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Sample records for activating notch1 mutations

  1. Cleaved NOTCH1 expression pattern in head and neck squamous cell carcinoma is associated with NOTCH1 mutation, HPV status and high-risk features

    PubMed Central

    Rettig, Eleni M; Chung, Christine H; Bishop, Justin A; Howard, Jason D; Sharma, Rajni; Li, Ryan J; Douville, Christopher; Karchin, Rachel; Izumchenko, Evgeny; Sidransky, David; Koch, Wayne; Califano, Joseph; Agrawal, Nishant; Fakhry, Carole

    2015-01-01

    The Notch pathway is frequently altered in HNSCCs, however the clinical significance of NOTCH1 dysregulation is poorly understood. This study was designed to characterize expression of the transcriptionally active NOTCH1 Intracellular Domain (NICD1) in HNSCCs and evaluate its association with NOTCH1 mutation status and clinical parameters. Immunohistochemistry for NICD1 was performed on 79 previously sequenced archival HNSCCs with known NOTCH1 mutation status. Three distinct immunohistochemical staining patterns were identified: positive/peripheral (47%), positive/non-peripheral (34%) and negative (19%). NICD1 expression was associated with NOTCH1 mutation status (p<0.001). Most NOTCH1-wild type tumors were peripheral (55%), while mutated NOTCH1 tumors were most commonly negative (47%). Non-peripheral tumors were more likely than peripheral tumors to have extracapsular spread (aOR 16.01, 95% CI=1.92–133.46, p=0.010) and poor differentiation (aOR 5.27, 95% CI=0.90–30.86, p=0.066). Negative staining tumors tended to be poorly differentiated (aOR 24.71, 95% CI=1.53–399.33, p=0.024) and were less likely to be HPV-positive (aOR 0.043, 95% CI=0.001–1.59, p=0.087). NOTCH1 mutagenesis was significantly associated with HPV status, with NOTCH1-wild-type tumors more likely to be HPV-positive than NOTCH1-mutated tumors (aOR 19.06, 95% CI=1.31–276.15, p=0.031). TP53 disruptive mutations were not associated with NICD1 expression or NOTCH1 mutation. In conclusion, NICD1 is expressed in three distinct patterns in HNSCC that are significantly associated with high-risk features. These findings further support a dual role for NOTCH1 as both tumor suppressor and oncogene in HNSCC. Further research is necessary to clarify the role of NOTCH1 in HNSCC and understand the clinical and therapeutic implications therein. PMID:25633867

  2. Mutations in NOTCH1 cause Adams-Oliver syndrome.

    PubMed

    Stittrich, Anna-Barbara; Lehman, Anna; Bodian, Dale L; Ashworth, Justin; Zong, Zheyuan; Li, Hong; Lam, Patricia; Khromykh, Alina; Iyer, Ramaswamy K; Vockley, Joseph G; Baveja, Rajiv; Silva, Ermelinda Santos; Dixon, Joanne; Leon, Eyby L; Solomon, Benjamin D; Glusman, Gustavo; Niederhuber, John E; Roach, Jared C; Patel, Millan S

    2014-09-01

    Notch signaling determines and reinforces cell fate in bilaterally symmetric multicellular eukaryotes. Despite the involvement of Notch in many key developmental systems, human mutations in Notch signaling components have mainly been described in disorders with vascular and bone effects. Here, we report five heterozygous NOTCH1 variants in unrelated individuals with Adams-Oliver syndrome (AOS), a rare disease with major features of aplasia cutis of the scalp and terminal transverse limb defects. Using whole-genome sequencing in a cohort of 11 families lacking mutations in the four genes with known roles in AOS pathology (ARHGAP31, RBPJ, DOCK6, and EOGT), we found a heterozygous de novo 85 kb deletion spanning the NOTCH1 5' region and three coding variants (c.1285T>C [p.Cys429Arg], c.4487G>A [p.Cys1496Tyr], and c.5965G>A [p.Asp1989Asn]), two of which are de novo, in four unrelated probands. In a fifth family, we identified a heterozygous canonical splice-site variant (c.743-1 G>T) in an affected father and daughter. These variants were not present in 5,077 in-house control genomes or in public databases. In keeping with the prominent developmental role described for Notch1 in mouse vasculature, we observed cardiac and multiple vascular defects in four of the five families. We propose that the limb and scalp defects might also be due to a vasculopathy in NOTCH1-related AOS. Our results suggest that mutations in NOTCH1 are the most common cause of AOS and add to a growing list of human diseases that have a vascular and/or bony component and are caused by alterations in the Notch signaling pathway. PMID:25132448

  3. Aspartate mutations in presenilin and gamma-secretase inhibitors both impair notch1 proteolysis and nuclear translocation with relative preservation of notch1 signaling.

    PubMed

    Berezovska, O; Jack, C; McLean, P; Aster, J C; Hicks, C; Xia, W; Wolfe, M S; Kimberly, W T; Weinmaster, G; Selkoe, D J; Hyman, B T

    2000-08-01

    It has been hypothesized that a presenilin 1 (PS1)-related enzymatic activity is responsible for proteolytic cleavage of the C-terminal intracellular protein of Notch1, in addition to its role in beta-amyloid protein (Abeta) formation from the amyloid precursor protein (APP). We developed an assay to monitor ligand-induced Notch1 proteolysis and nuclear translocation in individual cells : Treatment of full-length Notch1-enhanced green fluorescent protein-transfected Chinese hamster ovary (CHO) cells with a soluble preclustered form of the physiologic ligand Delta leads to rapid accumulation of the C terminus of Notch1 in the nucleus and to transcriptional activation of a C-promoter binding factor 1 (CBF1) reporter construct. Nuclear translocation was blocked by cotransfection with Notch's physiologic inhibitor Numb. Using this assay, we now confirm and extend the observation that PS1 is involved in Notch1 nuclear translocation and signaling in mammalian cells. We demonstrate that the D257A and the D385A PS1 mutations, which had been shown previously to block APP gamma-secretase activity, also prevent Notch1 cleavage and translocation to the nucleus but do not alter Notch1 trafficking to the cell surface. We also show that two APP gamma-secretase inhibitors block Notch1 nuclear translocation with an IC(50) similar to that reported for APP gamma-secretase. Notch1 signaling, assessed by measuring the activity of CBF1, a downstream transcription factor, was impaired but not abolished by the PS1 aspartate mutations or gamma-secretase inhibitors. Our results support the hypotheses that (a) PS1-dependent APP gamma-secretase-like enzymatic activity is critical for both APP and Notch processing and (b) the Notch1 signaling pathway remains partially activated even when Notch1 proteolytic processing and nuclear translocation are markedly inhibited. The latter is an important finding from the perspective of therapeutic treatment of Alzheimer's disease by targeting gamma

  4. PRDM14 promotes RAG-dependent Notch1 driver mutations in mouse T-ALL

    PubMed Central

    Carofino, Brandi L.; Ayanga, Bernard; Tracey, Lauren J.; Brooke-Bisschop, Travis

    2016-01-01

    ABSTRACT PRDM14 is an epigenetic regulator known for maintaining embryonic stem cell identity and resetting potency in primordial germ cells. However, hematopoietic expression of Prdm14 at supraphysiological levels results in fully penetrant and rapid-onset T-cell acute lymphoblastic leukemia (T-ALL) in the mouse. Here, we show that PRDM14-induced T-ALLs are driven by NOTCH1, a frequently mutated driver of human T-ALL. Notch1 is activated in this murine model via RAG-dependent promoter deletions and subsequent production of truncated, ligand-independent protein from downstream regions of the Notch1 locus. These T-ALLs also have focal changes in H3K4me3 deposition at the Notch1 locus and global increases in both H3K4me1 and H3K4me3. Using a PRDM14-FLAG mouse model, we show that PRDM14 binds within an intron of Notch1 prior to leukemia development. Our data support the idea that PRDM14 binding promotes a chromatin state that allows access of the RAG recombinase complex to cryptic RAG signal sequences embedded at the Notch1 locus. Indeed, breeding into a RAG recombination-deficient background abrogates T-ALL development and prevents Notch1 deletions, while allowing for transient hematopoietic stem cell (HSC)-like pre-leukemia cell expansion. Together, our data suggest that PRDM14 expands a progenitor cell population while promoting a permissive epigenetic state for the creation of driver mutations (here, in Notch1), enabling cancer development through the misappropriation of endogenous cellular DNA recombination machinery. PMID:27106930

  5. Notch1 Activation or Loss Promotes HPV-Induced Oral Tumorigenesis.

    PubMed

    Zhong, Rong; Bao, Riyue; Faber, Pieter W; Bindokas, Vytautas P; Bechill, John; Lingen, Mark W; Spiotto, Michael T

    2015-09-15

    Viral oncogene expression is insufficient for neoplastic transformation of human cells, so human papillomavirus (HPV)-associated cancers will also rely upon mutations in cellular oncogenes and tumor suppressors. However, it has been difficult so far to distinguish incidental mutations without phenotypic impact from causal mutations that drive the development of HPV-associated cancers. In this study, we addressed this issue by conducting a functional screen for genes that facilitate the formation of HPV E6/E7-induced squamous cell cancers in mice using a transposon-mediated insertional mutagenesis protocol. Overall, we identified 39 candidate driver genes, including Notch1, which unexpectedly was scored by gain- or loss-of-function mutations that were capable of promoting squamous cell carcinogenesis. Autochthonous HPV-positive oral tumors possessing an activated Notch1 allele exhibited high rates of cell proliferation and tumor growth. Conversely, Notch1 loss could accelerate the growth of invasive tumors in a manner associated with increased expression of matrix metalloproteinases and other proinvasive genes. HPV oncogenes clearly cooperated with loss of Notch1, insofar as its haploinsufficiency accelerated tumor growth only in HPV-positive tumors. In clinical specimens of various human cancers, there was a consistent pattern of NOTCH1 expression that correlated with invasive character, in support of our observations in mice. Although Notch1 acts as a tumor suppressor in mouse skin, we found that oncogenes enabling any perturbation in Notch1 expression promoted tumor growth, albeit via distinct pathways. Our findings suggest caution in interpreting the meaning of putative driver gene mutations in cancer, and therefore therapeutic efforts to target them, given the significant contextual differences in which such mutations may arise, including in virus-associated tumors.

  6. Notch1 activation or loss promotes HPV-induced oral tumorigenesis

    PubMed Central

    Faber, Pieter W.; Bindokas, Vytautas P.; Bechill, John; Lingen, Mark W.; Spiotto, Michael T.

    2015-01-01

    Viral oncogene expression is insufficient for neoplastic transformation of human cells, so HPV-associated cancers will also rely upon mutations in cellular oncogenes and tumor suppressors. However, it has been difficult so far to distinguish incidental mutations without phenotypic impact from causal mutations that drive the development of HPV-associated cancers. In this study, we addressed this issue by conducting a functional screen for genes that facilitate the formation of HPV E6/E7-induced squamous cell cancers in mice using a transposon-mediated insertional mutagenesis protocol. Overall, we identified 39 candidate driver genes including Notch1, which unexpectedly was scored by gain or loss of function mutations that were capable of promoting squamous cell carcinogenesis. Autochthonous HPV-positive oral tumors possessing an activated Notch1 allele exhibited high rates of cell proliferation and tumor growth. Conversely, Notch1 loss could accelerate the growth of invasive tumors in manner associated with increased expression of matrix metalloproteinases and other pro-invasive genes. HPV oncogenes clearly cooperated with loss of Notch1, insofar as its haploinsufficiency accelerated tumor growth only in HPV-positive tumors. In clinical specimens of various human cancers, there was a consistent pattern of NOTCH1 expression that correlated with invasive character, in support of our observations in mice. While Notch1 acts as a tumor suppressor in mouse skin, we found that oncogenes enabling any perturbation in Notch1 expression promoted tumor growth, albeit via distinct pathways. Our findings suggest caution in interpreting the meaning of putative driver gene mutations in cancer, and therefore therapeutic efforts to target them, given the significant contextual differences in which such mutations may arise, including in virus-associated tumors. PMID:26294213

  7. Elevated TRIB2 with NOTCH1 activation in paediatric/adult T-ALL.

    PubMed

    Hannon, Maura M; Lohan, Fiona; Erbilgin, Yucel; Sayitoglu, Muge; O'Hagan, Kathleen; Mills, Ken; Ozbek, Ugur; Keeshan, Karen

    2012-09-01

    TRIB2 is a potent oncogene, elevated in a subset of human acute myeloid leukaemias (AML) with a mixed myeloid/lymphoid phenotype and NOTCH1 mutations. Although rare in AML, activating NOTCH1 mutations occur in 50% of all T cell acute lymphoblastic leukaemias (T-ALL). TRIB2 is a NOTCH1 target gene that functions in the degradation of key proteins and modulation of MAPK signalling pathways, implicated in haematopoietic cell survival and proliferation. This study showed that TRIB2 expression level is highest in the lymphoid compartment of normal haematopoietic cells, specifically in T cells. Analysis of TRIB2 expression across 16 different subtypes of human leukaemia demonstrated that TRIB2 expression was higher in ALL phenotypes versus all other phenotypes including AML, chronic lymphocytic leukaemia (CLL), myelodysplastic syndrome (MDS) and chronic myeloid leukaemia (CML). A T cell profile was distinguished by high TRIB2 expression in normal and malignant haematopoiesis. High TRIB2 expression was seen in T-ALL with normal karyotype and correlated with NOTCH signalling pathways. High TRIB2 expression correlated with NOTCH1/FBXW7 mutations in a paediatric T-ALL cohort, strongly linking NOTCH1 activation and high TRIB2 expression in paediatric T-ALL. The relationship between TRIB2 and T cell signalling pathways uniquely identifies leukaemia subtypes and will be useful in the advancement of our understanding of T cell and ALL biology. PMID:22775572

  8. Elevated TRIB2 with NOTCH1 activation in paediatric/adult T-ALL.

    PubMed

    Hannon, Maura M; Lohan, Fiona; Erbilgin, Yucel; Sayitoglu, Muge; O'Hagan, Kathleen; Mills, Ken; Ozbek, Ugur; Keeshan, Karen

    2012-09-01

    TRIB2 is a potent oncogene, elevated in a subset of human acute myeloid leukaemias (AML) with a mixed myeloid/lymphoid phenotype and NOTCH1 mutations. Although rare in AML, activating NOTCH1 mutations occur in 50% of all T cell acute lymphoblastic leukaemias (T-ALL). TRIB2 is a NOTCH1 target gene that functions in the degradation of key proteins and modulation of MAPK signalling pathways, implicated in haematopoietic cell survival and proliferation. This study showed that TRIB2 expression level is highest in the lymphoid compartment of normal haematopoietic cells, specifically in T cells. Analysis of TRIB2 expression across 16 different subtypes of human leukaemia demonstrated that TRIB2 expression was higher in ALL phenotypes versus all other phenotypes including AML, chronic lymphocytic leukaemia (CLL), myelodysplastic syndrome (MDS) and chronic myeloid leukaemia (CML). A T cell profile was distinguished by high TRIB2 expression in normal and malignant haematopoiesis. High TRIB2 expression was seen in T-ALL with normal karyotype and correlated with NOTCH signalling pathways. High TRIB2 expression correlated with NOTCH1/FBXW7 mutations in a paediatric T-ALL cohort, strongly linking NOTCH1 activation and high TRIB2 expression in paediatric T-ALL. The relationship between TRIB2 and T cell signalling pathways uniquely identifies leukaemia subtypes and will be useful in the advancement of our understanding of T cell and ALL biology.

  9. Second-generation Notch1 activity-trap mouse line (N1IP::CreHI) provides a more comprehensive map of cells experiencing Notch1 activity.

    PubMed

    Liu, Zhenyi; Brunskill, Eric; Boyle, Scott; Chen, Shuang; Turkoz, Mustafa; Guo, Yuxuan; Grant, Rachel; Kopan, Raphael

    2015-03-15

    We have previously described the creation and analysis of a Notch1 activity-trap mouse line, Notch1 intramembrane proteolysis-Cre6MT or N1IP::Cre(LO), that marked cells experiencing relatively high levels of Notch1 activation. Here, we report and characterize a second line with improved sensitivity (N1IP::Cre(HI)) to mark cells experiencing lower levels of Notch1 activation. This improvement was achieved by increasing transcript stability and by restoring the native carboxy terminus of Cre, resulting in a five- to tenfold increase in Cre activity. The magnitude of this effect probably impacts Cre activity in strains with carboxy-terminal Ert2 fusion. These two trap lines and the related line N1IP::Cre(ERT2) form a complementary mapping tool kit to identify changes in Notch1 activation patterns in vivo as the consequence of genetic or pharmaceutical intervention, and illustrate the variation in Notch1 signal strength from one tissue to the next and across developmental time. PMID:25725069

  10. Second-generation Notch1 activity-trap mouse line (N1IP::CreHI) provides a more comprehensive map of cells experiencing Notch1 activity

    PubMed Central

    Liu, Zhenyi; Brunskill, Eric; Boyle, Scott; Chen, Shuang; Turkoz, Mustafa; Guo, Yuxuan; Grant, Rachel; Kopan, Raphael

    2015-01-01

    We have previously described the creation and analysis of a Notch1 activity-trap mouse line, Notch1 intramembrane proteolysis-Cre6MT or N1IP::CreLO, that marked cells experiencing relatively high levels of Notch1 activation. Here, we report and characterize a second line with improved sensitivity (N1IP::CreHI) to mark cells experiencing lower levels of Notch1 activation. This improvement was achieved by increasing transcript stability and by restoring the native carboxy terminus of Cre, resulting in a five- to tenfold increase in Cre activity. The magnitude of this effect probably impacts Cre activity in strains with carboxy-terminal Ert2 fusion. These two trap lines and the related line N1IP::CreERT2 form a complementary mapping tool kit to identify changes in Notch1 activation patterns in vivo as the consequence of genetic or pharmaceutical intervention, and illustrate the variation in Notch1 signal strength from one tissue to the next and across developmental time. PMID:25725069

  11. Notch1 Gene Mutations Target KRAS G12D-expressing CD8+ Cells and Contribute to Their Leukemogenic Transformation*

    PubMed Central

    Kong, Guangyao; Du, Juan; Liu, Yangang; Meline, Benjamin; Chang, Yuan-I; Ranheim, Erik A.; Wang, Jinyong; Zhang, Jing

    2013-01-01

    Acute T-cell lymphoblastic leukemia/lymphoma (T-ALL) is an aggressive hematopoietic malignancy affecting both children and adults. Previous studies of T-ALL mouse models induced by different genetic mutations have provided highly diverse results on the issues of T-cell leukemia/lymphoma-initiating cells (T-LICs) and potential mechanisms contributing to T-LIC transformation. Here, we show that oncogenic Kras (Kras G12D) expressed from its endogenous locus is a potent inducer of T-ALL even in a less sensitized BALB/c background. Notch1 mutations, including exon 34 mutations and recently characterized type 1 and 2 deletions, are detected in 100% of Kras G12D-induced T-ALL tumors. Although these mutations are not detected at the pre-leukemia stage, incremental up-regulation of NOTCH1 surface expression is observed at the pre-leukemia and leukemia stages. As secondary genetic hits in the Kras G12D model, Notch1 mutations target CD8+ T-cells but not hematopoietic stem cells to further promote T-ALL progression. Pre-leukemia T-cells without detectable Notch1 mutations do not induce T-ALL in secondary recipient mice compared with T-ALL tumor cells with Notch1 mutations. We found huge variations in T-LIC frequency and immunophenotypes of cells enriched for T-LICs. Unlike Pten deficiency-induced T-ALL, oncogenic Kras-initiated T-ALL is not associated with up-regulation of the Wnt/β-catenin pathway. Our results suggest that up-regulation of NOTCH1 signaling, through either overexpression of surface NOTCH1 or acquired gain-of-function mutations, is involved in both T-ALL initiation and progression. Notch1 mutations and Kras G12D contribute cooperatively to leukemogenic transformation of normal T-cells. PMID:23673656

  12. Notch1 signaling regulates chondrogenic lineage determination through Sox9 activation.

    PubMed

    Haller, R; Schwanbeck, R; Martini, S; Bernoth, K; Kramer, J; Just, U; Rohwedel, J

    2012-03-01

    Notch signaling is involved in several cell lineage determination processes during embryonic development. Recently, we have shown that Sox9 is most likely a primary target gene of Notch1 signaling in embryonic stem cells (ESCs). By using our in vitro differentiation protocol for chondrogenesis from ESCs through embryoid bodies (EBs) together with our tamoxifen-inducible system to activate Notch1, we analyzed the function of Notch signaling and its induction of Sox9 during EB differentiation towards the chondrogenic lineage. Temporary activation of Notch1 during early stages of EB, when lineage determination occurs, was accompanied by rapid and transient Sox9 upregulation and resulted in induction of chondrogenic differentiation during later stages of EB cultivation. Using siRNA targeting Sox9, we knocked down and adjusted this early Notch1-induced Sox9 expression peak to non-induced levels, which led to reversion of Notch1-induced chondrogenic differentiation. In contrast, continuous Notch1 activation during EB cultivation resulted in complete inhibition of chondrogenic differentiation. Furthermore, a reduction and delay of cardiac differentiation observed in EBs after early Notch1 activation was not reversed by siRNA-mediated Sox9 knockdown. Our data indicate that Notch1 signaling has an important role during early stages of chondrogenic lineage determination by regulation of Sox9 expression. PMID:21869831

  13. Effects of S1 Cleavage on the Structure, Surface Export, and Signaling Activity of Human Notch1 and Notch2

    SciTech Connect

    Gordon, Wendy R.; Vardar-Ulu, Didem; L'Heureux, Sarah; Ashworth, Todd; Malecki, Michael J.; Sanchez-Irizarry, Cheryll; McArthur, Debbie G.; Histen, Gavin; Mitchell, Jennifer L.; Aster, Jon C.; Blacklow, Stephen C.

    2009-09-25

    Notch receptors are normally cleaved during maturation by a furin-like protease at an extracellular site termed S1, creating a heterodimer of non-covalently associated subunits. The S1 site lies within a key negative regulatory region (NRR) of the receptor, which contains three highly conserved Lin12/Notch repeats and a heterodimerization domain (HD) that interact to prevent premature signaling in the absence of ligands. Because the role of S1 cleavage in Notch signaling remains unresolved, we investigated the effect of S1 cleavage on the structure, surface trafficking and ligand-mediated activation of human Notch1 and Notch2, as well as on ligand-independent activation of Notch1 by mutations found in human leukemia. The X-ray structure of the Notch1 NRR after furin cleavage shows little change when compared with that of an engineered Notch1 NRR lacking the S1-cleavage loop. Likewise, NMR studies of the Notch2 HD domain show that the loop containing the S1 site can be removed or cleaved without causing a substantial change in its structure. However, Notch1 and Notch2 receptors engineered to resist S1 cleavage exhibit unexpected differences in surface delivery and signaling competence: S1-resistant Notch1 receptors exhibit decreased, but detectable, surface expression and ligand-mediated receptor activation, whereas S1-resistant Notch2 receptors are fully competent for cell surface delivery and for activation by ligands. Variable dependence on S1 cleavage also extends to T-ALL-associated NRR mutations, as common class 1 mutations display variable decrements in ligand-independent activation when introduced into furin-resistant receptors, whereas a class 2 mutation exhibits increased signaling activity. S1 cleavage has distinct effects on the surface expression of Notch1 and Notch2, but is not generally required for physiologic or pathophysiologic activation of Notch proteins. These findings are consistent with models for receptor activation in which ligand-binding or

  14. Notch1 activity in the olfactory bulb is odour-dependent and contributes to olfactory behaviour.

    PubMed

    Brai, Emanuele; Marathe, Swananda; Zentilin, Lorena; Giacca, Mauro; Nimpf, Johannes; Kretz, Robert; Scotti, Alessandra; Alberi, Lavinia

    2014-11-01

    Notch signalling plays an important role in synaptic plasticity, learning and memory functions in both Drosophila and rodents. In this paper, we report that this feature is not restricted to hippocampal networks but also involves the olfactory bulb (OB). Odour discrimination and olfactory learning in rodents are essential for survival. Notch1 expression is enriched in mitral cells of the mouse OB. These principal neurons are responsive to specific input odorants and relay the signal to the olfactory cortex. Olfactory stimulation activates a subset of mitral cells, which show an increase in Notch activity. In Notch1cKOKln mice, the loss of Notch1 in mitral cells affects the magnitude of the neuronal response to olfactory stimuli. In addition, Notch1cKOKln mice display reduced olfactory aversion to propionic acid as compared to wildtype controls. This indicates, for the first time, that Notch1 is involved in olfactory processing and may contribute to olfactory behaviour.

  15. Activation of Notch1 signaling is required for β-catenin–mediated human primary melanoma progression

    PubMed Central

    Balint, Klara; Xiao, Min; Pinnix, Chelsea C.; Soma, Akinobu; Veres, Imre; Juhasz, Istvan; Brown, Eric J.; Capobianco, Anthony J.; Herlyn, Meenhard; Liu, Zhao-Jun

    2005-01-01

    Notch is a highly conserved transmembrane receptor that determines cell fate. Notch signaling denotes cleavage of the Notch intracellular domain, its translocation to the nucleus, and subsequent activation of target gene transcription. Involvement of Notch signaling in several cancers is well known, but its role in melanoma remains poorly characterized. Here we show that the Notch1 pathway is activated in human melanoma. Blocking Notch signaling suppressed whereas constitutive activation of the Notch1 pathway enhanced primary melanoma cell growth both in vitro and in vivo yet had little effect on metastatic melanoma cells. Activation of Notch1 signaling enabled primary melanoma cells to gain metastatic capability. Furthermore, the oncogenic effect of Notch1 on primary melanoma cells was mediated by β-catenin, which was upregulated following Notch1 activation. Inhibiting β-catenin expression reversed Notch1-enhanced tumor growth and metastasis. Our data therefore suggest a β-catenin–dependent, stage-specific role for Notch1 signaling in promoting the progression of primary melanoma. PMID:16239965

  16. EZH2 expands breast stem cells through activation of NOTCH1 signaling.

    PubMed

    Gonzalez, Maria E; Moore, Heather M; Li, Xin; Toy, Kathy A; Huang, Wei; Sabel, Michael S; Kidwell, Kelley M; Kleer, Celina G

    2014-02-25

    Breast cancer is the second-leading cause of cancer-related deaths in women, but the details of how it begins remain elusive. Increasing evidence supports the association of aggressive triple-negative (TN) breast cancer with heightened expression of the Polycomb group protein Enhancer of Zeste Homolog 2 (EZH2) and increased tumor-initiating cells (TICs). However, mechanistic links between EZH2 and TICs are unclear, and direct demonstration of a tumorigenic function of EZH2 in vivo is lacking. Here, we identify an unrecognized EZH2/NOTCH1 axis that controls breast TICs in TN breast carcinomas. EZH2 overexpression increases NOTCH1 expression and signaling, and inhibition of NOTCH1 activity prevents EZH2-mediated stem cell expansion in nontumorigenic breast cells. We uncover a unique role of EZH2 in activating, rather than repressing, NOTCH1 signaling through binding to the NOTCH1 promoter in TN breast cancer cells. EZH2 binding is independent of its catalytic histone H3 lysine 27 methyltransferase activity and of the Polycomb Repressive Complex 2 but corresponds instead to transcriptional activation marks. In vivo, EZH2 knockdown decreases the onset and volume of xenografts derived from TN breast TICs. Conversely, transgenic EZH2 overexpression accelerates mammary tumor initiation and increases NOTCH1 activation in mouse mammary tumor virus-neu mice. Consonant with these findings, in clinical samples, high levels of EZH2 are significantly associated with activated NOTCH1 protein and increased TICs in TN invasive carcinomas. These data reveal a functional and mechanistic link between EZH2 levels, NOTCH1 signaling activation, and TICs, and provide previously unidentified evidence that EZH2 enhances breast cancer initiation. PMID:24516139

  17. EZH2 expands breast stem cells through activation of NOTCH1 signaling

    PubMed Central

    Gonzalez, Maria E.; Moore, Heather M.; Li, Xin; Toy, Kathy A.; Huang, Wei; Sabel, Michael S.; Kidwell, Kelley M.; Kleer, Celina G.

    2014-01-01

    Breast cancer is the second-leading cause of cancer-related deaths in women, but the details of how it begins remain elusive. Increasing evidence supports the association of aggressive triple-negative (TN) breast cancer with heightened expression of the Polycomb group protein Enhancer of Zeste Homolog 2 (EZH2) and increased tumor-initiating cells (TICs). However, mechanistic links between EZH2 and TICs are unclear, and direct demonstration of a tumorigenic function of EZH2 in vivo is lacking. Here, we identify an unrecognized EZH2/NOTCH1 axis that controls breast TICs in TN breast carcinomas. EZH2 overexpression increases NOTCH1 expression and signaling, and inhibition of NOTCH1 activity prevents EZH2-mediated stem cell expansion in nontumorigenic breast cells. We uncover a unique role of EZH2 in activating, rather than repressing, NOTCH1 signaling through binding to the NOTCH1 promoter in TN breast cancer cells. EZH2 binding is independent of its catalytic histone H3 lysine 27 methyltransferase activity and of the Polycomb Repressive Complex 2 but corresponds instead to transcriptional activation marks. In vivo, EZH2 knockdown decreases the onset and volume of xenografts derived from TN breast TICs. Conversely, transgenic EZH2 overexpression accelerates mammary tumor initiation and increases NOTCH1 activation in mouse mammary tumor virus-neu mice. Consonant with these findings, in clinical samples, high levels of EZH2 are significantly associated with activated NOTCH1 protein and increased TICs in TN invasive carcinomas. These data reveal a functional and mechanistic link between EZH2 levels, NOTCH1 signaling activation, and TICs, and provide previously unidentified evidence that EZH2 enhances breast cancer initiation. PMID:24516139

  18. Epigenetic regulation of Delta-Like1 controls Notch1 activation in gastric cancer.

    PubMed

    Piazzi, Giulia; Fini, Lucia; Selgrad, Michael; Garcia, Melissa; Daoud, Yahya; Wex, Thomas; Malfertheiner, Peter; Gasbarrini, Antonio; Romano, Marco; Meyer, Richard L; Genta, Robert M; Fox, James G; Boland, C Richard; Bazzoli, Franco; Ricciardiello, Luigi

    2011-12-01

    The Notch signaling pathway drives proliferation, differentiation, apoptosis, cell fate, and maintenance of stem cells in several tissues. Aberrant activation of Notch signaling has been described in several tumours and in gastric cancer (GC), activated Notch1 has been associated with de-differentiation of lineage-committed stomach cells into stem progenitors and GC progression. However, the specific role of the Notch1 ligand DLL1 in GC has not yet been elucidated. To assess the role of DLL1 in GC cancer, the expression of Notch1 and its ligands DLL1 and Jagged1, was analyzed in 8 gastric cancer cell lines (KATOIII, SNU601, SNU719, AGS, SNU16, MKN1, MKN45, TMK1). DLL1 expression was absent in KATOIII, SNU601, SNU719 and AGS. The lack of DLL1 expression in these cells was associated with promoter hypermethylation and 5-aza-2'dC caused up-regulation of DLL1. The increase in DLL1 expression was associated with activation of Notch1 signalling, with an increase in cleaved Notch1 intracellular domain (NICD) and Hes1, and down-regulation in Hath1. Concordantly, Notch1 signalling was activated with the overexpression of DLL1. Moreover, Notch1 signalling together with DLL1 methylation were evaluated in samples from 52 GC patients and 21 healthy control as well as in INS-GAS mice infected with H. pylori and randomly treated with eradication therapy. In GC patients, we found a correlation between DLL1 and Hes1 expression, while DLL1 methylation and Hath1 expression were associated with the diffuse and mixed type of gastric cancer. Finally, none of the samples from INS-GAS mice infected with H. pylori, a model of intestinal-type gastric tumorigenesis, showed promoter methylation of DLL1. This study shows that Notch1 activity in gastric cancer is controlled by the epigenetic silencing of the ligand DLL1, and that Notch1 inhibition is associated with the diffuse type of gastric cancer. PMID:22249198

  19. Somatic Mutations and Genetic Variants of NOTCH1 in Head and Neck Squamous Cell Carcinoma Occurrence and Development

    PubMed Central

    Liu, Yu-Fan; Chiang, Shang-Lun; Lin, Chien-Yu; Chang, Jan-Gowth; Chung, Chia-Min; Ko, Albert Min-Shan; Lin, You-Zhe; Lee, Chien-Hung; Lee, Ka-Wo; Chen, Mu-Kuan; Hua, Chun-Hung; Tsai, Ming-Hsui; Chen, Yuan-Chien; Ko, Ying-Chin

    2016-01-01

    A number of genetic variants have been associated with cancer occurrence, however it may be the acquired somatic mutations (SMs) that drive cancer development. This study investigates the potential SMs and related genetic variants associated with the occurrence and development of head and neck squamous cell carcinoma (HNSCC). We identified several SMs in NOTCH1 from whole-exome sequencing and validated them in a 13-year cohort of 128 HNSCC patients using a high-resolution melting analysis and resequencing. Patients who have NOTCH1 SMs show higher 5-year relapse-free recurrence (P = 0.0013) and lower survival proportion (P = 0.0447) when the risk-associated SMs were analysed by Cox proportional hazard models. Interestingly, the NOTCH1 gene rs139994842 that shares linkage with SMs is associated with HNSCC risk (OR = 3.46), increasing when SMs in NOTCH1 are involved (OR = 7.74), and furthermore when there are SMs in conjunction to betel quid chewing (OR = 32.11), which is a related independent environmental risk factor after adjusting for substances use (alcohol, betel quid, cigarettes) and age. The findings indicate that betel quid chewing is highly associated with NOTCH1 SMs (especially with changes in EGF-like domains), and that rs139994842 may potentially serve as an early predictive and prognostic biomarker for the occurrence and development of HNSCC. PMID:27035284

  20. Notch1 Pathway Activity Determines the Regulatory Role of Cancer-Associated Fibroblasts in Melanoma Growth and Invasion

    PubMed Central

    Shao, Hongwei; Kong, Ranran; Ferrari, Massimiliano L.; Radtke, Freddy; Capobianco, Anthony J.; Liu, Zhao-Jun

    2015-01-01

    Cancer-associated fibroblasts (CAF) play a crucial role in regulating cancer progression, yet the molecular determinant that governs the tumor regulatory role of CAF remains unknown. Using a mouse melanoma model in which exogenous melanoma cells were grafted on the skin of two lines of mice where the genetic activation or inactivation of Notch1 signaling specifically occurs in natural host stromal fibroblasts, we demonstrated that Notch1 pathway activity could determine the tumor-promoting or tumor-suppressing phenotype in CAF. CAF carrying elevated Notch1 activity significantly inhibited melanoma growth and invasion, while those with a null Notch1 promoted melanoma invasion. These findings identify the Notch1 pathway as a molecular determinant that controls the regulatory role of CAF in melanoma skin growth and invasion, unveiling Notch1 signaling as a potential therapeutic target for melanoma and potentially other solid tumors. PMID:26562315

  1. Notch1 regulated autophagy controls survival and suppressor activity of activated murine T-regulatory cells

    PubMed Central

    Marcel, Nimi; Sarin, Apurva

    2016-01-01

    Cell survival is one of several processes regulated by the Notch pathway in mammalian cells. Here we report functional outcomes of non-nuclear Notch signaling to activate autophagy, a conserved cellular response to nutrient stress, regulating survival in murine natural T-regulatory cells (Tregs), an immune subset controlling tolerance and inflammation. Induction of autophagy required ligand-dependent, Notch intracellular domain (NIC) activity, which controlled mitochondrial organization and survival of activated Tregs. Consistently, NIC immune-precipitated Beclin and Atg14, constituents of the autophagy initiation complex. Further, ectopic expression of an effector of autophagy (Atg3) or recombinant NIC tagged to a nuclear export signal (NIC-NES), restored autophagy and suppressor function in Notch1-/- Tregs. Furthermore, Notch1 deficiency in the Treg lineage resulted in immune hyperactivity, implicating Notch activity in Treg homeostasis. Notch1 integration with autophagy, revealed in these experiments, holds implications for Notch regulated cell-fate decisions governing differentiation. DOI: http://dx.doi.org/10.7554/eLife.14023.001 PMID:27267497

  2. SF3B1 mutations correlated to cytogenetics and mutations in NOTCH1, FBXW7, MYD88, XPO1 and TP53 in 1160 untreated CLL patients.

    PubMed

    Jeromin, S; Weissmann, S; Haferlach, C; Dicker, F; Bayer, K; Grossmann, V; Alpermann, T; Roller, A; Kohlmann, A; Haferlach, T; Kern, W; Schnittger, S

    2014-01-01

    We analyzed a large cohort of 1160 untreated CLL patients for novel genetic markers (SF3B1, NOTCH1, FBXW7, MYD88, XPO1) in the context of molecular, immunophenotypic and cytogenetic data. NOTCH1 mutations (mut) (12.3%), SF3B1mut (9.0%) and TP53mut (7.1%) were more frequent than XPO1mut (3.4%), FBXW7mut (2.5%) and MYD88mut (1.5%). SF3B1mut, NOTCH1mut, TP53mut and XPO1mut were highly correlated to unmutated, whereas MYD88mut were associated with mutated IGHV status. Associations of diverse cytogenetic aberrations and mutations emerged: (1) SF3B1mut with del(11q), (2) NOTCH1mut and FBXW7mut with trisomy 12 and nearly exclusiveness of SF3B1mut, (3) MYD88mut with del(13q) sole and low frequencies of SF3B1mut, NOTCH1mut and FBXW7mut. In patients with normal karyotype only SF3B1mut were frequent, whereas NOTCH1mut rarely occurred. An adverse prognostic impact on time to treatment (TTT) and overall survival (OS) was observed for SF3B1mut, NOTCH1mut and TP53 disruption. In multivariate analyses SF3B1mut, IGHV mutational status and del(11q) were the only independent genetic markers for TTT, whereas for OS SF3B1mut, IGHV mutational status and TP53 disruption presented with significant impact. Finally, our data suggest that analysis of gene mutations refines the risk stratification of cytogenetic prognostic subgroups and confirms data of a recently proposed model integrating molecular and cytogenetic data. PMID:24113472

  3. Activation of Notch1 synergizes with multiple pathways in promoting castration-resistant prostate cancer

    PubMed Central

    Stoyanova, Tanya; Riedinger, Mireille; Lin, Shu; Faltermeier, Claire M.; Smith, Bryan A.; Zhang, Kelvin X.; Going, Catherine C.; Goldstein, Andrew S.; Lee, John K.; Drake, Justin M.; Rice, Meghan A.; Hsu, En-Chi; Nowroozizadeh, Behdokht; Castor, Brandon; Orellana, Sandra Y.; Blum, Steven M.; Cheng, Donghui; Pienta, Kenneth J.; Reiter, Robert E.; Pitteri, Sharon J.; Huang, Jiaoti; Witte, Owen N.

    2016-01-01

    Metastatic castration-resistant prostate cancer (CRPC) is the primary cause of prostate cancer-specific mortality. Defining new mechanisms that can predict recurrence and drive lethal CRPC is critical. Here, we demonstrate that localized high-risk prostate cancer and metastatic CRPC, but not benign prostate tissues or low/intermediate-risk prostate cancer, express high levels of nuclear Notch homolog 1, translocation-associated (Notch1) receptor intracellular domain. Chronic activation of Notch1 synergizes with multiple oncogenic pathways altered in early disease to promote the development of prostate adenocarcinoma. These tumors display features of epithelial-to-mesenchymal transition, a cellular state associated with increased tumor aggressiveness. Consistent with its activation in clinical CRPC, tumors driven by Notch1 intracellular domain in combination with multiple pathways altered in prostate cancer are metastatic and resistant to androgen deprivation. Our study provides functional evidence that the Notch1 signaling axis synergizes with alternative pathways in promoting metastatic CRPC and may represent a new therapeutic target for advanced prostate cancer. PMID:27694579

  4. Constitutively active Notch1 induces growth arrest of HPV-positive cervical cancer cells via separate signaling pathways

    SciTech Connect

    Talora, Claudio; Cialfi, Samantha; Segatto, Oreste; Morrone, Stefania; Kim Choi, John; Frati, Luigi; Paolo Dotto, Gian; Gulino, Alberto; Screpanti, Isabella . E-mail: isabella.screpanti@uniroma1.it

    2005-05-01

    Notch signaling plays a key role in cell-fate determination and differentiation in different organisms and cell types. Several reports suggest that Notch signaling may be involved in neoplastic transformation. However, in primary keratinocytes, Notch1 can function as a tumor suppressor. Similarly, in HPV-positive cervical cancer cells, constitutively active Notch1 signaling was found to cause growth suppression. Activated Notch1 in these cells represses viral E6/E7 expression through AP-1 down-modulation, resulting in increased p53 expression and a block of pRb hyperphosphorylation. Here we show that in cervical cancer cell lines in which Notch1 ability to repress AP-1 activity is impaired, Notch1-enforced expression elicits an alternative pathway leading to growth arrest. Indeed, activated Notch1 signaling suppresses activity of the helix-loop-helix transcription factor E47, via ERK1/2 activation, resulting in inhibition of cell cycle progression. Moreover, we found that RBP-J{kappa}-dependent Notch signaling is specifically repressed in cervical cancer cells and this repression could provide one such mechanism that needs to be activated for cervical carcinogenesis. Finally, we show that inhibition of endogenous Notch1 signaling, although results in a proliferative advantage, sensitizes cervical cancer cell lines to drug-induced apoptosis. Together, our results provide novel molecular insights into Notch1-dependent growth inhibitory effects, counteracting the transforming potential of HPV.

  5. Cellular senescence checkpoint function determines differential Notch1-dependent oncogenic and tumor suppressor activities

    PubMed Central

    Kagawa, Shingo; Natsuizaka, Mitsuteru; Whelan, Kelly A.; Facompre, Nicole; Naganuma, Seiji; Ohashi, Shinya; Kinugasa, Hideaki; Egloff, Ann Marie; Basu, Devraj; Gimotty, Phyllis A.; Klein-Szanto, Andres J; Bass, Adam; Wong, Kwok-Kin; Diehl, J. Alan; Rustgi, Anil K.; Nakagawa, Hiroshi

    2014-01-01

    Notch activity regulates tumor biology in a context-dependent and complex manner. Notch may act as an oncogene or a tumor suppressor gene even within the same tumor type. Recently, Notch signaling has been implicated in cellular senescence. Yet, it remains unclear as to how cellular senescence checkpoint functions may interact with Notch-mediated oncogenic and tumor suppressor activities. Herein, we used genetically engineered human esophageal keratinocytes and esophageal squamous cell carcinoma cells to delineate the functional consequences of Notch activation and inhibition along with pharmacological intervention and RNA interference (RNAi) experiments. When expressed in a tetracycline-inducible manner, the ectopically expressed activated form of Notch1 (ICN1) displayed oncogene-like characteristics inducing cellular senescence corroborated by the induction of G0/G1 cell-cycle arrest, Rb dephosphorylation, flat and enlarged cell morphology and senescence-associated β-galactosidase activity. Notch-induced senescence involves canonical CSL/RBPJ-dependent transcriptional activity and the p16INK4A-Rb pathway. Loss of p16INK4A or the presence of human papilloma virus (HPV) E6/E7 oncogene products not only prevented ICN1 from inducing senescence, but permitted ICN1 to facilitate anchorage-independent colony formation and xenograft tumor growth with increased cell proliferation and reduced squamous-cell differentiation. Moreover, Notch1 appears to mediate replicative senescence as well as TGF-β-induced cellular senescence in non-transformed cells and that HPV E6/E7 targets Notch1 for inactivation to prevent senescence, revealing a tumor suppressor attribute of endogenous Notch1. In aggregate, cellular senescence checkpoint functions may influence dichotomous Notch activities in the neoplastic context. PMID:24931169

  6. Cellular senescence checkpoint function determines differential Notch1-dependent oncogenic and tumor-suppressor activities.

    PubMed

    Kagawa, S; Natsuizaka, M; Whelan, K A; Facompre, N; Naganuma, S; Ohashi, S; Kinugasa, H; Egloff, A M; Basu, D; Gimotty, P A; Klein-Szanto, A J; Bass, A J; Wong, K-K; Diehl, J A; Rustgi, A K; Nakagawa, H

    2015-04-30

    Notch activity regulates tumor biology in a context-dependent and complex manner. Notch may act as an oncogene or a tumor-suppressor gene even within the same tumor type. Recently, Notch signaling has been implicated in cellular senescence. Yet, it remains unclear as to how cellular senescence checkpoint functions may interact with Notch-mediated oncogenic and tumor-suppressor activities. Herein, we used genetically engineered human esophageal keratinocytes and esophageal squamous cell carcinoma cells to delineate the functional consequences of Notch activation and inhibition along with pharmacological intervention and RNA interference experiments. When expressed in a tetracycline-inducible manner, the ectopically expressed activated form of Notch1 (ICN1) displayed oncogene-like characteristics inducing cellular senescence corroborated by the induction of G0/G1 cell-cycle arrest, Rb dephosphorylation, flat and enlarged cell morphology and senescence-associated β-galactosidase activity. Notch-induced senescence involves canonical CSL/RBPJ-dependent transcriptional activity and the p16(INK4A)-Rb pathway. Loss of p16(INK4A) or the presence of human papilloma virus (HPV) E6/E7 oncogene products not only prevented ICN1 from inducing senescence but permitted ICN1 to facilitate anchorage-independent colony formation and xenograft tumor growth with increased cell proliferation and reduced squamous-cell differentiation. Moreover, Notch1 appears to mediate replicative senescence as well as transforming growth factor-β-induced cellular senescence in non-transformed cells and that HPV E6/E7 targets Notch1 for inactivation to prevent senescence, revealing a tumor-suppressor attribute of endogenous Notch1. In aggregate, cellular senescence checkpoint functions may influence dichotomous Notch activities in the neoplastic context.

  7. Activation of Notch1 signaling in stromal fibroblasts inhibits melanoma growth by upregulating WISP-1.

    PubMed

    Shao, H; Cai, L; Grichnik, J M; Livingstone, A S; Velazquez, O C; Liu, Z-J

    2011-10-20

    The tumor microenvironment is emerging as an important target for cancer therapy. Fibroblasts (Fbs) within the tumor stroma are critically involved in promoting tumor growth and angiogenesis through secretion of soluble factors, synthesis of extracellular matrix and direct cell-cell interaction. In this work, we aim to alter the biological activity of stromal Fbs by modulating the Notch1 signaling pathway. We show that Fbs engineered to constitutively activate the Notch1 pathway significantly inhibit melanoma growth and tumor angiogenesis. We determine that the inhibitory effect of 'Notch-engineered' Fbs is mediated by increased secretion of Wnt-induced secreted protein-1 (WISP-1) as the effects of Notch1 activation in Fbs are reversed by shRNA-mediated blockade of WISP-1. When 'Notch-engineered' Fbs are co-grafted with melanoma cells in SCID mice, shRNA-mediated blockade of WISP-1 reverses the tumor-suppressive phenotype of the 'Notch-engineered' Fbs, significantly increases melanoma growth and tumor angiogenesis. Consistent with these findings, supplement of recombinant WISP-1 protein inhibits melanoma cell growth in vitro. In addition, WISP-1 is modestly expressed in melanoma-activated Fbs but highly expressed in inactivated Fbs. Evaluation of human melanoma skin biopsies indicates that expression of WISP-1 is significantly lower in melanoma nests and surrounding areas filled with infiltrated immune cells than in the adjacent dermis unaffected by the melanoma. Overall, our study shows that constitutive activation of the Notch1 pathway confers Fbs with a suppressive phenotype to melanoma growth, partially through WISP-1. Thus, targeting tumor stromal Fbs by activating Notch signaling and/or increasing WISP-1 may represent a novel therapeutic approach to combat melanoma.

  8. Tetrandrine induces autophagy and differentiation by activating ROS and Notch1 signaling in leukemia cells.

    PubMed

    Liu, Ting; Men, Qiuxu; Wu, Guixian; Yu, Chunrong; Huang, Zan; Liu, Xin; Li, Wenhua

    2015-04-10

    All-trans retinoic acid (ATRA) is a differentiating agent for the treatment of acute promyelocytic leukemia (APL). However, the therapeutic efficacy of ATRA has limitations. Tetrandrine is a traditional Chinese medicinal herb extract with antitumor effects. In this study, we investigated the effects of tetrandrine on human PML-RARα-positive acute promyelocytic leukemia cells. Tetrandrine inhibited tumors in vivo. It induced autophagy and differentiation by triggering ROS generation and activating Notch1 signaling. Tetrandrine induced autophagy and differentiation in M5 type patient primary leukemia cells. The in vivo results indicated that low concentrations of tetrandrine inhibited leukemia cells proliferation and induced autophagy and then facilitated their differentiation, by activating ROS and Notch1 signaling. We suggest that tetrandrine is a potential agent for the treatment of APL by inducing differentiation of leukemia cells. PMID:25797266

  9. Activation of an endothelial Notch1-Jagged1 circuit induces VCAM1 expression, an effect amplified by interleukin-1β.

    PubMed

    Verginelli, Federica; Adesso, Laura; Limon, Isabelle; Alisi, Anna; Gueguen, Marie; Panera, Nadia; Giorda, Ezio; Raimondi, Lavinia; Ciarapica, Roberta; Campese, Antonio F; Screpanti, Isabella; Stifani, Stefano; Kitajewski, Jan; Miele, Lucio; Rota, Rossella; Locatelli, Franco

    2015-12-22

    The Notch1 and Notch4 signaling pathways regulate endothelial cell homeostasis. Inflammatory cytokines induce the expression of endothelial adhesion molecules, including VCAM1, partly by downregulating Notch4 signaling. We investigated the role of endothelial Notch1 in this IL-1β-mediated process. Brief treatment with IL-1β upregulated endothelial VCAM1 and Notch ligand Jagged1. IL-1β decreased Notch1 mRNA levels, but levels of the active Notch1ICD protein remained constant. IL-1β-mediated VCAM1 induction was downregulated in endothelial cells subjected to pretreatment with a pharmacological inhibitor of the γ-secretase, which activates Notch receptors, producing NotchICD. It was also downregulated in cells in which Notch1 and/or Jagged1 were silenced.Conversely, the forced expression of Notch1ICD in naïve endothelial cells upregulated VCAM1 per se and amplified IL-1β-mediated VCAM1 induction. Jagged1 levels increased and Notch4 signaling was downregulated in parallel. Finally, Notch1ICD and Jagged1 expression was upregulated in the endothelium of the liver in a model of chronic liver inflammation.In conclusion, we describe here a cell-autonomous, pro-inflammatory endothelial Notch1-Jagged1 circuit (i) triggering the expression of VCAM1 even in the absence of inflammatory cytokines and (ii) enhancing the effects of IL-1β. Thus, IL-1β regulates Notch1 and Notch4 activity in opposite directions, consistent with a selective targeting of Notch1 in inflamed endothelium.

  10. Activation of an endothelial Notch1-Jagged1 circuit induces VCAM1 expression, an effect amplified by interleukin-1β.

    PubMed

    Verginelli, Federica; Adesso, Laura; Limon, Isabelle; Alisi, Anna; Gueguen, Marie; Panera, Nadia; Giorda, Ezio; Raimondi, Lavinia; Ciarapica, Roberta; Campese, Antonio F; Screpanti, Isabella; Stifani, Stefano; Kitajewski, Jan; Miele, Lucio; Rota, Rossella; Locatelli, Franco

    2015-12-22

    The Notch1 and Notch4 signaling pathways regulate endothelial cell homeostasis. Inflammatory cytokines induce the expression of endothelial adhesion molecules, including VCAM1, partly by downregulating Notch4 signaling. We investigated the role of endothelial Notch1 in this IL-1β-mediated process. Brief treatment with IL-1β upregulated endothelial VCAM1 and Notch ligand Jagged1. IL-1β decreased Notch1 mRNA levels, but levels of the active Notch1ICD protein remained constant. IL-1β-mediated VCAM1 induction was downregulated in endothelial cells subjected to pretreatment with a pharmacological inhibitor of the γ-secretase, which activates Notch receptors, producing NotchICD. It was also downregulated in cells in which Notch1 and/or Jagged1 were silenced.Conversely, the forced expression of Notch1ICD in naïve endothelial cells upregulated VCAM1 per se and amplified IL-1β-mediated VCAM1 induction. Jagged1 levels increased and Notch4 signaling was downregulated in parallel. Finally, Notch1ICD and Jagged1 expression was upregulated in the endothelium of the liver in a model of chronic liver inflammation.In conclusion, we describe here a cell-autonomous, pro-inflammatory endothelial Notch1-Jagged1 circuit (i) triggering the expression of VCAM1 even in the absence of inflammatory cytokines and (ii) enhancing the effects of IL-1β. Thus, IL-1β regulates Notch1 and Notch4 activity in opposite directions, consistent with a selective targeting of Notch1 in inflamed endothelium. PMID:26646450

  11. Co-existence of PHF6 and NOTCH1 mutations in adult T-cell acute lymphoblastic leukemia

    PubMed Central

    LI, MIN; XIAO, LICHAN; XU, JINGYAN; ZHANG, RUN; GUO, JINGJING; OLSON, JUSTIN; WU, YUJIE; LI, JIANYONG; SONG, CHUNHUA; GE, ZHENG

    2016-01-01

    T-cell acute lymphoblastic leukemia (T-ALL) results from the collaboration of multiple genetic abnormalities in the transformation of T-cell progenitors. Plant homeodomain finger protein 6 (PHF6) has recently been established as a key tumor suppressor, which is mutated in T-ALL; however, the clinical significance of PHF6 mutations has not been fully determined in adult T-ALL. In the present study, amplification of the PHF6 exons was performed, followed by DNA sequencing to identify the genomic mutations and examine the expression of PHF6 in adult patients with T-ALL. The correlation between PHF6 mutations and clinical features was also analyzed using a χ2 test, and between PHF6 mutations and survival curve using the Kaplan-Meier methods. PHF6 mutations were detected in 27.1% of the Chinese adults with T-ALL (16/59), 10 of which were found to be novel mutations. A significantly lower expression level of PHF6 was observed in T-ALL patients with PHF6 mutations compared with those without mutations. Of the observed mutations in PHF6, 6/16 were frame-shift mutations, indicating a PHF6 dysfunction in those patients. Of note, PHF6 mutations were found to be significantly associated with older age, lower hemoglobin levels, higher frequency of CD13 positivity and higher incidence of splenomegaly or lymphadenopathy. Furthermore, PHF6 mutations were found to be significantly correlated with Notch homolog 1, translocation-associated (Drosophila) (NOTCH1) mutations. The patients with T-ALL with co-existence of the two mutations had a significantly shorter event-free survival and a poor prognosis. The present results indicated that PHF6 is inactivated in adult T-ALL, due to its low expression and mutations. The present data indicated the synergistic effect of PHF6 and NOTCH1 mutations, as well as their co-existence, on the oncogenesis of adult T-ALL, and their potential as a prognostic marker for the disease. PMID:27347093

  12. Intracellular-activated Notch1 can reactivate Kaposi's sarcoma-associated herpesvirus from latency

    SciTech Connect

    Lan, Ke; Murakami, Masanao; Choudhuri, Tathagata; Kuppers, Daniel A.; Robertson, Erle S. . E-mail: erle@mail.med.upenn.edu

    2006-08-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) establishes a predominantly latent infection in the infected host. Importantly, during latency, only a small number of viral encoded genes are expressed. This viral gene expression pattern contributes to the establishment of long-term infection as well as the ability of the virus to evade the immune system. Previous studies have been shown that the replication and transcription activator (RTA) encoded by ORF50 activates it downstream genes and initiates viral lytic reactivation through functional interaction with RBP-J{kappa}, the major downstream effector of the Notch signaling pathway. This indicates that RTA can usurp the conserved Notch signaling pathway and mimic the activities of intracellular Notch1 to modulate gene expression. In this report, we show that the activated intracellular domain of Notch1 (ICN) is aberrantly accumulated in KSHV latently infected pleural effusion lymphoma (PEL) cells. ICN activated the RTA promoter in a dose-dependent manner, and forced expression of ICN in latently infected KSHV-positive cells initiated full blown lytic replication with the production of infectious viral progeny. However, latency-associated nuclear antigen (LANA) which is predominantly expressed during latency can specifically down-modulate ICN-mediated transactivation of RTA and so control KSHV for lytic reactivation. These results demonstrate that LANA can inhibit viral lytic replication by antagonizing ICN function and suggest that LANA is a critical component of the regulatory control mechanism for switching between viral latent and lytic replication by directly interacting with effectors of the conserved cellular Notch1 pathway.

  13. Trisomy 12 chronic lymphocytic leukemia cells exhibit upregulation of integrin signaling that is modulated by NOTCH1 mutations

    PubMed Central

    Riches, John C.; O’Donovan, Conor J.; Kingdon, Sarah J.; McClanahan, Fabienne; Clear, Andrew J.; Neuberg, Donna S.; Werner, Lillian; Croce, Carlo M.; Ramsay, Alan G.; Rassenti, Laura Z.; Kipps, Thomas J.; Gribben, John G.

    2014-01-01

    The leukocyte adhesion cascade is important in chronic lymphocytic leukemia (CLL), as it controls migration of malignant cells into the pro-survival lymph node microenvironment. Circulating trisomy 12 CLL cells have increased expression of the integrins CD11a and CD49d, as well as CD38, but the tissue expression of these and other molecules, and the functional and clinical sequelae of these changes have not been described. Here, we demonstrate that circulating trisomy 12 CLL cells also have increased expression of the integrins CD11b, CD18, CD29, and ITGB7, and the adhesion molecule CD323. Notably, there was reduced expression of CD11a, CD11b, and CD18 in trisomy 12 cases with NOTCH1 mutations compared with wild type. Trisomy 12 cells also exhibit upregulation of intracellular integrin signaling molecules CALDAG-GEFI, RAP1B, and Ras-related protein ligand, resulting in enhanced very late antigen-4 [VLA-4] directed adhesion and motility. CD38 expression in CLL has prognostic significance, but the increased CD38 expression in trisomy 12 CLL cells must be taken into account in this subgroup, and the threshold of CD38 positivity should be raised to 40% for this marker to retain its prognostic value. In conclusion, trisomy 12 CLL cells exhibit functional upregulation of integrin signaling, with β2-integrin expression being modulated by NOTCH1 mutation status. PMID:24829201

  14. In vivo consequences of deleting EGF repeats 8–12 including the ligand binding domain of mouse Notch1

    PubMed Central

    Ge, Changhui; Liu, Tongyi; Hou, Xinghua; Stanley, Pamela

    2008-01-01

    Background Notch signaling is highly conserved in the metazoa and is critical for many cell fate decisions. Notch activation occurs following ligand binding to Notch extracellular domain. In vitro binding assays have identified epidermal growth factor (EGF) repeats 11 and 12 as the ligand binding domain of Drosophila Notch. Here we show that an internal deletion in mouse Notch1 of EGF repeats 8–12, including the putative ligand binding domain (lbd), is an inactivating mutation in vivo. We also show that maternal and zygotic Notch1lbd/lbd mutant embryos develop through gastrulation to mid-gestation. Results Notch1lbd/lbd embryos died at mid-gestation with a phenotype indistinguishable from Notch1 null mutants. In embryonic stem (ES) cells, Notch1lbd was expressed on the cell surface at levels equivalent to wild type Notch1, but Delta1 binding was reduced to the same level as in Notch1 null cells. In an ES cell co-culture assay, Notch signaling induced by Jagged1 or Delta1 was reduced to a similar level in Notch1lbdand Notch1 null cells. However, the Notch1lbd/lbd allele was expressed similarly to wild type Notch1 in Notch1lbd/lbd ES cells and embryos at E8.75, indicating that Notch1 signaling is not essential for the Notch1 gene to be expressed. In addition, maternal and zygotic Notch1 mutant blastocysts developed through gastrulation. Conclusion Mouse Notch1 lacking the ligand binding domain is expressed at the cell surface but does not signal in response to the canonical Notch ligands Delta1 and Jagged1. Homozygous Notch1lbd/lbd mutant embryos die at ~E10 similar to Notch1 null embryos. While Notch1 is expressed in oocytes and blastocysts, Notch1 signaling via canonical ligands is dispensable during oogenesis, blastogenesis, implantation and gastrulation. PMID:18445292

  15. Requirement of HDAC6 for activation of Notch1 by TGF-β1

    PubMed Central

    Deskin, Brian; Lasky, Joseph; Zhuang, Yan; Shan, Bin

    2016-01-01

    TGF-β1 is enriched in the tumor microenvironment and acts as a key inducer of epithelial to mesenchymal transition (EMT) in lung cancer. The NOTCH signaling pathway is conserved across species and is an essential pathway for development, cell differentiation, and cancer biology. Dysregulation of Notch signaling is a common feature of non-small cell lung cancer (NSCLC) and is correlated with poor prognosis. Crosstalk exists between the NOTCH and TGF-β signaling pathways in EMT. Herein we report that histone deacetylase 6 (HDAC6) modulates TGF-β1-mediated activation of the Notch pathway. HDAC6, a primarily cytoplasmic deacetylase, mediates TGF-β1-induced EMT in human lung cancer cells. Inhibition of HDAC6 with a small molecule inhibitor, namely tubacin or with siRNA attenuated TGF-β1-induced Notch-1 signaling. We show that TGFβ-1-induced EMT is accompanied by rapid HDAC6-dependent deacetylation of heat shock protein 90 (HSP90). Consistently, inhibition of HSP90 with its small molecule inhibitor 17AAG attenuated expression of TGF-β1-induced Notch-1 target genes, HEY-1 and HES-1. These findings reveal a novel function of HDAC6 in EMT via mediating the TGF-β-Notch signaling cascade, and support HDAC6 as a key regulator of TGFβ-induced EMT in NSCLC. This work suggests that HDAC6 may be an attractive therapeutic target against tumor progression and metastasis. PMID:27499032

  16. Clinical impact of clonal and subclonal TP53, SF3B1, BIRC3, NOTCH1, and ATM mutations in chronic lymphocytic leukemia

    PubMed Central

    Nadeu, Ferran; Delgado, Julio; Royo, Cristina; Baumann, Tycho; Stankovic, Tatjana; Pinyol, Magda; Jares, Pedro; Navarro, Alba; Martín-García, David; Beà, Sílvia; Salaverria, Itziar; Oldreive, Ceri; Aymerich, Marta; Suárez-Cisneros, Helena; Rozman, Maria; Villamor, Neus; Colomer, Dolors; López-Guillermo, Armando; González, Marcos; Alcoceba, Miguel; Terol, Maria José; Colado, Enrique; Puente, Xose S.; López-Otín, Carlos; Enjuanes, Anna

    2016-01-01

    Genomic studies have revealed the complex clonal heterogeneity of chronic lymphocytic leukemia (CLL). The acquisition and selection of genomic aberrations may be critical to understanding the progression of this disease. In this study, we have extensively characterized the mutational status of TP53, SF3B1, BIRC3, NOTCH1, and ATM in 406 untreated CLL cases by ultra-deep next-generation sequencing, which detected subclonal mutations down to 0.3% allele frequency. Clonal dynamics were examined in longitudinal samples of 48 CLL patients. We identified a high proportion of subclonal mutations, isolated or associated with clonal aberrations. TP53 mutations were present in 10.6% of patients (6.4% clonal, 4.2% subclonal), ATM mutations in 11.1% (7.8% clonal, 1.3% subclonal, 2% germ line mutations considered pathogenic), SF3B1 mutations in 12.6% (7.4% clonal, 5.2% subclonal), NOTCH1 mutations in 21.8% (14.2% clonal, 7.6% subclonal), and BIRC3 mutations in 4.2% (2% clonal, 2.2% subclonal). ATM mutations, clonal SF3B1, and both clonal and subclonal NOTCH1 mutations predicted for shorter time to first treatment irrespective of the immunoglobulin heavy-chain variable-region gene (IGHV) mutational status. Clonal and subclonal TP53 and clonal NOTCH1 mutations predicted for shorter overall survival together with the IGHV mutational status. Clonal evolution in longitudinal samples mainly occurred in cases with mutations in the initial samples and was observed not only after chemotherapy but also in untreated patients. These findings suggest that the characterization of the subclonal architecture and its dynamics in the evolution of the disease may be relevant for the management of CLL patients. PMID:26837699

  17. Activated Notch1 Target Genes during Embryonic Cell Differentiation Depend on the Cellular Context and Include Lineage Determinants and Inhibitors

    PubMed Central

    Meier-Stiegen, Franziska; Schwanbeck, Ralf; Bernoth, Kristina; Martini, Simone; Hieronymus, Thomas; Ruau, David; Zenke, Martin; Just, Ursula

    2010-01-01

    Background Notch receptor signaling controls developmental cell fates in a cell-context dependent manner. Although Notch signaling directly regulates transcription via the RBP-J/CSL DNA binding protein, little is known about the target genes that are directly activated by Notch in the respective tissues. Methodology/Principal Findings To analyze how Notch signaling mediates its context dependent function(s), we utilized a Tamoxifen-inducible system to activate Notch1 in murine embryonic stem cells at different stages of mesodermal differentiation and performed global transcriptional analyses. We find that the majority of genes regulated by Notch1 are unique for the cell type and vary widely dependent on other signals. We further show that Notch1 signaling regulates expression of genes playing key roles in cell differentiation, cell cycle control and apoptosis in a context dependent manner. In addition to the known Notch1 targets of the Hes and Hey families of transcriptional repressors, Notch1 activates the expression of regulatory transcription factors such as Sox9, Pax6, Runx1, Myf5 and Id proteins that are critically involved in lineage decisions in the absence of protein synthesis. Conclusion/Significance We suggest that Notch signaling determines lineage decisions and expansion of stem cells by directly activating both key lineage specific transcription factors and their repressors (Id and Hes/Hey proteins) and propose a model by which Notch signaling regulates cell fate commitment and self renewal in dependence of the intrinsic and extrinsic cellular context. PMID:20628604

  18. Metabolic reprogramming induces resistance to anti-NOTCH1 therapies in acute lymphoblastic leukemia

    PubMed Central

    Herranz, Daniel; Ambesi-Impiombato, Alberto; Sudderth, Jessica; Sánchez-Martín, Marta; Belver, Laura; Tosello, Valeria; Xu, Luyao; Wendorff, Agnieszka A.; Castillo, Mireia; Haydu, J. Erika; Márquez, Javier; Matés, José M.; Kung, Andrew L.; Rayport, Stephen; Cordon-Cardo, Carlos; DeBerardinis, Ralph J.; Ferrando, Adolfo A.

    2015-01-01

    Activating mutations in NOTCH1 are common in T-cell acute lymphoblastic leukemia (TALL). Here we identify glutaminolysis as a critical pathway for leukemia cell growth downstream of NOTCH1 and a key determinant of clinical response to anti-NOTCH1 therapies. Mechanistically, inhibition of NOTCH1 signaling in T-ALL induces a metabolic shutdown with prominent inhibition of glutaminolysis and triggers autophagy as a salvage pathway supporting leukemia cell metabolism. Consequently, both inhibition of glutaminolysis and inhibition of autophagy strongly and synergistically enhance the antileukemic effects of anti-NOTCH1 therapies. Moreover, we demonstrate that Pten loss induces increased glycolysis and consequently rescues leukemic cell metabolism abrogating the antileukemic effects of NOTCH1 inhibition. Overall, these results identify glutaminolysis as a major node in cancer metabolism controlled by NOTCH1 and as therapeutic target for the treatment of T-ALL. PMID:26390244

  19. Metastasis-associated lung adenocarcinoma transcript 1 promotes the proliferation of chondrosarcoma cell via activating Notch-1 signaling pathway

    PubMed Central

    Xu, Fengqin; Zhang, Zhi-qiang; Fang, Yong-chao; Li, Xiao-lei; Sun, Yu; Xiong, Chuan-zhi; Yan, Lian-qi; Wang, Qiang

    2016-01-01

    Background Metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1) is identified to be overexpressed in several cancers. However, the role of MALAT-1 in chondrosarcoma is poorly understood. Methods The expression of MALAT-1 and Notch-1 signaling pathway was detected in chondrosarcoma tissues and chondrosarcoma cells by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was performed to examine the cell viability of chondrosarcoma cells transfected with si-MALAT-1 or pcDNA-MALAT-1. Then the expression of Notch-1 signaling pathway was detected when MALAT-1 was upregulated or downregulated in chondrosarcoma cells. A subcutaneous chondrosarcoma cells xenograft model was used to confirm the effect of MALAT-1 on tumor growth in vivo. Results We found the increased expression of MALAT-1 and Notch-1 signaling pathway in chondrosarcoma tissue and cells. MALAT-1 promoted the proliferation of chondrosarcoma cells. In addition, MALAT-1 activated the Notch-1 signaling pathway at posttranscriptional level in chondrosarcoma cells. Meanwhile, overexpression of Notch-1 reversed the effect of si-MALAT-1 on the proliferation of chondrosarcoma cells. Finally, we found that MALAT-1 promoted the tumor growth in a subcutaneous chondrosarcoma cells xenograft model, which confirmed the promoted effect of MALAT-1 on the tumor growth in vivo. Conclusion Taken together, our study demonstrated that MALAT-1 promoted the proliferation of chondrosarcoma cell via activating Notch-1 signaling pathway. PMID:27110130

  20. An activated Notch1 signaling pathway inhibits cell proliferation and induces apoptosis in human esophageal squamous cell carcinoma cell line EC9706.

    PubMed

    Lu, Zhaoming; Liu, Hongtao; Xue, Lexun; Xu, Peirong; Gong, Tianxiao; Hou, Guiqin

    2008-03-01

    Previous studies have demonstrated that Notch1 signaling pathway plays a major role in maintaining the balance of cell proliferation, differentiation and apoptosis, and is closely associated with tumorigenesis. However, roles of Notch1 signaling pathway in esophageal squamous cell carcinoma (ESCC), which is a common cause of mortality in China, remain poorly understood. Therefore, a novel strategy for seeking a rational molecular therapeutic target for ESCC is urgently needed. The purpose of this study is to examine the effect of the active Notch1 signaling pathway on the proliferation and apoptosis of ESCC cells and to investigate the underlying molecular mechanisms in carcinogenesis of the esophagus. The results revealed that a constitutively activated Notch1 signaling pathway was observed in ESCC cell line EC9706, through a pcNICD vector mediated expression system. Clearly, the activated Notch1 signaling pathway gave rise to proliferation suppression of the cells, accompanied with a cell cycle inhibition at the G0/G1 phase and apoptosis. In contrast to the expression of CDK2, cyclin D1 and cyclin E observed in EC9706 cells untreated and transfected with pcDNA3.1, there was a markedly decrease in the cells stably expressing Notch1 NICD. Up- and down-regulations of GSK3 beta and beta-catenin, respectively, indicated that Notch1 inhibited proliferation and induced apoptosis of EC9706 cells through Wnt-mediated signaling pathway. These findings suggest that Notch1 signaling pathway may participate in carcinogenesis of the esophagus.

  1. Notch1 Pathway Activation Results from the Epigenetic Abrogation of Notch-Related MicroRNAs in Mycosis Fungoides.

    PubMed

    Gallardo, Fernando; Sandoval, Juan; Díaz-Lagares, Angel; Garcia, Ricard; D'Altri, Teresa; González, Jessica; Alegre, Victor; Servitje, Octavio; Crujeiras, Ana-Belén; Stefánsson, Ólafur-Andri; Espinet, Blanca; Hernández, Maria-Inmaculada; Bellosillo, Beatriz; Esteller, Manel; Pujol, Ramon-Maria; Bigas, Anna; Espinosa, Lluis

    2015-12-01

    Notch is a family of transmembrane receptors that participate in the regulation of cell differentiation, proliferation, and stemness. Notch pathway activation has also been found associated with different human cancers including primary cutaneous T-cell lymphomas (CTCL). The elucidation of the mechanisms driving Notch activation in these particular diseases has remained elusive. Here we studied the possibility that DNA methylation at Notch pathway gene promoters and/or deregulation of Notch-associated microRNAs contribute to activate Notch in mycosis fungoides (MF). By genome-wide DNA methylation analysis, we failed to detect any consistent methylation at the Notch1, the Notch-ligand Jagged1, or the Notch-target Hes1 gene promoters, but found a significant methylation of the Notch-related microRNAs, in particular miR-200c and miR-124. Downregulation of miR-200c is associated with overexpression of Jagged1, concomitant to Notch1 activation. CTCL cell lines were infected with lentiviral vector encoding for miR-200c and ectopic expression of miR-200c in CTCL lines resulted in Jagged1 protein downregulation associated with a reduction in the levels of active Notch1. Our study deciphers an epigenetic mechanism regulating the Notch pathway in (MF) that might contribute to the future design of more specific therapeutic strategies. PMID:26302069

  2. Simultaneous targeted activation of Notch1 and Vhl-disruption in the kidney proximal epithelial tubular cells in mice

    PubMed Central

    Johansson, Elinn; Rönö, Birgitte; Johansson, Martin; Lindgren, David; Möller, Christina; Axelson, Håkan; Smith, Emma M. K.

    2016-01-01

    Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer, representing approximately 75% of all renal neoplasms. ccRCC is known to be strongly associated with silencing of the von Hippel Lindau (VHL) tumor suppressor gene, yet VHL deficiency alone does not seem to be sufficient to drive the oncogenic transformation of normal renal epithelium and induce renal tumorigenesis. We, and others, have previously suggested that constitutive activation of the Notch signaling pathway, alongside with VHL loss, contribute to the oncogenic features of ccRCC. Here we report a prevailing hyperactivation of the Notch1 receptor in human ccRCC relative to the healthy counterpart. To explore the consequences of the elevated Notch1 signaling observed in ccRCC patient material, we made use of a conditional mouse model based on concurrent ectopic expression of constitutively active Notch1 (NICD1) and deletion of the Vhl gene. Histological examination of the kidneys of the conditional mice demonstrate the existence of nests of dysplastic cells with a clear cytoplasm as a consequence of lipid accumulation, thus displaying a one important hallmark of human ccRCC. PMID:27491826

  3. Simultaneous targeted activation of Notch1 and Vhl-disruption in the kidney proximal epithelial tubular cells in mice.

    PubMed

    Johansson, Elinn; Rönö, Birgitte; Johansson, Martin; Lindgren, David; Möller, Christina; Axelson, Håkan; Smith, Emma M K

    2016-01-01

    Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer, representing approximately 75% of all renal neoplasms. ccRCC is known to be strongly associated with silencing of the von Hippel Lindau (VHL) tumor suppressor gene, yet VHL deficiency alone does not seem to be sufficient to drive the oncogenic transformation of normal renal epithelium and induce renal tumorigenesis. We, and others, have previously suggested that constitutive activation of the Notch signaling pathway, alongside with VHL loss, contribute to the oncogenic features of ccRCC. Here we report a prevailing hyperactivation of the Notch1 receptor in human ccRCC relative to the healthy counterpart. To explore the consequences of the elevated Notch1 signaling observed in ccRCC patient material, we made use of a conditional mouse model based on concurrent ectopic expression of constitutively active Notch1 (NICD1) and deletion of the Vhl gene. Histological examination of the kidneys of the conditional mice demonstrate the existence of nests of dysplastic cells with a clear cytoplasm as a consequence of lipid accumulation, thus displaying a one important hallmark of human ccRCC. PMID:27491826

  4. Inflammation increases NOTCH1 activity via MMP9 and is counteracted by Eicosapentaenoic Acid-free fatty acid in colon cancer cells

    PubMed Central

    Fazio, Chiara; Piazzi, Giulia; Vitaglione, Paola; Fogliano, Vincenzo; Munarini, Alessandra; Prossomariti, Anna; Milazzo, Maddalena; D’Angelo, Leonarda; Napolitano, Manuela; Chieco, Pasquale; Belluzzi, Andrea; Bazzoli, Franco; Ricciardiello, Luigi

    2016-01-01

    Aberrant NOTCH1 signalling is critically involved in multiple models of colorectal cancer (CRC) and a prominent role of NOTCH1 activity during inflammation has emerged. Epithelial to Mesenchymal Transition (EMT), a crucial event promoting malignant transformation, is regulated by inflammation and Metalloproteinase-9 (MMP9) plays an important role in this process. Eicosapentaenoic Acid (EPA), an omega-3 polyunsaturated fatty acid, was shown to prevent colonic tumors in different settings. We recently found that an extra-pure formulation of EPA as Free Fatty Acid (EPA-FFA) protects from colon cancer development in a mouse model of Colitis-Associated Cancer (CAC) through modulation of NOTCH1 signalling. In this study, we exposed colon cancer cells to an inflammatory stimulus represented by a cytokine-enriched Conditioned Medium (CM), obtained from THP1-differentiated macrophages. We found, for the first time, that CM strongly up-regulated NOTCH1 signalling and EMT markers, leading to increased invasiveness. Importantly, NOTCH1 signalling was dependent on MMP9 activity, upon CM exposure. We show that a non-cytotoxic pre-treatment with EPA-FFA antagonizes the effect of inflammation on NOTCH1 signalling, with reduction of MMP9 activity and invasiveness. In conclusion, our data suggest that, in CRC cells, inflammation induces NOTCH1 activity through MMP9 up-regulation and that this mechanism can be counteracted by EPA-FFA. PMID:26864323

  5. NOTCH1 Inhibits Activation of ATM by Impairing the Formation of an ATM-FOXO3a-KAT5/Tip60 Complex.

    PubMed

    Adamowicz, Marek; Vermezovic, Jelena; d'Adda di Fagagna, Fabrizio

    2016-08-23

    The DNA damage response (DDR) signal transduction pathway is responsible for sensing DNA damage and further relaying this signal into the cell. ATM is an apical DDR kinase that orchestrates the activation and the recruitment of downstream DDR factors to induce cell-cycle arrest and repair. We have previously shown that NOTCH1 inhibits ATM activation upon DNA damage, but the underlying mechanism remains unclear. Here, we show that NOTCH1 does not impair ATM recruitment to DNA double-strand breaks (DSBs). Rather, NOTCH1 prevents binding of FOXO3a and KAT5/Tip60 to ATM through a mechanism in which NOTCH1 competes with FOXO3a for ATM binding. Lack of FOXO3a binding to ATM leads to the loss of KAT5/Tip60 association with ATM. Moreover, expression of NOTCH1 or depletion of ATM impairs the formation of the FOXO3a-KAT5/Tip60 protein complex. Finally, we show that pharmacological induction of FOXO3a nuclear localization sensitizes NOTCH1-driven cancers to DNA-damage-induced cell death. PMID:27524627

  6. Insulin Growth Factor 1 Receptor Expression Is Associated with NOTCH1 Mutation, Trisomy 12 and Aggressive Clinical Course in Chronic Lymphocytic Leukaemia

    PubMed Central

    Maura, Francesco; Mosca, Laura; Fabris, Sonia; Cutrona, Giovanna; Matis, Serena; Lionetti, Marta; Agnelli, Luca; Barbieri, Marzia; D’Anca, Marianna; Manzoni, Martina; Colombo, Monica; Massucco, Carlotta; Reverberi, Daniele; Gentile, Massimo; Recchia, Anna Grazia; Bossio, Sabrina; Ilariucci, Fiorella; Musolino, Caterina; Di Raimondo, Francesco; Cortelezzi, Agostino; Morabito, Fortunato; Ferrarini, Manlio; Neri, Antonino

    2015-01-01

    IGF1R is emerging as an important gene in the pathogenesis of many solid and haematological cancers and its over-expression has been reported as frequently associated with aggressive disease and chemotherapy resistance. In this study we performed an investigation of the role of IGF1R expression in a large and representative prospective series of 217 chronic lymphocytic leukaemia (CLL) patients enrolled in the multicentre O-CLL1 protocol (clinicaltrial.gov #NCT00917540). High IGF1R gene expression was significantly associated with IGHV unmutated (IGHV-UM) status (p<0.0001), high CD38 expression (p<0.0001), trisomy 12 (p<0.0001), and del(11)(q23) (p=0.014). Interestingly, higher IGF1R expression (p=0.002) characterized patients with NOTCH1 mutation (c.7541_7542delCT), identified in 15.5% of cases of our series by next generation sequencing and ARMS-PCR. Furthermore, IGF1R expression has been proven as an independent prognostic factor associated with time to first treatment in our CLL prospective cohort. These data suggest that IGF1R may play an important role in CLL biology, in particular in aggressive CLL clones characterized by IGHV-UM, trisomy 12 and NOTCH1 mutation. PMID:25786252

  7. Temporal Notch activation through Notch1a and Notch3 is required for maintaining zebrafish rhombomere boundaries.

    PubMed

    Qiu, Xuehui; Lim, Chiaw-Hwee; Ho, Steven Hao-Kee; Lee, Kian-Hong; Jiang, Yun-Jin

    2009-07-01

    In vertebrates, hindbrain is subdivided into seven segments termed rhombomeres and the interface between each rhombomere forms the boundary. Similar to the D/V boundary formation in Drosophila, Notch activation has been shown to regulate the segregation of rhombomere boundary cells. Here we further explored the function of Notch signaling in the formation of rhombomere boundaries. By using bodipy ceramide cell-labeling technique, we found that the hindbrain boundary is formed initially in mib mutants but lost after 24 hours post-fertilization (hpf). This phenotype was more severe in mib(ta52b) allele than in mib(tfi91) allele. Similarly, injection of su(h)-MO led to boundary defects in a dosage-dependent manner. Boundary cells were recovered in mib(ta52b) mutants in the hdac1-deficient background, where neurogenesis is inhibited. Furthermore, boundary cells lost sensitivity to reduced Notch activation from 15 somite stage onwards. We also showed that knockdown of notch3 function in notch1a mutants leads to the loss of rhombomere boundary cells and causes neuronal hyperplasia, indicating that Notch1a and Notch3 play a redundant role in the maintenance of rhombomere boundary.

  8. NOTCH1 Regulates Matrix Gla Protein and Calcification Gene Networks in Human Valve Endothelium

    PubMed Central

    White, Mark P.; Theodoris, Christina V.; Liu, Lei; Collins, William J.; Blue, Kathleen W.; Lee, Joon Ho; Meng, Xianzhong; Robbins, Robert C.; Ivey, Kathryn N.; Srivastava, Deepak

    2015-01-01

    Valvular and vascular calcification are common causes of cardiovascular morbidity and mortality. Developing effective treatments requires understanding the molecular underpinnings of these processes. Shear stress is thought to play a role in inhibiting calcification. Furthermore, NOTCH1 regulates vascular and valvular endothelium, and human mutations in NOTCH1 can cause calcific aortic valve disease. Here, we determined the genome-wide impact of altering shear stress and NOTCH signaling on aortic valve endothelium. mRNA-sequencing of human aortic valve endothelial cells (HAVECs) with or without knockdown of NOTCH1, in the presence or absence of shear stress, revealed NOTCH1-dependency of the atherosclerosis-related gene connexin 40 (GJA5), and numerous repressors of endochondral ossification. Among these, Matrix GLA Protein (MGP) is highly expressed in aortic valve and vasculature, and inhibits soft tissue calcification by sequestering bone morphogenetic proteins (BMPs). Altering NOTCH1 levels affected MGP mRNA and protein in HAVECs. Furthermore, shear stress activated NOTCH signaling and MGP in a NOTCH1-dependent manner. NOTCH1 positively regulated endothelial MGP in vivo through specific binding motifs upstream of MGP. Our studies suggest that shear stress activates NOTCH1 in primary human aortic valve endothelial cells leading to downregulation of osteoblast-like gene networks that play a role in tissue calcification. PMID:25871831

  9. NOTCH1, SF3B1, BIRC3 and TP53 mutations in patients with chronic lymphocytic leukemia undergoing first-line treatment: correlation with biological parameters and response to treatment.

    PubMed

    Chiaretti, Sabina; Marinelli, Marilisa; Del Giudice, Ilaria; Bonina, Silvia; Piciocchi, Alfonso; Messina, Monica; Vignetti, Marco; Rossi, Davide; Di Maio, Valeria; Mauro, Francesca Romana; Guarini, Anna; Gaidano, Gianluca; Foà, Robin

    2014-12-01

    In chronic lymphocytic leukemia, NOTCH1, SF3B1, BIRC3 and TP53 disruptions are recurrent and affect survival. To define their incidence and clinical impact in patients undergoing first-line treatment, we evaluated 163 cases enrolled in the GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) LLC0405 protocol (fludarabine plus alemtuzumab or fludarabine plus cyclophosphamide), for young patients, or in the ML21445 protocol (chlorambucil plus rituximab), for elderly patients. NOTCH1, SF3B1, BIRC3 and TP53 disruptions were detected in 15.9%, 12.2%, 8.6% and 10.4% of cases. NOTCH1 mutations correlated with a shorter treatment-free interval (p = 0.058), an unmutated immunoglobulin heavy variable gene (IGHV) status (p < 0.0001), CD38 and ZAP-70 expression (p = 0.0025 and 0.026, respectively) and trisomy 12 (p = 0.0028), SF3B1 mutations with an unmutated IGHV status (p = 0.02), and BIRC3 disruptions with an unmutated IGHV configuration (p = 0.01) and 11q deletion (p < 0.0001). NOTCH1 and SF3B1 did not appear to impact on overall response, while an inferior response was observed for BIRC3- and TP53-disrupted cases in the LLC0405 and ML21445 protocols, respectively. Progression-free survival, evaluable in the LLC0405 protocol - not affected by NOTCH1, SF3B1 and TP53 - appeared inferior for BIRC3 disruption. NOTCH1 and SF3B1 mutations may be overcome by aggressive regimens, while BIRC3 might impact on outcome also in intensive regimens.

  10. DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur.

    PubMed

    Nichols, James T; Miyamoto, Alison; Olsen, Samantha L; D'Souza, Brendan; Yao, Christine; Weinmaster, Gerry

    2007-02-12

    Cleavage of Notch by furin is required to generate a mature, cell surface heterodimeric receptor that can be proteolytically activated to release its intracellular domain, which functions in signal transduction. Current models propose that ligand binding to heterodimeric Notch (hNotch) induces a disintegrin and metalloprotease (ADAM) proteolytic release of the Notch extracellular domain (NECD), which is subsequently shed and/or endocytosed by DSL ligand cells. We provide evidence for NECD release and internalization by DSL ligand cells, which, surprisingly, did not require ADAM activity. However, losses in either hNotch formation or ligand endocytosis significantly decreased NECD transfer to DSL ligand cells, as well as signaling in Notch cells. Because endocytosis-defective ligands bind hNotch, but do not dissociate it, additional forces beyond those produced through ligand binding must function to disrupt the intramolecular interactions that keep hNotch intact and inactive. Based on our findings, we propose that mechanical forces generated during DSL ligand endocytosis function to physically dissociate hNotch, and that dissociation is a necessary step in Notch activation.

  11. Function of Integrin-Linked Kinase in Modulating the Stemness of IL-6-Abundant Breast Cancer Cells by Regulating γ-Secretase-Mediated Notch1 Activation in Caveolae.

    PubMed

    Hsu, En-Chi; Kulp, Samuel K; Huang, Han-Li; Tu, Huang-Ju; Salunke, Santosh B; Sullivan, Nicholas J; Sun, Duxin; Wicha, Max S; Shapiro, Charles L; Chen, Ching-Shih

    2015-06-01

    Interleukin-6 (IL-6) and Notch signaling are important regulators of breast cancer stem cells (CSCs), which drive the malignant phenotype through self-renewal, differentiation, and development of therapeutic resistance. We investigated the role of integrin-linked kinase (ILK) in regulating IL-6-driven Notch1 activation and the ability to target breast CSCs through ILK inhibition. Ectopic expression/short hairpin RNA-mediated knockdown of ILK, pharmacological inhibition of ILK with the small molecule T315, Western blot analysis, immunofluorescence, and luciferase reporter assays were used to evaluate the regulation of IL-6-driven Notch1 activation by ILK in IL-6-producing triple-negative breast cancer cell lines (MDA-MB-231, SUM-159) and in MCF-7 and MCF-7(IL-6) cells. The effects of ILK on γ-secretase complex assembly and cellular localization were determined by immunofluorescence, Western blots of membrane fractions, and immunoprecipitation. In vivo effects of T315-induced ILK inhibition on CSCs in SUM-159 xenograft models were assessed by mammosphere assays, flow cytometry, and tumorigenicity assays. Results show that the genetic knockdown or pharmacological inhibition of ILK suppressed Notch1 activation and the abundance of the γ-secretase components presenilin-1, nicastrin, and presenilin enhancer 2 at the posttranscriptional level via inhibition of caveolin-1-dependent membrane assembly of the γ-secretase complex. Accordingly, knockdown of ILK inhibited breast CSC-like properties in vitro and the breast CSC subpopulation in vivo in xenograft tumor models. Based on these findings, we propose a novel function of ILK in regulating γ-secretase-mediated Notch1 activation, which suggests the targeting of ILK as a therapeutic approach to suppress IL-6-induced breast CSCs.

  12. Aberrant activation of canonical Notch1 signaling in the mouse uterus decreases progesterone receptor by hypermethylation and leads to infertility.

    PubMed

    Su, Ren-Wei; Strug, Michael R; Jeong, Jae-Wook; Miele, Lucio; Fazleabas, Asgerally T

    2016-02-23

    In mammalian reproduction, implantation is one of the most critical events. Failure of implantation and the subsequent decidualization contribute to more than 75% of pregnancy losses in women. Our laboratory has previously reported that inhibition of Notch signaling results in impaired decidualization in both women and a transgenic mouse model. In this study, we generated a Notch gain-of-function transgenic mouse by conditionally overexpressing the Notch1 intracellular domain (N1ICD) in the reproductive tract driven by a progesterone receptor (Pgr) -Cre. We show that the overexpression of N1ICD in the uterus results in complete infertility as a consequence of multiple developmental and physiological defects, including the absence of uterine glands and dysregulation of progesterone and estrogen signaling by a Recombination Signal Binding Protein Jκ-dependent signaling mechanism. We further show that the inhibition of progesterone signaling is caused by hypermethylation of its receptor Pgr by Notch1 overexpression through the transcription factor PU.1 and DNA methyltransferase 3b (Dnmt3b). We have generated a mouse model to study the consequence of increased Notch signaling in female reproduction and provide the first evidence, to our knowledge, that Notch signaling can regulate epigenetic modification of the Pgr.

  13. A Bovine Herpesvirus 1 Protein Expressed in Latently Infected Neurons (ORF2) Promotes Neurite Sprouting in the Presence of Activated Notch1 or Notch3

    PubMed Central

    Sinani, Devis; Frizzo da Silva, Leticia

    2013-01-01

    Bovine herpesvirus 1 (BHV-1) infection induces clinical symptoms in the upper respiratory tract, inhibits immune responses, and can lead to life-threatening secondary bacterial infections. Following acute infection, BHV-1 establishes latency in sensory neurons within trigeminal ganglia, but stress can induce reactivation from latency. The latency-related (LR) RNA is the only viral transcript abundantly expressed in latently infected sensory neurons. An LR mutant virus with stop codons at the amino terminus of the first open reading frame (ORF) in the LR gene (ORF2) is not reactivated from latency, in part because it induces higher levels of apoptosis in infected neurons. ORF2 inhibits apoptosis in transiently transfected cells, suggesting that it plays a crucial role in the latency-reactivation cycle. ORF2 also interacts with Notch1 or Notch3 and inhibits its ability to trans activate certain viral promoters. Notch3 RNA and protein levels are increased during reactivation from latency, suggesting that Notch may promote reactivation. Activated Notch signaling interferes with neuronal differentiation, in part because neurite and axon generation is blocked. In this study, we demonstrated that ORF2 promotes neurite formation in mouse neuroblastoma cells overexpressing Notch1 or Notch3. ORF2 also interfered with Notch-mediated trans activation of the promoter that regulates the expression of Hairy Enhancer of Split 5, an inhibitor of neurite formation. Additional studies provided evidence that ORF2 promotes the degradation of Notch3, but not that of Notch1, in a proteasome-dependent manner. In summary, these studies suggest that ORF2 promotes a mature neuronal phenotype that enhances the survival of infected neurons and consequently increases the pool of latently infected neurons. PMID:23152506

  14. Inhibitory Role of Notch1 in Calcific Aortic Valve Disease

    PubMed Central

    Koenig, Sara N.; Nichols, Haley A.; Galindo, Cristi L.; Garner, Harold R.; Merrill, Walter H.; Hinton, Robert B.; Garg, Vidu

    2011-01-01

    Aortic valve calcification is the most common form of valvular heart disease, but the mechanisms of calcific aortic valve disease (CAVD) are unknown. NOTCH1 mutations are associated with aortic valve malformations and adult-onset calcification in families with inherited disease. The Notch signaling pathway is critical for multiple cell differentiation processes, but its role in the development of CAVD is not well understood. The aim of this study was to investigate the molecular changes that occur with inhibition of Notch signaling in the aortic valve. Notch signaling pathway members are expressed in adult aortic valve cusps, and examination of diseased human aortic valves revealed decreased expression of NOTCH1 in areas of calcium deposition. To identify downstream mediators of Notch1, we examined gene expression changes that occur with chemical inhibition of Notch signaling in rat aortic valve interstitial cells (AVICs). We found significant downregulation of Sox9 along with several cartilage-specific genes that were direct targets of the transcription factor, Sox9. Loss of Sox9 expression has been published to be associated with aortic valve calcification. Utilizing an in vitro porcine aortic valve calcification model system, inhibition of Notch activity resulted in accelerated calcification while stimulation of Notch signaling attenuated the calcific process. Finally, the addition of Sox9 was able to prevent the calcification of porcine AVICs that occurs with Notch inhibition. In conclusion, loss of Notch signaling contributes to aortic valve calcification via a Sox9-dependent mechanism. PMID:22110751

  15. Activation of Notch1 inhibits medial edge epithelium apoptosis in all-trans retinoic acid-induced cleft palate in mice.

    PubMed

    Zhang, Yadong; Dong, Shiyi; Wang, Weicai; Wang, Jianning; Wang, Miao; Chen, Mu; Hou, Jinsong; Huang, Hongzhang

    2016-08-26

    Administration of all-trans retinoic acid (atRA) on E12.0 (embryonic day 12.0) leads to failure of medial edge epithelium (MEE) disappearance and cleft palate. However, the molecular mechanism underlying the relationship between atRA and MEE remains to be identified. In this study, atRA (200 mg/kg) administered by gavage induced a 75% incidence of cleft palate in C57BL/6 mice. Notch1 was up-regulated in MEE cells in the atRA-treated group compared with the controls at E15.0, together with reduced apoptosis and elevated proliferation. Next, we investigated the mechanisms underlying atRA, Notch1 and MEE degradation in palate organ culture. Our results revealed that down-regulation of Notch1 partially rescued the inhibition of atRA-induced palate fusion. Molecular analysis indicated that atRA increased the expression of Notch1 and Rbpj and decreased the expression of P21. In addition, depletion of Notch1 expression decreased the expression of Rbpj and increased the expression of P21. Moreover, inhibition of Rbpj expression partially reversed atRA-induced MEE persistence and increased P21 expression. These findings demonstrate that atRA inhibits MEE degradation, which in turn induces a cleft palate, possibly through the Notch1/RBPjk/P21 signaling pathway. PMID:27343556

  16. Involvement of Notch1/Hes signaling pathway in ankylosing spondylitis

    PubMed Central

    Xu, Wei; Liang, Chao-Ge; Li, Yi-Fan; Ji, Yun-Han; Qiu, Wen-Jun; Tang, Xian-Zhong

    2015-01-01

    We aimed to investigate the role of Notch1/Hes signaling pathway in the pathogenesis of abnormal ossification of hip ligament in patients with ankylosing spondylitis (AS). 22 AS patients scheduled for artificial hip arthroplasty were randomly chosen as AS group. As controls, we used 4 patients diagnosed with transcervical fracture who underwent hip replacement surgery. Notch1 and Hes mRNA expressions were detected by real-time fluorescent quantitative polymerase chain reaction (RFQ-PCR). Immunohistochemistry (IHC) was used to detect Notch1 and Hes protein expression. Correlation analyses of Notch-l and Hes with AS-related clinical factors were conducted with spearman’s correlation analysis and partial correlation analysis. RFQ-PCR results showed significant differences in Notch1 and Hes mRNA expressions between AS group and the control group (all P < 0.05). IHC analysis further indicated positive nuclear signals of Notch1 and Hes protein, indicating functional activation of the Notch1 and Hes pathways. Semi-quantitative IHC showed a higher Notch1 and Hes expression levels in AS group compared to the control group (all P < 0.05). Correlation analysis suggested that Hes protein expression was positively associated with the clinical course of the disease in AS patients. In conclusion, Notch1 and Hes overexpression was clearly detected in hip joint ligaments of AS patients, Hes protein expression was associated with the clinical course of AS. Taken together, we suggest that signaling pathways mediated by Notch1-Hes may contribute to ligament ossification of hip joints in AS patients. PMID:26045779

  17. Function of Integrin-Linked Kinase in Modulating the Stemness of IL-6–Abundant Breast Cancer Cells by Regulating γ-Secretase–Mediated Notch1 Activation in Caveolae12

    PubMed Central

    Hsu, En-Chi; Kulp, Samuel K.; Huang, Han-Li; Tu, Huang-Ju; Salunke, Santosh B.; Sullivan, Nicholas J.; Sun, Duxin; Wicha, Max S.; Shapiro, Charles L.; Chen, Ching-Shih

    2015-01-01

    Interleukin-6 (IL-6) and Notch signaling are important regulators of breast cancer stem cells (CSCs), which drive the malignant phenotype through self-renewal, differentiation, and development of therapeutic resistance. We investigated the role of integrin-linked kinase (ILK) in regulating IL-6–driven Notch1 activation and the ability to target breast CSCs through ILK inhibition. Ectopic expression/short hairpin RNA-mediated knockdown of ILK, pharmacological inhibition of ILK with the small molecule T315, Western blot analysis, immunofluorescence, and luciferase reporter assays were used to evaluate the regulation of IL-6–driven Notch1 activation by ILK in IL-6–producing triple-negative breast cancer cell lines (MDA-MB-231, SUM-159) and in MCF-7 and MCF-7IL-6 cells. The effects of ILK on γ-secretase complex assembly and cellular localization were determined by immunofluorescence, Western blots of membrane fractions, and immunoprecipitation. In vivo effects of T315-induced ILK inhibition on CSCs in SUM-159 xenograft models were assessed by mammosphere assays, flow cytometry, and tumorigenicity assays. Results show that the genetic knockdown or pharmacological inhibition of ILK suppressed Notch1 activation and the abundance of the γ-secretase components presenilin-1, nicastrin, and presenilin enhancer 2 at the posttranscriptional level via inhibition of caveolin-1-dependent membrane assembly of the γ-secretase complex. Accordingly, knockdown of ILK inhibited breast CSC-like properties in vitro and the breast CSC subpopulation in vivo in xenograft tumor models. Based on these findings, we propose a novel function of ILK in regulating γ-secretase–mediated Notch1 activation, which suggests the targeting of ILK as a therapeutic approach to suppress IL-6–induced breast CSCs. PMID:26152358

  18. Loss of Notch1-dependent p21Waf1/Cip1 expression influences the Notch1 outcome in tumorigenesis

    PubMed Central

    Cialfi, Samantha; Palermo, Rocco; Manca, Sonia; De Blasio, Carlo; Vargas Romero, Paula; Checquolo, Saula; Bellavia, Diana; Uccelletti, Daniela; Saliola, Michele; D'Alessandro, Angelo; Zolla, Lello; Gulino, Alberto; Screpanti, Isabella; Talora, Claudio

    2014-01-01

    Notch signaling plays a complex role in carcinogenesis, and its signaling pathway has both tumor-suppressor and oncogenic components. In this study we investigated the effects of reactive oxygen species (ROS) on Notch1 signaling outcome in keratinocyte biology. We demonstrate that Notch1 function contributes to the arsenic-induced keratinocyte transformation. We found that acute exposure to arsenite increases oxidative stress and inhibits proliferation of keratinocyte cells by upregulation of p21waf1/Cip1. The necessity of p21waf1/Cip1 for arsenite-induced cell death was demonstrated by targeted downregulation of p21waf1/Cip1 by using RNA interference. We further demonstrated that on acute exposure to arsenite, p21waf1/Cip1 is upregulated and Notch1 downmodulated, whereas on chronic exposure to arsenite, malignant progression of arsenite-treated keratinocytes cells was accompanied by regained expression and activity of Notch1. Notch1 activity in arsenite-transformed keratinocytes inhibits arsenite-induced upregulation of p21waf1/Cip1 by sustaining c-myc expression. We further demonstrated that c-myc collaborates with Nrf2, a key regulator for the maintenance of redox homeostasis, to promote metabolic activities that support cell proliferation and cytoprotection. Therefore, Notch1-mediated repression of p21waf1/Cip1 expression results in the inhibition of cell death and keratinocytes transformation. Our results not only demonstrate that sustained Notch1 expression is at least one key event implicated in the arsenite human skin carcinogenic effect, but also may provide mechanistic insights into the molecular aspects that determine whether Notch signaling will be either oncogenic or tumor suppressive. PMID:24801890

  19. The clerodane diterpene casearin J induces apoptosis of T-ALL cells through SERCA inhibition, oxidative stress, and interference with Notch1 signaling.

    PubMed

    De Ford, C; Heidersdorf, B; Haun, F; Murillo, R; Friedrich, T; Borner, C; Merfort, I

    2016-01-28

    T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that preferentially affects children and adolescents. Over 50% of human T-ALLs possess activating mutations of Notch1. The clerodane diterpene casearin J (CJ) is a natural product that inhibits the sarcoendoplasmatic reticulum calcium ATPase (SERCA) pump and induces cell death in leukemia cells, but the molecular mechanism of cytotoxicity remains poorly understood. Here we show that owing to SERCA pump inhibition, CJ induces depletion of the endoplasmic reticulum calcium pools, oxidative stress, and apoptosis via the intrinsic signaling pathway. Moreover, Notch1 signaling is reduced in T-ALL cells with auto-activating mutations in the HD-domain of Notch1, but not in cells that do not depend on Notch1 signaling. CJ also provoked a slight activation of NF-κB, and consistent with this notion a combined treatment of CJ and the NF-κB inhibitor parthenolide (Pt) led to a remarkable synergistic cell death in T-ALL cells. Altogether, our data support the concept that inhibition of the SERCA pump may be a novel strategy for the treatment of T-ALL with HD-domain-mutant Notch1 receptors and that additional treatment with the NF-κB inhibitor parthenolide may have further therapeutic benefits.

  20. The clerodane diterpene casearin J induces apoptosis of T-ALL cells through SERCA inhibition, oxidative stress, and interference with Notch1 signaling

    PubMed Central

    De Ford, C; Heidersdorf, B; Haun, F; Murillo, R; Friedrich, T; Borner, C; Merfort, I

    2016-01-01

    T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that preferentially affects children and adolescents. Over 50% of human T-ALLs possess activating mutations of Notch1. The clerodane diterpene casearin J (CJ) is a natural product that inhibits the sarcoendoplasmatic reticulum calcium ATPase (SERCA) pump and induces cell death in leukemia cells, but the molecular mechanism of cytotoxicity remains poorly understood. Here we show that owing to SERCA pump inhibition, CJ induces depletion of the endoplasmic reticulum calcium pools, oxidative stress, and apoptosis via the intrinsic signaling pathway. Moreover, Notch1 signaling is reduced in T-ALL cells with auto-activating mutations in the HD-domain of Notch1, but not in cells that do not depend on Notch1 signaling. CJ also provoked a slight activation of NF-κB, and consistent with this notion a combined treatment of CJ and the NF-κB inhibitor parthenolide (Pt) led to a remarkable synergistic cell death in T-ALL cells. Altogether, our data support the concept that inhibition of the SERCA pump may be a novel strategy for the treatment of T-ALL with HD-domain-mutant Notch1 receptors and that additional treatment with the NF-κB inhibitor parthenolide may have further therapeutic benefits. PMID:26821066

  1. K-Ras and cyclooxygenase-2 coactivation augments intraductal papillary mucinous neoplasm and Notch1 mimicking human pancreas lesions

    PubMed Central

    Chiblak, Sara; Steinbauer, Brigitte; Pohl-Arnold, Andrea; Kucher, Dagmar; Abdollahi, Amir; Schwager, Christian; Höft, Birgit; Esposito, Irene; Müller-Decker, Karin

    2016-01-01

    Mutational activation of K-Ras is an initiating event of pancreatic ductal adenocarcinomas (PDAC) that may develop either from pancreatic intraepithelial neoplasia (PanIN) or intraductal papillary mucinous neoplasms (IPMN). Cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) is causally related to pancreatic carcinogenesis. Here, we deciphered the impact of COX-2, a key modulator of inflammation, in concert with active mutant K-RasG12D on tumor burden and gene expression signature using compound mutant mouse lines. Concomitant activation of COX-2 and K-RasG12D accelerated the progression of pancreatic intraepithelial lesions predominantly with a cystic papillary phenotype resembling human IPMN. Transcriptomes derived from laser capture microdissected preneoplastic lesions of single and compound mutants revealed a signature that was significantly enriched in Notch1 signaling components. In vitro, Notch1 signaling was COX-2-dependent. In line with these findings, human IPMN stratified into intestinal, gastric and pancreatobillary types displayed Notch1 immunosignals with high prevalence, especially in the gastric lesions. In conclusion, a yet unknown link between activated Ras, protumorigenic COX-2 and Notch1 in IPMN onset was unraveled. PMID:27381829

  2. The relevance of PTEN-AKT in relation to NOTCH1-directed treatment strategies in T-cell acute lymphoblastic leukemia.

    PubMed

    Mendes, Rui D; Canté-Barrett, Kirsten; Pieters, Rob; Meijerink, Jules P P

    2016-09-01

    The tumor suppressor phosphatase and tensin homolog (PTEN) negatively regulates phosphatidylinositol 3-kinase (PI3K)-AKT signaling and is often inactivated by mutations (including deletions) in a variety of cancer types, including T-cell acute lymphoblastic leukemia. Here we review mutation-associated mechanisms that inactivate PTEN together with other molecular mechanisms that activate AKT and contribute to T-cell leukemogenesis. In addition, we discuss how Pten mutations in mouse models affect the efficacy of gamma-secretase inhibitors to block NOTCH1 signaling through activation of AKT. Based on these models and on observations in primary diagnostic samples from patients with T-cell acute lymphoblastic leukemia, we speculate that PTEN-deficient cells employ an intrinsic homeostatic mechanism in which PI3K-AKT signaling is dampened over time. As a result of this reduced PI3K-AKT signaling, the level of AKT activation may be insufficient to compensate for NOTCH1 inhibition, resulting in responsiveness to gamma-secretase inhibitors. On the other hand, de novo acquired PTEN-inactivating events in NOTCH1-dependent leukemia could result in temporary, strong activation of PI3K-AKT signaling, increased glycolysis and glutaminolysis, and consequently gamma-secretase inhibitor resistance. Due to the central role of PTEN-AKT signaling and in the resistance to NOTCH1 inhibition, AKT inhibitors may be a promising addition to current treatment protocols for T-cell acute lymphoblastic leukemia. PMID:27582570

  3. The relevance of PTEN-AKT in relation to NOTCH1-directed treatment strategies in T-cell acute lymphoblastic leukemia

    PubMed Central

    Mendes, Rui D.; Canté-Barrett, Kirsten; Pieters, Rob; Meijerink, Jules P.P.

    2016-01-01

    The tumor suppressor phosphatase and tensin homolog (PTEN) negatively regulates phosphatidylinositol 3-kinase (PI3K)-AKT signaling and is often inactivated by mutations (including deletions) in a variety of cancer types, including T-cell acute lymphoblastic leukemia. Here we review mutation-associated mechanisms that inactivate PTEN together with other molecular mechanisms that activate AKT and contribute to T-cell leukemogenesis. In addition, we discuss how Pten mutations in mouse models affect the efficacy of gamma-secretase inhibitors to block NOTCH1 signaling through activation of AKT. Based on these models and on observations in primary diagnostic samples from patients with T-cell acute lymphoblastic leukemia, we speculate that PTEN-deficient cells employ an intrinsic homeostatic mechanism in which PI3K-AKT signaling is dampened over time. As a result of this reduced PI3K-AKT signaling, the level of AKT activation may be insufficient to compensate for NOTCH1 inhibition, resulting in responsiveness to gamma-secretase inhibitors. On the other hand, de novo acquired PTEN-inactivating events in NOTCH1-dependent leukemia could result in temporary, strong activation of PI3K-AKT signaling, increased glycolysis and glutaminolysis, and consequently gamma-secretase inhibitor resistance. Due to the central role of PTEN-AKT signaling and in the resistance to NOTCH1 inhibition, AKT inhibitors may be a promising addition to current treatment protocols for T-cell acute lymphoblastic leukemia. PMID:27582570

  4. Inhibition of CK2α down-regulates Notch1 signalling in lung cancer cells

    PubMed Central

    Zhang, Shulin; Long, Hao; Yang, Yi-Lin; Wang, Yucheng; Hsieh, David; Li, Weiming; Au, Alfred; Stoppler, Hubert J; Xu, Zhidong; Jablons, David M; You, Liang

    2013-01-01

    Protein kinase CK2 is frequently elevated in a variety of human cancers. The Notch1 signalling pathway has been implicated in stem cell maintenance and its aberrant activation has been shown in several types of cancer including lung cancer. Here, we show, for the first time, that CK2α is a positive regulator of Notch1 signalling in lung cancer cell lines A549 and H1299. We found that Notch1 protein level was reduced after CK2α silencing. Down-regulation of Notch1 transcriptional activity was demonstrated after the silencing of CK2α in lung cancer cells. Furthermore, small-molecule CK2α inhibitor CX-4945 led to a dose-dependent inhibition of Notch1 transcriptional activity. Conversely, forced overexpression of CK2α resulted in an increase in Notch1 transcriptional activity. Finally, the inhibition of CK2α led to a reduced proportion of stem-like CD44 + /CD24− cell population. Thus, we report that the inhibition of CK2α down-regulates Notch1 signalling and subsequently reduces a cancer stem-like cell population in human lung cancer cells. Our data suggest that CK2α inhibitors may be beneficial to the lung cancer patients with activated Notch1 signalling. PMID:23651443

  5. Haploinsufficiency of the c-myc transcriptional repressor FIR, as a dominant negative-alternative splicing model, promoted p53-dependent T-cell acute lymphoblastic leukemia progression by activating Notch1.

    PubMed

    Matsushita, Kazuyuki; Kitamura, Kouichi; Rahmutulla, Bahityar; Tanaka, Nobuko; Ishige, Takayuki; Satoh, Mamoru; Hoshino, Tyuji; Miyagi, Satoru; Mori, Takeshi; Itoga, Sakae; Shimada, Hideaki; Tomonaga, Takeshi; Kito, Minoru; Nakajima-Takagi, Yaeko; Kubo, Shuji; Nakaseko, Chiaki; Hatano, Masahiko; Miki, Takashi; Matsuo, Masafumi; Fukuyo, Masaki; Kaneda, Atsushi; Iwama, Atsushi; Nomura, Fumio

    2015-03-10

    FUSE-binding protein (FBP)-interacting repressor (FIR) is a c-myc transcriptional suppressor. A splice variant of FIR that lacks exon 2 in the transcriptional repressor domain (FIRΔexon2) upregulates c-myc transcription by inactivating wild-type FIR. The ratio of FIRΔexon2/FIR mRNA was increased in human colorectal cancer and hepatocellular carcinoma tissues. Because FIRΔexon2 is considered to be a dominant negative regulator of FIR, FIR heterozygous knockout (FIR⁺/⁻) C57BL6 mice were generated. FIR complete knockout (FIR⁻/⁻) was embryonic lethal before E9.5; therefore, it is essential for embryogenesis. This strongly suggests that insufficiency of FIR is crucial for carcinogenesis. FIR⁺/⁻ mice exhibited prominent c-myc mRNA upregulation, particularly in the peripheral blood (PB), without any significant pathogenic phenotype. Furthermore, elevated FIRΔexon2/FIR mRNA expression was detected in human leukemia samples and cell lines. Because the single knockout of TP53 generates thymic lymphoma, FIR⁺/⁻TP53⁻/⁻ generated T-cell type acute lymphocytic/lymphoblastic leukemia (T-ALL) with increased organ or bone marrow invasion with poor prognosis. RNA-sequencing analysis of sorted thymic lymphoma cells revealed that the Notch signaling pathway was activated significantly in FIR⁺/⁻TP53⁻/⁻ compared with that in FIR⁺/⁺TP53⁻/⁻ mice. Notch1 mRNA expression in sorted thymic lymphoma cells was confirmed using qRT-PCR. In addition, flow cytometry revealed that c-myc mRNA was negatively correlated with FIR but positively correlated with Notch1 in sorted T-ALL/thymic lymphoma cells. Moreover, the knockdown of TP53 or c-myc using siRNA decreased Notch1 expression in cancer cells. In addition, an adenovirus vector encoding FIRΔexon2 cDNA increased bleomycin-induced DNA damage. Taken together, these data suggest that the altered expression of FIRΔexon2 increased Notch1 at least partially by activating c-Myc via a TP53-independent pathway. In

  6. Haploinsufficiency of the c-myc transcriptional repressor FIR, as a dominant negative-alternative splicing model, promoted p53-dependent T-cell acute lymphoblastic leukemia progression by activating Notch1

    PubMed Central

    Rahmutulla, Bahityar; Tanaka, Nobuko; Ishige, Takayuki; Satoh, Mamoru; Hoshino, Tyuji; Miyagi, Satoru; Mori, Takeshi; Itoga, Sakae; Shimada, Hideaki; Tomonaga, Takeshi; Kito, Minoru; Nakajima-Takagi, Yaeko; Kubo, Shuji; Nakaseko, Chiaki; Hatano, Masahiko; Miki, Takashi; Matsuo, Masafumi; Fukuyo, Masaki; Kaneda, Atsushi; Iwama, Atsushi; Nomura, Fumio

    2015-01-01

    FUSE-binding protein (FBP)-interacting repressor (FIR) is a c-myc transcriptional suppressor. A splice variant of FIR that lacks exon 2 in the transcriptional repressor domain (FIRΔexon2) upregulates c-myc transcription by inactivating wild-type FIR. The ratio of FIRΔexon2/FIR mRNA was increased in human colorectal cancer and hepatocellular carcinoma tissues. Because FIRΔexon2 is considered to be a dominant negative regulator of FIR, FIR heterozygous knockout (FIR+/−) C57BL6 mice were generated. FIR complete knockout (FIR−/−) was embryonic lethal before E9.5; therefore, it is essential for embryogenesis. This strongly suggests that insufficiency of FIR is crucial for carcinogenesis. FIR+/− mice exhibited prominent c-myc mRNA upregulation, particularly in the peripheral blood (PB), without any significant pathogenic phenotype. Furthermore, elevated FIRΔexon2/FIR mRNA expression was detected in human leukemia samples and cell lines. Because the single knockout of TP53 generates thymic lymphoma, FIR+/−TP53−/− generated T-cell type acute lymphocytic/lymphoblastic leukemia (T-ALL) with increased organ or bone marrow invasion with poor prognosis. RNA-sequencing analysis of sorted thymic lymphoma cells revealed that the Notch signaling pathway was activated significantly in FIR+/−TP53−/− compared with that in FIR+/+TP53−/− mice. Notch1 mRNA expression in sorted thymic lymphoma cells was confirmed using qRT-PCR. In addition, flow cytometry revealed that c-myc mRNA was negatively correlated with FIR but positively correlated with Notch1 in sorted T-ALL/thymic lymphoma cells. Moreover, the knockdown of TP53 or c-myc using siRNA decreased Notch1 expression in cancer cells. In addition, an adenovirus vector encoding FIRΔexon2 cDNA increased bleomycin-induced DNA damage. Taken together, these data suggest that the altered expression of FIRΔexon2 increased Notch1 at least partially by activating c-Myc via a TP53-independent pathway. In conclusion

  7. Cardioprotective actions of Notch1 against myocardial infarction via LKB1-dependent AMPK signaling pathway.

    PubMed

    Yang, Hui; Sun, Wanqing; Quan, Nanhu; Wang, Lin; Chu, Dongyang; Cates, Courtney; Liu, Quan; Zheng, Yang; Li, Ji

    2016-05-15

    AMP-activated protein kinase (AMPK) signaling pathway plays a pivotal role in intracellular adaptation to energy stress during myocardial ischemia. Notch1 signaling in the adult myocardium is also activated in response to ischemic stress. However, the relationship between Notch1 and AMPK signaling pathways during ischemia remains unclear. We hypothesize that Notch1 as an adaptive signaling pathway protects the heart from ischemic injury via modulating the cardioprotective AMPK signaling pathway. C57BL/6J mice were subjected to an in vivo ligation of left anterior descending coronary artery and the hearts from C57BL/6J mice were subjected to an ex vivo globe ischemia and reperfusion in the Langendorff perfusion system. The Notch1 signaling was activated during myocardial ischemia. A Notch1 γ-secretase inhibitor, dibenzazepine (DBZ), was intraperitoneally injected into mice to inhibit Notch1 signaling pathway by ischemia. The inhibition of Notch1 signaling by DBZ significantly augmented cardiac dysfunctions caused by myocardial infarction. Intriguingly, DBZ treatment also significantly blunted the activation of AMPK signaling pathway. The immunoprecipitation experiments demonstrated that an interaction between Notch1 and liver kinase beta1 (LKB1) modulated AMPK activation during myocardial ischemia. Furthermore, a ligand of Notch1 Jagged1 can significantly reduce cardiac damage caused by ischemia via activation of AMPK signaling pathway and modulation of glucose oxidation and fatty acid oxidation during ischemia and reperfusion. But Jagged1 did not have any cardioprotections on AMPK kinase dead transgenic hearts. Taken together, the results indicate that the cardioprotective effect of Notch1 against ischemic damage is mediated by AMPK signaling via an interaction with upstream LKB1.

  8. A novel Monoclonal Antibody against Notch1 Targets Leukemia-associated Mutant Notch1 and Depletes Therapy Resistant Cancer Stem Cells in Solid Tumors

    PubMed Central

    Sharma, Ankur; Gadkari, Rupali A; Ramakanth, Satthenapalli V; Padmanabhan, Krishnanand; Madhumathi, Davanam S; Devi, Lakshmi; Appaji, Lingappa; Aster, Jon C; Rangarajan, Annapoorni; Dighe, Rajan R

    2015-01-01

    Higher Notch signaling is known to be associated with hematological and solid cancers. We developed a potential immunotherapeutic monoclonal antibody (MAb) specific for the Negative Regulatory Region of Notch1 (NRR). The MAb604.107 exhibited higher affinity for the “Gain-of-function” mutants of Notch1 NRR associated with T Acute lymphoblastic Leukemia (T-ALL). Modeling of the mutant NRR with 12 amino-acid insertion demonstrated “opening” resulting in exposure of the S2-cleavage site leading to activated Notch1 signaling. The MAb, at low concentrations (1–2 μg/ml), inhibited elevated ligand-independent Notch1 signaling of NRR mutants, augmented effect of Thapsigargin, an inhibitor of mutant Notch1, but had no effect on the wild-type Notch1. The antibody decreased proliferation of the primary T-ALL cells and depleted leukemia initiating CD34/CD44 high population. At relatively high concentrations, (10–20 μg/ml), the MAb affected Notch1 signaling in the breast and colon cancer cell lines. The Notch-high cells sorted from solid-tumor cell lines exhibited characteristics of cancer stem cells, which were inhibited by the MAb. The antibody also increased the sensitivity to Doxorubucinirubicin. Further, the MAb impeded the growth of xenografts from breast and colon cancer cells potentiated regression of the tumors along with Doxorubucin. Thus, this antibody is potential immunotherapeutic tool for different cancers. PMID:26046801

  9. Notch1 endocytosis is induced by ligand and is required for signal transduction.

    PubMed

    Chapman, G; Major, J A; Iyer, K; James, A C; Pursglove, S E; Moreau, J L M; Dunwoodie, S L

    2016-01-01

    The Notch signalling pathway is widely utilised during embryogenesis in situations where cell-cell interactions are important for cell fate specification and differentiation. DSL ligand endocytosis into the ligand-expressing cell is an important aspect of Notch signalling because it is thought to supply the force needed to separate the Notch heterodimer to initiate signal transduction. A functional role for receptor endocytosis during Notch signal transduction is more controversial. Here we have used live-cell imaging to examine trafficking of the Notch1 receptor in response to ligand binding. Contact with cells expressing ligands induced internalisation and intracellular trafficking of Notch1. Notch1 endocytosis was accompanied by transendocytosis of ligand into the Notch1-expressing signal-receiving cell. Ligand caused Notch1 endocytosis into SARA-positive endosomes in a manner dependent on clathrin and dynamin function. Moreover, inhibition of endocytosis in the receptor-expressing cell impaired ligand-induced Notch1 signalling. Our findings resolve conflicting observations from mammalian and Drosophila studies by demonstrating that ligand-dependent activation of Notch1 signalling requires receptor endocytosis. Endocytosis of Notch1 may provide a force on the ligand:receptor complex that is important for potent signal transduction.

  10. Inhibition of fibroblast growth by Notch1 signaling is mediated by induction of Wnt11-dependent WISP-1.

    PubMed

    Liu, Zhao-Jun; Li, Yan; Tan, Yurong; Xiao, Min; Zhang, Jialin; Radtke, Freddy; Velazquez, Omaida C

    2012-01-01

    Fibroblasts are an integral component of stroma and important source of growth factors and extracellular matrix (ECM). They play a prominent role in maintaining tissue homeostasis and in wound healing and tumor growth. Notch signaling regulates biological function in a variety of cells. To elucidate the physiological function of Notch signaling in fibroblasts, we ablated Notch1 in mouse (Notch1(Flox/Flox)) embryonic fibroblasts (MEFs). Notch1-deficient (Notch1(-/-)) MEFs displayed faster growth and motility rate compared to Notch1(Flox/Flox) MEFs. Such phenotypic changes, however, were reversible by reconstitution of Notch1 activation via overexpression of the intracellular domain of Notch1 (NICD1) in Notch1-deficient MEFs. In contrast, constitutive activation of Notch1 signaling by introducing NICD1 into primary human dermal fibroblasts (FF2441), which caused pan-Notch activation, inhibited cell growth and motility, whereas cellular inhibition was relievable when the Notch activation was countered with dominant-negative mutant of Master-mind like 1 (DN-MAML-1). Functionally, "Notch-activated" stromal fibroblasts could inhibit tumor cell growth/invasion. Moreover, Notch activation induced expression of Wnt-induced secreted proteins-1 (WISP-1/CCN4) in FF2441 cells while deletion of Notch1 in MEFs resulted in an opposite effect. Notably, WISP-1 suppressed fibroblast proliferation, and was responsible for mediating Notch1's inhibitory effect since siRNA-mediated blockade of WISP-1 expression could relieve cell growth inhibition. Notch1-induced WISP-1 expression appeared to be Wnt11-dependent, but Wnt1-independent. Blockade of Wnt11 expression resulted in decreased WISP-1 expression and liberated Notch-induced cell growth inhibition. These findings indicated that inhibition of fibroblast proliferation by Notch pathway activation is mediated, at least in part, through regulating Wnt1-independent, but Wnt11-dependent WISP-1 expression.

  11. Notch1 signaling stimulates proliferation of immature cardiomyocytes.

    PubMed

    Collesi, Chiara; Zentilin, Lorena; Sinagra, Gianfranco; Giacca, Mauro

    2008-10-01

    The identification of the molecular mechanisms controlling cardiomyocyte proliferation during the embryonic, fetal, and early neonatal life appears of paramount interest in regard to exploiting this information to promote cardiac regeneration. Here, we show that the proliferative potential of neonatal rat cardiomyocytes is powerfully stimulated by the sustained activation of the Notch pathway. We found that Notch1 is expressed in proliferating ventricular immature cardiac myocytes (ICMs) both in vitro and in vivo, and that the number of Notch1-positive cells in the heart declines with age. Notch1 expression in ICMs paralleled the expression of its Jagged1 ligand on non-myocyte supporting cells. The inhibition of Notch signaling in ICMs blocked their proliferation and induced apoptosis; in contrast, its activation by Jagged1 or by the constitutive expression of its activated form using an adeno-associated virus markedly stimulated proliferative signaling and promoted ICM expansion. Maintenance or reactivation of Notch signaling in cardiac myocytes might represent an interesting target for innovative regenerative therapy. PMID:18824567

  12. Sox9 mediates Notch1-induced mesenchymal features in lung adenocarcinoma.

    PubMed

    Capaccione, Kathleen M; Hong, Xuehui; Morgan, Katherine M; Liu, Wenyu; Bishop, J Michael; Liu, LianXin; Markert, Elke; Deen, Malik; Minerowicz, Christine; Bertino, Joseph R; Allen, Thaddeus; Pine, Sharon R

    2014-06-15

    Sox9 has gained increasing importance both functionally and as a prognostic factor in cancer. We demonstrate a functional role for Sox9 in inducing a mesenchymal phenotype in lung ADC. We show that Sox9 mRNA and protein are overexpressed in lung ADC, particularly those with KRAS mutations. Sox9 expression correlated with the Notch target gene Hes1, and numerous other Notch pathway components. We observed that Sox9 is a potent inducer of lung cancer cell motility and invasion, and a negative regulator of E-cadherin, a key protein that is lost during epithelial-mesenchymal transition (EMT). Moreover, we show that Notch1 signaling directly regulates Sox9 expression through a SOX9 promoter binding site, independently of the TGF-β pathway, and that Sox9 participates in Notch-1 induced cell motility, cell invasion, and loss of E-cadherin expression. Together, the results identify a new functional role for a Notch1-Sox9 signaling axis in lung ADC that may explain the correlation of Sox9 with tumor progression, higher tumor grade, and poor lung cancer survival. In addition to Notch and TGF-β, Sox9 also acts downstream of NF-κB, BMP, EGFR, and Wnt/β-catenin signaling. Thus, Sox9 could potentially act as a hub to mediate cross-talk among key oncogenic pathways in lung ADC. Targeting Sox9 expression or transcriptional activity could potentially reduce resistance to targeted therapy for lung ADC caused by pathway redundancy. PMID:25004243

  13. Haploinsufficiency of the NOTCH1 Receptor as a Cause of Adams-Oliver Syndrome with Variable Cardiac Anomalies

    PubMed Central

    Southgate, Laura; Sukalo, Maja; Karountzos, Anastasios S.V.; Taylor, Edward J.; Collinson, Claire S.; Ruddy, Deborah; Snape, Katie M.; Dallapiccola, Bruno; Joss, Shelagh; Brancati, Francesco; Digilio, M. Cristina; Graul-Neumann, Luitgard M.; Salviati, Leonardo; Coerdt, Wiltrud; Jacquemin, Emmanuel; Wuyts, Wim; Zenker, Martin

    2015-01-01

    Background Adams-Oliver syndrome (AOS) is a rare disorder characterized by congenital limb defects and scalp cutis aplasia. In a proportion of cases, notable cardiac involvement is also apparent. Despite recent advances in the understanding of the genetic basis of AOS, for the majority of affected subjects the underlying molecular defect remains unresolved. This study aimed to identify novel genetic determinants of AOS. Methods and Results Whole-exome sequencing was performed for 12 probands, each with a clinical diagnosis of AOS. Analyses led to the identification of novel heterozygous truncating NOTCH1 mutations (c.1649dupA and c.6049_6050delTC) in two kindreds in which AOS was segregating as an autosomal dominant trait. Screening a cohort of 52 unrelated AOS subjects, we detected 8 additional unique NOTCH1 mutations, including three de novo amino-acid substitutions, all within the ligand-binding domain. Congenital heart anomalies were noted in 47% (8/17) of NOTCH1-positive probands and affected family members. In leucocyte-derived RNA from subjects harboring NOTCH1 extracellular domain mutations, we observed significant reduction of NOTCH1 expression, suggesting instability and degradation of mutant mRNA transcripts by the cellular machinery. Transient transfection of mutagenized NOTCH1 missense constructs also revealed significant reduction in gene expression. Mutant NOTCH1 expression was associated with down-regulation of the Notch target genes HEY1 and HES1, indicating that NOTCH1-related AOS arises through dysregulation of the Notch signaling pathway. Conclusions These findings highlight a key role for NOTCH1 across a range of developmental anomalies that include cardiac defects, and implicate NOTCH1 haploinsufficiency as a likely molecular mechanism for this group of disorders. PMID:25963545

  14. Notch1 Regulates Hippocampal Plasticity Through Interaction with the Reelin Pathway, Glutamatergic Transmission and CREB Signaling

    PubMed Central

    Brai, Emanuele; Marathe, Swananda; Astori, Simone; Fredj, Naila Ben; Perry, Elisabeth; Lamy, Christophe; Scotti, Alessandra; Alberi, Lavinia

    2015-01-01

    Notch signaling plays a crucial role in adult brain function such as synaptic plasticity, memory and olfaction. Several reports suggest an involvement of this pathway in neurodegenerative dementia. Yet, to date, the mechanism underlying Notch activity in mature neurons remains unresolved. In this work, we investigate how Notch regulates synaptic potentiation and contributes to the establishment of memory in mice. We observe that Notch1 is a postsynaptic receptor with functional interactions with the Reelin receptor, apolipoprotein E receptor 2 (ApoER2) and the ionotropic receptor, N-methyl-D-aspartate receptor (NMDAR). Targeted loss of Notch1 in the hippocampal CA fields affects Reelin signaling by influencing Dab1 expression and impairs the synaptic potentiation achieved through Reelin stimulation. Further analysis indicates that loss of Notch1 affects the expression and composition of the NMDAR but not AMPAR. Glutamatergic signaling is further compromised through downregulation of CamKII and its secondary and tertiary messengers resulting in reduced cAMP response element-binding (CREB) signaling. Our results identify Notch1 as an important regulator of mechanisms involved in synaptic plasticity and memory formation. These findings emphasize the possible involvement of this signaling receptor in dementia. Highlights In this paper, we propose a mechanism for Notch1-dependent plasticity that likely underlies the function of Notch1 in memory formation: Notch1 interacts with another important developmental pathway, the Reelin cascade. Notch1 regulates both NMDAR expression and composition. Notch1 influences a cascade of cellular events culminating in CREB activation. PMID:26635527

  15. Reduction of NOTCH1 expression pertains to maturation abnormalities of keratinocytes in squamous neoplasms.

    PubMed

    Sakamoto, Kei; Fujii, Takuma; Kawachi, Hiroshi; Miki, Yoshio; Omura, Ken; Morita, Kei-ichi; Kayamori, Kou; Katsube, Ken-ichi; Yamaguchi, Akira

    2012-05-01

    Notch is a transmembrane receptor functioning in the determination of cell fate. Abnormal Notch signaling promotes tumor development, showing either oncogenic or tumor suppressive activity. The uncertainty about the exact role of Notch signaling, partially, stems from inconsistencies in descriptions of Notch expression in human cancers. Here, we clarified basal-cell dominant expression of NOTCH1 in squamous epithelium. NOTCH1 was downregulated in squamous neoplasms of oral mucosa, esophagus and uterine cervix, compared with the normal basal cells, although the expression tended to be retained in cervical lesions. NOTCH1 downregulation was observed even in precancers, and there was little difference between cancers and high-grade precancerous lesions, suggesting its minor contribution to cancer-specific events such as invasion. In culture experiments, reduction of NOTCH1 expression resulted in downregulation of keratin 13 and keratin 15, and upregulation of keratin 17, and NOTCH1 knockdown cells formed a dysplastic stratified epithelium mimicking a precancerous lesion. The NOTCH1 downregulation and the concomitant alterations of those keratin expressions were confirmed in the squamous neoplasms both by immunohistochemical and cDNA microarray analyses. Our data indicate that reduction of NOTCH1 expression directs the basal cells to cease terminal differentiation and to form an immature epithelium, thereby playing a major role in the histopathogenesis of epithelial dysplasia. Furthermore, downregulation of NOTCH1 expression seems to be an inherent mechanism for switching the epithelium from a normal and mature state to an activated and immature state, suggesting its essential role in maintaining the epithelial integrity.

  16. Targeting Notch1 inhibits invasion and angiogenesis of human breast cancer cells via inhibition Nuclear Factor-κB signaling.

    PubMed

    Liu, Yuan; Su, Chuanfu; Shan, Yuqing; Yang, Shouxiang; Ma, Guifeng

    2016-01-01

    Notch-1, a type-1 transmembrane protein, plays critical roles in the pathogenesis and progression of human malignancies, including breast cancer; however, the precise mechanism by which Notch-1 causes tumor cell invasion and angiogenesis remain unclear. Nuclear factor-κB (NF-κB), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMP) are critically involved in the processes of tumor cell invasion and metastasis, we investigated whether targeting Notch-1 could be mechanistically associated with the down-regulation of NF-κB, IL-8, VEGF, and MMP-9, resulting in the inhibition of invasion and angiogenesis of breast cancer cells. Our data showed that down-regulation of Notch-1 leads to the inactivation of NF-κB activity and inhibits the expression of its target genes, such as IL-8, VEGF and MMP-9. We also found that down-regulation of Notch-1 decreased cell invasion, and vice versa Consistent with these results, we also found that the down-regulation of Notch-1 not only decreased MMP-9 mRNA and its protein expression but also inhibited MMP-9 active form. Moreover, conditioned medium from Notch-1 siRNA-transfected breast cancer cells showed reduced levels of IL-8 and VEGF and, in turn, inhibited the tube formation of HUVECs, suggesting that down-regulation of Notch-1 leads to the inhibition of angiogenesis. Furthermore, conditioned medium from Notch-1 cDNA-transfected breast cancer cells showed increased levels of IL-8 and VEGF and, in turn, promoted the tube formation of HUVECs, suggesting that Notch-1 overexpression leads to the promotion of angiogenesis.We therefore concluded that down-regulation of Notch-1 leads to the inactivation NF-κB and its target genes (IL-8, MMP-9 and VEGF), resulting in the inhibition of invasion and angiogenesis.

  17. Targeting Notch1 inhibits invasion and angiogenesis of human breast cancer cells via inhibition Nuclear Factor-κB signaling

    PubMed Central

    Liu, Yuan; Su, Chuanfu; Shan, Yuqing; Yang, Shouxiang; Ma, Guifeng

    2016-01-01

    Notch-1, a type-1 transmembrane protein, plays critical roles in the pathogenesis and progression of human malignancies, including breast cancer; however, the precise mechanism by which Notch-1 causes tumor cell invasion and angiogenesis remain unclear. Nuclear factor-κB (NF-κB), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMP) are critically involved in the processes of tumor cell invasion and metastasis, we investigated whether targeting Notch-1 could be mechanistically associated with the down-regulation of NF-κB, IL-8, VEGF, and MMP-9, resulting in the inhibition of invasion and angiogenesis of breast cancer cells. Our data showed that down-regulation of Notch-1 leads to the inactivation of NF-κB activity and inhibits the expression of its target genes, such as IL-8, VEGF and MMP-9. We also found that down-regulation of Notch-1 decreased cell invasion, and vice versa Consistent with these results, we also found that the down-regulation of Notch-1 not only decreased MMP-9 mRNA and its protein expression but also inhibited MMP-9 active form. Moreover, conditioned medium from Notch-1 siRNA-transfected breast cancer cells showed reduced levels of IL-8 and VEGF and, in turn, inhibited the tube formation of HUVECs, suggesting that down-regulation of Notch-1 leads to the inhibition of angiogenesis. Furthermore, conditioned medium from Notch-1 cDNA-transfected breast cancer cells showed increased levels of IL-8 and VEGF and, in turn, promoted the tube formation of HUVECs, suggesting that Notch-1 overexpression leads to the promotion of angiogenesis.We therefore concluded that down-regulation of Notch-1 leads to the inactivation NF-κB and its target genes (IL-8, MMP-9 and VEGF), resulting in the inhibition of invasion and angiogenesis. PMID:27398151

  18. Expression of Notch1 Correlates with Breast Cancer Progression and Prognosis

    PubMed Central

    Wu, Hua; Xu, Hanxiao; Han, Na; Chu, Qian; Yu, Shiying; Chen, Yuan; Wu, Kongming

    2015-01-01

    Various studies have evaluated the significance of Notch1 expression in breast cancer, but the results have ever been disputed. By using 21 studies involving 3867 patients, this meta-analysis revealed that the expression of Notch1 was significantly higher in breast cancer than in normal tissues (OR=7.21; 95%CI, 4.7-11.07) and that higher Notch1 expression was associated with transition from ductal carcinoma in situ (DCIS) to invasive cancer (OR=3.75; 95% CI, 1.8-7.78). Higher Notch1 activity was observed in the basal subtype of breast cancer (OR=2.53; 95% CI, 1.18-5.43). Moreover, patients with Notch1 overexpression exhibited significantly worse overall and recurrence-free survival. Our meta-analysis suggests that Notch inhibitors may be useful in blocking the early progression of DCIS and that the outcomes of clinical trials for Notch1-targeting therapeutics could be improved by the molecular stratification of breast cancer patients. PMID:26121683

  19. Heat shock protein 70 (Hsp70) interacts with the Notch1 intracellular domain and contributes to the activity of Notch signaling in myelin-reactive CD4 T cells.

    PubMed

    Juryńczyk, Maciej; Lewkowicz, Przemysław; Domowicz, Małgorzata; Mycko, Marcin P; Selmaj, Krzysztof W

    2015-10-15

    Notch receptors (Notch1-4) are involved in the differentiation of CD4 T cells and the development of autoimmunity. Mechanisms regulating Notch signaling in CD4 T cells are not fully elucidated. In this study we investigated potential crosstalk between Notch pathway molecules and heat shock protein 70 (Hsp70), the major intracellular chaperone involved in the protein transport during immune responses and other stress conditions. Using Hsp70(-/-) mice we found that Hsp70 is critical for up-regulation of NICD1 and induction of Notch target genes in Jagged1- and Delta-like1-stimulated CD4 T cells. Co-immunoprecipitation analysis of wild-type CD4 T cells stimulated with either Jagged1 or Delta-like1 showed a direct interaction between NICD1 and Hsp70. Both molecules co-localized within the nucleus of CD4 T cells stimulated with Notch ligands. Molecular interaction and nuclear colocalization of NICD1 and Hsp70 were also detected in CD4 T cells reactive against myelin oligodendrocyte glycoprotein (MOG)35-55, which showed Hsp70-dependent up-regulation of both NICD1 and Notch target genes. In conclusion, we demonstrate for the first time that Hsp70 interacts with NICD1 and contributes to the activity of Notch signaling in CD4 T cells. Interaction between Hsp70 and NICD1 may represent a novel mechanism regulating Notch signaling in activated CD4 T cells.

  20. Developmental Exposure To 2,3,7,8 Tetrachlorodibenzo-p-Dioxin Attenuates Later-Life Notch1-Mediated T Cell Development and Leukemogenesis

    PubMed Central

    Ahrenhoerster, Lori S.; Leuthner, Tess C.; Tate, Everett R.; Lakatos, Peter A.; Laiosa, Michael D.

    2015-01-01

    Over half of T-cell acute lymphoblastic leukemia (T-ALL) patients have activating mutations in the Notch gene. Moreover, the contaminant 2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD) is a known carcinogen that mediates its toxicity through the aryl hydrocarbon receptor (AHR), and crosstalk between activated AHR and Notch signaling pathways has previously been observed. Given the importance of Notch signaling in thymocyte development and T-ALL disease progression, we hypothesized that the activated AHR potentiates disease initiation and progression in an in vivo model of Notch1-induced thymoma. This hypothesis was tested utilizing adult and developmental exposure paradigms to TCDD in mice expressing a constitutively active Notch1 transgene (NotchICN-TG). Following exposure of adult NotchICN-TG mice to a single high dose of TCDD, we observed a significant increase in the efficiency of CD8 thymocyte generation. We next exposed pregnant mice to 3μg/kg of TCDD throughout gestation and lactation to elucidate effects of developmental AHR activation on later-life T cell development and T-ALL-like thymoma susceptibility induced by Notch1. We found that the vehicle-exposed NotchICN-TG offspring have a peripheral T-cell pool heavily biased toward the CD4 lineage, while TCDD-exposed NotchICN-TG offspring were biased toward the CD8 lineage. Furthermore, while the vehicle-exposed NotchICN-TG mice showed increased splenomegaly and B to T cell ratios indicative of disease, mice developmentally exposed to TCDD were largely protected from disease. These studies support a model where developmental AHR activation attenuates later-life Notch1-dependent impacts on thymocyte development and disease progression. PMID:25585350

  1. Notch1-mediated signaling regulates proliferation of porcine satellite cells (PSCs).

    PubMed

    Qin, Lili; Xu, Jian; Wu, Zhenfang; Zhang, Zhe; Li, Jiaqi; Wang, Chong; Long, Qiaoming

    2013-02-01

    Notch signaling is an evolutionarily conserved cell-cell communication mechanism involved in the regulation of cell proliferation, differentiation and fate decisions of mammalian cells. In the present study, we investigated the possible requirement for Notch signaling in the proliferation and differentiation of porcine satellite cells. We show that Notch1, 2 and 3 are expressed in cultured porcine satellite cells. Knock-down of NOTCH1, but not NOTCH2 and NOTCH3, decreases the proliferation of porcine satellite cells. In contrast, enhancement of NOTCH1 expression via treatment of porcine satellite cells with recombinant NF-κB increases the proliferation of porcine satellite cells. The alteration of porcine satellite cell proliferation is associated with significant changes in the expression of cell cycle related genes (cyclin B1, D1, D2, E1 and p21), myogenic regulatory factors (MyoD and myogenin) and the Notch effector Hes5. In addition, alteration of Notch1 expression in porcine satellite cells causes changes in the expression of GSK3β-3. Taken together, these findings suggest that of the four notch-related genes, Notch1is likely to be required for regulating the proliferation and therefore the maintenance of porcine satellite cells in vivo, and do so through activation of the Notch effector gene Hes5. PMID:23160004

  2. Notch1-mediated signaling regulates proliferation of porcine satellite cells (PSCs).

    PubMed

    Qin, Lili; Xu, Jian; Wu, Zhenfang; Zhang, Zhe; Li, Jiaqi; Wang, Chong; Long, Qiaoming

    2013-02-01

    Notch signaling is an evolutionarily conserved cell-cell communication mechanism involved in the regulation of cell proliferation, differentiation and fate decisions of mammalian cells. In the present study, we investigated the possible requirement for Notch signaling in the proliferation and differentiation of porcine satellite cells. We show that Notch1, 2 and 3 are expressed in cultured porcine satellite cells. Knock-down of NOTCH1, but not NOTCH2 and NOTCH3, decreases the proliferation of porcine satellite cells. In contrast, enhancement of NOTCH1 expression via treatment of porcine satellite cells with recombinant NF-κB increases the proliferation of porcine satellite cells. The alteration of porcine satellite cell proliferation is associated with significant changes in the expression of cell cycle related genes (cyclin B1, D1, D2, E1 and p21), myogenic regulatory factors (MyoD and myogenin) and the Notch effector Hes5. In addition, alteration of Notch1 expression in porcine satellite cells causes changes in the expression of GSK3β-3. Taken together, these findings suggest that of the four notch-related genes, Notch1is likely to be required for regulating the proliferation and therefore the maintenance of porcine satellite cells in vivo, and do so through activation of the Notch effector gene Hes5.

  3. Non-epigenetic function of HDAC8 in regulating breast cancer stem cells by maintaining Notch1 protein stability

    PubMed Central

    Chao, Min-Wu; Chu, Po-Chen; Chuang, Hsiao-Ching; Shen, Fang-Hsiu; Chou, Chih-Chien; Hsu, En-Chi; Himmel, Lauren E.; Huang, Han-Li; Tu, Huang-Ju; Kulp, Samuel K.; Teng, Che-Ming; Chen, Ching-Shih

    2016-01-01

    Here, we report a novel non-epigenetic function of histone deacetylase (HDAC) 8 in activating cancer stem cell (CSC)-like properties in breast cancer cells by enhancing the stability of Notch1 protein. The pan-HDAC inhibitors AR-42 and SAHA, and the class I HDAC inhibitor depsipeptide, suppressed mammosphere formation and other CSC markers by reducing Notch1 expression in MDA-MB-231 and SUM-159 cells. Interrogation of individual class I isoforms (HDAC1–3 and 8) using si/shRNA-mediated knockdown, ectopic expression and/or pharmacological inhibition revealed HDAC8 to be the primary mediator of this drug effect. This suppression of Notch1 in response to HDAC8 inhibition was abrogated by the proteasome inhibitor MG132 and siRNA-induced silencing of Fbwx7, indicating Notch1 suppression occurred through proteasomal degradation. However, co-immunoprecipitation analysis indicated that HDAC8 did not form complexes with Notch1 and HDAC inhibition had no effect on Notch1 acetylation. In a xenograft tumor model, the tumorigenicity of breast cancer cells was decreased by HDAC8 knockdown. These findings suggest the therapeutic potential of HDAC8 inhibition to suppress Notch1 signaling in breast cancer. PMID:26625202

  4. Non-epigenetic function of HDAC8 in regulating breast cancer stem cells by maintaining Notch1 protein stability.

    PubMed

    Chao, Min-Wu; Chu, Po-Chen; Chuang, Hsiao-Ching; Shen, Fang-Hsiu; Chou, Chih-Chien; Hsu, En-Chi; Himmel, Lauren E; Huang, Han-Li; Tu, Huang-Ju; Kulp, Samuel K; Teng, Che-Ming; Chen, Ching-Shih

    2016-01-12

    Here, we report a novel non-epigenetic function of histone deacetylase (HDAC) 8 in activating cancer stem cell (CSC)-like properties in breast cancer cells by enhancing the stability of Notch1 protein. The pan-HDAC inhibitors AR-42 and SAHA, and the class I HDAC inhibitor depsipeptide, suppressed mammosphere formation and other CSC markers by reducing Notch1 expression in MDA-MB-231 and SUM-159 cells. Interrogation of individual class I isoforms (HDAC1-3 and 8) using si/shRNA-mediated knockdown, ectopic expression and/or pharmacological inhibition revealed HDAC8 to be the primary mediator of this drug effect. This suppression of Notch1 in response to HDAC8 inhibition was abrogated by the proteasome inhibitor MG132 and siRNA-induced silencing of Fbwx7, indicating Notch1 suppression occurred through proteasomal degradation. However, co-immunoprecipitation analysis indicated that HDAC8 did not form complexes with Notch1 and HDAC inhibition had no effect on Notch1 acetylation. In a xenograft tumor model, the tumorigenicity of breast cancer cells was decreased by HDAC8 knockdown. These findings suggest the therapeutic potential of HDAC8 inhibition to suppress Notch1 signaling in breast cancer.

  5. Notch1 induces epithelial-mesenchymal transition and the cancer stem cell phenotype in breast cancer cells and STAT3 plays a key role.

    PubMed

    Zhang, Xiaojin; Zhao, Xiaoai; Shao, Shan; Zuo, Xiaoxiao; Ning, Qian; Luo, Minna; Gu, Shanzhi; Zhao, Xinhan

    2015-03-01

    Breast cancer is the most common malignancy in women. The Notch signaling pathway has been shown to be associated with the development and progression of many human cancers, including breast cancer, but the precise mechanism remains unknown. Here, the influence of Notch1 signaling in mammary epithelial cells was studied. We showed that Notch1 promotes proliferation in MCF7 and MCF10A cells. Transwell assay indicated that Notch1 overexpression promotes cell migration and the invasion of breast cancer cells. We showed that MCF7 and MCF10A cells overexpressing Notch1 acquired features of epithelial-mesenchymal transition (EMT) and displayed a cancer stem cell (CSC) phenotype. The expression levels of the epithelial markers E-cadherin and occludin were decreased, while the expression levels of the mesenchymal markers N-cadherin, vimentin and fibronectin were increased in cells overexpressing Notch1. We demonstrated that Notch1 induced phosphorylation of the signal transducer and activator of transcription 3 (STAT3) in breast cancer cells and increased the expression of p65 and interleukin (IL)-1β. Inhibition of STAT3 activity by JSI124 reduced the expression of p65 and IL-1. Treatment of MCF7-notch1 and MCF10A-notch1 cells with JSI124 also reduced the expression of N-cadherin, markers of epithelial mesenchymal transition and increased the expression of E-cadherin. Our results suggest that Notch1 promotes EMT and the CSC phenotype through induction of STAT3.

  6. Developmental exposure to 2,3,7,8 tetrachlorodibenzo-p-dioxin attenuates later-life Notch1-mediated T cell development and leukemogenesis

    SciTech Connect

    Ahrenhoerster, Lori S.; Leuthner, Tess C.; Tate, Everett R.; Lakatos, Peter A.; Laiosa, Michael D.

    2015-03-01

    Over half of T cell acute lymphoblastic leukemia (T-ALL) patients have activating mutations in the Notch gene. Moreover, the contaminant 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) is a known carcinogen that mediates its toxicity through the aryl hydrocarbon receptor (AHR), and crosstalk between activated AHR and Notch signaling pathways has previously been observed. Given the importance of Notch signaling in thymocyte development and T-ALL disease progression, we hypothesized that the activated AHR potentiates disease initiation and progression in an in vivo model of Notch1-induced thymoma. This hypothesis was tested utilizing adult and developmental exposure paradigms to TCDD in mice expressing a constitutively active Notch1 transgene (Notch{sup ICN-TG}). Following exposure of adult Notch{sup ICN-TG} mice to a single high dose of TCDD, we observed a significant increase in the efficiency of CD8 thymocyte generation. We next exposed pregnant mice to 3 μg/kg of TCDD throughout gestation and lactation to elucidate effects of developmental AHR activation on later-life T cell development and T-ALL-like thymoma susceptibility induced by Notch1. We found that the vehicle-exposed Notch{sup ICN-TG} offspring have a peripheral T cell pool heavily biased toward the CD4 lineage, while TCDD-exposed Notch{sup ICN-TG} offspring were biased toward the CD8 lineage. Furthermore, while the vehicle-exposed NotchICN-TG mice showed increased splenomegaly and B to T cell ratios indicative of disease, mice developmentally exposed to TCDD were largely protected from disease. These studies support a model where developmental AHR activation attenuates later-life Notch1-dependent impacts on thymocyte development and disease progression. - Highlights: • Adult mice exposed to 30 μg/kg TCDD have higher efficiency of CD8 thymocyte generation. • Mice carrying a constitutively active Notch transgene were exposed to 3 μg/kg TCDD throughout development. • Progression of Notch

  7. Immunohistochemical expression of aberrant Notch-1 signaling in vitiligo: an implication for pathogenesis.

    PubMed

    Seleit, Iman; Bakry, Ola Ahmed; Abdou, Asmaa Gaber; Dawoud, Noha Mohammed

    2014-06-01

    The etiopathogenetic mechanisms leading to pigment loss in vitiligo are not fully understood. Notch signaling is required for development and maintenance of melanocyte lineage and acts as a key component among keratinocyte-melanocyte interactions. The current study aimed to investigate the possible role of Notch signaling and its effect on the whole melanocyte lineage in vitiligo and correlating it with the different clinicopathologic parameters. Using immunohistochemical technique, Notch-1 expression was evaluated in 50 lesional and 20 perilesional biopsies of patients with vitiligo in comparison with 20 normal skin biopsies as a control group. Lesional biopsies were stained with human melanoma black-45 and tyrosinase-related protein-2 to demonstrate the melanocyte lineage. Membranous and/or nuclear expression of Notch-1 was in favor of control and perilesional skin, whereas cytoplasmic expression appeared only in vitiliginous lesions (P < .05). Membranous and/or nuclear expression of Notch-1 was significantly associated with epidermal human melanoma black-45 positivity (P = .01) and percentage of expression in both epidermis (P = .02) and hair follicles (P = .03) of lesional skin. Cytoplasmic pattern of Notch-1 expression in epidermis was significantly found in lesions with white hair (P = .04) and in cases with marked keratinocyte vacuolization (P = .03). Segmental and acrofacial vitiligo were associated with mild to moderate Notch-1 intensity, whereas generalized vitiligo was associated with strong intensity of expression (P = .02). In conclusion, Notch-1 signaling is inactivated in vitiligo with consequent loss of epidermal and/or follicular active melanocytes. Aberrant Notch signaling in vitiliginous white hair and acral and segmental vitiligo may be the cause of their treatment resistance.

  8. Notch-1 expression levels in 3T3-L1 cells influence ras signaling and transformation by oncogenic ras.

    PubMed

    Ruiz-Hidalgo, M J; Garcés, C; Laborda, J

    1999-04-01

    Notch proteins participate in interactions between several cell types involved on the specification of numerous cell fates during development. We previously showed that enforced downregulation of Notch-1 expression prevented adipogenesis of 3T3-L1 cells. Since adipogenesis of 3T3-L1 cells can be induced by oncogenic ras, we studied whether this was also the case in 3T3-L1 cells with decreased levels of Notch-1 expression. We found that oncogenic ras induces transformation and not differentiation of 3T3-L1 cells with diminished levels of Notch-1. This result suggests that Notch-1 is implicated in the interpretation of signals leading to activation of p21 Ras.

  9. Targeting of miR9/NOTCH1 interaction reduces metastatic behavior in triple-negative breast cancer.

    PubMed

    Mohammadi-Yeganeh, Samira; Mansouri, Ardalan; Paryan, Mahdi

    2015-11-01

    Many reports have indicated deregulation of a variety of microRNAs (miRNAs) in human cancers. In this study, we appraised miR-9 correlation with NOTCH1 involved in Notch signaling in metastatic breast cancer. The Notch signaling pathway has been approved to be associated with the development and progression of many human cancers, including breast cancer, but the precise mechanism has remained unknown. To the best of our knowledge, this is the first study that introduces miR-9 and NOTCH1 correlation as an effective factor in breast cancer. We found that miR-9 expression was decreased in MDA-MB-231 breast cancer cells compared with MCF-10A normal breast cell line. However, NOTCH1 was upregulated in the metastatic breast cancer cells. Furthermore, luciferase assay revealed a significant inverse correlation between miR-9 and NOTCH1. Overexpression of Notch signaling via Notch1 intracellular domain in MDA-MB-231 cell line was suppressed by lentiviruses expressing miR-9. Taken together, the results obtained by MTT, flow cytometry, migration, and wound healing assays showed that it is possible to inhibit metastasis and induce pro-apoptotic state by induction of miR-9 expression in MDA-MB-231 cells but with no effect on cell proliferation. These results shows that miR-9, by direct targeting of NOTCH1, can reveal a suppressor-like activity in metastatic breast cancer cells.

  10. MiR-433 inhibits retinoblastoma malignancy by suppressing Notch1 and PAX6 expression.

    PubMed

    Li, Xiaohua; Yang, Lan; Shuai, Tianjiao; Piao, Tianhua; Wang, Rui

    2016-08-01

    Retinoblastoma (RB) is the most frequent primary intraocular cancer. It has been demonstrated by previous studies that retinoblastoma is initiated primarily by the inactivation of the retinoblastoma Rb1 gene in retinal cells. However, additional genetic alterations than Rb1 mutation could play important roles in the process of transforming benign retinal cells into retinoblastoma tumor cells. In this study, we identified that microRNA miR-433 is one of such genetic factors. We found that the expression levels of miR-433 were downregulated in RB tissues. We also determined that miR-433 negatively regulated RB cell proliferation, migration and invasion, and induced cell cycle arrest and apoptosis of RB cells. We used bioinformatics method to predict and confirmed that Notch1 and PAX6 were miR-433 target genes in RB cells. Importantly, we demonstrated that restoration of Notch1 and PAX6 expression partially rescued the inhibition of cell proliferation and metastasis induced by miR-433 overexpression, suggesting that miR-433 regulates RB cell proliferation and metastasis through suppressing the expression of Notch1 and PAX6. PMID:27470361

  11. Notch1–STAT3–ETBR signaling axis controls reactive astrocyte proliferation after brain injury

    PubMed Central

    LeComte, Matthew D.; Shimada, Issei S.; Sherwin, Casey; Spees, Jeffrey L.

    2015-01-01

    Defining the signaling network that controls reactive astrogliosis may provide novel treatment targets for patients with diverse CNS injuries and pathologies. We report that the radial glial cell antigen RC2 identifies the majority of proliferating glial fibrillary acidic protein-positive (GFAP+) reactive astrocytes after stroke. These cells highly expressed endothelin receptor type B (ETBR) and Jagged1, a Notch1 receptor ligand. To study signaling in adult reactive astrocytes, we developed a model based on reactive astrocyte-derived neural stem cells isolated from GFAP-CreER-Notch1 conditional knockout (cKO) mice. By loss- and gain-of-function studies and promoter activity assays, we found that Jagged1/Notch1 signaling increased ETBR expression indirectly by raising the level of phosphorylated signal transducer and activator of transcription 3 (STAT3), a previously unidentified EDNRB transcriptional activator. Similar to inducible transgenic GFAP-CreER-Notch1-cKO mice, GFAP-CreER-ETBR-cKO mice exhibited a defect in reactive astrocyte proliferation after cerebral ischemia. Our results indicate that the Notch1–STAT3–ETBR axis connects a signaling network that promotes reactive astrocyte proliferation after brain injury. PMID:26124113

  12. High commitment of embryonic keratinocytes to terminal differentiation through a Notch1-caspase 3 regulatory mechanism.

    PubMed

    Okuyama, Ryuhei; Nguyen, Bach-Cuc; Talora, Claudio; Ogawa, Eisaku; Tommasi di Vignano, Alice; Lioumi, Maria; Chiorino, Giovanna; Tagami, Hachiro; Woo, Minna; Dotto, G Paolo

    2004-04-01

    Embryonic cells are expected to possess high growth/differentiation potential, required for organ morphogenesis and expansion during development. However, little is known about the intrinsic properties of embryonic epithelial cells due to difficulties in their isolation and cultivation. We report here that pure keratinocyte populations from E15.5 mouse embryos commit irreversibly to differentiation much earlier than newborn cells. Notch signaling, which promotes keratinocyte differentiation, is upregulated in embryonic keratinocyte and epidermis, and elevated caspase 3 expression, which we identify as a transcriptional Notch1 target, accounts in part for the high commitment of embryonic keratinocytes to terminal differentiation. In vivo, lack of caspase 3 results in increased proliferation and decreased differentiation of interfollicular embryonic keratinocytes, together with decreased activation of PKC-delta, a caspase 3 substrate which functions as a positive regulator of keratinocyte differentiation. Thus, a Notch1-caspase 3 regulatory mechanism underlies the intrinsically high commitment of embryonic keratinocytes to terminal differentiation.

  13. The matricellular protein CCN3 regulates NOTCH1 signalling in chronic myeloid leukaemia.

    PubMed

    Suresh, Sukanya; McCallum, Lynn; Crawford, Lisa J; Lu, Wan Hua; Sharpe, Daniel J; Irvine, Alexandra E

    2013-11-01

    Deregulated NOTCH1 has been reported in lymphoid leukaemia, although its role in chronic myeloid leukaemia (CML) is not well established. We previously reported BCR-ABL down-regulation of a novel haematopoietic regulator, CCN3, in CML; CCN3 is a non-canonical NOTCH1 ligand. This study characterizes the NOTCH1–CCN3 signalling axis in CML. In K562 cells, BCR-ABL silencing reduced full-length NOTCH1 (NOTCH1-FL) and inhibited the cleavage of NOTCH1 intracellular domain (NOTCH1-ICD), resulting in decreased expression of the NOTCH1 targets c-MYC and HES1. K562 cells stably overexpressing CCN3 (K562/CCN3) or treated with recombinant CCN3(rCCN3) showed a significant reduction in NOTCH1 signalling (> 50% reduction in NOTCH1-ICD, p < 0.05).Gamma secretase inhibitor (GSI), which blocks NOTCH1 signalling, reduced K562/CCN3 colony formation but increased that of K562/control cells. GSI combined with either rCCN3 or imatinib reduced K562 colony formation with enhanced reduction of NOTCH1 signalling observed with combination treatments. We demonstrate an oncogenic role for NOTCH1 in CML and suggest that BCR-ABL disruption of NOTCH1–CCN3 signalling contributes to the pathogenesis of CML.

  14. Anti-cancer effects of curcumin on lung cancer through the inhibition of EZH2 and NOTCH1

    PubMed Central

    Jiang, Hua; Wang, Xiao; Xue, Qian; Zheng, Ai-Hong; Zhou, Hong-Ying; Chen, Yun; Chen, Xiao-Chen; Xiao, Jian-Yong; Ying, Xu-Hua; Wang, Fu-Wei; Rui, Tao; Liao, Yi-Ji; Xie, Dan; Lu, Li-Qin; Huang, Dong-Sheng

    2016-01-01

    Curcumin is potentially therapeutic for malignant diseases. The mechanisms of this effect might involve a combination of antioxidant, immunomodulatory, proapoptotic, and antiangiogenic activities. However, the exact mechanisms are not fully understood. In the present study, we provided evidences that curcumin suppressed the expression of enhancer of zeste homolog 2 (EZH2) in lung cancer cells both transcriptionally and post-transcriptionally. Curcumin inhibited the expression of EZH2 through microRNA (miR)-let 7c and miR-101. Curcumin decreased the expression of NOTCH1 through the inhibition of EZH2. There was a reciprocal regulation between EZH2 and NOTCH1 in lung cancer cells. These observations suggest that curcumin inhibits lung cancer growth and metastasis at least partly through the inhibition of EZH2 and NOTCH1. PMID:27049834

  15. Role of Notch-1 signaling pathway in PC12 cell apoptosis induced by amyloid beta-peptide (25–35)

    PubMed Central

    Liang, Huimin; Zhang, Yaozhou; Shi, Xiaoyan; Wei, Tianxiang; Lou, Jiyu

    2014-01-01

    Recent studies have demonstrated that Notch-1 expression is increased in the hippocampus of Alzheimer's disease patients. We speculate that Notch-1 signaling may be involved in PC12 cell apoptosis induced by amyloid beta-peptide (25–35) (Aβ25–35). In the present study, PC12 cells were cultured with different doses (0, 0.1, 1.0, 10 and 100 nmol/L) of N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, a Notch-1 signaling pathway inhibitor, for 30 minutes. Then cultured cells were induced with Aβ25–35 for 48 hours. Pretreatment of PC12 cells with high doses of N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (> 10 nmol/L) prolonged the survival of PC12 cells after Aβ25–35 induction, decreased the expression of apoptosis-related proteins caspase-3, -8, -9, increased the activity of oxidative stress-related superoxide dismutase and catalase, inhibited the production of active oxygen, and reduced nuclear factor kappa B expression. This study indicates that the Notch-1 signaling pathway plays a pivotal role in Aβ25–35-induced PC12 apoptosis. PMID:25221582

  16. Differential control of Notch1 gene transcription by Klf4 and Sp3 transcription factors in normal versus cancer-derived keratinocytes.

    PubMed

    Lambertini, Chiara; Pantano, Serafino; Dotto, G Paolo

    2010-04-28

    In specific cell types like keratinocytes, Notch signaling plays an important pro-differentiation and tumor suppressing function, with down-modulation of the Notch1 gene being associated with cancer development. Besides being controlled by p53, little else is known on regulation of Notch1 gene expression in this context. We report here that transcription of this gene is driven by a TATA-less "sharp peak" promoter and that the minimal functional region of this promoter, which extends from the -342 bp position to the initiation codon, is differentially active in normal versus cancer cells. This GC rich region lacks p53 binding sites, but binds Klf4 and Sp3. This finding is likely to be of biological significance, as Klf4 and, to a lesser extent, Sp3 are up-regulated in a number of cancer cells where Notch1 expression is down-modulated, and Klf4 over-expression in normal cells is sufficient to down-modulate Notch1 gene transcription. The combined knock-down of Klf4 and Sp3 was necessary for the reverse effect of increasing Notch1 transcription, consistent with the two factors exerting an overlapping repressor function through their binding to the Notch1 promoter.

  17. MAST2 and NOTCH1 translocations in breast carcinoma and associated pre-invasive lesions.

    PubMed

    Clay, Michael R; Varma, Sushama; West, Robert B

    2013-12-01

    There are several mutations and structural variations common to breast cancer. Many of these genomic changes are thought to represent driver mutations in oncogenesis. Less well understood is how and when these changes take place in breast cancer development. Previous studies have identified gene rearrangements in the microtubule-associated serine-threonine kinase (MAST) and NOTCH gene families in 5% to 7% of invasive breast cancers. Some of these translocations can be detected by fluorescence in situ hybridization (FISH) allowing for examination of the correlation between these genomic changes and concurrent morphologic changes in early breast neoplasia. NOTCH and MAST gene rearrangements were identified by FISH in a large series of breast cancer cases organized on tissue microarrays (TMA). When translocations were identified by TMA, we performed full cross-section FISH to evaluate concurrent pre-invasive lesions. FISH break-apart assays were designed for NOTCH1 and MAST2 gene rearrangements. Translocations were identified in 16 cases of invasive carcinoma; 10 with MAST2 translocations (2.0%) and 6 cases with NOTCH1 translocations (1.2%). Whole section FISH analysis of these cases demonstrated that the translocations are present in the majority of concurrent ductal carcinoma in situ (DCIS) (6/8). When DCIS wasn't associated with an invasive component, it was never translocated (0/170, P=.0048). We have confirmed the presence of MAST and NOTCH family gene rearrangements in invasive breast carcinoma, and show that FISH studies can effectively be used with TMAs to screen normal, pre-invasive, and coexisting invasive disease. Our findings suggest that these translocations occur during the transition to DCIS and/or invasive carcinoma.

  18. The folding and structural integrity of the first LIN-12 module of human Notch1 are calcium-dependent.

    PubMed

    Aster, J C; Simms, W B; Zavala-Ruiz, Z; Patriub, V; North, C L; Blacklow, S C

    1999-04-13

    Notch1 is a member of a conserved family of large modular type 1 transmembrane receptors that control differentiation in multicellular animals. Notch function is mediated through a novel signal transduction pathway involving successive ligand-induced proteolytic cleavages that serve to release the intracellular domain of Notch, which then translocates to the nucleus and activates downstream transcription factors. The extracellular domain of all Notch receptors have three iterated LIN-12 modules that appear to act as negative regulatory domains, possibly by limiting proteolysis. Each LIN-12 module contains three disulfide bonds and three conserved aspartate (D) or asparagine (N) residues. To begin to understand the structural basis for LIN-12 function, the first LIN-12 module of human Notch1 (rLIN-12.1) has been expressed recombinantly in Escherichia coli and purified in a reduced form. In redox buffers, rLIN-12.1 forms only one disulfide isomer in the presence of millimolar Ca2+ concentrations, whereas multiple disulfide isomers are observed in the presence of Mg2+ and EDTA. Further, mutation of conserved residues N1460, D1475, and D1478 to alanine abolishes Ca2+-dependent folding of this module. Mass spectrometric analysis of partially reduced rLIN-12.1 has been used to deduce that disulfide bonds are formed between the first and fifth (C1449-C1472), second and fourth (C1454-C1467), and third and sixth (C1463-C1479) cysteines of this prototype module. This arrangement is distinct from that observed in other modules, such as EGF and LDL-A, that also contain three disulfide bonds. One-dimensional proton nuclear magnetic resonance shows that Ca2+ induces a dramatic increase in the extent of chemical shift dispersion of the native rLIN-12.1 amide protons, as seen for the Ca2+-binding LDL-A modules. We conclude that Ca2+ is required both for proper folding and for the maintenance of the structural integrity of Notch/LIN-12 modules.

  19. The matricellular protein CCN6 (WISP3) decreases Notch1 and suppresses breast cancer initiating cells.

    PubMed

    Huang, Wei; Martin, Emily E; Burman, Boris; Gonzalez, Maria E; Kleer, Celina G

    2016-05-01

    Increasing evidence supports that the epithelial to mesenchymal transition (EMT) in breast cancer cells generates tumor initiating cells (TICs) but the contribution of the tumor microenvironment to these programs needs further elucidation. CCN6 (WISP3) is a secreted matrix-associated protein (36.9 kDa) of the CCN family (named after CTGF, Cyr61 and Nov) that is reduced or lost in invasive carcinomas of the breast with lymph node metastasis and in inflammatory breast cancer. CCN6 exerts breast cancer growth and invasion inhibitory functions, but the mechanisms remain to be defined. In the present study we discovered that ectopic CCN6 overexpression in triple negative (TN) breast cancer cells and in cells derived from patients is sufficient to induce a mesenchymal to epithelial transition (MET) and to reduce TICs. In vivo, CCN6 overexpression in the TIC population of MDA-MB-231 cells delayed tumor initiation, reduced tumor volume, and inhibited the development of metastasis. Our studies reveal a novel CCN6/Slug signaling axis that regulates Notch1 signaling activation, epithelial cell phenotype and breast TICs, which requires the conserved thrombospondin type 1 (TSP1) motif of CCN6. The relevance of these data to human breast cancer is highlighted by the finding that CCN6 protein levels are inversely correlated with Notch1 intracellular activated form (NICD1) in 69.5% of invasive breast carcinomas. These results demonstrate that CCN6 regulates epithelial and mesenchymal states transition and TIC programs, and pinpoint one responsible mechanism.

  20. Epidermal Notch1 loss promotes skin tumorigenesis by impacting the stromal microenvironment

    PubMed Central

    Demehri, Shadmehr; Turkoz, Ahu; Kopan, Raphael

    2009-01-01

    Summary Notch1 is a proto-oncogene in several organs. In the skin, however, Notch1 deletion leads to tumor formation, suggesting that Notch1 is a “tumor suppressor” within this context. Here we demonstrate that, unlike classical tumor suppressors, Notch1 loss in epidermal keratinocytes promotes tumorigenesis non-cell autonomously by impairing skin-barrier integrity and creating a wound-like microenvironment in the skin. Using mice with a chimeric pattern of Notch1 deletion, we determined that Notch1-expressing keratinocytes in this microenvironment readily formed papillomas, showing that Notch1 was insufficient to suppress this tumor-promoting effect. Accordingly, loss of other Notch paralogs that impaired the skin barrier also predisposed Notch1-expressing skin to tumorigenesis, demonstrating that the tumor-promoting effect of Notch1 loss involves a crosstalk between barrier-defective epidermis and its stroma. Significance In contrast to the current dogma, we demonstrate unequivocally that the non-cell autonomous consequences of defective barrier formation are responsible for the tumor-promoting effects of Notch1 loss in mouse skin. Thus, individuals with sub-acute skin-barrier defects may also be prone to carcinogenesis upon exposure to initiating carcinogens like UV rays. As Notch1 deletion in skin tumors enhanced their progression to invasive arcinomas, patients with benign hyperplasic skin lesions receiving γ-secretase inhibitor therapy may, therefore, be at additional risk. More broadly, given that chronic injury causatively effects the development of several human carcinomas, Notch1-deficient mice with mild skin-barrier defects can serve as an experimental model in which to study the tumor-promoting elements of chronic injury/wound and develop relevant therapies. PMID:19573812

  1. Implications of the Notch1-Snail/Slug-epithelial to mesenchymal transition axis for lymph node metastasis in infiltrating ductal carcinoma.

    PubMed

    Cao, Yu-Wen; Wan, Guo-Xing; Sun, Jian-Ping; Cui, Xiao-Bin; Hu, Jian-Ming; Liang, Wei-Hua; Zheng, Yu-Qin; Li, Wen-Qin; Li, Feng

    2015-02-01

    Emerging evidence suggests that activation of the Notch1 signaling pathway inducing epithelial to mesenchymal transition (EMT) mediated by Snail/Slug promotes invasion and metastasis of breast cancer cells in vitro. However, the implication of the Notch1-Snail/Slug-EMT axis in breast cancer patients remains unclear. A total of 200 formalin-fixed paraffin-embedded samples of invasive ductal carcinoma (IDC), and 37 adjacent non-neoplastic tissue (ANNT) samples from patients who had not been treated with neoadjuvant therapy were examined. Expression of Notch1, Slug, Snail, E-cadherin, N-cadherin, and vimentin was determined by immunohistochemistry on tissue microarrays (TMAs). The correlation between protein expression and clinicopathological characteristics of breast cancer patients was also evaluated. Results showed that a significantly high percentage of cases with high expression of Notch1 (74%, 148/200), Slug (36%, 72/200), Snail (62%, 124/200), and N-cadherin (77%, 153/200) and a low percentage of cases with high expression of E-cadherin (27%, 54/200) were observed in IDC compared to those in ANNTs. High Notch1, Slug, Snail, and N-cadherin expression and low E-cadherin expression in patients with IDC were significantly correlated with lymph node metastasis. In addition, correlation analysis results revealed that high Notch1 expression was significantly associated with high Slug, Snail, and N-cadherin expression and low E-cadherin expression in IDC. Furthermore, a high Snail expression was significantly associated with low E-cadherin expression, and a high Slug expression was found to be significantly associated with increased N-cadherin expression in patients with IDC. Hence, our study suggested that the Notch1-Snail/Slug-EMT axis may be implicated in the lymph node metastasis affecting patients with IDC. PMID:25645984

  2. Expression patterns of Notch1, Notch2, and Notch3 suggest multiple functional roles for the Notch-DSL signaling system during brain development.

    PubMed

    Irvin, D K; Zurcher, S D; Nguyen, T; Weinmaster, G; Kornblum, H I

    2001-07-23

    The Notch-DSL signaling system consists of multiple receptors and ligands, and plays many roles in development. The function of Notch receptors and ligands in mammalian brain, however, is poorly understood. In the current study, we examined the expression patterns for three receptors of this system, Notch1, 2, and 3, in late embryonic and postnatal rat brain by in situ hybridization. The three receptors have overlapping but different patterns of expression. Messenger RNA for all three proteins is found in postnatal central nervous system (CNS) germinal zones and, in early postnatal life, within numerous cells throughout the CNS. Within zones of cellular proliferation of the postnatal brain, Notch1 mRNA is found in both the subventricular and the ventricular germinal zones, whereas Notch2 and Notch3 mRNAs are more highly localized to the ventricular zones. Both Notch1 and Notch3 mRNAs are expressed along the inner aspect of the dentate gyrus, a site of adult neurogenesis. Notch2 mRNA is expressed in the external granule cell layer of the developing cerebellum. In several brain areas, Notch1 and Notch2 mRNAs are relatively concentrated in white matter, whereas Notch3 mRNA is not. Neurosphere cultures (which contain CNS stem cells), purified astrocyte cultures, and striatal neuron-enriched cultures express Notch1 mRNA. However, in these latter cultures, Notch1 mRNA is produced by nestin-containing cells, rather than by postmitotic neurons. Taken together, these results support multiple roles for Notch1, 2, and 3 receptor activation during CNS development, particularly during gliogenesis.

  3. Mutations affecting enzymatic activity in liver arginase

    SciTech Connect

    Vockley, J.G.; Tabor, D.E.; Goodman, B.K.

    1994-09-01

    The hydrolysis of arginine to ornithine and urea is catalyzed by arginase in the last step of the urea cycle. We examined a group of arginase deficient patients by PCR-SSCP analysis to characterize the molecular basis of this disorder. A heterogeneous population of nonsense mutations, microdeletions, and missense mutations has been identified in our cohort. Microdeletions which introduce premature stop codons downstream of the deletion and nonsense mutations result in no arginase activity. These mutations occur randomly along the gene. The majority of missense mutations identified appear to occur in regions of high cross-species homology. To test the effect of these missense mutations on arginase activity, site-directed mutagenesis was used to re-create the patient mutations for in vivo expression studies in a prokaryotic fusion-protein expression system. Of 4 different missense mutations identified in 6 individuals, only one was located outside of a conserved region. The three substitution mutations within the conserved regions had a significant effect on enzymatic activity (0-3.1 nmole/30min, normal is 1300-1400 nmoles/30min, as determined by in vitro arginase assay), while the fourth mutation, a T to S substitution, did not. In addition, site-directed mutagenesis was utilized to create mutations not in residues postulated to play a significant role in the enzymatic function or active site formation in manganese-binding proteins such as arginase. We have determined that the substitution of glycine for a histidine residue, located in a very highly conserved region of exon 3, and the substitution of a histidine and an aspartic acid residue within a similarly conserved region in exon 4, totally abolishes enzymatic activity. Mutations substituting glycine for an additional histidine and aspartic acid residue in exon 4 and two aspartic acid residues in exon 7 have also been created. We are currently in the process of characterizing these mutations.

  4. Notch-1 promotes breast cancer cells proliferation by regulating LncRNA GAS5

    PubMed Central

    Pei, Jing; Wang, Benzhong

    2015-01-01

    Background: Notch signaling is indicated as novel therapeutic targets to prevent recurrence of breast cancer. LncRNAs were identified as downstream target of Notch pathway. However, the exact mechanisms involved in Notch signaling, lncRNAs and breast cancer remain to be explained. Objective: This original research aimed to determine the prognostic implications of Notch-1 for breast cancer, and explain mechanisms involved in regulation of lnRNA GAS5 by Notch-1, and identify the function of this mechanism on breast cancer. Method: Thirty breast cancer patients were included from The First Affiliated Hospital of Anhui Medical University (China) since January 2006 in this study. The mRNA level by RT-PCR and protein level of Notch-1 by western blot in tumor tissues and adjacent normal tissues were evaluated and 5-year survival analysis was applied to examine the significance of Notch-1. The levels of ten reported lncRNAs were determined by RT-PCR, and subsequently linear analysis was applied to analyze the relationship between these four unique lncRNAs and protein level of Notch-1, which identified the most relevant lncRNA GAS5 with Notch-1 in breast cancer. Subsequently, Notch1-siRNA was applied to influence the expression of Notch-1 in T47D, then the level of RSA5 was measured by RT-PCR, and CCK-8 assay was applied to measure the proliferation of T47D cells. Results: High level of Notch-1 provided a poor prognosis in breast cancer. Interference of Notch-1 significantly suppressed proliferation of T47D cell (P < 0.05), and significantly increased the level of GAS5. Conclusion: Notch-1 promotes breast cancer cells proliferation by regulating LncRNA GAS5. PMID:26550436

  5. Increased expression of Notch1 in temporal lobe epilepsy: animal models and clinical evidence

    PubMed Central

    Liu, Xijin; Yang, Zhiyong; Yin, Yaping; Deng, Xuejun

    2014-01-01

    Temporal lobe epilepsy is associated with astrogliosis. Notch1 signaling can induce astrogliosis in glioma. However, it remains unknown whether Notch1 signaling is involved in the pathogenesis of epilepsy. This study investigated the presence of Notch1, hairy and enhancer of split-1, and glial fibrillary acidic protein in the temporal neocortex and hippocampus of lithium-pilocarpine-treated rats. The presence of Notch1 and hairy and enhancer of split-1 was also explored in brain tissues of patients with intractable temporal lobe epilepsy. Quantitative electroencephalogram analysis and behavioral observations were used as auxiliary measures. Results revealed that the presence of Notch1, hairy and enhancer of split-1, and glial fibrillary acidic protein were enhanced in status epilepticus and vehicle-treated spontaneous recurrent seizures rats, but remain unchanged in the following groups: control, absence of either status epilepticus or spontaneous recurrent seizures, and zileuton-treated spontaneous recurrent seizures. Compared with patient control cases, the presences of Notch1 and hairy and enhancer of split-1 were upregulated in the temporal neocortex of patients with intractable temporal lobe epilepsy. Therefore, these results suggest that Notch1 signaling may play an important role in the onset of temporal lobe epilepsy via astrogliosis. Furthermore, zileuton may be a potential therapeutic strategy for temporal lobe epilepsy by blocking Notch1 signaling. PMID:25206850

  6. Efficient delivery of Notch1 siRNA to SKOV3 cells by cationic cholesterol derivative-based liposome

    PubMed Central

    Zhao, Yun-Chun; Zhang, Li; Feng, Shi-Sen; Hong, Lu; Zheng, Hai-Li; Chen, Li-Li; Zheng, Xiao-Ling; Ye, Yi-Qing; Zhao, Meng-Dan; Wang, Wen-Xi; Zheng, Cai-Hong

    2016-01-01

    A novel cationic cholesterol derivative-based small interfering RNA (siRNA) interference strategy was suggested to inhibit Notch1 activation in SKOV3 cells for the gene therapy of ovarian cancer. The cationic cholesterol derivative, N-(cholesterylhemisuccinoyl-amino-3-propyl)-N, N-dimethylamine (DMAPA-chems) liposome, was incubated with siRNA at different nitrogen-to-phosphate ratios to form stabilized, near-spherical siRNA/DMAPA-chems nanoparticles with sizes of 100–200 nm and zeta potentials of 40–50 mV. The siRNA/DMAPA-chems nanoparticles protected siRNA from nuclease degradation in 25% fetal bovine serum. The nanoparticles exhibited high cell uptake and Notch1 gene knockdown efficiency in SKOV3 cells at an nitrogen-to-phosphate ratio of 100 and an siRNA concentration of 50 nM. They also inhibited the growth and promoted the apoptosis of SKOV3 cells. These results may provide the potential for using cationic cholesterol derivatives as efficient nonviral siRNA carriers for the suppression of Notch1 activation in ovarian cancer cells. PMID:27799771

  7. Rac1 changes the substrate specificity of gamma-secretase between amyloid precursor protein and Notch1.

    PubMed

    Boo, Jung Hyun; Sohn, Ji Hoon; Kim, Ji Eun; Song, Hyundong; Mook-Jung, Inhee

    2008-08-01

    Beta amyloid peptide is generated from amyloid precursor protein (APP) by proteolytic cleavage of beta- and gamma-secretases, and plays a critical role in the pathogenesis of Alzheimer's disease. Since gamma-secretase cleaves several proteins including APP and Notch in a number of cell types, it is important to understand the conditions determining gamma-secretase substrate specificity. In the present study, inhibition of Rac1 attenuated gamma-secretase activity for APP, resulting in decreased production of the APP intracellular domain but accumulated C-terminal fragments (APP-CTF). In contrast, Rac1 inhibitor, NSC23766 increased production of the Notch1 intracellular domain but slightly decreased the ectodomain-shed form of Notch1 (NotchDeltaE). To elucidate the mechanism underlying these observations, we performed co-immunoprecipitation experiments to analyze the interaction between Rac1 and presenilin1 (PS1), a component of the gamma-secretase complex. Inhibition of Rac1 enhanced its interaction with PS1. Under the same condition, PS1 interacted more strongly with NotchDeltaE than with APP-CTF. Our results suggested that PS1 determines the preferred substrate for gamma-secretase between APP and Notch1, depending on the activation status of Rac1.

  8. Structural basis for Notch1 engagement of Delta-like 4

    SciTech Connect

    Luca, Vincent C.; Jude, Kevin M.; Pierce, Nathan W.; Nachury, Maxence V.; Fischer, Suzanne; Garcia, K. Christopher

    2015-02-20

    Notch receptors guide mammalian cell fate decisions by engaging the proteins Jagged and Delta-like (DLL). The 2.3 angstrom resolution crystal structure of the interacting regions of the Notch1-DLL4 complex reveals a two-site, antiparallel binding orientation assisted by Notch1 O-linked glycosylation. Notch1 epidermal growth factor–like repeats 11 and 12 interact with the DLL4 Delta/Serrate/Lag-2 (DSL) domain and module at the N-terminus of Notch ligands (MNNL) domains, respectively. Threonine and serine residues on Notch1 are functionalized with O-fucose and O-glucose, which act as surrogate amino acids by making specific, and essential, contacts to residues on DLL4. Lastly, the elucidation of a direct chemical role for O-glycans in Notch1 ligand engagement demonstrates how, by relying on posttranslational modifications of their ligand binding sites, Notch proteins have linked their functional capacity to developmentally regulated biosynthetic pathways.

  9. Decidual vascular endothelial cells promote maternal-fetal immune tolerance by inducing regulatory T cells through canonical Notch1 signaling.

    PubMed

    Yao, Yanyi; Song, Jieping; Wang, Weipeng; Liu, Nian

    2016-05-01

    Adaptation of the maternal immune response to accommodate the semiallogeneic fetus is necessary for pregnancy success. However, the mechanisms by which the fetus avoids rejection despite expression of paternal alloantigens remain incompletely understood. Regulatory T cells (Treg cells) are pivotal for maintaining immune homeostasis, preventing autoimmune disease and fetus rejection. In this study, we found that maternal decidual vascular endothelial cells (DVECs) sustained Foxp3 expression in resting Treg cells in vitro. Moreover, under in vitro Treg cell induction condition with agonistic antibodies and transforming growth factor (TGF)-β, DVECs promoted Treg cell differentiation from non-Treg conventional T cells. Consistent with the promotion of Treg cell maintenance and differentiation, Treg cell-associated gene expression such as TGF-β, Epstein-Barr-induced gene-3, CD39 and glucocorticoid-induced tumor necrosis factor receptor was also increased in the presence of DVECs. Further study revealed that DVECs expressed Notch ligands such as Jagged-1, Delta-like protein 1 (DLL-1) and DLL-4, while Treg cells expressed Notch1 on their surface. The effects of DVECs on Treg cells was inhibited by siRNA-induced knockdown of expression of Jagged-1 and DLL-1 in DVECs. Downregulation of Notch1 in Treg cells using lentiviral shRNA transduction decreased Foxp3 expression in Treg cells. Adoptive transfer of Notch1-deficient Treg cells increased abortion rate in a murine semiallogeneic pregnancy model. Taken together, our study suggests that maternal DVECs are able to maintain decidual Treg cell identity and promote Treg cell differentiation through activation of Notch1 signal pathway in Treg cells and subsequently inhibit the immune response against semiallogeneic fetuses and preventing spontaneous abortion. PMID:26714886

  10. All-trans-retinoic Acid Modulates the Plasticity and Inhibits the Motility of Breast Cancer Cells: ROLE OF NOTCH1 AND TRANSFORMING GROWTH FACTOR (TGFβ).

    PubMed

    Zanetti, Adriana; Affatato, Roberta; Centritto, Floriana; Fratelli, Maddalena; Kurosaki, Mami; Barzago, Maria Monica; Bolis, Marco; Terao, Mineko; Garattini, Enrico; Paroni, Gabriela

    2015-07-17

    All-trans-retinoic acid (ATRA) is a natural compound proposed for the treatment/chemoprevention of breast cancer. Increasing evidence indicates that aberrant regulation of epithelial-to-mesenchymal transition (EMT) is a determinant of the cancer cell invasive and metastatic behavior. The effects of ATRA on EMT are largely unknown. In HER2-positive SKBR3 and UACC812 cells, showing co-amplification of the ERBB2 and RARA genes, ATRA activates a RARα-dependent epithelial differentiation program. In SKBR3 cells, this causes the formation/reorganization of adherens and tight junctions. Epithelial differentiation and augmented cell-cell contacts underlie the anti-migratory action exerted by the retinoid in cells exposed to the EMT-inducing factors EGF and heregulin-β1. Down-regulation of NOTCH1, an emerging EMT modulator, is involved in the inhibition of motility by ATRA. Indeed, the retinoid blocks NOTCH1 up-regulation by EGF and/or heregulin-β1. Pharmacological inhibition of γ-secretase and NOTCH1 processing also abrogates SKBR3 cell migration. Stimulation of TGFβ contributes to the anti-migratory effect of ATRA. The retinoid switches TGFβ from an EMT-inducing and pro-migratory determinant to an anti-migratory mediator. Inhibition of the NOTCH1 pathway not only plays a role in the anti-migratory action of ATRA; it is relevant also for the anti-proliferative activity of the retinoid in HCC1599 breast cancer cells, which are addicted to NOTCH1 for growth/viability. This effect is enhanced by the combination of ATRA and the γ-secretase inhibitor N-(N-(3,5-difluorophenacetyl)-l-alanyl)-S-phenylglycine t-butyl ester, supporting the concept that the two compounds act at the transcriptional and post-translational levels along the NOTCH1 pathway. PMID:26018078

  11. All-trans-retinoic Acid Modulates the Plasticity and Inhibits the Motility of Breast Cancer Cells: ROLE OF NOTCH1 AND TRANSFORMING GROWTH FACTOR (TGFβ).

    PubMed

    Zanetti, Adriana; Affatato, Roberta; Centritto, Floriana; Fratelli, Maddalena; Kurosaki, Mami; Barzago, Maria Monica; Bolis, Marco; Terao, Mineko; Garattini, Enrico; Paroni, Gabriela

    2015-07-17

    All-trans-retinoic acid (ATRA) is a natural compound proposed for the treatment/chemoprevention of breast cancer. Increasing evidence indicates that aberrant regulation of epithelial-to-mesenchymal transition (EMT) is a determinant of the cancer cell invasive and metastatic behavior. The effects of ATRA on EMT are largely unknown. In HER2-positive SKBR3 and UACC812 cells, showing co-amplification of the ERBB2 and RARA genes, ATRA activates a RARα-dependent epithelial differentiation program. In SKBR3 cells, this causes the formation/reorganization of adherens and tight junctions. Epithelial differentiation and augmented cell-cell contacts underlie the anti-migratory action exerted by the retinoid in cells exposed to the EMT-inducing factors EGF and heregulin-β1. Down-regulation of NOTCH1, an emerging EMT modulator, is involved in the inhibition of motility by ATRA. Indeed, the retinoid blocks NOTCH1 up-regulation by EGF and/or heregulin-β1. Pharmacological inhibition of γ-secretase and NOTCH1 processing also abrogates SKBR3 cell migration. Stimulation of TGFβ contributes to the anti-migratory effect of ATRA. The retinoid switches TGFβ from an EMT-inducing and pro-migratory determinant to an anti-migratory mediator. Inhibition of the NOTCH1 pathway not only plays a role in the anti-migratory action of ATRA; it is relevant also for the anti-proliferative activity of the retinoid in HCC1599 breast cancer cells, which are addicted to NOTCH1 for growth/viability. This effect is enhanced by the combination of ATRA and the γ-secretase inhibitor N-(N-(3,5-difluorophenacetyl)-l-alanyl)-S-phenylglycine t-butyl ester, supporting the concept that the two compounds act at the transcriptional and post-translational levels along the NOTCH1 pathway.

  12. G protein-coupled receptor 183 facilitates endothelial-to-hematopoietic transition via Notch1 inhibition

    PubMed Central

    Zhang, Panpan; He, Qiuping; Chen, Dongbo; Liu, Weixiao; Wang, Lu; Zhang, Chunxia; Ma, Dongyuan; Li, Wei; Liu, Bing; Liu, Feng

    2015-01-01

    In vertebrates, embryonic hematopoietic stem and progenitor cells (HSPCs) are derived from a subset of endothelial cells, the hemogenic endothelium (HE), through the endothelial-to-hematopoietic transition (EHT). Notch signaling is essential for HSPC development during embryogenesis across vertebrates. However, whether and how it regulates EHT remains unclear. Here, we show that G protein-coupled receptor 183 (Gpr183) signaling serves as an indispensable switch for HSPC emergence by repressing Notch signaling before the onset of EHT. Inhibition of Gpr183 significantly upregulates Notch signaling and abolishes HSPC emergence. Upon activation by its ligand 7α-25-OHC, Gpr183 recruits β-arrestin1 and the E3 ligase Nedd4 to degrade Notch1 in specified HE cells and then facilitates the subsequent EHT. Importantly, 7α-25-OHC stimulation promotes HSPC emergence in vivo and in vitro, providing an attractive strategy for enhancing the in vitro generation of functional HSPCs. PMID:26358189

  13. The matricellular protein CCN6 (WISP3) decreases Notch1 and suppresses breast cancer initiating cells

    PubMed Central

    Huang, Wei; Martin, Emily E.; Burman, Boris; Gonzalez, Maria E.; Kleer, Celina G.

    2016-01-01

    Increasing evidence supports that the epithelial to mesenchymal transition (EMT) in breast cancer cells generates tumor initiating cells (TICs) but the contribution of the tumor microenvironment to these programs needs further elucidation. CCN6 (WISP3) is a secreted matrix-associated protein (36.9 kDa) of the CCN family (named after CTGF, Cyr61 and Nov) that is reduced or lost in invasive carcinomas of the breast with lymph node metastasis and in inflammatory breast cancer. CCN6 exerts breast cancer growth and invasion inhibitory functions, but the mechanisms remain to be defined. In the present study we discovered that ectopic CCN6 overexpression in triple negative (TN) breast cancer cells and in cells derived from patients is sufficient to induce a mesenchymal to epithelial transition (MET) and to reduce TICs. In vivo, CCN6 overexpression in the TIC population of MDA-MB-231 cells delayed tumor initiation, reduced tumor volume, and inhibited the development of metastasis. Our studies reveal a novel CCN6/Slug signaling axis that regulates Notch1 signaling activation, epithelial cell phenotype and breast TICs, which requires the conserved thrombospondin type 1 (TSP1) motif of CCN6. The relevance of these data to human breast cancer is highlighted by the finding that CCN6 protein levels are inversely correlated with Notch1 intracellular activated form (NICD1) in 69.5% of invasive breast carcinomas. These results demonstrate that CCN6 regulates epithelial and mesenchymal states transition and TIC programs, and pinpoint one responsible mechanism. PMID:26933820

  14. Relaxin Prevents Cardiac Fibroblast-Myofibroblast Transition via Notch-1-Mediated Inhibition of TGF-β/Smad3 Signaling

    PubMed Central

    Sassoli, Chiara; Chellini, Flaminia; Pini, Alessandro; Tani, Alessia; Nistri, Silvia; Nosi, Daniele; Zecchi-Orlandini, Sandra; Bani, Daniele; Formigli, Lucia

    2013-01-01

    The hormone relaxin (RLX) is produced by the heart and has beneficial actions on the cardiovascular system. We previously demonstrated that RLX stimulates mouse neonatal cardiomyocyte growth, suggesting its involvement in endogenous mechanisms of myocardial histogenesis and regeneration. In the present study, we extended the experimentation by evaluating the effects of RLX on primary cultures of neonatal cardiac stromal cells. RLX inhibited TGF-β1-induced fibroblast-myofibroblast transition, as judged by its ability to down-regulate α-smooth muscle actin and type I collagen expression. We also found that the hormone up-regulated metalloprotease (MMP)-2 and MMP-9 expression and downregulated the tissue inhibitor of metalloproteinases (TIMP)-2 in TGF-β1-stimulated cells. Interestingly, the effects of RLX on cardiac fibroblasts involved the activation of Notch-1 pathway. Indeed, Notch-1 expression was significantly decreased in TGF-β1-stimulatedfibroblasts as compared to the unstimulated controls; this reduction was prevented by the addition of RLX to TGF-β1-stimulated cells. Moreover, pharmacological inhibition of endogenous Notch-1 signaling by N-3,5-difluorophenyl acetyl-L-alanyl-2-phenylglycine-1,1-dimethylethyl ester (DAPT), a γ-secretase specific inhibitor, as well as the silencing of Notch-1 ligand, Jagged-1, potentiated TGF-β1-induced myofibroblast differentiation and abrogated the inhibitory effects of RLX. Interestingly, RLX and Notch-1 exerted their inhibitory effects by interfering with TGF-β1 signaling, since the addition of RLX to TGF-β1-stimulated cells caused a significant decrease in Smad3 phosphorylation, a typical downstream event of TGF-β1 receptor activation, while the treatment with a prevented this effect. These data suggest that Notch signaling can down-regulate TGF-β1/Smad3-induced fibroblast-myofibroblast transition and that RLX could exert its well known anti-fibrotic action through the up-regulation of this pathway. In conclusion

  15. Notch1 deficiency decreases hepatic lipid accumulation by induction of fatty acid oxidation.

    PubMed

    Song, No-Joon; Yun, Ui Jeong; Yang, Sunghee; Wu, Chunyan; Seo, Cho-Rong; Gwon, A-Ryeong; Baik, Sang-Ha; Choi, Yuri; Choi, Bo Youn; Bahn, Gahee; Kim, Suji; Kwon, So-Mi; Park, Jin Su; Baek, Seung Hyun; Park, Tae Joo; Yoon, Keejung; Kim, Byung-Joon; Mattson, Mark P; Lee, Sung-Joon; Jo, Dong-Gyu; Park, Kye Won

    2016-01-01

    Notch signaling pathways modulate various cellular processes, including cell proliferation, differentiation, adhesion, and communication. Recent studies have demonstrated that Notch1 signaling also regulates hepatic glucose production and lipid synthesis. However, the effect of Notch1 signaling on hepatic lipid oxidation has not yet been directly investigated. To define the function of Notch1 signaling in hepatic lipid metabolism, wild type mice and Notch1 deficient antisense transgenic (NAS) mice were fed a high-fat diet. High-fat diet -fed NAS mice exhibited a marked reduction in hepatic triacylglycerol accumulation compared with wild type obese mice. The improved fatty liver was associated with an increased expression of hepatic genes involved in fatty acid oxidation. However, lipogenic genes were not differentially expressed in the NAS liver, suggesting lipolytic-specific regulatory effects by Notch1 signaling. Expression of fatty acid oxidative genes and the rate of fatty acid oxidation were also increased by inhibition of Notch1 signaling in HepG2 cells. In addition, similar regulatory effects on lipid accumulation were observed in adipocytes. Taken together, these data show that inhibition of Notch1 signaling can regulate the expression of fatty acid oxidation genes and may provide therapeutic strategies in obesity-induced hepatic steatosis. PMID:26786165

  16. Notch1 deficiency decreases hepatic lipid accumulation by induction of fatty acid oxidation

    PubMed Central

    Song, No-Joon; Yun, Ui Jeong; Yang, Sunghee; Wu, Chunyan; Seo, Cho-Rong; Gwon, A-Ryeong; Baik, Sang-Ha; Choi, Yuri; Choi, Bo Youn; Bahn, Gahee; Kim, Suji; Kwon, So-Mi; Park, Jin Su; Baek, Seung Hyun; Park, Tae Joo; Yoon, Keejung; Kim, Byung-Joon; Mattson, Mark P.; Lee, Sung-Joon; Jo, Dong-Gyu; Park, Kye Won

    2016-01-01

    Notch signaling pathways modulate various cellular processes, including cell proliferation, differentiation, adhesion, and communication. Recent studies have demonstrated that Notch1 signaling also regulates hepatic glucose production and lipid synthesis. However, the effect of Notch1 signaling on hepatic lipid oxidation has not yet been directly investigated. To define the function of Notch1 signaling in hepatic lipid metabolism, wild type mice and Notch1 deficient antisense transgenic (NAS) mice were fed a high-fat diet. High-fat diet -fed NAS mice exhibited a marked reduction in hepatic triacylglycerol accumulation compared with wild type obese mice. The improved fatty liver was associated with an increased expression of hepatic genes involved in fatty acid oxidation. However, lipogenic genes were not differentially expressed in the NAS liver, suggesting lipolytic-specific regulatory effects by Notch1 signaling. Expression of fatty acid oxidative genes and the rate of fatty acid oxidation were also increased by inhibition of Notch1 signaling in HepG2 cells. In addition, similar regulatory effects on lipid accumulation were observed in adipocytes. Taken together, these data show that inhibition of Notch1 signaling can regulate the expression of fatty acid oxidation genes and may provide therapeutic strategies in obesity-induced hepatic steatosis. PMID:26786165

  17. Clinical significance of NOTCH1 intracellular cytoplasmic domain translocation into the nucleus in gastric cancer

    PubMed Central

    Saito, Shinichiro; Ishiguro, Hideyuki; Kimura, Masahiro; Ogawa, Ryo; Miyai, Hirotaka; Tanaka, Tatsuya; Mizoguchi, Koji; Takeyama, Hiromitsu

    2016-01-01

    Recent studies have shown constitutive activation of the Notch signaling pathway in various types of malignancies. However, it remains unclear whether this signaling pathway is activated in gastric cancer. In the present study, the aim was to investigate the role of Notch signaling in gastric cancer by investigating the subcellular localization of Notch-associated proteins in tissue samples from gastric cancer patients. Samples were obtained from 115 gastric cancer patients who had undergone surgery at the Department of Gastroenterological Surgery, Nagoya City University Graduate School of Medical Science without pre-operative chemotherapy or radiation. Subsequently the correlation between translocation of NOTCH1 intracellular cytoplasmic domain (NICD) into the nucleus (as measured by immunostaining) and survival in gastric cancer patients after surgery was investigated. The results were analyzed in reference to the patients' clinicopathological characteristics and the effects of these results on patient prognosis were determined. Significant correlations were observed between NICD nuclear localization and clinicopathological characteristics, such as tumor status (T factor), lymph node status (N factor), pathological stage and differentiation status. No significant correlations were observed between NICD nuclear localization and age, gender, tumor location, vein invasion or lymphatic invasion. Patients with >30% of cancer cell nuclei positively stained for NICD (as revealed by immunostaining) were associated with a significantly shorter survival following surgery than patients with <30% NICD-positive cancer cell nuclei (log-rank test, P=0.0194). Univariate analysis revealed that among the clinicopathological factors examined, T factor [risk rate (RR)=10.870; P=0.0016], N factor (RR=41.667; P=0.0003), lymphatic invasion (RR=13.158; P=0.0125), vein invasion (RR=25.000; P= 0.0019) and translocation of NICD to the nucleus (RR=3.937; P=0.0312) were all identified to be

  18. Clinical significance of NOTCH1 intracellular cytoplasmic domain translocation into the nucleus in gastric cancer

    PubMed Central

    Saito, Shinichiro; Ishiguro, Hideyuki; Kimura, Masahiro; Ogawa, Ryo; Miyai, Hirotaka; Tanaka, Tatsuya; Mizoguchi, Koji; Takeyama, Hiromitsu

    2016-01-01

    Recent studies have shown constitutive activation of the Notch signaling pathway in various types of malignancies. However, it remains unclear whether this signaling pathway is activated in gastric cancer. In the present study, the aim was to investigate the role of Notch signaling in gastric cancer by investigating the subcellular localization of Notch-associated proteins in tissue samples from gastric cancer patients. Samples were obtained from 115 gastric cancer patients who had undergone surgery at the Department of Gastroenterological Surgery, Nagoya City University Graduate School of Medical Science without pre-operative chemotherapy or radiation. Subsequently the correlation between translocation of NOTCH1 intracellular cytoplasmic domain (NICD) into the nucleus (as measured by immunostaining) and survival in gastric cancer patients after surgery was investigated. The results were analyzed in reference to the patients' clinicopathological characteristics and the effects of these results on patient prognosis were determined. Significant correlations were observed between NICD nuclear localization and clinicopathological characteristics, such as tumor status (T factor), lymph node status (N factor), pathological stage and differentiation status. No significant correlations were observed between NICD nuclear localization and age, gender, tumor location, vein invasion or lymphatic invasion. Patients with >30% of cancer cell nuclei positively stained for NICD (as revealed by immunostaining) were associated with a significantly shorter survival following surgery than patients with <30% NICD-positive cancer cell nuclei (log-rank test, P=0.0194). Univariate analysis revealed that among the clinicopathological factors examined, T factor [risk rate (RR)=10.870; P=0.0016], N factor (RR=41.667; P=0.0003), lymphatic invasion (RR=13.158; P=0.0125), vein invasion (RR=25.000; P= 0.0019) and translocation of NICD to the nucleus (RR=3.937; P=0.0312) were all identified to be

  19. Growth hormone treatment of premature ovarian failure in a mouse model via stimulation of the Notch-1 signaling pathway

    PubMed Central

    LIU, TE; WANG, SUWEI; ZHANG, LINA; GUO, LIHE; YU, ZHIHUA; CHEN, CHUAN; ZHENG, JIN

    2016-01-01

    Premature ovarian failure (POF) is a condition affecting 1% of women in the general population, causing amenorrhea, hypergonadotropism and hypoestrogenism before the age of 40. Currently, POF cannot be reversed and, although treatments are available, there is an urgent need for improved treatment strategies. Growth hormone (GH) is a pleiotropic hormone that affects a broad spectrum of physiological functions, from carbohydrate and lipid metabolism to the immune response. GH has previously been used to treat POF in non-transgenic preclinical trials, but the biochemical mechanism underlying these effects are unclear. In the present study, a mouse model of POF was generated using cyclophosphamide. Treatment of POF mice with recombinant mouse growth hormone (rmGH) was revealed to markedly reduce POF histopathology in ovarian tissue, relieve ovarian granulosa cell injury, reduce the number of atretic follicles and significantly increase the number of mature oocytes. Furthermore, an enzyme-linked immunosorbent assay revealed that plasma estradiol levels increased and plasma follicle stimulating hormone levels decreased with time in a group of mice treated with a medium dose of rmGH (0.8 mg/kg) when compared with the POF model group (P<0.05). In addition, reverse transcription-quantitative polymerase chain reaction and immunohistochemical analysis demonstrated elevated levels of Notch-1 signaling pathway factors (Notch1, CBF1, and HES1) in wild-type mice and those treated with medium and high doses of rmGH, but not in those treated with low doses of rmGH. In conclusion, GH may promote ovarian tissue repair, estrogen release and oocyte maturation via activation of the Notch-1 signaling pathway in ovarian tissue. PMID:27347041

  20. Structural basis for Notch1 engagement of Delta-like 4

    DOE PAGES

    Luca, Vincent C.; Jude, Kevin M.; Pierce, Nathan W.; Nachury, Maxence V.; Fischer, Suzanne; Garcia, K. Christopher

    2015-02-20

    Notch receptors guide mammalian cell fate decisions by engaging the proteins Jagged and Delta-like (DLL). The 2.3 angstrom resolution crystal structure of the interacting regions of the Notch1-DLL4 complex reveals a two-site, antiparallel binding orientation assisted by Notch1 O-linked glycosylation. Notch1 epidermal growth factor–like repeats 11 and 12 interact with the DLL4 Delta/Serrate/Lag-2 (DSL) domain and module at the N-terminus of Notch ligands (MNNL) domains, respectively. Threonine and serine residues on Notch1 are functionalized with O-fucose and O-glucose, which act as surrogate amino acids by making specific, and essential, contacts to residues on DLL4. Lastly, the elucidation of a directmore » chemical role for O-glycans in Notch1 ligand engagement demonstrates how, by relying on posttranslational modifications of their ligand binding sites, Notch proteins have linked their functional capacity to developmentally regulated biosynthetic pathways.« less

  1. Activation of the NOTCH pathway in head and neck cancer.

    PubMed

    Sun, Wenyue; Gaykalova, Daria A; Ochs, Michael F; Mambo, Elizabeth; Arnaoutakis, Demetri; Liu, Yan; Loyo, Myriam; Agrawal, Nishant; Howard, Jason; Li, Ryan; Ahn, Sun; Fertig, Elana; Sidransky, David; Houghton, Jeffery; Buddavarapu, Kalyan; Sanford, Tiffany; Choudhary, Ashish; Darden, Will; Adai, Alex; Latham, Gary; Bishop, Justin; Sharma, Rajni; Westra, William H; Hennessey, Patrick; Chung, Christine H; Califano, Joseph A

    2014-02-15

    NOTCH1 mutations have been reported to occur in 10% to 15% of head and neck squamous cell carcinomas (HNSCC). To determine the significance of these mutations, we embarked upon a comprehensive study of NOTCH signaling in a cohort of 44 HNSCC tumors and 25 normal mucosal samples through a set of expression, copy number, methylation, and mutation analyses. Copy number increases were identified in NOTCH pathway genes, including the NOTCH ligand JAG1. Gene set analysis defined a differential expression of the NOTCH signaling pathway in HNSCC relative to normal tissues. Analysis of individual pathway-related genes revealed overexpression of ligands JAG1 and JAG2 and receptor NOTCH3. In 32% of the HNSCC examined, activation of the downstream NOTCH effectors HES1/HEY1 was documented. Notably, exomic sequencing identified 5 novel inactivating NOTCH1 mutations in 4 of the 37 tumors analyzed, with none of these tumors exhibiting HES1/HEY1 overexpression. Our results revealed a bimodal pattern of NOTCH pathway alterations in HNSCC, with a smaller subset exhibiting inactivating NOTCH1 receptor mutations but a larger subset exhibiting other NOTCH1 pathway alterations, including increases in expression or gene copy number of the receptor or ligands as well as downstream pathway activation. Our results imply that therapies that target the NOTCH pathway may be more widely suitable for HNSCC treatment than appreciated currently.

  2. Activation of the NOTCH pathway in Head and Neck Cancer

    PubMed Central

    Sun, Wenyue; Gaykalova, Daria A.; Ochs, Michael F.; Mambo, Elizabeth; Arnaoutakis, Demetri; Liu, Yan; Loyo, Myriam; Agrawal, Nishant; Howard, Jason; Li, Ryan; Ahn, Sun; Fertig, Elana; Sidransky, David; Houghton, Jeffery; Buddavarapu, Kalyan; Sanford, Tiffany; Choudhary, Ashish; Darden, Will; Adai, Alex; Latham, Gary; Bishop, Justin; Sharma, Rajni; Westra, William H.; Hennessey, Patrick; Chung, Christine H.; Califano, Joseph A.

    2014-01-01

    NOTCH1 mutations have been reported to occur in 10 to 15% of head and neck squamous cell carcinomas (HNSCC). To determine the significance of these mutations, we embarked upon a comprehensive study of NOTCH signaling in a cohort of 44 HNSCC tumors and 25 normal mucosal samples through a set of expression, copy number, methylation and mutation analyses. Copy number increases were identified in NOTCH pathway genes including the NOTCH ligand JAG1. Gene set analysis defined a differential expression of the NOTCH signaling pathway in HNSCC relative to normal tissues. Analysis of individual pathway-related genes revealed overexpression of ligands JAG1 and JAG2 and receptor NOTCH3. In 32% of the HNSCC examined, activation of the downstream NOTCH effectors HES1/HEY1 was documented. Notably, exomic sequencing identified 5 novel inactivating NOTCH1 mutations in 4/37 of the tumors analyzed, with none of these tumors exhibiting HES1/HEY1 overexpression. Our results revealed a bimodal pattern of NOTCH pathway alterations in HNSCC, with a smaller subset exhibiting inactivating NOTCH1 receptors mutations but a larger subset exhibiting other NOTCH1 pathway alterations, including increases in expression or gene copy number of the receptor or ligands as well as downstream pathway activation. Our results imply that therapies that target the NOTCH pathway may be more widely suitable for HNSCC treatment than appreciated currently. PMID:24351288

  3. Effect of Tongxinluo on Nephrin Expression via Inhibition of Notch1/Snail Pathway in Diabetic Rats

    PubMed Central

    Cui, Fangqiang; Zou, Dawei; Gao, Yanbin; Zhang, Na; Wang, Jinyang; Xu, Liping; Geng, Jianguo; Li, Jiaoyang; Zhou, Shengnan; Wang, Xinyao

    2015-01-01

    Podocyte injury is an important mechanism of diabetic nephropathy (DN). Accumulating evidence suggests that nephrin expression is decreased in podocyte in DN. Moreover, it has been demonstrated that tongxinluo (TXL) can ameliorate renal structure disruption and dysfunction in DN. However, the effect of TXL on podocyte injury in DN and its molecular mechanism is unclear. In order to explore the effect of TXL on podocyte injury and its molecular mechanism in DN, our in vivo and in vitro studies were performed. Our results showed that TXL increased nephrin expression in diabetic rats and in high glucose cultured podocyte. Meanwhile, TXL decreased ICN1 (the intracellular domain of notch), HES1, and snail expression in podocyte in vivo and in vitro. More importantly, we found that TXL protected podocyte from injury in DN. The results demonstrated that TXL inhibited the activation of notch1/snail pathway and increased nephrin expression, which may be a mechanism of protecting effect on podocyte injury in DN. PMID:26417374

  4. γ-Secretase inhibitor DAPT attenuates intimal hyperplasia of vein grafts by inhibition of Notch1 signaling.

    PubMed

    Xiao, Yong Guang; Wang, Wei; Gong, Dan; Mao, Zhi Fu

    2014-06-01

    The proliferation and high plasticity of vascular smooth muscle cells (vSMCs) are the major reasons for restenosis of vein grafts. N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), specific inhibitor of γ-secretase, has been shown to regulate vSMC proliferation and differentiation through the Notch signaling pathway, but the pathophysiological importance of these findings in venous grafts has not yet been determined. A rat vein graft model was employed wherein the left jugular vein was surgically interposed into the left common carotid artery. Daily subcutaneous injections of DAPT or placebo (DMSO) were administered postoperatively (control animals received no treatment). We showed that DAPT can inhibit restenosis of vein grafts by inhibiting vSMC proliferation and increasing apoptosis in vivo. Notch1 signaling was highly active during the development of intima thickening. By blocking the Notch signaling pathway, the γ-secretase inhibitor DAPT can significantly attenuated intima thickening. These changes in vein grafts coincided with enhanced binding of myocardin to the smooth muscle-specific protein SM22 and smooth muscle myosin heavy chain at the promoters of vSMC differentiation-specific genes. These studies showed that DAPT can restore the vSMC phenotype and inhibit vSMC proliferation through suppression of the Notch1 signaling pathway, and thus opens a new avenue for the treatment of restenosis in vein grafts. PMID:24751889

  5. Activating STAT6 mutations in follicular lymphoma

    PubMed Central

    Yildiz, Mehmet; Li, Hongxiu; Bernard, Denzil; Amin, Nisar A.; Ouillette, Peter; Jones, Siân; Saiya-Cork, Kamlai; Parkin, Brian; Jacobi, Kathryn; Shedden, Kerby; Wang, Shaomeng; Chang, Alfred E.; Kaminski, Mark S.

    2015-01-01

    Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma in the Western world. FL cell-intrinsic and cell-extrinsic factors influence FL biology and clinical outcome. To further our understanding of the genetic basis of FL, we performed whole-exome sequencing of 23 highly purified FL cases and 1 transformed FL case and expanded findings to a combined total of 114 FLs. We report recurrent mutations in the transcription factor STAT6 in 11% of FLs and identified the STAT6 amino acid residue 419 as a novel STAT6 mutation hotspot (p.419D/G, p.419D/A, and p.419D/H). FL-associated STAT6 mutations were activating, as evidenced by increased transactivation in HEK293T cell–based transfection/luciferase reporter assays, heightened interleukin-4 (IL-4) –induced activation of target genes in stable STAT6 transfected lymphoma cell lines, and elevated baseline expression levels of STAT6 target genes in primary FL B cells harboring mutant STAT6. Mechanistically, FL-associated STAT6 mutations facilitated nuclear residency of STAT6, independent of IL-4–induced STAT6-Y641 phosphorylation. Structural modeling of STAT6 based on the structure of the STAT1-DNA complex revealed that most FL-associated STAT6 mutants locate to the STAT6-DNA interface, potentially facilitating heightened interactions. The genetic and functional data combined strengthen the recognition of the IL-4/JAK/STAT6 axis as a driver of FL pathogenesis. PMID:25428220

  6. Endothelial Jarid2/Jumonji is required for normal cardiac development and proper Notch1 expression.

    PubMed

    Mysliwiec, Matthew R; Bresnick, Emery H; Lee, Youngsook

    2011-05-13

    Jarid2/Jumonji critically regulates developmental processes including cardiovascular development. Jarid2 knock-out mice exhibit cardiac defects including hypertrabeculation with noncompaction of the ventricular wall. However, molecular mechanisms underlying Jarid2-mediated cardiac development remain unknown. To determine the cardiac lineage-specific roles of Jarid2, we generated myocardial, epicardial, cardiac neural crest, or endothelial conditional Jarid2 knock-out mice using Cre-loxP technology. Only mice with an endothelial deletion of Jarid2 recapitulate phenotypic defects observed in whole body mutants including hypertrabeculation and noncompaction of the ventricle. To identify potential targets of Jarid2, combinatorial approaches using microarray and candidate gene analyses were employed on Jarid2 knock-out embryonic hearts. Whole body or endothelial deletion of Jarid2 leads to increased endocardial Notch1 expression in the developing ventricle, resulting in increased Notch1-dependent signaling to the adjacent myocardium. Using quantitative chromatin immunoprecipitation analysis, Jarid2 was found to occupy a specific region on the endogenous Notch1 locus. We propose that failure to properly regulate Notch signaling in Jarid2 mutants likely leads to the defects in the developing ventricular chamber. The identification of Jarid2 as a potential regulator of Notch1 signaling has broad implications for many cellular processes including development, stem cell maintenance, and tumor formation.

  7. Notch1 Is Regulated by Chorionic Gonadotropin and Progesterone in Endometrial Stromal Cells and Modulates Decidualization in Primates

    PubMed Central

    Afshar, Yalda; Miele, Lucio

    2012-01-01

    No other tissue in the body undergoes such a vast and extensive growth and remodeling in a relatively short period of time as the primate endometrium. Endometrial integrity is coordinated by ovarian hormones, namely, estrogens, progesterone, and the embryonic hormone chorionic gonadotropin (CG). These regulated events modulate the menstrual cycle and decidualization. The Notch family of transmembrane receptors regulate cellular proliferation, differentiation, and apoptosis, cellular processes required to maintain endometrial integrity. In two primate models, the human and the simulated pregnant baboon model, we demonstrated that Notch1 is increased during the window of uterine receptivity, concomitant with CG. Furthermore, CG combined with estrogens and progesterone up-regulate the level of Notch1, whereas progesterone increases the intracellular transcriptionally competent Notch1, which binds in a complex with progesterone receptor. Inhibition of Notch1 prevented decidualization, and alternatively, when decidualization is biochemically recapitulated in vitro, Notch1 is down-regulated. A focused microarray demonstrated that the Notch inhibitor, Numb, dramatically increased when Notch1 decreased during decidualization. We propose that in the endometrium, Notch has a dual role during the window of uterine receptivity. Initially, Notch1 mediates a survival signal in the uterine endometrium in response to CG from the implanting blastocyst and progesterone, so that menstrual sloughing is averted. Subsequently, Notch1 down-regulation may be critical for the transition of stromal fibroblast to decidual cells, which is essential for the establishment of a successful pregnancy. PMID:22535768

  8. MiR-34a Promotes Osteogenic Differentiation of Human Adipose-Derived Stem Cells via the RBP2/NOTCH1/CYCLIN D1 Coregulatory Network.

    PubMed

    Fan, Cong; Jia, Lingfei; Zheng, Yunfei; Jin, Chanyuan; Liu, Yunsong; Liu, Hao; Zhou, Yongsheng

    2016-08-01

    MiR-34a was demonstrated to be upregulated during the osteogenic differentiation of human adipose-derived stem cells (hASCs). Overexpression of miR-34a significantly increased alkaline phosphatase activity, mineralization capacity, and the expression of osteogenesis-associated genes in hASCs in vitro. Enhanced heterotopic bone formation in vivo was also observed upon overexpression of miR-34a in hASCs. Mechanistic investigations revealed that miR-34a inhibited the expression of retinoblastoma binding protein 2 (RBP2) and reduced the luciferase activity of reporter gene construct comprising putative miR-34a binding sites in the 3' UTR of RBP2. Moreover, miR-34a downregulated the expression of NOTCH1 and CYCLIN D1 and upregulated the expression of RUNX2 by targeting RBP2, NOTCH1, and CYCLIN D1. Taken together, our results suggested that miR-34a promotes the osteogenic differentiation of hASCs via the RBP2/NOTCH1/CYCLIN D1 coregulatory network, indicating that miR-34a-targeted therapy could be a valuable approach to promote bone regeneration. PMID:27453008

  9. The crystal structure of a partial mouse Notch-1 ankyrin domain: Repeats 4 through 7 preserve an ankyrin fold

    SciTech Connect

    Lubman, Olga Y.; Kopan, Raphael; Waksman, Gabriel; Korolev, Sergey

    2010-07-20

    Folding and stability of proteins containing ankyrin repeats (ARs) is of great interest because they mediate numerous protein-protein interactions involved in a wide range of regulatory cellular processes. Notch, an ankyrin domain containing protein, signals by converting a transcriptional repression complex into an activation complex. The Notch ANK domain is essential for Notch function and contains seven ARs. Here, we present the 2.2 {angstrom} crystal structure of ARs 4-7 from mouse Notch 1 (m1ANK). These C-terminal repeats were resistant to degradation during crystallization, and their secondary and tertiary structures are maintained in the absence of repeats 1-3. The crystallized fragment adopts a typical ankyrin fold including the poorly conserved seventh AR, as seen in the Drosophila Notch ANK domain (dANK). The structural preservation and stability of the C-terminal repeats shed a new light onto the mechanism of hetero-oligomeric assembly during Notch-mediated transcriptional activation.

  10. NOTCH1 inhibition enhances the efficacy of conventional chemotherapeutic agents by targeting head neck cancer stem cell

    PubMed Central

    Zhao, Zhi-Li; Zhang, Lu; Huang, Cong-Fa; Ma, Si-Rui; Bu, Lin-Lin; Liu, Jian-Feng; Yu, Guang-Tao; Liu, Bing; Gutkind, J. Silvio; Kulkarni, Ashok B.; Zhang, Wen-Feng; Sun, Zhi-Jun

    2016-01-01

    Cancer stem cells (CSCs) are considered responsible for tumor initiation and chemoresistance. This study was aimed to investigate the possibility of targeting head neck squamous cell carcinoma (HNSCC) by NOTCH1 pathway inhibition and explore the synergistic effect of combining NOTCH inhibition with conventional chemotherapy. NOTCH1/HES1 elevation was found in human HNSCC, especially in tissue post chemotherapy and lymph node metastasis, which is correlated with CSCs markers. NOTCH1 inhibitor DAPT (GSI-IX) significantly reduces CSCs population and tumor self-renewal ability in vitro and in vivo. Flow cytometry analysis showed that NOTCH1 inhibition reduces CSCs frequency either alone or in combination with chemotherapeutic agents, namely, cisplatin, docetaxel, and 5-fluorouracil. The combined strategy of NOTCH1 blockade and chemotherapy synergistically attenuated chemotherapy-enriched CSC population, promising a potential therapeutic exploitation in future clinical trial. PMID:27108536

  11. A Disintegrin and Metalloproteinase Domain 17 Regulates Colorectal Cancer Stem Cells and Chemosensitivity Via Notch1 Signaling.

    PubMed

    Wang, Rui; Ye, Xiangcang; Bhattacharya, Rajat; Boulbes, Delphine R; Fan, Fan; Xia, Ling; Ellis, Lee M

    2016-03-01

    Evidence is accumulating for the role of cancer stem cells (CSCs) in mediating chemoresistance in patients with metastatic colorectal cancer (mCRC). A disintegrin and metalloproteinase domain 17 (ADAM17; also known as tumor necrosis factor-α-converting enzyme [TACE]) was shown to be overexpressed and to mediate cell proliferation and chemoresistance in CRC cells. However, its role in mediating the CSC phenotype in CRC has not been well-characterized. The objective of the present study was to determine whether ADAM17 regulates the CSC phenotype in CRC and to elucidate the downstream signaling mechanism that mediates cancer stemness. We treated established CRC cell lines and a newly established human CRC cell line HCP-1 with ADAM17-specific small interfering RNA (siRNA) or the synthetic peptide inhibitor TAPI-2. The effects of ADAM17 inhibition on the CSC phenotype and chemosensitivity to 5-fluorouracil (5-FU) in CRC cells were examined. siRNA knockdown and TAPI-2 decreased the protein levels of cleaved Notch1 (Notch1 intracellular domain) and HES-1 in CRC cells. A decrease in the CSC phenotype was determined by sphere formation and ALDEFLUOR assays. Moreover, TAPI-2 sensitized CRC cells to 5-FU by decreasing cell viability and the median lethal dose of 5-FU and increasing apoptosis. We also showed the cleavage and release of soluble Jagged-1 and -2 by ADAM17 in CRC cells. Our studies have elucidated a role of ADAM17 in regulating the CSC phenotype and chemoresistance in CRC cells. The use of drugs that inhibit ADAM17 activity might increase the therapeutic benefit to patients with mCRC and, potentially, those with other solid malignancies.

  12. The intracellular domains of Notch1 and Notch2 are functionally equivalent during development and carcinogenesis.

    PubMed

    Liu, Zhenyi; Brunskill, Eric; Varnum-Finney, Barbara; Zhang, Chi; Zhang, Andrew; Jay, Patrick Y; Bernstein, Irv; Morimoto, Mitsuru; Kopan, Raphael

    2015-07-15

    Although Notch1 and Notch2 are closely related paralogs and function through the same canonical signaling pathway, they contribute to different outcomes in some cell and disease contexts. To understand the basis for these differences, we examined in detail mice in which the Notch intracellular domains (N1ICD and N2ICD) were swapped. Our data indicate that strength (defined here as the ultimate number of intracellular domain molecules reaching the nucleus, integrating ligand-mediated release and nuclear translocation) and duration (half-life of NICD-RBPjk-MAML-DNA complexes, integrating cooperativity and stability dependent on shared sequence elements) are the factors that underlie many of the differences between Notch1 and Notch2 in all the contexts we examined, including T-cell development, skin differentiation and carcinogenesis, the inner ear, the lung and the retina. We were able to show that phenotypes in the heart, endothelium, and marginal zone B cells are attributed to haploinsufficiency but not to intracellular domain composition. Tissue-specific differences in NICD stability were most likely caused by alternative scissile bond choices by tissue-specific γ-secretase complexes following the intracellular domain swap. Reinterpretation of clinical findings based on our analyses suggests that differences in outcome segregating with Notch1 or Notch2 are likely to reflect outcomes dependent on the overall strength of Notch signals. PMID:26062937

  13. The intracellular domains of Notch1 and Notch2 are functionally equivalent during development and carcinogenesis

    PubMed Central

    Liu, Zhenyi; Brunskill, Eric; Varnum-Finney, Barbara; Zhang, Chi; Zhang, Andrew; Jay, Patrick Y.; Bernstein, Irv; Morimoto, Mitsuru; Kopan, Raphael

    2015-01-01

    Although Notch1 and Notch2 are closely related paralogs and function through the same canonical signaling pathway, they contribute to different outcomes in some cell and disease contexts. To understand the basis for these differences, we examined in detail mice in which the Notch intracellular domains (N1ICD and N2ICD) were swapped. Our data indicate that strength (defined here as the ultimate number of intracellular domain molecules reaching the nucleus, integrating ligand-mediated release and nuclear translocation) and duration (half-life of NICD-RBPjk-MAML-DNA complexes, integrating cooperativity and stability dependent on shared sequence elements) are the factors that underlie many of the differences between Notch1 and Notch2 in all the contexts we examined, including T-cell development, skin differentiation and carcinogenesis, the inner ear, the lung and the retina. We were able to show that phenotypes in the heart, endothelium, and marginal zone B cells are attributed to haploinsufficiency but not to intracellular domain composition. Tissue-specific differences in NICD stability were most likely caused by alternative scissile bond choices by tissue-specific γ-secretase complexes following the intracellular domain swap. Reinterpretation of clinical findings based on our analyses suggests that differences in outcome segregating with Notch1 or Notch2 are likely to reflect outcomes dependent on the overall strength of Notch signals. PMID:26062937

  14. Epidermal Notch1 recruits RORγ+ group 3 innate lymphoid cells to orchestrate normal skin repair

    PubMed Central

    Li, Zhi; Hodgkinson, Tom; Gothard, Elizabeth J.; Boroumand, Soulmaz; Lamb, Rebecca; Cummins, Ian; Narang, Priyanka; Sawtell, Amy; Coles, Jenny; Leonov, German; Reboldi, Andrea; Buckley, Christopher D.; Cupedo, Tom; Siebel, Christian; Bayat, Ardeshir; Coles, Mark C.; Ambler, Carrie A.

    2016-01-01

    Notch has a well-defined role in controlling cell fate decisions in the embryo and the adult epidermis and immune systems, yet emerging evidence suggests Notch also directs non-cell-autonomous signalling in adult tissues. Here, we show that Notch1 works as a damage response signal. Epidermal Notch induces recruitment of immune cell subsets including RORγ+ ILC3s into wounded dermis; RORγ+ ILC3s are potent sources of IL17F in wounds and control immunological and epidermal cell responses. Mice deficient for RORγ+ ILC3s heal wounds poorly resulting from delayed epidermal proliferation and macrophage recruitment in a CCL3-dependent process. Notch1 upregulates TNFα and the ILC3 recruitment chemokines CCL20 and CXCL13. TNFα, as a Notch1 effector, directs ILC3 localization and rates of wound healing. Altogether these findings suggest that Notch is a key stress/injury signal in skin epithelium driving innate immune cell recruitment and normal skin tissue repair. PMID:27099134

  15. Epidermal Notch1 recruits RORγ(+) group 3 innate lymphoid cells to orchestrate normal skin repair.

    PubMed

    Li, Zhi; Hodgkinson, Tom; Gothard, Elizabeth J; Boroumand, Soulmaz; Lamb, Rebecca; Cummins, Ian; Narang, Priyanka; Sawtell, Amy; Coles, Jenny; Leonov, German; Reboldi, Andrea; Buckley, Christopher D; Cupedo, Tom; Siebel, Christian; Bayat, Ardeshir; Coles, Mark C; Ambler, Carrie A

    2016-01-01

    Notch has a well-defined role in controlling cell fate decisions in the embryo and the adult epidermis and immune systems, yet emerging evidence suggests Notch also directs non-cell-autonomous signalling in adult tissues. Here, we show that Notch1 works as a damage response signal. Epidermal Notch induces recruitment of immune cell subsets including RORγ(+) ILC3s into wounded dermis; RORγ(+) ILC3s are potent sources of IL17F in wounds and control immunological and epidermal cell responses. Mice deficient for RORγ(+) ILC3s heal wounds poorly resulting from delayed epidermal proliferation and macrophage recruitment in a CCL3-dependent process. Notch1 upregulates TNFα and the ILC3 recruitment chemokines CCL20 and CXCL13. TNFα, as a Notch1 effector, directs ILC3 localization and rates of wound healing. Altogether these findings suggest that Notch is a key stress/injury signal in skin epithelium driving innate immune cell recruitment and normal skin tissue repair. PMID:27099134

  16. Notch1-Dll4 signalling and mechanical force regulate leader cell formation during collective cell migration.

    PubMed

    Riahi, Reza; Sun, Jian; Wang, Shue; Long, Min; Zhang, Donna D; Wong, Pak Kin

    2015-03-13

    At the onset of collective cell migration, a subset of cells within an initially homogenous population acquires a distinct 'leader' phenotype with characteristic morphology and motility. However, the factors driving the leader cell formation as well as the mechanisms regulating leader cell density during the migration process remain to be determined. Here we use single-cell gene expression analysis and computational modelling to show that the leader cell identity is dynamically regulated by Dll4 signalling through both Notch1 and cellular stress in a migrating epithelium. Time-lapse microscopy reveals that Dll4 is induced in leader cells after the creation of the cell-free region and leader cells are regulated via Notch1-Dll4 lateral inhibition. Furthermore, mechanical stress inhibits Dll4 expression and leader cell formation in the monolayer. Collectively, our findings suggest that a reduction of mechanical force near the boundary promotes Notch1-Dll4 signalling to dynamically regulate the density of leader cells during collective cell migration.

  17. Protective effect of curcumin on acute airway inflammation of allergic asthma in mice through Notch1-GATA3 signaling pathway.

    PubMed

    Chong, Lei; Zhang, Weixi; Nie, Ying; Yu, Gang; Liu, Liu; Lin, Li; Wen, Shunhang; Zhu, Lili; Li, Changchong

    2014-10-01

    Curcumin, a natural product derived from the plant Curcuma longa, has been found to have anti-inflammatory, antineoplastic and antifibrosis effects. It has been reported that curcumin attenuates allergic airway inflammation in mice through inhibiting NF-κB and its downstream transcription factor GATA3. It also has been proved the antineoplastic effect of curcumin through down-regulating Notch1 receptor and its downstream nuclear transcription factor NF-κB levels. In this study, we aimed to investigate the anti-inflammatory effect of curcumin on acute allergic asthma and its underlying mechanisms. 36 male BALB/c mice were randomly divided into four groups (normal, asthma, asthma+budesonide and asthma+curcumin groups). BALF (bronchoalveolar lavage fluid) and lung tissues were analyzed for airway inflammation and the expression of Notch1, Notch2, Notch3, Notch4 and the downstream transcription factor GATA3. Our findings showed that the levels of Notch1 and Notch2 receptors were up-regulated in asthma group, accompanied by the increased expression of GATA3. But the expression of Notch2 receptor was lower than Notch1 receptor. Curcumin pretreatment improved the airway inflammatory cells infiltration and reversed the increasing levels of Notch1/2 receptors and GATA3. Notch3 receptor was not expressed in all of the four groups. Notch4 receptor protein and mRNA expression level in the four groups had no significant differences. The results of the present study suggested that Notch1 and Notch2 receptor, major Notch1 receptor, played an important role in the development of allergic airway inflammation and the inhibition of Notch1-GATA3 signaling pathway by curcumin can prevent the development and deterioration of the allergic airway inflammation. This may be a possible therapeutic option of allergic asthma.

  18. Notch1 is a 5-fluorouracil resistant and poor survival marker in human esophagus squamous cell carcinomas.

    PubMed

    Liu, Jian; Fan, Huijie; Ma, Yuanyuan; Liang, Dongming; Huang, Ruixia; Wang, Junsheng; Zhou, Fuyou; Kan, Quancheng; Ming, Liang; Li, Huixiang; Giercksky, Karl-Erik; Nesland, Jahn Martin; Suo, Zhenhe

    2013-01-01

    Notch signaling involves the processes that govern cell proliferation, cell fate decision, cell differentiation and stem cell maintenance. Due to its fundamental role in stem cells, it has been speculated during the recent years that Notch family may have critical functions in cancer stem cells or cancer cells with a stem cell phenotype, therefore playing an important role in the process of oncogenesis. In this study, expression of Notch family in KYSE70, KYSE140 and KYSE450 squamous esophageal cancer cell lines and virus transformed squamous esophageal epithelial cell line Het-1A was examined by quantitative RT-PCR. Compared to the Het-1A cells, higher levels of Nocth1 and Notch3 expression in the cancer cell lines were identified. Due to the finding that NOTCH3 mainly mediates squamous cell differentiation, NOTCH1 expression was further studied in these cell lines. By Western blot analyses, the KYSE70 cell line which derived from a poorly differentiated tumor highly expressed Notch1, and the Notch1 expression in this cell line was hypoxia inducible, while the KYSE450 cell line which derived from a well differentiated tumor was always negative for Notch1, even in hypoxia. Additional studies demonstrated that the KYSE70 cell line was more 5-FU resistant than the KYSE450 cell line and such 5-FU resistance is correlated to Notch1 expression verified by Notch1 knockdown experiments. In clinical samples, Notch1 protein expression was detected in the basal cells of human esophagus epithelia, and its expression in squamous cell carcinomas was significantly associated with higher pathological grade and shorter overall survival. We conclude that Notch1 expression is associated with cell aggressiveness and 5-FU drug resistance in human esophageal squamous cell carcinoma cell lines in vitro and is significantly associated with a poor survival in human esophageal squamous cell carcinomas.

  19. Notch1 is associated with the multidrug resistance of hypoxic osteosarcoma by regulating MRP1 gene expression.

    PubMed

    Li, C; Guo, D; Tang, B; Zhang, Y; Zhang, K; Nie, L

    2016-01-01

    Hypoxia and Notch signaling pathway are closely related and both participate in cell proliferation and drug resistance of tumors. However, the molecular mechanisms of hypoxia and Notch signaling pathway in cell proliferation and drug resistance of osteosarcoma (OS) remain unclear. In this study, to further evaluate the role of hypoxia and Notch1 on drug resistance of OS, we investigated the influence of inhibiting Notch1 pathway by Notch1 small interference RNA (siRNA) on human MG-63 OS cells in hypoxia. Our data showed that hypoxia promoted OS cell proliferation, induced the G0/G1-S-G2/M phase transition, and increased multidrug resistance of human OS cells. Western blot analysis suggested that hypoxia increased the expression of HIF-1α, Notch1, and multidrug resistance protein-1 (MRP1) in human OS cells. Notch1 siRNA inhibits proliferation and increases apoptosis of hypoxic OS cells. Finally, these hypoxic OS cells can be sensitized to multidrug treatment through inhibition of the Notch protein expression by siRNA. Repression of the Notch protein expression resulted in down-regulation of MRP1 protein. These data support the conclusion that Notch signaling is up-regulated in human OS cells under hypoxia and Notch1 may represent a viable target to overcome chemoresistant OS cells in a hypoxic niche by regulating MRP1 gene expression. PMID:27468877

  20. Gene Mutation Analysis in EGFR Wild Type NSCLC Responsive to Erlotinib: Are There Features to Guide Patient Selection?

    PubMed Central

    Ulivi, Paola; Delmonte, Angelo; Chiadini, Elisa; Calistri, Daniele; Papi, Maximilian; Mariotti, Marita; Verlicchi, Alberto; Ragazzini, Angela; Capelli, Laura; Gamboni, Alessandro; Puccetti, Maurizio; Dubini, Alessandra; Burgio, Marco Angelo; Casanova, Claudia; Crinò, Lucio; Amadori, Dino; Dazzi, Claudio

    2014-01-01

    Tyrosine kinase inhibitors (TKIs) are very efficacious in non-small-cell lung cancer (NSCLC) patients harboring activating Epidermal Growth Factor Receptor (EGFR) mutations. However, about 10% of EGFR wild type (wt) patients respond to TKI, with unknown molecular mechanisms of sensitivity. We considered a case series of 34 EGFR wt NSCLC patients responsive to erlotinib after at least one line of therapy. Responsive patients were matched with an equal number of non-responsive EGFR wt patients. A panel of 26 genes, for a total of 214 somatic mutations, was analyzed by MassARRAY® System (Sequenom, San Diego, CA, USA). A 15% KRAS mutation was observed in both groups, with a prevalence of G12C in non-responders (80% vs. 40% in responders). NOTCH1, p53 and EGFR-resistance-related mutations were found more frequently in non-responders, whereas EGFR-sensitizing mutations and alterations in genes involved in proliferation pathways were more frequent in responders. In conclusion, our findings indicate that p53, NOTCH1 and exon 20 EGFR mutations seem to be related to TKI resistance. KRAS mutations do not appear to influence the TKI response, although G12C mutation is more frequent in non-responders. Finally, the use of highly sensitive methodologies could lead to the identification of under-represented EGFR mutations potentially associated with TKI sensitivity. PMID:25561229

  1. MicroRNA-449a Overexpression, Reduced NOTCH1 Signals and Scarce Goblet Cells Characterize the Small Intestine of Celiac Patients

    PubMed Central

    Tinto, Nadia; Montanaro, Donatella; Capobianco, Valentina; Izzo, Valentina; Tucci, Francesca; Troncone, Giancarlo; Greco, Luigi; Sacchetti, Lucia

    2011-01-01

    MiRNAs play a relevant role in regulating gene expression in a variety of physiological and pathological conditions including autoimmune disorders. MiRNAs are also important in the differentiation and function of the mouse intestinal epithelium. Our study was aimed to look for miRNA-based modulation of gene expression in celiac small intestine, and particularly for genes involved in cell intestinal differentiation/proliferation mechanisms. A cohort of 40 children (20 with active CD, 9 on a gluten-free diet (GFD), and 11 controls), were recruited at the Paediatrics Department (University of Naples Federico II). The expression of 365 human miRNAs was quantified by TaqMan low-density arrays. We used bioinformatics to predict putative target genes of miRNAs and to select biological pathways. The presence of NOTCH1, HES1, KLF4, MUC-2, Ki67 and beta-catenin proteins in the small intestine of CD and control children was tested by immunohistochemistry. The expression of about 20% of the miRNAs tested differed between CD and control children. We found that high miR-449a levels targeted and reduced both NOTCH1 and KLF4 in HEK-293 cells. NOTCH1, KLF4 signals and the number of goblet cells were lower in small intestine of children with active CD and in those on a GFD than in controls, whereas more nuclear beta-catenin staining, as a sign of the WNT pathway activation, and more Ki67 staining, as sign of proliferation, were present in crypts from CD patients than in controls. In conclusion we first demonstrate a miRNA mediated gene regulation in small intestine of CD patients. We also highlighted a reduced NOTCH1 pathway in our patients, irrespective of whether the disease was active or not. We suggest that NOTCH pathway could be constitutively altered in the celiac small intestine and could drive the increased proliferation and the decreased differentiation of intestinal cells towards the secretory goblet cell lineage. PMID:22194996

  2. Rho/ROCK-dependent inhibition of 3T3-L1 adipogenesis by G-protein-deamidating dermonecrotic toxins: differential regulation of Notch1, Pref1/Dlk1, and β-catenin signaling

    PubMed Central

    Bannai, Yuka; Aminova, Leila R.; Faulkner, Melinda J.; Ho, Mengfei; Wilson, Brenda A.

    2012-01-01

    The dermonecrotic toxins from Pasteurella multocida (PMT), Bordetella (DNT), Escherichia coli (CNF1-3), and Yersinia (CNFY) modulate their G-protein targets through deamidation and/or transglutamination of an active site Gln residue, which results in activation of the G protein and its cognate downstream signaling pathways. Whereas DNT and the CNFs act on small Rho GTPases, PMT acts on the α subunit of heterotrimeric Gq, Gi, and G12/13 proteins. We previously demonstrated that PMT potently blocks adipogenesis and adipocyte differentiation in a calcineurin-independent manner through downregulation of Notch1 and stabilization of β-catenin and Pref1/Dlk1, key proteins in signaling pathways strongly linked to cell fate decisions, including fat and bone development. Here, we report that similar to PMT, DNT, and CNF1 completely block adipogenesis and adipocyte differentiation by preventing upregulation of adipocyte markers, PPARγ and C/EBPα, while stabilizing the expression of Pref1/Dlk1 and β-catenin. We show that the Rho/ROCK inhibitor Y-27632 prevented or reversed these toxin-mediated effects, strongly supporting a role for Rho/ROCK signaling in dermonecrotic toxin-mediated inhibition of adipogenesis and adipocyte differentiation. Toxin treatment was also accompanied by downregulation of Notch1 expression, although this inhibition was independent of Rho/ROCK signaling. We further show that PMT-mediated downregulation of Notch1 expression occurs primarily through G12/13 signaling. Our results reveal new details of the pathways involved in dermonecrotic toxin action on adipocyte differentiation, and the role of Rho/ROCK signaling in mediating toxin effects on Wnt/β-catenin and Notch1 signaling, and in particular the role of Gq and G12/13 in mediating PMT effects on Rho/ROCK and Notch1 signaling. PMID:22919671

  3. Hepatic Notch1 deletion predisposes to diabetes and steatosis via glucose-6-phosphatase and perilipin-5 upregulation.

    PubMed

    Bernsmeier, Christine; Dill, Michael T; Provenzano, Angela; Makowska, Zuzanna; Krol, Ilona; Muscogiuri, Giovanna; Semela, David; Tornillo, Luigi; Marra, Fabio; Heim, Markus H; Duong, François H T

    2016-09-01

    Notch signaling pathways have recently been implicated in the pathogenesis of metabolic diseases. However, the role of hepatic Notch signaling in glucose and lipid metabolism remains unclear and needs further investigation as it might be a candidate therapeutic target in metabolic diseases such as nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD). We used hepatocyte-specific Notch1 knockout (KO) mice and liver biopsies from NASH and NAFLD patients to analyze the role of Notch1 in glucose and lipid metabolism. Hepatocyte-specific Notch1 KO mice were fed with a high fat diet (HFD) or a regular diet (RD). We assessed the metabolic phenotype, glucose and insulin tolerance tests, and liver histology. Hepatic mRNA expression was profiled by Affymetrix Mouse Gene arrays and validated by quantitative reverse transcription PCR (qPCR). Akt phosphorylation was visualized by immunoblotting. Gene expression was analyzed in liver biopsies from NASH, NAFLD, and control patients by qPCR. We found that Notch1 KO mice had elevated fasting glucose. Gene expression analysis showed an upregulation of glucose-6-phosphatase, involved in the final step of gluconeogenesis and glucose release from glycogenolysis, and perilipin-5, a regulator of hepatic lipid accumulation. When fed with an HFD KO mice developed overt diabetes and hepatic steatosis. Akt was highly phosphorylated in KO animals and the Foxo1 target gene expression was altered. Accordingly, a reduction in Notch1 and increase in glucose-6-phosphatase and perilipin-5 expression was observed in liver biopsies from NAFLD/NASH compared with controls. Notch1 is a regulator of hepatic glucose and lipid homeostasis. Hepatic impairment of Notch1 expression may be involved in the pathogenesis of human NAFLD/NASH. PMID:27428080

  4. Quantitative DNA hypomethylation of ligand Jagged1 and receptor Notch1 signifies occurrence and progression of breast carcinoma

    PubMed Central

    Cao, Yuwen; Li, Yixiao; Zhang, Na; Hu, Jianming; Yin, Liang; Pan, Zemin; Li, Yucong; Du, Xiaoming; Zhang, Wenjie; Li, Feng

    2015-01-01

    Methylation alterations of Jagged1 and Notch1 genes have been reported in non-tumor lesions and a few cancers. However, methylation profiles of Jagged1 promoter and Notch1 exon25 in breast cancer and matched normal tissue and the association of methylation with clinicopathological characteristics still remain unclear. To explore the potential effects of aberrant DNA methylation of Jagged1 and Notch1 on occurrence and progression of breast cancer, we detected the quantitative DNA methylation of Jagged1 and Notch1 in 73 breast cancer (BC) and 20 adjacent normal breast tissues (ANBT) by using MassARRAY spectrometry. The methylation level of overall and majority individual CpG sites of the two genes were synergistically significantly lower in BC than in ANBT. The overall hypomethylation of the two genes, particularly of Jagged1 CpG_8.9.10 and Notch1 CpG_14.15.16 in primary tumors, were markedly associated with lymph node metastasis, advanced stage and high grade. The protein expressions of the both genes were examined by immunohistochemical staining in same cohorts. The expression was significantly inverse correlation with methylation. The two proteins in primary tumor were synergistically up-regulated and dramatically related to lymph node metastasis, advanced stage and high grade. Our findings suggest that the synergetic hypomethylation of Jagged1 and Notch1 genes, especially of Jagged1 CpG_8.9.10 and Notch1 CpG_14.15.16, may involve tumorigenesis and development of breast cancer. The negative relationship between methylation and expression indicates methylation role for expression regulation. The synergetic overexpression of the two proteins further indicates the effects on occurrence and progression of breast cancer. PMID:26175933

  5. Quantitative DNA hypomethylation of ligand Jagged1 and receptor Notch1 signifies occurrence and progression of breast carcinoma

    PubMed Central

    Cao, Yuwen; Li, Yixiao; Zhang, Na; Hu, Jianming; Yin, Liang; Pan, Zemin; Li, Yucong; Du, Xiaoming; Zhang, Wenjie; Li, Feng

    2015-01-01

    Methylation alterations of Jagged1 and Notch1 genes have been reported in non-tumor lesions and a few cancers. However, methylation profiles of Jagged1 promoter and Notch1 exon25 in breast cancer and matched normal tissue and the association of methylation with clinicopathological characteristics still remain unclear. To explore the potential effects of aberrant DNA methylation of Jagged1 and Notch1 on occurrence and progression of breast cancer, we detected the quantitative DNA methylation of Jagged1 and Notch1 in 73 breast cancer (BC) and 20 adjacent normal breast tissues (ANBT) by using MassARRAY spectrometry. The methylation level of overall and majority individual CpG sites of the two genes were synergistically significantly lower in BC than in ANBT. The overall hypomethylation of the two genes, particularly of Jagged1 CpG_8.9.10 and Notch1 CpG_14.15.16 in primary tumors, were markedly associated with lymph node metastasis, advanced stage and high grade. The protein expressions of the both genes were examined by immunohistochemical staining in same cohorts. The expression was significantly inverse correlation with methylation. The two proteins in primary tumor were synergistically up-regulated and dramatically related to lymph node metastasis, advanced stage and high grade. Our findings suggest that the synergetic hypomethylation of Jagged1 and Notch1 genes, especially of Jagged1 CpG_8.9.10 and Notch1 CpG_14.15.16, may involve tumorigenesis and development of breast cancer. The negative relationship between methylation and expression indicates methylation role for expression regulation. The synergetic overexpression of the two proteins further indicates the effects on occurrence and progression of breast cancer. PMID:26269752

  6. An Aquaporin 3-Notch1 Axis in Keratinocyte Differentiation and Inflammation

    PubMed Central

    Guo, Liqiong; Chen, Hongxiang; Li, Yongsheng; Zhou, Qixing; Sui, Yang

    2013-01-01

    Aquaporin 3 (AQP3) is an aquaglyceroporin which transports water, glycerol and small solutes across the plasma membrane. Its functions are not limited to fluid transport but also involve the regulation of cell proliferation, migration, skin hydration, wound healing and tumorigenesis. While AQP3 has been reported to play an important role in keratinocyte proliferation, its role in differentiation remains controversial. Our study demonstrated that the expression of AQP3 was regulated during differentiation and that it participated in keratinocyte differentiation control. We further revealed that AQP3 was a transcriptional target of Notch signaling, a critical pathway regulating keratinocyte differentiation and tumor suppression, and it regulated differentiation through a reciprocal negative feedback loop with Notch1. When the expression level of AQP3 was elevated, impaired barrier integrity and increased pro-inflammatory cytokine production ensued, mimicking the pathological conditions in Notch deficient mice and in atopic dermatitis. Dysregulation of AQP3 and Notch receptors has been reported in several skin diseases, including skin cancer. Our discovery of the novel AQP3-Notch1 axis may provide insight into epidermal homeostasis control and possible translational applications, including its potential use as a biomarker for molecular diagnosis in environmental studies. PMID:24260356

  7. An aquaporin 3-notch1 axis in keratinocyte differentiation and inflammation.

    PubMed

    Guo, Liqiong; Chen, Hongxiang; Li, Yongsheng; Zhou, Qixing; Sui, Yang

    2013-01-01

    Aquaporin 3 (AQP3) is an aquaglyceroporin which transports water, glycerol and small solutes across the plasma membrane. Its functions are not limited to fluid transport but also involve the regulation of cell proliferation, migration, skin hydration, wound healing and tumorigenesis. While AQP3 has been reported to play an important role in keratinocyte proliferation, its role in differentiation remains controversial. Our study demonstrated that the expression of AQP3 was regulated during differentiation and that it participated in keratinocyte differentiation control. We further revealed that AQP3 was a transcriptional target of Notch signaling, a critical pathway regulating keratinocyte differentiation and tumor suppression, and it regulated differentiation through a reciprocal negative feedback loop with Notch1. When the expression level of AQP3 was elevated, impaired barrier integrity and increased pro-inflammatory cytokine production ensued, mimicking the pathological conditions in Notch deficient mice and in atopic dermatitis. Dysregulation of AQP3 and Notch receptors has been reported in several skin diseases, including skin cancer. Our discovery of the novel AQP3-Notch1 axis may provide insight into epidermal homeostasis control and possible translational applications, including its potential use as a biomarker for molecular diagnosis in environmental studies. PMID:24260356

  8. Celery Seed Extract Blocks Peroxide Injury in Macrophages via Notch1/NF-κB Pathway.

    PubMed

    Si, Yanhong; Guo, Shoudong; Fang, Yongqi; Qin, Shucun; Li, Furong; Zhang, Ying; Jiao, Peng; Zhang, Chunduo; Gao, Linlin

    2015-01-01

    Oxidized low-density lipoprotein (ox-LDL)-induced macrophage foam cell formation and injury is one of the major atherogenic factors. This study is aimed to investigate the protective effect of celery seed extract (CSE) on ox-LDL-induced injury of macrophages and the underlying signaling pathway. RAW264.7 macrophages were pre-incubated with CSE for 24 h, followed by stimulation with ox-LDL. Oil red O staining and enzymatic colorimetry indicated CSE significantly lessened lipid droplets and total cholesterol (TC) content in ox-LDL-injured macrophages. ELISA revealed that CSE decreased the secretion of inflammatory cytokine TNF-α and IL-6 by 12-27% and 5-15% respectively. MTT assay showed CSE promoted cell viability by 16-40%. Cell apoptosis was also analyzed by flow cytometry and laser scanning confocal microscope and the data indicated CSE inhibited ox-LDL-induced apoptosis of macrophages. Meanwhile, western blot analysis showed CSE suppressed NF-κBp65 and notch1 protein expressions stimulated by ox-LDL in macrophages. These results suggest that CSE inhibits ox-LDL-induced macrophages injury via notch1/NF-κB pathway. PMID:25916469

  9. Celery Seed Extract Blocks Peroxide Injury in Macrophages via Notch1/NF-κB Pathway.

    PubMed

    Si, Yanhong; Guo, Shoudong; Fang, Yongqi; Qin, Shucun; Li, Furong; Zhang, Ying; Jiao, Peng; Zhang, Chunduo; Gao, Linlin

    2015-01-01

    Oxidized low-density lipoprotein (ox-LDL)-induced macrophage foam cell formation and injury is one of the major atherogenic factors. This study is aimed to investigate the protective effect of celery seed extract (CSE) on ox-LDL-induced injury of macrophages and the underlying signaling pathway. RAW264.7 macrophages were pre-incubated with CSE for 24 h, followed by stimulation with ox-LDL. Oil red O staining and enzymatic colorimetry indicated CSE significantly lessened lipid droplets and total cholesterol (TC) content in ox-LDL-injured macrophages. ELISA revealed that CSE decreased the secretion of inflammatory cytokine TNF-α and IL-6 by 12-27% and 5-15% respectively. MTT assay showed CSE promoted cell viability by 16-40%. Cell apoptosis was also analyzed by flow cytometry and laser scanning confocal microscope and the data indicated CSE inhibited ox-LDL-induced apoptosis of macrophages. Meanwhile, western blot analysis showed CSE suppressed NF-κBp65 and notch1 protein expressions stimulated by ox-LDL in macrophages. These results suggest that CSE inhibits ox-LDL-induced macrophages injury via notch1/NF-κB pathway.

  10. Buyang Huanwu decoction up-regulates Notch1 gene expression in injured spinal cord.

    PubMed

    Guo, Zhan-Peng; Huang, Mi-Na; Liu, An-Qi; Yuan, Ya-Jiang; Zhao, Jian-Bo; Mei, Xi-Fan

    2015-08-01

    Expression of genes in the Notch signaling pathway is altered in the injured spinal cord, which indicates that Notch participates in repair after spinal cord injury. Buyang Huanwu decoction, a traditional Chinese herbal preparation, can promote the growth of nerve cells and nerve fibers; however, it is unclear whether Buyang Huanwu decoction affects the Notch signaling pathway in injured spinal cord. In this study, a rat model was established by injuring the T10 spinal cord. At 2 days after injury, rats were intragastrically administered 2 mL of 0.8 g/mL Buyang Huanwu decoction daily until sacrifice. Real-time reverse transcription polymerase chain reaction analysis demonstrated that at 7, 14 and 28 days after injury, the expression of Notch1 was increased in the Buyang Huanwu decoction group compared with controls. These findings confirm that Buyang Huanwu decoction can promote the expression of Notch1 in rats with incomplete spinal cord injury, and may indicate a mechanism to promote the repair of spinal cord injury.

  11. Notch 1 Receptor, Delta 1 Ligand and HES 1 Transcription Factor are Expressed in the Lining Epithelium of Periapical Cysts (Preliminary Study)

    PubMed Central

    Meliou, E; Kerezoudis, NP; Tosios, KI; Kiaris, H

    2010-01-01

    Periapical cyst is a chronic inflammatory disorder of periradicular tissues. The precise pathological mechanisms involved in periapical cyst enlargement remain unclear. Notch signaling is an evolutionarily conserved pathway with a regulatory role in cell fate decisions during development and in carcinogenesis. To date, there are no published data available on the expression of Notch signaling components in periapical cysts or any other jaw cyst. In this immunohistochemical study we have examined the expression of the receptor Notch 1, the ligand Delta 1 and the transcription factor HES 1 in the epithelium of well defined periapical cysts. Immunostaining reaction of Notch 1, Delta 1 and HES 1 was observed in the cytoplasm and/or the cytoplasmic membrane and occasionally in the nucleus in the majority of epithelial cells of all periapical cysts. The present observations indicate that Notch pathway is active in the epithelium of periapical cysts. It can be speculated that activation of epithelial cells of periapical cysts is associated with activation of Notch pathway and imply involvement of this pathway in periapical cyst growth and expansion. PMID:21116324

  12. Notch 1 Receptor, Delta 1 Ligand and HES 1 Transcription Factor are Expressed in the Lining Epithelium of Periapical Cysts (Preliminary Study).

    PubMed

    Meliou, E; Kerezoudis, Np; Tosios, Ki; Kiaris, H

    2010-01-01

    Periapical cyst is a chronic inflammatory disorder of periradicular tissues. The precise pathological mechanisms involved in periapical cyst enlargement remain unclear. Notch signaling is an evolutionarily conserved pathway with a regulatory role in cell fate decisions during development and in carcinogenesis. To date, there are no published data available on the expression of Notch signaling components in periapical cysts or any other jaw cyst. In this immunohistochemical study we have examined the expression of the receptor Notch 1, the ligand Delta 1 and the transcription factor HES 1 in the epithelium of well defined periapical cysts. Immunostaining reaction of Notch 1, Delta 1 and HES 1 was observed in the cytoplasm and/or the cytoplasmic membrane and occasionally in the nucleus in the majority of epithelial cells of all periapical cysts. The present observations indicate that Notch pathway is active in the epithelium of periapical cysts. It can be speculated that activation of epithelial cells of periapical cysts is associated with activation of Notch pathway and imply involvement of this pathway in periapical cyst growth and expansion.

  13. Co-delivery of platinum drug and siNotch1 with micelleplex for enhanced hepatocellular carcinoma therapy.

    PubMed

    Shen, Song; Sun, Chun-Yang; Du, Xiao-Jiao; Li, Hong-Jun; Liu, Yang; Xia, Jin-Xing; Zhu, Yan-Hua; Wang, Jun

    2015-11-01

    As part of HCC tumor cellularity, cancer stem cells (CSCs) are considered a major obstacle to eradicate hepatocellular carcinoma (HCC), which is the third most common cause of cancer-related death worldwide, and the accumulation of chemotherapeutic drug-resistant CSCs invariably accounts for poor prognosis and HCC relapse. In the present study, we explored the efficacy of co-delivery of platinum drug and siRNA targeting Notch1 to treat CSCs-harboring HCC. To overcome the challenging obstacles of platinum drug and siRNA in the systemic administration, we developed a micellar nanoparticle (MNP) to deliver platinum(IV) prodrug and siNotch1, hereafter referred to as (Pt(IV))MNP/siNotch1. We demonstrated that (Pt(IV))MNP/siNotch1 was able to efficiently deliver two drugs into both non-CSCs and CSCs of SMMC7721, a HCC cell line. We further found that siRNA-mediated inhibition of Notch1 suppression can increase the sensitivity of HCC cells to platinum drugs and decrease the percentage of HCC CSCs, and consequently resulting in enhanced proliferation inhibition and apoptosis induction in HCC cells in vitro. Moreover, our results indicated that the combined drug delivery system can remarkably augment drug enrichment in tumor tissues, substantially suppressing the tumor growth while avoiding the accumulation of CSCs in a synergistic manner in the SMMC7721 xenograft model.

  14. Activating mutations in CTNNB1 in aldosterone producing adenomas

    PubMed Central

    Åkerström, Tobias; Maharjan, Rajani; Sven Willenberg, Holger; Cupisti, Kenko; Ip, Julian; Moser, Ana; Stålberg, Peter; Robinson, Bruce; Alexander Iwen, K.; Dralle, Henning; Walz, Martin K.; Lehnert, Hendrik; Sidhu, Stan; Gomez-Sanchez, Celso; Hellman, Per; Björklund, Peyman

    2016-01-01

    Primary aldosteronism (PA) is the most common cause of secondary hypertension with a prevalence of 5–10% in unreferred hypertensive patients. Aldosterone producing adenomas (APAs) constitute a large proportion of PA cases and represent a surgically correctable form of the disease. The WNT signaling pathway is activated in APAs. In other tumors, a frequent cause of aberrant WNT signaling is mutation in the CTNNB1 gene coding for β-catenin. Our objective was to screen for CTNNB1 mutations in a well-characterized cohort of 198 APAs. Somatic CTNNB1 mutations were detected in 5.1% of the tumors, occurring mutually exclusive from mutations in KCNJ5, ATP1A1, ATP2B3 and CACNA1D. All of the observed mutations altered serine/threonine residues in the GSK3β binding domain in exon 3. The mutations were associated with stabilized β-catenin and increased AXIN2 expression, suggesting activation of WNT signaling. By CYP11B2 mRNA expression, CYP11B2 protein expression, and direct measurement of aldosterone in tumor tissue, we confirmed the ability for aldosterone production. This report provides compelling evidence that aberrant WNT signaling caused by mutations in CTNNB1 occur in APAs. This also suggests that other mechanisms that constitutively activate the WNT pathway may be important in APA formation. PMID:26815163

  15. Non-Linear and Flexible Regions of the Human Notch1 Extracellular Domain Revealed by High-Resolution Structural Studies

    PubMed Central

    Weisshuhn, Philip C.; Sheppard, Devon; Taylor, Paul; Whiteman, Pat; Lea, Susan M.; Handford, Penny A.; Redfield, Christina

    2016-01-01

    Summary The Notch receptor is a key component of a core metazoan signaling pathway activated by Delta/Serrate/Lag-2 ligands expressed on an adjacent cell. This results in a short-range signal with profound effects on cell-fate determination, cell proliferation, and cell death. Key to understanding receptor function is structural knowledge of the large extracellular portion of Notch which contains multiple repeats of epidermal growth factor (EGF)-like domains. Here we investigate the EGF4-13 region of human Notch1 (hN1) using a multidisciplinary approach. Ca2+-binding measurements, X-ray crystallography, {1H}-15N heteronuclear nuclear Overhauser effects, and residual dipolar couplings support a non-linear organization for the EGF4-13 region with a rigid, bent conformation for EGF4-7 and a single flexible linkage between EGF9 and EGF10. These data allow us to construct an informed model for EGF10-13 which, in conjunction with comparative binding studies, demonstrates that EGF10 has an important role in determining Notch receptor sensitivity to Dll-4. PMID:26996961

  16. Somatic Activating PIK3CA Mutations Cause Venous Malformation.

    PubMed

    Limaye, Nisha; Kangas, Jaakko; Mendola, Antonella; Godfraind, Catherine; Schlögel, Matthieu J; Helaers, Raphael; Eklund, Lauri; Boon, Laurence M; Vikkula, Miikka

    2015-12-01

    Somatic mutations in TEK, the gene encoding endothelial cell tyrosine kinase receptor TIE2, cause more than half of sporadically occurring unifocal venous malformations (VMs). Here, we report that somatic mutations in PIK3CA, the gene encoding the catalytic p110α subunit of PI3K, cause 54% (27 out of 50) of VMs with no detected TEK mutation. The hotspot mutations c.1624G>A, c.1633G>A, and c.3140A>G (p.Glu542Lys, p.Glu545Lys, and p.His1047Arg), frequent in PIK3CA-associated cancers, overgrowth syndromes, and lymphatic malformation (LM), account for >92% of individuals who carry mutations. Like VM-causative mutations in TEK, the PIK3CA mutations cause chronic activation of AKT, dysregulation of certain important angiogenic factors, and abnormal endothelial cell morphology when expressed in human umbilical vein endothelial cells (HUVECs). The p110α-specific inhibitor BYL719 restores all abnormal phenotypes tested, in PIK3CA- as well as TEK-mutant HUVECs, demonstrating that they operate via the same pathogenic pathways. Nevertheless, significant genotype-phenotype correlations in lesion localization and histology are observed between individuals with mutations in PIK3CA versus TEK, pointing to gene-specific effects. PMID:26637981

  17. HER2 activating mutations are targets for colorectal cancer treatment

    PubMed Central

    Kavuri, Shyam M.; Jain, Naveen; Galimi, Francesco; Cottino, Francesca; Leto, Simonetta M.; Migliardi, Giorgia; Searleman, Adam C.; Shen, Wei; Monsey, John; Trusolino, Livio; Jacobs, Samuel A.; Bertotti, Andrea; Bose, Ron

    2015-01-01

    The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of colorectal cancer patients. Introduction of the HER2 mutations, S310F, L755S, V777L, V842I, and L866M, into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutations are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors, neratinib and afatinib. HER2 gene sequencing of 48 cetuximab resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) WT colorectal cancer patient-derived xenografts (PDX’s) identified 4 PDX’s with HER2 mutations. HER2 targeted therapies were tested on two PDX’s. Treatment with a single HER2 targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2 targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2 mutated PDX’s. PMID:26243863

  18. Bi-Directional SIFT Predicts a Subset of Activating Mutations

    PubMed Central

    Lee, William; Lazarus, Robert A.; Zhang, Zemin

    2009-01-01

    Advancements in sequencing technologies have empowered recent efforts to identify polymorphisms and mutations on a global scale. The large number of variations and mutations found in these projects requires high-throughput tools to identify those that are most likely to have an impact on function. Numerous computational tools exist for predicting which mutations are likely to be functional, but none that specifically attempt to identify mutations that result in hyperactivation or gain-of-function. Here we present a modified version of the SIFT (Sorting Intolerant from Tolerant) algorithm that utilizes protein sequence alignments with homologous sequences to identify functional mutations based on evolutionary fitness. We show that this bi-directional SIFT (B-SIFT) is capable of identifying experimentally verified activating mutants from multiple datasets. B-SIFT analysis of large-scale cancer genotyping data identified potential activating mutations, some of which we have provided detailed structural evidence to support. B-SIFT could prove to be a valuable tool for efforts in protein engineering as well as in identification of functional mutations in cancer. PMID:20011534

  19. Complete noise analysis of a simple force spectroscopy AFM setup and its applications to study nanomechanics of mammalian Notch 1 protein

    NASA Astrophysics Data System (ADS)

    Dey, Ashim; Szoszkiewicz, Robert

    2012-05-01

    We describe a complete noise analysis and application of a custom made AFM force spectroscopy setup on pulling a recombinant protein with an NRR domain of mouse Notch 1. Our table top AFM setup is affordable, has an open architecture, and is easily transferable to other laboratories. Its calculated noise characteristics are dominated by the Brownian noise with 2% non-Brownian components integrated over the first thermally induced resonance of a typical cantilever. For a typical SiN cantilever with a force constant of ˜15 pN nm-1 and in water the force sensitivity and resolution are less than 10 pN, and the corresponding deflection sensitivities are less than 100 pm Hz-1/2. Also, we obtain a sub-ms time resolution in detecting the protein length change, and only few ms cantilever response times as measured in the force clamp mode on a well-known protein standard. Using this setup we investigate force-induced conformational transitions in the NRR region of a mouse Notch 1. Notch is an important protein related to leukemia and breast cancers in humans. We demonstrate that it is feasible to develop AFM-based studies of the force-induced conformational transitions in Notch. Our results match recent steered molecular dynamics simulations of the NRR unfolding and constitute a first step towards a detailed study of Notch activation with AFM.

  20. TERT promoter mutations and monoallelic activation of TERT in cancer

    PubMed Central

    Huang, F W; Bielski, C M; Rinne, M L; Hahn, W C; Sellers, W R; Stegmeier, F; Garraway, L A; Kryukov, G V

    2015-01-01

    Here we report that promoter mutations in telomerase (TERT), the most common noncoding mutations in cancer, give rise to monoallelic expression of TERT. Through deep RNA sequencing, we find that TERT activation in human cancer cell lines can occur in either mono- or biallelic manner. Without exception, hotspot TERT promoter mutations lead to the re-expression of only one allele, accounting for approximately half of the observed cases of monoallelic TERT expression. Furthermore, we show that monoallelic TERT expression is highly prevalent in certain tumor types and widespread across a broad spectrum of cancers. Taken together, these observations provide insights into the mechanisms of TERT activation and the ramifications of noncoding mutations in cancer. PMID:26657580

  1. Non-coding recurrent mutations in chronic lymphocytic leukaemia.

    PubMed

    Puente, Xose S; Beà, Silvia; Valdés-Mas, Rafael; Villamor, Neus; Gutiérrez-Abril, Jesús; Martín-Subero, José I; Munar, Marta; Rubio-Pérez, Carlota; Jares, Pedro; Aymerich, Marta; Baumann, Tycho; Beekman, Renée; Belver, Laura; Carrio, Anna; Castellano, Giancarlo; Clot, Guillem; Colado, Enrique; Colomer, Dolors; Costa, Dolors; Delgado, Julio; Enjuanes, Anna; Estivill, Xavier; Ferrando, Adolfo A; Gelpí, Josep L; González, Blanca; González, Santiago; González, Marcos; Gut, Marta; Hernández-Rivas, Jesús M; López-Guerra, Mónica; Martín-García, David; Navarro, Alba; Nicolás, Pilar; Orozco, Modesto; Payer, Ángel R; Pinyol, Magda; Pisano, David G; Puente, Diana A; Queirós, Ana C; Quesada, Víctor; Romeo-Casabona, Carlos M; Royo, Cristina; Royo, Romina; Rozman, María; Russiñol, Nuria; Salaverría, Itziar; Stamatopoulos, Kostas; Stunnenberg, Hendrik G; Tamborero, David; Terol, María J; Valencia, Alfonso; López-Bigas, Nuria; Torrents, David; Gut, Ivo; López-Guillermo, Armando; López-Otín, Carlos; Campo, Elías

    2015-10-22

    Chronic lymphocytic leukaemia (CLL) is a frequent disease in which the genetic alterations determining the clinicobiological behaviour are not fully understood. Here we describe a comprehensive evaluation of the genomic landscape of 452 CLL cases and 54 patients with monoclonal B-lymphocytosis, a precursor disorder. We extend the number of CLL driver alterations, including changes in ZNF292, ZMYM3, ARID1A and PTPN11. We also identify novel recurrent mutations in non-coding regions, including the 3' region of NOTCH1, which cause aberrant splicing events, increase NOTCH1 activity and result in a more aggressive disease. In addition, mutations in an enhancer located on chromosome 9p13 result in reduced expression of the B-cell-specific transcription factor PAX5. The accumulative number of driver alterations (0 to ≥4) discriminated between patients with differences in clinical behaviour. This study provides an integrated portrait of the CLL genomic landscape, identifies new recurrent driver mutations of the disease, and suggests clinical interventions that may improve the management of this neoplasia. PMID:26200345

  2. Non-coding recurrent mutations in chronic lymphocytic leukaemia.

    PubMed

    Puente, Xose S; Beà, Silvia; Valdés-Mas, Rafael; Villamor, Neus; Gutiérrez-Abril, Jesús; Martín-Subero, José I; Munar, Marta; Rubio-Pérez, Carlota; Jares, Pedro; Aymerich, Marta; Baumann, Tycho; Beekman, Renée; Belver, Laura; Carrio, Anna; Castellano, Giancarlo; Clot, Guillem; Colado, Enrique; Colomer, Dolors; Costa, Dolors; Delgado, Julio; Enjuanes, Anna; Estivill, Xavier; Ferrando, Adolfo A; Gelpí, Josep L; González, Blanca; González, Santiago; González, Marcos; Gut, Marta; Hernández-Rivas, Jesús M; López-Guerra, Mónica; Martín-García, David; Navarro, Alba; Nicolás, Pilar; Orozco, Modesto; Payer, Ángel R; Pinyol, Magda; Pisano, David G; Puente, Diana A; Queirós, Ana C; Quesada, Víctor; Romeo-Casabona, Carlos M; Royo, Cristina; Royo, Romina; Rozman, María; Russiñol, Nuria; Salaverría, Itziar; Stamatopoulos, Kostas; Stunnenberg, Hendrik G; Tamborero, David; Terol, María J; Valencia, Alfonso; López-Bigas, Nuria; Torrents, David; Gut, Ivo; López-Guillermo, Armando; López-Otín, Carlos; Campo, Elías

    2015-10-22

    Chronic lymphocytic leukaemia (CLL) is a frequent disease in which the genetic alterations determining the clinicobiological behaviour are not fully understood. Here we describe a comprehensive evaluation of the genomic landscape of 452 CLL cases and 54 patients with monoclonal B-lymphocytosis, a precursor disorder. We extend the number of CLL driver alterations, including changes in ZNF292, ZMYM3, ARID1A and PTPN11. We also identify novel recurrent mutations in non-coding regions, including the 3' region of NOTCH1, which cause aberrant splicing events, increase NOTCH1 activity and result in a more aggressive disease. In addition, mutations in an enhancer located on chromosome 9p13 result in reduced expression of the B-cell-specific transcription factor PAX5. The accumulative number of driver alterations (0 to ≥4) discriminated between patients with differences in clinical behaviour. This study provides an integrated portrait of the CLL genomic landscape, identifies new recurrent driver mutations of the disease, and suggests clinical interventions that may improve the management of this neoplasia.

  3. O-glucose trisaccharide is present at high but variable stoichiometry at multiple sites on mouse Notch1.

    PubMed

    Rana, Nadia A; Nita-Lazar, Aleksandra; Takeuchi, Hideyuki; Kakuda, Shinako; Luther, Kelvin B; Haltiwanger, Robert S

    2011-09-01

    Notch activity is regulated by both O-fucosylation and O-glucosylation, and Notch receptors contain multiple predicted sites for both. Here we examine the occupancy of the predicted O-glucose sites on mouse Notch1 (mN1) using the consensus sequence C(1)XSXPC(2). We show that all of the predicted sites are modified, although the efficiency of modifying O-glucose sites is site- and cell type-dependent. For instance, although most sites are modified at high stoichiometries, the site at EGF 27 is only partially glucosylated, and the occupancy of the site at EGF 4 varies with cell type. O-Glucose is also found at a novel, non-traditional consensus site at EGF 9. Based on this finding, we propose a revision of the consensus sequence for O-glucosylation to allow alanine N-terminal to cysteine 2: C(1)XSX(A/P)C(2). We also show through biochemical and mass spectral analyses that serine is the only hydroxyamino acid that is modified with O-glucose on EGF repeats. The O-glucose at all sites is efficiently elongated to the trisaccharide Xyl-Xyl-Glc. To establish the functional importance of individual O-glucose sites in mN1, we used a cell-based signaling assay. Elimination of most individual sites shows little or no effect on mN1 activation, suggesting that the major effects of O-glucose are mediated by modification of multiple sites. Interestingly, elimination of the site in EGF 28, found in the Abruptex region of Notch, does significantly reduce activity. These results demonstrate that, like O-fucose, the O-glucose modifications of EGF repeats occur extensively on mN1, and they play important roles in Notch function.

  4. Notch-1 regulates proliferation and differentiation of human bladder cancer cell lines by inhibiting expression of Krüppel-like factor 4.

    PubMed

    Ai, Xing; Jia, Zhuomin; Liu, Shuanglin; Wang, Jiajun; Zhang, Xu

    2014-10-01

    Inhibition of Notch signaling pathways, consisting of 4 highly conserved receptors (Notch 1-4), induces expression of Krüppel-like transcription factors (KLFs) linked to bladder cancer tumorigenesis and metastasis. Effects of Notch-1 knockdown on cell proliferation, differentiation and KLF4 levels in bladder cancer cell lines were investigated. PsiRNA1‑mediated Notch-1 and KLF4 knockdown models and control model without the psiRNA1 vector were constructed using bladder cancer cell lines T24 and BIU87. Cell proliferation, apoptosis and expression of Notch-1 and KLF4 were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry assay with Annexin V-FITC/PI staining, and reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analysis, respectively. Proliferation was assessed in Notch-1 and/or KLF4 knockdown. The results showed that Notch-1 mRNA and protein expression levels were significantly lower following psiRNA1 vector transfection in both cell lines (P<0.05). Growth and proliferation of both cell lines were significantly inhibited by Notch-1 knockdown (P<0.05), and more G0/G1 arrest and apoptosis were observed compared to those in the control groups (P<0.05). The effects were time-dependent, peaking between 24-48 h and declining by 72 h. KLF4 expression was significantly higher in the Notch-1 knockdown group than in control cells (P<0.05). Notch-1 knockdown cell proliferation was significantly lower than that of Notch-1 and KLF4 knockdown (P<0.05). In conclusion, Notch-1 may act as an oncogene, regulating the proliferation and differentiation of bladder cancer cells by inhibiting KLF4. Pending further exploration of pathway variations and crosstalk, these pathways may be useful targets for bladder cancer therapy.

  5. Glucocerebrosidase activity in Parkinson's disease with and without GBA mutations.

    PubMed

    Alcalay, Roy N; Levy, Oren A; Waters, Cheryl C; Fahn, Stanley; Ford, Blair; Kuo, Sheng-Han; Mazzoni, Pietro; Pauciulo, Michael W; Nichols, William C; Gan-Or, Ziv; Rouleau, Guy A; Chung, Wendy K; Wolf, Pavlina; Oliva, Petra; Keutzer, Joan; Marder, Karen; Zhang, Xiaokui

    2015-09-01

    Glucocerebrosidase (GBA) mutations have been associated with Parkinson's disease in numerous studies. However, it is unknown whether the increased risk of Parkinson's disease in GBA carriers is due to a loss of glucocerebrosidase enzymatic activity. We measured glucocerebrosidase enzymatic activity in dried blood spots in patients with Parkinson's disease (n = 517) and controls (n = 252) with and without GBA mutations. Participants were recruited from Columbia University, New York, and fully sequenced for GBA mutations and genotyped for the LRRK2 G2019S mutation, the most common autosomal dominant mutation in the Ashkenazi Jewish population. Glucocerebrosidase enzymatic activity in dried blood spots was measured by a mass spectrometry-based assay and compared among participants categorized by GBA mutation status and Parkinson's disease diagnosis. Parkinson's disease patients were more likely than controls to carry the LRRK2 G2019S mutation (n = 39, 7.5% versus n = 2, 0.8%, P < 0.001) and GBA mutations or variants (seven homozygotes and compound heterozygotes and 81 heterozygotes, 17.0% versus 17 heterozygotes, 6.7%, P < 0.001). GBA homozygotes/compound heterozygotes had lower enzymatic activity than GBA heterozygotes (0.85 µmol/l/h versus 7.88 µmol/l/h, P < 0.001), and GBA heterozygotes had lower enzymatic activity than GBA and LRRK2 non-carriers (7.88 µmol/l/h versus 11.93 µmol/l/h, P < 0.001). Glucocerebrosidase activity was reduced in heterozygotes compared to non-carriers when each mutation was compared independently (N370S, P < 0.001; L444P, P < 0.001; 84GG, P = 0.003; R496H, P = 0.018) and also reduced in GBA variants associated with Parkinson's risk but not with Gaucher disease (E326K, P = 0.009; T369M, P < 0.001). When all patients with Parkinson's disease were considered, they had lower mean glucocerebrosidase enzymatic activity than controls (11.14 µmol/l/h versus 11.85 µmol/l/h, P = 0.011). Difference compared to controls persisted in patients with

  6. Mutations Closer to the Active Site Improve the Promiscuous Aldolase Activity of 4-Oxalocrotonate Tautomerase More Effectively than Distant Mutations.

    PubMed

    Rahimi, Mehran; van der Meer, Jan-Ytzen; Geertsema, Edzard M; Poddar, Harshwardhan; Baas, Bert-Jan; Poelarends, Gerrit J

    2016-07-01

    The enzyme 4-oxalocrotonate tautomerase (4-OT), which catalyzes enol-keto tautomerization as part of a degradative pathway for aromatic hydrocarbons, promiscuously catalyzes various carbon-carbon bond-forming reactions. These include the aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde. Here, we demonstrate that 4-OT can be engineered into a more efficient aldolase for this condensation reaction, with a >5000-fold improvement in catalytic efficiency (kcat /Km ) and a >10(7) -fold change in reaction specificity, by exploring small libraries in which only "hotspots" are varied. The hotspots were identified by systematic mutagenesis (covering each residue), followed by a screen for single mutations that give a strong improvement in the desired aldolase activity. All beneficial mutations were near the active site of 4-OT, thus underpinning the notion that new catalytic activities of a promiscuous enzyme are more effectively enhanced by mutations close to the active site. PMID:27238293

  7. Gene mutation profiles and prognostic implications in Korean patients with T-lymphoblastic leukemia.

    PubMed

    Huh, Hee Jae; Lee, Soo Hyun; Yoo, Keon Hee; Sung, Ki Woong; Koo, Hong Hoe; Jang, Jun Ho; Kim, Kihyun; Kim, Seok Jin; Kim, Won Seog; Jung, Chul Won; Lee, Ki-O; Kim, Sun-Hee; Kim, Hee-Jin

    2013-05-01

    Genetic alterations implicated in the leukemogenesis of T cell acute lymphoblastic leukemia (T-ALL) have been identified in recent years. In this study, we investigated gene mutation profiles and prognostic implications in a series of Korean T-ALL patients. The study patients were 29 Korean patients with T-ALL; 13 adults (45 %) and 16 children (55 %; male-to-female ratio, 25:4). Clinical, hematologic, and cytogenetic findings were reviewed. We performed mutation analyses for NOTCH1, FBXW7, PHF6, and IL7R genes and survival analyses according to the mutational status. Gene mutations were identified in 66 % of the patients in our series (19/29). Eighteen patients (62 %) had NOTCH1/FBXW7 mutations. Sixteen patients (55 %) had NOTCH1 mutations including nine novel mutations, and eight patients (28 %) had known FBXW7 mutations. Eight patients (28 %; six males and two females) had PHF6 mutations including four novel mutations. Three patients (10 %) had IL7R mutations, which were all novel in-frame insertion or deletion-insertions. The gene mutation profile combined with cytogenetics and FISH study for the p16 gene detected genetic aberrations in 90 % of patients (26/29). There was no significant difference in the frequency of gene mutations between the pediatric and adult patients with T-ALL. Survival analyses suggested a favorable prognostic implication of NOTCH1 mutations in adult T-ALL. Gene mutation studies for NOTCH1, FBXW7, PHF6, and IL7R could detect genetic alterations in a majority of Korean T-ALL patients with novel mutations. We observed similar mutation profiles between adult and pediatric T-ALL, and a favorable prognostic implication of NOTCH1 mutations in adult T-ALL.

  8. A facile one-step strategy for the generation of conditional knockout mice to explore the role of Notch1 in oroesophageal tumorigenesis.

    PubMed

    Mandasari, Masita; Sawangarun, Wanlada; Katsube, Ken-ichi; Kayamori, Kou; Yamaguchi, Akira; Sakamoto, Kei

    2016-01-15

    NOTCH1 plays an important role in epithelial differentiation and carcinogenesis. To investigate the impact of Notch1 inactivation in oroesophageal epithelium, we generated conditional knockout (cKO) mice, using a combined construct which induces the expression of single guide RNA targeting Notch1 and Cas9 by the KRT14 promoter. The cKO mice exhibited patchy hair loss and multiple NOTCH1-negative areas in the tongue epithelium, indicative of heterogeneous knockout. The cKO mice showed susceptibility to esophageal tumorigenesis, underscoring Notch1 as a tumor suppressor. Our one-step strategy for generation of cKO mice provides a versatile method to examine a gene function in vivo. PMID:26682927

  9. MiR-129 triggers autophagic flux by regulating a novel Notch-1/ E2F7/Beclin-1 axis to impair the viability of human malignant glioma cells

    PubMed Central

    Shi, Yingying; Lian, Haiwei; Tu, Huilin; Han, Song; Yin, Jun; Peng, Biwen; Zhou, Beiyan; He, Xiaohua; Liu, Wanhong

    2016-01-01

    Abnormalities of autophagy have been implicated in an increasing number of human cancers, including glioma. To date, there is a wealth of evidence indicating that microRNAs (miRNAs) contribute significantly to autophagy in a variety of cancers. Previous studies have suggested that miR-129 functioned as an important inhibitor of the cell cycle and could promote the apoptosis of many cancer cell lines in vitro. Here, we reported that miR-129 acted as a potent inducer of autophagy. Forced expression of miR-129 could induce autophagic flux by targetedly suppressing Notch-1 in glioma cells. The autophagy induced by miR-129 could restrain the activity of mammalian target of rapamycin (mTOR) and upregulate Beclin-1. Moreover, we demonstrated that E2F transcription factor 7 (E2F7) could also trigger autophagic flux by upregulating Beclin-1 and mediating miR-129-induced autophagy. Additionally, knockdown of Notch-1 could upregulate the expression of E2F7, whereas downregulation of E2F7 alleviated shNotch-1-induced autophagic flux. In particular, knockdown of endogenous Beclin-1 could effectively reduce autophagic flux stimulated by miR-129 and E2F7. Interestingly, upon attenuation of miR-129- or E2F7-triggered autophagic flux rescued cell viability suppressed by them. More importantly, intratumoral injection of pHAGE-miR-129 lentivirus in a nude mouse xenograft model significantly restrained tumor growth and triggered autophagy. In conclusion, these findings identify a new function for miR-129 as a potent inducer of autophagy through a novel Notch-1/E2F7/Beclin-1 axis in glioma. PMID:26824182

  10. Identification of an active new mutator transposable element in maize.

    PubMed

    Tan, Bao-Cai; Chen, Zongliang; Shen, Yun; Zhang, Yafeng; Lai, Jinsheng; Sun, Samuel S M

    2011-09-01

    Robertson's Mutator (Mu) system has been used in large scale mutagenesis in maize, exploiting its high mutation frequency, controllability, preferential insertion in genes, and independence of donor location. Eight Mutator elements have been fully characterized (Mu1, Mu2 /Mu1.7, Mu3, Mu4, Mu5, Mu6/7, Mu8, MuDR), and three are defined by TIR (Mu10, Mu11 and Mu12). The genome sequencing revealed a complex family of Mu-like-elements (MULEs) in the B73 genome. In this article, we report the identification of a new Mu element, named Mu13. Mu13 showed typical Mu characteristics by having a ∼220 bp TIR, creating a 9 bp target site duplication upon insertion, yet the internal sequence is completely different from previously identified Mu elements. Mu13 is not present in the B73 genome or a Zea mays subsp. parviglumis accession, but in W22 and several inbreds that found the Robertson's Mutator line. Analysis of mutants isolated from the UniformMu mutagenic population indicated that the Mu13 element is active in transposition. Two novel insertions were found in expressed genes. To test other unknown Mu elements, we selected six new Mu elements from the B73 genome. Southern analysis indicated that most of these elements were present in the UniformMu lines. From these results, we conclude that Mu13 is a new and active Mu element that significantly contributed to the mutagenesis in the UniformMu population. The Robertson's Mutator line may harbor other unknown active Mu elements.

  11. Correlation of Notch1/Hes1 Genes Expression Levels in Egyptian Paediatric Patients with Newly Diagnosed and Persistent Primary Immune(Idiopathic) Thrombocytopenic Purpura.

    PubMed

    Gawdat, Rania Mohsen; Hammam, Amira Ahmed; Ezzat, Dina Ahmed

    2016-09-01

    Notch signalling is involved in the development of several autoimmune diseases, one of such diseases is ITP. The aim of this study was to investigate and compare the expression levels of Notch1 receptor and its target Hes1 gene in Egyptian paediatric ITP patients. Real-time quantitative reverse transcriptase polymerase chain reaction was used to analyse the expression levels of Notch1 and Hes1 in 42 children with primary ITP (22 newly diagnosed and 20 persistent) cases. Twenty age and sex matched non-ITP controls were included. The expression levels of Notch1 were higher in newly diagnosed and persistent cases than controls with high statistical significant difference (P value < 0.001, P < 0.001) respectively, similarly as regards the expression levels of HES1 (P value < 0.001, P < 0.007) respectively. A significant positive correlation was found between Notch1 and Hes1 expression levels in newly diagnosed cases (r = 0.587, P value = 0.004). There was an association between levels of both genes in most of ITP patients but Hes1 was markedly elevated than Notch1 in few cases. High expression levels of Notch1/Hes1 indicated the important role of Notch signalling in both newly diagnosed and persistent ITP. High expression levels of Hes1 than Notch1 may shed light on its value as a therapeutic target for future research in ITP. PMID:27429531

  12. miR-139-5p sensitizes colorectal cancer cells to 5-fluorouracil by targeting NOTCH-1.

    PubMed

    Liu, Heyong; Yin, Yuan; Hu, Yaling; Feng, Yuyang; Bian, Zehua; Yao, Surui; Li, Min; You, Qingjun; Huang, Zhaohui

    2016-07-01

    Multidrug resistance (MDR), a phenomenon that often occurs with drug treatment and is characterized by relapse or attenuation of drug efficacy, is almost unavoidable in colorectal cancer (CRC) patients receiving 5-fluorouracil (5-FU)-based chemotherapy. MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate gene expression. Our previous study has identified miR-139-5p as a potential tumor suppressor in CRC, but its role in chemoresistance of CRC has not been elucidated. In this study, we demonstrated that miR-139-5p was down-regulated either in CRC tumors receiving chemotherapy or in 5-FU-resistant CRC cell lines (HCT-8/5-FU and HCT-116/5-FU). Ectopic expression of miR-139-5p sensitized CRC cells to 5-FU by increasing 5-FU-induced apoptosis. In addition, miR-139-5p inhibited the expression of the miR-139-5p target gene NOTCH-1 and its downstream molecules MRP-1 and BCL-2, two key MDR-associated genes. Furthermore, silencing NOTCH-1 expression promoted the chemotherapeutic effects of 5-FU, and up-regulation of NOTCH-1 abrogated miR-139-5p-mediated sensitization to 5-FU in LoVo and HCT-116 cells. Taken together, our data indicate a new role of miR-139-5p/NOTCH-1 pathway in the drug resistance of CRC cells to 5-FU, which may be a promising therapeutic target for the anti-MDR treatment of CRC. PMID:27173050

  13. MicroRNA-101 regulates T-cell acute lymphoblastic leukemia progression and chemotherapeutic sensitivity by targeting Notch1

    PubMed Central

    Qian, Lu; Zhang, Wanggang; Lei, Bo; He, Aili; Ye, Lianhong; Li, Xingzhou; Dong, Xin

    2016-01-01

    The present study aimed to investigate the role of microRNA (miR)-101 in acute lymphoblastic leukemia progression and chemoresistance. Furthermore, a novel target gene of miR-101 was identified. Here, we confirmed that miR-101 was significantly downregulated in the blood samples of patients with T-cell acute lymphoblastic leukemia (T-ALL) compared with the healthy controls, as determined by reverse transcription quantitative polymerase chain reaction (RTqPCR) analysis. The in vitro experiments demonstrated that miR-101 significantly repressed the proliferation and invasion, and induced potent apoptosis in Jurkat cells, as determined by CCK-8, flow cytometer and cell invasion assays. Luciferase assay confirmed that Notch1 was a target gene of miR-101, and western blotting showed that miR-101 suppressed the expression of Notch1 at the protein level. Moreover, functional restoration assays revealed that Notch1 mediates the effects of miR-101 on Jurkat cell proliferation, apoptosis and invasion. miR-101 enhanced the sensitivity of Jurkat cells to the chemotherapeutic agent adriamycin. Taken together, our results show for the first time that miR-101 acts as a tumor suppressor in T-cell acute lymphoblastic leukaemia and it could enhance chemotherapeutic sensitivity. Furthermore, Notch1 was identified to be a novel target of miR-101. This study indicates that miR-101 may represent a potential therapeutic target for T-cell acute lymphoblastic leukemia intervention. PMID:27666896

  14. Jarid2 (Jumonji, AT rich interactive domain 2) regulates NOTCH1 expression via histone modification in the developing heart.

    PubMed

    Mysliwiec, Matthew R; Carlson, Clayton D; Tietjen, Josh; Hung, Holly; Ansari, Aseem Z; Lee, Youngsook

    2012-01-01

    Jarid2/Jumonji, the founding member of the Jmj factor family, critically regulates various developmental processes, including cardiovascular development. The Jmj family was identified as histone demethylases, indicating epigenetic regulation by Jmj proteins. Deletion of Jarid2 in mice resulted in cardiac malformation and increased endocardial Notch1 expression during development. Although Jarid2 has been shown to occupy the Notch1 locus in the developing heart, the precise molecular role of Jarid2 remains unknown. Here we show that deletion of Jarid2 results in reduced methylation of lysine 9 on histone H3 (H3K9) at the Notch1 genomic locus in embryonic hearts. Interestingly, SETDB1, a histone H3K9 methyltransferase, was identified as a putative cofactor of Jarid2 by yeast two-hybrid screening, and the physical interaction between Jarid2 and SETDB1 was confirmed by coimmunoprecipitation experiments. Concurrently, accumulation of SETDB1 at the site of Jarid2 occupancy was significantly reduced in Jarid2 knock out (KO) hearts. Employing genome-wide approaches, putative Jarid2 target genes regulated by SETDB1 via H3K9 methylation were identified in the developing heart by ChIP-chip. These targets are involved in biological processes that, when dysregulated, could manifest in the phenotypic defects observed in Jarid2 KO mice. Our data demonstrate that Jarid2 functions as a transcriptional repressor of target genes, including Notch1, through a novel process involving the modification of H3K9 methylation via specific interaction with SETDB1 during heart development. Therefore, our study provides new mechanistic insights into epigenetic regulation by Jarid2, which will enhance our understanding of the molecular basis of other organ development and biological processes.

  15. Oncogenically active MYD88 mutations in human lymphoma

    PubMed Central

    Ngo, Vu N.; Young, Ryan M.; Schmitz, Roland; Jhavar, Sameer; Xiao, Wenming; Lim, Kian-Huat; Kohlhammer, Holger; Xu, Weihong; Yang, Yandan; Zhao, Hong; Shaffer, Arthur L.; Romesser, Paul; Wright, George; Powell, John; Rosenwald, Andreas; Muller-Hermelink, Hans Konrad; Ott, German; Gascoyne, Randy D.; Connors, Joseph M.; Rimsza, Lisa M.; Campo, Elias; Jaffe, Elaine S.; Delabie, Jan; Smeland, Erlend B.; Fisher, Richard I.; Braziel, Rita M.; Tubbs, Raymond R.; Cook, J. R.; Weisenburger, Denny D.; Chan, Wing C.; Staudt, Louis M.

    2016-01-01

    The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) remains the least curable form of this malignancy despite recent advances in therapy1. Constitutive nuclear factor (NF)-κB and JAK kinase signalling promotes malignant cell survival in these lymphomas, but the genetic basis for this signalling is incompletely understood. Here we describe the dependence of ABC DLBCLs on MYD88, an adaptor protein that mediates toll and interleukin (IL)-1 receptor signalling2,3, and the discovery of highly recurrent oncogenic mutations affecting MYD88 in ABC DLBCL tumours. RNA interference screening revealed that MYD88 and the associated kinases IRAK1 and IRAK4 are essential for ABC DLBCL survival. High-throughput RNA resequencing uncovered MYD88 mutations in ABC DLBCL lines. Notably, 29% of ABC DLBCL tumours harboured the same amino acid substitution, L265P, in the MYD88 Toll/IL-1 receptor (TIR) domain at an evolutionarily invariant residue in its hydrophobic core. This mutation was rare or absent in other DLBCL subtypes and Burkitt’s lymphoma, but was observed in 9% of mucosa-associated lymphoid tissue lymphomas. At a lower frequency, additional mutations were observed in the MYD88 TIR domain, occurring in both the ABC and germinal centre B-cell-like (GCB) DLBCL subtypes. Survival of ABC DLBCL cells bearing the L265P mutation was sustained by the mutant but not the wild-type MYD88 isoform, demonstrating that L265P is a gain-of-function driver mutation. The L265P mutant promoted cell survival by spontaneously assembling a protein complex containing IRAK1 and IRAK4, leading to IRAK4 kinase activity, IRAK1 phosphorylation, NF-κB signalling, JAK kinase activation of STAT3, and secretion of IL-6, IL-10 and interferon-β. Hence, theMYD88 signalling pathway is integral to the pathogenesis of ABC DLBCL, supporting the development of inhibitors of IRAK4 kinase and other components of this pathway for the treatment of tumours bearing oncogenic MYD88 mutations

  16. Lysyl oxidase-like 2 represses Notch1 expression in the skin to promote squamous cell carcinoma progression.

    PubMed

    Martin, Alberto; Salvador, Fernando; Moreno-Bueno, Gema; Floristán, Alfredo; Ruiz-Herguido, Cristina; Cuevas, Eva P; Morales, Saleta; Santos, Vanesa; Csiszar, Katalin; Dubus, Pierre; Haigh, Jody J; Bigas, Anna; Portillo, Francisco; Cano, Amparo

    2015-04-15

    Lysyl oxidase-like 2 (LOXL2) is involved in a wide range of physiological and pathological processes, including fibrosis and tumor progression, implicating intracellular and extracellular functions. To explore the specific in vivo role of LOXL2 in physiological and tumor contexts, we generated conditional gain- and loss-of-function mouse models. Germ-line deletion of Loxl2 promotes lethality in half of newborn mice mainly associated to congenital heart defects, while Loxl2 overexpression triggers male sterility due to epididymal dysfunction caused by epithelial disorganization, fibrosis and acute inflammation. Remarkably, when challenged to chemical skin carcinogenesis, Loxl2-overexpressing mice increased tumor burden and malignant progression, while Loxl2-deficient mice exhibit the opposite phenotypes. Loxl2 levels in premalignant tumors negatively correlate with expression of epidermal differentiation markers and components of the Notch1 pathway. We show that LOXL2 is a direct repressor of NOTCH1. Additionally, we identify an exclusive expression pattern between LOXL2 and members of the canonical NOTCH1 pathway in human HNSCC. Our data identify for the first time novel LOXL2 roles in tissue homeostasis and support it as a target for SCC therapy.

  17. Notch-1 Signalling Is Activated in Brain Arteriovenous Malformations in Humans

    ERIC Educational Resources Information Center

    ZhuGe, Qichuan; Zhong, Ming; Zheng, WeiMing; Yang, Guo-Yuan; Mao, XiaoOu; Xie, Lin; Chen, Gourong; Chen, Yongmei; Lawton, Michael T.; Young, William L.; Greenberg, David A.; Jin, Kunlin

    2009-01-01

    A role for the Notch signalling pathway in the formation of arteriovenous malformations during development has been suggested. However, whether Notch signalling is involved in brain arteriovenous malformations in humans remains unclear. Here, we performed immunohistochemistry on surgically resected brain arteriovenous malformations and found that,…

  18. Clinical Impact of De-Regulated Notch-1 and Notch-3 in the Development and Progression of HPV-Associated Different Histological Subtypes of Precancerous and Cancerous Lesions of Human Uterine Cervix

    PubMed Central

    Tripathi, Richa; Rath, Gayatri; Jawanjal, Poonam; Sharma, Shweta; Singhal, Pallavi; Bhambhani, Suresh; Hussain, Showket; Bharadwaj, Mausumi

    2014-01-01

    Background Cervical cancer is the leading cause of cancer related deaths among women in India. Limited reports are available for Notch-1 and Notch-3 protein in cervical carcinoma, which play crucial role in cell proliferation, differentiation, and apoptosis. Methods This study was designed to evaluate the role of Notch-1 and Notch-3 with context to HPV infection in cervical carcinoma. A total of 168 tissue biopsy samples comprising of tumor specimens (n = 98), precancer (n = 30) and non-neoplastic cervical tissues (n = 40) were screened for HPV infection by PCR and expression of Notch-1 and Notch-3 protein by Immunohistochemistry and Immunoblotting. Results 80% (24/30) were found to be positive for HPV in precancer and 86.7% (85/98) in cancer patients. Notch-1 expression of precancer and cancer cases was found to be significantly down-regulated with severity of disease in nuclear (3.43±0.29; 2.04±0.19, p = 0.0001, p = 0.0001) and cytoplasm (3.07±0.29; 2.29±0.17, p = 0.0001, p = 0.0001) obtained from different stages as compared to normal cervix tissue (5.40±0.19, 4.97±0.15; p<0.001; p<0.001). However, Notch-3 expression of above cases was significantly up-regulated with severity of disease and showed intense nuclear (4.17±0.39; 4.74±0.18, p = 0.0001, p = 0.0001) and cytoplasm (3.67±0.36; 4.48±0.18, p = 0.0001, p = 0.0001) of different stages as compared to normal cervix tissue (0.95±0.20, 0.70±0.20; p<0.001; p<0.001) respectively. Conclusions These findings suggest that Notch-1 and Notch-3 may play an important role with synergistic effect of HPV in regulating development and proliferation of cervical cancer through the deregulation of Notch signalling. This study also shows the clinical utility of both proteins which may be used as predictable biomarkers in diagnosing different histological sub-types of HPV associated cervical cancer. Nevertheless, abnormal activation of this pathway may provide legitimate

  19. Mosaic Activating Mutations in FGFR1 Cause Encephalocraniocutaneous Lipomatosis.

    PubMed

    Bennett, James T; Tan, Tiong Yang; Alcantara, Diana; Tétrault, Martine; Timms, Andrew E; Jensen, Dana; Collins, Sarah; Nowaczyk, Malgorzata J M; Lindhurst, Marjorie J; Christensen, Katherine M; Braddock, Stephen R; Brandling-Bennett, Heather; Hennekam, Raoul C M; Chung, Brian; Lehman, Anna; Su, John; Ng, SuYuen; Amor, David J; Majewski, Jacek; Biesecker, Les G; Boycott, Kym M; Dobyns, William B; O'Driscoll, Mark; Moog, Ute; McDonell, Laura M

    2016-03-01

    Encephalocraniocutaneous lipomatosis (ECCL) is a sporadic condition characterized by ocular, cutaneous, and central nervous system anomalies. Key clinical features include a well-demarcated hairless fatty nevus on the scalp, benign ocular tumors, and central nervous system lipomas. Seizures, spasticity, and intellectual disability can be present, although affected individuals without seizures and with normal intellect have also been reported. Given the patchy and asymmetric nature of the malformations, ECCL has been hypothesized to be due to a post-zygotic, mosaic mutation. Despite phenotypic overlap with several other disorders associated with mutations in the RAS-MAPK and PI3K-AKT pathways, the molecular etiology of ECCL remains unknown. Using exome sequencing of DNA from multiple affected tissues from five unrelated individuals with ECCL, we identified two mosaic mutations, c.1638C>A (p.Asn546Lys) and c.1966A>G (p.Lys656Glu) within the tyrosine kinase domain of FGFR1, in two affected individuals each. These two residues are the most commonly mutated residues in FGFR1 in human cancers and are associated primarily with CNS tumors. Targeted resequencing of FGFR1 in multiple tissues from an independent cohort of individuals with ECCL identified one additional individual with a c.1638C>A (p.Asn546Lys) mutation in FGFR1. Functional studies of ECCL fibroblast cell lines show increased levels of phosphorylated FGFRs and phosphorylated FRS2, a direct substrate of FGFR1, as well as constitutive activation of RAS-MAPK signaling. In addition to identifying the molecular etiology of ECCL, our results support the emerging overlap between mosaic developmental disorders and tumorigenesis. PMID:26942290

  20. Mosaic Activating Mutations in FGFR1 Cause Encephalocraniocutaneous Lipomatosis

    PubMed Central

    Bennett, James T.; Tan, Tiong Yang; Alcantara, Diana; Tétrault, Martine; Timms, Andrew E.; Jensen, Dana; Collins, Sarah; Nowaczyk, Malgorzata J.M.; Lindhurst, Marjorie J.; Christensen, Katherine M.; Braddock, Stephen R.; Brandling-Bennett, Heather; Hennekam, Raoul C.M.; Chung, Brian; Lehman, Anna; Su, John; Ng, SuYuen; Amor, David J.; Majewski, Jacek; Biesecker, Les G.; Boycott, Kym M.; Dobyns, William B.; O’Driscoll, Mark; Moog, Ute; McDonell, Laura M.

    2016-01-01

    Encephalocraniocutaneous lipomatosis (ECCL) is a sporadic condition characterized by ocular, cutaneous, and central nervous system anomalies. Key clinical features include a well-demarcated hairless fatty nevus on the scalp, benign ocular tumors, and central nervous system lipomas. Seizures, spasticity, and intellectual disability can be present, although affected individuals without seizures and with normal intellect have also been reported. Given the patchy and asymmetric nature of the malformations, ECCL has been hypothesized to be due to a post-zygotic, mosaic mutation. Despite phenotypic overlap with several other disorders associated with mutations in the RAS-MAPK and PI3K-AKT pathways, the molecular etiology of ECCL remains unknown. Using exome sequencing of DNA from multiple affected tissues from five unrelated individuals with ECCL, we identified two mosaic mutations, c.1638C>A (p.Asn546Lys) and c.1966A>G (p.Lys656Glu) within the tyrosine kinase domain of FGFR1, in two affected individuals each. These two residues are the most commonly mutated residues in FGFR1 in human cancers and are associated primarily with CNS tumors. Targeted resequencing of FGFR1 in multiple tissues from an independent cohort of individuals with ECCL identified one additional individual with a c.1638C>A (p.Asn546Lys) mutation in FGFR1. Functional studies of ECCL fibroblast cell lines show increased levels of phosphorylated FGFRs and phosphorylated FRS2, a direct substrate of FGFR1, as well as constitutive activation of RAS-MAPK signaling. In addition to identifying the molecular etiology of ECCL, our results support the emerging overlap between mosaic developmental disorders and tumorigenesis. PMID:26942290

  1. Novel FOXC2 Mutation in Hereditary Distichiasis Impairs DNA-Binding Activity and Transcriptional Activation.

    PubMed

    Zhang, Leilei; He, Jie; Han, Bing; Lu, Linna; Fan, Jiayan; Zhang, He; Ge, Shengfang; Zhou, Yixiong; Jia, Renbing; Fan, Xianqun

    2016-01-01

    Distichiasis presents as double rows of eyelashes arising from aberrant differentiation of the meibomian glands of the eyelids, and it may be sporadic or hereditary. FOXC2 gene mutations in hereditary distichiasis are rarely reported. Here, we examined two generations of a Chinese family with hereditary distichiasis but without lymphedema or other features of LD syndrome. The FOXC2 gene was amplified and sequenced in all family members. Subcellular localization and luciferase assays were performed to assess the activity of the mutant FOXC2 protein. Clinical examinations showed distichiasis, lower eyelid ectropion, congenital ptosis and photophobia in all affected individuals. Sequence analysis revealed a novel frameshift mutation, c.964_965insG, in the coding region of the FOXC2 gene. This mutation caused protein truncation due to the presence of a premature stop codon. A fluorescence assay showed that this mutation did not change the nuclear localization of the protein. However, it impaired DNA-binding activity and decreased transcriptional activation. This is the first report of a FOXC2 mutation in hereditary distichiasis in the Chinese population. The findings of our study expand the FOXC2 mutation spectrum and contribute to the understanding of the genotype-phenotype correlation of this disease. PMID:27570485

  2. Novel FOXC2 Mutation in Hereditary Distichiasis Impairs DNA-Binding Activity and Transcriptional Activation

    PubMed Central

    Zhang, Leilei; He, Jie; Han, Bing; Lu, Linna; Fan, Jiayan; Zhang, He; Ge, Shengfang; Zhou, Yixiong; Jia, Renbing; Fan, Xianqun

    2016-01-01

    Distichiasis presents as double rows of eyelashes arising from aberrant differentiation of the meibomian glands of the eyelids, and it may be sporadic or hereditary. FOXC2 gene mutations in hereditary distichiasis are rarely reported. Here, we examined two generations of a Chinese family with hereditary distichiasis but without lymphedema or other features of LD syndrome. The FOXC2 gene was amplified and sequenced in all family members. Subcellular localization and luciferase assays were performed to assess the activity of the mutant FOXC2 protein. Clinical examinations showed distichiasis, lower eyelid ectropion, congenital ptosis and photophobia in all affected individuals. Sequence analysis revealed a novel frameshift mutation, c.964_965insG, in the coding region of the FOXC2 gene. This mutation caused protein truncation due to the presence of a premature stop codon. A fluorescence assay showed that this mutation did not change the nuclear localization of the protein. However, it impaired DNA-binding activity and decreased transcriptional activation. This is the first report of a FOXC2 mutation in hereditary distichiasis in the Chinese population. The findings of our study expand the FOXC2 mutation spectrum and contribute to the understanding of the genotype-phenotype correlation of this disease. PMID:27570485

  3. JAK-2 V617F mutation increases heparanase procoagulant activity.

    PubMed

    Kogan, Inna; Chap, Dafna; Hoffman, Ron; Axelman, Elena; Brenner, Benjamin; Nadir, Yona

    2016-01-01

    Patients with polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF) are at increased risk of arterial and venous thrombosis. In patients with ET a positive correlation was observed between JAK-2 V617F mutation, that facilitates erythropoietin receptor signalling, and thrombotic events, although the mechanism involved is not clear. We previously demonstrated that heparanase protein forms a complex and enhances the activity of the blood coagulation initiator tissue factor (TF) which leads to increased factor Xa production and subsequent activation of the coagulation system. The present study was aimed to evaluate heparanase procoagulant activity in myeloproliferative neoplasms. Forty bone marrow biopsies of patients with ET, PV, PMF and chronic myelogenous leukaemia (CML) were immunostained to heparanase, TF and TF pathway inhibitor (TFPI). Erythropoietin receptor positive cell lines U87 human glioma and MCF-7 human breast carcinoma were studied. Heparanase and TFPI staining were more prominent in ET, PV and PMF compared to CML. The strongest staining was in JAK-2 positive ET biopsies. Heparanase level and procoagulant activity were higher in U87 cells transfected to over express JAK-2 V617F mutation compared to control and the effect was reversed using JAK-2 inhibitors (Ruxolitinib, VZ3) and hydroxyurea, although the latter drug did not inhibit JAK-2 phosphorylation. Erythropoietin increased while JAK-2 inhibitors decreased the heparanase level and procoagulant activity in U87 and MCF-7 parental cells. In conclusion, JAK-2 is involved in heparanase up-regulation via the erythropoietin receptor. The present findings may potentially point to a new mechanism of thrombosis in JAK-2 positive ET patients. PMID:26489695

  4. Modulation of gene expression via overlapping binding sites exerted by ZNF143, Notch1 and THAP11.

    PubMed

    Ngondo-Mbongo, Richard Patryk; Myslinski, Evelyne; Aster, Jon C; Carbon, Philippe

    2013-04-01

    ZNF143 is a zinc-finger protein involved in the transcriptional regulation of both coding and non-coding genes from polymerase II and III promoters. Our study deciphers the genome-wide regulatory role of ZNF143 in relation with the two previously unrelated transcription factors Notch1/ICN1 and thanatos-associated protein 11 (THAP11) in several human and murine cells. We show that two distinct motifs, SBS1 and SBS2, are associated to ZNF143-binding events in promoters of >3000 genes. Without co-occupation, these sites are also bound by Notch1/ICN1 in T-lymphoblastic leukaemia cells as well as by THAP11, a factor involved in self-renewal of embryonic stem cells. We present evidence that ICN1 binding overlaps with ZNF143 binding events at the SBS1 and SBS2 motifs, whereas the overlap occurs only at SBS2 for THAP11. We demonstrate that the three factors modulate expression of common target genes through the mutually exclusive occupation of overlapping binding sites. The model we propose predicts that the binding competition between the three factors controls biological processes such as rapid cell growth of both neoplastic and stem cells. Overall, our study establishes a novel relationship between ZNF143, THAP11 and ICN1 and reveals important insights into ZNF143-mediated gene regulation. PMID:23408857

  5. Modulation of gene expression via overlapping binding sites exerted by ZNF143, Notch1 and THAP11

    PubMed Central

    Ngondo-Mbongo, Richard Patryk; Myslinski, Evelyne; Aster, Jon C.; Carbon, Philippe

    2013-01-01

    ZNF143 is a zinc-finger protein involved in the transcriptional regulation of both coding and non-coding genes from polymerase II and III promoters. Our study deciphers the genome-wide regulatory role of ZNF143 in relation with the two previously unrelated transcription factors Notch1/ICN1 and thanatos-associated protein 11 (THAP11) in several human and murine cells. We show that two distinct motifs, SBS1 and SBS2, are associated to ZNF143-binding events in promoters of >3000 genes. Without co-occupation, these sites are also bound by Notch1/ICN1 in T-lymphoblastic leukaemia cells as well as by THAP11, a factor involved in self-renewal of embryonic stem cells. We present evidence that ICN1 binding overlaps with ZNF143 binding events at the SBS1 and SBS2 motifs, whereas the overlap occurs only at SBS2 for THAP11. We demonstrate that the three factors modulate expression of common target genes through the mutually exclusive occupation of overlapping binding sites. The model we propose predicts that the binding competition between the three factors controls biological processes such as rapid cell growth of both neoplastic and stem cells. Overall, our study establishes a novel relationship between ZNF143, THAP11 and ICN1 and reveals important insights into ZNF143-mediated gene regulation. PMID:23408857

  6. Constitutive mutations of Agrobacterium tumefaciens transcriptional activator virG.

    PubMed Central

    Pazour, G J; Ta, C N; Das, A

    1992-01-01

    The virulence (vir) genes of Agrobacterium tumefaciens Ti plasmids are positively regulated by virG in conjunction with virA and plant-derived inducing molecules. A procedure that utilizes both genetic selection and a genetic screen was developed to isolate mutations in virG that led to elevated levels of vir gene expression in the absence of virA and plant phenolic inducers. Mutants were isolated at a frequency of 1 in 10(7) to 10(8). Substitution mutations at two positions in the virG coding region were found to result in the desired phenotype. One mutant had an asparagine-to-aspartic acid substitution at residue 54, and the other contained an isoleucine-to-leucine substitution at residue 106. In both cases, the mutant phenotype required the presence of the active-site aspartic acid residue at position 52. Further analysis showed that no other substitution at residue 54 resulted in a constitutive phenotype. In contrast, several substitutions at residue 106 led to a constitutive phenotype. The possible roles of the residues at positions 54 and 106 in VirG function are discussed. PMID:1597431

  7. Nobiletin inhibited hypoxia-induced epithelial-mesenchymal transition of lung cancer cells by inactivating of Notch-1 signaling and switching on miR-200b.

    PubMed

    Gao, Xue-Jun; Liu, Jian-Wei; Zhang, Qing-Guang; Zhang, Jing-Jing; Xu, Hai-Tao; Liu, Hong-Jian

    2015-04-01

    Epithelial-mesenchymal transition (EMT) is an early step in the process of tumor metastasis. It is well known that tumor microenvironment affects malignancy in various carcinomas; in particular, that hypoxia induces EMT. Deregulated notch signaling also contributes a lot to the development of EMT in lung cancer. In this study, we investigated the use of Notch-1-inhibiting compound as novel therapeutic candidates to regulate hypoxia-induced EMT in lung cancer cells. According to previous screening, nobiletin was selected as a Notch-1 inhibitor. Hypoxia-induced EMT was characteristic of increased N-cadherin & vimentin expressions and decreased E-cadherin expressions. Treatment with nobiletin notably attenuated hypoxia-induced EMT, invasion and migration in H1299 cells, accompanied with reduced Notch-1, Jagged1/2 expressions and its downstream genes Hey-1 and Hes-1. Nobiletin treatment also promoted tumorsuppressive miR-200b level. Moreover, notch-1 siRNA prevented hypoxia-mediated cell migration and decreased Twist1, Snail1, and ZEB1/2 expressions, which are key EMT markers. Re-expression of miR-200b blocked hypoxia-induced EMT and cell invasion. Our findings suggest that downregulation of Notch-1 and reexpression of miR-200b by nobiletin might be a novel remedy for the therapy of lung cancer.

  8. [Increasing activity of a monoamine oxidase by random mutation].

    PubMed

    Chen, Xuejun; Ma, Yuanhui; Shao, Jianhua; Lai, Dunyue; Wang, Zhiguo; Chen, Zhenming

    2014-01-01

    The monoamine oxidase mutant A-1 (F210V/L213C) from Aspergillus niger showed some catalytic activity on mexiletine. To futher improve its activity, the mutant was subjected to directed evolution with MegaWHOP PCR (Megaprimer PCR of Whole Plasmid) and selection employing a high-throughput agar plate-based colorimetric screen. This approach led to the identification of a mutant ep-1, which specific activity was 189% of that for A-1. The ep-1 also showed significantly improved enantioselectivity, with the E value increased from 101 to 282; its kinetic k(cat)/K(m) value increased from 0.001 51 mmol/(L x s) to 0.002 89 mmol/(L x s), suggesting that catalytic efficiency of ep-1 had been improved. The mutant showed obviously higher specific activities on 7 of all tested 11 amines substrates, and the others were comparable. Sequence analysis revealed that there was a new mutation T162A on ep-1. The molecular dynamics simulation indicated that T162A may affect the secondary structure of the substrate channel and expand the binding pocket. PMID:24818485

  9. Inhibition of γ-secretase activity synergistically enhances tumour necrosis factor-related apoptosis-inducing ligand induced apoptosis in T-cell acute lymphoblastic leukemia cells via upregulation of death receptor 5

    PubMed Central

    Greene, Lisa M.; Nathwani, Seema M.; Zisterer, Daniela M.

    2016-01-01

    T-cell acute lymphoblastic leukemia (T-ALL) is a rare and aggressive hematopoietic malignancy prone to relapse and drug resistance. Half of all T-ALL patients exhibit mutations in Notch1, which leads to aberrant Notch1 associated signaling cascades. Notch1 activation is mediated by the γ-secretase cleavage of the Notch1 receptor into the active intracellular domain of Notch1 (NCID). Clinical trials of γ-secretase small molecule inhibitors (GSIs) as single agents for the treatment of T-ALL have been unsuccessful. The present study demonstrated, using immunofluorescence and western blotting, that blocking γ-secretase activity in T-ALL cells with N-[(3,5-difluorophenyl) acetyl]-L-alanyl-2-phenyl] glycine-1,1-dimethylethyl ester (DAPT) downregulated NCID and upregulated the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 5 (DR5). Upregulation of DR5 restored the sensitivity of T-ALL cells to TRAIL. Combination index revealed that the combined treatment of DAPT and TRAIL synergistically enhanced apoptosis compared with treatment with either drug alone. TRAIL combined with the clinically evaluated γ-secretase inhibitor 3-[(1r, 4s)-4-(4-chlorophenylsulfonyl)-4-(2, 5-difluorophenyl) cyclohexyl] propanoic acid (MK-0752) also significantly enhanced TRAIL-induced cell death compared with either drug alone. DAPT/TRAIL apoptotic synergy was dependent on the extrinsic apoptotic pathway and was associated with a decrease in BH3 interacting-domain death agonist and x-linked inhibitor of apoptosis. In conclusion, γ-secretase inhibition represents a potential therapeutic strategy to overcome TRAIL resistance for the treatment of T-ALL.

  10. Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia

    PubMed Central

    Puente, Xose S.; Pinyol, Magda; Quesada, Víctor; Conde, Laura; Ordóñez, Gonzalo R.; Villamor, Neus; Escaramis, Georgia; Jares, Pedro; Beà, Sílvia; González-Díaz, Marcos; Bassaganyas, Laia; Baumann, Tycho; Juan, Manel; López-Guerra, Mónica; Colomer, Dolors; Tubío, José M. C.; López, Cristina; Navarro, Alba; Tornador, Cristian; Aymerich, Marta; Rozman, María; Hernández, Jesús M.; Puente, Diana A.; Freije, José M. P.; Velasco, Gloria; Gutiérrez-Fernández, Ana; Costa, Dolors; Carrió, Anna; Guijarro, Sara; Enjuanes, Anna; Hernández, Lluís; Yagüe, Jordi; Nicolás, Pilar; Romeo-Casabona, Carlos M.; Himmelbauer, Heinz; Castillo, Ester; Dohm, Juliane C.; de Sanjosé, Silvia; Piris, Miguel A.; de Alava, Enrique; Miguel, Jesús San; Royo, Romina; Gelpí, Josep L.; Torrents, David; Orozco, Modesto; Pisano, David G.; Valencia, Alfonso; Guigó, Roderic; Bayés, Mónica; Heath, Simon; Gut, Marta; Klatt, Peter; Marshall, John; Raine, Keiran; Stebbings, Lucy A.; Futreal, P. Andrew; Stratton, Michael R.; Campbell, Peter J.; Gut, Ivo; López-Guillermo, Armando; Estivill, Xavier; Montserrat, Emili; López-Otín, Carlos; Campo, Elías

    2012-01-01

    Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution1,2. Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes3,4. The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene function. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immunoglobulins. The patterns of somatic mutation, supported by functional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our knowledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the identification of clinically relevant mutations in cancer. PMID:21642962

  11. Augmented efficacy with the combination of blockade of the Notch-1 pathway, bortezomib and romidepsin in a murine MT-1 adult T-cell leukemia model.

    PubMed

    Yu, P; Petrus, M N; Ju, W; Zhang, M; Conlon, K C; Nakagawa, M; Maeda, M; Bamford, R N; Waldmann, T A

    2015-03-01

    Adult T-cell leukemia (ATL) is an aggressive malignancy caused by human T-cell lymphotropic virus-1. There is no accepted curative therapy for ATL. We have reported that certain ATL patients have increased Notch-1 signaling along with constitutive activation of the nuclear factor-κB pathway. Physical and functional interaction between these two pathways provides the rationale to combine the γ-secretase inhibitor compound E with the proteasome inhibitor bortezomib. Moreover, romidepsin, a histone deacetylase inhibitor, has demonstrated major antitumor action in leukemia/lymphoma. In this study, we investigated the therapeutic efficacy of the single agents and the combination of these agents in a murine model of human ATL, the MT-1 model. Single and double agents inhibited tumor growth as monitored by tumor size (P<0.05), and prolonged survival of leukemia-bearing mice (P<0.05) compared with the control group. The combination of three agents significantly enhanced the antitumor efficacy as assessed by tumor size, tumor markers in the serum (human soluble interleukin-2 receptor-α and β2-microglobulin) and survival of the MT-1 tumor-bearing mice, compared with all other treatment groups (P<0.05). Improved therapeutic efficacy obtained by combining compound E, bortezomib and romidepsin supports a clinical trial of this combination in the treatment of ATL.

  12. Fringe-mediated extension of O-linked fucose in the ligand-binding region of Notch1 increases binding to mammalian Notch ligands.

    PubMed

    Taylor, Paul; Takeuchi, Hideyuki; Sheppard, Devon; Chillakuri, Chandramouli; Lea, Susan M; Haltiwanger, Robert S; Handford, Penny A

    2014-05-20

    The Notch signaling pathway is essential for many aspects of development, cell fate determination, and tissue homeostasis. Notch signaling can be modulated by posttranslational modifications to the Notch receptor, which are known to alter both ligand binding and receptor activation. We have modified the ligand-binding region (EGF domains 11-13) of human Notch1 (hN1) with O-fucose and O-glucose glycans and shown by flow cytometry and surface plasmon resonance that the Fringe-catalyzed addition of GlcNAc to the O-fucose at T466 in EGF12 substantially increases binding to Jagged1 and Delta-like 1 (DLL1) ligands. We have subsequently determined the crystal structures of EGF domains 11-13 of hN1 modified with either the O-fucose monosaccharide or the GlcNAc-fucose disaccharide at T466 of EGF12 and observed no change in backbone structure for each variant. Collectively, these data demonstrate a role for GlcNAc in modulating the ligand-binding site in hN1 EGF12, resulting in an increased affinity of this region for ligands Jagged1 and DLL1. We propose that this finding explains the Fringe-catalyzed enhancement of Notch-Delta signaling observed in flies and humans, but suggest that the inhibitory effect of Fringe on Jagged/Serrate mediated signaling involves other regions of Notch.

  13. Multifunctional Core/Shell Nanoparticles Cross-linked Polyetherimide-folic Acid as Efficient Notch-1 siRNA Carrier for Targeted Killing of Breast Cancer

    PubMed Central

    Yang, Hong; Li, Ying; Li, Tingting; Xu, Min; Chen, Yin; Wu, Chunhui; Dang, Xitong; Liu, Yiyao

    2014-01-01

    In gene therapy, how genetic therapeutics can be efficiently and safely delivered into target tissues/cells remains a major obstacle to overcome. To address this issue, nanoparticles consisting of non-covalently coupled polyethyleneimine (PEI) and folic acid (FA) to the magnetic and fluorescent core/shell of Fe3O4@SiO2(FITC) was tested for their ability to deliver Notch-1 shRNA. Our results showed that Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanoparticles are 64 nm in diameter with well dispersed and superparamagnetic. These nanoparticles with on significant cytotoxicity are capable of delivering Notch-1 shRNA into human breast cancer MDA-MB-231 cells with high efficiency while effectively protected shRNA from degradation by exogenous DNaseI and nucleases. Magnetic resonance (MR) imaging and fluorescence microscopy showed significant preferential uptake of Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex by MDA-MB-231 cells. Transfected MDA-MB-231 cells exhibited significantly decreased expression of Notch-1, inhibited cell proliferation, and increased cell apoptosis, leading to the killing of MDA-MB-231 cells. In light of the magnetic targeting capabilities of Fe3O4@SiO2(FITC)/PEI-FA, our results show that by complexing with a second molecular targeting therapeutic, such as Notch-1 shRNA in this report, Fe3O4@SiO2(FITC)/PEI-FA can be exploited as a novel, non-viral, and concurrent targeting delivery system for targeted gene therapy as well as for MR imaging in cancer diagnosis. PMID:25400232

  14. Enhancing Human Spermine Synthase Activity by Engineered Mutations

    PubMed Central

    Zhang, Zhe; Zheng, Yueli; Petukh, Margo; Pegg, Anthony; Ikeguchi, Yoshihiko; Alexov, Emil

    2013-01-01

    Spermine synthase (SMS) is an enzyme which function is to convert spermidine into spermine. It was shown that gene defects resulting in amino acid changes of the wild type SMS cause Snyder-Robinson syndrome, which is a mild-to-moderate mental disability associated with osteoporosis, facial asymmetry, thin habitus, hypotonia, and a nonspecific movement disorder. These disease-causing missense mutations were demonstrated, both in silico and in vitro, to affect the wild type function of SMS by either destabilizing the SMS dimer/monomer or directly affecting the hydrogen bond network of the active site of SMS. In contrast to these studies, here we report an artificial engineering of a more efficient SMS variant by transferring sequence information from another organism. It is confirmed experimentally that the variant, bearing four amino acid substitutions, is catalytically more active than the wild type. The increased functionality is attributed to enhanced monomer stability, lowering the pKa of proton donor catalytic residue, optimized spatial distribution of the electrostatic potential around the SMS with respect to substrates, and increase of the frequency of mechanical vibration of the clefts presumed to be the gates toward the active sites. The study demonstrates that wild type SMS is not particularly evolutionarily optimized with respect to the reaction spermidine → spermine. Having in mind that currently there are no variations (non-synonymous single nucleotide polymorphism, nsSNP) detected in healthy individuals, it can be speculated that the human SMS function is precisely tuned toward its wild type and any deviation is unwanted and disease-causing. PMID:23468611

  15. Conformational Tinkering Drives Evolution of a Promiscuous Activity through Indirect Mutational Effects.

    PubMed

    Yang, Gloria; Hong, Nansook; Baier, Florian; Jackson, Colin J; Tokuriki, Nobuhiko

    2016-08-16

    How remote mutations can lead to changes in enzyme function at a molecular level is a central question in evolutionary biochemistry and biophysics. Here, we combine laboratory evolution with biochemical, structural, genetic, and computational analysis to dissect the molecular basis for the functional optimization of phosphotriesterase activity in a bacterial lactonase (AiiA) from the metallo-β-lactamase (MBL) superfamily. We show that a 1000-fold increase in phosphotriesterase activity is caused by a more favorable catalytic binding position of the paraoxon substrate in the evolved enzyme that resulted from conformational tinkering of the active site through peripheral mutations. A nonmutated active site residue, Phe68, was displaced by ∼3 Å through the indirect effects of two second-shell trajectory mutations, allowing molecular interactions between the residue and paraoxon. Comparative mutational scanning, i.e., examining the effects of alanine mutagenesis on different genetic backgrounds, revealed significant changes in the functional roles of Phe68 and other nonmutated active site residues caused by the indirect effects of trajectory mutations. Our work provides a quantitative measurement of the impact of second-shell mutations on the catalytic contributions of nonmutated residues and unveils the underlying intramolecular network of strong epistatic mutational relationships between active site residues and more remote residues. Defining these long-range conformational and functional epistatic relationships has allowed us to better understand the subtle, but cumulatively significant, role of second- and third-shell mutations in evolution.

  16. Conformational Tinkering Drives Evolution of a Promiscuous Activity through Indirect Mutational Effects.

    PubMed

    Yang, Gloria; Hong, Nansook; Baier, Florian; Jackson, Colin J; Tokuriki, Nobuhiko

    2016-08-16

    How remote mutations can lead to changes in enzyme function at a molecular level is a central question in evolutionary biochemistry and biophysics. Here, we combine laboratory evolution with biochemical, structural, genetic, and computational analysis to dissect the molecular basis for the functional optimization of phosphotriesterase activity in a bacterial lactonase (AiiA) from the metallo-β-lactamase (MBL) superfamily. We show that a 1000-fold increase in phosphotriesterase activity is caused by a more favorable catalytic binding position of the paraoxon substrate in the evolved enzyme that resulted from conformational tinkering of the active site through peripheral mutations. A nonmutated active site residue, Phe68, was displaced by ∼3 Å through the indirect effects of two second-shell trajectory mutations, allowing molecular interactions between the residue and paraoxon. Comparative mutational scanning, i.e., examining the effects of alanine mutagenesis on different genetic backgrounds, revealed significant changes in the functional roles of Phe68 and other nonmutated active site residues caused by the indirect effects of trajectory mutations. Our work provides a quantitative measurement of the impact of second-shell mutations on the catalytic contributions of nonmutated residues and unveils the underlying intramolecular network of strong epistatic mutational relationships between active site residues and more remote residues. Defining these long-range conformational and functional epistatic relationships has allowed us to better understand the subtle, but cumulatively significant, role of second- and third-shell mutations in evolution. PMID:27444875

  17. Error-prone polymerase activity causes multinucleotide mutations in humans

    PubMed Central

    Nielsen, Rasmus

    2014-01-01

    About 2% of human genetic polymorphisms have been hypothesized to arise via multinucleotide mutations (MNMs), complex events that generate SNPs at multiple sites in a single generation. MNMs have the potential to accelerate the pace at which single genes evolve and to confound studies of demography and selection that assume all SNPs arise independently. In this paper, we examine clustered mutations that are segregating in a set of 1092 human genomes, demonstrating that the signature of MNM becomes enriched as large numbers of individuals are sampled. We estimate the percentage of linked SNP pairs that were generated by simultaneous mutation as a function of the distance between affected sites and show that MNMs exhibit a high percentage of transversions relative to transitions, findings that are reproducible in data from multiple sequencing platforms and cannot be attributed to sequencing error. Among tandem mutations that occur simultaneously at adjacent sites, we find an especially skewed distribution of ancestral and derived alleles, with GC → AA, GA → TT, and their reverse complements making up 27% of the total. These mutations have been previously shown to dominate the spectrum of the error-prone polymerase Pol ζ, suggesting that low-fidelity DNA replication by Pol ζ is at least partly responsible for the MNMs that are segregating in the human population. We develop statistical estimates of MNM prevalence that can be used to correct phylogenetic and population genetic inferences for the presence of complex mutations. PMID:25079859

  18. Activating HER2 mutations in HER2 gene amplification negative breast cancer

    PubMed Central

    Bose, Ron; Kavuri, Shyam M.; Searleman, Adam C.; Shen, Wei; Shen, Dong; Koboldt, Daniel C.; Monsey, John; Goel, Nicholas; Aronson, Adam B.; Li, Shunqiang; Ma, Cynthia X.; Ding, Li; Mardis, Elaine R.; Ellis, Matthew J.

    2012-01-01

    Data from eight breast cancer genome sequencing projects identified 25 patients with HER2 somatic mutations in cancers lacking HER2 gene amplification. To determine the phenotype of these mutations, we functionally characterized thirteen HER2 mutations using in vitro kinase assays, protein structure analysis, cell culture and xenograft experiments. Seven of these mutations are activating mutations, including G309A, D769H, D769Y, V777L, P780ins, V842I, and R896C. HER2 in-frame deletion 755-759, which is homologous to EGFR exon 19 in-frame deletions, had a neomorphic phenotype with increased phosphorylation of EGFR or HER3. L755S produced lapatinib resistance, but was not an activating mutation in our experimental systems. All of these mutations were sensitive to the irreversible kinase inhibitor, neratinib. These findings demonstrate that HER2 somatic mutation is an alternative mechanism to activate HER2 in breast cancer and they validate HER2 somatic mutations as drug targets for breast cancer treatment. PMID:23220880

  19. Suppressor Mutations for Presenilin 1 Familial Alzheimer Disease Mutants Modulate γ-Secretase Activities.

    PubMed

    Futai, Eugene; Osawa, Satoko; Cai, Tetsuo; Fujisawa, Tomoya; Ishiura, Shoichi; Tomita, Taisuke

    2016-01-01

    γ-Secretase is a multisubunit membrane protein complex containing presenilin (PS1) as a catalytic subunit. Familial Alzheimer disease (FAD) mutations within PS1 were analyzed in yeast cells artificially expressing membrane-bound substrate, amyloid precursor protein, or Notch fused to Gal4 transcriptional activator. The FAD mutations, L166P and G384A (Leu-166 to Pro and Gly-384 to Ala substitution, respectively), were loss-of-function in yeast. We identified five amino acid substitutions that suppress the FAD mutations. The cleavage of amyloid precursor protein or Notch was recovered by the secondary mutations. We also found that secondary mutations alone activated the γ-secretase activity. FAD mutants with suppressor mutations, L432M or S438P within TMD9 together with a missense mutation in the second or sixth loops, regained γ-secretase activity when introduced into presenilin null mouse fibroblasts. Notably, the cells with suppressor mutants produced a decreased amount of Aβ42, which is responsible for Alzheimer disease. These results indicate that the yeast system is useful to screen for mutations and chemicals that modulate γ-secretase activity.

  20. Histopathological and molecular heterogeneity among individuals with dementia associated with Presenilin mutations

    PubMed Central

    Maarouf, Chera L; Daugs, Ian D; Spina, Salvatore; Vidal, Ruben; Kokjohn, Tyler A; Patton, R Lyle; Kalback, Walter M; Luehrs, Dean C; Walker, Douglas G; Castaño, Eduardo M; Beach, Thomas G; Ghetti, Bernardino; Roher, Alex E

    2008-01-01

    Background Mutations in the presenilin (PSEN) genes are associated with early-onset familial Alzheimer's disease (FAD). Biochemical characterizations and comparisons have revealed that many PSEN mutations alter γ-secretase activity to promote accumulation of toxic Aβ42 peptides. In this study, we compared the histopathologic and biochemical profiles of ten FAD cases expressing independent PSEN mutations and determined the degradation patterns of amyloid-β precursor protein (AβPP), Notch, N-cadherin and Erb-B4 by γ-secretase. In addition, the levels of Aβ40/42 peptides were quantified by ELISA. Results We observed a wide variation in type, number and distribution of amyloid deposits and neurofibrillary tangles. Four of the ten cases examined exhibited a substantial enrichment in the relative proportions of Aβ40 over Aβ42. The AβPP N-terminal and C-terminal fragments and Tau species, assessed by Western blots and scanning densitometry, also demonstrated a wide variation. The Notch-1 intracellular domain was negligible by Western blotting in seven PSEN cases. There was significant N-cadherin and Erb-B4 peptide heterogeneity among the different PSEN mutations. Conclusion These observations imply that missense mutations in PSEN genes can alter a range of key γ-secretase activities to produce an array of subtly different biochemical, neuropathological and clinical manifestations. Beyond the broad common features of dementia, plaques and tangles, the various PSEN mutations resulted in a wide heterogeneity and complexity and differed from sporadic AD. PMID:19021905

  1. G2385R and I2020T Mutations Increase LRRK2 GTPase Activity

    PubMed Central

    Jang, Jihoon; Joe, Eun-hye; Son, Ilhong; Seol, Wongi

    2016-01-01

    The LRRK2 mutation is a major causal mutation in familial Parkinson's disease. Although LRRK2 contains functional GTPase and kinase domains and their activities are altered by pathogenic mutations, most studies focused on LRRK2 kinase activity because the most prevalent mutant, G2019S, enhances kinase activity. However, the G2019S mutation is extremely rare in the Asian population. Instead, the G2385R mutation was reported as a major risk factor in the Asian population. Similar to other LRRK2 studies, G2385R studies have also focused on kinase activity. Here, we investigated GTPase activities of G2385R with other LRRK2 mutants, such as G2019S, R1441C, and I2020T, as well as wild type (WT). Our results suggest that both I2020T and G2385R contain GTPase activities stronger than that of WT. A kinase assay using the commercial recombinant proteins showed that I2020T harbored stronger activity, whereas G2385R had weaker activity than that of WT, as reported previously. This is the first report of LRRK2 I2020T and G2385R GTPase activities and shows that most of the LRRK2 mutations that are pathogenic or a risk factor altered either kinase or GTPase activity, suggesting that their physiological consequences are caused by altered enzyme activities. PMID:27314038

  2. Familial adult onset hyperinsulinism due to an activating glucokinase mutation: Implications for pharmacological glucokinase activation

    PubMed Central

    Challis, Benjamin G.; Harris, Julie; Sleigh, Alison; Isaac, Iona; Orme, Steve M.; Seevaratnam, Nandini; Dhatariya, Ketan; Simpson, Helen L.; Semple, Robert K.

    2016-01-01

    Context Glucokinase (GCK) phosphorylates and thereby “traps” glucose in cells, thus serving as a gatekeeper for cellular glucose metabolism, particularly in hepatocytes and pancreatic beta cells. In humans, activating GCK mutations cause familial hyperinsulinaemic hypoglycaemia (GCK-HH), leading to keen interest in the potential of small molecule glucokinase activators (GKAs) as treatments for diabetes mellitus. Many such agents have been developed, however observation of side effects including hypertriglyceridaemia and hepatic steatosis have delayed their clinical development. Objective To describe the clinical presentation and metabolic profiles of affected family members in a kindred with familial hyperinsulinism of adult presentation due to a known activating mutation in GCK. Design Clinical, biochemical and metabolic assessment, and GCK sequencing in affected family members. Results In the 60 year-old female proband, hyperinsulinaemic hypoglycaemia (blood glucose 2.1mmol/mol, insulin 18pmol/l) was confirmed following 34 hours of fasting, however abdominal computed tomography (CT), pancreatic MRI, endoscopic ultrasound, octreotide scintigraphy and selective arterial calcium stimulation failed to localise an insulinoma. A prolonged OGTT revealed fasting hypoglycaemia that was exacerbated after glucose challenge, consistent with dysregulated glucose-stimulated insulin release. A heterozygous activating mutation, p.Val389Leu, in the glucokinase gene (GCK) was found in the proband and four other family members. Of these, two had been investigated elsewhere for recurrent hypoglycaemia in adulthood, while the other two adult relatives were asymptomatic despite profound hypoglycaemia. All three of the available family members with the p.Val389Leu mutation had normal serum lipid profiles, normal rates of fasting hepatic de novo lipogenesis and had hepatic triglyceride levels commensurate with their degree of adiposity. Conclusion Activating GCK mutations may

  3. Alanine-Scanning Mutational Analysis of Durancin GL Reveals Residues Important for Its Antimicrobial Activity.

    PubMed

    Ju, Xingrong; Chen, Xinquan; Du, Lihui; Wu, Xueyou; Liu, Fang; Yuan, Jian

    2015-07-22

    Durancin GL is a novel class IIa bacteriocin with 43 residues produced by Enterococcus durans 41D. This bacteriocin demonstrates narrow inhibition spectrum and potent antimicrobial activity against several Listeria monocytogenes strains, including nisin-resistant L. monocytogenes NR30. A systematic alanine-scanning mutational analysis with site-directed mutagenesis was performed to analyze durancin GL residues important for antimicrobial activity and specificity. Results showed that three mutations lost their antimicrobial activity, ten mutations demonstrated a decreased effect on the activity, and seven mutations exhibited relatively high activity. With regard to inhibitory spectrum, four mutants demonstrated a narrower antimicrobial spectrum than wild-type durancin GL. Another four mutants displayed a broader target cell spectrum and increased potency relative to wild-type durancin GL. These findings broaden our understanding of durancin GL residues important for its antimicrobial activity and contribute to future rational design of variants with increased potency.

  4. Alanine-Scanning Mutational Analysis of Durancin GL Reveals Residues Important for Its Antimicrobial Activity.

    PubMed

    Ju, Xingrong; Chen, Xinquan; Du, Lihui; Wu, Xueyou; Liu, Fang; Yuan, Jian

    2015-07-22

    Durancin GL is a novel class IIa bacteriocin with 43 residues produced by Enterococcus durans 41D. This bacteriocin demonstrates narrow inhibition spectrum and potent antimicrobial activity against several Listeria monocytogenes strains, including nisin-resistant L. monocytogenes NR30. A systematic alanine-scanning mutational analysis with site-directed mutagenesis was performed to analyze durancin GL residues important for antimicrobial activity and specificity. Results showed that three mutations lost their antimicrobial activity, ten mutations demonstrated a decreased effect on the activity, and seven mutations exhibited relatively high activity. With regard to inhibitory spectrum, four mutants demonstrated a narrower antimicrobial spectrum than wild-type durancin GL. Another four mutants displayed a broader target cell spectrum and increased potency relative to wild-type durancin GL. These findings broaden our understanding of durancin GL residues important for its antimicrobial activity and contribute to future rational design of variants with increased potency. PMID:26168032

  5. MiR-146a modulates macrophage polarization by inhibiting Notch1 pathway in RAW264.7 macrophages.

    PubMed

    Huang, Cheng; Liu, Xue-jiao; QunZhou; Xie, Juan; Ma, Tao-tao; Meng, Xiao-ming; Li, Jun

    2016-03-01

    Macrophages are heterogeneous and plastic cells which are able to undergo dynamic transition between M1 and M2 polarized phenotypes in response to the microenvironment signals. However, the underlying molecular mechanisms of macrophage polarization are still obscure. In the current study, it was revealed that miR-146a might play a pivotal role in macrophage polarization. As our results indicated, miR-146a was highly expressed in M2 macrophages rather than M1 macrophages. Over-expression of miR-146a resulted in significantly decreased production of pro-inflammatory cytokines including iNOS and TNF-α in M1 macrophages, while increased production of M2 marker genes such as Arg1 and CD206 in M2 macrophages. In contrast, knockdown of miR-146a promoted M1 macrophage polarization but diminished M2 macrophage polarization. Mechanistically, it was revealed that miR-146a modulated macrophage polarization by targeting Notch1. Of note, PPARγ was responsible as another target for miR-146a-mediated macrophage polarization. Taken together, it was suggested that miR-146a might serve as a molecular regulator in macrophage polarization and is a potential therapeutic target for inflammatory diseases.

  6. Activation of nuclear PTEN by inhibition of Notch signaling induces G2/M cell cycle arrest in gastric cancer.

    PubMed

    Kim, S-J; Lee, H-W; Baek, J-H; Cho, Y-H; Kang, H G; Jeong, J S; Song, J; Park, H-S; Chun, K-H

    2016-01-14

    Mutation in PTEN has not yet been detected, but its function as a tumor suppressor is inactivated in many cancers. In this study we determined that, activated Notch signaling disables PTEN by phosphorylation and thereby contributes to gastric tumorigenesis. Notch inhibition by small interfering RNA or γ-secretase inhibitor (GSI) induced mitotic arrest and apoptosis in gastric cancer cells. Notch inhibition induced dephosphorylation in the C-terminal domain of PTEN, which led to PTEN nuclear localization. Overexpression of activated Notch1-induced phosphorylation of PTEN and reversed GSI-induced mitotic arrest. Dephosphorylated nuclear PTEN caused prometaphase arrest by interaction with the cyclin B1-CDK1 complex, resulting in their accumulation in the nucleus and subsequent apoptosis. We found a correlation between high expression levels of Notch1 and low survival rates and, similarly, between reduced nuclear PTEN expression and increasing the TNM classification of malignant tumours stages in malignant tissues from gastric cancer patients. The growth of Notch1-depleted gastric tumors was significantly retarded in xenografted mice, and in addition, PTEN deletion restored growth similar to control tumors. We also demonstrated that combination treatment with GSI and chemotherapeutic agents significantly reduced the orthotopically transplanted gastric tumors in mice without noticeable toxicity. Overall, our findings suggest that inhibition of Notch signaling can be employed as a PTEN activator, making it a potential target for gastric cancer therapy.

  7. Activation of Developmentally Mutated Human Globin Genes by Cell Fusion

    NASA Astrophysics Data System (ADS)

    Papayannopoulou, Thalia; Enver, Tariq; Takegawa, Susumu; Anagnou, Nicholas P.; Stamatoyannopoulos, George

    1988-11-01

    Human fetal globin genes are not expressed in hybrid cells produced by the fusion of normal human lymphocytes with mouse erythroleukemia cells. In contrast, when lymphocytes from persons with globin gene developmental mutations (hereditary persistence of fetal hemoglobin) are used for these fusions, fetal globin is expressed in the hybrid cells. Thus, mutations of developmental origin can be reconstituted in vitro by fusing mutant lymphoid cells with differentiated cell lines of the proper lineage. This system can readily be used for analyses, such as globin gene methylation, that normally require large numbers of pure nucleated erythroid cells, which are difficult to obtain.

  8. GNA14 Somatic Mutation Causes Congenital and Sporadic Vascular Tumors by MAPK Activation.

    PubMed

    Lim, Young H; Bacchiocchi, Antonella; Qiu, Jingyao; Straub, Robert; Bruckner, Anna; Bercovitch, Lionel; Narayan, Deepak; McNiff, Jennifer; Ko, Christine; Robinson-Bostom, Leslie; Antaya, Richard; Halaban, Ruth; Choate, Keith A

    2016-08-01

    Vascular tumors are among the most common neoplasms in infants and children; 5%-10% of newborns present with or develop lesions within the first 3 months of life. Most are benign infantile hemangiomas that typically regress by 5 years of age; other vascular tumors include congenital tufted angiomas (TAs), kaposiform hemangioendotheliomas (KHEs), and childhood lobular capillary hemangiomas (LCHs). Some of these lesions can become locally invasive and unresponsive to pharmacologic intervention, leading to significant complications. Recent investigation has revealed that activating mutations in HRAS, KRAS, NRAS, GNAQ, and GNA11 can cause certain types of rare childhood vascular tumors, and we have now identified causal recurrent somatic activating mutations in GNA14 by whole-exome and targeted sequencing. We found somatic activating GNA14 c.614A>T (p.Gln205Leu) mutations in one KHE, one TA, and one LCH and a GNA11 c.547C>T (p.Arg183Cys) mutation in two LCH lesions. We examined mutation pathobiology via expression of mutant GNA14 or GNA11 in primary human endothelial cells and melanocytes. GNA14 and GNA11 mutations induced changes in cellular morphology and rendered cells growth-factor independent by upregulating the MAPK pathway. Our findings identify GNA14 mutations as a cause of childhood vascular tumors, offer insight into mechanisms of oncogenic transformation by mutations affecting Gaq family members, and identify potential targets for therapeutic intervention. PMID:27476652

  9. Balancing Protein Stability and Activity in Cancer: A New Approach for Identifying Driver Mutations Affecting CBL Ubiquitin Ligase Activation.

    PubMed

    Li, Minghui; Kales, Stephen C; Ma, Ke; Shoemaker, Benjamin A; Crespo-Barreto, Juan; Cangelosi, Andrew L; Lipkowitz, Stanley; Panchenko, Anna R

    2016-02-01

    Oncogenic mutations in the monomeric Casitas B-lineage lymphoma (Cbl) gene have been found in many tumors, but their significance remains largely unknown. Several human c-Cbl (CBL) structures have recently been solved, depicting the protein at different stages of its activation cycle and thus providing mechanistic insight underlying how stability-activity tradeoffs in cancer-related proteins-may influence disease onset and progression. In this study, we computationally modeled the effects of missense cancer mutations on structures representing four stages of the CBL activation cycle to identify driver mutations that affect CBL stability, binding, and activity. We found that recurrent, homozygous, and leukemia-specific mutations had greater destabilizing effects on CBL states than random noncancer mutations. We further tested the ability of these computational models, assessing the changes in CBL stability and its binding to ubiquitin-conjugating enzyme E2, by performing blind CBL-mediated EGFR ubiquitination assays in cells. Experimental CBL ubiquitin ligase activity was in agreement with the predicted changes in CBL stability and, to a lesser extent, with CBL-E2 binding affinity. Two thirds of all experimentally tested mutations affected the ubiquitin ligase activity by either destabilizing CBL or disrupting CBL-E2 binding, whereas about one-third of tested mutations were found to be neutral. Collectively, our findings demonstrate that computational methods incorporating multiple protein conformations and stability and binding affinity evaluations can successfully predict the functional consequences of cancer mutations on protein activity, and provide a proof of concept for mutations in CBL. PMID:26676746

  10. Clustered mutations in hominid genome evolution are consistent with APOBEC3G enzymatic activity

    PubMed Central

    Pinto, Yishay; Gabay, Orshay; Arbiza, Leonardo; Sams, Aaron J.; Keinan, Alon

    2016-01-01

    The gradual accumulation of mutations by any of a number of mutational processes is a major driving force of divergence and evolution. Here, we investigate a potentially novel mutational process that is based on the activity of members of the AID/APOBEC family of deaminases. This gene family has been recently shown to introduce—in multiple types of cancer—enzyme-induced clusters of co-occurring somatic mutations caused by cytosine deamination. Going beyond somatic mutations, we hypothesized that APOBEC3—following its rapid expansion in primates—can introduce unique germline mutation clusters that can play a role in primate evolution. In this study, we tested this hypothesis by performing a comprehensive comparative genomic screen for APOBEC3-induced mutagenesis patterns across different hominids. We detected thousands of mutation clusters introduced along primate evolution which exhibit features that strongly fit the known patterns of APOBEC3G mutagenesis. These results suggest that APOBEC3G-induced mutations have contributed to the evolution of all genomes we studied. This is the first indication of site-directed, enzyme-induced genome evolution, which played a role in the evolution of both modern and archaic humans. This novel mutational mechanism exhibits several unique features, such as its higher tendency to mutate transcribed regions and regulatory elements and its ability to generate clusters of concurrent point mutations that all occur in a single generation. Our discovery demonstrates the exaptation of an anti-viral mechanism as a new source of genomic variation in hominids with a strong potential for functional consequences. PMID:27056836

  11. Complex N-Glycans Are Essential, but Core 1 and 2 Mucin O-Glycans, O-Fucose Glycans, and NOTCH1 Are Dispensable, for Mammalian Spermatogenesis1

    PubMed Central

    Batista, Frank; Lu, Linchao; Williams, Suzannah A.; Stanley, Pamela

    2012-01-01

    ABSTRACT To identify roles in spermatogenesis for major subclasses of N- and O-glycans and Notch signaling, male mice carrying floxed C1galt1, Pofut1, Notch1 or Mgat1 alleles and a testis-specific Cre recombinase transgene were generated. T-synthase (C1GALT1) transfers Gal to generate core 1 and core 2 mucin O-glycans; POFUT1 transfers O-fucose to particular epidermal growth factor-like repeats and is essential for canonical Notch signaling; and MGAT1 (GlcNAcT-I) transfers GlcNAc to initiate hybrid and complex N-glycan synthesis. Cre recombinase transgenes driven by various promoters were investigated, including Stra8-iCre expressed in spermatogonia, Sycp1-Cre expressed in spermatocytes, Prm1-Cre expressed in spermatids, and AMH-Cre expressed in Sertoli cells. All Cre transgenes deleted floxed alleles, but efficiencies varied widely. Stra8-iCre was the most effective, deleting floxed Notch1 and Mgat1 alleles with 100% efficiency and floxed C1galt1 and Pofut1 alleles with ∼80% efficiency, based on transmission of deleted alleles. Removal of C1galt1, Pofut1, or Notch1 in spermatogonia had no effect on testicular weight, histology, or fertility. However, males in which the synthesis of complex N-glycans was blocked by deletion of Mgat1 in spermatogonia did not produce sperm. Spermatogonia, spermatocytes, and spermatids were generated, but most spermatids formed giant multinucleated cells or symplasts, and apoptosis was increased. Therefore, although core 1 and 2 mucin O-glycans, NOTCH1, POFUT1, O-fucose glycans, and Notch signaling are dispensable, MGAT1 and complex N-glycans are essential for spermatogenesis. PMID:22492969

  12. Mutation-Independent Activation of the Anaplastic Lymphoma Kinase in Neuroblastoma.

    PubMed

    Regairaz, Marie; Munier, Fabienne; Sartelet, Hervé; Castaing, Marine; Marty, Virginie; Renauleaud, Céline; Doux, Camille; Delbé, Jean; Courty, José; Fabre, Monique; Ohta, Shigeru; Viehl, Philippe; Michiels, Stefan; Valteau-Couanet, Dominique; Vassal, Gilles

    2016-02-01

    Activating mutations of anaplastic lymphoma kinase (ALK) have been identified as important players in neuroblastoma development. Our goal was to evaluate the significance of overall ALK activation in neuroblastoma. Expression of phosphorylated ALK, ALK, and its putative ligands, pleiotrophin and midkine, was screened in 289 neuroblastomas and 56 paired normal tissues. ALK was expressed in 99% of tumors and phosphorylated in 48% of cases. Pleiotrophin and midkine were expressed in 58% and 79% of tumors, respectively. ALK activation was significantly higher in tumors than in paired normal tissues, together with ALK and midkine expression. ALK activation was largely independent of mutations and correlated with midkine expression in tumors. ALK activation in tumors was associated with favorable features, including a younger age at diagnosis, hyperdiploidy, and detection by mass screening. Antitumor activity of the ALK inhibitor TAE684 was evaluated in wild-type or mutated ALK neuroblastoma cell lines and xenografts. TAE684 was cytotoxic in vitro in all cell lines, especially those harboring an ALK mutation. TAE684 efficiently inhibited ALK phosphorylation in vivo in both F1174I and R1275Q xenografts but demonstrated antitumor activity only against the R1275Q xenograft. In conclusion, ALK activation occurs frequently during neuroblastoma oncogenesis, mainly through mutation-independent mechanisms. However, ALK activation is not associated with a poor outcome and is not always a driver of cell proliferation and/or survival in neuroblastoma. PMID:26687816

  13. Utility of LRF/Pokemon and NOTCH1 protein expression in the distinction between nodular lymphocyte-predominant Hodgkin lymphoma and classical Hodgkin lymphoma.

    PubMed

    Bohn, Olga; Maeda, Takahiro; Filatov, Alexander; Lunardi, Andrea; Pandolfi, Pier Paolo; Teruya-Feldstein, Julie

    2014-02-01

    Classical Hodgkin lymphoma (CHL) and nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) are considered separate entities with different prognosis and treatment. However, morphologic features can be similar and immunohistochemical studies are essential in the distinction; thus, determination of additional biomarkers is of utmost importance. LRF/Pokemon is a proto-oncogene, an interacting partner co-expressed with BCL6 in germinal centers and highly expressed in diffuse large B-cell lymphoma and follicular lymphoma. Conversely, loss of the LRF gene in mouse hematopoietic stem cells results in complete block of early B cell development with concomitant Notch de-repression, indicating its critical role in B versus T cell fate decision at the hematopoietic stem cell stage. For the first time, we show that LRF/Pokemon is predominantly expressed in NLPHL cases as is BCL6 with low to absent NOTCH1 protein expression; while Hodgkin Reed-Sternberg (HRS) cells in CHL show low to absent BCL6 and LRF/Pokemon expression with higher NOTCH1 expression. We illustrate a potential functional interaction between LRF and BCL6 in NLPHL pathogenesis, and differential expression of LRF/Pokemon and NOTCH1 proteins in CHL thus showing differential expression, making for an additional diagnostic marker and therapeutic target. PMID:24326827

  14. Utility of LRF/Pokemon and NOTCH1 Protein Expression in the Distinction of Nodular Lymphocyte-Predominant Hodgkin Lymphoma and Classical Hodgkin Lymphoma

    PubMed Central

    Bohn, Olga; Maeda, Takahiro; Filatov, Alexander; Lunardi, Andrea; Pandolfi, Pier Paolo; Teruya-Feldstein, Julie

    2014-01-01

    Classical Hodgkin lymphoma (CHL) and nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) are considered separate entities with different prognosis and treatment. However, morphologic features can be similar and immunohistochemical studies are essential in the distinction; thus, determination of additional biomarkers is of utmost importance. LRF/Pokemon is a protooncogene, an interacting partner co-expressed with BCL6 in germinal centers and highly expressed in diffuse large B-cell lymphoma and follicular lymphoma. Conversely, loss of the LRF gene in mouse hematopoietic stem cells results in complete block of early B cell development with concomitant Notch derepression, indicating its critical role in B versus T cell fate decision at the hematopoietic stem cell stage. For the first time, we show that LRF/Pokemon is predominantly expressed in NLPHL cases as is BCL6 with low to absent NOTCH1 protein expression; while Hodgkin Reed-Sternberg (HRS) cells in CHL show low to absent BCL6 and LRF/Pokemon expression with higher NOTCH1 expression. We illustrate a potential functional interaction between LRF and BCL6 in NLPHL pathogenesis, and differential expression of LRF/Pokemon and NOTCH1 proteins in CHL thus showing differential expression, making for an additional diagnostic marker and therapeutic target. PMID:24326827

  15. MicroRNA-10b regulates epithelial-mesenchymal transition by modulating KLF4/Notch1/E-cadherin in cisplatin-resistant nasopharyngeal carcinoma cells

    PubMed Central

    Zhang, Pei; Hong, Haiyu; Sun, Xiaojin; Jiang, Hao; Ma, Shiyin; Zhao, Surong; Zhang, Mengxiao; Wang, Zhiwei; Jiang, Chenchen; Liu, Hao

    2016-01-01

    Epithelial-mesenchymal transition (EMT) is an initiating event in tumor cell invasion and metastasis that contributes to therapeutic resistance to compounds including cisplatin. MicroRNAs (miRNAs) have been associated with EMT as well as resistance to standard therapies. However, the underlying mechanisms by which miRNAs control the development of resistance to cisplatin (DDP), and the accompanying EMT-like properties are required to elucidate. Here we show that microRNA-10b (miR-10b) is up-regulated in HNE1/DDP cells, and inhibition of miR-10b expression reversed the EMT phenotype. However, over-expression of miR-10b was able to promote the acquisition of an EMT phenotype in HNE1 cells. Additionally, we identified that miR-10b expression inversely correlates with KLF4, which then controls expression of Notch1. Knock-down of Notch1 inhibited cell migration, invasion, and reversed EMT in HNE1/DDP cells, which was dependent on miR-10b. In summary, our results reveal that miR-10b regulates EMT by modulating KLF4/Notch1/E-cadherin expression, which promotes invasion and migration of nasal pharyngeal carcinoma cells. PMID:27186392

  16. Utility of LRF/Pokemon and NOTCH1 protein expression in the distinction between nodular lymphocyte-predominant Hodgkin lymphoma and classical Hodgkin lymphoma.

    PubMed

    Bohn, Olga; Maeda, Takahiro; Filatov, Alexander; Lunardi, Andrea; Pandolfi, Pier Paolo; Teruya-Feldstein, Julie

    2014-02-01

    Classical Hodgkin lymphoma (CHL) and nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) are considered separate entities with different prognosis and treatment. However, morphologic features can be similar and immunohistochemical studies are essential in the distinction; thus, determination of additional biomarkers is of utmost importance. LRF/Pokemon is a proto-oncogene, an interacting partner co-expressed with BCL6 in germinal centers and highly expressed in diffuse large B-cell lymphoma and follicular lymphoma. Conversely, loss of the LRF gene in mouse hematopoietic stem cells results in complete block of early B cell development with concomitant Notch de-repression, indicating its critical role in B versus T cell fate decision at the hematopoietic stem cell stage. For the first time, we show that LRF/Pokemon is predominantly expressed in NLPHL cases as is BCL6 with low to absent NOTCH1 protein expression; while Hodgkin Reed-Sternberg (HRS) cells in CHL show low to absent BCL6 and LRF/Pokemon expression with higher NOTCH1 expression. We illustrate a potential functional interaction between LRF and BCL6 in NLPHL pathogenesis, and differential expression of LRF/Pokemon and NOTCH1 proteins in CHL thus showing differential expression, making for an additional diagnostic marker and therapeutic target.

  17. Activating PI3Kδ mutations in a cohort of 669 patients with primary immunodeficiency.

    PubMed

    Elgizouli, M; Lowe, D M; Speckmann, C; Schubert, D; Hülsdünker, J; Eskandarian, Z; Dudek, A; Schmitt-Graeff, A; Wanders, J; Jørgensen, S F; Fevang, B; Salzer, U; Nieters, A; Burns, S; Grimbacher, B

    2016-02-01

    The gene PIK3CD codes for the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ), and is expressed solely in leucocytes. Activating mutations of PIK3CD have been described to cause an autosomal dominant immunodeficiency that shares clinical features with common variable immunodeficiency (CVID). We screened a cohort of 669 molecularly undefined primary immunodeficiency patients for five reported mutations (four gain-of-function mutations in PIK3CD and a loss of function mutation in PIK3R1) using pyrosequencing. PIK3CD mutations were identified in three siblings diagnosed with CVID and two sporadic cases with a combined immunodeficiency (CID). The PIK3R1 mutation was not identified in the cohort. Our patients with activated PI3Kδ syndrome (APDS) showed a range of clinical and immunological findings, even within a single family, but shared a reduction in naive T cells. PIK3CD gain of function mutations are more likely to occur in patients with defective B and T cell responses and should be screened for in CVID and CID, but are less likely in patients with a pure B cell/hypogammaglobulinaemia phenotype. PMID:26437962

  18. Recurrent mTORC1-activating RRAGC mutations in follicular lymphoma.

    PubMed

    Okosun, Jessica; Wolfson, Rachel L; Wang, Jun; Araf, Shamzah; Wilkins, Lucy; Castellano, Brian M; Escudero-Ibarz, Leire; Al Seraihi, Ahad Fahad; Richter, Julia; Bernhart, Stephan H; Efeyan, Alejo; Iqbal, Sameena; Matthews, Janet; Clear, Andrew; Guerra-Assunção, José Afonso; Bödör, Csaba; Quentmeier, Hilmar; Mansbridge, Christopher; Johnson, Peter; Davies, Andrew; Strefford, Jonathan C; Packham, Graham; Barrans, Sharon; Jack, Andrew; Du, Ming-Qing; Calaminici, Maria; Lister, T Andrew; Auer, Rebecca; Montoto, Silvia; Gribben, John G; Siebert, Reiner; Chelala, Claude; Zoncu, Roberto; Sabatini, David M; Fitzgibbon, Jude

    2016-02-01

    Follicular lymphoma is an incurable B cell malignancy characterized by the t(14;18) translocation and mutations affecting the epigenome. Although frequent gene mutations in key signaling pathways, including JAK-STAT, NOTCH and NF-κB, have also been defined, the spectrum of these mutations typically overlaps with that in the closely related diffuse large B cell lymphoma (DLBCL). Using a combination of discovery exome and extended targeted sequencing, we identified recurrent somatic mutations in RRAGC uniquely enriched in patients with follicular lymphoma (17%). More than half of the mutations preferentially co-occurred with mutations in ATP6V1B2 and ATP6AP1, which encode components of the vacuolar H(+)-ATP ATPase (V-ATPase) known to be necessary for amino acid-induced activation of mTORC1. The RagC variants increased raptor binding while rendering mTORC1 signaling resistant to amino acid deprivation. The activating nature of the RRAGC mutations, their existence in the dominant clone and their stability during disease progression support their potential as an excellent candidate for therapeutic targeting. PMID:26691987

  19. Recurrent mTORC1-activating RRAGC mutations in follicular lymphoma

    PubMed Central

    Wang, Jun; Araf, Shamzah; Wilkins, Lucy; Castellano, Brian M.; Escudero-Ibarz, Leire; Al Seraihi, Ahad Fahad; Richter, Julia; Bernhart, Stephan H.; Efeyan, Alejo; Iqbal, Sameena; Matthews, Janet; Clear, Andrew; Guerra-Assunção, José Afonso; Bödör, Csaba; Quentmeier, Hilmar; Mansbridge, Christopher; Johnson, Peter; Davies, Andrew; Strefford, Jonathan C.; Packham, Graham; Barrans, Sharon; Jack, Andrew; Du, Ming-Qing; Calaminici, Maria; Lister, T. Andrew; Auer, Rebecca; Montoto, Silvia; Gribben, John G.; Siebert, Reiner; Chelala, Claude; Zoncu, Roberto; Sabatini, David M.; Fitzgibbon, Jude

    2015-01-01

    Follicular lymphoma is an incurable B-cell malignancy1 characterized by the t(14;18) and mutations in one or more components of the epigenome2,3. Whilst frequent gene mutations in signaling pathways, including JAK-STAT, NOTCH and NF-κB, have also been defined2-7, the spectrum of these mutations typically overlap with the closely-related diffuse large B cell lymphoma (DLBCL)6-13. A combination of discovery exome and extended targeted sequencing revealed recurrent somatic mutations in RRAGC uniquely enriched in FL patients (17%). More than half of the mutations preferentially co-occurred with ATP6V1B2 and ATP6AP1 mutations, components of the vacuolar H+-adenosine triphosphate ATPase (v-ATPase) known to be necessary for amino acid-induced mTORC1 activation. The RagC mutants increased raptor binding whilst rendering mTORC1 signaling resistant to amino acid deprivation. Collectively, the activating nature of the RRAGC mutations, their existence within the dominant clone and stability during disease progression supports their potential as an excellent candidate to be therapeutically exploited. PMID:26691987

  20. Active-to-absorbing-state phase transition in an evolving population with mutation

    NASA Astrophysics Data System (ADS)

    Sarkar, Niladri

    2015-10-01

    We study the active to absorbing phase transition (AAPT) in a simple two-component model system for a species and its mutant. We uncover the nontrivial critical scaling behavior and weak dynamic scaling near the AAPT that shows the significance of mutation and highlights the connection of this model with the well-known directed percolation universality class. Our model should be a useful starting point to study how mutation may affect extinction or survival of a species.

  1. Characterization of LEF1 High Expression and Novel Mutations in Adult Acute Lymphoblastic Leukemia

    PubMed Central

    Liu, Juan; Li, Min; Song, Chunhua; Dovat, Sinisa; Li, Jianyong; Ge, Zheng

    2015-01-01

    Aberrant activation of the Wnt pathway plays a pathogenetic role in tumors and has been associated with adverse outcome in acute lymphoblastic leukemia (ALL). Lymphoid enhancer binding factor 1 (LEF1), a key mediator of Wnt signaling, has been linked to leukemic transformation, and LEF1 mutations have been identified in T-ALL. Here we found LEF1 is highly expressed in 25.0% adult ALL patients and LEF1 high expression was associated with high-risk leukemia factors (high WBC, Philadelphia chromosome positive, complex karyotype), shorter event-free survival (EFS), and high relapse rates in patients with B-ALL. LEF1 high expression is also associated with high mutation rate of Notch1 and JAK1 in T-ALL. We identified 2 novel LEF1 mutations (K86E and P106L) in 4 of 131 patients with ALL, and those patients with high-risk ALL (high WBC, complex karyotype). These results suggest a role for LEF1 mutations in leukemogenesis. We further explored the effect of the mutations on cell proliferation and found both mutations significantly promoted the proliferation of ALL cells. We also observed the effect of LEF1 and its mutations on the transcription of its targets, c-MYC and Cyclin D1. We found LEF1 increased the promoter activity of its targets c-MYC and Cyclin D1, and LEF1 K86E and P106L mutants further significantly enhanced this effect. We also observed that the c-MYC and Cyclin D1 mRNA levels were significantly increased in patients with LEF1 high expression compared with those with low expression. Taken together, our findings indicate high LEF1 expression and mutation are associated with high-risk leukemia and our results also revealed that LEF1 high expression and/or gain-of-function mutations are involved in leukemogenesis of ALL. PMID:25942645

  2. Impact of kinase activating and inactivating patient mutations on binary PKA interactions

    PubMed Central

    Röck, Ruth; Mayrhofer, Johanna E.; Bachmann, Verena; Stefan, Eduard

    2015-01-01

    The second messenger molecule cAMP links extracellular signals to intracellular responses. The main cellular cAMP effector is the compartmentalized protein kinase A (PKA). Upon receptor initiated cAMP-mobilization, PKA regulatory subunits (R) bind cAMP thereby triggering dissociation and activation of bound PKA catalytic subunits (PKAc). Mutations in PKAc or RIa subunits manipulate PKA dynamics and activities which contribute to specific disease patterns. Mutations activating cAMP/PKA signaling contribute to carcinogenesis or hormone excess, while inactivating mutations cause hormone deficiency or resistance. Here we extended the application spectrum of a Protein-fragment Complementation Assay based on the Renilla Luciferase to determine binary protein:protein interactions (PPIs) of the PKA network. We compared time- and dose-dependent influences of cAMP-elevation on mutually exclusive PPIs of PKAc with the phosphotransferase inhibiting RIIb and RIa subunits and the protein kinase inhibitor peptide (PKI). We analyzed PKA dynamics following integration of patient mutations into PKAc and RIa. We observed that oncogenic modifications of PKAc(L206R) and RIa(Δ184-236) as well as rare disease mutations in RIa(R368X) affect complex formation of PKA and its responsiveness to cAMP elevation. With the cell-based PKA PPI reporter platform we precisely quantified the mechanistic details how inhibitory PKA interactions and defined patient mutations contribute to PKA functions. PMID:26347651

  3. Promoter-dependent activity on androgen receptor N-terminal domain mutations in androgen insensitivity syndrome.

    PubMed

    Tadokoro-Cuccaro, Rieko; Davies, John; Mongan, Nigel P; Bunch, Trevor; Brown, Rosalind S; Audi, Laura; Watt, Kate; McEwan, Iain J; Hughes, Ieuan A

    2014-01-01

    Androgen receptor (AR) mutations are associated with androgen insensitivity syndrome (AIS). Missense mutations identified in the AR-N-terminal domain (AR-NTD) are rare, and clinical phenotypes are typically mild. We investigated 7 missense mutations and 2 insertion/deletions located in the AR-NTD. This study aimed to elucidate the pathogenic role of AR-NTD mutants in AIS and to use this knowledge to further define AR-NTD function. AR-NTD mutations (Q120E, A159T, G216R, N235K, G248V, L272F, and P380R) were introduced into AR-expression plasmids. Stably expressing cell lines were established for del57L and ins58L. Transactivation was measured using luciferase reporter constructs under the control of GRE and Pem promoters. Intrinsic fluorescence spectroscopy and partial proteolysis studies were performed for mutations which showed reduced activities by using a purified AR-AF1 protein. Pem-luciferase reporter activation was reduced for A159T, N235K, and G248V but not the GRE-luciferase reporter. Protein structure analysis detected no significant change in the AR-AF1 region for these mutations. Reduced cellular expression and transactivation activity were observed for ins58L. The mutations Q120E, G216R, L272F, P380R, and del57L showed small or no detectable changes in function. Thus, clinical and experimental analyses have identified novel AR-signalling defects associated with mutations in the structurally disordered AR-NTD domain in patients with AIS.

  4. Analysis of PIK3CA Mutations and Activation Pathways in Triple Negative Breast Cancer

    PubMed Central

    Muroni, Maria Rosaria; Sanges, Francesca; Sotgiu, Giovanni; Ena, Sara; Pira, Giovanna; Murgia, Luciano; Manca, Alessandra; Uras, Maria Gabriela; Sarobba, Maria Giuseppina; Urru, Silvana; De Miglio, Maria Rosaria

    2015-01-01

    Background Triple Negative Breast Cancer (TNBC) accounts for 12–24% of all breast carcinomas, and shows worse prognosis compared to other breast cancer subtypes. Molecular studies demonstrated that TNBCs are a heterogeneous group of tumors with different clinical and pathologic features, prognosis, genetic-molecular alterations and treatment responsivity. The PI3K/AKT is a major pathway involved in the regulation of cell survival and proliferation, and is the most frequently altered pathway in breast cancer, apparently with different biologic impact on specific cancer subtypes. The most common genetic abnormality is represented by PIK3CA gene activating mutations, with an overall frequency of 20–40%. The aims of our study were to investigate PIK3CA gene mutations on a large series of TNBC, to perform a wider analysis on genetic alterations involving PI3K/AKT and BRAF/RAS/MAPK pathways and to correlate the results with clinical-pathologic data. Materials and Methods PIK3CA mutation analysis was performed by using cobas® PIK3CA Mutation Test. EGFR, AKT1, BRAF, and KRAS genes were analyzed by sequencing. Immunohistochemistry was carried out to identify PTEN loss and to investigate for PI3K/AKT pathways components. Results PIK3CA mutations were detected in 23.7% of TNBC, whereas no mutations were identified in EGFR, AKT1, BRAF, and KRAS genes. Moreover, we observed PTEN loss in 11.3% of tumors. Deregulation of PI3K/AKT pathways was revealed by consistent activation of pAKT and p-p44/42 MAPK in all PIK3CA mutated TNBC. Conclusions Our data shows that PIK3CA mutations and PI3K/AKT pathway activation are common events in TNBC. A deeper investigation on specific TNBC genomic abnormalities might be helpful in order to select patients who would benefit from current targeted therapy strategies. PMID:26540293

  5. Activation Mechanism of Oncogenic Deletion Mutations in BRAF, EGFR, and HER2.

    PubMed

    Foster, Scott A; Whalen, Daniel M; Özen, Ayşegül; Wongchenko, Matthew J; Yin, JianPing; Yen, Ivana; Schaefer, Gabriele; Mayfield, John D; Chmielecki, Juliann; Stephens, Philip J; Albacker, Lee A; Yan, Yibing; Song, Kyung; Hatzivassiliou, Georgia; Eigenbrot, Charles; Yu, Christine; Shaw, Andrey S; Manning, Gerard; Skelton, Nicholas J; Hymowitz, Sarah G; Malek, Shiva

    2016-04-11

    Activating mutations in protein kinases drive many cancers. While how recurring point mutations affect kinase activity has been described, the effect of in-frame deletions is not well understood. We show that oncogenic deletions within the β3-αC loop of HER2 and BRAF are analogous to the recurrent EGFR exon 19 deletions. We identify pancreatic carcinomas with BRAF deletions mutually exclusive with KRAS mutations. Crystal structures of BRAF deletions reveal the truncated loop restrains αC in an active "in" conformation, imparting resistance to inhibitors like vemurafenib that bind the αC "out" conformation. Characterization of loop length explains the prevalence of five amino acid deletions in BRAF, EGFR, and HER2 and highlights the importance of this region for kinase activity and inhibitor efficacy. PMID:26996308

  6. Lethal Factor Active-Site Mutations Affect Catalytic Activity In Vitro

    PubMed Central

    Hammond, S. E.; Hanna, P. C.

    1998-01-01

    The lethal factor (LF) protein of Bacillus anthracis lethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093–1100, 1994). LF is hypothesized to act as a Zn2+ metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block LF activity on macrophages, did not block LF activity in vitro. These data provide the first direct evidence that LF may act as an endopeptidase. PMID:9573135

  7. Activation-Induced Cytidine Deaminase Contributes to Pancreatic Tumorigenesis by Inducing Tumor-Related Gene Mutations.

    PubMed

    Sawai, Yugo; Kodama, Yuzo; Shimizu, Takahiro; Ota, Yuji; Maruno, Takahisa; Eso, Yuji; Kurita, Akira; Shiokawa, Masahiro; Tsuji, Yoshihisa; Uza, Norimitsu; Matsumoto, Yuko; Masui, Toshihiko; Uemoto, Shinji; Marusawa, Hiroyuki; Chiba, Tsutomu

    2015-08-15

    Pancreatic ductal adenocarcinoma (PDAC) develops via an accumulation of various gene mutations. The mechanism underlying the mutations in PDAC development, however, is not fully understood. Recent insight into the close association between the mutation pattern of various cancers and specific mutagens led us to investigate the possible involvement of activation-induced cytidine deaminase (AID), a DNA editing enzyme, in pancreatic tumorigenesis. Our immunohistochemical findings revealed AID protein expression in human acinar ductal metaplasia, pancreatic intraepithelial neoplasia, and PDAC. Both the amount and intensity of the AID protein expression increased with the progression from precancerous to cancerous lesions in human PDAC tissues. To further assess the significance of ectopic epithelial AID expression in pancreatic tumorigenesis, we analyzed the phenotype of AID transgenic (AID Tg) mice. Consistent with our hypothesis that AID is involved in the mechanism of the mutations underlying pancreatic tumorigenesis, we found precancerous lesions developing in the pancreas of AID Tg mice. Using deep sequencing, we also detected Kras and c-Myc mutations in our analysis of the whole pancreas of AID Tg mice. In addition, Sanger sequencing confirmed the presence of Kras, c-Myc, and Smad4 mutations, with the typical mutational footprint of AID in precancerous lesions in AID Tg mice separated by laser capture microdissection. Taken together, our findings suggest that AID contributes to the development of pancreatic precancerous lesions by inducing tumor-related gene mutations. Our new mouse model without intentional manipulation of specific tumor-related genes provides a powerful system for analyzing the mutations involved in PDAC.

  8. Frequencies, Laboratory Features, and Granulocyte Activation in Chinese Patients with CALR-Mutated Myeloproliferative Neoplasms

    PubMed Central

    Tian, Ruiyuan; Chang, Jianmei; Li, Jianlan; Tan, Yanhong; Xu, Zhifang; Ren, Fanggang; Zhao, Junxia; Pan, Jie; Zhang, Na; Wang, Xiaojuan; He, Jianxia; Yang, Wanfang; Wang, Hongwei

    2015-01-01

    Somatic mutations in the CALR gene have been recently identified as acquired alterations in myeloproliferative neoplasms (MPNs). In this study, we evaluated mutation frequencies, laboratory features, and granulocyte activation in Chinese patients with MPNs. A combination of qualitative allele-specific polymerase chain reaction and Sanger sequencing was used to detect three driver mutations (i.e., CALR, JAK2V617F, and MPL). CALR mutations were identified in 8.4% of cases with essential thrombocythemia (ET) and 5.3% of cases with primary myelofibrosis (PMF). Moreover, 25% of polycythemia vera, 29.5% of ET, and 48.1% of PMF were negative for all three mutations (JAK2V617F, MPL, and CALR). Compared with those patients with JAK2V617F mutation, CALR-mutated ET patients displayed unique hematological phenotypes, including higher platelet counts, and lower leukocyte counts and hemoglobin levels. Significant differences were not found between Chinese PMF patients with mutants CALR and JAK2V617F in terms of laboratory features. Interestingly, patients with CALR mutations showed markedly decreased levels of leukocyte alkaline phosphatase (LAP) expression, whereas those with JAK2V617F mutation presented with elevated levels. Overall, a lower mutant rate of CALR gene and a higher triple-negative rate were identified in the cohort of Chinese patients with MPNs. This result indicates that an undiscovered mutant gene may have a significant role in these patients. Moreover, these pathological features further imply that the disease biology varies considerably between mutants CALR and JAK2V617F. PMID:26375990

  9. Segmental basal cell naevus syndrome caused by an activating mutation in smoothened.

    PubMed

    Khamaysi, Z; Bochner, R; Indelman, M; Magal, L; Avitan-Hersh, E; Sarig, O; Sprecher, E; Bergman, R

    2016-07-01

    Aberrant sonic hedgehog signalling, mostly due to PTCH1 mutations, has been shown to play a central role in the pathogenesis of basal cell carcinoma (BCC), as well as in basal cell naevus syndrome (BCNS). Mutations in smoothened (SMO) encoding a receptor for sonic hedgehog have been reported in sporadic BCCs but not in BCNS. We report a case with multiple BCCs, pits and comedones in a segmental distribution over the upper part of the body, along with other findings compatible with BCNS. Histopathologically, there were different types of BCC. A heterozygous mutation (c.1234C>T, p.L412F) in SMO was detected in three BCCs but not in peripheral blood lymphocytes or the uninvolved skin. These were compatible with the type 1 mosaic form of BCNS. The p.L412F mutation was found experimentally to result in increased SMO transactivating activity, and the patient responded to vismodegib therapy. Activating mutations in SMO may cause BCNS. The identification of a gain-of-function mutation in SMO causing a type 1 mosaic form of BCNS further expands our understanding of the pathogenesis of BCC, with implications for the treatment of these tumours, whether sporadic or inherited. PMID:26822128

  10. Activation of Antibiotic Production in Bacillus spp. by Cumulative Drug Resistance Mutations.

    PubMed

    Tojo, Shigeo; Tanaka, Yukinori; Ochi, Kozo

    2015-12-01

    Bacillus subtilis strains produce a wide range of antibiotics, including ribosomal and nonribosomal peptide antibiotics, as well as bacilysocin and neotrehalosadiamine. Mutations in B. subtilis strain 168 that conferred resistance to drugs such as streptomycin and rifampin resulted in overproduction of the dipeptide antibiotic bacilysin. Cumulative drug resistance mutations, such as mutations in the mthA and rpsL genes, which confer low- and high-level resistance, respectively, to streptomycin, and mutations in rpoB, which confer resistance to rifampin, resulted in cells that overproduced bacilysin. Transcriptional analysis demonstrated that the enhanced transcription of biosynthesis genes was responsible for the overproduction of bacilysin. This approach was effective also in activating the cryptic genes of Bacillus amyloliquefaciens, leading to actual production of antibiotic(s).

  11. Antitumor effects and molecular mechanisms of ponatinib on endometrial cancer cells harboring activating FGFR2 mutations.

    PubMed

    Kim, Do-Hee; Kwak, Yeonui; Kim, Nam Doo; Sim, Taebo

    2016-01-01

    Aberrant mutational activation of FGFR2 is associated with endometrial cancers (ECs). AP24534 (ponatinib) currently undergoing clinical trials has been known to be an orally available multi-targeted tyrosine kinase inhibitor. Our biochemical kinase assay showed that AP24534 is potent against wild-type FGFR1-4 and 5 mutant FGFRs (V561M-FGFR1, N549H-FGFR2, K650E-FGFR3, G697C-FGFR3, N535K-FGFR4) and possesses the strongest kinase-inhibitory activity on N549H-FGFR2 (IC50 of 0.5 nM) among all FGFRs tested. We therefore investigated the effects of AP24534 on endometrial cancer cells harboring activating FGFR2 mutations and explored the underlying molecular mechanisms. AP24534 significantly inhibited the proliferation of endometrial cancer cells bearing activating FGFR2 mutations (N549K, K310R/N549K, S252W) and mainly induced G1/S cell cycle arrest leading to apoptosis. AP24534 also diminished the kinase activity of immunoprecipitated FGFR2 derived from MFE-296 and MFE-280 cells and reduced the phosphorylation of FGFR2 and FRS2 on MFE-296 and AN3CA cells. AP24534 caused substantial reductions in ERK phosphorylation, PLCγ signaling and STAT5 signal transduction on ECs bearing FGFR2 activating mutations. Akt signaling pathway was also deactivated by AP24534. AP24534 causes the chemotherapeutic effect through mainly the blockade of ERK, PLCγ and STAT5 signal transduction on ECs. Moreover, AP24534 inhibited migration and invasion of endometrial cancer cells with FGFR2 mutations. In addition, AP24534 significantly blocked anchorage-independent growth of endometrial cancer cells. We, for the first time, report the molecular mechanisms by which AP24534 exerts antitumor effects on ECs with FGFR2 activating mutations, which would provide mechanistic insight into ongoing clinical investigations of AP24534 for ECs.

  12. Novel mutations in dihydrolipoamide dehydrogenase deficiency in two cousins with borderline-normal PDH complex activity.

    PubMed

    Cameron, Jessie M; Levandovskiy, Valeriy; Mackay, Neviana; Raiman, Julian; Renaud, Deborah L; Clarke, Joe T R; Feigenbaum, Annette; Elpeleg, Orly; Robinson, Brian H

    2006-07-15

    We have diagnosed dihydrolipoamide dehydrogenase (DLD) deficiency in two male second cousins, who presented with markedly different clinical phenotypes. Patient 1 had a recurrent encephalopathy, and patient 2 had microcephaly and lactic acidosis. Their presentation is unusual, in that the DLD subunit deficiency had little effect on pyruvate dehydrogenase complex activity, but caused a severe reduction in the activities of other enzymes that utilize this subunit. We have identified two mutations in the DLD gene in each patient. The second cousins have one novel mutation in common resulting in a substitution of isoleucine for threonine (I47T), which has not been previously reported in the literature. Patient 1 has a second mutation that has been reported to be common in the Ashkenazi Jewish population, G229C. Patient 2 has a second mutation, E375K, which has also been previously reported in the literature. Enzyme kinetic measurements on patient fibroblasts show that under certain conditions, one heteroallelic mutation may have a higher K(m). This may account for the differing clinical phenotypes. These findings have important repercussions for other patients with similar clinical phenotypes, as DLD activity is not normally measured in cases with normal PDHc activity.

  13. Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site.

    PubMed Central

    Weaver, T.; Lees, M.; Banaszak, L.

    1997-01-01

    Two mutant forms of fumarase C from E. coli have been made using PCR and recombinant DNA. The recombinant form of the protein included a histidine arm on the C-terminal facilitating purification. Based on earlier studies, two different carboxylic acid binding sites, labeled A- and B-, were observed in crystal structures of the wild type and inhibited forms of the enzyme. A histidine at each of the sites was mutated to an asparagine. H188N at the A-site resulted in a large decrease in specific activity, while the H129N mutation at the B-site had essentially no effect. From the results, we conclude that the A-site is indeed the active site, and a dual role for H188 as a potential catalytic base is proposed. Crystal structures of the two mutant proteins produced some unexpected results. Both mutations reduced the affinity for the carboxylic acids at their respective sites. The H129N mutant should be particularly useful in future kinetic studies because it sterically blocks the B-site with the carboxyamide of asparagine assuming the position of the ligand's carboxylate. In the H188N mutation at the active site, the new asparagine side chain still interacts with an active site water that appears to have moved slightly as a result of the mutation. PMID:9098893

  14. Functional impact of mutational activation on the Listeria monocytogenes central virulence regulator PrfA

    PubMed Central

    Miner, Maurine D.; Port, Gary C.; Freitag, Nancy E.

    2009-01-01

    SUMMARY The transcriptional activator PrfA is required for the expression of virulence factors necessary for Listeria monocytogenes pathogenesis. PrfA is believed to become activated following L. monocytogenes entry into the cytosol of infected host cells resulting in the induction of target genes whose products are required for bacterial intracellular growth and cell-to-cell spread. Several mutations have been identified that appear to lock PrfA into its highly activated cytosolic form (known as prfA* mutations). In this study PrfA and five PrfA* mutant proteins exhibiting differing degrees of activity were purified and analyzed to define the influences of the mutations on distinct aspects of PrfA activity. Based on limited proteolytic digestion conformational changes were detected for the PrfA* mutant proteins in comparison to wild type PrfA. For all but one mutant (PrfA Y63C), the DNA binding affinity as measured by electophoretic mobility shift assay (EMSA) appeared to directly correlate with levels of PrfA mutational activation such that the high activity mutants exhibited the largest increases in DNA binding affinity and moderately activated mutants exhibited more moderate increases. Surprisingly, the ability of PrfA and PrfA* mutants to form dimers in solution appeared to inversely correlate with levels of PrfA-dependent gene expression. Based on comparisons of protein activity and structural similarities with PrfA family members Crp and CooA, the prfA* mutations modify distinct aspects of PrfA activity that include DNA binding and protein-protein interactions. PMID:18957610

  15. A single point mutation leading to loss of catalytic activity in human thiopurine S-methyltransferase.

    PubMed Central

    Krynetski, E Y; Schuetz, J D; Galpin, A J; Pui, C H; Relling, M V; Evans, W E

    1995-01-01

    Thiopurine S-methyltransferase (TPMT; S-adenosyl-L-methionine:thiopurine S-methyltransferase, EC 2.1.1.67) activity exhibits genetic polymorphism, with approximately 0.33% of Caucasians and African-Americans inheriting TPMT deficiency as an autosomal recessive trait. To determine the molecular genetic basis for this polymorphism, we cloned the TPMT cDNA from a TPMT-deficient patient who had developed severe hematopoietic toxicity during mercaptopurine therapy. Northern blot analysis of RNA isolated from leukocytes of the deficient patient demonstrated the presence of TPMT mRNAs of comparable size to that in subjects with high TPMT activity. Sequencing of the mutant TPMT cDNA revealed a single point mutation (G238-->C), leading to an amino acid substitution at codon 80 (Ala80-->Pro). When assessed in a yeast heterologous expression system, this mutation led to a 100-fold reduction in TPMT catalytic activity relative to the wild-type cDNA, despite a comparable level of mRNA expression. A mutation-specific PCR amplification method was developed and used to detect the G238-->C mutation in genomic DNA of the propositus and her mother. This inactivating mutation in the human TPMT gene provides insights into the genetic basis for this inherited polymorphism in drug metabolism. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:7862671

  16. SCN10A Mutation in a Patient with Erythromelalgia Enhances C-Fiber Activity Dependent Slowing.

    PubMed

    Kist, Andreas M; Sagafos, Dagrun; Rush, Anthony M; Neacsu, Cristian; Eberhardt, Esther; Schmidt, Roland; Lunden, Lars Kristian; Ørstavik, Kristin; Kaluza, Luisa; Meents, Jannis; Zhang, Zhiping; Carr, Thomas Hedley; Salter, Hugh; Malinowsky, David; Wollberg, Patrik; Krupp, Johannes; Kleggetveit, Inge Petter; Schmelz, Martin; Jørum, Ellen; Lampert, Angelika; Namer, Barbara

    2016-01-01

    Gain-of-function mutations in the tetrodotoxin (TTX) sensitive voltage-gated sodium channel (Nav) Nav1.7 have been identified as a key mechanism underlying chronic pain in inherited erythromelalgia. Mutations in TTX resistant channels, such as Nav1.8 or Nav1.9, were recently connected with inherited chronic pain syndromes. Here, we investigated the effects of the p.M650K mutation in Nav1.8 in a 53 year old patient with erythromelalgia by microneurography and patch-clamp techniques. Recordings of the patient's peripheral nerve fibers showed increased activity dependent slowing (ADS) in CMi and less spontaneous firing compared to a control group of erythromelalgia patients without Nav mutations. To evaluate the impact of the p.M650K mutation on neuronal firing and channel gating, we performed current and voltage-clamp recordings on transfected sensory neurons (DRGs) and neuroblastoma cells. The p.M650K mutation shifted steady-state fast inactivation of Nav1.8 to more hyperpolarized potentials and did not significantly alter any other tested gating behaviors. The AP half-width was significantly broader and the stimulated action potential firing rate was reduced for M650K transfected DRGs compared to WT. We discuss the potential link between enhanced steady state fast inactivation, broader action potential width and the potential physiological consequences.

  17. Recurrent activating mutations of CD28 in peripheral T-cell lymphomas.

    PubMed

    Rohr, J; Guo, S; Huo, J; Bouska, A; Lachel, C; Li, Y; Simone, P D; Zhang, W; Gong, Q; Wang, C; Cannon, A; Heavican, T; Mottok, A; Hung, S; Rosenwald, A; Gascoyne, R; Fu, K; Greiner, T C; Weisenburger, D D; Vose, J M; Staudt, L M; Xiao, W; Borgstahl, G E O; Davis, S; Steidl, C; McKeithan, T; Iqbal, J; Chan, W C

    2016-05-01

    Peripheral T-cell lymphomas (PTCLs) comprise a heterogeneous group of mature T-cell neoplasms with a poor prognosis. Recently, mutations in TET2 and other epigenetic modifiers as well as RHOA have been identified in these diseases, particularly in angioimmunoblastic T-cell lymphoma (AITL). CD28 is the major co-stimulatory receptor in T cells which, upon binding ligand, induces sustained T-cell proliferation and cytokine production when combined with T-cell receptor stimulation. We have identified recurrent mutations in CD28 in PTCLs. Two residues-D124 and T195-were recurrently mutated in 11.3% of cases of AITL and in one case of PTCL, not otherwise specified (PTCL-NOS). Surface plasmon resonance analysis of mutations at these residues with predicted differential partner interactions showed increased affinity for ligand CD86 (residue D124) and increased affinity for intracellular adaptor proteins GRB2 and GADS/GRAP2 (residue T195). Molecular modeling studies on each of these mutations suggested how these mutants result in increased affinities. We found increased transcription of the CD28-responsive genes CD226 and TNFA in cells expressing the T195P mutant in response to CD3 and CD86 co-stimulation and increased downstream activation of NF-κB by both D124V and T195P mutants, suggesting a potential therapeutic target in CD28-mutated PTCLs. PMID:26719098

  18. Recurrent activating mutation in PRKACA in cortisol-producing adrenal tumors.

    PubMed

    Goh, Gerald; Scholl, Ute I; Healy, James M; Choi, Murim; Prasad, Manju L; Nelson-Williams, Carol; Kunstman, John W; Kuntsman, John W; Korah, Reju; Suttorp, Anna-Carinna; Dietrich, Dimo; Haase, Matthias; Willenberg, Holger S; Stålberg, Peter; Hellman, Per; Akerström, Göran; Björklund, Peyman; Carling, Tobias; Lifton, Richard P

    2014-06-01

    Adrenal tumors autonomously producing cortisol cause Cushing's syndrome. We performed exome sequencing of 25 tumor-normal pairs and identified 2 subgroups. Eight tumors (including three carcinomas) had many somatic copy number variants (CNVs) with frequent deletion of CDC42 and CDKN2A, amplification of 5q31.2 and protein-altering mutations in TP53 and RB1. Seventeen tumors (all adenomas) had no somatic CNVs or TP53 or RB1 mutations. Six of these had known gain-of-function mutations in CTNNB1 (β-catenin) or GNAS (Gαs). Six others had somatic mutations in PRKACA (protein kinase A (PKA) catalytic subunit) resulting in a p.Leu206Arg substitution. Further sequencing identified this mutation in 13 of 63 tumors (35% of adenomas with overt Cushing's syndrome). PRKACA, GNAS and CTNNB1 mutations were mutually exclusive. Leu206 directly interacts with the regulatory subunit of PKA, PRKAR1A. Leu206Arg PRKACA loses PRKAR1A binding, increasing the phosphorylation of downstream targets. PKA activity induces cortisol production and cell proliferation, providing a mechanism for tumor development. These findings define distinct mechanisms underlying adrenal cortisol-producing tumors. PMID:24747643

  19. SCN10A Mutation in a Patient with Erythromelalgia Enhances C-Fiber Activity Dependent Slowing

    PubMed Central

    Neacsu, Cristian; Eberhardt, Esther; Schmidt, Roland; Lunden, Lars Kristian; Ørstavik, Kristin; Kaluza, Luisa; Meents, Jannis; Zhang, Zhiping; Carr, Thomas Hedley; Salter, Hugh; Malinowsky, David; Wollberg, Patrik; Krupp, Johannes; Kleggetveit, Inge Petter; Schmelz, Martin; Jørum, Ellen; Namer, Barbara

    2016-01-01

    Gain-of-function mutations in the tetrodotoxin (TTX) sensitive voltage-gated sodium channel (Nav) Nav1.7 have been identified as a key mechanism underlying chronic pain in inherited erythromelalgia. Mutations in TTX resistant channels, such as Nav1.8 or Nav1.9, were recently connected with inherited chronic pain syndromes. Here, we investigated the effects of the p.M650K mutation in Nav1.8 in a 53 year old patient with erythromelalgia by microneurography and patch-clamp techniques. Recordings of the patient’s peripheral nerve fibers showed increased activity dependent slowing (ADS) in CMi and less spontaneous firing compared to a control group of erythromelalgia patients without Nav mutations. To evaluate the impact of the p.M650K mutation on neuronal firing and channel gating, we performed current and voltage-clamp recordings on transfected sensory neurons (DRGs) and neuroblastoma cells. The p.M650K mutation shifted steady-state fast inactivation of Nav1.8 to more hyperpolarized potentials and did not significantly alter any other tested gating behaviors. The AP half-width was significantly broader and the stimulated action potential firing rate was reduced for M650K transfected DRGs compared to WT. We discuss the potential link between enhanced steady state fast inactivation, broader action potential width and the potential physiological consequences. PMID:27598514

  20. SCN10A Mutation in a Patient with Erythromelalgia Enhances C-Fiber Activity Dependent Slowing.

    PubMed

    Kist, Andreas M; Sagafos, Dagrun; Rush, Anthony M; Neacsu, Cristian; Eberhardt, Esther; Schmidt, Roland; Lunden, Lars Kristian; Ørstavik, Kristin; Kaluza, Luisa; Meents, Jannis; Zhang, Zhiping; Carr, Thomas Hedley; Salter, Hugh; Malinowsky, David; Wollberg, Patrik; Krupp, Johannes; Kleggetveit, Inge Petter; Schmelz, Martin; Jørum, Ellen; Lampert, Angelika; Namer, Barbara

    2016-01-01

    Gain-of-function mutations in the tetrodotoxin (TTX) sensitive voltage-gated sodium channel (Nav) Nav1.7 have been identified as a key mechanism underlying chronic pain in inherited erythromelalgia. Mutations in TTX resistant channels, such as Nav1.8 or Nav1.9, were recently connected with inherited chronic pain syndromes. Here, we investigated the effects of the p.M650K mutation in Nav1.8 in a 53 year old patient with erythromelalgia by microneurography and patch-clamp techniques. Recordings of the patient's peripheral nerve fibers showed increased activity dependent slowing (ADS) in CMi and less spontaneous firing compared to a control group of erythromelalgia patients without Nav mutations. To evaluate the impact of the p.M650K mutation on neuronal firing and channel gating, we performed current and voltage-clamp recordings on transfected sensory neurons (DRGs) and neuroblastoma cells. The p.M650K mutation shifted steady-state fast inactivation of Nav1.8 to more hyperpolarized potentials and did not significantly alter any other tested gating behaviors. The AP half-width was significantly broader and the stimulated action potential firing rate was reduced for M650K transfected DRGs compared to WT. We discuss the potential link between enhanced steady state fast inactivation, broader action potential width and the potential physiological consequences. PMID:27598514

  1. A novel human autoimmune syndrome caused by combined hypomorphic and activating mutations in ZAP-70.

    PubMed

    Chan, Alice Y; Punwani, Divya; Kadlecek, Theresa A; Cowan, Morton J; Olson, Jean L; Mathes, Erin F; Sunderam, Uma; Fu, Shu Man; Srinivasan, Rajgopal; Kuriyan, John; Brenner, Steven E; Weiss, Arthur; Puck, Jennifer M

    2016-02-01

    A brother and sister developed a previously undescribed constellation of autoimmune manifestations within their first year of life, with uncontrollable bullous pemphigoid, colitis, and proteinuria. The boy had hemophilia due to a factor VIII autoantibody and nephrotic syndrome. Both children required allogeneic hematopoietic cell transplantation (HCT), which resolved their autoimmunity. The early onset, severity, and distinctive findings suggested a single gene disorder underlying the phenotype. Whole-exome sequencing performed on five family members revealed the affected siblings to be compound heterozygous for two unique missense mutations in the 70-kD T cell receptor ζ-chain associated protein (ZAP-70). Healthy relatives were heterozygous mutation carriers. Although pre-HCT patient T cells were not available, mutation effects were determined using transfected cell lines and peripheral blood from carriers and controls. Mutation R192W in the C-SH2 domain exhibited reduced binding to phosphorylated ζ-chain, whereas mutation R360P in the N lobe of the catalytic domain disrupted an autoinhibitory mechanism, producing a weakly hyperactive ZAP-70 protein. Although human ZAP-70 deficiency can have dysregulated T cells, and autoreactive mouse thymocytes with weak Zap-70 signaling can escape tolerance, our patients' combination of hypomorphic and activating mutations suggested a new disease mechanism and produced previously undescribed human ZAP-70-associated autoimmune disease. PMID:26783323

  2. Synthetic Human NOTCH1 EGF Modules Unraveled Molecular Mechanisms for the Structural and Functional Roles of Calcium Ions and O-Glycans in the Ligand-Binding Region.

    PubMed

    Hayakawa, Shun; Koide, Ryosuke; Hinou, Hiroshi; Nishimura, Shin-Ichiro

    2016-02-01

    The Notch signaling pathway is an evolutionarily highly conserved mechanism that operates across multicellular organisms and is critical for cell-fate decisions during development and homeostasis in most tissues. Notch signaling is modified by posttranslational glycosylations of the Notch extracellular EGF-like domain. To evaluate the structural and functional roles of various glycoforms at multiple EGF domains in the human Notch transmembrane receptor, we established a universal method for the construction of NOTCH1 EGF modules displaying the desired O-glycans at the designated glycosylation sites. The versatility of this strategy was demonstrated by the rapid and highly efficient synthesis of NOTCH1 EGF12 concurrently having a β-D-glucopyranose-initiated glycan (Xylα1 → 3Xylα1 → 3Glcβ1 →) at Ser458 and α-L-fucopyranose-initiated glycan (Neu5Acα2 → 3Galβ1 → 4GlcNAcβ1 → 3Fucα1 →) at Thr466. The efficiency of the proper folding of the glycosylated EGF12 was markedly enhanced in the presence of 5 mM CaCl2. A nuclear magnetic resonance study revealed the existence of strong nuclear Overhauser effects between key sugar moieties and neighboring amino acid residues, indicating that both O-glycans contribute independently to the intramolecular stabilization of the antiparallel β-sheet structure in the ligand-binding region of EGF12. A preliminary test using synthetic human NOTCH1 EGF modules showed significant inhibitory effects on the proliferation and adhesiveness of human breast cancer cell line MCF-7 and lung adenocarcinoma epithelial cell line A549, demonstrating for the first time evidence that exogenously applied synthetic EGF modules have the ability to interact with intrinsic Notch ligands on the surface of cancer cells.

  3. Transforming activity of the c-Ha-ras oncogene having two point mutations in codons 12 and 61.

    PubMed

    Sekiya, T; Prassolov, V S; Fushimi, M; Nishimura, S

    1985-09-01

    A recombinant plasmid carrying the human c-Ha-ras gene with two point mutations in codons 12 and 61 was constructed and its transforming activity on mouse NIH 3T3 cells was compared with those of genes with a single mutation in either codon 12 or 61. Quantitative analyses revealed that the gene with two mutations had essentially the same transforming activity as the genes with single mutations. These results indicate that a single mutation of the c-Ha-ras gene in either codon 12 or 61 is sufficient to activate the gene and that neither of the two mutation sites involved in activation of the gene needs to be intact for transforming activity.

  4. [Astragalus induces human amniotic epithelial cells (WISH) to differentiate toward neurons, inhibits the expression of Notch1 and promotes cell survival].

    PubMed

    Chen, Xu-Dong; Wang, Jian-Guo

    2012-12-25

    The aim of the study was to investigate the effect of astragalus on differentiation of human amniotic epithelial cell line WISH into neurons, the expression of Notch1 gene and cell viability. WISH were randomly divided into astragalus group (4 subgroups), alltransretinoic acid (RA) group and control group. Astragalus group and RA group were induced to differentiate into neurocytes by using chemical inducer RA and astragalus, respectively. The expression of neuron-specific enolase (NSE), microtubule associated protein 2 (MAP-2), Nestin and GFAP of induced cells in three groups were detected using immunocytochemical method. RT- PCR was further used to detect the expression of Oct4, Notch1, Hes1, Nestin and NSE. The cell viability was measured by methyl thiazolyl tetrazolium methods. Under the convert microscope it was observed that WISH cells started to change their shape, and there were several axon or dendrite-like processes out from the cell body induced by astragalus for 24 h or RA for 12 h. The positive cell rates of NSE and MAP-2 in 100 μL/mL astragalus-induced group were less than those in RA-induced group at 48 h (P < 0.05), but higher than those in control group. Cell viability in astragalus group was higher than that of RA group (P < 0.05). While the positive cell rates of Nestin and GFAP in 100 μL/mL astragalus-induced group were higher than those in RA-induced group at 48 h (P < 0.05). The positive cell rates of Nestin in the two induced groups were lower than those in control group. RT-PCR showed that the expressions of Oct4, Notch1 and Hes1 in RA and astragalus (100 μL/mL) groups were less than those in control group, but the expression of NSE was higher than that in control group. These results suggest that astragalus (especially at 100 μL/mL, 48 h) and RA can both induce human amniotic epithelial cell line WISH cells into neuron-like cells, but astragalus induction has a higher cell survival rate than RA induction, and the expression of Notch1

  5. Directed differentiation of patient-specific induced pluripotent stem cells identifies the transcriptional repression and epigenetic modification of NKX2-5, HAND1, and NOTCH1 in hypoplastic left heart syndrome.

    PubMed

    Kobayashi, Junko; Yoshida, Masashi; Tarui, Suguru; Hirata, Masataka; Nagai, Yusuke; Kasahara, Shingo; Naruse, Keiji; Ito, Hiroshi; Sano, Shunji; Oh, Hidemasa

    2014-01-01

    The genetic basis of hypoplastic left heart syndrome (HLHS) remains unknown, and the lack of animal models to reconstitute the cardiac maldevelopment has hampered the study of this disease. This study investigated the altered control of transcriptional and epigenetic programs that may affect the development of HLHS by using disease-specific induced pluripotent stem (iPS) cells. Cardiac progenitor cells (CPCs) were isolated from patients with congenital heart diseases to generate patient-specific iPS cells. Comparative gene expression analysis of HLHS- and biventricle (BV) heart-derived iPS cells was performed to dissect the complex genetic circuits that may promote the disease phenotype. Both HLHS- and BV heart-derived CPCs were reprogrammed to generate disease-specific iPS cells, which showed characteristic human embryonic stem cell signatures, expressed pluripotency markers, and could give rise to cardiomyocytes. However, HLHS-iPS cells exhibited lower cardiomyogenic differentiation potential than BV-iPS cells. Quantitative gene expression analysis demonstrated that HLHS-derived iPS cells showed transcriptional repression of NKX2-5, reduced levels of TBX2 and NOTCH/HEY signaling, and inhibited HAND1/2 transcripts compared with control cells. Although both HLHS-derived CPCs and iPS cells showed reduced SRE and TNNT2 transcriptional activation compared with BV-derived cells, co-transfection of NKX2-5, HAND1, and NOTCH1 into HLHS-derived cells resulted in synergistic restoration of these promoters activation. Notably, gain- and loss-of-function studies revealed that NKX2-5 had a predominant impact on NPPA transcriptional activation. Moreover, differentiated HLHS-derived iPS cells showed reduced H3K4 dimethylation as well as histone H3 acetylation but increased H3K27 trimethylation to inhibit transcriptional activation on the NKX2-5 promoter. These findings suggest that patient-specific iPS cells may provide molecular insights into complex transcriptional and

  6. Recurrent activating mutations of G-protein-coupled receptor CYSLTR2 in uveal melanoma

    PubMed Central

    Moore, Amanda R; Ceraudo, Emilie; Sher, Jessica J; Guan, Youxin; Shoushtari, Alexander N; Chang, Matthew T; Zhang, Jenny Q; Walczak, Edward G; Kazmi, Manija A; Taylor, Barry S; Huber, Thomas; Chi, Ping; Sakmar, Thomas P; Chen, Yu

    2016-01-01

    Uveal melanomas are molecularly distinct from cutaneous melanomas and lack mutations in BRAF, NRAS, KIT, and NF1. Instead, they are characterized by activating mutations in GNAQ and GNA11, two highly homologous α subunits of Gαq/11 heterotrimeric G proteins, and in PLCB4 (phospholipase C β4), the downstream effector of Gαq signaling 1–3. We analyzed genomics data from 136 uveal melanoma samples and found a recurrent mutation in CYSLTR2 (cysteinyl leukotriene receptor 2) encoding a p.Leu129Gln substitution in 4 of 9 samples that lacked mutations in GNAQ, GNA11, and PLCB4 but in 0 of 127 samples that harbored mutations in these genes. The Leu129Gln CysLT2R mutant protein constitutively activates endogenous Gαq and is unresponsive to stimulation by leukotriene. Expression of Leu129Gln CysLT2R in melanocytes enforces expression of a melanocyte-lineage signature, drives phorbol ester–independent growth in vitro, and promotes tumorigenesis in vivo. Our findings implicate CYSLTR2 as a uveal melanoma oncogene and highlight the critical role of Gαq signaling in uveal melanoma pathogenesis. PMID:27089179

  7. Dehydrated Hereditary Stomatocytosislinked to gain-of-function mutations in mechanically activated PIEZO1 ion channels

    PubMed Central

    Albuisson, Juliette; Murthy, Swetha E.; Bandell, Michael; Coste, Bertrand; Louis-dit-Picard, Hélène; Mathur, Jayanti; Fénéant-Thibault, Madeleine; Tertian, Gérard; de Jaureguiberry, Jean-Pierre; Syfuss, Pierre-Yves; Cahalan, Stuart; Garçon, Loic; Toutain, Fabienne; Rohrlich, Pierre Simon; Delaunay, Jean; Picard, Véronique; Jeunemaitre, Xavier; Patapoutian, Ardem

    2013-01-01

    Dehydrated hereditary stomatocytosis (DHS) is a genetic condition with defective red blood cell (RBC) membrane properties that causes an imbalance in intracellular cation concentrations. Recently, two missense mutations inthe mechanically activated PIEZO1(FAM38A) ion channel were associated with DHS. However, it is not known how these mutations affect PIEZO1 function. Here, by combining linkage analysis and whole-exome sequencing in a large pedigree and Sanger sequencing in two additional kindreds and 11 unrelated DHS cases, we identifythree novel missense mutations and one recurrent duplication in PIEZO1, demonstrating that it is the major gene for DHS. All the DHS-associated mutations locate at C-terminal half of PIEZO1. Remarkably, we find that all PIEZO1 mutations give rise to mechanically activated currents that inactivate more slowly than wild-type currents. This gain-of-function PIEZO1 phenotype provides insight that helps to explain the increased permeability of cations in RBCs of DHS patients. Our findings also suggest a new role for mechanotransduction in RBC biology and pathophysiology. PMID:23695678

  8. A limited spectrum of mutations causes constitutive activation of the yeast alpha-factor receptor.

    PubMed

    Sommers, C M; Martin, N P; Akal-Strader, A; Becker, J M; Naider, F; Dumont, M E

    2000-06-13

    Activation of G protein coupled receptors (GPCRs) by binding of ligand is the initial event in diverse cellular signaling pathways. To examine the frequency and diversity of mutations that cause constitutive activation of one particular GPCR, the yeast alpha-factor receptor, we screened libraries of random mutations for constitutive alleles. In initial screens for mutant receptor alleles that exhibit signaling in the absence of added ligand, 14 different point mutations were isolated. All of these 14 mutants could be further activated by alpha-factor. Ten of the mutants also acquired the ability to signal in response to binding of desTrp(1)¿Ala(3)ălpha-factor, a peptide that acts as an antagonist toward normal alpha-factor receptors. Of these 10 mutants, at least eight alleles residing in the third, fifth, sixth, and seventh transmembrane segments exhibit bona fide constitutive signaling. The remaining alleles are hypersensitive to alpha-factor rather than constitutive. They can be activated by low concentrations of endogenous alpha-factor present in MATa cells. The strongest constitutively active receptor alleles were recovered multiple times from the mutational libraries, and extensive mutagenesis of certain regions of the alpha-factor receptor did not lead to recovery of any additional constitutive alleles. Thus, only a limited number of mutations is capable of causing constitutive activation of this receptor. Constitutive and hypersensitive signaling by the mutant receptors is partially suppressed by coexpression of normal receptors, consistent with preferential association of the G protein with unactivated receptors. PMID:10841771

  9. Activating Mutations in PIK3CA Lead to Widespread Modulation of the Tyrosine Phosphoproteome

    PubMed Central

    Blair, Brian G.; Pinto, Sneha M.; Nirujogi, Raja S.; Jelinek, Christine A.; Malhotra, Radhika; Kim, Min-Sik; Park, Ben Ho; Pandey, Akhilesh

    2015-01-01

    The human oncogene PIK3CA is frequently mutated in human cancers. Two hotspot mutations in PIK3CA, E545K and H1047R, have been shown to regulate widespread signaling events downstream of AKT, leading to increased cell proliferation, growth, survival, and motility. We used quantitative mass spectrometry to profile the global phosphotyrosine proteome of isogenic knock-in cell lines containing these activating mutations, where we identified 824 unique phosphopeptides. Although it is well understood that these mutations result in hyperactivation of the serine/threonine kinase AKT, we found a surprisingly widespread modulation of tyrosine phosphorylation levels of proteins in the mutant cells. In the tyrosine kinome alone, 29 tyrosine kinases were altered in their phosphorylation status. Many of the regulated phosphosites that we identified were located in the kinase domain or the canonical activation sites, indicating that these kinases and their downstream signaling pathways were activated. Our study demonstrates that there is frequent and unexpected cross-talk that occurs between tyrosine signaling pathways and serine/threonine signaling pathways activated by the canonical PI3K-AKT axis. PMID:26267517

  10. XPD Helicase Structures and Activities: Insights into the Cancer and Aging Phenotypes from XPD Mutations

    SciTech Connect

    Tainer, John; Fan, Li; Fuss, Jill O.; Cheng, Quen J.; Arvai, Andrew S.; Hammel, Michal; Roberts, Victoria A.; Cooper, Priscilla K.; Tainer, John A.

    2008-06-02

    Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.

  11. XPD Helicase Structures And Activities: Insights Into the Cancer And Aging Phenotypes From XPD Mutations

    SciTech Connect

    Fan, L.; Fuss, J.O.; Cheng, Q.J.; Arvai, A.S.; Hammel, M.; Roberts, V.A.; Cooper, P.K.; Tainer, J.A.

    2009-05-18

    Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.

  12. Hotspot activating PRKD1 somatic mutations in polymorphous low-grade adenocarcinomas of the salivary glands.

    PubMed

    Weinreb, Ilan; Piscuoglio, Salvatore; Martelotto, Luciano G; Waggott, Daryl; Ng, Charlotte K Y; Perez-Ordonez, Bayardo; Harding, Nicholas J; Alfaro, Javier; Chu, Kenneth C; Viale, Agnes; Fusco, Nicola; da Cruz Paula, Arnaud; Marchio, Caterina; Sakr, Rita A; Lim, Raymond; Thompson, Lester D R; Chiosea, Simion I; Seethala, Raja R; Skalova, Alena; Stelow, Edward B; Fonseca, Isabel; Assaad, Adel; How, Christine; Wang, Jianxin; de Borja, Richard; Chan-Seng-Yue, Michelle; Howlett, Christopher J; Nichols, Anthony C; Wen, Y Hannah; Katabi, Nora; Buchner, Nicholas; Mullen, Laura; Kislinger, Thomas; Wouters, Bradly G; Liu, Fei-Fei; Norton, Larry; McPherson, John D; Rubin, Brian P; Clarke, Blaise A; Weigelt, Britta; Boutros, Paul C; Reis-Filho, Jorge S

    2014-11-01

    Polymorphous low-grade adenocarcinoma (PLGA) is the second most frequent type of malignant tumor of the minor salivary glands. We identified PRKD1 hotspot mutations encoding p.Glu710Asp in 72.9% of PLGAs but not in other salivary gland tumors. Functional studies demonstrated that this kinase-activating alteration likely constitutes a driver of PLGA.

  13. The prognostic IDH1( R132 ) mutation is associated with reduced NADP+-dependent IDH activity in glioblastoma.

    PubMed

    Bleeker, Fonnet E; Atai, Nadia A; Lamba, Simona; Jonker, Ard; Rijkeboer, Denise; Bosch, Klazien S; Tigchelaar, Wikky; Troost, Dirk; Vandertop, W Peter; Bardelli, Alberto; Van Noorden, Cornelis J F

    2010-04-01

    Somatic mutations in the isocitrate dehydrogenase 1 gene (IDH1) occur at high frequency in gliomas and seem to be a prognostic factor for survival in glioblastoma patients. In our set of 98 glioblastoma patients, IDH1 ( R132 ) mutations were associated with improved survival of 1 year on average, after correcting for age and other variables with Cox proportional hazards models. Patients with IDH1 mutations were on average 17 years younger than patients without mutation. Mutated IDH1 has a gain of function to produce 2-hydroxyglutarate by NADPH-dependent reduction of alpha-ketoglutarate, but it is unknown whether NADPH production in gliomas is affected by IDH1 mutations. We assessed the effect of IDH1 (R132 ) mutations on IDH-mediated NADPH production in glioblastomas in situ. Metabolic mapping and image analysis was applied to 51 glioblastoma samples of which 16 carried an IDH1 (R132 ) mutation. NADP+-dependent IDH activity was determined in comparison with activity of NAD+-dependent IDH and all other NADPH-producing dehydrogenases, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, malate dehydrogenase, and hexose-6-phosphate dehydrogenase. The occurrence of IDH1 mutations correlated with approx. twofold diminished NADP+-dependent IDH activity, whereas activity of NAD+-dependent IDH and the other NADP+-dependent dehydrogenases was not affected in situ in glioblastoma. The total NADPH production capacity in glioblastoma was provided for 65% by IDH activity and the occurrence of IDH1 (R132 ) mutation reduced this capacity by 38%. It is concluded that NADPH production is hampered in glioblastoma with IDH1 (R132 ) mutation. Moreover, mutated IDH1 consumes rather than produces NADPH, thus likely lowering NADPH levels even further. The low NADPH levels may sensitize glioblastoma to irradiation and chemotherapy, thus explaining the prolonged survival of patients with mutated glioblastoma.

  14. Notch1, Notch2, and Epstein-Barr virus-encoded nuclear antigen 2 signaling differentially affects proliferation and survival of Epstein-Barr virus-infected B cells.

    PubMed

    Kohlhof, Hella; Hampel, Franziska; Hoffmann, Reinhard; Burtscher, Helmut; Weidle, Ulrich H; Hölzel, Michael; Eick, Dirk; Zimber-Strobl, Ursula; Strobl, Lothar J

    2009-05-28

    The canonical mode of transcriptional activation by both the Epstein-Barr viral protein, Epstein-Barr virus-encoded nuclear antigen 2 (EBNA2), and an activated Notch receptor (Notch-IC) requires their recruitment to RBPJ, suggesting that EBNA2 uses the Notch pathway to achieve B-cell immortalization. To gain further insight into the biologic equivalence between Notch-IC and EBNA2, we performed a genome-wide expression analysis, revealing that Notch-IC and EBNA2 exhibit profound differences in the regulation of target genes. Whereas Notch-IC is more potent in regulating genes associated with differentiation and development, EBNA2 is more potent in inducing viral and cellular genes involved in proliferation, survival, and chemotaxis. Because both EBNA2 and Notch-IC induced the expression of cell cycle-associated genes, we analyzed whether Notch1-IC or Notch2-IC can replace EBNA2 in B-cell immortalization. Although Notch-IC could drive quiescent B cells into the cell cycle, B-cell immortalization was not maintained, partially due to an increased apoptosis rate in Notch-IC-expressing cells. Expression analysis revealed that both EBNA2 and Notch-IC induced the expression of proapoptotic genes, but only in EBNA2-expressing cells were antiapoptotic genes strongly up-regulated. These findings suggest that Notch signaling in B cells and B-cell lymphomas is only compatible with proliferation if pathways leading to antiapototic signals are active. PMID:19339697

  15. Mutational analysis of amino acid residues involved in catalytic activity of a family 18 chitinase from tulip bulbs.

    PubMed

    Suzukawa, Keisuke; Yamagami, Takeshi; Ohnuma, Takayuki; Hirakawa, Hideki; Kuhara, Satoru; Aso, Yoichi; Ishiguro, Masatsune

    2003-02-01

    We expressed chitinase-1 (TBC-1) from tulip bulbs (Tulipa bakeri) in E. coli cells and used site-directed mutagenesis to identify amino acid residues essential for catalytic activity. Mutations at Glu-125 and Trp-251 completely abolished enzyme activity, and activity decreased with mutations at Asp-123 and Trp-172 when glycolchitin was the substrate. Activity changed with the mutations of Trp-251 to one of several amino acids with side-chains of little hydrophobicity, suggesting that hydrophobic interaction of Trp-251 is important for the activity. Molecular dynamics (MD) simulation analysis with hevamine as the model compound showed that the distance between Asp-123 and Glu-125 was extended by mutation of Trp-251. Kinetic studies of Trp-251-mutated chitinases confirmed these various phenomena. The results suggested that Glu-125 and Trp-251 are essential for enzyme activity and that Trp-251 had a direct role in ligand binding.

  16. Structural mutations that probe the interactions between the catalytic and dianion activation sites of triosephosphate isomerase.

    PubMed

    Zhai, Xiang; Amyes, Tina L; Wierenga, Rik K; Loria, J Patrick; Richard, John P

    2013-08-27

    Triosephosphate isomerase (TIM) catalyzes the isomerization of dihydroxyacetone phosphate to form d-glyceraldehyde 3-phosphate. The effects of two structural mutations in TIM on the kinetic parameters for catalysis of the reaction of the truncated substrate glycolaldehyde (GA) and the activation of this reaction by phosphite dianion are reported. The P168A mutation results in similar 50- and 80-fold decreases in (kcat/Km)E and (kcat/Km)E·HPi, respectively, for deprotonation of GA catalyzed by free TIM and by the TIM·HPO(3)(2-) complex. The mutation has little effect on the observed and intrinsic phosphite dianion binding energy or the magnitude of phosphite dianion activation of TIM for catalysis of deprotonation of GA. A loop 7 replacement mutant (L7RM) of TIM from chicken muscle was prepared by substitution of the archaeal sequence 208-TGAG with 208-YGGS. L7RM exhibits a 25-fold decrease in (kcat/Km)E and a larger 170-fold decrease in (kcat/Km)E·HPi for reactions of GA. The mutation has little effect on the observed and intrinsic phosphodianion binding energy and only a modest effect on phosphite dianion activation of TIM. The observation that both the P168A and loop 7 replacement mutations affect mainly the kinetic parameters for TIM-catalyzed deprotonation but result in much smaller changes in the parameters for enzyme activation by phosphite dianion provides support for the conclusion that catalysis of proton transfer and dianion activation of TIM take place at separate, weakly interacting, sites in the protein catalyst.

  17. DNA transposon activity is associated with increased mutation rates in genes of rice and other grasses

    PubMed Central

    Wicker, Thomas; Yu, Yeisoo; Haberer, Georg; Mayer, Klaus F. X.; Marri, Pradeep Reddy; Rounsley, Steve; Chen, Mingsheng; Zuccolo, Andrea; Panaud, Olivier; Wing, Rod A.; Roffler, Stefan

    2016-01-01

    DNA (class 2) transposons are mobile genetic elements which move within their ‘host' genome through excising and re-inserting elsewhere. Although the rice genome contains tens of thousands of such elements, their actual role in evolution is still unclear. Analysing over 650 transposon polymorphisms in the rice species Oryza sativa and Oryza glaberrima, we find that DNA repair following transposon excisions is associated with an increased number of mutations in the sequences neighbouring the transposon. Indeed, the 3,000 bp flanking the excised transposons can contain over 10 times more mutations than the genome-wide average. Since DNA transposons preferably insert near genes, this is correlated with increases in mutation rates in coding sequences and regulatory regions. Most importantly, we find this phenomenon also in maize, wheat and barley. Thus, these findings suggest that DNA transposon activity is a major evolutionary force in grasses which provide the basis of most food consumed by humankind. PMID:27599761

  18. DNA transposon activity is associated with increased mutation rates in genes of rice and other grasses.

    PubMed

    Wicker, Thomas; Yu, Yeisoo; Haberer, Georg; Mayer, Klaus F X; Marri, Pradeep Reddy; Rounsley, Steve; Chen, Mingsheng; Zuccolo, Andrea; Panaud, Olivier; Wing, Rod A; Roffler, Stefan

    2016-01-01

    DNA (class 2) transposons are mobile genetic elements which move within their 'host' genome through excising and re-inserting elsewhere. Although the rice genome contains tens of thousands of such elements, their actual role in evolution is still unclear. Analysing over 650 transposon polymorphisms in the rice species Oryza sativa and Oryza glaberrima, we find that DNA repair following transposon excisions is associated with an increased number of mutations in the sequences neighbouring the transposon. Indeed, the 3,000 bp flanking the excised transposons can contain over 10 times more mutations than the genome-wide average. Since DNA transposons preferably insert near genes, this is correlated with increases in mutation rates in coding sequences and regulatory regions. Most importantly, we find this phenomenon also in maize, wheat and barley. Thus, these findings suggest that DNA transposon activity is a major evolutionary force in grasses which provide the basis of most food consumed by humankind. PMID:27599761

  19. Rosai-Dorfman Disease Harboring an Activating KRAS K117N Missense Mutation.

    PubMed

    Shanmugam, Vignesh; Margolskee, Elizabeth; Kluk, Michael; Giorgadze, Tamara; Orazi, Attilio

    2016-09-01

    Rosai-Dorfman disease (RDD) or sinus histiocytosis with massive lymphadenopathy is a rare histiocytic proliferation that is generally considered to be reactive with a benign clinical course. The etiology of RDD is very poorly understood. Recent studies have shown frequent BRAF, NRAS, KRAS, and PIK3CA activating mutations in several histiocytic neoplasms highlighting the emerging importance of the RAF/MEK/ERK pathway in the pathogenesis of these diseases. Here we report a case of Rosai-Dorfman disease involving the submandibular salivary gland with a KRAS K117N missense mutation discovered by next-generation sequencing. These results suggest that at least a subset of RDD cases may be clonal processes. Further mutational studies on this rare histiocytic disease should be undertaken to better characterize its pathogenesis as well as open up potential avenues for therapy.

  20. Genetic Studies on the Loss of Mu Mutator Activity in Maize

    PubMed Central

    Robertson, Donald S.

    1986-01-01

    Mutator activity of the Mu mutator system of maize can be lost by either outcrossing or inbreeding Mu stocks. The nature of these two kinds of Mu-loss phenomena was analyzed by testing the results of crossing Mu-loss stocks by active Mu lines. Outcross- Mu-loss stocks are capable of supporting Mu activity if crossed by an active mutator line. Inbred-Mu-loss stocks, however, inactivate the active Mu system contributed by a Mu line. Also, inbred- Mu-loss lines do not regain Mu activity after at least three generations of outcrossing to non-Mu stocks. These results suggest that, once the Mu system is inactivated by inbreeding, it remains inactivated for at least three generations of outcrossing. Further, once the system responsible for inactivation is established, it will, in turn, inactivate an active Mu system contributed by crossing with Mu plants. The outcross-Mu-loss does not seem to involve such an inactivation system. These results are interpreted in the light of recent evidence that Mu inactivation results from the modification of Mu 1 transposable elements involved in the Mu phenotype. PMID:17246337

  1. Neonatal diabetes mellitus: description of two Puerto Rican children with KCNJ11 activating gene mutation.

    PubMed

    Nieves-Rivera, Francisco; González-Pijem, Lilliam

    2011-06-01

    Neonatal diabetes mellitus (NDM) is a rare disorder. A one-month-old boy presented with vomiting, hyperglycemia (968 mg/dl [53.8 mmol/L]), severe acetonemia, and metabolic acidosis (pH 6.95, HCO3-4.2 mmol/L). A second child (three months of age) presented with upper respiratory tract symptoms and a plasma glucose level of 835 mg/dl, without acetonemia or acidosis. Both were hospitalized and managed with intravenous fluids and then discharged on insulin. Genetic testing identified the presence of the de nova V59M and E322K activating mutations in the KCNJ11 gene encoding the sulphonylurea/potassium channel (Kir6.2 subunit) of the insulin beta cell. Both patients were switched to glibenclamide and remain off insulin. To our knowledge, these are the first children in Puerto Rico identified with NDM secondary to a KCNJ11 activating mutation. We conclude that NDM secondary to KCNJ11/Kir6.2 activating mutations, although unusual, should be considered in similar cases since patients with these mutations could come off insulin.

  2. Analysis of phenotype, enzyme activity and genotype of Chinese patients with POMT1 mutation.

    PubMed

    Yang, Haipo; Manya, Hiroshi; Kobayashi, Kazuhiro; Jiao, Hui; Fu, Xiaona; Xiao, Jiangxi; Li, Xiaoqing; Wang, Jingmin; Jiang, Yuwu; Toda, Tatsushi; Endo, Tamao; Wu, Xiru; Xiong, Hui

    2016-08-01

    Protein O-mannosyltransferase 1 (POMT1) is a glycosyltransferase involved in α-dystroglycan glycosylation. POMT1 mutations cause a wide spectrum of clinical conditions from Walker-Warburg syndrome (WWS), which involves muscle, eye and brain abnormalities, to mild forms of limb-girdle muscular dystrophy with mental retardation. We aimed to elucidate the impact of different POMT1 mutations on the clinical phenotype. We report five Chinese patients with POMT1 mutations: one had a typical clinical manifestation of WWS, and the other four were diagnosed with congenital muscular dystrophy with mental retardation of varying severity. We analyzed the influence of the POMT1 mutations on POMT activity by assaying the patients' muscles and cultured skin fibroblasts. We demonstrated different levels of decreased POMT activity that correlated highly with decreased α-dystroglycan glycosylation. Our results suggest that POMT activity is inversely proportional to clinical severity, and demonstrate that skin fibroblasts can be used for differential diagnosis of patients with α-dystroglycanopathies. We have provided clinical, histological, enzymatic and genetic evidence of POMT1 involvement in five unrelated Chinese patients.

  3. Increased sleep spindle activity in patients with Costello syndrome (HRAS gene mutation).

    PubMed

    Della Marca, Giacomo; Leoni, Chiara; Dittoni, Serena; Battaglia, Domenica; Losurdo, Anna; Testani, Elisa; Colicchio, Salvatore; Gnoni, Valentina; Gambardella, Maria L; Mariotti, Paolo; Alfieri, Paolo; Tartaglia, Marco; Zampino, Giuseppe

    2011-06-01

    Costello syndrome is a congenital disorder because of HRAS gene mutation, frequently associated with neurologic impairment and sleep disorders. The aims of the study were to evaluate the sleep EEG, and particularly the sleep spindles, in a population of patients with Costello syndrome and to compare them with those characterizing unaffected subjects. Eleven subjects (5 men and 6 women) with Costello syndrome were included in the study; age ranged between 18 months and 31 years (mean, 9.6 ± 9.4 years). The diagnosis was posed on the basis of established clinical criteria and confirmed molecularly. Sleep EEG was studied by means of full-night, laboratory-based video-polysomnography, performed overnight, during hospitalization. Sleep activity was quantified by means of power spectral analysis. Patients heterozygous for an HRAS mutation exhibited increased EEG power in 12- to 15-Hz activity band compared with age-matched control subjects. In conclusion, the authors observed a consistent increase in the amplitude of cortical sleep spindles in all our subjects with an HRAS mutation. These "giant" spindles were not associated with any evidence of structural damage of the cortex or the thalami and should be considered as phenotypic feature of sleep EEG activity in Costello syndrome because of HRAS mutation.

  4. Altered Activation of Protein Kinase PKR and Enhanced Apoptosis in Dystonia Cells Carrying a Mutation in PKR Activator Protein PACT*

    PubMed Central

    Vaughn, Lauren S; Bragg, D. Cristopher; Sharma, Nutan; Camargos, Sarah; Cardoso, Francisco; Patel, Rekha C

    2015-01-01

    PACT is a stress-modulated activator of the interferon-induced double-stranded RNA-activated protein kinase (PKR). Stress-induced phosphorylation of PACT is essential for PACT's association with PKR leading to PKR activation. PKR activation leads to phosphorylation of translation initiation factor eIF2α inhibition of protein synthesis and apoptosis. A recessively inherited form of early-onset dystonia DYT16 has been recently identified to arise due to a homozygous missense mutation P222L in PACT. To examine if the mutant P222L protein alters the stress-response pathway, we examined the ability of mutant P222L to interact with and activate PKR. Our results indicate that the substitution mutant P222L activates PKR more robustly and for longer duration albeit with slower kinetics in response to the endoplasmic reticulum stress. In addition, the affinity of PACT-PACT and PACT-PKR interactions is enhanced in dystonia patient lymphoblasts, thereby leading to intensified PKR activation and enhanced cellular death. P222L mutation also changes the affinity of PACT-TRBP interaction after cellular stress, thereby offering a mechanism for the delayed PKR activation in response to stress. Our results demonstrate the impact of a dystonia-causing substitution mutation on stress-induced cellular apoptosis. PMID:26231208

  5. Quantitative metabolome analysis profiles activation of glutaminolysis in glioma with IDH1 mutation.

    PubMed

    Ohka, Fumiharu; Ito, Maki; Ranjit, Melissa; Senga, Takeshi; Motomura, Ayako; Motomura, Kazuya; Saito, Kaori; Kato, Keiko; Kato, Yukinari; Wakabayashi, Toshihiko; Soga, Tomoyoshi; Natsume, Atsushi

    2014-06-01

    Isocitrate dehydrogenase 1 (IDH1), which localizes to the cytosol and peroxisomes, catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) and in parallel converts NADP(+) to NADPH. IDH1 mutations are frequently detected in grades 2-4 gliomas and in acute myeloid leukemias (AML). Mutations of IDH1 have been identified at codon 132, with arginine being replaced with histidine in most cases. Mutant IDH1 gains novel enzyme activity converting α-KG to D-2-hydroxyglutarate (2-HG) which acts as a competitive inhibitor of α-KG. As a result, the activity of α-KG-dependent enzyme is reduced. Based on these findings, 2-HG has been proposed to be an oncometabolite. In this study, we established HEK293 and U87 cells that stably expressed IDH1-WT and IDH1-R132H and investigated the effect of glutaminase inhibition on cell proliferation with 6-diazo-5-oxo-L-norleucine (DON). We found that cell proliferation was suppressed in IDH1-R132H cells. The addition of α-KG restored cell proliferation. The metabolic features of 33 gliomas with wild type IDH1 (IDH1-WT) and with IDH1-R132H mutation were examined by global metabolome analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). We showed that the 2-HG levels were highly elevated in gliomas with IDH1-R132H mutation. Intriguingly, in gliomas with IDH1-R132H, glutamine and glutamate levels were significantly reduced which implies replenishment of α-KG by glutaminolysis. Based on these results, we concluded that glutaminolysis is activated in gliomas with IDH1-R132H mutation and that development of novel therapeutic approaches targeting activated glutaminolysis is warranted.

  6. Impaired dNTPase Activity of SAMHD1 by Phosphomimetic Mutation of Thr-592*♦

    PubMed Central

    Tang, Chenxiang; Ji, Xiaoyun; Wu, Li; Xiong, Yong

    2015-01-01

    SAMHD1 is a cellular protein that plays key roles in HIV-1 restriction and regulation of cellular dNTP levels. Mutations in SAMHD1 are also implicated in the pathogenesis of chronic lymphocytic leukemia and Aicardi-Goutières syndrome. The anti-HIV-1 activity of SAMHD1 is negatively modulated by phosphorylation at residue Thr-592. The mechanism underlying the effect of phosphorylation on anti-HIV-1 activity remains unclear. SAMHD1 forms tetramers that possess deoxyribonucleotide triphosphate triphosphohydrolase (dNTPase) activity, which is allosterically controlled by the combined action of GTP and all four dNTPs. Here we demonstrate that the phosphomimetic mutation T592E reduces the stability of the SAMHD1 tetramer and the dNTPase activity of the enzyme. To better understand the underlying mechanisms, we determined the crystal structures of SAMHD1 variants T592E and T592V. Although the neutral substitution T592V does not perturb the structure, the charged T592E induces large conformational changes, likely triggered by electrostatic repulsion from a distinct negatively charged environment surrounding Thr-592. The phosphomimetic mutation results in a significant decrease in the population of active SAMHD1 tetramers, and hence the dNTPase activity is substantially decreased. These results provide a mechanistic understanding of how SAMHD1 phosphorylation at residue Thr-592 may modulate its cellular and antiviral functions. PMID:26294762

  7. Mutations in MAP3K7 that Alter the Activity of the TAK1 Signaling Complex Cause Frontometaphyseal Dysplasia.

    PubMed

    Wade, Emma M; Daniel, Philip B; Jenkins, Zandra A; McInerney-Leo, Aideen; Leo, Paul; Morgan, Tim; Addor, Marie Claude; Adès, Lesley C; Bertola, Debora; Bohring, Axel; Carter, Erin; Cho, Tae-Joon; Duba, Hans-Christoph; Fletcher, Elaine; Kim, Chong A; Krakow, Deborah; Morava, Eva; Neuhann, Teresa; Superti-Furga, Andrea; Veenstra-Knol, Irma; Wieczorek, Dagmar; Wilson, Louise C; Hennekam, Raoul C M; Sutherland-Smith, Andrew J; Strom, Tim M; Wilkie, Andrew O M; Brown, Matthew A; Duncan, Emma L; Markie, David M; Robertson, Stephen P

    2016-08-01

    Frontometaphyseal dysplasia (FMD) is a progressive sclerosing skeletal dysplasia affecting the long bones and skull. The cause of FMD in some individuals is gain-of-function mutations in FLNA, although how these mutations result in a hyperostotic phenotype remains unknown. Approximately one half of individuals with FMD have no identified mutation in FLNA and are phenotypically very similar to individuals with FLNA mutations, except for an increased tendency to form keloid scars. Using whole-exome sequencing and targeted Sanger sequencing in 19 FMD-affected individuals with no identifiable FLNA mutation, we identified mutations in two genes-MAP3K7, encoding transforming growth factor β (TGF-β)-activated kinase (TAK1), and TAB2, encoding TAK1-associated binding protein 2 (TAB2). Four mutations were found in MAP3K7, including one highly recurrent (n = 15) de novo mutation (c.1454C>T [ p.Pro485Leu]) proximal to the coiled-coil domain of TAK1 and three missense mutations affecting the kinase domain (c.208G>C [p.Glu70Gln], c.299T>A [p.Val100Glu], and c.502G>C [p.Gly168Arg]). Notably, the subjects with the latter three mutations had a milder FMD phenotype. An additional de novo mutation was found in TAB2 (c.1705G>A, p.Glu569Lys). The recurrent mutation does not destabilize TAK1, or impair its ability to homodimerize or bind TAB2, but it does increase TAK1 autophosphorylation and alter the activity of more than one signaling pathway regulated by the TAK1 kinase complex. These findings show that dysregulation of the TAK1 complex produces a close phenocopy of FMD caused by FLNA mutations. Furthermore, they suggest that the pathogenesis of some of the filaminopathies caused by FLNA mutations might be mediated by misregulation of signaling coordinated through the TAK1 signaling complex. PMID:27426733

  8. PAK4 kinase activity and somatic mutation promote carcinoma cell motility and influence inhibitor sensitivity

    PubMed Central

    Whale, Andrew D.; Dart, Anna; Holt, Mark; Jones, Gareth E.; Wells, Claire M.

    2012-01-01

    Hepatocyte growth factor (HGF) and its receptor (c-Met) are associated with cancer cell motility and invasiveness. p21-activated kinase 4 (PAK4), a potential therapeutic target, is recruited to and activated by c-Met. In response, PAK4 phosphorylates LIM kinase 1 (LIMK1) in an HGF-dependent manner in metastatic prostate carcinoma cells. PAK4 overexpression is known to induce increased cell migration speed but the requirement for kinase activity has not been established. We have used a panel of PAK4 truncations and mutations in a combination of over-expression and RNAi rescue experiments to determine the requirement for PAK4 kinase activity during carcinoma cell motility downstream of HGF. We find that neither the kinase domain alone nor a PAK4 mutant unable to bind Cdc42 is able to fully rescue cell motility in a PAK4-deficient background. Nevertheless, we find that PAK4 kinase activity and associated LIMK1 activity are essential for carcinoma cell motility, highlighting PAK4 as a potential anti-metastatic therapeutic target. We also show here that overexpression of PAK4 harboring a somatic mutation, E329K, increased the HGF-driven motility of metastatic prostate carcinoma cells. E329 lies within the G-loop region of the kinase. Our data suggest E329K mutation leads to a modest increase in kinase activity conferring resistance to competitive ATP inhibitors in addition to promoting cell migration. The existence of such a mutation may have implications for the development of PAK4-specific competitive ATP inhibitors should PAK4 be further explored for clinical inhibition. PMID:22689056

  9. TERT Promoter Mutations Lead to High Transcriptional Activity under Hypoxia and Temozolomide Treatment and Predict Poor Prognosis in Gliomas

    PubMed Central

    Meng, Lingxuan; Li, Zhonghua; Zhang, Xue; Wu, Anhua

    2014-01-01

    Objective This study explored the effects of telomerase reverse transcriptase (TERT) promoter mutations on transcriptional activity of the TERT gene under hypoxic and temozolomide (TMZ) treatment conditions, and investigated the status and prognostic value of these mutations in gliomas. Methods The effect of TERT promoter mutations on the transcriptional activity of the TERT gene under hypoxic and TMZ treatment conditions was investigated in glioma cells using the luciferase assay. TERT promoter mutations were detected in 101 glioma samples (grades I–IV) and 49 other brain tumors by sequencing. TERT mRNA expression in gliomas was examined by real-time PCR. Hazard ratios from survival analysis of glioma patients were determined relative to the presence of TERT promoter mutations. Results Mutations in the TERT promoter enhanced gene transcription even under hypoxic and TMZ treatment conditions, inducing upregulation of TERT mRNA expression. Mutations were detected in gliomas, but not in meningiomas, pituitary adenomas, cavernomas, intracranial metastases, normal brain tissues, or peripheral blood of glioma patients. Patients with TERT promoter mutations had lower survival rates, even after adjusting for other known or potential risk factors, and the incidence of mutation was correlated with patient age. Conclusion TERT promoter mutations were specific to gliomas. TERT promoter mutations maintained its ability of inducing high transcriptional activity even under hypoxic and TMZ treatment conditions, and the presence of mutations was associated with poor prognosis in glioma patients. These findings demonstrate that TERT promoter mutations are novel prognostic markers for gliomas that can inform prospective therapeutic strategies. PMID:24937153

  10. An activating Pik3ca mutation coupled with Pten loss is sufficient to initiate ovarian tumorigenesis in mice.

    PubMed

    Kinross, Kathryn M; Montgomery, Karen G; Kleinschmidt, Margarete; Waring, Paul; Ivetac, Ivan; Tikoo, Anjali; Saad, Mirette; Hare, Lauren; Roh, Vincent; Mantamadiotis, Theo; Sheppard, Karen E; Ryland, Georgina L; Campbell, Ian G; Gorringe, Kylie L; Christensen, James G; Cullinane, Carleen; Hicks, Rodney J; Pearson, Richard B; Johnstone, Ricky W; McArthur, Grant A; Phillips, Wayne A

    2012-02-01

    Mutations in the gene encoding the p110α subunit of PI3K (PIK3CA) that result in enhanced PI3K activity are frequently observed in human cancers. To better understand the role of mutant PIK3CA in the initiation or progression of tumorigenesis, we generated mice in which a PIK3CA mutation commonly detected in human cancers (the H1047R mutation) could be conditionally knocked into the endogenous Pik3ca locus. Activation of this mutation in the mouse ovary revealed that alone, Pik3caH1047R induced premalignant hyperplasia of the ovarian surface epithelium but no tumors. Concomitantly, we analyzed several human ovarian cancers and found PIK3CA mutations coexistent with KRAS and/or PTEN mutations, raising the possibility that a secondary defect in a co-regulator of PI3K activity may be required for mutant PIK3CA to promote transformation. Consistent with this notion, we found that Pik3caH1047R mutation plus Pten deletion in the mouse ovary led to the development of ovarian serous adenocarcinomas and granulosa cell tumors. Both mutational events were required for early, robust Akt activation. Pharmacological inhibition of PI3K/mTOR in these mice delayed tumor growth and prolonged survival. These results demonstrate that the Pik3caH1047R mutation with loss of Pten is enough to promote ovarian cell transformation and that we have developed a model system for studying possible therapies.

  11. A novel TMPRSS6 mutation that prevents protease auto-activation causes IRIDA

    PubMed Central

    Altamura, Sandro; D'Alessio, Flavia; Selle, Barbara; Muckenthaler, Martina U.

    2010-01-01

    IRIDA (iron-refractory iron-deficiency anaemia) is a rare autosomal-recessive disorder hallmarked by hypochromic microcytic anaemia, low transferrin saturation and high levels of the iron-regulated hormone hepcidin. The disease is caused by mutations in the transmembrane serine protease TMPRSS6 (transmembrane protease serine 6) that prevent inactivation of HJV (haemojuvelin), an activator of hepcidin transcription. In the present paper, we describe a patient with IRIDA who carries a novel mutation (Y141C) in the SEA domain of the TMPRSS6 gene. Functional characterization of the TMPRSS6(Y141C) mutant protein in cultured cells showed that it localizes to similar subcellular compartments as wild-type TMPRSS6 and binds HJV, but fails to auto-catalytically activate itself. As a consequence, hepcidin mRNA expression is increased, causing the clinical symptoms observed in this IRIDA patient. The present study provides important mechanistic insight into how TMPRSS6 is activated. PMID:20704562

  12. Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma

    PubMed Central

    Lissanu Deribe, Yonathan; Shi, Yanxia; Rai, Kunal; Nezi, Luigi; Amin, Samir B.; Wu, Chia-Chin; Akdemir, Kadir C.; Mahdavi, Mozhdeh; Peng, Qian; Chang, Qing Edward; Hornigold, Kirsti; Arold, Stefan T.; Welch, Heidi C. E.; Garraway, Levi A.; Chin, Lynda

    2016-01-01

    PREX2 (phosphatidylinositol-3,4,5-triphosphate-dependent Rac-exchange factor 2) is a PTEN (phosphatase and tensin homolog deleted on chromosome 10) binding protein that is significantly mutated in cutaneous melanoma and pancreatic ductal adenocarcinoma. Here, genetic and biochemical analyses were conducted to elucidate the nature and mechanistic basis of PREX2 mutation in melanoma development. By generating an inducible transgenic mouse model we showed an oncogenic role for a truncating PREX2 mutation (PREX2E824*) in vivo in the context of mutant NRAS. Using integrative cross-species gene expression analysis, we identified deregulated cell cycle and cytoskeleton organization as significantly perturbed biological pathways in PREX2 mutant tumors. Mechanistically, truncation of PREX2 activated its Rac1 guanine nucleotide exchange factor activity, abolished binding to PTEN and activated the PI3K (phosphatidyl inositol 3 kinase)/Akt signaling pathway. We further showed that PREX2 truncating mutations or PTEN deletion induces down-regulation of the tumor suppressor and cell cycle regulator CDKN1C (also known as p57KIP2). This down-regulation occurs, at least partially, through DNA hypomethylation of a differentially methylated region in chromosome 11 that is a known regulatory region for expression of the CDKN1C gene. Together, these findings identify PREX2 as a mediator of NRAS-mutant melanoma development that acts through the PI3K/PTEN/Akt pathway to regulate gene expression of a cell cycle regulator. PMID:26884185

  13. Intragenic suppression of an active site mutation in the human apurinic/apyrimidinic endonuclease.

    PubMed

    Izumi, T; Malecki, J; Chaudhry, M A; Weinfeld, M; Hill, J H; Lee, J C; Mitra, S

    1999-03-19

    The apurinic/apyrimidinic endonucleases (APE) contain several highly conserved sequence motifs. The glutamic acid residue in a consensus motif, LQE96TK98 in human APE (hAPE-1), is crucial because of its role in coordinating Mg2+, an essential cofactor. Random mutagenesis of the inactive E96A mutant cDNA, followed by phenotypic screening in Escherichia coli, led to isolation of an intragenic suppressor with a second site mutation, K98R. Although the Km of the suppressor mutant was about sixfold higher than that of the wild-type enzyme, their kcat values were similar for AP endonuclease activity. These results suggest that the E96A mutation affects only the DNA-binding step, but not the catalytic step of the enzyme. The 3' DNA phosphoesterase activities of the wild-type and the suppressor mutant were also comparable. No global change of the protein conformation is induced by the single or double mutations, but a local perturbation in the structural environment of tryptophan residues may be induced by the K98R mutation. The wild-type and suppressor mutant proteins have similar Mg2+ requirement for activity. These results suggest a minor perturbation in conformation of the suppressor mutant enabling an unidentified Asp or Glu residue to substitute for Glu96 in positioning Mg2+ during catalysis. The possibility that Asp70 is such a residue, based on its observed proximity to the metal-binding site in the wild-type protein, was excluded by site-specific mutation studies. It thus appears that another acidic residue coordinates with Mg2+ in the mutant protein. These results suggest a rather flexible conformation of the region surrounding the metal binding site in hAPE-1 which is not obvious from the X-ray crystallographic structure. PMID:10074406

  14. The protist Trichomonas vaginalis harbors multiple lineages of transcriptionally active Mutator-like elements

    PubMed Central

    Lopes, Fabrício R; Silva, Joana C; Benchimol, Marlene; Costa, Gustavo GL; Pereira, Gonçalo AG; Carareto, Claudia MA

    2009-01-01

    Background For three decades the Mutator system was thought to be exclusive of plants, until the first homolog representatives were characterized in fungi and in early-diverging amoebas earlier in this decade. Results Here, we describe and characterize four families of Mutator-like elements in a new eukaryotic group, the Parabasalids. These Trichomonas vaginalis Mutator- like elements, or TvMULEs, are active in T. vaginalis and patchily distributed among 12 trichomonad species and isolates. Despite their relatively distinctive amino acid composition, the inclusion of the repeats TvMULE1, TvMULE2, TvMULE3 and TvMULE4 into the Mutator superfamily is justified by sequence, structural and phylogenetic analyses. In addition, we identified three new TvMULE-related sequences in the genome sequence of Candida albicans. While TvMULE1 is a member of the MuDR clade, predominantly from plants, the other three TvMULEs, together with the C. albicans elements, represent a new and quite distinct Mutator lineage, which we named TvCaMULEs. The finding of TvMULE1 sequence inserted into other putative repeat suggests the occurrence a novel TE family not yet described. Conclusion These findings expand the taxonomic distribution and the range of functional motif of MULEs among eukaryotes. The characterization of the dynamics of TvMULEs and other transposons in this organism is of particular interest because it is atypical for an asexual species to have such an extreme level of TE activity; this genetic landscape makes an interesting case study for causes and consequences of such activity. Finally, the extreme repetitiveness of the T. vaginalis genome and the remarkable degree of sequence identity within its repeat families highlights this species as an ideal system to characterize new transposable elements. PMID:19622157

  15. Activation of initiation factor 2 by ligands and mutations for rapid docking of ribosomal subunits.

    PubMed

    Pavlov, Michael Y; Zorzet, Anna; Andersson, Dan I; Ehrenberg, Måns

    2011-01-19

    We previously identified mutations in the GTPase initiation factor 2 (IF2), located outside its tRNA-binding domain, compensating strongly (A-type) or weakly (B-type) for initiator tRNA formylation deficiency. We show here that rapid docking of 30S with 50S subunits in initiation of translation depends on switching 30S subunit-bound IF2 from its inactive to active form. Activation of wild-type IF2 requires GTP and formylated initiator tRNA (fMet-tRNA(i)). In contrast, extensive activation of A-type IF2 occurs with only GTP or with GDP and fMet-tRNA(i), implying a passive role for initiator tRNA as activator of IF2 in subunit docking. The theory of conditional switching of GTPases quantitatively accounts for all our experimental data. We find that GTP, GDP, fMet-tRNA(i) and A-type mutations multiplicatively increase the equilibrium ratio, K, between active and inactive forms of IF2 from a value of 4 × 10(-4) for wild-type apo-IF2 by factors of 300, 8, 80 and 20, respectively. Functional characterization of the A-type mutations provides keys to structural interpretation of conditional switching of IF2 and other multidomain GTPases. PMID:21151095

  16. A three-dimensional model of mammalian tyrosinase active site accounting for loss of function mutations.

    PubMed

    Schweikardt, Thorsten; Olivares, Concepción; Solano, Francisco; Jaenicke, Elmar; García-Borrón, José Carlos; Decker, Heinz

    2007-10-01

    Tyrosinases are the first and rate-limiting enzymes in the synthesis of melanin pigments responsible for colouring hair, skin and eyes. Mutation of tyrosinases often decreases melanin production resulting in albinism, but the effects are not always understood at the molecular level. Homology modelling of mouse tyrosinase based on recently published crystal structures of non-mammalian tyrosinases provides an active site model accounting for loss-of-function mutations. According to the model, the copper-binding histidines are located in a helix bundle comprising four densely packed helices. A loop containing residues M374, S375 and V377 connects the CuA and CuB centres, with the peptide oxygens of M374 and V377 serving as hydrogen acceptors for the NH-groups of the imidazole rings of the copper-binding His367 and His180. Therefore, this loop is essential for the stability of the active site architecture. A double substitution (374)MS(375) --> (374)GG(375) or a single M374G mutation lead to a local perturbation of the protein matrix at the active site affecting the orientation of the H367 side chain, that may be unable to bind CuB reliably, resulting in loss of activity. The model also accounts for loss of function in two naturally occurring albino mutations, S380P and V393F. The hydroxyl group in S380 contributes to the correct orientation of M374, and the substitution of V393 for a bulkier phenylalanine sterically impedes correct side chain packing at the active site. Therefore, our model explains the mechanistic necessity for conservation of not only active site histidines but also adjacent amino acids in tyrosinase. PMID:17850513

  17. Disease Mutations in Rab7 Result in Unregulated Nucleotide Exchange and Inappropriate Activation

    SciTech Connect

    B McCray; E Skordalakes; J Taylor

    2011-12-31

    Rab GTPases are molecular switches that orchestrate vesicular trafficking, maturation and fusion by cycling between an active, GTP-bound form, and an inactive, GDP-bound form. The activity cycle is coupled to GTP hydrolysis and is tightly controlled by regulatory proteins. Missense mutations of the GTPase Rab7 cause a dominantly inherited axonal degeneration known as Charcot-Marie-Tooth type 2B through an unknown mechanism. We present the 2.8 A crystal structure of GTP-bound L129F mutant Rab7 which reveals normal conformations of the effector binding regions and catalytic site, but an alteration to the nucleotide binding pocket that is predicted to alter GTP binding. Through extensive biochemical analysis, we demonstrate that disease-associated mutations in Rab7 do not lead to an intrinsic GTPase defect, but permit unregulated nucleotide exchange leading to both excessive activation and hydrolysis-independent inactivation. Consistent with augmented activity, mutant Rab7 shows significantly enhanced interaction with a subset of effector proteins. In addition, dynamic imaging demonstrates that mutant Rab7 is abnormally retained on target membranes. However, we show that the increased activation of mutant Rab7 is counterbalanced by unregulated, GTP hydrolysis-independent membrane cycling. Notably, disease mutations are able to rescue the membrane cycling of a GTPase-deficient mutant. Thus, we demonstrate that disease mutations uncouple Rab7 from the spatial and temporal control normally imposed by regulatory proteins and cause disease not by a gain of novel toxic function, but by misregulation of native Rab7 activity.

  18. Disease-associated mutations inactivate AMP-lysine hydrolase activity of Aprataxin.

    PubMed

    Seidle, Heather F; Bieganowski, Pawel; Brenner, Charles

    2005-06-01

    Ataxia-oculomotor apraxia syndrome 1 is an early onset cerebellar ataxia that results from loss of function mutations in the APTX gene, encoding Aprataxin, which contains three conserved domains. The forkhead-associated domain of Aprataxin mediates protein-protein interactions with molecules that respond to DNA damage, but the cellular phenotype of the disease does not appear to be consistent with a major loss in DNA damage responses. Disease-associated mutations in Aprataxin target a histidine triad domain that is similar to Hint, a universally conserved AMP-lysine hydrolase, or truncate the protein NH2-terminal to a zinc finger. With novel fluorigenic substrates, we demonstrate that Aprataxin possesses an active-site-dependent AMP-lysine and GMP-lysine hydrolase activity that depends additionally on the zinc finger for protein stability and on the forkhead associated domain for enzymatic activity. Alleles carrying any of eight recessive mutations associated with ataxia and oculomotor apraxia encode proteins with huge losses in protein stability and enzymatic activity, consistent with a null phenotype. The mild presentation allele, APTX-K197Q, associated with ataxia but not oculomotor apraxia, encodes a protein with a mild defect in stability and activity, while enzyme encoded by the atypical presentation allele, APTX-R199H, retained substantial function, consistent with altered and not loss of activity. The data suggest that the essential function of Aprataxin is reversal of nucleotidylylated protein modifications, that all three domains contribute to formation of a stable enzyme, and that the in vitro behavior of cloned APTX alleles can score disease-associated mutations. PMID:15790557

  19. The STAT3 HIES mutation is a gain-of-function mutation that activates genes via AGG-element carrying promoters

    PubMed Central

    Xu, Li; Ji, Jin-Jun; Le, Wangping; Xu, Yan S.; Dou, Dandan; Pan, Jieli; Jiao, Yifeng; Zhong, Tianfei; Wu, Dehong; Wang, Yumei; Wen, Chengping; Xie, Guan-Qun; Yao, Feng; Zhao, Heng; Fan, Yong-Sheng; Chin, Y. Eugene

    2015-01-01

    Cytokine or growth factor activated STAT3 undergoes multiple post-translational modifications, dimerization and translocation into nuclei, where it binds to serum-inducible element (SIE, ‘TTC(N3)GAA’)-bearing promoters to activate transcription. The STAT3 DNA binding domain (DBD, 320–494) mutation in hyper immunoglobulin E syndrome (HIES), called the HIES mutation (R382Q, R382W or V463Δ), which elevates IgE synthesis, inhibits SIE binding activity and sensitizes genes such as TNF-α for expression. However, the mechanism by which the HIES mutation sensitizes STAT3 in gene induction remains elusive. Here, we report that STAT3 binds directly to the AGG-element with the consensus sequence ‘AGG(N3)AGG’. Surprisingly, the helical N-terminal region (1–355), rather than the canonical STAT3 DBD, is responsible for AGG-element binding. The HIES mutation markedly enhances STAT3 AGG-element binding and AGG-promoter activation activity. Thus, STAT3 is a dual specificity transcription factor that promotes gene expression not only via SIE- but also AGG-promoter activity. PMID:26384563

  20. Retinitis Pigmentosa Mutations in Bad Response to Refrigeration 2 (Brr2) Impair ATPase and Helicase Activity.

    PubMed

    Ledoux, Sarah; Guthrie, Christine

    2016-06-01

    Brr2 is an RNA-dependent ATPase required to unwind the U4/U6 snRNA duplex during spliceosome assembly. Mutations within the ratchet helix of the Brr2 RNA binding channel result in a form of degenerative human blindness known as retinitis pigmentosa (RP). The biochemical consequences of these mutations on Brr2's RNA binding, helicase, and ATPase activity have not yet been characterized. Therefore, we identified the largest construct of Brr2 that is soluble in vitro, which truncates the first 247 amino acids of the N terminus (Δ247-Brr2), to characterize the effects of the RP mutations on Brr2 activity. The Δ247-Brr2 RP mutants exhibit a gradient of severity of weakened RNA binding, reduced helicase activity, and reduced ATPase activity compared with wild type Δ247-Brr2. The globular C-terminal Jab1/Mpn1-like domain of Prp8 increases the ability of Δ247-Brr2 to bind the U4/U6 snRNA duplex at high pH and increases Δ247-Brr2's RNA-dependent ATPase activity and the extent of RNA unwinding. However, this domain of Prp8 does not differentially affect the Δ247-Brr2 RP mutants compared with the wild type Δ247-Brr2. When stimulated by Prp8, wild type Δ247-Brr2 is able to unwind long stable duplexes in vitro, and even the RP mutants capable of binding RNA with tight affinity are incapable of fully unwinding short duplex RNAs. Our data suggest that the RP mutations within the ratchet helix impair Brr2 translocation through RNA helices. PMID:27072132

  1. The Splicing Efficiency of Activating HRAS Mutations Can Determine Costello Syndrome Phenotype and Frequency in Cancer.

    PubMed

    Hartung, Anne-Mette; Swensen, Jeff; Uriz, Inaki E; Lapin, Morten; Kristjansdottir, Karen; Petersen, Ulrika S S; Bang, Jeanne Mari V; Guerra, Barbara; Andersen, Henriette Skovgaard; Dobrowolski, Steven F; Carey, John C; Yu, Ping; Vaughn, Cecily; Calhoun, Amy; Larsen, Martin R; Dyrskjøt, Lars; Stevenson, David A; Andresen, Brage S

    2016-05-01

    Costello syndrome (CS) may be caused by activating mutations in codon 12/13 of the HRAS proto-oncogene. HRAS p.Gly12Val mutations have the highest transforming activity, are very frequent in cancers, but very rare in CS, where they are reported to cause a severe, early lethal, phenotype. We identified an unusual, new germline p.Gly12Val mutation, c.35_36GC>TG, in a 12-year-old boy with attenuated CS. Analysis of his HRAS cDNA showed high levels of exon 2 skipping. Using wild type and mutant HRAS minigenes, we confirmed that c.35_36GC>TG results in exon 2 skipping by simultaneously disrupting the function of a critical Exonic Splicing Enhancer (ESE) and creation of an Exonic Splicing Silencer (ESS). We show that this vulnerability of HRAS exon 2 is caused by a weak 3' splice site, which makes exon 2 inclusion dependent on binding of splicing stimulatory proteins, like SRSF2, to the critical ESE. Because the majority of cancer- and CS- causing mutations are located here, they affect splicing differently. Therefore, our results also demonstrate that the phenotype in CS and somatic cancers is not only determined by the different transforming potentials of mutant HRAS proteins, but also by the efficiency of exon 2 inclusion resulting from the different HRAS mutations. Finally, we show that a splice switching oligonucleotide (SSO) that blocks access to the critical ESE causes exon 2 skipping and halts proliferation of cancer cells. This unravels a potential for development of new anti-cancer therapies based on SSO-mediated HRAS exon 2 skipping.

  2. The Splicing Efficiency of Activating HRAS Mutations Can Determine Costello Syndrome Phenotype and Frequency in Cancer

    PubMed Central

    Kristjansdottir, Karen; Petersen, Ulrika S. S.; Bang, Jeanne Mari V.; Guerra, Barbara; Andersen, Henriette Skovgaard; Dobrowolski, Steven F.; Carey, John C.; Yu, Ping; Calhoun, Amy; Larsen, Martin R.; Dyrskjøt, Lars; Stevenson, David A.; Andresen, Brage S.

    2016-01-01

    Costello syndrome (CS) may be caused by activating mutations in codon 12/13 of the HRAS proto-oncogene. HRAS p.Gly12Val mutations have the highest transforming activity, are very frequent in cancers, but very rare in CS, where they are reported to cause a severe, early lethal, phenotype. We identified an unusual, new germline p.Gly12Val mutation, c.35_36GC>TG, in a 12-year-old boy with attenuated CS. Analysis of his HRAS cDNA showed high levels of exon 2 skipping. Using wild type and mutant HRAS minigenes, we confirmed that c.35_36GC>TG results in exon 2 skipping by simultaneously disrupting the function of a critical Exonic Splicing Enhancer (ESE) and creation of an Exonic Splicing Silencer (ESS). We show that this vulnerability of HRAS exon 2 is caused by a weak 3’ splice site, which makes exon 2 inclusion dependent on binding of splicing stimulatory proteins, like SRSF2, to the critical ESE. Because the majority of cancer- and CS- causing mutations are located here, they affect splicing differently. Therefore, our results also demonstrate that the phenotype in CS and somatic cancers is not only determined by the different transforming potentials of mutant HRAS proteins, but also by the efficiency of exon 2 inclusion resulting from the different HRAS mutations. Finally, we show that a splice switching oligonucleotide (SSO) that blocks access to the critical ESE causes exon 2 skipping and halts proliferation of cancer cells. This unravels a potential for development of new anti-cancer therapies based on SSO-mediated HRAS exon 2 skipping. PMID:27195699

  3. Impact of cofactor-binding loop mutations on thermotolerance and activity of E. coli transketolase.

    PubMed

    Morris, P; Rios-Solis, L; García-Arrazola, R; Lye, G J; Dalby, P A

    2016-07-01

    Improvement of thermostability in engineered enzymes can allow biocatalysis on substrates with poor aqueous solubility. Denaturation of the cofactor-binding loops of Escherichia coli transketolase (TK) was previously linked to the loss of enzyme activity under conditions of high pH or urea. Incubation at temperatures just below the thermal melting transition, above which the protein aggregates, was also found to anneal the enzyme to give an increased specific activity. The potential role of cofactor-binding loop instability in this process remained unclear. In this work, the two cofactor-binding loops (residues 185-192 and 382-392) were progressively mutated towards the equivalent sequence from the thermostable Thermus thermophilus TK and variants assessed for their impact on both thermostability and activity. Cofactor-binding loop 2 variants had detrimental effects on specific activity at elevated temperatures, whereas the H192P mutation in cofactor-binding loop 1 resulted in a two-fold improved stability to inactivation at elevated temperatures, and increased the critical onset temperature for aggregation. The specific activity of H192P was 3-fold and 19-fold higher than that for wild-type at 60°C and 65°C respectively, and also remained 2.7-4 fold higher after re-cooling from pre-incubations at either 55°C or 60°C for 1h. Interestingly, H192P was also 2-times more active than wild-type TK at 25°C. Optimal activity was achieved at 60°C for H192P compared to 55°C for wild type. These results show that cofactor-binding loop 1, plays a pivotal role in partial denaturation and aggregation at elevated temperatures. Furthermore, a single rigidifying mutation within this loop can significantly improve the enzyme specific activity, as well as the stability to thermal denaturation and aggregation, to give an increased temperature optimum for activity.

  4. Mutational analysis of the active center of plant fructosyltransferases: Festuca 1-SST and barley 6-SFT.

    PubMed

    Altenbach, Denise; Nüesch, Eveline; Ritsema, Tita; Boller, Thomas; Wiemken, Andres

    2005-08-29

    The active center of the glycoside hydrolase family 32 contains the three characteristic motifs (N/S)DPNG, RDP, and EC. We replaced the N-terminal region including the (N/S)DPNG motif of barley 6-SFT (sucrose:fructan 6-fructosyltransferase) by the corresponding region of Festuca 1-SST (sucrose:sucrose 1-fructosyltransferase). The chimeric enzyme, expressed in Pichia, retained the specificity of 6-SFT. Attempts to replace a larger piece at the N-terminus including also the RDP motif failed. A point mutation introduced in the RDP motif of 1-SST abolished enzymatic activity. Interestingly, point mutations of the EC-motif resulted in an enzyme which had lost the capability to form 1-kestose and glucose from sucrose but still accepted 1-kestose, producing fructose and sucrose as well as nystose.

  5. A novel SCARB2 mutation in progressive myoclonus epilepsy indicated by reduced β-glucocerebrosidase activity.

    PubMed

    Zeigler, Marsha; Meiner, Vardiella; Newman, J P; Steiner-Birmanns, Bettina; Bargal, Ruth; Sury, Vivi; Mengistu, Getu; Kakhlon, Or; Leykin, Ina; Argov, Zohar; Abramsky, Oded; Lossos, Alexander

    2014-04-15

    Action myoclonus renal failure (AMRF) syndrome is a rare form of progressive myoclonus epilepsy with renal dysfunction related to mutations in the SCARB2 gene. This gene is involved in lysosomal mannose-6-phosphate-independent trafficking of β-glucocerebrosidase (GC), an enzyme deficient in Gaucher disease. We report a family with myoclonic epilepsy, ataxia and skeletal muscle atrophy but without cognitive impairment or overt renal disease. A novel SCARB2 mutation was indicated by a striking discrepancy between lymphocyte and fibroblast GC activity in the proband evaluated for possible Gaucher disease. Our findings expand the genetic and phenotypic diversity of AMRF and suggest that low GC activity may present an important biochemical clue to the diagnosis of AMRF.

  6. Progranulin Mutations Affects Brain Oscillatory Activity in Fronto-Temporal Dementia

    PubMed Central

    Moretti, Davide V.; Benussi, Luisa; Fostinelli, Silvia; Ciani, Miriam; Binetti, Giuliano; Ghidoni, Roberta

    2016-01-01

    Background: Mild cognitive impairment (MCI) is a clinical stage indicating a prodromal phase of dementia. This practical concept could be used also for fronto-temporal dementia (FTD). Progranulin (PGRN) has been recently recognized as a useful diagnostic biomarker for fronto-temporal lobe degeneration (FTLD) due to GRN null mutations. Electroencephalography (EEG) is a reliable tool in detecting brain networks changes. The working hypothesis of the present study is that EEG oscillations could detect different modifications among FTLD stages (FTD-MCI versus overt FTD) as well as differences between GRN mutation carriers versus non-carriers in patients with overt FTD. Materials and Methods: EEG in all patients and PGRN dosage in patients with a clear FTD were detected. The cognitive state has been investigated through mini mental state examination (MMSE). Results: MCI-FTD showed a significant lower spectral power in both alpha and theta oscillations as compared to overt FTD. GRN mutations carriers affected by FTLD show an increase in high alpha and decrease in theta oscillations as compared to non-carriers. Conclusion: EEG frequency rhythms are sensible to different stage of FTD and could detect changes in brain oscillatory activity affected by GRN mutations. PMID:26973510

  7. Somatic Activating Mutations in GNAQ and GNA11 Are Associated with Congenital Hemangioma.

    PubMed

    Ayturk, Ugur M; Couto, Javier A; Hann, Steven; Mulliken, John B; Williams, Kaitlin L; Huang, August Yue; Fishman, Steven J; Boyd, Theonia K; Kozakewich, Harry P W; Bischoff, Joyce; Greene, Arin K; Warman, Matthew L

    2016-04-01

    Congenital hemangioma is a rare vascular tumor that forms in utero. Postnatally, the tumor either involutes quickly (i.e., rapidly involuting congenital hemangioma [RICH]) or partially regresses and stabilizes (i.e., non-involuting congenital hemangioma [NICH]). We hypothesized that congenital hemangiomas arise due to somatic mutation and performed massively parallel mRNA sequencing on affected tissue from eight participants. We identified mutually exclusive, mosaic missense mutations that alter glutamine at amino acid 209 (Glu209) in GNAQ or GNA11 in all tested samples, at variant allele frequencies (VAF) ranging from 3% to 33%. We verified the presence of the mutations in genomic DNA using a combination of molecular inversion probe sequencing (MIP-seq) and digital droplet PCR (ddPCR). The Glu209 GNAQ and GNA11 missense variants we identified are common in uveal melanoma and have been shown to constitutively activate MAPK and/or YAP signaling. When we screened additional archival formalin-fixed paraffin-embedded (FFPE) congenital cutaneous and hepatic hemangiomas, 4/8 had GNAQ or GNA11 Glu209 variants. The same GNAQ or GNA11 mutation is found in both NICH and RICH, so other factors must account for these tumors' different postnatal behaviors.

  8. Elastase Activity in Aspergillus fumigatus Can Arise by Random, Spontaneous Mutations

    PubMed Central

    Álvarez-Pérez, Sergio; Blanco, Jose L.; López-Rodas, Victoria; Flores-Moya, Antonio; Costas, Eduardo; García, Marta E.

    2010-01-01

    Aspergillus fumigatus Fresenius has the capacity to degrade elastin (the principal protein of the lungs) and it is considered that elastase activity (EA) is among the most important pathogenicity factors of this mold. In particular, there is a strong correlation between EA in A. fumigatus and invasive aspergillosis. However, EA is not universal in this mold, and it is unknown whether the capacity to degrade elastin is the consequence of physiological mechanisms and/or genetic changes (putative adaptive mutations) induced after the exposure to this substrate or, on the contrary, it is due to random spontaneous mutations that occur under nonselective conditions. In order to discriminate between these possibilities, a Luria-Delbrück fluctuation analysis was carried out on an elastase-negative (EA−) A. fumigatus strain, using as selective factor a culture medium containing elastin as the sole source of nitrogen. Here we show that the EA− → EA+ transformation in A. fumigatus appears by rare, random mutations before the exposure of the strain to selective conditions. This work represents the first experimental evidence of pathogenicity factor acquisition in mycelial fungi by preselective mutation. PMID:21350652

  9. Somatic Activating Mutations in GNAQ and GNA11 Are Associated with Congenital Hemangioma

    PubMed Central

    Ayturk, Ugur M.; Couto, Javier A.; Hann, Steven; Mulliken, John B.; Williams, Kaitlin L.; Huang, August Yue; Fishman, Steven J.; Boyd, Theonia K.; Kozakewich, Harry P.W.; Bischoff, Joyce; Greene, Arin K.; Warman, Matthew L.

    2016-01-01

    Congenital hemangioma is a rare vascular tumor that forms in utero. Postnatally, the tumor either involutes quickly (i.e., rapidly involuting congenital hemangioma [RICH]) or partially regresses and stabilizes (i.e., non-involuting congenital hemangioma [NICH]). We hypothesized that congenital hemangiomas arise due to somatic mutation and performed massively parallel mRNA sequencing on affected tissue from eight participants. We identified mutually exclusive, mosaic missense mutations that alter glutamine at amino acid 209 (Glu209) in GNAQ or GNA11 in all tested samples, at variant allele frequencies (VAF) ranging from 3% to 33%. We verified the presence of the mutations in genomic DNA using a combination of molecular inversion probe sequencing (MIP-seq) and digital droplet PCR (ddPCR). The Glu209 GNAQ and GNA11 missense variants we identified are common in uveal melanoma and have been shown to constitutively activate MAPK and/or YAP signaling. When we screened additional archival formalin-fixed paraffin-embedded (FFPE) congenital cutaneous and hepatic hemangiomas, 4/8 had GNAQ or GNA11 Glu209 variants. The same GNAQ or GNA11 mutation is found in both NICH and RICH, so other factors must account for these tumors’ different postnatal behaviors. PMID:27058448

  10. Novel Mutations in the Transcriptional Activator Domain of the Human TBX20 in Patients with Atrial Septal Defect

    PubMed Central

    Monroy-Muñoz, Irma Eloisa; Rodríguez-Pérez, José Manuel; Muñoz-Medina, José Esteban; Angeles-Martínez, Javier; García-Trejo, José J.; Morales-Ríos, Edgar; Massó, Felipe; Sandoval-Jones, Juan Pablo; Cervantes-Salazar, Jorge; García-Montes, José Antonio; Calderón-Colmenero, Juan; Vargas-Alarcón, Gilberto

    2015-01-01

    Background. The relevance of TBX20 gene in heart development has been demonstrated in many animal models, but there are few works that try to elucidate the effect of TBX20 mutations in human congenital heart diseases. In these studies, all missense mutations associated with atrial septal defect (ASD) were found in the DNA-binding T-box domain, none in the transcriptional activator domain. Methods. We search for TBX20 mutations in a group of patients with ASD or ventricular septal defect (VSD) using the High Resolution Melting (HRM) method and DNA sequencing. Results. We report three missense mutations (Y309D, T370O, and M395R) within the transcriptional activator domain of human TBX20 that were associated with ASD. Conclusions. This is the first association of TBX20 transcriptional activator domain missense mutations with ASD. These findings could have implications for diagnosis, genetic screening, and patient follow-up. PMID:25834824

  11. Activating JAK1 mutation may predict the sensitivity of JAK-STAT inhibition in hepatocellular carcinoma.

    PubMed

    Yang, Shuqun; Luo, Chonglin; Gu, Qingyang; Xu, Qiang; Wang, Guan; Sun, Hongye; Qian, Ziliang; Tan, Yexiong; Qin, Yuxin; Shen, Yuhong; Xu, Xiaowei; Chen, Shu-Hui; Chan, Chi-Chung; Wang, Hongyang; Mao, Mao; Fang, Douglas D

    2016-02-01

    Hepatocellular carcinoma (HCC) is the fifth most common type of cancers worldwide. However, current therapeutic approaches for this epidemic disease are limited, and its 5-year survival rate hasn't been improved in the past decades. Patient-derived xenograft (PDX) tumor models have become an excellent in vivo system for understanding of disease biology and drug discovery. In order to identify new therapeutic targets for HCC, whole-exome sequencing (WES) was performed on more than 60 HCC PDX models. Among them, four models exhibited protein-altering mutations in JAK1 (Janus Kinase 1) gene. To explore the transforming capability, these mutations were then introduced into HEK293FT and Ba/F3 cells. The results demonstrated that JAK1S703I mutation was able to activate JAK-STAT (Signal Transducer and Activator of Transcription) signaling pathway and drive cell proliferation in the absence of cytokine stimulation in vitro. Furthermore,the sensitivity to the treatment of a JAK1/2 inhibitor, ruxolitinib, was observed in JAK1S703I mutant PDX model, but not in other non-activating mutant or wild type models. Pharmacodynamic analysis showed that phosphorylation of STAT3 in the Ruxolitinib-treated tumor tissues was significantly suppressed. Collectively, our results suggested that JAK1S703I is an activating mutation for JAK-STAT signaling pathway in vitro and in vivo, and JAK-STAT pathway might represent a new therapeutic approach for HCC treatment. Monotherapy using a more potent and specific JAK1 inhibitor and combinatory therapy should be further explored in JAK1 mutant PDX models. PMID:26701727

  12. Structural Characterization of Human 8-Oxoguanine DNA Glycosylase Variants Bearing Active Site Mutations

    SciTech Connect

    Radom,C.; Banerjee, A.; Verdine, G.

    2007-01-01

    The human 8-oxoguanine DNA glycosylase (hOGG1) protein is responsible for initiating base excision DNA repair of the endogenous mutagen 8-oxoguanine. Like nearly all DNA glycosylases, hOGG1 extrudes its substrate from the DNA helix and inserts it into an extrahelical enzyme active site pocket lined with residues that participate in lesion recognition and catalysis. Structural analysis has been performed on mutant versions of hOGG1 having changes in catalytic residues but not on variants having altered 7,8-dihydro-8-oxoguanine (oxoG) contact residues. Here we report high resolution structural analysis of such recognition variants. We found that Ala substitution at residues that contact the phosphate 5 to the lesion (H270A mutation) and its Watson-Crick face (Q315A mutation) simply removed key functionality from the contact interface but otherwise had no effect on structure. Ala substitution at the only residue making an oxoG-specific contact (G42A mutation) introduced torsional stress into the DNA contact surface of hOGG1, but this was overcome by local interactions within the folded protein, indicating that this oxoG recognition motif is 'hardwired'. Introduction of a side chain intended to sterically obstruct the active site pocket (Q315F mutation) led to two different structures, one of which (Q315F{sup *149}) has the oxoG lesion in an exosite flanking the active site and the other of which (Q315F{sup *292}) has the oxoG inserted nearly completely into the lesion recognition pocket. The latter structure offers a view of the latest stage in the base extrusion pathway yet observed, and its lack of catalytic activity demonstrates that the transition state for displacement of the lesion base is geometrically demanding.

  13. Activating JAK1 mutation may predict the sensitivity of JAK-STAT inhibition in hepatocellular carcinoma.

    PubMed

    Yang, Shuqun; Luo, Chonglin; Gu, Qingyang; Xu, Qiang; Wang, Guan; Sun, Hongye; Qian, Ziliang; Tan, Yexiong; Qin, Yuxin; Shen, Yuhong; Xu, Xiaowei; Chen, Shu-Hui; Chan, Chi-Chung; Wang, Hongyang; Mao, Mao; Fang, Douglas D

    2016-02-01

    Hepatocellular carcinoma (HCC) is the fifth most common type of cancers worldwide. However, current therapeutic approaches for this epidemic disease are limited, and its 5-year survival rate hasn't been improved in the past decades. Patient-derived xenograft (PDX) tumor models have become an excellent in vivo system for understanding of disease biology and drug discovery. In order to identify new therapeutic targets for HCC, whole-exome sequencing (WES) was performed on more than 60 HCC PDX models. Among them, four models exhibited protein-altering mutations in JAK1 (Janus Kinase 1) gene. To explore the transforming capability, these mutations were then introduced into HEK293FT and Ba/F3 cells. The results demonstrated that JAK1S703I mutation was able to activate JAK-STAT (Signal Transducer and Activator of Transcription) signaling pathway and drive cell proliferation in the absence of cytokine stimulation in vitro. Furthermore,the sensitivity to the treatment of a JAK1/2 inhibitor, ruxolitinib, was observed in JAK1S703I mutant PDX model, but not in other non-activating mutant or wild type models. Pharmacodynamic analysis showed that phosphorylation of STAT3 in the Ruxolitinib-treated tumor tissues was significantly suppressed. Collectively, our results suggested that JAK1S703I is an activating mutation for JAK-STAT signaling pathway in vitro and in vivo, and JAK-STAT pathway might represent a new therapeutic approach for HCC treatment. Monotherapy using a more potent and specific JAK1 inhibitor and combinatory therapy should be further explored in JAK1 mutant PDX models.

  14. Activating JAK1 mutation may predict the sensitivity of JAK-STAT inhibition in hepatocellular carcinoma

    PubMed Central

    Yang, Shuqun; Luo, Chonglin; Gu, Qingyang; Xu, Qiang; Wang, Guan; Sun, Hongye; Qian, Ziliang; Tan, Yexiong; Qin, Yuxin; Shen, Yuhong; Xu, Xiaowei; Chen, Shu-Hui; Chan, Chi-Chung; Wang, Hongyang; Mao, Mao; Fang, Douglas D.

    2016-01-01

    Hepatocellular carcinoma (HCC) is the fifth most common type of cancers worldwide. However, current therapeutic approaches for this epidemic disease are limited, and its 5-year survival rate hasn't been improved in the past decades. Patient-derived xenograft (PDX) tumor models have become an excellent in vivo system for understanding of disease biology and drug discovery. In order to identify new therapeutic targets for HCC, whole-exome sequencing (WES) was performed on more than 60 HCC PDX models. Among them, four models exhibited protein-altering mutations in JAK1 (Janus Kinase 1) gene. To explore the transforming capability, these mutations were then introduced into HEK293FT and Ba/F3 cells. The results demonstrated that JAK1S703I mutation was able to activate JAK-STAT (Signal Transducer and Activator of Transcription) signaling pathway and drive cell proliferation in the absence of cytokine stimulation in vitro. Furthermore, the sensitivity to the treatment of a JAK1/2 inhibitor, ruxolitinib, was observed in JAK1S703I mutant PDX model, but not in other non-activating mutant or wild type models. Pharmacodynamic analysis showed that phosphorylation of STAT3 in the Ruxolitinib-treated tumor tissues was significantly suppressed. Collectively, our results suggested that JAK1S703I is an activating mutation for JAK-STAT signaling pathway in vitro and in vivo, and JAK-STAT pathway might represent a new therapeutic approach for HCC treatment. Monotherapy using a more potent and specific JAK1 inhibitor and combinatory therapy should be further explored in JAK1 mutant PDX models. PMID:26701727

  15. Binding of AID to DNA does not correlate with mutator activity.

    PubMed

    Matthews, Allysia J; Husain, Solomon; Chaudhuri, Jayanta

    2014-07-01

    The DNA deaminase activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) by deaminating cytidines to uridines at V region (V) genes and switch (S) regions. The mechanism by which AID is recruited to V genes and S region DNA is poorly understood. In this study, we used the CH12 B lymphoma line to demonstrate that, although S regions can efficiently recruit AID and undergo mutations and deletions, AID neither binds to nor mutates the V gene, thus clearly demonstrating intraimmunoglobulin locus specificity. Depletion of the RNA-binding protein polypyrimidine tract binding protein-2, previously shown to promote recruitment of AID to S regions, enables stable association of AID with the V gene. Surprisingly, AID binding to the V gene does not induce SHM. These results unmask a striking lack of correlation between AID binding and its mutator activity, providing evidence for the presence of factors required downstream of AID binding to effect SHM. Furthermore, our findings suggest that S regions are preferred targets for AID and, aided by polypyrimidine tract binding protein-2, act as "sinks" to sequester AID activity from other genomic regions.

  16. Nuclear Localization of the Autism Candidate Gene Neurobeachin and Functional Interaction with the NOTCH1 Intracellular Domain Indicate a Role in Regulating Transcription

    PubMed Central

    Tuand, Krizia; Stijnen, Pieter; Volders, Karolien; Declercq, Jeroen; Nuytens, Kim; Meulemans, Sandra; Creemers, John

    2016-01-01

    Background Neurobeachin (NBEA) is an autism spectrum disorders (ASD) candidate gene. NBEA deficiency affects regulated secretion, receptor trafficking, synaptic architecture and protein kinase A (PKA)-mediated phosphorylation. NBEA is a large multidomain scaffolding protein. From N- to C-terminus, NBEA has a concanavalin A-like lectin domain flanked by armadillo repeats (ACA), an A-kinase anchoring protein domain that can bind to PKA, a domain of unknown function (DUF1088) and a BEACH domain, preceded by a pleckstrin homology-like domain and followed by WD40 repeats (PBW). Although most of these domains mediate protein-protein interactions, no interaction screen has yet been performed. Methods Yeast two-hybrid screens with the ACA and PBW domain modules of NBEA gave a list of interaction partners, which were analyzed for Gene Ontology (GO) enrichment. Neuro-2a cells were used for confocal microscopy and nuclear extraction analysis. NOTCH-mediated transcription was studied with luciferase reporter assays and qRT-PCR, combined with NBEA knockdown or overexpression. Results Both domain modules showed a GO enrichment for the nucleus. PBW almost exclusively interacted with transcription regulators, while ACA interacted with a number of PKA substrates. NBEA was partially localized in the nucleus of Neuro-2a cells, albeit much less than in the cytoplasm. A nuclear localization signal was found in the DUF1088 domain, which was shown to contribute to the nuclear localization of an EGFP-DPBW fusion protein. Yeast two-hybrid identified the Notch1 intracellular domain as a physical interactor of the PBW domain and a role for NBEA as a negative regulator in Notch-mediated transcription was demonstrated. Conclusion Defining novel interaction partners of conserved NBEA domain modules identified a role for NBEA as transcriptional regulator in the nucleus. The physical interaction of NBEA with NOTCH1 is most relevant for ASD pathogenesis because NOTCH signaling is essential for

  17. Mutation Analysis of the LH Receptor Gene in Leydig Cell Adenoma and Hyperplasia and Functional and Biochemical Studies of Activating Mutations of the LH Receptor Gene

    PubMed Central

    Lumbroso, Serge; Verhoef-Post, Miriam; Richter-Unruh, Annette; Looijenga, Leendert H. J.; Funaro, Ada; Beishuizen, Auke; van Marle, André; Drop, Stenvert L. S.; Themmen, Axel P. N.

    2011-01-01

    Context: Germline and somatic activating mutations in the LH receptor (LHR) gene have been reported. Objective: Our objective was to perform mutation analysis of the LHR gene of patients with Leydig cell adenoma or hyperplasia. Functional studies were conducted to compare the D578H-LHR mutant with the wild-type (WT)-LHR and the D578G-LHR mutant, a classic cause of testotoxicosis. The three main signal transduction pathways in which LHR is involved were studied. Patients: We describe eight male patients with gonadotropin-independent precocious puberty due to Leydig cell adenoma or hyperplasia. Results: The D578H-LHR mutation was found in the adenoma or nodule with hyperplasia in all but two patients. D578H-LHR displayed a constitutively increased but noninducible production of cAMP, led to a very high production of inositol phosphates, and induced a slight phosphorylation of p44/42 MAPK in the absence of human chorionic gonadotropin. The D578G-LHR showed a response intermediate between WT-LHR and the D578H-LHR. Subcellular localization studies showed that the WT-LHR was almost exclusively located at the cell membrane, whereas the D578H-LHR showed signs of internalization. D578H-LHR was the only receptor to colocalize with early endosomes in the absence of human chorionic gonadotropin. Conclusions: Although several LHR mutations have been reported in testotoxicosis, the D578H-LHR mutation, which has been found only as a somatic mutation, appears up until now to be specifically responsible for Leydig cell adenomas. This is reflected by the different activation of the signal transduction pathways, when compared with the WT-LHR or D578G-LHR, which may explain the tumorigenesis in the D578H mutant. PMID:21490077

  18. Mutational analysis of Agrobacterium tumefaciens virD2: tyrosine 29 is essential for endonuclease activity.

    PubMed Central

    Vogel, A M; Das, A

    1992-01-01

    Agrobacterium tumefaciens VirD2 polypeptide, in the presence of VirD1, catalyzes a site- and strand-specific nicking reaction at the T-DNA border sequences. VirD2 is found tightly attached to the 5' end of the nicked DNA. The protein-DNA complex is presumably formed via a tyrosine residue of VirD2 (F. Durrenberger, A. Crameri, B. Hohn, and Z. Koukolikova-Nicola, Proc. Natl. Acad. Sci. USA 86:9154-9158, 1989). A mutational approach was used to study whether a tyrosine residue(s) of VirD2 is required for its activity. By site-specific mutagenesis, a tyrosine (Y) residue at position 29, 68, 99, 119, 121, 160, or 195 of the octopine Ti plasmid pTiA6 VirD2 was altered to phenylalanine (F). The Y-29-F or Y-121-F mutation completely abolished nicking activity of VirD2 in vivo in Escherichia coli. Two other substitutions, Y-68-F and Y-160-F, drastically reduced VirD2 activity. A substitution at position 99, 119, or 195 had no effect on VirD2 activity. Additional mutagenesis experiments showed that at position 29, no other amino acid could substitute for tyrosine without destroying VirD2 activity. At position 121, only a tryptophan (W) residue could be substituted. This, however, yielded a mutant protein with significantly reduced VirD2 activity. The nicked DNA from strains bearing a Y-68-F, Y-99-F, Y-119-F, Y-160-F, Y-195-F, or Y-121-W mutation in VirD2 was always found to contain a tightly linked protein. Images PMID:1309520

  19. Effect of lysine to alanine mutations on the phosphate activation and BPTES inhibition of glutaminase.

    PubMed

    McDonald, Charles J; Acheff, Eric; Kennedy, Ryan; Taylor, Lynn; Curthoys, Norman P

    2015-09-01

    The GLS1 gene encodes a mitochondrial glutaminase that is highly expressed in brain, kidney, small intestine and many transformed cells. Recent studies have identified multiple lysine residues in glutaminase that are sites of N-acetylation. Interestingly, these sites are located within either a loop segment that regulates access of glutamine to the active site or the dimer:dimer interface that participates in the phosphate-dependent oligomerization and activation of the enzyme. These two segments also contain the binding sites for bis-2[5-phenylacetamido-1,2,4-thiadiazol-2-yl]ethylsulfide (BPTES), a highly specific and potent uncompetitive inhibitor of this glutaminase. BPTES is also the lead compound for development of novel cancer chemotherapeutic agents. To provide a preliminary assessment of the potential effects of N-acetylation, the corresponding lysine to alanine mutations were constructed in the hGACΔ1 plasmid. The wild type and mutated proteins were purified by Ni(+)-affinity chromatography and their phosphate activation and BPTES inhibition profiles were analyzed. Two of the alanine substitutions in the loop segment (K311A and K328A) and the one in the dimer:dimer interface (K396A) form enzymes that require greater concentrations of phosphate to produce half-maximal activation and exhibit greater sensitivity to BPTES inhibition. By contrast, the K320A mutation results in a glutaminase that exhibits near maximal activity in the absence of phosphate and is not inhibited by BPTES. Thus, lysine N-acetylation may contribute to the acute regulation of glutaminase activity in various tissues and alter the efficacy of BPTES-type inhibitors.

  20. Germline NLRP1 Mutations Cause Skin Inflammatory and Cancer Susceptibility Syndromes via Inflammasome Activation.

    PubMed

    Zhong, Franklin L; Mamaï, Ons; Sborgi, Lorenzo; Boussofara, Lobna; Hopkins, Richard; Robinson, Kim; Szeverényi, Ildikó; Takeichi, Takuya; Balaji, Reshmaa; Lau, Aristotle; Tye, Hazel; Roy, Keya; Bonnard, Carine; Ahl, Patricia J; Jones, Leigh Ann; Baker, Paul; Lacina, Lukas; Otsuka, Atsushi; Fournie, Pierre R; Malecaze, François; Lane, E Birgitte; Akiyama, Masashi; Kabashima, Kenji; Connolly, John E; Masters, Seth L; Soler, Vincent J; Omar, Salma Samir; McGrath, John A; Nedelcu, Roxana; Gribaa, Moez; Denguezli, Mohamed; Saad, Ali; Hiller, Sebastian; Reversade, Bruno

    2016-09-22

    Inflammasome complexes function as key innate immune effectors that trigger inflammation in response to pathogen- and danger-associated signals. Here, we report that germline mutations in the inflammasome sensor NLRP1 cause two overlapping skin disorders: multiple self-healing palmoplantar carcinoma (MSPC) and familial keratosis lichenoides chronica (FKLC). We find that NLRP1 is the most prominent inflammasome sensor in human skin, and all pathogenic NLRP1 mutations are gain-of-function alleles that predispose to inflammasome activation. Mechanistically, NLRP1 mutations lead to increased self-oligomerization by disrupting the PYD and LRR domains, which are essential in maintaining NLRP1 as an inactive monomer. Primary keratinocytes from patients experience spontaneous inflammasome activation and paracrine IL-1 signaling, which is sufficient to cause skin inflammation and epidermal hyperplasia. Our findings establish a group of non-fever inflammasome disorders, uncover an unexpected auto-inhibitory function for the pyrin domain, and provide the first genetic evidence linking NLRP1 to skin inflammatory syndromes and skin cancer predisposition. PMID:27662089

  1. Target DNA sequence directly regulates the frequency of activation-induced deaminase-dependent mutations.

    PubMed

    Chen, Zhangguo; Viboolsittiseri, Sawanee S; O'Connor, Brian P; Wang, Jing H

    2012-10-15

    Activation-induced deaminase (AID) catalyses class switch recombination (CSR) and somatic hypermutation (SHM) in B lymphocytes to enhance Ab diversity. CSR involves breaking and rejoining highly repetitive switch (S) regions in the IgH (Igh) locus. S regions appear to be preferential targets of AID. To determine whether S region sequence per se, independent of Igh cis regulatory elements, can influence AID targeting efficiency and mutation frequency, we established a knock-in mouse model by inserting a core Sγ1 region into the first intron of proto-oncogene Bcl6, which is a non-Ig target of SHM. We found that the mutation frequency of the inserted Sγ1 region was dramatically higher than that of the adjacent Bcl6 endogenous sequence. Mechanistically, S region-enhanced SHM was associated with increased recruitment of AID and RNA polymerase II, together with Spt5, albeit to a lesser extent. Our studies demonstrate that target DNA sequences influence mutation frequency via regulating AID recruitment. We propose that the nucleotide sequence preference may serve as an additional layer of AID regulation by restricting its mutagenic activity to specific sequences despite the observation that AID has the potential to access the genome widely.

  2. Deep Intronic Mutation and Pseudo Exon Activation as a Novel Muscular Hypertrophy Modifier in Cattle

    PubMed Central

    Bouyer, Claire; Forestier, Lionel; Renand, Gilles; Oulmouden, Ahmad

    2014-01-01

    Myostatin is essential for proper regulation of myogenesis, and inactivation of Myostatin results in muscle hypertrophy. Here, we identified an unexpected mutation in the myostatin gene which is almost fixed in Blonde d'Aquitaine cattle. In skeletal muscle, the mutant allele was highly expressed leading to an abnormal transcript consisting of a 41-bp inclusion and premature termination codons and to residual levels of a correctly spliced transcript. This expression pattern, caused by a leaky intronic mutation with regard to spliceosome activity and its apparent stability with regard to surveillance mechanisms, could contribute to the moderate muscle hypertrophy in this cattle breed. This finding is of importance for genetic counseling for meat quantity and quality in livestock production and possibly to manipulate myostatin pre-mRNA in human muscle diseases. PMID:24827585

  3. A GPR54-activating mutation in a patient with central precocious puberty.

    PubMed

    Teles, Milena Gurgel; Bianco, Suzy D C; Brito, Vinicius Nahime; Trarbach, Ericka B; Kuohung, Wendy; Xu, Shuyun; Seminara, Stephanie B; Mendonca, Berenice B; Kaiser, Ursula B; Latronico, Ana Claudia

    2008-02-14

    Gonadotropin-dependent, or central, precocious puberty is caused by early maturation of the hypothalamic-pituitary-gonadal axis. In girls, this condition is most often idiopathic. Recently, a G protein-coupled receptor, GPR54, and its ligand, kisspeptin, were described as an excitatory neuroregulator system for the secretion of gonadotropin-releasing hormone (GnRH). In this study, we have identified an autosomal dominant GPR54 mutation--the substitution of proline for arginine at codon 386 (Arg386Pro)--in an adopted girl with idiopathic central precocious puberty (whose biologic family was not available for genetic studies). In vitro studies have shown that this mutation leads to prolonged activation of intracellular signaling pathways in response to kisspeptin. The Arg386Pro mutant appears to be associated with central precocious puberty.

  4. A comparison of ARMS and mutation specific IHC for common activating EGFR mutations analysis in small biopsy and cytology specimens of advanced non small cell lung cancer.

    PubMed

    Wang, Xueqing; Wang, Guoqing; Hao, Yueyue; Xu, Yinhong; Zhang, Lihua

    2014-01-01

    We have compared mutation analysis by Amplification Refractory Mutation System (ARMS) and epidermal growth factor receptor (EGFR) mutant-specific antibodies for their ability to detect two common activating EGFR mutations in a cohort of 115 advanced non-small cell lung cancer (NSCLC), including cytology material, core biopsy, and bronchoscopic biopsies. Assessment of EGFR mutation status was performed by using antibodies and ARMS assay specific to the two major forms of mutant EGFR, exon 19 deletion E746-A750 (c.2235_2249del15 or c.2236_2250del15, p. Glu746_Ala750 del) and exon 21 L858R point mutation (c.2573T>G, p.Leu858Arg). In this study the optimal buffer for antigen retrieval was sodium citrate (pH 6.0). Q score was used to evaluate the specific mutant EGFR proteins expression. Validation using clinical material showed deletions in exon 19 were detected in 19.1% and L858R mutation in 20% of all cases by ARMS assay. A cutoff value of score 1 was used as positive by IHC. No wild type cases were immuno-reactive. The antibodies performed well in cytology, core biopsies and bronchoscopic biopsies. There were only one false positive case using L858R IHC (sensitivity 100%, specificity 98.5%, positive predictive value 96%, negative predictive value 100%). All 23 E746-A750 exon 19 deletions identified by mutation analysis were positive by IHC. The sensitivity of exon 19 IHC for E746-A750 was 100%, specificity 100%, positive predictive value 100% and negative predictive value 100%. The result of the IHC stains was finely correlated with mutations status determined by ARMS assay. Although inferior to molecular genetic analysis of the EGFR gene, IHC is highly specific and sensitive for the targeted EGFR mutations. The antibodies are likely to be of clinical value in cases especially where limited tumor material is available, or in situations where molecular genetic analysis is not readily available.

  5. Myopathic lamin mutations cause reductive stress and activate the nrf2/keap-1 pathway.

    PubMed

    Dialynas, George; Shrestha, Om K; Ponce, Jessica M; Zwerger, Monika; Thiemann, Dylan A; Young, Grant H; Moore, Steven A; Yu, Liping; Lammerding, Jan; Wallrath, Lori L

    2015-05-01

    Mutations in the human LMNA gene cause muscular dystrophy by mechanisms that are incompletely understood. The LMNA gene encodes A-type lamins, intermediate filaments that form a network underlying the inner nuclear membrane, providing structural support for the nucleus and organizing the genome. To better understand the pathogenesis caused by mutant lamins, we performed a structural and functional analysis on LMNA missense mutations identified in muscular dystrophy patients. These mutations perturb the tertiary structure of the conserved A-type lamin Ig-fold domain. To identify the effects of these structural perturbations on lamin function, we modeled these mutations in Drosophila Lamin C and expressed the mutant lamins in muscle. We found that the structural perturbations had minimal dominant effects on nuclear stiffness, suggesting that the muscle pathology was not accompanied by major structural disruption of the peripheral nuclear lamina. However, subtle alterations in the lamina network and subnuclear reorganization of lamins remain possible. Affected muscles had cytoplasmic aggregation of lamins and additional nuclear envelope proteins. Transcription profiling revealed upregulation of many Nrf2 target genes. Nrf2 is normally sequestered in the cytoplasm by Keap-1. Under oxidative stress Nrf2 dissociates from Keap-1, translocates into the nucleus, and activates gene expression. Unexpectedly, biochemical analyses revealed high levels of reducing agents, indicative of reductive stress. The accumulation of cytoplasmic lamin aggregates correlated with elevated levels of the autophagy adaptor p62/SQSTM1, which also binds Keap-1, abrogating Nrf2 cytoplasmic sequestration, allowing Nrf2 nuclear translocation and target gene activation. Elevated p62/SQSTM1 and nuclear enrichment of Nrf2 were identified in muscle biopsies from the corresponding muscular dystrophy patients, validating the disease relevance of our Drosophila model. Thus, novel connections were made

  6. Myopathic Lamin Mutations Cause Reductive Stress and Activate the Nrf2/Keap-1 Pathway

    PubMed Central

    Dialynas, George; Shrestha, Om K.; Ponce, Jessica M.; Zwerger, Monika; Thiemann, Dylan A.; Young, Grant H.; Moore, Steven A.; Yu, Liping; Lammerding, Jan; Wallrath, Lori L.

    2015-01-01

    Mutations in the human LMNA gene cause muscular dystrophy by mechanisms that are incompletely understood. The LMNA gene encodes A-type lamins, intermediate filaments that form a network underlying the inner nuclear membrane, providing structural support for the nucleus and organizing the genome. To better understand the pathogenesis caused by mutant lamins, we performed a structural and functional analysis on LMNA missense mutations identified in muscular dystrophy patients. These mutations perturb the tertiary structure of the conserved A-type lamin Ig-fold domain. To identify the effects of these structural perturbations on lamin function, we modeled these mutations in Drosophila Lamin C and expressed the mutant lamins in muscle. We found that the structural perturbations had minimal dominant effects on nuclear stiffness, suggesting that the muscle pathology was not accompanied by major structural disruption of the peripheral nuclear lamina. However, subtle alterations in the lamina network and subnuclear reorganization of lamins remain possible. Affected muscles had cytoplasmic aggregation of lamins and additional nuclear envelope proteins. Transcription profiling revealed upregulation of many Nrf2 target genes. Nrf2 is normally sequestered in the cytoplasm by Keap-1. Under oxidative stress Nrf2 dissociates from Keap-1, translocates into the nucleus, and activates gene expression. Unexpectedly, biochemical analyses revealed high levels of reducing agents, indicative of reductive stress. The accumulation of cytoplasmic lamin aggregates correlated with elevated levels of the autophagy adaptor p62/SQSTM1, which also binds Keap-1, abrogating Nrf2 cytoplasmic sequestration, allowing Nrf2 nuclear translocation and target gene activation. Elevated p62/SQSTM1 and nuclear enrichment of Nrf2 were identified in muscle biopsies from the corresponding muscular dystrophy patients, validating the disease relevance of our Drosophila model. Thus, novel connections were made

  7. Identification of FGFR4-activating mutations in human rhabdomyosarcomas that promote metastasis in xenotransplanted models

    PubMed Central

    VI, James G. Taylor; Cheuk, Adam T.; Tsang, Patricia S.; Chung, Joon-Yong; Song, Young K.; Desai, Krupa; Yu, Yanlin; Chen, Qing-Rong; Shah, Kushal; Youngblood, Victoria; Fang, Jun; Kim, Su Young; Yeung, Choh; Helman, Lee J.; Mendoza, Arnulfo; Ngo, Vu; Staudt, Louis M.; Wei, Jun S.; Khanna, Chand; Catchpoole, Daniel; Qualman, Stephen J.; Hewitt, Stephen M.; Merlino, Glenn; Chanock, Stephen J.; Khan, Javed

    2009-01-01

    Rhabdomyosarcoma (RMS) is a childhood cancer originating from skeletal muscle, and patient survival is poor in the presence of metastatic disease. Few determinants that regulate metastasis development have been identified. The receptor tyrosine kinase FGFR4 is highly expressed in RMS tissue, suggesting a role in tumorigenesis, although its functional importance has not been defined. Here, we report the identification of mutations in FGFR4 in human RMS tumors that lead to its activation and present evidence that it functions as an oncogene in RMS. Higher FGFR4 expression in RMS tumors was associated with advanced-stage cancer and poor survival, while FGFR4 knockdown in a human RMS cell line reduced tumor growth and experimental lung metastases when the cells were transplanted into mice. Moreover, 6 FGFR4 tyrosine kinase domain mutations were found among 7 of 94 (7.5%) primary human RMS tumors. The mutants K535 and E550 increased autophosphorylation, Stat3 signaling, tumor proliferation, and metastatic potential when expressed in a murine RMS cell line. These mutants also transformed NIH 3T3 cells and led to an enhanced metastatic phenotype. Finally, murine RMS cell lines expressing the K535 and E550 FGFR4 mutants were substantially more susceptible to apoptosis in the presence of a pharmacologic FGFR inhibitor than the control cell lines expressing the empty vector or wild-type FGFR4. Together, our results demonstrate that mutationally activated FGFR4 acts as an oncogene, and these are what we believe to be the first known mutations in a receptor tyrosine kinase in RMS. These findings support the potential therapeutic targeting of FGFR4 in RMS. PMID:19809159

  8. Mutations in the diastrophic dysplasia sulfate transporter (DTDST) gene: correlation between sulfate transport activity and chondrodysplasia phenotype.

    PubMed

    Karniski, L P

    2001-07-01

    The diastrophic dysplasia sulfate transporter (DTDST) gene encodes a transmembrane protein that transports sulfate into chondrocytes to maintain adequate sulfation of proteoglycans. Mutations in this gene are responsible for four recessively inherited chondrodysplasias that include diastrophic dysplasia, multiple epiphyseal dysplasia, atelosteogenesis type 2 and achondrogenesis 1B (ACG-1B). To determine whether the DTDST mutations found in individuals with these chondrodysplasias differ functionally from each other, we compared the sulfate transport activity of 11 reported DTDST mutations. Five mutations, G255E, Delta a1751, L483P, R178X and N425D, had minimal sulfate transport function following expression in Xenopus laevis oocytes. Two mutations, Delta V340 and R279W, transported sulfate at rates of 17 and 32%, respectively, of wild-type DTDST. Four mutations, A715V, C653S, Q454P and G678V, had rates of sulfate transport nearly equal to that of wild-type DTDST. Transport kinetics were not different among the four mutations with near-normal sulfate transport function and wild-type DTDST. When the sulfate transport function of the different DTDST mutations are grouped according to the general phenotypes, individuals with the most severe form, ACG-1B, tend to be homozygous for null mutations, individuals with the moderately severe atelosteogenesis type 2 have at least one allele with a loss-of-function mutation, and individuals with the mildest forms are typically homozygous for mutations with residual sulfate transport function. However, in the X.laevis oocyte expression system, the correlation between residual transport function and the severity of phenotype was not absolute, suggesting that factors in addition to the intrinsic sulfate transport properties of the DTDST protein may influence the phenotype in individuals with DTDST mutations. PMID:11448940

  9. Disruption of dopamine neuron activity pattern regulation through selective expression of a human KCNN3 mutation.

    PubMed

    Soden, Marta E; Jones, Graham L; Sanford, Christina A; Chung, Amanda S; Güler, Ali D; Chavkin, Charles; Luján, Rafael; Zweifel, Larry S

    2013-11-20

    The calcium-activated small conductance potassium channel SK3 plays an essential role in the regulation of dopamine neuron activity patterns. Here we demonstrate that expression of a human disease-related SK3 mutation (hSK3Δ) in dopamine neurons of mice disrupts the balance between tonic and phasic dopamine neuron activity. Expression of hSK3Δ suppressed endogenous SK currents, reducing coupling between SK channels and NMDA receptors (NMDARs) and increasing permissiveness for burst firing. Consistent with enhanced excitability of dopamine neurons, hSK3Δ increased evoked calcium signals in dopamine neurons in vivo and potentiated evoked dopamine release. Specific expression of hSK3Δ led to deficits in attention and sensory gating and heightened sensitivity to a psychomimetic drug. Sensory-motor alterations and psychomimetic sensitivity were recapitulated in a mouse model of transient, reversible dopamine neuron activation. These results demonstrate the cell-autonomous effects of a human ion channel mutation on dopamine neuron physiology and the impact of activity pattern disruption on behavior. PMID:24206670

  10. Disruption of dopamine neuron activity pattern regulation through selective expression of a human KCNN3 mutation.

    PubMed

    Soden, Marta E; Jones, Graham L; Sanford, Christina A; Chung, Amanda S; Güler, Ali D; Chavkin, Charles; Luján, Rafael; Zweifel, Larry S

    2013-11-20

    The calcium-activated small conductance potassium channel SK3 plays an essential role in the regulation of dopamine neuron activity patterns. Here we demonstrate that expression of a human disease-related SK3 mutation (hSK3Δ) in dopamine neurons of mice disrupts the balance between tonic and phasic dopamine neuron activity. Expression of hSK3Δ suppressed endogenous SK currents, reducing coupling between SK channels and NMDA receptors (NMDARs) and increasing permissiveness for burst firing. Consistent with enhanced excitability of dopamine neurons, hSK3Δ increased evoked calcium signals in dopamine neurons in vivo and potentiated evoked dopamine release. Specific expression of hSK3Δ led to deficits in attention and sensory gating and heightened sensitivity to a psychomimetic drug. Sensory-motor alterations and psychomimetic sensitivity were recapitulated in a mouse model of transient, reversible dopamine neuron activation. These results demonstrate the cell-autonomous effects of a human ion channel mutation on dopamine neuron physiology and the impact of activity pattern disruption on behavior.

  11. Mutation analysis of PobR and PcaU, closely related transcriptional activators in acinetobacter.

    PubMed

    Kok, R G; D'Argenio, D A; Ornston, L N

    1998-10-01

    Acinetobacter PobR and PcaU are transcriptional activators that closely resemble each other in primary structure, DNA-binding sites, metabolic modulators, and physiological function. PobR responds to the inducer-metabolite p-hydroxybenzoate and activates transcription of pobA, the structural gene for the enzyme that converts p-hydroxybenzoate to protocatechuate. This compound, differing from p-hydroxybenzoate only in that it contains an additional oxygen atom, binds to PcaU and thereby specifically activates transcription of the full set of genes for protocatechuate catabolism. Particular experimental attention has been paid to PobR and PcaU from Acinetobacter strain ADP1, which exhibits exceptional competence for natural transformation. This trait allowed selection of mutant strains in which pobR function had been impaired by nucleotide substitutions introduced by PCR replication errors. Contrary to expectation, the spectrum of amino acids whose substitution led to loss of function in PobR shows no marked similarity to the spectrum of amino acids conserved by the demand for continued function during evolutionary divergence of PobR, PcaU, and related proteins. Surface plasmon resonance was used to determine the ability of mutant PobR proteins to bind to DNA in the pobA-pobR intergenic region. Deleterious mutations that strongly affect DNA binding all cluster in and around the PobR region that contains a helix-turn-helix motif, whereas mutations causing defects in the central portion of the PobR primary sequence do not seem to have a significant effect on operator binding. PCR-generated mutations allowing PobR to mimic PcaU function invariably caused a T57A amino acid substitution, making the helix-turn-helix sequence of PobR more like that of PcaU. The mutant PobR depended on p-hydroxybenzoate for its activity, but this dependence could be relieved by any of six amino acid substitutions in the center of the PobR primary sequence. Independent mutations allowing Pca

  12. Novel gene mutations in patients with 1alpha-hydroxylase deficiency that confer partial enzyme activity in vitro.

    PubMed

    Wang, Xuemei; Zhang, Martin Y H; Miller, Walter L; Portale, Anthony A

    2002-06-01

    The rate-limiting, hormonally regulated step in the biological activation of vitamin D is its 1alpha-hydroxylation to 1,25-dihydroxyvitamin D [1,25-(OH)(2)D] in the kidney, catalyzed by the mitochondrial cytochrome P450 enzyme, P450c1alpha. We previously cloned the human P450c1alpha cDNA and gene, and identified 14 different mutations, including 7 missense, in 19 patients with 1alpha-hydroxylase deficiency, also known as vitamin D-dependent rickets type 1. None of the missense mutations encoded a protein with detectable enzymatic activity in vitro. Although there is phenotypic variation among such patients, the molecular basis of this variation is unknown. We analyzed 6 additional patients with clinical and radiographic features of rickets; in 4 patients the laboratory abnormalities were typical of 1alpha-hydroxylase deficiency, but in 2 they were unusually mild [mild hypocalcemia and normal serum 1,25-(OH)(2)D concentration]. Direct sequencing revealed that all patients had P450c1alpha mutations on both alleles. Five new and 2 known mutations were identified. The new mutations included a 5-bp deletion with a 6-bp novel insertion causing a frameshift in exon 2, and a G to A change at +1 of intron 2; a minigene experiment proved that this intronic mutation prevented proper splicing. Three new missense mutations were found and tested by expressing the mutant cDNA in mouse Leydig MA-10 cells. The R389G mutant was totally inactive, but mutant L343F retained 2.3% of wild-type activity, and mutant E189G retained 22% of wild-type activity. The two mutations that confer partial enzyme activity in vitro were found in the 2 patents with mild laboratory abnormalities, suggesting that such mutations contribute to the phenotypic variation observed in patients with 1alpha-hydroxylase deficiency.

  13. Selective disruption of high sensitivity heat activation but not capsaicin activation of TRPV1 channels by pore turret mutations.

    PubMed

    Cui, Yuanyuan; Yang, Fan; Cao, Xu; Yarov-Yarovoy, Vladimir; Wang, KeWei; Zheng, Jie

    2012-04-01

    The capsaicin receptor transient receptor potential vanilloid (TRPV)1 is a highly heat-sensitive ion channel. Although chemical activation and heat activation of TRPV1 elicit similar pungent, painful sensation, the molecular mechanism underlying synergistic activation remains mysterious. In particular, where the temperature sensor is located and whether heat and capsaicin share a common activation pathway are debated. To address these fundamental issues, we searched for channel mutations that selectively affected one form of activation. We found that deletion of the first 10 amino acids of the pore turret significantly reduced the heat response amplitude and shifted the heat activation threshold, whereas capsaicin activation remained unchanged. Removing larger portions of the turret disrupted channel function. Introducing an artificial sequence to replace the deleted region restored sensitive capsaicin activation in these nonfunctional channels. The heat activation, however, remained significantly impaired, with the current exhibiting diminishing heat sensitivity to a level indistinguishable from that of a voltage-gated potassium channel, Kv7.4. Our results demonstrate that heat and capsaicin activation of TRPV1 are structurally and mechanistically distinct processes, and the pore turret is an indispensible channel structure involved in the heat activation process but is not part of the capsaicin activation pathway. Synergistic effect of heat and capsaicin on TRPV1 activation may originate from convergence of the two pathways on a common activation gate.

  14. VEGF neutralizing aerosol therapy in primary pulmonary adenocarcinoma with K-ras activating-mutations.

    PubMed

    Hervé, Virginie; Rabbe, Nathalie; Guilleminault, Laurent; Paul, Flora; Schlick, Laurène; Azzopardi, Nicolas; Duruisseaux, Michael; Fouquenet, Delphine; Montharu, Jérôme; Redini, Françoise; Paintaud, Gilles; Lemarié, Etienne; Cadranel, Jacques; Wislez, Marie; Heuzé-Vourc'h, Nathalie

    2014-01-01

    K-ras mutations promote angiogenesis in lung cancer and contribute to the drug resistance of cancer cells. It is not clear whether K-ras mutated adenocarcinomas are sensitive to anti-angiogenic therapy with monoclonal antibodies (mAbs) that target vascular endothelial growth factor (VEGF). Anti-angiogenic mAbs are usually delivered systemically, but only a small proportion reaches the lung after intravenous injection. We investigated the relevance of a non-invasive pulmonary route for the delivery of anti-VEGF mAbs in the mouse K-ras(LA1) model. We found that pulmonary delivery of these mAbs significantly reduced the number of tumor lesions and inhibited malignant progression. The antitumor effect involves the VEGFR2-dependent inhibition of blood vessel growth, which impairs tumor proliferation. Pharmacokinetic analysis of aerosolized anti-VEGF showed its low rate of passage into the bloodstream, suggesting that this delivery route is associated with reduced systemic side effects. Our findings highlight the value of the aerosol route for administration of anti-angiogenic mAbs in pulmonary adenocarcinoma with K-ras activating-mutations. PMID:25484066

  15. KA1-targeted regulatory domain mutations activate Chk1 in the absence of DNA damage.

    PubMed

    Gong, Eun-Yeung; Smits, Veronique A J; Fumagallo, Felipe; Piscitello, Desiree; Morrice, Nick; Freire, Raimundo; Gillespie, David A

    2015-01-01

    The Chk1 protein kinase is activated in response to DNA damage through ATR-mediated phosphorylation at multiple serine-glutamine (SQ) residues within the C-terminal regulatory domain, however the molecular mechanism is not understood. Modelling indicates a high probability that this region of Chk1 contains a kinase-associated 1 (KA1) domain, a small, compact protein fold found in multiple protein kinases including SOS2, AMPK and MARK3. We introduced mutations into Chk1 designed to disrupt specific structural elements of the predicted KA1 domain. Remarkably, six of seven Chk1 KA1 mutants exhibit constitutive biological activity (Chk1-CA) in the absence of DNA damage, profoundly arresting cells in G2 phase of the cell cycle. Cell cycle arrest induced by selected Chk1-CA mutants depends on kinase catalytic activity, which is increased several-fold compared to wild-type, however phosphorylation of the key ATR regulatory site serine 345 (S345) is not required. Thus, mutations targeting the putative Chk1 KA1 domain confer constitutive biological activity by circumventing the need for ATR-mediated positive regulatory phosphorylation. PMID:26039276

  16. Gene mutations in chronic lymphocytic leukemia.

    PubMed

    Amin, Nisar A; Malek, Sami N

    2016-04-01

    The recent discovery of genes mutated in chronic lymphocytic leukemia (CLL) has stimulated new research into the role of these genes in CLL pathogenesis. CLL cases carry approximately 5-20 mutated genes per exome, a lower number than detected in many human tumors. Of the recurrently mutated genes in CLL, all are mutated in 10% or less of patients when assayed in unselected CLL cohorts at diagnosis. Mutations in TP53 are of major clinical relevance, are often associated with del17p and gain in frequency over time. TP53 mutated and associated del17p states substantially lower response rates, remission duration, and survival in CLL. Mutations in NOTCH1 and SF3B1 are recurrent, often associated with progressive CLL that is also IgVH unmutated and ZAP70-positive and are under investigation as targets for novel therapies and as factors influencing CLL outcome. There are an estimated 20-50 additional mutated genes with frequencies of 1%-5% in CLL; more work is needed to identify these and to study their significance. Finally, of the major biological aberration categories influencing CLL as a disease, gene mutations will need to be placed into context with regard to their ultimate role and importance. Such calibrated appreciation necessitates studies incorporating multiple CLL driver aberrations into biological and clinical analyses. PMID:27040699

  17. Characterization of two MODY2 mutations with different susceptibility to activation

    SciTech Connect

    Langer, Sara; Platz, Christian; Waterstradt, Rica; Baltrusch, Simone

    2015-09-04

    Glucokinase plays a key role in glucose sensing in pancreatic beta cells and in liver metabolism. Heterozygous inactivating glucokinase mutations cause the autosomal dominantly inherited MODY2 subtype of maturity-onset diabetes of the young. The goal of this study was to elucidate the pathogenicity of the recently described glucokinase mutants L304P and L315H, located in an alpha-helix and connecting region, respectively, at the outer region of the large domain of glucokinase. Both mutants showed wild-type-like cytosolic localization, but faster protein degradation in insulin-secreting MIN6 cells. However, strongly reduced nuclear/cytoplasmic localization of the mutants was observed in primary hepatocytes suggesting reduced interaction with the liver specific glucokinase regulatory protein. Both mutants displayed a significantly lowered glucokinase activity compared to the wild-type protein. Even though the L315H protein showed the lowest enzymatic activity, this mutant was very sensitive to allosteric activation. The endogenous activator fructose-2,6-bisphosphatase evoked an increase in glucokinase activity for both mutants, but much stronger for L315H compared to L304P. The synthetic activator RO281675 was ineffective against the L304P mutant. Expression of the mutant proteins evoked loss of glucose-induced insulin secretion in MIN6 cells. Administration of RO281675 increased insulin secretion, however, only for the L315H mutant. Thus, a glucokinase activator drug therapy may help MODY2 patients not in general, but seems to be a useful strategy for carriers of the L315H glucokinase mutation. - Highlights: • The GK mutants L304P and L315H display a highly reduced enzymatic activity. • In hepatocytes both mutations lower the nuclear/cytoplasmic localization ratio of GK. • Both mutants inhibit stimulus-secretion coupling in insulin-producing cells. • Activation by fructose-2,6-bisphosphatase and by RO281675 is stronger for L315H. • RO281675 stimulates

  18. Mutational landscape of MCPyV-positive and MCPyV-negative Merkel cell carcinomas with implications for immunotherapy.

    PubMed

    Goh, Gerald; Walradt, Trent; Markarov, Vladimir; Blom, Astrid; Riaz, Nadeem; Doumani, Ryan; Stafstrom, Krista; Moshiri, Ata; Yelistratova, Lola; Levinsohn, Jonathan; Chan, Timothy A; Nghiem, Paul; Lifton, Richard P; Choi, Jaehyuk

    2016-01-19

    Merkel cell carcinoma (MCC) is a rare but highly aggressive cutaneous neuroendocrine carcinoma, associated with the Merkel cell polyomavirus (MCPyV) in 80% of cases. To define the genetic basis of MCCs, we performed exome sequencing of 49 MCCs. We show that MCPyV-negative MCCs have a high mutation burden (median of 1121 somatic single nucleotide variants (SSNVs) per-exome with frequent mutations in RB1 and TP53 and additional damaging mutations in genes in the chromatin modification (ASXL1, MLL2, and MLL3), JNK (MAP3K1 and TRAF7), and DNA-damage pathways (ATM, MSH2, and BRCA1). In contrast, MCPyV-positive MCCs harbor few SSNVs (median of 12.5 SSNVs/tumor) with none in the genes listed above. In both subgroups, there are rare cancer-promoting mutations predicted to activate the PI3K pathway (HRAS, KRAS, PIK3CA, PTEN, and TSC1) and to inactivate the Notch pathway (Notch1 and Notch2). TP53 mutations appear to be clinically relevant in virus-negative MCCs as 37% of these tumors harbor potentially targetable gain-of-function mutations in TP53 at p.R248 and p.P278. Moreover, TP53 mutational status predicts death in early stage MCC (5-year survival in TP53 mutant vs wild-type stage I and II MCCs is 20% vs. 92%, respectively; P = 0.0036). Lastly, we identified the tumor neoantigens in MCPyV-negative and MCPyV-positive MCCs. We found that virus-negative MCCs harbor more tumor neoantigens than melanomas or non-small cell lung cancers (median of 173, 65, and 111 neoantigens/sample, respectively), two cancers for which immune checkpoint blockade can produce durable clinical responses. Collectively, these data support the use of immunotherapies for virus-negative MCCs. PMID:26655088

  19. Mutational landscape of MCPyV-positive and MCPyV-negative Merkel cell carcinomas with implications for immunotherapy

    PubMed Central

    Goh, Gerald; Walradt, Trent; Markarov, Vladimir; Blom, Astrid; Riaz, Nadeem; Doumani, Ryan; Stafstrom, Krista; Moshiri, Ata; Yelistratova, Lola; Levinsohn, Jonathan; Chan, Timothy A.; Nghiem, Paul; Lifton, Richard P.; Choi, Jaehyuk

    2016-01-01

    Merkel cell carcinoma (MCC) is a rare but highly aggressive cutaneous neuroendocrine carcinoma, associated with the Merkel cell polyomavirus (MCPyV) in 80% of cases. To define the genetic basis of MCCs, we performed exome sequencing of 49 MCCs. We show that MCPyV-negative MCCs have a high mutation burden (median of 1121 somatic single nucleotide variants (SSNVs) per-exome with frequent mutations in RB1 and TP53 and additional damaging mutations in genes in the chromatin modification (ASXL1, MLL2, and MLL3), JNK (MAP3K1 and TRAF7), and DNA-damage pathways (ATM, MSH2, and BRCA1). In contrast, MCPyV-positive MCCs harbor few SSNVs (median of 12.5 SSNVs/tumor) with none in the genes listed above. In both subgroups, there are rare cancer-promoting mutations predicted to activate the PI3K pathway (HRAS, KRAS, PIK3CA, PTEN, and TSC1) and to inactivate the Notch pathway (Notch1 and Notch2). TP53 mutations appear to be clinically relevant in virus-negative MCCs as 37% of these tumors harbor potentially targetable gain-of-function mutations in TP53 at p.R248 and p.P278. Moreover, TP53 mutational status predicts death in early stage MCC (5-year survival in TP53 mutant vs wild-type stage I and II MCCs is 20% vs. 92%, respectively; P = 0.0036). Lastly, we identified the tumor neoantigens in MCPyV-negative and MCPyV-positive MCCs. We found that virus-negative MCCs harbor more tumor neoantigens than melanomas or non-small cell lung cancers (median of 173, 65, and 111 neoantigens/sample, respectively), two cancers for which immune checkpoint blockade can produce durable clinical responses. Collectively, these data support the use of immunotherapies for virus-negative MCCs. PMID:26655088

  20. Identification of a Tumor Specific, Active-Site Mutation in Casein Kinase 1α by Chemical Proteomics.

    PubMed

    Okerberg, Eric S; Hainley, Anna; Brown, Heidi; Aban, Arwin; Alemayehu, Senait; Shih, Ann; Wu, Jane; Patricelli, Matthew P; Kozarich, John W; Nomanbhoy, Tyzoon; Rosenblum, Jonathan S

    2016-01-01

    We describe the identification of a novel, tumor-specific missense mutation in the active site of casein kinase 1α (CSNK1A1) using activity-based proteomics. Matched normal and tumor colon samples were analyzed using an ATP acyl phosphate probe in a kinase-targeted LC-MS2 platform. An anomaly in the active-site peptide from CSNK1A1 was observed in a tumor sample that was consistent with an altered catalytic aspartic acid. Expression and analysis of the suspected mutant verified the presence of asparagine in the probe-labeled, active-site peptide for CSNK1A1. Genomic sequencing of the colon tumor samples confirmed the presence of a missense mutation in the catalytic aspartic acid of CSNK1A1 (GAC→AAC). To our knowledge, the D163N mutation in CSNK1A1 is a newly defined mutation to the conserved, catalytic aspartic acid of a protein kinase and the first missense mutation identified using activity-based proteomics. The tumorigenic potential of this mutation remains to be determined. PMID:27031502

  1. Identification of a Tumor Specific, Active-Site Mutation in Casein Kinase 1α by Chemical Proteomics

    PubMed Central

    Okerberg, Eric S.; Hainley, Anna; Brown, Heidi; Aban, Arwin; Alemayehu, Senait; Shih, Ann; Wu, Jane; Patricelli, Matthew P.; Kozarich, John W.; Nomanbhoy, Tyzoon; Rosenblum, Jonathan S.

    2016-01-01

    We describe the identification of a novel, tumor-specific missense mutation in the active site of casein kinase 1α (CSNK1A1) using activity-based proteomics. Matched normal and tumor colon samples were analyzed using an ATP acyl phosphate probe in a kinase-targeted LC-MS2 platform. An anomaly in the active-site peptide from CSNK1A1 was observed in a tumor sample that was consistent with an altered catalytic aspartic acid. Expression and analysis of the suspected mutant verified the presence of asparagine in the probe-labeled, active-site peptide for CSNK1A1. Genomic sequencing of the colon tumor samples confirmed the presence of a missense mutation in the catalytic aspartic acid of CSNK1A1 (GAC→AAC). To our knowledge, the D163N mutation in CSNK1A1 is a newly defined mutation to the conserved, catalytic aspartic acid of a protein kinase and the first missense mutation identified using activity-based proteomics. The tumorigenic potential of this mutation remains to be determined. PMID:27031502

  2. Mutations, kataegis, and translocations in B lymphocytes: towards a mechanistic understanding of AID promiscuous activity

    PubMed Central

    Casellas, Rafael; Basu, Uttiya; Yewdell, William T.; Chaudhuri, Jayanta; Robbiani, Davide F.; Di Noia, Javier M.

    2016-01-01

    As B cells engage in the immune response they express the deaminase AID to initiate the hypermutation and recombination of immunoglobulin genes, which are crucial processes for the efficient recognition and disposal of pathogens, However, AID must be tightly controlled in B cells to minimize off-targeting mutations, which can drive chromosomal translocations and the development of B cell malignancies, such as lymphomas. Recent genomic and biochemical analyses have begun to unravel the crucial question of how AID-mediated deamination is targeted outside immunoglobulin genes. Here, we discuss the transcriptional and topological features that are emerging as key drivers of AID promiscuous activity. PMID:26898111

  3. The H29D Mutation Does Not Enhance Cytosolic Ca2+ Activation of the Cardiac Ryanodine Receptor

    PubMed Central

    Xiao, Zhichao; Guo, Wenting; Yuen, Siobhan M. Wong King; Wang, Ruiwu; Zhang, Lin; Van Petegem, Filip; Chen, S. R. Wayne

    2015-01-01

    The N-terminal domain of the cardiac ryanodine receptor (RyR2) harbors a large number of naturally occurring mutations that are associated with stress-induced ventricular tachyarrhythmia and sudden death. Nearly all these disease-associated N-terminal mutations are located at domain interfaces or buried within domains. Mutations at these locations would alter domain-domain interactions or the stability/folding of domains. Recently, a novel RyR2 mutation H29D associated with ventricular arrhythmia at rest was found to enhance the activation of single RyR2 channels by diastolic levels of cytosolic Ca2+. Unlike other N-terminal disease-associated mutations, the H29D mutation is located on the surface of the N-terminal domain. It is unclear how this surface-exposed H29D mutation that does not appear to interact with other parts of the RyR2 structure could alter the intrinsic properties of the channel. Here we carried out detailed functional characterization of the RyR2-H29D mutant at the molecular and cellular levels. We found that the H29D mutation has no effect on the basal level or the Ca2+ dependent activation of [3H]ryanodine binding to RyR2, the cytosolic Ca2+ activation of single RyR2 channels, or the cytosolic Ca2+- or caffeine-induced Ca2+ release in HEK293 cells. In addition, the H29D mutation does not alter the propensity for spontaneous Ca2+ release or the thresholds for Ca2+ release activation or termination. Furthermore, the H29D mutation does not have significant impact on the thermal stability of the N-terminal region (residues 1–547) of RyR2. Collectively, our data show that the H29D mutation exerts little or no effect on the function of RyR2 or on the folding stability of the N-terminal region. Thus, our results provide no evidence that the H29D mutation enhances the cytosolic Ca2+ activation of RyR2. PMID:26405799

  4. The H29D Mutation Does Not Enhance Cytosolic Ca2+ Activation of the Cardiac Ryanodine Receptor.

    PubMed

    Xiao, Zhichao; Guo, Wenting; Yuen, Siobhan M Wong King; Wang, Ruiwu; Zhang, Lin; Van Petegem, Filip; Chen, S R Wayne

    2015-01-01

    The N-terminal domain of the cardiac ryanodine receptor (RyR2) harbors a large number of naturally occurring mutations that are associated with stress-induced ventricular tachyarrhythmia and sudden death. Nearly all these disease-associated N-terminal mutations are located at domain interfaces or buried within domains. Mutations at these locations would alter domain-domain interactions or the stability/folding of domains. Recently, a novel RyR2 mutation H29D associated with ventricular arrhythmia at rest was found to enhance the activation of single RyR2 channels by diastolic levels of cytosolic Ca2+. Unlike other N-terminal disease-associated mutations, the H29D mutation is located on the surface of the N-terminal domain. It is unclear how this surface-exposed H29D mutation that does not appear to interact with other parts of the RyR2 structure could alter the intrinsic properties of the channel. Here we carried out detailed functional characterization of the RyR2-H29D mutant at the molecular and cellular levels. We found that the H29D mutation has no effect on the basal level or the Ca2+ dependent activation of [3H]ryanodine binding to RyR2, the cytosolic Ca2+ activation of single RyR2 channels, or the cytosolic Ca2+- or caffeine-induced Ca2+ release in HEK293 cells. In addition, the H29D mutation does not alter the propensity for spontaneous Ca2+ release or the thresholds for Ca2+ release activation or termination. Furthermore, the H29D mutation does not have significant impact on the thermal stability of the N-terminal region (residues 1-547) of RyR2. Collectively, our data show that the H29D mutation exerts little or no effect on the function of RyR2 or on the folding stability of the N-terminal region. Thus, our results provide no evidence that the H29D mutation enhances the cytosolic Ca2+ activation of RyR2.

  5. M-CSF receptor mutations in hereditary diffuse leukoencephalopathy with spheroids impair not only kinase activity but also surface expression

    SciTech Connect

    Hiyoshi, Masateru; Hashimoto, Michihiro; Yukihara, Mamiko; Bhuyan, Farzana; Suzu, Shinya

    2013-11-01

    Highlights: •Many mutations were identified in Fms as a putative genetic cause of HDLS. •All of the mutations tested severely impair the kinase activity. •Most of the mutations also impair the trafficking to the cell surface. •These defects further suggest that HDLS is caused by a loss of Fms function. -- Abstract: The tyrosine kinase Fms, the cell surface receptor for M-CSF and IL-34, is critical for microglial proliferation and differentiation in the brain. Recently, a number of mutations have been identified in Fms as a putative genetic cause of hereditary diffuse leukoencephalopathy with spheroids (HDLS), implying an important role of microglial dysfunction in HDLS pathogenesis. In this study, we initially confirmed that 11 mutations, which reside within the ATP-binding or major tyrosine kinase domain, caused a severe impairment of ligand-induced Fms auto-phosphorylation. Intriguingly, we found that 10 of the 11 mutants also showed a weak cell surface expression, which was associated with a concomitant increase in the low molecular weight hypo-N-glycosylated immature gp130Fms-like species. Indeed, the mutant proteins heavily accumulated to the Golgi-like perinuclear regions. These results indicate that all of the Fms mutations tested severely impair the kinase activity and most of the mutations also impair the trafficking to the cell surface, further suggesting that HDLS is caused by the loss of Fms function.

  6. Catch-and-Hold Activation of Muscle Acetylcholine Receptors Having Transmitter Binding Site Mutations

    PubMed Central

    Purohit, Prasad; Bruhova, Iva; Gupta, Shaweta; Auerbach, Anthony

    2014-01-01

    Agonists turn on receptors because their target sites have a higher affinity in the active versus resting conformation of the protein. We used single-channel electrophysiology to measure the lower-affinity (LA) and higher-affinity (HA) equilibrium dissociation constants for acetylcholine in adult-type muscle mouse nicotinic receptors (AChRs) having mutations of agonist binding site amino acids. For a series of agonists and for all mutations of αY93, αG147, αW149, αY190, αY198, εW55, and δW57, the change in LA binding energy was approximately half that in HA binding energy. The results were analyzed as a linear free energy relationship between LA and HA agonist binding, the slope of which (κ) gives the fraction of the overall binding chemical potential where the LA complex is established. The linear correlation between LA and HA binding energies suggests that the overall binding process is by an integrated mechanism (catch-and-hold). For the agonist and the above mutations, κ ∼ 0.5, but side-chain substitutions of two residues had a slope that was significantly higher (0.90; αG153) or lower (0.25; εP121). The results suggest that backbone rearrangements in loop B, loop C, and the non-α surface participate in both LA binding and the LA ↔ HA affinity switch. It appears that all of the intermediate steps in AChR activation comprise a single, energetically coupled process. PMID:24988344

  7. LYN-activating mutations mediate antiestrogen resistance in estrogen receptor-positive breast cancer.

    PubMed

    Schwarz, Luis J; Fox, Emily M; Balko, Justin M; Garrett, Joan T; Kuba, María Gabriela; Estrada, Mónica Valeria; González-Angulo, Ana María; Mills, Gordon B; Red-Brewer, Monica; Mayer, Ingrid A; Abramson, Vandana; Rizzo, Monica; Kelley, Mark C; Meszoely, Ingrid M; Arteaga, Carlos L

    2014-12-01

    Estrogen receptor-positive (ER(+)) breast cancers adapt to hormone deprivation and become resistant to antiestrogen therapy. Here, we performed deep sequencing on ER(+) tumors that remained highly proliferative after treatment with the aromatase inhibitor letrozole and identified a D189Y mutation in the inhibitory SH2 domain of the SRC family kinase (SFK) LYN. Evaluation of 463 breast tumors in The Cancer Genome Atlas revealed four LYN mutations, two of which affected the SH2 domain. In addition, LYN was upregulated in multiple ER(+) breast cancer lines resistant to long-term estrogen deprivation (LTED). An RNAi-based kinome screen revealed that LYN is required for growth of ER(+) LTED breast cancer cells. Kinase assays and immunoblot analyses of SRC substrates in transfected cells indicated that LYN(D189Y) has higher catalytic activity than WT protein. Further, LYN(D189Y) exhibited reduced phosphorylation at the inhibitory Y507 site compared with LYN(WT). Other SH2 domain LYN mutants, E159K and K209N, also exhibited higher catalytic activity and reduced inhibitory site phosphorylation. LYN(D189Y) overexpression abrogated growth inhibition by fulvestrant and/or the PI3K inhibitor BKM120 in 3 ER(+) breast cancer cell lines. The SFK inhibitor dasatinib enhanced the antitumor effect of BKM120 and fulvestrant against estrogen-deprived ER(+) xenografts but not LYN(D189Y)-expressing xenografts. These results suggest that LYN mutations mediate escape from antiestrogens in a subset of ER(+) breast cancers.

  8. The effects of R683S (G) genetic mutations on the JAK2 activity, structure and stability.

    PubMed

    Li, Feng; Guo, Hua-Yan; Wang, Man; Geng, Hong-Li; Bian, Mei-Ru; Cao, Jiang; Chen, Chong; Zeng, Ling-Yu; Wang, Xiao-Yun; Wu, Qing-Yun

    2013-09-01

    Janus kinase 2 (JAK2) is an important mediator of cytokine receptor signaling and plays key roles in the hematopoietic and immune response. The acquired JAK2 R683S (G) mutations are presumed to be a biomarker for B-cell acute lymphoblastic leukemia (B-ALL). However, how these mutations leading to the B-ALL is still unclear. The crystal structure of JAK2 JH2 domain suggests that the residue R683 locating in the linker between the N and C lobes of JH2 domain is important for keeping the compact structure, activity and structural stability of this domain. Mutations R683S, R683G and R683E significantly increase JAK2 activity and decrease its structural stability. While the R683K and R683H mutations almost have no effects on the JAK2 activity and structural stability. Furthermore, the spectroscopic experiments imply that mutations R683S, R683G and R683E impair the structure of JAK2 JH2 domain, and lead JAK2 to partially unfolded state. It may be this partially unfolded state that caused JAK2 R683S (G) constitutive activation. This study provides clues in understanding the mechanism of JAK2 R683S (G) mutations caused B-ALL.

  9. Targeting Mitogen-Activated Protein Kinase Signaling in Mouse Models of Cardiomyopathy Caused by Lamin A/C Gene Mutations.

    PubMed

    Muchir, Antoine; Worman, Howard J

    2016-01-01

    The most frequently occurring mutations in the gene encoding nuclear lamin A and nuclear lamin C cause striated muscle diseases virtually always involving the heart. In this review, we describe the approaches and methods used to discover that cardiomyopathy-causing lamin A/C gene mutations increase MAP kinase signaling in the heart and that this plays a role in disease pathogenesis. We review different mouse models of cardiomyopathy caused by lamin A/C gene mutations and how transcriptomic analysis of one model identified increased cardiac activity of the ERK1/2, JNK, and p38α MAP kinases. We describe methods used to measure the activity of these MAP kinases in mouse hearts and then discuss preclinical treatment protocols using pharmacological inhibitors to demonstrate their role in pathogenesis. Several of these kinase inhibitors are in clinical development and could potentially be used to treat human subjects with cardiomyopathy caused by lamin A/C gene mutations.

  10. Lack of SOD1 gene mutations and activity alterations in two Italian families with amyotrophic lateral sclerosis.

    PubMed

    Gestri, D; Cecchi, C; Tedde, A; Latorraca, S; Orlacchio, A; Grassi, E; Massaro, A M; Liguri, G; St George-Hyslop, P H; Sorbi, S

    2000-08-11

    Amyotrophic lateral sclerosis (ALS) is a progressive fatal disorder, which results from the degeneration of motor neurons in the brain and spinal cord. Approximately 20% of the inherited autosomal dominant cases are due to mutations within the gene coding for Cu/Zn superoxide dismutase 1 (SOD1), a cytosolic homodimeric enzyme that catalyzes the dismutation of toxic superoxide anion. We investigated the presence of SOD1 gene mutations and activity alterations in two unrelated families of ALS patients from Elba, an island of central Italy. No mutation in SOD1 exon 1 to 5 and no activity alteration were observed in all members of the two analyzed ALS families (FALS). These data show an apparent heterogeneous distribution of ALS patients with SOD1 gene mutations among different populations and suggest that another genetic locus could be involved in the disease. PMID:10961653

  11. Functional Trade-Offs in Promiscuous Enzymes Cannot Be Explained by Intrinsic Mutational Robustness of the Native Activity

    PubMed Central

    Kaltenbach, Miriam; Emond, Stephane; Tokuriki, Nobuhiko

    2016-01-01

    The extent to which an emerging new function trades off with the original function is a key characteristic of the dynamics of enzyme evolution. Various cases of laboratory evolution have unveiled a characteristic trend; a large increase in a new, promiscuous activity is often accompanied by only a mild reduction of the native, original activity. A model that associates weak trade-offs with “evolvability” was put forward, which proposed that enzymes possess mutational robustness in the native activity and plasticity in promiscuous activities. This would enable the acquisition of a new function without compromising the original one, reducing the benefit of early gene duplication and therefore the selection pressure thereon. Yet, to date, no experimental study has examined this hypothesis directly. Here, we investigate the causes of weak trade-offs by systematically characterizing adaptive mutations that occurred in two cases of evolutionary transitions in enzyme function: (1) from phosphotriesterase to arylesterase, and (2) from atrazine chlorohydrolase to melamine deaminase. Mutational analyses in various genetic backgrounds revealed that, in contrast to the prevailing model, the native activity is less robust to mutations than the promiscuous activity. For example, in phosphotriesterase, the deleterious effect of individual mutations on the native phosphotriesterase activity is much larger than their positive effect on the promiscuous arylesterase activity. Our observations suggest a revision of the established model: weak trade-offs are not caused by an intrinsic robustness of the native activity and plasticity of the promiscuous activity. We propose that upon strong adaptive pressure for the new activity without selection against the original one, selected mutations will lead to the largest possible increases in the new function, but whether and to what extent they decrease the old function is irrelevant, creating a bias towards initially weak trade-offs and

  12. Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL.

    PubMed

    Tzoneva, Gannie; Perez-Garcia, Arianne; Carpenter, Zachary; Khiabanian, Hossein; Tosello, Valeria; Allegretta, Maddalena; Paietta, Elisabeth; Racevskis, Janis; Rowe, Jacob M; Tallman, Martin S; Paganin, Maddalena; Basso, Giuseppe; Hof, Jana; Kirschner-Schwabe, Renate; Palomero, Teresa; Rabadan, Raul; Ferrando, Adolfo

    2013-03-01

    Acute lymphoblastic leukemia (ALL) is an aggressive hematological tumor resulting from the malignant transformation of lymphoid progenitors. Despite intensive chemotherapy, 20% of pediatric patients and over 50% of adult patients with ALL do not achieve a complete remission or relapse after intensified chemotherapy, making disease relapse and resistance to therapy the most substantial challenge in the treatment of this disease. Using whole-exome sequencing, we identify mutations in the cytosolic 5'-nucleotidase II gene (NT5C2), which encodes a 5'-nucleotidase enzyme that is responsible for the inactivation of nucleoside-analog chemotherapy drugs, in 20/103 (19%) relapse T cell ALLs and 1/35 (3%) relapse B-precursor ALLs. NT5C2 mutant proteins show increased nucleotidase activity in vitro and conferred resistance to chemotherapy with 6-mercaptopurine and 6-thioguanine when expressed in ALL lymphoblasts. These results support a prominent role for activating mutations in NT5C2 and increased nucleoside-analog metabolism in disease progression and chemotherapy resistance in ALL.

  13. Theaflavin-3, 3′-digallate decreases human ovarian carcinoma OVCAR-3 cell-induced angiogenesis via Akt and Notch-1 pathways, not via MAPK pathways

    PubMed Central

    GAO, YING; RANKIN, GARY O.; TU, YOUYING; CHEN, YI CHARLIE

    2016-01-01

    Theaflavin-3, 3′-digallate (TF3) is a black tea poly-phenol produced from polymerization and oxidization of the green tea ployphenols epicatechin gallate and (−)-epigallocatechin-3-gallate (EGCG) during fermentation of fresh tea leaves. TF3 has been reported to have anticancer properties. However, the effect of TF3 on tumor angiogenesis and the underlying mechanisms are not clear. In the present study, TF3 was verified to inhibit tumor angiogenesis. Compared with EGCG, TF3 was more potent. TF3 inhibited human ovarian carcinoma OVCAR-3 cell-induced angiogenesis in human umbilical vein endothelial cell model and in chick chorioallantoic membrane model. TF3 reduced tumor angiogenesis by downregulating HIF-1α and VEGF. One of the mechanisms was TF3 inactivated Akt/mTOR/p70S6K/4E-BP1 pathway and Akt/c-Myc pathway. Besides, TF3 suppressed the cleavage of Notch-1, subsequently decreased the expression of c-Myc, HIF-1α and VEGF, and finally the impaired cancer cells induced angiogenesis. Nevertheless, TF3 did not have any influence on the MAPK pathways. Taken together, these findings suggest that TF3 might serve as a potential anti-angiogenic agent for cancer treatment. PMID:26648098

  14. Different spectra of recurrent gene mutations in subsets of chronic lymphocytic leukemia harboring stereotyped B-cell receptors.

    PubMed

    Sutton, Lesley-Ann; Young, Emma; Baliakas, Panagiotis; Hadzidimitriou, Anastasia; Moysiadis, Theodoros; Plevova, Karla; Rossi, Davide; Kminkova, Jana; Stalika, Evangelia; Pedersen, Lone Bredo; Malcikova, Jitka; Agathangelidis, Andreas; Davis, Zadie; Mansouri, Larry; Scarfò, Lydia; Boudjoghra, Myriam; Navarro, Alba; Muggen, Alice F; Yan, Xiao-Jie; Nguyen-Khac, Florence; Larrayoz, Marta; Panagiotidis, Panagiotis; Chiorazzi, Nicholas; Niemann, Carsten Utoft; Belessi, Chrysoula; Campo, Elias; Strefford, Jonathan C; Langerak, Anton W; Oscier, David; Gaidano, Gianluca; Pospisilova, Sarka; Davi, Frederic; Ghia, Paolo; Stamatopoulos, Kostas; Rosenquist, Richard

    2016-08-01

    We report on markedly different frequencies of genetic lesions within subsets of chronic lymphocytic leukemia patients carrying mutated or unmutated stereotyped B-cell receptor immunoglobulins in the largest cohort (n=565) studied for this purpose. By combining data on recurrent gene mutations (BIRC3, MYD88, NOTCH1, SF3B1 and TP53) and cytogenetic aberrations, we reveal a subset-biased acquisition of gene mutations. More specifically, the frequency of NOTCH1 mutations was found to be enriched in subsets expressing unmutated immunoglobulin genes, i.e. #1, #6, #8 and #59 (22-34%), often in association with trisomy 12, and was significantly different (P<0.001) to the frequency observed in subset #2 (4%, aggressive disease, variable somatic hypermutation status) and subset #4 (1%, indolent disease, mutated immunoglobulin genes). Interestingly, subsets harboring a high frequency of NOTCH1 mutations were found to carry few (if any) SF3B1 mutations. This starkly contrasts with subsets #2 and #3 where, despite their immunogenetic differences, SF3B1 mutations occurred in 45% and 46% of cases, respectively. In addition, mutations within TP53, whilst enriched in subset #1 (16%), were rare in subsets #2 and #8 (both 2%), despite all being clinically aggressive. All subsets were negative for MYD88 mutations, whereas BIRC3 mutations were infrequent. Collectively, this striking bias and skewed distribution of mutations and cytogenetic aberrations within specific chronic lymphocytic leukemia subsets implies that the mechanisms underlying clinical aggressiveness are not uniform, but rather support the existence of distinct genetic pathways of clonal evolution governed by a particular stereotyped B-cell receptor selecting a certain molecular lesion(s). PMID:27198719

  15. Different spectra of recurrent gene mutations in subsets of chronic lymphocytic leukemia harboring stereotyped B-cell receptors

    PubMed Central

    Sutton, Lesley-Ann; Young, Emma; Baliakas, Panagiotis; Hadzidimitriou, Anastasia; Moysiadis, Theodoros; Plevova, Karla; Rossi, Davide; Kminkova, Jana; Stalika, Evangelia; Pedersen, Lone Bredo; Malcikova, Jitka; Agathangelidis, Andreas; Davis, Zadie; Mansouri, Larry; Scarfò, Lydia; Boudjoghra, Myriam; Navarro, Alba; Muggen, Alice F.; Yan, Xiao-Jie; Nguyen-Khac, Florence; Larrayoz, Marta; Panagiotidis, Panagiotis; Chiorazzi, Nicholas; Niemann, Carsten Utoft; Belessi, Chrysoula; Campo, Elias; Strefford, Jonathan C.; Langerak, Anton W.; Oscier, David; Gaidano, Gianluca; Pospisilova, Sarka; Davi, Frederic; Ghia, Paolo; Stamatopoulos, Kostas; Rosenquist, Richard

    2016-01-01

    We report on markedly different frequencies of genetic lesions within subsets of chronic lymphocytic leukemia patients carrying mutated or unmutated stereotyped B-cell receptor immunoglobulins in the largest cohort (n=565) studied for this purpose. By combining data on recurrent gene mutations (BIRC3, MYD88, NOTCH1, SF3B1 and TP53) and cytogenetic aberrations, we reveal a subset-biased acquisition of gene mutations. More specifically, the frequency of NOTCH1 mutations was found to be enriched in subsets expressing unmutated immunoglobulin genes, i.e. #1, #6, #8 and #59 (22–34%), often in association with trisomy 12, and was significantly different (P<0.001) to the frequency observed in subset #2 (4%, aggressive disease, variable somatic hypermutation status) and subset #4 (1%, indolent disease, mutated immunoglobulin genes). Interestingly, subsets harboring a high frequency of NOTCH1 mutations were found to carry few (if any) SF3B1 mutations. This starkly contrasts with subsets #2 and #3 where, despite their immunogenetic differences, SF3B1 mutations occurred in 45% and 46% of cases, respectively. In addition, mutations within TP53, whilst enriched in subset #1 (16%), were rare in subsets #2 and #8 (both 2%), despite all being clinically aggressive. All subsets were negative for MYD88 mutations, whereas BIRC3 mutations were infrequent. Collectively, this striking bias and skewed distribution of mutations and cytogenetic aberrations within specific chronic lymphocytic leukemia subsets implies that the mechanisms underlying clinical aggressiveness are not uniform, but rather support the existence of distinct genetic pathways of clonal evolution governed by a particular stereotyped B-cell receptor selecting a certain molecular lesion(s). PMID:27198719

  16. Mutations in POFUT1, Encoding Protein O-fucosyltransferase 1, Cause Generalized Dowling-Degos Disease

    PubMed Central

    Li, Ming; Cheng, Ruhong; Liang, Jianying; Yan, Heng; Zhang, Hui; Yang, Lijia; Li, Chengrang; Jiao, Qingqing; Lu, Zhiyong; He, Jianhui; Ji, Jin; Shen, Zhu; Li, Chunqi; Hao, Fei; Yu, Hong; Yao, Zhirong

    2013-01-01

    Dowling-Degos disease (DDD), or reticular pigmented anomaly of the flexures, is a type of rare autosomal-dominant genodermatosis characterized by reticular hyperpigmentation and hypopigmentation of the flexures, such as the neck, axilla, and areas below the breasts and groin, and shows considerable heterogeneity. Loss-of-function mutations of keratin 5 (KRT5) have been identified in DDD individuals. In this study, we collected DNA samples from a large Chinese family affected by generalized DDD and found no mutation of KRT5. We performed a genome-wide linkage analysis of this family and mapped generalized DDD to a region between rs1293713 and rs244123 on chromosome 20. By exome sequencing, we identified nonsense mutation c.430G>T (p.Glu144∗) in POFUT1, which encodes protein O-fucosyltransferase 1, in the family. Study of an additional generalized DDD individual revealed the heterozygous deletion mutation c.482delA (p.Lys161Serfs∗42) in POFUT1. Knockdown of POFUT1 reduces the expression of NOTCH1, NOTCH2, HES1, and KRT5 in HaCaT cells. Using zebrafish, we showed that pofut1 is expressed in the skin and other organs. Morpholino knockdown of pofut1 in zebrafish produced a phenotype characteristic of hypopigmentation at 48 hr postfertilization (hpf) and abnormal melanin distribution at 72 hpf, replicating the clinical phenotype observed in our DDD individuals. At 48 and 72 hpf, tyrosinase activities decreased by 33% and 45%, respectively, and melanin protein contents decreased by 20% and 25%, respectively. Our findings demonstrate that POFUT1 mutations cause generalized DDD. These results strongly suggest that the protein product of POFUT1 plays a significant and conserved role in melanin synthesis and transport. PMID:23684010

  17. Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities

    NASA Astrophysics Data System (ADS)

    Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

    2016-01-01

    APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1.

  18. Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities.

    PubMed

    Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

    2016-01-01

    APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1. PMID:26738439

  19. Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities

    PubMed Central

    Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

    2016-01-01

    APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1. PMID:26738439

  20. A constitutive thiamine metabolism mutation, thi80, causing reduced thiamine pyrophosphokinase activity in Saccharomyces cerevisiae.

    PubMed Central

    Nishimura, H; Kawasaki, Y; Nosaka, K; Kaneko, Y; Iwashima, A

    1991-01-01

    We identified a strain carrying a recessive constitutive mutation (thi80-1) with an altered thiamine transport system, thiamine-repressible acid phosphatase, and several enzymes of thiamine synthesis from 2-methyl-4-amino-5-hydroxymethylpyrimidine and 4-methyl-5-beta-hydroxyethylthiazole. The mutant shows markedly reduced activity of thiamine pyrophosphokinase (EC 2.7.6.2) and high resistance to oxythiamine, a thiamine antagonist whose potency depends on thiamine pyrophosphokinase activity. The intracellular thiamine pyrophosphate content of the mutant cells grown with exogenous thiamine (2 x 10(-7) M) was found to be about half that of the wild-type strain under the same conditions. These results suggest that the utilization and synthesis of thiamine in Saccharomyces cerevisiae is controlled negatively by the intracellular thiamine pyrophosphate level. PMID:1849514

  1. Mutation in the Plasmodium falciparum CRT protein determines the stereospecific activity of antimalarial cinchona alkaloids.

    PubMed

    Griffin, Carol E; Hoke, Jonathan M; Samarakoon, Upeka; Duan, Junhui; Mu, Jianbing; Ferdig, Michael T; Warhurst, David C; Cooper, Roland A

    2012-10-01

    The Cinchona alkaloids are quinoline aminoalcohols that occur as diastereomer pairs, typified by (-)-quinine and (+)-quinidine. The potency of (+)-isomers is greater than the (-)-isomers in vitro and in vivo against Plasmodium falciparum malaria parasites. They may act by the inhibition of heme crystallization within the parasite digestive vacuole in a manner similar to chloroquine. Earlier studies showed that a K76I mutation in the digestive vacuole-associated protein, PfCRT (P. falciparum chloroquine resistance transporter), reversed the normal potency order of quinine and quinidine toward P. falciparum. To further explore PfCRT-alkaloid interactions in the malaria parasite, we measured the in vitro susceptibility of eight clonal lines of P. falciparum derived from the 106/1 strain, each containing a unique pfcrt allele, to four Cinchona stereoisomer pairs: quinine and quinidine; cinchonidine and cinchonine; hydroquinine and hydroquinidine; 9-epiquinine and 9-epiquinidine. Stereospecific potency of the Cinchona alkaloids was associated with changes in charge and hydrophobicity of mutable PfCRT amino acids. In isogenic chloroquine-resistant lines, the IC(50) ratio of (-)/(+) CA pairs correlated with side chain hydrophobicity of the position 76 residue. Second-site PfCRT mutations negated the K76I stereospecific effects: charge-change mutations C72R or Q352K/R restored potency patterns similar to the parent K76 line, while V369F increased susceptibility to the alkaloids and nullified stereospecific differences between alkaloid pairs. Interactions between key residues of the PfCRT channel/transporter with (-) and (+) alkaloids are stereospecifically determined, suggesting that PfCRT binding plays an important role in the antimalarial activity of quinine and other Cinchona alkaloids.

  2. Mutation in the Plasmodium falciparum CRT Protein Determines the Stereospecific Activity of Antimalarial Cinchona Alkaloids

    PubMed Central

    Griffin, Carol E.; Hoke, Jonathan M.; Samarakoon, Upeka; Duan, Junhui; Mu, Jianbing; Ferdig, Michael T.; Warhurst, David C.

    2012-01-01

    The Cinchona alkaloids are quinoline aminoalcohols that occur as diastereomer pairs, typified by (−)-quinine and (+)-quinidine. The potency of (+)-isomers is greater than the (−)-isomers in vitro and in vivo against Plasmodium falciparum malaria parasites. They may act by the inhibition of heme crystallization within the parasite digestive vacuole in a manner similar to chloroquine. Earlier studies showed that a K76I mutation in the digestive vacuole-associated protein, PfCRT (P. falciparum chloroquine resistance transporter), reversed the normal potency order of quinine and quinidine toward P. falciparum. To further explore PfCRT-alkaloid interactions in the malaria parasite, we measured the in vitro susceptibility of eight clonal lines of P. falciparum derived from the 106/1 strain, each containing a unique pfcrt allele, to four Cinchona stereoisomer pairs: quinine and quinidine; cinchonidine and cinchonine; hydroquinine and hydroquinidine; 9-epiquinine and 9-epiquinidine. Stereospecific potency of the Cinchona alkaloids was associated with changes in charge and hydrophobicity of mutable PfCRT amino acids. In isogenic chloroquine-resistant lines, the IC50 ratio of (−)/(+) CA pairs correlated with side chain hydrophobicity of the position 76 residue. Second-site PfCRT mutations negated the K76I stereospecific effects: charge-change mutations C72R or Q352K/R restored potency patterns similar to the parent K76 line, while V369F increased susceptibility to the alkaloids and nullified stereospecific differences between alkaloid pairs. Interactions between key residues of the PfCRT channel/transporter with (−) and (+) alkaloids are stereospecifically determined, suggesting that PfCRT binding plays an important role in the antimalarial activity of quinine and other Cinchona alkaloids. PMID:22869567

  3. Activating Mutations of the TRPML1 Channel Revealed by Proline-scanning Mutagenesis*

    PubMed Central

    Dong, Xian-ping; Wang, Xiang; Shen, Dongbiao; Chen, Su; Liu, Meiling; Wang, Yanbin; Mills, Eric; Cheng, Xiping; Delling, Markus; Xu, Haoxing

    2009-01-01

    The mucolipin TRP (TRPML) proteins are a family of endolysosomal cation channels with genetically established importance in humans and rodent. Mutations of human TRPML1 cause type IV mucolipidosis, a devastating pediatric neurodegenerative disease. Our recent electrophysiological studies revealed that, although a TRPML1-mediated current can only be recorded in late endosome and lysosome (LEL) using the lysosome patch clamp technique, a proline substitution in TRPML1 (TRPML1V432P) results in a large whole cell current. Thus, it remains unknown whether the large TRPML1V432P-mediated current results from an increased surface expression (trafficking), elevated channel activity (gating), or both. Here we performed systemic Pro substitutions in a region previously implicated in the gating of various 6 transmembrane cation channels. We found that several Pro substitutions displayed gain-of-function (GOF) constitutive activities at both the plasma membrane (PM) and endolysosomal membranes. Although wild-type TRPML1 and non-GOF Pro substitutions localized exclusively in LEL and were barely detectable in the PM, the GOF mutations with high constitutive activities were not restricted to LEL compartments, and most significantly, exhibited significant surface expression. Because lysosomal exocytosis is Ca2+-dependent, constitutive Ca2+ permeability due to Pro substitutions may have resulted in stimulus-independent intralysosomal Ca2+ release, hence the surface expression and whole cell current of TRPML1. Indeed, surface staining of lysosome-associated membrane protein-1 (Lamp-1) was dramatically increased in cells expressing GOF TRPML1 channels. We conclude that TRPML1 is an inwardly rectifying, proton-impermeable, Ca2+ and Fe2+/Mn2+ dually permeable cation channel that may be gated by unidentified cellular mechanisms through a conformational change in the cytoplasmic face of the transmembrane 5 (TM5). Furthermore, activation of TRPML1 in LEL may lead to the appearance of TRPML

  4. The antimutagenic activity of Lavandula angustifolia (lavender) essential oil in the bacterial reverse mutation assay.

    PubMed

    Evandri, M G; Battinelli, L; Daniele, C; Mastrangelo, S; Bolle, P; Mazzanti, G

    2005-09-01

    Essential oils from Melaleuca alternifolia (tea-tree oil) and Lavandula angustifolia (lavender oil) are commonly used to treat minor health problems. Tea-tree oil possesses broad-spectrum antimicrobial activity, and is increasingly used for skin problems. Lavender oil, traditionally used as an antiseptic agent, is now predominantly used as a relaxant, carminative, and sedative in aromatherapy. Despite their growing use no data are available on their mutagenic potential. In this study, after determining the chemical composition of tea-tree oil and lavender oil, by gas-chromatography and mass spectrometry, we investigated their mutagenic and antimutagenic activities by the bacterial reverse mutation assay in Salmonella typhimurium TA98 and TA100 strains and in Escherichia coli WP2 uvrA strain, with and without an extrinsic metabolic activation system. Neither essential oil had mutagenic activity on the two tested Salmonella strains or on E. coli, with or without the metabolic activation system. Conversely, lavender oil exerted strong antimutagenic activity, reducing mutant colonies in the TA98 strain exposed to the direct mutagen 2-nitrofluorene. Antimutagenicity was concentration-dependent: the maximal concentration (0.80 mg/plate) reduced the number of histidine-independent revertant colonies by 66.4%. Lavender oil (0.80 mg/plate) also showed moderate antimutagenicity against the TA98 strain exposed to the direct mutagen 1-nitropyrene. Its antimutagenic property makes lavender oil a promising candidate for new applications in human healthcare. PMID:15907354

  5. The antimutagenic activity of Lavandula angustifolia (lavender) essential oil in the bacterial reverse mutation assay.

    PubMed

    Evandri, M G; Battinelli, L; Daniele, C; Mastrangelo, S; Bolle, P; Mazzanti, G

    2005-09-01

    Essential oils from Melaleuca alternifolia (tea-tree oil) and Lavandula angustifolia (lavender oil) are commonly used to treat minor health problems. Tea-tree oil possesses broad-spectrum antimicrobial activity, and is increasingly used for skin problems. Lavender oil, traditionally used as an antiseptic agent, is now predominantly used as a relaxant, carminative, and sedative in aromatherapy. Despite their growing use no data are available on their mutagenic potential. In this study, after determining the chemical composition of tea-tree oil and lavender oil, by gas-chromatography and mass spectrometry, we investigated their mutagenic and antimutagenic activities by the bacterial reverse mutation assay in Salmonella typhimurium TA98 and TA100 strains and in Escherichia coli WP2 uvrA strain, with and without an extrinsic metabolic activation system. Neither essential oil had mutagenic activity on the two tested Salmonella strains or on E. coli, with or without the metabolic activation system. Conversely, lavender oil exerted strong antimutagenic activity, reducing mutant colonies in the TA98 strain exposed to the direct mutagen 2-nitrofluorene. Antimutagenicity was concentration-dependent: the maximal concentration (0.80 mg/plate) reduced the number of histidine-independent revertant colonies by 66.4%. Lavender oil (0.80 mg/plate) also showed moderate antimutagenicity against the TA98 strain exposed to the direct mutagen 1-nitropyrene. Its antimutagenic property makes lavender oil a promising candidate for new applications in human healthcare.

  6. A recurrent activating PLCG1 mutation in cardiac angiosarcomas increases apoptosis resistance and invasiveness of endothelial cells.

    PubMed

    Kunze, Kristin; Spieker, Tilmann; Gamerdinger, Ulrike; Nau, Kerstin; Berger, Johannes; Dreyer, Thomas; Sindermann, Jürgen R; Hoffmeier, Andreas; Gattenlöhner, Stefan; Bräuninger, Andreas

    2014-11-01

    Primary cardiac angiosarcomas are rare tumors with unfavorable prognosis. Pathogenic driver mutations are largely unknown. We therefore analyzed a collection of cases for genomic aberrations using SNP arrays and targeted next-generation sequencing (tNGS) of oncogenes and tumor-suppressor genes. Recurrent gains of chromosome 1q and a small region of chromosome 4 encompassing KDR and KIT were identified by SNP array analysis. Repeatedly mutated genes identified by tNGS were KDR with different nonsynonymous mutations, MLL2 with different nonsense mutations, and PLCG1 with a recurrent nonsynonymous mutation (R707Q) in the highly conserved autoinhibitory SH2 domain in three of 10 cases. PLCγ1 is usually activated by Y783 phosphorylation and activates protein kinase C and Ca(2+)-dependent second messengers, with effects on cellular proliferation, migration, and invasiveness. Ectopic expression of the PLCγ1-R707Q mutant in endothelial cells revealed reduced PLCγ1-Y783 phosphorylation with concomitant increased c-RAF/MEK/ERK1/2 phosphorylation, increased IP3 amounts, and increased Ca(2+)-dependent calcineurin activation compared with ectopic expressed PLCγ1-wild-type. Furthermore, cofilin, whose activation is associated with actin skeleton reorganization, showed decreased phosphorylation, and thus activation after expression of PLCγ1-R707Q compared with PLCγ1-wild-type. At the cellular level, expression of PLCγ1-R707Q in endothelial cells had no influence on proliferation rate, but increased apoptosis resistance and migration and invasiveness in in vitro assays. Together, these findings indicate that the PLCγ1-R707Q mutation causes constitutive activation of PLCγ1 and may represent an alternative way of activation of KDR/PLCγ1 signaling besides KDR activation in angiosarcomas, with implications for VEGF/KDR targeted therapies. PMID:25252913

  7. The Role of Distant Mutations and Allosteric Regulation on LovD Active Site Dynamics

    PubMed Central

    Jiménez-Osés, Gonzalo; Osuna, Sílvia; Gao, Xue; Sawaya, Michael R.; Gilson, Lynne; Collier, Steven J.; Huisman, Gjalt W.; Yeates, Todd O.; Tang, Yi; Houk, K. N.

    2014-01-01

    Natural enzymes have evolved to perform their cellular functions under complex selective pressures, which often require their catalytic activities to be regulated by other proteins. We contrasted a natural enzyme, LovD, which acts on a protein-bound (LovF) acyl substrate, with a laboratory-generated variant that was transformed by directed evolution to accept instead a small free acyl thioester, and no longer requires the acyl carrier protein. The resulting 29-mutant variant is 1000-fold more efficient in the synthesis of the drug simvastatin than the wild-type LovD. This is the first non-patent report of the enzyme currently used for the manufacture of simvastatin, as well as the intermediate evolved variants. Crystal structures and microsecond molecular dynamics simulations revealed the mechanism by which the laboratory-generated mutations free LovD from dependence on protein-protein interactions. Mutations dramatically altered conformational dynamics of the catalytic residues, obviating the need for allosteric modulation by the acyl carrier LovF. PMID:24727900

  8. Mutation in the SH1 helix reduces the activation energy of the ATP-induced conformational transition of myosin.

    PubMed

    Iwai, Sosuke; Chaen, Shigeru

    2007-05-25

    The SH1 helix is a joint that links the converter subdomain to the rest of the myosin motor domain. Recently, we showed that a mutation within the SH1 helix in Dictyostelium myosin II (R689H) reduced the elasticity and thermal stability of the protein. To reveal the involvement of the SH1 helix in ATP-dependent conformational changes of the motor domain, we have investigated the effects of the R689H mutation on the conformational changes of the converter, using a GFP-based fluorescence resonance energy transfer method. Although the mutation does not seem to strongly affect conformations, we found that it significantly reduced the activation energy required for the ATP-induced conformational transition corresponding to the recovery stroke. Given the effects of the mutation on the mechanical properties of myosin, we propose that the SH1 helix plays an important role in the mechanochemical energy conversion underlying the conformational change of the myosin motor domain.

  9. Mutation in E1, the ubiquitin activating enzyme, reduces Drosophila lifespan and results in motor impairment.

    PubMed

    Liu, Hsiu-Yu; Pfleger, Cathie M

    2013-01-01

    Neurodegenerative diseases cause tremendous suffering for those afflicted and their families. Many of these diseases involve accumulation of mis-folded or aggregated proteins thought to play a causal role in disease pathology. Ubiquitinated proteins are often found in these protein aggregates, and the aggregates themselves have been shown to inhibit the activity of the proteasome. These and other alterations in the Ubiquitin Pathway observed in neurodegenerative diseases have led to the question of whether impairment of the Ubiquitin Pathway on its own can increase mortality or if ongoing neurodegeneration alters Ubiquitin Pathway function as a side-effect. To address the role of the Ubiquitin Pathway in vivo, we studied loss-of-function mutations in the Drosophila Ubiquitin Activating Enzyme, Uba1 or E1, the most upstream enzyme in the Ubiquitin Pathway. Loss of only one functional copy of E1 caused a significant reduction in adult lifespan. Rare homozygous hypomorphic E1 mutants reached adulthood. These mutants exhibited further reduced lifespan and showed inappropriate Ras activation in the brain. Removing just one functional copy of Ras restored the lifespan of heterozygous E1 mutants to that of wild-type flies and increased the survival of homozygous E1 mutants. E1 homozygous mutants also showed severe motor impairment. Our findings suggest that processes that impair the Ubiquitin Pathway are sufficient to cause early mortality. Reduced lifespan and motor impairment are seen in the human disease X-linked Infantile Spinal Muscular Atrophy, which is associated with mutation in human E1 warranting further analysis of these mutants as a potential animal model for study of this disease.

  10. Psoriasis mutations disrupt CARD14 autoinhibition promoting BCL10-MALT1-dependent NF-κB activation.

    PubMed

    Howes, Ashleigh; O'Sullivan, Paul A; Breyer, Felix; Ghose, Ashavari; Cao, Li; Krappmann, Daniel; Bowcock, Anne M; Ley, Steven C

    2016-06-15

    Inherited and de novo mutations in the CARD14 gene promote the development of psoriasis, an inflammatory disease of the skin. Caspase recruitment domain-containing protein 14 (CARD14) is a member of the CARMA protein family that includes the structurally related CARD11 adaptor that mediates NF-κB activation by antigen receptors. We investigated the mechanism by which CARD14 mutation in psoriasis activates NF-κB. In contrast with wild-type CARD14, CARD14(E138A) and CARD14(G117S) psoriasis mutants interacted constitutively with BCL10 and MALT1, and triggered BCL10- and MALT1-dependent activation of NF-κB in keratinocytes. These alterations disrupted the inhibitory effect of the CARD14 linker region (LR) on NF-κB activation by facilitating BCL10 binding. Therefore, psoriasis mutations activated CARD14 by a mechanism analogous to oncogenic CARD11 mutations in non-Hodgkin B cell lymphomas. CARD14(E138A) also stimulated MALT1 paracaspase activity and activated both ERK1/2 and p38α MAP kinases. Inhibition of MALT1 with mepazine reduced CARD14(E138A)-induced expression of specific psoriasis-associated transcripts in keratinocytes. Our results establish the mechanism whereby gain-of-function CARD14 variants, which induce psoriatic disease in affected individuals, activate pro-inflammatory signalling. PMID:27071417

  11. Intrinsic resistance to EGFR tyrosine kinase inhibitors in advanced non-small-cell lung cancer with activating EGFR mutations

    PubMed Central

    Wang, Jun; Wang, Baocheng; Chu, Huili; Yao, Yunfeng

    2016-01-01

    Identifying activating EGFR mutations is a useful predictive strategy that helps select a population of advanced non-small-cell lung cancer (NSCLC) patients for treatment with EGFR tyrosine kinase inhibitors (TKIs). Patients with sensitizing EGFR mutations (predominantly an in-frame deletion in exon 19 and an L858R substitution) are highly responsive to first-generation EGFR TKIs, such as gefitinib and erlotinib, and show improved progression-free survival without serious side effects. However, all patients with activating EGFR mutations who are initially responsive to EGFR TKIs eventually develop acquired resistance after a median progression-free survival of 10–16 months, followed by disease progression. Moreover, ~20%–30% of NSCLC patients have no objective tumor regression on initial EGFR TKI treatment, although they harbor an activating EGFR mutation. These patients represent an NSCLC subgroup that is defined as having intrinsic or primary resistance to EGFR TKIs. Different mechanisms of acquired EGFR TKI resistance have been identified, and several novel compounds have been developed to reverse acquired resistance, but little is known about EGFR TKI intrinsic resistance. In this review, we summarize the latest findings involving mechanisms of intrinsic resistance to EGFR TKIs in advanced NSCLC with activating EGFR mutations and present possible therapeutic strategies to overcome this resistance. PMID:27382309

  12. Mutation at Glu23 eliminates the neuron growth inhibitory activity of human metallothionein-3

    SciTech Connect

    Ding Zhichun; Teng Xinchen; Cai Bin; Wang Hui; Zheng Qi; Wang Yang; Zhou Guoming; Zhang Mingjie; Wu Houming; Sun Hongzhe . E-mail: hsun@hku.hk; Huang Zhongxian . E-mail: zxhuang@fudan.edu.cn

    2006-10-20

    Human metallothionein-3 (hMT3), first isolated and identified as a neuronal growth inhibitory factor (GIF), is a metalloprotein expressed predominantly in brain. However, untill now, the exact mechanism of the bioactivity of hMT3 is still unknown. In order to study the influence of acid-base catalysis on S-nitrosylation of hMT3, we constructed the E23K mutant of hMT3. During the course of bioassay, we found out unexpectedly that mutation at E23 of hMT3 eliminates the neuronal growth inhibitory activity completely. To the best of our knowledge, it is First report that other residues, besides the TCPCP motif, in the {beta}-domain can alter the bioactivity of hMT3. In order to figure out the causes for the loss of bioactivity of the E23K mutant, the biochemical properties were characterized by UV-vis spectroscopy, CD spectroscopy, pH titration, DTNB reaction, EDTA reaction, and SNOC reaction. All data demonstrated that stability of the metal-thiolate cluster and overall structure of the E23K mutant were not altered too much. However, the reaction of the E23K mutant with SNOC exhibited biphasic kinetics and the mutant protein released zinc ions much faster than hMT3 in the initial step, while hMT3 exhibited single kinetic process. The 2D [{sup 1}H-{sup 15}N] HSQC was also employed to characterize structural changes during the reaction of hMT3 with varying mounts of nitric oxide. It was shown that the resonance of Glu23 disappeared at a molar ratio of NO to protein of 4. Based on these results, we suggest that mutation at Glu23 may alter the NO metabolism and/or affect zinc homeostasis in brain, thus altering the neuronal growth inhibitory activity.

  13. Rhabdomyolysis-Associated Mutations in Human LPIN1 Lead to Loss of Phosphatidic Acid Phosphohydrolase Activity.

    PubMed

    Schweitzer, George G; Collier, Sara L; Chen, Zhouji; Eaton, James M; Connolly, Anne M; Bucelli, Robert C; Pestronk, Alan; Harris, Thurl E; Finck, Brian N

    2015-01-01

    Rhabdomyolysis is an acute syndrome due to extensive injury of skeletal muscle. Recurrent rhabdomyolysis is often caused by inborn errors in intermediary metabolism, and recent work has suggested that mutations in the human gene encoding lipin 1 (LPIN1) may be a common cause of recurrent rhabdomyolysis in children. Lipin 1 dephosphorylates phosphatidic acid to form diacylglycerol (phosphatidic acid phosphohydrolase; PAP) and acts as a transcriptional regulatory protein to control metabolic gene expression. Herein, a 3-year-old boy with severe recurrent rhabdomyolysis was determined to be a compound heterozygote for a novel c.1904T>C (p.Leu635Pro) substitution and a previously reported genomic deletion of exons 18-19 (E766-S838_del) in LPIN1. Western blotting with patient muscle biopsy lysates demonstrated a marked reduction in lipin 1 protein, while immunohistochemical staining for lipin 1 showed abnormal subcellular localization. We cloned cDNAs to express recombinant lipin 1 proteins harboring pathogenic mutations and showed that the E766-S838_del allele was not expressed at the RNA or protein level. Lipin 1 p.Leu635Pro was expressed, but the protein was less stable, was aggregated in the cytosol, and was targeted for proteosomal degradation. Another pathogenic single amino acid substitution, lipin 1 p.Arg725His, was well expressed and retained its transcriptional regulatory function. However, both p.Leu635Pro and p.Arg725His proteins were found to be deficient in PAP activity. Kinetic analyses demonstrated a loss of catalysis rather than diminished substrate binding. These data suggest that loss of lipin 1-mediated PAP activity may be involved in the pathogenesis of rhabdomyolysis in lipin 1 deficiency. PMID:25967228

  14. Evidence for activation of mutated p53 by apigenin in human pancreatic cancer

    PubMed Central

    King, Jonathan C; Lu, Qing-Yi; Li, Gang; Moro, Aune; Takahashi, Hiroki; Chen, Monica; Go, Vay Liang W; Reber, Howard A; Eibl, Guido; Hines, O. Joe

    2012-01-01

    Pancreatic cancer is an exceedingly lethal disease with a five-year survival that ranks among the lowest of gastrointestinal malignancies. Part of its lethality is attributable to a generally poor response to existing chemotherapeutic regimens. New therapeutic approaches are urgently needed. We aimed to elucidate the anti-neoplastic mechanisms of apigenin-an abundant, naturally-occurring plant flavonoid-with a particular focus on p53 function. Pancreatic cancer cells (BxPC-3, MiaPaCa-2) experienced dose and time-dependent growth inhibition and increased apoptosis with apigenin treatment. p53 post-translational modification, nuclear translocation, DNA binding, and upregulation of p21 and PUMA were all enhanced by apigenin treatment despite mutated p53 in both cell lines. Transcription-dependent p53 activity was reversed by pifithrin-α, a specific DNA binding inhibitor of p53, but not growth inhibition or apoptosis suggesting transcription-independent p53 activity. This was supported by immunoprecipitation assays which demonstrated disassociation of p53/BclXL and PUMA/BclXL and formation of complexes with Bak followed by Cytochrome c release. Treated animals grew smaller tumors with increased cellular apoptosis than those fed control diet. These results suggest that despite deactivating mutation, p53 retains some of its function which is augmented following treatment with apigenin. Cell cycle arrest and apoptosis induction may be mediated by transcription-independent p53 function via interactions with BclXL and PUMA. Further study of flavonoids as chemotherapeutics is warranted PMID:22227579

  15. Rhabdomyolysis-Associated Mutations in Human LPIN1 Lead to Loss of Phosphatidic Acid Phosphohydrolase Activity.

    PubMed

    Schweitzer, George G; Collier, Sara L; Chen, Zhouji; Eaton, James M; Connolly, Anne M; Bucelli, Robert C; Pestronk, Alan; Harris, Thurl E; Finck, Brian N

    2015-01-01

    Rhabdomyolysis is an acute syndrome due to extensive injury of skeletal muscle. Recurrent rhabdomyolysis is often caused by inborn errors in intermediary metabolism, and recent work has suggested that mutations in the human gene encoding lipin 1 (LPIN1) may be a common cause of recurrent rhabdomyolysis in children. Lipin 1 dephosphorylates phosphatidic acid to form diacylglycerol (phosphatidic acid phosphohydrolase; PAP) and acts as a transcriptional regulatory protein to control metabolic gene expression. Herein, a 3-year-old boy with severe recurrent rhabdomyolysis was determined to be a compound heterozygote for a novel c.1904T>C (p.Leu635Pro) substitution and a previously reported genomic deletion of exons 18-19 (E766-S838_del) in LPIN1. Western blotting with patient muscle biopsy lysates demonstrated a marked reduction in lipin 1 protein, while immunohistochemical staining for lipin 1 showed abnormal subcellular localization. We cloned cDNAs to express recombinant lipin 1 proteins harboring pathogenic mutations and showed that the E766-S838_del allele was not expressed at the RNA or protein level. Lipin 1 p.Leu635Pro was expressed, but the protein was less stable, was aggregated in the cytosol, and was targeted for proteosomal degradation. Another pathogenic single amino acid substitution, lipin 1 p.Arg725His, was well expressed and retained its transcriptional regulatory function. However, both p.Leu635Pro and p.Arg725His proteins were found to be deficient in PAP activity. Kinetic analyses demonstrated a loss of catalysis rather than diminished substrate binding. These data suggest that loss of lipin 1-mediated PAP activity may be involved in the pathogenesis of rhabdomyolysis in lipin 1 deficiency.

  16. Signal transducer and activator of transcription 1 (STAT1) gain-of-function mutations and disseminated coccidioidomycosis and histoplasmosis

    PubMed Central

    Sampaio, Elizabeth P.; Hsu, Amy P.; Pechacek, Joseph; Bax, Hannelore I.; Dias, Dalton L.; Paulson, Michelle L.; Chandrasekaran, Prabha; Rosen, Lindsey B.; Carvalho, Daniel S.; Ding, Li; Vinh, Donald C.; Browne, Sarah K.; Datta, Shrimati; Milner, Joshua D.; Kuhns, Douglas B.; Long Priel, Debra A.; Sadat, Mohammed A.; Shiloh, Michael; De Marco, Brendan; Alvares, Michael; Gillman, Jason W.; Ramarathnam, Vivek; de la Morena, Maite; Bezrodnik, Liliana; Moreira, Ileana; Uzel, Gulbu; Johnson, Daniel; Spalding, Christine; Zerbe, Christa S.; Wiley, Henry; Greenberg, David E.; Hoover, Susan E.; Rosenzweig, Sergio D.; Galgiani, John N.; Holland, Steven M.

    2013-01-01

    Background Impaired signaling in the IFN-γ/IL-12 pathway causes susceptibility to severe disseminated infections with mycobacteria and dimorphic yeasts. Dominant gain-of-function mutations in signal transducer and activator of transcription 1 (STAT1) have been associated with chronic mucocutaneous candidiasis. Objective We sought to identify the molecular defect in patients with disseminated dimorphic yeast infections. Methods PBMCs, EBV-transformed B cells, and transfected U3A cell lines were studied for IFN-γ/IL-12 pathway function. STAT1 was sequenced in probands and available relatives. Interferon-induced STAT1 phosphorylation, transcriptional responses, protein-protein interactions, target gene activation, and function were investigated. Results We identified 5 patients with disseminated Coccidioides immitis or Histoplasma capsulatum with heterozygous missense mutations in the STAT1 coiled-coil or DNA-binding domains. These are dominant gain-of-function mutations causing enhanced STAT1 phosphorylation, delayed dephosphorylation, enhanced DNA binding and transactivation, and enhanced interaction with protein inhibitor of activated STAT1. The mutations caused enhanced IFN-γ–induced gene expression, but we found impaired responses to IFN-γ restimulation. Conclusion Gain-of-function mutations in STAT1 predispose to invasive, severe, disseminated dimorphic yeast infections, likely through aberrant regulation of IFN-γ–mediated inflammation. PMID:23541320

  17. Active RNAP pre-initiation sites are highly mutated by cytidine deaminases in yeast, with AID targeting small RNA genes

    PubMed Central

    Taylor, Benjamin JM; Wu, Yee Ling; Rada, Cristina

    2014-01-01

    Cytidine deaminases are single stranded DNA mutators diversifying antibodies and restricting viral infection. Improper access to the genome leads to translocations and mutations in B cells and contributes to the mutation landscape in cancer, such as kataegis. It remains unclear how deaminases access double stranded genomes and whether off-target mutations favor certain loci, although transcription and opportunistic access during DNA repair are thought to play a role. In yeast, AID and the catalytic domain of APOBEC3G preferentially mutate transcriptionally active genes within narrow regions, 110 base pairs in width, fixed at RNA polymerase initiation sites. Unlike APOBEC3G, AID shows enhanced mutational preference for small RNA genes (tRNAs, snoRNAs and snRNAs) suggesting a putative role for RNA in its recruitment. We uncover the high affinity of the deaminases for the single stranded DNA exposed by initiating RNA polymerases (a DNA configuration reproduced at stalled polymerases) without a requirement for specific cofactors. DOI: http://dx.doi.org/10.7554/eLife.03553.001 PMID:25237741

  18. Mutations in Recombination Activating Gene 1 and 2 in patients with severe combined immunodeficiency disorders in Egypt.

    PubMed

    Meshaal, Safa; El Hawary, Rabab; Elsharkawy, Marwa; Mousa, Reem K; Farid, Reem J; Abd Elaziz, Dalia; Alkady, Radwa; Galal, Nermeen; Massaad, Michel J; Boutros, Jeannette; Elmarsafy, Aisha

    2015-06-01

    The Recombination Activating Genes (RAG) 1/2 are important for the development and function of T and B cells. Loss of RAG1/2 function results in severe combined immunodeficiency (SCID), which could lead to early death. We studied the prevalence of RAG1/2 mutations in ten SCID patients in Egypt. We identified two novel homozygous nonsense mutations in RAG1, a novel homozygous deletion, and a previously reported homozygous missense mutation from four patients, as well as two homozygous mutations in RAG2 from the same patient. Prenatal diagnosis performed in the mother of a patient with RAG1 deficiency determined that the fetus was heterozygous for the same mutation. This represents the first report on RAG1/2 mutations in SCID patients in Egypt. The early diagnosis dramatically affects the outcome of the disease by allowing bone marrow transplantation at an early age, and providing prenatal diagnosis and genetic counseling for families with a history of SCID.

  19. Potent and selective activation of abscisic acid receptors in vivo by mutational stabilization of their agonist-bound conformation

    PubMed Central

    Mosquna, Assaf; Peterson, Francis C.; Park, Sang-Youl; Lozano-Juste, Jorge; Volkman, Brian F.; Cutler, Sean R.

    2011-01-01

    Pyrabactin resistance (PYR) 1 and its relatives belong to a family of soluble abscisic acid (ABA) receptors that inhibit type 2C protein phosphatases (PP2C) when in their agonist-stabilized conformation. Given their switch-like properties, we envisioned that mutations that stabilize their agonist-bound conformation could be used to activate signaling in vivo. To identify such mutations, we subjected PYR1 to site-saturation mutagenesis at 39 highly conserved residues that participate in ABA or PP2C contacts. All 741 possible single amino acid substitutions at these sites were tested to identify variants that increase basal PYR1-PP2C interactions, which uncovered activating mutations in 10 residues that preferentially cluster in PYR1's gate loop and C-terminal helix. The mutations cause measurable but incomplete receptor activation in vitro; however, specific triple and quadruple mutant combinations were constructed that promote an agonist-bound conformation, as measured by heteronuclear single quantum coherence NMR, and lead to full receptor activation. Moreover, these mutations retain functionality when introduced into divergent family members, and can therefore be used to dissect individual receptor function in vivo, which has been problematic because of redundancy and family size. Expression of activated PYL2 in Arabidopsis seeds activates ABA signaling by a number of measures: modulation of ABA-regulated gene expression, induction of hyperdormancy, and suppression of ABA deficiency phenotypes in the aba2-1 mutant. Our results set the stage for systematic gain-of-function studies of PYR1 and related ABA receptors and reveal that, despite the large number of receptors, activation of a single receptor is sufficient to activate signaling in planta. PMID:22139369

  20. T396I Mutation of Mouse Sufu Reduces the Stability and Activity of Gli3 Repressor

    PubMed Central

    Makino, Shigeru; Zhulyn, Olena; Mo, Rong; Puviindran, Vijitha; Zhang, Xiaoyun; Murata, Takuya; Fukumura, Ryutaro; Ishitsuka, Yuichi; Kotaki, Hayato; Matsumaru, Daisuke; Ishii, Shunsuke; Hui, Chi-Chung; Gondo, Yoichi

    2015-01-01

    Hedgehog signaling is primarily transduced by two transcription factors: Gli2, which mainly acts as a full-length activator, and Gli3, which tends to be proteolytically processed from a full-length form (Gli3FL) to an N-terminal repressor (Gli3REP). Recent studies using a Sufu knockout mouse have indicated that Sufu is involved in regulating Gli2 and Gli3 activator and repressor activity at multiple steps of the signaling cascade; however, the mechanism of specific Gli2 and Gli3 regulation remains to be elucidated. In this study, we established an allelic series of ENU-induced mouse strains. Analysis of one of the missense alleles, SufuT396I, showed that Thr396 residue of Sufu played a key role in regulation of Gli3 activity. SufuT396I/T396I embryos exhibited severe polydactyly, which is indicative of compromised Gli3 activity. Concomitantly, significant quantitative reductions of unprocessed Gli3 (Gli3FL) and processed Gli3 (Gli3REP) were observed in vivo as well as in vitro. Genetic experiments showed that patterning defects in the limb buds of SufuT396I/T396I were rescued by a constitutive Gli3REP allele (Gli3∆699), strongly suggesting that SufuT396I reduced the truncated Gli3 repressor. In contrast, SufuT396I qualitatively exhibited no mutational effects on Gli2 regulation. Taken together, the results of this study show that the Thr396 residue of Sufu is specifically required for regulation of Gli3 but not Gli2. This implies a novel Sufu-mediated mechanism in which Gli2 activator and Gli3 repressor are differentially regulated. PMID:25760946

  1. Antimutagenic and mutagenic activities of some terpenes in the bacterial reverse mutation assay.

    PubMed

    Di Sotto, Antonella; Evandri, Maria Grazia; Mazzanti, Gabriela

    2008-05-31

    The mutagenic and antimutagenic effects of linalool, linalyl acetate and beta-caryophyllene were evaluated by the bacterial reverse mutation assay on Salmonella typhimurium TA 98 and TA 100, and on Escherichia coli WP2uvrA strains. Neither linalool nor beta-caryophyllene showed mutagenicity, but linalyl acetate induced a statistically significant increase in the number of revertant colonies in WP2uvrA, both with and without S9 mixture. Linalool was devoid of antimutagenic activity against 2-nitrofluorene (2NF), sodium azide (SA), methyl methane sulfonate (MMS) and 2-aminoanthracene (2AA). In contrast, beta-caryophyllene showed a strong antimutagenic activity against 2NF: at the maximum concentration tested (6.40mg/plate) the number of 2NF-induced revertant colonies was reduced by 83.9%. beta-Caryophyllene also showed to counteract the mutagenicity of SA (in TA 100), MMS and 2AA (in WP2uvrA): the effect was weak against SA (inhibition lower than 25%) and moderate against MMS and 2AA (up to 30.5%). The antimutagenic activity of beta-caryophyllene observed here suggests further studies to evaluate its possible chemopreventive properties. PMID:18514567

  2. Mechanistic study of CuZn-SOD from Ipomoea carnea mutated at dimer interface: enhancement of peroxidase activity upon monomerization.

    PubMed

    Mishra, Panchanand; Dixit, Anshuman; Ray, Mamata; Sabat, Surendra Chandra

    2014-02-01

    The enzymatically active monomeric form of CuZn-superoxide dismutase has always been of interest to decipher the structure-function relationship in this class of enzymes. In the present study, spectroscopic and enzymatic characteristics of the dimeric and monomeric forms of recombinant Ipomoea carnea CuZn-superoxide dismutase were made to decipher their stability and altered catalytic properties. The monomeric form of protein was produced through site directed mutagenesis by replacing a conserved hydrophobic leucine with a polar lysine residue at the dimer-interface. Spectral characteristics of both the forms (monomer and dimer) showed the presence of novel electronic transitions. Superoxide scavenging activity of the mutated form was reduced to nearly half of the activity found in the native enzyme. Concomitantly, compared to native form the mutated enzyme showed an increase in peroxidase activity. High temperature dependent circular dichroism spectral analysis, differential scanning calorimetric profile, and the measurement of temperature dependent superoxide scavenging activity indicated an increased susceptibility of the mutated form to higher temperature as compared to the native form. The inhibitor studies like hydrogen peroxide, diethyldithiocarbamate and phenylglyoxal also indicate higher susceptibility, which might be due to, altered arrangement of active site residues as a consequence of the mutation. Molecular modeling and MD simulation studies further indicated that this specific mutation induces loss of hydrophobic interaction at dimer interface, resulting in the observed instability of the dimeric form. Increased peroxidative activity of the enzyme, upon monomerization may have physiological implication essentially in presence of high concentration of H2O2, as in case of plant cells specifically under stress conditions. PMID:24513093

  3. Mutational analysis of the active site of indoleglycerol phosphate synthase from Escherichia coli.

    PubMed Central

    Darimont, B.; Stehlin, C.; Szadkowski, H.; Kirschner, K.

    1998-01-01

    Indoleglycerol phosphate synthase catalyzes the ring closure of 1-(2-carboxyphenylamino)-1-deoxyribulose 5'-phosphate to indoleglycerol phosphate, the fifth step in the pathway of tryptophan biosynthesis from chorismate. Because chemical synthesis of indole derivatives from arylamino ketones requires drastic solvent conditions, it is interesting by what mechanism the enzyme catalyzes the same condensation reaction. Seven invariant polar residues in the active site of the enzyme from Escherichia coli have been mutated directly or randomly, to identify the catalytically essential ones. A strain of E. coli suitable for selecting and classifying active mutants by functional complementation was constructed by precise deletion of the trpC gene from the genome. Judged by growth rates of transformants on selective media, mutants with either S58 or S60 replaced by alanine were indistinguishable from the wild-type, but R186 replaced by alanine was still partially active. Saturation random mutagenesis of individual codons showed that E53 was partially replaceable by aspartate and cysteine, whereas K114, E163, and N184 could not be replaced by any other residue. Partially active mutant proteins were purified and their steady-state kinetic and inhibitor binding constants determined. Their relative catalytic efficiencies paralleled their relative complementation efficiencies. These results are compatible with the location of the essential residues in the active site of the enzyme and support a chemically plausible catalytic mechanism. It involves two enzyme-bound intermediates and general acid-base catalysis by K114 and E163 with the support of E53 and N184. PMID:9605328

  4. Substrate activation of brewers' yeast pyruvate decarboxylase is abolished by mutation of cysteine 221 to serine.

    PubMed

    Baburina, I; Gao, Y; Hu, Z; Jordan, F; Hohmann, S; Furey, W

    1994-05-10

    Brewers' yeast pyruvate decarboxylase (EC 4.1.1.1), a thiamin diphosphate and Mg(II)-dependent enzyme, isolated from Saccharomyces cerevisiae possesses four cysteines/subunit at positions 69, 152, 221, and 222. Earlier studies conducted on a variant of the enzyme with a single Cys at position 221 (derived from a gene that was the product of spontaneous fusion) showed that this enzyme is still subject to substrate activation [Zeng, X., Farrenkopf, B., Hohmann, S., Jordan, F., Dyda, F., & Furey, W. (1993) Biochemistry 32, 2704-2709], indicating that if Cys was responsible for this activation, it had to be C221. To further test the hypothesis, the C221S and C222S single and the C221S-C222S double mutants were constructed. It is clearly shown that the mutation at C221, but not at C222, leads to abolished substrate activation according to a number of kinetic criteria, both steady state and pre steady state. On the basis of the three-dimensional structure of the enzyme [Dyda, F., Furey, W., Swaminathan, S., Sax, M., Farrenkopf, B., Jordan, F. (1993) Biochemistry 32, 6165-6170], it is obvious that while C221 is located on the beta domain, whereas thiamin diphosphate is wedged at the interface of the alpha and gamma domains, addition of pyruvate or pyruvamide as a hemiketal adduct to the sulfur of C221 can easily bridge the gap between the beta and alpha domains. In fact, residues in one or both domains must be dislocated by this adduct formation. It is very likely that regulation as expressed in substrate activation is transmitted via this direct contact made between the two domains in the presence of the activator.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Catalytically Active Guanylyl Cyclase B Requires Endoplasmic Reticulum-mediated Glycosylation, and Mutations That Inhibit This Process Cause Dwarfism.

    PubMed

    Dickey, Deborah M; Edmund, Aaron B; Otto, Neil M; Chaffee, Thomas S; Robinson, Jerid W; Potter, Lincoln R

    2016-05-20

    C-type natriuretic peptide activation of guanylyl cyclase B (GC-B), also known as natriuretic peptide receptor B or NPR2, stimulates long bone growth, and missense mutations in GC-B cause dwarfism. Four such mutants (L658F, Y708C, R776W, and G959A) bound (125)I-C-type natriuretic peptide on the surface of cells but failed to synthesize cGMP in membrane GC assays. Immunofluorescence microscopy also indicated that the mutant receptors were on the cell surface. All mutant proteins were dephosphorylated and incompletely glycosylated, but dephosphorylation did not explain the inactivation because the mutations inactivated a "constitutively phosphorylated" enzyme. Tunicamycin inhibition of glycosylation in the endoplasmic reticulum or mutation of the Asn-24 glycosylation site decreased GC activity, but neither inhibition of glycosylation in the Golgi by N-acetylglucosaminyltransferase I gene inactivation nor PNGase F deglycosylation of fully processed GC-B reduced GC activity. We conclude that endoplasmic reticulum-mediated glycosylation is required for the formation of an active catalytic, but not ligand-binding domain, and that mutations that inhibit this process cause dwarfism. PMID:26980729

  6. Catalytically Active Guanylyl Cyclase B Requires Endoplasmic Reticulum-mediated Glycosylation, and Mutations That Inhibit This Process Cause Dwarfism.

    PubMed

    Dickey, Deborah M; Edmund, Aaron B; Otto, Neil M; Chaffee, Thomas S; Robinson, Jerid W; Potter, Lincoln R

    2016-05-20

    C-type natriuretic peptide activation of guanylyl cyclase B (GC-B), also known as natriuretic peptide receptor B or NPR2, stimulates long bone growth, and missense mutations in GC-B cause dwarfism. Four such mutants (L658F, Y708C, R776W, and G959A) bound (125)I-C-type natriuretic peptide on the surface of cells but failed to synthesize cGMP in membrane GC assays. Immunofluorescence microscopy also indicated that the mutant receptors were on the cell surface. All mutant proteins were dephosphorylated and incompletely glycosylated, but dephosphorylation did not explain the inactivation because the mutations inactivated a "constitutively phosphorylated" enzyme. Tunicamycin inhibition of glycosylation in the endoplasmic reticulum or mutation of the Asn-24 glycosylation site decreased GC activity, but neither inhibition of glycosylation in the Golgi by N-acetylglucosaminyltransferase I gene inactivation nor PNGase F deglycosylation of fully processed GC-B reduced GC activity. We conclude that endoplasmic reticulum-mediated glycosylation is required for the formation of an active catalytic, but not ligand-binding domain, and that mutations that inhibit this process cause dwarfism.

  7. Identifying activating mutations in the EGFR gene: prognostic and therapeutic implications in non-small cell lung cancer *

    PubMed Central

    Lopes, Gabriel Lima; Vattimo, Edoardo Filippo de Queiroz; de Castro, Gilberto

    2015-01-01

    Abstract Lung cancer is the leading cause of cancer-related deaths worldwide. Promising new therapies have recently emerged from the development of molecular targeted drugs; particularly promising are those blocking the signal transduction machinery of cancer cells. One of the most widely studied cell signaling pathways is that of EGFR, which leads to uncontrolled cell proliferation, increased cell angiogenesis, and greater cell invasiveness. Activating mutations in the EGFR gene (deletions in exon 19 and mutation L858R in exon 21), first described in 2004, have been detected in approximately 10% of all non-squamous non-small cell lung cancer (NSCLC) patients in Western countries and are the most important predictors of a response to EGFR tyrosine-kinase inhibitors (EGFR-TKIs). Studies of the EGFR-TKIs gefitinib, erlotinib, and afatinib, in comparison with platinum-based regimens, as first-line treatments in chemotherapy-naïve patients have shown that the EGFR-TKIs produce gains in progression-free survival and overall response rates, although only in patients whose tumors harbor activating mutations in the EGFR gene. Clinical trials have also shown EGFR-TKIs to be effective as second- and third-line therapies in advanced NSCLC. Here, we review the main aspects of EGFR pathway activation in NSCLC, underscore the importance of correctly identifying activating mutations in the EGFR gene, and discuss the main outcomes of EGFR-TKI treatment in NSCLC. PMID:26398757

  8. Mutational spectrum of adult T-ALL.

    PubMed

    Neumann, Martin; Vosberg, Sebastian; Schlee, Cornelia; Heesch, Sandra; Schwartz, Stefan; Gökbuget, Nicola; Hoelzer, Dieter; Graf, Alexander; Krebs, Stefan; Bartram, Isabelle; Blum, Helmut; Brüggemann, Monika; Hecht, Jochen; Bohlander, Stefan K; Greif, Philipp A; Baldus, Claudia D

    2015-02-20

    Novel target discovery is warranted to improve treatment in adult T-cell acute lymphoblastic leukemia (T-ALL) patients. We provide a comprehensive study on mutations to enhance the understanding of therapeutic targets and studied 81 adult T-ALL patients. NOTCH1 exhibitedthe highest mutation rate (53%). Mutation frequencies of FBXW7 (10%), WT1 (10%), JAK3 (12%), PHF6 (11%), and BCL11B (10%) were in line with previous reports. We identified recurrent alterations in transcription factors DNM2, and RELN, the WNT pathway associated cadherin FAT1, and in epigenetic regulators (MLL2, EZH2). Interestingly, we discovered novel recurrent mutations in the DNA repair complex member HERC1, in NOTCH2, and in the splicing factor ZRSR2. A frequently affected pathway was the JAK/STAT pathway (18%) and a significant proportion of T-ALL patients harboured mutations in epigenetic regulators (33%), both predominantly found in the unfavourable subgroup of early T-ALL. Importantly, adult T-ALL patients not only showed a highly heterogeneous mutational spectrum, but also variable subclonal allele frequencies implicated in therapy resistance and evolution of relapse. In conclusion, we provide novel insights in genetic alterations of signalling pathways (e.g. druggable by γ-secretase inhibitors, JAK inhibitors or EZH2 inhibitors), present in over 80% of all adult T-ALL patients, that could guide novel therapeutic approaches.

  9. Mutational spectrum of adult T-ALL.

    PubMed

    Neumann, Martin; Vosberg, Sebastian; Schlee, Cornelia; Heesch, Sandra; Schwartz, Stefan; Gökbuget, Nicola; Hoelzer, Dieter; Graf, Alexander; Krebs, Stefan; Bartram, Isabelle; Blum, Helmut; Brüggemann, Monika; Hecht, Jochen; Bohlander, Stefan K; Greif, Philipp A; Baldus, Claudia D

    2015-02-20

    Novel target discovery is warranted to improve treatment in adult T-cell acute lymphoblastic leukemia (T-ALL) patients. We provide a comprehensive study on mutations to enhance the understanding of therapeutic targets and studied 81 adult T-ALL patients. NOTCH1 exhibitedthe highest mutation rate (53%). Mutation frequencies of FBXW7 (10%), WT1 (10%), JAK3 (12%), PHF6 (11%), and BCL11B (10%) were in line with previous reports. We identified recurrent alterations in transcription factors DNM2, and RELN, the WNT pathway associated cadherin FAT1, and in epigenetic regulators (MLL2, EZH2). Interestingly, we discovered novel recurrent mutations in the DNA repair complex member HERC1, in NOTCH2, and in the splicing factor ZRSR2. A frequently affected pathway was the JAK/STAT pathway (18%) and a significant proportion of T-ALL patients harboured mutations in epigenetic regulators (33%), both predominantly found in the unfavourable subgroup of early T-ALL. Importantly, adult T-ALL patients not only showed a highly heterogeneous mutational spectrum, but also variable subclonal allele frequencies implicated in therapy resistance and evolution of relapse. In conclusion, we provide novel insights in genetic alterations of signalling pathways (e.g. druggable by γ-secretase inhibitors, JAK inhibitors or EZH2 inhibitors), present in over 80% of all adult T-ALL patients, that could guide novel therapeutic approaches. PMID:25595890

  10. Influence of Drug Resistance Mutations on the Activity of HIV-1 Subtypes A and B Integrases: a Comparative Study.

    PubMed

    Shadrina, O A; Zatsepin, T S; Agapkina, Yu Yu; Isaguliants, M G; Gottikh, M B

    2015-01-01

    Integration of human immunodeficiency virus (HIV-1) DNA into the genome of an infected cell is one of the key steps in the viral replication cycle. The viral enzyme integrase (IN), which catalyzes the integration, is an attractive target for the development of new antiviral drugs. However, the HIV-1 therapy often results in the IN gene mutations inducing viral resistance to integration inhibitors. To assess the impact of drug resistance mutations on the activity of IN of HIV-1 subtype A strain FSU-A, which is dominant in Russia, variants of the consensus IN of this subtype containing the primary resistance mutations G118R and Q148K and secondary compensatory substitutions E138K and G140S were prepared and characterized. Comparative study of these enzymes with the corresponding mutants of IN of HIV-1 subtype B strains HXB-2 was performed. The mutation Q148K almost equally reduced the activity of integrases of both subtypes. Its negative effect was partially compensated by the secondary mutations E138K and G140S. Primary substitution G118R had different influence on the activity of proteins of the subtypes A and B, and the compensatory effect of the secondary substitution E138K also depended on the viral subtype. Comparison of the mutants resistance to the known strand transfer inhibitors raltegravir and elvitegravir, and a new inhibitor XZ-259 (a dihydro-1H-isoindol derivative), showed that integrases of both subtypes with the Q148K mutation were insensitive to raltegravir and elvitegravir but were effectively inhibited by XZ-259. The substitution G118R slightly reduced the efficiency of IN inhibition by raltegravir and elvitegravir and caused no resistance to XZ_259.

  11. Mutational Analysis of Substrate Interactions with the Active Site of Dialkylglycine Decarboxylase

    PubMed Central

    Fogle, Emily J.; Toney, Michael D.

    2010-01-01

    Pyridoxal phosphate (PLP) dependent enzymes catalyze many different types of reactions at the α-, β-, and γ-carbons of amine and amino acid substrates. Dialkylglycine decarboxylase (DGD) is an unusual PLP dependent enzyme that catalyzes two reaction types, decarboxylation and transamination, in the same active site. A structurally-based, functional model has been proposed for the DGD active site, which maintains that R406 is important in determining substrate specificity through interactions with the substrate carboxylate while W138 provides specificity for short-chain alkyl groups. The mechanistic roles of R406 and W138 were investigated using site directed mutagenesis, alternate substrates, and analysis of steady-state and half-reaction kinetics. Experiments on the R406M and R406K mutants confirm the importance of R406 in substrate binding. Surprisingly, this work also shows that the positive charge of R406 facilitates catalysis of decarboxylation. The W138F mutant demonstrates that W138 indeed acts to limit the size of the subsite C binding pocket, determining specificity for 2,2-dialkylglycines with small side chains as predicted by the model. Finally, work with the double mutant W138F/M141R shows that these mutations expand substrate specificity to include L-glutamate and lead to an increase in specificity for L-glutamate over 2-aminoisobutyrate of approximately eight orders of magnitude compared to WT DGD. PMID:20540501

  12. Mutational Analysis of Escherichia coli MoeA: Two Functional Activities Map to the Active Site Cleft

    SciTech Connect

    Nichols,J.; Xiang, S.; Schindelin, H.; Rajagopalan, K.

    2007-01-01

    The molybdenum cofactor is ubiquitous in nature, and the pathway for Moco biosynthesis is conserved in all three domains of life. Recent work has helped to illuminate one of the most enigmatic steps in Moco biosynthesis, ligation of metal to molybdopterin (the organic component of the cofactor) to form the active cofactor. In Escherichia coli, the MoeA protein mediates ligation of Mo to molybdopterin while the MogA protein enhances this process in an ATP-dependent manner. The X-ray crystal structures for both proteins have been previously described as well as two essential MogA residues, Asp49 and Asp82. Here we describe a detailed mutational analysis of the MoeA protein. Variants of conserved residues at the putative active site of MoeA were analyzed for a loss of function in two different, previously described assays, one employing moeA{sup -} crude extracts and the other utilizing a defined system. Oddly, no correlation was observed between the activity in the two assays. In fact, our results showed a general trend toward an inverse relationship between the activity in each assay. Moco binding studies indicated a strong correlation between a variant's ability to bind Moco and its activity in the purified component assay. Crystal structures of the functionally characterized MoeA variants revealed no major structural changes, indicating that the functional differences observed are not due to disruption of the protein structure. On the basis of these results, two different functional areas were assigned to regions at or near the MoeA active site cleft.

  13. Pacemaker activity of the human sinoatrial node: effects of HCN4 mutations on the hyperpolarization-activated current.

    PubMed

    Verkerk, Arie O; Wilders, Ronald

    2014-03-01

    The hyperpolarization-activated 'funny' current, If, plays an important modulating role in the pacemaker activity of the human sinoatrial node (SAN). If is carried by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are tetramers built of four HCN subunits. In human SAN, HCN4 is the most abundant of the four isoforms of the HCN family. Since 2003, several loss-of-function mutations in the HCN4 gene, which encodes the HCN4 protein, or in the KCNE2 gene, which encodes the MiRP1 accessory β-subunit, have been associated with sinus node dysfunction. Voltage-clamp experiments on HCN4 channels expressed in COS-7 cells, Xenopus oocytes, or HEK-293 cells have revealed changes in the expression and kinetics of mutant channels, but the extent to which these changes would affect If flowing during a human SAN action potential is unresolved. Here, we review the changes in expression and kinetics of HCN4 mutant channels and provide an overview of their effects on If during the time course of a human SAN action potential, both under resting conditions and upon adrenergic stimulation. These effects are assessed in simulated action potential clamp experiments, with action potentials recorded from isolated human SAN pacemaker cells as command potential and kinetics of If based on voltage-clamp data from these cells. Results from in vitro and in silico experiments show several inconsistencies with clinical observations, pointing to challenges for future research.

  14. Mutational spectra of PTEN/MMAC1 gene: a tumor suppressor with lipid phosphatase activity.

    PubMed

    Ali, I U; Schriml, L M; Dean, M

    1999-11-17

    PTEN/MMAC1 (phosphatase, tensin homologue/mutated in multiple advanced cancers) is a tumor suppressor protein that has sequence homology with dual-specificity phosphatases, which are capable of dephosphorylating both tyrosine phosphate and serine/threonine phosphate residues on proteins. The in vivo function of PTEN/MMAC1 appears to be dephosphorylation of phosphotidylinositol 3,4, 5-triphosphate. The PTEN/MMAC1 gene is mutated in the germline of patients with rare autosomal dominant cancer syndromes and in subsets of specific cancers. Here we review the mutational spectra of the PTEN/MMAC1 gene in tumors from various tissues, especially endometrium, brain, prostate, and ovary, in which the gene is inactivated very frequently. Germline and somatic mutations in the PTEN/MMAC1 gene occur mostly in the protein coding region and involve the phosphatase domain and poly(A)(6) stretches. Compared with germline alterations found in the PTEN/MMAC1 gene, there is a substantially increased frequency of frameshift mutations in tumors. Glioblastomas and endometrial carcinomas appear to have distinct mutational spectra, probably reflecting differences in the underlying mechanisms of inactivation of the PTEN/MMAC1 gene in the two tissue types. Also, depending on the tissue type, the gene appears to be involved in the initiation or the progression of cancers. Further understanding of PTEN/MMAC1 gene mutations in different tumors and the physiologic consequences of these mutations is likely to open up new therapeutic opportunities for targeting this critical gene.

  15. Targeting EZH2 methyltransferase activity in ARID1A mutated cancer cells is synthetic lethal

    PubMed Central

    Biter, Benjamin G.; Aird, Katherine M.; Garipov, Azat; Li, Hua; Amatangelo, Michael; Kossenkov, Andrew V.; Schultz, David C.; Liu, Qin; Shih, Ie-Ming; Conejo-Garcia, Jose R.; Speicher, David W.; Zhang, Rugang

    2015-01-01

    ARID1A, a chromatin remodeler, shows one of the highest mutation rates across many cancer types. Notably, ARID1A is mutated in over 50% of ovarian clear cell carcinomas, which currently has no effective therapy. To date, clinically applicable targeted cancer therapy based on ARID1A mutational status has not been described. Here we show that inhibition of the EZH2 methyltransferase acts in a synthetic lethal manner in ARID1A mutated ovarian cancer cells. ARID1A mutational status correlates with response to the EZH2 inhibitor. We identified PIK3IP1 as a direct ARID1A/EZH2 target, which is upregulated by EZH2 inhibition and contributes to the observed synthetic lethality by inhibiting PI3K/AKT signaling. Significantly, EZH2 inhibition causes regression of ARID1A mutated ovarian tumors in vivo. Together, these data demonstrate for the first time a synthetic lethality between ARID1A mutation and EZH2 inhibition. They indicate that pharmacological inhibition of EZH2 represents a novel treatment strategy for ARID1A mutated cancers. PMID:25686104

  16. Diiron centre mutations in Ciona intestinalis alternative oxidase abolish enzymatic activity and prevent rescue of cytochrome oxidase deficiency in flies

    PubMed Central

    Andjelković, Ana; Oliveira, Marcos T.; Cannino, Giuseppe; Yalgin, Cagri; Dhandapani, Praveen K.; Dufour, Eric; Rustin, Pierre; Szibor, Marten; Jacobs, Howard T.

    2015-01-01

    The mitochondrial alternative oxidase, AOX, carries out the non proton-motive re-oxidation of ubiquinol by oxygen in lower eukaryotes, plants and some animals. Here we created a modified version of AOX from Ciona instestinalis, carrying mutations at conserved residues predicted to be required for chelation of the diiron prosthetic group. The modified protein was stably expressed in mammalian cells or flies, but lacked enzymatic activity and was unable to rescue the phenotypes of flies knocked down for a subunit of cytochrome oxidase. The mutated AOX transgene is thus a potentially useful tool in studies of the physiological effects of AOX expression. PMID:26672986

  17. Achievement of Cure with Gefitinib in Advanced Lung Adenocarcinoma Harboring an Activating EGFR Mutation: A Case Report

    PubMed Central

    Kuwata, Taiji; Yoneda, Kazue; Kobayashi, Kenichi; Oyama, Rintarou; Matumiya, Hiroki; Shinohara, Shuichi; Takenaka, Masaru; Oka, Soichi; Chikaishi, Yasuhiro; Imanishi, Naoko; Kuroda, Koji; Tanaka, Fumihiro

    2016-01-01

    Tyrosine kinase inhibitors (TKIs) of the epidermal growth factor receptor (EGFR) may achieve long-term survival in selected cases with advanced non-small cell lung cancer harboring activating mutations in the EGFR gene, but a cured case has not been reported yet. Here, we present the first case of EGFR-mutated lung adenocarcinoma cured with an EGFR-TKI, as the 75-year-old Japanese man has achieved complete response with gefitinib treatment and has survived without tumor 10 years after termination of gefitinib treatment.

  18. Network Analysis of Genome-Wide Selective Constraint Reveals a Gene Network Active in Early Fetal Brain Intolerant of Mutation

    PubMed Central

    Choi, Jinmyung; Samocha, Kaitlin E.; Daly, Mark J.

    2016-01-01

    Using robust, integrated analysis of multiple genomic datasets, we show that genes depleted for non-synonymous de novo mutations form a subnetwork of 72 members under strong selective constraint. We further show this subnetwork is preferentially expressed in the early development of the human hippocampus and is enriched for genes mutated in neurological Mendelian disorders. We thus conclude that carefully orchestrated developmental processes are under strong constraint in early brain development, and perturbations caused by mutation have adverse outcomes subject to strong purifying selection. Our findings demonstrate that selective forces can act on groups of genes involved in the same process, supporting the notion that purifying selection can act coordinately on multiple genes. Our approach provides a statistically robust, interpretable way to identify the tissues and developmental times where groups of disease genes are active. PMID:27305007

  19. Identification of a novel HER3 activating mutation homologous to EGFR-L858R in lung cancer

    PubMed Central

    Umelo, Ijeoma; Noeparast, Amir; Chen, Gang; Renard, Marleen; Geers, Caroline; Vansteenkiste, Johan; Giron, Philippe; De Wever, Olivier; Teugels, Erik; De Grève, Jacques

    2016-01-01

    Somatic mutations found within the tyrosine kinase domain (TKD) of the human epidermal growth factor (HER) family of receptors have been implicated in the development and progression of non-small cell lung cancer (NSCLC). However, no conclusive reports have described pathogenic mutations in kinase-impaired HER3. Here, we report a case of an advanced chemotherapy-resistant NSCLC, harboring a novel HER3V855A somatic mutation homologous to the EGFRL858Ractivating mutation. Co-expression of HER3V855A and wild-type HER2 enhances ligand-induced transformation of murine and human cell lines, while HER-targeted inhibitors potently suppress mutant HER3 activity. Consistent with these observations, in silico computational modeling predicts that mutant V855A alters the kinase domain and c-terminal end of the HER3 protein. Taken together, these findings provide a basis for the clinical exploration of targeted therapies in HER3 mutant NSCLC and by extrapolation, in other cancers that more frequently carry somatic HER3 mutations. PMID:26689995

  20. Evidence in Latin America of recurrence of V388M, a phenylketonuria mutation with high in vitro residual activity

    SciTech Connect

    Desviat, L.R.; Perez, B.; De Lucca, M.

    1995-08-01

    Phenylketonuria mutation V388M is frequent in the Iberian Peninsula. In vitro, the V388M mutant enzyme has similar immunoreactive protein and phenylalanine hydroxylase mRNA and had 43% residual activity, which correlates well with the mild phenotype exhibited by the homozygous patients. In Spain it has been detected in 5.7% of the mutant alleles and is always associated with haplotype 1.7. This mutation is also present in high frequency in some Latin American countries (Brazil, 9% Chile, 13%). It is interesting that in Chile most of the alleles bearing this mutation carry haplotype 4.3, although in Brazil it is found only on the background of haplotype 1.7. The origin of V388M in Spain on haplotype 1.7 and in Chile on haplotype 4.3 is clearly different. Recurrence is the most plausible explanation, because the mutation involves a CpG dinucleotide, and a recombination event transferring the mutation from haplotype 1 to 4 is unlikely. 29 refs., 2 figs., 3 tabs.

  1. Probing impact of active site residue mutations on stability and activity of Neisseria polysaccharea amylosucrase.

    PubMed

    Daudé, David; Topham, Christopher M; Remaud-Siméon, Magali; André, Isabelle

    2013-12-01

    The amylosucrase from Neisseria polysaccharea is a transglucosidase from the GH13 family of glycoside-hydrolases that naturally catalyzes the synthesis of α-glucans from the widely available donor sucrose. Interestingly, natural molecular evolution has modeled a dense hydrogen bond network at subsite -1 responsible for the specific recognition of sucrose and conversely, it has loosened interactions at the subsite +1 creating a highly promiscuous subsite +1. The residues forming these subsites are considered to be likely involved in the activity as well as the overall stability of the enzyme. To assess their role, a structure-based approach was followed to reshape the subsite -1. A strategy based on stability change predictions, using the FoldX algorithm, was considered to identify the best candidates for site-directed mutagenesis and guide the construction of a small targeted library. A miniaturized purification protocol was developed and both mutant stability and substrate promiscuity were explored. A range of 8 °C between extreme melting temperature values was observed and some variants were able to synthesize series of oligosaccharides with distributions differing from that of the parental enzyme. The crucial role of subsite -1 was thus highlighted and the biocatalysts generated can now be considered as starting points for further engineering purposes.

  2. An activating mutation of the follicle-stimulating hormone receptor autonomously sustains spermatogenesis in a hypophysectomized man

    SciTech Connect

    Gromoll, J.; Simoni, M.; Nieschlag, E.

    1996-04-01

    As both gonadotropins, LH and FSH, are required for normal spermatogenesis, patients with pituitary insufficiency need hCG plus human menopausal gonadotropin therapy to induce spermatogenesis and establish fertility. In a patient hypophysectomized because of a pituitary tumor, who, despite undetectable serum gonadotropin levels, had normal testis volume and semen parameters and fathered three children under testosterone substitution alone, we hypothesized an activating mutation of the FSH receptor. Exon 10 of the FSH receptor gene was amplified from genomic DNA by PCR, screened by single stranded conformation polymorphism gel electrophoresis, and sequenced. We identified a heterozygous A{r_arrow}G base change at nucleotide position 1700, leading to an Asp,Gly transition in codon 567 in the third intracytoplasmatic loop. COS-7 cells transiently transfected with the mutated receptor displayed a 1.5-fold increase in basal cAMP production compared to wild-type receptor, indicating that this mutation leads to ligand-independent constitutive activation of the FSH receptor. We conclude that this activating mutation of the FSH receptor, the first ever described, autonomously sustains spermatogenesis in the absence of gonadotropins. 31 refs., 5 figs., 1 tab.

  3. A Mutational Analysis of the Active Site Loop Residues in cis-3-Chloroacrylic Acid Dehalogenase

    PubMed Central

    Schroeder, Gottfried K.; Huddleston, Jamison P.; Johnson, William H.; Whitman, Christian P.

    2013-01-01

    cis -3-Chloroacrylic acid dehalogenase (cis-CaaD) from Pseudomonas pavonaceae 170 and a homologue from Corynebacterium glutamicum designated Cg10062 share 34% sequence identity (54% similarity). The former catalyzes a key step in a bacterial catabolic pathway for the nematocide 1,3-dichloropropene, whereas the latter has no known biological activity. Although Cg10062 has the six active site residues (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, Glu-114) that are critical for cis-CaaD activity, it shows only a low level cis-CaaD activity and lacks the specificity of cis-CaaD: Cg10062 processes both isomers of 3-chloroacrylate with a preference for the cis-isomer. Although the basis for these differences is unknown, a comparison of the crystal structures of the enzymes covalently modified by an adduct resulting from their incubation with the same inhibitor offers a possible explanation. A 6-residue active site loop in cis-CaaD shows a strikingly different conformation from that observed in Cg10062: the loop closes down on the active site of cis-CaaD, but not on that of Cg10062. In order to examine what this loop might contribute to cis-CaaD catalysis and specificity, the residues were changed individually to those found in Cg10062. Subsequent kinetic and mechanistic analysis suggests that the T34A mutant of cis-CaaD is more Cg10062-like. The mutant enzyme shows a 4-fold increase in Km (using cis-3-bromoacrylate), but not to the degree observed for Cg10062 (687-fold). The mutation also causes a 4-fold decrease in the burst rate (compared to the wild type cis-CaaD), whereas Cg10062 shows no burst rate. More telling is the reaction of the T34A mutant of cis-CaaD with the alternate substrate, 2,3-butadienoate. In the presence of NaBH4 and the allene, cis-CaaD is completely inactivated after one turnover due to the covalent modification of Pro-1. The same experiment with Cg10062 does not result in the covalent modification of Pro-1. The different outcomes are attributed to

  4. The influence of allosteric modulators and transmembrane mutations on desensitisation and activation of α7 nicotinic acetylcholine receptors

    PubMed Central

    Chatzidaki, Anna; D'Oyley, Jarryl M.; Gill-Thind, JasKiran K.; Sheppard, Tom D.; Millar, Neil S.

    2015-01-01

    Acetylcholine activates nicotinic acetylcholine receptors (nAChRs) by binding at an extracellular orthosteric site. Previous studies have described several positive allosteric modulators (PAMs) that are selective for homomeric α7 nAChRs. These include type I PAMs, which exert little or no effect on the rate of receptor desensitisation, and type II PAMs, which cause a dramatic loss of agonist-induced desensitisation. Here we report evidence that transmembrane mutations in α7 nAChRs have diverse effects on receptor activation and desensitisation by allosteric ligands. It has been reported previously that the L247T mutation, located toward the middle of the second transmembrane domain (at the 9′ position), confers reduced levels of desensitisation. In contrast, the M260L mutation, located higher up in the TM2 domain (at the 22′ position), does not show any difference in desensitisation compared to wild-type receptors. We have found that in receptors containing the L247T mutation, both type I PAMs and type II PAMs are converted into non-desensitising agonists. In contrast, in receptors containing the M260L mutation, this effect is seen only with type II PAMs. These findings, indicating that the M260L mutation has a selective effect on type II PAMs, have been confirmed both with previously described PAMs and also with a series of novel α7-selective PAMs. The novel PAMs examined in this study have close chemical similarity but diverse pharmacological properties. For example, they include compounds displaying effects on receptor desensitisation that are typical of classical type I and type II PAMs but, in addition, they include compounds with intermediate properties. PMID:25998276

  5. [Antirestriction and antimodification activities of the T7 Ocr protein: effect of mutations in interface].

    PubMed

    Zavil'gel'skiĭ, G B; Kotova, V Iu; Rastorguev, S M

    2009-01-01

    Antirestriction protein Ocr (bacteriophage T7) is specific inhibitor of the type I restriction-modification enzymes. The bacteriophage T7 0.3 (ocr) gene is cloned in pUC18 vector. It was shown that T7 Ocr protein inhibits both restriction and modification activities of the type I restriction-modification enzyme (EcoKI) in Escherichia coli K12 cells. The mutation form of Ocr-Ocr F53D A57E, which inhibits only the restriction activity of EcoKI-enzyme, was constructed. The T7 0.3 (ocr) and the Photorhabdus luminescens luxCDABE genes were cloned in pZ-series vectors with the P(ltet0-1) promoter which is tightly repressible by the TetR repressor. Controlling the expression of the lux-genes encoding bacterial luciferase demonstrates that the P(ltet0-1) promoter can be regulated over and up to 5000 fold range by supplying anhydrotetracycline (aTc) to the E. coli MG1655Z1 tetR+ cells. It was determined the dependence of the effectiveness of the antirestriction activity of the Ocr and Ocr F53D A57E proteins on the intracellular concentration. It was shown that the values of the dissociation constants K(d) for Ocr and Ocr F53D A57E proteins with EcoKI enzyme differ in 1000 times: Kd (Ocr) = 10(-10) M, K(d) (Ocr F53D A57E) = 10(-7) M. PMID:19334532

  6. Mutation of Asn28 Disrupts the Dimerization and Enzymatic Activity of SARS 3CL

    SciTech Connect

    Barrila, J.; Gabelli, S; Bacha, U; Amzel, M; Freire, E

    2010-01-01

    Coronaviruses are responsible for a significant proportion of annual respiratory and enteric infections in humans and other mammals. The most prominent of these viruses is the severe acute respiratory syndrome coronavirus (SARS-CoV) which causes acute respiratory and gastrointestinal infection in humans. The coronavirus main protease, 3CL{sup pro}, is a key target for broad-spectrum antiviral development because of its critical role in viral maturation and high degree of structural conservation among coronaviruses. Dimerization is an indispensable requirement for the function of SARS 3CL{sup pro} and is regulated through mechanisms involving both direct and long-range interactions in the enzyme. While many of the binding interactions at the dimerization interface have been extensively studied, those that are important for long-range control are not well-understood. Characterization of these dimerization mechanisms is important for the structure-based design of new treatments targeting coronavirus-based infections. Here we report that Asn28, a residue 11 {angstrom} from the closest residue in the opposing monomer, is essential for the enzymatic activity and dimerization of SARS 3CLpro. Mutation of this residue to alanine almost completely inactivates the enzyme and results in a 19.2-fold decrease in the dimerization K{sub d}. The crystallographic structure of the N28A mutant determined at 2.35 {angstrom} resolution reveals the critical role of Asn28 in maintaining the structural integrity of the active site and in orienting key residues involved in binding at the dimer interface and substrate catalysis. These findings provide deeper insight into complex mechanisms regulating the activity and dimerization of SARS 3CL{sup pro}.

  7. Absence of missense mutations in activated c-myc genes in avian leukosis virus-induced B-cell lymphomas

    SciTech Connect

    Hahn, M.; Hayward, W.S.

    1988-06-01

    The authors determined the nucleotide sequences of two independent DNA clones which contained the activated c-myc genes from avian leukosis virus-induced B-cell lymphomas. Neither of these c-myce genes contained missense mutations. This strongly supports the notion that the c-myc photo-oncogene in avian leukosis virus-induced B-cell lymphomas can be oncogenically activated by altered expression of the gene without a change in the primary structure of the gene product.

  8. Anti-tumor activity of ESX1 on cancer cells harboring oncogenic K-ras mutation

    SciTech Connect

    Nakajima, Junta; Ishikawa, Susumu; Hamada, Jun-Ichi; Yanagihara, Masatomo; Koike, Takao; Hatakeyama, Masanori

    2008-05-23

    Human ESX1 is a 65-kilodalton (kDa) paired-like homeoprotein that is proteolytically processed into N-terminal 45-kDa and C-terminal 20-kDa fragments. The N-terminal ESX1 fragment, which contains the homeodomain, localizes to the nucleus and represses mRNA transcription from the K-ras gene. When we inoculated human colorectal carcinoma HCT116 constitutive expressing N-terminal region of ESX1 (N-ESX1) into nude mice, transfectant cells uniformly showed decreased tumor-forming activity compared with that of the parental cells. Furthermore, pretreatment of HCT116 carcinoma cells with a fusion protein consisting of N-ESX1 and the protein-transduction domain derived from the human immunodeficiency virus type-1 TAT protein gave rise to a dramatic reduction in the tumorigenicity of HCT116 cells in nude mice. Our results provide first in vivo evidence for the molecular targeting therapeutic application of the K-ras repressor ESX1, especially TAT-mediated transduction of N-ESX1, in the treatment of human cancers having oncogenic K-ras mutations.

  9. Characterization and genetic mapping of a mutation affecting apurinic endonuclease activity in Staphylococcus aureus.

    PubMed Central

    Tam, J E; Pattee, P A

    1986-01-01

    Protoplast fusion between the Rec- mutant RN981 (L. Wyman, R. V. Goering, and R. P. Novick, Genetics 76:681-702, 1974) of Staphylococcus aureus NCTC 8325 and a Rec+ NCTC 8325 derivative yielded Rec+ recombinants that exhibited the increased sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine characteristic of RN981. Transformation analyses identified a specific mutation, designated ngr-374, that was responsible not only for N-methyl-N'-nitro-N-nitrosoguanidine sensitivity, but also sensitivity to methyl methanesulfonate, ethyl methanesulfonate, nitrous acid, and UV irradiation. However, ngr-374-carrying recombinants showed no significant increase in their sensitivity to mitomycin C or 4-nitroquinoline 1-oxide and were unaffected in recombination proficiency. In vitro assays showed that ngr-374-carrying strains had lower apurinic/apyrimidinic endonuclease activities than the wild type. The chromosomal locus occupied by ngr-374 was shown to exist in the gene order omega(Chr::Tn551)40-ngr-374-thrB106. PMID:2430940

  10. Mutational analysis of the active site residues of a D: -psicose 3-epimerase from Agrobacterium tumefaciens.

    PubMed

    Kim, Hye-Jung; Yeom, Soo-Jin; Kim, Kwangsoo; Rhee, Sangkee; Kim, Dooil; Oh, Deok-Kun

    2010-02-01

    D-Psicose 3-epimerase from Agrobacterium tumefacience catalyzes the conversion of D: -fructose to D-psicose. According to mutational analysis, the ring at position 112, the negative charge at position 156, and the positive charge at position 215 were essential components for enzyme activity and for binding fructose and psicose. The surface contact area and distance to the bound substrate by molecular modeling suggest that the positive charge of Arg215 was involved in stabilization of cis-endiol intermediate. The distances between the catalytic residues (Glu150 and Glu244) and Mn(2+) are critical to the catalysis, and the negative charges of the metal-binding residues are important for interaction with metal ion. The kinetic parameters of the D183E and H209A mutants for metal-binding residues with substrate and the near-UV circular dichroism spectra indicate that the metal ion bound to Asp183 and His209 is involved not only in catalysis but also in substrate binding.

  11. Human NR5A1/SF-1 Mutations Show Decreased Activity on BDNF (Brain-Derived Neurotrophic Factor), an Important Regulator of Energy Balance: Testing Impact of Novel SF-1 Mutations Beyond Steroidogenesis

    PubMed Central

    Malikova, Jana; Camats, Núria; Fernández-Cancio, Mónica; Heath, Karen; González, Isabel; Caimarí, María; del Campo, Miguel; Albisu, Marian; Kolouskova, Stanislava; Audí, Laura; Flück, Christa E.

    2014-01-01

    Context Human NR5A1/SF-1 mutations cause 46,XY disorder of sex development (DSD) with broad phenotypic variability, and rarely cause adrenal insufficiency although SF-1 is an important transcription factor for many genes involved in steroidogenesis. In addition, the Sf-1 knockout mouse develops obesity with age. Obesity might be mediated through Sf-1 regulating activity of brain-derived neurotrophic factor (BDNF), an important regulator of energy balance in the ventromedial hypothalamus. Objective To characterize novel SF-1 gene variants in 4 families, clinical, genetic and functional studies were performed with respect to steroidogenesis and energy balance. Patients 5 patients with 46,XY DSD were found to harbor NR5A1/SF-1 mutations including 2 novel variations. One patient harboring a novel mutation also suffered from adrenal insufficiency. Methods SF-1 mutations were studied in cell systems (HEK293, JEG3) for impact on transcription of genes involved in steroidogenesis (CYP11A1, CYP17A1, HSD3B2) and in energy balance (BDNF). BDNF regulation by SF-1 was studied by promoter assays (JEG3). Results Two novel NR5A1/SF-1 mutations (Glu7Stop, His408Profs*159) were confirmed. Glu7Stop is the 4th reported SF-1 mutation causing DSD and adrenal insufficiency. In vitro studies revealed that transcription of the BDNF gene is regulated by SF-1, and that mutant SF-1 decreased BDNF promoter activation (similar to steroid enzyme promoters). However, clinical data from 16 subjects carrying SF-1 mutations showed normal birth weight and BMI. Conclusions Glu7Stop and His408Profs*159 are novel SF-1 mutations identified in patients with 46,XY DSD and adrenal insufficiency (Glu7Stop). In vitro, SF-1 mutations affect not only steroidogenesis but also transcription of BDNF which is involved in energy balance. However, in contrast to mice, consequences on weight were not found in humans with SF-1 mutations. PMID:25122490

  12. Impact of nonsynonymous mutations of factor X on the functions of factor X and anticoagulant activity of edoxaban.

    PubMed

    Noguchi, Kengo; Morishima, Yoshiyuki; Takahashi, Shinichi; Ishihara, Hiroaki; Shibano, Toshiro; Murata, Mitsuru

    2015-03-01

    Edoxaban is an oral direct factor Xa (FXa) inhibitor and its efficacy as an oral anticoagulant is less subject to drug-food and drug-drug interaction than existing vitamin K antagonists. Although this profile of edoxaban suggests it is well suited for clinical use, it is not clear whether genetic variations of factor X influence the activity of edoxaban. Our aim was to investigate a possible impact of single-nucleotide polymorphisms (SNPs) in the factor X gene on the functions of factor X and the activity of edoxaban. Two nonsynonymous SNPs within mature factor X, Ala152Thr and Gly192Arg, were selected as possible candidates that might affect the functions of FXa and the activity of edoxaban. We measured catalytic activities of wild type and mutant FXas in a chromogenic assay using S-2222 and coagulation times including prothrombin time (PT) and activated partial thrombin time (aPTT) of plasma-containing recombinant FXs in the presence and absence of edoxaban. Michaelis-Menten kinetic parameters of FXas, Km and Vmax values, PT and aPTT were not influenced by either mutation indicating these mutations do not affect the FXa catalytic and coagulation activities. The Ki values of edoxaban for the FXas and the concentrations of edoxaban required to double PT and aPTT were not different between wild type and mutated FXas indicating that both mutations have little impact on the activity of edoxaban. In conclusion, these data suggest that edoxaban has little interpatient variability stemming from SNPs in the factor X gene. PMID:24911450

  13. Detection of one single mutation predicts thiopurine S-methyltransferase activity in a population of Saami in northern Norway.

    PubMed

    Loennechen, T; Utsi, E; Hartz, I; Lysaa, R; Kildalsen, H; Aarbakke, J

    2001-08-01

    Thiopurine S-methyltransferase (TPMT) activity exhibits genetic polymorphism. The purpose of this investigation was to identify TPMT mutant alleles in the Saami population as a basis of developing genotyping tests for prediction of TPMT activity. The most predominant allele in Saamis (n = 194) was the TPMT*3C allele (A719G mutation) representing 92% of the mutant alleles, with an estimated allelic frequency of 3.3%. The most frequent allele in Caucasians (n = 66) living in the same geographic area was the TPMT*3A (A719G and G460A mutations) representing 91% of the mutant alleles, with an estimated allelic frequency of 3.4%. A test for one mutation, A719G, may prospectively identify more than 90% of the Saami individuals who require reduction in thiopurine dose to avoid hematopoietic toxicity. In a Norwegian population, comprising both the major Caucasian population and a minor Saami population, the same genotyping tests (eg, tests for the A719G and G460A mutations) may be used.

  14. Incomplete activation of Escherichia coli hemolysin (HlyA) due to mutations in the 3' region of hlyC.

    PubMed Central

    Guzmán-Verri, C; García, F; Arvidson, S

    1997-01-01

    Mutational analysis of the carboxy-terminal region of Escherichia coli HlyC was performed by site-directed mutagenesis. Replacement of residue Val-127 or Lys-129 reduced the activity of HlyC to about 30 or 60%, respectively, of that of the wild type, while replacement of Gly-128 reduced the activity to less than 1% of the wild-type level. Complete inactivation of HlyC was caused by a double mutation, replacement of Gly-128 with valine and of Lys-129 with isoleucine. Analysis of culture supernatants from mutants with reduced hemolytic activity by two-dimensional gel electrophoresis revealed the production and simultaneous secretion of nonacylated, monoacylated, and fully acylated HlyA forms, demonstrating impairment of the acylation reaction, possibly due to a decreased affinity of HlyC for the individual HlyA acylation sites. PMID:9294460

  15. Toxic effects of nanoparticles on bioluminescence activity, seed germination, and gene mutation.

    PubMed

    Ko, Kyung-Seok; Kong, In Chul

    2014-04-01

    The potential environmental toxicities of several metal oxide nanoparticles (NPs; CuO, TiO2, NiO, Fe2O3, ZnO, and Co3O4) were evaluated in the context of bioluminescence activity, seed germination, and bacterial gene mutation. The bioassays exhibited different sensitivities, i.e., each kind of NP exhibited a different level of toxicity in each of the bioassays. However, with a few exceptions, CuO and ZnO NPs had most toxic for germination of Lactuca seed (EC50 0.46 mg CuO/l) and bioluminescence (EC50 1.05 mg ZnO/l). Three NPs (Co3O4, TiO2, and Fe2O3) among all tested concentrations (max. 1,000 mg/l) showed no inhibitory effects on the tested organisms, except for Co3O4 NPs on bioluminescence activity (EC50 62.04 mg/l). The sensitivity of Lactuca seeds was greater than that of Raphanus seeds (EC50 0.46 mg CuO/l versus 26.84 mg CuO /l ). The ranking of metal toxicity levels on bioluminescence was in the order of ZnO > CuO > Co3O4 > NiO > Fe2O3, TiO2, while CuO > ZnO > NiO > Co3O4, Fe2O3, TiO2 on germination. No revertant mutagenic ratio (greater than 2.0) of Salmonella typhimurium TA 98 was observed under any tested condition. These findings demonstrate that several bioassays, as opposed to any single one, are needed for the accurate assessment of NP toxicity on ecosystems. PMID:24297479

  16. Toxic effects of nanoparticles on bioluminescence activity, seed germination, and gene mutation.

    PubMed

    Ko, Kyung-Seok; Kong, In Chul

    2014-04-01

    The potential environmental toxicities of several metal oxide nanoparticles (NPs; CuO, TiO2, NiO, Fe2O3, ZnO, and Co3O4) were evaluated in the context of bioluminescence activity, seed germination, and bacterial gene mutation. The bioassays exhibited different sensitivities, i.e., each kind of NP exhibited a different level of toxicity in each of the bioassays. However, with a few exceptions, CuO and ZnO NPs had most toxic for germination of Lactuca seed (EC50 0.46 mg CuO/l) and bioluminescence (EC50 1.05 mg ZnO/l). Three NPs (Co3O4, TiO2, and Fe2O3) among all tested concentrations (max. 1,000 mg/l) showed no inhibitory effects on the tested organisms, except for Co3O4 NPs on bioluminescence activity (EC50 62.04 mg/l). The sensitivity of Lactuca seeds was greater than that of Raphanus seeds (EC50 0.46 mg CuO/l versus 26.84 mg CuO /l ). The ranking of metal toxicity levels on bioluminescence was in the order of ZnO > CuO > Co3O4 > NiO > Fe2O3, TiO2, while CuO > ZnO > NiO > Co3O4, Fe2O3, TiO2 on germination. No revertant mutagenic ratio (greater than 2.0) of Salmonella typhimurium TA 98 was observed under any tested condition. These findings demonstrate that several bioassays, as opposed to any single one, are needed for the accurate assessment of NP toxicity on ecosystems.

  17. Activity of the Escherichia coli mutT mutator allele in an anaerobic environment.

    PubMed Central

    Fowler, R G; Erickson, J A; Isbell, R J

    1994-01-01

    Mutation frequencies for an Escherichia coli mutT strain were measured in both aerobic and anaerobic environments. When cells were grown in a rich medium (L broth), mutation frequencies were similar in both aerobic and anaerobic conditions. In contrast, when grown in a minimal medium, mutT anaerobic mutation frequencies were reduced dramatically compared with aerobic values, which were similar to L broth frequencies. L broth mutT cultures treated with a commercial enzyme complex that reduces free oxygen in the medium also showed strongly reduced anaerobic mutation frequencies. These results indicate that the biological role of the MutT protein is to prevent oxidative damage from becoming mutagenic. PMID:8002599

  18. Novel activating JAK2 mutation in a patient with Down syndrome and B-cell precursor acute lymphoblastic leukemia.

    PubMed

    Malinge, Sebastien; Ben-Abdelali, Raouf; Settegrana, Catherine; Radford-Weiss, Isabelle; Debre, Marianne; Beldjord, Kheira; Macintyre, Elizabeth A; Villeval, Jean-Luc; Vainchenker, William; Berger, Roland; Bernard, Olivier A; Delabesse, Eric; Penard-Lacronique, Virginie

    2007-03-01

    Activation of tyrosine kinase genes is a frequent event in human hematologic malignancies. Because gene activation could be associated with gene dysregulation, we attempted to screen for activating gene mutation based on high-level gene expression. We focused our study on the Janus kinase 2 (JAK2) gene in 90 cases of acute leukemia. This strategy led to the identification of a novel JAK2-acquired mutation in a patient with Down syndrome (DS) with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). This mutation involves a 5-amino acid deletion within the JH2 pseudokinase domain (JAK2DeltaIREED). Expression of JAK2DeltaIREED in Ba/F3 cells induced constitutive activation of the JAK-STAT pathway and growth factor-independent cell proliferation. These results highlight the JAK2 pseudokinase domain as an oncogenic hot spot and indicate that activation of the JAK-STAT pathway may contribute to lymphoid malignancies and hematologic disorders observed in children with DS.

  19. Mutational analysis of the redox-sensitive transcriptional regulator OxyR: regions important for oxidation and transcriptional activation.

    PubMed Central

    Kullik, I; Toledano, M B; Tartaglia, L A; Storz, G

    1995-01-01

    OxyR is a redox-sensitive transcriptional regulator of the LysR family which activates the expression of genes important for the defense against hydrogen peroxide in Escherichia coli and Samonella typhimurium. OxyR is sensitive to oxidation and reduction, and only oxidized OxyR is able to activate transcription of its target genes. Using site-directed mutagenesis, we found that one cysteine residue (C-199) is critical for the redox sensitivity of OxyR, and a C-199-->S mutation appears to lock the OxyR protein in the reduced form. We also used a random mutagenesis approach to isolate eight constitutively active mutants. All of the mutations are located in the C-terminal half of the protein, and four of the mutations map near the critical C-199 residue. In vivo as well as in vitro transcription experiments showed that the constitutive mutant proteins were able to activate transcription under both oxidizing and reducing conditions, and DNase I footprints showed that this activation is due to the ability of the mutant proteins to induce cooperative binding of RNA polymerase. Unexpectedly, RNA polymerase was also found to reciprocally affect OxyR binding. PMID:7868602

  20. Landscape of activating cancer mutations in FGFR kinases and their differential responses to inhibitors in clinical use

    PubMed Central

    Thiyagarajan, Nethaji; Norman, Richard A.; Ogg, Derek; Breed, Jason; Ashford, Paul; Potterton, Andrew; Edwards, Mina; Williams, Sarah V.; Thomson, Gary S.; Pang, Camilla S.M.; Knowles, Margaret A.; Breeze, Alexander L.; Orengo, Christine; Phillips, Chris; Katan, Matilda

    2016-01-01

    Frequent genetic alterations discovered in FGFRs and evidence implicating some as drivers in diverse tumors has been accompanied by rapid progress in targeting FGFRs for anticancer treatments. Wider assessment of the impact of genetic changes on the activation state and drug responses is needed to better link the genomic data and treatment options. We here apply a direct comparative and comprehensive analysis of FGFR3 kinase domain variants representing the diversity of point-mutations reported in this domain. We reinforce the importance of N540K and K650E and establish that not all highly activating mutations (for example R669G) occur at high-frequency and conversely, that some “hotspots” may not be linked to activation. Further structural characterization consolidates a mechanistic view of FGFR kinase activation and extends insights into drug binding. Importantly, using several inhibitors of particular clinical interest (AZD4547, BGJ-398, TKI258, JNJ42756493 and AP24534), we find that some activating mutations (including different replacements of the same residue) result in distinct changes in their efficacy. Considering that there is no approved inhibitor for anticancer treatments based on FGFR-targeting, this information will be immediately translatable to ongoing clinical trials. PMID:26992226

  1. Arylamine N-acetyltransferase (NAT2) mutations and their allelic linkage in unrelated caucasian individuals: Correlation with phenotypic activity

    SciTech Connect

    Cascorbi, I.; Drakoulis, N.; Brockmoeller, J.

    1995-09-01

    The polymorphic arylamine N-acetyltransferase (NAT2; EC2.3.1.5) is supposed to be a susceptibility factor for several drug side effects and certain malignancies. A group of 844 unrelated German subjects was genotyped for their acetylation type, and 563 of them were also phenotyped. Seven mutations of the NAT2 gene were evaluated by allele-specific PCR (mutation 341C to T) and PCR-RFLP for mutations at nt positions 191, 282, 481, 590, 803, and 857. From the mutation pattern eight different alleles, including the wild type coding for rapid acetylation and seven alleles coding for slow phenotype, were determined. Four hundred ninety-seven subjects had a genotype of slow acetylation (58.9%; 95% confidence limits 55.5%-62.2%). Phenotypic acetylation capacity was expressed as the ratio of 5-acetylamino-6-formylamino-3-methyluracil and 1-methylxanthine in urine after caffeine intake. Some 6.7% of the cases deviated in genotype and phenotype, but sequencing DNA of these probands revealed no new mutations. Furthermore, linkage pattern of the mutations was always confirmed, as tested in 533 subjects. In vivo acetylation capacity of homozygous wild-type subjects (NAT2{sup *}4/{sup *}4) was significantly higher than in heterozygous genotypes (P = .001). All mutant alleles showed low in vivo acetylation capacities, including the previously not-yet-defined alleles {sup *}5A, {sup *}5C, and {sup *}13. Moreover, distinct slow genotypes differed significantly among each other, as reflected in lower acetylation capacity of {sup *}6A, {sup *}7B, and {sup *}13 alleles than the group of {sup *}5 alleles. The study demonstrated differential phenotypic activity of various NAT2 genes and gives a solid basis for clinical and molecular-epidemiological investigations. 34 refs., 4 figs., 7 tabs.

  2. Emergence of constitutively active estrogen receptor-α mutations in pretreated advanced estrogen receptor positive breast cancer

    PubMed Central

    Meric-Bernstam, Funda; Gonzalez-Angulo, Ana Maria; Ferrer-Lozano, Jaime; Perez-Fidalgo, Jose A.; Cristofanilli, Massimo; Gómez, Henry; Arteaga, Carlos L.; Giltnane, Jennifer; Balko, Justin M.; Cronin, Maureen T; Jarosz, Mirna; Sun, James; Hawryluk, Matthew; Lipson, Doron; Otto, Geoff; Ross, Jeffrey S; Dvir, Addie; Soussan-Gutman, Lior; Wolf, Ido; Rubinek, Tamar; Gilmore, Lauren; Schnitt, Stuart; Come, Steven E.; Pusztai, Lajos; Stephens, Philip; Brown, Myles; Miller, Vincent A.

    2014-01-01

    Purpose We undertook this study to determine the prevalence of estrogen receptor (ER) α (ESR1) mutations throughout the natural history of hormone dependent breast cancer and to delineate the functional roles of the most commonly detected alterations. Experimental Design We studied a total of 249 tumor specimens from 208 patients. The specimens include 134 ER positive (ER+/HER2–) and, as controls, 115 ER negative (ER−) tumors. The ER+ samples consist of 58 primary breast cancers and 76 metastatic samples. All tumors were sequenced to high unique coverage using next generation sequencing targeting the coding sequence of the estrogen receptor and an additional 182 cancer-related genes. Results Recurring somatic mutations in codons 537 and 538 within the ligand-binding domain of ER were detected in ER+ metastatic disease. Overall, the frequency of these mutations was 12% (9/76, 95% CI 6%-21%) in metastatic tumors and in a subgroup of patients who received an average of 7 lines of treatment the frequency was 20% (5/25, 95% CI 7%-41%). These mutations were not detected in primary or treatment naïve ER+ cancer or in any stage of ER− disease. Functional studies in cell line models demonstrate that these mutations render estrogen receptor constitutive activity and confer partial resistance to currently available endocrine treatments. Conclusions In this study we show evidence for the temporal selection of functional ESR1 mutations as potential drivers of endocrine resistance during the progression of ER positive breast cancer. PMID:24398047

  3. Notch and Wnt/β-catenin signaling pathway play important roles in activating liver cancer stem cells

    PubMed Central

    Wang, Ronghua; Sun, Qian; Wang, Peng; Liu, Man; Xiong, Si; Luo, Jing; Huang, Hai; Du, Qiang; Geller, David A.; Cheng, Bin

    2016-01-01

    Human hepatocellular carcinoma (HCC) is driven and maintained by liver cancer stem cells (LCSCs) that display stem cell properties. These LCSCs are promoted by the intersecting of Notch and Wnt/β-Catenin signaling pathways. In this study, we demonstrate that LCSCs with markers CD90, CD24, CD13, and CD133 possess stem properties of self-renewal and tumorigenicity in NOD/SCID mice. The increased expression of these markers was correlated with advanced disease stage, larger tumors, and worse overall survival in 61 HCC cases. We also found that both Notch and Wnt/β-catenin signaling pathways played important roles in increasing the stem-ness characteristics of LCSCs. Our data suggested that Notch1 was downstream of Wnt/β-catenin. The active form of Notch1 intracellular domain (NICD) expression depended on Wnt/β-catenin pathway activation. Moreover, Notch1 negatively contributed to Wnt/β-catenin signaling modulation. Knock down of Notch1 with lentivirus N1ShRNA up-regulated the active form of β-catenin. Ectopic expression of NICD with LV-Notch1 in LCSCs attenuated β-catenin/TCF dependent luciferase activity significantly. In addition, there was a non-proteasome mediated feedback loop between Notch1 and Wnt/β-catenin signaling in LCSCs. The central role of Notch and the Wnt/β-catenin signaling pathway in LCSCs may provide an attractive therapeutic strategy against HCC. PMID:26735577

  4. A single mutation Gln142Lys doubles the catalytic activity of VPR, a cold adapted subtilisin-like serine proteinase.

    PubMed

    Óskarsson, Kristinn R; Nygaard, Mads; Ellertsson, Brynjar Ö; Thorbjarnardottir, Sigríður H; Papaleo, Elena; Kristjánsson, Magnús M

    2016-10-01

    Structural comparisons of the cold adapted subtilase VPR and its thermophilic homologue, aqualysin I (AQUI) indicated the presence of additional salt bridges in the latter. Few of those appear to contribute significantly to thermal stability of AQUI. This includes a putative salt bridge between residues Lys142 and Glu172 as its deletion did not have any significant effect on its stability or activity (Jónsdóttir et al. (2014)). Insertion of this putative salt bridge into the structure of VPR, in a double mutant (VPRΔC_Q142K/S172E), however was detrimental to the stability of the enzyme. Incorporation of either the Q142K or S172E mutations into VPR, were found to significantly affect the catalytic properties of the enzyme. The single mutation Q142K was highly effective, as it increased the kcat and kcat/Km more than twofold. When the Q142K mutation was inserted into a thermostabilized, but a low activity mutant of VPR (VPRΔC_N3P/I5P), the activity increased about tenfold in terms of kcat and kcat/Km, while retaining the stability of the mutant. Molecular dynamics simulations of the single mutants were carried out to provide structural rationale for these experimental observations. Based on root mean square fluctuation (RMSF) profiles, the two mutants were more flexible in certain regions of the structure and the Q142K mutant had the highest overall flexibility of the three enzymes. The results suggest that weakening of specific H-bonds resulting from the mutations may be propagated over some distance giving rise to higher flexibility in the active site regions of the enzyme, causing higher catalytic activity in the mutants. PMID:27456266

  5. Comparison of benzo(a)pyrene metabolism and mutation induction in CHO cells using rat liver homogenate (S9) or Syrian hamster embryonic cell-mediated activation systems

    SciTech Connect

    Chen, D.J.; Okinaka, R.T.; Strniste, G.F.

    1981-01-01

    Mutagenesis in CHO cells has been studied by the addition of an ezymatically active liver homogenate (S9) fraction. However, the metabolism of procarcinogens, such as benzo(a)pyrene (B(a)P), by rat liver homogenate differs from that in intact cellular activation systems. Consequently, B(a)P-induced mutation frequencies in mammalian cells may vary when different activation systems are used. This study attempts to compare B(a)P metabolism and conjugation in rat liver homogenate (S9 preparation) and in Syrian hamster embryonic (SHE) cells. Furthermore, a CHO mutation assay incorporating either of the activation systems is being used to measure the mutation induction frequency.

  6. Multiple cryptic splice sites can be activated by IDS point mutations generating misspliced transcripts.

    PubMed

    Lualdi, Susanna; Pittis, Maria G; Regis, Stefano; Parini, Rossella; Allegri, Anna E; Furlan, Francesca; Bembi, Bruno; Filocamo, Mirella

    2006-08-01

    Mutations in the gene encoding the enzyme iduronate-2-sulfatase (IDS) were reported as the cause of the X-linked recessive lysosomal disease, mucopolysaccharidosis II (MPS II). Amongst the different mutations, it emerges that nearly 10% are nucleotide substitutions causing splicing mutations. We now report the molecular characterisation of three MPS II patients with multiple aberrant transcripts due to three different point mutations. The c.418+1G>C that occurred in the invariant splice-site motif, produced only aberrantly spliced transcripts. Whilst the mutations affecting variant motifs (c.419G>T) or coding regions (c.245C>T) led to aberrantly spliced transcripts in addition to correctly spliced transcripts with the respective predicted missense mutation, p.G140V or p.A82V. A combination of experimental tests and computational approaches were used to understand the molecular basis underlying the altered transcription patterns. In addition, by using real-time reverse transcriptase polymerase chain reaction, the reduction of mRNA amount in two patients observed was likely due to nonsense-mediated mRNA decay pathway. Overall, our results further emphasised the importance of cloning and sequencing independent transcripts to reveal less abundant, aberrant products, which often could not be detected by direct sequencing. Moreover, the different splicing patterns observed in the three patients as a consequence of point mutations show how sensitive the balance is between constitutive and cryptic splice sites in the IDS gene. The generation of such diverse transcripts, together with their level of expression, could contribute to the profound phenotypic variability reported in MPS II.

  7. New Tricks for Old Proteins: Single Mutations in a Nonenzymatic Protein Give Rise to Various Enzymatic Activities.

    PubMed

    Moroz, Yurii S; Dunston, Tiffany T; Makhlynets, Olga V; Moroz, Olesia V; Wu, Yibing; Yoon, Jennifer H; Olsen, Alissa B; McLaughlin, Jaclyn M; Mack, Korrie L; Gosavi, Pallavi M; van Nuland, Nico A J; Korendovych, Ivan V

    2015-12-01

    Design of a new catalytic function in proteins, apart from its inherent practical value, is important for fundamental understanding of enzymatic activity. Using a computationally inexpensive, minimalistic approach that focuses on introducing a single highly reactive residue into proteins to achieve catalysis we converted a 74-residue-long C-terminal domain of calmodulin into an efficient esterase. The catalytic efficiency of the resulting stereoselective, allosterically regulated catalyst, nicknamed AlleyCatE, is higher than that of any previously reported de novo designed esterases. The simplicity of our design protocol should complement and expand the capabilities of current state-of-art approaches to protein design. These results show that even a small nonenzymatic protein can efficiently attain catalytic activities in various reactions (Kemp elimination, ester hydrolysis, retroaldol reaction) as a result of a single mutation. In other words, proteins can be just one mutation away from becoming entry points for subsequent evolution.

  8. New Tricks for Old Proteins: Single Mutations in a Nonenzymatic Protein Give Rise to Various Enzymatic Activities.

    PubMed

    Moroz, Yurii S; Dunston, Tiffany T; Makhlynets, Olga V; Moroz, Olesia V; Wu, Yibing; Yoon, Jennifer H; Olsen, Alissa B; McLaughlin, Jaclyn M; Mack, Korrie L; Gosavi, Pallavi M; van Nuland, Nico A J; Korendovych, Ivan V

    2015-12-01

    Design of a new catalytic function in proteins, apart from its inherent practical value, is important for fundamental understanding of enzymatic activity. Using a computationally inexpensive, minimalistic approach that focuses on introducing a single highly reactive residue into proteins to achieve catalysis we converted a 74-residue-long C-terminal domain of calmodulin into an efficient esterase. The catalytic efficiency of the resulting stereoselective, allosterically regulated catalyst, nicknamed AlleyCatE, is higher than that of any previously reported de novo designed esterases. The simplicity of our design protocol should complement and expand the capabilities of current state-of-art approaches to protein design. These results show that even a small nonenzymatic protein can efficiently attain catalytic activities in various reactions (Kemp elimination, ester hydrolysis, retroaldol reaction) as a result of a single mutation. In other words, proteins can be just one mutation away from becoming entry points for subsequent evolution. PMID:26555770

  9. Stage-specific effects of Notch activation during skeletal myogenesis

    PubMed Central

    Bi, Pengpeng; Yue, Feng; Sato, Yusuke; Wirbisky, Sara; Liu, Weiyi; Shan, Tizhong; Wen, Yefei; Zhou, Daoguo; Freeman, Jennifer; Kuang, Shihuan

    2016-01-01

    Skeletal myogenesis involves sequential activation, proliferation, self-renewal/differentiation and fusion of myogenic stem cells (satellite cells). Notch signaling is known to be essential for the maintenance of satellite cells, but its function in late-stage myogenesis, i.e. post-differentiation myocytes and post-fusion myotubes, is unknown. Using stage-specific Cre alleles, we uncovered distinct roles of Notch1 in mononucleated myocytes and multinucleated myotubes. Specifically, constitutive Notch1 activation dedifferentiates myocytes into Pax7 quiescent satellite cells, leading to severe defects in muscle growth and regeneration, and postnatal lethality. By contrast, myotube-specific Notch1 activation improves the regeneration and exercise performance of aged and dystrophic muscles. Mechanistically, Notch1 activation in myotubes upregulates the expression of Notch ligands, which modulate Notch signaling in the adjacent satellite cells to enhance their regenerative capacity. These results highlight context-dependent effects of Notch activation during myogenesis, and demonstrate that Notch1 activity improves myotube’s function as a stem cell niche. DOI: http://dx.doi.org/10.7554/eLife.17355.001 PMID:27644105

  10. Complete androgen insensitivity syndrome caused by a deep intronic pseudoexon-activating mutation in the androgen receptor gene.

    PubMed

    Känsäkoski, Johanna; Jääskeläinen, Jarmo; Jääskeläinen, Tiina; Tommiska, Johanna; Saarinen, Lilli; Lehtonen, Rainer; Hautaniemi, Sampsa; Frilander, Mikko J; Palvimo, Jorma J; Toppari, Jorma; Raivio, Taneli

    2016-01-01

    Mutations in the X-linked androgen receptor (AR) gene underlie complete androgen insensitivity syndrome (CAIS), the most common cause of 46,XY sex reversal. Molecular genetic diagnosis of CAIS, however, remains uncertain in patients who show normal coding region of AR. Here, we describe a novel mechanism of AR disruption leading to CAIS in two 46,XY sisters. We analyzed whole-genome sequencing data of the patients for pathogenic variants outside the AR coding region. Patient fibroblasts from the genital area were used for AR cDNA analysis and protein quantification. Analysis of the cDNA revealed aberrant splicing of the mRNA caused by a deep intronic mutation (c.2450-118A>G) in the intron 6 of AR. The mutation creates a de novo 5' splice site and a putative exonic splicing enhancer motif, which leads to the preferential formation of two aberrantly spliced mRNAs (predicted to include a premature stop codon). Patient fibroblasts contained no detectable AR protein. Our results show that patients with CAIS and normal AR coding region need to be examined for deep intronic mutations that can lead to pseudoexon activation. PMID:27609317

  11. An arginine to glutamine mutation in residue 109 of human ornithine transcarbamylase completely abolishes enzymatic activity in Cos1 cells.

    PubMed Central

    Lee, J T; Nussbaum, R L

    1989-01-01

    Ornithine transcarbamylase (OTC) is an important enzyme in the detoxification of ammonia to urea, and its deficiency is the most common inborn error of ureagenesis in humans. Among 24 cases of OTC deficiency previously examined, three unrelated individuals all showed loss of a Taq I site in the OTC gene corresponding to codon 109, suggesting that this Taq I site may be prone to mutation. Two of these patients demonstrated the same C----T transition (in antisense strand) converting Arg109 to Gln. Although these studies implied a strong association between the missense mutation and OTC-deficient phenotype, a causal relationship could not be firmly established. We have investigated this relationship by reconstructing the mutation in vitro. A full-length human OTC cDNA was cloned into an SV40-based expression vector and has been reproducibly expressed at high levels in the cell line Cos1. By site-directed mutagenesis of this wild type sequence, we constructed a missense mutation which contains the C----T transition. Electroporation and transient assay in Cos1 indicated that the specific activity of mutant OTC was 100-fold lower than that of wild type. This result confirms that the Taq I alteration leading to the Gln missense is responsible for the OTC deficiency affecting the above patients. PMID:2556444

  12. Complete androgen insensitivity syndrome caused by a deep intronic pseudoexon-activating mutation in the androgen receptor gene

    PubMed Central

    Känsäkoski, Johanna; Jääskeläinen, Jarmo; Jääskeläinen, Tiina; Tommiska, Johanna; Saarinen, Lilli; Lehtonen, Rainer; Hautaniemi, Sampsa; Frilander, Mikko J.; Palvimo, Jorma J.; Toppari, Jorma; Raivio, Taneli

    2016-01-01

    Mutations in the X-linked androgen receptor (AR) gene underlie complete androgen insensitivity syndrome (CAIS), the most common cause of 46,XY sex reversal. Molecular genetic diagnosis of CAIS, however, remains uncertain in patients who show normal coding region of AR. Here, we describe a novel mechanism of AR disruption leading to CAIS in two 46,XY sisters. We analyzed whole-genome sequencing data of the patients for pathogenic variants outside the AR coding region. Patient fibroblasts from the genital area were used for AR cDNA analysis and protein quantification. Analysis of the cDNA revealed aberrant splicing of the mRNA caused by a deep intronic mutation (c.2450-118A>G) in the intron 6 of AR. The mutation creates a de novo 5′ splice site and a putative exonic splicing enhancer motif, which leads to the preferential formation of two aberrantly spliced mRNAs (predicted to include a premature stop codon). Patient fibroblasts contained no detectable AR protein. Our results show that patients with CAIS and normal AR coding region need to be examined for deep intronic mutations that can lead to pseudoexon activation. PMID:27609317

  13. Outside-binding site mutations modify the active site's shapes in neuraminidase from influenza A H1N1.

    PubMed

    Tolentino-Lopez, Luis; Segura-Cabrera, Aldo; Reyes-Loyola, Paola; Zimic, Mirko; Quiliano, Miguel; Briz, Veronica; Muñoz-Fernández, Angeles; Rodríguez-Pérez, Mario; Ilizaliturri-Flores, Ian; Correa-Basurto, Jose

    2013-01-01

    The recent occurrence of 2009 influenza A (H1N1) pandemic as well as others has raised concern of a far more dangerous outcome should this virus becomes resistant to current drug therapies. The number of clinical cases that are resistant to oseltamivir (Tamiflu®) is larger than the limited number of neuraminidase (NA) mutations (H275Y, N295S, and I223R) that have been identified at the active site and that are associated to oseltamivir resistance. In this study, we have performed a comparative analysis between a set of NAs that have the most representative mutations located outside the active site. The recently crystallized NA-oseltamivir complex (PDB ID: 3NSS) was used as a wild-type structure. After selecting the target NA sequences, their three-dimensional (3D) structure was built using 3NSS as a template by homology modeling. The 3D NA models were refined by molecular dynamics (MD) simulations. The refined models were used to perform a docking study, using oseltamivir as a ligand. Furthermore, the docking results were refined by free-energy analysis using the MM-PBSA method. The analysis of the MD simulation results showed that the NA models reached convergence during the first 10 ns. Visual inspection and structural measures showed that the mutated NA active sites show structural variations. The docking and MM-PBSA results from the complexes showed different binding modes and free energy values. These results suggest that distant mutations located outside the active site of NA affect its structure and could be considered to be a new source of resistance to oseltamivir, which agrees with reports in the clinical literature.

  14. Vertebrate opsins belonging to different classes vary in constitutively active properties resulting from salt-bridge mutations.

    PubMed

    Nickle, Benjamin; Wilkie, Susan E; Cowing, Jill A; Hunt, David M; Robinson, Phyllis R

    2006-06-13

    Vertebrate opsins are classified into one of five classes on the basis of amino acid similarity. These classes are short wavelength sensitive 1 and 2 (SWS1, SWS2), medium/long wavelength sensitive (M/LWS), and rod opsin like 1 and 2 (RH1, RH2). In bovine rod opsin (RH1), two critical amino acids form a salt bridge in the apoprotein that maintains the opsin in an inactive state. These residues are K296, which functions as the chromophore binding site, and E113, which functions as the counterion to the protonated Schiff base. Corresponding residues in each of the other vertebrate opsin classes are believed to play similar roles. Previous reports have demonstrated that mutations in these critical residues result in constitutive activation of transducin by RH1 class opsins in the absence of chromophore. Additionally, recent reports have shown that an E113Q mutation in SWS1 opsin is constitutively active. Here we ask if the other classes of vertebrate opsins maintain activation characteristics similar to that of bovine RH1 opsin. We approach this question by making the corresponding substitutions which disrupt the K296/E113 salt bridge in opsins belonging to the other vertebrate opsin classes. The mutant opsins are tested for their ability to constitutively activate bovine transducin. We demonstrate that mutations disrupting this key salt bridge produce constitutive activation in all classes. However, the mutant opsins differ in their ability to be quenched in the dark state by the addition of chromophore as well as in their level of constitutive activation. The differences in constitutive activation profiles suggest that structural differences exist among the opsin classes that may translate into a difference in activation properties.

  15. Identification of a recently active Prunus-specific non-autonomous Mutator element with considerable genome shaping force.

    PubMed

    Halász, Júlia; Kodad, Ossama; Hegedűs, Attila

    2014-07-01

    Miniature inverted-repeat transposable elements (MITEs) are known to contribute to the evolution of plants, but only limited information is available for MITEs in the Prunus genome. We identified a MITE that has been named Falling Stones, FaSt. All structural features (349-bp size, 82-bp terminal inverted repeats and 9-bp target site duplications) are consistent with this MITE being a putative member of the Mutator transposase superfamily. FaSt showed a preferential accumulation in the short AT-rich segments of the euchromatin region of the peach genome. DNA sequencing and pollination experiments have been performed to confirm that the nested insertion of FaSt into the S-haplotype-specific F-box gene of apricot resulted in the breakdown of self-incompatibility (SI). A bioinformatics-based survey of the known Rosaceae and other genomes and a newly designed polymerase chain reaction (PCR) assay verified the Prunoideae-specific occurrence of FaSt elements. Phylogenetic analysis suggested a recent activity of FaSt in the Prunus genome. The occurrence of a nested insertion in the apricot genome further supports the recent activity of FaSt in response to abiotic stress conditions. This study reports on a presumably active non-autonomous Mutator element in Prunus that exhibits a major indirect genome shaping force through inducing loss-of-function mutation in the SI locus.

  16. The nemaline myopathy-causing E117K mutation in β-tropomyosin reduces thin filament activation.

    PubMed

    Karpicheva, Olga E; Robinson, Paul; Piers, Adam; Borovikov, Yurii S; Redwood, Charles S

    2013-08-01

    The effect of the nemaline myopathy-causing E117K mutation in β-tropomyosin (TM) on the structure and function of this regulatory protein was studied. The E117K mutant was found to have indistinguishable actin affinity compared with wild-type (WT) and similar secondary structure as measured by circular dichroism. However the E117K mutation significantly lowered maximum activation of actomyosin ATPase. To explain the molecular mechanism of impaired ATPase activation, WT and E117K TMs were covalently labeled at Cys-36 with 5-iodoacetimido-fluorescein and incorporated into ghost muscle fibers. The changes in the position and flexibility of tropomyosin strands on the thin filaments were observed at simulation of weak and strong binding states of actomyosin at high or low Ca(2+) by polarized fluorescence techniques. The E117K mutation was found to shift the tropomyosin strands towards the closed position and restrict the tropomyosin displacement during the transformation of actomyosin from weak to strong binding state thus leading to a reduction in thin filament activation. PMID:23689010

  17. Meconium Ileus Caused by Mutations in GUCY2C, Encoding the CFTR-Activating Guanylate Cyclase 2C

    PubMed Central

    Romi, Hila; Cohen, Idan; Landau, Daniella; Alkrinawi, Suliman; Yerushalmi, Baruch; Hershkovitz, Reli; Newman-Heiman, Nitza; Cutting, Garry R.; Ofir, Rivka; Sivan, Sara; Birk, Ohad S.

    2012-01-01

    Meconium ileus, intestinal obstruction in the newborn, is caused in most cases by CFTR mutations modulated by yet-unidentified modifier genes. We now show that in two unrelated consanguineous Bedouin kindreds, an autosomal-recessive phenotype of meconium ileus that is not associated with cystic fibrosis (CF) is caused by different homozygous mutations in GUCY2C, leading to a dramatic reduction or fully abrogating the enzymatic activity of the encoded guanlyl cyclase 2C. GUCY2C is a transmembrane receptor whose extracellular domain is activated by either the endogenous ligands, guanylin and related peptide uroguanylin, or by an external ligand, Escherichia coli (E. coli) heat-stable enterotoxin STa. GUCY2C is expressed in the human intestine, and the encoded protein activates the CFTR protein through local generation of cGMP. Thus, GUCY2C is a likely candidate modifier of the meconi