogenous STAT1 in vivo. Importantly, a small-interfering RNA-mediated reduction in KAP1 expression enhanced IFN-induced STAT1-dependent IRF-1 gene expression. These results indicate that KAP1 may act as an endogenous regulatormore » of the IFN/STAT1 signaling pathway.« less

Epigenetic repression of the Igk locus by STAT5-mediated Ezh2 recruitment

PubMed Central

Mandal, Malay; Powers, Sarah E.; Maienschein-Cline, Mark; Bartom, Elizabeth T.; Hamel, Keith M.; Kee, Barbara L.; Dinner, Aaron R.; Clark, Marcus R.

2011-01-01

During B lymphopoiesis, Igk recombination requires pre-B cell receptor (pre-BCR) expression and escape from interleukin 7 receptor (IL-7R) signaling. By activating the transcription factor STAT5, IL-7R signaling maintains proliferation and represses Igk germline transcription by unknown mechanisms. We demonstrate that STAT5 tetramer bound the Igk intronic enhancer (Eκi), leading to recruitment of the histone methyltransferase Ezh2. Ezh2 marked H3K27me3 throughout Jκ to Cκ. In the absence of Ezh2, IL-7 failed to repress Igk germline transcription. H3K27me3 modifications were lost after termination of IL-7R–STAT5 signaling and E2A bound Eκi, resulting in acquisition of H3K4me1 and H4Ac. Genome-wide analyses revealed a STAT5 tetrameric binding motif associated with transcriptional repression. These data demonstrate how IL-7R signaling represses Igk germline transcription and provide a general model for STAT5-mediated epigenetic transcriptional repression. PMID:22037603

Activation of Stat3 Transcription Factor by Herpesvirus Saimiri STP-A Oncoprotein

PubMed Central

Chung, Young-Hwa; Cho, Nam-hyuk; Garcia, Maria Ines; Lee, Sun-Hwa; Feng, Pinghui; Jung, Jae U.

2004-01-01

The saimiri transforming protein (STP) oncogene of Herpesvirus saimiri subgroup A strain 11 (STP-A11) is not required for viral replication but is required for lymphoid cell immortalization in culture and lymphoma induction in primates. We previously showed that STP-A11 interacts with cellular Src kinase through its SH2 binding motif and that this interaction elicits Src signal transduction. Here we demonstrate that STP-A11 interacts with signal transducer and activator of transcription 3 (Stat3) independently of Src association and that the amino-terminal short proline-rich motif of STP-A11 and the central linker region of Stat3 are necessary for their interaction. STP-A11 formed a triple complex with Src kinase and Stat3 where Src kinase phosphorylated Stat3, resulting in the nuclear localization and transcriptional activation of Stat3. Consequently, the constitutively active Stat3 induced by STP-A11 elicited cellular signal transduction, which ultimately induced cell survival and proliferation upon serum deprivation. Furthermore, this activity was strongly correlated with the induction of Fos, cyclin D1, and Bcl-XL expression. These results demonstrate that STP-A11 independently targets two important cellular signaling molecules, Src and Stat3, and that these proteins cooperate efficiently to induce STP-A11-mediated transformation. PMID:15163742

TNFα-senescence initiates a STAT-dependent positive feedback loop, leading to a sustained interferon signature, DNA damage, and cytokine secretion

PubMed Central

Kandhaya-Pillai, Renuka; Miro-Mur, Francesc; Alijotas-Reig, Jaume; Tchkonia, Tamara; Kirkland, James L.; Schwartz, Simo

2017-01-01

Cellular senescence is a cell fate program that entails essentially irreversible proliferative arrest in response to damage signals. Tumor necrosis factor-alpha (TNFα), an important pro-inflammatory cytokine secreted by some types of senescent cells, can induce senescence in mouse and human cells. However, downstream signaling pathways linking TNFα-related inflammation to senescence are not fully characterized. Using human umbilical vein endothelial cells (HUVECs) as a model, we show that TNFα induces permanent growth arrest and increases p21CIP1, p16INK4A, and SA-β-gal, accompanied by persistent DNA damage and ROS production. By gene expression profiling, we identified the crucial involvement of inflammatory and JAK/STAT pathways in TNFα-mediated senescence. We found that TNFα activates a STAT-dependent autocrine loop that sustains cytokine secretion and an interferon signature to lock cells into senescence. Furthermore, we show STAT1/3 activation is necessary for cytokine and ROS production during TNFα-induced senescence. However, inhibition of STAT1/3 did not rescue cells from proliferative arrest, but rather suppressed cell cycle regulatory genes and altered TNFα-induced senescence. Our findings suggest a positive feedback mechanism via the STAT pathway that sustains cytokine production and reveal a reciprocal regulatory role of JAK/STAT in TNFα-mediated senescence. PMID:29176033

Severe Early-Onset Combined Immunodeficiency due to Heterozygous Gain-of-Function Mutations in STAT1.

PubMed

Baris, Safa; Alroqi, Fayhan; Kiykim, Ayca; Karakoc-Aydiner, Elif; Ogulur, Ismail; Ozen, Ahmet; Charbonnier, Louis-Marie; Bakır, Mustafa; Boztug, Kaan; Chatila, Talal A; Barlan, Isil B

2016-10-01

Loss and gain-of-function (GOF) mutations in human signal transducer and activator of transcription 1 (STAT1) lead to distinct phenotypes. Although recurrent infections are common to both types of STAT1 mutations, GOF mutations are distinguished by chronic mucocutaneous candidiasis and autoimmunity. However, the clinical spectra of STAT1 GOF mutations continue to expand. We here describe two patients with STAT1 GOF mutations presenting early in life with combined immunodeficiency (CID). Clinical data and laboratory findings including immunophenotyping, level of interferon (IFN)-γ/IL-17(+) T cells, interferon-induced STAT1 phosphorylation, and JAK inhibitor assays were evaluated. Sequencing of STAT1 gene was performed by Sanger sequencer. Patient 1 (P1) had persistent oral candidiasis and cytomegalovirus (CMV) infection since 2 months of age and later developed cavitary lung lesions due to Mycobacterium tuberculosis. Patient 2 (P2) presented with oral candidiasis and recurrent pneumonia at 4 months of age and subsequently developed CMV pneumonitis. Both patients suffered heterozygous missense mutations in STAT1, leading to deleterious amino acid substitutions in the DNA binding domain (P1: c.1154C > T; p.T385M; P2. c.971G > T; p.C324F). Circulating CD4(+) T cells of both patients exhibited increased interferon-γ and decreased IL-17 expression as compared to controls. They also exhibited increased IFN-β and -γ-induced STAT1 phosphorylation that was reversed upon treatment with the JAK kinase inhibitor ruxolitinib. STAT1 GOF mutations may present early in life with CID, consistent with the clinical heterogeneity of the disease. JAK kinase inhibitors may potentially be useful in some patients as adjunct therapy pending definitive treatment with bone marrow transplantation.

Lipopolysaccharide potentiates the effect of hepatocyte growth factor on hepatocyte replication in rats by augmenting AP-1 activity.

PubMed

Gao, C; Jokerst, R; Gondipalli, P; Cai, S R; Kennedy, S; Flye, M W; Ponder, K P

1999-12-01

The liver regenerates by replication of differentiated hepatocytes after damage or removal of part of the liver. Although several growth factors and signaling pathways are activated during regeneration, it is unclear as to which of these are essential for hepatocyte replication. We show here that low- (1 mg/kg) and high- (10 mg/kg) dose hepatocyte growth factor (HGF) induced replication of 2.1% and 11.1% of hepatocytes in rats, respectively. Lipopolysaccharide (LPS), an inducer of the acute phase response, augmented hepatocyte replication in response to low- and high-dose HGF by 4- and 2-fold, respectively. HGF alone induced moderate levels of c-Jun-N-terminal kinase (JNK) and p44/p42 mitogen-activated protein kinase (MAPK), resulting in moderate levels of AP-1-DNA binding activity. The combination of LPS + HGF increased JNK and AP-1-DNA binding activity more than levels seen with LPS or HGF alone. The activation of Stat3 that was observed after administration of LPS + HGF, but not HGF alone, could contribute to increased transcription of AP-1 components. Because phosphorylation of the c-Jun component of AP-1 by JNK increases its ability to activate transcription, the AP-1 in hepatocytes from animals treated with LPS + HGF may be more active than in rats treated with LPS or HGF alone. LPS may contribute to hepatocyte replication by potentiating the effect of HGF on the activation of both AP-1-DNA binding and transcriptional activity.

Genetics Home Reference: autosomal dominant hyper-IgE syndrome

MedlinePlus

... binding domain of STAT3 cause hyper-IgE syndrome. Nature. 2007 Aug 30;448(7157):1058-62. Epub 2007 Aug 5. Citation on PubMed Renner ED, Torgerson TR, Rylaarsdam S, Añover-Sombke S, Golob K, LaFlam T, Zhu Q, Ochs HD. STAT3 mutation in the original patient with Job's syndrome. N Engl J Med. 2007 Oct 18; ...

Inhibition of Oncogenic functionality of STAT3 Protein by Membrane Anchoring

NASA Astrophysics Data System (ADS)

Liu, Baoxu; Fletcher, Steven; Gunning, Patrick; Gradinaru, Claudiu

2009-03-01

Signal Transducer and Activator of Transcription 3 (STAT3) protein plays an important role in oncogenic processes. A novel molecular therapeutic approach to inhibit the oncogenic functionality of STAT3 is to design a prenylated small peptide sequence which could sequester STAT3 to the plasma membrane. We have also developed a novel fluorescein derivative label (F-NAc), which is much more photostable compared to the popular fluorescein label FITC. Remarkably, the new dye shows fluorescent properties that are invariant over a wide pH range, which is advantageous for our application. We have shown that F-NAc is suitable for single-molecule measurements and its properties are not affected by ligation to biomolecules. The membrane localization via high-affinity prenylated small-molecule binding agents is studied by encapsulating FNAc-labeled STAT3 and inhibitors within a liposome model cell system. The dynamics of the interaction between the protein and the prenylated ligands is investigated at single molecule level. The efficiency and stability of the STAT3 anchoring in lipid membranes are addressed via quantitative confocal imaging and single-molecule spectroscopy using a custom-built multiparameter fluorescence microscope.

Deficiency of activated STAT1 in head and neck cancer cells mediates TAP1-dependent escape from cytotoxic T lymphocytes

PubMed Central

Leibowitz, Michael S.; Filho, Pedro A. Andrade; Ferrone, Soldano; Ferris, Robert L.

2012-01-01

Squamous cell carcinoma of the head and neck (SCCHN) cells can escape recognition by tumor antigen (TA)-specific cytotoxic T lymphocytes (CTL) by downregulation of antigen processing machinery (APM) components, such as the transporter associated with antigen processing (TAP)-1/2 heterodimer. APM component upregulation by interferon gamma (IFN-γ) restores SCCHN cell recognition and susceptibility to lysis by CTL, but the mechanism underlying TAP1/2 downregulation in SCCHN cells is not known. Because IFN-γ activates signal transducer and activator of transcription (STAT)-1, we investigated phosphorylated (p)-STAT1 as a mediator of low basal TAP1/2 expression in SCCHN cells. SCCHN cells were found to express basal total STAT1 but low to undetectable levels of activated STAT1. The association of increased pSTAT1 levels and APM components likely reflects a cause–effect relationship, since STAT1 knockdown significantly reduced both IFN-γ-mediated APM component expression and TA-specific CTL recognition of IFN-γ-treated SCCHN cells. On the other hand, since oncogenic pSTAT3 is overexpressed in SCCHN cells and was found to heterodimerize with pSTAT1, we also tested whether pSTAT3 and pSTAT1:pSTAT3 heterodimers inhibited IFN-γ-induced STAT1 activation and APM component expression. First, STAT3 activation or depletion did not affect basal or IFN-γ-induced expression of pSTAT1 and APM components or recognition of SCCHN cells by TA-specific CTL. Second, pSTAT1:pSTAT3 heterodimers did not interfere with IFN-γ-induced STAT1 binding to the TAP1 promoter or APM protein expression. These findings demonstrate that APM component downregulation is regulated primarily by an IFN-γ-pSTAT1-mediated signaling pathway, independent of oncogenic STAT3 overexpression in SCCHN cells. PMID:21207025

Fanconi Anemia Core Complex Gene Promoters Harbor Conserved Transcription Regulatory Elements

PubMed Central

Meier, Daniel; Schindler, Detlev

2011-01-01

The Fanconi anemia (FA) gene family is a recent addition to the complex network of proteins that respond to and repair certain types of DNA damage in the human genome. Since little is known about the regulation of this novel group of genes at the DNA level, we characterized the promoters of the eight genes (FANCA, B, C, E, F, G, L and M) that compose the FA core complex. The promoters of these genes show the characteristic attributes of housekeeping genes, such as a high GC content and CpG islands, a lack of TATA boxes and a low conservation. The promoters functioned in a monodirectional way and were, in their most active regions, comparable in strength to the SV40 promoter in our reporter plasmids. They were also marked by a distinctive transcriptional start site (TSS). In the 5′ region of each promoter, we identified a region that was able to negatively regulate the promoter activity in HeLa and HEK 293 cells in isolation. The central and 3′ regions of the promoter sequences harbor binding sites for several common and rare transcription factors, including STAT, SMAD, E2F, AP1 and YY1, which indicates that there may be cross-connections to several established regulatory pathways. Electrophoretic mobility shift assays and siRNA experiments confirmed the shared regulatory responses between the prominent members of the TGF-β and JAK/STAT pathways and members of the FA core complex. Although the promoters are not well conserved, they share region and sequence specific regulatory motifs and transcription factor binding sites (TBFs), and we identified a bi-partite nature to these promoters. These results support a hypothesis based on the co-evolution of the FA core complex genes that was expanded to include their promoters. PMID:21826217

Fanconi anemia core complex gene promoters harbor conserved transcription regulatory elements.

PubMed

Meier, Daniel; Schindler, Detlev

2011-01-01

The Fanconi anemia (FA) gene family is a recent addition to the complex network of proteins that respond to and repair certain types of DNA damage in the human genome. Since little is known about the regulation of this novel group of genes at the DNA level, we characterized the promoters of the eight genes (FANCA, B, C, E, F, G, L and M) that compose the FA core complex. The promoters of these genes show the characteristic attributes of housekeeping genes, such as a high GC content and CpG islands, a lack of TATA boxes and a low conservation. The promoters functioned in a monodirectional way and were, in their most active regions, comparable in strength to the SV40 promoter in our reporter plasmids. They were also marked by a distinctive transcriptional start site (TSS). In the 5' region of each promoter, we identified a region that was able to negatively regulate the promoter activity in HeLa and HEK 293 cells in isolation. The central and 3' regions of the promoter sequences harbor binding sites for several common and rare transcription factors, including STAT, SMAD, E2F, AP1 and YY1, which indicates that there may be cross-connections to several established regulatory pathways. Electrophoretic mobility shift assays and siRNA experiments confirmed the shared regulatory responses between the prominent members of the TGF-β and JAK/STAT pathways and members of the FA core complex. Although the promoters are not well conserved, they share region and sequence specific regulatory motifs and transcription factor binding sites (TBFs), and we identified a bi-partite nature to these promoters. These results support a hypothesis based on the co-evolution of the FA core complex genes that was expanded to include their promoters.

Caveolin-1-deficient mice show accelerated mammary gland development during pregnancy, premature lactation, and hyperactivation of the Jak-2/STAT5a signaling cascade.

PubMed

Park, David S; Lee, Hyangkyu; Frank, Philippe G; Razani, Babak; Nguyen, Andrew V; Parlow, Albert F; Russell, Robert G; Hulit, James; Pestell, Richard G; Lisanti, Michael P

2002-10-01

It is well established that mammary gland development and lactation are tightly controlled by prolactin signaling. Binding of prolactin to its cognate receptor (Prl-R) leads to activation of the Jak-2 tyrosine kinase and the recruitment/tyrosine phosphorylation of STAT5a. However, the mechanisms for attenuating the Prl-R/Jak-2/STAT5a signaling cascade are just now being elucidated. Here, we present evidence that caveolin-1 functions as a novel suppressor of cytokine signaling in the mammary gland, akin to the SOCS family of proteins. Specifically, we show that caveolin-1 expression blocks prolactin-induced activation of a STAT5a-responsive luciferase reporter in mammary epithelial cells. Furthermore, caveolin-1 expression inhibited prolactin-induced STAT5a tyrosine phosphorylation and DNA binding activity, suggesting that caveolin-1 may negatively regulate the Jak-2 tyrosine kinase. Because the caveolin-scaffolding domain bears a striking resemblance to the SOCS pseudosubstrate domain, we examined whether Jak-2 associates with caveolin-1. In accordance with this homology, we demonstrate that Jak-2 cofractionates and coimmunoprecipitates with caveolin-1. We next tested the in vivo relevance of these findings using female Cav-1 (-/-) null mice. If caveolin-1 normally functions as a suppressor of cytokine signaling in the mammary gland, then Cav-1 null mice should show premature development of the lobuloalveolar compartment because of hyperactivation of the prolactin signaling cascade via disinhibition of Jak-2. In accordance with this prediction, Cav-1 null mice show accelerated development of the lobuloalveolar compartment, premature milk production, and hyperphosphorylation of STAT5a (pY694) at its Jak-2 phosphorylation site. In addition, the Ras-p42/44 MAPK cascade is hyper-activated. Because a similar premature lactation phenotype is observed in SOCS1 (-/-) null mice, we conclude that caveolin-1 is a novel suppressor of cytokine signaling.

miR-125b suppresses the proliferation and migration of osteosarcoma cells through down-regulation of STAT3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Li-hong; Li, Hui; Li, Jin-ping

2011-12-09

Highlights: Black-Right-Pointing-Pointer miR-125b is frequently down-regulated in osteosarcoma samples and human osteosarcoma cell lines. Black-Right-Pointing-Pointer Ectopic restoration of miR-125b suppresses cell proliferation and migration in vitro. Black-Right-Pointing-Pointer STAT3 is the direct and functional downstream target of miR-125b. Black-Right-Pointing-Pointer STAT3 can bind to the promoter region of miR-125b and serves as a transactivator. -- Abstract: There is accumulating evidence that microRNAs are involved in multiple processes in development and tumor progression. Abnormally expressed miR-125b was found to play a fundamental role in several types of cancer; however, whether miR-125b participates in regulating the initiation and progress of osteosarcoma still remains unclear.more » Here we demonstrate that miR-125b is frequently down-regulated in osteosarcoma samples and human osteosarcoma cell lines. The ectopic restoration of miR-125b expression in human osteosarcoma cells suppresses proliferation and migration in vitro and inhibits tumor formation in vivo. We further identified signal transducer and activator of transcription 3 (STAT3) as the direct and functional downstream target of miR-125b. Interestingly, we discovered that the expression of miR-125b is regulated by STAT3 at the level of transcription. STAT3 binds to the promoter region of miR-125b in vitro and serves as a transactivator. Taken together, our findings point to an important role in the molecular etiology of osteosarcoma and suggest that miR-125b is a potential target in the treatment of osteosarcoma.« less

The SH3BGR/STAT3 Pathway Regulates Cell Migration and Angiogenesis Induced by a Gammaherpesvirus MicroRNA

PubMed Central

Ding, Xiangya; Shen, Chenyou; Hu, Minmin; Zhu, Ying; Qin, Di; Lu, Hongmei; Krueger, Brian J.; Renne, Rolf; Gao, Shou-Jiang; Lu, Chun

2016-01-01

Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) is a gammaherpesvirus etiologically associated with KS, a highly disseminated angiogenic tumor of hyperproliferative spindle endothelial cells. KSHV encodes 25 mature microRNAs but their roles in KSHV-induced tumor dissemination and angiogenesis remain unknown. Here, we investigated KSHV-encoded miR-K12-6-3p (miR-K6-3p) promotion of endothelial cell migration and angiogenesis, which are the underlying mechanisms of tumor dissemination and angiogenesis. We found that ectopic expression of miR-K6-3p promoted endothelial cell migration and angiogenesis. Mass spectrometry, bioinformatics and luciferase reporter analyses revealed that miR-K6-3p directly targeted sequence in the 3’ untranslated region (UTR) of SH3 domain binding glutamate-rich protein (SH3BGR). Overexpression of SH3BGR reversed miR-K6-3p induction of cell migration and angiogenesis. Mechanistically, miR-K6-3p downregulated SH3BGR, hence relieved STAT3 from SH3BGR direct binding and inhibition, which was required for miR-K6-3p maximum activation of STAT3 and induction of cell migration and angiogenesis. Finally, deletion of miR-K6 from the KSHV genome abrogated its effect on the SH3BGR/STAT3 pathway, and KSHV-induced migration and angiogenesis. Our results illustrated that, by inhibiting SH3BGR, miR-K6-3p enhances cell migration and angiogenesis by activating the STAT3 pathway, and thus contributes to the dissemination and angiogenesis of KSHV-induced malignancies. PMID:27128969

Upregulation of survivin by leptin/STAT3 signaling in MCF-7 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang Haiping; Tianjin Medical University Cancer Hospital, Tianjin; Yu Jinming

2008-03-28

Leptin and its receptors are overexpressed in breast cancer tissues and correlate with poor prognosis. Survivin, a member of the inhibitor of apoptosis protein (IAP) gene family, is generally upregulated in tumor tissues and prevents tumor cells from apoptosis. Here we showed that leptin upregulated survivin mRNA and protein expression in MCF-7 breast cancer cells. Meanwhile, leptin suppressed docetaxel-induced apoptosis by inhibiting caspase activity. Knockdown of signal transducer and activator transcription 3 (STAT3) expression by small interfering RNA (siRNA) blocked leptin-induced upregulation of survivin. TransAM ELISA showed that leptin increased nuclear translocation of active STAT3. In addition, chromatin immunoprecipitation (ChIP)more » assay detected an enhanced binding of STAT3 to survivin promoter in MCF-7 cells after treatment by leptin. Further studies showed that leptin enhanced the transcriptional activity of survivin promoter. Collectively, our findings identify leptin/STAT3 signaling as a novel pathway for survivin expression in breast cancer cells.« less

Hypothyroidism reduces ObRb-STAT3 leptin signalling in the hypothalamus and pituitary of rats associated with resistance to leptin acute anorectic action.

PubMed

Calvino, Camila; Souza, Luana L; Costa-e-Sousa, Ricardo H; Almeida, Norma A S; Trevenzoli, Isis H; Pazos-Moura, Carmen C

2012-10-01

Leptin has been shown to regulate the hypothalamus-pituitary-thyroid axis, acting primarily through the STAT3 pathway triggered through the binding of leptin to the long-chain isoform of the leptin receptor, ObRb. We previously demonstrated that although hyperthyroid rats presented leptin effects on TSH secretion, those effects were abolished in hypothyroid rats. We addressed the hypothesis that changes in the STAT3 pathway might explain the lack of TSH response to leptin in hypothyroidism by evaluating the protein content of components of leptin signalling via the STAT3 pathway in the hypothalamus and pituitary of hypothyroid (0·03% methimazole in the drinking water/21 days) and hyperthyroid (thyroxine 5 μg/100 g body weight /5 days) rats. Hypothyroid rats exhibited decreased ObRb and phosphorylated STAT3 (pSTAT3) protein in the hypothalamus, and in the pituitary gland they exhibited decreased ObRb, total STAT3, pSTAT3 and SOCS3 (P<0·05). Except for a modest decrease in pituitary STAT3, no other alterations were observed in hyperthyroid rats. Moreover, unlike euthyroid rats, the hypothyroid rats did not exhibit a reduction in food ingestion after a single injection of leptin (0·5 mg/kg body weight). Therefore, hypothyroidism decreased ObRb-STAT3 signalling in the hypothalamus and pituitary gland, which likely contributes to the loss of leptin action on food intake and TSH secretion, as previously observed in hypothyroid rats.

Apatinib promotes autophagy and apoptosis through VEGFR2/STAT3/BCL-2 signaling in osteosarcoma

PubMed Central

Liu, Kuisheng; Ren, Tingting; Huang, Yi; Sun, Kunkun; Bao, Xing; Wang, Shidong; Zheng, Bingxin; Guo, Wei

2017-01-01

The cure rate of osteosarcoma has not improved in the past 30 years. The search for new treatments and drugs is urgently needed. Apatinib is a high selectivity inhibitor of vascular endothelial growth factor receptor-2 (VEGFR2) tyrosine kinase, exerting promising antitumoral effect in various tumors. The antitumor effect of Apatinib in human osteosarcoma has never been reported. We investigated the effects of Apatinib in osteosarcoma in vitro and in vivo. Osteosarcoma patients with high levels of VEGFR2 have poor prognosis. Apatinib can inhibit cell growth of osteosarcoma cells. In addition to cycle arrest and apoptosis, Apatinib induces autophagy. Interestingly, inhibition of autophagy increased Apatinib-induced apoptosis in osteosarcoma cells. Immunoprecipitation confirmed direct binding between VEGFR2 and signal transducer and activator of transcription 3 (STAT3). Downregulation of VEGFR2 by siRNA resulted in STAT3 inhibition in KHOS cells. VEGFR2 and STAT3 are inhibited by Apatinib in KHOS cells, and STAT3 act downstream of VEGFR2. STAT3 and BCL-2 were downregulated by Apatinib. STAT3 knockdown by siRNA reinforced autophagy and apoptosis induced by Apatinib. BCL-2 inhibits autophagy and was apoptosis restrained by Apatinib too. Overexpression of BCL-2 decreased Apatinib-induced apoptosis and autophagy. Apatinib repressed the expression of STAT3 and BCL-2 and suppressed the growth of osteosarcoma in vivo. To sum up, deactivation of VEGFR2/STAT3/BCL-2 signal pathway leads to Apatinib-induced growth inhibition of osteosarcoma. PMID:28837148

Apatinib promotes autophagy and apoptosis through VEGFR2/STAT3/BCL-2 signaling in osteosarcoma.

PubMed

Liu, Kuisheng; Ren, Tingting; Huang, Yi; Sun, Kunkun; Bao, Xing; Wang, Shidong; Zheng, Bingxin; Guo, Wei

2017-08-24

The cure rate of osteosarcoma has not improved in the past 30 years. The search for new treatments and drugs is urgently needed. Apatinib is a high selectivity inhibitor of vascular endothelial growth factor receptor-2 (VEGFR2) tyrosine kinase, exerting promising antitumoral effect in various tumors. The antitumor effect of Apatinib in human osteosarcoma has never been reported. We investigated the effects of Apatinib in osteosarcoma in vitro and in vivo. Osteosarcoma patients with high levels of VEGFR2 have poor prognosis. Apatinib can inhibit cell growth of osteosarcoma cells. In addition to cycle arrest and apoptosis, Apatinib induces autophagy. Interestingly, inhibition of autophagy increased Apatinib-induced apoptosis in osteosarcoma cells. Immunoprecipitation confirmed direct binding between VEGFR2 and signal transducer and activator of transcription 3 (STAT3). Downregulation of VEGFR2 by siRNA resulted in STAT3 inhibition in KHOS cells. VEGFR2 and STAT3 are inhibited by Apatinib in KHOS cells, and STAT3 act downstream of VEGFR2. STAT3 and BCL-2 were downregulated by Apatinib. STAT3 knockdown by siRNA reinforced autophagy and apoptosis induced by Apatinib. BCL-2 inhibits autophagy and was apoptosis restrained by Apatinib too. Overexpression of BCL-2 decreased Apatinib-induced apoptosis and autophagy. Apatinib repressed the expression of STAT3 and BCL-2 and suppressed the growth of osteosarcoma in vivo. To sum up, deactivation of VEGFR2/STAT3/BCL-2 signal pathway leads to Apatinib-induced growth inhibition of osteosarcoma.

Activation of peroxisome proliferator-activated receptor beta/delta inhibits lipopolysaccharide-induced cytokine production in adipocytes by lowering nuclear factor-kappaB activity via extracellular signal-related kinase 1/2.

PubMed

Rodríguez-Calvo, Ricardo; Serrano, Lucía; Coll, Teresa; Moullan, Norman; Sánchez, Rosa M; Merlos, Manuel; Palomer, Xavier; Laguna, Juan C; Michalik, Liliane; Wahli, Walter; Vázquez-Carrera, Manuel

2008-08-01

Chronic activation of the nuclear factor-kappaB (NF-kappaB) in white adipose tissue leads to increased production of pro-inflammatory cytokines, which are involved in the development of insulin resistance. It is presently unknown whether peroxisome proliferator-activated receptor (PPAR) beta/delta activation prevents inflammation in adipocytes. First, we examined whether the PPARbeta/delta agonist GW501516 prevents lipopolysaccharide (LPS)-induced cytokine production in differentiated 3T3-L1 adipocytes. Treatment with GW501516 blocked LPS-induced IL-6 expression and secretion by adipocytes and the subsequent activation of the signal transducer and activator of transcription 3 (STAT3)-Suppressor of cytokine signaling 3 (SOCS3) pathway. This effect was associated with the capacity of GW501516 to impede LPS-induced NF-kappaB activation. Second, in in vivo studies, white adipose tissue from Zucker diabetic fatty (ZDF) rats, compared with that of lean rats, showed reduced PPARbeta/delta expression and PPAR DNA-binding activity, which was accompanied by enhanced IL-6 expression and NF-kappaB DNA-binding activity. Furthermore, IL-6 expression and NF-kappaB DNA-binding activity was higher in white adipose tissue from PPARbeta/delta-null mice than in wild-type mice. Because mitogen-activated protein kinase-extracellular signal-related kinase (ERK)1/2 (MEK1/2) is involved in LPS-induced NF-kappaB activation in adipocytes, we explored whether PPARbeta/delta prevented NF-kappaB activation by inhibiting this pathway. Interestingly, GW501516 prevented ERK1/2 phosphorylation by LPS. Furthermore, white adipose tissue from animal showing constitutively increased NF-kappaB activity, such as ZDF rats and PPARbeta/delta-null mice, also showed enhanced phospho-ERK1/2 levels. These findings indicate that activation of PPARbeta/delta inhibits enhanced cytokine production in adipocytes by preventing NF-kappaB activation via ERK1/2, an effect that may help prevent insulin resistance.

Biologically active leptin-related synthetic peptides activate STAT3 via phosphorylation of ERK1/2 and PI-3K.

PubMed

Lin, Hung-Yun; Yang, Sheng-Huei; Tang, Heng-Yuan; Cheng, Guei-Yun; Davis, Paul J; Grasso, Patricia

2014-07-01

The effects of leptin-related synthetic peptides [d-Leu-4]-OB3 and OB3 on energy balance and glucose homeostasis in ob/ob and db/db mice have been confirmed. The molecular basis of these effects, however, remains unclear. In the present study, we examined the ability of these peptides to activate signal transduction pathways known to be involved in transduction of the leptin signal. In a specific and concentration-dependent manner, [d-Leu-4]-OB3 induced phosphorylation of ERK1/2, PI-3K, Ser-727 STAT3, and Tyr-705 of STAT3. OB3 also induced activation of STAT3 via phosphorylation of ERK1/2, STAT3 Ser-727, STAT3 Tyr-705 and PI-3K p85, but to a lesser degree. Using PD98059 and LY294002, specific inhibitors of MEK and PI-3K, respectively, we were able to identify the signal transduction pathways involved in peptide-induced STAT3 activation. [d-Leu-4]-OB3 induced serine phosphorylation of STAT3 primarily through activation of ERK1/2. Tyrosine phosphorylation of STAT3, however, was induced primarily through activation of PI-3K. Our data suggest that in db/db mice, [d-Leu-4]-OB3 binding to short isoforms of the leptin receptor induces intracellular signaling cascades which do not require OB-Rb activation. These signals may ultimately result in peptide effects on transcriptional and translational events associated with energy balance and glycemic regulation. In summary, we have shown for the first time that, similar to leptin, bioactive leptin-related synthetic peptide analogs activate STAT3 via phosphorylation of serine and tyrosine residues by multiple signal transduction pathways. Copyright © 2014 Elsevier Inc. All rights reserved.

Metformin ameliorates IL-6-induced hepatic insulin resistance via induction of orphan nuclear receptor small heterodimer partner (SHP) in mouse models.

PubMed

Kim, Y D; Kim, Y H; Cho, Y M; Kim, D K; Ahn, S W; Lee, J M; Chanda, D; Shong, M; Lee, C H; Choi, H S

2012-05-01

IL-6 is a proinflammatory cytokine associated with the pathogenesis of hepatic diseases. Metformin is an anti-diabetic drug used for the treatment of type 2 diabetes, and orphan nuclear receptor small heterodimer partner (SHP, also known as NR0B2), a transcriptional co-repressor, plays an important role in maintaining metabolic homeostasis. Here, we demonstrate that metformin-mediated activation of AMP-activated protein kinase (AMPK) increases SHP protein production and regulates IL-6-induced hepatic insulin resistance. We investigated metformin-mediated SHP production improved insulin resistance through the regulation of an IL-6-dependent pathway (involving signal transducer and activator of transcription 3 [STAT3] and suppressor of cytokine signalling 3 [SOCS3]) in both Shp knockdown and Shp null mice. IL-6-induced STAT3 transactivation and SOCS3 production were significantly repressed by metformin, adenoviral constitutively active AMPK (Ad-CA-AMPK), and adenoviral SHP (Ad-SHP), but not in Shp knockdown, or with the adenoviral dominant negative form of AMPK (Ad-DN-AMPK). Chromatin immunoprecipitation (ChIP), co-immunoprecipitation (Co-IP) and protein localisation studies showed that SHP inhibits DNA binding of STAT3 on the Socs3 gene promoter via interaction and colocalisation within the nucleus. Upregulation of inflammatory genes and downregulation of hepatic insulin signalling by acute IL-6 treatment were observed in wild-type mice but not in Shp null mice. Finally, chronic IL-6 exposure caused hepatic insulin resistance, leading to impaired insulin tolerance and elevated gluconeogenesis, and these phenomena were aggravated in Shp null mice. Our results demonstrate that SHP upregulation by metformin may prevent hepatic disorders by regulating the IL-6-dependent pathway, and that this pathway can help to ameliorate the pathogenesis of cytokine-mediated metabolic dysfunction.

Target specificity, in vivo pharmacokinetics, and efficacy of the putative STAT3 inhibitor LY5 in osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma.

PubMed

Yu, Peter Y; Gardner, Heather L; Roberts, Ryan; Cam, Hakan; Hariharan, Seethalakshmi; Ren, Ling; LeBlanc, Amy K; Xiao, Hui; Lin, Jiayuh; Guttridge, Denis C; Mo, Xiaokui; Bennett, Chad E; Coss, Christopher C; Ling, Yonghua; Phelps, Mitch A; Houghton, Peter; London, Cheryl A

2017-01-01

STAT3 is a transcription factor involved in cytokine and receptor kinase signal transduction that is aberrantly activated in a variety of sarcomas, promoting metastasis and chemotherapy resistance. The purpose of this work was to develop and test a novel putative STAT3 inhibitor, LY5. An in silico fragment-based drug design strategy was used to create LY5, a small molecule inhibitor that blocks the STAT3 SH2 domain phosphotyrosine binding site, inhibiting homodimerization. LY5 was evaluated in vitro demonstrating good biologic activity against rhabdomyosarcoma, osteosarcoma and Ewing's sarcoma cell lines at high nanomolar/low micromolar concentrations, as well as specific inhibition of STAT3 phosphorylation without effects on other STAT3 family members. LY5 exhibited excellent oral bioavailability in both mice and healthy dogs, and drug absorption was enhanced in the fasted state with tolerable dosing in mice at 40 mg/kg BID. However, RNAi-mediated knockdown of STAT3 did not phenocopy the biologic effects of LY5 in sarcoma cell lines. Moreover, concentrations needed to inhibit ex vivo metastasis growth using the PuMA assay were significantly higher than those needed to inhibit STAT3 phosphorylation in vitro. Lastly, LY5 treatment did not inhibit the growth of sarcoma xenografts or prevent pulmonary metastasis in mice. LY5 is a novel small molecule inhibitor that effectively inhibits STAT3 phosphorylation and cell proliferation at nanomolar concentrations. LY5 demonstrates good oral bioavailability in mice and dogs. However LY5 did not decrease tumor growth in xenograft mouse models and STAT3 knockdown did not induce concordant biologic effects. These data suggest that the anti-cancer effects of LY5 identified in vitro were not mediated through STAT3 inhibition.

Regulation of rat heme oxygenase-1 expression by interleukin-6 via the Jak/STAT pathway in hepatocytes.

PubMed

Tron, Kyrylo; Samoylenko, Anatoly; Musikowski, Gernot; Kobe, Fritz; Immenschuh, Stephan; Schaper, Fred; Ramadori, Giuliano; Kietzmann, Thomas

2006-07-01

Heme oxygenase-1 (HO-1) can be induced by various stimuli, one of which is interleukin-6 (IL-6). Therefore, the aim of this study was to elucidate the molecular mechanisms responsible for IL-6-dependent HO-1 induction in the liver. The IL-6-dependent HO-1 regulation in rat primary hepatocytes and HepG2 hepatoma cells was studied by Northern and Western blot analyses, HO-1 promoter reporter gene assays and EMSA. The HO-1 expression was transcriptionally induced by IL-6 in a time- and dose-dependent manner. Activation of signal transducers and activators of transcription (STAT) factors by the IL-6 receptor was crucial for HO-1 induction. By contrast, negative regulation of HO-1 expression appeared to be mediated through the SH2-domain-containing tyrosine phosphatase-2 (SHP2)/ suppressors of cytokine signaling-3 (SOCS3) binding site within the gp130 IL-6 receptor subunit. Among the three putative STAT binding elements (SBE) in the HO-1 promoter, only the distal one was functional and when deleted, the remaining Luc induction was completely obliterated by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. The HO-1 SBE3 mediates HO-1 gene induction by IL-6 mainly via activation of the Jak/STAT pathway.

Inhibition of Stat3 activation suppresses caspase-3 and the ubiquitin-proteasome system, leading to preservation of muscle mass in cancer cachexia.

PubMed

Silva, Kleiton Augusto Santos; Dong, Jiangling; Dong, Yanjun; Dong, Yanlan; Schor, Nestor; Tweardy, David J; Zhang, Liping; Mitch, William E

2015-04-24

Cachexia occurs in patients with advanced cancers. Despite the adverse clinical impact of cancer-induced muscle wasting, pathways causing cachexia are controversial, and clinically reliable therapies are not available. A trigger of muscle protein loss is the Jak/Stat pathway, and indeed, we found that conditioned medium from C26 colon carcinoma (C26) or Lewis lung carcinoma cells activates Stat3 (p-Stat3) in C2C12 myotubes. We identified two proteolytic pathways that are activated in muscle by p-Stat3; one is activation of caspase-3, and the other is p-Stat3 to myostatin, MAFbx/Atrogin-1, and MuRF-1 via CAAT/enhancer-binding protein δ (C/EBPδ). Using sequential deletions of the caspase-3 promoter and CHIP assays, we determined that Stat3 activation increases caspase-3 expression in C2C12 cells. Caspase-3 expression and proteolytic activity were stimulated by p-Stat3 in muscles of tumor-bearing mice. In mice with cachexia caused by Lewis lung carcinoma or C26 tumors, knock-out of p-Stat3 in muscle or with a small chemical inhibitor of p-Stat3 suppressed muscle mass losses, improved protein synthesis and degradation in muscle, and increased body weight and grip strength. Activation of p-Stat3 stimulates a pathway from C/EBPδ to myostatin and expression of MAFbx/Atrogin-1 and increases the ubiquitin-proteasome system. Indeed, C/EBPδ KO decreases the expression of MAFbx/Atrogin-1 and myostatin, while increasing muscle mass and grip strength. In conclusion, cancer stimulates p-Stat3 in muscle, activating protein loss by stimulating caspase-3, myostatin, and the ubiquitin-proteasome system. These results could lead to novel strategies for preventing cancer-induced muscle wasting. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Inhibition of Stat3 Activation Suppresses Caspase-3 and the Ubiquitin-Proteasome System, Leading to Preservation of Muscle Mass in Cancer Cachexia*

PubMed Central

Silva, Kleiton Augusto Santos; Dong, Jiangling; Dong, Yanjun; Dong, Yanlan; Schor, Nestor; Tweardy, David J.; Zhang, Liping; Mitch, William E.

2015-01-01

Cachexia occurs in patients with advanced cancers. Despite the adverse clinical impact of cancer-induced muscle wasting, pathways causing cachexia are controversial, and clinically reliable therapies are not available. A trigger of muscle protein loss is the Jak/Stat pathway, and indeed, we found that conditioned medium from C26 colon carcinoma (C26) or Lewis lung carcinoma cells activates Stat3 (p-Stat3) in C2C12 myotubes. We identified two proteolytic pathways that are activated in muscle by p-Stat3; one is activation of caspase-3, and the other is p-Stat3 to myostatin, MAFbx/Atrogin-1, and MuRF-1 via CAAT/enhancer-binding protein δ (C/EBPδ). Using sequential deletions of the caspase-3 promoter and CHIP assays, we determined that Stat3 activation increases caspase-3 expression in C2C12 cells. Caspase-3 expression and proteolytic activity were stimulated by p-Stat3 in muscles of tumor-bearing mice. In mice with cachexia caused by Lewis lung carcinoma or C26 tumors, knock-out of p-Stat3 in muscle or with a small chemical inhibitor of p-Stat3 suppressed muscle mass losses, improved protein synthesis and degradation in muscle, and increased body weight and grip strength. Activation of p-Stat3 stimulates a pathway from C/EBPδ to myostatin and expression of MAFbx/Atrogin-1 and increases the ubiquitin-proteasome system. Indeed, C/EBPδ KO decreases the expression of MAFbx/Atrogin-1 and myostatin, while increasing muscle mass and grip strength. In conclusion, cancer stimulates p-Stat3 in muscle, activating protein loss by stimulating caspase-3, myostatin, and the ubiquitin-proteasome system. These results could lead to novel strategies for preventing cancer-induced muscle wasting. PMID:25787076

Vaccinia Virus Protein C6 Inhibits Type I IFN Signalling in the Nucleus and Binds to the Transactivation Domain of STAT2.

PubMed

Stuart, Jennifer H; Sumner, Rebecca P; Lu, Yongxu; Snowden, Joseph S; Smith, Geoffrey L

2016-12-01

The type I interferon (IFN) response is a crucial innate immune signalling pathway required for defense against viral infection. Accordingly, the great majority of mammalian viruses possess means to inhibit this important host immune response. Here we show that vaccinia virus (VACV) strain Western Reserve protein C6, is a dual function protein that inhibits the cellular response to type I IFNs in addition to its published function as an inhibitor of IRF-3 activation, thereby restricting type I IFN production from infected cells. Ectopic expression of C6 inhibits the induction of interferon stimulated genes (ISGs) in response to IFNα treatment at both the mRNA and protein level. C6 inhibits the IFNα-induced Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathway at a late stage, downstream of STAT1 and STAT2 phosphorylation, nuclear translocation and binding of the interferon stimulated gene factor 3 (ISGF3) complex to the interferon stimulated response element (ISRE). Mechanistically, C6 associates with the transactivation domain of STAT2 and this might explain how C6 inhibits the type I IFN signalling very late in the pathway. During virus infection C6 reduces ISRE-dependent gene expression despite the presence of the viral protein phosphatase VH1 that dephosphorylates STAT1 and STAT2. The ability of a cytoplasmic replicating virus to dampen the immune response within the nucleus, and the ability of viral immunomodulators such as C6 to inhibit multiple stages of the innate immune response by distinct mechanisms, emphasizes the intricacies of host-pathogen interactions and viral immune evasion.

JAK and STAT members of yellow catfish Pelteobagrus fulvidraco and their roles in leptin affecting lipid metabolism.

PubMed

Wu, Kun; Tan, Xiao-Ying; Xu, Yi-Huan; Chen, Qi-Liang; Pan, Ya-Xiong

2016-01-15

The present study clones and characterizes the full-length cDNA sequences of members in JAK-STAT pathway, explores their mRNA tissue expression and the biological role in leptin influencing lipid metabolism in yellow catfish Pelteobagrus fulvidraco. Full-length cDNA sequences of five JAKs and seven STAT members, including some splicing variants, were obtained from yellow catfish. Compared to mammals, more members of the JAKs and STATs family were found in yellow catfish, which provided evidence that the JAK and STAT family members had arisen by the whole genome duplications during vertebrate evolution. All of these members were widely expressed across the eleven tissues (liver, white muscle, spleen, brain, gill, mesenteric fat, anterior intestine, heart, mid-kidney, testis and ovary) but at the variable levels. Intraperitoneal injection in vivo and incubation in vitro of recombinant human leptin changed triglyceride content and mRNA expression of several JAKs and STATs members, and genes involved in lipid metabolism. AG490, a specific inhibitor of JAK2-STAT pathway, partially reversed leptin-induced effects, indicating that the JAK2a/b-STAT3 pathway exerts main regulating actions of leptin on lipid metabolism at transcriptional level. Meanwhile, the different splicing variants were differentially regulated by leptin incubation. Thus, our data suggest that leptin activated the JAK/STAT pathway and increases the expression of target genes, which partially accounts for the leptin-induced changes in lipid metabolism in yellow catfish. Copyright © 2015 Elsevier Inc. All rights reserved.

Rapid Molecular Analysis of the STAT3 Gene in Job Syndrome of Hyper-IgE and Recurrent Infectious Diseases

PubMed Central

Kumánovics, Attila; Wittwer, Carl T.; Pryor, Robert J.; Augustine, Nancy H.; Leppert, Mark F.; Carey, John C.; Ochs, Hans D.; Wedgwood, Ralph J.; Faville, Ralph J.; Quie, Paul G.; Hill, Harry R.

2010-01-01

With the recent discovery of mutations in the STAT3 gene in the majority of patients with classic Hyper-IgE syndrome, it is now possible to make a molecular diagnosis in most of these cases. We have developed a PCR-based high-resolution DNA-melting assay to scan selected exons of the STAT3 gene for mutations responsible for Hyper-IgE syndrome, which is then followed by targeted sequencing. We scanned for mutations in 10 unrelated pedigrees, which include 16 patients with classic Hyper-IgE syndrome. These pedigrees include both sporadic and familial cases and their relatives, and we have found STAT3 mutations in all affected individuals. High-resolution melting analysis allows a single day turn-around time for mutation scanning and targeted sequencing of the STAT3 gene, which will greatly facilitate the rapid diagnosis of the Hyper-IgE syndrome, allowing prompt and appropriate therapy, prophylaxis, improved clinical outcome, and accurate genetic counseling. PMID:20093388

Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

PubMed

Gaviglio, Angela L; Knelson, Erik H; Blobe, Gerard C

2017-05-01

High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor-like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.-Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation. © FASEB.

Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

PubMed Central

Gaviglio, Angela L.; Knelson, Erik H.; Blobe, Gerard C.

2017-01-01

High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor–like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.—Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation. PMID:28174207

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Yanxin; Biomedical Research Institute, Shenzhen-PKU-HKUST Medical Center, Guangdong Province, Shenzhen 518036; Yue, Xupeng

Highlights: •miR-124 is down-regulated in hepatocellular carcinoma HepG2 cells. •Over-expression of miR-124 suppresses proliferation and induces apoptosis in HepG2 cells. •miR-124 inhibits xenograft tumor growth in nude mice implanted with HepG2 cells by reducing STAT3 expression. •STATs function as a novel target of miR-124 in HCC HepG2 cells. -- Abstract: The aberrant expression of microRNAs is associated with development and progression of cancers. Down-regulation of miR-124 has been demonstrated in the hepatocellular carcinoma (HCC), but the underlying mechanism by which miR-124 suppresses tumorigenesis in HCC remains elusive. In this study, we found that miR-124 suppresses the tumor growth of HCCmore » through targeting the signal transducers and activators of transcription 3 (STAT3). Overexpression of miR-124 suppressed proliferation and induced apoptosis in HepG-2 cells. Luciferase assay confirmed that miR-124 binding to the 3′-UTR region of STAT3 inhibited the expression of STAT3 and phosphorylated STAT3 proteins in HepG-2 cells. Knockdown of STAT3 by siRNA in HepG-2 cells mimicked the effect induced by miR-124. Overexpression of STAT3 in miR-124-transfected HepG-2 cells effectively rescued the inhibition of cell proliferation caused by miR-124. Furthermore, miR-124 suppressed xenograft tumor growth in nude mice implanted with HepG-2 cells by reducing STAT3 expression. Taken together, our findings show that miR-124 functions as tumor suppressor in HCC by targeting STAT3, and miR-124 may therefore serve as a biomarker for diagnosis and therapeutics in HCC.« less

Altered IFN-γ-mediated immunity and transcriptional expression patterns in N-Ethyl-N-nitrosourea-induced STAT4 mutants confer susceptibility to acute typhoid-like disease.

PubMed

Eva, Megan M; Yuki, Kyoko E; Dauphinee, Shauna M; Schwartzentruber, Jeremy A; Pyzik, Michal; Paquet, Marilène; Lathrop, Mark; Majewski, Jacek; Vidal, Silvia M; Malo, Danielle

2014-01-01

Salmonella enterica is a ubiquitous Gram-negative intracellular bacterium that continues to pose a global challenge to human health. The etiology of Salmonella pathogenesis is complex and controlled by pathogen, environmental, and host genetic factors. In fact, patients immunodeficient in genes in the IL-12, IL-23/IFN-γ pathway are predisposed to invasive nontyphoidal Salmonella infection. Using a forward genomics approach by N-ethyl-N-nitrosourea (ENU) germline mutagenesis in mice, we identified the Ity14 (Immunity to Typhimurium locus 14) pedigree exhibiting increased susceptibility following in vivo Salmonella challenge. A DNA-binding domain mutation (p.G418_E445) in Stat4 (Signal Transducer and Activator of Transcription Factor 4) was the causative mutation. STAT4 signals downstream of IL-12 to mediate transcriptional regulation of inflammatory immune responses. In mutant Ity14 mice, the increased splenic and hepatic bacterial load resulted from an intrinsic defect in innate cell function, IFN-γ-mediated immunity, and disorganized granuloma formation. We further show that NK and NKT cells play an important role in mediating control of Salmonella in Stat4(Ity14/Ity14) mice. Stat4(Ity14/Ity14) mice had increased expression of genes involved in cell-cell interactions and communication, as well as increased CD11b expression on a subset of splenic myeloid dendritic cells, resulting in compromised recruitment of inflammatory cells to the spleen during Salmonella infection. Stat4(Ity14/Ity14) presented upregulated compensatory mechanisms, although inefficient and ultimately Stat4(Ity14/Ity14) mice develop fatal bacteremia. The following study further elucidates the pathophysiological impact of STAT4 during Salmonella infection.

Defective interleukin-4/Stat6 activity correlates with increased constitutive expression of negative regulators SOCS-3, SOCS-7, and CISH in colon cancer cells.

PubMed

Liu, Xiao Hong; Xu, Shuang Bing; Yuan, Jia; Li, Ben Hui; Zhang, Yan; Yuan, Qin; Li, Pin Dong; Li, Feng; Zhang, Wen Jie

2009-12-01

Interleukin-4 (IL-4)-induced Stat6 activities (phenotypes) vary among human cancer cells, of which the HT-29 cell line carries an active Stat6(high) phenotype, while Caco-2 carries a defective Stat6(null) phenotype, respectively. Cancer cells with Stat6(high) show resistance to apoptosis and exaggerated metastasis, suggesting the clinical significance of Stat6 phenotypes. We previously showed that Stat6(high) HT-29 cells exhibited low constitutive expression of Stat6-negative regulators SOCS-1 and SHP-1 because of gene hypermethylation. This study further examined the constitutive expression of other closely related SOCS family numbers including SOCS-3, SOCS-5, SOCS-7, and CISH using RT-PCR. Similar to SOCS-1 and SHP-1, Stat6(high) HT-29 cells expressed low constitutive mRNA of SOCS-3, SOCS-7, and CISH than Stat6(null) Caco-2 cells. Interestingly, DNA demethylation using 5-aza-2'-deoxycytidine in HT-29 cells up-regulated mRNA expression of the above genes, indicating a hypermethylation status, which was confirmed by methylation-specific sequencing in selected SOCS-3 gene. Furthermore, defective Stat6(null) Caco-2 exhibited impaired phosphorylation of Stat6 after IL-4 stimulation by flow cytometry, in keeping with the notion of an over-performed negative regulation. The findings that IL-4/Stat6 phenotypes show differential expression of multiple negative regulators suggest a model that a collective force of powerful negative regulators, directly and indirectly, acts on Stat6 activation, which may result in differential Stat6 phenotypes.

A genetic and developmental pathway from STAT3 to the OCT4–NANOG circuit is essential for maintenance of ICM lineages in vivo

PubMed Central

Do, Dang Vinh; Ueda, Jun; Messerschmidt, Daniel M.; Lorthongpanich, Chanchao; Zhou, Yi; Feng, Bo; Guo, Guoji; Lin, Peiyu J.; Hossain, Md Zakir; Zhang, Wenjun; Moh, Akira; Wu, Qiang; Robson, Paul; Ng, Huck Hui; Poellinger, Lorenz; Knowles, Barbara B.; Solter, Davor; Fu, Xin-Yuan

2013-01-01

Although it is known that OCT4–NANOG are required for maintenance of pluripotent cells in vitro, the upstream signals that regulate this circuit during early development in vivo have not been identified. Here we demonstrate, for the first time, signal transducers and activators of transcription 3 (STAT3)-dependent regulation of the OCT4–NANOG circuitry necessary to maintain the pluripotent inner cell mass (ICM), the source of in vitro-derived embryonic stem cells (ESCs). We show that STAT3 is highly expressed in mouse oocytes and becomes phosphorylated and translocates to the nucleus in the four-cell and later stage embryos. Using leukemia inhibitory factor (Lif)-null embryos, we found that STAT3 phosphorylation is dependent on LIF in four-cell stage embryos. In blastocysts, interleukin 6 (IL-6) acts in an autocrine fashion to ensure STAT3 phosphorylation, mediated by janus kinase 1 (JAK1), a LIF- and IL-6-dependent kinase. Using genetically engineered mouse strains to eliminate Stat3 in oocytes and embryos, we firmly establish that STAT3 is essential for maintenance of ICM lineages but not for ICM and trophectoderm formation. Indeed, STAT3 directly binds to the Oct4 and Nanog distal enhancers, modulating their expression to maintain pluripotency of mouse embryonic and induced pluripotent stem cells. These results provide a novel genetic model of cell fate determination operating through STAT3 in the preimplantation embryo and pluripotent stem cells in vivo. PMID:23788624

FL3, a Synthetic Flavagline and Ligand of Prohibitins, Protects Cardiomyocytes via STAT3 from Doxorubicin Toxicity

PubMed Central

Gasser, Adeline; Basmadjian, Christine; Zhao, Qian; Wilmet, Jean-Philippe; Désaubry, Laurent; Nebigil, Canan G.

2015-01-01

Aims The clinical use of doxorubicin for the treatment of cancer is limited by its cardiotoxicity. Flavaglines are natural products that have both potent anticancer and cardioprotective properties. A synthetic analog of flavaglines, FL3, efficiently protects mice from the cardiotoxicity of doxorubicin. The mechanism underlying this cardioprotective effect has yet to be elucidated. Methods and Results Here, we show that FL3 binds to the scaffold proteins prohibitins (PHBs) and thus promotes their translocation to mitochondria in the H9c2 cardiomyocytes. FL3 induces heterodimerization of PHB1 with STAT3, thereby ensuring cardioprotection from doxorubicin toxicity. This interaction is associated with phosphorylation of STAT3. A JAK2 inhibitor, WP1066, suppresses both the phosphorylation of STAT3 and the protective effect of FL3 in cardiomyocytes. The involvement of PHBs in the FL3-mediated cardioprotection was confirmed by means of small interfering RNAs (siRNAs) targeting PHB1 and PHB2. The siRNA knockdown of PHBs inhibits both phosphorylation of STAT3 and the cardioprotective effect of FL3. Conclusion Activation of mitochondrial STAT3/PHB1 complex by PHB ligands may be a new strategy against doxorubicin-induced cardiotoxicity and possibly other cardiac problems. PMID:26536361

Hierarchy within the mammary STAT5-driven Wap super-enhancer

PubMed Central

Zeng, Xianke; Wang, Chaochen; Metser, Gil; Hennighausen, Lothar

2016-01-01

Super-enhancers comprise of dense transcription factor platforms highly enriched for active chromatin marks. A paucity of functional data led us to investigate their role in the mammary gland, an organ characterized by exceptional gene regulatory dynamics during pregnancy. ChIP-Seq for the master regulator STAT5, the glucocorticoid receptor, H3K27ac and MED1, identified 440 mammary-specific super-enhancers, half of which were associated with genes activated during pregnancy. We interrogated the Wap super-enhancer, generating mice carrying mutations in STAT5 binding sites within its three constituent enhancers. Individually, only the most distal site displayed significant enhancer activity. However, combinatorial mutations showed that the 1,000-fold gene induction relied on all enhancers. Disabling the binding sites of STAT5, NFIB and ELF5 in the proximal enhancer incapacitated the entire super-enhancer, suggesting an enhancer hierarchy. The identification of mammary-specific super-enhancers and the mechanistic exploration of the Wap locus provide insight into the complexity of cell-specific and hormone-regulated genes. PMID:27376239

Parathyroid hormone inhibition of Na{sup +}/H{sup +} exchanger 3 transcription: Intracellular signaling pathways and transcription factor expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Neri, Elida Adalgisa; Bezerra, Camila Nogueira Alves, E-mail: camilab@icb.usp.br; Queiroz-Leite, Gabriella Duarte

2015-06-12

The main transport mechanism of reabsorption of sodium bicarbonate and fluid in the renal proximal tubules involves Na{sup +}/H{sup +} exchanger 3 (NHE3), which is acutely and chronically downregulated by parathyroid hormone (PTH). Although PTH is known to exert an inhibitory effect on NHE3 expression and transcription, the molecular mechanisms involved remain unclear. Here, we demonstrated that, in opossum kidney proximal tubule (OKP) cells, PTH-induced inhibition of Nhe3 gene promoter occurs even in the core promoter that controls expression of the reporter gene. We found that inhibition of the protein kinase A (PKA) and Janus kinase/signal transducer and activator ofmore » transcription (JAK/STAT) pathways transformed PTH from an inhibitor of promoter activity into an activator of that same activity, as did point mutations in the EGR1, Sp1, and Sp3 binding consensus elements in the promoter. In nuclear extracts of PTH-treated OKP cells, we also observed increased expression of EGR1 mRNA and of some Sp3 isoforms. Electrophoretic mobility shift assay showed a supershift of the −61 to −42-bp probe with an anti-EGR1 antibody in PTH-treated cells, suggesting that EGR1 binding is relevant for the inhibitory activity of PTH. We conclude that PTH-induced inhibition of NHE3 transcription is related to higher EGR1 expression; to EGR1 binding to the proximal and core promoters; and to PKA and JAK/STAT pathway activation. This mechanism might be responsible, at least in part, for lower NHE3 expression and sodium reabsorption in renal proximal tubules in the presence of high PTH levels. - Highlights: • PTH regulation of Nhe3 promoter depends on EGR1 binding. • EGR1, PKA and JAK/STAT are involved in PTH inhibition of the Nhe3 promoter. • PTH alters expression of EGR1 and Sp3. • PTH inhibits the Nhe3 promoter by regulating PKA and JAK/STAT signaling.« less

Reciprocal activation of α5-nAChR and STAT3 in nicotine-induced human lung cancer cell proliferation.

PubMed

Zhang, Yao; Jia, Yanfei; Li, Ping; Li, Huanjie; Xiao, Dongjie; Wang, Yunshan; Ma, Xiaoli

2017-07-20

Cigarette smoking is the top environmental risk factor for lung cancer. Nicotine, the addictive component of cigarettes, induces lung cancer cell proliferation, invasion and migration via the activation of nicotinic acetylcholine receptors (nAChRs). Genome-wide association studies (GWAS) show that CHRNA5 gene encoding α5-nAChR is especially relevant to lung cancer. However, the mechanism of this subunit in lung cancer is not clear. In the present study, we demonstrate that the expression of α5-nAChR is correlated with phosphorylated STAT3 (pSTAT3) expression, smoking history and lower survival of non-small cell lung cancer (NSCLC) samples. Nicotine increased the levels of α5-nAChR mRNA and protein in NSCLC cell lines and activated the JAK2/STAT3 signaling cascade. Nicotine-induced activation of JAK2/STAT3 signaling was inhibited by the silencing of α5-nAChR. Characterization of the CHRNA5 promoter revealed four STAT3-response elements. ChIP assays confirmed that the CHRNA5 promoter contains STAT3 binding sites. By silencing STAT3 expression, nicotine-induced upregulation of α5-nAChR was suppressed. Downregulation of α5-nAChR and/or STAT3 expression inhibited nicotine-induced lung cancer cell proliferation. These results suggest that there is a feedback loop between α5-nAChR and STAT3 that contributes to the nicotine-induced tumor cell proliferation, which indicates that α5-nAChR is an important therapeutic target involved in tobacco-associated lung carcinogenesis. Copyright © 2017 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Identification of novel kynurenine production-inhibiting benzenesulfonamide derivatives in cancer cells.

PubMed

Nakano, Shintaro; Takai, Kazushige; Isaka, Yoshinobu; Takahashi, Susumu; Unno, Yuka; Ogo, Naohisa; Matsuno, Kenji; Takikawa, Osamu; Asai, Akira

2012-03-16

Kynurenine (Kyn), a metabolite of tryptophan (Trp), is known to be a key regulator of human immune responses including cancer immune tolerance. Therefore, abrogation of Kyn production from cancer cells by small molecules may be a promising approach to anticancer therapy. Indeed, several small molecule inhibitors of indoleamine 2,3-dioxygenase (IDO), a rate-limiting enzyme in the catabolism of Trp to Kyn, exert antitumor effects in animal models. We screened our chemical libraries using a cell-based Kyn production assay to identify a new type of small molecules that regulate Kyn production, and for the first time identified a benzenesulfonamide derivative (compound 1) as a hit with the ability to inhibit Kyn production in interferon-γ (IFN-γ)-stimulated A431 and HeLa cells. Unlike the previously identified S-benzylisothiourea derivative, compound 2, compound 1 had little effect on the enzymatic activity of recombinant human IDO in vitro but suppressed the expression of IDO at the mRNA level in cells. Furthermore, compound 1 suppressed STAT1-dependent transcriptional activity and DNA binding, whereas no decrement in either the expression or phosphorylation level of STAT1 was observed. The inhibition of IDO expression by several benzenesulfonamide derivatives is associated with the suppression of STAT1. Thus, compound 1 and its analogs might be useful for analyzing the regulation of IDO activation, and STAT1-targeting could be an alternative to the IDO-directed approach for the regulation of Kyn levels by small molecules in the tumor microenvironment. Copyright © 2012 Elsevier Inc. All rights reserved.

Disruption of Stat3 reveals a critical role in both the initiation and the promotion stages of epithelial carcinogenesis

PubMed Central

Chan, Keith Syson; Sano, Shigetoshi; Kiguchi, Kaoru; Anders, Joanne; Komazawa, Nobuyasu; Takeda, Junji; DiGiovanni, John

2004-01-01

Constitutive activation of signal transducer and activator of transcription 3 (Stat3) has been found in a wide spectrum of human malignancies. Here, we have assessed the effect of Stat3 deficiency on skin tumor development using the 2-stage chemical carcinogenesis model. The epidermis of Stat3-deficient mice showed a significantly reduced proliferative response following treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) because of a defect in G1-to-S-phase cell cycle progression. Treatment with the tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA) resulted in a significant increase in the number of keratinocyte stem cells undergoing apoptosis in the bulge region of hair follicles of Stat3-deficient mice compared with nontransgenic littermates. Notably, Stat3-deficient mice were completely resistant to skin tumor development when DMBA was used as the initiator and TPA as the promoter. Abrogation of Stat3 function using a decoy oligonucleotide inhibited the growth of initiated keratinocytes possessing an activated Ha-ras gene, both in vitro and in vivo. In addition, injection of Stat3 decoy into skin tumors inhibited their growth. To our knowledge, these data provide the first evidence that Stat3 is required for de novo epithelial carcinogenesis, through maintaining the survival of DNA-damaged stem cells and through mediating and maintaining the proliferation necessary for clonal expansion of initiated cells during tumor promotion. Collectively, these data suggest that, in addition to its emerging role as a target for cancer therapy, Stat3 may also be a target for cancer prevention strategies. PMID:15343391

Activated STAT5 proteins induce activation of the PI 3-kinase/Akt and Ras/MAPK pathways via the Gab2 scaffolding adapter.

PubMed

Nyga, Rémy; Pecquet, Christian; Harir, Noria; Gu, Haihua; Dhennin-Duthille, Isabelle; Régnier, Aline; Gouilleux-Gruart, Valérie; Lassoued, Kaïss; Gouilleux, Fabrice

2005-08-15

The active forms of STAT5A (signal transducer and activator of transcription 5A) and STAT5B are able to relieve the cytokine dependence of haematopoietic cells and to induce leukaemia in mice. We have demonstrated previously that activation of the PI3K (phosphoinositide 3-kinase) signalling cascade plays a major role in cell growth and survival induced by these proteins. Interaction between STAT5 and p85, the regulatory subunit of the PI3K, has been suggested to be required for this activation. We show in the present study that the scaffolding protein Gab2 [Grb2 (growth-factor-receptor-bound protein 2)-associated binder-2] is an essential component of this interaction. Gab2 is persistently tyrosine-phosphorylated in Ba/F3 cells expressing caSTAT5 (constitutively activated STAT5), independent of JAK2 (Janus kinase 2) activation where it interacts with STAT5, p85 and Grb2, but not with Shp2 [SH2 (Src homology 2)-domain-containing tyrosine phosphatase] proteins. Interaction of STAT5 with Gab2 was also observed in Ba/F3 cells stimulated with interleukin-3 or expressing the oncogenic fusion protein Tel-JAK2. The MAPKs (mitogen-activated protein kinases) ERK1 (extracellular-signal-regulated kinase 1) and ERK2 were constitutively activated in the caSTAT5-expressing cells and were found to be required for caSTAT5-induced cell proliferation. Overexpression of Gab2-3YF, a mutant of Gab2 incapable of binding PI3K, inhibited the proliferation and survival of caSTAT5-expressing cells as well as ERK1/2 and Akt/protein kinase B phosphorylation. Taken together, our results indicate that Gab2 is required for caSTAT5-induced cell proliferation by regulating both the PI3K/Akt and the Ras/MAPK pathways.

Activated STAT5 proteins induce activation of the PI 3-kinase/Akt and Ras/MAPK pathways via the Gab2 scaffolding adapter

PubMed Central

2005-01-01

The active forms of STAT5A (signal transducer and activator of transcription 5A) and STAT5B are able to relieve the cytokine dependence of haematopoietic cells and to induce leukaemia in mice. We have demonstrated previously that activation of the PI3K (phosphoinositide 3-kinase) signalling cascade plays a major role in cell growth and survival induced by these proteins. Interaction between STAT5 and p85, the regulatory subunit of the PI3K, has been suggested to be required for this activation. We show in the present study that the scaffolding protein Gab2 [Grb2 (growth-factor-receptor-bound protein 2)-associated binder-2] is an essential component of this interaction. Gab2 is persistently tyrosine-phosphorylated in Ba/F3 cells expressing caSTAT5 (constitutively activated STAT5), independent of JAK2 (Janus kinase 2) activation where it interacts with STAT5, p85 and Grb2, but not with Shp2 [SH2 (Src homology 2)-domain-containing tyrosine phosphatase] proteins. Interaction of STAT5 with Gab2 was also observed in Ba/F3 cells stimulated with interleukin-3 or expressing the oncogenic fusion protein Tel–JAK2. The MAPKs (mitogen-activated protein kinases) ERK1 (extracellular-signal-regulated kinase 1) and ERK2 were constitutively activated in the caSTAT5-expressing cells and were found to be required for caSTAT5-induced cell proliferation. Overexpression of Gab2-3YF, a mutant of Gab2 incapable of binding PI3K, inhibited the proliferation and survival of caSTAT5-expressing cells as well as ERK1/2 and Akt/protein kinase B phosphorylation. Taken together, our results indicate that Gab2 is required for caSTAT5-induced cell proliferation by regulating both the PI3K/Akt and the Ras/MAPK pathways. PMID:15833084

New and recurrent gain-of-function STAT1 mutations in patients with chronic mucocutaneous candidiasis from Eastern and Central Europe.

PubMed

Soltész, Beáta; Tóth, Beáta; Shabashova, Nadejda; Bondarenko, Anastasia; Okada, Satoshi; Cypowyj, Sophie; Abhyankar, Avinash; Csorba, Gabriella; Taskó, Szilvia; Sarkadi, Adrien Katalin; Méhes, Leonóra; Rozsíval, Pavel; Neumann, David; Chernyshova, Liudmyla; Tulassay, Zsolt; Puel, Anne; Casanova, Jean-Laurent; Sediva, Anna; Litzman, Jiri; Maródi, László

2013-09-01

Chronic mucocutaneous candidiasis disease (CMCD) may result from various inborn errors of interleukin (IL)-17-mediated immunity. Twelve of the 13 causal mutations described to date affect the coiled-coil domain (CCD) of STAT1. Several mutations, including R274W in particular, are recurrent, but the underlying mechanism is unclear. To investigate and describe nine patients with CMCD in Eastern and Central Europe, to assess the biochemical impact of STAT1 mutations, to determine cytokines in supernatants of Candida-exposed blood cells, to determine IL-17-producing T cell subsets and to determine STAT1 haplotypes in a family with the c.820C>T (R274W) mutation. The novel c.537C>A (N179K) STAT1 mutation was gain-of-function (GOF) for γ-activated factor (GAF)-dependent cellular responses. In a Russian patient, the cause of CMCD was the newly identified c.854 A>G (Q285R) STAT1 mutation, which was also GOF for GAF-dependent responses. The c.1154C>T (T385M) mutation affecting the DNA-binding domain (DBD) resulted in a gain of STAT1 phosphorylation in a Ukrainian patient. Impaired Candida-induced IL-17A and IL-22 secretion by leucocytes and lower levels of intracellular IL-17 and IL-22 production by T cells were found in several patients. Haplotype studies indicated that the c.820C>T (R274W) mutation was recurrent due to a hotspot rather than a founder effect. Severe clinical phenotypes, including intracranial aneurysm, are presented. The c.537C>A and c.854A>G mutations affecting the CCD and the c.1154C>T mutation affecting the DBD of STAT1 are GOF. The c.820C>T mutation of STAT1 in patients with CMCD is recurrent due to a hotspot. Patients carrying GOF mutations of STAT1 may develop multiple intracranial aneurysms by hitherto unknown mechanisms.

Farm exposure and time trends in early childhood may influence DNA methylation in genes related to asthma and allergy.

PubMed

Michel, S; Busato, F; Genuneit, J; Pekkanen, J; Dalphin, J-C; Riedler, J; Mazaleyrat, N; Weber, J; Karvonen, A M; Hirvonen, M-R; Braun-Fahrländer, C; Lauener, R; von Mutius, E; Kabesch, M; Tost, J

2013-03-01

Genetic susceptibility and environmental influences are important contributors to the development of asthma and atopic diseases. Epigenetic mechanisms may facilitate gene by environment interactions in these diseases. We studied the rural birth cohort PASTURE (Protection against allergy: study in rural environments) to investigate (a) whether epigenetic patterns in asthma candidate genes are influenced by farm exposure in general, (b) change over the first years of life, and (c) whether these changes may contribute to the development of asthma. DNA was extracted from cord blood and whole blood collected at the age of 4.5 years in 46 samples per time point. DNA methylation in 23 regions in ten candidate genes (ORMDL1, ORMDL2, ORMDL3, CHI3L1, RAD50, IL13, IL4, STAT6, FOXP3, and RUNX3) was assessed by pyrosequencing, and differences between strata were analyzed by nonparametric Wilcoxon-Mann-Whitney tests. In cord blood, regions in ORMDL1 and STAT6 were hypomethylated in DNA from farmers' as compared to nonfarmers' children, while regions in RAD50 and IL13 were hypermethylated (lowest P-value (STAT6) = 0.001). Changes in methylation over time occurred in 15 gene regions (lowest P-value (IL13) = 1.57*10(-8)). Interestingly, these differences clustered in the genes highly associated with asthma (ORMDL family) and IgE regulation (RAD50, IL13, and IL4), but not in the T-regulatory genes (FOXP3, RUNX3). In this first pilot study, DNA methylation patterns change significantly in early childhood in specific asthma- and allergy-related genes in peripheral blood cells, and early exposure to farm environment seems to influence methylation patterns in distinct genes. © 2013 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

New sulfurated derivatives of cinnamic acids and rosmaricine as inhibitors of STAT3 and NF-κB transcription factors.

PubMed

Gabriele, Elena; Brambilla, Dario; Ricci, Chiara; Regazzoni, Luca; Taguchi, Kyoko; Ferri, Nicola; Asai, Akira; Sparatore, Anna

2017-12-01

A set of new sulfurated drug hybrids, mainly derived from caffeic and ferulic acids and rosmaricine, has been synthesized and their ability to inhibit both STAT3 and NF-κB transcription factors have been evaluated. Results showed that most of the new hybrid compounds were able to strongly and selectively bind to STAT3, whereas the parent drugs were devoid of this ability at the tested concentrations. Some of them were also able to inhibit the NF-κB transcriptional activity in HCT-116 cell line and inhibited HCT-116 cell proliferation in vitro with IC 50 in micromolar range, thus suggesting a potential anticancer activity. Taken together, our study described the identification of new derivatives with dual STAT3/NF-κB inhibitory activity, which may represent hit compounds for developing multi-target anticancer agents.

Propofol enhances the cisplatin-induced apoptosis on cervical cancer cells via EGFR/JAK2/STAT3 pathway.

PubMed

Li, Haoran; Lu, Yan; Pang, Yangyang; Li, Mengjiao; Cheng, Xi; Chen, Jiawei

2017-02-01

The main purpose of this study was to evaluate propofol and its combined effect with cisplatin on apoptosis of cervical cancer cells and molecular mechanisms of this phenomenon. The effects of propofol and cisplatin on cell viability and apoptosis were detected by cell counting kit-8 (CCK-8) assay, colony formation assay and flow cytometry assay. Besides, protein expression of EGFR/JAK2/STAT3 pathway was determined by western blot. STAT3 was over-expressed in cervical cancer cells by STAT3 cDNA. Expression of EGFR and STAT3 protein of human tissues was evaluated by immunohistochemistry (IHC) assay. In this study, we found that not only propofol alone could inhibit cervical cancer cells viability but also could increase the inhibitory effect of cisplatin on cervical cancer cells growth. Meanwhile, propofol sensitized cervical cancer cells to cisplatin-induced apoptosis but not affected normal cervical cells. In genetic level, propofol could enhance the anti-tumor effect of cisplatin through EGFR/JAK2/STAT3 pathway. Further studies indicated that overexpression of EGFR and STAT3 is related to poor prognoses in cervical cancer patients, which contributed to confirm the clinical role of combined application of propofol and cisplatin. Propofol enhances the cisplatin-induced cell apoptosis cervical cancer cells via EGFR/JAK2/STAT3 pathway and may be developed as a potential therapeutic agent to treat cervical cancer. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Uterine Deletion of Gp130 or Stat3 Shows Implantation Failure with Increased Estrogenic Responses

PubMed Central

Sun, Xiaofei; Bartos, Amanda; Whitsett, Jeffrey A.

2013-01-01

Leukemia inhibitory factor (LIF), a downstream target of estrogen, is essential for implantation in mice. LIF function is thought to be mediated by its binding to LIF receptor (LIFR) and recruitment of coreceptor GP130 (glycoprotein 130), and this receptor complex then activates signal transducer and activator of transcription (STAT)1/3. However, the importance of LIFR and GP130 acting via STAT3 in implantation remains uncertain, because constitutive inactivation of Lifr, Gp130, or Stat3 shows embryonic lethality in mice. To address this issue, we generated mice with conditional deletion of uterine Gp130 or Stat3 and show that both GP130 and STAT3 are critical for uterine receptivity and implantation. Implantation failure in these deleted mice is associated with higher uterine estrogenic responses prior to the time of implantation. These heightened estrogenic responses are not due to changes in ovarian hormone levels or expression of their nuclear receptors. In the deleted mice, estrogen-responsive gene, Lactoferrin (Ltf), and Mucin 1 protein, were up-regulated in the uterus. In addition, progesterone-responsive genes, Hoxa10 and Indian hedgehog (Ihh), were markedly down-regulated in STAT3-inactivated uteri. These changes in uteri of deleted mice were reflected by the failure of differentiation of the luminal epithelium, which is essential for blastocyst attachment. PMID:23885093

STAT3 activation in pressure-overloaded feline myocardium: role for integrins and the tyrosine kinase BMX.

PubMed

Willey, Christopher D; Palanisamy, Arun P; Johnston, Rebecca K; Mani, Santhosh K; Shiraishi, Hirokazu; Tuxworth, William J; Zile, Michael R; Balasubramanian, Sundaravadivel; Kuppuswamy, Dhandapani

2008-06-27

Growth, survival and cytoskeletal rearrangement of cardiomyocytes are critical for cardiac hypertrophy. Signal transducer and activator of transcription-3 (STAT3) activation is an important cardioprotective factor associated with cardiac hypertrophy. Although STAT3 activation has been reported via signaling through Janus Kinase 2 (JAK2) in several cardiac models of hypertrophy, the importance of other nonreceptor tyrosine kinases (NTKs) has not been explored. Utilizing an in vivo feline right ventricular pressure-overload (RVPO) model of hypertrophy, we demonstrate that in 48 h pressure-overload (PO) myocardium, STAT3 becomes phosphorylated and redistributed to detergent-insoluble fractions with no accompanying JAK2 activation. PO also caused increased levels of phosphorylated STAT3 in both cytoplasmic and nuclear fractions. To investigate the role of other NTKs, we used our established in vitro cell culture model of hypertrophy where adult feline cardiomyocytes are embedded three-dimensionally (3D) in type-I collagen and stimulated with an integrin binding peptide containing an Arg-Gly-Asp (RGD) motif that we have previously shown to recapitulate the focal adhesion complex (FAC) formation of 48 h RVPO. RGD stimulation of adult cardiomyocytes in vitro caused both STAT3 redistribution and activation that were accompanied by the activation and redistribution of c-Src and the TEC family kinase, BMX, but not JAK2. However, infection with dominant negative c-Src adenovirus was unable to block RGD-stimulated changes on either STAT3 or BMX. Further analysis in vivo in 48 h PO myocardium showed the presence of both STAT3 and BMX in the detergent-insoluble fraction with their complex formation and phosphorylation. Therefore, these studies indicate a novel mechanism of BMX-mediated STAT3 activation within a PO model of cardiac hypertrophy that might contribute to cardiomyocyte growth and survival.

Vorinostat Enhances Cytotoxicity of SN-38 and Temozolomide in Ewing Sarcoma Cells and Activates STAT3/AKT/MAPK Pathways

PubMed Central

Sampson, Valerie B.; Vetter, Nancy S.; Kamara, Davida F.; Collier, Anderson B.; Gresh, Renee C.; Kolb, E. Anders

2015-01-01

Histone deacetylase inhibitors (HDACi) have been evaluated in patients with Ewing sarcoma (EWS) but demonstrated limited activity. To better understand the potential for HDACi in EWS, we evaluated the combination of the HDACi vorinostat, with DNA damaging agents SN-38 (the active metabolite of irinotecan and topoisomerase 1 inhibitor) plus the alkylating agent temozolomide (ST). Drugs were evaluated in sequential and simultaneous combinations in two EWS cell lines. Results demonstrate that cell viability, DNA damage and reactive oxygen species (ROS) production are dependent on the sequence of drug administration. Enhanced cytotoxicity is exhibited in vitro in EWS cell lines treated with ST administered before vorinostat, which was modestly higher than concomitant treatment and superior to vorinostat administered before ST. Drug combinations downregulate cyclin D1 to induce G0/G1 arrest and promote apoptosis by cleavage of caspase-3 and PARP. When ST is administered before or concomitantly with vorinostat there is activation of STAT3, MAPK and the p53 pathway. In contrast, when vorinostat is administered before ST, there is DNA repair, increased AKT phosphorylation and reduced H2B acetylation. Inhibition of AKT using the small molecule inhibitor MK-2206 did not restore H2B acetylation. Combining ST with the dual ALK and IGF-1R inhibitor, AZD3463 simultaneously inhibited STAT3 and AKT to enhance the cytotoxic effects of ST and further reduce cell growth suggesting that STAT3 and AKT activation were in part mediated by ALK and IGF-1R signaling. In summary, potent antiproliferative and proapoptotic activity were demonstrated for ST induced DNA damage before or simultaneous with HDAC inhibition and cell death was mediated through the p53 pathway. These observations may aid in designing new protocols for treating pediatric patients with high-risk EWS. PMID:26571493

Activation of the protein tyrosine phosphatase SHP2 via the interleukin-6 signal transducing receptor protein gp130 requires tyrosine kinase Jak1 and limits acute-phase protein expression.

PubMed

Schaper, F; Gendo, C; Eck, M; Schmitz, J; Grimm, C; Anhuf, D; Kerr, I M; Heinrich, P C

1998-11-01

Stimulation of the interleukin-6 (IL-6) signalling pathway occurs via the IL-6 receptor-glycoprotein 130 (IL-6R-gp130) receptor complex and results in the regulation of acute-phase protein genes in liver cells. Ligand binding to the receptor complex leads to tyrosine phosphorylation and activation of Janus kinases (Jak), phosphorylation of the signal transducing subunit gp130, followed by recruitment and phosphorylation of the signal transducer and activator of transcription factors STAT3 and STAT1 and the src homology domain (SH2)-containing protein tyrosine phosphatase (SHP2). The tyrosine phosphorylated STAT factors dissociate from the receptor, dimerize and translocate to the nucleus where they bind to enhancer sequences of IL-6 target genes. Phosphorylated SHP2 is able to bind growth factor receptor bound protein (grb2) and thus might link the Jak/STAT pathway to the ras/raf/mitogen-activated protein kinase pathway. Here we present data on the dose-dependence, kinetics and kinase requirements for SHP2 phosphorylation after the activation of the signal transducer, gp130, of the IL-6-type family receptor complex. When human fibrosarcoma cell lines deficient in Jak1, Jak2 or tyrosine kinase 2 (Tyk2) were stimulated with IL-6-soluble IL-6R complexes it was found that only in Jak1-, but not in Jak 2- or Tyk2-deficient cells, SHP2 activation was greatly impaired. It is concluded that Jak1 is required for the tyrosine phosphorylation of SHP2. This phosphorylation depends on Tyr-759 in the cytoplasmatic domain of gp130, since a Tyr-759-->Phe exchange abrogates SHP2 activation and in turn leads to elevated and prolonged STAT3 and STAT1 activation as well as enhanced acute-phase protein gene induction. Therefore, SHP2 plays an important role in acute-phase gene regulation.

Regulation of Spi 2.1 and 2.2 gene expression after turpentine inflammation: discordant responses to IL-6.

PubMed

Berry, S A; Bergad, P L; Stolz, A M; Towle, H C; Schwarzenberg, S J

1999-06-01

The rat serine protease inhibitor (Spi) 2 gene family includes both positive (Spi 2.2) and negative (Spi 2.1) acute phase reactants, facilitating modeling of regulation of hepatic acute phase response (APR). To examine the role of signal transducer and activation of transcription (STAT) proteins in the divergent regulation of these model genes after induction of APR, we evaluated the proximal promoters of the genes, focusing on STAT binding sites contained in these promoter elements. Induction of APR by turpentine injection includes activation of a STAT3 complex that can bind to a gamma-activated sequence (GAS) in the Spi 2.2 gene promoter, although the Spi 2.2 GAS site can bind STAT1 or STAT5 as well. To create an in vitro model of APR, primary hepatocytes were treated with combinations of cytokines and hormones to mimic the hormonal milieu of the whole animal after APR induction. Incubation of primary rat hepatocytes with interleukin (IL)-6, a critical APR cytokine, leads to activation of STAT3 and a 28-fold induction of a chloramphenicol acetyltransferase reporter construct containing the -319 to +85 region of the Spi 2.2 promoter. This suggests the turpentine-induced increase of Spi 2.2 is mediated primarily by IL-6. In contrast, although turpentine treatment reduces Spi 2.1 mRNA in vivo and IL-6 does not increase Spi 2.1 mRNA in primary rat hepatocytes, treatment of hepatocytes with IL-6 results in a 5. 4-fold induction of Spi 2.1 promoter activity mediated through the paired GAS elements in this promoter. Differential regulation of Spi 2.1 and 2.2 genes is due in part to differences in the promoters of these genes at the GAS sites. IL-6 alone fails to reproduce the pattern of rat Spi 2 gene expression that results from turpentine-induced inflammation.

Reciprocal activation between STAT3 and miR-181b regulates the proliferation of esophageal cancer stem-like cells via the CYLD pathway.

PubMed

Xu, Dan-Dan; Zhou, Peng-Jun; Wang, Ying; Zhang, Li; Fu, Wu-Yu; Ruan, Bi-Bo; Xu, Hai-Peng; Hu, Chao-Zhi; Tian, Lu; Qin, Jin-Hong; Wang, Sheng; Wang, Xiao; Li, Yi-Cheng; Liu, Qiu-Ying; Ren, Zhe; Zhang, Rong; Wang, Yi-Fei

2016-05-17

Recent studies have suggested that cancer cells contain subpopulations that can initiate tumor growth, self-renew, and maintain tumor cell growth. However, for esophageal cancer cells, the relationship between STAT3, microRNAs and cancer stem cells remains unclear. Serum-free culture was used to enrich esophageal cancer stem-like cells (ECSLC). Flow cytometry determined the proportion of ECSLC. qPCR were performed to examine expression level of stemness factors, mesenchymal markers, ATP-binding cassette (ABC) transporters, STAT3, miR-181b, CYLD. Western blot were performed to analyze the expression of STAT3, p-STAT3 and CYLD (cylindromatosis). BALB/c mice xenograft studies were conducted to evaluate the tumorigenicity of enriched ECSLC. Sphere formation assay and colony formation assays were employed to analyze the relationship between STAT3 and miR-181b. Luciferase assays were used to evaluate activity which CYLD is a target of miR-181b. Sphere formation cells (SFCs) with properties of ECSLC were enriched. Enriched SFCs in serum-free suspension culture exhibited cancer stem-like cell properties and increased single-positive CD44 + CD24-, stemness factor, mesenchymal marker expression ABC transporters and tumorigenicity in vivo compared with the parental cells. Additionally, we found that reciprocal activation between STAT3 and miR-181b regulated SFCs proliferation. Moreover, STAT3 directly activated miR-181b transcription in SFCs and miR-181b then potentiated p-STAT3 activity. Luciferase assays indicated that CYLD was a direct and functional target of miR-181b. The mutual regulation between STAT3 and miR-181b in SFCs was required for proliferation and apoptosis resistance. STAT3 and miR-181b control each other's expression in a positive feedback loop that regulates SFCs via CYLD pathway. These findings maybe is helpful for targeting ECSLC and providing approach for esophageal cancer treatments.

Epigenetic and SP1-mediated regulation is involved in the repression of galactokinase 1 gene in the liver of neonatal piglets born to betaine-supplemented sows.

PubMed

Cai, Demin; Yuan, Mengjie; Liu, Haoyu; Han, Zhengqiang; Pan, Shifeng; Yang, Yang; Zhao, Ruqian

2017-08-01

In this study, we sought to investigate the effects of maternal betaine supplementation on the expression and regulation of GALK1 gene in the liver of neonatal piglets. Sixteen sows of two groups were fed control or betaine-supplemented diets (3 g/kg), respectively, throughout the pregnancy. Newborn piglets were individually weighed immediately after birth, and one male piglet close to mean body weight from the same litter was selected and killed before suckling. Serum samples of newborn piglets were analyzed for biochemical indexes, hormone and amino acid levels. Liver samples were analyzed for GALK1 expression by real-time PCR and western blotting, while GALK1 regulational mechanism was analyzed by methylated DNA immunoprecipitation, chromatin immunoprecipitation and microRNAs expression. Betaine-exposed neonatal piglets had lower serum concentration of galactose, which was associated with significantly down-regulated hepatic GALK1 expression. The repression of GALK1 mRNA expression was associated with DNA hypermethylation and more enriched repression histone mark H3K27me3 on its promoter. Binding sites of SP1, GR and STAT3 were predicted on GALK1 promoter, and decreased SP1 protein content and lower SP1 binding to GALK1 promoter were detected in the liver of betaine-exposed piglets. Furthermore, the expression of miRNA-149 targeting GALK1 was up-regulated in the liver of betaine-exposed piglets, along with elevated miRNAs-processing enzymes Dicer and Ago2. Our results suggest that maternal dietary betaine supplementation during gestation suppresses GALK1 expression in the liver of neonatal piglets, which involves complex gene regulation mechanisms including DNA methylation, histone modification, miRNAs expression and SP1-mediated transcriptional modulation.

Arctigenin inhibits lipopolysaccharide-induced iNOS expression in RAW264.7 cells through suppressing JAK-STAT signal pathway.

PubMed

Kou, Xianjuan; Qi, Shimei; Dai, Wuxing; Luo, Lan; Yin, Zhimin

2011-08-01

Arctigenin has been demonstrated to have an anti-inflammatory function, but the precise mechanisms of its action remain to be fully defined. In the present study, we determined the effects of arctigenin on lipopolysaccharide (LPS)-induced production of proinflammatory mediators and the underlying mechanisms involved in RAW264.7 cells. Our results indicated that arctigenin exerted its anti-inflammatory effect by inhibiting ROS-dependent STAT signaling through its antioxidant activity. Arctigenin also significantly reduced the phosphorylation of STAT1 and STAT 3 as well as JAK2 in LPS-stimulated RAW264.7 cells. The inhibitions of STAT1 and STAT 3 by arctigenin prevented their translocation to the nucleus and consequently inhibited expression of iNOS, thereby suppressing the expression of inflammation-associated genes, such as IL-1β, IL-6 and MCP-1, whose promoters contain STAT-binding elements. However, COX-2 expression was slightly inhibited at higher drug concentrations (50 μM). Our data demonstrate that arctigenin inhibits iNOS expression via suppressing JAK-STAT signaling pathway in macrophages. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

Signal Transducer and Activator of Transcription 1 (STAT1) is Essential for Chromium Silencing of Gene Induction in Human Airway Epithelial Cells

PubMed Central

Nemec, Antonia A.; Barchowsky, Aaron

2009-01-01

Hexavalent chromium (Cr(VI)) promotes lung injury and pulmonary diseases through poorly defined mechanisms that may involve the silencing of inducible protective genes. The current study investigated the hypothesis that Cr(VI) actively signals through a signal transducer and activator of transcription 1 (STAT1)–dependent pathway to silence nickel (Ni)–induced expression of vascular endothelial cell growth factor A (VEGFA), an important mediator of lung injury and repair. In human bronchial airway epithelial (BEAS-2B) cells, Ni-induced VEGFA transcription by stimulating an extracellular regulated kinase (ERK) signaling cascade that involved Src kinase–activated Sp1 transactivation, as well as increased hypoxia-inducible factor-1α (HIF-1α) stabilization and DNA binding. Ni-stimulated ERK, Src, and HIF-1α activities, as well as Ni-induced VEGFA transcript levels were inhibited in Cr(VI)-exposed cells. We previously demonstrated that Cr(VI) stimulates STAT1 to suppress VEGFA expression. In BEAS-2B cells stably expressing STAT1 short hairpin RNA, Cr(VI) increased VEGFA transcript levels and Sp1 transactivation. Moreover, in the absence of STAT1, Cr(VI), and Ni coexposures positively interacted to further increase VEGFA transcripts. This study demonstrates that metal-stimulated signaling cascades interact to regulate transcription and induction of adaptive or repair responses in airway cells. In addition, the data implicate STAT1 as a rate limiting mediator of Cr(VI)-stimulated gene regulation and suggest that cells lacking STAT1, such as many tumor cell lines, have opposite responses to Cr(VI) relative to normal cells. PMID:19403854

Disseminated Tuberculosis and Chronic Mucocutaneous Candidiasis in a Patient with a Gain-of-Function Mutation in Signal Transduction and Activator of Transcription 1.

PubMed

Pedraza-Sánchez, Sigifredo; Lezana-Fernández, Jose Luis; Gonzalez, Yolanda; Martínez-Robles, Luis; Ventura-Ayala, María Laura; Sadowinski-Pine, Stanislaw; Nava-Frías, Margarita; Moreno-Espinosa, Sarbelio; Casanova, Jean-Laurent; Puel, Anne; Boisson-Dupuis, Stephanie; Torres, Martha

2017-01-01

In humans, recessive loss-of-function mutations in STAT1 are associated with mycobacterial and viral infections, whereas gain-of-function (GOF) mutations in STAT1 are associated with a type of primary immunodeficiency related mainly, but not exclusively, to chronic mucocutaneous candidiasis (CMC). We studied and established a molecular diagnosis in a pediatric patient with mycobacterial infections, associated with CMC. The patient, daughter of a non-consanguineous mestizo Mexican family, had axillary adenitis secondary to BCG vaccination and was cured with resection of the abscess at 1-year old. At the age of 4 years, she had a supraclavicular abscess with acid-fast-staining bacilli identified in the soft tissue and bone, with clinical signs of disseminated infection and a positive Gene-X-pert test, which responded to anti-mycobacterial drugs. Laboratory tests of the IL-12/interferon gamma (IFN-γ) circuit showed a higher production of IL-12p70 in the whole blood from the patient compared to healthy controls, when stimulated with BCG and BCG + IFN-γ. The whole blood of the patient produced 35% less IFN-γ compared to controls assessed by ELISA and flow cytometry, but IL-17 producing T cells from patient were almost absent in PBMC stimulated with PMA plus ionomycin. Signal transduction and activator of transcription 1 (STAT1) was hyperphosphorylated at tyrosine 701 in response to IFN-γ and -α, as demonstrated by flow cytometry and Western blotting in fresh blood mononuclear cells and in Epstein-Barr virus lymphoblastoid cell lines (EBV-LCLs); phosphorylation of STAT1 in EBV-LCLs from the patient was resistant to inhibition by staurosporine but sensitive to ruxolitinib, a Jak phosphorylation inhibitor. Genomic DNA sequencing showed a de novo mutation in STAT1 in cells from the patient, absent in her parents and brother; a known T385M missense mutation in the DNA-binding domain of the transcription factor was identified, and it is a GOF mutation. Therefore, GOF mutations in STAT1 can induce susceptibility not only to fungal but also to mycobacterial infections by mechanisms to be determined.

Disseminated Tuberculosis and Chronic Mucocutaneous Candidiasis in a Patient with a Gain-of-Function Mutation in Signal Transduction and Activator of Transcription 1

PubMed Central

Pedraza-Sánchez, Sigifredo; Lezana-Fernández, Jose Luis; Gonzalez, Yolanda; Martínez-Robles, Luis; Ventura-Ayala, María Laura; Sadowinski-Pine, Stanislaw; Nava-Frías, Margarita; Moreno-Espinosa, Sarbelio; Casanova, Jean-Laurent; Puel, Anne; Boisson-Dupuis, Stephanie; Torres, Martha

2017-01-01

In humans, recessive loss-of-function mutations in STAT1 are associated with mycobacterial and viral infections, whereas gain-of-function (GOF) mutations in STAT1 are associated with a type of primary immunodeficiency related mainly, but not exclusively, to chronic mucocutaneous candidiasis (CMC). We studied and established a molecular diagnosis in a pediatric patient with mycobacterial infections, associated with CMC. The patient, daughter of a non-consanguineous mestizo Mexican family, had axillary adenitis secondary to BCG vaccination and was cured with resection of the abscess at 1-year old. At the age of 4 years, she had a supraclavicular abscess with acid-fast-staining bacilli identified in the soft tissue and bone, with clinical signs of disseminated infection and a positive Gene-X-pert test, which responded to anti-mycobacterial drugs. Laboratory tests of the IL-12/interferon gamma (IFN-γ) circuit showed a higher production of IL-12p70 in the whole blood from the patient compared to healthy controls, when stimulated with BCG and BCG + IFN-γ. The whole blood of the patient produced 35% less IFN-γ compared to controls assessed by ELISA and flow cytometry, but IL-17 producing T cells from patient were almost absent in PBMC stimulated with PMA plus ionomycin. Signal transduction and activator of transcription 1 (STAT1) was hyperphosphorylated at tyrosine 701 in response to IFN-γ and -α, as demonstrated by flow cytometry and Western blotting in fresh blood mononuclear cells and in Epstein-Barr virus lymphoblastoid cell lines (EBV-LCLs); phosphorylation of STAT1 in EBV-LCLs from the patient was resistant to inhibition by staurosporine but sensitive to ruxolitinib, a Jak phosphorylation inhibitor. Genomic DNA sequencing showed a de novo mutation in STAT1 in cells from the patient, absent in her parents and brother; a known T385M missense mutation in the DNA-binding domain of the transcription factor was identified, and it is a GOF mutation. Therefore, GOF mutations in STAT1 can induce susceptibility not only to fungal but also to mycobacterial infections by mechanisms to be determined. PMID:29270166

Bioluminescent Imaging Reveals Divergent Viral Pathogenesis in Two Strains of Stat1-Deficient Mice, and in αßγ Interferon Receptor-Deficient Mice

PubMed Central

Pasieka, Tracy Jo; Collins, Lynne; O'Connor, Megan A.; Chen, Yufei; Parker, Zachary M.; Berwin, Brent L.; Piwnica-Worms, David R.; Leib, David A.

2011-01-01

Pivotal components of the IFN response to virus infection include the IFN receptors (IFNR), and the downstream factor signal transducer and activator of transcription 1 (Stat1). Mice deficient for Stat1 and IFNR (Stat1−/− and IFNαßγR−/− mice) lack responsiveness to IFN and exhibit high sensitivity to various pathogens. Here we examined herpes simplex virus type 1 (HSV-1) pathogenesis in Stat1−/− mice and in IFNαßγR−/− mice following corneal infection and bioluminescent imaging. Two divergent and paradoxical patterns of infection were observed. Mice with an N-terminal deletion in Stat1 (129Stat1−/− (N-term)) had transient infection of the liver and spleen, but succumbed to encephalitis by day 10 post-infection. In stark contrast, infection of IFNαßγR−/− mice was rapidly fatal, with associated viremia and fulminant infection of the liver and spleen, with infected infiltrating cells being primarily of the monocyte/macrophage lineage. To resolve the surprising difference between Stat1−/− and IFNαßγR−/− mice, we infected an additional Stat1−/− strain deleted in the DNA-binding domain (129Stat1−/− (DBD)). These 129Stat1−/− (DBD) mice recapitulated the lethal pattern of liver and spleen infection seen following infection of IFNαßγR−/− mice. This lethal pattern was also observed when 129Stat1−/− (N-term) mice were infected and treated with a Type I IFN-blocking antibody, and immune cells derived from 129Stat1−/− (N-term) mice were shown to be responsive to Type I IFN. These data therefore show significant differences in viral pathogenesis between two commonly-used Stat1−/− mouse strains. The data are consistent with the hypothesis that Stat1−/− (N-term) mice have residual Type I IFN receptor-dependent IFN responses. Complete loss of IFN signaling pathways allows viremia and rapid viral spread with a fatal infection of the liver. This study underscores the importance of careful comparisons between knockout mouse strains in viral pathogenesis, and may also be relevant to the causation of HSV hepatitis in humans, a rare but frequently fatal infection. PMID:21915277

Direct Interaction of Jak1 and v-Abl Is Required for v-Abl-Induced Activation of STATs and Proliferation

PubMed Central

Danial, Nika N.; Losman, Julie A.; Lu, Tianhong; Yip, Natalie; Krishnan, Kartik; Krolewski, John; Goff, Stephen P.; Wang, Jean Y. J.; Rothman, Paul B.

1998-01-01

In Abelson murine leukemia virus (A-MuLV)-transformed cells, members of the Janus kinase (Jak) family of non-receptor tyrosine kinases and the signal transducers and activators of transcription (STAT) family of signaling proteins are constitutively activated. In these cells, the v-Abl oncoprotein and the Jak proteins physically associate. To define the molecular mechanism of constitutive Jak-STAT signaling in these cells, the functional significance of the v-Abl–Jak association was examined. Mapping the Jak1 interaction domain in v-Abl demonstrates that amino acids 858 to 1080 within the carboxyl-terminal region of v-Abl bind Jak1 through a direct interaction. A mutant of v-Abl lacking this region exhibits a significant defect in Jak1 binding in vivo, fails to activate Jak1 and STAT proteins, and does not support either the proliferation or the survival of BAF/3 cells in the absence of cytokine. Cells expressing this v-Abl mutant show extended latency and decreased frequency in generating tumors in nude mice. In addition, inducible expression of a kinase-inactive mutant of Jak1 protein inhibits the ability of v-Abl to activate STATs and to induce cytokine-independent proliferation, indicating that an active Jak1 is required for these v-Abl-induced signaling pathways in vivo. We propose that Jak1 is a mediator of v-Abl-induced STAT activation and v-Abl induced proliferation in BAF/3 cells, and may be important for efficient transformation of immature B cells by the v-abl oncogene. PMID:9774693

The ebolavirus VP24 interferon antagonist: know your enemy.

PubMed

Zhang, Adrianna P P; Abelson, Dafna M; Bornholdt, Zachary A; Liu, Tong; Woods, Virgil L; Saphire, Erica Ollmann

2012-08-15

Suppression during the early phases of the immune system often correlates directly with a fatal outcome for the host. The ebolaviruses, some of the most lethal viruses known, appear to cripple initial stages of the host defense network via multiple distinct paths. Two of the eight viral proteins are critical for immunosuppression. One of these proteins is VP35, which binds double-stranded RNA and antagonizes several antiviral signaling pathways. The other protein is VP24, which binds transporter molecules to prevent STAT1 translocation. A more recent discovery is that VP24 also binds STAT1 directly, suggesting that VP24 may operate in at least two separate branches of the interferon pathway. New crystal structures of VP24 derived from pathogenic and nonpathogenic ebolaviruses reveal its novel, pyramidal fold, upon which can be mapped sites required for virulence and for STAT1 binding. These structures of VP24, and new information about its direct binding to STAT1, provide avenues by which we may explore its many roles in the viral life cycle, and reasons for differences in pathogenesis among the ebolaviruses.

DNA binding by the ribosomal DNA transcription factor rrn3 is essential for ribosomal DNA transcription.

PubMed

Stepanchick, Ann; Zhi, Huijun; Cavanaugh, Alice H; Rothblum, Katrina; Schneider, David A; Rothblum, Lawrence I

2013-03-29

The human homologue of yeast Rrn3 is an RNA polymerase I-associated transcription factor that is essential for ribosomal DNA (rDNA) transcription. The generally accepted model is that Rrn3 functions as a bridge between RNA polymerase I and the transcription factors bound to the committed template. In this model Rrn3 would mediate an interaction between the mammalian Rrn3-polymerase I complex and SL1, the rDNA transcription factor that binds to the core promoter element of the rDNA. In the course of studying the role of Rrn3 in recruitment, we found that Rrn3 was in fact a DNA-binding protein. Analysis of the sequence of Rrn3 identified a domain with sequence similarity to the DNA binding domain of heat shock transcription factor 2. Randomization, or deletion, of the amino acids in this region in Rrn3, amino acids 382-400, abrogated its ability to bind DNA, indicating that this domain was an important contributor to DNA binding by Rrn3. Control experiments demonstrated that these mutant Rrn3 constructs were capable of interacting with both rpa43 and SL1, two other activities demonstrated to be essential for Rrn3 function. However, neither of these Rrn3 mutants was capable of functioning in transcription in vitro. Moreover, although wild-type human Rrn3 complemented a yeast rrn3-ts mutant, the DNA-binding site mutant did not. These results demonstrate that DNA binding by Rrn3 is essential for transcription by RNA polymerase I.

DNA Binding by the Ribosomal DNA Transcription Factor Rrn3 Is Essential for Ribosomal DNA Transcription*

PubMed Central

Stepanchick, Ann; Zhi, Huijun; Cavanaugh, Alice H.; Rothblum, Katrina; Schneider, David A.; Rothblum, Lawrence I.

2013-01-01

The human homologue of yeast Rrn3 is an RNA polymerase I-associated transcription factor that is essential for ribosomal DNA (rDNA) transcription. The generally accepted model is that Rrn3 functions as a bridge between RNA polymerase I and the transcription factors bound to the committed template. In this model Rrn3 would mediate an interaction between the mammalian Rrn3-polymerase I complex and SL1, the rDNA transcription factor that binds to the core promoter element of the rDNA. In the course of studying the role of Rrn3 in recruitment, we found that Rrn3 was in fact a DNA-binding protein. Analysis of the sequence of Rrn3 identified a domain with sequence similarity to the DNA binding domain of heat shock transcription factor 2. Randomization, or deletion, of the amino acids in this region in Rrn3, amino acids 382–400, abrogated its ability to bind DNA, indicating that this domain was an important contributor to DNA binding by Rrn3. Control experiments demonstrated that these mutant Rrn3 constructs were capable of interacting with both rpa43 and SL1, two other activities demonstrated to be essential for Rrn3 function. However, neither of these Rrn3 mutants was capable of functioning in transcription in vitro. Moreover, although wild-type human Rrn3 complemented a yeast rrn3-ts mutant, the DNA-binding site mutant did not. These results demonstrate that DNA binding by Rrn3 is essential for transcription by RNA polymerase I. PMID:23393135

Single-molecule fluorescence study of the inhibition of the oncogenic functionality of STAT3

NASA Astrophysics Data System (ADS)

Liu, Baoxu; Badali, Daniel; Fletcher, Steven; Avadisian, Miriam; Gunning, Patrick; Gradinaru, Claudiu

2009-06-01

Signal-Transducer-and-Activator-of-Transcription 3 (STAT3) protein plays an important role in the onset of cancers such as leukemia and lymphoma. In this study, we aim to test the effectiveness of a novel peptide drug designed to tether STAT3 to the phospholipid bilayer of the cell membrane and thus inhibit unwanted transcription. As a first step, STAT3 proteins were successfully labelled with tetramethylrhodamine (TMR), a fluorescent dye with suitable photostability for single molecule studies. The effectiveness of labelling was determined using fluorescence correlation spectroscopy in a custom built confocal microscope, from which diffusion times and hydrodynamic radii of individual proteins were determined. A newly developed fluorescein derivative label (F-NAc) has been designed to be incorporated into the structure of the peptide drug so that peptide-STAT3 interactions can be examined. This dye is spectrally characterized and is found to be well suited for its application to this project, as well as other single-molecule studies. The membrane localization via high-affinity cholesterol-bound small-molecule binding agents can be demonstrated by encapsulating TMR-labeled STAT3 and inhibitors within a vesicle model cell system. To this end, unilaminar lipid vesicles were examined for size and encapsulation ability. Preliminary results of the efficiency and stability of the STAT3 anchoring in lipid membranes obtained via quantitative confocal imaging and single-molecule spectroscopy using a custom-built multiparameter fluorescence microscope are reported here.

The interferon signaling antagonist function of yellow fever virus NS5 protein is activated by type I interferon.

PubMed

Laurent-Rolle, Maudry; Morrison, Juliet; Rajsbaum, Ricardo; Macleod, Jesica M Levingston; Pisanelli, Giuseppe; Pham, Alissa; Ayllon, Juan; Miorin, Lisa; Martinez, Carles; tenOever, Benjamin R; García-Sastre, Adolfo

2014-09-10

To successfully establish infection, flaviviruses have to overcome the antiviral state induced by type I interferon (IFN-I). The nonstructural NS5 proteins of several flaviviruses antagonize IFN-I signaling. Here we show that yellow fever virus (YFV) inhibits IFN-I signaling through a unique mechanism that involves binding of YFV NS5 to the IFN-activated transcription factor STAT2 only in cells that have been stimulated with IFN-I. This NS5-STAT2 interaction requires IFN-I-induced tyrosine phosphorylation of STAT1 and the K63-linked polyubiquitination at a lysine in the N-terminal region of YFV NS5. We identified TRIM23 as the E3 ligase that interacts with and polyubiquitinates YFV NS5 to promote its binding to STAT2 and trigger IFN-I signaling inhibition. Our results demonstrate the importance of YFV NS5 in overcoming the antiviral action of IFN-I and offer a unique example of a viral protein that is activated by the same host pathway that it inhibits. Copyright © 2014 Elsevier Inc. All rights reserved.

Hierarchy within the mammary STAT5-driven Wap super-enhancer.

PubMed

Shin, Ha Youn; Willi, Michaela; HyunYoo, Kyung; Zeng, Xianke; Wang, Chaochen; Metser, Gil; Hennighausen, Lothar

2016-08-01

Super-enhancers comprise dense transcription factor platforms highly enriched for active chromatin marks. A paucity of functional data led us to investigate the role of super-enhancers in the mammary gland, an organ characterized by exceptional gene regulatory dynamics during pregnancy. ChIP-seq analysis for the master regulator STAT5A, the glucocorticoid receptor, H3K27ac and MED1 identified 440 mammary-specific super-enhancers, half of which were associated with genes activated during pregnancy. We interrogated the Wap super-enhancer, generating mice carrying mutations in STAT5-binding sites within its constituent enhancers. Individually, the most distal site displayed the greatest enhancer activity. However, combinatorial mutation analysis showed that the 1,000-fold induction in gene expression during pregnancy relied on all enhancers. Disabling the binding sites of STAT5, NFIB and ELF5 in the proximal enhancer incapacitated the entire super-enhancer. Altogether, these data suggest a temporal and functional enhancer hierarchy. The identification of mammary-specific super-enhancers and the mechanistic exploration of the Wap locus provide insights into the regulation of cell-type-specific expression of hormone-sensing genes.

The interferon signaling antagonist function of yellow fever virus NS5 protein is activated by Type I interferon

PubMed Central

Rajsbaum, Ricardo; Macleod, Jesica M. Levingston; Pisanelli, Giuseppe; Pham, Alissa; Ayllon, Juan; Miorin, Lisa; Martinez, Carles; tenOever, Benjamin R; García-Sastre, Adolfo

2014-01-01

Summary To successfully establish infection Flaviviruses have to overcome the antiviral state induced by type I interferon (IFN-I). The nonstructural NS5 proteins of several flaviviruses antagonize IFN-I signaling. Here we show that yellow fever virus (YFV) inhibits IFN-I signaling through a unique mechanism that involves binding of YFV NS5 to the IFN-activated transcription factor STAT2 only in cells that have been stimulated with IFN-I. This NS5-STAT2 interaction requires IFN-I-induced tyrosine phosphorylation of STAT1 and the K63-linked polyubiquitination at a lysine in the N-terminal region of YFV NS5. We identified TRIM23 as the E3 ligase that interacts with and polyubiquitinates YFV NS5 to promote its binding to STAT2 and trigger IFN-I signaling inhibition. Our results demonstrate the importance of YFV NS5 in overcoming the antiviral action of IFN-I and offer a unique example of a viral protein that is activated by the same host pathway that it inhibits. PMID:25211074

Increasing Maternal Body Mass Index Is Associated with Systemic Inflammation in the Mother and the Activation of Distinct Placental Inflammatory Pathways1

PubMed Central

Aye, Irving L.M.H.; Lager, Susanne; Ramirez, Vanessa I.; Gaccioli, Francesca; Dudley, Donald J.; Jansson, Thomas; Powell, Theresa L.

2014-01-01

ABSTRACT Obese pregnant women have increased levels of proinflammatory cytokines in maternal circulation and placental tissues. However, the pathways contributing to placental inflammation in obesity are largely unknown. We tested the hypothesis that maternal body mass index (BMI) was associated with elevated proinflammatory cytokines in maternal and fetal circulations and increased activation of placental inflammatory pathways. A total of 60 women of varying pre-/early pregnancy BMI, undergoing delivery by Cesarean section at term, were studied. Maternal and fetal (cord) plasma were collected for analysis of insulin, leptin, IL-1beta, IL-6, IL-8, monocyte chemoattractant protein (MCP) 1, and TNFalpha by multiplex ELISA. Activation of the inflammatory pathways in the placenta was investigated by measuring the phosphorylated and total protein expression of p38-mitogen-activated protein kinase (MAPK), c-Jun-N-terminal kinase (JNK)-MAPK, signal transducer-activated transcription factor (STAT) 3, caspase-1, IL-1beta, IkappaB-alpha protein, and p65 DNA-binding activity. To determine the link between activated placental inflammatory pathways and elevated maternal cytokines, cultured primary human trophoblast (PHT) cells were treated with physiological concentrations of insulin, MCP-1, and TNFalpha, and inflammatory signaling analyzed by Western blot. Maternal BMI was positively correlated with maternal insulin, leptin, MCP-1, and TNFalpha, whereas only fetal leptin was increased with BMI. Placental phosphorylation of p38-MAPK and STAT3, and the expression of IL-1beta protein, were increased with maternal BMI; phosphorylation of p38-MAPK was also correlated with birth weight. In contrast, placental NFkappaB, JNK and caspase-1 signaling, and fetal cytokine levels were unaffected by maternal BMI. In PHT cells, p38-MAPK was activated by MCP-1 and TNFalpha, whereas STAT3 phosphorylation was increased following TNFalpha treatment. Maternal BMI is associated with elevated maternal cytokines and activation of placental p38-MAPK and STAT3 inflammatory pathways, without changes in fetal systemic inflammatory profile. Activation of p38-MAPK by MCP-1 and TNFalpha, and STAT3 by TNFalpha, suggests a link between elevated proinflammatory cytokines in maternal plasma and activation of placental inflammatory pathways. We suggest that inflammatory processes associated with elevated maternal BMI may influence fetal growth by altering placental function. PMID:24759787

Increasing maternal body mass index is associated with systemic inflammation in the mother and the activation of distinct placental inflammatory pathways.

PubMed

Aye, Irving L M H; Lager, Susanne; Ramirez, Vanessa I; Gaccioli, Francesca; Dudley, Donald J; Jansson, Thomas; Powell, Theresa L

2014-06-01

Obese pregnant women have increased levels of proinflammatory cytokines in maternal circulation and placental tissues. However, the pathways contributing to placental inflammation in obesity are largely unknown. We tested the hypothesis that maternal body mass index (BMI) was associated with elevated proinflammatory cytokines in maternal and fetal circulations and increased activation of placental inflammatory pathways. A total of 60 women of varying pre-/early pregnancy BMI, undergoing delivery by Cesarean section at term, were studied. Maternal and fetal (cord) plasma were collected for analysis of insulin, leptin, IL-1beta, IL-6, IL-8, monocyte chemoattractant protein (MCP) 1, and TNFalpha by multiplex ELISA. Activation of the inflammatory pathways in the placenta was investigated by measuring the phosphorylated and total protein expression of p38-mitogen-activated protein kinase (MAPK), c-Jun-N-terminal kinase (JNK)-MAPK, signal transducer-activated transcription factor (STAT) 3, caspase-1, IL-1beta, IkappaB-alpha protein, and p65 DNA-binding activity. To determine the link between activated placental inflammatory pathways and elevated maternal cytokines, cultured primary human trophoblast (PHT) cells were treated with physiological concentrations of insulin, MCP-1, and TNFalpha, and inflammatory signaling analyzed by Western blot. Maternal BMI was positively correlated with maternal insulin, leptin, MCP-1, and TNFalpha, whereas only fetal leptin was increased with BMI. Placental phosphorylation of p38-MAPK and STAT3, and the expression of IL-1beta protein, were increased with maternal BMI; phosphorylation of p38-MAPK was also correlated with birth weight. In contrast, placental NFkappaB, JNK and caspase-1 signaling, and fetal cytokine levels were unaffected by maternal BMI. In PHT cells, p38-MAPK was activated by MCP-1 and TNFalpha, whereas STAT3 phosphorylation was increased following TNFalpha treatment. Maternal BMI is associated with elevated maternal cytokines and activation of placental p38-MAPK and STAT3 inflammatory pathways, without changes in fetal systemic inflammatory profile. Activation of p38-MAPK by MCP-1 and TNFalpha, and STAT3 by TNFalpha, suggests a link between elevated proinflammatory cytokines in maternal plasma and activation of placental inflammatory pathways. We suggest that inflammatory processes associated with elevated maternal BMI may influence fetal growth by altering placental function. © 2014 by the Society for the Study of Reproduction, Inc.

Obatoclax analog SC-2001 inhibits STAT3 phosphorylation through enhancing SHP-1 expression and induces apoptosis in human breast cancer cells.

PubMed

Liu, Chun-Yu; Su, Jung-Chen; Ni, Mei-Huei; Tseng, Ling-Ming; Chu, Pei-Yi; Wang, Duen-Shian; Tai, Wei-Tien; Kao, Yuan-Ping; Hung, Man-Hsin; Shiau, Chung-Wai; Chen, Kuen-Feng

2014-07-01

Interfering oncogenic STAT3 signaling is a promising anti-cancer strategy. We examined the efficacy and drug mechanism of an obatoclax analog SC-2001, a novel STAT3 inhibitor, in human breast cancer cells. Human breast cancer cell lines were used for in vitro studies. Apoptosis was examined by both flow cytometry and western blot. Signaling pathways were assessed by western blot. In vivo efficacy of SC-2001 was tested in xenograft nude mice. SC-2001 inhibited cell growth and induced apoptosis in association with downregulation of p-STAT3 (Tyr 705) in breast cancer cells. STAT3-regulated proteins, including Mcl-1, survivin, and cyclin D1, were repressed by SC-2001. Over-expression of STAT3 in MDA-MB-468 cells protected cells from SC-2001-induced apoptosis. Moreover, SC-2001 enhanced the expression of protein tyrosine phosphatase SHP-1, a negative regulator of STAT3. Furthermore, the enhanced SHP-1 expression, in conjunction with increased SHP-1 phosphatase activity, was mediated by upregulated transcription by RFX-1. Chromatin immunoprecipitation assay revealed that SC-2001 increased the binding capacity of RFX-1 to the SHP-1 promoter. Knockdown of either RFX-1 or SHP-1 reduced SC-2001-induced apoptosis, whereas ectopic expression of RFX-1 increased SHP-1 expression and enhanced the apoptotic effect of SC-2001. Importantly, SC-2001 suppressed tumor growth in association with enhanced RFX-1 and SHP-1 expression and p-STAT3 downregulation in MDA-MB-468 xenograft tumors. SC-2001 induced apoptosis in breast cancer cells, an effect that was mediated by RFX-1 upregulated SHP-1 expression and SHP-1-dependent STAT3 inactivation. Our study indicates targeting STAT3 signaling pathway may be a useful approach for the development of targeted agents for anti-breast cancer.

SOCS3, a Major Regulator of Infection and Inflammation

PubMed Central

Carow, Berit; Rottenberg, Martin E.

2014-01-01

In this review, we describe the role of suppressor of cytokine signaling-3 (SOCS3) in modulating the outcome of infections and autoimmune diseases as well as the underlying mechanisms. SOCS3 regulates cytokine or hormone signaling usually preventing, but in some cases aggravating, a variety of diseases. A main role of SOCS3 results from its binding to both the JAK kinase and the cytokine receptor, which results in the inhibition of STAT3 activation. Available data also indicate that SOCS3 can regulate signaling via other STATs than STAT3 and also controls cellular pathways unrelated to STAT activation. SOCS3 might either act directly by hampering JAK activation or by mediating the ubiquitination and subsequent proteasome degradation of the cytokine/growth factor/hormone receptor. Inflammation and infection stimulate SOCS3 expression in different myeloid and lymphoid cell populations as well as in diverse non-hematopoietic cells. The accumulated data suggest a relevant program coordinated by SOCS3 in different cell populations, devoted to the control of immune homeostasis in physiological and pathological conditions such as infection and autoimmunity. PMID:24600449

CTLA4 aptamer delivers STAT3 siRNA to tumor-associated and malignant T cells

PubMed Central

Herrmann, Andreas; Priceman, Saul J.; Kujawski, Maciej; Xin, Hong; Cherryholmes, Gregory A.; Zhang, Wang; Zhang, Chunyan; Lahtz, Christoph; Kowolik, Claudia; Forman, Steve J.; Kortylewski, Marcin; Yu, Hua

2014-01-01

Intracellular therapeutic targets that define tumor immunosuppression in both tumor cells and T cells remain intractable. Here, we have shown that administration of a covalently linked siRNA to an aptamer (apt) that selectively binds cytotoxic T lymphocyte–associated antigen 4 (CTLA4apt) allows gene silencing in exhausted CD8+ T cells and Tregs in tumors as well as CTLA4-expressing malignant T cells. CTLA4 expression was upregulated in CD8+ T cells in the tumor milieu; therefore, CTLA4apt fused to a STAT3-targeting siRNA (CTLA4apt–STAT3 siRNA) resulted in internalization into tumor-associated CD8+ T cells and silencing of STAT3, which activated tumor antigen–specific T cells in murine models. Both local and systemic administration of CTLA4apt–STAT3 siRNA dramatically reduced tumor-associated Tregs. Furthermore, CTLA4apt–STAT3 siRNA potently inhibited tumor growth and metastasis in various mouse tumor models. Importantly, CTLA4 expression is observed in T cells of patients with blood malignancies, and CTLA4apt–STAT3 siRNA treatment of immunodeficient mice bearing human T cell lymphomas promoted tumor cell apoptosis and tumor growth inhibition. These data demonstrate that a CTLA4apt-based siRNA delivery strategy allows gene silencing in both tumor-associated T cells and tumor cells and inhibits tumor growth and metastasis. PMID:24892807

Cr(3+) Binding to DNA Backbone Phosphate and Bases: Slow Ligand Exchange Rates and Metal Hydrolysis.

PubMed

Zhou, Wenhu; Yu, Tianmeng; Vazin, Mahsa; Ding, Jinsong; Liu, Juewen

2016-08-15

The interaction between chromium ions and DNA is of great interest in inorganic chemistry, toxicology, and analytical chemistry. Most previous studies focused on in situ reduction of Cr(VI), producing Cr(3+) for DNA binding. Recently, Cr(3+) was reported to activate the Ce13d DNAzyme for RNA cleavage. Herein, the Ce13d is used to study two types of Cr(3+) and DNA interactions. First, Cr(3+) binds to the DNA phosphate backbone weakly through reversible electrostatic interactions, which is weakened by adding competing inorganic phosphate. However, Cr(3+) coordinates with DNA nucleobases forming stable cross-links that can survive denaturing gel electrophoresis condition. The binding of Cr(3+) to different nucleobases was further studied in terms of binding kinetics and affinity by exploiting carboxyfluorescein-labeled DNA homopolymers. Once binding takes place, the stable Cr(3+)/DNA complex cannot be dissociated by EDTA, attributable to the ultraslow ligand exchange rate of Cr(3+). The binding rate follows the order of G > C > T ≈ A. Finally, Cr(3+) gradually loses its DNA binding ability after being stored at neutral or high pH, attributable to hydrolysis. This hydrolysis can be reversed by lowering the pH. This work provides a deeper insight into the bioinorganic chemistry of Cr(3+) coordination with DNA, clarifies some inconsistency in the previous literature, and offers practically useful information for generating reproducible results.

Activation of Peroxisome Proliferator–Activated Receptor β/δ Inhibits Lipopolysaccharide-Induced Cytokine Production in Adipocytes by Lowering Nuclear Factor-κB Activity via Extracellular Signal–Related Kinase 1/2

PubMed Central

Rodríguez-Calvo, Ricardo; Serrano, Lucía; Coll, Teresa; Moullan, Norman; Sánchez, Rosa M.; Merlos, Manuel; Palomer, Xavier; Laguna, Juan C.; Michalik, Liliane; Wahli, Walter; Vázquez-Carrera, Manuel

2008-01-01

OBJECTIVE—Chronic activation of the nuclear factor-κB (NF-κB) in white adipose tissue leads to increased production of pro-inflammatory cytokines, which are involved in the development of insulin resistance. It is presently unknown whether peroxisome proliferator–activated receptor (PPAR) β/δ activation prevents inflammation in adipocytes. RESEARCH DESIGN AND METHODS AND RESULTS—First, we examined whether the PPARβ/δ agonist GW501516 prevents lipopolysaccharide (LPS)-induced cytokine production in differentiated 3T3-L1 adipocytes. Treatment with GW501516 blocked LPS-induced IL-6 expression and secretion by adipocytes and the subsequent activation of the signal transducer and activator of transcription 3 (STAT3)–Suppressor of cytokine signaling 3 (SOCS3) pathway. This effect was associated with the capacity of GW501516 to impede LPS-induced NF-κB activation. Second, in in vivo studies, white adipose tissue from Zucker diabetic fatty (ZDF) rats, compared with that of lean rats, showed reduced PPARβ/δ expression and PPAR DNA-binding activity, which was accompanied by enhanced IL-6 expression and NF-κB DNA-binding activity. Furthermore, IL-6 expression and NF-κB DNA-binding activity was higher in white adipose tissue from PPARβ/δ-null mice than in wild-type mice. Because mitogen-activated protein kinase–extracellular signal–related kinase (ERK)1/2 (MEK1/2) is involved in LPS-induced NF-κB activation in adipocytes, we explored whether PPARβ/δ prevented NF-κB activation by inhibiting this pathway. Interestingly, GW501516 prevented ERK1/2 phosphorylation by LPS. Furthermore, white adipose tissue from animal showing constitutively increased NF-κB activity, such as ZDF rats and PPARβ/δ-null mice, also showed enhanced phospho-ERK1/2 levels. CONCLUSIONS—These findings indicate that activation of PPARβ/δ inhibits enhanced cytokine production in adipocytes by preventing NF-κB activation via ERK1/2, an effect that may help prevent insulin resistance. PMID:18443198

Controlling nuclear JAKs and STATs for specific gene activation by IFNγ.

PubMed

Noon-Song, Ezra N; Ahmed, Chulbul M; Dabelic, Rea; Canton, Johnathan; Johnson, Howard M

2011-07-08

We previously showed that gamma interferon (IFNγ) and its receptor subunit, IFNGR1, interacted with the promoter region of IFNγ-activated genes along with transcription factor STAT1α. Recent studies have suggested that activated Janus kinases pJAK2 and pJAK1 also played a role in gene activation by phosphorylation of histone H3 on tyrosine 41. This study addresses the question of the role of activated JAKs in specific gene activation by IFNγ. We carried out chromatin immunoprecipitation (ChIP) followed by PCR in IFNγ treated WISH cells and showed association of pJAK1, pJAK2, IFNGR1, and STAT1 on the same DNA sequence of the IRF-1 gene promoter. The β-actin gene, which is not activated by IFNγ, did not show this association. The movement of activated JAK to the nucleus and the IRF-1 promoter was confirmed by the combination of nuclear fractionation, confocal microscopy and DNA precipitation analysis using the biotinylated GAS promoter. Activated JAKs in the nucleus was associated with phosphorylated tyrosine 41 on histone H3 in the region of the GAS promoter. Unphosphorylated JAK2 was found to be constitutively present in the nucleus and was capable of undergoing activation in IFNγ treated cells, most likely via nuclear IFNGR1. Association of pJAK2 and IFNGR1 with histone H3 in IFNγ treated cells was demonstrated by histone H3 immunoprecipitation. Unphosphorylated STAT1 protein was associated with histone H3 of untreated cells. IFNγ treatment resulted in its disassociation and then re-association as pSTAT1. The results suggest a novel role for activated JAKs in epigenetic events for specific gene activation. Copyright © 2011 Elsevier Inc. All rights reserved.

The substrate binding interface of alkylpurine DNA glycosylase AlkD.

PubMed

Mullins, Elwood A; Rubinson, Emily H; Eichman, Brandt F

2014-01-01

Tandem helical repeats have emerged as an important DNA binding architecture. DNA glycosylase AlkD, which excises N3- and N7-alkylated nucleobases, uses repeating helical motifs to bind duplex DNA and to selectively pause at non-Watson-Crick base pairs. Remodeling of the DNA backbone promotes nucleotide flipping of the lesion and the complementary base into the solvent and toward the protein surface, respectively. The important features of this new DNA binding architecture that allow AlkD to distinguish between damaged and normal DNA without contacting the lesion are poorly understood. Here, we show through extensive mutational analysis that DNA binding and N3-methyladenine (3mA) and N7-methylguanine (7mG) excision are dependent upon each residue lining the DNA binding interface. Disrupting electrostatic or hydrophobic interactions with the DNA backbone substantially reduced binding affinity and catalytic activity. These results demonstrate that residues seemingly only involved in general DNA binding are important for catalytic activity and imply that base excision is driven by binding energy provided by the entire substrate interface of this novel DNA binding architecture. Copyright © 2013 Elsevier B.V. All rights reserved.

Statistical error propagation in ab initio no-core full configuration calculations of light nuclei

DOE PAGES

Navarro Pérez, R.; Amaro, J. E.; Ruiz Arriola, E.; ...

2015-12-28

We propagate the statistical uncertainty of experimental N N scattering data into the binding energy of 3H and 4He. Here, we also study the sensitivity of the magnetic moment and proton radius of the 3 H to changes in the N N interaction. The calculations are made with the no-core full configuration method in a sufficiently large harmonic oscillator basis. For those light nuclei we obtain Δ E stat (3H) = 0.015 MeV and Δ E stat ( 4He) = 0.055 MeV .

Agomelatine, a MT1/MT2 melatonergic receptor agonist with serotonin 5-HT2C receptor antagonistic properties, suppresses Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages.

PubMed

Hyeon, Jin-Yi; Choi, Eun-Young; Choe, So-Hui; Park, Hae Ryoun; Choi, Jeom-Il; Choi, In Soon; Kim, Sung-Jo

2017-10-01

This study was performed in an attempt to examine the influence of agomelatine in mitigating the generation of proinflammatory mediators in RAW264.7 murine macrophages exposed to lipopolysaccharide (LPS) obtained from Prevotella intermedia, a gram-negative anaerobic bacterium that is related with various types of periodontal diseases, and the molecular mechanisms behind its effects. LPS from P. intermedia strain ATCC 25611 was prepared employing the conventional phenol-water procedure. Conditioned culture media were analyzed for the levels of nitric oxide (NO), interleukin-1β (IL-1β) and IL-6. Real-time PCR analysis was carried out to determine the mRNA levels of inducible NO synthase (iNOS), IL-1β, IL-6 and SOCS1. Protein expression levels were evaluated by immunoblot test. NF-κB-dependent SEAP reporter assay was performed using a reporter cell line. DNA-binding activities of NF-κB subunits were analyzed utilizing the ELISA-based kits. Agomelatine was found to down-regulate significantly the generation of iNOS-derived NO, IL-1β and IL-6 as well as the expression of their mRNAs in cells activated with P. intermedia LPS. Agomelatine decreased NF-κB-dependent SEAP release caused by P. intermedia LPS. Agomelatine did not inhibit NF-κB transcription induced by LPS at the level of IκB-α degradation. Instead, LPS-induced nuclear translocation and DNA binding of NF-κB p50 subunit was blocked by agomelatine. P. intermedia LPS-elicited activation of STAT1 and STAT3 was reduced notably by co-treatment with agomelatine. Agomelatine showed a tendency to enhance mRNA level of SOCS1 in LPS-activated cells as well. Agomelatine merits further evaluation to reveal its usefulness on the host modulation of periodontal disease. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fatty Acid Binding Protein FABP4 Mechanistically Links Obesity with Aggressive AML by Enhancing Aberrant DNA Methylation in AML Cells

PubMed Central

Yan, F; Shen, N; Pang, JX; Zhang, YW; Rao, EY; Bode, AM; Al-Kali, A; Zhang, DE; Litzow, MR; Li, B; Liu, SJ

2016-01-01

Obesity is becoming more prevalent worldwide and is a major risk factor for cancer development. Acute myeloid leukemia (AML), the most common acute leukemia in adults, remains a frequently fatal disease. Here, we investigated the molecular mechanisms by which obesity favors AML growth and uncovered the fatty acid binding protein 4 (FABP4) and DNA methyltransferase 1 (DNMT1) regulatory axis that mediates aggressive AML in obesity. We showed that leukemia burden was much higher in high-fat diet-induced obese mice, which had higher levels of FABP4 and IL-6 in sera. Upregulation of environmental and cellular FABP4 accelerated AML cell growth in both a cell-autonomous and cell-non-autonomous manner. Genetic disruption of FABP4 in AML cells or in mice blocked cell proliferation in vitro and induced leukemia regression in vivo. Mechanistic investigations showed that FABP4 upregulation increased IL-6 expression and STAT3 phosphorylation leading to DNMT1 overexpression and further silencing of the p15INK4B tumor suppressor gene in AML cells. Conversely, FABP4 ablation reduced DNMT1-dependent DNA methylation and restored p15INK4B expression, thus conferring substantial protection against AML growth. Our findings reveal the FABP4/DNMT1 axis in the control of AML cell fate in obesity, and suggest that interference with the FABP4/DNMT1 axis might be a new strategy to treat leukemia. PMID:27885273

Asiatic acid attenuates methamphetamine-induced neuroinflammation and neurotoxicity through blocking of NF-kB/STAT3/ERK and mitochondria-mediated apoptosis pathway.

PubMed

Park, Ji-Hyun; Seo, Young Ho; Jang, Jung-Hee; Jeong, Chul-Ho; Lee, Sooyeun; Park, Byoungduck

2017-12-11

Methamphetamine (METH) is a commonly abused drug that may result in neurotoxic effects. Recent studies have suggested that involvement of neuroinflammatory processes in brain dysfunction is induced by misuse of this drug. However, the mechanism underlying METH-induced inflammation and neurotoxicity in neurons is still unclear. In this study, we investigated whether asiatic acid (AA) effected METH-mediated neuroinflammation and neurotoxicity in dopaminergic neuronal cells. And we further determined whether the effect involved in the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription (STAT)3 and extracellular signal-regulated kinase (ERK) pathway. We used the human dopaminergic neuroblastoma SH-SY5Y cell line, murine microglial BV2 cell line, and primary culture of rat embryo mesencephalic neurons. Pro-inflammatory cytokine production was monitored by ELISA and RT/real-time PCR. The cell cycle distribution and mitochondrial membrane integrity was analyzed by flow cytometry. We used immunoblotting, DNA-binding activity, and immunofluorescence staining to analyze the effect of AA on activation of the NF-κB, STAT3, MAPK-ERK, and apoptosis signaling pathways. METH induced TNF receptor (TNFR) expression and led to morphological changes of cells. Additionally, this drug increased pro-inflammatory cytokine (TNFα and IL-6) expression. AA significantly suppressed METH-induced TNFR expression in concentration dependent. Increased secretion of TNFα and IL-6 was inhibited in METH-stimulated neuronal cells by AA administration. AA showed significant protection against METH-induced translocation of NF-κB/STAT3 and ERK phosphorylation. AA inhibited METH-induced proteolytic fragmentation of caspase-3 and PARP. The pro-apoptotic protein Bax was significantly decreased, while the anti-apoptotic protein Bcl-xL was increased by AA treatment in METH-stimulated cells. A similar protective effect of AA on mitochondrial membrane integrity was also confirmed by flow cytometry and immunofluorescence staining. Based on the literatures and our findings, AA is a promising candidate for an anti-neurotoxic agent, and it can potentially be used for the prevention and treatment of various neurological disorders.

Cooperative STAT/NF-κB signaling regulates lymphoma metabolic reprogramming and aberrant GOT2 expression.

PubMed

Feist, Maren; Schwarzfischer, Philipp; Heinrich, Paul; Sun, Xueni; Kemper, Judith; von Bonin, Frederike; Perez-Rubio, Paula; Taruttis, Franziska; Rehberg, Thorsten; Dettmer, Katja; Gronwald, Wolfram; Reinders, Jörg; Engelmann, Julia C; Dudek, Jan; Klapper, Wolfram; Trümper, Lorenz; Spang, Rainer; Oefner, Peter J; Kube, Dieter

2018-04-17

Knowledge of stromal factors that have a role in the transcriptional regulation of metabolic pathways aside from c-Myc is fundamental to improvements in lymphoma therapy. Using a MYC-inducible human B-cell line, we observed the cooperative activation of STAT3 and NF-κB by IL10 and CpG stimulation. We show that IL10 + CpG-mediated cell proliferation of MYC low cells depends on glutaminolysis. By 13 C- and 15 N-tracing of glutamine metabolism and metabolite rescue experiments, we demonstrate that GOT2 provides aspartate and nucleotides to cells with activated or aberrant Jak/STAT and NF-κB signaling. A model of GOT2 transcriptional regulation is proposed, in which the cooperative phosphorylation of STAT3 and direct joint binding of STAT3 and p65/NF-κB to the proximal GOT2 promoter are important. Furthermore, high aberrant GOT2 expression is prognostic in diffuse large B-cell lymphoma underscoring the current findings and importance of stromal factors in lymphoma biology.

Characterization of the DNA binding properties of polyomavirus capsid protein

NASA Technical Reports Server (NTRS)

Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1993-01-01

The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.

Structural and mechanistic insights into nuclear transport and delivery of the critical pluripotency factor Oct4 to DNA.

PubMed

Okuyama, Takahide; Yamagishi, Ryosuke; Shimada, Jiro; Ikeda, Masaaki; Maruoka, Yayoi; Kaneko, Hiroki

2018-02-01

Oct4 is a master regulator of the induction and maintenance of cellular pluripotency, and has crucial roles in early stages of differentiation. It is the only factor that cannot be substituted by other members of the same protein family to induce pluripotency. However, although Oct4 nuclear transport and delivery to target DNA are critical events for reprogramming to pluripotency, little is known about the molecular mechanism. Oct4 is imported to the nucleus by the classical nuclear transport mechanism, which requires importin α as an adaptor to bind the nuclear localization signal (NLS). Although there are structures of complexes of the NLS of transcription factors (TFs) in complex with importin α, there are no structures available for complexes involving intact TFs. We have therefore modeled the structure of the complex of the whole Oct4 POU domain and importin α2 using protein-protein docking and molecular dynamics. The model explains how the Ebola virus VP24 protein has a negative effect on the nuclear import of STAT1 by importin α but not on Oct4, and how Nup 50 facilitates cargo release from importin α. The model demonstrates the structural differences between the Oct4 importin α bound and DNA bound crystal states. We propose that the 'expanded linker' between the two DNA-binding domains of Oct4 is an intrinsically disordered region and that its conformational changes have a key role in the recognition/binding to both DNA and importin α. Moreover, we propose that this structural change enables efficient delivery to DNA after release from importin α.

Mechanisms of JAK/STAT pathway negative regulation by the short coreceptor Eye Transformer/Latran.

PubMed

Fisher, Katherine H; Stec, Wojciech; Brown, Stephen; Zeidler, Martin P

2016-02-01

Transmembrane receptors interact with extracellular ligands to transduce intracellular signaling cascades, modulate target gene expression, and regulate processes such as proliferation, apoptosis, differentiation, and homeostasis. As a consequence, aberrant signaling events often underlie human disease. Whereas the vertebrate JAK/STAT signaling cascade is transduced via multiple receptor combinations, the Drosophila pathway has only one full-length signaling receptor, Domeless (Dome), and a single negatively acting receptor, Eye Transformer/Latran (Et/Lat). Here we investigate the molecular mechanisms underlying Et/Lat activity. We demonstrate that Et/Lat negatively regulates the JAK/STAT pathway activity and can bind to Dome, thus reducing Dome:Dome homodimerization by creating signaling-incompetent Dome:Et/Lat heterodimers. Surprisingly, we find that Et/Lat is able to bind to both JAK and STAT92E but, despite the presence of putative cytokine-binding motifs, does not detectably interact with pathway ligands. We find that Et/Lat is trafficked through the endocytic machinery for lysosomal degradation but at a much slower rate than Dome, a difference that may enhance its ability to sequester Dome into signaling-incompetent complexes. Our data offer new insights into the molecular mechanism and regulation of Et/Lat in Drosophila that may inform our understanding of how short receptors function in other organisms. © 2016 Fisher et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Isolation and Expression Analysis of STAT Members from Synechogobius hasta and Their Roles in Leptin Affecting Lipid Metabolism

PubMed Central

Wu, Kun; Tan, Xiao-Ying; Wei, Chuan-Chuan; You, Wen-Jing; Zhuo, Mei-Qin; Song, Yu-Feng

2016-01-01

Signal transducers and activators of transcription proteins (STATs) act as important mediators in multiple biological processes induced by a large number of cytokines. In the present study, full-length cDNA sequences of seven STAT members, including some splicing variants different from those in mammals, were obtained from Synechogobius hasta. The phylogenetic analysis revealed that the seven STAT members were derived from paralogous genes that might have arisen by whole genome duplication (WGD) events during vertebrate evolution. All of these members share similar domain structure compared with those of mammals, and were widely expressed across the tested tissues (brain, gill, heart, intestine, liver, muscle and spleen), but at variable levels. Incubation in vitro of recombinant human leptin changed the intracellular triglyceride (TG) content and mRNA levels of several STATs members, as well as expressions and activities of genes involved in lipid metabolism. Furthermore, Tyrphostin B42 (AG490), a specific inhibitor of the Janus Kinase 2(JAK2)-STAT pathway, partially reversed leptin-induced change on STAT3 and its two spliced isoforms expression, as well as expressions and activities of genes involved in lipid metabolism. As a consequence, the decrease of TG content was also reversed. Thus, our study suggests that STAT3 is the requisite for the leptin signal and the activation of the STAT3 member may account for the leptin-induced changes in lipid metabolism in S. hasta. PMID:27011172

Isolation and Expression Analysis of STAT Members from Synechogobius hasta and Their Roles in Leptin Affecting Lipid Metabolism.

PubMed

Wu, Kun; Tan, Xiao-Ying; Wei, Chuan-Chuan; You, Wen-Jing; Zhuo, Mei-Qin; Song, Yu-Feng

2016-03-22

Signal transducers and activators of transcription proteins (STATs) act as important mediators in multiple biological processes induced by a large number of cytokines. In the present study, full-length cDNA sequences of seven STAT members, including some splicing variants different from those in mammals, were obtained from Synechogobius hasta. The phylogenetic analysis revealed that the seven STAT members were derived from paralogous genes that might have arisen by whole genome duplication (WGD) events during vertebrate evolution. All of these members share similar domain structure compared with those of mammals, and were widely expressed across the tested tissues (brain, gill, heart, intestine, liver, muscle and spleen), but at variable levels. Incubation in vitro of recombinant human leptin changed the intracellular triglyceride (TG) content and mRNA levels of several STATs members, as well as expressions and activities of genes involved in lipid metabolism. Furthermore, Tyrphostin B42 (AG490), a specific inhibitor of the Janus Kinase 2(JAK2)-STAT pathway, partially reversed leptin-induced change on STAT3 and its two spliced isoforms expression, as well as expressions and activities of genes involved in lipid metabolism. As a consequence, the decrease of TG content was also reversed. Thus, our study suggests that STAT3 is the requisite for the leptin signal and the activation of the STAT3 member may account for the leptin-induced changes in lipid metabolism in S. hasta.

Silibinin downregulates MMP2 expression via Jak2/STAT3 pathway and inhibits the migration and invasive potential in MDA-MB-231 cells.

PubMed

Byun, Hyo Joo; Darvin, Pramod; Kang, Dong Young; Sp, Nipin; Joung, Youn Hee; Park, Jong Hwan; Kim, Sun Jin; Yang, Young Mok

2017-06-01

Worldwide, breast cancer (BCa) is the most common cancer in women. Among its subtypes, triple-negative breast cancer (TNBC) is an aggressive form associated with diminished survival. TNBCs are characterized by their absence, or minimal expression, of the estrogen and progesterone receptors, as well as the human epidermal growth factor receptor 2 (i.e. ER-/-, PR-/-, Her2-/Low). Consequently, treatment for this subtype of BCa remains problematic. Silibinin, a derivative of the flavonoid silymarin, is reported to have anticancer activities against hepatic and non-small cell lung cancers. We hypothesized that silibinin might inhibit cell-extracellular matrix interactions via the regulation, expression, and activation of STAT3 in TNBCs, which could directly inhibit metastasis in silibinin-treated BCa cells. Using proliferation assays, we found that exposure to silibinin at a concentration of 200 µM inhibited the proliferation of breast cancer (BCa) cells; this concentration also inhibited phosphorylation of STAT3 and its principal upstream kinase, Jak2. Furthermore, we found that silibinin inhibited the nuclear translocation of STAT3, as well as its binding to the MMP2 gene promoter. The ability of silibinin to inhibit metastasis was further studied using an in vitro invasion assay. The results confirm the role of STAT3 as a critical mediator in the invasive potential of BCa cells, and STAT3 knock-down resulted in inhibition of invasion. The invasion ability of silibinin-treated BCa cells was studied in detail with the expression of MMP2. Prevention of STAT3 activation also resulted in the inhibition of MMP2 expression. Use of a small interfering RNA to knock down STAT3 (siSTAT3) allowed us to confirm the role of STAT3 in regulating MMP2 expression, as well as the mechanism of action of silibinin in inhibiting MMP2. Taken together, we found that silibinin inhibits the Jak2/STAT3/MMP2 signaling pathway, and inhibits the proliferation, migration, and invasion of triple-negative BCa cells.

La Piedad Michoacán Mexico Virus V protein antagonizes type I interferon response by binding STAT2 protein and preventing STATs nuclear translocation.

PubMed

Pisanelli, Giuseppe; Laurent-Rolle, Maudry; Manicassamy, Balaji; Belicha-Villanueva, Alan; Morrison, Juliet; Lozano-Dubernard, Bernardo; Castro-Peralta, Felipa; Iovane, Giuseppe; García-Sastre, Adolfo

2016-02-02

La Piedad Michoacán Mexico Virus (LPMV) is a member of the Rubulavirus genus within the Paramyxoviridae family. LPMV is the etiologic agent of "blue eye disease", causing a significant disease burden in swine in Mexico with long-term implications for the agricultural industry. This virus mainly affects piglets and is characterized by meningoencephalitis and respiratory distress. It also affects adult pigs, causing reduced fertility and abortions in females, and orchitis and epididymitis in males. Viruses of the Paramyxoviridae family evade the innate immune response by targeting components of the interferon (IFN) signaling pathway. The V protein, expressed by most paramyxoviruses, is a well-characterized IFN signaling antagonist. Until now, there were no reports on the role of the LPMV-V protein in inhibiting the IFN response. In this study we demonstrate that LPMV-V protein antagonizes type I but not type II IFN signaling by binding STAT2, a component of the type I IFN cascade. Our results indicate that the last 18 amino acids of LPMV-V protein are required for binding to STAT2 in human and swine cells. While LPMV-V protein does not affect the protein levels of STAT1 or STAT2, it does prevent the IFN-induced phosphorylation and nuclear translocation of STAT1 and STAT2 thereby inhibiting cellular responses to IFN α/β. Copyright © 2015 Elsevier B.V. All rights reserved.

La Piedad Michoacán Mexico Virus V protein antagonizes type I interferon response by binding STAT2 protein and preventing STATs nuclear translocation

PubMed Central

Pisanelli, Giuseppe; Laurent-Rolle, Maudry; Manicassamy, Balaji; Belicha-Villanueva, Alan; Morrison, Juliet; Lozano-Dubernard, Bernardo; Castro-Peralta, Felipa; Iovane, Giuseppe; García-Sastre, Adolfo

2017-01-01

La Piedad Michoacán Mexico Virus (LPMV) is a member of the Rubulavirus genus within the Paramyxoviridae family. LPMV is the etiologic agent of “blue eye disease”, causing a significant disease burden in swine in Mexico with long-term implications for the agricultural industry. This virus mainly affects piglets and is characterized by meningoencephalitis and respiratory distress. It also affects adult pigs, causing reduced fertility and abortions in females, and orchitis and epididymitis in males. Viruses of the Paramyxoviridae family evade the innate immune response by targeting components of the interferon (IFN) signaling pathway. The V protein, expressed by most paramyxoviruses, is a well-characterized IFN signaling antagonist. Until now, there were no reports on the role of the LPMV-V protein in inhibiting the IFN response. In this study we demonstrate that LPMV-V protein antagonizes type I but not type II IFN signaling by binding STAT2, a component of the type I IFN cascade. Our results indicate that the last 18 amino acids of LPMV-V protein are required for binding to STAT2 in human and swine cells. While LPMV-V protein does not affect the protein levels of STAT1 or STAT2, it does prevent the IFN-induced phosphorylation and nuclear translocation of STAT1 and STAT2 thereby inhibiting cellular responses to IFN α/β PMID:26546155

NCX 4040, a nitric oxide-donating aspirin derivative, inhibits Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages.

PubMed

Choi, Eun-Young; Choe, So-Hui; Hyeon, Jin-Yi; Park, Hae Ryoun; Choi, Jeom-Il; Choi, In Soon; Kim, Sung-Jo

2015-12-05

In this study, the effects and underlying mechanisms of NCX 4040, a nitric oxide (NO)-donating aspirin derivative, on the production of proinflammatory mediators were examined using murine macrophages exposed to lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in the etiology of periodontal disease. NCX 4040 significantly reduced P. intermedia LPS-induced production of inducible NO synthase (iNOS)-derived NO, IL-1β and IL-6 as well as their mRNA expression in RAW264.7 cells. Notably, NCX 4040 was much more effective than the parental compound aspirin in reducing LPS-induced production of inflammatory mediators. NCX 4040 induced the expression of heme oxygenase-1 (HO-1) in cells treated with P. intermedia LPS, and the suppressive effect of NCX 4040 on LPS-induced NO production was significantly reversed by SnPP, a competitive HO-1 inhibitor. NCX 4040 did not influence LPS-induced phosphorylation of JNK and p38. IκB-α degradation as well as nuclear translocation and DNA-binding activities of NF-κB p65 and p50 subunits induced by P. intermedia LPS were significantly reduced by NCX 4040. Besides, LPS-induced phosphorylation of STAT1 and STAT3 was significantly down-regulated by NCX 4040. Further, NCX 4040 elevated the SOCS1 mRNA in cells stimulated with LPS. This study indicates that NCX 4040 inhibits P. intermedia LPS-induced production of NO, IL-1β and IL-6 in murine macrophages through anti-inflammatory HO-1 induction and suppression of NF-κB, STAT1 and STAT3 activation, which is associated with the activation of SOCS1 signaling. NCX 4040 could potentially be a promising tool in the treatment of periodontal disease, although further studies are required to verify this. Copyright © 2015 Elsevier B.V. All rights reserved.

STAT3 Activation in Pressure-Overloaded Feline Myocardium: Role for Integrins and the Tyrosine Kinase BMX

PubMed Central

Willey, Christopher D.; Palanisamy, Arun P.; Johnston, Rebecca K.; Mani, Santhosh K.; Shiraishi, Hirokazu; Tuxworth, William J.; Zile, Michael R.; Balasubramanian, Sundaravadivel; Kuppuswamy, Dhandapani

2008-01-01

Growth, survival and cytoskeletal rearrangement of cardiomyocytes are critical for cardiac hypertrophy. Signal transducer and activator of transcription-3 (STAT3) activation is an important cardioprotective factor associated with cardiac hypertrophy. Although STAT3 activation has been reported via signaling through Janus Kinase 2 (JAK2) in several cardiac models of hypertrophy, the importance of other nonreceptor tyrosine kinases (NTKs) has not been explored. Utilizing an in vivo feline right ventricular pressure-overload (RVPO) model of hypertrophy, we demonstrate that in 48 h pressure-overload (PO) myocardium, STAT3 becomes phosphorylated and redistributed to detergent-insoluble fractions with no accompanying JAK2 activation. PO also caused increased levels of phosphorylated STAT3 in both cytoplasmic and nuclear fractions. To investigate the role of other NTKs, we used our established in vitro cell culture model of hypertrophy where adult feline cardiomyocytes are embedded three-dimensionally (3D) in type-I collagen and stimulated with an integrin binding peptide containing an Arg-Gly-Asp (RGD) motif that we have previously shown to recapitulate the focal adhesion complex (FAC) formation of 48 h RVPO. RGD stimulation of adult cardiomyocytes in vitro caused both STAT3 redistribution and activation that were accompanied by the activation and redistribution of c-Src and the TEC family kinase, BMX, but not JAK2. However, infection with dominant negative c-Src adenovirus was unable to block RGD-stimulated changes on either STAT3 or BMX. Further analysis in vivo in 48 h PO myocardium showed the presence of both STAT3 and BMX in the detergent-insoluble fraction with their complex formation and phosphorylation. Therefore, these studies indicate a novel mechanism of BMX-mediated STAT3 activation within a PO model of cardiac hypertrophy that might contribute to cardiomyocyte growth and survival. PMID:18612371

Acetylation within the N- and C-Terminal Domains of Src Regulates Distinct Roles of STAT3-Mediated Tumorigenesis.

PubMed

Huang, Chao; Zhang, Zhe; Chen, Lihan; Lee, Hank W; Ayrapetov, Marina K; Zhao, Ting C; Hao, Yimei; Gao, Jinsong; Yang, Chunzhang; Mehta, Gautam U; Zhuang, Zhengping; Zhang, Xiaoren; Hu, Guohong; Chin, Y Eugene

2018-06-01

Posttranslational modifications of mammalian c-Src N-terminal and C-terminal domains regulate distinct functions. Myristoylation of G 2 controls its cell membrane association and phosphorylation of Y419/Y527 controls its activation or inactivation, respectively. We provide evidence that Src-cell membrane association-dissociation and catalytic activation-inactivation are both regulated by acetylation. In EGF-treated cells, CREB binding protein (CBP) acetylates an N-terminal lysine cluster (K5, K7, and K9) of c-Src to promote dissociation from the cell membrane. CBP also acetylates the C-terminal K401, K423, and K427 of c-Src to activate intrinsic kinase activity for STAT3 recruitment and activation. N-terminal domain phosphorylation (Y14, Y45, and Y68) of STAT3 by c-Src activates transcriptionally active dimers of STAT3. Moreover, acetyl-Src translocates into nuclei, where it forms the Src-STAT3 enhanceosome for gene regulation and cancer cell proliferation. Thus, c-Src acetylation in the N-terminal and C-terminal domains play distinct roles in Src activity and regulation. Significance: CBP-mediated acetylation of lysine clusters in both the N-terminal and C-terminal regions of c-Src provides additional levels of control over STAT3 transcriptional activity. Cancer Res; 78(11); 2825-38. ©2018 AACR . ©2018 American Association for Cancer Research.

New and recurrent gain-of-function STAT1 mutations in patients with chronic mucocutaneous candidiasis from Eastern and Central Europe

PubMed Central

Soltész, Beáta; Tóth, Beáta; Shabashova, Nadejda; Bondarenko, Anastasia; Okada, Satoshi; Cypowyj, Sophie; Abhyankar, Avinash; Csorba, Gabriella; Taskó, Szilvia; Sarkadi, Adrien Katalin; Méhes, Leonóra; Rozsíval, Pavel; Neumann, David; Chernyshova, Liudmyla; Tulassay, Zsolt; Puel, Anne; Casanova, Jean-Laurent; Sediva, Anna; Litzman, Jiri; Maródi, László

2013-01-01

Background Chronic mucocutaneous candidiasis disease (CMCD) may result from various inborn errors of interleukin (IL)-17-mediated immunity. Twelve of the 13 causal mutations described to date affect the coiled-coil domain (CCD) of STAT1. Several mutations, including R274W in particular, are recurrent, but the underlying mechanism is unclear. Objective To investigate and describe nine patients with CMCD in Eastern and Central Europe, to assess the biochemical impact of STAT1 mutations, to determine cytokines in supernatants of Candida-exposed blood cells, to determine IL-17-producing T cell subsets and to determine STAT1 haplotypes in a family with the c.820C>T (R274W) mutation. Results The novel c.537C>A (N179K) STAT1 mutation was gain-of-function (GOF) for γ-activated factor (GAF)-dependent cellular responses. In a Russian patient, the cause of CMCD was the newly identified c.854 A>G (Q285R) STAT1 mutation, which was also GOF for GAF-dependent responses. The c.1154C>T (T385M) mutation affecting the DNA-binding domain (DBD) resulted in a gain of STAT1 phosphorylation in a Ukrainian patient. Impaired Candida-induced IL-17A and IL-22 secretion by leucocytes and lower levels of intracellular IL-17 and IL-22 production by T cells were found in several patients. Haplotype studies indicated that the c.820C>T (R274W) mutation was recurrent due to a hotspot rather than a founder effect. Severe clinical phenotypes, including intracranial aneurysm, are presented. Conclusions The c.537C>A and c.854A>G mutations affecting the CCD and the c.1154C>T mutation affecting the DBD of STAT1 are GOF. The c.820C>T mutation of STAT1 in patients with CMCD is recurrent due to a hotspot. Patients carrying GOF mutations of STAT1 may develop multiple intracranial aneurysms by hitherto unknown mechanisms. PMID:23709754

Evaluation of STAT5A Gene Expression in Aflatoxin B1 Treated Bovine Mammary Epithelial Cells

PubMed Central

Forouharmehr, Ali; Harkinezhad, Taher; Qasemi-Panahi, Babak

2013-01-01

Purpose: Aflatoxin B1 (AFB1) is a potent mycotoxin which has been produced by fungi such as Aspergillus flavus and Aspergillus parasiticus as secondary metabolites due to their growth on food stuffs and induces hepatocellular carcinoma in many animal species, including humans. In the present study, the effect of AFB1 on STAT5A gene expression was investigated in bovine mammary epithelial cells using real time RT-PCR. Methods: Bovine mammary epithelial cells were seeded in a 24-well culture plate for three-dimensional (3D) culture in Matrigel matrix. After 21 days of 3D culture and reaching the required number of cells, cells were treated with AFB1 and incubated for 8 h. For real time PCR reaction, total RNA from the cultured and treated cells was extracted and used for complementary DNA synthesis. Results: The expression of STAT5A gene was significantly down regulated by AFB1 in dose- dependent manner and led to the reduction of proliferation and differentiation of epithelial cells, which has direct effect in milk protein quantity and quality. Conclusion: According to the results, it seems that down regulation of STAT5A gene in AFB1-treated cells maybe due to DNA damage induced by AFB1 in bovine mammary epithelial cells. PMID:24312879

Stoichiometry of DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein TF1.

PubMed

Schneider, G J; Geiduschek, E P

1990-06-25

The stoichiometry of DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein TF1 has been determined. 3H-Labeled TF1 was allowed to bind to a 32P-labeled DNA fragment containing a TF1 binding site. Multiple TF1-DNA complexes were resolved from each other and from unbound DNA by native gel electrophoresis. DNA-protein complexes were cut from polyacrylamide gels, and the amounts of 3H and 32P contained in each slice were measured. A ratio of 1.12 +/- 0.06 TF1 dimer/DNA molecule was calculated for the fastest-migrating TF1-DNA complex. We conclude that TF1 has a DNA-binding unit of one dimer. More slowly migrating complexes are apparently formed by serial addition of single TF1 dimers.

α-Lipoic Acid Inhibits Expression of IL-8 by Suppressing Activation of MAPK, Jak/Stat, and NF-κB in H. pylori-Infected Gastric Epithelial AGS Cells.

PubMed

Choi, Ji Hyun; Cho, Soon Ok; Kim, Hyeyoung

2016-01-01

The epithelial cytokine response, associated with reactive oxygen species (ROS), is important in Helicobacter pylori (H. pylori)-induced inflammation. H. pylori induces the production of ROS, which may be involved in the activation of mitogen-activated protein kinases (MAPK), janus kinase/signal transducers and activators of transcription (Jak/Stat), and oxidant-sensitive transcription factor, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and thus, expression of interleukin-8 (IL-8) in gastric epithelial cells. α-lipoic acid, a naturally occurring thiol compound, is a potential antioxidant. It shows beneficial effects in treatment of oxidant-associated diseases including diabetes. The present study is purposed to investigate whether α-lipoic acid inhibits expression of inflammatory cytokine IL-8 by suppressing activation of MAPK, Jak/Stat, and NF-κB in H. pylori-infected gastric epithelial cells. Gastric epithelial AGS cells were pretreated with or without α-lipoic acid for 2 h and infected with H. pylori in a Korean isolate (HP99) at a ratio of 300:1. IL-8 mRNA expression was analyzed by RT-PCR analysis. IL-8 levels in the medium were determined by enzyme-linked immunosorbent assay. NF-κB-DNA binding activity was determined by electrophoretic mobility shift assay. Phospho-specific and total forms of MAPK and Jak/Stat were assessed by Western blot analysis. ROS levels were determined using dichlorofluorescein fluorescence. As a result, H. pylori induced increases in ROS levels, mRNA, and protein levels of IL-8, as well as the activation of MAPK [extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinase 1/2 (JNK1/2), p38], Jak/Stat (Jak1/2, Stat3), and NF-κB in AGS cells, which was inhibited by α-lipoic acid. In conclusion, α-lipoic acid may be beneficial for prevention and/or treatment of H. pylori infection-associated gastric inflammation.

Curcumin suppresses the production of interleukin-6 in Prevotella intermedia lipopolysaccharide-activated RAW 264.7 cells

PubMed Central

2011-01-01

Purpose Curcumin is known to exert numerous biological effects including anti-inflammatory activity. In this study, we investigated the effects of curcumin on the production of interleukin-6 (IL-6) by murine macrophage-like RAW 264.7 cells stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a major cause of inflammatory periodontal disease, and sought to determine the underlying mechanisms of action. Methods LPS was prepared from lyophilized P. intermedia ATCC 25611 cells by the standard hot phenol-water method. Culture supernatants were collected and assayed for IL-6. We used real-time polymerase chain reaction to detect IL-6 mRNA expression. IκB-α degradation, nuclear translocation of NF-κB subunits, and STAT1 phosphorylation were characterized via immunoblotting. DNA-binding of NF-κB was also analyzed. Results Curcumin strongly suppressed the production of IL-6 at both gene transcription and translation levels in P. intermedia LPS-activated RAW 264.7 cells. Curcumin did not inhibit the degradation of IκB-α induced by P. intermedia LPS. Curcumin blocked NF-κB signaling through the inhibition of nuclear translocation of NF-κB p50 subunit. Curcumin also attenuated DNA binding activity of p50 and p65 subunits and suppressed STAT1 phosphorylation. Conclusions Although further study is required to explore the detailed mechanism of action, curcumin may contribute to blockade of the host-destructive processes mediated by IL-6 and appears to have potential therapeutic values in the treatment of inflammatory periodontal disease. PMID:21811692

Visual detection of STAT5B gene expression in living cell using the hairpin DNA modified gold nanoparticle beacon.

PubMed

Xue, Jianpeng; Shan, Lingling; Chen, Haiyan; Li, Yang; Zhu, Hongyan; Deng, Dawei; Qian, Zhiyu; Achilefu, Samuel; Gu, Yueqing

2013-03-15

Signal transducer and activator of transcription 5B (STAT5B) is an important protein in JAK-STAT signaling pathway that is responsible for the metastasis and proliferation of tumor cells. Determination of the STAT5B messenger Ribonucleic Acid (mRNA) relating to the STAT5B expression provides insight into the mechanism of tumor progression. In this study, we designed and used a special hairpin deoxyribonucleic acid (DNA) for human STAT5B mRNA to functionalize gold nanoparticles, which served as a beacon for detecting human STAT5B expression. Up to 90% quenching efficiency was achieved. Upon hybridizing with the target mRNA, the hairpin DNA modified gold nanoparticle beacons (hDAuNP beacons) release the fluorophores attached at 5' end of the oligonucleotide sequence. The fluorescence properties of the beacon before and after the hybridization with the complementary DNA were confirmed in vitro. The stability of hDAuNP beacons against degradation by DNase I and GSH indicated that the prepared beacon is stable inside cells. The detected fluorescence in MCF-7 cancer cells correlates with the specific STAT5B mRNA expression, which is consistent with the result from PCR measurement. Fluorescence microscopy showed that the hDAuNP beacons internalized in cells without using transfection agents, with intracellular distribution in the cytoplasm rather than the nucleus. The results demonstrated that this beacon could directly provide quantitative measurement of the intracellular STAT5B mRNA in living cells. Compared to the previous approaches, this beacon has advantages of higher target to background ratio of detection and an increased resistance to nuclease degradation. The strategy reported in this study is a promising approach for the intracellular measurement of RNA or protein expression in living cells, and has great potential in the study of drug screening and discovery. Copyright © 2012 Elsevier B.V. All rights reserved.

Smad7 enables STAT3 activation and promotes pluripotency independent of TGF-β signaling

PubMed Central

Yu, Yi; Gu, Shuchen; Li, Wenjian; Sun, Chuang; Chen, Fenfang; Xiao, Mu; Wang, Lei; Xu, Dewei; Li, Ye; Ding, Chen; Xia, Zongping; Li, Yi; Ye, Sheng; Xu, Pinglong; Zhao, Bin; Qin, Jun; Chen, Ye-Guang; Lin, Xia; Feng, Xin-Hua

2017-01-01

Smad7 is a negative feedback product of TGF-β superfamily signaling and fine tunes a plethora of pleiotropic responses induced by TGF-β ligands. However, its noncanonical functions independent of TGF-β signaling remain to be elucidated. Here, we show that Smad7 activates signal transducers and activators of transcription 3 (STAT3) signaling in maintaining mouse embryonic stem cell pluripotency in a manner independent of the TGF-β receptors, yet dependent on the leukemia inhibitory factor (LIF) coreceptor glycoprotein 130 (gp130). Smad7 directly binds to the intracellular domain of gp130 and disrupts the SHP2–gp130 or SOCS3–gp130 complex, thereby amplifying STAT3 activation. Consequently, Smad7 facilitates LIF-mediated self-renewal of mouse ESCs and is also critical for induced pluripotent stem cell reprogramming. This finding illustrates an uncovered role of the Smad7–STAT3 interplay in maintaining cell pluripotency and also implicates a mechanism involving Smad7 underlying cytokine-dependent regulation of cancer and inflammation. PMID:28874583

RNA binding to APOBEC3G induces the disassembly of functional deaminase complexes by displacing single-stranded DNA substrates

PubMed Central

Polevoda, Bogdan; McDougall, William M.; Tun, Bradley N.; Cheung, Michael; Salter, Jason D.; Friedman, Alan E.; Smith, Harold C.

2015-01-01

APOBEC3G (A3G) DNA deaminase activity requires a holoenzyme complex whose assembly on nascent viral reverse transcripts initiates with A3G dimers binding to ssDNA followed by formation of higher-order A3G homo oligomers. Catalytic activity is inhibited when A3G binds to RNA. Our prior studies suggested that RNA inhibited A3G binding to ssDNA. In this report, near equilibrium binding and gel shift analyses showed that A3G assembly and disassembly on ssDNA was an ordered process involving A3G dimers and multimers thereof. Although, fluorescence anisotropy showed that A3G had similar nanomolar affinity for RNA and ssDNA, RNA stochastically dissociated A3G dimers and higher-order oligomers from ssDNA, suggesting a different modality for RNA binding. Mass spectrometry mapping of A3G peptides cross-linked to nucleic acid suggested ssDNA only bound to three peptides, amino acids (aa) 181–194 in the N-terminus and aa 314–320 and 345–374 in the C-terminus that were part of a continuous exposed surface. RNA bound to these peptides and uniquely associated with three additional peptides in the N- terminus, aa 15–29, 41–52 and 83–99, that formed a continuous surface area adjacent to the ssDNA binding surface. The data predict a mechanistic model of RNA inhibition of ssDNA binding to A3G in which competitive and allosteric interactions determine RNA-bound versus ssDNA-bound conformational states. PMID:26424853

Evaluation of simultaneous binding of Chromomycin A3 to the multiple sites of DNA by the new restriction enzyme assay.

PubMed

Murase, Hirotaka; Noguchi, Tomoharu; Sasaki, Shigeki

2018-06-01

Chromomycin A3 (CMA3) is an aureolic acid-type antitumor antibiotic. CMA3 forms dimeric complexes with divalent cations, such as Mg 2+ , which strongly binds to the GC rich sequence of DNA to inhibit DNA replication and transcription. In this study, the binding property of CMA3 to the DNA sequence containing multiple GC-rich binding sites was investigated by measuring the protection from hydrolysis by the restriction enzymes, AccII and Fnu4HI, for the center of the CGCG site and the 5'-GC↓GGC site, respectively. In contrast to the standard DNase I footprinting method, the DNA substrates are fully hydrolyzed by the restriction enzymes, therefore, the full protection of DNA at all the cleavable sites indicates that CMA3 simultaneously binds to all the binding sites. The restriction enzyme assay has suggested that CMA3 has a high tendency to bind the successive CGCG sites and the CGG repeat. Copyright © 2018 Elsevier Ltd. All rights reserved.

Interaction of the Transactivation Domain of B-Myb with the TAZ2 Domain of the Coactivator p300: Molecular Features and Properties of the Complex

PubMed Central

Oka, Ojore; Waters, Lorna C.; Strong, Sarah L.; Dosanjh, Nuvjeevan S.; Veverka, Vaclav; Muskett, Frederick W.; Renshaw, Philip S.; Klempnauer, Karl-Heinz; Carr, Mark D.

2012-01-01

The transcription factor B-Myb is a key regulator of the cell cycle in vertebrates, with activation of transcription involving the recognition of specific DNA target sites and the recruitment of functional partner proteins, including the coactivators p300 and CBP. Here we report the results of detailed studies of the interaction between the transactivation domain of B-Myb (B-Myb TAD) and the TAZ2 domain of p300. The B-Myb TAD was characterized using circular dichroism, fluorescence and NMR spectroscopy, which revealed that the isolated domain exists as a random coil polypeptide. Pull-down and spectroscopic experiments clearly showed that the B-Myb TAD binds to p300 TAZ2 to form a moderately tight (Kd ∼1.0–10 µM) complex, which results in at least partial folding of the B-Myb TAD. Significant changes in NMR spectra of p300 TAZ2 suggest that the B-Myb TAD binds to a relatively large patch on the surface of the domain (∼1200 Å2). The apparent B-Myb TAD binding site on p300 TAZ2 shows striking similarity to the surface of CBP TAZ2 involved in binding to the transactivation domain of the transcription factor signal transducer and activator of transcription 1 (STAT1), which suggests that the structure of the B-Myb TAD-p300 TAZ2 complex may share many features with that reported for STAT1 TAD-p300 TAZ2. PMID:23300815

Native Hydrophobic Binding Interactions at the Transition State for Association between the TAZ1 Domain of CBP and the Disordered TAD-STAT2 Are Not a Requirement.

PubMed

Lindström, Ida; Dogan, Jakob

2017-08-15

A significant fraction of the eukaryotic proteome consists of proteins that are either partially or completely disordered under native-like conditions. Intrinsically disordered proteins (IDPs) are common in protein-protein interactions and are involved in numerous cellular processes. Although many proteins have been identified as disordered, much less is known about the binding mechanisms of the coupled binding and folding reactions involving IDPs. Here we have analyzed the rate-limiting transition state for binding between the TAZ1 domain of CREB binding protein and the intrinsically disordered transactivation domain of STAT2 (TAD-STAT2) by site-directed mutagenesis and kinetic experiments (Φ-value analysis) and found that the native protein-protein binding interface is not formed at the transition state for binding. Instead, native hydrophobic binding interactions form late, after the rate-limiting barrier has been crossed. The association rate constant in the absence of electrostatic enhancement was determined to be rather high. This is consistent with the Φ-value analysis, which showed that there are few or no obligatory native contacts. Also, linear free energy relationships clearly demonstrate that native interactions are cooperatively formed, a scenario that has usually been observed for proteins that fold according to the so-called nucleation-condensation mechanism. Thus, native hydrophobic binding interactions at the rate-limiting transition state for association between TAD-STAT2 and TAZ1 are not a requirement, which is generally in agreement with previous findings on other IDP systems and might be a common mechanism for IDPs.

Protective effects of calycosin against CCl4-induced liver injury with activation of FXR and STAT3 in mice.

PubMed

Chen, Xinli; Meng, Qiang; Wang, Changyuan; Liu, Qi; Sun, Huijun; Huo, Xiaokui; Sun, Pengyuan; Yang, Xiaobo; Peng, Jinyong; Liu, Kexin

2015-02-01

Investigating the hepatoprotective effect of calycosin against acute liver injury in association with FXR activation and STAT3 phosphorylation. The acute liver injury model was established by intraperitoneal injection of CCl4 in C57BL/6 mice. Serum alanine aminotransferase, aspartate aminotransferase, HE staining and TUNEL assay were used to identify the amelioration of the liver histopathological changes and hepatocytes apoptosis after calycosin treatment. ELISA kit and 5-bromo-2-deoxyuridine immunohistochemistry were used to measure the liver bile acid concentration and hepatocyte mitotic rate in vivo. The relation between calycosin and activation of FXR and STAT3 was comfirmed using the Luciferase assay, Molecular docking, Real-time PCR and Western Blot in vitro. The liver histopathological changes, hepatocytes apoptosis, liver bile acid overload and hepatocyte mitosis showed significant changes after calycosin treatment. Calycosin promoted the expression of FXR target genes such as FoxM1B and SHP but the effect was reversed by FXR suppressor guggulsterone. Molecular docking results indicated that calycosin could be embedded into the binding pocket of FXR, thereby increasing the expressions of STAT3 tyrosine phosphorylation and its target genes, Bcl-xl and SOCS3. Calycosin plays a critical role in hepatoprotection against liver injury in association with FXR activation and STAT3 phosphorylation.

Regulation of normal B-cell differentiation and malignant B-cell survival by OCT2

PubMed Central

Hodson, Daniel J.; Shaffer, Arthur L.; Xiao, Wenming; Wright, George W.; Schmitz, Roland; Phelan, James D.; Yang, Yandan; Webster, Daniel E.; Rui, Lixin; Kohlhammer, Holger; Nakagawa, Masao; Waldmann, Thomas A.; Staudt, Louis M.

2016-01-01

The requirement for the B-cell transcription factor OCT2 (octamer-binding protein 2, encoded by Pou2f2) in germinal center B cells has proved controversial. Here, we report that germinal center B cells are formed normally after depletion of OCT2 in a conditional knockout mouse, but their proliferation is reduced and in vivo differentiation to antibody-secreting plasma cells is blocked. This finding led us to examine the role of OCT2 in germinal center-derived lymphomas. shRNA knockdown showed that almost all diffuse large B-cell lymphoma (DLBCL) cell lines are addicted to the expression of OCT2 and its coactivator OCA-B. Genome-wide chromatin immunoprecipitation (ChIP) analysis and gene-expression profiling revealed the broad transcriptional program regulated by OCT2 that includes the expression of STAT3, IL-10, ELL2, XBP1, MYC, TERT, and ADA. Importantly, genetic alteration of OCT2 is not a requirement for cellular addiction in DLBCL. However, we detected amplifications of the POU2F2 locus in DLBCL tumor biopsies and a recurrent mutation of threonine 223 in the DNA-binding domain of OCT2. This neomorphic mutation subtly alters the DNA-binding preference of OCT2, leading to the transactivation of noncanonical target genes including HIF1a and FCRL3. Finally, by introducing mutations designed to disrupt the OCT2–OCA-B interface, we reveal a requirement for this protein–protein interface that ultimately might be exploited therapeutically. Our findings, combined with the predominantly B-cell–restricted expression of OCT2 and the absence of a systemic phenotype in our knockout mice, suggest that an OCT2-targeted therapeutic strategy would be efficacious in both major subtypes of DLBCL while avoiding systemic toxicity. PMID:26993806

Binding of galectin-1 to integrin β1 potentiates drug resistance by promoting survivin expression in breast cancer cells.

PubMed

Nam, KeeSoo; Son, Seog-Ho; Oh, Sunhwa; Jeon, Donghwan; Kim, Hyungjoo; Noh, Dong-Young; Kim, Sangmin; Shin, Incheol

2017-05-30

Galectin-1 is a β-galactoside binding protein secreted by many types of aggressive cancer cells. Although many studies have focused on the role of galectin-1 in cancer progression, relatively little attention has been paid to galectin-1 as an extracellular therapeutic target. To elucidate the molecular mechanisms underlying galectin-1-mediated cancer progression, we established galectin-1 knock-down cells via retroviral delivery of short hairpin RNA (shRNA) against galectin-1 in two triple-negative breast cancer (TNBC) cell lines, MDA-MB-231 and Hs578T. Ablation of galectin-1 expression decreased cell proliferation, migration, invasion, and doxorubicin resistance. We found that these effects were caused by decreased galectin-1-integrin β1 interactions and suppression of the downstream focal adhesion kinase (FAK)/c-Src pathway. We also found that silencing of galectin-1 inhibited extracellular signal-regulated kinase (ERK)/signal transducer and activator of transcription 3 (STAT3) signaling, thereby down-regulating survivin expression. This finding implicates STAT3 as a transcription factor for survivin. Finally, rescue of endogenous galectin-1 knock-down and recombinant galectin-1 treatment both recovered signaling through the FAK/c-Src/ERK/STAT3/survivin pathway. Taken together, these results suggest that extracellular galectin-1 contributes to cancer progression and doxorubicin resistance in TNBC cells. These effects appear to be mediated by galectin-1-induced up-regulation of the integrin β1/FAK/c-Src/ERK/STAT3/survivin pathway. Our results imply that extracellular galectin-1 has potential as a therapeutic target for triple-negative breast cancer.

Janus kinase (JAK) inhibitors in the treatment of inflammatory and neoplastic diseases.

PubMed

Roskoski, Robert

2016-09-01

The Janus kinase (JAK) family of non-receptor protein-tyrosine kinases consists of JAK1, JAK2, JAK3, and TYK2 (tyrosine kinase-2). Each of these proteins contains a JAK homology pseudokinase (JH2) domain that regulates the adjacent protein kinase domain (JH1). JAK1/2 and TYK2 are ubiquitously expressed whereas JAK3 is found predominantly in hematopoietic cells. The Janus kinase family is regulated by numerous cytokines including interleukins, interferons, and hormones such as erythropoietin, thrombopoietin, and growth hormone. Ligand binding to cytokine and hormone receptors leads to the activation of associated Janus kinases, which then mediate the phosphorylation of the receptors. The SH2 domain of STATs (signal transducers and activators of transcription) binds to the receptor phosphotyrosines thereby promoting STAT phosphorylation by the Janus kinases and consequent activation. STAT dimers are translocated to the nucleus where they participate in the regulation of the expression of thousands of proteins. JAK-STAT dysregulation results in autoimmune disorders such as rheumatoid arthritis, ulcerative colitis, and Crohn disease. JAK-STAT dysregulation also plays a role in the pathogenesis of myelofibrosis, polycythemia vera, and other myeloproliferative illnesses. An activating JAK2 V617F mutation occurs in 95% of people with polycythemia vera and in a lower percentage of people with other neoplasms. JAK1/3 signaling participates in the pathogenesis of inflammatory afflictions while JAK1/2 signaling participates in the development of several malignancies including leukemias and lymphomas as well as myeloproliferative neoplasms. Tofacitinib is a pan-JAK inhibitor that is approved by the FDA for the treatment of rheumatoid arthritis and ruxolitinib is a JAK1/2 inhibitor that is approved for the treatment of polycythemia vera and myelofibrosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Detection of Z DNA binding proteins in tissue culture cells.

PubMed Central

Leith, I R; Hay, R T; Russell, W C

1988-01-01

A gel electrophoresis DNA binding assay to detect Z DNA binding proteins has been developed utilising [32P] labelled poly [d(G-C)] which was converted to the Z form by incubation in 100 microM Co(NH3)6Cl3. The parameters of the assay were established using a Z DNA antibody as a model system and then applied to extracts of Hela and BHK21 cells. Using an anti-Z DNA antibody conditions were established which allowed resolution of antibody-DNA complexes and free DNA in the presence of 100 microM Co(NH3)6Cl3. The inclusion of unlabelled complementary homopolymers eliminated non-specific binding to the labelled Z-DNA probe. Competition experiments demonstrated that the assay was highly specific for double stranded non-B DNA. Application of the technique to extracts of mammalian cells demonstrated that human and hamster cells contain Z-DNA binding proteins; further characterisation by a blotting technique indicated that a 56,000 molecular weight cell protein preferentially binds Z-DNA. Images PMID:3419919

Binding characteristics and protective capacity of cyanidin-3-glucoside and its aglycon to calf thymus DNA.

PubMed

Zhang, Chao; Guo, Xiaofei; Cai, Wenqian; Ma, Yue; Zhao, Xiaoyan

2015-04-01

The binding characteristics and protective capacity of cyanidin (Cy) and cyanidin-3-glucoside (C3G) to calf thymus DNA were explored for the first time. The Cy and C3G gave a bathochromic shift to the ultraviolet-visible spectra of the DNA, indicating the formation of the DNA-Cy and DNA-C3G complexes. The complexes were formed by an intercalative binding mode based on the results of the fluorescence spectra and competitive binding analysis. Meanwhile, the Cy and C3G protected the DNA from the damage induced by the hydroxyl radical. The binding capacity and protective capacity of the C3G were stronger than that of the Cy. Furthermore, the formation of the DNA-anthocyanin complexes was spontaneous when the hydrogen bond and hydrophobic force played a key role. Hence, the Cy and C3G could protect the DNA automatically from the damage induced by the hydroxyl radical. © 2015 Institute of Food Technologists®

Spatio-temporal kinetics of growth hormone receptor signaling in single cells using FRET microscopy.

PubMed

Biener-Ramanujan, Eva; Ramanujan, V Krishnan; Herman, Brian; Gertler, Arieh

2006-08-01

The growth hormone (GH) receptor (R)-mediated JAK2 (Janus kinase-2)-STAT5 (signaling transducer and activator of transcription-5) pathway involves a cascade of protein-protein interactions and tyrosine phosphorylations that occur in a spatially and temporally sensitive manner in cells. To study GHR dimerization or GH-induced conformational change of predimerized GHRs and STAT5 activation kinetics in intact cells, fluorescence resonance energy transfer (FRET) and live-cell imaging methods were employed. FRET measurements at the membrane of HEK-293T cells co-expressing GHRs tagged at the C-terminus with cyan (C) and yellow (Y) fluorescent proteins (FPs) revealed transient GHR dimerization lasting 2-3 min, with a maximum at 3 min after GH stimulation, which was sufficient to induce STAT5 activation. The transient nature of the dimerization or GH-induced conformational change of predimerized GHRs kinetics was not a result of GHR internalization, as neither potassium- nor cholesterol-depletion treatments prolonged the FRET signal. YFP-tagged STAT5 recruitment to the membrane, binding to GHR-CFP, and phosphorylation, occurred within minutes of GH stimulation. Activated STAT5a-YFP did not show nuclear accumulation, despite nuclear pSTAT5 increase, suggesting high turnover of STAT5 nuclear shuttling. Although GHR dimerization and STAT5 activation have been reported previously, this is the first spatially resolved demonstration of GHR-signaling kinetics in intact cells.

DNA methylation and transcription in a distal region upstream from the bovine AlphaS1 casein gene after once or twice daily milking.

PubMed

Nguyen, Minh; Boutinaud, Marion; Pétridou, Barbara; Gabory, Anne; Pannetier, Maëlle; Chat, Sophie; Bouet, Stephan; Jouneau, Luc; Jaffrezic, Florence; Laloë, Denis; Klopp, Christophe; Brun, Nicolas; Kress, Clémence; Jammes, Hélène; Charlier, Madia; Devinoy, Eve

2014-01-01

Once daily milking (ODM) induces a reduction in milk production when compared to twice daily milking (TDM). Unilateral ODM of one udder half and TDM of the other half, enables the study of underlying mechanisms independently of inter-individual variability (same genetic background) and of environmental factors. Our results show that in first-calf heifers three CpG, located 10 kb upstream from the CSN1S1 gene were methylated to 33, 34 and 28%, respectively, after TDM but these levels were higher after ODM, 38, 38 and 33%, respectively. These methylation levels were much lower than those observed in the mammary gland during pregnancy (57, 59 and 50%, respectively) or in the liver (74, 78 and 61%, respectively). The methylation level of a fourth CpG (CpG4), located close by (29% during TDM) was not altered after ODM. CpG4 methylation reached 39.7% and 59.5%, during pregnancy or in the liver, respectively. CpG4 is located within a weak STAT5 binding element, arranged in tandem with a second high affinity STAT5 element. STAT5 binding is only marginally modulated by CpG4 methylation, but it may be altered by the methylation levels of the three other CpG nearby. Our results therefore shed light on mechanisms that help to explain how milk production is almost, but not fully, restored when TDM is resumed (15.1 ± 0.2 kg/day instead of 16.2 ± 0.2 kg/day, p<0.01). The STAT5 elements are 100 bp away from a region transcribed in the antisense orientation, in the mammary gland during lactation, but not during pregnancy or in other reproductive organs (ovary or testes). We now need to clarify whether the transcription of this novel RNA is a consequence of STAT5 interacting with the CSN1S1 distal region, or whether it plays a role in the chromatin structure of this region.

15-deoxy-Delta12,14-prostaglandin J2 inhibits INF-gamma-induced JAK/STAT1 signalling pathway activation and IP-10/CXCL10 expression in mesangial cells.

PubMed

Panzer, Ulf; Zahner, Gunther; Wienberg, Ulrike; Steinmetz, Oliver M; Peters, Anett; Turner, Jan-Eric; Paust, Hans-Joachim; Wolf, Gunter; Stahl, Rolf A K; Schneider, André

2008-12-01

Activators of the peroxisome proliferator-activated receptor gamma (PPARgamma), originally found to be implicated in lipid metabolism and glucose homeostasis, have been shown to modulate inflammatory responses through interference with cytokine and chemokine production. Given the central role of mesangial cell-derived chemokines in glomerular leukocyte recruitment in human and experimental glomerulonephritis, we studied the influence of natural and synthetic PPARgamma activators on INF-gamma-induced expression of the T cell-attracting chemokines IP-10/CXCL10, Mig/CXCL9 and I-TAC/CXCL11 in mouse mesangial cells. INF-gamma-treated mesangial cells were cultured in the presence or absence of either the naturally occurring PPARgamma ligand 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) or synthetic PPARgamma activators of the glitazone group. Chemokine mRNA and protein expression and activation of the JAK/STAT signalling pathway were analysed. The 15d-PGJ(2), but not synthetic PPARgamma ligands, dose-dependently inhibited INF-gamma-induced chemokine gene (mRNA and protein) expression. Combined results from EMSA and western blot analysis revealed the inhibitory ability of 15d-PGJ(2), but not of synthetic PPARgamma ligands, on IFN-gamma-induced tyrosine phosphorylation of JAK1, JAK2, STAT1 and nuclear STAT1 translocation and DNA binding. Our results demonstrate that 15d-PGJ(2) inhibits INF-gamma-induced chemokine expression in mesangial cells by targeting the JAK/STAT signalling pathway. This effect is independent of an interference with PPARgamma.

Nuclear matrix protein SMAR1 control regulatory T-cell fate during inflammatory bowel disease (IBD)

PubMed Central

Mirlekar, B; Ghorai, S; Khetmalas, M; Bopanna, R; Chattopadhyay, S

2015-01-01

Regulatory T (Treg) cells are essential for self-tolerance and immune homeostasis. Transcription factor Foxp3, a positive regulator of Treg cell differentiation, has been studied to some extent. Signal transducer and activator of transcription factor 3 (STAT3) is known to negatively regulate Foxp3. It is not clear how STAT3 is regulated during Treg differentiation. We show that SMAR1, a known transcription factor and tumor suppressor, is directly involved in maintaining Treg cell fate decision. T-cell-specific conditional knockdown of SMAR1 exhibits increased susceptibility towards inflammatory disorders, such as colitis. The suppressive function of Treg cells is compromised in the absence of SMAR1 leading to increased T helper type 17 (Th17) differentiation and inflammation. Compared with wild-type, the SMAR1−/− Treg cells showed increased susceptibility of inflammatory bowel disease in Rag1−/− mice, indicating the role of SMAR1 in compromising Treg cell differentiation resulting in severe colitis. We show that SMAR1 negatively regulate STAT3 expression favoring Foxp3 expression and Treg cell differentiation. SMAR1 binds to the MAR element of STAT3 promoter, present adjacent to interleukin-6 response elements. Thus Foxp3, a major driver of Treg cell differentiation, is regulated by SMAR1 via STAT3 and a fine-tune balance between Treg and Th17 phenotype is maintained. PMID:25993445

Transcription factor-dependent chromatin remodeling of Il18r1 during Th1 and Th2 differentiation 1

PubMed Central

Yu, Qing; Chang, Hua-Chen; Ahyi, Ayele-Nati N.; Kaplan, Mark H.

2008-01-01

The IL-18Rα chain is expressed on Th1 but not Th2 cells. We have recently shown that Stat4 is an important component of programming the Il18r1 locus (encoding IL-18Rα) for maximal expression in Th1 cells. Il18r1 is reciprocally repressed during Th2 development. In this report we demonstrate that the establishment of DNase hypersensitivity patterns that are distinct among undifferentiated CD4 T cells, Th1 and Th2 cells. Stat6 is required for the repression of Il18r1 expression and in Stat6-deficient Th2 cultures, mRNA levels, histone acetylation and H3K4 methylation levels are intermediate between levels observed in Th1 and Th2 cells. Despite the repressive effects of IL-4 during Th2 differentiation, we observed only modest binding of Stat6 to the Il18r1 locus. In contrast, we observed robust GATA-3 binding to a central region of the locus where DNase hypersensitivity sites overlapped with conserved non-coding sequences in Il18r1 introns. Ectopic expression of GATA-3 in differentiated Th1 cells repressed Il18r1 mRNA and surface expression of IL-18Rα. These data provide further mechanistic insight into transcription factor dependent establishment of Th subset-specific patterns of gene expression. PMID:18714006

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Yu-Kyoung; Lee, Tae-Yoon; Choi, Jong-Soon

Highlights: • Compound 7b, a meridianin C derivative, inhibits adipogenesis. • Compound 7b inhibits C/EBP-α, PPAR-γ, FAS, STAT-3, and STAT-5 in 3T3-L1 adipocytes. • Compound 7b inhibits leptin, but not adiponectin, expression in 3T3-L1 adipocytes. • Compound 7b thus may have therapeutic potential against obesity. - Abstract: Meridianin C, a marine alkaloid, is a potent protein kinase inhibitor and has anti-cancer activity. We have recently developed a series of meridianin C derivatives (compound 7a–7j) and reported their proviral integration Moloney Murine Leukemia Virus (pim) kinases’ inhibitory and anti-proliferative effects on human leukemia cells. Here we investigated the effect of thesemore » meridianin C derivatives on adipogenesis. Strikingly, among the derivatives tested, compound 7b most strongly inhibited lipid accumulation during the differentiation of 3T3-L1 preadipocytes into adipocytes. However, meridianin C treatment was largely cytotoxic to 3T3-L1 adipocytes. On mechanistic levels, compound 7b reduced not only the expressions of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), and fatty acid synthase (FAS) but also the phosphorylation levels of signal transducer and activator of transcription-3 (STAT-3) and STAT-5 during adipocyte differentiation. Moreover, compound 7b repressed leptin, but not adiponectin, expression during adipocyte differentiation. Collectively, these findings demonstrate that a meridianin C derivative inhibits adipogenesis by down-regulating expressions and/or phosphorylations of C/EBP-α, PPAR-γ, FAS, STAT-3 and STAT-5.« less

(CAG)(n)-hairpin DNA binds to Msh2-Msh3 and changes properties of mismatch recognition.

PubMed

Owen, Barbara A L; Yang, Zungyoon; Lai, Maoyi; Gajec, Maciej; Gajek, Maciez; Badger, John D; Hayes, Jeffrey J; Edelmann, Winfried; Kucherlapati, Raju; Wilson, Teresa M; McMurray, Cynthia T

2005-08-01

Cells have evolved sophisticated DNA repair systems to correct damaged DNA. However, the human DNA mismatch repair protein Msh2-Msh3 is involved in the process of trinucleotide (CNG) DNA expansion rather than repair. Using purified protein and synthetic DNA substrates, we show that Msh2-Msh3 binds to CAG-hairpin DNA, a prime candidate for an expansion intermediate. CAG-hairpin binding inhibits the ATPase activity of Msh2-Msh3 and alters both nucleotide (ADP and ATP) affinity and binding interfaces between protein and DNA. These changes in Msh2-Msh3 function depend on the presence of A.A mispaired bases in the stem of the hairpin and on the hairpin DNA structure per se. These studies identify critical functional defects in the Msh2-Msh3-CAG hairpin complex that could misdirect the DNA repair process.

α-Mangostin: a dietary antioxidant derived from the pericarp of Garcinia mangostana L. inhibits pancreatic tumor growth in xenograft mouse model.

PubMed

Hafeez, Bilal Bin; Mustafa, Ala; Fischer, Joseph W; Singh, Ashok; Zhong, Weixiong; Shekhani, Mohammed Ozair; Meske, Louise; Havighurst, Thomas; Kim, KyungMann; Verma, Ajit Kumar

2014-08-10

Pancreatic cancer (PC) is the most aggressive malignant disease, ranking as the fourth most leading cause of cancer-related death among men and women in the United States. In this study, we provide evidence of chemotherapeutic effects of α-mangostin, a dietary antioxidant isolated from the pericarp of Garcinia mangostana L. against human PC. The chemotherapeutic effect of α-mangostin was determined using four human PC cells (PL-45, PANC1, BxPC3, and ASPC1). α-Mangostin resulted in a significant inhibition of PC cells viability without having any effects on normal human pancreatic duct epithelial cells. α-Mangostin showed a dose-dependent increase of apoptosis in PC cells. Also, α-mangostin inhibited the expression levels of pNF-κB/p65Ser552, pStat3Ser727, and pStat3Tyr705. α-Mangostin inhibited DNA binding activity of nuclear factor kappa B (NF-κB) and signal transducer and activator 3 (Stat3). α-Mangostin inhibited the expression levels of matrix metallopeptidase 9 (MMP9), cyclin D1, and gp130; however, increased expression of tissue inhibitor of metalloproteinase 1 (TIMP1) was observed in PC cells. In addition, i.p. administration of α-mangostin (6 mg/kg body weight, 5 days a week) resulted in a significant inhibition of both primary (PL-45) and secondary (ASPC1) human PC cell-derived orthotopic and ectopic xenograft tumors in athymic nude mice. No sign of toxicity was observed in any of the mice administered with α-mangostin. α-Mangostin treatment inhibited the biomarkers of cell proliferation (Ki-67 and proliferating cell nuclear antigen [PCNA]) in the xenograft tumor tissues. We present, for the first time, that dietary antioxidant α-mangostin inhibits the growth of PC cells in vitro and in vivo. These results suggest the potential therapeutic efficacy of α-mangostin against human PC.

Liver Inflammation and Metabolic Signaling in ApcMin/+ Mice: The Role of Cachexia Progression

PubMed Central

Narsale, Aditi A.; Enos, Reilly T.; Puppa, Melissa J.; Chatterjee, Saurabh; Murphy, E. Angela; Fayad, Raja; Pena, Majorette O’; Durstine, J. Larry; Carson, James A.

2015-01-01

The ApcMin/+ mouse exhibits an intestinal tumor associated loss of muscle and fat that is accompanied by chronic inflammation, insulin resistance and hyperlipidemia. Since the liver governs systemic energy demands through regulation of glucose and lipid metabolism, it is likely that the liver is a pathological target of cachexia progression in the ApcMin/+ mouse. The purpose of this study was to determine if cancer and the progression of cachexia affected liver endoplasmic reticulum (ER)-stress, inflammation, metabolism, and protein synthesis signaling. The effect of cancer (without cachexia) was examined in wild-type and weight-stable ApcMin/+ mice. Cachexia progression was examined in weight-stable, pre-cachectic, and severely-cachectic ApcMin/+ mice. Livers were analyzed for morphology, glycogen content, ER-stress, inflammation, and metabolic changes. Cancer induced hepatic expression of ER-stress markers BiP (binding immunoglobulin protein), IRE-1α (endoplasmic reticulum to nucleus signaling 1), and inflammatory intermediate STAT-3 (signal transducer and activator of transcription 3). While gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression was suppressed by cancer, glycogen content or protein synthesis signaling remained unaffected. Cachexia progression depleted liver glycogen content and increased mRNA expression of glycolytic enzyme PFK (phosphofrucktokinase) and gluconeogenic enzyme PEPCK. Cachexia progression further increased pSTAT-3 but suppressed p-65 and JNK (c-Jun NH2-terminal kinase) activation. Interestingly, progression of cachexia suppressed upstream ER-stress markers BiP and IRE-1α, while inducing its downstream target CHOP (DNA-damage inducible transcript 3). Cachectic mice exhibited a dysregulation of protein synthesis signaling, with an induction of p-mTOR (mechanistic target of rapamycin), despite a suppression of Akt (thymoma viral proto-oncogene 1) and S6 (ribosomal protein S6) phosphorylation. Thus, cancer induced ER-stress markers in the liver, however cachexia progression further deteriorated liver ER-stress, disrupted protein synthesis regulation and caused a differential inflammatory response related to STAT-3 and NF-κB (Nuclear factor—κB) signaling. PMID:25789991

Molecular mechanisms of cellular transformation by HTLV-1 Tax.

PubMed

Grassmann, Ralph; Aboud, Mordechai; Jeang, Kuan-Teh

2005-09-05

The HTLV Tax protein is crucial for viral replication and for initiating malignant transformation leading to the development of adult T-cell leukemia. Tax has been shown to be oncogenic, since it transforms and immortalizes rodent fibroblasts and human T-lymphocytes. Through CREB, NF-kappaB and SRF pathways Tax transactivates cellular promoters including those of cytokines (IL-13, IL-15), cytokine receptors (IL-2Ralpha) and costimulatory surface receptors (OX40/OX40L) leading to upregulated protein expression and activated signaling cascades (e.g. Jak/STAT, PI3Kinase, JNK). Tax also stimulates cell growth by direct binding to cyclin-dependent kinase holenzymes and/or inactivating tumor suppressors (e.g. p53, DLG). Moreover, Tax silences cellular checkpoints, which guard against DNA structural damage and chromosomal missegregation, thereby favoring the manifestation of a mutator phenotype in cells.

Dynamics, Conformational Entropy, and Frustration in Protein-Protein Interactions Involving an Intrinsically Disordered Protein Domain.

PubMed

Lindström, Ida; Dogan, Jakob

2018-05-18

Intrinsically disordered proteins (IDPs) are abundant in the eukaryotic proteome. However, little is known about the role of subnanosecond dynamics and the conformational entropy that it represents in protein-protein interactions involving IDPs. Using nuclear magnetic resonance side chain and backbone relaxation, stopped-flow kinetics, isothermal titration calorimetry, and computational studies, we have characterized the interaction between the globular TAZ1 domain of the CREB binding protein and the intrinsically disordered transactivation domain of STAT2 (TAD-STAT2). We show that the TAZ1/TAD-STAT2 complex retains considerable subnanosecond motions, with TAD-STAT2 undergoing only a partial disorder-to-order transition. We report here the first experimental determination of the conformational entropy change for both binding partners in an IDP binding interaction and find that the total change even exceeds in magnitude the binding enthalpy and is comparable to the contribution from the hydrophobic effect, demonstrating its importance in the binding energetics. Furthermore, we show that the conformational entropy change for TAZ1 is also instrumental in maintaining a biologically meaningful binding affinity. Strikingly, a spatial clustering of very high amplitude motions and a cluster of more rigid sites in the complex exist, which through computational studies we found to overlap with regions that experience energetic frustration and are less frustrated, respectively. Thus, the residual dynamics in the bound state could be necessary for faster dissociation, which is important for proteins that interact with multiple binding partners.

Characterisation of a DNA sequence element that directs Dictyostelium stalk cell-specific gene expression.

PubMed

Ceccarelli, A; Zhukovskaya, N; Kawata, T; Bozzaro, S; Williams, J

2000-12-01

The ecmB gene of Dictyostelium is expressed at culmination both in the prestalk cells that enter the stalk tube and in ancillary stalk cell structures such as the basal disc. Stalk tube-specific expression is regulated by sequence elements within the cap-site proximal part of the promoter, the stalk tube (ST) promoter region. Dd-STATa, a member of the STAT transcription factor family, binds to elements present in the ST promoter-region and represses transcription prior to entry into the stalk tube. We have characterised an activatory DNA sequence element, that lies distal to the repressor elements and that is both necessary and sufficient for expression within the stalk tube. We have mapped this activator to a 28 nucleotide region (the 28-mer) within which we have identified a GA-containing sequence element that is required for efficient gene transcription. The Dd-STATa protein binds to the 28-mer in an in vitro binding assay, and binding is dependent upon the GA-containing sequence. However, the ecmB gene is expressed in a Dd-STATa null mutant, therefore Dd-STATa cannot be responsible for activating the 28-mer in vivo. Instead, we identified a distinct 28-mer binding activity in nuclear extracts from the Dd-STATa null mutant, the activity of this GA binding activity being largely masked in wild type extracts by the high affinity binding of the Dd-STATa protein. We suggest, that in addition to the long range repression exerted by binding to the two known repressor sites, Dd-STATa inhibits transcription by direct competition with this putative activator for binding to the GA sequence.

Structures of apo IRF-3 and IRF-7 DNA binding domains: effect of loop L1 on DNA binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

De Ioannes, Pablo; Escalante, Carlos R.; Aggarwal, Aneel K.

2013-11-20

Interferon regulatory factors IRF-3 and IRF-7 are transcription factors essential in the activation of interferon-{beta} (IFN-{beta}) gene in response to viral infections. Although, both proteins recognize the same consensus IRF binding site AANNGAAA, they have distinct DNA binding preferences for sites in vivo. The X-ray structures of IRF-3 and IRF-7 DNA binding domains (DBDs) bound to IFN-{beta} promoter elements revealed flexibility in the loops (L1-L3) and the residues that make contacts with the target sequence. To characterize the conformational changes that occur on DNA binding and how they differ between IRF family members, we have solved the X-ray structures ofmore » IRF-3 and IRF-7 DBDs in the absence of DNA. We found that loop L1, carrying the conserved histidine that interacts with the DNA minor groove, is disordered in apo IRF-3 but is ordered in apo IRF-7. This is reflected in differences in DNA binding affinities when the conserved histidine in loop L1 is mutated to alanine in the two proteins. The stability of loop L1 in IRF-7 derives from a unique combination of hydrophobic residues that pack against the protein core. Together, our data show that differences in flexibility of loop L1 are an important determinant of differential IRF-DNA binding.« less

APPL1-mediated activation of STAT3 contributes to inhibitory effect of adiponectin on hepatic gluconeogenesis.

PubMed

Ding, Youming; Zhang, Deling; Wang, Bin; Zhang, Yemin; Wang, Lei; Chen, Xiaoyan; Li, Mingxin; Tang, Zhao; Wang, Changhua

2016-09-15

Adiponectin has been shown to suppress hepatic gluconeogenesis. However, the signaling pathways underlying its action remain ill-defined. The purpose of this study was to examine the potential role of APPL1 in mediating anti-gluconeogenic ability of adiponectin. Primary hepatocytes were isolated from male C57BL/6 mice. Western blot and RT-PCR were performed to detect protein expression and mRNA level, respectively. The protein-protein association was determined by immunoprecipitation and GST pull-down assay. We found that APPL1 protein levels were negatively associated with expressions of proteins and mRNAs of gluconeogenesis enzymes under stimulation with adiponectin. In addition, adiponectin-stimulated STAT3 phosphorylation and acetylation were positively regulated by APPL1 and negative regulated by SirT1. Pharmacological and genetic inhibition of STAT3 mitigated impact of adiponectin on hepatic gluconeogenesis. Furthermore, adiponectin administration facilitated the binding of APPL1 to SirT1 and suppressed the association of SirT1 with STAT3. Taken together, our study showed that APPL1-SirT1-STAT3 pathway mediated adiponectin signaling in primary hepatocytes. This new finding provides a novel mechanism by which adiponectin suppresses hepatic gluconeogenesis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Herbal remedy magnolol suppresses IL-6-induced STAT3 activation and gene expression in endothelial cells

PubMed Central

Chen, Shih-Chung; Chang, Ying-Ling; Wang, Danny Ling; Cheng, Jing-Jy

2006-01-01

Magnolol (Mag), an active constituent isolated from the Chinese herb Hou p'u (Magnolia officinalis) has long been used to suppress inflammatory processes. Chronic inflammation is well known to be involved in vascular injuries such as atherosclerosis in which interleukin (IL)-6 may participate. Signal transducer and activator of transcription protein 3 (STAT3), a transcription factor involved in inflammation and the cell cycle, is activated by IL-6. In this study, we evaluated whether Mag can serve as an anti-inflammatory agent during endothelial injuries. The effects of Mag on IL-6-induced STAT3 activation and downstream target gene induction in endothelial cells (ECs) were examined. Pretreatment of ECs with Mag dose dependently inhibited IL-6-induced Tyr705 and Ser727 phosphorylation in STAT3 without affecting the phosphorylation of JAK1, JAK2, and ERK1/2. Mag pretreatment of these ECs dose dependently suppressed IL-6-induced promoter activity of intracellular cell adhesion molecule (ICAM)-1 that contains functional IL-6 response elements (IREs). An electrophoretic mobility shift assay (EMSA) revealed that Mag treatment significantly reduced STAT3 binding to the IRE region. Consistently, Mag treatment markedly inhibited ICAM-1 expression on the endothelial surface. As a result, reduced monocyte adhesion to IL-6-activated ECs was observed. Furthermore, Mag suppressed IL-6-induced promoter activity of cyclin D1 and monocyte chemotactic protein (MCP)-1 for which STAT3 activation plays a role. In conclusion, our results indicate that Mag inhibits IL-6-induced STAT3 activation and subsequently results in the suppression of downstream target gene expression in ECs. These results provide a therapeutic basis for the development of Mag as an anti-inflammatory agent for vascular disorders including atherosclerosis. PMID:16520748

Long non-coding RNA H19 suppresses retinoblastoma progression via counteracting miR-17-92 cluster.

PubMed

Zhang, Aihui; Shang, Weiwei; Nie, Qiaoli; Li, Ting; Li, Suhui

2018-04-01

Long non-coding RNAs (lncRNAs) are frequently dysregulated and play important roles in many cancers. lncRNA H19 is one of the earliest discovered lncRNAs which has diverse roles in different cancers. However, the expression, roles, and action mechanisms of H19 in retinoblastoma are still largely unknown. In this study, we found that H19 is downregulated in retinoblastoma tissues and cell lines. Gain-of-function and loss-of-function assays showed that H19 inhibits retinoblastoma cell proliferation, induces retinoblastoma cell cycle arrest and cell apoptosis. Mechanistically, we identified seven miR-17-92 cluster binding sites on H19, and found that H19 directly bound to miR-17-92 cluster via these seven binding sites. Through binding to miR-17-92 cluster, H19 relieves the suppressing roles of miR-17-92 cluster on p21. Furthermore, H19 represses STAT3 activation induced by miR-17-92 cluster. Hence, our results revealed that H19 upregulates p21 expression, inhibits STAT3 phosphorylation, and downregulates the expression of STAT3 target genes BCL2, BCL2L1, and BIRC5. In addition, functional assays demonstrated that the mutation of miR-17-92 cluster binding sites on H19 abolished the proliferation inhibiting, cell cycle arrest and cell apoptosis inducing roles of H19 in retinoblastoma. In conclusion, our data suggested that H19 inhibits retinoblastoma progression via counteracting the roles of miR-17-92 cluster, and implied that enhancing the action of H19 may be a promising therapeutic strategy for retinoblastoma. © 2017 Wiley Periodicals, Inc.

Effect of angiotensin II on proliferation and differentiation of mouse induced pluripotent stem cells into mesodermal progenitor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ishizuka, Toshiaki, E-mail: tishizu@ndmc.ac.jp; Goshima, Hazuki; Ozawa, Ayako

2012-03-30

Highlights: Black-Right-Pointing-Pointer Treatment with angiotensin II enhanced LIF-induced DNA synthesis of mouse iPS cells. Black-Right-Pointing-Pointer Angiotensin II may enhance the DNA synthesis via induction of superoxide. Black-Right-Pointing-Pointer Treatment with angiotensin II significantly increased JAK/STAT3 phosphorylation. Black-Right-Pointing-Pointer Angiotensin II enhanced differentiation into mesodermal progenitor cells. Black-Right-Pointing-Pointer Angiotensin II may enhance the differentiation via activation of p38 MAPK. -- Abstract: Previous studies suggest that angiotensin receptor stimulation may enhance not only proliferation but also differentiation of undifferentiated stem/progenitor cells. Therefore, in the present study, we determined the involvement of the angiotensin receptor in the proliferation and differentiation of mouse induced pluripotent stemmore » (iPS) cells. Stimulation with angiotensin II (Ang II) significantly increased DNA synthesis in mouse iPS cells cultured in a medium with leukemia inhibitory factor (LIF). Pretreatment of the cells with either candesartan (a selective Ang II type 1 receptor [AT{sub 1}R] antagonist) or Tempol (a cell-permeable superoxide scavenger) significantly inhibited Ang II-induced DNA synthesis. Treatment with Ang II significantly increased JAK/STAT3 phosphorylation. Pretreatment with candesartan significantly inhibited Ang II- induced JAK/STAT3 phosphorylation. In contrast, induction of mouse iPS cell differentiation into Flk-1-positive mesodermal progenitor cells was performed in type IV collagen (Col IV)- coated dishes in a differentiation medium without LIF. When Col IV-exposed iPS cells were treated with Ang II for 5 days, the expression of Flk-1 was significantly increased compared with that in the cells treated with the vehicle alone. Pretreatment of the cells with both candesartan and SB203580 (a p38 MAPK inhibitor) significantly inhibited the Ang II- induced increase in Flk-1 expression. Treatment with Ang II enhanced the phosphorylation of p38 MAPK in Col IV- exposed iPS cells. These results suggest that the stimulation of mouse iPS cells with AT{sub 1}R may enhance LIF-induced DNA synthesis, by augmenting the generation of superoxide and activating JAK/STAT3, and that AT{sub 1}R stimulation may enhance Col IV-induced differentiation into mesodermal progenitor cells via p38 MAPK activation.« less

Disruption of mTORC1 in Macrophages Decreases Chemokine Gene Expression and Atherosclerosis

PubMed Central

Ai, Ding; Jiang, Hongfeng; Westerterp, Marit; Murphy, Andrew J.; Wang, Mi; Ganda, Anjali; Abramowicz, Sandra; Welch, Carrie; Almazan, Felicidad; Zhu, Yi; Miller, Yury I; Tall, Alan R.

2014-01-01

Rationale The mammalian target of rapamycin complex 1 (mTORC1) inhibitor, rapamycin, has been shown to decrease atherosclerosis, even while increasing plasma LDL levels. This suggests an anti-atherogenic effect possibly mediated by modulation of inflammatory responses in atherosclerotic plaques. Objective To assess the role of macrophage mTORC1 in atherogenesis. Methods and Results We transplanted bone marrow from mice in which a key mTORC1 adaptor, Raptor, was deleted in macrophages by Cre/loxP recombination (Mac-RapKO mice) into Ldlr-/- mice and then fed them the Western-type diet (WTD). Atherosclerotic lesions from Mac-RapKO mice showed decreased infiltration of macrophages, lesion size and chemokine gene expression compared with control mice. Treatment of macrophages with minimally modified LDL (mmLDL) resulted in increased levels of chemokine mRNAs and STAT3 phosphorylation; these effects were reduced in Mac-RapKO macrophages. While wild-type and Mac-RapKO macrophages showed similar STAT3 phosphorylation on Tyr705, Mac-RapKO macrophages showed decreased STAT3 Ser727 phosphorylation in response to mmLDL treatment and decreased Ccl2 promoter binding of STAT3. Conclusions The results demonstrate cross-talk between nutritionally-induced mTORC1 signaling and mmLDL-mediated inflammatory signaling via combinatorial phosphorylation of STAT3 in macrophages, leading to increased STAT3 activity on the CCL2 (MCP-1)promoter with pro-atherogenic consequences. PMID:24687132

Cellular STAT3 functions via PCBP2 to restrain Epstein-Barr Virus lytic activation in B lymphocytes.

PubMed

Koganti, Siva; Clark, Carissa; Zhi, Jizu; Li, Xiaofan; Chen, Emily I; Chakrabortty, Sharmistha; Hill, Erik R; Bhaduri-McIntosh, Sumita

2015-05-01

A major hurdle to killing Epstein-Barr virus (EBV)-infected tumor cells using oncolytic therapy is the presence of a substantial fraction of EBV-infected cells that does not support the lytic phase of EBV despite exposure to lytic cycle-promoting agents. To determine the mechanism(s) underlying this refractory state, we developed a strategy to separate lytic from refractory EBV-positive (EBV(+)) cells. By examining the cellular transcriptome in separated cells, we previously discovered that high levels of host STAT3 (signal transducer and activator of transcription 3) curtail the susceptibility of latently infected cells to lytic cycle activation signals. The goals of the present study were 2-fold: (i) to determine the mechanism of STAT3-mediated resistance to lytic activation and (ii) to exploit our findings to enhance susceptibility to lytic activation. We therefore analyzed our microarray data set, cellular proteomes of separated lytic and refractory cells, and a publically available STAT3 chromatin immunoprecipitation sequencing (ChIP-Seq) data set to identify cellular PCBP2 [poly(C)-binding protein 2], an RNA-binding protein, as a transcriptional target of STAT3 in refractory cells. Using Burkitt lymphoma cells and EBV(+) cell lines from patients with hypomorphic STAT3 mutations, we demonstrate that single cells expressing high levels of PCBP2 are refractory to spontaneous and induced EBV lytic activation, STAT3 functions via cellular PCBP2 to regulate lytic susceptibility, and suppression of PCBP2 levels is sufficient to increase the number of EBV lytic cells. We expect that these findings and the genome-wide resources that they provide will accelerate our understanding of a longstanding mystery in EBV biology and guide efforts to improve oncolytic therapy for EBV-associated cancers. Most humans are infected with Epstein-Barr virus (EBV), a cancer-causing virus. While EBV generally persists silently in B lymphocytes, periodic lytic (re)activation of latent virus is central to its life cycle and to most EBV-related diseases. However, a substantial fraction of EBV-infected B cells and tumor cells in a population is refractory to lytic activation. This resistance to lytic activation directly and profoundly impacts viral persistence and the effectiveness of oncolytic therapy for EBV(+) cancers. To identify the mechanisms that underlie susceptibility to EBV lytic activation, we used host gene and protein expression profiling of separated lytic and refractory cells. We find that STAT3, a transcription factor overactive in many cancers, regulates PCBP2, a protein important in RNA biogenesis, to regulate susceptibility to lytic cycle activation signals. These findings advance our understanding of EBV persistence and provide important leads on devising methods to improve viral oncolytic therapies. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Hemi-methylated DNA regulates DNA methylation inheritance through allosteric activation of H3 ubiquitylation by UHRF1.

PubMed

Harrison, Joseph S; Cornett, Evan M; Goldfarb, Dennis; DaRosa, Paul A; Li, Zimeng M; Yan, Feng; Dickson, Bradley M; Guo, Angela H; Cantu, Daniel V; Kaustov, Lilia; Brown, Peter J; Arrowsmith, Cheryl H; Erie, Dorothy A; Major, Michael B; Klevit, Rachel E; Krajewski, Krzysztof; Kuhlman, Brian; Strahl, Brian D; Rothbart, Scott B

2016-09-06

The epigenetic inheritance of DNA methylation requires UHRF1, a histone- and DNA-binding RING E3 ubiquitin ligase that recruits DNMT1 to sites of newly replicated DNA through ubiquitylation of histone H3. UHRF1 binds DNA with selectivity towards hemi-methylated CpGs (HeDNA); however, the contribution of HeDNA sensing to UHRF1 function remains elusive. Here, we reveal that the interaction of UHRF1 with HeDNA is required for DNA methylation but is dispensable for chromatin interaction, which is governed by reciprocal positive cooperativity between the UHRF1 histone- and DNA-binding domains. HeDNA recognition activates UHRF1 ubiquitylation towards multiple lysines on the H3 tail adjacent to the UHRF1 histone-binding site. Collectively, our studies are the first demonstrations of a DNA-protein interaction and an epigenetic modification directly regulating E3 ubiquitin ligase activity. They also define an orchestrated epigenetic control mechanism involving modifications both to histones and DNA that facilitate UHRF1 chromatin targeting, H3 ubiquitylation, and DNA methylation inheritance.

Th17 cytokine differentiation and loss of plasticity after SOCS1 inactivation in a cutaneous T-cell lymphoma.

PubMed

Ehrentraut, Stefan; Schneider, Björn; Nagel, Stefan; Pommerenke, Claudia; Quentmeier, Hilmar; Geffers, Robert; Feist, Maren; Kaufmann, Maren; Meyer, Corinna; Kadin, Marshall E; Drexler, Hans G; MacLeod, Roderick A F

2016-06-07

We propose that deregulated T-helper-cell (Th) signaling underlies evolving Th17 cytokine expression seen during progression of cutaneous T-cell lymphoma (CTCL). Accordingly, we developed a lymphoma progression model comprising cell lines established at indolent (MAC-1) and aggressive (MAC-2A) CTCL stages. We discovered activating JAK3 (V722I) mutations present at indolent disease, reinforced in aggressive disease by novel compound heterozygous SOCS1 (G78R/D105N) JAK-binding domain inactivating mutations. Though isogenic, indolent and aggressive-stage cell lines had diverged phenotypically, the latter expressing multiple Th17 related cytokines, the former a narrower profile. Importantly, indolent stage cells remained poised for Th17 cytokine expression, readily inducible by treatment with IL-2 - a cytokine which mitigates Th17 differentiation in mice. In indolent stage cells JAK3 expression was boosted by IL-2 treatment. Th17 conversion of MAC-1 cells by IL-2 was blocked by pharmacological inhibition of JAK3 or STAT5, implicating IL2RG - JAK3 - STAT5 signaling in plasticity responses. Like IL-2 treatment, SOCS1 knockdown drove indolent stage cells to mimic key aggressive stage properties, notably IL17F upregulation. Co-immunoprecipitation experiments showed that SOCS1 mutations abolished JAK3 binding, revealing a key role for SOCS1 in regulating JAK3/STAT5 signaling. Collectively, our results show how JAK/STAT pathway mutations contribute to disease progression in CTCL cells by potentiating inflammatory cytokine signaling, widening the potential therapeutic target range for this intractable entity. MAC-1/2A cells also provide a candidate human Th17 laboratory model for identifying potentally actionable CTCL markers or targets and testing their druggability in vitro.

Therapeutic effect of Cryptotanshinone on experimental rheumatoid arthritis through downregulating p300 mediated-STAT3 acetylation.

PubMed

Wang, Ying; Zhou, Chun; Gao, Hui; Li, Cuixian; Li, Dong; Liu, Peiqing; Huang, Min; Shen, Xiaoyan; Liu, Liang

2017-08-15

The balance between T helper 17 (Th17) cells and regulatory T (Treg) cells, plays a critical role in rheumatoid arthritis (RA). The differentiation of Th17 cells requires the activation of STAT3, which determines the balance of Th17/Treg. Here, we investigated the therapeutic effect of Cryptotanshinone (CTS) on collagen induced mouse arthritis and explored the underlying mechanisms. Arthritis was induced in DBA/1 mice with bovine collagen type II and complete Freund's adjuvant. CTS was given at 20mgkg -1 d -1 or 60mgkg -1 d -1 by gavage for 6weeks. The immuno-inflammation and joint destruction were evaluated and the balance of Th17/Treg was determined. STAT3 acetylation and phosphorylation were detected by western blotting, and the involvement of p300 was investigated by siRNA and plasmid overexpression. CTS at a dose of 60mgkg -1 d -1 ameliorated the inflammation and joint destruction in CIA mice. It improved Th17/Treg imbalance, and inhibited both acetylation and phosphorylation of STAT3. CTS reduced p300 expression and its binding to STAT3, but increased phosphorylated AMPK. Knockdown of p300 mimicked the inhibitory effect of CTS on STAT3 acetylation and phosphorylation, which could be partially rescued by overexpression of p300-WT, but not p300-dominant negative (DN) construct. Our study suggested that the anti-arthritis effects of CTS were attained through suppression of p300-mediated STAT3 acetylation. Our data suggest that CTS might be a potential immune modulator for RA treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

6-Shogaol exerts anti-proliferative and pro-apoptotic effects through the modulation of STAT3 and MAPKs signaling pathways.

PubMed

Kim, Sung-Moo; Kim, Chulwon; Bae, Hang; Lee, Jong Hyun; Baek, Seung Ho; Nam, Dongwoo; Chung, Won-Seok; Shim, Bum Sang; Lee, Seok-Geun; Kim, Sung-Hoon; Sethi, Gautam; Ahn, Kwang Seok

2015-10-01

6-shogaol (6SG), one of active ingredients in ginger (Zingiber officinale), is known to exhibit anti-proliferative, anti-metastatic, and pro-apoptotic activities through a mechanism that is not fully elucidated. Because the aberrant activation of STAT3 and MAPKs have been associated with regulation of proliferation, invasion, and metastasis of tumors, we hypothesized that 6SG modulates the activation of STAT3 and MAPKs activation in tumor cells. We found that 6SG strongly inhibited constitutive phosphorylation of STAT3 through inhibition of the activation of upstream JAK2 and c-Src kinases and nuclear translocation of STAT3 on both MDA-MB231 and DU145 cells. Also, 6SG caused the activation of JNK, p38 MAPK, and ERK. Inhibition of ROS generation by N-acetylcysteine (NAC) significantly prevented 6SG-induced apoptosis. 6SG induced apoptosis as characterized by cleavage of PARP, accumulation of cells in subG1 phase, positive Annexin V binding, down-regulation of STAT3-regulated proteins, and activation of caspase-8, -9, -3 in both MDA-MB231 cells. Compared with other analogues of 6SG, such as 6-gingerol (6G), 8-gingerol (8G), and 10-gingerol (10G), 6SG was found to be the most potent blocker of STAT3 activation. We observed that the administration of 6SG alone significantly suppressed the growth of the tumor. As compared to the vehicle control, 6SG also suppressed the expression of STAT3-regulated gene products such as Bcl-2, Bcl-xL, and Survivin in tumor tissues. Overall, these findings suggest that 6SG can interfere with multiple signaling cascades involved in tumorigenesis and can be used as a potential therapeutic candidate for both the prevention and treatment of cancer. © 2014 Wiley Periodicals, Inc.

Footprinting of Chlorella virus DNA ligase bound at a nick in duplex DNA.

PubMed

Odell, M; Shuman, S

1999-05-14

The 298-amino acid ATP-dependent DNA ligase of Chlorella virus PBCV-1 is the smallest eukaryotic DNA ligase known. The enzyme has intrinsic specificity for binding to nicked duplex DNA. To delineate the ligase-DNA interface, we have footprinted the enzyme binding site on DNA and the DNA binding site on ligase. The size of the exonuclease III footprint of ligase bound a single nick in duplex DNA is 19-21 nucleotides. The footprint is asymmetric, extending 8-9 nucleotides on the 3'-OH side of the nick and 11-12 nucleotides on the 5'-phosphate side. The 5'-phosphate moiety is essential for the binding of Chlorella virus ligase to nicked DNA. Here we show that the 3'-OH moiety is not required for nick recognition. The Chlorella virus ligase binds to a nicked ligand containing 2',3'-dideoxy and 5'-phosphate termini, but cannot catalyze adenylation of the 5'-end. Hence, the 3'-OH is important for step 2 chemistry even though it is not itself chemically transformed during DNA-adenylate formation. A 2'-OH cannot substitute for the essential 3'-OH in adenylation at a nick or even in strand closure at a preadenylated nick. The protein side of the ligase-DNA interface was probed by limited proteolysis of ligase with trypsin and chymotrypsin in the presence and absence of nicked DNA. Protease accessible sites are clustered within a short segment from amino acids 210-225 located distal to conserved motif V. The ligase is protected from proteolysis by nicked DNA. Protease cleavage of the native enzyme prior to DNA addition results in loss of DNA binding. These results suggest a bipartite domain structure in which the interdomain segment either comprises part of the DNA binding site or undergoes a conformational change upon DNA binding. The domain structure of Chlorella virus ligase inferred from the solution experiments is consistent with the structure of T7 DNA ligase determined by x-ray crystallography.

Analysis of the activation status of Akt, NFkappaB, and Stat3 in human diffuse gliomas.

PubMed

Wang, Huamin; Wang, Hua; Zhang, Wei; Huang, Helen J; Liao, Warren S L; Fuller, Gregory N

2004-08-01

Loss of phosphatase and tensin homolog (PTEN) and amplification of the epidermal growth factor receptor (EGFR) gene contribute to the progression of gliomas. As downstream targets of the PTEN and EGFR signaling pathways, Akt, NFkappaB, and signal transducer and activator of transcription-3 (Stat3) have been shown to play important roles in the control of cell proliferation, apoptosis, and oncogenesis. We examined the activation status of Akt, NFkappaB, and Stat3 in 259 diffuse gliomas using tissue microarrays and immunohistochemistry, and evaluated their association with glioma grade. We observed significant positive correlations between the activation status of Akt and NFkappaB and glioma grade. In contrast, only focal immunoreactivity for phospho-Stat3 was observed in < 9% of high-grade gliomas. In addition, we observed a significant correlation between the activation of Akt and NFkappaB. Functional correlation between Akt activation and the activation of NFkappaB was confirmed in U251MG GBM cells in which inhibition of Akt activation either by stable expression of PTEN or by the PI3-kinase inhibitors, wortmannin and LY294002, led to a concomitant decrease in NFkappaB-binding activity. Thus, our results demonstrate that constitutive activation of Akt and NFkappaB, but not Stat3, contributes significantly to the progression of diffuse gliomas, and activation of Akt may lead to NFkappaB activation in high-grade gliomas.

Inhibition of STAT3/VEGF/CDK2 axis signaling is critically involved in the antiangiogenic and apoptotic effects of arsenic herbal mixture PROS in non-small lung cancer cells

PubMed Central

Lee, Hyemin; Lee, Hyo-Jung; Bae, Ill Ju; Kim, Jeong Jin; Kim, Sung-Hoon

2017-01-01

Despite the antitumor effects of asrsenic trioxide (As2O3), tetraarsenic hexoxide (As4O6 or PR) and tetraarsenic tetrasulfide (As4S4) in several cancers, their adverse poisoning, toxicity and resistance are still hot issues for effective cancer therapy. Here, antitumor mechanism of arsenic herbal mixture PROS including PR and OS (Oldenlandia diffusa and Salvia miltiorrhiza extract) was elucidated in non-small cell lung cancer cells (NSCLCs), since PR alone showed resistant cytotoxicity in NSCLCs compared to other cancers. PROS exerted significant cytotoxicity, induced sub-G1 phase and S phase arrest, increased apoptotic bodies, and attenuated the expression of pro-PARP, Bcl-2, Cyclin E, Cyclin A, CDK2, E2F1, p-Src, p-STAT3, p-ERK, p-AKT, COX-2 and SOCS-1 in A549 and H460 cells along with disrupted binding of STAT3 with CDK2 or VEGF. Notably, PROS inhibited VEGF induced proliferation, migration and tube formation in HUVECs and suppressed angiogenesis in chorioallantoic membrane (CAM) assay via reduced phosphorylation of VEGFR2, Src and STAT3. Consistently, PROS reduced the growth of H460 cells implanted in BALB/c athymic nude mice via inhibition of STAT3, and VEGF and activation of caspase 3. Overall, these findings suggest that PROS exerts antiangiogenic and apoptotic effects via inhibition of STAT3/ VEGF/ CDK2 axis signaling as a potent anticancer agent for lung cancer treatment. PMID:29254203

Parturition failure in mice lacking Mamld1

PubMed Central

Miyado, Mami; Miyado, Kenji; Katsumi, Momori; Saito, Kazuki; Nakamura, Akihiro; Shihara, Daizou; Ogata, Tsutomu; Fukami, Maki

2015-01-01

In mice, the onset of parturition is triggered by a rapid decline in circulating progesterone. Progesterone withdrawal occurs as a result of functional luteolysis, which is characterized by an increase in the enzymatic activity of 20α-hydroxysteroid dehydrogenase (20α-HSD) in the corpus luteum and is mediated by the prostaglandin F2α (PGF2α) signaling. Here, we report that the genetic knockout (KO) of Mamld1, which encodes a putative non-DNA-binding regulator of testicular steroidogenesis, caused defective functional luteolysis and subsequent parturition failure and neonatal deaths. Progesterone receptor inhibition induced the onset of parturition in pregnant KO mice, and MAMLD1 regulated the expression of Akr1c18, the gene encoding 20α-HSD, in cultured cells. Ovaries of KO mice at late gestation were morphologically unremarkable; however, Akr1c18 expression was reduced and expression of its suppressor Stat5b was markedly increased. Several other genes including Prlr, Cyp19a1, Oxtr, and Lgals3 were also dysregulated in the KO ovaries, whereas PGF2α signaling genes remained unaffected. These results highlight the role of MAMLD1 in labour initiation. MAMLD1 likely participates in functional luteolysis by regulating Stat5b and other genes, independent of the PGF2α signaling pathway. PMID:26435405

[Methylation Status of the SOCS3 Gene Promoter in H2228 Cells and 
EML4-ALK-positive Lung Cancer Tissues].

PubMed

Liu, Chunlai; Li, Yongwen; Dong, Yunlong; Zhang, Hongbing; Li, Ying; Liu, Hongyu; Chen, Jun

2016-09-20

The EML4-ALK fusion gene is a newly discovered driver gene of non-small cell lung cancer and exhibits special clinical and pathological features. The JAK-STAT signaling pathway, an important downstream signaling pathway of EML4-ALK, is aberrantly sustained and activated in EML4-ALK-positive lung cancer cells fusion gene, but the underlying reason remains unknown. The suppressor of cytokine signaling (SOCS) is a negative regulatory factor that mainly inhibits the proliferation, differentiation, and induction of apoptotic cells by inhibiting the JAK-STAT signaling pathway. The aberrant methylation of the SOCS gene leads to inactivation of tumors and abnormal activation of the JAK2-STAT signaling pathway. The aim of this study is to investigate the methylation status of the SOCS3 promoter in EML4-ALK-positive H2228 cells and lung cancer tissues. The methylation status of the SOCS3 promoter in EML4-ALK-positive H2228 lung cancer cells and lung cancer tissues was detected by methylation-specific PCR (MSP) analysis and verified by DNA sequencing. The expression levels of SOCS3 in H2228 cells were detected by Western blot and Real-time PCR analyses after treatment with the DNA methyltransferase inhibitor 5'-Aza-dC. MSP and DNA sequencing assay results indicated the presence of SOCS3 promoter methylation in H2228 cells as well as in three cases of seven EML4-ALK-positive lung cancer tissues. The expression level of SOCS3 significantly increased in H2228 cells after 5'-Aza-dC treatment. The aerrant methylation of the SOCS3 promoter region in EML4-ALK (+) H2228 cells and lung cancer tissues may be significantly involved in the pathogenesis of EML4-ALK-positive lung cancer.

APOBEC3G Interacts with ssDNA by Two Modes: AFM Studies

NASA Astrophysics Data System (ADS)

Shlyakhtenko, Luda S.; Dutta, Samrat; Banga, Jaspreet; Li, Ming; Harris, Reuben S.; Lyubchenko, Yuri L.

2015-10-01

APOBEC3G (A3G) protein has antiviral activity against HIV and other pathogenic retroviruses. A3G has two domains: a catalytic C-terminal domain (CTD) that deaminates cytidine, and a N-terminal domain (NTD) that binds to ssDNA. Although abundant information exists about the biological activities of A3G protein, the interplay between sequence specific deaminase activity and A3G binding to ssDNA remains controversial. We used the topographic imaging and force spectroscopy modalities of Atomic Force Spectroscopy (AFM) to characterize the interaction of A3G protein with deaminase specific and nonspecific ssDNA substrates. AFM imaging demonstrated that A3G has elevated affinity for deaminase specific ssDNA than for nonspecific ssDNA. AFM force spectroscopy revealed two distinct binding modes by which A3G interacts with ssDNA. One mode requires sequence specificity, as demonstrated by stronger and more stable complexes with deaminase specific ssDNA than with nonspecific ssDNA. Overall these observations enforce prior studies suggesting that both domains of A3G contribute to the sequence specific binding of ssDNA.

APOBEC3G Interacts with ssDNA by Two Modes: AFM Studies.

PubMed

Shlyakhtenko, Luda S; Dutta, Samrat; Banga, Jaspreet; Li, Ming; Harris, Reuben S; Lyubchenko, Yuri L

2015-10-27

APOBEC3G (A3G) protein has antiviral activity against HIV and other pathogenic retroviruses. A3G has two domains: a catalytic C-terminal domain (CTD) that deaminates cytidine, and a N-terminal domain (NTD) that binds to ssDNA. Although abundant information exists about the biological activities of A3G protein, the interplay between sequence specific deaminase activity and A3G binding to ssDNA remains controversial. We used the topographic imaging and force spectroscopy modalities of Atomic Force Spectroscopy (AFM) to characterize the interaction of A3G protein with deaminase specific and nonspecific ssDNA substrates. AFM imaging demonstrated that A3G has elevated affinity for deaminase specific ssDNA than for nonspecific ssDNA. AFM force spectroscopy revealed two distinct binding modes by which A3G interacts with ssDNA. One mode requires sequence specificity, as demonstrated by stronger and more stable complexes with deaminase specific ssDNA than with nonspecific ssDNA. Overall these observations enforce prior studies suggesting that both domains of A3G contribute to the sequence specific binding of ssDNA.

Pleiotrophin (PTN) is expressed in vascularized human atherosclerotic plaques: IFN-γ/JAK/STAT1 signaling is critical for the expression of PTN in macrophages

PubMed Central

Li, Fuqiang; Tian, Fang; Wang, Lai; Williamson, Ian K.; Sharifi, Behrooz G.; Shah, Prediman K.

2010-01-01

Neovascularization is critical to destabilization of atheroma. We previously reported that the angiogenic growth factor pleiotrophin (PTN) coaxes monocytes to assume the phenotype of functional endothelial cells in vitro and in vivo. In this study we show that PTN expression is colocalized with capillaries of human atherosclerotic plaques. Among the various reagents that are critical to the pathogenesis of atherosclerosis, interferon (IFN)-γ was found to markedly induce PTN mRNA expression in a dose-dependent manner in macrophages. Mechanistic studies revealed that the Janus kinase inhibitors, WHI-P154 and ATA, efficiently blocked STAT1 phosphorylation in a concentration- and time-dependent manner. Notably, the level of phosphorylated STAT1 was found to correlate directly with the PTN mRNA levels. In addition, STAT1/STAT3/p44/42 signaling molecules were found to be phosphorylated by IFN-γ in macrophages, and they were translocated into the nucleus. Further, PTN promoter analysis showed that a gamma-activated sequence (GAS) located at −2086 to −2078 bp is essential for IFN-γ-regulated promoter activity. Moreover, electrophoretic mobility shift, supershift, and chromatin immunoprecipitation analyses revealed that both STAT1 and STAT3 bind to the GAS at the chromatin level in the IFN-γ stimulated cells. Finally, to test whether the combined effect of STAT1/STAT3/p44/42 signaling is required for the expression of PTN in macrophages, gene knockdowns of these transcription factors were performed using siRNA. Cells lacking STAT1, but not STAT3 or p42, have markedly reduced PTN mRNA levels. These data suggest that PTN expression in the human plaques may be in part regulated by IFN-γ and that PTN is involved in the adaptive immunity.—Li, F., Tian, F., Wang, L., Williamson, I. K., Sharifi, B. G., Shah, P. K. Pleiotrophin (PTN) is expressed in vascularized human atherosclerotic plaques: IFN-γ/JAK/STAT1 signaling is critical for the expression of PTN in macrophages PMID:19917672

Labor Inhibits Placental Mechanistic Target of Rapamycin Complex 1 Signaling

PubMed Central

LAGER, Susanne; AYE, Irving L.M.H.; GACCIOLI, Francesca; RAMIREZ, Vanessa I.; JANSSON, Thomas; POWELL, Theresa L.

2014-01-01

Introduction Labor induces a myriad of changes in placental gene expression. These changes may represent a physiological adaptation inhibiting placental cellular processes associated with a high demand for oxygen and energy (e.g., protein synthesis and active transport) thereby promoting oxygen and glucose transfer to the fetus. We hypothesized that mechanistic target of rapamycin complex 1 (mTORC1) signaling, a positive regulator of trophoblast protein synthesis and amino acid transport, is inhibited by labor. Methods Placental tissue was collected from healthy, term pregnancies (n=15 no-labor; n=12 labor). Activation of Caspase-1, IRS1/Akt, STAT, mTOR, and inflammatory signaling pathways was determined by Western blot. NFκB p65 and PPARγ DNA binding activity was measured in isolated nuclei. Results Labor increased Caspase-1 activation and mTOR complex 2 signaling, as measured by phosphorylation of Akt (S473). However, mTORC1 signaling was inhibited in response to labor as evidenced by decreased phosphorylation of mTOR (S2448) and 4EBP1 (T37/46 and T70). Labor also decreased NFκB and PPARγ DNA binding activity, while having no effect on IRS1 or STAT signaling pathway. Discussion and conclusion Several placental signaling pathways are affected by labor, which has implications for experimental design in studies of placental signaling. Inhibition of placental mTORC1 signaling in response to labor may serve to down-regulate protein synthesis and amino acid transport, processes that account for a large share of placental oxygen and glucose consumption. We speculate that this response preserves glucose and oxygen for transfer to the fetus during the stressful events of labor. PMID:25454472

Centchroman inhibits proliferation of head and neck cancer cells through the modulation of PI3K/mTOR Pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Srivastava, Vikas Kumar; Gara, Rishi Kumar; Bhatt, M.L.B.

Research highlights: {yields} Centchroman (CC) inhibits cellular proliferation in HNSCC cells through the dual inhibition of PI3/mTOR pathway. {yields} CC treatment also inhibits STAT3 activation and alters expression of proteins involved in cell cycle regulation and DNA repair response in HNSCC cells. {yields} CC exhibits anti-proliferative activity in a variety of non-HNSCC cancer cell lines and is devoid of cytotoxicity to normal cell types of diverse origins. -- Abstract: Centchroman (CC; 67/20; INN: Ormeloxifene) is a non-steroidal antiestrogen extensively used as a female contraceptive in India. In the present study, we report the anti-proliferative effect of CC in head andmore » neck squamous cell carcinoma (HNSCC) cells. CC inhibited cell proliferation in a dose dependent manner at 24 h of treatment. Further studies showed that CC treatment induced apoptosis, inhibited Akt/mTOR and signal transducers and activators of transcription protein 3 (STAT3) signaling, altered proteins associated with cell cycle regulation and DNA damage and inhibited colony forming efficiency of HNSCC cells. In addition, CC displayed anti-proliferative activity against a variety of non-HNSCC cell lines of diverse origin. The ability of CC to serve as a dual-inhibitor of Akt/mTOR and STAT3 signaling warrants further studies into its role as a therapeutic strategy against HNSCC.« less

Saccharomyces cerevisiae MSH2-MSH3 and MSH2-MSH6 complexes display distinct requirements for DNA binding Domain I in mismatch recognition.

PubMed Central

Lee, Susan D.; Surtees, Jennifer A.; Alani, Eric

2007-01-01

In eukaryotic mismatch repair (MMR) MSH2-MSH6 initiates the repair of base-base and small insertion/deletion mismatches while MSH2-MSH3 repairs larger insertion/deletion mismatches. In this study we showed that the msh2Δ1 mutation, containing a complete deletion of the conserved mismatch recognition Domain I of MSH2, conferred a separation of function phenotype with respect to MSH2-MSH3 and MSH2-MSH6 functions. Strains bearing the msh2Δ1 mutation were nearly wild-type in MSH2-MSH6-mediated MMR and in suppressing recombination between DNA sequences predicted to form mismatches recognized by MSH2-MSH6. However, these strains were completely defective in MSH2-MSH3-mediated MMR and recombination functions. This information encouraged us to analyze the contributions of Domain I to the mismatch binding specificity of MSH2-MSH3 in genetic and biochemical assays. We found that Domain I in MSH2 contributed a non-specific DNA binding activity while Domain I of MSH3 appeared important for mismatch binding specificity and for suppressing non-specific DNA-binding. These observations reveal distinct requirements for the MSH2 DNA binding Domain I in the repair of DNA mismatches and suggest that the binding of MSH2-MSH3 to mismatch DNA involves protein-DNA contacts that appear very different from those required for MSH2-MSH6 mismatch binding. PMID:17157869

Saccharomyces cerevisiae MSH2-MSH3 and MSH2-MSH6 complexes display distinct requirements for DNA binding domain I in mismatch recognition.

PubMed

Lee, Susan D; Surtees, Jennifer A; Alani, Eric

2007-02-09

In eukaryotic mismatch repair (MMR) MSH2-MSH6 initiates the repair of base-base and small insertion/deletion mismatches while MSH2-MSH3 repairs larger insertion/deletion mismatches. Here, we show that the msh2Delta1 mutation, containing a complete deletion of the conserved mismatch recognition domain I of MSH2, conferred a separation of function phenotype with respect to MSH2-MSH3 and MSH2-MSH6 functions. Strains bearing the msh2Delta1 mutation were nearly wild-type in MSH2-MSH6-mediated MMR and in suppressing recombination between DNA sequences predicted to form mismatches recognized by MSH2-MSH6. However, these strains were completely defective in MSH2-MSH3-mediated MMR and recombination functions. This information encouraged us to analyze the contributions of domain I to the mismatch binding specificity of MSH2-MSH3 in genetic and biochemical assays. We found that domain I in MSH2 contributed a non-specific DNA binding activity while domain I of MSH3 appeared important for mismatch binding specificity and for suppressing non-specific DNA binding. These observations reveal distinct requirements for the MSH2 DNA binding domain I in the repair of DNA mismatches and suggest that the binding of MSH2-MSH3 to mismatch DNA involves protein-DNA contacts that appear very different from those required for MSH2-MSH6 mismatch binding.

Fas Promotes T Helper 17 Cell Differentiation and Inhibits T Helper 1 Cell Development by Binding and Sequestering Transcription Factor STAT1.

PubMed

Meyer Zu Horste, Gerd; Przybylski, Dariusz; Schramm, Markus A; Wang, Chao; Schnell, Alexandra; Lee, Youjin; Sobel, Raymond; Regev, Aviv; Kuchroo, Vijay K

2018-03-20

The death receptor Fas removes activated lymphocytes through apoptosis. Previous transcriptional profiling predicted that Fas positively regulates interleukin-17 (IL-17)-producing T helper 17 (Th17) cells. Here, we demonstrate that Fas promoted the generation and stability of Th17 cells and prevented their differentiation into Th1 cells. Mice with T-cell- and Th17-cell-specific deletion of Fas were protected from induced autoimmunity, and Th17 cell differentiation and stability were impaired. Fas-deficient Th17 cells instead developed a Th1-cell-like transcriptional profile, which a new algorithm predicted to depend on STAT1. Experimentally, Fas indeed bound and sequestered STAT1, and Fas deficiency enhanced IL-6-induced STAT1 activation and nuclear translocation, whereas deficiency of STAT1 reversed the transcriptional changes induced by Fas deficiency. Thus, our computational and experimental approach identified Fas as a regulator of the Th17-to-Th1 cell balance by controlling the availability of opposing STAT1 and STAT3 to have a direct impact on autoimmunity. Copyright © 2018. Published by Elsevier Inc.

DNA mutagenic activity and capacity for HIV-1 restriction of the cytidine deaminase APOBEC3G depend on whether DNA or RNA binds to tyrosine 315

PubMed Central

Polevoda, Bogdan; Joseph, Rebecca; Friedman, Alan E.; Bennett, Ryan P.; Greiner, Rebecca; De Zoysa, Thareendra; Stewart, Ryan A.; Smith, Harold C.

2017-01-01

APOBEC3G (A3G) belongs to the AID/APOBEC protein family of cytidine deaminases (CDA) that bind to nucleic acids. A3G mutates the HIV genome by deamination of dC to dU, leading to accumulation of virus-inactivating mutations. Binding to cellular RNAs inhibits A3G binding to substrate single-stranded (ss) DNA and CDA activity. Bulk RNA and substrate ssDNA bind to the same three A3G tryptic peptides (amino acids 181–194, 314–320, and 345–374) that form parts of a continuously exposed protein surface extending from the catalytic domain in the C terminus of A3G to its N terminus. We show here that the A3G tyrosines 181 and 315 directly cross-linked ssDNA. Binding experiments showed that a Y315A mutation alone significantly reduced A3G binding to both ssDNA and RNA, whereas Y181A and Y182A mutations only moderately affected A3G nucleic acid binding. Consistent with these findings, the Y315A mutant exhibited little to no deaminase activity in an Escherichia coli DNA mutator reporter, whereas Y181A and Y182A mutants retained ∼50% of wild-type A3G activity. The Y315A mutant also showed a markedly reduced ability to assemble into viral particles and had reduced antiviral activity. In uninfected cells, the impaired RNA-binding capacity of Y315A was evident by a shift of A3G from high-molecular-mass ribonucleoprotein complexes to low-molecular-mass complexes. We conclude that Tyr-315 is essential for coordinating ssDNA interaction with or entry to the deaminase domain and hypothesize that RNA bound to Tyr-315 may be sufficient to competitively inhibit ssDNA deaminase-dependent antiviral activity. PMID:28381554

The Growth Hormone Receptor: Mechanism of Receptor Activation, Cell Signaling, and Physiological Aspects

PubMed Central

Dehkhoda, Farhad; Lee, Christine M. M.; Medina, Johan; Brooks, Andrew J.

2018-01-01

The growth hormone receptor (GHR), although most well known for regulating growth, has many other important biological functions including regulating metabolism and controlling physiological processes related to the hepatobiliary, cardiovascular, renal, gastrointestinal, and reproductive systems. In addition, growth hormone signaling is an important regulator of aging and plays a significant role in cancer development. Growth hormone activates the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) signaling pathway, and recent studies have provided a new understanding of the mechanism of JAK2 activation by growth hormone binding to its receptor. JAK2 activation is required for growth hormone-mediated activation of STAT1, STAT3, and STAT5, and the negative regulation of JAK–STAT signaling comprises an important step in the control of this signaling pathway. The GHR also activates the Src family kinase signaling pathway independent of JAK2. This review covers the molecular mechanisms of GHR activation and signal transduction as well as the physiological consequences of growth hormone signaling. PMID:29487568

Synthesis, crystal structure, DFT calculation and DNA binding studies of new water-soluble derivatives of dppz

NASA Astrophysics Data System (ADS)

Aminzadeh, Mohammad; Eslami, Abbas; Kia, Reza; Aleeshah, Roghayeh

2017-10-01

Diquaternarization of dipyrido-[2,3-a:2‧,3‧-c]-phenazine,(dppz) and its analogous dipyrido-[2,3-a:2‧,3‧-c]-dimethylphenazine,(dppx) using 1,3-dibromopropane afford new water-soluble derivatives of phenazine, propylene-bipyridyldiylium-phenazine (1) and propylene-bipyridyldiylium-dimethylphenazine (2). The compounds have been characterized by means of FT-IR, NMR, elemental analysis and conductometric measurements and their structure were determined by X-ray crystallography. The experimental studies on the compounds have been accompanied computationally by Density Functional Theory (DFT) calculations. The DNA binding properties of both compounds to calf thymus DNA (ctDNA) were investigated by UV-Vis absorption and emission methods. The expanded UV-Vis spectral data matrix was analyzed by multivariate curve resolution-alternating least squares (MCR-ALS) technique to obtain the concentration profile and pure spectra of all reaction species which existed in the interaction procedure. Multivariate curve resolution may help us to give a better understanding of the 1(Cl)2-ctDNA and 2(Cl)2-ctDNA interaction mechanism. The results suggest that both compounds bind tightly to DNA through intercalation mechanism and the DNA binding affinity of 2 is slightly lower than that of 1 due to steric hindrance of the methyl group. Also, thermal denaturation studies reveal that these compounds show strong affinity for binding with calf thymus DNA. The thermodynamic parameters of the DNA binding process were obtained from the temperature dependence of the binding constants and the results showed that binding of both compounds to DNA is an enthalpically driven process that is in agreement with proposed DNA intercalation capability of these compounds.

Analysis of gene expression profile microarray data in complex regional pain syndrome.

PubMed

Tan, Wulin; Song, Yiyan; Mo, Chengqiang; Jiang, Shuangjian; Wang, Zhongxing

2017-09-01

The aim of the present study was to predict key genes and proteins associated with complex regional pain syndrome (CRPS) using bioinformatics analysis. The gene expression profiling microarray data, GSE47603, which included peripheral blood samples from 4 patients with CRPS and 5 healthy controls, was obtained from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) in CRPS patients compared with healthy controls were identified using the GEO2R online tool. Functional enrichment analysis was then performed using The Database for Annotation Visualization and Integrated Discovery online tool. Protein‑protein interaction (PPI) network analysis was subsequently performed using Search Tool for the Retrieval of Interaction Genes database and analyzed with Cytoscape software. A total of 257 DEGs were identified, including 243 upregulated genes and 14 downregulated ones. Genes in the human leukocyte antigen (HLA) family were most significantly differentially expressed. Enrichment analysis demonstrated that signaling pathways, including immune response, cell motion, adhesion and angiogenesis were associated with CRPS. PPI network analysis revealed that key genes, including early region 1A binding protein p300 (EP300), CREB‑binding protein (CREBBP), signal transducer and activator of transcription (STAT)3, STAT5A and integrin α M were associated with CRPS. The results suggest that the immune response may therefore serve an important role in CRPS development. In addition, genes in the HLA family, such as HLA‑DQB1 and HLA‑DRB1, may present potential biomarkers for the diagnosis of CRPS. Furthermore, EP300, its paralog CREBBP, and the STAT family genes, STAT3 and STAT5 may be important in the development of CRPS.

IbeA and OmpA of Escherichia coli K1 Exploit Rac1 Activation for Invasion of Human Brain Microvascular Endothelial Cells

PubMed Central

Maruvada, Ravi

2012-01-01

Meningitis-causing Escherichia coli K1 internalization of the blood-brain barrier is required for penetration into the brain, but the host-microbial interactions involved in E. coli entry of the blood-brain barrier remain incompletely understood. We show here that a meningitis-causing E. coli K1 strain RS218 activates Rac1 (GTP-Rac1) of human brain microvascular endothelial cells (HBMEC) in a time-dependent manner. Both activation and bacterial invasion were significantly inhibited in the presence of a Rac1 inhibitor. We further showed that the guanine nucleotide exchange factor Vav2, not β-Pix, was involved in E. coli K1-mediated Rac1 activation. Since activated STAT3 is known to bind GTP-Rac1, the relationship between STAT3 and Rac1 was examined in E. coli K1 invasion of HBMEC. Downregulation of STAT3 resulted in significantly decreased E. coli invasion compared to control HBMEC, as well as a corresponding decrease in GTP-Rac1, suggesting that Rac1 activation in response to E. coli is under the control of STAT3. More importantly, two E. coli determinants contributing to HBMEC invasion, IbeA and OmpA, were shown to affect both Rac1 activation and their association with STAT3. These findings demonstrate for the first time that specific E. coli determinants regulate a novel mechanism of STAT3 cross talk with Rac1 in E. coli K1 invasion of HBMEC. PMID:22451524

IbeA and OmpA of Escherichia coli K1 exploit Rac1 activation for invasion of human brain microvascular endothelial cells.

PubMed

Maruvada, Ravi; Kim, Kwang Sik

2012-06-01

Meningitis-causing Escherichia coli K1 internalization of the blood-brain barrier is required for penetration into the brain, but the host-microbial interactions involved in E. coli entry of the blood-brain barrier remain incompletely understood. We show here that a meningitis-causing E. coli K1 strain RS218 activates Rac1 (GTP-Rac1) of human brain microvascular endothelial cells (HBMEC) in a time-dependent manner. Both activation and bacterial invasion were significantly inhibited in the presence of a Rac1 inhibitor. We further showed that the guanine nucleotide exchange factor Vav2, not β-Pix, was involved in E. coli K1-mediated Rac1 activation. Since activated STAT3 is known to bind GTP-Rac1, the relationship between STAT3 and Rac1 was examined in E. coli K1 invasion of HBMEC. Downregulation of STAT3 resulted in significantly decreased E. coli invasion compared to control HBMEC, as well as a corresponding decrease in GTP-Rac1, suggesting that Rac1 activation in response to E. coli is under the control of STAT3. More importantly, two E. coli determinants contributing to HBMEC invasion, IbeA and OmpA, were shown to affect both Rac1 activation and their association with STAT3. These findings demonstrate for the first time that specific E. coli determinants regulate a novel mechanism of STAT3 cross talk with Rac1 in E. coli K1 invasion of HBMEC.

Transcriptional regulation of the novel monoamine oxidase renalase: Crucial roles of transcription factors Sp1, STAT3, and ZBP89.

PubMed

Sonawane, Parshuram J; Gupta, Vinayak; Sasi, Binu K; Kalyani, Ananthamohan; Natarajan, Bhargavi; Khan, Abrar A; Sahu, Bhavani S; Mahapatra, Nitish R

2014-11-11

Renalase, a novel monoamine oxidase, is emerging as an important regulator of cardiovascular, metabolic, and renal diseases. However, the mechanism of transcriptional regulation of this enzyme remains largely unknown. We undertook a systematic analysis of the renalase gene to identify regulatory promoter elements and transcription factors. Computational analysis coupled with transfection of human renalase promoter/luciferase reporter plasmids (5'-promoter-deletion constructs) into various cell types (HEK-293, IMR32, and HepG2) identified two crucial promoter domains at base pairs -485 to -399 and -252 to -150. Electrophoretic mobility shift assays using renalase promoter oligonucleotides with and without potential binding sites for transcription factors Sp1, STAT3, and ZBP89 displayed formation of specific complexes with HEK-293 nuclear proteins. Consistently, overexpression of Sp1, STAT3, and ZBP89 augmented renalase promoter activity; additionally, siRNA-mediated downregulation of Sp1, STAT3, and ZBP89 reduced the level of endogenous renalase transcription as well as the transfected renalase promoter activity. In addition, chromatin immunoprecipitation assays showed in vivo interactions of these transcription factors with renalase promoter. Interestingly, renalase promoter activity was augmented by nicotine and catecholamines; while Sp1 and STAT3 synergistically activated the nicotine-induced effect, Sp1 appeared to enhance epinephrine-evoked renalase transcription. Moreover, renalase transcript levels in mouse models of human essential hypertension were concomitantly associated with endogenous STAT3 and ZBP89 levels, suggesting crucial roles for these transcription factors in regulating renalase gene expression in cardiovascular pathological conditions.

Targeting the PD-L1/DNMT1 axis in acquired resistance to sorafenib in human hepatocellular carcinoma

PubMed Central

Liu, Jianhua; Liu, Yahui; Meng, Lingyu; Liu, Kai; Ji, Bai

2017-01-01

Molecule-targeted therapy, such as sorafenib, is one of the effectively therapeutic options for advanced hepatocellular carcinoma (HCC). However, acquired resistance to sorafenib has been found in some HCC patients, resulting in poor prognosis. It is reported that PD-L1 and DNA methyltransferases (DNMTs) contribute to drug resistance. In this study, by inducing sorafenib-resistant HCC cell lines, we investigated their molecular and functional characteristics. Our data indicated that highly upregulated DNMT1 was positively correlated with PD-L1 overexpression in sorafenib-resistant HCC cells. We demonstrate that PD-L1 regulate DNMT1 through STAT3 signaling pathway. Knockdown of PD-L1 induced DNMT1-dependent DNA hypomethylation and restored the expression of methylation-silenced CDH1. Moreover, inactivation of NFκB blocked PD-L1/STAT3/DNMT1 pathway in sorafenib-resistant HCC cells. Functionally, genetic or pharmacological disruption of PD-L1 or/and DNMT1 sensitize HCC resistance to sorafenib. Importantly, dual inactivation of PD-L1 and DNMT1 by their inhibitor synergistically disrupts the colony formation of sorafenib-resistant HCC cells. These results demonstrate that targeting NFκB/PDL1/STAT3/DNMT1 axis is a new therapeutic strategy for preventing or overcoming the acquired resistance to sorafenib in HCC patients. PMID:28627705

Hemi-methylated DNA opens a closed conformation of UHRF1 to facilitate its histone recognition

NASA Astrophysics Data System (ADS)

Fang, Jian; Cheng, Jingdong; Wang, Jiaolong; Zhang, Qiao; Liu, Mengjie; Gong, Rui; Wang, Ping; Zhang, Xiaodan; Feng, Yangyang; Lan, Wenxian; Gong, Zhou; Tang, Chun; Wong, Jiemin; Yang, Huirong; Cao, Chunyang; Xu, Yanhui

2016-04-01

UHRF1 is an important epigenetic regulator for maintenance DNA methylation. UHRF1 recognizes hemi-methylated DNA (hm-DNA) and trimethylation of histone H3K9 (H3K9me3), but the regulatory mechanism remains unknown. Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. Hm-DNA impairs the intramolecular interactions and promotes H3K9me3 recognition by TTD-PHD. The Spacer also facilitates UHRF1-DNMT1 interaction and enhances hm-DNA-binding affinity of the SRA. When TTD-PHD binds to H3K9me3, SRA-Spacer may exist in a dynamic equilibrium: either recognizes hm-DNA or recruits DNMT1 to chromatin. Our study reveals the mechanism for regulation of H3K9me3 and hm-DNA recognition by URHF1.

APOBEC3G Interacts with ssDNA by Two Modes: AFM Studies

PubMed Central

Shlyakhtenko, Luda S.; Dutta, Samrat; Banga, Jaspreet; Li, Ming; Harris, Reuben S.; Lyubchenko, Yuri L.

2015-01-01

APOBEC3G (A3G) protein has antiviral activity against HIV and other pathogenic retroviruses. A3G has two domains: a catalytic C-terminal domain (CTD) that deaminates cytidine, and a N-terminal domain (NTD) that binds to ssDNA. Although abundant information exists about the biological activities of A3G protein, the interplay between sequence specific deaminase activity and A3G binding to ssDNA remains controversial. We used the topographic imaging and force spectroscopy modalities of Atomic Force Spectroscopy (AFM) to characterize the interaction of A3G protein with deaminase specific and nonspecific ssDNA substrates. AFM imaging demonstrated that A3G has elevated affinity for deaminase specific ssDNA than for nonspecific ssDNA. AFM force spectroscopy revealed two distinct binding modes by which A3G interacts with ssDNA. One mode requires sequence specificity, as demonstrated by stronger and more stable complexes with deaminase specific ssDNA than with nonspecific ssDNA. Overall these observations enforce prior studies suggesting that both domains of A3G contribute to the sequence specific binding of ssDNA. PMID:26503602

The PPARdelta agonist GW501516 suppresses interleukin-6-mediated hepatocyte acute phase reaction via STAT3 inhibition.

PubMed

Kino, T; Rice, K C; Chrousos, G P

2007-05-01

Interleukin-6 and downstream liver effectors acute phase reactants are implicated in the systemic inflammatory reaction. Peroxisome proliferator-activated receptor delta (PPARdelta), which binds to and is activated by a variety of fatty acids, was recently shown to have anti-inflammatory actions. We examined the ability of the synthetic PPARdelta agonist GW501516 to suppress interleukin-6-induced expression of acute phase proteins in human hepatoma HepG2 cells and rat primary hepatocytes. Results GW501516 dose-dependently suppressed interleukin-6-induced mRNA expression of the acute phase protein alpha1-antichymotrypsin in HepG2 cells. The compound also suppressed interleukin-6-induced mRNA expression of alpha2-acid glycoprotein, beta-fibrinogen and alpha2-macroglobulin in and the secretion of C-reactive protein by rat primary hepatocytes. Depletion of the PPARdelta receptor, but not of PPARalpha or gamma, attenuated the suppressive effect of GW501516 on interleukin-6-induced alpha1-antichymotrypsin mRNA expression, indicating that PPARdelta specifically mediated this effect. Since interleukin-6 stimulates the transcriptional activity of the alpha1-antichymotrypsin promoter by activating the signal transducer and activator of transcription (STAT) 3, we examined functional interaction of this transcription factor and PPARdelta on this promoter. Overexpression of PPARdelta enhanced the suppressive effect of GW501516 on STAT3-activated transcriptional activity of the alpha1-antichymotrypsin promoter, while GW501516 suppressed interleukin-6-induced binding of this transcription factor to this promoter. These findings indicate that agonist-activated PPARdelta interferes with interleukin-6-induced acute phase reaction in the liver by inhibiting the transcriptional activity of STAT3. PPARdelta agonists might be useful for the suppression of systemic inflammatory reactions in which IL-6 plays a central role.

Interleukin-23 promotes the migration and invasion of gastric cancer cells by inducing epithelial-to-mesenchymal transition via the STAT3 pathway.

PubMed

Xu, Xianhui; Yang, Changdong; Chen, Jun; Liu, Junyan; Li, Ping'ang; Shi, Yan; Yu, Peiwu

2018-05-05

Chronic inflammation is associated with all stages of cancer development. Moreover, a proinflammatory microenvironment resulted from chronic inflammation is considered to be an essential component of cancer. Interleukin-23 (IL-23) is a general proinflammatory factor; and is involved in tumor-associated inflammation in gastric cancer (GC). However, the direct effect of IL-23 on GC cells has been rarely reported. The aim of the study was to clarify the direct role of IL-23 in regulating GC progression, and to identify the underlying mechanism. In this study, Positive expression of IL-23R was observed in GC tissues and cell lines by using immunohistochemistry or immunofluorescence. In western blots, the expression of IL-23R was higher in GC tissues compared with adjacent normal tissues. Furthermore, IL-23R positive GC tissues were closely related with larger tumor size and worse T stage and clinical stage. By performing in vitro experiments, we found that IL-23 binding to its receptor promoted the migration and invasion of BGC-823 cells in vitro. Moreover, IL-23 induced the activation of STAT3 and epithelial-to-mesenchymal transition (EMT) in BGC-823 cells. Knocking down STAT3 in BGC-823 cells attenuated the effect of IL-23 on EMT and cell migration and invasion. Taken together, our study has firstly demonstrated the positive expression of IL-23R in human GC tissues and cell lines. IL-23 binding to its receptor promotes the migration and invasion of GC cells by inducing EMT through the STAT3 signaling pathway. This work provides a new mechanism for the oncogenic role of IL-23 on GC progression. Copyright © 2018. Published by Elsevier Inc.

RPA binds histone H3-H4 and functions in DNA replication-coupled nucleosome assembly.

PubMed

Liu, Shaofeng; Xu, Zhiyun; Leng, He; Zheng, Pu; Yang, Jiayi; Chen, Kaifu; Feng, Jianxun; Li, Qing

2017-01-27

DNA replication-coupled nucleosome assembly is essential to maintain genome integrity and retain epigenetic information. Multiple involved histone chaperones have been identified, but how nucleosome assembly is coupled to DNA replication remains elusive. Here we show that replication protein A (RPA), an essential replisome component that binds single-stranded DNA, has a role in replication-coupled nucleosome assembly. RPA directly binds free H3-H4. Assays using a synthetic sequence that mimics freshly unwound single-stranded DNA at replication fork showed that RPA promotes DNA-(H3-H4) complex formation immediately adjacent to double-stranded DNA. Further, an RPA mutant defective in H3-H4 binding exhibited attenuated nucleosome assembly on nascent chromatin. Thus, we propose that RPA functions as a platform for targeting histone deposition to replication fork, through which RPA couples nucleosome assembly with ongoing DNA replication. Copyright © 2017, American Association for the Advancement of Science.

Bortezomib inhibits STAT5-dependent degradation of LEF-1, inducing granulocytic differentiation in congenital neutropenia CD34+ cells

PubMed Central

Gupta, Kshama; Kuznetsova, Inna; Klimenkova, Olga; Klimiankou, Maksim; Meyer, Johann; Moore, Malcolm A. S.; Zeidler, Cornelia; Welte, Karl

2014-01-01

The transcription factor lymphoid enhancer–binding factor 1 (LEF-1), which plays a definitive role in granulocyte colony-stimulating factor (G-CSF) receptor-triggered granulopoiesis, is downregulated in granulocytic progenitors of severe congenital neutropenia (CN) patients. However, the exact mechanism of LEF-1 downregulation is unclear. CN patients are responsive to therapeutically high doses of G-CSF and are at increased risk of developing acute myeloid leukemia. The normal expression of LEF-1 in monocytes and lymphocytes, whose differentiation is unaffected in CN, suggests the presence of a granulopoiesis-specific mechanism downstream of G-CSF receptor signaling that leads to LEF-1 downregulation. Signal transducer and activator of transcription 5 (STAT5) is activated by G-CSF and is hyperactivated in acute myeloid leukemia. Here, we investigated the effects of activated STAT5 on LEF-1 expression and functions in hematopoietic progenitor cells. We demonstrated that constitutively active STAT5a (caSTAT5a) inhibited LEF-1–dependent autoregulation of the LEF-1 gene promoter by binding to the LEF-1 protein, recruiting Nemo-like kinase and the E3 ubiquitin-ligase NARF to LEF-1, leading to LEF-1 ubiquitination and a reduction in LEF-1 protein levels. The proteasome inhibitor bortezomib reversed the defective G-CSF–triggered granulocytic differentiation of CD34+ cells from CN patients in vitro, an effect that was accompanied by restoration of LEF-1 protein levels and LEF-1 messenger RNA autoregulation. Taken together, our data define a novel mechanism of LEF-1 downregulation in CN patients via enhanced ubiquitination and degradation of LEF-1 protein by hyperactivated STAT5. PMID:24394665

Analytical evaluation of the automated galectin-3 assay on the Abbott ARCHITECT immunoassay instruments.

PubMed

Gaze, David C; Prante, Christian; Dreier, Jens; Knabbe, Cornelius; Collet, Corinne; Launay, Jean-Marie; Franekova, Janka; Jabor, Antonin; Lennartz, Lieselotte; Shih, Jessie; del Rey, Jose Manuel; Zaninotto, Martina; Plebani, Mario; Collinson, Paul O

2014-06-01

Galectin-3 is secreted from macrophages and binds and activates fibroblasts forming collagen. Tissue fibrosis is central to the progression of chronic heart failure (CHF). We performed a European multicentered evaluation of the analytical performance of the two-step routine and Short Turn-Around-Time (STAT) galectin-3 immunoassay on the ARCHITECT i1000SR, i2000SR, and i4000SR (Abbott Laboratories). We evaluated the assay precision and dilution linearity for both routine and STAT assays and compared serum and plasma, and fresh vs. frozen samples. The reference interval and biological variability were also assessed. Measurable samples were compared between ARCHITECT instruments and between the routine and STAT assays and also to a galectin-3 ELISA (BG Medicine). The total assay coefficient of variation (CV%) was 2.3%-6.2% and 1.7%-7.4% for the routine and STAT assays, respectively. Both assays demonstrated linearity up to 120 ng/mL. Galectin-3 concentrations were higher in plasma samples than in serum samples and correlated well between fresh and frozen samples (R=0.997), between the routine and STAT assays, between the ARCHITECT i1000 and i2000 instruments and with the galectin-3 ELISA. The reference interval on 627 apparently healthy individuals (53% male) yielded upper 95th and 97.5th percentiles of 25.2 and 28.4 ng/mL, respectively. Values were significantly lower in subjects younger than 50 years. The galectin-3 routine and STAT assays on the Abbott ARCHITECT instruments demonstrated good analytical performance. Further clinical studies are required to demonstrate the diagnostic and prognostic potential of this novel marker in patients with CHF.

Quaking Is a Key Regulator of Endothelial Cell Differentiation, Neovascularization, and Angiogenesis

PubMed Central

Cochrane, Amy; Kelaini, Sophia; Tsifaki, Marianna; Bojdo, James; Vilà‐González, Marta; Drehmer, Daiana; Caines, Rachel; Magee, Corey; Eleftheriadou, Magdalini; Hu, Yanhua; Grieve, David; Stitt, Alan W.; Zeng, Lingfang; Xu, Qingbo

2017-01-01

Abstract The capability to derive endothelial cell (ECs) from induced pluripotent stem cells (iPSCs) holds huge therapeutic potential for cardiovascular disease. This study elucidates the precise role of the RNA‐binding protein Quaking isoform 5 (QKI‐5) during EC differentiation from both mouse and human iPSCs (hiPSCs) and dissects how RNA‐binding proteins can improve differentiation efficiency toward cell therapy for important vascular diseases. iPSCs represent an attractive cellular approach for regenerative medicine today as they can be used to generate patient‐specific therapeutic cells toward autologous cell therapy. In this study, using the model of iPSCs differentiation toward ECs, the QKI‐5 was found to be an important regulator of STAT3 stabilization and vascular endothelial growth factor receptor 2 (VEGFR2) activation during the EC differentiation process. QKI‐5 was induced during EC differentiation, resulting in stabilization of STAT3 expression and modulation of VEGFR2 transcriptional activation as well as VEGF secretion through direct binding to the 3′ UTR of STAT3. Importantly, mouse iPS‐ECs overexpressing QKI‐5 significantly improved angiogenesis and neovascularization and blood flow recovery in experimental hind limb ischemia. Notably, hiPSCs overexpressing QKI‐5, induced angiogenesis on Matrigel plug assays in vivo only 7 days after subcutaneous injection in SCID mice. These results highlight a clear functional benefit of QKI‐5 in neovascularization, blood flow recovery, and angiogenesis. Thus, they provide support to the growing consensus that elucidation of the molecular mechanisms underlying EC differentiation will ultimately advance stem cell regenerative therapy and eventually make the treatment of cardiovascular disease a reality. The RNA binding protein QKI‐5 is induced during EC differentiation from iPSCs. RNA binding protein QKI‐5 was induced during EC differentiation in parallel with the EC marker CD144. Immunofluorescence staining showing that QKI‐5 is localized in the nucleus and stained in parallel with CD144 in differentiated ECs (scale bar = 50 µm). stem cells 2017 Stem Cells 2017;35:952–966 PMID:28207177

Controlling nuclear JAKs and STATs for specific gene activation by IFN{gamma}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Noon-Song, Ezra N.; Ahmed, Chulbul M.; Dabelic, Rea

2011-07-08

Highlights: {yields} Gamma interferon (IFN{gamma}) and its receptor subunit, IFNGR1, interact with the promoter region of IFN{gamma}-associated genes along with transcription factor STAT1{alpha}. {yields} We show that activated Janus kinases pJAK2 and pJAK1 also associate with IFNGR1 in the nucleus. {yields} The activated Janus kinases are responsible for phosphorylation of tyrosine 41 on histone H3, an important epigenetic event for specific gene activation. -- Abstract: We previously showed that gamma interferon (IFN{gamma}) and its receptor subunit, IFNGR1, interacted with the promoter region of IFN{gamma}-activated genes along with transcription factor STAT1{alpha}. Recent studies have suggested that activated Janus kinases pJAK2 andmore » pJAK1 also played a role in gene activation by phosphorylation of histone H3 on tyrosine 41. This study addresses the question of the role of activated JAKs in specific gene activation by IFN{gamma}. We carried out chromatin immunoprecipitation (ChIP) followed by PCR in IFN{gamma} treated WISH cells and showed association of pJAK1, pJAK2, IFNGR1, and STAT1 on the same DNA sequence of the IRF-1 gene promoter. The {beta}-actin gene, which is not activated by IFN{gamma}, did not show this association. The movement of activated JAK to the nucleus and the IRF-1 promoter was confirmed by the combination of nuclear fractionation, confocal microscopy and DNA precipitation analysis using the biotinylated GAS promoter. Activated JAKs in the nucleus was associated with phosphorylated tyrosine 41 on histone H3 in the region of the GAS promoter. Unphosphorylated JAK2 was found to be constitutively present in the nucleus and was capable of undergoing activation in IFN{gamma} treated cells, most likely via nuclear IFNGR1. Association of pJAK2 and IFNGR1 with histone H3 in IFN{gamma} treated cells was demonstrated by histone H3 immunoprecipitation. Unphosphorylated STAT1 protein was associated with histone H3 of untreated cells. IFN{gamma} treatment resulted in its disassociation and then re-association as pSTAT1. The results suggest a novel role for activated JAKs in epigenetic events for specific gene activation.« less

ROCK2 signaling is required to induce a subset of T follicular helper cells through opposing effects on STATs in autoimmune settings.

PubMed

Weiss, Jonathan M; Chen, Wei; Nyuydzefe, Melanie S; Trzeciak, Alissa; Flynn, Ryan; Tonra, James R; Marusic, Suzana; Blazar, Bruce R; Waksal, Samuel D; Zanin-Zhorov, Alexandra

2016-07-19

Rho-associated kinase 2 (ROCK2) determines the balance between human T helper 17 (TH17) cells and regulatory T (Treg) cells. We investigated its role in the generation of T follicular helper (TFH) cells, which help to generate antibody-producing B cells under normal and autoimmune conditions. Inhibiting ROCK2 in normal human T cells or peripheral blood mononuclear cells from patients with active systemic lupus erythematosus (SLE) decreased the number and function of TFH cells induced by activation ex vivo. Moreover, inhibition of ROCK2 activity decreased the abundance of the transcriptional regulator Bcl6 (B cell lymphoma 6) and increased that of Blimp1 by reducing the binding of signal transducer and activator of transcription 3 (STAT3) and increasing that of STAT5 to the promoters of the genes Bcl6 and PRDM1, respectively. In the MRL/lpr murine model of SLE, oral administration of the selective ROCK2 inhibitor KD025 resulted in a twofold reduction in the numbers of TFH cells and antibody-producing plasma cells in the spleen, as well as a decrease in the size of splenic germinal centers, which are the sites of interaction between TFH cells and B cells. KD025-treated mice showed a substantial improvement in both histological and clinical scores compared to those of untreated mice and had reduced amounts of Bcl6 and phosphorylated STAT3, as well as increased STAT5 phosphorylation. Together, these data suggest that ROCK2 signaling plays a critical role in controlling the development of TFH cells induced by autoimmune conditions through reciprocal regulation of STAT3 and STAT5 activation. Copyright © 2016, American Association for the Advancement of Science.

5-Hydroxymethylcytosine in E-box motifs ACAT|GTG and ACAC|GTG increases DNA-binding of the B-HLH transcription factor TCF4.

PubMed

Khund-Sayeed, Syed; He, Ximiao; Holzberg, Timothy; Wang, Jun; Rajagopal, Divya; Upadhyay, Shriyash; Durell, Stewart R; Mukherjee, Sanjit; Weirauch, Matthew T; Rose, Robert; Vinson, Charles

2016-09-12

We evaluated DNA binding of the B-HLH family members TCF4 and USF1 using protein binding microarrays (PBMs) containing double-stranded DNA probes with cytosine on both strands or 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) on one DNA strand and cytosine on the second strand. TCF4 preferentially bound the E-box motif (CAN|NTG) with strongest binding to the 8-mer CAG|GTGGT. 5mC uniformly decreases DNA binding of both TCF4 and USF1. The bulkier 5hmC also inhibited USF1 binding to DNA. In contrast, 5hmC dramatically enhanced TCF4 binding to E-box motifs ACAT|GTG and ACAC|GTG, being better bound than any 8-mer containing cytosine. Examination of X-ray structures of the closely related TCF3 and USF1 bound to DNA suggests TCF3 can undergo a conformational shift to preferentially bind to 5hmC while the USF1 basic region is bulkier and rigid precluding a conformation shift to bind 5hmC. These results greatly expand the regulatory DNA sequence landscape bound by TCF4.

Evasion of interferon responses by Ebola and Marburg viruses.

PubMed

Basler, Christopher F; Amarasinghe, Gaya K

2009-09-01

The filoviruses, Ebola virus (EBOV) and Marburg virus (MARV), cause frequently lethal viral hemorrhagic fever. These infections induce potent cytokine production, yet these host responses fail to prevent systemic virus replication. Consistent with this, filoviruses have been found to encode proteins VP35 and VP24 that block host interferon (IFN)-alpha/beta production and inhibit signaling downstream of the IFN-alpha/beta and the IFN-gamma receptors, respectively. VP35, which is a component of the viral nucleocapsid complex and plays an essential role in viral RNA synthesis, acts as a pseudosubstrate for the cellular kinases IKK-epsilon and TBK-1, which phosphorylate and activate interferon regulatory factor 3 (IRF-3) and interferon regulatory factor 7 (IRF-7). VP35 also promotes SUMOylation of IRF-7, repressing IFN gene transcription. In addition, VP35 is a dsRNA-binding protein, and mutations that disrupt dsRNA binding impair VP35 IFN-antagonist activity while leaving its RNA replication functions intact. The phenotypes of recombinant EBOV bearing mutant VP35s unable to inhibit IFN-alpha/beta demonstrate that VP35 IFN-antagonist activity is critical for full virulence of these lethal pathogens. The structure of the VP35 dsRNA-binding domain, which has recently become available, is expected to provide insight into how VP35 IFN-antagonist and dsRNA-binding functions are related. The EBOV VP24 protein inhibits IFN signaling through an interaction with select host cell karyopherin-alpha proteins, preventing the nuclear import of otherwise activated STAT1. It remains to be determined to what extent VP24 may also modulate the nuclear import of other host cell factors and to what extent this may influence the outcome of infection. Notably, the Marburg virus VP24 protein does not detectably block STAT1 nuclear import, and, unlike EBOV, MARV infection inhibits STAT1 and STAT2 phosphorylation. Thus, despite their similarities, there are fundamental differences by which these deadly viruses counteract the IFN system. It will be of interest to determine how these differences influence pathogenesis.

The RhoU/Wrch1 Rho GTPase gene is a common transcriptional target of both the gp130/STAT3 and Wnt-1 pathways

PubMed Central

SCHIAVONE, Davide; DEWILDE, Sarah; VALLANIA, Francesco; TURKSON, James; CUNTO, Ferdinando DI; POLI, Valeria

2010-01-01

STAT3 (signal transducer and activator of transcription 3) is a transcription factor activated by cytokines, growth factors and oncogenes, whose activity is required for cell survival/proliferation of a wide variety of primary tumours and tumour cell lines. Prominent among its multiple effects on tumour cells is the stimulation of cell migration and metastasis, whose functional mechanisms are however not completely characterized. RhoU/Wrch1 (Wnt-responsive Cdc42 homologue) is an atypical Rho GTPase thought to be constitutively bound to GTP. RhoU was first identified as a Wnt-1-inducible mRNA and subsequently shown to act on the actin cytoskeleton by stimulating filopodia formation and stress fibre dissolution. It was in addition recently shown to localize to focal adhesions and to Src-induced podosomes and enhance cell migration. RhoU overexpression in mammary epithelial cells stimulates quiescent cells to re-enter the cell cycle and morphologically phenocopies Wnt-1-dependent transformation. In the present study we show that Wnt-1-mediated RhoU induction occurs at the transcriptional level. Moreover, we demonstrate that RhoU can also be induced by gp130 cytokines via STAT3, and we identify two functional STAT3-binding sites on the mouse RhoU promoter. RhoU induction by Wnt-1 is independent of β-catenin, but does not involve STAT3. Rather, it is mediated by the Wnt/planar cell polarity pathway through the activation of JNK (c-Jun N-terminal kinase). Both the so-called non-canonical Wnt pathway and STAT3 are therefore able to induce RhoU, which in turn may be involved in mediating their effects on cell migration. PMID:19397496

MPLW515L Is a Novel Somatic Activating Mutation in Myelofibrosis with Myeloid Metaplasia

PubMed Central

Pikman, Yana; Lee, Benjamin H; Mercher, Thomas; McDowell, Elizabeth; Ebert, Benjamin L; Gozo, Maricel; Cuker, Adam; Wernig, Gerlinde; Moore, Sandra; Galinsky, Ilene; DeAngelo, Daniel J; Clark, Jennifer J; Lee, Stephanie J; Golub, Todd R; Wadleigh, Martha; Gilliland, D. Gary; Levine, Ross L

2006-01-01

Background The JAK2V617F allele has recently been identified in patients with polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis with myeloid metaplasia (MF). Subsequent analysis has shown that constitutive activation of the JAK-STAT signal transduction pathway is an important pathogenetic event in these patients, and that enzymatic inhibition of JAK2V617F may be of therapeutic benefit in this context. However, a significant proportion of patients with ET or MF are JAK2V617F-negative. We hypothesized that activation of the JAK-STAT pathway might also occur as a consequence of activating mutations in certain hematopoietic-specific cytokine receptors, including the erythropoietin receptor (EPOR), the thrombopoietin receptor (MPL), or the granulocyte-colony stimulating factor receptor (GCSFR). Methods and Findings DNA sequence analysis of the exons encoding the transmembrane and juxtamembrane domains of EPOR, MPL, and GCSFR, and comparison with germline DNA derived from buccal swabs, identified a somatic activating mutation in the transmembrane domain of MPL (W515L) in 9% (4/45) of JAKV617F-negative MF. Expression of MPLW515L in 32D, UT7, or Ba/F3 cells conferred cytokine-independent growth and thrombopoietin hypersensitivity, and resulted in constitutive phosphorylation of JAK2, STAT3, STAT5, AKT, and ERK. Furthermore, a small molecule JAK kinase inhibitor inhibited MPLW515L-mediated proliferation and JAK-STAT signaling in vitro. In a murine bone marrow transplant assay, expression of MPLW515L, but not wild-type MPL, resulted in a fully penetrant myeloproliferative disorder characterized by marked thrombocytosis (Plt count 1.9–4.0 × 10 12/L), marked splenomegaly due to extramedullary hematopoiesis, and increased reticulin fibrosis. Conclusions Activation of JAK-STAT signaling via MPLW515L is an important pathogenetic event in patients with JAK2V617F-negative MF. The bone marrow transplant model of MPLW515L-mediated myeloproliferative disorders (MPD) exhibits certain features of human MF, including extramedullary hematopoiesis, splenomegaly, and megakaryocytic proliferation. Further analysis of positive and negative regulators of the JAK-STAT pathway is warranted in JAK2V617F-negative MPD. PMID:16834459

MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia.

PubMed

Pikman, Yana; Lee, Benjamin H; Mercher, Thomas; McDowell, Elizabeth; Ebert, Benjamin L; Gozo, Maricel; Cuker, Adam; Wernig, Gerlinde; Moore, Sandra; Galinsky, Ilene; DeAngelo, Daniel J; Clark, Jennifer J; Lee, Stephanie J; Golub, Todd R; Wadleigh, Martha; Gilliland, D Gary; Levine, Ross L

2006-07-01

The JAK2V617F allele has recently been identified in patients with polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis with myeloid metaplasia (MF). Subsequent analysis has shown that constitutive activation of the JAK-STAT signal transduction pathway is an important pathogenetic event in these patients, and that enzymatic inhibition of JAK2V617F may be of therapeutic benefit in this context. However, a significant proportion of patients with ET or MF are JAK2V617F-negative. We hypothesized that activation of the JAK-STAT pathway might also occur as a consequence of activating mutations in certain hematopoietic-specific cytokine receptors, including the erythropoietin receptor (EPOR), the thrombopoietin receptor (MPL), or the granulocyte-colony stimulating factor receptor (GCSFR). DNA sequence analysis of the exons encoding the transmembrane and juxtamembrane domains of EPOR, MPL, and GCSFR, and comparison with germline DNA derived from buccal swabs, identified a somatic activating mutation in the transmembrane domain of MPL (W515L) in 9% (4/45) of JAKV617F-negative MF. Expression of MPLW515L in 32D, UT7, or Ba/F3 cells conferred cytokine-independent growth and thrombopoietin hypersensitivity, and resulted in constitutive phosphorylation of JAK2, STAT3, STAT5, AKT, and ERK. Furthermore, a small molecule JAK kinase inhibitor inhibited MPLW515L-mediated proliferation and JAK-STAT signaling in vitro. In a murine bone marrow transplant assay, expression of MPLW515L, but not wild-type MPL, resulted in a fully penetrant myeloproliferative disorder characterized by marked thrombocytosis (Plt count 1.9-4.0 x 10(12)/L), marked splenomegaly due to extramedullary hematopoiesis, and increased reticulin fibrosis. Activation of JAK-STAT signaling via MPLW515L is an important pathogenetic event in patients with JAK2V617F-negative MF. The bone marrow transplant model of MPLW515L-mediated myeloproliferative disorders (MPD) exhibits certain features of human MF, including extramedullary hematopoiesis, splenomegaly, and megakaryocytic proliferation. Further analysis of positive and negative regulators of the JAK-STAT pathway is warranted in JAK2V617F-negative MPD.

DNA as a Target for Anticancer Phen-Imidazole Pd(II) Complexes.

PubMed

Heydari, Maryam; Moghadam, Mahboube Eslami; Tarlani, AliAkbar; Farhangian, Hossein

2017-05-01

Imidazole ring is a known structure in many natural or synthetic drug molecules and its metal complexes can interact with DNA and do the cleavage. Hence, to study the influence of the structure and size of the ligand on biological behavior of metal complexes, two water-soluble Pd(II) complexes of phen and FIP ligands (where phen is 1,10-phenanthroline and FIP is 2-(Furan-2-yl)-1H-Imidazo[4,5-f][1, 10]phenanthroline) with the formula of [Pd(phen)(FIP)](NO 3 ) 2 and [Pd(FIP) 2 ]Cl 2 , that were activated against chronic myelogenous leukemia cell line, K562, were selected. Also, the interaction of these anticancer Pd(II) complexes with highly polymerized calf thymus DNA was extensively studied by means of electronic absorption, fluorescence, and circular dichroism in Tris-buffer. The results showed that the binding was positive cooperation and [Pd(phen)(FIP)](NO 3 ) 2 (K f  = 127 M -1 G = 1.2) exhibited higher binding constant and number of binding sites than [Pd(FIP) 2 ]Cl 2 (K f  = 13 M -1 G = 1.03) upon binding to DNA. The fluorescence data indicates that quenching effect for [Pd(phen)(FIP)](NO 3 ) 2 (K SV  = 58 mM -1 ) was higher than [Pd(FIP) 2 ]Cl 2 (K SV  = 12 mM -1 ). Also, [Pd(FIP) 2 ]Cl 2 interacts with ethidium bromide-DNA, as non-competitive inhibition, and can bind to DNA via groove binding and [Pd(phen)(FIP)](NO 3 ) 2 can intercalate in DNA. These results were confirmed by circular dichroism spectra. Docking data revealed that longer complexes have higher interaction energy and bind to DNA via groove binding. Graphical Abstract Two anticancer Pd(II) complexes of imidazole derivative have been synthesized and interacted with calf thymus DNA. Modes of binding have been studied by electronic absorption, fluorescence, and CD measurements. [Pd(FIP) 2 ]Cl 2 can bind to DNA via groove binding while intercalation mode of binding is observed for [Pd(phen)(FIP)](NO 3 ) 2 .

Effects of nucleoside analog incorporation on DNA binding to the DNA binding domain of the GATA-1 erythroid transcription factor.

PubMed

Foti, M; Omichinski, J G; Stahl, S; Maloney, D; West, J; Schweitzer, B I

1999-02-05

We investigate here the effects of the incorporation of the nucleoside analogs araC (1-beta-D-arabinofuranosylcytosine) and ganciclovir (9-[(1,3-dihydroxy-2-propoxy)methyl] guanine) into the DNA binding recognition sequence for the GATA-1 erythroid transcription factor. A 10-fold decrease in binding affinity was observed for the ganciclovir-substituted DNA complex in comparison to an unmodified DNA of the same sequence composition. AraC substitution did not result in any changes in binding affinity. 1H-15N HSQC and NOESY NMR experiments revealed a number of chemical shift changes in both DNA and protein in the ganciclovir-modified DNA-protein complex when compared to the unmodified DNA-protein complex. These changes in chemical shift and binding affinity suggest a change in the binding mode of the complex when ganciclovir is incorporated into the GATA DNA binding site.

STAT3 precedes HIF1α transcriptional responses to oxygen and oxygen and glucose deprivation in human brain pericytes.

PubMed

Carlsson, Robert; Özen, Ilknur; Barbariga, Marco; Gaceb, Abderahim; Roth, Michaela; Paul, Gesine

2018-01-01

Brain pericytes are important to maintain vascular integrity of the neurovascular unit under both physiological and ischemic conditions. Ischemic stroke is known to induce an inflammatory and hypoxic response due to the lack of oxygen and glucose in the brain tissue. How this early response to ischemia is molecularly regulated in pericytes is largely unknown and may be of importance for future therapeutic targets. Here we evaluate the transcriptional responses in in vitro cultured human brain pericytes after oxygen and/or glucose deprivation. Hypoxia has been widely known to stabilise the transcription factor hypoxia inducible factor 1-alpha (HIF1α) and mediate the induction of hypoxic transcriptional programs after ischemia. However, we find that the transcription factors Jun Proto-Oncogene (c-JUN), Nuclear Factor Of Kappa Light Polypeptide Gene Enhancer In B-Cells (NFκB) and signal transducer and activator of transcription 3 (STAT3) bind genes regulated after 2hours (hs) of omitted glucose and oxygen before HIF1α. Potent HIF1α responses require 6hs of hypoxia to substantiate transcriptional regulation comparable to either c-JUN or STAT3. Phosphorylated STAT3 protein is at its highest after 5 min of oxygen and glucose (OGD) deprivation, whereas maximum HIF1α stabilisation requires 120 min. We show that STAT3 regulates angiogenic and metabolic pathways before HIF1α, suggesting that HIF1α is not the initiating trans-acting factor in the response of pericytes to ischemia.

Integrated genomic sequencing reveals mutational landscape of T-cell prolymphocytic leukemia

PubMed Central

Kiel, Mark J.; Velusamy, Thirunavukkarasu; Rolland, Delphine; Sahasrabuddhe, Anagh A.; Chung, Fuzon; Bailey, Nathanael G.; Schrader, Alexandra; Li, Bo; Li, Jun Z.; Ozel, Ayse B.; Betz, Bryan L.; Miranda, Roberto N.; Medeiros, L. Jeffrey; Zhao, Lili; Herling, Marco

2014-01-01

The comprehensive genetic alterations underlying the pathogenesis of T-cell prolymphocytic leukemia (T-PLL) are unknown. To address this, we performed whole-genome sequencing (WGS), whole-exome sequencing (WES), high-resolution copy-number analysis, and Sanger resequencing of a large cohort of T-PLL. WGS and WES identified novel mutations in recurrently altered genes not previously implicated in T-PLL including EZH2, FBXW10, and CHEK2. Strikingly, WGS and/or WES showed largely mutually exclusive mutations affecting IL2RG, JAK1, JAK3, or STAT5B in 38 of 50 T-PLL genomes (76.0%). Notably, gain-of-function IL2RG mutations are novel and have not been reported in any form of cancer. Further, high-frequency mutations in STAT5B have not been previously reported in T-PLL. Functionally, IL2RG-JAK1-JAK3-STAT5B mutations led to signal transducer and activator of transcription 5 (STAT5) hyperactivation, transformed Ba/F3 cells resulting in cytokine-independent growth, and/or enhanced colony formation in Jurkat T cells. Importantly, primary T-PLL cells exhibited constitutive activation of STAT5, and targeted pharmacologic inhibition of STAT5 with pimozide induced apoptosis in primary T-PLL cells. These results for the first time provide a portrait of the mutational landscape of T-PLL and implicate deregulation of DNA repair and epigenetic modulators as well as high-frequency mutational activation of the IL2RG-JAK1-JAK3-STAT5B axis in the pathogenesis of T-PLL. These findings offer opportunities for novel targeted therapies in this aggressive leukemia. PMID:24825865

Integrated genomic sequencing reveals mutational landscape of T-cell prolymphocytic leukemia.

PubMed

Kiel, Mark J; Velusamy, Thirunavukkarasu; Rolland, Delphine; Sahasrabuddhe, Anagh A; Chung, Fuzon; Bailey, Nathanael G; Schrader, Alexandra; Li, Bo; Li, Jun Z; Ozel, Ayse B; Betz, Bryan L; Miranda, Roberto N; Medeiros, L Jeffrey; Zhao, Lili; Herling, Marco; Lim, Megan S; Elenitoba-Johnson, Kojo S J

2014-08-28

The comprehensive genetic alterations underlying the pathogenesis of T-cell prolymphocytic leukemia (T-PLL) are unknown. To address this, we performed whole-genome sequencing (WGS), whole-exome sequencing (WES), high-resolution copy-number analysis, and Sanger resequencing of a large cohort of T-PLL. WGS and WES identified novel mutations in recurrently altered genes not previously implicated in T-PLL including EZH2, FBXW10, and CHEK2. Strikingly, WGS and/or WES showed largely mutually exclusive mutations affecting IL2RG, JAK1, JAK3, or STAT5B in 38 of 50 T-PLL genomes (76.0%). Notably, gain-of-function IL2RG mutations are novel and have not been reported in any form of cancer. Further, high-frequency mutations in STAT5B have not been previously reported in T-PLL. Functionally, IL2RG-JAK1-JAK3-STAT5B mutations led to signal transducer and activator of transcription 5 (STAT5) hyperactivation, transformed Ba/F3 cells resulting in cytokine-independent growth, and/or enhanced colony formation in Jurkat T cells. Importantly, primary T-PLL cells exhibited constitutive activation of STAT5, and targeted pharmacologic inhibition of STAT5 with pimozide induced apoptosis in primary T-PLL cells. These results for the first time provide a portrait of the mutational landscape of T-PLL and implicate deregulation of DNA repair and epigenetic modulators as well as high-frequency mutational activation of the IL2RG-JAK1-JAK3-STAT5B axis in the pathogenesis of T-PLL. These findings offer opportunities for novel targeted therapies in this aggressive leukemia. © 2014 by The American Society of Hematology.

Synthesis, DNA Binding, and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives

PubMed Central

de Almeida, Sinara Mônica Vitalino; Lafayette, Elizabeth Almeida; Gomes da Silva, Lúcia Patrícia Bezerra; Amorim, Cézar Augusto da Cruz; de Oliveira, Tiago Bento; Gois Ruiz, Ana Lucia Tasca; de Carvalho, João Ernesto; de Moura, Ricardo Olímpio; Beltrão, Eduardo Isidoro Carneiro; de Lima, Maria do Carmo Alves; de Carvalho Júnior, Luiz Bezerra

2015-01-01

In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide derivatives (3a–h) were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 104 to 1.0 × 106 M−1 and quenching constants from −0.2 × 104 to 2.18 × 104 M−1 indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z)-2-(acridin-9-ylmethylene)-N-(4-chlorophenyl) hydrazinecarbothioamide (3f), while the most active compound in antiproliferative assay was (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (3a). There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties. PMID:26068233

Interactions of vitamin K3 with herring-sperm DNA using spectroscopy and electrochemistry.

PubMed

Huang, Jianhang; Wang, Xingming; Fei, Dan; Ding, Lisheng

2010-10-01

By means of ultraviolet-visible (UV-Vis) and fluorescence spectra, the binding ratio between vitamin K(3) and herring-sperm DNA in a physiological pH environment (pH = 7.40) was determined as n(K3):n(DNA) = 2:1, and the binding constants of vitamin K(3) binding to DNA at different temperatures were determined as K(θ)(298K) = 1.28 × 10(5) L·mol(-1) and K(θ)(310K) = 7.19 × 10(4) L·mol(-1), which were confirmed using the double reciprocal method are Δ(r)H(m)(θ) = -3.57 × 10(4) J·mol(-1), Δ(r)G(m)(θ) = -2.92 × 10(4) J·mol(-1), and Δ(r)S(m)(θ) = 217.67 J·mol(-1)K(-1). The driving power of this process was enthalpy. An intercalation binding of the vitamin K(3) with DNA was supported by a competitive experiment using acridine orange (AO) as a spectral probe. By combination analysis of the Scatchard method and cyclic voltammetry, we suggested that the interaction mode between vitamin K(3) and herring-sperm DNA would be a mixed mode. The quinonoid, duality fused-ring of vitamin K(3) can intercalate into the base pairs of DNA, and there is an electrostatic binding along with intercalation binding.

Nuclear envelope proteins Nesprin2 and LaminA regulate proliferation and apoptosis of vascular endothelial cells in response to shear stress.

PubMed

Han, Yue; Wang, Lu; Yao, Qing-Ping; Zhang, Ping; Liu, Bo; Wang, Guo-Liang; Shen, Bao-Rong; Cheng, Binbin; Wang, Yingxiao; Jiang, Zong-Lai; Qi, Ying-Xin

2015-05-01

The dysfunction of vascular endothelial cells (ECs) influenced by flow shear stress is crucial for vascular remodeling. However, the roles of nuclear envelope (NE) proteins in shear stress-induced EC dysfunction are still unknown. Our results indicated that, compared with normal shear stress (NSS), low shear stress (LowSS) suppressed the expression of two types of NE proteins, Nesprin2 and LaminA, and increased the proliferation and apoptosis of ECs. Targeted small interfering RNA (siRNA) and gene overexpression plasmid transfection revealed that Nesprin2 and LaminA participate in the regulation of EC proliferation and apoptosis. A protein/DNA array was further used to detect the activation of transcription factors in ECs following transfection with target siRNAs and overexpression plasmids. The regulation of AP-2 and TFIID mediated by Nesprin2 and the activation of Stat-1, Stat-3, Stat-5 and Stat-6 by LaminA were verified under shear stress. Furthermore, using Ingenuity Pathway Analysis software and real-time RT-PCR, the effects of Nesprin2 or LaminA on the downstream target genes of AP-2, TFIID, and Stat-1, Stat-3, Stat-5 and Stat-6, respectively, were investigated under LowSS. Our study has revealed that NE proteins are novel mechano-sensitive molecules in ECs. LowSS suppresses the expression of Nesprin2 and LaminA, which may subsequently modulate the activation of important transcription factors and eventually lead to EC dysfunction. Copyright © 2015 Elsevier B.V. All rights reserved.

DNA-binding regulates site-specific ubiquitination of IRF-1.

PubMed

Landré, Vivien; Pion, Emmanuelle; Narayan, Vikram; Xirodimas, Dimitris P; Ball, Kathryn L

2013-02-01

Understanding the determinants for site-specific ubiquitination by E3 ligase components of the ubiquitin machinery is proving to be a challenge. In the present study we investigate the role of an E3 ligase docking site (Mf2 domain) in an intrinsically disordered domain of IRF-1 [IFN (interferon) regulatory factor-1], a short-lived IFNγ-regulated transcription factor, in ubiquitination of the protein. Ubiquitin modification of full-length IRF-1 by E3 ligases such as CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] and MDM2 (murine double minute 2), which dock to the Mf2 domain, was specific for lysine residues found predominantly in loop structures that extend from the DNA-binding domain, whereas no modification was detected in the more conformationally flexible C-terminal half of the protein. The E3 docking site was not available when IRF-1 was in its DNA-bound conformation and cognate DNA-binding sequences strongly suppressed ubiquitination, highlighting a strict relationship between ligase binding and site-specific modification at residues in the DNA-binding domain. Hyperubiquitination of a non-DNA-binding mutant supports a mechanism where an active DNA-bound pool of IRF-1 is protected from polyubiquitination and degradation.

Leptin activates STAT and ERK2 pathways and induces gastric cancer cell proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pai, Rama; Lin Cal; Tran, Teresa

2005-06-17

Although leptin is known to induce proliferative response in gastric cancer cells, the mechanism(s) underlying this action remains poorly understood. Here, we provide evidence that leptin-induced gastric cancer cell proliferation involves activation of STAT and ERK2 signaling pathways. Leptin-induced STAT3 phosphorylation is independent of ERK2 activation. Leptin increases SHP2 phosphorylation and enhances binding of Grb2 to SHP2. Inhibition of SHP2 expression with siRNA but not SHP2 phosphatase activity abolished leptin-induced ERK2 activation. While JAK inhibition with AG490 significantly reduced leptin-induced ERK2, STAT3 phosphorylation, and cell proliferation, SHP2 inhibition only partially reduced cancer cell proliferation. Immunostaining of gastric cancer tissues displayedmore » local overexpression of leptin and its receptor indicating that leptin might be produced and act locally in a paracrine or autocrine manner. These findings indicate that leptin promotes cancer growth by activating multiple signaling pathways and therefore blocking its action at the receptor level could be a rational therapeutic strategy.« less

Interaction of antitumor drug Sn(CH 3) 2Cl 2 with DNA and RNA

NASA Astrophysics Data System (ADS)

Nafisi, Shohreh; Sobhanmanesh, Amir; Esm-Hosseini, Majid; Alimoghaddam, Kamran; Tajmir-Riahi, Heidar Ali

2005-08-01

Sn(CH3)2Cl2 exerts its antitumor activity in a specific way. Unlike anticancer cis-Pt(NH3)2Cl2 drug which binds strongly to the nitrogen atoms of DNA bases, Sn(CH3)2Cl2 shows no major affinity towards base binding. Thus, the mechanism of action by which tinorganometallic compounds exert antitumor activity would be different from that of the cisplatin drug. The aim of this study was to examine the binding of Sn(CH3)2Cl2 with calf thymus DNA and yeast RNA in aqueous solutions at pH 7.1-6.6 with constant concentrations of DNA and RNA and various molar ratios of Sn(CH3)2Cl2/DNA (phosphate) and Sn(CH3)2Cl2/RNA of 1/40, 1/20, 1/10, 1/5. Fourier transform infrared (FTIR) and UV-visible difference spectroscopic methods were used to determine the Sn(CH3)2Cl2 binding mode, binding constant, sequence selectivity and structural variations of Sn(CH3)2Cl2/DNA and Sn(CH3)2Cl2/RNA complexes in aqueous solution. Sn(CH3)2Cl2 hydrolyzes in water to give Sn(CH3)2(OH)2 and [Sn(CH3)2(OH)(H2O)n]+ species. Spectroscopic evidence showed that interaction occurred mainly through (CH3)2Sn(IV) hydroxide and polynucleotide backbone phosphate group with overall binding constant of K(Sn(CH3)2Cl2-DNA)=1.47×105 M-1 and K(Sn(CH3)2Cl2-RNA)=7.33×105 M-1. Sn(CH3)2Cl2 induced no biopolymer conformational changes with DNA remaining in the B-family structure and RNA in A-conformation upon drug complexation.

Molecular basis of splotch and Waardenburg Pax-3 mutations.

PubMed Central

Chalepakis, G; Goulding, M; Read, A; Strachan, T; Gruss, P

1994-01-01

Pax genes control certain aspects of development, as mutations result in (semi)dominant defects apparent during embryogenesis. Pax-3 has been associated with the mouse mutant splotch (Sp) and the human Waardenburg syndrome type 1 (WS1). We have examined the molecular basis of splotch and WS1 by studying the effect of mutations on DNA binding, using a defined target sequence. Pax-3 contains two different types of functional DNA-binding domains, a paired domain and a homeodomain. Mutational analysis of Pax-3 reveals different modes of DNA binding depending on the presence of these domains. A segment of Pax-3 located between the two DNA-binding domains, including a conserved octapeptide, participates in protein homodimerization. Pax-3 mutations found in splotch alleles and WS1 individuals change DNA binding and, in the case of a protein product of the Sp allele, dimerization. These findings were taken as a basis to define the molecular nature of the mutants. Images PMID:7909605

Dual DNA binding property of ABA insensitive 3 like factors targeted to promoters responsive to ABA and auxin.

PubMed

Nag, Ronita; Maity, Manas Kanti; Dasgupta, Maitrayee

2005-11-01

The ABA responsive ABI3 and the auxin responsive ARF family of transcription factors bind the CATGCATG (Sph) and TGTCTC core motifs in ABA and auxin response elements (ABRE and AuxRE), respectively. Several evidences indicate ABI3s to act downstream to auxin too. Because DNA binding domain of ABI3s shows significant overlap with ARFs we enquired whether auxin responsiveness through ABI3s could be mediated by their binding to canonical AuxREs. Investigations were undertaken through in vitro gel mobility shift assays (GMSA) using the DNA binding domain B3 of PvAlf (Phaseolus vulgaris ABI3 like factor) and upstream regions of auxin responsive gene GH3 (-267 to -141) and ABA responsive gene Em (-316 to -146) harboring AuxRE and ABRE, respectively. We demonstrate that B3 domain of PvAlf could bind AuxRE only when B3 was associated with its flanking domain B2 (B2B3). Such strict requirement of B2 domain was not observed with ABRE, where B3 could bind with or without being associated with B2. This dual specificity in DNA binding of ABI3s was also demonstrated with nuclear extracts of cultured cells of Arachis hypogea. Supershift analysis of ABRE and AuxRE bound nuclear proteins with antibodies raised against B2B3 domains of PvAlf revealed that ABI3 associated complexes were detectable in association with both cis elements. Competition GMSA confirmed the same complexes to bind ABRE and AuxRE. This dual specificity of ABI3 like factors in DNA binding targeted to natural promoters responsive to ABA and auxin suggests them to have a potential role in conferring crosstalk between these two phytohormones.

The retinol esterifying enzyme LRAT supports cell signaling by retinol-binding protein and its receptor STRA6.

PubMed

Marwarha, Gurdeep; Berry, Daniel C; Croniger, Colleen M; Noy, Noa

2014-01-01

Vitamin A, retinol, circulates in blood bound to retinol-binding protein (RBP). At some tissues, holo-RBP is recognized by a plasma membrane receptor termed STRA6, which serves a dual role: it mediates transport of retinol from RBP into cells, and it functions as a cytokine receptor that, on binding holo-RBP, activates JAK2/STAT5 signaling. As STAT target genes include SOCS3, an inhibitor of insulin receptor, holo-RBP suppresses insulin responses in STRA6-expressing cells. We have shown previously that the two functions of STRA6 are interdependent. These observations suggest factors that regulate STRA6-mediated retinol transport may also control STRA6-mediated cell signaling. One such factor is retinol metabolism, which enables cellular uptake of retinol by maintaining an inward-directed concentration gradient. We show here that lecithin:retinol acyl transferase (LRAT), which catalyzes esterification of retinol to its storage species retinyl esters, is necessary for activation of the STRA6/JAK2/STAT5 cascade by holo-RBP. In accordance, LRAT-null mice are protected from holo-RBP-induced suppression of insulin responses. Hence, STRA6 signaling, which requires STRA6-mediated retinol transport, is supported by LRAT-catalyzed retinol metabolism. The observations demonstrate that STRA6 regulates key cellular processes by coupling circulating holo-RBP levels and intracellular retinol metabolism to cell signaling.

Protein Cofactors Are Essential for High-Affinity DNA Binding by the Nuclear Factor κB RelA Subunit.

PubMed

Mulero, Maria Carmen; Shahabi, Shandy; Ko, Myung Soo; Schiffer, Jamie M; Huang, De-Bin; Wang, Vivien Ya-Fan; Amaro, Rommie E; Huxford, Tom; Ghosh, Gourisankar

2018-05-22

Transcription activator proteins typically contain two functional domains: a DNA binding domain (DBD) that binds to DNA with sequence specificity and an activation domain (AD) whose established function is to recruit RNA polymerase. In this report, we show that purified recombinant nuclear factor κB (NF-κB) RelA dimers bind specific κB DNA sites with an affinity significantly lower than that of the same dimers from nuclear extracts of activated cells, suggesting that additional nuclear cofactors might facilitate DNA binding by the RelA dimers. Additionally, recombinant RelA binds DNA with relatively low affinity at a physiological salt concentration in vitro. The addition of p53 or RPS3 (ribosomal protein S3) increases RelA:DNA binding affinity 2- to >50-fold depending on the protein and ionic conditions. These cofactor proteins do not form stable ternary complexes, suggesting that they stabilize the RelA:DNA complex through dynamic interactions. Surprisingly, the RelA-DBD alone fails to bind DNA under the same solution conditions even in the presence of cofactors, suggesting an important role of the RelA-AD in DNA binding. Reduced RelA:DNA binding at a physiological ionic strength suggests that multiple cofactors might be acting simultaneously to mitigate the electrolyte effect and stabilize the RelA:DNA complex in vivo. Overall, our observations suggest that the RelA-AD and multiple cofactor proteins function cooperatively to prime the RelA-DBD and stabilize the RelA:DNA complex in cells. Our study provides a mechanism for nuclear cofactor proteins in NF-κB-dependent gene regulation.

DNA Binding Mode Transitions of Escherichia coli HUαβ: Evidence for Formation of a Bent DNA – Protein Complex on Intact, Linear Duplex DNA

PubMed Central

Koh, Junseock; Saecker, Ruth M.; Record, M. Thomas

2008-01-01

Escherichia coli HUαβ, a major nucleoid associated protein (NAP), organizes the DNA chromosome and facilitates numerous DNA transactions. Using isothermal titration calorimetry (ITC), fluorescence resonance energy transfer (FRET) and a series of DNA lengths (8, 15, 34, 38 and 160 base pairs) we establish that HUαβ interacts with duplex DNA using three different nonspecific binding modes. Both the HU to DNA mole ratio ([HU]/[DNA]) and DNA length dictate the dominant HU binding mode. On sufficiently long DNA (≥ 34 base pairs), at low [HU]/[DNA], HU populates a noncooperative 34 bp binding mode with a binding constant of 2.1 (± 0.4) × 106 M−1, and a binding enthalpy of +7.7 (± 0.6) kcal/mol at 15 °C and 0.15 M Na+. With increasing [HU]/[DNA], HU bound in the noncooperative 34 bp mode progressively converts to two cooperative (ω ~ 20) modes with site sizes of 10 bp and 6 bp. These latter modes exhibit smaller binding constants (1.1 (± 0.2) × 105 M−1 for the 10 bp mode, 3.5 (± 1.4) × 104 M−1 for the 6 bp mode) and binding enthalpies (4.2 (± 0.3) kcal/mol for the 10 bp mode, −1.6 (±0.3) kcal/mol for the 6 bp mode). As DNA length increases to 34 bp or more at low [HU]/[DNA], the small modes are replaced by the 34 bp binding mode. FRET data demonstrate that the 34 bp mode bends DNA by 143 ± 6° whereas the 6 and 10 bp modes do not. The model proposed in this study provides a novel quantitative and comprehensive framework for reconciling previous structural and solution studies of HU, including single molecule (force extension measurement, AFM), fluorescence, and electrophoretic gel mobility shift assays. In particular, it explains how HU condenses or extends DNA depending on the relative concentrations of HU and DNA. PMID:18657548

Dpb11 may function with RPA and DNA to initiate DNA replication.

PubMed

Bruck, Irina; Dhingra, Nalini; Martinez, Matthew P; Kaplan, Daniel L

2017-01-01

Dpb11 is required for the initiation of DNA replication in budding yeast. We found that Dpb11 binds tightly to single-stranded DNA (ssDNA) or branched DNA structures, while its human homolog, TopBP1, binds tightly to branched-DNA structures. We also found that Dpb11 binds stably to CDK-phosphorylated RPA, the eukaryotic ssDNA binding protein, in the presence of branched DNA. A Dpb11 mutant specifically defective for DNA binding did not exhibit tight binding to RPA in the presence of DNA, suggesting that Dpb11-interaction with DNA may promote the recruitment of RPA to melted DNA. We then characterized a mutant of Dpb11 that is specifically defective in DNA binding in budding yeast cells. Expression of dpb11-m1,2,3,5,ΔC results in a substantial decrease in RPA recruitment to origins, suggesting that Dpb11 interaction with DNA may be required for RPA recruitment to origins. Expression of dpb11-m1,2,3,5,ΔC also results in diminished GINS interaction with Mcm2-7 during S phase, while Cdc45 interaction with Mcm2-7 is like wild-type. The reduced GINS interaction with Mcm2-7 may be an indirect consequence of diminished origin melting. We propose that the tight interaction between Dpb11, CDK-phosphorylated RPA, and branched-DNA may be required for the essential function of stabilizing melted origin DNA in vivo. We also propose an alternative model, wherein Dpb11-DNA interaction is required for some other function in DNA replication initiation, such as helicase activation.

[Features of binding of proflavine to DNA at different DNA-ligand concentration ratios].

PubMed

Berezniak, E G; gladkovskaia, N A; Khrebtova, A S; Dukhopel'nikov, E V; Zinchenko, A V

2009-01-01

The binding of proflavine to calf thymus DNA has been studied using the methods of differential scanning calorimetry and spectrophotometry. It was shown that proflavine can interact with DNA by at least 3 binding modes. At high DNA-ligand concentration ratios (P/D), proflavine intercalates into both GC- and AT-sites, with a preference to GC-rich sequences. At low P/D ratios proflavine interacts with DNA by the external binding mode. From spectrophotometric concentration dependences, the parameters of complexing of proflavine with DNA were calculated. Thermodynamic parameters of DNA melting were calculated from differential scanning calorimetry data.

Dynamic DNA binding licenses a repair factor to bypass roadblocks in search of DNA lesions.

PubMed

Brown, Maxwell W; Kim, Yoori; Williams, Gregory M; Huck, John D; Surtees, Jennifer A; Finkelstein, Ilya J

2016-02-03

DNA-binding proteins search for specific targets via facilitated diffusion along a crowded genome. However, little is known about how crowded DNA modulates facilitated diffusion and target recognition. Here we use DNA curtains and single-molecule fluorescence imaging to investigate how Msh2-Msh3, a eukaryotic mismatch repair complex, navigates on crowded DNA. Msh2-Msh3 hops over nucleosomes and other protein roadblocks, but maintains sufficient contact with DNA to recognize a single lesion. In contrast, Msh2-Msh6 slides without hopping and is largely blocked by protein roadblocks. Remarkably, the Msh3-specific mispair-binding domain (MBD) licences a chimeric Msh2-Msh6(3MBD) to bypass nucleosomes. Our studies contrast how Msh2-Msh3 and Msh2-Msh6 navigate on a crowded genome and suggest how Msh2-Msh3 locates DNA lesions outside of replication-coupled repair. These results also provide insights into how DNA repair factors search for DNA lesions in the context of chromatin.

Electrostatic study of Alanine mutational effects on transcription: application to GATA-3:DNA interaction complex.

PubMed

El-Assaad, Atlal; Dawy, Zaher; Nemer, Georges

2015-01-01

Protein-DNA interaction is of fundamental importance in molecular biology, playing roles in functions as diverse as DNA transcription, DNA structure formation, and DNA repair. Protein-DNA association is also important in medicine; understanding Protein-DNA binding kinetics can assist in identifying disease root causes which can contribute to drug development. In this perspective, this work focuses on the transcription process by the GATA Transcription Factor (TF). GATA TF binds to DNA promoter region represented by `G,A,T,A' nucleotides sequence, and initiates transcription of target genes. When proper regulation fails due to some mutations on the GATA TF protein sequence or on the DNA promoter sequence (weak promoter), deregulation of the target genes might lead to various disorders. In this study, we aim to understand the electrostatic mechanism behind GATA TF and DNA promoter interactions, in order to predict Protein-DNA binding in the presence of mutations, while elaborating on non-covalent binding kinetics. To generate a family of mutants for the GATA:DNA complex, we replaced every charged amino acid, one at a time, with a neutral amino acid like Alanine (Ala). We then applied Poisson-Boltzmann electrostatic calculations feeding into free energy calculations, for each mutation. These calculations delineate the contribution to binding from each Ala-replaced amino acid in the GATA:DNA interaction. After analyzing the obtained data in view of a two-step model, we are able to identify potential key amino acids in binding. Finally, we applied the model to GATA-3:DNA (crystal structure with PDB-ID: 3DFV) binding complex and validated it against experimental results from the literature.

p38 Mitogen-Activated Protein Kinase/Signal Transducer and Activator of Transcription-3 Pathway Signaling Regulates Expression of Inhibitory Molecules in T Cells Activated by HIV-1–Exposed Dendritic Cells

PubMed Central

Che, Karlhans Fru; Shankar, Esaki Muthu; Muthu, Sundaram; Zandi, Sasan; Sigvardsson, Mikael; Hinkula, Jorma; Messmer, Davorka; Larsson, Marie

2012-01-01

Human immunodeficiency virus type 1 (HIV-1) infection enhances the expression of inhibitory molecules on T cells, leading to T-cell impairment. The signaling pathways underlying the regulation of inhibitory molecules and subsequent onset of T-cell impairment remain elusive. We showed that both autologous and allogeneic T cells exposed to HIV-pulsed dendritic cells (DCs) upregulated cytotoxic T-lymphocyte antigen (CTLA-4), tumor-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), lymphocyte-activation gene-3 (LAG3), T-cell immunoglobulin mucin-3 (TIM-3), CD160 and certain suppression-associated transcription factors, such as B-lymphocyte induced maturation protein-1 (BLIMP-1), deltex homolog 1 protein (DTX1) and forkhead box P3 (FOXP3), leading to T-cell suppression. This induction was regulated by p38 mitogen-activated protein kinase/signal transducer and activator of transcription-3 (P38MAPK/STAT3) pathways, because their blockade significantly abrogated expression of all the inhibitory molecules studied and a subsequent recovery in T-cell proliferation. Neither interleukin-6 (IL-6) nor IL-10 nor growth factors known to activate STAT3 signaling events were responsible for STAT3 activation. Involvement of the P38MAPK/STAT3 pathways was evident because these proteins had a higher level of phosphorylation in the HIV-1–primed cells. Furthermore, blockade of viral CD4 binding and fusion significantly reduced the negative effects DCs imposed on primed T cells. In conclusion, HIV-1 interaction with DCs modulated their functionality, causing them to trigger the activation of the P38MAPK/STAT3 pathway in T cells, which was responsible for the upregulation of inhibitory molecules. PMID:22777388

Mismatch repair factor MSH2-MSH3 binds and alters the conformation of branched DNA structures predicted to form during genetic recombination.

PubMed

Surtees, Jennifer A; Alani, Eric

2006-07-14

Genetic studies in Saccharomyces cerevisiae predict that the mismatch repair (MMR) factor MSH2-MSH3 binds and stabilizes branched recombination intermediates that form during single strand annealing and gene conversion. To test this model, we constructed a series of DNA substrates that are predicted to form during these recombination events. We show in an electrophoretic mobility shift assay that S. cerevisiae MSH2-MSH3 specifically binds branched DNA substrates containing 3' single-stranded DNA and that ATP stimulates its release from these substrates. Chemical footprinting analyses indicate that MSH2-MSH3 specifically binds at the double-strand/single-strand junction of branched substrates, alters its conformation and opens up the junction. Therefore, MSH2-MSH3 binding to its substrates creates a unique nucleoprotein structure that may signal downstream steps in repair that include interactions with MMR and nucleotide excision repair factors.

Transcriptional deregulation of oncogenic myocyte enhancer factor 2C in T-cell acute lymphoblastic leukemia.

PubMed

Nagel, Stefan; Venturini, Letizia; Meyer, Corinna; Kaufmann, Maren; Scherr, Michaela; Drexler, Hans G; Macleod, Roderick A F

2011-02-01

Myocyte enhancer factor 2C (MEF2C) encodes a transcription factor which is ectopically expressed in T-cell acute lymphoblastic leukemia (T-ALL) cell lines, deregulated directly by ectopically expressed homeodomain protein NKX2-5 or by loss of promoter regions via del(5)(q14). Here, we analyzed the MEF2C 5'-region, thus identifying potential regulatory binding sites for GFI1B, basic helix-loop-helix proteins, STAT5, and HOXA9/HOXA10. Chromatin immunoprecipitation and overexpression analyses demonstrated direct activation by GFI1B and LYL1 and inhibition by STAT5. HOXA9/HOXA10 activated expression of NMYC which in turn mediated MEF2C repression, indicating an indirect mode of regulation via NMYC interactor (NMI) and STAT5. Lacking comma: Chromosomal deletion of the STAT5 binding site in LOUCY cells reduced protein levels of STAT5 in some MEF2C-positve T-ALL cell lines, and the presence of inhibitory IL7-JAK-STAT5 signaling highlighted the repressive impact of this factor in MEF2C regulation. Taken together, our results indicate that the expression of MEF2C in T-ALL cells is principally deregulated via activating leukemic transcription factors GFI1B or NKX2-5 and by escaping inhibitory developmental STAT5 signaling.

α-Mangostin: A Dietary Antioxidant Derived from the Pericarp of Garcinia mangostana L. Inhibits Pancreatic Tumor Growth in Xenograft Mouse Model

PubMed Central

Mustafa, Ala; Fischer, Joseph W.; Singh, Ashok; Zhong, Weixiong; Shekhani, Mohammed Ozair; Meske, Louise; Havighurst, Thomas; Kim, KyungMann; Verma, Ajit Kumar

2014-01-01

Abstract Aims: Pancreatic cancer (PC) is the most aggressive malignant disease, ranking as the fourth most leading cause of cancer-related death among men and women in the United States. In this study, we provide evidence of chemotherapeutic effects of α-mangostin, a dietary antioxidant isolated from the pericarp of Garcinia mangostana L. against human PC. Results: The chemotherapeutic effect of α-mangostin was determined using four human PC cells (PL-45, PANC1, BxPC3, and ASPC1). α-Mangostin resulted in a significant inhibition of PC cells viability without having any effects on normal human pancreatic duct epithelial cells. α-Mangostin showed a dose-dependent increase of apoptosis in PC cells. Also, α-mangostin inhibited the expression levels of pNF-κB/p65Ser552, pStat3Ser727, and pStat3Tyr705. α-Mangostin inhibited DNA binding activity of nuclear factor kappa B (NF-κB) and signal transducer and activator 3 (Stat3). α-Mangostin inhibited the expression levels of matrix metallopeptidase 9 (MMP9), cyclin D1, and gp130; however, increased expression of tissue inhibitor of metalloproteinase 1 (TIMP1) was observed in PC cells. In addition, i.p. administration of α-mangostin (6 mg/kg body weight, 5 days a week) resulted in a significant inhibition of both primary (PL-45) and secondary (ASPC1) human PC cell-derived orthotopic and ectopic xenograft tumors in athymic nude mice. No sign of toxicity was observed in any of the mice administered with α-mangostin. α-Mangostin treatment inhibited the biomarkers of cell proliferation (Ki-67 and proliferating cell nuclear antigen [PCNA]) in the xenograft tumor tissues. Innovation: We present, for the first time, that dietary antioxidant α-mangostin inhibits the growth of PC cells in vitro and in vivo. Conclusion: These results suggest the potential therapeutic efficacy of α-mangostin against human PC. Antioxid. Redox Signal. 21, 682–699. PMID:24295217

A Bayesian mixture model for chromatin interaction data.

PubMed

Niu, Liang; Lin, Shili

2015-02-01

Chromatin interactions mediated by a particular protein are of interest for studying gene regulation, especially the regulation of genes that are associated with, or known to be causative of, a disease. A recent molecular technique, Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET), that uses chromatin immunoprecipitation (ChIP) and high throughput paired-end sequencing, is able to detect such chromatin interactions genomewide. However, ChIA-PET may generate noise (i.e., pairings of DNA fragments by random chance) in addition to true signal (i.e., pairings of DNA fragments by interactions). In this paper, we propose MC_DIST based on a mixture modeling framework to identify true chromatin interactions from ChIA-PET count data (counts of DNA fragment pairs). The model is cast into a Bayesian framework to take into account the dependency among the data and the available information on protein binding sites and gene promoters to reduce false positives. A simulation study showed that MC_DIST outperforms the previously proposed hypergeometric model in terms of both power and type I error rate. A real data study showed that MC_DIST may identify potential chromatin interactions between protein binding sites and gene promoters that may be missed by the hypergeometric model. An R package implementing the MC_DIST model is available at http://www.stat.osu.edu/~statgen/SOFTWARE/MDM.

The Jak-STAT pathway stimulated by interferon alpha or interferon beta.

PubMed

Horvath, Curt M

2004-11-23

Type I interferons, such as interferon alpha and interferon beta (IFN-alpha and beta), signal through a Janus kinase (Jak) to signal transduction and activator of transcription (STAT) pathway to stimulate gene expression. In response to ligand binding, the receptors dimerize, Jaks phosphorylate STAT1 and STAT2, which then dimerize and interact with a third transcriptional regulator IFN regulatory factor 9 (IRF9) to stimulate gene expression. IFN-alpha is the main innate antiviral cytokine and is essential for effective immune response to viral infection. The animation shows activation of STAT-responsive gene expression in response to type I IFNs.

The effects of humanin and its analogues on male germ cell apoptosis induced by chemotherapeutic drugs.

PubMed

Jia, Yue; Ohanyan, Aikoui; Lue, Yan-He; Swerdloff, Ronald S; Liu, Peter Y; Cohen, Pinchas; Wang, Christina

2015-04-01

Human (HN) prevents stress-induced apoptosis in many cells/tissues. In this study we showed that HN ameliorated chemotherapy [cyclophosphamide (CP) and Doxorubicin (DOX)]-induced male germ cell apoptosis both ex vivo in seminiferous tubule cultures and in vivo in the testis. HN acts by several putative mechanisms via binding to: an IL-12 like trimeric membrane receptor; BAX; or insulin-like growth factor binding protein-3 (IGFBP-3, a proapoptotic factor). To understand the mechanisms of HN on male germ cell apoptosis, we studied five HN analogues including: HNG (HN-S14G, a potent agonist), HNG-F6A (no binding to IGFBP-3), HN-S7A (no self-dimerization), HN-C8P (no binding to BAX), and HN-L12A (a HN antagonist) on CP-induced male germ cell apoptosis in mice. CP-induced germ cell apoptosis was inhibited by HN, HNG, HNG-F6A, HN-S7A, and HN-C8P (less effective); but not by HN-L12A. HN-L12A, but not HN-S7A or HN-C8P, blocked the protective effect of HN against CP-induced male germ cell apoptosis. HN, HN-S7A, and HN-C8P restored CP-suppressed STAT3 phosphorylation. These results suggest that HN: (1) decreases DOX (ex vivo) and CP (in vivo) induced male germ cell apoptosis; (2) action is mediated by the membrane receptor/STAT3 with minor contribution by BAX-binding pathway; (3) self-dimerization or binding to IGFBP-3 may not be involved in HN's effect in testis. HN is an important molecule in the regulation of germ cell homeostasis after injury and agonistic analogues may be developed for treating male infertility or protection against chemotherapy side effects.

The Effects of Humanin and Its Analogues on Male Germ Cell Apoptosis Induced by Chemotherapeutic Drugs

PubMed Central

Jia, Yue; Ohanyan, Aikoui; Lue, Yan-He; Swerdloff, Ronald S.; Liu, Peter Y.; Cohen, Pinchas; Wang, Christina

2015-01-01

Human (HN) prevents stress-induced apoptosis in many cells/tissues. In this study we showed that HN ameliorated chemotherapy (Cyclophosphamide, CP and Doxorubicin, DOX)-induced male germ cell apoptosis both ex vivo in seminiferous tubule cultures and in vivo in the testis. HN acts by several putative mechanisms via binding to: an IL-12 like trimeric membrane receptor; BAX; or Insulin-Like Growth Factor Binding Protein-3 (IGFBP-3, a proapoptotic factor). To understand the mechanisms of HN on male germ cell apoptosis, we studied five HN analogues including: HNG (HN-S14G, a potent agonist), HNG-F6A (no binding to IGFBP-3), HN-S7A (no self-dimerization), HN-C8P (no binding to BAX), and HN-L12A (a HN antagonist) on CP-induced male germ cell apoptosis in mice. CP-induced germ cell apoptosis was inhibited by HN, HNG, HNG-F6A, HN-S7A, and HN-C8P (less effective); but not by HN-L12A. HN-L12A, but not HN-S7A or HN-C8P, blocked the protective effect of HN against CP-induced male germ cell apoptosis. HN, HN-S7A, and HN-C8P restored CP-suppressed STAT3 phosphorylation. These results suggest that HN: 1) decreases DOX (ex vivo) and CP (in vivo) induced male germ cell apoptosis; 2) action is mediated by the membrane receptor/STAT3 with minor contribution by BAX-binding pathway; 3) self-dimerization or binding to IGFBP-3 may not be involved in HN’s effect in testis. HN is an important molecule in the regulation of germ cell homeostasis after injury and agonistic analogues may be developed for treating male infertility or protection against chemotherapy side effects. PMID:25666707

E-cadherin and, in its absence, N-cadherin promotes Nanog expression in mouse embryonic stem cells via STAT3 phosphorylation.

PubMed

Hawkins, Kate; Mohamet, Lisa; Ritson, Sarah; Merry, Catherine L R; Ward, Christopher M

2012-09-01

We have recently shown that loss of E-cadherin in mouse embryonic stem cells (mESCs) results in significant alterations to both the transcriptome and hierarchy of pluripotency-associated signaling pathways. Here, we show that E-cadherin promotes kruppel-like factor 4 (Klf4) and Nanog transcript and protein expression in mESCs via STAT3 phosphorylation and that β-catenin, and its binding region in E-cadherin, is required for this function. To further investigate the role of E-cadherin in leukemia inhibitory factor (LIF)-dependent pluripotency, E-cadherin null (Ecad(-/-)) mESCs were cultured in LIF/bone morphogenetic protein supplemented medium. Under these conditions, Ecad(-/-) mESCs exhibited partial restoration of cell-cell contact and STAT3 phosphorylation and upregulated Klf4, Nanog, and N-cadherin transcripts and protein. Abrogation of N-cadherin using an inhibitory peptide caused loss of phospho STAT3, Klf4, and Nanog in these cells, demonstrating that N-cadherin supports LIF-dependent pluripotency in this context. We therefore identify a novel molecular mechanism linking E- and N-cadherin to the core circuitry of pluripotency in mESCs. This mechanism may explain the recently documented role of E-cadherin in efficient induced pluripotent stem cell reprogramming. Copyright © 2012 AlphaMed Press.

Tumor-associated mesenchymal stem cells inhibit naïve T cell expansion by blocking cysteine export from dendritic cells.

PubMed

Ghosh, Tithi; Barik, Subhasis; Bhuniya, Avishek; Dhar, Jesmita; Dasgupta, Shayani; Ghosh, Sarbari; Sarkar, Madhurima; Guha, Ipsita; Sarkar, Koustav; Chakrabarti, Pinak; Saha, Bhaskar; Storkus, Walter J; Baral, Rathindranath; Bose, Anamika

2016-11-01

Mesenchymal stem cells (MSCs) represent an important cellular constituent of the tumor microenvironment, which along with tumor cells themselves, serve to regulate protective immune responses in support of progressive disease. We report that tumor MSCs prevent the ability of dendritic cells (DC) to promote naïve CD4(+) and CD8(+) T cell expansion, interferon gamma secretion and cytotoxicity against tumor cells, which are critical to immune-mediated tumor eradication. Notably, tumor MSCs fail to prevent DC-mediated early T cell activation events or the ability of responder T cells to produce IL-2. The immunoregulatory activity of tumor MSCs is IL-10- and STAT3-dependent, with STAT3 repressing DC expression of cystathionase, a critical enzyme that converts methionine-to-cysteine. Under cysteine-deficient priming conditions, naïve T cells exhibit defective cellular metabolism and proliferation. Bioinformatics analyses as well as in vitro observations suggest that STAT3 may directly bind to a GAS-like motif within the cystathionase promoter (-269 to -261) leading to IL-10-STAT3 mediated repression of cystathionase gene transcription. Our collective results provide evidence for a novel mechanism of tumor MSC-mediated T cell inhibition within tumor microenvironment. © 2016 UICC.

Quantification of transcription factor-DNA binding affinity in a living cell

PubMed Central

Belikov, Sergey; Berg, Otto G.; Wrange, Örjan

2016-01-01

The apparent dissociation constant (Kd) for specific binding of glucocorticoid receptor (GR) and androgen receptor (AR) to DNA was determined in vivo in Xenopus oocytes. The total nuclear receptor concentration was quantified as specifically retained [3H]-hormone in manually isolated oocyte nuclei. DNA was introduced by nuclear microinjection of single stranded phagemid DNA, chromatin is then formed during second strand synthesis. The fraction of DNA sites occupied by the expressed receptor was determined by dimethylsulphate in vivo footprinting and used for calculation of the receptor-DNA binding affinity. The forkhead transcription factor FoxA1 enhanced the DNA binding by GR with an apparent Kd of ∼1 μM and dramatically stimulated DNA binding by AR with an apparent Kd of ∼0.13 μM at a composite androgen responsive DNA element containing one FoxA1 binding site and one palindromic hormone receptor binding site known to bind one receptor homodimer. FoxA1 exerted a weak constitutive- and strongly cooperative DNA binding together with AR but had a less prominent effect with GR, the difference reflecting the licensing function of FoxA1 at this androgen responsive DNA element. PMID:26657626

A verotoxin 1 B subunit-lambda CRO chimeric protein specifically binds both DNA and globotriaosylceramide (Gb(3)) to effect nuclear targeting of exogenous DNA in Gb(3) positive cells.

PubMed

Facchini, L M; Lingwood, C A

2001-09-10

Inefficient nuclear incorporation of foreign DNA remains a critical roadblock in the development of effective nonviral gene delivery systems. DNA delivered by traditional protocols remains within endosomal/lysosomal vesicles, or is rapidly degraded in the cytoplasm. Verotoxin I (VT), an AB(5) subunit toxin produced by enterohaemorrhagic Escherichia coli, binds to the cell surface glycolipid, globotriaosylceramide (Gb(3)) and is internalized into preendosomes. VT is then retrograde transported to the Golgi, endoplasmic reticulum (ER), and nucleus of highly VT-sensitive cells. We have utilized this nuclear targeting of VT to design a unique delivery system which transports exogenous DNA via vesicular traffic to the nucleus. The nontoxic VT binding subunit (VTB) was fused to the lambda Cro DNA-binding repressor, generating a 14-kDa VTB-Cro chimera. VTB-Cro binds specifically via the Cro domain to a 25-bp DNA fragment containing the consensus Cro operator. VTB-Cro demonstrates simultaneous specific binding to Gb(3). Treatment of Vero cells with fluorescent-labeled Cro operator DNA in the presence of VTB-Cro, results in DNA internalization to the Golgi, ER, and nucleus, whereas fluorescent DNA alone is incorporated poorly and randomly within the cytoplasm. VTB-Cro mediated nuclear DNA transport is prevented by brefeldin A, consistent with Golgi/ER intracellular routing. Pretreatment with filipin had no effect, indicating that caveoli are not involved. This novel VTB-Cro shuttle protein may find practical applications in the fields of intracellular targeting, gene delivery, and gene therapy. Copyright 2001 Academic Press.

MicroRNA-203 Modulates the Radiation Sensitivity of Human Malignant Glioma Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Ji Hyun; Hwang, Yeo Hyun; Lee, David J.

Purpose: We investigated whether miR-203 could modulate the radiation sensitivity of glioblastoma (GBM) cells and which target gene(s) could be involved. Methods and Materials: Three human malignant glioma (MG) cell lines and normal human astrocytes were transfected with control microRNA, pre-miR-203, or antisense miR-203. Real-time PCR (RT-PCR), clonogenic assays, immunofluorescence, and invasion/migration assays were performed. To predict the target(s), bioinformatics analyses using microRNA target databases were performed. Results: Overexpression of miR-203 increased the radiation sensitivity of all 3 human MG cell lines and prolonged radiation-induced γ-H2AX foci formation. Bioinformatics analyses suggested that miR-203 could be involved in post-transcriptional control of DNAmore » repair, PI3K/AKT, SRC, and JAK/STAT3 and the vascular signaling pathway. Western blot analysis validated the fact that miR-203 downregulated ATM, RAD51, SRC, PLD2, PI3K-AKT, JAK-STAT3, VEGF, HIF-1α, and MMP2. Overexpression of miR-203 inhibited invasion and migration potentials, downregulated SLUG and Vimentin, and upregulated Claudin-1 and ZO1. Conclusions: These data demonstrate that miR-203 potentially controls DNA damage repair via the PI3K/AKT and JAK/STAT3 pathways and may collectively contribute to the modulation of radiation sensitivity in MG cells by inhibiting DNA damage repair, prosurvival signaling, and epithelium-mesenchyme transition. Taken together, these findings demonstrate that miR-203 could be a target for overcoming the radiation resistance of GBM.« less

Influence of Maternal Undernutrition and Overfeeding on Cardiac Ciliary Neurotrophic Factor Receptor and Ventricular Size in Fetal Sheep

PubMed Central

Dong, Feng; Ford, Stephen P.; Nijland, Mark J.; Nathanielsz, Peter W.; Ren, Jun

2008-01-01

Intrauterine nutrition status is reported to correlate with risk of cardiovascular diseases in adulthood. Either under- or over-nutrition during early to mid gestation contributes to altered fetal growth and ventricular geometry. This study was designed to examine myocardial expression of ciliary neurotrophic factor receptor α (CTNFRα) and its down-stream mediator signal transducer and activator of transcription 3 (STAT3) on maternal under- or over-nutrition-induced changes in fetal heart weight. Multiparous ewes were fed with 50% (nutrient-restricted, NR), 100% (control) or 150% (overfed, OF) of NRC requirements from 28 to 78 days of gestation (dG; Term 148 dG). Ewes were euthanized on day 78, and the gravid uteri and fetuses recovered. Ventricular protein expression of CTNFRα, STAT3, phosphorylated STAT3, insulin-like growth factor I receptor (IGF-1R) and IGF binding protein 3 (IGFBP3) were quantitated using western blot. Plasma cortisol levels were higher in both NR and OF fetuses whereas plasma IGF-1 levels were lower and higher, in NR and OF fetuses. Fetal weights were reduced by 29.9% in NR ewes and were increased by 22.2% in fetuses from OF ewes compared to control group. Nutrient restriction did not affect fetal heart or ventricular weights whereas overfeeding increased heart and ventricular weights. Protein expression of CTNFRα in fetal ventricular tissue was reduced in OF group whereas STAT3 and pSTAT3 levels were reduced in both NR and OF groups. Expression of IGF-1R and IGFBP3 was unaffected in either NR or OF group. These data suggested that compared with maternal undernutrition, intrauterine overfeeding during early to mid gestation is associated with increases fetal blood concentrations of cortisol and IGF-1 in association with ventricular hypertrophy where reduced expression of CNTFRα and STAT3 may play a role. PMID:17869083

Leptin upregulates telomerase activity and transcription of human telomerase reverse transcriptase in MCF-7 breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ren, He, E-mail: herenrh@yahoo.com.cn; Zhao, Tiansuo; Wang, Xiuchao

2010-03-26

The aim was to analyze the mechanism of leptin-induced activity of telomerase in MCF-7 breast cancer cells. We found that leptin activated telomerase in a dose-dependent manner; leptin upregulated the expression of Human Telomerase Reverse Transcriptase (hTERT) at mRNA and protein levels; blockade of signal transducer and activator of transcription 3 (STAT3) phosphorylation significantly counteracted leptin-induced hTERT transcription and protein expression; chromatin immunoprecipitation analysis showed that leptin enhanced the binding of STAT3 to the hTERT promoter. This study uncovers a new mechanism of the proliferative effect of leptin on breast cancer cells and provides a new explanation of obesity-related breastmore » cancer.« less

DNA sensing by a Eu-binding peptide containing a proflavine unit.

PubMed

Ancel, Laetitia; Gateau, Christelle; Lebrun, Colette; Delangle, Pascale

2013-01-18

Synthesis of a lanthanide-binding peptide (LBP) for the detection of double-stranded DNA is presented. A proflavine moiety was introduced into a high affinity LBP involving two unnatural chelating amino acids in the Ln ion coordination. The Eu(3+)-LBP complex is demonstrated to bind to ct-DNA and to sensitize Eu luminescence. The DNA binding process is effectively detected via the Eu-centered luminescence thanks to the intimate coupling between the LBP scaffold and DNA intercalating unit.

IL-4 function can be transferred to the IL-2 receptor by tyrosine containing sequences found in the IL-4 receptor alpha chain.

PubMed

Wang, H Y; Paul, W E; Keegan, A D

1996-02-01

IL-4 binds to a cell surface receptor complex that consists of the IL-4 binding protein (IL-4R alpha) and the gamma chain of the IL-2 receptor complex (gamma c). The receptors for IL-4 and IL-2 have several features in common; both use the gamma c as a receptor component, and both activate the Janus kinases JAK-1 and JAK-3. In spite of these similarities, IL-4 evokes specific responses, including the tyrosine phosphorylation of 4PS/IRS-2 and the induction of CD23. To determine whether sequences within the cytoplasmic domain of the IL-4R alpha specify these IL-4-specific responses, we transplanted the insulin IL-4 receptor motif (I4R motif) of the huIL-4R alpha to the cytoplasmic domain of a truncated IL-2R beta. In addition, we transplanted a region that contains peptide sequences shown to block Stat6 binding to DNA. We analyzed the ability of cells expressing these IL-2R-IL-4R chimeric constructs to respond to IL-2. We found that IL-4 function could be transplanted to the IL-2 receptor by these regions and that proliferative and differentiative functions can be induced by different receptor sequences.

Clusterin/ApoJ enhances central leptin signaling through Lrp2-mediated endocytosis.

PubMed

Byun, Kyunghee; Gil, So Young; Namkoong, Churl; Youn, Byung-Soo; Huang, Hu; Shin, Mi-Seon; Kang, Gil Myoung; Kim, Hyun-Kyong; Lee, Bonghee; Kim, Young-Bum; Kim, Min-Seon

2014-07-01

Hypothalamic leptin signaling plays a central role in maintaining body weight homeostasis. Here, we show that clusterin/ApoJ, recently identified as an anorexigenic neuropeptide, is an important regulator in the hypothalamic leptin signaling pathway. Coadministration of clusterin potentiates the anorexigenic effect of leptin and boosts leptin-induced hypothalamic Stat3 activation. In cultured neurons, clusterin enhances receptor binding and subsequent endocytosis of leptin. These effects are mainly mediated through the LDL receptor-related protein-2 (Lrp2). Notably, inhibition of hypothalamic clusterin, Lrp2 or endocytosis abrogates anorexia and hypothalamic Stat3 activation caused by leptin. These findings propose a novel regulatory mechanism in central leptin signaling pathways. © 2014 The Authors.

MCM ring hexamerization is a prerequisite for DNA-binding

DOE PAGES

Froelich, Clifford A.; Nourse, Amanda; Enemark, Eric J.

2015-09-13

The hexameric Minichromosome Maintenance (MCM) protein complex forms a ring that unwinds DNA at the replication fork in eukaryotes and archaea. Our recent crystal structure of an archaeal MCM N-terminal domain bound to single-stranded DNA (ssDNA) revealed ssDNA associating across tight subunit interfaces but not at the loose interfaces, indicating that DNA-binding is governed not only by the DNA-binding residues of the subunits (MCM ssDNA-binding motif, MSSB) but also by the relative orientation of the subunits. We now extend these findings to show that DNA-binding by the MCM N-terminal domain of the archaeal organism Pyrococcus furiosus occurs specifically in themore » hexameric oligomeric form. We show that mutants defective for hexamerization are defective in binding ssDNA despite retaining all the residues observed to interact with ssDNA in the crystal structure. One mutation that exhibits severely defective hexamerization and ssDNA-binding is at a conserved phenylalanine that aligns with the mouse Mcm4(Chaos3) mutation associated with chromosomal instability, cancer, and decreased intersubunit association.« less

Dpb11 may function with RPA and DNA to initiate DNA replication

PubMed Central

Bruck, Irina; Dhingra, Nalini; Martinez, Matthew P.

2017-01-01

Dpb11 is required for the initiation of DNA replication in budding yeast. We found that Dpb11 binds tightly to single-stranded DNA (ssDNA) or branched DNA structures, while its human homolog, TopBP1, binds tightly to branched-DNA structures. We also found that Dpb11 binds stably to CDK-phosphorylated RPA, the eukaryotic ssDNA binding protein, in the presence of branched DNA. A Dpb11 mutant specifically defective for DNA binding did not exhibit tight binding to RPA in the presence of DNA, suggesting that Dpb11-interaction with DNA may promote the recruitment of RPA to melted DNA. We then characterized a mutant of Dpb11 that is specifically defective in DNA binding in budding yeast cells. Expression of dpb11-m1,2,3,5,ΔC results in a substantial decrease in RPA recruitment to origins, suggesting that Dpb11 interaction with DNA may be required for RPA recruitment to origins. Expression of dpb11-m1,2,3,5,ΔC also results in diminished GINS interaction with Mcm2-7 during S phase, while Cdc45 interaction with Mcm2-7 is like wild-type. The reduced GINS interaction with Mcm2-7 may be an indirect consequence of diminished origin melting. We propose that the tight interaction between Dpb11, CDK-phosphorylated RPA, and branched-DNA may be required for the essential function of stabilizing melted origin DNA in vivo. We also propose an alternative model, wherein Dpb11-DNA interaction is required for some other function in DNA replication initiation, such as helicase activation. PMID:28467467

Evidence for glucocorticoid receptor binding to a site(s) in a remote region of the 5' flanking sequences of the human proopiomelanocortin gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tully, D.B.; Hillman, D.; Herbert, E.

1986-05-01

Glucocorticoids negatively regulate expression of the human proopiomelanocortin (POMC) gene. It has been postulated that this effect may be modulated by a direct interaction of the glucocorticoid receptor (GR) with DNA in the vicinity of the POMC promoter. In order to investigate interactions of GR with POMC DNA, DNA-cellulose competitive binding assays have been performed using isolated fragments of cloned POMC DNA to compete with calf thymus DNA-cellulose for binding of triamcinolone acetonide affinity-labelled GR prepared from HeLa S/sub 3/ cells. In these assays, two fragments isolated from the 5' flanking sequences of POMC DNA (Fragment 3,-1765 to -677 andmore » Fragment 4, -676 to +125 with respect to the mRNA cap site) have competed favorably, with Fragment 3 consistently competing more strongly than Fragment 4. Additional studies have been conducted utilizing a newly developed South-western Blot procedure in which specific /sup 32/P-labelled DNA fragments are allowed to bind to dexamethasone mesylate labelled GR immobilized on nitrocellulose filters. Results from these studies have also shown preferential binding by POMC DNA fragments 3 and 4. DNA footprinting and gene transfer experiments are now being conducted to further characterize the nature of GR interaction with POMC DNA.« less

An oncogenic axis of STAT-mediated BATF3 upregulation causing MYC activity in classical Hodgkin lymphoma and anaplastic large cell lymphoma.

PubMed

Lollies, A; Hartmann, S; Schneider, M; Bracht, T; Weiß, A L; Arnolds, J; Klein-Hitpass, L; Sitek, B; Hansmann, M-L; Küppers, R; Weniger, M A

2018-01-01

Classical Hodgkin lymphoma (cHL) and anaplastic large cell lymphoma (ALCL) feature high expression of activator protein-1 (AP-1) transcription factors, which regulate various physiological processes but also promote lymphomagenesis. The AP-1 factor basic leucine zipper transcription factor, ATF-like 3 (BATF3), is highly transcribed in cHL and ALCL; however, its functional importance in lymphomagenesis is unknown. Here we show that proto-typical CD30 + lymphomas, namely cHL (21/30) and primary mediastinal B-cell lymphoma (8/9), but also CD30 + diffuse large B-cell lymphoma (15/20) frequently express BATF3 protein. Mass spectrometry and co-immunoprecipitation established interactions of BATF3 with JUN and JUNB in cHL and ALCL lines. BATF3 knockdown using short hairpin RNAs was toxic for cHL and ALCL lines, reducing their proliferation and survival. We identified MYC as a critical BATF3 target and confirmed binding of BATF3 to the MYC promoter. JAK/STAT signaling regulated BATF3 expression, as chemical JAK2 inhibition reduced and interleukin 13 stimulation induced BATF3 expression in cHL lines. Chromatin immunoprecipitation substantiated a direct regulation of BATF3 by STAT proteins in cHL and ALCL lines. In conclusion, we identified STAT-mediated BATF3 expression that is essential for lymphoma cell survival and promoted MYC activity in cHL and ALCL, hence we recognized a new oncogenic axis in these lymphomas.

Response of brain oxygenation and metabolism to deep hypothermic circulatory arrest in newborn piglets: comparison of pH-stat and alpha-stat strategies.

PubMed

Markowitz, Scott D; Mendoza-Paredes, Alberto; Liu, Huiping; Pastuszko, Peter; Schultz, Steven P; Schears, Gregory J; Greeley, William J; Wilson, David F; Pastuszko, Anna

2007-07-01

To determine the effect of pH-stat as compared with alpha-stat management on brain oxygenation, level of striatal extracellular dopamine, phosphorylation, and levels of protein kinase B (Akt) and cyclic adenosine 3', 5'-monophosphate response element-binding protein (CREB), and levels of extracellular signal-regulated kinase (ERK)1/2, Bcl-2, and Bax in a piglet model of deep hypothermic circulatory arrest (DHCA). The piglets were placed on cardiopulmonary bypass (CPB), cooled with pH-stat or alpha-stat to 18 degrees C, subjected to 90 minutes of DHCA, rewarmed, weaned from CPB, and maintained for two hours recovery. The cortical oxygen was measured by: quenching of phosphorescence; dopamine by microdialysis; phosphorylation of CREB (p-CREB), ERK (p-ERK) 1/2, Akt (p-Akt), and level of Bcl-2, Bax by Western blots. Oxygen pressure histograms for the microvasculature of the cortex show substantially higher oxygen levels during cooling and during the oxygen depletion period after cardiac arrest (up to 15 minutes) when using pH-stat compared with alpha-stat management. Significant increases in dopamine occurred at 45 minutes and 60 minutes of DHCA in the alpha-stat and pH-stat groups, respectively. The p-CREB and p-Akt in the pH-stat group were significantly higher than in the alpha-stat group (140 +/- 9%, p < 0.05 and 125 +/- 6%, p < 0.05, respectively). There was no significant difference in p-ERK1/2 and Bax. The Bcl-2 increased in the pH-stat group to 121 +/- 4% (p < 0.05) compared with the alpha-stat group. The ratio Bcl-2:Bax increased in the pH-stat group compared with the alpha-stat group. The increase in p-CREB, p-Akt, Bcl-2, Bcl-2/Bax, and delay in increase of dopamine indicated that pH-stat, in the piglet model, prolongs "safe" time of DHCA and provides some brain protection against ischemic injury.

Gene structure, cDNA characterization and RNAi-based functional analysis of a myeloid differentiation factor 88 homolog in Tenebrio molitor larvae exposed to Staphylococcus aureus infection.

PubMed

Patnaik, Bharat Bhusan; Patnaik, Hongray Howrelia; Seo, Gi Won; Jo, Yong Hun; Lee, Yong Seok; Lee, Bok Luel; Han, Yeon Soo

2014-10-01

Myeloid differentiation factor 88 (MyD88), an intracellular adaptor protein involved in Toll/Toll-like receptor (TLR) signal processing, triggers activation of nuclear factor-kappaB (NF-κB) transcription factors. In the present study, we analyzed the gene structure and biological function of MyD88 in a coleopteran insect, Tenebrio molitor (TmMyD88). The TmMyD88 gene was 1380 bp in length and consisted of five exons and four introns. The 5'-flanking sequence revealed several putative transcription factor binding sites, such as STAT-4, AP-1, cJun, cfos, NF-1 and many heat shock factor binding elements. The cDNA contained a typical death domain, a conservative Toll-like interleukin-1 receptor (TIR) domain, and a C-terminal extension (CTE). The TmMyD88 TIR domain showed three significantly conserved motifs for interacting with the TIR domain of TLRs. TmMyD88 was grouped within the invertebrate cluster of the phylogenetic tree and shared 75% sequence identity with the TIR domain of Tribolium castaneum MyD88. Homology modeling of the TmMyD88 TIR domain revealed five parallel β-strands surrounded by five α-helices that adopted loop conformations to function as an adaptor. TmMyD88 expression was upregulated 7.3- and 4.79-fold after 12 and 6h, respectively, of challenge with Staphylococcus aureus and fungal β-1,3 glucan. Silencing of the TmMyD88 transcript by RNA interference led to reduced resistance of the host to infection by S. aureus. These results indicate that TmMyD88 is required for survival against Staphylococcus infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

Absence of suppressor of cytokine signalling 3 reduces self-renewal and promotes differentiation in murine embryonic stem cells.

PubMed

Forrai, Ariel; Boyle, Kristy; Hart, Adam H; Hartley, Lynne; Rakar, Steven; Willson, Tracy A; Simpson, Ken M; Roberts, Andrew W; Alexander, Warren S; Voss, Anne K; Robb, Lorraine

2006-03-01

Leukemia inhibitory factor (LIF) is required to maintain pluripotency and permit self-renewal of murine embryonic stem (ES) cells. LIF binds to a receptor complex of LIFR-beta and gp130 and signals via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, with signalling attenuated by suppressor of cytokine signalling (SOCS) proteins. Recent in vivo studies have highlighted the role of SOCS-3 in the negative regulation of signalling via gp130. To determine the role of SOCS-3 in ES cell biology, SOCS-3-null ES cell lines were generated. When cultured in LIF levels that sustain self-renewal of wild-type cells, SOCS-3-null ES cell lines exhibited less self-renewal and greater differentiation into primitive endoderm. The absence of SOCS-3 enhanced JAK-STAT and extracellular signal-related kinase 1/2 (ERK-1/2)-mitogen-activated protein kinase (MAPK) signal transduction via gp130, with higher levels of phosphorylated STAT-1, STAT-3, SH-2 domain-containing cytoplasmic protein tyrosine phosphatase 2 (SHP-2), and ERK-1/2 in steady state and in response to LIF stimulation. Attenuation of ERK signalling by the addition of MAPK/ERK kinase (MEK) inhibitors to SOCS-3-null ES cell cultures rescued the differentiation phenotype, but did not restore proliferation to wild-type levels. In summary, SOCS-3 plays a crucial role in the regulation of the LIF signalling pathway in murine ES cells. Its absence perturbs the balance between activation of the JAK-STAT and SHP-2-ERK-1/2-MAPK pathways, resulting in less self-renewal and a greater potential for differentiation into the primitive endoderm lineage.

Quantitative characterization of conformational-specific protein-DNA binding using a dual-spectral interferometric imaging biosensor

NASA Astrophysics Data System (ADS)

Zhang, Xirui; Daaboul, George G.; Spuhler, Philipp S.; Dröge, Peter; Ünlü, M. Selim

2016-03-01

DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions.DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions. Electronic supplementary information (ESI) available: DNA sequences and nomenclature (Table 1S); SDS-PAGE assay of IHF stock solution (Fig. 1S); determination of the concentration of IHF stock solution by Bradford assay (Fig. 2S); equilibrium binding isotherm fitting results of other DNA sequences (Table 2S); calculation of dissociation constants (Fig. 3S, 4S; Table 2S); geometric model for quantitation of DNA bending angle induced by specific IHF binding (Fig. 4S); customized flow cell assembly (Fig. 5S); real-time measurement of average fluorophore height change by SSFM (Fig. 6S); summary of binding parameters obtained from additive isotherm model fitting (Table 3S); average surface densities of 10 dsDNA spots and bound IHF at equilibrium (Table 4S); effects of surface densities on the binding and bending of dsDNA (Tables 5S, 6S and Fig. 7S-10S). See DOI: 10.1039/c5nr06785e

Effective targeting of STAT5-mediated survival in myeloproliferative neoplasms using ABT-737 combined with rapamycin

PubMed Central

Li, Geqiang; Miskimen, Kristy L.; Wang, Zhengqi; Xie, Xiu Yan; Tse, William; Gouilleux, Fabrice; Moriggl, Richard; Bunting, Kevin D.

2010-01-01

Signal transducer and activator of transcription-5 (STAT5) is a critical transcription factor for normal hematopoiesis and its sustained activation is associated with hematologic malignancy. A persistently active mutant of STAT5 (STAT5aS711F) associates with Grb2 associated binding protein 2 (Gab2) in myeloid leukemias and promotes growth in vitro through AKT activation. Here we have retrovirally transduced wild-type or Gab2−/− mouse bone marrow cells expressing STAT5aS711F and transplanted into irradiated recipient mice to test an in vivo myeloproliferative disease (MPD) model. To target Gab2-independent AKT/mTOR activation, wild-type mice were treated separately with rapamycin. In either case, mice lacking Gab2 or treated with rapamycin displayed attenuated myeloid hyperplasia and modestly improved survival, but the effects were not cytotoxic and were reversible. To improve upon this approach, in vitro targeting of STAT5-mediated AKT/mTOR using rapamycin was combined with inhibition of the STAT5 direct target genes bcl-2 and bcl-XL using ABT-737. Striking synergy with both drugs was observed in mouse BaF3 cells expressing STAT5aS711F, TEL-JAK2, or BCR-ABL and in the relatively single agent-resistant human BCR-ABL positive K562 cell line. Therefore, targeting distinct STAT5 mediated survival signals, e.g. bcl-2/bcl-XL and AKT/mTOR may be an effective therapeutic approach for human myeloproliferative neoplasms. PMID:20535152

Structural insight into the specificity of the B3 DNA-binding domains provided by the co-crystal structure of the C-terminal fragment of BfiI restriction enzyme

PubMed Central

Golovenko, Dmitrij; Manakova, Elena; Zakrys, Linas; Zaremba, Mindaugas; Sasnauskas, Giedrius; Gražulis, Saulius; Siksnys, Virginijus

2014-01-01

The B3 DNA-binding domains (DBDs) of plant transcription factors (TF) and DBDs of EcoRII and BfiI restriction endonucleases (EcoRII-N and BfiI-C) share a common structural fold, classified as the DNA-binding pseudobarrel. The B3 DBDs in the plant TFs recognize a diverse set of target sequences. The only available co-crystal structure of the B3-like DBD is that of EcoRII-N (recognition sequence 5′-CCTGG-3′). In order to understand the structural and molecular mechanisms of specificity of B3 DBDs, we have solved the crystal structure of BfiI-C (recognition sequence 5′-ACTGGG-3′) complexed with 12-bp cognate oligoduplex. Structural comparison of BfiI-C–DNA and EcoRII-N–DNA complexes reveals a conserved DNA-binding mode and a conserved pattern of interactions with the phosphodiester backbone. The determinants of the target specificity are located in the loops that emanate from the conserved structural core. The BfiI-C–DNA structure presented here expands a range of templates for modeling of the DNA-bound complexes of the B3 family of plant TFs. PMID:24423868

Modeling the interactions of the nucleotide excision repair UvrA(2) dimer with DNA.

PubMed

Gantchev, Tsvetan G; Hunting, Darel J

2010-12-28

The UvrA protein initiates the DNA damage recognition process by the bacterial nucleotide excision repair (NER) system. Recently, crystallographic structures of holo-UvrA(2) dimers from two different microorganisms have been released (Protein Data Bank entries 2r6f , 2vf7 , and 2vf8 ). However, the details of the DNA binding by UvrA(2) and other peculiarities involved in the damage recognition process remain unknown. We have undertaken a molecular modeling approach to appraise the possible modes of DNA-UvrA(2) interaction using molecular docking and short-scale guided molecular dynamics [continuum field, constrained, and/or unrestricted simulated annealing (SA)], taking into account the three-dimensional location of a series of mutation-identified UvrA residues implicated in DNA binding. The molecular docking was based on the assumptions that the UvrA(2) dimer is preformed prior to DNA binding and that no major protein conformational rearrangements, except moderate domain reorientations, are required for binding of undamaged DNA. As a first approximation, DNA was treated as a rigid ligand. From the electrostatic relief of the ventral surface of UvrA(2), we initially identified three, noncollinear DNA binding paths. Each of the three resulting nucleoprotein complexes (C1, C2, and C3) was analyzed separately, including calculation of binding energies, the number and type of interaction residues (including mutated ones), and the predominant mode of translational and rotational motion of specific protein domains after SA to ensure improved DNA binding. The UvrA(2) dimer can accommodate DNA in all three orientations, albeit with different binding strengths. One of the UvrA(2)-DNA complexes (C1) fulfilled most of the requirements (high interaction energy, proximity of DNA to mutated residues, etc.) expected for a natural, high-affinity DNA binding site. This nucleoprotein presents a structural organization that is designed to clamp and bend double-stranded DNA. We examined the binding site in more detail by docking DNAs of significantly different (AT- vs CG-enriched) sequences and by submitting the complexes to DNA-unrestricted SA. It was found that in a manner independent of the DNA sequence and applied MD protocols, UvrA(2) favors binding of a bent and unwound undamaged DNA, with a kink positioned in the proximity of the Zn3 hairpins, anticollinearly aligned at the bottom of the ventral protein surface. It is further hypothesized that the Zn3 modules play an essential role in the damage recognition process and that the apparent existence of a family of DNA binding sites might be biologically relevant. Our data should prove to be useful in rational (structure-based) mutation studies.

Selective affinity chromatography of DNA polymerases with associated 3' to 5' exonuclease activities.

PubMed

Lee, M Y; Whyte, W A

1984-05-01

The use of 5'-AMP as a ligand for the affinity chromatography of DNA polymerases with intrinsic 3' to 5' exonuclease activities was investigated. The basis for this is that 5'-AMP would be expected to act as a ligand for the associated 3' to 5' exonuclease. The requirements for binding of Escherichia coli DNA polymerase I, T4 DNA polymerase, and calf thymus DNA polymerase delta, all of which have associated 3' to 5' exonuclease activities, to several commercially available 5'-AMP supports with different linkages of 5'-AMP to either agarose or cellulose were examined. The DNA polymerases which possessed 3' to 5' exonuclease activities were bound to agarose types in which the 5'-phosphoryl group and the 3'-hydroxyl group of the AMP were unsubstituted. Bound enzyme could be eluted by either an increase in ionic strength or competitive binding of nucleoside 5'-monophosphates. Magnesium was found to reinforce the binding of the enzyme to these affinity supports. DNA polymerase alpha, which does not have an associated 3' to 5' exonuclease activity, did not bind to any of these columns. These differences can be used to advantage for the purification of DNA polymerases that have associated 3' to 5' exonuclease activities, as well as a means for establishing the association of 3' to 5' exonuclease activities with DNA polymerases.

Hospicells promote upregulation of the ATP-binding cassette genes by insulin-like growth factor-I via the JAK2/STAT3 signaling pathway in an ovarian cancer cell line.

PubMed

Benabbou, Nadia; Mirshahi, Pezhman; Cadillon, Mélodie; Soria, Jeannette; Therwath, Amu; Mirshahi, Massoud

2013-09-01

Interaction between tumor cells and their micro-environment has a crucial role in the development, progression and drug resistance of cancer. Our objective was to confirm the role of Hospicells, which are stromal cells from the cancer microenvironment, in drug resistance and tumor cell growth. We demonstrated that soluble factors secreted by Hospicells activate several genes and upregulate the JAK/STAT signaling pathway in ovarian cancer cell lines. Hospicells express all insulin-like growth factor (IGF) family as detected by gene array, RT-PCR, protein array and immunocytochemistry. While focusing attention on the microenvironment, we considered the role of IGF-I in proliferation and survival of ovarian cancer cells. Indeed, IGF-I is a major regulator of different stages of cancer development. We studied the effect of exogenously added IGF-I on the regulation of ATP-binding cassette (ABC) genes (MDR1, MRP1, MRP2, MRP3, MRP5 and BCRP) in the ovarian cancer cell line OVCAR3 and validated the results obtained using the IGF-IR antagonist picropodophyllin. IGF-I regulates the expression of ABC genes in OVCAR3 cells via the PI3-kinase, MEK and JAK2/STAT3 signaling pathways. The OVCAR3 cell line when co-cultured with Hospicells showed a marked degree of drug resistance. The drug resistance observed could be amplified with exogenous IGF-I. Addition of IGF-IR inhibitor, however, reduced the degree of resistance in these exposed cells. Cells that were treated with anticancer drugs and then exposed to IGF-I showed an increase in drug resistance and, thereby, an increase in cell survival. This observation indicates that drug resistance of OVCAR3 cells increases when there is synergy between OVCAR3 cells and Hospicells and it is amplified when IGF-I was exogenously added. In conclusion, inhibition of IGF-IR and targeting of the JAK2/STAT3 signaling pathway can be a target for ovarian cancer therapy.

Hospicells promote upregulation of the ATP-binding cassette genes by insulin-like growth factor-I via the JAK2/STAT3 signaling pathway in an ovarian cancer cell line

PubMed Central

BENABBOU, NADIA; MIRSHAHI, PEZHMAN; CADILLON, MÉLODIE; SORIA, JEANNETTE; THERWATH, AMU; MIRSHAHI, MASSOUD

2013-01-01

Interaction between tumor cells and their microenvironment has a crucial role in the development, progression and drug resistance of cancer. Our objective was to confirm the role of Hospicells, which are stromal cells from the cancer microenvironment, in drug resistance and tumor cell growth. We demonstrated that soluble factors secreted by Hospicells activate several genes and upregulate the JAK/STAT signaling pathway in ovarian cancer cell lines. Hospicells express all insulin-like growth factor (IGF) family as detected by gene array, RT-PCR, protein array and immunocytochemistry. While focusing attention on the microenvironment, we considered the role of IGF-I in proliferation and survival of ovarian cancer cells. Indeed, IGF-I is a major regulator of different stages of cancer development. We studied the effect of exogenously added IGF-I on the regulation of ATP-binding cassette (ABC) genes (MDR1, MRP1, MRP2, MRP3, MRP5 and BCRP) in the ovarian cancer cell line OVCAR3 and validated the results obtained using the IGF-IR antagonist picropodophyllin. IGF-I regulates the expression of ABC genes in OVCAR3 cells via the PI3-kinase, MEK and JAK2/STAT3 signaling pathways. The OVCAR3 cell line when co-cultured with Hospicells showed a marked degree of drug resistance. The drug resistance observed could be amplified with exogenous IGF-I. Addition of IGF-IR inhibitor, however, reduced the degree of resistance in these exposed cells. Cells that were treated with anticancer drugs and then exposed to IGF-I showed an increase in drug resistance and, thereby, an increase in cell survival. This observation indicates that drug resistance of OVCAR3 cells increases when there is synergy between OVCAR3 cells and Hospicells and it is amplified when IGF-I was exogenously added. In conclusion, inhibition of IGF-IR and targeting of the JAK2/STAT3 signaling pathway can be a target for ovarian cancer therapy. PMID:23857432

Interaction of Zn(II)bleomycin-A2 and Zn(II)peplomycin with a DNA hairpin containing the 5'-GT-3' binding site in comparison with the 5'-GC-3' binding site studied by NMR spectroscopy.

PubMed

Follett, Shelby E; Ingersoll, Azure D; Murray, Sally A; Reilly, Teresa M; Lehmann, Teresa E

2017-10-01

Bleomycins are a group of glycopeptide antibiotics synthesized by Streptomyces verticillus that are widely used for the treatment of various neoplastic diseases. These antibiotics have the ability to chelate a metal center, mainly Fe(II), and cause site-specific DNA cleavage. Bleomycins are differentiated by their C-terminal regions. Although this antibiotic family is a successful course of treatment for some types of cancers, it is known to cause pulmonary fibrosis. Previous studies have identified that bleomycin-related pulmonary toxicity is linked to the C-terminal region of these drugs. This region has been shown to closely interact with DNA. We examined the binding of Zn(II)peplomycin and Zn(II)bleomycin-A 2 to a DNA hairpin of sequence 5'-CCAGTATTTTTACTGG-3', containing the binding site 5'-GT-3', and compared the results with those obtained from our studies of the same MBLMs bound to a DNA hairpin containing the binding site 5'-GC-3'. We provide evidence that the DNA base sequence has a strong impact in the final structure of the drug-target complex.

Dihydroartemisinin induces autophagy-dependent death in human tongue squamous cell carcinoma cells through DNA double-strand break-mediated oxidative stress

PubMed Central

Li, Xiaoming; Bai, Jing; Li, Jianchun; Li, Shenghao; Wang, Zeming; Zhou, Mingrui

2017-01-01

Dihydroartemisinin is an effective antimalarial agent with multiple biological activities. In the present investigation, we elucidated its therapeutic potential and working mechanism on human tongue squamous cell carcinoma (TSCC). It was demonstrated that dihydroartemisinin could significantly inhibit cell growth in a dose- and time-dependent manner by the Cell Counting Kit-8 and colony formation assay in vitro. Meanwhile, autophagy was promoted in the Cal-27 cells treated by dihydroartemisinin, evidenced by increased LC3B-II level, increased autophagosome formation, and increased Beclin-1 level compared to dihydroartemisinin-untreated cells. Importantly, dihydroartemisinin caused DNA double-strand break with simultaneously increased γH2AX foci and oxidative stress; this inhibited the nuclear localization of phosphorylated signal transducer and activator of transcription 3 (p-STAT3), finally leading to autophagic cell death. Furthermore, the antitumor effect of dihydroartemisinin-monotherapy was confirmed with a mouse xenograft model, and no kidney injury associated with toxic effect was observed after intraperitoneal injection with dihydroartemisinin for 3 weeks in vivo. In the present study, it was revealed that dihydroartemisinin-induced DNA double-strand break promoted oxidative stress, which decreased p-STAT3 (Tyr705) nuclear localization, and successively increased autophagic cell death in the Cal-27 cells. Thus, dihydroartemisinin alone may represent an effective and safe therapeutic agent for human TSCC. PMID:28526807

Dynamic DNA binding licenses a repair factor to bypass roadblocks in search of DNA lesions

PubMed Central

Brown, Maxwell W.; Kim, Yoori; Williams, Gregory M.; Huck, John D.; Surtees, Jennifer A.; Finkelstein, Ilya J.

2016-01-01

DNA-binding proteins search for specific targets via facilitated diffusion along a crowded genome. However, little is known about how crowded DNA modulates facilitated diffusion and target recognition. Here we use DNA curtains and single-molecule fluorescence imaging to investigate how Msh2–Msh3, a eukaryotic mismatch repair complex, navigates on crowded DNA. Msh2–Msh3 hops over nucleosomes and other protein roadblocks, but maintains sufficient contact with DNA to recognize a single lesion. In contrast, Msh2–Msh6 slides without hopping and is largely blocked by protein roadblocks. Remarkably, the Msh3-specific mispair-binding domain (MBD) licences a chimeric Msh2–Msh6(3MBD) to bypass nucleosomes. Our studies contrast how Msh2–Msh3 and Msh2–Msh6 navigate on a crowded genome and suggest how Msh2–Msh3 locates DNA lesions outside of replication-coupled repair. These results also provide insights into how DNA repair factors search for DNA lesions in the context of chromatin. PMID:26837705

NMR and computational methods applied to the 3- dimensional structure determination of DNA and ligand-DNA complexes in solution

NASA Astrophysics Data System (ADS)

Smith, Jarrod Anson

2D homonuclear 1H NMR methods and restrained molecular dynamics (rMD) calculations have been applied to determining the three-dimensional structures of DNA and minor groove-binding ligand-DNA complexes in solution. The structure of the DNA decamer sequence d(GCGTTAACGC)2 has been solved both with a distance-based rMD protocol and an NOE relaxation matrix backcalculation-based protocol in order to probe the relative merits of the different refinement methods. In addition, three minor groove binding ligand-DNA complexes have been examined. The solution structure of the oligosaccharide moiety of the antitumor DNA scission agent calicheamicin γ1I has been determined in complex with a decamer duplex containing its high affinity 5'-TCCT- 3' binding sequence. The structure of the complex reinforces the belief that the oligosaccharide moiety is responsible for the sequence selective minor-groove binding activity of the agent, and critical intermolecular contacts are revealed. The solution structures of both the (+) and (-) enantiomers of the minor groove binding DNA alkylating agent duocarmycin SA have been determined in covalent complex with the undecamer DNA duplex d(GACTAATTGTC).d(GAC AATTAGTC). The results support the proposal that the alkylation activity of the duocarmycin antitumor antibiotics is catalyzed by a binding-induced conformational change in the ligand which activates the cyclopropyl group for reaction with the DNA. Comparisons between the structures of the two enantiomers covalently bound to the same DNA sequence at the same 5'-AATTA-3 ' site have provided insight into the binding orientation and site selectivity, as well as the relative rates of reactivity of these two agents.

The DNA-recognition mode shared by archaeal feast/famine-regulatory proteins revealed by the DNA-binding specificities of TvFL3, FL10, FL11 and Ss-LrpB

PubMed Central

Yokoyama, Katsushi; Nogami, Hideki; Kabasawa, Mamiko; Ebihara, Sonomi; Shimowasa, Ai; Hashimoto, Keiko; Kawashima, Tsuyoshi; Ishijima, Sanae A.; Suzuki, Masashi

2009-01-01

The DNA-binding mode of archaeal feast/famine-regulatory proteins (FFRPs), i.e. paralogs of the Esherichia coli leucine-responsive regulatory protein (Lrp), was studied. Using the method of systematic evolution of ligands by exponential enrichment (SELEX), optimal DNA duplexes for interacting with TvFL3, FL10, FL11 and Ss-LrpB were identified as TACGA[AAT/ATT]TCGTA, GTTCGA[AAT/ATT]TCGAAC, CCGAAA[AAT/ATT]TTTCGG and TTGCAA[AAT/ATT]TTGCAA, respectively, all fitting into the form abcdeWWWedcba. Here W is A or T, and e.g. a and a are bases complementary to each other. Apparent equilibrium binding constants of the FFRPs and various DNA duplexes were determined, thereby confirming the DNA-binding specificities of the FFRPs. It is likely that these FFRPs recognize DNA in essentially the same way, since their DNA-binding specificities were all explained by the same pattern of relationship between amino-acid positions and base positions to form chemical interactions. As predicted from this relationship, when Gly36 of TvFL3 was replaced by Thr, the b base in the optimal DNA duplex changed from A to T, and, when Thr36 of FL10 was replaced by Ser, the b base changed from T to G/A. DNA-binding characteristics of other archaeal FFRPs, Ptr1, Ptr2, Ss-Lrp and LysM, are also consistent with the relationship. PMID:19468044

Differences in DNA Binding Specificity of Floral Homeotic Protein Complexes Predict Organ-Specific Target Genes.

PubMed

Smaczniak, Cezary; Muiño, Jose M; Chen, Dijun; Angenent, Gerco C; Kaufmann, Kerstin

2017-08-01

Floral organ identities in plants are specified by the combinatorial action of homeotic master regulatory transcription factors. However, how these factors achieve their regulatory specificities is still largely unclear. Genome-wide in vivo DNA binding data show that homeotic MADS domain proteins recognize partly distinct genomic regions, suggesting that DNA binding specificity contributes to functional differences of homeotic protein complexes. We used in vitro systematic evolution of ligands by exponential enrichment followed by high-throughput DNA sequencing (SELEX-seq) on several floral MADS domain protein homo- and heterodimers to measure their DNA binding specificities. We show that specification of reproductive organs is associated with distinct binding preferences of a complex formed by SEPALLATA3 and AGAMOUS. Binding specificity is further modulated by different binding site spacing preferences. Combination of SELEX-seq and genome-wide DNA binding data allows differentiation between targets in specification of reproductive versus perianth organs in the flower. We validate the importance of DNA binding specificity for organ-specific gene regulation by modulating promoter activity through targeted mutagenesis. Our study shows that intrafamily protein interactions affect DNA binding specificity of floral MADS domain proteins. Differential DNA binding of MADS domain protein complexes plays a role in the specificity of target gene regulation. © 2017 American Society of Plant Biologists. All rights reserved.

Optical tweezers reveal how proteins alter replication

NASA Astrophysics Data System (ADS)

Chaurasiya, Kathy

Single molecule force spectroscopy is a powerful method that explores the DNA interaction properties of proteins involved in a wide range of fundamental biological processes such as DNA replication, transcription, and repair. We use optical tweezers to capture and stretch a single DNA molecule in the presence of proteins that bind DNA and alter its mechanical properties. We quantitatively characterize the DNA binding mechanisms of proteins in order to provide a detailed understanding of their function. In this work, we focus on proteins involved in replication of Escherichia coli (E. coli ), endogenous eukaryotic retrotransposons Ty3 and LINE-1, and human immunodeficiency virus (HIV). DNA polymerases replicate the entire genome of the cell, and bind both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) during DNA replication. The replicative DNA polymerase in the widely-studied model system E. coli is the DNA polymerase III subunit alpha (DNA pol III alpha). We use optical tweezers to determine that UmuD, a protein that regulates bacterial mutagenesis through its interactions with DNA polymerases, specifically disrupts alpha binding to ssDNA. This suggests that UmuD removes alpha from its ssDNA template to allow DNA repair proteins access to the damaged DNA, and to facilitate exchange of the replicative polymerase for an error-prone translesion synthesis (TLS) polymerase that inserts nucleotides opposite the lesions, so that bacterial DNA replication may proceed. This work demonstrates a biophysical mechanism by which E. coli cells tolerate DNA damage. Retroviruses and retrotransposons reproduce by copying their RNA genome into the nuclear DNA of their eukaryotic hosts. Retroelements encode proteins called nucleic acid chaperones, which rearrange nucleic acid secondary structure and are therefore required for successful replication. The chaperone activity of these proteins requires strong binding affinity for both single- and double-stranded nucleic acids. We use single molecule DNA stretching to show that the nucleocapsid protein (NC) of the yeast retrotransposon Ty3, which is likely to be an ancestor of HIV NC, has optimal nucleic acid chaperone activity with only a single zinc finger. We also show that the chaperone activity of the ORF1 protein is responsible for successful replication of the mouse LINE-1 retrotransposon. LINE-1 is also 17% of the human genome, where it generates insertion mutations and alters gene expression. Retrotransposons such as LINE-1 and Ty3 are likely to be ancestors of retroviruses such as HIV. Human APOBEC3G (A3G) inhibits HIV-1 replication via cytidine deamination of the viral ssDNA genome, as well as via a distinct deamination-independent mechanism. Efficient deamination requires rapid on-off binding kinetics, but a slow dissociation rate is required for the proposed deaminase-independent mechanism. We resolve this apparent contradiction with a new quantitative single molecule method, which shows that A3G initially binds ssDNA with fast on-off rates and subsequently converts to a slow binding mode. This suggests that oligomerization transforms A3G from a fast enzyme to a slow binding protein, which is the biophysical mechanism that allows A3G to inhibit HIV replication. A complete understanding of the mechanism of A3G-mediated antiviral activity is required to design drugs that disrupt the viral response to A3G, enhance A3G packaging inside the viral core, and other potential strategies for long-term treatment of HIV infection. We use single molecule biophysics to explore the function of proteins involved in bacterial DNA replication, endogenous retrotransposition of retroelements in eukaryotic hosts such yeast and mice, and HIV replication in human cells. Our quantitative results provide insight into protein function in a range of complex biological systems and have wide-ranging implications for human health.

Study binding of Al-curcumin complex to ds-DNA, monitoring by multispectroscopic and voltammetric techniques

NASA Astrophysics Data System (ADS)

Ahmadi, F.; Alizadeh, A. A.; Shahabadi, N.; Rahimi-Nasrabadi, M.

2011-09-01

In this work a complex of Al 3+ with curcumin ([Al(curcumin) (EtOH) 2](NO 3) 2) was synthesized and characterized by UV-vis, FT-IR, elemental analysis and spectrophotometric titration techniques. The mole ratio plot revealed a 1:1 complex between Al 3+ and curcumin in solution. For binding studies of this complex to calf thymus-DNA various methods such as: UV-vis, fluorescence, circular dichroism (CD), FT-IR spectroscopy and cyclic voltammetry were used. The intrinsic binding constant of ACC with DNA at 25 °C was calculated by UV-vis and cyclic voltammetry as 2.1 × 10 4 and 2.6 × 10 4, respectively. The thermodynamic studies showed that the reaction is enthalpy and entropy favored. The CD results showed that only the Δ-ACC interacts with DNA and the Δ-ACC form has not any tendency to interact with DNA, also the pure curcumin has not any stereoselective interaction with CT-DNA. Fluorimetric studies showed that fluorescence enhancement was initiated by a static process in the ground state. The cyclic voltammetry showed that ACC interact with DNA with a binding site size of 2. From the FT-IR we concluded that the Δ-ACC interacts with DNA via partial electrostatic and minor groove binding. In comparison with previous works it was concluded that curcumin significantly reduced the affinity of Al 3+ to the DNA.

The clinical value of HPV E6/E7 and STAT3 mRNA detection in cervical cancer screening.

PubMed

Fan, Yibing; Shen, Zongji

2018-05-01

To explore the value of human papillomavirus (HPV) E6/E7 and signal transducer and activator of transcription 3 (STAT3) mRNA detection in the screening of cervical lesions. 192 patients with abnormal ThinPrep cytology test (TCT) results and/or high-risk HPV infection were screened to identify possible cervical lesions in cases. Diagnoses were confirmed by histopathology. Fluorescence in situ hybridization (FISH) was performed to detect and qualify the mRNAs of HPV E6/E7, STAT3, and Survivin in cervical exfoliated cells. In addition, the performance of separate and combined mRNA detection methods were compared with TCT, HR-HPV DNA schemes respectively. 1. Compared with HPVE6/E7 and STAT3 mRNA methods, Survivin mRNA assay had poor specificity (Sp), Youden index (YI) and concordance rate. 2. HPV E6/E7, STAT3, and STAT3 + HR-HPV methods had the best Sp, concordance rate and positive predictive value (PPV) for cervical lesions screening and atypical squamous cells of undetermined significance (ASCUS) triage. For screening of high grade squamous intraepithelial lesions or greater (HSILs+), no difference was observed in the Se of mRNA detection methods in comparison with that of TCT, HR-HPV and TCT + HR-HPV, whereas the false positive rate (FPR) decreased by 41.48%/55.99%/17.19% and the colposcopy referral rate reduced by about 20.00%/25.00%/11.17%. For triage of women with ASCUS, no difference was observed in the Se of mRNA detection methods as compared to that of HR-HPV (χ 2  = 1.05, P > 0.75), while the FPR decreased by 45.83%/37.50%/41.66% and the colposcopy referral rate reduced by 32.42%/22.60%/25.28%, respectively. The Se, YI, and PPV of the combined methods increased in comparison to each method alone. 3. Compared with the TCT + HR-HPV method, HPV E6/E7 + STAT3 method had perfect Sp (95.92%) and PPV (95.40%) for screening HSILs+, the FPR and colposcopy referral rate decreased by 31.06% and 22.48% respectively. 1. The expression of HPV E6/E7 and STAT3 mRNA confirmed using FISH assay is expected to be a new method and molecular marker for cervical lesions screening. Survivin mRNA was excluded due to its poor performance. 2. HPV E6/E7, STAT3, and STAT3 +HR-HPV assays could be new approaches for cervical cancer screening and ASCUS triage, and the efficiency of combined screening program was better than that of a separate one. 3. HPV E6/E7 + STAT3 regimen is expected to be a diagnostic strategy for cervical lesions. Copyright © 2018 Elsevier GmbH. All rights reserved.

The ebola virus interferon antagonist VP24 directly binds STAT1 and has a novel, pyramidal fold.

PubMed

Zhang, Adrianna P P; Bornholdt, Zachary A; Liu, Tong; Abelson, Dafna M; Lee, David E; Li, Sheng; Woods, Virgil L; Saphire, Erica Ollmann

2012-02-01

Ebolaviruses cause hemorrhagic fever with up to 90% lethality and in fatal cases, are characterized by early suppression of the host innate immune system. One of the proteins likely responsible for this effect is VP24. VP24 is known to antagonize interferon signaling by binding host karyopherin α proteins, thereby preventing them from transporting the tyrosine-phosphorylated transcription factor STAT1 to the nucleus. Here, we report that VP24 binds STAT1 directly, suggesting that VP24 can suppress at least two distinct branches of the interferon pathway. Here, we also report the first crystal structures of VP24, derived from different species of ebolavirus that are pathogenic (Sudan) and nonpathogenic to humans (Reston). These structures reveal that VP24 has a novel, pyramidal fold. A site on a particular face of the pyramid exhibits reduced solvent exchange when in complex with STAT1. This site is above two highly conserved pockets in VP24 that contain key residues previously implicated in virulence. These crystal structures and accompanying biochemical analysis map differences between pathogenic and nonpathogenic viruses, offer templates for drug design, and provide the three-dimensional framework necessary for biological dissection of the many functions of VP24 in the virus life cycle.

Epigenetic Signatures at AQP3 and SOCS3 Engage in Low-Grade Inflammation across Different Tissues

PubMed Central

Marzi, Carola; Holdt, Lesca M; Fiorito, Giovanni; Tsai, Pei-Chien; Kretschmer, Anja; Wahl, Simone; Guarrera, Simonetta; Teupser, Daniel; Spector, Tim D.; Iacoviello, Licia; Sacerdote, Carlotta; Strauch, Konstantin; Lee, Serene; Thasler, Wolfgang E.; Peters, Annette; Thorand, Barbara; Wolf, Petra; Prokisch, Holger; Tumino, Rosario; Gieger, Christian; Krogh, Vittorio; Panico, Salvatore; Bell, Jordana T.; Matullo, Giuseppe

2016-01-01

Background Elevated levels of C-reactive protein (CRP, determined by a high-sensitivity assay) indicate low-grade inflammation which is implicated in many age-related disorders. Epigenetic studies on CRP might discover molecular mechanisms underlying CRP regulation. We aimed to identify DNA methylation sites related to CRP concentrations in cells and tissues regulating low-grade inflammation. Results Genome-wide DNA methylation was measured in peripheral blood in 1,741 participants of the KORA F4 study using Illumina HumanMethylation450 BeadChip arrays. Four CpG sites (located at BCL3, AQP3, SOCS3, and cg19821297 intergenic at chromosome 19p13.2, P ≤ 1.01E-07) were significantly hypomethylated at high CRP concentrations independent of various confounders including age, sex, BMI, smoking, and white blood cell composition. Findings were not sex-specific. CRP-related top genes were enriched in JAK/STAT pathways (Benjamini-Hochberg corrected P < 0.05). Results were followed-up in three studies using DNA from peripheral blood (EPICOR, n = 503) and adipose tissue (TwinsUK, n = 368) measured as described above and from liver tissue (LMU liver cohort, n = 286) measured by MALDI-TOF mass spectrometry using EpiTYPER. CpG sites at the AQP3 locus (significant p-values in peripheral blood = 1.72E-03 and liver tissue = 1.51E-03) and the SOCS3 locus (p-values in liver < 2.82E-05) were associated with CRP in the validation panels. Conclusions Epigenetic modifications seem to engage in low-grade inflammation, possibly via JAK/STAT mediated pathways. Results suggest a shared relevance across different tissues at the AQP3 locus and highlight a role of DNA methylation for CRP regulation at the SOCS3 locus. PMID:27824951

Amino acids 16-275 of minute virus of mice NS1 include a domain that specifically binds (ACCA)2-3-containing DNA.

PubMed

Mouw, M; Pintel, D J

1998-11-10

GST-NS1 purified from Escherichia coli and insect cells binds double-strand DNA in an (ACCA)2-3-dependent fashion under similar ionic conditions, independent of the presence of anti-NS1 antisera or exogenously supplied ATP and interacts with single-strand DNA and RNA in a sequence-independent manner. An amino-terminal domain (amino acids 1-275) of NS1 [GST-NS1(1-275)], representing 41% of the full-length NS1 molecule, includes a domain that binds double-strand DNA in a sequence-specific manner at levels comparable to full-length GST-NS1, as well as single-strand DNA and RNA in a sequence-independent manner. The deletion of 15 additional amino-terminal amino acids yielded a molecule [GST-NS1(1-275)] that maintained (ACCA)2-3-specific double-strand DNA binding; however, this molecule was more sensitive to increasing ionic conditions than full-length GST-NS1 and GST-NS1(1-275) and could not be demonstrated to bind single-strand nucleic acids. A quantitative filter binding assay showed that E. coli- and baculovirus-expressed GST-NS1 and E. coli GST-NS1(1-275) specifically bound double-strand DNA with similar equilibrium kinetics [as measured by their apparent equilibrium DNA binding constants (KD)], whereas GST-NS1(16-275) bound 4- to 8-fold less well. Copyright 1998 Academic Press.

Self-Assembled Multivalent (SAMul) Polyanion Binding-Impact of Hydrophobic Modifications in the Micellar Core on DNA and Heparin Binding at the Peripheral Cationic Ligands.

PubMed

Albanyan, Buthaina; Laurini, Erik; Posocco, Paola; Pricl, Sabrina; Smith, David K

2017-05-05

This paper reports a small family of cationic surfactants designed to bind polyanions such as DNA and heparin. Each molecule has the same hydrophilic cationic ligand and a hydrophobic aliphatic group with eighteen carbon atoms with one, two, or three alkene groups within the hydrophobic chain (C18-1, C18-2 and C18-3). Dynamic light scattering indicates that more alkenes lead to geometric distortion, giving rise to larger self-assembled multivalent (SAMul) nanostructures. Mallard Blue and Ethidium Bromide dye displacement assays demonstrate that heparin and DNA have markedly different binding preferences, with heparin binding most effectively to C18-1, and DNA to C18-3, even though the molecular structural differences of these SAMul systems are buried in the hydrophobic core. Multiscale modelling suggests that adaptive heparin maximises enthalpically favourable interactions with C18-1, while shape-persistent DNA forms a similar number of interactions with each ligand display, but with slightly less entropic cost for binding to C18-3-fundamental thermodynamic differences in SAMul binding of heparin or DNA. This study therefore provides unique insight into electrostatic molecular recognition between highly charged nanoscale surfaces in biologically relevant systems. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Elevated levels of STAT1 in Fanconi anemia group A lymphoblasts correlate with the cells’ sensitivity to DNA interstrand crosslinking drugs

PubMed Central

Prieto-Remón, Inés; Sánchez-Carrera, Dámaso; López-Duarte, Mónica; Richard, Carlos; Pipaón, Carlos

2013-01-01

Progressive bone marrow failure starting in the first decade of life is one of the main characteristics of Fanconi anemia. Along with the bone marrow failure, this pathology is characterized by congenital malformations, endocrine dysfunction and an extraordinary predisposition to develop cancer. The fact that hematopoietic progenitor cells from subjects with Fanconi anemia are sensitive to both DNA-interstrand crosslinking agents and inflammatory cytokines, which are aberrantly overproduced in these patients, has led to different explanations for the causes of the bone marrow failure. We analyzed STAT1 expression in lymphoblastoid cell lines derived from patients with Fanconi anemia group A and correlated this with aspects of the Fanconi anemia phenotype such as sensitivity to genotoxic agents or to inhibitory cytokines. We provide evidence of overexpression of STAT1 in FANCA-deficient cells which has both transcriptional and post-translational components, and is related to the constitutive activation of ERK in Fanconi anemia group A cells, since it can be reverted by treatment with U0126. STAT1 phosphorylation was not defective in the lymphoblasts, so these cells accumulated higher levels of active STAT1 in response to interferon gamma, probably in relation to their greater sensitivity to this cytokine. On the other hand, inhibition of STAT1 by genetic or chemical means reverted the hypersensitivity of Fanconi anemia group A lymphoblasts to DNA interstrand crosslinking agents. Our data provide an explanation for the mixed sensitivity of Fanconi anemia group A cells to both genotoxic stress and inflammatory cytokines and indicate new targets for the treatment of bone marrow failure in these patients. PMID:23585528

Thermodynamic characterization of binding Oxytricha nova single strand telomere DNA with the alpha protein N-terminal domain.

PubMed

Buczek, Pawel; Horvath, Martin P

2006-06-23

The Oxytricha nova telemere binding protein alpha subunit binds single strand DNA and participates in a nucleoprotein complex that protects the very ends of chromosomes. To understand how the N-terminal, DNA binding domain of alpha interacts with DNA we measured the stoichiometry, enthalpy (DeltaH), entropy (DeltaS), and dissociation constant (K(D-DNA)) for binding telomere DNA fragments at different temperatures and salt concentrations using native gel electrophoresis and isothermal titration calorimetry (ITC). About 85% of the total free energy of binding corresponded with non-electrostatic interactions for all DNAs. Telomere DNA fragments d(T(2)G(4)), d(T(4)G(4)), d(G(3)T(4)G(4)), and d(G(4)T(4)G(4)) each formed monovalent protein complexes. In the case of d(T(4)G(4)T(4)G(4)), which has two tandemly repeated d(TTTTTGGGG) telomere motifs, two binding sites were observed. The high-affinity "A site" has a dissociation constant, K(D-DNA(A)) = 13(+/-4) nM, while the low-affinity "B site" is characterized by K(D-DNA(B)) = 5600(+/-600) nM at 25 degrees C. Nucleotide substitution variants verified that the A site corresponds principally with the 3'-terminal portion of d(T(4)G(4)T(4)G(4)). The relative contributions of entropy (DeltaS) and enthalpy (DeltaH) for binding reactions were DNA length-dependent as was heat capacity (DeltaCp). These trends with respect to DNA length likely reflect structural transitions in the DNA molecule that are coupled with DNA-protein association. Results presented here are important for understanding early intermediates and subsequent stages in the assembly of the full telomere nucleoprotein complex and how binding events can prepare the telomere DNA for extension by telomerase, a critical event in telomere biology.

High density genotyping of STAT4 gene reveals multiple haplotypic associations with Systemic Lupus Erythematosus in different racial groups

PubMed Central

Namjou, Bahram; Sestak, Andrea L.; Armstrong, Don L.; Zidovetzki, Raphael; Kelly, Jennifer A.; Jacob, Noam; Ciobanu, Voicu; Kaufman, Kenneth M.; Ojwang, Joshua O.; Ziegler, Julie; Quismorio, Francesco; Reiff, Andreas; Myones, Barry L.; Guthridge, Joel M.; Nath, Swapan K.; Bruner, Gail R.; Mehrian-Shai, Ruth; Silverman, Earl; Klein-Gitelman, Marisa; McCurdy, Deborah; Wagner-Weiner, Linda; Nocton, James J.; Putterman, Chaim; Bae, Sang-Cheol; Kim, Yun Jung; Petri, Michelle; Reveille, John D.; Vyse, Timothy J.; Gilkeson, Gary S.; Kamen, Diane L.; Alarcón-Riquelme, Marta E.; Gaffney, Patrick M.; Moser, Kathy L; Merrill, Joan T.; Scofield, R. Hal; James, Judith A.; Langefeld, Carl D.; Harley, John B.; Jacob, Chaim O.

2009-01-01

Objective Systemic lupus erythematosus (SLE) is the prototypic systemic autoimmune disorder with complex etiology and a strong genetic component. Recently, gene products involved in the interferon pathway have been under intense investigation in SLE pathogenesis. STAT1 and STAT4 are transcription factors that play key roles in the interferon and Th1 signaling pathways, making them attractive candidates for SLE susceptibility. Methods Fifty-six single-nucleotide polymorphisms (SNPs) across STAT1 and STAT4 genes on chromosome 2 were genotyped using Illumina platform as a part of extensive association study in a large collection of 9923 lupus cases and controls from different racial groups. DNA from patients and controls was obtained from peripheral blood. Principal component analyses and population based case-control association analyses were performed and the p values, FDR q values and Odds ratios with 95% confidence intervals (95% CIs) were calculated. Results We observed strong genetic associations with SLE and multiple SNPs located within the STAT4 gene in different ethnicities (Fisher combined p= 7.02×10−25). In addition to strong confirmation of the association in the 3rd intronic region of this gene reported previously, we identified additional haplotypic association across STAT4 gene and in particular a common risk haplotype that is found in multiple racial groups. In contrast, only a relatively weak suggestive association was observed with STAT1, probably due to the proximity to STAT4. Conclusion Our findings indicate that the STAT4 gene is likely to be a crucial component in SLE pathogenesis among multiple racial groups. The functional effects of this association, when revealed, might improve our understanding of the disease and provide new therapeutic targets. PMID:19333953

Functionalized bioengineered spider silk spheres improve nuclease resistance and activity of oligonucleotide therapeutics providing a strategy for cancer treatment.

PubMed

Kozlowska, Anna Karolina; Florczak, Anna; Smialek, Maciej; Dondajewska, Ewelina; Mackiewicz, Andrzej; Kortylewski, Marcin; Dams-Kozlowska, Hanna

2017-09-01

Cell-selective delivery and sensitivity to serum nucleases remain major hurdles to the clinical application of RNA-based oligonucleotide therapeutics, such as siRNA. Spider silk shows great potential as a biomaterial due to its biocompatibility and biodegradability. Self-assembling properties of silk proteins allow for processing into several different morphologies such as fibers, scaffolds, films, hydrogels, capsules and spheres. Moreover, bioengineering of spider silk protein sequences can functionalize silk by adding peptide moieties with specific features including binding or cell recognition domains. We demonstrated that modification of silk protein by adding the nucleic acid binding domain enabled the development of a novel oligonucleotide delivery system that can be utilized to improve pharmacokinetics of RNA-based therapeutics, such as CpG-siRNA. The MS2 bioengineered silk was functionalized with poly-lysine domain (KN) to generate hybrid silk MS2KN. CpG-siRNA efficiently bound to MS2KN in contrary to control MS2. Both MS2KN complexes and spheres protected CpG-siRNA from degradation by serum nucleases. CpG-siRNA molecules encapsulated into MS2KN spheres were efficiently internalized and processed by TLR9-positive macrophages. Importantly, CpG-STAT3siRNA loaded in silk spheres showed delayed and extended target gene silencing compared to naked oligonucleotides. The prolonged Stat3 silencing resulted in the more pronounced downregulation of interleukin 6 (IL-6), a proinflammatory cytokine and upstream activator of STAT3, which limits the efficacy of TLR9 immunostimulation. Our results demonstrate the feasibility of using spider silk spheres as a carrier of therapeutic nucleic acids. Moreover, the modified kinetic and activity of the CpG-STAT3siRNA embedded into silk spheres is likely to improve immunotherapeutic effects in vivo. We demonstrated that modification of silk protein by adding the nucleic acid binding domain enabled the development of a novel oligonucleotide delivery system that can be utilized to improve pharmacokinetics of RNA-based therapeutics. Although, the siRNA constructs have already given very promising results in the cancer therapy, the in vivo application of RNA-based oligonucleotide therapeutics still is limited due to their sensitivity to serum nucleases and some toxicity. We propose a carrier for RNA-based therapeutics that is made of bioengineered spider silk. We showed that functionalized bioengineered spider silk spheres not only protected RNA-based therapeutics from degradation by serum nucleases, but what is more important the embedding of siRNA into silk spheres delayed and extended target gene silencing compared with naked oligonucleotides. Moreover, we showed that plain silk spheres did not have unspecific effect on target gene levels proving not only to be non-cytotoxic but also very neutral vehicles in terms of TLR9/STAT3 activation in macrophages. We demonstrated advantages of novel delivery technology in safety and efficacy comparing with delivery of naked CpG-STAT3siRNA therapeutics. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

DNA condensing effects and sequence selectivity of DNA binding of antitumor noncovalent polynuclear platinum complexes.

PubMed

Malina, Jaroslav; Farrell, Nicholas P; Brabec, Viktor

2014-02-03

The noncovalent analogues of antitumor polynuclear platinum complexes represent a structurally discrete class of platinum drugs. Their chemical and biological properties differ significantly from those of most platinum chemotherapeutics, which bind to DNA in a covalent manner by formation of Pt-DNA adducts. In spite of the fact that these noncovalent polynuclear platinum complexes contain no leaving groups, they have been shown to bind to DNA with high affinity. We report here on the DNA condensation properties of a series of noncovalent analogues of antitumor polynuclear platinum complexes described by biophysical and biochemical methods. The results demonstrate that these polynuclear platinum compounds are capable of inducing DNA condensation at more than 1 order of magnitude lower concentrations than conventional spermine. Atomic force microscopy studies of DNA condensation confined to a mica substrate have revealed that the DNA morphologies become more compact with increasing concentration of the platinum complexes. Moreover, we also found that the noncovalent polynuclear platinum complex [{Pt(NH3)3}2-μ-{trans-Pt(NH3)2(NH2(CH2)6NH2)2}](6+) (TriplatinNC-A) binds to DNA in a sequence-dependent manner, namely, to A/T-rich sequences and A-tract regions, and that noncovalent polynuclear platinum complexes protect DNA from enzymatic cleavage by DNase I. The results suggest that mechanisms of antitumor and cytotoxic activities of these complexes may be associated with their unique ability to condense DNA along with their sequence-specific DNA binding. Owing to their high cellular accumulation, it is also reasonable to suggest that their mechanism of action is based on the competition with naturally occurring DNA condensing agents, such as polyamines spermine, spermidine, and putrescine, for intracellular binding sites, resulting in the disturbance of the correct binding of regulatory proteins initiating the onset of apoptosis.

Hyperglycaemia Exacerbates Choroidal Neovascularisation in Mice via the Oxidative Stress-Induced Activation of STAT3 Signalling in RPE Cells

PubMed Central

Wang, Yu-Sheng; Shi, Yuan-Yuan; Hou, Wei; Xu, Chun-Sheng; Wang, Hai-Yan; Ye, Zi; Yao, Li-Bo; Zhang, Jian

2012-01-01

Choroidal neovascularisation (CNV) that occurs as a result of age-related macular degeneration (AMD) causes severe vision loss among elderly patients. The relationship between diabetes and CNV remains controversial. However, oxidative stress plays a critical role in the pathogenesis of both AMD and diabetes. In the present study, we investigated the influence of diabetes on experimentally induced CNV and on the underlying molecular mechanisms of CNV. CNV was induced via photocoagulation in the ocular fundi of mice with streptozotocin-induced diabetes. The effect of diabetes on the severity of CNV was measured. An immunofluorescence technique was used to determine the levels of oxidative DNA damage by anti-8-hydroxy-2-deoxyguanosine (8-OHdG) antibody, the protein expression of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) and vascular endothelial growth factor (VEGF), in mice with CNV. The production of reactive oxygen species (ROS) in retinal pigment epithelial (RPE) cells that had been cultured under high glucose was quantitated using the 2′,7′-dichlorofluorescein diacetate (DCFH-DA) method. p-STAT3 expression was examined using Western blot analysis. RT-PCR and ELISA processes were used to detect VEGF expression. Hyperglycaemia exacerbated the development of CNV in mice. Oxidative stress levels and the expression of p-STAT3 and VEGF were highly elevated both in mice and in cultured RPE cells. Treatment with the antioxidant compound N-acetyl-cysteine (NAC) rescued the severity of CNV in diabetic mice. NAC also inhibited the overexpression of p-STAT3 and VEGF in CNV and in RPE cells. The JAK-2/STAT3 pathway inhibitor AG490 blocked VEGF expression but had no effect on the production of ROS in vitro. These results suggest that hyperglycaemia promotes the development of CNV by inducing oxidative stress, which in turn activates STAT3 signalling in RPE cells. Antioxidant supplementation helped attenuate the development of CNV. Thus, our results reveal a potential strategy for the treatment and prevention of diseases involving CNV. PMID:23094067

Analysis of the DNA-Binding Activities of the Arabidopsis R2R3-MYB Transcription Factor Family by One-Hybrid Experiments in Yeast

PubMed Central

Kelemen, Zsolt; Sebastian, Alvaro; Xu, Wenjia; Grain, Damaris; Salsac, Fabien; Avon, Alexandra; Berger, Nathalie; Tran, Joseph; Dubreucq, Bertrand; Lurin, Claire; Lepiniec, Loïc; Contreras-Moreira, Bruno; Dubos, Christian

2015-01-01

The control of growth and development of all living organisms is a complex and dynamic process that requires the harmonious expression of numerous genes. Gene expression is mainly controlled by the activity of sequence-specific DNA binding proteins called transcription factors (TFs). Amongst the various classes of eukaryotic TFs, the MYB superfamily is one of the largest and most diverse, and it has considerably expanded in the plant kingdom. R2R3-MYBs have been extensively studied over the last 15 years. However, DNA-binding specificity has been characterized for only a small subset of these proteins. Therefore, one of the remaining challenges is the exhaustive characterization of the DNA-binding specificity of all R2R3-MYB proteins. In this study, we have developed a library of Arabidopsis thaliana R2R3-MYB open reading frames, whose DNA-binding activities were assayed in vivo (yeast one-hybrid experiments) with a pool of selected cis-regulatory elements. Altogether 1904 interactions were assayed leading to the discovery of specific patterns of interactions between the various R2R3-MYB subgroups and their DNA target sequences and to the identification of key features that govern these interactions. The present work provides a comprehensive in vivo analysis of R2R3-MYB binding activities that should help in predicting new DNA motifs and identifying new putative target genes for each member of this very large family of TFs. In a broader perspective, the generated data will help to better understand how TF interact with their target DNA sequences. PMID:26484765

Pluripotency transcription factors and Tet1/2 maintain Brd4-independent stem cell identity.

PubMed

Finley, Lydia W S; Vardhana, Santosha A; Carey, Bryce W; Alonso-Curbelo, Direna; Koche, Richard; Chen, Yanyang; Wen, Duancheng; King, Bryan; Radler, Megan R; Rafii, Shahin; Lowe, Scott W; Allis, C David; Thompson, Craig B

2018-05-01

A robust network of transcription factors and an open chromatin landscape are hallmarks of the naive pluripotent state. Recently, the acetyllysine reader Brd4 has been implicated in stem cell maintenance, but the relative contribution of Brd4 to pluripotency remains unclear. Here, we show that Brd4 is dispensable for self-renewal and pluripotency of embryonic stem cells (ESCs). When maintained in their ground state, ESCs retain transcription factor binding and chromatin accessibility independent of Brd4 function or expression. In metastable ESCs, Brd4 independence can be achieved by increased expression of pluripotency transcription factors, including STAT3, Nanog or Klf4, so long as the DNA methylcytosine oxidases Tet1 and Tet2 are present. These data reveal that Brd4 is not essential for ESC self-renewal. Rather, the levels of pluripotency transcription factor abundance and Tet1/2 function determine the extent to which bromodomain recognition of protein acetylation contributes to the maintenance of gene expression and cell identity.

Thermodynamic Characterization of Binding Oxytricha nova Single Strand Telomere DNA with the Alpha Protein N-terminal Domain

PubMed Central

Buczek, Pawel; Horvath, Martin P.

2010-01-01

The Oxytricha nova telomere binding protein alpha subunit binds single strand DNA and participates in a nucleoprotein complex that protects the very ends of chromosomes. To understand how the N-terminal, DNA binding domain of alpha interacts with DNA we measured the stoichiometry, enthalpy (ΔH), entropy (ΔS), and dissociation constant (KD-DNA) for binding telomere DNA fragments at different temperatures and salt concentrations using native gel electrophoresis and isothermal titration calorimetry (ITC). About 85% of the total free energy of binding corresponded with non-electrostatic interactions for all DNAs. Telomere DNA fragments d(T2G4), d(T4G4), d(G3T4G4), and d(G4T4G4) each formed monovalent protein complexes. In the case of d(T4G4T4G4), which has two tandemly repeated d(TTTTTGGGG) telomere motifs, two binding sites were observed. The high-affinity “A site” has a dissociation constant, KD-DNA(A)=13(±4) nM, while the low-affinity “B site” is characterized by KD-DNA(B)=5600(±600) nM at 25 °C. Nucleotide substitution variants verified that the A site corresponds principally with the 3′-terminal portion of d(T4G4T4G4). The relative contributions of entropy (ΔS) and enthalpy (ΔH) for binding reactions were DNA length-dependent as was heat capacity (ΔCp). These trends with respect to DNA length likely reflect structural transitions in the DNA molecule that are coupled with DNA–protein association. Results presented here are important for understanding early intermediates and subsequent stages in the assembly of the full telomere nucleoprotein complex and how binding events can prepare the telomere DNA for extension by telomerase, a critical event in telomere biology. PMID:16678852

Sliding of proteins non-specifically bound to DNA: Brownian dynamics studies with coarse-grained protein and DNA models.

PubMed

Ando, Tadashi; Skolnick, Jeffrey

2014-12-01

DNA binding proteins efficiently search for their cognitive sites on long genomic DNA by combining 3D diffusion and 1D diffusion (sliding) along the DNA. Recent experimental results and theoretical analyses revealed that the proteins show a rotation-coupled sliding along DNA helical pitch. Here, we performed Brownian dynamics simulations using newly developed coarse-grained protein and DNA models for evaluating how hydrodynamic interactions between the protein and DNA molecules, binding affinity of the protein to DNA, and DNA fluctuations affect the one dimensional diffusion of the protein on the DNA. Our results indicate that intermolecular hydrodynamic interactions reduce 1D diffusivity by 30%. On the other hand, structural fluctuations of DNA give rise to steric collisions between the CG-proteins and DNA, resulting in faster 1D sliding of the protein. Proteins with low binding affinities consistent with experimental estimates of non-specific DNA binding show hopping along the CG-DNA. This hopping significantly increases sliding speed. These simulation studies provide additional insights into the mechanism of how DNA binding proteins find their target sites on the genome.

Multivalent DNA-binding properties of the HMG-1 proteins.

PubMed Central

Maher, J F; Nathans, D

1996-01-01

HMG-I proteins are DNA-binding proteins thought to affect the formation and function of transcription complexes. Each protein contains three DNA-binding motifs, known as AT-hooks, that bind in the minor groove of AT tracts in DNA. Multiple AT-hooks within a polypeptide chain should contact multiple AT tracts, but the rules governing these interactions have not been defined. In this study, we demonstrate that high-affinity binding uses two or three appropriately spaced AT tracts as a single multivalent binding site. These principles have implications for binding to regulatory elements such as the interferon beta enhancer, TATA boxes, and serum response elements. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8692884

IL-15 regulates Bcl-2 family members Bim and Mcl-1 through JAK/STAT and PI3K/AKT pathways in T cells.

PubMed

Shenoy, Aparna R; Kirschnek, Susanne; Häcker, Georg

2014-08-01

Maintenance of T cells is determined by their survival capacity, which is regulated by Bcl-2 proteins. Cytokines signalling through the common gamma chains such as IL-2, IL-7 and IL-15 are important for T-cell survival but how these cytokines determine the expression of Bcl-2-family proteins is not clear. We report signalling events of cytokines that regulate expression of two key Bcl-2 proteins, pro-apoptotic Bim and anti-apoptotic Mcl-1, in resting C57BL/6 mouse T cells. IL-2, IL-7 and IL-15 inhibited apoptosis but paradoxically induced the expression of Bim, countered by concomitant induction of Mcl-1. Bim induction by IL-15 was found at the mRNA and protein levels and depended on both JAK/STAT and PI3K signals. A new STAT5-binding site was identified in the Bim promoter, which was occupied by STAT5 upon IL-15 stimulation. Although it also depended on JAK/STAT- and PI3K signalling, Mcl-1 regulation was independent of Mcl-1 mRNA levels and of regulation of protein stability, suggesting translational regulation. Concurrent CD3 signals inhibited some of the IL-7 effect but not the IL-15 effect on Bcl-2 proteins. The data suggest that cytokines induce Bim and prime T cells for apoptosis, but also inhibit apoptosis by stabilising Mcl-1. Later downregulation of short-lived Mcl-1 may induce efficient, Bim-dependent apoptosis. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inactivation of JAK2/STAT3 Signaling Axis and Downregulation of M1 mAChR Cause Cognitive Impairment in klotho Mutant Mice, a Genetic Model of Aging

PubMed Central

Park, Seok-Joo; Shin, Eun-Joo; Min, Sun Seek; An, Jihua; Li, Zhengyi; Hee Chung, Yoon; Hoon Jeong, Ji; Bach, Jae-Hyung; Nah, Seung-Yeol; Kim, Won-Ki; Jang, Choon-Gon; Kim, Yong-Sun; Nabeshima, Yo-ichi; Nabeshima, Toshitaka; Kim, Hyoung-Chun

2013-01-01

We previously reported cognitive dysfunction in klotho mutant mice. In the present study, we further examined novel mechanisms involved in cognitive impairment in these mice. Significantly decreased janus kinase 2 (JAK2) and signal transducer and activator of transcription3 (STAT3) phosphorylation were observed in the hippocampus of klotho mutant mice. A selective decrease in protein expression and binding density of the M1 muscarinic cholinergic receptor (M1 mAChR) was observed in these mice. Cholinergic parameters (ie, acetylcholine (ACh), choline acetyltransferase (ChAT), and acetylcholinesterase (AChE)) and NMDAR-dependent long-term potentiation (LTP) were significantly impaired in klotho mutant mice. McN-A-343 (McN), an M1 mAChR agonist, significantly attenuated these impairments. AG490 (AG), a JAK2 inhibitor, counteracted the attenuating effects of McN, although AG did not significantly alter the McN-induced effect on AChE. Furthermore, AG significantly inhibited the attenuating effects of McN on decreased NMDAR-dependent LTP, protein kinase C βII, p-ERK, p-CREB, BDNF, and p-JAK2/p-STAT3-expression in klotho mutant mice. In addition, k252a, a BDNF receptor tyrosine kinase B (TrkB) inhibitor, significantly counteracted McN effects on decreased ChAT, ACh, and M1 mAChR and p-JAK2/p-STAT3 expression. McN-induced effects on cognitive impairment in klotho mutant mice were consistently counteracted by either AG or k252a. Our results suggest that inactivation of the JAK2/STAT3 signaling axis and M1 mAChR downregulation play a critical role in cognitive impairment observed in klotho mutant mice. PMID:23389690

Identification of Nucleic Acid Binding Sites on Translin-Associated Factor X (TRAX) Protein

PubMed Central

Gupta, Gagan Deep; Kumar, Vinay

2012-01-01

Translin and TRAX proteins play roles in very important cellular processes such as DNA recombination, spatial and temporal expression of mRNA, and in siRNA processing. Translin forms a homomeric nucleic acid binding complex and binds to ssDNA and RNA. However, a mutant translin construct that forms homomeric complex lacking nucleic acid binding activity is able to form fully active heteromeric translin-TRAX complex when co-expressed with TRAX. A substantial progress has been made in identifying translin sites that mediate its binding activity, while TRAX was thought not to bind DNA or RNA on its own. We here for the first time demonstrate nucleic acid binding to TRAX by crosslinking radiolabeled ssDNA to heteromeric translin-TRAX complex using UV-laser. The TRAX and translin, photochemically crosslinked with ssDNA, were individually detected on SDS-PAGE. We mutated two motifs in TRAX and translin, designated B2 and B3, to help define the nucleic acid binding sites in the TRAX sequence. The most pronounced effect was observed in the mutants of B3 motif that impaired nucleic acid binding activity of the heteromeric complexes. We suggest that both translin and TRAX are binding competent and contribute to the nucleic acid binding activity. PMID:22427937

A novel class of plant-specific zinc-dependent DNA-binding protein that binds to A/T-rich DNA sequences

PubMed Central

Nagano, Yukio; Furuhashi, Hirofumi; Inaba, Takehito; Sasaki, Yukiko

2001-01-01

Complementary DNA encoding a DNA-binding protein, designated PLATZ1 (plant AT-rich sequence- and zinc-binding protein 1), was isolated from peas. The amino acid sequence of the protein is similar to those of other uncharacterized proteins predicted from the genome sequences of higher plants. However, no paralogous sequences have been found outside the plant kingdom. Multiple alignments among these paralogous proteins show that several cysteine and histidine residues are invariant, suggesting that these proteins are a novel class of zinc-dependent DNA-binding proteins with two distantly located regions, C-x2-H-x11-C-x2-C-x(4–5)-C-x2-C-x(3–7)-H-x2-H and C-x2-C-x(10–11)-C-x3-C. In an electrophoretic mobility shift assay, the zinc chelator 1,10-o-phenanthroline inhibited DNA binding, and two distant zinc-binding regions were required for DNA binding. A protein blot with 65ZnCl2 showed that both regions are required for zinc-binding activity. The PLATZ1 protein non-specifically binds to A/T-rich sequences, including the upstream region of the pea GTPase pra2 and plastocyanin petE genes. Expression of the PLATZ1 repressed those of the reporter constructs containing the coding sequence of luciferase gene driven by the cauliflower mosaic virus (CaMV) 35S90 promoter fused to the tandem repeat of the A/T-rich sequences. These results indicate that PLATZ1 is a novel class of plant-specific zinc-dependent DNA-binding protein responsible for A/T-rich sequence-mediated transcriptional repression. PMID:11600698

Growth-hormone-induced signal transducer and activator of transcription 5 signaling causes gigantism, inflammation, and premature death but protects mice from aggressive liver cancer.

PubMed

Friedbichler, Katrin; Themanns, Madeleine; Mueller, Kristina M; Schlederer, Michaela; Kornfeld, Jan-Wilhelm; Terracciano, Luigi M; Kozlov, Andrey V; Haindl, Susanne; Kenner, Lukas; Kolbe, Thomas; Mueller, Mathias; Snibson, Kenneth J; Heim, Markus H; Moriggl, Richard

2012-03-01

Persistently high levels of growth hormone (GH) can cause liver cancer. GH activates multiple signal-transduction pathways, among them janus kinase (JAK) 2-signal transducer and activator of transcription (STAT) 5 (signal transducer and activator of transcription 5). Both hyperactivation and deletion of STAT5 in hepatocytes have been implicated in the development of hepatocellular carcinoma (HCC); nevertheless, the role of STAT5 in the development of HCC as a result of high GH levels remains enigmatic. Thus, we crossed a mouse model of gigantism and inflammatory liver cancer caused by hyperactivated GH signaling (GH(tg) ) to mice with hepatic deletion of STAT5 (STAT5(Δhep) ). Unlike GH(tg) mice, GH(tg) STAT5(Δhep) animals did not display gigantism. Moreover, the premature mortality, which was associated with chronic inflammation, as well as the pathologic alterations of hepatocytes observed in GH(tg) mice, were not observed in GH(tg) animals lacking STAT5. Strikingly, loss of hepatic STAT5 proteins led to enhanced HCC development in GH(tg) mice. Despite reduced chronic inflammation, GH(tg) STAT5(Δhep) mice displayed earlier and more advanced HCC than GH(tg) animals. This may be attributed to the combination of increased peripheral lipolysis, hepatic lipid synthesis, loss of hepatoprotective mediators accompanied by aberrant activation of tumor-promoting c-JUN and STAT3 signaling cascades, and accumulation of DNA damage secondary to loss of cell-cycle control. Thus, HCC was never observed in STAT5(Δhep) mice. As a result of their hepatoprotective functions, STAT5 proteins prevent progressive fatty liver disease and the formation of aggressive HCC in the setting of hyperactivated GH signaling. At the same time, they play a key role in controlling systemic inflammation and regulating organ and body size. Copyright © 2011 American Association for the Study of Liver Diseases.

A Comparison of Two Single-Stranded DNA Binding Models by Mutational Analysis of APOBEC3G

PubMed Central

Shindo, Keisuke; Li, Ming; Gross, Phillip J.; Brown, William L.; Harjes, Elena; Lu, Yongjian; Matsuo, Hiroshi; Harris, Reuben S.

2012-01-01

APOBEC3G is the best known of several DNA cytosine deaminases that function to inhibit the replication of parasitic genetic elements including the lentivirus HIV. Several high-resolution structures of the APOBEC3G catalytic domain have been generated, but none reveal how this enzyme binds to substrate single-stranded DNA. Here, we constructed a panel of APOBEC3G amino acid substitution mutants and performed a series of biochemical, genetic, and structural assays to distinguish between “Brim” and “Kink” models for single-strand DNA binding. Each model predicts distinct sets of interactions between surface arginines and negatively charged phosphates in the DNA backbone. Concordant with both models, changing the conserved arginine at position 313 to glutamate abolished both catalytic and restriction activities. In support of the Brim model, arginine to glutamate substitutions at positions 213, 215, and 320 also compromised these APOBEC3G activities. Arginine to glutamate substitutions at Kink model residues 374 and 376 had smaller effects. These observations were supported by A3G catalytic domain-ssDNA chemical shift perturbation experiments. The overall data set is most consistent with the Brim model for single-stranded DNA binding by APOBEC3G. PMID:24832226

RTVP-1 promotes mesenchymal transformation of glioma via a STAT-3/IL-6-dependent positive feedback loop

PubMed Central

Giladi, Nis David; Ziv-Av, Amotz; Lee, Hae Kyung; Finniss, Susan; Cazacu, Simona; Xiang, Cunli; Ben-Asher, Hiba Waldman; deCarvalho, Ana; Mikkelsen, Tom; Poisson, Laila; Brodie, Chaya

2015-01-01

Glioblastomas (GBMs), the most aggressive primary brain tumors, exhibit increased invasiveness and resistance to anti-tumor treatments. We explored the role of RTVP-1, a glioma-associated protein that promotes glioma cell migration, in the mesenchymal transformation of GBM. Analysis of The Cancer Genome Atlas (TCGA) demonstrated that RTVP-1 expression was higher in mesenchymal GBM and predicted tumor recurrence and poor clinical outcome. ChiP analysis revealed that the RTVP-1 promoter binds STAT3 and C/EBPβ, two master transcription factors that regulate mesenchymal transformation of GBM. In addition, IL-6 induced RTVP-1 expression in a STAT3-dependent manner. RTVP-1 increased the migration and mesenchymal transformation of glioma cells. Similarly, overexpression of RTVP-1 in human neural stem cells induced mesenchymal differentiation, whereas silencing of RTVP-1 in glioma stem cells (GSCs) decreased the mesenchymal transformation and stemness of these cells. Silencing of RTVP-1 also increased the survival of mice bearing GSC-derived xenografts. Using gene array analysis of RTVP-1 silenced glioma cells we identified IL-6 as a mediator of RTVP-1 effects on the mesenchymal transformation and migration of GSCs, therefore acting in a positive feedback loop by upregulating RTVP-1 expression via the STAT3 pathway. Collectively, these results implicate RTVP-1 as a novel prognostic marker and therapeutic target in GBM. PMID:26267319

EGFR inhibition by pentacyclic triterpenes exhibit cell cycle and growth arrest in breast cancer cells.

PubMed

Sathya, Shanmugaraj; Sudhagar, Selvaraj; Sarathkumar, Baskaran; Lakshmi, Baddireddi Subhadra

2014-01-24

Pentacyclic triterpenes are a group of molecules with promising anticancer potential, although their precise molecular target remains elusive. The current work aims to investigate the antiproliferative and associated mechanisms of triterpenes in breast cancer cells in vitro. Effect of triterpenes on cell cycle distribution, ROS and key regulatory proteins were analyzed in three breast cancer cells in vitro. Growth inhibition, new DNA synthesis, colony formation assays and Western blot analysis were performed to assess the EGFR inhibitory effect of triterpenes. Molecular docking was performed to study the interaction between EGFR and triterpenes. We have demonstrated the ability of dimethyl melaleucate (DMM), a pentacyclic triterpene to exhibit cell cycle arrest at G0/G1 phase by down-regulation of cyclin D1 through PI3K/AKT inhibition. Further, to identify the upstream target of DMM, potential EGFR inhibitory activity of DMM and three structurally related pentacyclic triterpenes, ursolic acid, 18α-glycyrrhetinic acid and carbenoxolone was investigated. Interestingly, pentacyclic triterpenes limit EGF mediated breast cancer proliferation through sustained inhibition of EGFR and its downstream effectors STAT3 and cyclin D1 in breast cancer lines. We also show pentacyclic triterpenes to bind at the ATP binding pocket of tyrosine kinase domain of EGFR leading to the hypothesis that pentacyclic triterpenes could be a novel class of EGFR inhibitors. In conclusion, pentacyclic triterpenes inhibit EGFR activation through binding with tyrosine kinase domain thereby suppressing breast cancer proliferation. Pentacyclic triterpenes may serve as a potential platform for development of novel drugs against breast cancer. Copyright © 2013 Elsevier Inc. All rights reserved.

Binding the Mammalian High Mobility Group Protein AT-hook 2 to AT-Rich Deoxyoligonucleotides: Enthalpy-Entropy Compensation

PubMed Central

Joynt, Suzanne; Morillo, Victor; Leng, Fenfei

2009-01-01

HMGA2 is a DNA minor-groove binding protein. We previously demonstrated that HMGA2 binds to AT-rich DNA with very high binding affinity where the binding of HMGA2 to poly(dA-dT)2 is enthalpy-driven and to poly(dA)poly(dT) is entropy-driven. This is a typical example of enthalpy-entropy compensation. To further study enthalpy-entropy compensation of HMGA2, we used isothermal-titration-calorimetry to examine the interactions of HMGA2 with two AT-rich DNA hairpins: 5′-CCAAAAAAAAAAAAAAAGCCCCCGCTTTTTTTTTTTTTTTGG-3′ (FL-AT-1) and 5′-CCATATATATATATATAGCCCCCGCTATATATATATATATGG-3′ (FL-AT-2). Surprisingly, we observed an atypical isothermal-titration-calorimetry-binding curve at low-salt aqueous solutions whereby the apparent binding-enthalpy decreased dramatically as the titration approached the end. This unusual behavior can be attributed to the DNA-annealing coupled to the ligand DNA-binding and is eliminated by increasing the salt concentration to ∼200 mM. At this condition, HMGA2 binding to FL-AT-1 is entropy-driven and to FL-AT-2 is enthalpy-driven. Interestingly, the DNA-binding free energies for HMGA2 binding to both hairpins are almost temperature independent; however, the enthalpy-entropy changes are dependent on temperature, which is another aspect of enthalpy-entropy compensation. The heat capacity change for HMGA2 binding to FL-AT-1 and FL-AT-2 are almost identical, indicating that the solvent displacement and charge-charge interaction in the coupled folding/binding processes for both binding reactions are similar. PMID:19450485

CHD3 and CHD4 recruitment and chromatin remodeling activity at DNA breaks is promoted by early poly(ADP-ribose)-dependent chromatin relaxation.

PubMed

Smith, Rebecca; Sellou, Hafida; Chapuis, Catherine; Huet, Sébastien; Timinszky, Gyula

2018-05-04

One of the first events to occur upon DNA damage is the local opening of the compact chromatin architecture, facilitating access of repair proteins to DNA lesions. This early relaxation is triggered by poly(ADP-ribosyl)ation by PARP1 in addition to ATP-dependent chromatin remodeling. CHD4 recruits to DNA breaks in a PAR-dependent manner, although it lacks any recognizable PAR-binding domain, and has the ability to relax chromatin structure. However, its role in chromatin relaxation at the site of DNA damage has not been explored. Using a live cell fluorescence three-hybrid assay, we demonstrate that the recruitment of CHD4 to DNA damage, while being poly(ADP-ribosyl)ation-dependent, is not through binding poly(ADP-ribose). Additionally, we show that CHD3 is recruited to DNA breaks in the same manner as CHD4 and that both CHD3 and CHD4 play active roles in chromatin remodeling at DNA breaks. Together, our findings reveal a two-step mechanism for DNA damage induced chromatin relaxation in which PARP1 and the PAR-binding remodeler activities of Alc1/CHD1L induce an initial chromatin relaxation phase that promotes the subsequent recruitment of CHD3 and CHD4 via binding to DNA for further chromatin remodeling at DNA breaks.

SPARC expression induces cell cycle arrest via STAT3 signaling pathway in medulloblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chetty, Chandramu; Dontula, Ranadheer; Ganji, Purnachandra Nagaraju

2012-01-13

Highlights: Black-Right-Pointing-Pointer Ectopic expression of SPARC impaired cell proliferation in medulloblastoma cells. Black-Right-Pointing-Pointer SPARC expression induces STAT3 mediated cell cycle arrest in medulloblastoma cells. Black-Right-Pointing-Pointer SPARC expression significantly inhibited pre-established tumor growth in nude-mice. -- Abstract: Dynamic cell interaction with ECM components has profound influence in cancer progression. SPARC is a component of the ECM, impairs the proliferation of different cell types and modulates tumor cell aggressive features. We previously reported that SPARC expression significantly impairs medulloblastoma tumor growth in vivo. In this study, we demonstrate that expression of SPARC inhibits medulloblastoma cell proliferation. MTT assay indicated a dose-dependent reductionmore » in tumor cell proliferation in adenoviral mediated expression of SPARC full length cDNA (Ad-DsRed-SP) in D425 and UW228 cells. Flow cytometric analysis showed that Ad-DsRed-SP-infected cells accumulate in the G2/M phase of cell cycle. Further, immunoblot and immunoprecipitation analyses revealed that SPARC induced G2/M cell cycle arrest was mediated through inhibition of the Cyclin-B-regulated signaling pathway involving p21 and Cdc2 expression. Additionally, expression of SPARC decreased STAT3 phosphorylation at Tyr-705; constitutively active STAT3 expression reversed SPARC induced G2/M arrest. Ad-DsRed-SP significantly inhibited the pre-established orthotopic tumor growth and tumor volume in nude-mice. Immunohistochemical analysis of tumor sections from mice treated with Ad-DsRed-SP showed decreased immunoreactivity for pSTAT3 and increased immunoreactivity for p21 compared to tumor section from mice treated with mock and Ad-DsRed. Taken together our studies further reveal that STAT3 plays a key role in SPARC induced G2/M arrest in medulloblastoma cells. These new findings provide a molecular basis for the mechanistic understanding of the effects of SPARC on medulloblastoma tumor cell proliferation.« less

JAK inhibitor has the amelioration effect in lupus-prone mice: the involvement of IFN signature gene downregulation.

PubMed

Ikeda, Keigo; Hayakawa, Kunihiro; Fujishiro, Maki; Kawasaki, Mikiko; Hirai, Takuya; Tsushima, Hiroshi; Miyashita, Tomoko; Suzuki, Satoshi; Morimoto, Shinji; Tamura, Naoto; Takamori, Kenji; Ogawa, Hideoki; Sekigawa, Iwao

2017-08-22

We previously reported that JAK-STAT-pathway mediated regulation of IFN-regulatory factor genes could play an important role in SLE pathogenesis. Here, we evaluated the efficacy of the JAK inhibitor tofacitinib (TOFA) for controlling IFN signalling via the JAK-STAT pathway and as a therapeutic for SLE. We treated NZB/NZW F1 mice with TOFA and assessed alterations in their disease, pathological, and immunological conditions. Gene-expression results obtained from CD4 + T cells (SLE mice) and CD3 + T cells (human SLE patients) were measured by DNA microarray and qRT-PCR. TOFA treatment resulted in reduced levels of anti-dsDNA antibodies, decreased proteinuria, and amelioration of nephritis as compared with those observed in control animals. Moreover, we observed the rebalance in the populations of naïve CD4 + T cells and effector/memory cells in TOFA-treated mice; however, treatment with a combination of TOFA and dexamethasone (DEXA) elicited a stronger inhibitory effect toward the effector/memory cells than did TOFA or DEXA monotherapy. We also detected decreased expression of several IFN-signature genes Ifit3 and Isg15 in CD4 + from SLE-prone mice following TOFA and DEXA treatment, and IFIT3 in CD3 + T cells from human patients following immunosuppressant therapy including steroid, respectively. Modulation of type I IFN signalling via JAK-STAT inhibition may exert a beneficial effect in SLE patients, and our results suggest that TOFA could be utilised for the development of new SLE-specific therapeutic strategies.

Repeated administration of CGP 46381, a gamma-aminobutyric acidB antagonist, and ethosuximide suppresses seizure-associated cyclic adenosine 3'5' monophosphate response element- and activator protein-1 DNA-binding activities in lethargic (lh/lh) mice.

PubMed

Ishige, K; Endo, H; Saito, H; Ito, Y

2001-01-19

To characterize seizure-associated increases in cerebral cortical and thalamic cyclic AMP responsive element (CRE)- and activator protein 1 (AP-1) DNA-binding activities in lethargic (lh/lh) mice, a genetic model of absence seizures, we examined the effects of ethosuximide and CGP 46381 on these DNA-binding activities. Repeated administration (twice a day for 5 days) of ethosuximide (200 mg/kg) or CGP 46381 (60 mg/kg) attenuated both seizure behavior and the increased DNA-binding activities, and was more effective than a single administration of these drugs. These treatments did not affect either normal behavior or basal DNA-binding activities in non-epileptic control (+/+) mice. Gel supershift assays revealed that the increased CRE-binding activity was attributable to activation of the binding activity of CREB, and that the c-Fos-c-Jun complex was a component of the increased AP-1 DNA-binding activity.

Molecular characterization and immunological roles of avian IL-22 and its soluble receptor IL-22 binding protein

USDA-ARS?s Scientific Manuscript database

As a member of the interleukin (IL)-10 family, IL-22 is an important mediator in modulating tissue responses during inflammation. Through activation of STAT3-signaling cascades, IL-22 induces proliferative and anti-apoptotic pathways, as well as antimicrobial peptides (AMPs), that help prevent tissu...

Sequence-based prediction of protein-binding sites in DNA: comparative study of two SVM models.

PubMed

Park, Byungkyu; Im, Jinyong; Tuvshinjargal, Narankhuu; Lee, Wook; Han, Kyungsook

2014-11-01

As many structures of protein-DNA complexes have been known in the past years, several computational methods have been developed to predict DNA-binding sites in proteins. However, its inverse problem (i.e., predicting protein-binding sites in DNA) has received much less attention. One of the reasons is that the differences between the interaction propensities of nucleotides are much smaller than those between amino acids. Another reason is that DNA exhibits less diverse sequence patterns than protein. Therefore, predicting protein-binding DNA nucleotides is much harder than predicting DNA-binding amino acids. We computed the interaction propensity (IP) of nucleotide triplets with amino acids using an extensive dataset of protein-DNA complexes, and developed two support vector machine (SVM) models that predict protein-binding nucleotides from sequence data alone. One SVM model predicts protein-binding nucleotides using DNA sequence data alone, and the other SVM model predicts protein-binding nucleotides using both DNA and protein sequences. In a 10-fold cross-validation with 1519 DNA sequences, the SVM model that uses DNA sequence data only predicted protein-binding nucleotides with an accuracy of 67.0%, an F-measure of 67.1%, and a Matthews correlation coefficient (MCC) of 0.340. With an independent dataset of 181 DNAs that were not used in training, it achieved an accuracy of 66.2%, an F-measure 66.3% and a MCC of 0.324. Another SVM model that uses both DNA and protein sequences achieved an accuracy of 69.6%, an F-measure of 69.6%, and a MCC of 0.383 in a 10-fold cross-validation with 1519 DNA sequences and 859 protein sequences. With an independent dataset of 181 DNAs and 143 proteins, it showed an accuracy of 67.3%, an F-measure of 66.5% and a MCC of 0.329. Both in cross-validation and independent testing, the second SVM model that used both DNA and protein sequence data showed better performance than the first model that used DNA sequence data. To the best of our knowledge, this is the first attempt to predict protein-binding nucleotides in a given DNA sequence from the sequence data alone. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Detection of DNA damage in individual cells by flow cytometric analysis using anti-DNA monoclonal antibody

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frankfurt, O.S.

A new method for the measurement of DNA damage in individual cells treated with alkylating agents is described. The method is based on the binding of anti-DNA monoclonal antibody to DNA in situ. Binding of antibody was evaluated by flow cytometry with indirect immunofluorescence. No binding of antibody to DNA in non-treated HeLa S3 cells was detected. Treatment of cells with HN2 or L-phenylalanine mustard induced binding of antibody to DNA in situ. Binding of antibody was observed after treating cells with doses of drugs which reduced the surviving fraction below 20%. Intensity of binding increased in proportion to themore » drug dose. In HN2-treated cells a cell subset with the lowest antibody binding was observed among cells in G1 phase. Binding of antibody to DNA in HN2-treated cells was eliminated by single-strand (ss) specific S1 nuclease. In competition assay, antibody was inhibited by thermally denatured DNA, but not by native double-stranded (ds) DNA, RNA, nucleosides and deoxyribohomopolymers. Immunoreactivity of cells with the monoclonal antibody F7-26 may be a useful probe for the assessment of cell damage induced by alkylating agents, especially in heterogeneous cell populations.« less

Spectrophotometric study on binding of 2-thioxanthone acetic acid with ct-DNA.

PubMed

Ataci, Nese; Ozcelik, Elif; Arsu, Nergis

2018-06-02

Thioxanthone and its derivatives are the most remarkable molecules due to their vast variety of application such as radiation curing that is, until using them as a therapeutic drug. Therefore, in this study it was intended to use 2-Thioxanthone acetic acid with and without NaCl in Tris HCl buffer solution (pH:7.0) to represent the interaction with ct-DNA. The UV-vis absorption spectra of TXCH 2 COOH in the presence of ct-DNA showed hypochromism and the intrinstic binding constant (K b ) was determined as 6 × 10 3  L mol -1 . The fluoresence intensity of TXCH 2 COOH with ct-DNA clearly increased up to 101% which indicated that the fluorescence intensity was very sensitive to ct-DNA concentration. The binding constant (K) and the values of number of binding sites (n) and were calculated as 1.8 × 10 3  L mol -1 and 0.69, respectively. When the quenching constants (K sv ) of free TXCH 2 COOH and TXCH 2 COOH, which were bonded with ct-DNA were compared, slightly changed values of Ksv were seen. Moreover, displacement assay with Hoechst 33,258 and viscosity measurements in the presence and absence of NaCl salt also confirmed the binding mode which noted the electrostatic interaction following groove binding between TXCH 2 COOH and ct-DNA. Last but not least, the salt effect was examined on ct-DNA binding with TXCH 2 COOH. The results of the experiments indicated that the groove binding was strengthened by NaCl whereas in the high NaCl concentration, the binding ability of TXCH 2 COOH to ct-DNA was inversely affected. Copyright © 2018 Elsevier B.V. All rights reserved.

Excited State Proton Transfer of Natural Flavonoids and Their Chromophores in Duplex and Tetraplex DNAs

PubMed Central

2015-01-01

Fisetin (3,7,3′,4′-tetrahydroxyflavone) and quercetin (3,5,7,3′,4′-pentahydroxyflavone) are the bioactive plant flavonoids that are potentially useful therapeutic drugs for the treatment of a broad spectrum of diseases, including atherosclerosis, cardiovascular disease, obesity, hypertension, and cancer. 3-Hydroxyflavone (3HF) and 7-hydroxyflavone (7HF) are the synthetic chromophores of fisetin and quercetin. We have exploited dual luminescence properties of fisetin and quercetin along with 3-HF and 7HF to examine their efficacy of binding and compare their interactions with DNA, which is one of the macromolecular targets of flavonoids in physiological systems. Following the sequence of the human telomeric DNA 5′-d (CCCTAA-)n/(-TTAGGG)n-5′, two single-stranded DNA oligonucleotides, 5′-d(C3TA2)3C3-3′ and 5′-d(T2AG3)4-3′, and their duplex were used as receptors to study binding by the ligands quercetin, fisetin, and their chromophores. Circular dichroism, differential absorption, UV thermal melting, and size exclusion chromatographic studies indicated the formation of unusual DNA structures (such as C4 and G4 tetraplexes) for both the C- and G-rich single-stranded DNAs. Upon binding to DNA, dramatic changes were observed in the intrinsic fluorescence behavior of the flavonoids. Molecular docking studies were performed to describe the likely binding sites for the ligands. The spectroscopic studies on flavonoid–DNA interactions described herein demonstrate a powerful approach for examining their DNA binding through exploiting the highly sensitive intrinsic fluorescence properties of the flavonoids as their own “reporter” for their interactions with macromolecular targets. PMID:25393681

Lenalidomide induces lipid raft assembly to enhance erythropoietin receptor signaling in myelodysplastic syndrome progenitors.

PubMed

McGraw, Kathy L; Basiorka, Ashley A; Johnson, Joseph O; Clark, Justine; Caceres, Gisela; Padron, Eric; Heaton, Ruth; Ozawa, Yukiyasu; Wei, Sheng; Sokol, Lubomir; List, Alan F

2014-01-01

Anemia remains the principal management challenge for patients with lower risk Myelodysplastic Syndromes (MDS). Despite appropriate cytokine production and cellular receptor display, erythropoietin receptor (EpoR) signaling is impaired. We reported that EpoR signaling is dependent upon receptor localization within lipid raft microdomains, and that disruption of raft integrity abolishes signaling capacity. Here, we show that MDS erythroid progenitors display markedly diminished raft assembly and smaller raft aggregates compared to normal controls (p = 0.005, raft number; p = 0.023, raft size). Because lenalidomide triggers raft coalescence in T-lymphocytes promoting immune synapse formation, we assessed effects of lenalidomide on raft assembly in MDS erythroid precursors and UT7 cells. Lenalidomide treatment rapidly induced lipid raft formation accompanied by EpoR recruitment into raft fractions together with STAT5, JAK2, and Lyn kinase. The JAK2 phosphatase, CD45, a key negative regulator of EpoR signaling, was displaced from raft fractions. Lenalidomide treatment prior to Epo stimulation enhanced both JAK2 and STAT5 phosphorylation in UT7 and primary MDS erythroid progenitors, accompanied by increased STAT5 DNA binding in UT7 cells, and increased erythroid colony forming capacity in both UT7 and primary cells. Raft induction was associated with F-actin polymerization, which was blocked by Rho kinase inhibition. These data indicate that deficient raft integrity impairs EpoR signaling, and provides a novel strategy to enhance EpoR signal fidelity in non-del(5q) MDS.

Lenalidomide Induces Lipid Raft Assembly to Enhance Erythropoietin Receptor Signaling in Myelodysplastic Syndrome Progenitors

PubMed Central

McGraw, Kathy L.; Basiorka, Ashley A.; Johnson, Joseph O.; Clark, Justine; Caceres, Gisela; Padron, Eric; Heaton, Ruth; Ozawa, Yukiyasu; Wei, Sheng; Sokol, Lubomir; List, Alan F.

2014-01-01

Anemia remains the principal management challenge for patients with lower risk Myelodysplastic Syndromes (MDS). Despite appropriate cytokine production and cellular receptor display, erythropoietin receptor (EpoR) signaling is impaired. We reported that EpoR signaling is dependent upon receptor localization within lipid raft microdomains, and that disruption of raft integrity abolishes signaling capacity. Here, we show that MDS erythroid progenitors display markedly diminished raft assembly and smaller raft aggregates compared to normal controls (p = 0.005, raft number; p = 0.023, raft size). Because lenalidomide triggers raft coalescence in T-lymphocytes promoting immune synapse formation, we assessed effects of lenalidomide on raft assembly in MDS erythroid precursors and UT7 cells. Lenalidomide treatment rapidly induced lipid raft formation accompanied by EpoR recruitment into raft fractions together with STAT5, JAK2, and Lyn kinase. The JAK2 phosphatase, CD45, a key negative regulator of EpoR signaling, was displaced from raft fractions. Lenalidomide treatment prior to Epo stimulation enhanced both JAK2 and STAT5 phosphorylation in UT7 and primary MDS erythroid progenitors, accompanied by increased STAT5 DNA binding in UT7 cells, and increased erythroid colony forming capacity in both UT7 and primary cells. Raft induction was associated with F-actin polymerization, which was blocked by Rho kinase inhibition. These data indicate that deficient raft integrity impairs EpoR signaling, and provides a novel strategy to enhance EpoR signal fidelity in non-del(5q) MDS. PMID:25469886

CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference.

PubMed

Hochstrasser, Megan L; Taylor, David W; Bhat, Prashant; Guegler, Chantal K; Sternberg, Samuel H; Nogales, Eva; Doudna, Jennifer A

2014-05-06

In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA-E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage.

Pyrimidine tract-binding protein 1 mediates pyruvate kinase M2-dependent phosphorylation of signal transducer and activator of transcription 3 and oncogenesis in anaplastic large cell lymphoma.

PubMed

Hwang, Steven R; Murga-Zamalloa, Carlos; Brown, Noah; Basappa, Johnvesly; McDonnell, Scott Rp; Mendoza-Reinoso, Veronica; Basrur, Venkatesha; Wilcox, Ryan; Elenitoba-Johnson, Kojo; Lim, Megan S

2017-08-01

PKM2 (pyruvate kinase M2), a critical regulator of glycolysis, is phosphorylated by numerous growth factor receptors and oncogenic tyrosine kinases including NPM-ALK which is expressed in a subset of aggressive T-cell non-Hodgkin lymphomas known as anaplastic large cell lymphoma, ALK-positive. Our previous work demonstrated that phosphorylation of Y105-PKM2 by NPM-ALK regulates a major metabolic shift to promote lymphomagenesis. In addition to its role in metabolism, recent studies have shown that PKM2 promotes oncogenesis by phosphorylating nuclear STAT3 (signal transducer and activator of transcription 3) and regulating transcription of genes involved in cell survival and proliferation. We hypothesized that identification of novel PKM2 interactors could provide additional insights into its expanding functional role in cancer. To this end, immunocomplexes of FLAG-tagged PKM2 were isolated from NPM-ALK-positive ALCL (anaplastic large cell lymphoma) cells and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) which led to the identification of polypyrimidine tract-binding protein (PTBP1) as a novel interactor of PKM2. The interaction between PTBP1 and PKM2 was restricted to the nucleus and was dependent on NPM-ALK mediated Y105 phosphorylation of PKM2. Stable shRNA-mediated silencing of PTBP1 resulted in a marked decrease in pY105-PKM2 and pY705-STAT3 which led to decreased ALCL cell proliferation and colony formation. Overall, our data demonstrate that PTBP1 interacts with PKM2 and promotes ALCL oncogenesis by facilitating PKM2-dependent activation of STAT3 within the nucleus.

New cobalt(II) and nickel(II) complexes of benzyl carbazate Schiff bases: Syntheses, crystal structures, in vitro DNA and HSA binding studies.

PubMed

Nithya, Palanivelu; Helena, Sannasi; Simpson, Jim; Ilanchelian, Malaichamy; Muthusankar, Aathi; Govindarajan, Subbiah

2016-12-01

In the present study, new Schiff base complexes with the composition [M(NCS) 2 (L1) 2 ]·nH 2 O, where M=Co (n=0) (1) and Ni (n=2) (2); [M(NCS) 2 (L2) 2 ], M=Co (3) and Ni (4) as well as [M(NCS) 2 (L3) 2 ], M=Co (5) and Ni (6); (L1=benzyl 2-(propan-2-ylidene)hydrazinecarboxylate, L2=benzyl 2-(butan-2-ylidene)hydrazinecarboxylate and L3=benzyl 2-(pentan-3-ylidene)hydrazinecarboxylate) have been synthesized by a template method. The complexes were characterised by analytical methods, spectroscopic studies, thermal and X-ray diffraction techniques. The structures of all the complexes explore that the metal(II) cation has a trans-planar coordination environment, the monomeric units containing a six-coordinated metal center in octahedral geometry with N-bound isothiocyanate anions coordinated as terminal ligands. Furthermore, the binding of the two Schiff base ligands to the metal centers involves the azomethine nitrogen and the carbonyl oxygen in mutually trans configuration. The binding interactions of all the complexes with Calf thymus-deoxyribonucleic acid (CT-DNA) and human serum albumin (HSA) have been investigated using absorption and emission spectral techniques. The CT-DNA binding properties of these complexes reveal that they bind to CT-DNA through a partial intercalation mode and the binding constant values were calculated using the absorption and emission spectral data. The binding constant values (~10×10 6 moldm -3 ) indicate strong binding of metal complexes with CT-DNA. HSA binding interaction studies showed that the cobalt and nickel complexes can quench the intrinsic fluorescence of HSA through static quenching process. Also, molecular docking studies were supported out to apprehend the binding interactions of these complexes with DNA and HSA which offer new understandings into the experimental model observations. Copyright © 2016 Elsevier B.V. All rights reserved.

Naturally occurring phenolic acids modulate TPA-induced activation of EGFR, AP-1, and STATs in mouse epidermis.

PubMed

Cichocki, Michał; Dałek, Miłosz; Szamałek, Mateusz; Baer-Dubowska, Wanda

2014-01-01

Epidermal growth factor receptor (EGFR) plays an important role in epithelial carcinogenesis and appears to be involved in STATs activation. In this study we investigated the possible interference of naturally occurring phenolic acids with EGFR, activator protein-1 (AP-1), and signal transducers and activators of transcription (STATs) pathways activated by topical application of tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in Balb/c mice epidermis. Pretreatment with tannic or chlorogenic acid resulted in a significant decrease in the phosphorylation of EGFR Y-1068 and Y-1173 tyrosine residues, which was accompanied by reduced activation of AP-1. Tannic acid decreased also the c-Jun AP-1 subunit level and binding to TPA response element (TRE) (3- and 2-fold in comparison with TPA-treated group respectively). Simultaneous reduction of JNK activity might be responsible for reduced activation of AP-1. In contrast to these more complex phenolics, protocatechuic acid increased the activity of JNK and was also the most efficient inhibitor of STATs activation. These results indicate that naturally occurring phenolic acids, by decreasing EGFR, AP-1, and STATs activation, may modulate other elements both upstream and downstream in these pathways and thus inhibit the tumor development. Although more complex phenolics affect mainly the EGFR/AP-1 pathway, STATs seem to be the most important targets for simple compounds, such as protocatechuic acid.

Induction of Interferon-Stimulated Genes by Simian Virus 40 T Antigens

PubMed Central

Rathi, Abhilasha V.; Cantalupo, Paul G.; Sarkar, Saumendra N.; Pipas, James M.

2010-01-01

Simian virus 40 (SV40) large T antigen (TAg) is a multifunctional oncoprotein essential for productive viral infection and for cellular transformation. We have used microarray analysis to examine the global changes in cellular gene expression induced by wild-type T antigen (TAgwt) and TAg-mutants in mouse embryo fibroblasts (MEFs). The expression profile of approximately 800 cellular genes was altered by TAgwt and a truncated TAg (TAgN136), including many genes that influence cell cycle, DNA-replication, transcription, chromatin structure and DNA repair. Unexpectedly, we found a significant number of immune response genes upregulated by TAgwt including many interferon stimulated genes (ISGs) such as ISG56, OAS, Rsad2, Ifi27 and Mx1. Additionally, we also observed activation of STAT1 by TAgwt. Our genetic studies using several TAg mutants reveal an unexplored function of TAg and indicate that the LXCXE motif and p53 binding are required for the upregulation of ISGs. PMID:20692676

Oligomerization transforms human APOBEC3G from an efficient enzyme to a slowly dissociating nucleic acid-binding protein

NASA Astrophysics Data System (ADS)

Chaurasiya, Kathy R.; McCauley, Micah J.; Wang, Wei; Qualley, Dominic F.; Wu, Tiyun; Kitamura, Shingo; Geertsema, Hylkje; Chan, Denise S. B.; Hertz, Amber; Iwatani, Yasumasa; Levin, Judith G.; Musier-Forsyth, Karin; Rouzina, Ioulia; Williams, Mark C.

2014-01-01

The human APOBEC3 proteins are a family of DNA-editing enzymes that play an important role in the innate immune response against retroviruses and retrotransposons. APOBEC3G is a member of this family that inhibits HIV-1 replication in the absence of the viral infectivity factor Vif. Inhibition of HIV replication occurs by both deamination of viral single-stranded DNA and a deamination-independent mechanism. Efficient deamination requires rapid binding to and dissociation from ssDNA. However, a relatively slow dissociation rate is required for the proposed deaminase-independent roadblock mechanism in which APOBEC3G binds the viral template strand and blocks reverse transcriptase-catalysed DNA elongation. Here, we show that APOBEC3G initially binds ssDNA with rapid on-off rates and subsequently converts to a slowly dissociating mode. In contrast, an oligomerization-deficient APOBEC3G mutant did not exhibit a slow off rate. We propose that catalytically active monomers or dimers slowly oligomerize on the viral genome and inhibit reverse transcription.

Metal Ion Binding at the Catalytic Site Induces Widely Distributed Changes in a Sequence Specific Protein–DNA Complex

PubMed Central

2016-01-01

Metal ion cofactors can alter the energetics and specificity of sequence specific protein–DNA interactions, but it is unknown if the underlying effects on structure and dynamics are local or dispersed throughout the protein–DNA complex. This work uses EcoRV endonuclease as a model, and catalytically inactive lanthanide ions, which replace the Mg2+ cofactor. Nuclear magnetic resonance (NMR) titrations indicate that four Lu3+ or two La3+ cations bind, and two new crystal structures confirm that Lu3+ binding is confined to the active sites. NMR spectra show that the metal-free EcoRV complex with cognate (GATATC) DNA is structurally distinct from the nonspecific complex, and that metal ion binding sites are not assembled in the nonspecific complex. NMR chemical shift perturbations were determined for 1H–15N amide resonances, for 1H–13C Ile-δ-CH3 resonances, and for stereospecifically assigned Leu-δ-CH3 and Val-γ-CH3 resonances. Many chemical shifts throughout the cognate complex are unperturbed, so metal binding does not induce major conformational changes. However, some large perturbations of amide and side chain methyl resonances occur as far as 34 Å from the metal ions. Concerted changes in specific residues imply that local effects of metal binding are propagated via a β-sheet and an α-helix. Both amide and methyl resonance perturbations indicate changes in the interface between subunits of the EcoRV homodimer. Bound metal ions also affect amide hydrogen exchange rates for distant residues, including a distant subdomain that contacts DNA phosphates and promotes DNA bending, showing that metal ions in the active sites, which relieve electrostatic repulsion between protein and DNA, cause changes in slow dynamics throughout the complex. PMID:27786446

Interaction of Sulforaphane with DNA and RNA

PubMed Central

Abassi Joozdani, Farzaneh; Yari, Faramarz; Abassi Joozdani, Parvaneh; Nafisi, Shohreh

2015-01-01

Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables with anti-inflammatory, anti-oxidant and anti-cancer activities. However, the antioxidant and anticancer mechanism of sulforaphane is not well understood. In the present research, we reported binding modes, binding constants and stability of SFN–DNA and -RNA complexes by Fourier transform infrared (FTIR) and UV–Visible spectroscopic methods. Spectroscopic evidence showed DNA intercalation with some degree of groove binding. SFN binds minor and major grooves of DNA and backbone phosphate (PO2), while RNA binding is through G, U, A bases with some degree of SFN–phosphate (PO2) interaction. Overall binding constants were estimated to be K(SFN–DNA)=3.01 (± 0.035)×104 M-1 and K(SFN–RNA)= 6.63 (±0.042)×103 M-1. At high SFN concentration (SFN/RNA = 1/1), DNA conformation changed from B to A occurred, while RNA remained in A-family structure. PMID:26030290

DNA-binding studies of a tetraalkyl-substituted porphyrin and the mutually adaptive distortion principle.

PubMed

Ghimire, Srijana; Fanwick, Phillip E; McMillin, David R

2014-10-20

This investigation explores DNA-binding interactions of various forms of an alkyl-substituted cationic porphyrin, H2TC3 (5,10,15,20-tetra[3-(3'-methylimidazolium-1'-yl)]porphyrin). The motivating idea is that incorporating alkyl rather than aryl substituents in the meso positions will enhance the prospects for intercalative as well as external binding to DNA hosts. The ligands may also be applicable for photodynamic and/or anticancer therapy. Methods employed include absorbance, circular dichroism, and emission spectroscopies, as well as viscometry and X-ray crystallography. By comparison with the classical H2T4 system, H2TC3 exhibits a higher molar extinction coefficient but is more prone to self-association. Findings of note include that the copper(II)-containing form Cu(TC3) is adept at internalizing into single-stranded as well as B-form DNA, regardless of the base composition. Surprisingly, however, external binding of H2TC3 occurs within domains that are rich in adenine-thymine base pairs. The difference in the deformability of H2TC3 versus Cu(TC3) probably accounts for the reactivity difference. Finally, Zn(TC3) binds externally, as the metal center remains five-coordinate.

Tetrandrine has anti-adipogenic effect on 3T3-L1 preadipocytes through the reduced expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, perilipin A, and STAT-3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jang, Byeong-Churl, E-mail: jangbc123@gw.kmu.ac.kr

Tetrandrine is a bisbenzylisoquinoline alkaloid isolated from the roots of Stephania tetrandra S. Moore and has been shown to possess anti-inflammatory and anti-cancerous activities. In this study, the effect of tetrandrine on adipogenesis in 3T3-L1 preadipocytes was investigated. Tetrandrine at 10 μM concentration strongly inhibited lipid accumulation and triglyceride (TG) synthesis during the differentiation of 3T3-L1 preadipocytes into adipocytes. On mechanistic levels, tetrandrine reduced not only the expressions of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), and perilipin A but also the phosphorylation levels of signal transducer and activator of transcription-3 (STAT-3) during 3T3-L1 adipocyte differentiation. Tetrandrinemore » also reduced the mRNA expression of leptin, but not adiponectin, during 3T3-L1 adipocyte differentiation. Collectively, these findings show that tetrandrine has strong anti-adipogenic effect on 3T3-L1 preadipocytes and the effect is largely attributable to the reduced expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, perilipin A, and STAT-3. - Highlights: • Tetrandrine, a bisbenzylisoquinoline alkaloid, inhibits adipogenesis. • Tetrandrine inhibits C/EBP-α, PPAR-γ, FAS, perilipin A, and STAT-3 in 3T3-L1 adipocytes. • Tetrandrine reduces leptin, but not adiponectin, expression in 3T3-L1 adipocytes. • Tetrandrine may thus have therapeutic potential against obesity.« less

Fatty acid binding protein 5 promotes tumor angiogenesis and activates the IL6/STAT3/VEGFA pathway in hepatocellular carcinoma.

PubMed

Pan, Long; Xiao, Heng; Liao, Rui; Chen, Qingsong; Peng, Chong; Zhang, Yuchi; Mu, Tong; Wu, Zhongjun

2018-06-25

Tumor angiogenesis is an essential process for facilitating tumor growth and metastasis. Fatty acid binding protein 5(FABP5)is highly expressed in hepatocellular carcinoma (HCC). Thus, we investigated the role of FABP5 in tumor angiogenesis during HCC development. In this study, the protein and mRNA levels of FABP5 in matched HCC and adjacent noncancerous liver tissues from 43 patients were determined using immunohistochemistry and real-time quantitative PCR, respectively. Two HCC cell lines (Huh7 and SMMC-7721) and human umbilical vein endothelial cells (HUVECS) were used to investigate the pro-angiogenic effect of FABP5 by tube formation, CCK8 and Transwell migration assays. The expression levels of interleukin 6 (IL6) and vascular endothelial growth factor A (VEGFA) secreted from HCC cells were detected by enzyme-linked immunosorbent assay (ELISA). In 43 HCC patients, the expression of FABP5 mRNA was positively correlated with intratumoral VEGFA mRNA expression. FABP5 mRNA expression was also associated with adverse HCC characteristics. In vitro, cell viability, cell migration and tube formation in HUVECs were enhanced with increasing expression of FABP5 in HCC cells. Downregulation of FABP5 expression inhibited the IL6/STAT3/VEGFA pathway in HCC cells and inhibited tumor angiogenesis. FABP5 was shown to promote angiogenesis and activate the IL6/STAT3/VEGFA pathway in HCC. FABP5 may be a potential antiangiogenic target in the treatment of HCC. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Reduced DNA repair in mouse satellite DNA after treatment with methylmethanesulfonate, and N-methyl-N-nitrosourea.

PubMed Central

Bodell, W J; Banerjee, M R

1976-01-01

We have measured DNA repair in mouse satellite and main band DNA as resolved by Ag+-Cs2SO4 centrifugation in response to treatment with the alkylating agents, methyl methanesulfonate, and N-methyl-N-nitrosourea. We find that there is a statistically significant lower incorporation of 3H-Tdr into the satellite DNA as compared to the main band at varying periods after treatment with the alkylating agents. This suggests a reduced repair activity in the satellite DNA. We have measured the extent of binding of 14C-methyl methanesulfonate to the satellite, and main band DNA, and no difference in binding was observed, indicating that the reduced repair activity of satellite DNA is not due to a difference in binding of alkylating agents. We believe that the reduced incorporation of 3H-Tdr into satellite DNA may be due to its location in the condensed chromatin fraction. PMID:184436

Toxicological effect of TiO2 nanoparticle-induced myocarditis in mice

NASA Astrophysics Data System (ADS)

Hong, Fashui; Wang, Ling; Yu, Xiaohong; Zhou, Yingjun; Hong, Jie; Sheng, Lei

2015-08-01

Currently, impacts of exposure to TiO2 nanoparticles (NPs) on the cardiovascular system are not well understood. The aim of this study was to investigate whether TiO2 NPs induce myocarditis and its underlying molecular mechanism in the cardiac inflammation in mice. Mice were exposed to TiO2 NPs for 6 months; biochemical parameters of serum and expression of Th1-related and Th2-related cytokines in the heart were investigated. The results showed that TiO2 NP exposure resulted in cardiac lesions coupling with pulmonary inflammation; increases of aspartate aminotransferase (AST), creatine kinase (CK), C-reaction protein (CRP), lactate dehydrogenase (LDH), alpha-hydroxybutyrate dehydrogenase (HBDH), adhesion molecule-1 (ICAM-1), and monocyte chemoattractant protein-1 (MCP-1) levels; and a reduction of nitric oxide (NOx) level in the serum. These were associated with increases of nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNF-α), interleukin (IL)-4, IL-6, transforming growth factor-β (TGF-β), creatine kinase, CRP, adhesion molecule-1, and monocyte chemoattractant protein-1, interferon-γ (IFN-γ), signal transducers and activators of transcription (STAT)1, STAT3, or STAT6, GATA-binding domain-3, GATA-binding domain-4, endothelin-1 expression levels, and T-box expressed in T cells expression level that is the master regulator of pro-inflammatory cytokines and transcription factors in the heart. These findings imply that TiO2 NP exposure may increase the occurrence and development of cardiovascular diseases.

Interferon Independent Non-Canonical STAT Activation and Virus Induced Inflammation

PubMed Central

Wu, Chunyan

2018-01-01

Interferons (IFNs) are a group of secreted proteins that play critical roles in antiviral immunity, antitumor activity, activation of cytotoxic T cells, and modulation of host immune responses. IFNs are cytokines, and bind receptors on cell surfaces to trigger signal transduction. The major signaling pathway activated by IFNs is the JAK/STAT (Janus kinase/signal transducer and activator of transcription) pathway, a complex pathway involved in both viral and host survival strategies. On the one hand, viruses have evolved strategies to escape from antiviral host defenses evoked by IFN-activated JAK/STAT signaling. On the other hand, viruses have also evolved to exploit the JAK/STAT pathway to evoke activation of certain STATs that somehow promote viral pathogenesis. In this review, recent progress in our understanding of the virus-induced IFN-independent STAT signaling and its potential roles in viral induced inflammation and pathogenesis are summarized in detail, and perspectives are provided. PMID:29662014

Ligand binding to telomeric G-quadruplex DNA investigated by funnel-metadynamics simulations

PubMed Central

Moraca, Federica; Amato, Jussara; Ortuso, Francesco; Artese, Anna; Novellino, Ettore; Alcaro, Stefano; Parrinello, Michele; Limongelli, Vittorio

2017-01-01

G-quadruplexes (G4s) are higher-order DNA structures typically present at promoter regions of genes and telomeres. Here, the G4 formation decreases the replicative DNA at each cell cycle, finally leading to apoptosis. The ability to control this mitotic clock, particularly in cancer cells, is fascinating and passes through a rational understanding of the ligand/G4 interaction. We demonstrate that an accurate description of the ligand/G4 binding mechanism is possible using an innovative free-energy method called funnel-metadynamics (FM), which we have recently developed to investigate ligand/protein interaction. Using FM simulations, we have elucidated the binding mechanism of the anticancer alkaloid berberine to the human telomeric G4 (d[AG3(T2AG3)3]), computing also the binding free-energy landscape. Two ligand binding modes have been identified as the lowest energy states. Furthermore, we have found prebinding sites, which are preparatory to reach the final binding mode. In our simulations, the ions and the water molecules have been explicitly represented and the energetic contribution of the solvent during ligand binding evaluated. Our theoretical results provide an accurate estimate of the absolute ligand/DNA binding free energy (ΔGb0 = −10.3 ± 0.5 kcal/mol) that we validated through steady-state fluorescence binding assays. The good agreement between the theoretical and experimental value demonstrates that FM is a most powerful method to investigate ligand/DNA interaction and can be a useful tool for the rational design also of G4 ligands. PMID:28232513

STAT1 is essential for the inhibition of hepatitis C virus replication by interferon-λ but not by interferon-α.

PubMed

Yamauchi, Shota; Takeuchi, Kenji; Chihara, Kazuyasu; Honjoh, Chisato; Kato, Yuji; Yoshiki, Hatsumi; Hotta, Hak; Sada, Kiyonao

2016-12-08

Interferon-α (IFN-α) and IFN-λ are structurally distinct cytokines that bind to different receptors, but induce expression of similar sets of genes through Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathways. The difference between IFN-α and IFN-λ signaling remains poorly understood. Here, using the CRISPR/Cas9 system, we examine the role of STAT1 and STAT2 in the inhibition of hepatitis C virus (HCV) replication by IFN-α and IFN-λ. Treatment with IFN-α increases expression of IFN-stimulated genes (ISGs) such as double-stranded RNA-activated protein kinase (PKR) and decreases viral RNA and protein levels in HCV-infected Huh-7.5 human hepatoma cells. These responses are only partially attenuated by knockout of STAT1 but are abolished by knockout of STAT2. In contrast, the inhibition of HCV replication by IFN-λ is abolished by knockout of STAT1 or STAT2. Microarray analysis reveals that IFN-α but not IFN-λ can induce expression of the majority of ISGs in STAT1 knockout cells. These findings suggest that IFN-α can inhibit HCV replication through a STAT2-dependent but STAT1-independent pathway, whereas IFN-λ induces ISG expression and inhibits HCV replication exclusively through a STAT1- and STAT2-dependent pathway.

STAT1 is essential for the inhibition of hepatitis C virus replication by interferon-λ but not by interferon-α

PubMed Central

Yamauchi, Shota; Takeuchi, Kenji; Chihara, Kazuyasu; Honjoh, Chisato; Kato, Yuji; Yoshiki, Hatsumi; Hotta, Hak; Sada, Kiyonao

2016-01-01

Interferon-α (IFN-α) and IFN-λ are structurally distinct cytokines that bind to different receptors, but induce expression of similar sets of genes through Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathways. The difference between IFN-α and IFN-λ signaling remains poorly understood. Here, using the CRISPR/Cas9 system, we examine the role of STAT1 and STAT2 in the inhibition of hepatitis C virus (HCV) replication by IFN-α and IFN-λ. Treatment with IFN-α increases expression of IFN-stimulated genes (ISGs) such as double-stranded RNA-activated protein kinase (PKR) and decreases viral RNA and protein levels in HCV-infected Huh-7.5 human hepatoma cells. These responses are only partially attenuated by knockout of STAT1 but are abolished by knockout of STAT2. In contrast, the inhibition of HCV replication by IFN-λ is abolished by knockout of STAT1 or STAT2. Microarray analysis reveals that IFN-α but not IFN-λ can induce expression of the majority of ISGs in STAT1 knockout cells. These findings suggest that IFN-α can inhibit HCV replication through a STAT2-dependent but STAT1-independent pathway, whereas IFN-λ induces ISG expression and inhibits HCV replication exclusively through a STAT1- and STAT2-dependent pathway. PMID:27929099

Analytical methods to determine the comparative DNA binding studies of curcumin-Cu(II) complexes

NASA Astrophysics Data System (ADS)

Rajesh, Jegathalaprathaban; Rajasekaran, Marichamy; Rajagopal, Gurusamy; Athappan, Periakaruppan

2012-11-01

DNA interaction studies of two mononuclear [1:1(1); 1:2(2)] copper(II) complexes of curcumin have been studied. The interaction of these complexes with CT-DNA has been explored by physical methods to propose modes of DNA binding of the complexes. Absorption spectral titrations of complex 1 with CT-DNA shows a red-shift of 3 nm with the DNA binding affinity of Kb, 5.21 × 104 M-1 that are higher than that obtained for 2 (red-shift, 2 nm; Kb, 1.73 × 104 M-1) reveal that the binding occurs in grooves as a result of the interaction is via exterior phosphates. The CD spectra of these Cu(II) complexes show a red shift of 3-10 nm in the positive band with increase in intensities. This spectral change of induced CD due to the hydrophobic interaction of copper complexes with DNA is the characteristic of B to A conformational change. The EB displacement assay also reveals the same trend as observed in UV-Vis spectral titration. The addition of complexes 1 and 2 to the DNA bound ethidium bromide (EB) solutions causes an obvious reduction in emission intensities indicating that these complexes competitively bind to DNA with EB. The positive shift of both the Epc and E0' accompanied by reduction of peak currents in differential pulse voltammogram (DPV), upon adding different concentrations of DNA to the metal complexes, are obviously in favor of strong binding to DNA. The super coiled plasmid pUC18 DNA cleavage ability of Cu(II) complexes in the presence of reducing agent reveals the single strand DNA cleavage (ssDNA) is observed. The hydroxyl radical (HOrad ) and the singlet oxygen are believed to be the reactive species responsible for the cleavage.

Artesunate inhibits adipogeneis in 3T3-L1 preadipocytes by reducing the expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, perilipin A, and STAT-3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jang, Byeong-Churl, E-mail: jangbc123@gw.kmu.ac.kr

Differentiation of preadipocyte, also called adipogenesis, leads to the phenotype of mature adipocyte. However, excessive adipogenesis is closely linked to the development of obesity. Artesunate, one of artemisinin-type sesquiterpene lactones from Artemisia annua L., is known for anti-malarial and anti-cancerous activities. In this study, we investigated the effect of artesunate on adipogenesis in 3T3-L1 preadipocytes. Artesunate strongly inhibited lipid accumulation and triglyceride (TG) synthesis during the differentiation of 3T3-L1 preadipocytes into adipocytes at 5 μM concentration. Artesunate at 5 μM also reduced not only the expressions of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), and perilipin A butmore » also the phosphorylation levels of signal transducer and activator of transcription-3 (STAT-3) during adipocyte differentiation. Moreover, artesunate at 5 μM reduced leptin, but not adiponectin, mRNA expression during adipocyte differentiation. Taken together, these findings demonstrate that artesunate inhibits adipogenesis in 3T3-L1 preadipoytes through the reduced expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, perilipin A, and STAT-3. -- Highlights: •Artesunate, an artemisinin derivative, inhibits adipogenesis. •Artesunate inhibits C/EBP-α, PPAR-γ, FAS, perilipin A, and STAT-3 in 3T3-L1 adipocytes. •Artesunate reduces leptin, but not adiponectin, expression in 3T3-L1 adipocytes. •Artesunate thus may have therapeutic potential against obesity.« less

Preliminary crystallographic analysis of mouse Elf3 C-terminal DNA-binding domain in complex with type II TGF-[beta] receptor promoter DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Agarkar, Vinod B.; Babayeva, Nigar D.; Rizzino, Angie

2010-10-08

Ets proteins are transcription factors that activate or repress the expression of genes that are involved in various biological processes, including cellular proliferation, differentiation, development, transformation and apoptosis. Like other Ets-family members, Elf3 functions as a sequence-specific DNA-binding transcriptional factor. A mouse Elf3 C-terminal fragment (amino-acid residues 269-371) containing the DNA-binding domain has been crystallized in complex with mouse type II TGF-{beta} receptor promoter (TR-II) DNA. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 42.66, b = 52, c = 99.78 {angstrom}, and diffracted to a resolution of 2.2 {angstrom}.

RING finger and WD repeat domain 3 (RFWD3) associates with replication protein A (RPA) and facilitates RPA-mediated DNA damage response.

PubMed

Liu, Shangfeng; Chu, Jessica; Yucer, Nur; Leng, Mei; Wang, Shih-Ya; Chen, Benjamin P C; Hittelman, Walter N; Wang, Yi

2011-06-24

DNA damage response is crucial for maintaining genomic integrity and preventing cancer by coordinating the activation of checkpoints and the repair of damaged DNA. Central to DNA damage response are the two checkpoint kinases ATM and ATR that phosphorylate a wide range of substrates. RING finger and WD repeat domain 3 (RFWD3) was initially identified as a substrate of ATM/ATR from a proteomic screen. Subsequent studies showed that RFWD3 is an E3 ubiquitin ligase that ubiquitinates p53 in vitro and positively regulates p53 levels in response to DNA damage. We report here that RFWD3 associates with replication protein A (RPA), a single-stranded DNA-binding protein that plays essential roles in DNA replication, recombination, and repair. Binding of RPA to single-stranded DNA (ssDNA), which is generated by DNA damage and repair, is essential for the recruitment of DNA repair factors to damaged sites and the activation of checkpoint signaling. We show that RFWD3 is physically associated with RPA and rapidly localizes to sites of DNA damage in a RPA-dependent manner. In vitro experiments suggest that the C terminus of RFWD3, which encompass the coiled-coil domain and the WD40 domain, is necessary for binding to RPA. Furthermore, DNA damage-induced phosphorylation of RPA and RFWD3 is dependent upon each other. Consequently, loss of RFWD3 results in the persistent foci of DNA damage marker γH2AX and the repair protein Rad51 in damaged cells. These findings suggest that RFWD3 is recruited to sites of DNA damage and facilitates RPA-mediated DNA damage signaling and repair.

Synthesis and characterization of DNA minor groove binding alkylating agents.

PubMed

Iyer, Prema; Srinivasan, Ajay; Singh, Sreelekha K; Mascara, Gerard P; Zayitova, Sevara; Sidone, Brian; Fouquerel, Elise; Svilar, David; Sobol, Robert W; Bobola, Michael S; Silber, John R; Gold, Barry

2013-01-18

Derivatives of methyl 3-(1-methyl-5-(1-methyl-5-(propylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-ylamino)-3-oxopropane-1-sulfonate (1), a peptide-based DNA minor groove binding methylating agent, were synthesized and characterized. In all cases, the N-terminus was appended with an O-methyl sulfonate ester, while the C-terminus group was varied with nonpolar and polar side chains. In addition, the number of pyrrole rings was varied from 2 (dipeptide) to 3 (tripeptide). The ability of the different analogues to efficiently generate N3-methyladenine was demonstrated as was their selectivity for minor groove (N3-methyladenine) versus major groove (N7-methylguanine) methylation. Induced circular dichroism studies were used to measure the DNA equilibrium binding properties of the stable sulfone analogues; the tripeptide binds with affinity that is >10-fold higher than that of the dipeptide. The toxicities of the compounds were evaluated in alkA/tag glycosylase mutant E. coli and in human WT glioma cells and in cells overexpressing and under-expressing N-methylpurine-DNA glycosylase, which excises N3-methyladenine from DNA. The results show that equilibrium binding correlates with the levels of N3-methyladenine produced and cellular toxicity. The toxicity of 1 was inversely related to the expression of MPG in both the bacterial and mammalian cell lines. The enhanced toxicity parallels the reduced activation of PARP and the diminished rate of formation of aldehyde reactive sites observed in the MPG knockdown cells. It is proposed that unrepaired N3-methyladenine is toxic due to its ability to directly block DNA polymerization.

Neisseria conserved protein DMP19 is a DNA mimic protein that prevents DNA binding to a hypothetical nitrogen-response transcription factor

PubMed Central

Wang, Hao-Ching; Ko, Tzu-Ping; Wu, Mao-Lun; Ku, Shan-Chi; Wu, Hsing-Ju; Wang, Andrew H.-J.

2012-01-01

DNA mimic proteins occupy the DNA binding sites of DNA-binding proteins, and prevent these sites from being accessed by DNA. We show here that the Neisseria conserved hypothetical protein DMP19 acts as a DNA mimic. The crystal structure of DMP19 shows a dsDNA-like negative charge distribution on the surface, suggesting that this protein should be added to the short list of known DNA mimic proteins. The crystal structure of another related protein, NHTF (Neisseria hypothetical transcription factor), provides evidence that it is a member of the xenobiotic-response element (XRE) family of transcriptional factors. NHTF binds to a palindromic DNA sequence containing a 5′-TGTNAN11TNACA-3′ recognition box that controls the expression of an NHTF-related operon in which the conserved nitrogen-response protein [i.e. (Protein-PII) uridylyltransferase] is encoded. The complementary surface charges between DMP19 and NHTF suggest specific charge–charge interaction. In a DNA-binding assay, we found that DMP19 can prevent NHTF from binding to its DNA-binding sites. Finally, we used an in situ gene regulation assay to provide evidence that NHTF is a repressor of its down-stream genes and that DMP19 can neutralize this effect. We therefore conclude that the interaction of DMP19 and NHTF provides a novel gene regulation mechanism in Neisseria spps. PMID:22373915

Unveiling Stability Criteria of DNA-Carbon Nanotubes Constructs by Scanning Tunneling Microscopy and Computational Modeling

DOE PAGES

Kilina, Svetlana; Yarotski, Dzmitry A.; Talin, A. Alec; ...

2011-01-01

We present a combined approach that relies on computational simulations and scanning tunneling microscopy (STM) measurements to reveal morphological properties and stability criteria of carbon nanotube-DNA (CNT-DNA) constructs. Application of STM allows direct observation of very stable CNT-DNA hybrid structures with the well-defined DNA wrapping angle of 63.4 ° and a coiling period of 3.3 nm. Using force field simulations, we determine how the DNA-CNT binding energy depends on the sequence and binding geometry of a single strand DNA. This dependence allows us to quantitatively characterize the stability of a hybrid structure with an optimal π-stacking between DNA nucleotides and themore » tube surface and better interpret STM data. Our simulations clearly demonstrate the existence of a very stable DNA binding geometry for (6,5) CNT as evidenced by the presence of a well-defined minimum in the binding energy as a function of an angle between DNA strand and the nanotube chiral vector. This novel approach demonstrates the feasibility of CNT-DNA geometry studies with subnanometer resolution and paves the way towards complete characterization of the structural and electronic properties of drug-delivering systems based on DNA-CNT hybrids as a function of DNA sequence and a nanotube chirality.« less

PHF1 Tudor and N-terminal domains synergistically target partially unwrapped nucleosomes to increase DNA accessibility

PubMed Central

Gibson, Matthew D.; Gatchalian, Jovylyn; Slater, Andrew; Kutateladze, Tatiana G.

2017-01-01

Abstract The Tudor domain of human PHF1 recognizes trimethylated lysine 36 on histone H3 (H3K36me3). PHF1 relies on this interaction to regulate PRC2 methyltransferase activity, localize to DNA double strand breaks and mediate nucleosome accessibility. Here, we investigate the impact of the PHF1 N-terminal domain (NTD) on the Tudor domain interaction with the nucleosome. We show that the NTD is partially ordered when it is natively attached to the Tudor domain. Through a combination of FRET and single molecule studies, we find that the increase of DNA accessibility within the H3K36me3-containing nucleosome, instigated by the Tudor binding to H3K36me3, is dramatically enhanced by the NTD. We demonstrate that this nearly order of magnitude increase is due to preferential binding of PHF1 to partially unwrapped nucleosomes, and that PHF1 alters DNA–protein binding within the nucleosome by decreasing dissociation rates. These results highlight the potency of a PTM-binding protein to regulate DNA accessibility and underscores the role of the novel mechanism by which nucleosomes control DNA–protein binding through increasing protein dissociation rates. PMID:28082396

Strip biosensor for amplified detection of nerve growth factor-beta based on a molecular translator and catalytic DNA circuit.

PubMed

Liu, Jun; Lai, Ting; Mu, Kejie; Zhou, Zheng

2014-10-07

We have demonstrated a new visual detection approach based on a molecular translator and a catalytic DNA circuit for the detection of nerve growth factor-beta (NGF-β). In this assay, a molecular translator based on the binding-induced DNA strand-displacement reaction was employed to convert the input protein to an output DNA signal. The molecular translator is composed of a target recognition element and a signal output element. Target recognition is achieved by the binding of the anti-NGF-β antibody to the target protein. Polyclonal anti-NGF-β antibody is conjugated to DNA1 and DNA2. The antibody conjugated DNA1 is initially hybridized to DNA3 to form a stable DNA1/DNA3 duplex. In the presence of NGF-β, the binding of the same target protein brings DNA1 and DNA2 into close proximity, resulting in an increase in their local effective concentration. This process triggers the strand-displacement reaction between DNA2 and DNA3 and releases the output DNA3. The released DNA3 is further amplified by a catalytic DNA circuit. The product of the catalytic DNA circuit is detected by a strip biosensor. This proposed assay has high sensitivity and selectivity with a dynamic response ranging from 10 fM to 10 pM, and its detection limit is 10 fM of NGF-β. This work provides a sensitive, enzyme-free, and universal strategy for the detection of other proteins.

Correlation of Local Effects of DNA Sequence and Position of Beta-Alanine Inserts with Polyamide-DNA Complex Binding Affinities and Kinetics

PubMed Central

Wang, Shuo; Nanjunda, Rupesh; Aston, Karl; Bashkin, James K.; Wilson, W. David

2012-01-01

In order to better understand the effects of β-alanine (β) substitution and the number of heterocycles on DNA binding affinity and selectivity, the interactions of an eight-ring hairpin polyamide (PA) and two β derivatives as well as a six-heterocycle analog have been investigated with their cognate DNA sequence, 5′-TGGCTT-3′. Binding selectivity and the effects of β have been investigated with the cognate and five mutant DNAs. A set of powerful and complementary methods have been employed for both energetic and structural evaluations: UV-melting, biosensor-surface plasmon resonance, isothermal titration calorimetry, circular dichroism and a DNA ligation ladder global structure assay. The reduced number of heterocycles in the six-ring PA weakens the binding affinity; however, the smaller PA aggregates significantly less than the larger PAs, and allows us to obtain the binding thermodynamics. The PA-DNA binding enthalpy is large and negative with a large negative ΔCp, and is the primary driving component of the Gibbs free energy. The complete SPR binding results clearly show that β substitutions can substantially weaken the binding affinity of hairpin PAs in a position-dependent manner. More importantly, the changes in PA binding to the mutant DNAs further confirm the position-dependent effects on PA-DNA interaction affinity. Comparison of mutant DNA sequences also shows a different effect in recognition of T•A versus A•T base pairs. The effects of DNA mutations on binding of a single PA as well as the effects of the position of β substitution on binding tell a clear and very important story about sequence dependent binding of PAs to DNA. PMID:23167504

Structure-Based Mutational Analysis of the C-Terminal DNA-Binding Domain of Human Immunodeficiency Virus Type 1 Integrase: Critical Residues for Protein Oligomerization and DNA Binding

PubMed Central

Lutzke, Ramon A. Puras; Plasterk, Ronald H. A.

1998-01-01

The C-terminal domain of human immunodeficiency virus type 1 (HIV-1) integrase (IN) is a dimer that binds to DNA in a nonspecific manner. The structure of the minimal region required for DNA binding (IN220–270) has been solved by nuclear magnetic resonance spectroscopy. The overall fold of the C-terminal domain of HIV-1 IN is similar to those of Src homology region 3 domains. Based on the structure of IN220–270, we studied the role of 15 amino acid residues potentially involved in DNA binding and oligomerization by mutational analysis. We found that two amino acid residues, arginine 262 and leucine 234, contribute to DNA binding in the context of IN220–270, as indicated by protein-DNA UV cross-link analysis. We also analyzed mutant proteins representing portions of the full-length IN protein. Amino acid substitution of residues located in the hydrophobic dimer interface, such as L241A and L242A, results in the loss of oligomerization of IN; consequently, the levels of 3′ processing, DNA strand transfer, and intramolecular disintegration are strongly reduced. These results suggest that dimerization of the C-terminal domain of IN is important for correct multimerization of IN. PMID:9573250

Isolation and characterization of the DNA-binding protein (DBP) of the Autographa californica multiple nucleopolyhedrovirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mikhailov, Victor S.; N. K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow 117808; Vanarsdall, Adam L.

2008-01-20

DNA-binding protein (DBP) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was expressed as an N-terminal His{sub 6}-tag fusion using a recombinant baculovirus and purified to near homogeneity. Purified DBP formed oligomers that were crosslinked by redox reagents resulting in predominantly protein dimers and tetramers. In gel retardation assays, DBP showed a high affinity for single-stranded oligonucleotides and was able to compete with another baculovirus SSB protein, LEF-3, for binding sites. DBP binding protected ssDNA against hydrolysis by a baculovirus alkaline nuclease AN/LEF-3 complex. Partial proteolysis by trypsin revealed a domain structure of DBP that is required for interaction with DNA andmore » that can be disrupted by thermal treatment. Binding to ssDNA, but not to dsDNA, changed the pattern of proteolytic fragments of DBP indicating adjustments in protein structure upon interaction with ssDNA. DBP was capable of unwinding short DNA duplexes and also promoted the renaturation of long complementary strands of ssDNA into duplexes. The unwinding and renaturation activities of DBP, as well as the DNA binding activity, were sensitive to sulfhydryl reagents and were inhibited by oxidation of thiol groups with diamide or by alkylation with N-ethylmaleimide. A high affinity of DBP for ssDNA and its unwinding and renaturation activities confirmed identification of DBP as a member of the SSB/recombinase family. These activities and a tight association with subnuclear structures suggests that DBP is a component of the virogenic stroma that is involved in the processing of replicative intermediates.« less

Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides

PubMed Central

Yoga, Yano M. K.; Traore, Daouda A. K.; Sidiqi, Mahjooba; Szeto, Chris; Pendini, Nicole R.; Barker, Andrew; Leedman, Peter J.; Wilce, Jacqueline A.; Wilce, Matthew C. J.

2012-01-01

Poly-C-binding proteins are triple KH (hnRNP K homology) domain proteins with specificity for single stranded C-rich RNA and DNA. They play diverse roles in the regulation of protein expression at both transcriptional and translational levels. Here, we analyse the contributions of individual αCP1 KH domains to binding C-rich oligonucleotides using biophysical and structural methods. Using surface plasmon resonance (SPR), we demonstrate that KH1 makes the most stable interactions with both RNA and DNA, KH3 binds with intermediate affinity and KH2 only interacts detectibly with DNA. The crystal structure of KH1 bound to a 5′-CCCTCCCT-3′ DNA sequence shows a 2:1 protein:DNA stoichiometry and demonstrates a molecular arrangement of KH domains bound to immediately adjacent oligonucleotide target sites. SPR experiments, with a series of poly-C-sequences reveals that cytosine is preferred at all four positions in the oligonucleotide binding cleft and that a C-tetrad binds KH1 with 10 times higher affinity than a C-triplet. The basis for this high affinity interaction is finally detailed with the structure determination of a KH1.W.C54S mutant bound to 5′-ACCCCA-3′ DNA sequence. Together, these data establish the lead role of KH1 in oligonucleotide binding by αCP1 and reveal the molecular basis of its specificity for a C-rich tetrad. PMID:22344691

Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides.

PubMed

Yoga, Yano M K; Traore, Daouda A K; Sidiqi, Mahjooba; Szeto, Chris; Pendini, Nicole R; Barker, Andrew; Leedman, Peter J; Wilce, Jacqueline A; Wilce, Matthew C J

2012-06-01

Poly-C-binding proteins are triple KH (hnRNP K homology) domain proteins with specificity for single stranded C-rich RNA and DNA. They play diverse roles in the regulation of protein expression at both transcriptional and translational levels. Here, we analyse the contributions of individual αCP1 KH domains to binding C-rich oligonucleotides using biophysical and structural methods. Using surface plasmon resonance (SPR), we demonstrate that KH1 makes the most stable interactions with both RNA and DNA, KH3 binds with intermediate affinity and KH2 only interacts detectibly with DNA. The crystal structure of KH1 bound to a 5'-CCCTCCCT-3' DNA sequence shows a 2:1 protein:DNA stoichiometry and demonstrates a molecular arrangement of KH domains bound to immediately adjacent oligonucleotide target sites. SPR experiments, with a series of poly-C-sequences reveals that cytosine is preferred at all four positions in the oligonucleotide binding cleft and that a C-tetrad binds KH1 with 10 times higher affinity than a C-triplet. The basis for this high affinity interaction is finally detailed with the structure determination of a KH1.W.C54S mutant bound to 5'-ACCCCA-3' DNA sequence. Together, these data establish the lead role of KH1 in oligonucleotide binding by αCP1 and reveal the molecular basis of its specificity for a C-rich tetrad.

Berberine-induced Inactivation of Signal Transducer and Activator of Transcription 5 Signaling Promotes Male-specific Expression of a Bile Acid Uptake Transporter*

PubMed Central

Bu, Pengli; Le, Yuan; Zhang, Yue; Zhang, Youcai; Cheng, Xingguo

2017-01-01

Sodium-taurocholate co-transporting polypeptide (Ntcp/NTCP) is the major uptake transporter of bile salts in mouse and human livers. In certain diseases, including endotoxemia, cholestasis, diabetes, and hepatocarcinoma, Ntcp/NTCP expression is markedly reduced, which interferes with enterohepatic circulation of bile salts, impairing the absorption of lipophilic compounds. Therefore, normal Ntcp/NTCP expression in the liver is physiologically important. Berberine is an herbal medicine used historically to improve liver function and has recently been shown to repress STAT signaling. However, berberine effects on Ntcp/NTCP expression are unknown, prompting use to investigate this possible connection. Our results showed that berberine dose-dependently increased Ntcp expression in male mouse liver and decreased taurocholic acid levels in serum but increased them in the liver. In mouse and human hepatoma cells, berberine induced Ntcp/NTCP mRNA and protein expression and increased cellular uptake of [3H] taurocholate. Mechanistically, berberine decreased nuclear protein levels of phospho-JAK2 and phospho-STAT5, thus disrupting the JAK2-STAT5 signaling. Moreover, berberine stimulated luciferase reporter expression from the mouse Ntcp promoter when one putative STAT5 response element (RE) (−1137 bp) was deleted and from the human NTCP promoter when three putative STAT5REs (−2898, −2164, and −691 bp) were deleted. Chromatin immunoprecipitation demonstrated that berberine decreased binding of phospho-STAT5 protein to the−2164 and −691 bp STAT5REs in the human NTCP promoter. In summary, berberine-disrupted STAT5 signaling promoted mouse and human Ntcp/NTCP expression, resulting in enhanced bile acid uptake. Therefore, berberine may be a therapeutic candidate compound for maintaining bile acid homeostasis. PMID:28154180

Berberine-induced Inactivation of Signal Transducer and Activator of Transcription 5 Signaling Promotes Male-specific Expression of a Bile Acid Uptake Transporter.

PubMed

Bu, Pengli; Le, Yuan; Zhang, Yue; Zhang, Youcai; Cheng, Xingguo

2017-03-17

Sodium-taurocholate co-transporting polypeptide (Ntcp/NTCP) is the major uptake transporter of bile salts in mouse and human livers. In certain diseases, including endotoxemia, cholestasis, diabetes, and hepatocarcinoma, Ntcp/NTCP expression is markedly reduced, which interferes with enterohepatic circulation of bile salts, impairing the absorption of lipophilic compounds. Therefore, normal Ntcp/NTCP expression in the liver is physiologically important. Berberine is an herbal medicine used historically to improve liver function and has recently been shown to repress STAT signaling. However, berberine effects on Ntcp/NTCP expression are unknown, prompting use to investigate this possible connection. Our results showed that berberine dose-dependently increased Ntcp expression in male mouse liver and decreased taurocholic acid levels in serum but increased them in the liver. In mouse and human hepatoma cells, berberine induced Ntcp/NTCP mRNA and protein expression and increased cellular uptake of [3H] taurocholate. Mechanistically, berberine decreased nuclear protein levels of phospho-JAK2 and phospho-STAT5, thus disrupting the JAK2-STAT5 signaling. Moreover, berberine stimulated luciferase reporter expression from the mouse Ntcp promoter when one putative STAT5 response element (RE) (-1137 bp) was deleted and from the human NTCP promoter when three putative STAT5REs (-2898, -2164, and -691 bp) were deleted. Chromatin immunoprecipitation demonstrated that berberine decreased binding of phospho-STAT5 protein to the-2164 and -691 bp STAT5REs in the human NTCP promoter. In summary, berberine-disrupted STAT5 signaling promoted mouse and human Ntcp/NTCP expression, resulting in enhanced bile acid uptake. Therefore, berberine may be a therapeutic candidate compound for maintaining bile acid homeostasis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Photochemical and DFT studies on DNA-binding ability and antibacterial activity of lanthanum(III)-phenanthroline complex

NASA Astrophysics Data System (ADS)

Niroomand, Sona; Khorasani-Motlagh, Mozhgan; Noroozifar, Meissam; Jahani, Shohreh; Moodi, Asieh

2017-02-01

The binding of the lanthanum(III) complex containing 1,10-phenanthroline (phen), [La(phen)3Cl3·OH2], to DNA is investigated by absorption and emission methods. This complex shows absorption decreasing in a charge transfer band, and fluorescence decrement when it binds to DNA. Electronic absorption spectroscopy (UV-Vis), fluorescence spectra, iodide quenching experiments, salt effect and viscosity measurements, ethidium bromide (EB) competition test, circular dichroism (CD) spectra as well as variable temperature experiments indicate that the La(III) complex binds to fish salmon (FS) DNA, presumably via groove binding mode. The binding constants (Kb) of the La(III) complex with DNA is (2.55 ± 0.02) × 106 M-1. Furthermore, the binding site size, n, the Stern-Volmer constant KSV and thermodynamic parameters; enthalpy change (ΔH0) and entropy change (ΔS0) and Gibb's free energy (ΔG0), are calculated according to relevant fluorescent data and the Van't Hoff equation. The La(III) complex has been screened for its antibacterial activities by the disc diffusion method. Also, in order to supplement the experimental findings, DFT computation and NBO analysis are carried out.

On the connection between inherent DNA flexure and preferred binding of hydroxymethyluracil-containing DNA by the type II DNA-binding protein TF1.

PubMed

Grove, A; Galeone, A; Mayol, L; Geiduschek, E P

1996-07-12

TF1 is a member of the family of type II DNA-binding proteins, which also includes the bacterial HU proteins and the Escherichia coli integration host factor (IHF). Distinctive to TF1, which is encoded by the Bacillus subtilis bacteriophage SPO1, is its preferential binding to DNA in which thymine is replaced by 5-hydroxymethyluracil (hmU), as it is in the phage genome. TF1 binds to preferred sites within the phage genome and generates pronounced DNA bending. The extent to which DNA flexibility contributes to the sequence-specific binding of TF1, and the connection between hmU preference and DNA flexibility has been examined. Model flexible sites, consisting of consecutive mismatches, increase the affinity of thymine-containing DNA for TF1. In particular, tandem mismatches separated by nine base-pairs generate an increase, by orders of magnitude, in the affinity of TF1 for T-containing DNA with the sequence of a preferred TF1 binding site, and fully match the affinity of TF1 for this cognate site in hmU-containing DNA (Kd approximately 3 nM). Other placements of loops generate suboptimal binding. This is consistent with a significant contribution of site-specific DNA flexibility to complex formation. Analysis of complexes with hmU-DNA of decreasing length shows that a major part of the binding affinity is generated within a central 19 bp segment (delta G0 = 41.7 kJ mol-1) with more-distal DNA contributing modestly to the affinity (delta delta G = -0.42 kJ mol-1 bp-1 on increasing duplex length to 37 bp). However, a previously characterised thermostable and more tightly binding mutant TF1, TF1(E15G/T32I), derives most of its extra affinity from interaction with flanking DNA. We propose that inherent but sequence-dependent deformability of hmU-containing DNA underlies the preferential binding of TF1 and that TF1-induced DNA bendings is a result of distortions at two distinct sites separated by 9 bp of duplex DNA.

Spectroscopic studies of the interaction of aspirin and its important metabolite, salicylate ion, with DNA, A·T and G·C rich sequences

NASA Astrophysics Data System (ADS)

Bathaie, S. Z.; Nikfarjam, L.; Rahmanpour, R.; Moosavi-Movahedi, A. A.

2010-12-01

Among different biological effects of acetylsalicylic acid (ASA), its anticancer property is controversial. Since ASA hydrolyzes rapidly to salicylic acid (SA), especially in the blood, interaction of both ASA and SA (as the small molecules) with ctDNA, oligo(dA·dT) 15 and oligo(dG·dC) 15, as a possible mechanism of their action, is investigated here. The results show that the rate of ASA hydrolysis in the absence and presence of ctDNA is similar. The spectrophotometric results indicate that both ASA and SA cooperatively bind to ctDNA. The binding constants ( K) are (1.7 ± 0.7) × 10 3 M -1 and (6.7 ± 0.2) × 10 3 M -1 for ASA and SA, respectively. Both ligands quench the fluorescence emission of ethidium bromide (Et)-ctDNA complex. The Scatchard plots indicate the non-displacement based quenching (non-intercalative binding). The circular dichroism (CD) spectra of ASA- or SA-ctDsNA complexes show the minor distortion of ctDNA structure, with no characteristic peaks for intercalation of ligands. Tm of ctDNA is decreased up to 3 °C upon ASA binding. The CD results also indicate more distortions on oligo(dG·dC) 15 structure due to the binding of both ASA and SA in comparison with oligo(dA·dT) 15. All data indicate the more affinity for SA binding with DNA minor groove in comparison with ASA which has more hydrophobic character.

pUL34 binding near the human cytomegalovirus origin of lytic replication enhances DNA replication and viral growth.

PubMed

Slayton, Mark; Hossain, Tanvir; Biegalke, Bonita J

2018-05-01

The human cytomegalovirus (HCMV) UL34 gene encodes sequence-specific DNA-binding proteins (pUL34) which are required for viral replication. Interactions of pUL34 with DNA binding sites represses transcription of two viral immune evasion genes, US3 and US9. 12 additional predicted pUL34-binding sites are present in the HCMV genome (strain AD169) with three binding sites concentrated near the HCMV origin of lytic replication (oriLyt). We used ChIP-seq analysis of pUL34-DNA interactions to confirm that pUL34 binds to the oriLyt region during infection. Mutagenesis of the UL34-binding sites in an oriLyt-containing plasmid significantly reduced viral-mediated oriLyt-dependent DNA replication. Mutagenesis of these sites in the HCMV genome reduced the replication efficiencies of the resulting viruses. Protein-protein interaction analyses demonstrated that pUL34 interacts with the viral proteins IE2, UL44, and UL84, that are essential for viral DNA replication, suggesting that pUL34-DNA interactions in the oriLyt region are involved in the DNA replication cascade. Copyright © 2018 Elsevier Inc. All rights reserved.

Genome-wide survey of DNA-binding proteins in Arabidopsis thaliana: analysis of distribution and functions.

PubMed

Malhotra, Sony; Sowdhamini, Ramanathan

2013-08-01

The interaction of proteins with their respective DNA targets is known to control many high-fidelity cellular processes. Performing a comprehensive survey of the sequenced genomes for DNA-binding proteins (DBPs) will help in understanding their distribution and the associated functions in a particular genome. Availability of fully sequenced genome of Arabidopsis thaliana enables the review of distribution of DBPs in this model plant genome. We used profiles of both structure and sequence-based DNA-binding families, derived from PDB and PFam databases, to perform the survey. This resulted in 4471 proteins, identified as DNA-binding in Arabidopsis genome, which are distributed across 300 different PFam families. Apart from several plant-specific DNA-binding families, certain RING fingers and leucine zippers also had high representation. Our search protocol helped to assign DNA-binding property to several proteins that were previously marked as unknown, putative or hypothetical in function. The distribution of Arabidopsis genes having a role in plant DNA repair were particularly studied and noted for their functional mapping. The functions observed to be overrepresented in the plant genome harbour DNA-3-methyladenine glycosylase activity, alkylbase DNA N-glycosylase activity and DNA-(apurinic or apyrimidinic site) lyase activity, suggesting their role in specialized functions such as gene regulation and DNA repair.

Molecular cloning, subcelluar location and expression profile of signal transducer and activator of transcription 2 (STAT2) from turbot, Scophthalmus maximus.

PubMed

Wang, Na; Wang, Xian-Li; Yang, Chang-Geng; Chen, Song-Lin

2013-10-01

Signal transducer and activator of transcription 2 (STAT2) is an important molecule involved in the type I interferon signalling pathway. To date, little STAT2 homologue is available in fish except Atlantic salmon and goldfish. In this paper, STAT2 was firstly cloned and characterized from turbot, a marine flatfish with high economic value. Briefly, turbot STAT2 cDNA is 3206 bp in length encoding a predicted protein of 793 amino acids. The phylogenetic tree shows that turbot STAT2 protein shared the closest relationship with Atlantic salmon. Analysis of subcellular distribution indicates that STAT2 is mainly present in the cytoplasm of TK cells. Stat2 mRNA is constitutively expressed in widespread tissues and induced by several folds in turbot tissues and TK cells after stimulation with Vibrio anguillarum and lymphocystis disease virus (LCDV). Unlike the higher vertebrate STAT2, turbot STAT2 nuclear export signal (NES) exists not in the C-terminal 79 amino acids but in N-terminal 137-312 amino acids (STAT_alpha domain). The nuclear translocation of turbot STAT2 after Poly(I:C) treatment proved its transcription activity in TK cells. All these results suggested that STAT2 may be involved in the immune response in turbot as a transcription factor. Copyright © 2013 Elsevier Ltd. All rights reserved.

An Evolutionary/Biochemical Connection Between Promoter- and Primer-Dependent Polymerases Revealed by Selective Evolution of Ligands by Exponential Enrichment (SELEX).

PubMed

Fenstermacher, Katherine J; Achuthan, Vasudevan; Schneider, Thomas D; DeStefano, Jeffrey J

2018-01-16

DNA polymerases (DNAPs) recognize 3' recessed termini on duplex DNA and carry out nucleotide catalysis. Unlike promoter-specific RNA polymerases (RNAPs), no sequence specificity is required for binding or initiation of catalysis. Despite this, previous results indicate that viral reverse transcriptases bind much more tightly to DNA primers that mimic the polypurine tract. In the current report, primer sequences that bind with high affinity to Taq and Klenow polymerases were identified using a modified Selective Evolution of Ligands by Exponential Enrichment (SELEX) approach. Two Taq -specific primers that bound ∼10 (Taq1) and over 100 (Taq2) times more stably than controls to Taq were identified. Taq1 contained 8 nucleotides (5' -CACTAAAG-3') that matched the phage T3 RNAP "core" promoter. Both primers dramatically outcompeted primers with similar binding thermodynamics in PCR reactions. Similarly, exonuclease minus Klenow polymerase also selected a high affinity primer that contained a related core promoter sequence from phage T7 RNAP (5' -ACTATAG-3'). For both Taq and Klenow, even small modifications to the sequence resulted in large losses in binding affinity suggesting that binding was highly sequence-specific. The results are discussed in the context of possible effects on multi-primer (multiplex) PCR assays, molecular information theory, and the evolution of RNAPs and DNAPs. Importance This work further demonstrates that primer-dependent DNA polymerases can have strong sequence biases leading to dramatically tighter binding to specific sequences. These may be related to biological function, or be a consequences of the structural architecture of the enzyme. New sequence specificity for Taq and Klenow polymerases were uncovered and among them were sequences that contained the core promoter elements from T3 and T7 phage RNA polymerase promoters. This suggests the intriguing possibility that phage RNA polymerases exploited intrinsic binding affinities of ancestral DNA polymerases to develop their promotors. Conversely, DNA polymerases could have evolved from related RNA polymerases and retained the intrinsic binding preference despite there being no clear function for such a preference in DNA biology. Copyright © 2018 American Society for Microbiology.

The nucleotide binding dynamics of human MSH2-MSH3 are lesion dependent.

PubMed

Owen, Barbara A L; H Lang, Walter; McMurray, Cynthia T

2009-05-01

Here we report that the human DNA mismatch complex MSH2-MSH3 recognizes small loops by a mechanism different from that of MSH2-MSH6 for single-base mismatches. The subunits MSH2 and MSH3 can bind either ADP or ATP with similar affinities. Upon binding to a DNA loop, however, MSH2-MSH3 adopts a single 'nucleotide signature', in which the MSH2 subunit is occupied by an ADP molecule and the MSH3 subunit is empty. Subsequent ATP binding and hydrolysis in the MSH3 subunit promote ADP-ATP exchange in the MSH2 subunit to yield a hydrolysis-independent ATP-MSH2-MSH3-ADP intermediate. Human MSH2-MSH3 and yeast Msh2-Msh6 both undergo ADP-ATP exchange in the Msh2 subunit but, apparently, have opposite requirements for ATP hydrolysis: ADP release from DNA-bound Msh2-Msh6 requires ATP stabilization in the Msh6 subunit, whereas ADP release from DNA-bound MSH2-MSH3 requires ATP hydrolysis in the MSH3 subunit. We propose a model in which lesion binding converts MSH2-MSH3 into a distinct nucleotide-bound form that is poised to be a molecular sensor for lesion specificity.

Evidence for the binding of the carcinogen 3-methylcholanthrene to both the purine and the pyrimidine bases of hamster fibroblast deoxyribonucleic acid (Short Communication)

PubMed Central

Jones, Peter A.; Gevers, Wieland; Hawtrey, Arthur O.

1973-01-01

The binding of [3H]3-methylcholanthrene to the DNA of hamster fibroblasts was studied by using chemical methods for DNA degradation. DNA depurinated by mild acid hydrolysis released approximately half of the radioactivity at the same rate as the purine bases, but the resulting apurinic acid still contained radioactive carcinogen. PMID:4797167

DNA Binding Peptide Directed Synthesis of Continuous DNA Nanowires for Analysis of Large DNA Molecules by Scanning Electron Microscope.

PubMed

Kim, Kyung-Il; Lee, Seonghyun; Jin, Xuelin; Kim, Su Ji; Jo, Kyubong; Lee, Jung Heon

2017-01-01

Synthesis of smooth and continuous DNA nanowires, preserving the original structure of native DNA, and allowing its analysis by scanning electron microscope (SEM), is demonstrated. Gold nanoparticles densely assembled on the DNA backbone via thiol-tagged DNA binding peptides work as seeds for metallization of DNA. This method allows whole analysis of DNA molecules with entangled 3D features. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Solution structure of telomere binding domain of AtTRB2 derived from Arabidopsis thaliana

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yun, Ji-Hye; Lee, Won Kyung; Kim, Heeyoun

Highlights: • We have determined solution structure of Myb domain of AtTRB2. • The Myb domain of AtTRB2 is located in the N-terminal region. • The Myb domain of AtTRB2 binds to plant telomeric DNA without fourth helix. • Helix 2 and 3 of the Myb domain of AtTRB2 are involved in DNA recognition. • AtTRB2 is a novel protein distinguished from other known plant TBP. - Abstract: Telomere homeostasis is regulated by telomere-associated proteins, and the Myb domain is well conserved for telomere binding. AtTRB2 is a member of the SMH (Single-Myb-Histone)-like family in Arabidopsis thaliana, having an N-terminalmore » Myb domain, which is responsible for DNA binding. The Myb domain of AtTRB2 contains three α-helices and loops for DNA binding, which is unusual given that other plant telomere-binding proteins have an additional fourth helix that is essential for DNA binding. To understand the structural role for telomeric DNA binding of AtTRB2, we determined the solution structure of the Myb domain of AtTRB2 (AtTRB2{sub 1–64}) using nuclear magnetic resonance (NMR) spectroscopy. In addition, the inter-molecular interaction between AtTRB2{sub 1–64} and telomeric DNA has been characterized by the electrophoretic mobility shift assay (EMSA) and NMR titration analyses for both plant (TTTAGGG)n and human (TTAGGG)n telomere sequences. Data revealed that Trp28, Arg29, and Val47 residues located in Helix 2 and Helix 3 are crucial for DNA binding, which are well conserved among other plant telomere binding proteins. We concluded that although AtTRB2 is devoid of the additional fourth helix in the Myb-extension domain, it is able to bind to plant telomeric repeat sequences as well as human telomeric repeat sequences.« less

Inhibition of DNA binding of Sox2 by the SUMO conjugation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsuruzoe, Shu; Ishihara, Ko; Uchimura, Yasuhiro

2006-12-29

Sox2 is a member of the high mobility group (HMG) domain DNA-binding proteins for transcriptional control and chromatin architecture. The HMG domain of Sox2 binds the DNA to facilitate transactivation by the cooperative transcription factors such as Oct3/4. We report that mouse Sox2 is modified by SUMO at lysine 247. Substitution of the target lysine to arginine lost the sumoylation but little affected transcriptional potential or nuclear localization of Sox2. By contrast with the unmodified form, Sox2 fused to SUMO-1 did not augment transcription via the Fgf4 enhancer in the presence of Oct3/4. Further, SUMO-1-conjugated Sox2 at the lysine 247more » or at the carboxyl terminus reduced the binding to the Fgf4 enhancer. These indicate that Sox2 sumoylation negatively regulates its transcriptional role through impairing the DNA binding.« less

Non-uniform binding of single-stranded DNA binding proteins to hybrids of single-stranded DNA and single-walled carbon nanotubes observed by atomic force microscopy in air and in liquid

NASA Astrophysics Data System (ADS)

Umemura, Kazuo; Ishizaka, Kei; Nii, Daisuke; Izumi, Katsuki

2016-12-01

Using atomic force spectroscopy (AFM), we observed hybrids of single-stranded DNA (ssDNA) and single-walled carbon nanotubes (SWNTs) with or without protein molecules in air and in an aqueous solution. This is the first report of ssDNA-SWNT hybrids with proteins in solution analyzed by AFM. In the absence of protein, the height of the ssDNA-SWNT hybrids was 1.1 ± 0.3 nm and 2.4 ± 0.6 nm in air and liquid, respectively, suggesting that the ssDNA molecules adopted a flexible structure on the SWNT surface. In the presence of single-stranded DNA binding (SSB) proteins, the heights of the hybrids in air and liquid increased to 6.4 ± 3.1 nm and 10.0 ± 4.5 nm, respectively. The AFM images clearly showed binding of the SSB proteins to the ssDNA-SWNT hybrids. The morphology of the SSB-ssDNA-SWNT hybrids was non-uniform, particularly in aqueous solution. The variance of hybrid height was quantitatively estimated by cross-section analysis along the long-axis of each hybrid. The SSB-ssDNA-SWNT hybrids showed much larger variance than the ssDNA-SWNT hybrids.

Cooperative DNA binding and protein/DNA fiber formation increases the activity of the Dnmt3a DNA methyltransferase.

PubMed

Emperle, Max; Rajavelu, Arumugam; Reinhardt, Richard; Jurkowska, Renata Z; Jeltsch, Albert

2014-10-24

The Dnmt3a DNA methyltransferase has been shown to bind cooperatively to DNA and to form large multimeric protein/DNA fibers. However, it has also been reported to methylate DNA in a processive manner, a property that is incompatible with protein/DNA fiber formation. We show here that the DNA methylation rate of Dnmt3a increases more than linearly with increasing enzyme concentration on a long DNA substrate, but not on a short 30-mer oligonucleotide substrate. We also show that addition of a catalytically inactive Dnmt3a mutant, which carries an amino acid exchange in the catalytic center, increases the DNA methylation rate by wild type Dnmt3a on the long substrate but not on the short one. In agreement with this finding, preincubation experiments indicate that stable protein/DNA fibers are formed on the long, but not on the short substrate. In addition, methylation experiments with substrates containing one or two CpG sites did not provide evidence for a processive mechanism over a wide range of enzyme concentrations. These data clearly indicate that Dnmt3a binds to DNA in a cooperative reaction and that the formation of stable protein/DNA fibers increases the DNA methylation rate. Fiber formation occurs at low μm concentrations of Dnmt3a, which are in the range of Dnmt3a concentrations in the nucleus of embryonic stem cells. Understanding the mechanism of Dnmt3a is of vital importance because Dnmt3a is a hotspot of somatic cancer mutations one of which has been implicated in changing Dnmt3a processivity. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Variola virus E3L Zα domain, but not its Z-DNA binding activity, is required for PKR inhibition.

PubMed

Thakur, Meghna; Seo, Eun Joo; Dever, Thomas E

2014-02-01

Responding to viral infection, the interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase PKR phosphorylates translation initiation factor eIF2α to inhibit cellular and viral protein synthesis. To overcome this host defense mechanism, many poxviruses express the protein E3L, containing an N-terminal Z-DNA binding (Zα) domain and a C-terminal dsRNA-binding domain (dsRBD). While E3L is thought to inhibit PKR activation by sequestering dsRNA activators and by directly binding the kinase, the role of the Zα domain in PKR inhibition remains unclear. Here, we show that the E3L Zα domain is required to suppress the growth-inhibitory properties associated with expression of human PKR in yeast, to inhibit PKR kinase activity in vitro, and to reverse the inhibitory effects of PKR on reporter gene expression in mammalian cells treated with dsRNA. Whereas previous studies revealed that the Z-DNA binding activity of E3L is critical for viral pathogenesis, we identified point mutations in E3L that functionally uncouple Z-DNA binding and PKR inhibition. Thus, our studies reveal a molecular distinction between the nucleic acid binding and PKR inhibitory functions of the E3L Zα domain, and they support the notion that E3L contributes to viral pathogenesis by targeting PKR and other components of the cellular anti-viral defense pathway.

Mutations in the C-terminal fragment of DnaK affecting peptide binding.

PubMed Central

Burkholder, W F; Zhao, X; Zhu, X; Hendrickson, W A; Gragerov, A; Gottesman, M E

1996-01-01

Escherichia coli DnaK acts as a molecular chaperone through its ATP-regulated binding and release of polypeptide substrates. Overexpressing a C-terminal fragment (CTF) of DnaK (Gly-384 to Lys-638) containing the polypeptide substrate binding domain is lethal in wild-type E. coli. This dominant-negative phenotype may result from the nonproductive binding of CTF to cellular polypeptide targets of DnaK. Mutations affecting DnaK substrate binding were identified by selecting noncytotoxic CTF mutants followed by in vitro screening. The clustering of such mutations in the three-dimensional structure of CTF suggests the model that loops L1,2 and L4,5 form a rigid core structure critical for interactions with substrate. Images Fig. 1 Fig. 2 Fig. 3 PMID:8855230

Design, synthesis and DNA-binding study of some novel morpholine linked thiazolidinone derivatives

NASA Astrophysics Data System (ADS)

War, Javeed Ahmad; Srivastava, Santosh Kumar; Srivastava, Savitri Devi

2017-02-01

The emergence of multiple drug resistance amongst bacterial strains resulted in many clinical drugs to be ineffective. Being vulnerable to bacterial infections any lack in the development of new antimicrobial drugs could pose a serious threat to public health. Here we report design and synthesis of a novel class of morpholine linked thiazolidinone hybrid molecules. The compounds were characterized by FT-IR, NMR and HRMS techniques. Susceptibility tests showed that most of the synthesized molecules were highly active against multiple bacterial strains. Compound 3f displayed MIC values which were better than the standard drug for most of the tested strains. DNA being a well defined target for many antimicrobial drugs was probed as possible target for these synthetic molecules. DNA-binding study of 3f with sm-DNA was probed through UV-vis absorption, fluorescence quenching, gel electrophoresis and molecular docking techniques. The studies revealed that compound 3f has strong affinity towards DNA and binds at the minor groove. The docking studies revealed that the compound 3f shows preferential binding towards A/T residues.

Design, synthesis and DNA-binding study of some novel morpholine linked thiazolidinone derivatives.

PubMed

War, Javeed Ahmad; Srivastava, Santosh Kumar; Srivastava, Savitri Devi

2017-02-15

The emergence of multiple drug resistance amongst bacterial strains resulted in many clinical drugs to be ineffective. Being vulnerable to bacterial infections any lack in the development of new antimicrobial drugs could pose a serious threat to public health. Here we report design and synthesis of a novel class of morpholine linked thiazolidinone hybrid molecules. The compounds were characterized by FT-IR, NMR and HRMS techniques. Susceptibility tests showed that most of the synthesized molecules were highly active against multiple bacterial strains. Compound 3f displayed MIC values which were better than the standard drug for most of the tested strains. DNA being a well defined target for many antimicrobial drugs was probed as possible target for these synthetic molecules. DNA-binding study of 3f with sm-DNA was probed through UV-vis absorption, fluorescence quenching, gel electrophoresis and molecular docking techniques. The studies revealed that compound 3f has strong affinity towards DNA and binds at the minor groove. The docking studies revealed that the compound 3f shows preferential binding towards A/T residues. Copyright © 2016 Elsevier B.V. All rights reserved.

Oct4 suppresses IR‑induced premature senescence in breast cancer cells through STAT3- and NF‑κB-mediated IL‑24 production.

PubMed

Kim, Jeong-Yub; Kim, Jeong-Chul; Lee, Ji-Yun; Park, Myung-Jin

2018-07-01

Breast cancer stem cells (BCSCs) are a small subpopulation of breast cancer cells that have been proposed to be a primary cause of failure of therapies, including ionizing radiation (IR). Their embryonic stem-like signature is associated with poor clinical outcome. In the present study, the function of octamer-binding transcription factor 4 (Oct4), an embryonic stem cell factor, in the resistance of BCSCs to IR was investigated. Mammosphere cells exhibited increased expression of stemness-associated genes, including Oct4 and sex‑determining region Y‑box 2 (Sox2), and were more resistant to IR compared with serum-cultured monolayer cells. IR‑resistant MCF7 cells also exhibited significantly increased expression of Oct4. To investigate the possible involvement of Oct4 in IR resistance of breast cancer cells, cells were transfected with Oct4. Ectopic expression of Oct4 increased the clonogenic survival of MCF7 cells following IR, which was reversed by treatment with small interfering RNA (siRNA) targeting Oct4. Oct4 expression decreased phosphorylated histone H2AX (γ-H2AX) focus formation and suppressed IR‑induced premature senescence in these cells. Mammosphere, IR‑resistant and Oct4‑overexpressing MCF7 cells exhibited enhanced phosphorylation of signal transducer and activation of transcription 3 (STAT3) (Tyr705) and inhibitor of nuclear factor κB (NF‑κB), and blockade of these pathways with siRNA against STAT3 and/or specific inhibitors of STAT3 and NF‑κB significantly increased IR‑induced senescence. Secretome analysis revealed that Oct4 upregulated interleukin 24 (IL‑24) expression through STAT3 and NF‑κB signaling, and siRNA against IL‑24 increased IR‑induced senescence, whereas recombinant human IL‑24 suppressed it. The results of the present study indicated that Oct4 confers IR resistance on breast cancer cells by suppressing IR‑induced premature senescence through STAT3- and NF‑κB-mediated IL‑24 production.

Cyclic perylene diimide: Selective ligand for tetraplex DNA binding over double stranded DNA.

PubMed

Vasimalla, Suresh; Sato, Shinobu; Takenaka, Fuminori; Kurose, Yui; Takenaka, Shigeori

2017-12-15

Synthesized cyclic perylene diimide, cPDI, showed the binding constant of 6.3 × 10 6  M -1 with binding number of n = 2 with TA-core as a tetraplex DNA in 50 mM Tris-HCl buffer (pH = 7.4) containing 100 mM KCl using Schatchard analysis and showed a higher preference for tetraplex DNA than for double stranded DNA with over 10 3 times. CD spectra showed that TA-core induced its antiparallel conformation upon addition of cPDI in the absence or presence of K + or Na + ions. The cPDI inhibits the telomerase activity with IC 50 of 0.3 µM using TRAP assay which is potential anti-cancer drug with low side effect. Copyright © 2017 Elsevier Ltd. All rights reserved.

Preparation and DNA-binding properties of substituted triostin antibiotics.

PubMed

Cornish, A; Fox, K R; Waring, M J

1983-02-01

Novel derivatives of the triostin group of antibiotics were prepared by supplementing cultures of the producing organism Streptomyces triostinicus with a variety of aromatic carboxylic acids. Five new antibiotics, each having both the natural quinoxaline chromophores replaced by a substituted ring system, were purified to homogeneity and characterized by high-pressure liquid chromatography and nuclear magnetic resonance. Their antibacterial activities and DNA-binding properties were investigated. Addition of a halogen atom at position 6 of the quinoxaline ring or an amino group at position 3 had little effect on either the biological activity or the DNA-binding characteristics. The bis-3-amino derivative is fluorescent, and its fluorescence is strongly quenched by calf thymus DNA and polydeoxyguanylate-polydeoxycytidylate but not by polydeoxyadenylate-polydeoxythymidylate, suggesting that it binds preferentially to guanosine-cytosine-rich sequences in natural DNA. Binding constants for the bis-6-chloro and bis-3-amino derivatives do not differ greatly from those of unsubstituted triostin A. The analogs having two quinoline chromophores or a chlorine atom in position 7 of the quinoxaline ring display little or no detectable antibacterial activity, in marked contrast to the other congeners. Bis-7-chloro-triostin A binds conspicuously more tightly to polydeoxyadenylate-polydeoxythymidylate than to any other polynucleotide tested.

ATP binding and hydrolysis by Saccharomyces cerevisiae Msh2-Msh3 are differentially modulated by mismatch and double-strand break repair DNA substrates.

PubMed

Kumar, Charanya; Eichmiller, Robin; Wang, Bangchen; Williams, Gregory M; Bianco, Piero R; Surtees, Jennifer A

2014-06-01

In Saccharomyces cerevisiae, Msh2-Msh3-mediated mismatch repair (MMR) recognizes and targets insertion/deletion loops for repair. Msh2-Msh3 is also required for 3' non-homologous tail removal (3'NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, we recently demonstrated that the two pathways have distinct requirements with respect to Msh2-Msh3 activities. We identified a set of aromatic residues in the nucleotide binding pocket (FLY motif) of Msh3 that, when mutated, disrupted MMR, but left 3'NHTR largely intact. One of these mutations, msh3Y942A, was predicted to disrupt the nucleotide sandwich and allow altered positioning of ATP within the pocket. To develop a mechanistic understanding of the differential requirements for ATP binding and/or hydrolysis in the two pathways, we characterized Msh2-Msh3 and Msh2-msh3Y942A ATP binding and hydrolysis activities in the presence of MMR and 3'NHTR DNA substrates. We observed distinct, substrate-dependent ATP hydrolysis and nucleotide turnover by Msh2-Msh3, indicating that the MMR and 3'NHTR DNA substrates differentially modify the ATP binding/hydrolysis activities of Msh2-Msh3. Msh2-msh3Y942A retained the ability to bind DNA and ATP but exhibited altered ATP hydrolysis and nucleotide turnover. We propose that both ATP and structure-specific repair substrates cooperate to direct Msh2-Msh3-mediated repair and suggest an explanation for the msh3Y942A separation-of-function phenotype. Copyright © 2014 Elsevier B.V. All rights reserved.

ATP binding and hydrolysis by Saccharomyces cerevisiae Msh2-Msh3 are differentially modulated by Mismatch and Double-strand Break Repair DNA substrates

PubMed Central

Kumar, Charanya; Eichmiller, Robin; Wang, Bangchen; Williams, Gregory M.; Bianco, Piero R.; Surtees, Jennifer A.

2014-01-01

In Saccharomyces cerevisiae, Msh2-Msh3-mediated mismatch repair (MMR) recognizes and targets insertion/deletion loops for repair. Msh2-Msh3 is also required for 3′ non-homologous tail removal (3′NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, we recently demonstrated that the two pathways have distinct requirements with respect to Msh2-Msh3 activities. We identified a set of aromatic residues in the nucleotide binding pocket (FLY motif) of Msh3 that, when mutated, disrupted MMR, but left 3′ NHTR largely intact. One of these mutations, msh3Y942A, was predicted to disrupt the nucleotide sandwich and allow altered positioning of ATP within the pocket. To develop a mechanistic understanding of the differential requirements for ATP binding and/or hydrolysis in the two pathways, we characterized Msh2-Msh3 and Msh2-msh3Y942A ATP binding and hydrolysis activities in the presence of MMR and 3′ NHTR DNA substrates. We observed distinct, substrate-dependent ATP hydrolysis and nucleotide turnover by Msh2-Msh3, indicating that the MMR and 3′ NHTR DNA substrates differentially modify the ATP binding/hydrolysis activities of Msh2-Msh3. Msh2-msh3Y942A retained the ability to bind DNA and ATP but exhibited altered ATP hydrolysis and nucleotide turnover. We propose that both ATP and structure-specific repair substrates cooperate to direct Msh2-Msh3-mediated repair and suggest an explanation for the msh3Y942A separation-of-function phenotype. PMID:24746922

Binding polarity of RPA to telomeric sequences and influence of G-quadruplex stability.

PubMed

Safa, Layal; Delagoutte, Emmanuelle; Petruseva, Irina; Alberti, Patrizia; Lavrik, Olga; Riou, Jean-François; Saintomé, Carole

2014-08-01

Replication protein A (RPA) is a single-stranded DNA binding protein that plays an essential role in telomere maintenance. RPA binds to and unfolds G-quadruplex (G4) structures formed in telomeric DNA, thus facilitating lagging strand DNA replication and telomerase activity. To investigate the effect of G4 stability on the interactions with human RPA (hRPA), we used a combination of biochemical and biophysical approaches. Our data revealed an inverse relationship between G4 stability and ability of hRPA to bind to telomeric DNA; notably small G4 ligands that enhance G4 stability strongly impaired G4 unfolding by hRPA. To gain more insight into the mechanism of binding and unfolding of telomeric G4 structures by RPA, we carried out photo-crosslinking experiments to elucidate the spatial arrangement of the RPA subunits along the DNA strands. Our results showed that RPA1 and RPA2 are arranged from 5' to 3' along the unfolded telomeric G4, as already described for unstructured single-stranded DNA, while no contact is possible with RPA3 on this short oligonucleotide. In addition, these data are compatible with a 5' to 3' directionality in G4 unfolding by hRPA. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Synthesis and structure elucidation of a copper(II) Schiff-base complex: in vitro DNA binding, pBR322 plasmid cleavage and HSA binding studies.

PubMed

Tabassum, Sartaj; Ahmad, Musheer; Afzal, Mohd; Zaki, Mehvash; Bharadwaj, Parimal K

2014-11-01

New copper(II) complex with Schiff base ligand 4-[(2-Hydroxy-3-methoxy-benzylidene)-amino]-benzoic acid (H₂L) was synthesized and characterized by spectroscopic and analytical and single crystal X-ray diffraction studies which revealed that the complex 1 exist in a distorted octahedral environment. In vitro CT-DNA binding studies were performed by employing different biophysical technique which indicated that the 1 strongly binds to DNA in comparison to ligand via electrostatic binding mode. Complex 1 cleaves pBR322 DNA via hydrolytic pathway and recognizes minor groove of DNA double helix. The HSA binding results showed that ligand and complex 1 has ability to quench the fluorescence emission intensity of Trp 214 residue available in the subdomain IIA of HSA. Copyright © 2014 Elsevier B.V. All rights reserved.

Substitutional impact on biological activity of new water soluble Ni(II) complexes: Preparation, spectral characterization, X-ray crystallography, DNA/protein binding, antibacterial activity and in vitro cytotoxicity.

PubMed

Umadevi, C; Kalaivani, P; Puschmann, H; Murugan, S; Mohan, P S; Prabhakaran, R

2017-02-01

A series of new water soluble nickel(II) complexes containing triphenylphosphine and 4-methoxysalicylaldehyde-4(N)-substituted thiosemicarbazones were synthesized and characterized. Crystallographic investigations confirmed the structure of the complexes (1-4) having the general structure [Ni(4-Msal-Rtsc)(PPh 3 )] (Where R=H (1); CH 3 (2); C 2 H 5 (3); C 6 H 5 (4)) which showed that thiosemicarbazone ligands coordinated to nickel(II) ion as ONS tridentate bibasic donor. DNA/BSA protein binding ability of the ligands and their new complexes were studied by taking calf-thymus DNA (CT-DNA) and Bovine serum albumin (BSA) through absorption and emission titrations. Ethidium bromide (EB) displacement study showed the intercalative binding trend of the complexes to DNA. From the albumin binding studies, the mechanism of quenching was found as static and the alterations in the secondary structure of BSA by the compounds were confirmed with synchronous spectral studies. The binding affinity of the complexes to CT-DNA and BSA has the order of [Ni(4-Msal-etsc)(PPh 3 )] (3) >[Ni(4-Msal-mtsc)(PPh 3 )] (2) >[Ni(4-Msal-tsc)(PPh 3 )] (1) >[Ni(4-Msal-ptsc)(PPh 3 )] (4). In vitro cytotoxicity of the complexes was tested on human lung cancer cells (A549), human cervical cancer cells (HeLa), human liver carcinoma cells (Hep G2). All the complexes exhibited significant activity against three cancer cells. Among them, complex 4 exhibited almost 2.5 fold activity than cisplatin in A549 and HepG2 cell lines. In HeLa cell line, the complexes exhibited significant activity which is less than cisplatin. While comparing the activity of the complexes in A549 and HepG2 cell lines it falls in the order 4>1>2>3>cisplatin. The results obtained from DNA, protein binding and cytotoxicity studies, it is concluded that the cytotoxicity of the complexes as determined by MTT assay were not unduly influenced by the complexes having different binding efficiency with DNA and protein. The complexes exhibited good spectrum of antibacterial activity against four pathogenic bacteria such as E. faecalis (gram +ve), S. aureus (gram +ve), E. coli (gram -ve) and P. aeruginosa (gram -ve). Copyright © 2016 Elsevier B.V. All rights reserved.

Determination of the DNA-binding characteristics of ethidium bromide, proflavine, and cisplatin by flow injection analysis: usefulness in studies on antitumor drugs.

PubMed

Alonso, A; Almendral, M J; Curto, Y; Criado, J J; Rodríguez, E; Manzano, J L

2006-08-15

Flow injection analysis was used to study the reactions occurring between DNA and certain compounds that bind to its double helix, deforming this and even breaking it, such that some of them (e.g., cisplatin) are endowed with antitumoral activity. Use of this technique in the merging zones and stopped-flow modes afforded data on the binding parameters and the kinetic characteristics of the process. The first compound studied was ethidium bromide (EtdBr), used as a fluorescent marker because its fluorescence is enhanced when it binds to DNA. The DNA-EtdBr binding parameters, the apparent intrinsic binding constant (0.31+/-0.02 microM(-1)), and the maximum number of binding sites per nucleotide (0.327+/-0.009) were determined. The modification introduced in these parameters by the presence of proflavine (Prf), a classic competitive inhibitor of the binding of EtdBr to the DNA double helix, was also studied, determining the value of the intrinsic binding constant of Prf (K(Prf) = 0.119+/-9x10(-3) microM(-1)). Finally, we determined the binding parameters between DNA and EtdBr in the presence of the antitumor agent cisplatin, a noncompetitive inhibitor of such binding. This provided information about the binding mechanism as well as the duration and activity of the binding of the compound in its pharmacological use.

Pt(IV) complexes as prodrugs for cisplatin.

PubMed

Shi, Yi; Liu, Shu-An; Kerwood, Deborah J; Goodisman, Jerry; Dabrowiak, James C

2012-02-01

The antitumor effects of platinum(IV) complexes, considered prodrugs for cisplatin, are believed to be due to biological reduction of Pt(IV) to Pt(II), with the reduction products binding to DNA and other cellular targets. In this work we used pBR322 DNA to capture the products of reduction of oxoplatin, c,t,c-[PtCl(2)(OH)(2)(NH(3))(2)], 3, and a carboxylate-modified analog, c,t,c-[PtCl(2)(OH)(O(2)CCH(2)CH(2)CO(2)H)(NH(3))(2)], 4, by ascorbic acid (AsA) or glutathione (GSH). Since carbonate plays a significant role in the speciation of platinum complexes in solution, we also investigated the effects of carbonate on the reduction/DNA-binding process. In pH 7.4 buffer in the absence of carbonate, both 3 and 4 are reduced by AsA to cisplatin (confirmed using ((195))Pt NMR), which binds to and unwinds closed circular DNA in a manner consistent with the formation of the well-known 1, 2 intrastrand DNA crosslink. However, when GSH is used as the reducing agent for 3 and 4, ((195))Pt NMR shows that cisplatin is not produced in the reaction medium. Although the Pt(II) products bind to closed circular DNA, their effect on the mobility of Form I DNA is different from that produced by cisplatin. When physiological carbonate is present in the reduction medium, ((13))C NMR shows that Pt(II) carbonato complexes form which block or impede platinum binding to DNA. The results of the study vis-à-vis the ability of the Pt(IV) complexes to act as prodrugs for cisplatin are discussed. Copyright © 2011 Elsevier Inc. All rights reserved.

Probing the DNA kink structure induced by the hyperthermophilic chromosomal protein Sac7d

PubMed Central

Chen, Chin-Yu; Ko, Tzu-Ping; Lin, Ting-Wan; Chou, Chia-Cheng; Chen, Chun-Jung; Wang, Andrew H.-J.

2005-01-01

Sac7d, a small, abundant, sequence-general DNA-binding protein from the hyperthermophilic archaeon Sulfolobus acidocaldarius, causes a single-step sharp kink in DNA (∼60°) via the intercalation of both Val26 and Met29. These two amino acids were systematically changed in size to probe their effects on DNA kinking. Eight crystal structures of five Sac7d mutant–DNA complexes have been analyzed. The DNA-binding pattern of the V26A and M29A single mutants is similar to that of the wild-type, whereas the V26A/M29A protein binds DNA without side chain intercalation, resulting in a smaller overall bending (∼50°). The M29F mutant inserts the Phe29 side chain orthogonally to the C2pG3 step without stacking with base pairs, inducing a sharp kink (∼80°). In the V26F/M29F-GCGATCGC complex, Phe26 intercalates deeply into DNA bases by stacking with the G3 base, whereas Phe29 is stacked on the G15 deoxyribose, in a way similar to those used by the TATA box-binding proteins. All mutants have reduced DNA-stabilizing ability, as indicated by their lower Tm values. The DNA kink patterns caused by different combinations of hydrophobic side chains may be relevant in understanding the manner by which other minor groove-binding proteins interact with DNA. PMID:15653643

mom identifies a receptor for the Drosophila JAK/STAT signal transduction pathway and encodes a protein distantly related to the mammalian cytokine receptor family

PubMed Central

Chen, Hua-Wei; Chen, Xiu; Oh, Su-Wan; Marinissen, Maria J.; Gutkind, J. Silvio; Hou, Steven X.

2002-01-01

The JAK/STAT signal transduction pathway controls numerous events in Drosophila melanogaster development. Receptors for the pathway have yet to be identified. Here we have identified a Drosophila gene that shows embryonic mutant phenotypes identical to those in the hopscotch (hop)/JAK kinase and marelle (mrl)/Stat92e mutations. We named this gene master of marelle (mom). Genetic analyses place mom's function between upd (the ligand) and hop. We further show that cultured cells transfected with the mom gene bind UPD and activate the HOP/STAT92E signal transduction pathway. mom encodes a protein distantly related to the mammalian cytokine receptor family. These data show that mom functions as a receptor of the Drosophila JAK/STAT signal transduction pathway. PMID:11825879

The transformed glucocorticoid receptor has a lower steroid-binding affinity than the nontransformed receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nemoto, Takayuki; Ohara-Nemoto, Yuko; Denis, M.

1990-02-20

High-salt treatment of cytosolic glucocorticoid receptor (GR) preparations reduces the steroid-binding ability of the receptor and induces the conversion of the receptor from a nontransformed (non-DNA-binding) 9S form to a transformed (DNA-binding) 4S entity. Therefore, the authors decided to investigate the possible relationship between these two phenomena. The binding of ({sup 3}H)triamcinolone acetonide (({sup 3}H)TA) to the 9S form was almost saturated at a concentration of 20 nM, whereas ({sup 3}H)TA was hardly bound to the 4S form at this concentration. The 4S form was efficiently labeled at 200 nM. Scatchard analysis of the GR showed the presence of twomore » types of binding sites. In the absence of molybdate, the ratio of the lower affinity site was increased, but the total number of binding sites was not modified. The GR with the low ({sup 3}H)TA-binding affinity bound to DNA-cellulose even in its unliganded state, whereas the form with the high affinity did not. These results indicate that the transformed GR has a reduced ({sup 3}H)TA-binding affinity as compared to the nontransformed GR. The steroid-binding domain (amino acids 477-777) and the DNA- and steroid-binding domains (amino acids 415-777) of the human GR were expressed in Escherichia coli as protein A fused proteins. Taken together, these results suggest that the component(s) associating with the nontransformed GR, possibly the heat shock protein hsp 90, play(s) an important role in stabilizing the GR in a high-affinity state for steroids.« less

Molecular dynamics simulations show altered secondary structure of clawless in binary complex with DNA providing insights into aristaless-clawless-DNA ternary complex formation.

PubMed

Kachhap, Sangita; Priyadarshini, Pragya; Singh, Balvinder

2017-05-01

Aristaless (Al) and clawless (Cll) homeodomains that are involved in leg development in Drosophila melanogaster are known to bind cooperatively to 5'-(T/C)TAATTAA(T/A)(T/A)G-3' DNA sequence, but the mechanism of their binding to DNA is unknown. Molecular dynamics (MD) studies have been carried out on binary, ternary, and reconstructed protein-DNA complexes involving Al, Cll, and DNA along with binding free energy analysis of these complexes. Analysis of MD trajectories of Cll-3A01, binary complex reveals that C-terminal end of helixIII of Cll, unwind in the absence of Al and remains so in reconstructed ternary complex, Cll-3A01-Al. In addition, this change in secondary structure of Cll does not allow it to form protein-protein interactions with Al in the ternary reconstructed complex. However, secondary structure of Cll and its interactions are maintained in other reconstructed ternary complex, Al-3A01-Cll where Cll binds to Al-3A01, binary complex to form ternary complex. These interactions as observed during MD simulations compare well with those observed in ternary crystal structure. Thus, this study highlights the role of helixIII of Cll and protein-protein interactions while proposing likely mechanism of recognition in ternary complex, Al-Cll-DNA.

Generation of mice deficient in RNA-binding motif protein 3 (RBM3) and characterization of its role in innate immune responses and cell growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsuda, Atsushi; Core Research for Evolution Science and Technology, Japan Science and Technology Agency, Chiyoda-ku, Tokyo 102-0075; Ogawa, Masahiro

Highlights: {yields} We identified RNA-binding motif protein 3 (RBM3) as CpG-B DNA-binding protein. {yields} RBM3 translocates from the nucleus to the cytoplasm and co-localized with CpG-B DNA. {yields} We newly generated Rbm3-deficient (Rbm3{sup -/-}) mice. {yields} DNA-mediated cytokine gene induction was normally occured in Rbm3{sup -/-} cells. {yields}Rbm3{sup -/-} MEFs showed poorer proliferation rate and increased number of G2-phase cells. -- Abstract: The activation of innate immune responses is critical to host defense against microbial infections, wherein nucleic acid-sensing pattern recognition receptors recognize DNA or RNA from viruses or bacteria and activate downstream signaling pathways. In a search for newmore » DNA-sensing molecules that regulate innate immune responses, we identified RNA-binding motif protein 3 (RBM3), whose role has been implicated in the regulation of cell growth. In this study, we generated Rbm3-deficient (Rbm3{sup -/-}) mice to study the role of RBM3 in immune responses and cell growth. Despite evidence for its interaction with immunogenic DNA in a cell, no overt phenotypic abnormalities were found in cells from Rbm3{sup -/-} mice for the DNA-mediated induction of cytokine genes. Interestingly, however, Rbm3{sup -/-} mouse embryonic fibroblasts (MEFs) showed poorer proliferation rates as compared to control MEFs. Further cell cycle analysis revealed that Rbm3{sup -/-} MEFs have markedly increased number of G2-phase cells, suggesting a hitherto unknown role of RBM3 in the G2-phase control. Thus, these mutant mice and cells may provide new tools with which to study the mechanisms underlying the regulation of cell cycle and oncogenesis.« less

The H3-H4 N-Terminal Tail Domains Are the Primary Mediators of Transcription Factor IIIA Access to 5S DNA within a Nucleosome

PubMed Central

Vitolo, Joseph M.; Thiriet, Christophe; Hayes, Jeffrey J.

2000-01-01

Reconstitution of a DNA fragment containing a Xenopus borealis somatic type 5S rRNA gene into a nucleosome greatly restricts the binding of transcription factor IIIA (TFIIIA) to its cognate DNA sequence within the internal promoter of the gene. Removal of all core histone tail domains by limited trypsin proteolysis or acetylation of the core histone tails significantly relieves this inhibition and allows TFIIIA to exhibit high-affinity binding to nucleosomal DNA. Since only a single tail or a subset of tails may be primarily responsible for this effect, we determined whether removal of the individual tail domains of the H2A-H2B dimer or the H3-H4 tetramer affects TFIIIA binding to its cognate DNA site within the 5S nucleosome in vitro. The results show that the tail domains of H3 and H4, but not those of H2A and/or H2B, directly modulate the ability of TFIIIA to bind nucleosomal DNA. In vitro transcription assays carried out with nucleosomal templates lacking individual tail domains show that transcription efficiency parallels the binding of TFIIIA. In addition, we show that the stoichiometry of core histones within the 5S DNA-core histone-TFIIIA triple complex is not changed upon TFIIIA association. Thus, TFIIIA binding occurs by displacement of H2A-H2B–DNA contacts but without complete loss of the dimer from the nucleoprotein complex. These data, coupled with previous reports (M. Vettese-Dadey, P. A. Grant, T. R. Hebbes, C. Crane-Robinson, C. D. Allis, and J. L. Workman, EMBO J. 15:2508–2518, 1996; L. Howe, T. A. Ranalli, C. D. Allis, and J. Ausio, J. Biol. Chem. 273:20693–20696, 1998), suggest that the H3/H4 tails are the primary arbiters of transcription factor access to intranucleosomal DNA. PMID:10688663

Structure and DNA-binding of meiosis-specific protein Hop2

NASA Astrophysics Data System (ADS)

Zhou, Donghua; Moktan, Hem; Pezza, Roberto

2014-03-01

Here we report structure elucidation of the DNA binding domain of homologous pairing protein 2 (Hop2), which is important to gene diversity when sperms and eggs are produced. Together with another protein Mnd1, Hop2 enhances the strand invasion activity of recombinase Dmc1 by over 30 times, facilitating proper synapsis of homologous chromosomes. However, the structural and biochemical bases for the function of Hop2 and Mnd1 have not been well understood. As a first step toward such understanding, we recently solved the structure for the N-terminus of Hop2 (1-84) using solution NMR. This fragment shows a typical winged-head conformation with recognized DNA binding activity. DNA interacting sites were then investigated by chemical shift perturbations in a titration experiment. Information of these sites was used to guide protein-DNA docking with MD simulation, revealing that helix 3 is stably lodged in the DNA major groove and that wing 1 (connecting strands 2 and 3) transiently comes in contact with the minor groove in nanosecond time scale. Mutagenesis analysis further confirmed the DNA binding sites in this fragment of the protein.

Structural and Thermodynamic Signatures of DNA Recognition by Mycobacterium tuberculosis DnaA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsodikov, Oleg V.; Biswas, Tapan

An essential protein, DnaA, binds to 9-bp DNA sites within the origin of replication oriC. These binding events are prerequisite to forming an enigmatic nucleoprotein scaffold that initiates replication. The number, sequences, positions, and orientations of these short DNA sites, or DnaA boxes, within the oriCs of different bacteria vary considerably. To investigate features of DnaA boxes that are important for binding Mycobacterium tuberculosis DnaA (MtDnaA), we have determined the crystal structures of the DNA binding domain (DBD) of MtDnaA bound to a cognate MtDnaA-box (at 2.0 {angstrom} resolution) and to a consensus Escherichia coli DnaA-box (at 2.3 {angstrom}). Thesemore » structures, complemented by calorimetric equilibrium binding studies of MtDnaA DBD in a series of DnaA-box variants, reveal the main determinants of DNA recognition and establish the [T/C][T/A][G/A]TCCACA sequence as a high-affinity MtDnaA-box. Bioinformatic and calorimetric analyses indicate that DnaA-box sequences in mycobacterial oriCs generally differ from the optimal binding sequence. This sequence variation occurs commonly at the first 2 bp, making an in vivo mycobacterial DnaA-box effectively a 7-mer and not a 9-mer. We demonstrate that the decrease in the affinity of these MtDnaA-box variants for MtDnaA DBD relative to that of the highest-affinity box TTGTCCACA is less than 10-fold. The understanding of DnaA-box recognition by MtDnaA and E. coli DnaA enables one to map DnaA-box sequences in the genomes of M. tuberculosis and other eubacteria.« less

Intramuscular injection of exogenous leptin induces adiposity, glucose intolerance and fatty liver by repressing the JAK2-STAT3/PI3K pathway in a rat model.

PubMed

Wu, Lihong; Chen, Guoxiong; Liu, Wen; Yang, Xuechao; Gao, Jie; Huang, Liwen; Guan, Hongbing; Li, Zhengmao; Zheng, Zhichao; Li, Meiling; Gu, Weiwang; Ge, Linhu

2017-10-01

Obesity, diabetes and fatty liver disease are extremely common in leptin-resistant patients. Dysfunction of leptin or its receptor is associated with obesity. The present study aimed to assess the effects of intramuscular injection of exogenous leptin or its receptor on fat deposition and leptin-insulin feedback regulation. Forty-five 40-day old female Sprague Dawley (SD) rats were injected thrice with leptin or its receptor intramuscularly. Adiposity and fat deposition were assessed by assessing the Lee's index, body weight, food intake, and total cholesterol, high density lipoprotein, low density lipoprotein, and triglyceride levels, as well as histological properties (liver and adipose tissue). Serum glucose, leptin, and insulin amounts were evaluated, and glucose tolerance assessed to monitor glucose metabolism in SD rats; pancreas specimens were analyzed immunohistochemically. Hypothalamic phosphorylated Janus kinase 2 (p-JAK2), phosphorylated signal transducer and activator of transcription 3 (p-STAT3), and phosphatidylinositol-3-kinase (PI3K) signaling, and hepatic sterol regulatory element binding protein-1 (SREBP-1) were qualified by Western blotting. Leptin receptor immunogen reduced fat deposition, increased appetite, and lowered serum leptin levels, enhancing STAT3 signaling in hypothalamus and down-regulating hepatic SREBP-1. In contrast, SD rats administered leptin immunogen displayed significantly increased body weight and fat deposition, with up-regulated SREBP-1, indicating adiposity occurrence. SD rats administered leptin immunogen also showed glucose intolerance, β- cell reduction in the pancreas, and deregulation of JAK2-STAT3/PI3K signaling, indicating that Lep rats were at risk of diabetes. In conclusion, intramuscular injection of exogenous leptin or its receptor, a novel rat model approach, can be used in obesity pathogenesis and therapeutic studies. Copyright © 2017. Published by Elsevier Inc.

Role of protein structure and the role of individual fingers in zinc finger protein-DNA recognition: a molecular dynamics simulation study and free energy calculations

NASA Astrophysics Data System (ADS)

Hamed, Mazen Y.

2018-05-01

Molecular dynamics and MM_GBSA energy calculations on various zinc finger proteins containing three and four fingers bound to their target DNA gave insights into the role of each finger in the DNA binding process as part of the protein structure. The wild type Zif 268 (PDB code: 1AAY) gave a ΔG value of - 76.1 (14) kcal/mol. Zinc fingers ZF1, ZF2 and ZF3 were mutated in one experiment and in another experiment one finger was cut and the rest of the protein was studied for binding. The ΔΔG values for the Zinc Finger protein with both ZF1 and ZF2 mutated was + 80 kcal/mol, while mutating only ZF1 the ΔΔG value was + 52 kcal/mol (relative to the wild type). Cutting ZF3 and studying the protein consisting only of ZF1 linked to ZF2 gave a ΔΔG value of + 68 kcal/mol. Upon cutting ZF1, the resulting ZF2 linked to ZF3 protein gave a ΔΔG value of + 41 kcal/mol. The above results shed light on the importance of each finger in the binding process, especially the role of ZF1 as the anchoring finger followed in importance by ZF2 and ZF3. The energy difference between the binding of the wild type protein Zif268 (1AAY) and that for individual finger binding to DNA according to the formula: ΔΔGlinkers, otherstructuralfactors = ΔGzif268 - (ΔGF1+F2+F3) gave a value = - 44.5 kcal/mol. This stabilization can be attributed to the contribution of linkers and other structural factors in the intact protein in the DNA binding process. DNA binding energies of variant proteins of the wild type Zif268 which differ in their ZF1 amino acid sequence gave evidence of a good relationship between binding energy and recognition and specificity, this finding confirms the reported vital role of ZF1 in the ZF protein scanning and anchoring to the target DNA sequence. The role of hydrogen bonds in both specific and nonspecific amino acid-DNA contacts is discussed in relation to mutations. The binding energies of variant Zinc Finger proteins confirmed the role of ZF1 in the recognition, specificity and anchoring of the zinc finger protein to DNA.

Role of protein structure and the role of individual fingers in zinc finger protein-DNA recognition: a molecular dynamics simulation study and free energy calculations.

PubMed

Hamed, Mazen Y

2018-05-03

Molecular dynamics and MM_GBSA energy calculations on various zinc finger proteins containing three and four fingers bound to their target DNA gave insights into the role of each finger in the DNA binding process as part of the protein structure. The wild type Zif 268 (PDB code: 1AAY) gave a ΔG value of - 76.1 (14) kcal/mol. Zinc fingers ZF1, ZF2 and ZF3 were mutated in one experiment and in another experiment one finger was cut and the rest of the protein was studied for binding. The ΔΔG values for the Zinc Finger protein with both ZF1 and ZF2 mutated was + 80 kcal/mol, while mutating only ZF1 the ΔΔG value was + 52 kcal/mol (relative to the wild type). Cutting ZF3 and studying the protein consisting only of ZF1 linked to ZF2 gave a ΔΔG value of + 68 kcal/mol. Upon cutting ZF1, the resulting ZF2 linked to ZF3 protein gave a ΔΔG value of + 41 kcal/mol. The above results shed light on the importance of each finger in the binding process, especially the role of ZF1 as the anchoring finger followed in importance by ZF2 and ZF3. The energy difference between the binding of the wild type protein Zif268 (1AAY) and that for individual finger binding to DNA according to the formula: ΔΔG linkers, otherstructuralfactors  = ΔG zif268  - (ΔG F1+F2+F3 ) gave a value = - 44.5 kcal/mol. This stabilization can be attributed to the contribution of linkers and other structural factors in the intact protein in the DNA binding process. DNA binding energies of variant proteins of the wild type Zif268 which differ in their ZF1 amino acid sequence gave evidence of a good relationship between binding energy and recognition and specificity, this finding confirms the reported vital role of ZF1 in the ZF protein scanning and anchoring to the target DNA sequence. The role of hydrogen bonds in both specific and nonspecific amino acid-DNA contacts is discussed in relation to mutations. The binding energies of variant Zinc Finger proteins confirmed the role of ZF1 in the recognition, specificity and anchoring of the zinc finger protein to DNA.

The NS1 polypeptide of the murine parvovirus minute virus of mice binds to DNA sequences containing the motif [ACCA]2-3.

PubMed Central

Cotmore, S F; Christensen, J; Nüesch, J P; Tattersall, P

1995-01-01

A DNA fragment containing the minute virus of mice 3' replication origin was specifically coprecipitated in immune complexes containing the virally coded NS1, but not the NS2, polypeptide. Antibodies directed against the amino- or carboxy-terminal regions of NS1 precipitated the NS1-origin complexes, but antibodies directed against NS1 amino acids 284 to 459 blocked complex formation. Using affinity-purified histidine-tagged NS1 preparations, we have shown that the specific protein-DNA interaction is of moderate affinity, being stable in 0.1 M salt but rapidly lost at higher salt concentrations. In contrast, generalized (or nonspecific) DNA binding by NS1 could be demonstrated only in low salt. Addition of ATP or gamma S-ATP enhanced specific DNA binding by wild-type NS1 severalfold, but binding was lost under conditions which favored ATP hydrolysis. NS1 molecules with mutations in a critical lysine residue (amino acid 405) in the consensus ATP-binding site bound to the origin, but this binding could not be enhanced by ATP addition. DNase I protection assays carried out with wild-type NS1 in the presence of gamma S-ATP gave footprints which extended over 43 nucleotides on both DNA strands, from the middle of the origin bubble sequence to a position some 14 bp beyond the nick site. The DNA-binding site for NS1 was mapped to a 22-bp fragment from the middle of the 3' replication origin which contains the sequence ACCAACCA. This conforms to a reiterated motif (ACCA)2-3, which occurs, in more or less degenerate form, at many sites throughout the minute virus of mice genome (J. W. Bodner, Virus Genes 2:167-182, 1989). Insertion of a single copy of the sequence (ACCA)3 was shown to be sufficient to confer NS1 binding on an otherwise unrecognized plasmid fragment. The functions of NS1 in the viral life cycle are reevaluated in the light of this result. PMID:7853501

The NS1 polypeptide of the murine parvovirus minute virus of mice binds to DNA sequences containing the motif [ACCA]2-3.

PubMed

Cotmore, S F; Christensen, J; Nüesch, J P; Tattersall, P

1995-03-01

A DNA fragment containing the minute virus of mice 3' replication origin was specifically coprecipitated in immune complexes containing the virally coded NS1, but not the NS2, polypeptide. Antibodies directed against the amino- or carboxy-terminal regions of NS1 precipitated the NS1-origin complexes, but antibodies directed against NS1 amino acids 284 to 459 blocked complex formation. Using affinity-purified histidine-tagged NS1 preparations, we have shown that the specific protein-DNA interaction is of moderate affinity, being stable in 0.1 M salt but rapidly lost at higher salt concentrations. In contrast, generalized (or nonspecific) DNA binding by NS1 could be demonstrated only in low salt. Addition of ATP or gamma S-ATP enhanced specific DNA binding by wild-type NS1 severalfold, but binding was lost under conditions which favored ATP hydrolysis. NS1 molecules with mutations in a critical lysine residue (amino acid 405) in the consensus ATP-binding site bound to the origin, but this binding could not be enhanced by ATP addition. DNase I protection assays carried out with wild-type NS1 in the presence of gamma S-ATP gave footprints which extended over 43 nucleotides on both DNA strands, from the middle of the origin bubble sequence to a position some 14 bp beyond the nick site. The DNA-binding site for NS1 was mapped to a 22-bp fragment from the middle of the 3' replication origin which contains the sequence ACCAACCA. This conforms to a reiterated motif (ACCA)2-3, which occurs, in more or less degenerate form, at many sites throughout the minute virus of mice genome (J. W. Bodner, Virus Genes 2:167-182, 1989). Insertion of a single copy of the sequence (ACCA)3 was shown to be sufficient to confer NS1 binding on an otherwise unrecognized plasmid fragment. The functions of NS1 in the viral life cycle are reevaluated in the light of this result.

Modulation of DNA-Polyamide Interaction by β-alanine Substitutions: A Study of Positional Effects on Binding Affinity, Kinetics and Thermodynamics

PubMed Central

Wang, Shuo; Aston, Karl; Koeller, Kevin J.; Harris, G. Davis; Rath, Nigam P.

2014-01-01

Hairpin polyamides (PAs) are an important class of sequence-specific DNA minor groove binders, and frequently employ a flexible motif, β-alanine (β), to reduce the molecular rigidity to maintain the DNA recognition register. To better understand the diverse effects β can have on DNA-PA binding affinity, selectivity, and especially kinetics, which have rarely been reported, we have initiated a detailed study for an eight-heterocyclic hairpin PA and its β derivatives with their cognate and mutant sequences. With these derivatives, all internal pyrroles of the parent PA are systematically substituted with single or double βs. A set of complementary experiments have been conducted to evaluate the molecular interactions in detail: UV-melting, biosensor-surface plasmon resonance, circular dichroism and isothermal titration calorimetry. The β substitutions generally weaken the binding affinities of these PAs with cognate DNA, and have large and diverse influences on PA binding kinetics in a position- and number-dependent manner. The DNA base mutations have also shown positional effects on binding of a single PA. Besides the β substitutions, the monocationic Dp group [3-(dimethylamino) propylamine] in parent PA has been modified into a dicationic Ta group (3, 3'-Diamino-N-methyldipropylamine) to minimize the frequently observed PA aggregation with ITC experiments. The results clearly show that the Ta modification not only maintains the DNA binding mode and affinity of PA, but also significantly reduces PA aggregation and allows the complete thermodynamic signature of eight-ring hairpin PA to be determined for the first time. This combined set of results significantly extends our understanding of the energetic basis of specific DNA recognition by PAs. PMID:25141096

Programmable Oligomers Targeting 5′-GGGG-3′ in the Minor Groove of DNA and NF-κB Binding Inhibition

PubMed Central

Chenoweth, David M.; Poposki, Julie A.; Marques, Michael A.; Dervan, Peter B.

2009-01-01

A series of hairpin oligomers containing benzimidazole (Bi) and imidazopyridine (Ip) rings were synthesized and screened to target 5′-WGGGGW-3′, a core sequence in the DNA binding site of NF-κB, a prolific transcription factor important in biology and disease. Five Bi and Ip containing oligomers bound to the 5′-WGGGGW-3′ site with high affinity. One of the oligomers (Im-Im-Im-Im-γ-PyBi-PyBi-β-Dp) was able to inhibit DNA binding by the transcription factor NF-κB. PMID:17095230

DNA binding properties and biological evaluation of dihydropyrimidinones derivatives as potential antitumor agents.

PubMed

Wang, Gongke; Li, Xiangrong; Gou, Yaping; Chen, Yuhan; Yan, Changling; Lu, Yan

2013-10-01

The binding properties of two medicinally important dihydropyrimidinones derivatives 5-(Ethoxycarbonyl)-6-methyl-4-phenyl-3,4-dihydropyrimidin-2(1H)-one (EMPD) and 5-(Ethoxycarbonyl)-6-methyl-4-(4-chlorophenyl)-3,4-dihydropyrimidin-2(1H)-one (EMCD) with calf-thymus DNA (ctDNA) were investigated by spectroscopy, viscosity, isothermal titration calorimetry (ITC) and molecular modeling techniques. Simultaneously, their biological activities were evaluated with MTT assay method. The binding constants determined with spectroscopic titration and ITC were found to be in the same order of 10(4)M(-1). According to the results of viscosity studies, fluorescence competitive binding experiment and ITC investigations, intercalative binding was evaluated as the dominant binding modes between the two compounds and ctDNA. Furthermore, the results of molecular modeling corroborated those obtained from spectroscopic, viscosimetric and ITC investigations. Evaluation of the antitumor activities of the two derivatives against different tumor cell lines proved that they exhibited significant tumor cell inhibition rate, accordingly blocking DNA transcription and replication. The present results favor the development of potential drugs related with dihydropyrimidinones derivatives in the treatment of some diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

New modulated design and synthesis of chiral CuII/SnIV bimetallic potential anticancer drug entity: In vitro DNA binding and pBR322 DNA cleavage activity

NASA Astrophysics Data System (ADS)

Tabassum, Sartaj; Sharma, Girish Chandra; Arjmand, Farukh

2012-05-01

A new chiral ligand scaffold L derived from (R)-2-amino-2-phenyl ethanol and diethyl oxalate was isolated and thoroughly characterized by various spectroscopic methods. The ligand L was allowed to react with CuCl2·2H2O and NiCl2·6H2O to achieve monometallic complexes 1 and 2, respectively. Subsequently modulation of 1 and 2 was carried out in the presence of SnCl4·5H2O to obtain heterobimetallic potential drug candidates 3 and 4 possessing (CuII/SnIV and NiII/SnIV) metallic cores, respectively and characterized by elemental analysis and spectroscopic data including 1H, 13C and 119Sn NMR in case of 3 and 4. In vitro DNA binding studies revealed that complex 3 avidly binds to DNA as quantified by Kb and Ksv values. Complex 3 exhibits a remarkable DNA cleavage activity (concentration dependent) with pBR322 DNA and the cleavage activity of 3 was significantly enhanced in the presence of activators and follows the order H2O2 > Asc > MPA > GSH. Complex 3 cleave pBR322 DNA via hydrolytic pathway and accessible to major groove of DNA.

Regulation of apoptosis by resveratrol through JAK/STAT and mitochondria mediated pathway in human epidermoid carcinoma A431 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Madan, Esha; Prasad, Sahdeo; Roy, Preeti

2008-12-26

Resveratrol (trans-3,4',5-trihydroxystilbene), a polyphenolic phytoalexin present mainly in grapes, red wine and berries, is known to possess strong chemopreventive and anticancer properties. Here, we demonstrated the anti-proliferative and apoptosis-inducing activities of resveratrol in human epidermoid carcinoma A431 cells. Resveratrol has cytotoxic effects through inhibiting cellular proliferation of A431 cells, which leads to the induction of apoptosis, as evident by an increase in the fraction of cells in the sub-G{sub 1} phase of the cell cycle and Annexin-V binding of externalized phosphatidylserine. Results revealed that inhibition of proliferation is associated with regulation of the JAK/STAT pathway, where resveratrol prevents phosphorylation ofmore » JAK, thereby inhibiting STAT1 phosphorylation. Furthermore, resveratrol treatment actively stimulated reactive oxygen species (ROS) and mitochondrial membrane depolarization. Consequently, an imbalance in the Bax/Bcl-2 ratio triggered the caspase cascade and subsequent cleavage of PARP, thereby shifting the balance in favor of apoptosis. These observations indicate that resveratrol treatment inhibits JAK/STAT-mediated gene transcription and induce the mitochondrial cell death pathway.« less

Chlorella virus DNA ligase: nick recognition and mutational analysis.

PubMed

Sriskanda, V; Shuman, S

1998-01-15

Chlorella virus PBCV-1 DNA ligase seals nicked DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging DNA template strand. The enzyme discriminates at the DNA binding step between substrates containing a 5'-phosphate versus a 5'-hydroxyl at the nick. Mutational analysis of the active site motif KxDGxR (residues 27-32) illuminates essential roles for the conserved Lys, Asp and Arg moieties at different steps of the ligase reaction. Mutant K27A is unable to form the covalent ligase-(Lys-straightepsilonN-P)-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K27A catalyzes phosphodiester bond formation at a pre-adenylated nick. This shows that the active site lysine is not required for the strand closure reaction. K27A binds to nicked DNA-adenylate, but not to a standard DNA nick. This suggests that occupancy of the AMP binding pocket of DNA ligase is important for nick recognition. Mutant D29A is active in enzyme-adenylate formation and binds readily to nicked DNA, but is inert in DNA-adenylate formation. R32A is unable to catalyze any of the three reactions of the ligation pathway and does not bind to nicked DNA.

Cdc6-Induced Conformational Changes in ORC Bound to Origin DNA Revealed by Cryo-Electron Microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun J.; Li H.; Kawakami, H.

2012-03-07

The eukaryotic origin recognition complex (ORC) interacts with and remodels origins of DNA replication prior to initiation in S phase. Here, we report a single-particle cryo-EM-derived structure of the supramolecular assembly comprising Saccharomyces cerevisiae ORC, the replication initiation factor Cdc6, and double-stranded ARS1 origin DNA in the presence of ATP{gamma}S. The six subunits of ORC are arranged as Orc1:Orc4:Orc5:Orc2:Orc3, with Orc6 binding to Orc2. Cdc6 binding changes the conformation of ORC, in particular reorienting the Orc1 N-terminal BAH domain. Segmentation of the 3D map of ORC-Cdc6 on DNA and docking with the crystal structure of the homologous archaeal Orc1/Cdc6 proteinmore » suggest an origin DNA binding model in which the DNA tracks along the interior surface of the crescent-like ORC. Thus, ORC bends and wraps the DNA. This model is consistent with the observation that binding of a single Cdc6 extends the ORC footprint on origin DNA from both ends.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Xusheng, E-mail: maxushengtt@163.com; Yang, Xing; Nian, Xiaofeng

Peste des petits ruminants virus (PPRV) causes a fatal disease in small ruminants. V protein of PPRV plays a pivotal role in interfering with host innate immunity by blocking IFNs signaling through interacting with STAT1 and STAT2. In the present study, the results demonstrated that PPRV V protein blocks IFN actions in a dose dependent manner and restrains the translocation of STAT1/2 proteins. We speculate that the translocation inhibition might be caused by the interfering of the downstream of STAT protein. Mutagenesis defines that Cys cluster and Trp motif of PPRV V protein are essential for STAT-mediated IFN signaling. Thesemore » findings give a new sight for the further studies to understand the delicate mechanism of PPRV to escape the IFN signaling. - Highlights: • PPRV V protein inhibits type I IFN production and blocks its activation. • PPRV V protein negatively regulates activation of ISRE and GAS promoter. • PPRV V protein inhibits nuclear translocation of STAT protein by non-degradation. • PNT and VCT domain of PPRV V protein inhibit IFN transduction. • PPRV V protein binds with STAT protein via some conserved motifs.« less

Novel fused oxazepino-indoles (FOIs) attenuate liver carcinogenesis via IL-6/JAK2/STAT3 signaling blockade as evidenced through data-based mathematical modeling.

PubMed

Singh, Ashok K; Bhadauria, Archana Singh; Kumar, Umesh; Raj, Vinit; Maurya, Vimal; Kumar, Dinesh; Maity, Biswanath; Prakash, Anand; De, Arnab; Samanta, Amalesh; Saha, Sudipta

2018-05-15

To potentiate the well-documented tumor protecting ability of paullones, literatures demand for rational modifications in paullone ring structure and exploration of a precise mechanism underlying their antitumor effects. Thus, recently we synthesized novel paullone-like scaffold, 5H-benzo [2, 3][1,4]oxazepino[5,6-b]indoles, where compounds 13a and 14a attenuated the growth of liver cancer specific Hep-G2 cells in vitro and formed stable binding complex with IL-6. Henceforth, we hypothesized that this action is probably due to the blockade of IL-6 mediated JAK2/STAT3 signaling cascade. A preclinical study was conducted using NDEA-induced HCC rat model by oral administration of FOIs at 10 mg/kg dose for 15 days. The molecular insights were confirmed through ELISA, qRT-PCR, western blot analyses. The study was further confirmed by data-based mathematical modeling using the quantitative data obtained from western blot analysis. 1 H NMR based metabolomics study was also performed to unveil metabolite discriminations among various studied groups. We identified that the HCC condition was produced due to the IL-6 induced activation of JAK2 and STAT3 which, in turn, was due to enhanced phosphorylation of JAK2 and STAT3. The treatment with FOIs led to the significant blockade of the IL-6 mediated JAK2/STAT3 signaling pathway. Besides, FOIs showed their potential ability in restoring perturbed metabolites linked to HCC. In particular, the anticancer efficacy of compound 13a was comparable or somewhat better than marketed chemotherapeutics, 5-flurouracil. These findings altogether opened up possibilities of developing fused oxazepino-indoles (FOIs) as new candidate molecule for plausible alternative of paullones to treat liver cancer. Copyright © 2018 Elsevier Inc. All rights reserved.

Synthesis, characterization and DNA-binding studies of mono and heterobimetallic complexes Cu sbnd Sn 2/Zn sbnd Sn 2 and their DNA cleavage activity

NASA Astrophysics Data System (ADS)

Arjmand, Farukh; Sayeed, Fatima

2010-02-01

Heterobimetallic complexes C 6H 24N 4O 6CuSn 2Cl 63, C 6H 24N 4O 6ZnSn 2Cl 64 have been synthesized from their monometallic analogs C 6H 16N 4O 2CuCl 21, C 6H 16N 4O 2ZnCl 22, and were characterized by various spectroscopic and analytical methods. The complexes 1-4 reveal an octahedral geometry for both central metal ions Cu/Zn as well as for Sn metal ion. The interaction of complexes 1-4 with CT-DNA, were investigated by using absorption, emission, cyclic voltammetry, viscometry and DNA cleavage studies. The emission quenching of 3 and 4 by [Fe(CN) 6] 4- depressed greatly when bound to CT-DNA. The results of spectroscopic, viscometric and cyclic voltammetry of complexes 3 and 4 revealed electrostatic mode of binding of the complexes with CT-DNA. These results revealed that 4 bind more avidly in comparison to 3 with CT-DNA. Gel electrophoresis of DNA with complexes 3 and 4 demonstrated that the complexes exhibit excellent cleavage activity under physiological conditions.

Substrate sequence selectivity of APOBEC3A implicates intra-DNA interactions.

PubMed

Silvas, Tania V; Hou, Shurong; Myint, Wazo; Nalivaika, Ellen; Somasundaran, Mohan; Kelch, Brian A; Matsuo, Hiroshi; Kurt Yilmaz, Nese; Schiffer, Celia A

2018-05-14

The APOBEC3 (A3) family of human cytidine deaminases is renowned for providing a first line of defense against many exogenous and endogenous retroviruses. However, the ability of these proteins to deaminate deoxycytidines in ssDNA makes A3s a double-edged sword. When overexpressed, A3s can mutate endogenous genomic DNA resulting in a variety of cancers. Although the sequence context for mutating DNA varies among A3s, the mechanism for substrate sequence specificity is not well understood. To characterize substrate specificity of A3A, a systematic approach was used to quantify the affinity for substrate as a function of sequence context, length, secondary structure, and solution pH. We identified the A3A ssDNA binding motif as (T/C)TC(A/G), which correlated with enzymatic activity. We also validated that A3A binds RNA in a sequence specific manner. A3A bound tighter to substrate binding motif within a hairpin loop compared to linear oligonucleotide, suggesting A3A affinity is modulated by substrate structure. Based on these findings and previously published A3A-ssDNA co-crystal structures, we propose a new model with intra-DNA interactions for the molecular mechanism underlying A3A sequence preference. Overall, the sequence and structural preferences identified for A3A leads to a new paradigm for identifying A3A's involvement in mutation of endogenous or exogenous DNA.

The Orphan Nuclear Receptor TLX Is an Enhancer of STAT1-Mediated Transcription and Immunity to Toxoplasma gondii.

PubMed

Beiting, Daniel P; Hidano, Shinya; Baggs, Julie E; Geskes, Jeanne M; Fang, Qun; Wherry, E John; Hunter, Christopher A; Roos, David S; Cherry, Sara

2015-07-01

The protozoan parasite, Toxoplasma, like many intracellular pathogens, suppresses interferon gamma (IFN-γ)-induced signal transducer and activator of transcription 1 (STAT1) activity. We exploited this well-defined host-pathogen interaction as the basis for a high-throughput screen, identifying nine transcription factors that enhance STAT1 function in the nucleus, including the orphan nuclear hormone receptor TLX. Expression profiling revealed that upon IFN-γ treatment TLX enhances the output of a subset of IFN-γ target genes, which we found is dependent on TLX binding at those loci. Moreover, infection of TLX deficient mice with the intracellular parasite Toxoplasma results in impaired production of the STAT1-dependent cytokine interleukin-12 by dendritic cells and increased parasite burden in the brain during chronic infection. These results demonstrate a previously unrecognized role for this orphan nuclear hormone receptor in regulating STAT1 signaling and host defense and reveal that STAT1 activity can be modulated in a context-specific manner by such "modifiers."

CD95/Fas Increases Stemness in Cancer Cells by Inducing a STAT1-Dependent Type I Interferon Response.

PubMed

Qadir, Abdul S; Ceppi, Paolo; Brockway, Sonia; Law, Calvin; Mu, Liang; Khodarev, Nikolai N; Kim, Jung; Zhao, Jonathan C; Putzbach, William; Murmann, Andrea E; Chen, Zhuo; Chen, Wenjing; Liu, Xia; Salomon, Arthur R; Liu, Huiping; Weichselbaum, Ralph R; Yu, Jindan; Peter, Marcus E

2017-03-07

Stimulation of CD95/Fas drives and maintains cancer stem cells (CSCs). We now report that this involves activation of signal transducer and activator of transcription 1 (STAT1) and induction of STAT1-regulated genes and that this process is inhibited by active caspases. STAT1 is enriched in CSCs in cancer cell lines, patient-derived human breast cancer, and CD95 high -expressing glioblastoma neurospheres. CD95 stimulation of cancer cells induced secretion of type I interferons (IFNs) that bind to type I IFN receptors, resulting in activation of Janus-activated kinases, activation of STAT1, and induction of a number of STAT1-regulated genes that are part of a gene signature recently linked to therapy resistance in five primary human cancers. Consequently, we identified type I IFNs as drivers of cancer stemness. Knockdown or knockout of STAT1 resulted in a strongly reduced ability of CD95L or type I IFN to increase cancer stemness. This identifies STAT1 as a key regulator of the CSC-inducing activity of CD95. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Spectroscopy of the neutron-rich hypernucleus He7Λ from electron scattering

NASA Astrophysics Data System (ADS)

Gogami, T.; Chen, C.; Kawama, D.; Achenbach, P.; Ahmidouch, A.; Albayrak, I.; Androic, D.; Asaturyan, A.; Asaturyan, R.; Ates, O.; Baturin, P.; Badui, R.; Boeglin, W.; Bono, J.; Brash, E.; Carter, P.; Chiba, A.; Christy, E.; Danagoulian, S.; De Leo, R.; Doi, D.; Elaasar, M.; Ent, R.; Fujii, Y.; Fujita, M.; Furic, M.; Gabrielyan, M.; Gan, L.; Garibaldi, F.; Gaskell, D.; Gasparian, A.; Han, Y.; Hashimoto, O.; Horn, T.; Hu, B.; Hungerford, Ed. V.; Jones, M.; Kanda, H.; Kaneta, M.; Kato, S.; Kawai, M.; Khanal, H.; Kohl, M.; Liyanage, A.; Luo, W.; Maeda, K.; Margaryan, A.; Markowitz, P.; Maruta, T.; Matsumura, A.; Maxwell, V.; Mkrtchyan, A.; Mkrtchyan, H.; Nagao, S.; Nakamura, S. N.; Narayan, A.; Neville, C.; Niculescu, G.; Niculescu, M. I.; Nunez, A.; Nuruzzaman, Okayasu, Y.; Petkovic, T.; Pochodzalla, J.; Qiu, X.; Reinhold, J.; Rodriguez, V. M.; Samanta, C.; Sawatzky, B.; Seva, T.; Shichijo, A.; Tadevosyan, V.; Tang, L.; Taniya, N.; Tsukada, K.; Veilleux, M.; Vulcan, W.; Wesselmann, F. R.; Wood, S. A.; Yamamoto, T.; Ya, L.; Ye, Z.; Yokota, K.; Yuan, L.; Zhamkochyan, S.; Zhu, L.; HKS JLab E05-115 Collaboration

2016-08-01

The missing mass spectroscopy of the He7Λ hypernucleus was performed using the 7Li(e ,e'K+) He7Λ reaction at the Thomas Jefferson National Accelerator Facility Hall C. The Λ -binding energy of the ground-state (1 /2+ ) was determined with a smaller error than that of the previous measurement, being BΛ=5.55 ±0 .10stat .±0 .11sys .MeV . The experiment also provided new insight into charge symmetry breaking in p -shell hypernuclear systems. Finally, a peak at BΛ=3.65 ±0 .20stat .±0 .11sys .MeV was observed and assigned as a mixture of 3 /2+ and 5 /2+ states, confirming the "gluelike" behavior of Λ , which makes an unstable state in 6He stable against neutron emission.

Cooperative Assembly of Co-Smad4 MH1 with R-Smad1/3 MH1 on DNA: A Molecular Dynamics Simulation Study

PubMed Central

Wang, Guihong; Li, Chaoqun; Wang, Yan; Chen, Guangju

2013-01-01

Background Smads, the homologs of Sma and MAD proteins, play a key role in gene expression regulation in the transforming growth factor-β (TGF-β) signaling pathway. Recent experimental studies have revealed that Smad4/R-Smad heterodimers bound on DNA are energetically more favorable than homodimeric R-Smad/R-Smad complexes bound on DNA, which indicates that Smad4 might act as binding vehicle to cooperatively assemble with activated R-Smads on DNA in the nucleus. However, the details of interaction mechanism for cooperative recruitment of Smad4 protein to R-Smad proteins on DNA, and allosteric communication between the Smad4-DNA and R-Smad-DNA interfaces via DNA mediating are not yet clear so far. Methodology In the present work, we have constructed a series of Smadn+DNA+Smadn (n = 1, 3, 4) models and carried out molecular dynamics simulations, free energy calculations and DNA dynamics analysis for them to study the interaction properties of Smadn (n = 1, 3, 4) with DNA molecule. Results The results revealed that the binding of Smad4 protein to DNA molecule facilitates energetically the formation of the heteromeric Smad4+DNA+Smad1/3 complex by increasing the affinity of Smad1/3 with DNA molecule. Further investigations through the residue/base motion correlation and DNA dynamics analyses predicted that the binding of Smad4 protein to DNA molecule in the heteromeric Smad4+DNA+Smad1/3 model induces an allosteric communication from the Smad4-DNA interface to Smad1/Smad3-DNA interface via DNA base-pair helical motions, surface conformation changes and new hydrogen bond formations. The present work theoretically explains the mechanism of cooperative recruitment of Smad4 protein to Smad1/3 protein via DNA-mediated indirect readout mode in the nucleus. PMID:23326519

STAT3, p-STAT3 and HIF-1α are associated with vasculogenic mimicry and impact on survival in gastric adenocarcinoma

PubMed Central

SONG, YAN-YAN; SUN, LI-DAN; LIU, MIN-LI; LIU, ZHONG-LIANG; CHEN, FEI; ZHANG, YING-ZHE; ZHENG, YAN; ZHANG, JIAN-PING

2014-01-01

Vasculogenic mimicry (VM) formation is important for invasion and metastasis of tumor cells in gastric adenocarcinoma (GAC). The present study aimed to investigate the association between signal transducer and activator of transcription-3 (STAT3), phosphor-STAT3 (p-STAT3), hypoxia-inducible factor-1α (HIF-1α) and VM formation in GAC, and discuss their clinical significance and correlation with the prognosis of patients with GAC. The expression levels of STAT3, p-STAT3, HIF-1α and VM were assessed in 60 cases of patients with GAC and 20 cases of patients with gastritis on tissue microarrays by immunohistochemical methods. The expression levels of STAT3, p-STAT3, HIF-1α and VM were higher in patients with GAC (particularly in poorly differentiated GAC) than in those with gastritis (P<0.05). The expression levels of STAT3, p-STAT3 and HIF-1α were higher in VM tissues compared with non-VM tissues (P<0.05). Positive correlations existed between STAT3, p-STAT3, HIF-1α and VM expression (P<0.05). The expression levels of STAT3, p-STAT3 and HIF-1α, VM, status of lymph node metastasis and tumor differentiation degree were associated with the overall survival time of patients with GAC (P<0.05). However, only p-STAT3 and VM expression were identified as the independent risk factors of GAC OS when analyzed with multivariate analysis. p-STAT3 and VM play a significant role in indicating the prognosis of patients with GAC. STAT3 activation may play a positive role in VM formation of GAC by the STAT3-p-STAT3-HIF-1α-VM effect axis. PMID:24959290

Sanguinarine interacts with chromatin, modulates epigenetic modifications, and transcription in the context of chromatin.

PubMed

Selvi B, Ruthrotha; Pradhan, Suman Kalyan; Shandilya, Jayasha; Das, Chandrima; Sailaja, Badi Sri; Shankar G, Naga; Gadad, Shrikanth S; Reddy, Ashok; Dasgupta, Dipak; Kundu, Tapas K

2009-02-27

DNA-binding anticancer agents cause alteration in chromatin structure and dynamics. We report the dynamic interaction of the DNA intercalator and potential anticancer plant alkaloid, sanguinarine (SGR), with chromatin. Association of SGR with different levels of chromatin structure was enthalpy driven with micromolar dissociation constant. Apart from DNA, it binds with comparable affinity with core histones and induces chromatin aggregation. The dual binding property of SGR leads to inhibition of core histone modifications. Although it potently inhibits H3K9 methylation by G9a in vitro, H3K4 and H3R17 methylation are more profoundly inhibited in cells. SGR inhibits histone acetylation both in vitro and in vivo. It does not affect the in vitro transcription from DNA template but significantly represses acetylation-dependent chromatin transcription. SGR-mediated repression of epigenetic marks and the alteration of chromatin geography (nucleography) also result in the modulation of global gene expression. These data, conclusively, show an anticancer DNA binding intercalator as a modulator of chromatin modifications and transcription in the chromatin context.

The monomeric form of Neisseria DNA mimic protein DMP19 prevents DNA from binding to the histone-like HU protein

PubMed Central

Ko, Tzu-Ping; Liao, Yi-Ting; Hsu, Kai-Cheng

2017-01-01

DNA mimicry is a direct and effective strategy by which the mimic competes with DNA for the DNA binding sites on other proteins. Until now, only about a dozen proteins have been shown to function via this strategy, including the DNA mimic protein DMP19 from Neisseria meningitides. We have shown previously that DMP19 dimer prevents the operator DNA from binding to the transcription factor NHTF. Here, we provide new evidence that DMP19 monomer can also interact with the Neisseria nucleoid-associated protein HU. Using BS3 crosslinking, gel filtration and isothermal titration calorimetry assays, we found that DMP19 uses its monomeric form to interact with the Neisseria HU dimer. Crosslinking conjugated mass spectrometry was used to investigate the binding mode of DMP19 monomer and HU dimer. Finally, an electrophoretic mobility shift assay (EMSA) confirmed that the DNA binding affinity of HU is affected by DMP19. These results showed that DMP19 is bifunctional in the gene regulation of Neisseria through its variable oligomeric forms. PMID:29220372

Wing 1 of protein HOP2 is as important as helix 3 in DNA binding by MD simulation.

PubMed

Moktan, Hem; Zhou, Donghua H

2018-05-01

The repair of programmed DNA double-strand breaks through recombination is required for proper association and disjunction of the meiotic homologous chromosomes. Meiosis-specific protein HOP2 plays essential roles in recombination by promoting recombinase activities. The N-terminal domain of HOP2 interacts with DNA through helix 3 (H3) and wing 1 (W1). Mutations in wing 1 (Y65A/K67A/Q68A) slightly weakened the binding but mutations in helices 2 and 3 (Q30A/K44A/K49A) nearly abolished the binding. To better understand such differential effects at atomic level, molecular dynamics simulations were employed. Despite losing some hydrogen bonds, the W1-mutant DNA complex was rescued by stronger hydrophobic interactions. For the wild type and W1-mutant, the protein was found to slide along the DNA grooves as the DNA rolls along its double-helix axis. This motion could be functionally important to facilitate the precise positioning of the single-stranded DNA with the homologous double-stranded DNA. The sliding motion was reduced in the W1-mutant. The H-mutant nearly lost all intermolecular interactions. Moreover, an additional mutation in wing 1 (Y65A/K67A/Q68A/K69A) also caused complete complex dissociation. Therefore, both wing 1 and helix 3 make important contribution to the DNA binding, which could be important to the strand invasion function of HOP2 homodimer and HOP2-MND1 heterodimer. Similar to cocking a medieval crossbow with the archer's foot placed in the stirrup, wing 1 may push the minor groove to cause distortion while helix 3 grabs the major groove.

Structural Reorganization and the Cooperative Binding of Single-stranded Telomere DNA in Sterkiella nova*

PubMed Central

Buczek, Pawel; Horvath, Martin P.

2009-01-01

In Sterkiella nova, α and β telomere proteins bind cooperatively with single-stranded DNA to form a ternary α·β·DNA complex. Association of telomere protein subunits is DNA-dependent, and α-β association enhances DNA affinity. To further understand the molecular basis for binding cooperativity, we characterized several possible stepwise assembly pathways using isothermal titration calorimetry. In one path, α and DNA first form a stable α·DNA complex followed by addition of β in a second step. Binding energy accumulates with nearly equal free energy of association for each of these steps. Heat capacity is nonetheless dramatically different with ΔCp = −305 ± 3 cal mol−1 K−1 for α binding with DNA and ΔCp = −2010 ± 20 cal mol−1 K−1 for addition of β to complete the α·β·DNA complex. By examining alternate routes including titration of single-stranded DNA with a preformed α·β complex, a significant portion of binding energy and heat capacity could be assigned to structural reorganization involving protein-protein interactions and repositioning of the DNA. Structural reorganization probably affords a mechanism to regulate high affinity binding of telomere single-stranded DNA with important implications for telomere biology. Regulation of telomere complex dissociation is thought to involve post-translational modifications in the lysine-rich C-terminal portion of β. We observed no difference in binding energetics or crystal structure when comparing complexes prepared with full-length β or a C-terminally truncated form, supporting interesting parallels between the intrinsically disordered regions of histones and this portion of β. PMID:17082188

Stopped-flow kinetic studies of poly(amidoamine) dendrimer-calf thymus DNA to form dendriplexes.

PubMed

Dey, Debabrata; Kumar, Santosh; Maiti, Souvik; Dhara, Dibakar

2013-11-07

Poly(amidoamine) (PAMAM) dendrimers are known to be highly efficient nonviral carriers in gene delivery. Dendrimer-mediated transfection is known to be a function of the dendrimer to DNA charge ratio as well as the size of the dendrimer. In the present study, the binding kinetics of four PAMAM dendrimers (G1, G2, G3, and G4) with calf thymus DNA (CT-DNA) has been studied using stopped-flow fluorescence spectroscopy. The effect of dendrimer-to-DNA charge ratio and dendrimer generation on the binding kinetics was investigated. In most cases, the results of dendrimer-CT-DNA binding can be explained by a two-step reaction mechanism: a rapid electrostatic binding between the dendrimer and DNA, followed by a conformational change of the dendrimer-DNA complex that ultimately leads to DNA condensation. It was observed that the charge ratio on the dendrimer and the DNA phosphate groups, as well as the dendrimer generation (size), has a marked effect on the kinetics of binding between the DNA and the dendrimers. The rate constant (k'1) of the first step was much higher compared to that of the second step (k'2), and both were found to increase with an increase in dendrimer concentration. Among the four generations of dendrimers, G4 exhibited significantly faster binding kinetics compared to the three smaller generation dendrimers.

The critical role that STAT3 plays in glioma-initiating cells: STAT3 addiction in glioma

PubMed Central

Ganguly, Debolina; Fan, Meiyun; Yang, Chuan He; Zbytek, Blazej; Finkelstein, David; Roussel, Martine F.; Pfeffer, Lawrence M.

2018-01-01

Glioma-Initiating Cells (GICs) are thought to be responsible for tumor initiation, progression and recurrence in glioblastoma (GBM). In previous studies, we reported the constitutive phosphorylation of the STAT3 transcription factor in GICs derived from GBM patient-derived xenografts, and that STAT3 played a critical role in GBM tumorigenesis. In this study, we show that CRISPR/Cas9-mediated deletion of STAT3 in an established GBM cell line markedly inhibited tumorigenesis by intracranial injection but had little effect on cell proliferation in vitro. Tumorigenesis was rescued by the enforced expression of wild-type STAT3 in cells lacking STAT3. In contrast, GICs were highly addicted to STAT3 and upon STAT3 deletion GICs were non-viable. Moreover, we found that STAT3 was constitutively activated in GICs by phosphorylation on both tyrosine (Y705) and serine (S727) residues. Therefore, to study STAT3 function in GICs we established an inducible system to knockdown STAT3 expression (iSTAT3-KD). Using this approach, we demonstrated that Y705-STAT3 phosphorylation was critical and indispensable for GIC-induced tumor formation. Both phosphorylation sites in STAT3 promoted GIC proliferation in vitro. We further showed that S727-STAT3 phosphorylation was Y705-dependent. Targeted microarray and RNA sequencing revealed that STAT3 activated cell-cycle regulator genes, and downregulated genes involved in the interferon response, the hypoxia response, the TGFβ pathway, and remodeling of the extracellular matrix. Since STAT3 is an important oncogenic driver of GBM, the identification of these STAT3 regulated pathways in GICs will inform the development of better targeted therapies against STAT3 in GBM and other cancers. PMID:29774125

Structure-Function Correlation of G6, a Novel Small Molecule Inhibitor of Jak2

PubMed Central

Majumder, Anurima; Govindasamy, Lakshmanan; Magis, Andrew; Kiss, Róbert; Polgár, Tímea; Baskin, Rebekah; Allan, Robert W.; Agbandje-McKenna, Mavis; Reuther, Gary W.; Keserű, György M.; Bisht, Kirpal S.; Sayeski, Peter P.

2010-01-01

Somatic mutations in the Jak2 protein, such as V617F, cause aberrant Jak/STAT signaling and can lead to the development of myeloproliferative neoplasms. This discovery has led to the search for small molecule inhibitors that target Jak2. Using structure-based virtual screening, our group recently identified a novel small molecule inhibitor of Jak2 named G6. Here, we identified a structure-function correlation of this compound. Specifically, five derivative compounds of G6 having structural similarity to the original lead compound were obtained and analyzed for their ability to (i) inhibit Jak2-V617F-mediated cell growth, (ii) inhibit the levels of phospho-Jak2, phospho-STAT3, and phospho-STAT5; (iii) induce apoptosis in human erythroleukemia cells; and (iv) suppress pathologic cell growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Additionally, we computationally examined the interactions of these compounds with the ATP-binding pocket of the Jak2 kinase domain. We found that the stilbenoid core-containing derivatives of G6 significantly inhibited Jak2-V617F-mediated cell proliferation in a time- and dose-dependent manner. They also inhibited phosphorylation of Jak2, STAT3, and STAT5 proteins within cells, resulting in higher levels of apoptosis via the intrinsic apoptotic pathway. Finally, the stilbenoid derivatives inhibited the pathologic growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Collectively, our data demonstrate that G6 has a stilbenoid core that is indispensable for maintaining its Jak2 inhibitory potential. PMID:20667821

DTX3L and ARTD9 inhibit IRF1 expression and mediate in cooperation with ARTD8 survival and proliferation of metastatic prostate cancer cells

PubMed Central

2014-01-01

Background Prostate cancer (PCa) is one of the leading causes of cancer-related mortality and morbidity in the aging male population and represents the most frequently diagnosed malignancy in men around the world. The Deltex (DTX)-3-like E3 ubiquitin ligase (DTX3L), also known as B-lymphoma and BAL-associated protein (BBAP), was originally identified as a binding partner of the diphtheria-toxin-like macrodomain containing ADP-ribosyltransferase-9 (ARTD9), also known as BAL1 and PARP9. We have previously demonstrated that ARTD9 acts as a novel oncogenic survival factor in high-risk, chemo-resistant, diffuse large B cell lymphoma (DLBCL). The mono-ADP-ribosyltransferase ARTD8, also known as PARP14 functions as a STAT6-specific co-regulator of IL4-mediated proliferation and survival in B cells. Methods Co-expression of DTX3L, ARTD8, ARTD9 and STAT1 was analyzed in the metastatic PCa (mPCa) cell lines PC3, DU145, LNCaP and in the normal prostate luminal epithelial cell lines HPE and RWPE1. Effects on cell proliferation, survival and cell migration were determined in PC3, DU145 and/or LNCaP cells depleted of DTX3L, ARTD8, ARTD9, STAT1 and/or IRF1 compared to their proficient control cells, respectively. In further experiments, real-time RT-PCR, Western blot, immunofluorescence and co-immunoprecipitations were conducted to evaluate the physical and functional interactions between DTX3L, ARTD8 and ARTD9. Results Here we could identify DTX3L, ARTD9 and ARTD8 as novel oncogenic survival factors in mPCa cells. Our studies revealed that DTX3L forms a complex with ARTD8 and mediates together with ARTD8 and ARTD9 proliferation, chemo-resistance and survival of mPCa cells. In addition, DTX3L, ARTD8 and ARTD9 form complexes with each other. Our study provides first evidence that the enzymatic activity of ARTD8 is required for survival of mPCa cells. DTX3L and ARTD9 act together as repressors of the tumor suppressor IRF1 in mPCa cells. Furthermore, the present study shows that DTX3L together with STAT1 and STAT3 is implicated in cell migration of mPCa cells. Conclusions Our data strongly indicate that a crosstalk between STAT1, DTX3L and ARTD-like mono-ADP-ribosyltransferases mediates proliferation and survival of mPCa cells. The present study further suggests that the combined targeted inhibition of STAT1, ARTD8, ARTD9 and/or DTX3L could increase the efficacy of chemotherapy or radiation treatment in prostate and other high-risk tumor types with an increased STAT1 signaling. PMID:24886089

Generalized theory on the mechanism of site-specific DNA-protein interactions

NASA Astrophysics Data System (ADS)

Niranjani, G.; Murugan, R.

2016-05-01

We develop a generalized theoretical framework on the binding of transcription factor proteins (TFs) with specific sites on DNA that takes into account the interplay of various factors regarding overall electrostatic potential at the DNA-protein interface, occurrence of kinetic traps along the DNA sequence, presence of other roadblock protein molecules along DNA and crowded environment, conformational fluctuations in the DNA binding domains (DBDs) of TFs, and the conformational state of the DNA. Starting from a Smolochowski type theoretical framework on site-specific binding of TFs we logically build our model by adding the effects of these factors one by one. Our generalized two-step model suggests that the electrostatic attractive forces present inbetween the positively charged DBDs of TFs and the negatively charged phosphate backbone of DNA, along with the counteracting shielding effects of solvent ions, is the core factor that creates a fluidic type environment at the DNA-protein interface. This in turn facilitates various one-dimensional diffusion (1Dd) processes such as sliding, hopping and intersegmental transfers. These facilitating processes as well as flipping dynamics of conformational states of DBDs of TFs between stationary and mobile states can enhance the 1Dd coefficient on a par with three-dimensional diffusion (3Dd). The random coil conformation of DNA also plays critical roles in enhancing the site-specific association rate. The extent of enhancement over the 3Dd controlled rate seems to be directly proportional to the maximum possible 1Dd length. We show that the overall site-specific binding rate scales with the length of DNA in an asymptotic way. For relaxed DNA, the specific binding rate will be independent of the length of DNA as length increases towards infinity. For condensed DNA as in in vivo conditions, the specific binding rate depends on the length of DNA in a turnover way with a maximum. This maximum rate seems to scale with the maximum possible 1Dd length of TFs in a square root manner. Results suggest that 1Dd processes contribute much less to the enhancement of specific binding rate under in vivo conditions for condensed DNA. There exists a critical length of binding stretch of TFs beyond which the probability associated with the random occurrence of similar specific binding sites will be close to zero. TFs in natural systems from prokaryotes to eukaryotes seem to handle sequence-mediated kinetic traps via increasing the length of their recognition stretch or combinatorial binding. TFs overcome the hurdles of roadblocks via switching efficiently between sliding, hopping and intersegmental transfer modes. The site-specific binding rate as well as the maximum possible 1Dd length seem to be directly proportional to the square root of the probability (p R) of finding a nonspecific binding site to be free from dynamic roadblocks. Here p R seems to be a function of the number of nsbs available per DNA binding protein (ϕ) inside the living cell. It seems that p R  >  0.8 when ϕ  >  10 which is true for the Escherichia coli cell system.

Selection and identification of a DNA aptamer targeted to Vibrio parahemolyticus.

PubMed

Duan, Nuo; Wu, Shijia; Chen, Xiujuan; Huang, Yukun; Wang, Zhouping

2012-04-25

A whole-bacterium systemic evolution of ligands by exponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules to identify DNA aptamers demonstrating specific binding to Vibrio parahemolyticus . FAM-labeled aptamer sequences with high binding affinity to V. parahemolyticus were identified by flow cytometric analysis. Aptamer A3P, which showed a particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K(d)) of 16.88 ± 1.92 nM. Binding assays to assess the specificity of aptamer A3P showed a high binding affinity (76%) for V. parahemolyticus and a low apparent binding affinity (4%) for other bacteria. Whole-bacterium SELEX is a promising technique for the design of aptamer-based molecular probes for microbial pathogens that does not require the labor-intensive steps of isolating and purifying complex markers or targets.

Carboxylation of cytosine (5caC) in the CG dinucleotide in the E-box motif (CGCAG|GTG) increases binding of the Tcf3|Ascl1 helix-loop-helix heterodimer 10-fold.

PubMed

Golla, Jaya Prakash; Zhao, Jianfei; Mann, Ishminder K; Sayeed, Syed K; Mandal, Ajeet; Rose, Robert B; Vinson, Charles

2014-06-27

Three oxidative products of 5-methylcytosine (5mC) occur in mammalian genomes. We evaluated if these cytosine modifications in a CG dinucleotide altered DNA binding of four B-HLH homodimers and three heterodimers to the E-Box motif CGCAG|GTG. We examined 25 DNA probes containing all combinations of cytosine in a CG dinucleotide and none changed binding except for carboxylation of cytosine (5caC) in the strand CGCAG|GTG. 5caC enhanced binding of all examined B-HLH homodimers and heterodimers, particularly the Tcf3|Ascl1 heterodimer which increased binding ~10-fold. These results highlight a potential function of the oxidative products of 5mC, changing the DNA binding of sequence-specific transcription factors. Published by Elsevier Inc.

Alternative activation of STAT1 and STAT3 in response to interferon-gamma.

PubMed

Qing, Yulan; Stark, George R

2004-10-01

Interferon-gamma (IFNgamma) is a pluripotent cytokine whose major biological effects are mediated through a pathway in which STAT1 is the predominant and essential transcription factor. STAT3 can also be activated weakly by IFNgamma, but the mechanism of activation and function of STAT3 as a part of the interferon response are not known. Here we show that STAT3 activation is much stronger and more prolonged in STAT1-null mouse embryo fibroblasts than in wild-type cells. In response to IFNgamma, SRC-family kinases are required to activate STAT3 (but not STAT1) through tyrosine phosphorylation, whereas the receptor-bound kinases JAK1 and JAK2 are required to activate both STATs. Tyrosine 419 of the IFNgamma receptor subunit 1 (IFNGR1) is required to activate both STATs, suggesting that STAT1 and STAT3 compete with each other for the same receptor phosphotyrosine motif. Activated STAT3 can replace STAT1 in STAT1-null cells to drive the transcription of certain genes, for example, socs-3 and c/ebpdelta, which have gamma-activated sequence motifs in their promoters. Work from Ian Kerr's laboratory reveals that the gp130-linked interleukin-6 receptor, which usually activates STAT3 predominantly, activates STAT1 efficiently when STAT3 is absent. Because STAT1 and STAT3 have opposing biological effects (STAT3 is an oncogene, and STAT1 is a tumor suppressor), the reciprocal activation of these two transcription factors in response to IFNgamma or interleukin-6 suggests that their relative abundance, which may vary substantially in different normal cell types, under different conditions or in tumors is likely to have a major impact on how cells behave in response to different cytokines.