Proliferating Cell Nuclear Antigen-dependent Rapid Recruitment of Cdt1 and CRL4Cdt2 at DNA-damaged Sites after UV Irradiation in HeLa Cells*

Science.gov (United States)

Ishii, Takashi; Shiomi, Yasushi; Takami, Toshihiro; Murakami, Yusuke; Ohnishi, Naho; Nishitani, Hideo

2010-01-01

The licensing factor Cdt1 is degraded by CRL4Cdt2 ubiquitin ligase dependent on proliferating cell nuclear antigen (PCNA) during S phase and when DNA damage is induced in G1 phase. Association of both Cdt2 and PCNA with chromatin was observed in S phase and after UV irradiation. Here we used a micropore UV irradiation assay to examine Cdt2 accumulation at cyclobutane pyrimidine dimer-containing DNA-damaged sites in the process of Cdt1 degradation in HeLa cells. Cdt2, present in the nucleus throughout the cell cycle, accumulated rapidly at damaged DNA sites during G1 phase. The recruitment of Cdt2 is dependent on prior PCNA chromatin binding because Cdt2 association was prevented when PCNA was silenced. Cdt1 was also recruited to damaged sites soon after UV irradiation through its PIP-box. As Cdt1 was degraded, the Cdt2 signal at damaged sites was reduced, but PCNA, cyclobutane pyrimidine dimer, and XPA (xeroderma pigmentosum, complementation group A) signals remained at the same levels. These findings suggest that Cdt1 degradation following UV irradiation occurs rapidly at damaged sites due to PCNA chromatin loading and the recruitment of Cdt1 and CRL4Cdt2, before DNA damage repair is completed. PMID:20929861

The Copenhagen Dependency Treebank (CDT)

DEFF Research Database (Denmark)

Høeg Müller, Henrik; Korzen, Iørn

2014-01-01

the fundamentals of how CDT is marked up with semantic relations in accordance with the dependency principles governing the annotation on the other levels of CDT. Specifically, focus will be on how Generative Lexicon (GL) theory has been incorporated into the unitary theoretical dependency framework of CDT....... An annotation scheme for lexical semantics has been designed so as to account for the lexico-semantic structure of complex NPs, and the four GL qualia also appear in some of the CDT discourse relation labels as a description of parallel semantic relations at this level....

Non-canonical CRL4A/4B(CDT2 interacts with RAD18 to modulate post replication repair and cell survival.

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Sarah Sertic

Full Text Available The Cullin-4(CDT2 E3 ubiquitin ligase plays an essential role in DNA replication origin licensing directing degradation of several licensing factors at the G1/S transition in order to prevent DNA re-replication. Recently a RAD18-independent role of Cullin-4(CDT2 in PCNA monoubiquitylation has been proposed. In an effort to better understand the function of Cullin-4(CDT2 E3 ubiquitin ligase in mammalian Post-Replication Repair during an unperturbed S-phase, we show that down-regulation of Cullin-4(CDT2 leads to two distinguishable independent phenotypes in human cells that unveil at least two independent roles of Cullin-4(CDT2 in S-phase. Apart from the re-replication preventing activity, we identified a non-canonical Cullin-4(CDT2 complex, containing both CUL4A and CUL4B, associated to the COP9 signalosome, that controls a RAD18-dependent damage avoidance pathway essential during an unperturbed S-phase. Indeed, we show that the non-canonical Cullin-4A/4B(CDT2 complex binds to RAD18 and it is required to modulate RAD18 protein levels onto chromatin and the consequent dynamics of PCNA monoubiquitylation during a normal S-phase. This function prevents replication stress, ATR hyper-signaling and, ultimately, apoptosis. A very similar PRR regulatory mechanism has been recently described for Spartan. Our findings uncover a finely regulated process in mammalian cells involving Post-Replication Repair factors, COP9 signalosome and a non-canonical Cullin4-based E3 ligase which is essential to tolerate spontaneous damage and for cell survival during physiological DNA replication.

Distinct Roles for CdtA and CdtC during Intoxication by Cytolethal Distending Toxins.

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Shandee D Dixon

Full Text Available Cytolethal distending toxins (CDTs are heterotrimeric protein exotoxins produced by a diverse array of Gram-negative pathogens. The enzymatic subunit, CdtB, possesses DNase and phosphatidylinositol 3-4-5 trisphosphate phosphatase activities that induce host cell cycle arrest, cellular distension and apoptosis. To exert cyclomodulatory and cytotoxic effects CDTs must be taken up from the host cell surface and transported intracellularly in a manner that ultimately results in localization of CdtB to the nucleus. However, the molecular details and mechanism by which CDTs bind to host cells and exploit existing uptake and transport pathways to gain access to the nucleus are poorly understood. Here, we report that CdtA and CdtC subunits of CDTs derived from Haemophilus ducreyi (Hd-CDT and enteropathogenic E. coli (Ec-CDT are independently sufficient to support intoxication by their respective CdtB subunits. CdtA supported CdtB-mediated killing of T-cells and epithelial cells that was nearly as efficient as that observed with holotoxin. In contrast, the efficiency by which CdtC supported intoxication was dependent on the source of the toxin as well as the target cell type. Further, CdtC was found to alter the subcellular trafficking of Ec-CDT as determined by sensitivity to EGA, an inhibitor of endosomal trafficking, colocalization with markers of early and late endosomes, and the kinetics of DNA damage response. Finally, host cellular cholesterol was found to influence sensitivity to intoxication mediated by Ec-CdtA, revealing a role for cholesterol or cholesterol-rich membrane domains in intoxication mediated by this subunit. In summary, data presented here support a model in which CdtA and CdtC each bind distinct receptors on host cell surfaces that direct alternate intracellular uptake and/or trafficking pathways.

Break-induced ATR and Ddb1-Cul4(Cdt)² ubiquitin ligase-dependent nucleotide synthesis promotes homologous recombination repair in fission yeast

DEFF Research Database (Denmark)

Moss, Jennifer; Tinline-Purvis, Helen; Walker, Carol A

2010-01-01

Nucleotide synthesis is a universal response to DNA damage, but how this response facilitates DNA repair and cell survival is unclear. Here we establish a role for DNA damage-induced nucleotide synthesis in homologous recombination (HR) repair in fission yeast. Using a genetic screen, we found...... the Ddb1-Cul4(Cdt)² ubiquitin ligase complex and ribonucleotide reductase (RNR) to be required for HR repair of a DNA double-strand break (DSB). The Ddb1-Cul4(Cdt)² ubiquitin ligase complex is required for degradation of Spd1, an inhibitor of RNR in fission yeast. Accordingly, deleting spd1(+) suppressed...

Geminin Participates in Differentiation Decisions of Adult Neural Stem Cells Transplanted in the Hemiparkinsonian Mouse Brain.

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Taouki, Ioanna; Tasiudi, Eve; Lalioti, Maria-Eleni; Kyrousi, Christina; Skavatsou, Eleni; Kaplani, Konstantina; Lygerou, Zoi; Kouvelas, Elias D; Mitsacos, Adamantia; Giompres, Panagiotis; Taraviras, Stavros

2017-08-15

Neural stem cells have been considered as a source of stem cells that can be used for cell replacement therapies in neurodegenerative diseases, as they can be isolated and expanded in vitro and can be used for autologous grafting. However, due to low percentages of survival and varying patterns of differentiation, strategies that will enhance the efficacy of transplantation are under scrutiny. In this article, we have examined whether alterations in Geminin's expression, a protein that coordinates the balance between self-renewal and differentiation, can improve the properties of stem cells transplanted in 6-OHDA hemiparkinsonian mouse model. Our results indicate that, in the absence of Geminin, grafted cells differentiating into dopaminergic neurons were decreased, while an increased number of oligodendrocytes were detected. The number of proliferating multipotent cells was not modified by the absence of Geminin. These findings encourage research related to the impact of Geminin on transplantations for neurodegenerative disorders, as an important molecule in influencing differentiation decisions of the cells composing the graft.

A new functional site W115 in CdtA is critical for Aggregatibacter actinomycetemcomitans cytolethal distending toxin.

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Lu Li

Full Text Available Aggregatibacter actinomycetemcomitans, a specific pathogen of localized aggressive periodontitis, produces a cytolethal distending toxin (CDT that arrests eukaryotic cells irreversibly in G0/G1 or G2/M phase of the cell cycle. Although structural studies show that the aromatic patch region of CdtA plays an important role in its biological activity, the functional sites of CdtA have not been firmly established. In this study, site-specific mutagenesis strategy was employed for cdtA point mutations construction so as to examine the contributions of individual amino acids to receptor binding and the biological activity of holotoxin. The binding ability was reduced in CdtA(Y181ABC holotoxin and the biological function of CDT was not weaken in CdtA(Y105ABC, CdtA(Y125ABC, CdtA(F109ABC and CdtA(S106NBC holotoxin suggesting that these sites were not critical to CDT. But the binding activity and cell cycle arrest ability of holotoxin complexes were inhibited in CdtA(W115GBC. And this site did not affect the holotoxin assembly by size exclusion chromatography. Therefore, W115 might be a critical site of CdtA binding ability. These findings suggest that the functional sites of CdtA are not only in the aromatic patch region. W115, the new functional site is critical for receptor binding and cell cycle arrest, which provides potential targets for pharmacological disruption of CDT activity.

CDT meets Horava-Lifshitz gravity

International Nuclear Information System (INIS)

Ambjorn, J.; Goerlich, A.; Jordan, S.; Jurkiewicz, J.; Loll, R.

2010-01-01

The theory of causal dynamical triangulations (CDT) attempts to define a nonperturbative theory of quantum gravity as a sum over spacetime geometries. One of the ingredients of the CDT framework is a global time foliation, which also plays a central role in the quantum gravity theory recently formulated by Horava. We show that the phase diagram of CDT bears a striking resemblance with the generic Lifshitz phase diagram appealed to by Horava. We argue that CDT might provide a unifying nonperturbative framework for anisotropic as well as isotropic theories of quantum gravity.

Synthesis of Cyclododecatriene from 1,3-Butadiene by Trimerization over Amine-Titanium Complex Catalyst

International Nuclear Information System (INIS)

Park, Da Min; Kim, Gye Ryung; Lee, Ju Hyun; Kim, Geon-Joong; Cho, Deuk Hee

2013-01-01

The new complex catalysts were synthesized by the reaction of titanium compounds (titanium chloride or titanium butoxide) and diamines in this work, and they showed very high catalytic activities for the cyclododecatriene (CDT) synthesis from 1,3-butadiene through trimerization. CDT synthetic reaction was performed in an autoclave reactor, and the effects of reaction temperature, type of catalyst, catalyst amount added into the system, the mole ratio of Al/Ti and immobilization method were investigated on the yield of product CDT. The titanium complex catalyst combined to diamine with 1:1 ratio showed high selectivity to CDT more than 90%. The ratio of TTT-CDT/TTC-CDT isomers in the product revealed as different values, depending on the type of diamine combined to titanium and Ti/diamine ratios. Those homogeneous complexes could be used as a heterogenized catalyst after anchoring on the supports, and the immobilized titanium catalyst retained the catalytic activities for several times in the recycled reactions without leaching. The carbon support containing titanium has exhibited superior activity to the silica support. Especially, when the titanium complex was anchored on the support which was fabricated by the hydrolysis of tripropylaminosilane itself, the resulting titanium catalyst showed the highest BD conversion and CDT selectivity

SCF^{Cyclin F}-dependent degradation of CDC6 suppresses DNA re-replication

DEFF Research Database (Denmark)

Walter, David; Hoffmann, Saskia; Komseli, Eirini-Stavroula

2016-01-01

interact through defined sequence motifs that promote CDC6 ubiquitylation and degradation. Absence of Cyclin F or expression of a stable mutant of CDC6 promotes re-replication and genome instability in cells lacking the CDT1 inhibitor Geminin. Together, our work reveals a novel SCF(Cyclin F...

CDT and the Big Bang

OpenAIRE

Ambjorn, J.; Watabiki, Y.

2017-01-01

We describe a CDT-like model where breaking of W3 symmetry will lead to the emergence of time and subsequently of space. Surprisingly the simplest such models which lead to higher dimensional spacetimes are based on the four "magical" Jordan algebras of 3x3 Hermitian matrices with real, complex, quaternion and octonion entries, respectively. The simplest symmetry breaking leads to universes with spacetime dimensions 3, 4, 6, and 10.

Inhibition of NEDD8-activating enzyme induces rereplication and apoptosis in human tumor cells consistent with deregulating CDT1 turnover.

Science.gov (United States)

Milhollen, Michael A; Narayanan, Usha; Soucy, Teresa A; Veiby, Petter O; Smith, Peter G; Amidon, Benjamin

2011-04-15

Loss of NEDD8-activating enzyme (NAE) function by siRNA knockdown or inhibition by the small molecule NAE inhibitor MLN4924 leads to increased steady-state levels of direct Cullin-RING ligase (CRL) substrates by preventing their ubiquitination and proteasome-dependent degradation. Many of these CRL substrates are involved in cell cycle progression, including a critical DNA replication licensing factor CDT1. Cell cycle analysis of asynchronous and synchronous cultures after NAE inhibition revealed effects on cell cycle distribution and activation of DNA break repair signaling pathways similar to that reported for CDT1 overexpression. The siRNA knockdown of cullins critical for the turnover of CDT1 recapitulated the aberrant rereplication phenotype while CDT1 knockdown was suppressing. Although NAE inhibition leads to deregulation of many CRL substrates, these data demonstrate that CDT1 accumulation mediates the DNA rereplication phenotype resulting from loss of NAE function. DNA rereplication is an unrecoverable cellular insult and the small molecule inhibitor MLN4924, currently in phase I trials, represents an unprecedented opportunity to explore this mechanism of cytotoxicity for the treatment of cancer. ©2011 AACR.

APC/C--the master controller of origin licensing?

Science.gov (United States)

Sivaprasad, Umasundari; Machida, Yuichi J; Dutta, Anindya

2007-02-23

DNA replication must be tightly controlled to prevent initiation of a second round of replication until mitosis is complete. So far, components of the pre-replicative complex (Cdt1, Cdc6 and geminin) were considered key players in this regulation. In a new study, Machida and Dutta have shown that depletion of Emi1 caused cells to replicate their DNA more than once per cell cycle 1. This effect was dependent on the ability of Emi1 to inhibit the APC/C. In addition to its role in regulating entry into mitosis, oscillation of APC/C activity regulates pre-RC formation: high APC/C activity in late M/G1 allows pre-RC formation and low APC/C activity in S/G2 prevents pre-RC formation for a second time thereby preventing rereplication. Each redundant pathway to prevent rereplication is dependent on regulating one of the pre-RC components, and all of the pathways are co-regulated by Emi1 through the APC/C. In this commentary we discuss how this new role of Emi1 adds to our understanding of the regulation of replication initiation. We also review the literature to analyze whether APC/C has a role in regulating endoreduplication (a normal state of polyploidy in some differentiated cells). Similarly a role of premature APC/C activation in genomic instability of tumors is discussed.

APC/C – the master controller of origin licensing?

Directory of Open Access Journals (Sweden)

Dutta Anindya

2007-02-01

Full Text Available Abstract DNA replication must be tightly controlled to prevent initiation of a second round of replication until mitosis is complete. So far, components of the pre-replicative complex (Cdt1, Cdc6 and geminin were considered key players in this regulation. In a new study, Machida and Dutta have shown that depletion of Emi1 caused cells to replicate their DNA more than once per cell cycle 1. This effect was dependent on the ability of Emi1 to inhibit the APC/C. In addition to its role in regulating entry into mitosis, oscillation of APC/C activity regulates pre-RC formation: high APC/C activity in late M/G1 allows pre-RC formation and low APC/C activity in S/G2 prevents pre-RC formation for a second time thereby preventing rereplication. Each redundant pathway to prevent rereplication is dependent on regulating one of the pre-RC components, and all of the pathways are co-regulated by Emi1 through the APC/C. In this commentary we discuss how this new role of Emi1 adds to our understanding of the regulation of replication initiation. We also review the literature to analyze whether APC/C has a role in regulating endoreduplication (a normal state of polyploidy in some differentiated cells. Similarly a role of premature APC/C activation in genomic instability of tumors is discussed.

PIP degron proteins, substrates of CRL4Cdt2, and not PIP boxes, interfere with DNA polymerase η and κ focus formation on UV damage.

Science.gov (United States)

Tsanov, Nikolay; Kermi, Chames; Coulombe, Philippe; Van der Laan, Siem; Hodroj, Dana; Maiorano, Domenico

2014-04-01

Proliferating cell nuclear antigen (PCNA) is a well-known scaffold for many DNA replication and repair proteins, but how the switch between partners is regulated is currently unclear. Interaction with PCNA occurs via a domain known as a PCNA-Interacting Protein motif (PIP box). More recently, an additional specialized PIP box has been described, the « PIP degron », that targets PCNA-interacting proteins for proteasomal degradation via the E3 ubiquitin ligase CRL4(Cdt2). Here we provide evidence that CRL4(Cdt2)-dependent degradation of PIP degron proteins plays a role in the switch of PCNA partners during the DNA damage response by facilitating accumulation of translesion synthesis DNA polymerases into nuclear foci. We show that expression of a nondegradable PIP degron (Cdt1) impairs both Pol η and Pol κ focus formation on ultraviolet irradiation and reduces cell viability, while canonical PIP box-containing proteins have no effect. Furthermore, we identify PIP degron-containing peptides from several substrates of CRL4(Cdt2) as efficient inhibitors of Pol η foci formation. By site-directed mutagenesis we show that inhibition depends on a conserved threonine residue that confers high affinity for PCNA-binding. Altogether these findings reveal an important regulative role for the CRL4(Cdt2) pathway in the switch of PCNA partners on DNA damage.

Optimization of CDT-1 and XYL1 Expression for Balanced Co-Production of Ethanol and Xylitol from Cellobiose and Xylose by Engineered Saccharomyces cerevisiae

Science.gov (United States)

Zha, Jian; Li, Bing-Zhi; Shen, Ming-Hua; Hu, Meng-Long; Song, Hao; Yuan, Ying-Jin

2013-01-01

Production of ethanol and xylitol from lignocellulosic hydrolysates is an alternative to the traditional production of ethanol in utilizing biomass. However, the conversion efficiency of xylose to xylitol is restricted by glucose repression, causing a low xylitol titer. To this end, we cloned genes CDT-1 (encoding a cellodextrin transporter) and gh1-1 (encoding an intracellular β-glucosidase) from Neurospora crassa and XYL1 (encoding a xylose reductase that converts xylose into xylitol) from Scheffersomyces stipitis into Saccharomyces cerevisiae, enabling simultaneous production of ethanol and xylitol from a mixture of cellobiose and xylose (main components of lignocellulosic hydrolysates). We further optimized the expression levels of CDT-1 and XYL1 by manipulating their promoters and copy-numbers, and constructed an engineered S. cerevisiae strain (carrying one copy of PGK1p-CDT1 and two copies of TDH3p-XYL1), which showed an 85.7% increase in xylitol production from the mixture of cellobiose and xylose than that from the mixture of glucose and xylose. Thus, we achieved a balanced co-fermentation of cellobiose (0.165 g/L/h) and xylose (0.162 g/L/h) at similar rates to co-produce ethanol (0.36 g/g) and xylitol (1.00 g/g). PMID:23844185

Optimization of CDT-1 and XYL1 expression for balanced co-production of ethanol and xylitol from cellobiose and xylose by engineered Saccharomyces cerevisiae.

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Jian Zha

Full Text Available Production of ethanol and xylitol from lignocellulosic hydrolysates is an alternative to the traditional production of ethanol in utilizing biomass. However, the conversion efficiency of xylose to xylitol is restricted by glucose repression, causing a low xylitol titer. To this end, we cloned genes CDT-1 (encoding a cellodextrin transporter and gh1-1 (encoding an intracellular β-glucosidase from Neurospora crassa and XYL1 (encoding a xylose reductase that converts xylose into xylitol from Scheffersomyces stipitis into Saccharomyces cerevisiae, enabling simultaneous production of ethanol and xylitol from a mixture of cellobiose and xylose (main components of lignocellulosic hydrolysates. We further optimized the expression levels of CDT-1 and XYL1 by manipulating their promoters and copy-numbers, and constructed an engineered S. cerevisiae strain (carrying one copy of PGK1p-CDT1 and two copies of TDH3p-XYL1, which showed an 85.7% increase in xylitol production from the mixture of cellobiose and xylose than that from the mixture of glucose and xylose. Thus, we achieved a balanced co-fermentation of cellobiose (0.165 g/L/h and xylose (0.162 g/L/h at similar rates to co-produce ethanol (0.36 g/g and xylitol (1.00 g/g.

De Novo GMNN Mutations Cause Autosomal-Dominant Primordial Dwarfism Associated with Meier-Gorlin Syndrome.

Science.gov (United States)

Burrage, Lindsay C; Charng, Wu-Lin; Eldomery, Mohammad K; Willer, Jason R; Davis, Erica E; Lugtenberg, Dorien; Zhu, Wenmiao; Leduc, Magalie S; Akdemir, Zeynep C; Azamian, Mahshid; Zapata, Gladys; Hernandez, Patricia P; Schoots, Jeroen; de Munnik, Sonja A; Roepman, Ronald; Pearring, Jillian N; Jhangiani, Shalini; Katsanis, Nicholas; Vissers, Lisenka E L M; Brunner, Han G; Beaudet, Arthur L; Rosenfeld, Jill A; Muzny, Donna M; Gibbs, Richard A; Eng, Christine M; Xia, Fan; Lalani, Seema R; Lupski, James R; Bongers, Ernie M H F; Yang, Yaping

2015-12-03

Meier-Gorlin syndrome (MGS) is a genetically heterogeneous primordial dwarfism syndrome known to be caused by biallelic loss-of-function mutations in one of five genes encoding pre-replication complex proteins: ORC1, ORC4, ORC6, CDT1, and CDC6. Mutations in these genes cause disruption of the origin of DNA replication initiation. To date, only an autosomal-recessive inheritance pattern has been described in individuals with this disorder, with a molecular etiology established in about three-fourths of cases. Here, we report three subjects with MGS and de novo heterozygous mutations in the 5' end of GMNN, encoding the DNA replication inhibitor geminin. We identified two truncating mutations in exon 2 (the 1(st) coding exon), c.16A>T (p.Lys6(∗)) and c.35_38delTCAA (p.Ile12Lysfs(∗)4), and one missense mutation, c.50A>G (p.Lys17Arg), affecting the second-to-last nucleotide of exon 2 and possibly RNA splicing. Geminin is present during the S, G2, and M phases of the cell cycle and is degraded during the metaphase-anaphase transition by the anaphase-promoting complex (APC), which recognizes the destruction box sequence near the 5' end of the geminin protein. All three GMNN mutations identified alter sites 5' to residue Met28 of the protein, which is located within the destruction box. We present data supporting a gain-of-function mechanism, in which the GMNN mutations result in proteins lacking the destruction box and hence increased protein stability and prolonged inhibition of replication leading to autosomal-dominant MGS. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

The Role of the CRL4Cdt2 Target Spd1 in Chromosome Segregation in Fission Yeast

DEFF Research Database (Denmark)

Landvad, Katrine

Ddb1, a component of the E3 ubiquitin ligase CRL4Cdt2, is needed for proper chromosome segregation in fission yeast as ddb1 deleted cells show unequal distribution of DNA to daughter cells and sensitivity to the microtubule destabilising drug TBZ. In this study we show that Δddb1 cells have...

Loss of Geminin induces rereplication in the presence of functional p53

DEFF Research Database (Denmark)

Melixetian, Marina; Ballabeni, Andrea; Masiero, Laura

2004-01-01

nuclear foci. Abrogation of the checkpoint leads to abortive mitosis and death of rereplicated cells. In addition, we demonstrate that the induction of rereplication is dependent on the replication initiation factors CDT1 and CDC6, and independent of the functional status of p53. These data show...

2d CDT is 2d Horava-Lifshitz quantum gravity

DEFF Research Database (Denmark)

Ambjørn, J.; Glaser, L.; Sato, Y.

2013-01-01

Causal Dynamical Triangulations (CDT) is a lattice theory where aspects of quantum gravity can be studied. Two-dimensional CDT can be solved analytically and the continuum (quantum) Hamiltonian obtained. In this Letter we show that this continuum Hamiltonian is the one obtained by quantizing two......-dimensional projectable Horava-Lifshitz gravity....

Suppressor Analysis of CRL4Cdt2 Defective and cdc48-353 Temperature Sensitive Mutants in Fission Yeast

DEFF Research Database (Denmark)

Marinova, Irina Nikolaeva

chaperone-like complex involved in numerous cellular processes, including protein degradation, cell cycle control, DNA repair, and vesicle fusion. The cdc48 gene is essential in fission yeast and mutations or changes in Cdc48/p97 protein expression have been linked to neurological disorders and cancer......SummaryPart 1CRL4Cdt2 E3 ligase is a key regulator of cellular proliferation and genome integrity, as it promotes the degradation of proteins involved in cell cycle progression, DNA replication and repair. In fission yeast the small intrinsically disordered protein Spd1 is targeted for degradation...... that these mutations alleviate the checkpoint dependency, the DNA damage sensitivity and the meiotic defects associated with Spd1 accumulation. Further analysis showed that whereas the V40G and S43L substitutions do not have a significant impact on Suc22R2 nuclear import function of Spd1, they affect the interaction...

Soaking RNAi in Bombyx mori BmN4-SID1 Cells Arrests Cell Cycle Progression

Science.gov (United States)

Mon, Hiroaki; Li, Zhiqing; Kobayashi, Isao; Tomita, Shuichiro; Lee, JaeMan; Sezutsu, Hideki; Tamura, Toshiki; Kusakabe, Takahiro

2013-01-01

RNA interference (RNAi) is an evolutionarily conserved mechanism for sequence-specific gene silencing. Previously, the BmN4-SID1 cell expressing Caenorhabditis ele gans SID-1 was established, in which soaking RNAi could induce effective gene silencing. To establish its utility, 6 cell cycle progression related cDNAs, CDK1, MYC, MYB, RNRS, CDT1, and GEMININ, were isolated from the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), and their expressions were further silenced by soaking RNAi in the BmN4-SID1 cells. The cell cycle progression analysis using flow cytometer demonstrated that the small amount of double stranded RNA was enough to arrest cell cycle progression at the specific cell phases. These data suggest that RNAi in the BmN4-SID1 cells can be used as a powerful tool for loss-of-function analysis of B. mori genes. PMID:24773378

Initiation and termination of DNA replication during S phase in relation to cyclins D1, E and A, p21WAF1, Cdt1 and the p12 subunit of DNA polymerase δ revealed in individual cells by cytometry.

Science.gov (United States)

Darzynkiewicz, Zbigniew; Zhao, Hong; Zhang, Sufang; Lee, Marietta Y W T; Lee, Ernest Y C; Zhang, Zhongtao

2015-05-20

During our recent studies on mechanism of the regulation of human DNA polymerase δ in preparation for DNA replication or repair, multiparameter imaging cytometry as exemplified by laser scanning cytometry (LSC) has been used to assess changes in expression of the following nuclear proteins associated with initiation of DNA replication: cyclin A, PCNA, Ki-67, p21(WAF1), DNA replication factor Cdt1 and the smallest subunit of DNA polymerase δ, p12. In the present review, rather than focusing on Pol δ, we emphasize the application of LSC in these studies and outline possibilities offered by the concurrent differential analysis of DNA replication in conjunction with expression of the nuclear proteins. A more extensive analysis of the data on a correlation between rates of EdU incorporation, likely reporting DNA replication, and expression of these proteins, is presently provided. New data, specifically on the expression of cyclin D1 and cyclin E with respect to EdU incorporation as well as on a relationship between expression of cyclin A vs. p21(WAF1) and Ki-67 vs. Cdt1, are also reported. Of particular interest is the observation that this approach makes it possible to assess the temporal sequence of degradation of cyclin D1, p21(WAF1), Cdt1 and p12, each with respect to initiation of DNA replication and with respect to each other. Also the sequence or reappearance of these proteins in G2 after termination of DNA replication is assessed. The reviewed data provide a more comprehensive presentation of potential markers, whose presence or absence marks the DNA replicating cells. Discussed is also usefulness of these markers as indicators of proliferative activity in cancer tissues that may bear information on tumor progression and have a prognostic value.

Clostridium difficile toxin CDT induces formation of microtubule-based protrusions and increases adherence of bacteria.

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Carsten Schwan

2009-10-01

Full Text Available Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis by production of the Rho GTPase-glucosylating toxins A and B. Recently emerging hypervirulent Clostridium difficile strains additionally produce the binary ADP-ribosyltransferase toxin CDT (Clostridium difficile transferase, which ADP-ribosylates actin and inhibits actin polymerization. Thus far, the role of CDT as a virulence factor is not understood. Here we report by using time-lapse- and immunofluorescence microscopy that CDT and other binary actin-ADP-ribosylating toxins, including Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin, induce redistribution of microtubules and formation of long (up to >150 microm microtubule-based protrusions at the surface of intestinal epithelial cells. The toxins increase the length of decoration of microtubule plus-ends by EB1/3, CLIP-170 and CLIP-115 proteins and cause redistribution of the capture proteins CLASP2 and ACF7 from microtubules at the cell cortex into the cell interior. The CDT-induced microtubule protrusions form a dense meshwork at the cell surface, which wrap and embed bacterial cells, thereby largely increasing the adherence of Clostridia. The study describes a novel type of microtubule structure caused by less efficient microtubule capture and offers a new perspective for the pathogenetic role of CDT and other binary actin-ADP-ribosylating toxins in host-pathogen interactions.

MMSET is dynamically regulated during cell-cycle progression and promotes normal DNA replication.

Science.gov (United States)

Evans, Debra L; Zhang, Haoxing; Ham, Hyoungjun; Pei, Huadong; Lee, SeungBaek; Kim, JungJin; Billadeau, Daniel D; Lou, Zhenkun

2016-01-01

The timely and precise duplication of cellular DNA is essential for maintaining genome integrity and is thus tightly-regulated. During mitosis and G1, the Origin Recognition Complex (ORC) binds to future replication origins, coordinating with multiple factors to load the minichromosome maintenance (MCM) complex onto future replication origins as part of the pre-replication complex (pre-RC). The pre-RC machinery, in turn, remains inactive until the subsequent S phase when it is required for replication fork formation, thereby initiating DNA replication. Multiple myeloma SET domain-containing protein (MMSET, a.k.a. WHSC1, NSD2) is a histone methyltransferase that is frequently overexpressed in aggressive cancers and is essential for normal human development. Several studies have suggested a role for MMSET in cell-cycle regulation; however, whether MMSET is itself regulated during cell-cycle progression has not been examined. In this study, we report that MMSET is degraded during S phase in a cullin-ring ligase 4-Cdt2 (CRL4(Cdt2)) and proteasome-dependent manner. Notably, we also report defects in DNA replication and a decreased association of pre-RC factors with chromatin in MMSET-depleted cells. Taken together, our results suggest a dynamic regulation of MMSET levels throughout the cell cycle, and further characterize the role of MMSET in DNA replication and cell-cycle progression.

Trees and spatial topology change in CDT

DEFF Research Database (Denmark)

Ambjorn, Jan; Budd, Timothy George

2013-01-01

Generalized causal dynamical triangulations (generalized CDT) is a model of two-dimensional quantum gravity in which a limited number of spatial topology changes is allowed to occur. We solve the model at the discretized level using bijections between quadrangulations and trees. In the continuum...

UV Damage-Induced Phosphorylation of HBO1 Triggers CRL4DDB2-Mediated Degradation To Regulate Cell Proliferation

Science.gov (United States)

Matsunuma, Ryoichi; Ohhata, Tatsuya; Kitagawa, Kyoko; Sakai, Satoshi; Uchida, Chiharu; Shiotani, Bunsyo; Matsumoto, Masaki; Nakayama, Keiichi I.; Ogura, Hiroyuki; Shiiya, Norihiko; Kitagawa, Masatoshi

2015-01-01

Histone acetyltransferase binding to ORC-1 (HBO1) is a critically important histone acetyltransferase for forming the prereplicative complex (pre-RC) at the replication origin. Pre-RC formation is completed by loading of the MCM2-7 heterohexameric complex, which functions as a helicase in DNA replication. HBO1 recruited to the replication origin by CDT1 acetylates histone H4 to relax the chromatin conformation and facilitates loading of the MCM complex onto replication origins. However, the acetylation status and mechanism of regulation of histone H3 at replication origins remain elusive. HBO1 positively regulates cell proliferation under normal cell growth conditions. Whether HBO1 regulates proliferation in response to DNA damage is poorly understood. In this study, we demonstrated that HBO1 was degraded after DNA damage to suppress cell proliferation. Ser50 and Ser53 of HBO1 were phosphorylated in an ATM/ATR DNA damage sensor-dependent manner after UV treatment. ATM/ATR-dependently phosphorylated HBO1 preferentially interacted with DDB2 and was ubiquitylated by CRL4DDB2. Replacement of endogenous HBO1 in Ser50/53Ala mutants maintained acetylation of histone H3K14 and impaired cell cycle regulation in response to UV irradiation. Our findings demonstrate that HBO1 is one of the targets in the DNA damage checkpoint. These results show that ubiquitin-dependent control of the HBO1 protein contributes to cell survival during UV irradiation. PMID:26572825

Use of finger-prick dried blood spots (fpDBS) and capillary electrophoresis for carbohydrate deficient transferrin (CDT) screening in forensic toxicology.

Science.gov (United States)

Bertaso, Anna; Sorio, Daniela; Vandoros, Anthula; De Palo, Elio F; Bortolotti, Federica; Tagliaro, Franco

2016-10-01

Continued progress in chronic alcohol abuse investigation requires the development of less invasive procedures for screening purposes. The application of finger-prick and related dried blood spots (fpDBS) for carbohydrate deficient transferrin (CDT) detection appears suitable for this aim. Therefore, the goal of this project was to develop a screening method for CDT using fpDBS with CZE analysis. Blood samples prepared by finger-prick were placed on DBS cards and left to air dry; each dried fpDBS disc was shredded into small pieces and suspended in acid solution (60 μL of HCl 120 mmol/L). After centrifugation (10 min at 1500 × g), the collected sample was adjusted to pH 3.5. After an overnight incubation, the pH was neutralised and an iron rich solution was added. After 1 h, CZE analysis was carried out. A group of 47 individuals was studied. Parallel serum samples were collected from each investigated subject and the %CDT for each sample was measured using HPLC and CZE techniques. The fpDBS transferrin sialo isoform electropherograms were similar to those obtained with serum. Moreover, fpDBS CZE CDT percentage levels demonstrated significant statistical correlation with those obtained from serum for both HPLC and CZE %CDT (p < 0.01; r 2 = 0.8913 and 0.8976, respectively), with %CDT from 0.8 to 13.7% for fpDBS and from 0.7 to 12.7% for serum. The newly developed fpDBS procedure for CDT analysis provides a simple and inexpensive tool for use in population screening. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PyCDT: A Python toolkit for modeling point defects in semiconductors and insulators

Science.gov (United States)

Broberg, Danny; Medasani, Bharat; Zimmermann, Nils E. R.; Yu, Guodong; Canning, Andrew; Haranczyk, Maciej; Asta, Mark; Hautier, Geoffroy

2018-05-01

Point defects have a strong impact on the performance of semiconductor and insulator materials used in technological applications, spanning microelectronics to energy conversion and storage. The nature of the dominant defect types, how they vary with processing conditions, and their impact on materials properties are central aspects that determine the performance of a material in a certain application. This information is, however, difficult to access directly from experimental measurements. Consequently, computational methods, based on electronic density functional theory (DFT), have found widespread use in the calculation of point-defect properties. Here we have developed the Python Charged Defect Toolkit (PyCDT) to expedite the setup and post-processing of defect calculations with widely used DFT software. PyCDT has a user-friendly command-line interface and provides a direct interface with the Materials Project database. This allows for setting up many charged defect calculations for any material of interest, as well as post-processing and applying state-of-the-art electrostatic correction terms. Our paper serves as a documentation for PyCDT, and demonstrates its use in an application to the well-studied GaAs compound semiconductor. We anticipate that the PyCDT code will be useful as a framework for undertaking readily reproducible calculations of charged point-defect properties, and that it will provide a foundation for automated, high-throughput calculations.

The Cytolethal Distending Toxin Subunit CdtB of Helicobacter hepaticus Promotes Senescence and Endoreplication in Xenograft Mouse Models of Hepatic and Intestinal Cell Lines

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Christelle Péré-Védrenne

2017-06-01

Full Text Available Cytolethal distending toxins (CDTs are common among pathogenic bacteria of the human and animal microbiota. CDTs exert cytopathic effets, via their active CdtB subunit. No clear description of those cytopathic effects has been reported at the cellular level in the target organs in vivo. In the present study, xenograft mouse models of colon and liver cell lines were set up to study the effects of the CdtB subunit of Helicobacter hepaticus. Conditional transgenic cell lines were established, validated in vitro and then engrafted into immunodeficient mice. After successful engraftment, mice were treated with doxycyclin to induce the expression of transgenes (red fluorescent protein, CdtB, and mutated CdtB. For both engrafted cell lines, results revealed a delayed tumor growth and a reduced tumor weight in CdtB-expressing tumors compared to controls. CdtB-derived tumors showed γ-H2AX foci formation, an increase in apoptosis, senescence, p21 and Ki-67 nuclear antigen expression. No difference in proliferating cells undergoing mitosis (phospho-histone H3 was observed. CdtB intoxication was also associated with an overexpression of cytokeratins in cells at the invasive front of the tumor as well as an increase in ploidy. All these features are hallmarks of endoreplication, as well as aggressiveness in cancer. These effects were dependent on the histidine residue at position 265 of the CdtB, underlying the importance of this residue in CdtB catalytic activity. Taken together, these data indicate that the CdtB triggers senescence and cell endoreplication leading to giant polyploid cells in these xenograft mouse models.

Regulation of hepatic lipogenesis by the transcription complex Prep1-Pbx1

OpenAIRE

Cabaro, Serena

2011-01-01

Prep1 is an homeodomain transcription factor belonging to the TALE proteins, including also Pbx1, which plays an essential role in hematopoiesis, organogenesis and development. Prep1 forms transcriptionally active complexes with Pbx1 and regulates the activity of several genes. The Prep1 null mutation leads to embryonic death at a very early stage. Therefore, Prep1 hypomorphic (Prep1i/i) mice have been generated. Prep1 heterozygous (Prep1i/+) mice, which express only 55-57% of protein, have a...

Pilot Study: The Effectiveness of Complex Decongestive Therapy for Lymphedema in Palliative Care Patients with Advanced Cancer.

LENUS (Irish Health Repository)

Cobbe, Sinead

2017-12-05

Complex decongestive therapy (CDT) is a regimen of physical treatment for lymphedema. Its effectiveness is unknown in advanced cancer patients. This study evaluates effectiveness of CDT in this population.

Alteration of light-dependent gene regulation by the absence of the RCO-1/RCM-1 repressor complex in the fungus Neurospora crassa.

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Carmen Ruger-Herreros

Full Text Available The activation of transcription by light in the fungus Neurospora crassa requires the White Collar Complex (WCC, a photoreceptor and transcription factor complex. After light reception two WCCs interact and bind the promoters of light-regulated genes to activate transcription. This process is regulated by VVD, a small photoreceptor that disrupts the interaction between WCCs and leads to a reduction in transcription after long exposures to light. The N. crassa RCO-1/RCM-1 repressor complex is the homolog of the Tup1-Ssn6 repressor complex in yeast, and its absence modifies photoadaptation. We show that the absence of the RCO-1/RCM-1 repressor complex leads to several alterations in transcription that are gene-specific: an increase in the accumulation of mRNAs in the dark, a repression of transcription, and a derepression of transcription after long exposures to light. The absence of the RCO-1/RCM-1 repressor complex leads to lower VVD levels that are available for the regulation of the activity of the WCC. The reduction in the amount of VVD results in increased WCC binding to the promoters of light-regulated genes in the dark and after long exposures to light, leading to the modification of photoadaptation that has been observed in rco-1 and rcm-1 mutants. Our results show that the photoadaptation phenotype of mutants in the RCO-1/RCM-1 repressor complex is, at least in part, an indirect consequence of the reduction of vvd transcription, and the resulting modification in the regulation of transcription by the WCC.

Neurexin-Neuroligin Synaptic Complex Regulates Schizophrenia-Related DISC1/Kal-7/Rac1 “Signalosome”

DEFF Research Database (Denmark)

Jacobsen, Sylwia Owczarek; Bang, Marie Louise; Berezin, Vladimir

2015-01-01

Neurexins (NXs) and neuroligins (NLs) are cell adhesion molecules that are localized at opposite sites of synaptic membranes. They interact with each other to promote the assembly, maintenance, and function of synapses in the central nervous system. Both NX and NL are cleaved from a membrane......-attached intracellular domain in an activity-dependent manner, generating the soluble ectodomain of NX or NL. Expression of the NX1 and NX3 genes in the brain appears to be regulated by a schizophrenia-related protein, DISC1. Here, we show that soluble ecto-NX1β can regulate the expression of DISC1 and induce signaling...... downstream of DISC1. We also show that NL1 binds to a well-characterized DISC1 interaction partner, Kal-7, and this interaction can be compromised by DISC1. Our results indicate that the NX/NL synaptic complex is intrinsically involved in the regulation of DISC1 function, thus contributing to a better...

Small G proteins Rac1 and Ras regulate serine/threonine protein phosphatase 5 (PP5)·extracellular signal-regulated kinase (ERK) complexes involved in the feedback regulation of Raf1.

Science.gov (United States)

Mazalouskas, Matthew D; Godoy-Ruiz, Raquel; Weber, David J; Zimmer, Danna B; Honkanen, Richard E; Wadzinski, Brian E

2014-02-14

Serine/threonine protein phosphatase 5 (PP5, PPP5C) is known to interact with the chaperonin heat shock protein 90 (HSP90) and is involved in the regulation of multiple cellular signaling cascades that control diverse cellular processes, such as cell growth, differentiation, proliferation, motility, and apoptosis. Here, we identify PP5 in stable complexes with extracellular signal-regulated kinases (ERKs). Studies using mutant proteins reveal that the formation of PP5·ERK1 and PP5·ERK2 complexes partially depends on HSP90 binding to PP5 but does not require PP5 or ERK1/2 activity. However, PP5 and ERK activity regulates the phosphorylation state of Raf1 kinase, an upstream activator of ERK signaling. Whereas expression of constitutively active Rac1 promotes the assembly of PP5·ERK1/2 complexes, acute activation of ERK1/2 fails to influence the phosphatase-kinase interaction. Introduction of oncogenic HRas (HRas(V12)) has no effect on PP5-ERK1 binding but selectively decreases the interaction of PP5 with ERK2, in a manner that is independent of PP5 and MAPK/ERK kinase (MEK) activity, yet paradoxically requires ERK2 activity. Additional studies conducted with oncogenic variants of KRas4B reveal that KRas(L61), but not KRas(V12), also decreases the PP5-ERK2 interaction. The expression of wild type HRas or KRas proteins fails to reduce PP5-ERK2 binding, indicating that the effect is specific to HRas(V12) and KRas(L61) gain-of-function mutations. These findings reveal a novel, differential responsiveness of PP5-ERK1 and PP5-ERK2 interactions to select oncogenic Ras variants and also support a role for PP5·ERK complexes in regulating the feedback phosphorylation of PP5-associated Raf1.

Cytolethal Distending Toxin Enhances Radiosensitivity in Prostate Cancer Cells by Regulating Autophagy

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Hwai-Jeng Lin

2017-06-01

Full Text Available Cytolethal distending toxin (CDT produced by Campylobacter jejuni contains three subunits: CdtA, CdtB, and CdtC. Among these three toxin subunits, CdtB is the toxic moiety of CDT with DNase I activity, resulting in DNA double-strand breaks (DSB and, consequently, cell cycle arrest at the G2/M stage and apoptosis. Radiation therapy is an effective modality for the treatment of localized prostate cancer (PCa. However, patients often develop radioresistance. Owing to its particular biochemical properties, we previously employed CdtB as a therapeutic agent for sensitizing radioresistant PCa cells to ionizing radiation (IR. In this study, we further demonstrated that CDT suppresses the IR-induced autophagy pathway in PCa cells by attenuating c-Myc expression and therefore sensitizes PCa cells to radiation. We further showed that CDT prevents the formation of autophagosomes via decreased high-mobility group box 1 (HMGB1 expression and the inhibition of acidic vesicular organelle (AVO formation, which are associated with enhanced radiosensitivity in PCa cells. The results of this study reveal the detailed mechanism of CDT for the treatment of radioresistant PCa.

Deoxynucleoside salvage in fission yeast allows rescue of ribonucleotide reductase deficiency but not Spd1-mediated inhibition of replication

DEFF Research Database (Denmark)

Fleck, Oliver; Fahnøe, Ulrik; Løvschal, Katrine Vyff

2017-01-01

In fission yeast, the small, intrinsically disordered protein S-phase delaying protein 1 (Spd1) blocks DNA replication and causes checkpoint activation at least in part, by inhibiting the enzyme ribonucleotide reductase, which is responsible for the synthesis of DNA. The CRL4(Cdt2) E3 ubiquitin...... ligase mediates degradation of Spd1 and the related protein Spd2 at S phase of the cell cycle. We have generated a conditional allele of CRL4(Cdt2), by expressing the highly unstable substrate-recruiting protein Cdt2 from a repressible promoter. Unlike Spd1, Spd2 does not regulate deoxynucleotide...... triphosphate (dNTP) pools; yet we find that Spd1 and Spd2 together inhibit DNA replication upon Cdt2 depletion. To directly test whether this block of replication was solely due to insufficient dNTP levels, we established a deoxy-nucleotide salvage pathway in fission yeast by expressing the human nucleoside...

PIP degron proteins, substrates of CRL4Cdt2, and not PIP boxes, interfere with DNA polymerase η and κ focus formation on UV damage

OpenAIRE

Tsanov, Nikolay; Kermi, Chames; Coulombe, Philippe; Van der Laan, Siem; Hodroj, Dana; Maiorano, Domenico

2014-01-01

Proliferating cell nuclear antigen (PCNA) is a well-known scaffold for many DNA replication and repair proteins, but how the switch between partners is regulated is currently unclear. Interaction with PCNA occurs via a domain known as a PCNA-Interacting Protein motif (PIP box). More recently, an additional specialized PIP box has been described, the « PIP degron », that targets PCNA-interacting proteins for proteasomal degradation via the E3 ubiquitin ligase CRL4Cdt2. Here we provide evidence...

ATF1 Modulates the Heat Shock Response by Regulating the Stress-Inducible Heat Shock Factor 1 Transcription Complex

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Takii, Ryosuke; Fujimoto, Mitsuaki; Tan, Ke; Takaki, Eiichi; Hayashida, Naoki; Nakato, Ryuichiro; Shirahige, Katsuhiko

2014-01-01

The heat shock response is an evolutionally conserved adaptive response to high temperatures that controls proteostasis capacity and is regulated mainly by an ancient heat shock factor (HSF). However, the regulation of target genes by the stress-inducible HSF1 transcription complex has not yet been examined in detail in mammalian cells. In the present study, we demonstrated that HSF1 interacted with members of the ATF1/CREB family involved in metabolic homeostasis and recruited them on the HSP70 promoter in response to heat shock. The HSF1 transcription complex, including the chromatin-remodeling factor BRG1 and lysine acetyltransferases p300 and CREB-binding protein (CBP), was formed in a manner that was dependent on the phosphorylation of ATF1. ATF1-BRG1 promoted the establishment of an active chromatin state and HSP70 expression during heat shock, whereas ATF1-p300/CBP accelerated the shutdown of HSF1 DNA-binding activity during recovery from acute stress, possibly through the acetylation of HSF1. Furthermore, ATF1 markedly affected the resistance to heat shock. These results revealed the unanticipated complexity of the primitive heat shock response mechanism, which is connected to metabolic adaptation. PMID:25312646

Regulators of cyclin-dependent kinases are crucial for maintaining genome integrity in S phase

DEFF Research Database (Denmark)

Beck, Halfdan; Nähse, Viola; Larsen, Marie Sofie Yoo

2010-01-01

are important negative regulators of CDK1 and -2. Strikingly, WEE1 depletion rapidly induced DNA damage in S phase in newly replicated DNA, which was accompanied by a marked increase in single-stranded DNA. This DNA damage is dependent on CDK1 and -2 as well as the replication proteins MCM2 and CDT1 but not CDC...

Modulation of chromatin structure by the FACT histone chaperone complex regulates HIV-1 integration.

Science.gov (United States)

Matysiak, Julien; Lesbats, Paul; Mauro, Eric; Lapaillerie, Delphine; Dupuy, Jean-William; Lopez, Angelica P; Benleulmi, Mohamed Salah; Calmels, Christina; Andreola, Marie-Line; Ruff, Marc; Llano, Manuel; Delelis, Olivier; Lavigne, Marc; Parissi, Vincent

2017-07-28

Insertion of retroviral genome DNA occurs in the chromatin of the host cell. This step is modulated by chromatin structure as nucleosomes compaction was shown to prevent HIV-1 integration and chromatin remodeling has been reported to affect integration efficiency. LEDGF/p75-mediated targeting of the integration complex toward RNA polymerase II (polII) transcribed regions ensures optimal access to dynamic regions that are suitable for integration. Consequently, we have investigated the involvement of polII-associated factors in the regulation of HIV-1 integration. Using a pull down approach coupled with mass spectrometry, we have selected the FACT (FAcilitates Chromatin Transcription) complex as a new potential cofactor of HIV-1 integration. FACT is a histone chaperone complex associated with the polII transcription machinery and recently shown to bind LEDGF/p75. We report here that a tripartite complex can be formed between HIV-1 integrase, LEDGF/p75 and FACT in vitro and in cells. Biochemical analyzes show that FACT-dependent nucleosome disassembly promotes HIV-1 integration into chromatinized templates, and generates highly favored nucleosomal structures in vitro. This effect was found to be amplified by LEDGF/p75. Promotion of this FACT-mediated chromatin remodeling in cells both increases chromatin accessibility and stimulates HIV-1 infectivity and integration. Altogether, our data indicate that FACT regulates HIV-1 integration by inducing local nucleosomes dissociation that modulates the functional association between the incoming intasome and the targeted nucleosome.

TOR complex 2-Ypk1 signaling is an essential positive regulator of the general amino acid control response and autophagy.

Science.gov (United States)

Vlahakis, Ariadne; Graef, Martin; Nunnari, Jodi; Powers, Ted

2014-07-22

The highly conserved Target of Rapamycin (TOR) kinase is a central regulator of cell growth and metabolism in response to nutrient availability. TOR functions in two structurally and functionally distinct complexes, TOR Complex 1 (TORC1) and TOR Complex 2 (TORC2). Through TORC1, TOR negatively regulates autophagy, a conserved process that functions in quality control and cellular homeostasis and, in this capacity, is part of an adaptive nutrient deprivation response. Here we demonstrate that during amino acid starvation TOR also operates independently as a positive regulator of autophagy through the conserved TORC2 and its downstream target protein kinase, Ypk1. Under these conditions, TORC2-Ypk1 signaling negatively regulates the Ca(2+)/calmodulin-dependent phosphatase, calcineurin, to enable the activation of the amino acid-sensing eIF2α kinase, Gcn2, and to promote autophagy. Our work reveals that the TORC2 pathway regulates autophagy in an opposing manner to TORC1 to provide a tunable response to cellular metabolic status.

Application of a high throughput method of biomarker discovery to improvement of the EarlyCDT(®-Lung Test.

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Isabel K Macdonald

Full Text Available BACKGROUND: The National Lung Screening Trial showed that CT screening for lung cancer led to a 20% reduction in mortality. However, CT screening has a number of disadvantages including low specificity. A validated autoantibody assay is available commercially (EarlyCDT®-Lung to aid in the early detection of lung cancer and risk stratification in patients with pulmonary nodules detected by CT. Recent advances in high throughput (HTP cloning and expression methods have been developed into a discovery pipeline to identify biomarkers that detect autoantibodies. The aim of this study was to demonstrate the successful clinical application of this strategy to add to the EarlyCDT-Lung panel in order to improve its sensitivity and specificity (and hence positive predictive value, (PPV. METHODS AND FINDINGS: Serum from two matched independent cohorts of lung cancer patients were used (n = 100 and n = 165. Sixty nine proteins were initially screened on an abridged HTP version of the autoantibody ELISA using protein prepared on small scale by a HTP expression and purification screen. Promising leads were produced in shake flask culture and tested on the full assay. These results were analyzed in combination with those from the EarlyCDT-Lung panel in order to provide a set of re-optimized cut-offs. Five proteins that still displayed cancer/normal differentiation were tested for reproducibility and validation on a second batch of protein and a separate patient cohort. Addition of these proteins resulted in an improvement in the sensitivity and specificity of the test from 38% and 86% to 49% and 93% respectively (PPV improvement from 1 in 16 to 1 in 7. CONCLUSION: This is a practical example of the value of investing resources to develop a HTP technology. Such technology may lead to improvement in the clinical utility of the EarlyCDT--Lung test, and so further aid the early detection of lung cancer.

Ethylene Regulates Levels of Ethylene Receptor/CTR1 Signaling Complexes in Arabidopsis thaliana*

Science.gov (United States)

Shakeel, Samina N.; Gao, Zhiyong; Amir, Madiha; Chen, Yi-Feng; Rai, Muneeza Iqbal; Haq, Noor Ul; Schaller, G. Eric

2015-01-01

The plant hormone ethylene is perceived by a five-member family of receptors in Arabidopsis thaliana. The receptors function in conjunction with the Raf-like kinase CTR1 to negatively regulate ethylene signal transduction. CTR1 interacts with multiple members of the receptor family based on co-purification analysis, interacting more strongly with receptors containing a receiver domain. Levels of membrane-associated CTR1 vary in response to ethylene, doing so in a post-transcriptional manner that correlates with ethylene-mediated changes in levels of the ethylene receptors ERS1, ERS2, EIN4, and ETR2. Interactions between CTR1 and the receptor ETR1 protect ETR1 from ethylene-induced turnover. Kinetic and dose-response analyses support a model in which two opposing factors control levels of the ethylene receptor/CTR1 complexes. Ethylene stimulates the production of new complexes largely through transcriptional induction of the receptors. However, ethylene also induces turnover of receptors, such that levels of ethylene receptor/CTR1 complexes decrease at higher ethylene concentrations. Implications of this model for ethylene signaling are discussed. PMID:25814663

A CDT-Based Heuristic Zone Design Approach for Economic Census Investigators

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Changixu Cheng

2015-01-01

Full Text Available This paper addresses a special zone design problem for economic census investigators that is motivated by a real-world application. This paper presented a heuristic multikernel growth approach via Constrained Delaunay Triangulation (CDT. This approach not only solved the barriers problem but also dealt with the polygon data in zoning procedure. In addition, it uses a new heuristic method to speed up the zoning process greatly on the premise of the required quality of zoning. At last, two special instances for economic census were performed, highlighting the performance of this approach.

Regulation of actomyosin ATPase activity by troponin-tropomyosin: effect of the binding of the myosin subfragment 1 (S-1) ATP complex

International Nuclear Information System (INIS)

Greene, L.E.; Williams, D.L. Jr.; Eisenberg, E.

1987-01-01

In the authors' model of regulation, the observed lack of cooperativity in the binding of myosin subfragment 1 (S-1) with bound ATP to the troponin-tropomyosin-actin complex (regulated actin) is explained by S-1 ATP having about the same affinity for the conformation of the regulated actin that activates the myosin ATPase activity (turned-on form) and the conformation that does not activate the myosin ATPase activity (turned-off form). This predicts that, in the absence of Ca 2+ , S-1 ATP should not turn on the regulated actin filament. In the present study, they tested this prediction by using either unmodified S-1 or S-1 chemically modified with N,N'-p-phenylenedimaleimide (pPDM S-1) so that functionally it acts like S-1 ATP, although it does not hydrolyze ATP. [ 14 C]pPDM and [ 32 P]ATP were used as tracers. They found that, in the absence of Ca 2+ , neither S-1 ATP nor pPDM S-1 ATP significantly turns on the ATPase activity of the regulated complex of actin and S-1 (acto S-1). In contrast, in the presence of Ca 2+ , pPDM S-1 ATP binding almost completely turns on the regulated acto S-1 ATPase activity. These results can be explained by their original cooperativity model, with pPDM S-1 ATP binding only ≅ 2 fold more strongly to the turned-on form that to the turned-off form of regulated actin. However, the results are not consistent with our alternative model, which predicts that if pPDM S-1 ATP binds to actin in the absence of Ca 2+ but does not turn on the ATPase activity, then it should also turn on the ATPase activity in the presence of Ca 2+

The mechanism of Jurkat cells apoptosis induced by Aggregatibacter actinomycetemcomitans cytolethal distending toxin.

Science.gov (United States)

Chen, Hui-Ping; Li, Lu; Chen, Xu; Yang, Mi-Fang; Ye, Yu; Wang, Xiao-Qian; Xu, Yan

2017-06-01

Cytolethal distending toxin (CDT) which is produced by Aggregatibacter actinomycetemcomitans causes apoptosis in lymphocytes. But the specific mechanism is not clear. The aim of our research was to investigate the effect and mechanism during this process. The wild-type CdtA, CdtB, CdtC (CdtA W , CdtB W , CdtC W ) and mutant CdtB (CdtB M ) were expressed and purified respectively and the purity of each subunit was examined by BandScan software. And the type I deoxyribonuclease and PI-3,4,5-triphosphate (PI-3,4,5-P3, PIP3) phosphatase activity were detected by DNA agarose gel electrophoresis and enzyme-linked immunosorbent assay respectively. The cell apoptosis rates were analyzed by flow cytometry. The morphological changes of apoptosis cells were observed by confocal laser scanning microscopy. The protein expression of Bax and Bcl-2 was examined by western blot. Differentially expressed apoptosis-related proteins were identified based on isobaric tags for relative and absolute quantitation technology. In the present study we found that: (i) recombinant wild-type CdtA, CdtB and CdtC (CdtA W , CdtB W , CdtC W ) and mutant CdtB (CdtB M ) were correctly expressed and the purity of each protein was higher than 80%, (ii) the average apoptosis rate in wild-type CDT (CDT W ) treated groups was 50.54%, which was significantly higher than the controls (4.71%) and mutant CDT (CDT M ) treated groups (5.58%) (p apoptosis were observed in CDT W treated cells, (iv) the expression of Bax protein was significantly increased in CDT W treated cells, while Bcl-2 protein expression was significantly decreased, (v) 17 apoptosis-related proteins were expressed differentially, among which 10 proteins (SMNDC1, TNFRSF10B, UBE2I, ITM2A, CASP3, P53, EIF1, TCF3, HMGN5, CASP8) were up-regulated and 7 proteins (RRM2, TPX2, KIF11, NUCKS1, TOP2A, XRCC1, PTPLAD1, RRM2) were down-regulated, (vi) one possible apoptotic pathway [Ubc9 (UBE2I)/P53/DR5 (TNFRSF10B)/Caspase-8 (CASP8)/ Caspase-3 (CASP3

The human CTC1/STN1/TEN1 complex regulates telomere maintenance in ALT cancer cells

Energy Technology Data Exchange (ETDEWEB)

Huang, Chenhui; Jia, Pingping; Chastain, Megan; Shiva, Olga; Chai, Weihang, E-mail: wchai@wsu.edu

2017-06-15

Maintaining functional telomeres is important for long-term proliferation of cells. About 15% of cancer cells are telomerase-negative and activate the alternative-lengthening of telomeres (ALT) pathway to maintain their telomeres. Recent studies have shown that the human CTC1/STN1/TEN1 complex (CST) plays a multi-faceted role in telomere maintenance in telomerase-expressing cancer cells. However, the role of CST in telomere maintenance in ALT cells is unclear. Here, we report that human CST forms a functional complex localizing in the ALT-associated PML bodies (APBs) in ALT cells throughout the cell cycle. Suppression of CST induces telomere instabilities including telomere fragility and elevates telomeric DNA recombination, leading to telomere dysfunction. In addition, CST deficiency significantly diminishes the abundance of extrachromosomal circular telomere DNA known as C-circles and t-circles. Suppression of CST also results in multinucleation in ALT cells and impairs cell proliferation. Our findings imply that the CST complex plays an important role in regulating telomere maintenance in ALT cells. - Highlights: • CST localizes at telomeres and ALT-associated PML bodies in ALT cells throughout the cell cycle. • CST is important for promoting telomeric DNA replication in ALT cells. • CST deficiency decreases ECTR formation and increases T-SCE. • CST deficiency impairs ALT cell proliferation and results in multinucleation.

The Adhesion Molecule KAL-1/anosmin-1 Regulates Neurite Branching through a SAX-7/L1CAM–EGL-15/FGFR Receptor Complex

Directory of Open Access Journals (Sweden)

Carlos A. Díaz-Balzac

2015-06-01

Full Text Available Neurite branching is essential for correct assembly of neural circuits, yet it remains a poorly understood process. For example, the neural cell adhesion molecule KAL-1/anosmin-1, which is mutated in Kallmann syndrome, regulates neurite branching through mechanisms largely unknown. Here, we show that KAL-1/anosmin-1 mediates neurite branching as an autocrine co-factor with EGL-17/FGF through a receptor complex consisting of the conserved cell adhesion molecule SAX-7/L1CAM and the fibroblast growth factor receptor EGL-15/FGFR. This protein complex, which appears conserved in humans, requires the immunoglobulin (Ig domains of SAX-7/L1CAM and the FN(III domains of KAL-1/anosmin-1 for formation in vitro as well as function in vivo. The kinase domain of the EGL-15/FGFR is required for branching, and genetic evidence suggests that ras-mediated signaling downstream of EGL-15/FGFR is necessary to effect branching. Our studies establish a molecular pathway that regulates neurite branching during development of the nervous system.

A new sample treatment for asialo-Tf determination with capillary electrophoresis: an added value to the analysis of CDT.

Science.gov (United States)

Porpiglia, Nadia Maria; De Palo, Elio Franco; Savchuk, Sergey Alexandrovich; Appolonova, Svetlana Alexandrovna; Bortolotti, Federica; Tagliaro, Franco

2018-05-10

The non-glycosylated glycoform of transferrin (Tf), known as asialo-Tf, was not selected (in favor of disialo-Tf) as the measurand for the standardization of carbohydrate deficient transferrin (CDT) determination because of a lower diagnostic sensitivity provided with the currently available analytical procedures for sera. However, asialo-Tf could provide an additional value to disialo-Tf in the CDT analysis employed in forensic toxicology contexts. The present work aimed at developing an easy sample preparation based on PEG precipitation in order to improve the detectability of asialo-Tf in capillary electrophoresis (CE). Equal volumes (35 μL) of serum and of 30% PEG-8000 were mixed and briefly vortexed. After centrifugation, the supernatant was iron saturated with a ferric solution (1:1, v/v). The mixture was analyzed in CE for asialo-Tf and disialo-Tf determination. PEG-8000 precipitation allowed the improvement of the baseline in the electropherograms in terms of interferences reduction particularly in the asialo-Tf migration region. The detection of asialo-Tf was possible in 89% of samples with disialo-Tf above the cut-off limit, whereas only 16% of them showed asialo-Tf by employing the traditional sample preteatment. Asialo-Tf represents an additional value to disialo-Tf as a biomarker of alcohol abuse in forensic toxicology. Copyright © 2018. Published by Elsevier B.V.

Prevalence, antibiogram, and cdt genes of toxigenic Campylobacter jejuni in salad style vegetables (ulam) at farms and retail outlets in Terengganu.

Science.gov (United States)

Khalid, Mohd Ikhsan; Tang, John Yew Huat; Baharuddin, Nabila Huda; Rahman, Nasiha Shakina; Rahimi, Nurul Faizzah; Radu, Son

2015-01-01

The present study was conducted to investigate the prevalence and antibiotic resistance among Campylobacter jejuni in ulam at farms and retail outlets located in Kuala Terengganu, Malaysia. A total of 526 samples (ulam, soil, and fertilizer) were investigated for the presence of C. jejuni and the gene for cytolethal distending toxin (cdt) by using a multiplex PCR method. Antibiotic susceptibility to 10 types of antibiotics was determined using the disk diffusion method for 33 C. jejuni isolates. The average prevalence of contaminated samples from farms, wet markets, and supermarkets was 35.29, 52.66, and 69.88%, respectively. The cdt gene was not detected in 24 of the 33 C. jejuni isolates, but 9 isolates harbored cdtC. Antibiotic resistance in C. jejuni isolates was highest to penicillin G (96.97% of isolates) followed by vancomycin (87.88%), ampicillin (75.76%), erythromycin (60.61%), tetracycline (9.09%), amikacin (6.06%), and norfloxacin (3.03%); none of the isolates were resistant to ciprofloxacin, enrofloxacin, and gentamicin. In this study, C. jejuni was present in ulam, and some isolates were highly resistant to some antibiotics but not to quinolones. Thus, appropriate attention and measures are required to prevent C. jejuni contamination on farms and at retail outlets.

Extracellular Na+ levels regulate formation and activity of the NaX/alpha1-Na+/K+-ATPase complex in neuronal cells.

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Emmanuelle eBerret

2014-12-01

Full Text Available MnPO neurons play a critical role in hydromineral homeostasis regulation by acting as sensors of extracellular sodium concentration ([Na+]out. The mechanism underlying Na+-sensing involves Na+-flow through the NaX channel, directly regulated by the Na+/K+-ATPase α1-isoform which controls Na+-influx by modulating channel permeability. Together, these two partners form a complex involved in the regulation of intracellular sodium ([Na+]in. Here we aim to determine whether environmental changes in Na+ could actively modulate the NaX/Na+/K+-ATPase complex activity.We investigated the complex activity using patch-clamp recordings from rat MnPO neurons and Neuro2a cells. When the rats were fed with a high-salt-diet, or the [Na+] in the culture medium was increased, the activity of the complex was up-regulated. In contrast, drop in environmental [Na+] decreased the activity of the complex. Interestingly under hypernatremic condition, the colocalization rate and protein level of both partners were up-regulated. Under hyponatremic condition, only NaX protein expression was increased and the level of NaX/Na+/K+-ATPase remained unaltered. This unbalance between NaX and Na+/K+-ATPase pump proportion would induce a bigger portion of Na+/K+-ATPase-control-free NaX channel. Thus we suggest that hypernatremic environment increases NaX/Na+/K+-ATPase α1-isoform activity by increasing the number of both partners and their colocalization rate, whereas hyponatremic environment down-regulates complex activity via a decrease in the relative number of NaX channels controlled by the pump.

m-AAA and i-AAA complexes coordinate to regulate OMA1, the stress-activated supervisor of mitochondrial dynamics.

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Consolato, Francesco; Maltecca, Francesca; Tulli, Susanna; Sambri, Irene; Casari, Giorgio

2018-04-09

The proteolytic processing of dynamin-like GTPase OPA1, mediated by the activity of both YME1L1 [intermembrane (i)-AAA protease complex] and OMA1, is a crucial step in the regulation of mitochondrial dynamics. OMA1 is a zinc metallopeptidase of the inner mitochondrial membrane that undergoes pre-activating proteolytic and auto-proteolytic cleavage after mitochondrial import. Here, we identify AFG3L2 [matrix (m) - AAA complex] as the major protease mediating this event, which acts by maturing the 60 kDa pre-pro-OMA1 to the 40 kDa pro-OMA1 form by severing the N-terminal portion without recognizing a specific consensus sequence. Therefore, m - AAA and i - AAA complexes coordinately regulate OMA1 processing and turnover, and consequently control which OPA1 isoforms are present, thus adding new information on the molecular mechanisms of mitochondrial dynamics and neurodegenerative diseases affected by these phenomena.This article has an associated First Person interview with the first author of the paper. © 2018. Published by The Company of Biologists Ltd.

The NSL Complex Regulates Housekeeping Genes in Drosophila

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Raja, Sunil Jayaramaiah; Holz, Herbert; Luscombe, Nicholas M.; Manke, Thomas; Akhtar, Asifa

2012-01-01

MOF is the major histone H4 lysine 16-specific (H4K16) acetyltransferase in mammals and Drosophila. In flies, it is involved in the regulation of X-chromosomal and autosomal genes as part of the MSL and the NSL complexes, respectively. While the function of the MSL complex as a dosage compensation regulator is fairly well understood, the role of the NSL complex in gene regulation is still poorly characterized. Here we report a comprehensive ChIP–seq analysis of four NSL complex members (NSL1, NSL3, MBD-R2, and MCRS2) throughout the Drosophila melanogaster genome. Strikingly, the majority (85.5%) of NSL-bound genes are constitutively expressed across different cell types. We find that an increased abundance of the histone modifications H4K16ac, H3K4me2, H3K4me3, and H3K9ac in gene promoter regions is characteristic of NSL-targeted genes. Furthermore, we show that these genes have a well-defined nucleosome free region and broad transcription initiation patterns. Finally, by performing ChIP–seq analyses of RNA polymerase II (Pol II) in NSL1- and NSL3-depleted cells, we demonstrate that both NSL proteins are required for efficient recruitment of Pol II to NSL target gene promoters. The observed Pol II reduction coincides with compromised binding of TBP and TFIIB to target promoters, indicating that the NSL complex is required for optimal recruitment of the pre-initiation complex on target genes. Moreover, genes that undergo the most dramatic loss of Pol II upon NSL knockdowns tend to be enriched in DNA Replication–related Element (DRE). Taken together, our findings show that the MOF-containing NSL complex acts as a major regulator of housekeeping genes in flies by modulating initiation of Pol II transcription. PMID:22723752

CYLD Limits Lys63- and Met1-Linked Ubiquitin at Receptor Complexes to Regulate Innate Immune Signaling

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Matous Hrdinka

2016-03-01

Full Text Available Innate immune signaling relies on the deposition of non-degradative polyubiquitin at receptor-signaling complexes, but how these ubiquitin modifications are regulated by deubiquitinases remains incompletely understood. Met1-linked ubiquitin (Met1-Ub is assembled by the linear ubiquitin assembly complex (LUBAC, and this is counteracted by the Met1-Ub-specific deubiquitinase OTULIN, which binds to the catalytic LUBAC subunit HOIP. In this study, we report that HOIP also interacts with the deubiquitinase CYLD but that CYLD does not regulate ubiquitination of LUBAC components. Instead, CYLD limits extension of Lys63-Ub and Met1-Ub conjugated to RIPK2 to restrict signaling and cytokine production. Accordingly, Met1-Ub and Lys63-Ub were individually required for productive NOD2 signaling. Our study thus suggests that LUBAC, through its associated deubiquitinases, coordinates the deposition of not only Met1-Ub but also Lys63-Ub to ensure an appropriate response to innate immune receptor activation.

Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings.

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Gonzalez, Antonio; Zhao, Mingzhe; Leavitt, John M; Lloyd, Alan M

2008-03-01

In all higher plants studied to date, the anthocyanin pigment pathway is regulated by a suite of transcription factors that include Myb, bHLH and WD-repeat proteins. However, in Arabidopsis thaliana, the Myb regulators remain to be conclusively identified, and little is known about anthocyanin pathway regulation by TTG1-dependent transcriptional complexes. Previous overexpression of the PAP1 Myb suggested that genes from the entire phenylpropanoid pathway are targets of regulation by Myb/bHLH/WD-repeat complexes in Arabidopsis, in contrast to other plants. Here we demonstrate that overexpression of Myb113 or Myb114 results in substantial increases in pigment production similar to those previously seen as a result of over-expression of PAP1, and pigment production in these overexpressors remains TTG1- and bHLH-dependent. Also, plants harboring an RNAi construct targeting PAP1 and three Myb candidates (PAP2, Myb113 and Myb114) showed downregulated Myb gene expression and obvious anthocyanin deficiencies. Correlated with these anthocyanin deficiencies is downregulation of the same late anthocyanin structural genes that are downregulated in ttg1 and bHLH anthocyanin mutants. Expression studies using GL3:GR and TTG1:GR fusions revealed direct regulation of the late biosynthetic genes only. Functional diversification between GL3 and EGL3 with regard to activation of gene targets was revealed by GL3:GR studies in single and double bHLH mutant seedlings. Expression profiles for Myb and bHLH regulators are also presented in the context of pigment production in young seedlings.

The Mediator co-activator complex regulates Ty1 retromobility by controlling the balance between Ty1i and Ty1 promoters.

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Salinero, Alicia C; Knoll, Elisabeth R; Zhu, Z Iris; Landsman, David; Curcio, M Joan; Morse, Randall H

2018-02-01

The Ty1 retrotransposons present in the genome of Saccharomyces cerevisiae belong to the large class of mobile genetic elements that replicate via an RNA intermediary and constitute a significant portion of most eukaryotic genomes. The retromobility of Ty1 is regulated by numerous host factors, including several subunits of the Mediator transcriptional co-activator complex. In spite of its known function in the nucleus, previous studies have implicated Mediator in the regulation of post-translational steps in Ty1 retromobility. To resolve this paradox, we systematically examined the effects of deleting non-essential Mediator subunits on the frequency of Ty1 retromobility and levels of retromobility intermediates. Our findings reveal that loss of distinct Mediator subunits alters Ty1 retromobility positively or negatively over a >10,000-fold range by regulating the ratio of an internal transcript, Ty1i, to the genomic Ty1 transcript. Ty1i RNA encodes a dominant negative inhibitor of Ty1 retromobility that blocks virus-like particle maturation and cDNA synthesis. These results resolve the conundrum of Mediator exerting sweeping control of Ty1 retromobility with only minor effects on the levels of Ty1 genomic RNA and the capsid protein, Gag. Since the majority of characterized intrinsic and extrinsic regulators of Ty1 retromobility do not appear to effect genomic Ty1 RNA levels, Mediator could play a central role in integrating signals that influence Ty1i expression to modulate retromobility.

Regulation of metabolism by the Mediator complex.

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Youn, Dou Yeon; Xiaoli, Alus M; Pessin, Jeffrey E; Yang, Fajun

2016-01-01

The Mediator complex was originally discovered in yeast, but it is conserved in all eukaryotes. Its best-known function is to regulate RNA polymerase II-dependent gene transcription. Although the mechanisms by which the Mediator complex regulates transcription are often complicated by the context-dependent regulation, this transcription cofactor complex plays a pivotal role in numerous biological pathways. Biochemical, molecular, and physiological studies using cancer cell lines or model organisms have established the current paradigm of the Mediator functions. However, the physiological roles of the mammalian Mediator complex remain poorly defined, but have attracted a great interest in recent years. In this short review, we will summarize some of the reported functions of selective Mediator subunits in the regulation of metabolism. These intriguing findings suggest that the Mediator complex may be an important player in nutrient sensing and energy balance in mammals.

Deoxynucleoside Salvage in Fission Yeast Allows Rescue of Ribonucleotide Reductase Deficiency but Not Spd1-Mediated Inhibition of Replication

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Oliver Fleck

2017-04-01

Full Text Available In fission yeast, the small, intrinsically disordered protein S-phase delaying protein 1 (Spd1 blocks DNA replication and causes checkpoint activation at least in part, by inhibiting the enzyme ribonucleotide reductase, which is responsible for the synthesis of DNA. The CRL4Cdt2 E3 ubiquitin ligase mediates degradation of Spd1 and the related protein Spd2 at S phase of the cell cycle. We have generated a conditional allele of CRL4Cdt2, by expressing the highly unstable substrate-recruiting protein Cdt2 from a repressible promoter. Unlike Spd1, Spd2 does not regulate deoxynucleotide triphosphate (dNTP pools; yet we find that Spd1 and Spd2 together inhibit DNA replication upon Cdt2 depletion. To directly test whether this block of replication was solely due to insufficient dNTP levels, we established a deoxy-nucleotide salvage pathway in fission yeast by expressing the human nucleoside transporter human equilibrative nucleoside transporter 1 (hENT1 and the Drosophila deoxynucleoside kinase. We present evidence that this salvage pathway is functional, as 2 µM of deoxynucleosides in the culture medium is able to rescue the growth of two different temperature-sensitive alleles controlling ribonucleotide reductase. However, salvage completely failed to rescue S phase delay, checkpoint activation, and damage sensitivity, which was caused by CRL4Cdt2 inactivation, suggesting that Spd1—in addition to repressing dNTP synthesis—together with Spd2, can inhibit other replication functions. We propose that this inhibition works at the point of the replication clamp proliferating cell nuclear antigen, a co-factor for DNA replication.

Klotho Regulates 14-3-3ζ Monomerization and Binding to the ASK1 Signaling Complex in Response to Oxidative Stress.

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Reynolds K Brobey

Full Text Available The reactive oxygen species (ROS-sensitive apoptosis signal-regulating kinase 1 (ASK1 signaling complex is a key regulator of p38 MAPK activity, a major modulator of stress-associated with aging disorders. We recently reported that the ratio of free ASK1 to the complex-bound ASK1 is significantly decreased in Klotho-responsive manner and that Klotho-deficient tissues have elevated levels of free ASK1 which coincides with increased oxidative stress. Here, we tested the hypothesis that: 1 covalent interactions exist among three identified proteins constituting the ASK1 signaling complex; 2 in normal unstressed cells the ASK1, 14-3-3ζ and thioredoxin (Trx proteins simultaneously engage in a tripartite complex formation; 3 Klotho's stabilizing effect on the complex relied solely on 14-3-3ζ expression and its apparent phosphorylation and dimerization changes. To verify the hypothesis, we performed 14-3-3ζ siRNA knock-down experiments in conjunction with cell-based assays to measure ASK1-client protein interactions in the presence and absence of Klotho, and with or without an oxidant such as rotenone. Our results show that Klotho activity induces posttranslational modifications in the complex targeting 14-3-3ζ monomer/dimer changes to effectively protect against ASK1 oxidation and dissociation. This is the first observation implicating all three proteins constituting the ASK1 signaling complex in close proximity.

The KRAB Zinc Finger Protein Roma/Zfp157 Is a Critical Regulator of Cell-Cycle Progression and Genomic Stability

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Teresa L.F. Ho

2016-04-01

Full Text Available Regulation of DNA replication and cell division is essential for tissue growth and maintenance of genomic integrity and is particularly important in tissues that undergo continuous regeneration such as mammary glands. We have previously shown that disruption of the KRAB-domain zinc finger protein Roma/Zfp157 results in hyperproliferation of mammary epithelial cells (MECs during pregnancy. Here, we delineate the mechanism by which Roma engenders this phenotype. Ablation of Roma in MECs leads to unscheduled proliferation, replication stress, DNA damage, and genomic instability. Furthermore, mouse embryonic fibroblasts (MEFs depleted for Roma exhibit downregulation of p21Cip1 and geminin and have accelerated replication fork velocities, which is accompanied by a high rate of mitotic errors and polyploidy. In contrast, overexpression of Roma in MECs halts cell-cycle progression, whereas siRNA-mediated p21Cip1 knockdown ameliorates, in part, this phenotype. Thus, Roma is an essential regulator of the cell cycle and is required to maintain genomic stability.

The E2F-DP1 Transcription Factor Complex Regulates Centriole Duplication in Caenorhabditis elegans

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Jacqueline G. Miller

2016-03-01

Full Text Available Centrioles play critical roles in the organization of microtubule-based structures, from the mitotic spindle to cilia and flagella. In order to properly execute their various functions, centrioles are subjected to stringent copy number control. Central to this control mechanism is a precise duplication event that takes place during S phase of the cell cycle and involves the assembly of a single daughter centriole in association with each mother centriole . Recent studies have revealed that posttranslational control of the master regulator Plk4/ZYG-1 kinase and its downstream effector SAS-6 is key to ensuring production of a single daughter centriole. In contrast, relatively little is known about how centriole duplication is regulated at a transcriptional level. Here we show that the transcription factor complex EFL-1-DPL-1 both positively and negatively controls centriole duplication in the Caenorhabditis elegans embryo. Specifically, we find that down regulation of EFL-1-DPL-1 can restore centriole duplication in a zyg-1 hypomorphic mutant and that suppression of the zyg-1 mutant phenotype is accompanied by an increase in SAS-6 protein levels. Further, we find evidence that EFL-1-DPL-1 promotes the transcription of zyg-1 and other centriole duplication genes. Our results provide evidence that in a single tissue type, EFL-1-DPL-1 sets the balance between positive and negative regulators of centriole assembly and thus may be part of a homeostatic mechanism that governs centriole assembly.

TORC1 regulates Pah1 phosphatidate phosphatase activity via the Nem1/Spo7 protein phosphatase complex.

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Emmanuelle Dubots

Full Text Available The evolutionarily conserved target of rapamycin complex 1 (TORC1 controls growth-related processes such as protein, nucleotide, and lipid metabolism in response to growth hormones, energy/ATP levels, and amino acids. Its deregulation is associated with cancer, type 2 diabetes, and obesity. Among other substrates, mammalian TORC1 directly phosphorylates and inhibits the phosphatidate phosphatase lipin-1, a central enzyme in lipid metabolism that provides diacylglycerol for the synthesis of membrane phospholipids and/or triacylglycerol as neutral lipid reserve. Here, we show that yeast TORC1 inhibits the function of the respective lipin, Pah1, to prevent the accumulation of triacylglycerol. Surprisingly, TORC1 regulates Pah1 in part indirectly by controlling the phosphorylation status of Nem1 within the Pah1-activating, heterodimeric Nem1-Spo7 protein phosphatase module. Our results delineate a hitherto unknown TORC1 effector branch that controls lipin function in yeast, which, given the recent discovery of Nem1-Spo7 orthologous proteins in humans, may be conserved.

Characteristics of the new phase in CDT

Energy Technology Data Exchange (ETDEWEB)

Ambjoern, J. [Copenhagen University, The Niels Bohr Institute, Copenhagen Oe (Denmark); Radboud University, Institute for Mathematics, Astrophysics and Particle Physics (IMAPP), Nijmegen (Netherlands); Gizbert-Studnicki, J.; Jurkiewicz, J. [Jagiellonian University, Institute of Physics, Krakow (Poland); Goerlich, A. [Copenhagen University, The Niels Bohr Institute, Copenhagen Oe (Denmark); Jagiellonian University, Institute of Physics, Krakow (Poland); Klitgaard, N.; Loll, R. [Radboud University, Institute for Mathematics, Astrophysics and Particle Physics (IMAPP), Nijmegen (Netherlands)

2017-03-15

The approach of Causal Dynamical Triangulations (CDT), a candidate theory of nonperturbative quantum gravity in 4D, turns out to have a rich phase structure. We investigate the recently discovered bifurcation phase C{sub b} and relate some of its characteristics to the presence of singular vertices of very high order. The transition lines separating this phase from the ''time-collapsed'' B-phase and the de Sitter phase C{sub dS} are of great interest when searching for physical scaling limits. The work presented here sheds light on the mechanisms behind these transitions. First, we study how the B-C{sub b} transition signal depends on the volume fixing implemented in the simulations, and find results compatible with the previously determined second-order character of the transition. The transition persists in a transfer matrix formulation, where the system's time extension is taken to be minimal. Second, we relate the new C{sub b}-C{sub dS} transition to the appearance of singular vertices, which leads to a direct physical interpretation in terms of a breaking of the homogeneity and isotropy observed in the de Sitter phase when crossing from C{sub dS} to the bifurcation phase C{sub b}. (orig.)

Differential roles of the glycogen-binding domains of beta subunits in regulation of the Snf1 kinase complex.

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Mangat, Simmanjeet; Chandrashekarappa, Dakshayini; McCartney, Rhonda R; Elbing, Karin; Schmidt, Martin C

2010-01-01

Members of the AMP-activated protein kinase family, including the Snf1 kinase of Saccharomyces cerevisiae, are activated under conditions of nutrient stress. AMP-activated protein kinases are heterotrimeric complexes composed of a catalytic alpha subunit and regulatory beta and gamma subunits. In this study, the role of the beta subunits in the regulation of Snf1 activity was examined. Yeasts express three isoforms of the AMP-activated protein kinase consisting of Snf1 (alpha), Snf4 (gamma), and one of three alternative beta subunits, either Sip1, Sip2, or Gal83. The Gal83 isoform of the Snf1 complex is the most abundant and was analyzed in the greatest detail. All three beta subunits contain a conserved domain referred to as the glycogen-binding domain. The deletion of this domain from Gal83 results in a deregulation of the Snf1 kinase, as judged by a constitutive activity independent of glucose availability. In contrast, the deletion of this homologous domain from the Sip1 and Sip2 subunits had little effect on Snf1 kinase regulation. Therefore, the different Snf1 kinase isoforms are regulated through distinct mechanisms, which may contribute to their specialized roles in different stress response pathways. In addition, the beta subunits are subjected to phosphorylation. The responsible kinases were identified as being Snf1 and casein kinase II. The significance of the phosphorylation is unclear since the deletion of the region containing the phosphorylation sites in Gal83 had little effect on the regulation of Snf1 in response to glucose limitation.

The Binary Toxin CDT of Clostridium difficile as a Tool for Intracellular Delivery of Bacterial Glucosyltransferase Domains

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Lara-Antonia Beer

2018-06-01

Full Text Available Binary toxins are produced by several pathogenic bacteria. Examples are the C2 toxin from Clostridium botulinum, the iota toxin from Clostridium perfringens, and the CDT from Clostridium difficile. All these binary toxins have ADP-ribosyltransferases (ADPRT as their enzymatically active component that modify monomeric actin in their target cells. The binary C2 toxin was intensively described as a tool for intracellular delivery of allogenic ADPRTs. Here, we firstly describe the binary toxin CDT from C. difficile as an effective tool for heterologous intracellular delivery. Even 60 kDa glucosyltransferase domains of large clostridial glucosyltransferases can be delivered into cells. The glucosyltransferase domains of five tested large clostridial glucosyltransferases were successfully introduced into cells as chimeric fusions to the CDTa adapter domain (CDTaN. Cell uptake was demonstrated by the analysis of cell morphology, cytoskeleton staining, and intracellular substrate glucosylation. The fusion toxins were functional only when the adapter domain of CDTa was N-terminally located, according to its native orientation. Thus, like other binary toxins, the CDTaN/b system can be used for standardized delivery systems not only for bacterial ADPRTs but also for a variety of bacterial glucosyltransferase domains.

Rac1 regulates neuronal polarization through the WAVE complex

DEFF Research Database (Denmark)

Tahirovic, Sabina; Hellal, Farida; Neukirchen, Dorothee

2010-01-01

the physiological function of Rac1 in neuronal development, we have generated a conditional knock-out mouse, in which Rac1 is ablated in the whole brain. Rac1-deficient cerebellar granule neurons, which do not express other Rac isoforms, showed impaired neuronal migration and axon formation both in vivo...... and in vitro. In addition, Rac1 ablation disrupts lamellipodia formation in growth cones. The analysis of Rac1 effectors revealed the absence of the Wiskott-Aldrich syndrome protein (WASP) family verprolin-homologous protein (WAVE) complex from the plasma membrane of knock-out growth cones. Loss of WAVE...... function inhibited axon growth, whereas overexpression of a membrane-tethered WAVE mutant partially rescued axon growth in Rac1-knock-out neurons. In addition, pharmacological inhibition of the WAVE complex effector Arp2/3 also reduced axon growth. We propose that Rac1 recruits the WAVE complex...

Hem-1 complexes are essential for Rac activation, actin polymerization, and myosin regulation during neutrophil chemotaxis.

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Orion D Weiner

2006-02-01

Full Text Available Migrating cells need to make different actin assemblies at the cell's leading and trailing edges and to maintain physical separation of signals for these assemblies. This asymmetric control of activities represents one important form of cell polarity. There are significant gaps in our understanding of the components involved in generating and maintaining polarity during chemotaxis. Here we characterize a family of complexes (which we term leading edge complexes, scaffolded by hematopoietic protein 1 (Hem-1, that organize the neutrophil's leading edge. The Wiskott-Aldrich syndrome protein family Verprolin-homologous protein (WAVE2 complex, which mediates activation of actin polymerization by Rac, is only one member of this family. A subset of these leading edge complexes are biochemically separable from the WAVE2 complex and contain a diverse set of potential polarity-regulating proteins. RNA interference-mediated knockdown of Hem-1-containing complexes in neutrophil-like cells: (a dramatically impairs attractant-induced actin polymerization, polarity, and chemotaxis; (b substantially weakens Rac activation and phosphatidylinositol-(3,4,5-tris-phosphate production, disrupting the (phosphatidylinositol-(3,4,5-tris-phosphate/Rac/F-actin-mediated feedback circuit that organizes the leading edge; and (c prevents exclusion of activated myosin from the leading edge, perhaps by misregulating leading edge complexes that contain inhibitors of the Rho-actomyosin pathway. Taken together, these observations show that versatile Hem-1-containing complexes coordinate diverse regulatory signals at the leading edge of polarized neutrophils, including but not confined to those involving WAVE2-dependent actin polymerization.

Role of the BAHD1 Chromatin-Repressive Complex in Placental Development and Regulation of Steroid Metabolism.

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Goran Lakisic

2016-03-01

Full Text Available BAHD1 is a vertebrate protein that promotes heterochromatin formation and gene repression in association with several epigenetic regulators. However, its physiological roles remain unknown. Here, we demonstrate that ablation of the Bahd1 gene results in hypocholesterolemia, hypoglycemia and decreased body fat in mice. It also causes placental growth restriction with a drop of trophoblast glycogen cells, a reduction of fetal weight and a high neonatal mortality rate. By intersecting transcriptome data from murine Bahd1 knockout (KO placentas at stages E16.5 and E18.5 of gestation, Bahd1-KO embryonic fibroblasts, and human cells stably expressing BAHD1, we also show that changes in BAHD1 levels alter expression of steroid/lipid metabolism genes. Biochemical analysis of the BAHD1-associated multiprotein complex identifies MIER proteins as novel partners of BAHD1 and suggests that BAHD1-MIER interaction forms a hub for histone deacetylases and methyltransferases, chromatin readers and transcription factors. We further show that overexpression of BAHD1 leads to an increase of MIER1 enrichment on the inactive X chromosome (Xi. In addition, BAHD1 and MIER1/3 repress expression of the steroid hormone receptor genes ESR1 and PGR, both playing important roles in placental development and energy metabolism. Moreover, modulation of BAHD1 expression in HEK293 cells triggers epigenetic changes at the ESR1 locus. Together, these results identify BAHD1 as a core component of a chromatin-repressive complex regulating placental morphogenesis and body fat storage and suggest that its dysfunction may contribute to several human diseases.

Transcription regulation by the Mediator complex.

Science.gov (United States)

Soutourina, Julie

2018-04-01

Alterations in the regulation of gene expression are frequently associated with developmental diseases or cancer. Transcription activation is a key phenomenon in the regulation of gene expression. In all eukaryotes, mediator of RNA polymerase II transcription (Mediator), a large complex with modular organization, is generally required for transcription by RNA polymerase II, and it regulates various steps of this process. The main function of Mediator is to transduce signals from the transcription activators bound to enhancer regions to the transcription machinery, which is assembled at promoters as the preinitiation complex (PIC) to control transcription initiation. Recent functional studies of Mediator with the use of structural biology approaches and functional genomics have revealed new insights into Mediator activity and its regulation during transcription initiation, including how Mediator is recruited to transcription regulatory regions and how it interacts and cooperates with PIC components to assist in PIC assembly. Novel roles of Mediator in the control of gene expression have also been revealed by showing its connection to the nuclear pore and linking Mediator to the regulation of gene positioning in the nuclear space. Clear links between Mediator subunits and disease have also encouraged studies to explore targeting of this complex as a potential therapeutic approach in cancer and fungal infections.

Pregnancy and variations of carbohydrate-deficient transferrin levels measured by the candidate reference HPLC method.

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Bianchi, Vincenza; Ivaldi, Alessandra; Raspagni, Alessia; Arfini, Carlo; Vidali, Matteo

2011-01-01

Contrasting data are available on the diagnostic accuracy of carbohydrate-deficient transferrin (CDT) during pregnancy. These differences may depend in part on how CDT was evaluated and expressed. Here, we report on variations of CDT levels in pregnant women using the high performance liquid chromatography (HPLC) candidate reference method. Alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, mean corpuscular volume, serum transferrin, urine and serum ethyl glucuronide and CDT were measured in 64 women, self-reporting as non-alcohol abusers (age: median 34, IQR: 28-38), at different stages of normal pregnancy (gestational weeks: median 28, IQR: 8-33). CDT was expressed as percentage of disialotransferrin to total transferrin (%CDT). Transferrin was associated with both %CDT (r = 0.66; P pregnancy trimester (first trimester: mean 1.01% (SD 0.19); second trimester: 1.30% (SD 0.14); third trimester: 1.53% (SD 0.22); ANOVA P pregnancy trimesters (P pregnancy and CDT could be more complex. The diagnostic accuracy of CDT for detecting alcohol abuse in a legal context may be limited in pregnant women and the effect of gestational age should be considered.

SUMOylation of the ING1b tumor suppressor regulates gene transcription

DEFF Research Database (Denmark)

Satpathy, Shankha; Guérillon, Claire; Kim, Tae-Sun

2014-01-01

members of histone deacetylase complexes, whereas ING3-5 are stoichiometric components of different histone acetyltransferase complexes. The INGs target these complexes to histone marks, thus acting as epigenetic regulators. ING proteins affect angiogenesis, apoptosis, DNA repair, metastasis......1b E195A), we further demonstrate that ING1b SUMOylation regulates the binding of ING1b to the ISG15 and DGCR8 promoters, consequently regulating ISG15 and DGCR8 transcription. These results suggest a role for ING1b SUMOylation in the regulation of gene transcription....

Regulation of protease-activated receptor 1 signaling by the adaptor protein complex 2 and R4 subfamily of regulator of G protein signaling proteins.

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Chen, Buxin; Siderovski, David P; Neubig, Richard R; Lawson, Mark A; Trejo, Joann

2014-01-17

The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of "regulator of G protein signaling" (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 (420)AKKAA(424) mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.

Molecular chaperone complexes with antagonizing activities regulate stability and activity of the tumor suppressor LKB1.

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Gaude, H; Aznar, N; Delay, A; Bres, A; Buchet-Poyau, K; Caillat, C; Vigouroux, A; Rogon, C; Woods, A; Vanacker, J-M; Höhfeld, J; Perret, C; Meyer, P; Billaud, M; Forcet, C

2012-03-22

LKB1 is a tumor suppressor that is constitutionally mutated in a cancer-prone condition, called Peutz-Jeghers syndrome, as well as somatically inactivated in a sizeable fraction of lung and cervical neoplasms. The LKB1 gene encodes a serine/threonine kinase that associates with the pseudokinase STRAD (STE-20-related pseudokinase) and the scaffolding protein MO25, the formation of this heterotrimeric complex promotes allosteric activation of LKB1. We have previously reported that the molecular chaperone heat shock protein 90 (Hsp90) binds to and stabilizes LKB1. Combining pharmacological studies and RNA interference approaches, we now provide evidence that the co-chaperone Cdc37 participates to the regulation of LKB1 stability. It is known that the Hsp90-Cdc37 complex recognizes a surface within the N-terminal catalytic lobe of client protein kinases. In agreement with this finding, we found that the chaperones Hsp90 and Cdc37 interact with an LKB1 isoform that differs in the C-terminal region, but not with a novel LKB1 variant that lacks a portion of the kinase N-terminal lobe domain. Reconstitution of the two complexes LKB1-STRAD and LKB1-Hsp90-Cdc37 with recombinant proteins revealed that the former is catalytically active whereas the latter is inactive. Furthermore, consistent with a documented repressor function of Hsp90, LKB1 kinase activity was transiently stimulated upon dissociation of Hsp90. Finally, disruption of the LKB1-Hsp90 complex favors the recruitment of both Hsp/Hsc70 and the U-box dependent E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70-interacting protein) that triggers LKB1 degradation. Taken together, our results establish that the Hsp90-Cdc37 complex controls both the stability and activity of the LKB1 kinase. This study further shows that two chaperone complexes with antagonizing activities, Hsp90-Cdc37 and Hsp/Hsc70-CHIP, finely control the cellular level of LKB1 protein.

LHX3 interacts with inhibitor of histone acetyltransferase complex subunits LANP and TAF-1β to modulate pituitary gene regulation.

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Hunter, Chad S; Malik, Raleigh E; Witzmann, Frank A; Rhodes, Simon J

2013-01-01

LIM-homeodomain 3 (LHX3) is a transcription factor required for mammalian pituitary gland and nervous system development. Human patients and animal models with LHX3 gene mutations present with severe pediatric syndromes that feature hormone deficiencies and symptoms associated with nervous system dysfunction. The carboxyl terminus of the LHX3 protein is required for pituitary gene regulation, but the mechanism by which this domain operates is unknown. In order to better understand LHX3-dependent pituitary hormone gene transcription, we used biochemical and mass spectrometry approaches to identify and characterize proteins that interact with the LHX3 carboxyl terminus. This approach identified the LANP/pp32 and TAF-1β/SET proteins, which are components of the inhibitor of histone acetyltransferase (INHAT) multi-subunit complex that serves as a multifunctional repressor to inhibit histone acetylation and modulate chromatin structure. The protein domains of LANP and TAF-1β that interact with LHX3 were mapped using biochemical techniques. Chromatin immunoprecipitation experiments demonstrated that LANP and TAF-1β are associated with LHX3 target genes in pituitary cells, and experimental alterations of LANP and TAF-1β levels affected LHX3-mediated pituitary gene regulation. Together, these data suggest that transcriptional regulation of pituitary genes by LHX3 involves regulated interactions with the INHAT complex.

Phosphorylation of the Synaptonemal Complex Protein Zip1 Regulates the Crossover/Noncrossover Decision during Yeast Meiosis.

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Xiangyu Chen

2015-12-01

Full Text Available Interhomolog crossovers promote proper chromosome segregation during meiosis and are formed by the regulated repair of programmed double-strand breaks. This regulation requires components of the synaptonemal complex (SC, a proteinaceous structure formed between homologous chromosomes. In yeast, SC formation requires the "ZMM" genes, which encode a functionally diverse set of proteins, including the transverse filament protein, Zip1. In wild-type meiosis, Zmm proteins promote the biased resolution of recombination intermediates into crossovers that are distributed throughout the genome by interference. In contrast, noncrossovers are formed primarily through synthesis-dependent strand annealing mediated by the Sgs1 helicase. This work identifies a conserved region on the C terminus of Zip1 (called Zip1 4S, whose phosphorylation is required for the ZMM pathway of crossover formation. Zip1 4S phosphorylation is promoted both by double-strand breaks (DSBs and the meiosis-specific kinase, MEK1/MRE4, demonstrating a role for MEK1 in the regulation of interhomolog crossover formation, as well as interhomolog bias. Failure to phosphorylate Zip1 4S results in meiotic prophase arrest, specifically in the absence of SGS1. This gain of function meiotic arrest phenotype is suppressed by spo11Δ, suggesting that it is due to unrepaired breaks triggering the meiotic recombination checkpoint. Epistasis experiments combining deletions of individual ZMM genes with sgs1-md zip1-4A indicate that Zip1 4S phosphorylation functions prior to the other ZMMs. These results suggest that phosphorylation of Zip1 at DSBs commits those breaks to repair via the ZMM pathway and provides a mechanism by which the crossover/noncrossover decision can be dynamically regulated during yeast meiosis.

Comparison of the effectiveness of complex decongestive therapy and compression bandaging as a method of treatment of lymphedema in the elderly

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Zasadzka E

2018-05-01

Full Text Available Ewa Zasadzka,1 Tomasz Trzmiel,1 Maria Kleczewska,2 Mariola Pawlaczyk1 1Department of Geriatric Medicine and Gerontology, Karol Marcinkowski University of Medical Sciences, Poznan, Poland; 2Day Rehabilitation Center, Hospicjum Palium, Poznań, Poland Background: Lymphedema is a chronic condition which significantly lowers the quality of patient life, particularly among elderly populations, whose mobility and physical function are often reduced. Objectives: The aim of the study was to compare the effectiveness of multi-layer compression bandaging (MCB and complex decongestive therapy (CDT, and to show that MCB is a cheaper, more accessible and less labor intensive method of treating lymphedema in elderly patients. Patients and methods: The study included 103 patients (85 women and 18 men aged ≥60 years, with unilateral lower limb lymphedema. The subjects were divided into two groups: 50 treated with CDT and 53 with MCB. Pre- and post-treatment BMI, and average and maximum circumference of the edematous extremities were analyzed. Results: Reduction in swelling in both groups was achieved after 15 interventions. Both therapies demonstrated similar efficacy in reducing limb volume and circumference, but MCB showed greater efficacy in reducing the maximum circumference. Conclusion: Compression bandaging is a vital component of CDT. Maximum lymphedema reduction during therapy and maintaining its effect cannot be achieved without it. It also demonstrates its effectiveness as an independent method, which can reduce therapy cost and accessibility. Keywords: lymphedema, elderly, therapy, compression bandaging

Platelet-derived growth factor regulates vascular smooth muscle phenotype via mammalian target of rapamycin complex 1

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Ha, Jung Min; Yun, Sung Ji; Kim, Young Whan; Jin, Seo Yeon; Lee, Hye Sun; Song, Sang Heon; Shin, Hwa Kyoung; Bae, Sun Sik

2015-01-01

Mammalian target of rapamycin complex (mTORC) regulates various cellular processes including proliferation, growth, migration and differentiation. In this study, we showed that mTORC1 regulates platelet-derived growth factor (PDGF)-induced phenotypic conversion of vascular smooth muscle cells (VSMCs). Stimulation of contractile VSMCs with PDGF significantly reduced the expression of contractile marker proteins in a time- and dose-dependent manner. In addition, angiotensin II (AngII)-induced contraction of VSMCs was completely blocked by the stimulation of VSMCs with PDGF. PDGF-dependent suppression of VSMC marker gene expression was significantly blocked by inhibition of phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and mTOR whereas inhibition of p38 MAPK had no effect. In particular, inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked the PDGF-dependent phenotypic change of VSMCs whereas silencing of Rictor had no effect. In addition, loss of AngII-dependent contraction by PDGF was significantly retained by silencing of Raptor. Inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked PDGF-induced proliferation of VSMCs. Taken together, we suggest that mTORC1 plays an essential role in PDGF-dependent phenotypic changes of VSMCs. - Graphical abstract: Regulation of VSMC phenotype by PDGF-dependent activation of mTORC1. - Highlights: • The expression of contractile marker proteins was reduced by PDGF stimulation. • PDGF-dependent phenotypic conversion of VSMCs was blocked by inhibition of mTOR. • PDGF-induced proliferation of VSMCs was attenuated by inhibition of mTORC1. • mTORC1 plays a critical role in PDGF-dependent phenotypic conversion of VSMCs

Platelet-derived growth factor regulates vascular smooth muscle phenotype via mammalian target of rapamycin complex 1

Energy Technology Data Exchange (ETDEWEB)

Ha, Jung Min; Yun, Sung Ji; Kim, Young Whan; Jin, Seo Yeon; Lee, Hye Sun [Medical Research Institute, Department of Pharmacology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Song, Sang Heon [Department of Internal Medicine, Pusan National University Hospital, Busan (Korea, Republic of); Shin, Hwa Kyoung [Department of Anatomy, Pusan National University School of Korean Medicine, Yangsan (Korea, Republic of); Bae, Sun Sik, E-mail: sunsik@pusan.ac.kr [Medical Research Institute, Department of Pharmacology, Pusan National University School of Medicine, Yangsan (Korea, Republic of)

2015-08-14

Mammalian target of rapamycin complex (mTORC) regulates various cellular processes including proliferation, growth, migration and differentiation. In this study, we showed that mTORC1 regulates platelet-derived growth factor (PDGF)-induced phenotypic conversion of vascular smooth muscle cells (VSMCs). Stimulation of contractile VSMCs with PDGF significantly reduced the expression of contractile marker proteins in a time- and dose-dependent manner. In addition, angiotensin II (AngII)-induced contraction of VSMCs was completely blocked by the stimulation of VSMCs with PDGF. PDGF-dependent suppression of VSMC marker gene expression was significantly blocked by inhibition of phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and mTOR whereas inhibition of p38 MAPK had no effect. In particular, inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked the PDGF-dependent phenotypic change of VSMCs whereas silencing of Rictor had no effect. In addition, loss of AngII-dependent contraction by PDGF was significantly retained by silencing of Raptor. Inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked PDGF-induced proliferation of VSMCs. Taken together, we suggest that mTORC1 plays an essential role in PDGF-dependent phenotypic changes of VSMCs. - Graphical abstract: Regulation of VSMC phenotype by PDGF-dependent activation of mTORC1. - Highlights: • The expression of contractile marker proteins was reduced by PDGF stimulation. • PDGF-dependent phenotypic conversion of VSMCs was blocked by inhibition of mTOR. • PDGF-induced proliferation of VSMCs was attenuated by inhibition of mTORC1. • mTORC1 plays a critical role in PDGF-dependent phenotypic conversion of VSMCs.

Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

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Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald, E-mail: gerald.thiel@uks.eu

2015-03-01

Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.

Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

International Nuclear Information System (INIS)

Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald

2015-01-01

Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified

Knockdown of SCF(Skp2 function causes double-parked accumulation in the nucleus and DNA re-replication in Drosophila plasmatocytes.

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Paul T Kroeger

Full Text Available In Drosophila, circulating hemocytes are derived from the cephalic mesoderm during the embryonic wave of hematopoiesis. These cells are contributed to the larva and persist through metamorphosis into the adult. To analyze this population of hemocytes, we considered data from a previously published RNAi screen in the hematopoietic niche, which suggested several members of the SCF complex play a role in lymph gland development. eater-Gal4;UAS-GFP flies were crossed to UAS-RNAi lines to knockdown the function of all known SCF complex members in a plasmatocyte-specific fashion, in order to identify which members are novel regulators of plasmatocytes. This specific SCF complex contains five core members: Lin-19-like, SkpA, Skp2, Roc1a and complex activator Nedd8. The complex was identified by its very distinctive large cell phenotype. Furthermore, these large cells stained for anti-P1, a plasmatocyte-specific antibody. It was also noted that the DNA in these cells appeared to be over-replicated. Gamma-tubulin and DAPI staining suggest the cells are undergoing re-replication as they had multiple centrioles and excessive DNA content. Further experimentation determined enlarged cells were BrdU-positive indicating they have progressed through S-phase. To determine how these cells become enlarged and undergo re-replication, cell cycle proteins were analyzed by immunofluorescence. This analysis identified three proteins that had altered subcellular localization in these enlarged cells: Cyclin E, Geminin and Double-parked. Previous research has shown that Double-parked must be degraded to exit S-phase, otherwise the DNA will undergo re-replication. When Double-parked was titrated from the nucleus by an excess of its inhibitor, geminin, the enlarged cells and aberrant protein localization phenotypes were partially rescued. The data in this report suggests that the SCF(Skp2 complex is necessary to ubiquitinate Double-parked during plasmatocyte cell division

Inositol hexakisphosphate kinase-1 mediates assembly/disassembly of the CRL4–signalosome complex to regulate DNA repair and cell death

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Rao, Feng; Xu, Jing; Khan, A. Basit; Gadalla, Moataz M.; Cha, Jiyoung Y.; Xu, Risheng; Tyagi, Richa; Dang, Yongjun; Chakraborty, Anutosh; Snyder, Solomon H.

2014-01-01

Inositol polyphosphates containing an energetic pyrophosphate bond are formed primarily by a family of three inositol hexakisphosphate (IP6) kinases (IP6K1–3). The Cullin-RING ubiquitin ligases (CRLs) regulate diverse biological processes through substrate ubiquitylation. CRL4, comprising the scaffold Cullin 4A/B, the E2-interacting Roc1/2, and the adaptor protein damage-specific DNA-binding protein 1, is activated by DNA damage. Basal CRL4 activity is inhibited by binding to the COP9 signalosome (CSN). UV radiation and other stressors dissociate the complex, leading to E3 ligase activation, but signaling events that trigger signalosome dissociation from CRL4 have been unclear. In the present study, we show that, under basal conditions, IP6K1 forms a ternary complex with CSN and CRL4 in which IP6K1 and CRL4 are inactive. UV dissociates IP6K1 to generate IP7, which then dissociates CSN–CRL4 to activate CRL4. Thus, IP6K1 is a novel CRL4 subunit that transduces UV signals to mediate disassembly of the CRL4–CSN complex, thereby regulating nucleotide excision repair and cell death. PMID:25349427

Detecção dos genes codificantes da toxina CDT, e pesquisa de fatores que influenciam na produção de hemolisinas em amostras de Campylobacter jejuni de origem avícola

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Michele M. Trindade

2015-08-01

Full Text Available Resumo: Membros termofílicos do gênero Campylobacter são reconhecidos como importantes enteropatógenos para o ser humano e animais. A grande diversidade ecológica destes micro-organismos em diferentes habitats tais como água, animais e alimentos predispõem ao aparecimento de novos fatores de virulência. Este trabalho teve por objetivo detectar os genes codificantes da Toxina Distensiva Citoletal (CDT por meio da técnica de PCR, pesquisar a atividade de hemolisinas e a influência de soluções quelantes e de íons nesta atividade. Foram utilizadas 45 amostras de Campylobacter jejuni de origem avícola para pesquisa de atividade hemolítica, cultivadas em Caldo Triptona de Soja (TSB. Após o crescimento bacteriano, as amostras foram semeadas em Ágar tríptico de soja (TSA contendo 5% de sangue de ovino. Para verificar a influência de agentes quelantes e solução de íons na atividade hemolítica, as amostras de C. jejuni foram cultivadas em TSB contendo separadamente os quelantes EDTA, ácido acético, soluções de íons CaCl2, MgCl2 e FeCl3, em atmosfera de microaerofilia. Quanto à atividade de hemolisina de C. jejuni em placas de TSA - sangue ovino foi possível observar que houve hemólise em 40% das amostras analisadas apenas com caldo TSB. Somente o ácido acético apresentou ação quelante sobre a atividade de hemolisinas em amostras de C. jejuni semeadas em placas de TSA - sangue ovino. Para detecção dos genes cdtA, cdtB e cdtC através da técnica da Reação em Cadeia da Polimerase (PCR foram utilizadas 119 amostras de C. jejuni de origem avícola. Foi possível observar que 37,8% possuíam o perfil de genes cdtABC. Os resultados demonstraram em amostras avícolas a presença de cepas de C. jejuni com potencial virulento, devido à presença dos genes da toxina CDT e potencial hemolítico, que apresentou ação reduzida in vitro com ácido acético.

The Mediator complex and transcription regulation

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Poss, Zachary C.; Ebmeier, Christopher C.

2013-01-01

The Mediator complex is a multi-subunit assembly that appears to be required for regulating expression of most RNA polymerase II (pol II) transcripts, which include protein-coding and most non-coding RNA genes. Mediator and pol II function within the pre-initiation complex (PIC), which consists of Mediator, pol II, TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH and is approximately 4.0 MDa in size. Mediator serves as a central scaffold within the PIC and helps regulate pol II activity in ways that remain poorly understood. Mediator is also generally targeted by sequence-specific, DNA-binding transcription factors (TFs) that work to control gene expression programs in response to developmental or environmental cues. At a basic level, Mediator functions by relaying signals from TFs directly to the pol II enzyme, thereby facilitating TF-dependent regulation of gene expression. Thus, Mediator is essential for converting biological inputs (communicated by TFs) to physiological responses (via changes in gene expression). In this review, we summarize an expansive body of research on the Mediator complex, with an emphasis on yeast and mammalian complexes. We focus on the basics that underlie Mediator function, such as its structure and subunit composition, and describe its broad regulatory influence on gene expression, ranging from chromatin architecture to transcription initiation and elongation, to mRNA processing. We also describe factors that influence Mediator structure and activity, including TFs, non-coding RNAs and the CDK8 module. PMID:24088064

Proteolytic regulation of metabolic enzymes by E3 ubiquitin ligase complexes: lessons from yeast.

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Nakatsukasa, Kunio; Okumura, Fumihiko; Kamura, Takumi

2015-01-01

Eukaryotic organisms use diverse mechanisms to control metabolic rates in response to changes in the internal and/or external environment. Fine metabolic control is a highly responsive, energy-saving process that is mediated by allosteric inhibition/activation and/or reversible modification of preexisting metabolic enzymes. In contrast, coarse metabolic control is a relatively long-term and expensive process that involves modulating the level of metabolic enzymes. Coarse metabolic control can be achieved through the degradation of metabolic enzymes by the ubiquitin-proteasome system (UPS), in which substrates are specifically ubiquitinated by an E3 ubiquitin ligase and targeted for proteasomal degradation. Here, we review select multi-protein E3 ligase complexes that directly regulate metabolic enzymes in Saccharomyces cerevisiae. The first part of the review focuses on the endoplasmic reticulum (ER) membrane-associated Hrd1 and Doa10 E3 ligase complexes. In addition to their primary roles in the ER-associated degradation pathway that eliminates misfolded proteins, recent quantitative proteomic analyses identified native substrates of Hrd1 and Doa10 in the sterol synthesis pathway. The second part focuses on the SCF (Skp1-Cul1-F-box protein) complex, an abundant prototypical multi-protein E3 ligase complex. While the best-known roles of the SCF complex are in the regulation of the cell cycle and transcription, accumulating evidence indicates that the SCF complex also modulates carbon metabolism pathways. The increasing number of metabolic enzymes whose stability is directly regulated by the UPS underscores the importance of the proteolytic regulation of metabolic processes for the acclimation of cells to environmental changes.

The E3 ubiquitin ligase protein associated with Myc (Pam) regulates mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling in vivo through N- and C-terminal domains.

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Han, Sangyeul; Kim, Sun; Bahl, Samira; Li, Lin; Burande, Clara F; Smith, Nicole; James, Marianne; Beauchamp, Roberta L; Bhide, Pradeep; DiAntonio, Aaron; Ramesh, Vijaya

2012-08-31

Pam and its homologs (the PHR protein family) are large E3 ubiquitin ligases that function to regulate synapse formation and growth in mammals, zebrafish, Drosophila, and Caenorhabditis elegans. Phr1-deficient mouse models (Phr1(Δ8,9) and Phr1(Magellan), with deletions in the N-terminal putative guanine exchange factor region and the C-terminal ubiquitin ligase region, respectively) exhibit axon guidance/outgrowth defects and striking defects of major axon tracts in the CNS. Our earlier studies identified Pam to be associated with tuberous sclerosis complex (TSC) proteins, ubiquitinating TSC2 and regulating mammalian/mechanistic target of rapamycin (mTOR) signaling. Here, we examine the potential involvement of the TSC/mTOR complex 1(mTORC1) signaling pathway in Phr1-deficient mouse models. We observed attenuation of mTORC1 signaling in the brains of both Phr1(Δ8,9) and Phr1(Magellan) mouse models. Our results establish that Pam regulates TSC/mTOR signaling in vitro and in vivo through two distinct domains. To further address whether Pam regulates mTORC1 through two functionally independent domains, we undertook heterozygous mutant crossing between Phr1(Δ8,9) and Phr1(Magellan) mice to generate a compound heterozygous model to determine whether these two domains can complement each other. mTORC1 signaling was not attenuated in the brains of double mutants (Phr1(Δ8,9/Mag)), confirming that Pam displays dual regulation of the mTORC1 pathway through two functional domains. Our results also suggest that although dysregulation of mTORC1 signaling may be responsible for the corpus callosum defects, other neurodevelopmental defects observed with Phr1 deficiency are independent of mTORC1 signaling. The ubiquitin ligase complex containing Pam-Fbxo45 likely targets additional synaptic and axonal proteins, which may explain the overlapping neurodevelopmental defects observed in Phr1 and Fbxo45 deficiency.

EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin.

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Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; Azarnia Tehran, Domenico; Montecucco, Cesare; Barth, Holger

2016-04-01

The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins.

EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin

Science.gov (United States)

Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R.; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; Tehran, Domenico Azarnia; Montecucco, Cesare; Barth, Holger

2016-01-01

The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins. PMID:27043629

Candida albicans Swi/Snf and Mediator Complexes Differentially Regulate Mrr1-Induced MDR1 Expression and Fluconazole Resistance.

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Liu, Zhongle; Myers, Lawrence C

2017-11-01

Long-term azole treatment of patients with chronic Candida albicans infections can lead to drug resistance. Gain-of-function (GOF) mutations in the transcription factor Mrr1 and the consequent transcriptional activation of MDR1 , a drug efflux coding gene, is a common pathway by which this human fungal pathogen acquires fluconazole resistance. This work elucidates the previously unknown downstream transcription mechanisms utilized by hyperactive Mrr1. We identified the Swi/Snf chromatin remodeling complex as a key coactivator for Mrr1, which is required to maintain basal and induced open chromatin, and Mrr1 occupancy, at the MDR1 promoter. Deletion of snf2 , the catalytic subunit of Swi/Snf, largely abrogates the increases in MDR1 expression and fluconazole MIC observed in MRR1 GOF mutant strains. Mediator positively and negatively regulates key Mrr1 target promoters. Deletion of the Mediator tail module med3 subunit reduces, but does not eliminate, the increased MDR1 expression and fluconazole MIC conferred by MRR1 GOF mutations. Eliminating the kinase activity of the Mediator Ssn3 subunit suppresses the decreased MDR1 expression and fluconazole MIC of the snf2 null mutation in MRR1 GOF strains. Ssn3 deletion also suppresses MDR1 promoter histone displacement defects in snf2 null mutants. The combination of this work with studies on other hyperactive zinc cluster transcription factors that confer azole resistance in fungal pathogens reveals a complex picture where the induction of drug efflux pump expression requires the coordination of multiple coactivators. The observed variations in transcription factor and target promoter dependence of this process may make the search for azole sensitivity-restoring small molecules more complicated. Copyright © 2017 American Society for Microbiology.

Specificity of antibodies directed against the cytolethal distending toxin of Haemophilus ducreyi in patients with chancroid.

Science.gov (United States)

Mbwana, Judica; Ahmed, Hinda J; Ahlman, Karin; Sundaeus, Vivian; Dahlén, Gunnar; Lyamuya, Eligius; Lagergård, Teresa

2003-09-01

Antibodies specific for the cytolethal-distending toxin of Haemophilus ducreyi (HdCDT) complex and for the CdtA, CdtB, and CdtC components were measured by ELISA in the sera of 50 patients with culture and/or PCR proven chancroid, 42 patients with periodontitis, 50 blood donors from Tanzania, 50 blood donors from Sweden. In addition, the biological activity e.g. neutralization capacity of the sera were tested. Our results demonstrate that majority of chancroid patients and healthy individuals had detectable levels of serum antibodies to HdCDT complex and to separate toxin components. However, high levels (> or =100 units) of antibodies to HdCDT complex were significantly more prevalent in the sera of patients with both chancroid and periodontitis than in the sera of the corresponding controls (P=0.001 and P=0.04, respectively). In the sera of the 50 patients with chancroid, antibodies to CdtA, CdtB, and CdtC were detected in 50, 35, and 34 individuals, respectively. Antibodies to CdtC, being less frequently detected than the antibodies to other components, show a good correlation with the neutralizing capacity of sera. High levels of neutralizing antibodies (> or =160) were detected in only 22 and 2% of the patients with chancroid and periodontitis, respectively. The data suggest that the low levels of anti-HdCDT antibodies, which include neutralizing antibodies, may contribute to limited protection in chancroid and since anti-HdCDT antibodies, may be detected in healthy individuals and in patients with certain disease conditions (e.g. periodontitis), they may not be specific markers for chancroid infection.

TOR Complex 2-Ypk1 Signaling Maintains Sphingolipid Homeostasis by Sensing and Regulating ROS Accumulation

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Brad J. Niles

2014-02-01

Full Text Available Reactive oxygen species (ROS are produced during normal metabolism and can function as signaling molecules. However, ROS at elevated levels can damage cells. Here, we identify the conserved target of rapamycin complex 2 (TORC2/Ypk1 signaling module as an important regulator of ROS in the model eukaryotic organism, S. cerevisiae. We show that TORC2/Ypk1 suppresses ROS produced both by mitochondria as well as by nonmitochondrial sources, including changes in acidification of the vacuole. Furthermore, we link vacuole-related ROS to sphingolipids, essential components of cellular membranes, whose synthesis is also controlled by TORC2/Ypk1 signaling. In total, our data reveal that TORC2/Ypk1 act within a homeostatic feedback loop to maintain sphingolipid levels and that ROS are a critical regulatory signal within this system. Thus, ROS sensing and signaling by TORC2/Ypk1 play a central physiological role in sphingolipid biosynthesis and in the maintenance of cell growth and viability.

Med1 subunit of the mediator complex in nuclear receptor-regulated energy metabolism, liver regeneration, and hepatocarcinogenesis.

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Jia, Yuzhi; Viswakarma, Navin; Reddy, Janardan K

2014-01-01

Several nuclear receptors regulate diverse metabolic functions that impact on critical biological processes, such as development, differentiation, cellular regeneration, and neoplastic conversion. In the liver, some members of the nuclear receptor family, such as peroxisome proliferator-activated receptors (PPARs), constitutive androstane receptor (CAR), farnesoid X receptor (FXR), liver X receptor (LXR), pregnane X receptor (PXR), glucocorticoid receptor (GR), and others, regulate energy homeostasis, the formation and excretion of bile acids, and detoxification of xenobiotics. Excess energy burning resulting from increases in fatty acid oxidation systems in liver generates reactive oxygen species, and the resulting oxidative damage influences liver regeneration and liver tumor development. These nuclear receptors are important sensors of exogenous activators as well as receptor-specific endogenous ligands. In this regard, gene knockout mouse models revealed that some lipid-metabolizing enzymes generate PPARα-activating ligands, while others such as ACOX1 (fatty acyl-CoA oxidase1) inactivate these endogenous PPARα activators. In the absence of ACOX1, the unmetabolized ACOX1 substrates cause sustained activation of PPARα, and the resulting increase in energy burning leads to hepatocarcinogenesis. Ligand-activated nuclear receptors recruit the multisubunit Mediator complex for RNA polymerase II-dependent gene transcription. Evidence indicates that the Med1 subunit of the Mediator is essential for PPARα, PPARγ, CAR, and GR signaling in liver. Med1 null hepatocytes fail to respond to PPARα activators in that these cells do not show induction of peroxisome proliferation and increases in fatty acid oxidation enzymes. Med1-deficient hepatocytes show no increase in cell proliferation and do not give rise to liver tumors. Identification of nuclear receptor-specific coactivators and Mediator subunits should further our understanding of the complexities of metabolic

The Chromatin Regulator Brpf1 Regulates Embryo Development and Cell Proliferation*

Science.gov (United States)

You, Linya; Yan, Kezhi; Zou, Jinfeng; Zhao, Hong; Bertos, Nicholas R.; Park, Morag; Wang, Edwin; Yang, Xiang-Jiao

2015-01-01

With hundreds of chromatin regulators identified in mammals, an emerging issue is how they modulate biological and pathological processes. BRPF1 (bromodomain- and PHD finger-containing protein 1) is a unique chromatin regulator possessing two PHD fingers, one bromodomain and a PWWP domain for recognizing multiple histone modifications. In addition, it binds to the acetyltransferases MOZ, MORF, and HBO1 (also known as KAT6A, KAT6B, and KAT7, respectively) to promote complex formation, restrict substrate specificity, and enhance enzymatic activity. We have recently showed that ablation of the mouse Brpf1 gene causes embryonic lethality at E9.5. Here we present systematic analyses of the mutant animals and demonstrate that the ablation leads to vascular defects in the placenta, yolk sac, and embryo proper, as well as abnormal neural tube closure. At the cellular level, Brpf1 loss inhibits proliferation of embryonic fibroblasts and hematopoietic progenitors. Molecularly, the loss reduces transcription of a ribosomal protein L10 (Rpl10)-like gene and the cell cycle inhibitor p27, and increases expression of the cell-cycle inhibitor p16 and a novel protein homologous to Scp3, a synaptonemal complex protein critical for chromosome association and embryo survival. These results uncover a crucial role of Brpf1 in controlling mouse embryo development and regulating cellular and gene expression programs. PMID:25773539

The putative cellodextrin transporter-like protein CLP1 is involved in cellulase induction in Neurospora crassa.

Science.gov (United States)

Cai, Pengli; Wang, Bang; Ji, Jingxiao; Jiang, Yongsheng; Wan, Li; Tian, Chaoguang; Ma, Yanhe

2015-01-09

Neurospora crassa recently has become a novel system to investigate cellulase induction. Here, we discovered a novel membrane protein, cellodextrin transporter-like protein 1 (CLP1; NCU05853), a putative cellodextrin transporter-like protein that is a critical component of the cellulase induction pathway in N. crassa. Although CLP1 protein cannot transport cellodextrin, the suppression of cellulase induction by this protein was discovered on both cellobiose and Avicel. The co-disruption of the cellodextrin transporters cdt2 and clp1 in strain Δ3βG formed strain CPL7. With induction by cellobiose, cellulase production was enhanced 6.9-fold in CPL7 compared with Δ3βG. We also showed that the suppression of cellulase expression by CLP1 occurred by repressing the expression of cellodextrin transporters, particularly cdt1 expression. Transcriptome analysis of the hypercellulase-producing strain CPL7 showed that the cellulase expression machinery was dramatically stimulated, as were the cellulase enzyme genes including the inducer transporters and the major transcriptional regulators. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The Putative Cellodextrin Transporter-like Protein CLP1 Is Involved in Cellulase Induction in Neurospora crassa*

Science.gov (United States)

Cai, Pengli; Wang, Bang; Ji, Jingxiao; Jiang, Yongsheng; Wan, Li; Tian, Chaoguang; Ma, Yanhe

2015-01-01

Neurospora crassa recently has become a novel system to investigate cellulase induction. Here, we discovered a novel membrane protein, cellodextrin transporter-like protein 1 (CLP1; NCU05853), a putative cellodextrin transporter-like protein that is a critical component of the cellulase induction pathway in N. crassa. Although CLP1 protein cannot transport cellodextrin, the suppression of cellulase induction by this protein was discovered on both cellobiose and Avicel. The co-disruption of the cellodextrin transporters cdt2 and clp1 in strain Δ3βG formed strain CPL7. With induction by cellobiose, cellulase production was enhanced 6.9-fold in CPL7 compared with Δ3βG. We also showed that the suppression of cellulase expression by CLP1 occurred by repressing the expression of cellodextrin transporters, particularly cdt1 expression. Transcriptome analysis of the hypercellulase-producing strain CPL7 showed that the cellulase expression machinery was dramatically stimulated, as were the cellulase enzyme genes including the inducer transporters and the major transcriptional regulators. PMID:25398875

Defective replication initiation results in locus specific chromosome breakage and a ribosomal RNA deficiency in yeast.

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Joseph C Sanchez

2017-10-01

Full Text Available A form of dwarfism known as Meier-Gorlin syndrome (MGS is caused by recessive mutations in one of six different genes (ORC1, ORC4, ORC6, CDC6, CDT1, and MCM5. These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and CDC45. The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human ORC4 alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (orc4Y232C. We find that yeast cells with the orc4Y232C allele have a prolonged S-phase, due to compromised replication initiation at the ribosomal DNA (rDNA locus located on chromosome XII. The inability to initiate replication at the rDNA locus results in chromosome breakage and a severely reduced rDNA copy number in the survivors, presumably helping to ensure complete replication of chromosome XII. Although reducing rDNA copy number may help ensure complete chromosome replication, orc4Y232C cells struggle to meet the high demand for ribosomal RNA synthesis. This finding provides additional evidence linking two essential cellular pathways-DNA replication and ribosome biogenesis.

Defective replication initiation results in locus specific chromosome breakage and a ribosomal RNA deficiency in yeast.

Science.gov (United States)

Sanchez, Joseph C; Kwan, Elizabeth X; Pohl, Thomas J; Amemiya, Haley M; Raghuraman, M K; Brewer, Bonita J

2017-10-01

A form of dwarfism known as Meier-Gorlin syndrome (MGS) is caused by recessive mutations in one of six different genes (ORC1, ORC4, ORC6, CDC6, CDT1, and MCM5). These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and CDC45). The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human ORC4 alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (orc4Y232C). We find that yeast cells with the orc4Y232C allele have a prolonged S-phase, due to compromised replication initiation at the ribosomal DNA (rDNA) locus located on chromosome XII. The inability to initiate replication at the rDNA locus results in chromosome breakage and a severely reduced rDNA copy number in the survivors, presumably helping to ensure complete replication of chromosome XII. Although reducing rDNA copy number may help ensure complete chromosome replication, orc4Y232C cells struggle to meet the high demand for ribosomal RNA synthesis. This finding provides additional evidence linking two essential cellular pathways-DNA replication and ribosome biogenesis.

Ctr9, a Protein in the Transcription Complex Paf1, Regulates Dopamine Transporter Activity at the Plasma Membrane.

Science.gov (United States)

De Gois, Stéphanie; Slama, Patrick; Pietrancosta, Nicolas; Erdozain, Amaia M; Louis, Franck; Bouvrais-Veret, Caroline; Daviet, Laurent; Giros, Bruno

2015-07-17

Dopamine (DA) is a major regulator of sensorimotor and cognitive functions. The DA transporter (DAT) is the key protein that regulates the spatial and temporal activity of DA release into the synaptic cleft via the rapid reuptake of DA into presynaptic termini. Several lines of evidence have suggested that transporter-interacting proteins may play a role in DAT function and regulation. Here, we identified the tetratricopeptide repeat domain-containing protein Ctr9 as a novel DAT binding partner using a yeast two-hybrid system. We showed that Ctr9 is expressed in dopaminergic neurons and forms a stable complex with DAT in vivo via GST pulldown and co-immunoprecipitation assays. In mammalian cells co-expressing both proteins, Ctr9 partially colocalizes with DAT at the plasma membrane. This interaction between DAT and Ctr9 results in a dramatic enhancement of DAT-mediated DA uptake due to an increased number of DAT transporters at the plasma membrane. We determined that the binding of Ctr9 to DAT requires residues YKF in the first half of the DAT C terminus. In addition, we characterized Ctr9, providing new insight into this protein. Using three-dimensional modeling, we identified three novel tetratricopeptide repeat domains in the Ctr9 sequence, and based on deletion mutation experiments, we demonstrated the role of the SH2 domain of Ctr9 in nuclear localization. Our results demonstrate that Ctr9 localization is not restricted to the nucleus, as previously described for the transcription complex Paf1. Taken together, our data provide evidence that Ctr9 modulates DAT function by regulating its trafficking. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

GEMC1 is a TopBP1-interacting protein required for chromosomal DNA replication.

Science.gov (United States)

Balestrini, Alessia; Cosentino, Claudia; Errico, Alessia; Garner, Elizabeth; Costanzo, Vincenzo

2010-05-01

Many of the factors required for chromosomal DNA replication have been identified in unicellular eukaryotes. However, DNA replication is poorly understood in multicellular organisms. Here, we report the identification of GEMC1 (geminin coiled-coil containing protein 1), a novel vertebrate protein required for chromosomal DNA replication. GEMC1 is highly conserved in vertebrates and is preferentially expressed in proliferating cells. Using Xenopus laevis egg extract we show that Xenopus GEMC1 (xGEMC1) binds to the checkpoint and replication factor TopBP1, which promotes binding of xGEMC1 to chromatin during pre-replication complex (pre-RC) formation. We demonstrate that xGEMC1 interacts directly with replication factors such as Cdc45 and the kinase Cdk2-CyclinE, through which it is heavily phosphorylated. Phosphorylated xGEMC1 stimulates initiation of DNA replication, whereas depletion of xGEMC1 prevents the onset of DNA replication owing to the impairment of Cdc45 loading onto chromatin. Similarly, inhibition of GEMC1 expression with morpholino and siRNA oligos prevents DNA replication in embryonic and somatic vertebrate cells. These data suggest that GEMC1 promotes initiation of chromosomal DNA replication in multicellular organisms by mediating TopBP1- and Cdk2-dependent recruitment of Cdc45 onto replication origins.

Expression, purification and spectroscopic characterization of the Regulator complex

Energy Technology Data Exchange (ETDEWEB)

Nogueira, M.L.C.; Silva, A.L.S.; Camilotti, D.; Silva, C.A.; Sforca, M.L.; Smetana, J.H.C.; Zeri, A.C. [Laboratorio Nacional de Biociencias - LNBIO, Campinas, SP (Brazil); Ospina-Bedoya, M. [Universidad de Antioquia, Medellin (Colombia)

2012-07-01

Full text: The mammalian target of rapamycin (mTOR) signaling pathway integrates both intracellular and extracellular signals, serves as a central regulator of cell metabolism in humans and its deregulation is linked to diseases like cancer and diabetes. The small GTPases Rag are mediators of signaling by amino acid (leucine). These GT-Pases are anchored on the surface of the lysosome through an interaction with a complex of three proteins, p18, MP1 and p14, called Ragulator. The p18 protein is responsible for interaction with the lysosomal membrane through its N terminal post translational modification. The objective of this project is to study the interaction of p18 and other components of the Ragulator complex. The p18 protein was expressed in inclusion bodies, which were isolated and solubilized in urea. p18 was renatured with its partners MP1/p14 and this complex, the Ragulator, was subjected to spectroscopic characterization using circular dichroism and dynamic light scattering. (author)

Expression, purification and spectroscopic characterization of the Regulator complex

International Nuclear Information System (INIS)

Nogueira, M.L.C.; Silva, A.L.S.; Camilotti, D.; Silva, C.A.; Sforca, M.L.; Smetana, J.H.C.; Zeri, A.C.; Ospina-Bedoya, M.

2012-01-01

Full text: The mammalian target of rapamycin (mTOR) signaling pathway integrates both intracellular and extracellular signals, serves as a central regulator of cell metabolism in humans and its deregulation is linked to diseases like cancer and diabetes. The small GTPases Rag are mediators of signaling by amino acid (leucine). These GT-Pases are anchored on the surface of the lysosome through an interaction with a complex of three proteins, p18, MP1 and p14, called Ragulator. The p18 protein is responsible for interaction with the lysosomal membrane through its N terminal post translational modification. The objective of this project is to study the interaction of p18 and other components of the Ragulator complex. The p18 protein was expressed in inclusion bodies, which were isolated and solubilized in urea. p18 was renatured with its partners MP1/p14 and this complex, the Ragulator, was subjected to spectroscopic characterization using circular dichroism and dynamic light scattering. (author)

Fostering Self-Regulation in Training Complex Cognitive Tasks

Science.gov (United States)

van Meeuwen, Ludo W.; Brand-Gruwel, Saskia; Kirschner, Paul A.; de Bock, Jeano J. P. R.; van Merriënboer, Jeroen J. G.

2018-01-01

In complex cognitive domains such as air traffic control, professionals must be able to adapt to and act upon continuing changes in a highly advanced technological work environment. To function optimally in such an environment, the controllers must be able to regulate their learning. Although these regulation skills should be part of their…

Dynamic Regulation of a Cell Adhesion Protein Complex Including CADM1 by Combinatorial Analysis of FRAP with Exponential Curve-Fitting

Science.gov (United States)

Sakurai-Yageta, Mika; Maruyama, Tomoko; Suzuki, Takashi; Ichikawa, Kazuhisa; Murakami, Yoshinori

2015-01-01

Protein components of cell adhesion machinery show continuous renewal even in the static state of epithelial cells and participate in the formation and maintenance of normal epithelial architecture and tumor suppression. CADM1 is a tumor suppressor belonging to the immunoglobulin superfamily of cell adhesion molecule and forms a cell adhesion complex with an actin-binding protein, 4.1B, and a scaffold protein, MPP3, in the cytoplasm. Here, we investigate dynamic regulation of the CADM1-4.1B-MPP3 complex in mature cell adhesion by fluorescence recovery after photobleaching (FRAP) analysis. Traditional FRAP analysis were performed for relatively short period of around 10min. Here, thanks to recent advances in the sensitive laser detector systems, we examine FRAP of CADM1 complex for longer period of 60 min and analyze the recovery with exponential curve-fitting to distinguish the fractions with different diffusion constants. This approach reveals that the fluorescence recovery of CADM1 is fitted to a single exponential function with a time constant (τ) of approximately 16 min, whereas 4.1B and MPP3 are fitted to a double exponential function with two τs of approximately 40-60 sec and 16 min. The longer τ is similar to that of CADM1, suggesting that 4.1B and MPP3 have two distinct fractions, one forming a complex with CADM1 and the other present as a free pool. Fluorescence loss in photobleaching analysis supports the presence of a free pool of these proteins near the plasma membrane. Furthermore, double exponential fitting makes it possible to estimate the ratio of 4.1B and MPP3 present as a free pool and as a complex with CADM1 as approximately 3:2 and 3:1, respectively. Our analyses reveal a central role of CADM1 in stabilizing the complex with 4.1B and MPP3 and provide insight in the dynamics of adhesion complex formation. PMID:25780926

Compound C prevents Hypoxia-Inducible Factor-1α protein stabilization by regulating the cellular oxygen availability via interaction with Mitochondrial Complex I

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Hagen Thilo

2011-04-01

Full Text Available Abstract The transcription factor Hypoxia-Inducible Factor-1α is a master regulator of the cellular response to low oxygen concentration. Compound C, an inhibitor of AMP-activated kinase, has been reported to inhibit hypoxia dependent Hypoxia-Inducible Factor-1α activation via a mechanism that is independent of AMP-activated kinase but dependent on its interaction with the mitochondrial electron transport chain. The objective of this study is to characterize the interaction of Compound C with the mitochondrial electron transport chain and to determine the mechanism through which the drug influences the stability of the Hypoxia-Inducible Factor-1α protein. We found that Compound C functions as an inhibitor of complex I of the mitochondrial electron transport chain as demonstrated by its effect on mitochondrial respiration. It also prevents hypoxia-induced Hypoxia-Inducible Factor-1α stabilization in a dose dependent manner. In addition, Compound C does not have significant effects on reactive oxygen species production from complex I via both forward and reverse electron flux. This study provides evidence that similar to other mitochondrial electron transport chain inhibitors, Compound C regulates Hypoxia-Inducible Factor-1α stability by controlling the cellular oxygen concentration.

HACE1 Negatively Regulates Virus-Triggered Type I IFN Signaling by Impeding the Formation of the MAVS-TRAF3 Complex

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He-Ting Mao

2016-05-01

Full Text Available During virus infection, the cascade signaling pathway that leads to the production of proinflammatory cytokines is controlled at multiple levels to avoid detrimental overreaction. HACE1 has been characterized as an important tumor suppressor. Here, we identified HACE1 as an important negative regulator of virus-triggered type I IFN signaling. Overexpression of HACE1 inhibited Sendai virus- or poly (I:C-induced signaling and resulted in reduced IFNB1 production and enhanced virus replication. Knockdown of HACE1 expression exhibited the opposite effects. Ubiquitin E3 ligase activity of the dead mutant HACE1/C876A had a comparable inhibitory function as WT HACE1, suggesting that the suppressive function of HACE1 on virus-induced signaling is independent of its E3 ligase activity. Further study indicated that HACE1 acted downstream of MAVS and upstream of TBK1. Mechanistic studies showed that HACE1 exerts its inhibitory role on virus-induced signaling by disrupting the MAVS-TRAF3 complex. Therefore, we uncovered a novel function of HACE1 in innate immunity regulation.

Novel Functions for TAF7, a Regulator of TAF1-independent Transcription

OpenAIRE

Devaiah, Ballachanda N.; Lu, Hanxin; Gegonne, Anne; Sercan, Zeynep; Zhang, Hongen; Clifford, Robert J.; Lee, Maxwell P.; Singer, Dinah S.

2010-01-01

The transcription factor TFIID components TAF7 and TAF1 regulate eukaryotic transcription initiation. TAF7 regulates transcription initiation of TAF1-dependent genes by binding to the acetyltransferase (AT) domain of TAF1 and inhibiting the enzymatic activity that is essential for transcription. TAF7 is released from the TAF1-TFIID complex upon completion of preinitiation complex assembly, allowing transcription to initiate. However, not all transcription is TAF1-dependent, and the role of TA...

The human RNA polymerase II-associated factor 1 (hPaf1: a new regulator of cell-cycle progression.

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Nicolas Moniaux

2009-09-01

Full Text Available The human PAF (hPAF complex is part of the RNA polymerase II transcription apparatus and regulates multiple steps in gene expression. Further, the yeast homolog of hPaf1 has a role in regulating the expression of a subset of genes involved in the cell-cycle. We therefore investigated the role of hPaf1 during progression of the cell-cycle.Herein, we report that the expression of hPaf1, a subunit of the hPAF complex, increases with cell-cycle progression and is regulated in a cell-cycle dependant manner. hPaf1 specifically regulates a subclass of genes directly implicated in cell-cycle progression during G1/S, S/G2, and G2/M. In prophase, hPaf1 aligns in filament-like structures, whereas in metaphase it is present within the pole forming a crown-like structure, surrounding the centrosomes. Moreover, hPaf1 is degraded during the metaphase to anaphase transition. In the nucleus, hPaf1 regulates the expression of cyclins A1, A2, D1, E1, B1, and Cdk1. In addition, expression of hPaf1 delays DNA replication but favors the G2/M transition, in part through microtubule assembly and mitotic spindle formation.Our results identify hPaf1 and the hPAF complex as key regulators of cell-cycle progression. Mutation or loss of stoichiometry of at least one of the members may potentially lead to cancer development.

Time-dependent, glucose-regulated Arabidopsis Regulator of G-protein Signaling 1 network

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Dinesh Kumar Jaiswal

2016-04-01

Full Text Available Plants lack 7-transmembrane, G-protein coupled receptors (GPCRs because the G alpha subunit of the heterotrimeric G protein complex is “self-activating”—meaning that it spontaneously exchanges bound GDP for GTP without the need of a GPCR. In lieu of GPCRs, most plants have a seven transmembrane receptor-like regulator of G-protein signaling (RGS protein, a component of the complex that keeps G-protein signaling in its non-activated state. The addition of glucose physically uncouples AtRGS1 from the complex through specific endocytosis leaving the activated G protein at the plasma membrane. The complement of proteins in the AtRGS1/G-protein complex over time from glucose-induced endocytosis was profiled by immunoprecipitation coupled to mass spectrometry (IP-MS. A total of 119 proteins in the AtRGS1 complex were identified. Several known interactors of the complex were identified, thus validating the approach, but the vast majority (93/119 were not known previously. AtRGS1 protein interactions were dynamically modulated by d-glucose. At low glucose levels, the AtRGS1 complex is comprised of proteins involved in transport, stress and metabolism. After glucose application, the AtRGS1 complex rapidly sheds many of these proteins and recruits other proteins involved in vesicular trafficking and signal transduction. The profile of the AtRGS1 components answers several questions about the type of coat protein and vesicular trafficking GTPases used in AtRGS1 endocytosis and the function of endocytic AtRGS1.

Nucleophosmin1 is a negative regulator of the small GTPase Rac1

NARCIS (Netherlands)

Zoughlami, Younes; van Stalborgh, Anne M.; van Hennik, Paula B.; Hordijk, Peter L.

2013-01-01

The Rac1 GTPase is a critical regulator of cytoskeletal dynamics and controls many biological processes, such as cell migration, cell-cell contacts, cellular growth and cell division. These complex processes are controlled by Rac1 signaling through effector proteins. We have previously identified

SKR-1, a homolog of Skp1 and a member of the SCFSEL-10 complex, regulates sex-determination and LIN-12/Notch signaling in C. elegans

Science.gov (United States)

Killian, Darrell J.; Harvey, Elizabeth; Johnson, Peter; Otori, Muneyoshi; Mitani, Shohei; Xue, Ding

2008-01-01

Sex-determination in C. elegans requires regulation of gene transcription and protein activity and stability. sel-10 encodes a WD40-repeat-containing F-box protein that likely mediates the ubiquitin-mediated degradation of important sex-determination factors. Loss of sel-10 results in a mild masculinization of hermaphrodites, whereas dominant alleles of sel-10, such as sel-10(n1074), cause a more severe masculinization, including a reversal of the life versus death decision in sex-specific neurons. To investigate about how sel-10 regulates sex-determination, we conducted a sel-10(n1074) suppressor screen and isolated a weak loss-of-function allele of skr-1, one of 21 Skp1-related genes in C. elegans. Skp1, Cullin, and F-box proteins, such as SEL-10, are components of the SCF E3 ubiquitin ligase complex. We present genetic evidence that the sel-10(n1074) masculinization phenotype is dependent upon skr-1 and cul-1 activity. Furthermore, we show that the SKR-1(M140I) weak loss-of-function mutation interferes with SKR-1/SEL-10 binding. Unexpectedly, we found that the G567E substitution in SEL-10 caused by the n1074 allele impairs the binding of SEL-10 to SKR-1 and the dimerization of SEL-10, which may be important for SEL-10 function. Our results suggest that SKR-1, CUL-1 and SEL-10 constitute an SCF E3 ligase complex that plays an important role in modulating sex-determination and LIN-12/Notch signaling in C. elegans. PMID:18718460

The altered complexity of cardiovascular regulation in depressed patients

International Nuclear Information System (INIS)

Schulz, Steffen; Voss, Andreas; Koschke, Mandy; Bär, Karl-Jürgen

2010-01-01

Major depressive disorders (MDD) are associated with an increased risk for cardiovascular morbidity and mortality. Even if it is known that MDD are accompanied by an autonomic dysbalance with increased sympathetic and/or reduced parasympathetic activity, to date only limited information is available about the degree and complexity of cardiovascular regulation. The aim of this study was to investigate the influence of MDD on the autonomous nervous system and cardiovascular complexity by means of linear and nonlinear indices from heart rate and blood pressure variability (HRV, BPV). From 57 non-medicated patients and 57 matched healthy controls with respect to age and gender HRV and BPV in time and frequency domain, symbolic dynamics, compression entropy, multiscale entropy, detrended fluctuation analysis, Poincaré plot analysis and baroreflex sensitivity were analysed from 30 min short-term recordings. Complexity indices from nonlinear dynamics demonstrated considerable changes in autonomous regulation due to MDD. For the first time we could show that non-medicated depressed patients who were matched with respect to age and gender reveal a significantly changed short-term as well as long-term complexity of cardiovascular regulation. These results suggest substantial changes in autonomic control probably due to a change of interactions between different physiological control loops in MDD

SUMO regulates p21Cip1 intracellular distribution and with p21Cip1 facilitates multiprotein complex formation in the nucleolus upon DNA damage.

Science.gov (United States)

Brun, Sonia; Abella, Neus; Berciano, Maria T; Tapia, Olga; Jaumot, Montserrat; Freire, Raimundo; Lafarga, Miguel; Agell, Neus

2017-01-01

We previously showed that p21Cip1 transits through the nucleolus on its way from the nucleus to the cytoplasm and that DNA damage inhibits this transit and induces the formation of p21Cip1-containing intranucleolar bodies (INoBs). Here, we demonstrate that these INoBs also contain SUMO-1 and UBC9, the E2 SUMO-conjugating enzyme. Furthermore, whereas wild type SUMO-1 localized in INoBs, a SUMO-1 mutant, which is unable to conjugate with proteins, does not, suggesting the presence of SUMOylated proteins at INoBs. Moreover, depletion of the SUMO-conjugating enzyme UBC9 or the sumo hydrolase SENP2 changed p21Cip1 intracellular distribution. In addition to SUMO-1 and p21Cip1, cell cycle regulators and DNA damage checkpoint proteins, including Cdk2, Cyclin E, PCNA, p53 and Mdm2, and PML were also detected in INoBs. Importantly, depletion of UBC9 or p21Cip1 impacted INoB biogenesis and the nucleolar accumulation of the cell cycle regulators and DNA damage checkpoint proteins following DNA damage. The impact of p21Cip1 and SUMO-1 on the accumulation of proteins in INoBs extends also to CRM1, a nuclear exportin that is also important for protein translocation from the cytoplasm to the nucleolus. Thus, SUMO and p21Cip1 regulate the transit of proteins through the nucleolus, and that disruption of nucleolar export by DNA damage induces SUMO and p21Cip1 to act as hub proteins to form a multiprotein complex in the nucleolus.

Detecting coordinated regulation of multi-protein complexes using logic analysis of gene expression

Directory of Open Access Journals (Sweden)

Yeates Todd O

2009-12-01

Full Text Available Abstract Background Many of the functional units in cells are multi-protein complexes such as RNA polymerase, the ribosome, and the proteasome. For such units to work together, one might expect a high level of regulation to enable co-appearance or repression of sets of complexes at the required time. However, this type of coordinated regulation between whole complexes is difficult to detect by existing methods for analyzing mRNA co-expression. We propose a new methodology that is able to detect such higher order relationships. Results We detect coordinated regulation of multiple protein complexes using logic analysis of gene expression data. Specifically, we identify gene triplets composed of genes whose expression profiles are found to be related by various types of logic functions. In order to focus on complexes, we associate the members of a gene triplet with the distinct protein complexes to which they belong. In this way, we identify complexes related by specific kinds of regulatory relationships. For example, we may find that the transcription of complex C is increased only if the transcription of both complex A AND complex B is repressed. We identify hundreds of examples of coordinated regulation among complexes under various stress conditions. Many of these examples involve the ribosome. Some of our examples have been previously identified in the literature, while others are novel. One notable example is the relationship between the transcription of the ribosome, RNA polymerase and mannosyltransferase II, which is involved in N-linked glycan processing in the Golgi. Conclusions The analysis proposed here focuses on relationships among triplets of genes that are not evident when genes are examined in a pairwise fashion as in typical clustering methods. By grouping gene triplets, we are able to decipher coordinated regulation among sets of three complexes. Moreover, using all triplets that involve coordinated regulation with the ribosome

Cell cycle regulation of the BRCA1/acetyl-CoA-carboxylase complex.

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Ray, H; Suau, F; Vincent, A; Dalla Venezia, N

2009-01-16

Germ-line alterations in BRCA1 are associated with an increased susceptibility to breast and ovarian cancer. The BRCA1 protein has been implicated in multiple cellular functions. We have recently demonstrated that BRCA1 reduces acetyl-CoA-carboxylase alpha (ACCA) activity through its phospho-dependent binding to ACCA, and further established that the phosphorylation of the Ser1263 of ACCA is required for this interaction. Here, to gain more insight into the cellular conditions that trigger the BRCA1/ACCA interaction, we designed an anti-pSer1263 antibody and demonstrated that the Ser1263 of ACCA is phosphorylated in vivo, in a cell cycle-dependent manner. We further showed that the interaction between BRCA1 and ACCA is regulated during cell cycle progression. Taken together, our findings reveal a novel mechanism of regulation of ACCA distinct from the previously described phosphorylation of Ser79, and provide new insights into the control of lipogenesis through the cell cycle.

Sestrin regulation of TORC1: Is Sestrin a leucine sensor?

Energy Technology Data Exchange (ETDEWEB)

Lee, Jun Hee; Cho, Uhn-Soo; Karin, Michael (Michigan); (UCSD)

2016-06-07

Sestrins are highly conserved, stress-inducible proteins that inhibit target of rapamycin complex 1 (TORC1) signaling. After their transcriptional induction, both vertebrate and invertebrate Sestrins turn on the adenosine monophosphate (AMP)–activated protein kinase (AMPK), which activates the tuberous sclerosis complex (TSC), a key inhibitor of TORC1 activation. However, Sestrin overexpression, on occasion, can result in TORC1 inhibition even in AMPK-deficient cells. This effect has been attributed to Sestrin’s ability to bind the TORC1-regulating GATOR2 protein complex, which was postulated to control trafficking of TORC1 to lysosomes. How the binding of Sestrins to GATOR2 is regulated and how it contributes to TORC1 inhibition are unknown. New findings suggest that the amino acid leucine specifically disrupts the association of Sestrin2 with GATOR2, thus explaining how leucine and related amino acids stimulate TORC1 activity. We discuss whether and how these findings fit what has already been learned about Sestrin-mediated TORC1 inhibition from genetic studies conducted in fruit flies and mammals.

Interaction of hyperalgesia and sensory loss in complex regional pain syndrome type I (CRPS I).

Science.gov (United States)

Huge, Volker; Lauchart, Meike; Förderreuther, Stefanie; Kaufhold, Wibke; Valet, Michael; Azad, Shahnaz Christina; Beyer, Antje; Magerl, Walter

2008-07-23

Sensory abnormalities are a key feature of Complex Regional Pain Syndrome (CRPS). In order to characterise these changes in patients suffering from acute or chronic CRPS I, we used Quantitative Sensory Testing (QST) in comparison to an age and gender matched control group. 61 patients presenting with CRPS I of the upper extremity and 56 healthy subjects were prospectively assessed using QST. The patients' warm and cold detection thresholds (WDT; CDT), the heat and cold pain thresholds (HPT; CPT) and the occurrence of paradoxical heat sensation (PHS) were observed. In acute CRPS I, patients showed warm and cold hyperalgesia, indicated by significant changes in HPT and CPT. WDT and CDT were significantly increased as well, indicating warm and cold hypoaesthesia. In chronic CRPS, thermal hyperalgesia declined, but CDT as well as WDT further deteriorated. Solely patients with acute CRPS displayed PHS. To a minor degree, all QST changes were also present on the contralateral limb. We propose three pathomechanisms of CRPS I, which follow a distinct time course: Thermal hyperalgesia, observed in acute CRPS, indicates an ongoing aseptic peripheral inflammation. Thermal hypoaesthesia, as detected in acute and chronic CRPS, signals a degeneration of A-delta and C-fibres, which further deteriorates in chronic CRPS. PHS in acute CRPS I indicates that both inflammation and degeneration are present, whilst in chronic CRPS I, the pathomechanism of degeneration dominates, signalled by the absence of PHS. The contralateral changes observed strongly suggest the involvement of the central nervous system.

The USP1-UAF1 complex interacts with RAD51AP1 to promote homologous recombination repair.

Science.gov (United States)

Cukras, Scott; Lee, Euiho; Palumbo, Emily; Benavidez, Pamela; Moldovan, George-Lucian; Kee, Younghoon

2016-10-01

USP1 deubiquitinating enzyme and its stoichiometric binding partner UAF1 play an essential role in promoting DNA homologous recombination (HR) repair in response to various types of DNA damaging agents. Deubiquitination of FANCD2 may be attributed to the key role of USP1-UAF1 complex in regulating HR repair, however whether USP1-UAF1 promotes HR repair independently of FANCD2 deubiquitination is not known. Here we show evidence that the USP1-UAF1 complex has a FANCD2-independent function in promoting HR repair. Proteomic search of UAF1-interacting proteins revealed that UAF1 associates with RAD51AP1, a RAD51-interacting protein implicated in HR repair. We show that UAF1 mediates the interaction between USP1 and RAD51AP1, and that depletion of USP1 or UAF1 led to a decreased stability of RAD51AP1. Protein interaction mapping analysis identified some key residues within RAD51AP1 required for interacting with the USP1-UAF1 complex. Cells expressing the UAF1 interaction-deficient mutant of RAD51AP1 show increased chromosomal aberrations in response to Mitomycin C treatment. Moreover, similar to the RAD51AP1 depleted cells, the cells expressing UAF1-interaction deficient RAD51AP1 display persistent RAD51 foci following DNA damage exposure, indicating that these factors regulate a later step during the HR repair. These data altogether suggest that the USP1-UAF1 complex promotes HR repair via multiple mechanisms: through FANCD2 deubiquitination, as well as by interacting with RAD51AP1.

Complex fluids with mobile charge-regulating macro-ions

Science.gov (United States)

Markovich, Tomer; Andelman, David; Podgornik, Rudi

2017-10-01

We generalize the concept of charge regulation of ionic solutions, and apply it to complex fluids with mobile macro-ions having internal non-electrostatic degrees of freedom. The suggested framework provides a convenient tool for investigating systems where mobile macro-ions can self-regulate their charge (e.g., proteins). We show that even within a simplified charge-regulation model, the charge dissociation equilibrium results in different and notable properties. Consequences of the charge regulation include a positional dependence of the effective charge of the macro-ions, a non-monotonic dependence of the effective Debye screening length on the concentration of the monovalent salt, a modification of the electric double-layer structure, and buffering by the macro-ions of the background electrolyte.

Regulation of glycogen metabolism by the CRE-1, RCO-1 and RCM-1 proteins in Neurospora crassa. The role of CRE-1 as the central transcriptional regulator.

Science.gov (United States)

Cupertino, Fernanda Barbosa; Virgilio, Stela; Freitas, Fernanda Zanolli; Candido, Thiago de Souza; Bertolini, Maria Célia

2015-04-01

The transcription factor CreA/Mig1/CRE-1 is a repressor protein that regulates the use of alternative carbon sources via a mechanism known as Carbon Catabolite Repression (CCR). In Saccharomyces cerevisiae, Mig1 recruits the complex Ssn6-Tup1, the Neurospora crassa RCM-1 and RCO-1 orthologous proteins, respectively, to bind to promoters of glucose-repressible genes. We have been studying the regulation of glycogen metabolism in N. crassa and the identification of the RCO-1 corepressor as a regulator led us to investigate the regulatory role of CRE-1 in this process. Glycogen content is misregulated in the rco-1(KO), rcm-1(RIP) and cre-1(KO) strains, and the glycogen synthase phosphorylation is decreased in all strains, showing that CRE-1, RCO-1 and RCM-1 proteins are involved in glycogen accumulation and in the regulation of GSN activity by phosphorylation. We also confirmed the regulatory role of CRE-1 in CCR and its nuclear localization under repressing condition in N. crassa. The expression of all glycogenic genes is misregulated in the cre-1(KO) strain, suggesting that CRE-1 also controls glycogen metabolism by regulating gene expression. The existence of a high number of the Aspergillus nidulans CreA motif (5'-SYGGRG-3') in the glycogenic gene promoters led us to analyze the binding of CRE-1 to some DNA motifs both in vitro by DNA gel shift and in vivo by ChIP-qPCR analysis. CRE-1 bound in vivo to all motifs analyzed demonstrating that it down-regulates glycogen metabolism by controlling gene expression and GSN phosphorylation. Copyright © 2015 Elsevier Inc. All rights reserved.

A designated centre for people with disabilities operated by St Michael's House, Co. Dublin

LENUS (Irish Health Repository)

Cobbe, Sinead

2017-12-05

Complex decongestive therapy (CDT) is a regimen of physical treatment for lymphedema. Its effectiveness is unknown in advanced cancer patients. This study evaluates effectiveness of CDT in this population.

Pro- and Anti-Inflammatory Cytokines Release in Mice Injected with Crotalus durissus terrificus Venom

Directory of Open Access Journals (Sweden)

A. Hernández Cruz

2008-01-01

Full Text Available The effects of Crotalus durissus terrificus venom (Cdt were analyzed with respect to the susceptibility and the inflammatory mediators in an experimental model of severe envenomation. BALB/c female mice injected intraperitoneally presented sensibility to Cdt, with changes in specific signs, blood biochemical and inflammatory mediators. The venom induced reduction of glucose and urea levels and an increment of creatinine levels in serum from mice. Significant differences were observed in the time-course of mediator levels in sera from mice injected with Cdt. The maximum levels of IL-6, NO, IL-5, TNF, IL-4 and IL-10 were observed 15 min, 30 min, 1, 2 and 4 hours post-injection, respectively. No difference was observed for levels of IFN-γ. Taken together, these data indicate that the envenomation by Cdt is regulated both pro- and anti-inflammatory cytokine responses at time-dependent manner. In serum from mice injected with Cdt at the two first hours revealed of pro-inflammatory dominance. However, with an increment of time an increase of anti-inflammatory cytokines was observed and the balance toward to anti-inflammatory dominance. In conclusion, the observation that Cdt affects the production of pro- and anti-inflammatory cytokines provides further evidence for the role played by Cdt in modulating pro/anti-inflammatory cytokine balance.

MCM interference during licensing of DNA replication in Xenopus egg extracts-Possible Role of a C-terminal region of MCM3.

Science.gov (United States)

Mimura, Satoru; Kubota, Yumiko; Takisawa, Haruhiko

2018-01-01

The minichromosome maintenance (MCM) complex, consisting of six subunits, Mcm2-7, is loaded onto replication origins through loading factors (origin recognition complex [ORC], Cdc6, and Cdt1) and forms an MCM double hexamer that licenses the initiation of DNA replication. Previous studies with Xenopus egg extracts showed that loading factors, especially Cdc6, dissociate from chromatin on MCM loading, but the molecular mechanism and physiological significance remain largely unknown. Using a cell-free system for MCM loading onto plasmid DNA in Xenopus egg extracts, we found that MCM loaded onto DNA prevents DNA binding of the loading factors ORC, Cdc6, and Cdt1. We further report that a peptide of the C-terminal region of MCM3 (MCM3-C), previously implicated in the initial association with ORC/Cdc6 in budding yeast, prevents ORC/Cdc6/Cdt1 binding to DNA in the absence of MCM loading. ATP-γ-S suppresses inhibitory activities of both the MCM loaded onto DNA and the MCM3-C peptide. Other soluble factors in the extract, but neither MCM nor Cdt1, are required for the activity. Conservation of the amino acid sequences of MCM3-C and its activity in vertebrates implies a novel negative autoregulatory mechanism that interferes with MCM loading in the vicinity of licensed origins to ensure proper origin licensing.

Shikonin regulates C-MYC and GLUT1 expression through the MST1-YAP1-TEAD1 axis

Energy Technology Data Exchange (ETDEWEB)

Vališ, Karel, E-mail: karel.valis@biomed.cas.cz [Laboratory of Structural Biology and Cell Signaling, Institute of Microbiology, v.v.i., The Czech Academy of Sciences, Prague (Czech Republic); Faculty of Science, Charles University, Prague (Czech Republic); Talacko, Pavel; Grobárová, Valéria [Laboratory of Structural Biology and Cell Signaling, Institute of Microbiology, v.v.i., The Czech Academy of Sciences, Prague (Czech Republic); Faculty of Science, Charles University, Prague (Czech Republic); Černý, Jan [Faculty of Science, Charles University, Prague (Czech Republic); Novák, Petr, E-mail: pnovak@biomed.cas.cz [Laboratory of Structural Biology and Cell Signaling, Institute of Microbiology, v.v.i., The Czech Academy of Sciences, Prague (Czech Republic); Faculty of Science, Charles University, Prague (Czech Republic)

2016-12-10

The general mechanism underlying the tumor suppressor activity of the Hippo signaling pathway remains unclear. In this study, we explore the molecular mechanisms connecting the Hippo signaling pathway with glucose metabolism. We have found that two key regulators of glycolysis, C-MYC and GLUT1, are targets of the Hippo signaling pathway in human leukemia cells. Our results revealed that activation of MST1 by the natural compound shikonin inhibited the expression of GLUT1 and C-MYC. Furthermore, RNAi experiments confirmed the regulation of GLUT1 and C-MYC expression via the MST1-YAP1-TEAD1 axis. Surprisingly, YAP1 was found to positively regulate C-MYC mRNA levels in complex with TEAD1, while it negatively regulates C-MYC levels in cooperation with MST1. Hence, YAP1 serves as a rheostat for C-MYC, which is regulated by MST1. In addition, depletion of MST1 stimulates lactate production, whereas the specific depletion of TEAD1 has an opposite effect. The inhibition of lactate production and cellular proliferation induced by shikonin also depends on the Hippo pathway activity. Finally, a bioinformatic analysis revealed conserved TEAD-binding motifs in the C-MYC and GLUT1 promoters providing another molecular data supporting our observations. In summary, regulation of glucose metabolism could serve as a new tumor suppressor mechanism orchestrated by the Hippo signaling pathway. - Highlights: • Shikonin inhibits C-MYC and GLUT1 expression in MST1 and YAP1 dependent manner. • YAP1-TEAD1 interaction activates C-MYC and GLUT1 expression. • MST1 in cooperation with YAP1 inhibits C-MYC and GLUT1 expression. • MST1-YAP1-TEAD1 axis regulates lactate production by leukemic cells. • MST1 and YAP1 proteins block proliferation of leukemic cells.

Networks, complexity and internet regulation scale-free law

OpenAIRE

Guadamuz, Andres

2013-01-01

This book, then, starts with a general statement: that regulators should try, wherever possible, to use the physical methodological tools presently available in order to draft better legislation. While such an assertion may be applied to the law in general, this work will concentrate on the much narrower area of Internet regulation and the science of complex networks The Internet is the subject of this book not only because it is my main area of research, but also because –without...

The secret life of tethers: the role of tethering factors in SNARE complex regulation

Directory of Open Access Journals (Sweden)

Michelle L Dubuke

2016-05-01

Full Text Available Trafficking in eukaryotic cells is a tightly regulated process to ensure correct cargo delivery to the proper destination organelle or plasma membrane. In this review, we focus on how the vesicle fusion machinery, the SNARE complex, is regulated by the interplay of the multisubunit tethering complexes (MTC with the SNAREs and Sec1/Munc18 (SM proteins. Although these factors are used in different stages of membrane trafficking, e.g. Golgi to plasma membrane transport vs vacuolar fusion, and in a variety of diverse eukaryotic cell types, many commonalities between their functions are being revealed. We explore the various protein-protein interactions and findings from functional reconstitution studies in order to highlight both their common features and the differences in their modes of regulation. These studies serve as a starting point for mechanistic explorations in other systems.

Methylation-regulated decommissioning of multimeric PP2A complexes

Energy Technology Data Exchange (ETDEWEB)

Wu, Cheng-Guo; Zheng, Aiping; Jiang, Li; Rowse, Michael; Stanevich, Vitali; Chen, Hui; Li, Yitong; Satyshur, Kenneth A.; Johnson, Benjamin; Gu, Ting-Jia; Liu, Zuojia; Xing, Yongna

2017-12-01

Dynamic assembly/disassembly of signaling complexes are crucial for cellular functions. Specialized latency and activation chaperones control the biogenesis of protein phosphatase 2A (PP2A) holoenzymes that contain a common scaffold and catalytic subunits and a variable regulatory subunit. Here we show that the butterfly-shaped TIPRL (TOR signaling pathway regulator) makes highly integrative multibranching contacts with the PP2A catalytic subunit, selective for the unmethylated tail and perturbing/inactivating the phosphatase active site. TIPRL also makes unusual wobble contacts with the scaffold subunit, allowing TIPRL, but not the overlapping regulatory subunits, to tolerate disease-associated PP2A mutations, resulting in reduced holoenzyme assembly and enhanced inactivation of mutant PP2A. Strikingly, TIPRL and the latency chaperone, α4, coordinate to disassemble active holoenzymes into latent PP2A, strictly controlled by methylation. Our study reveals a mechanism for methylation-responsive inactivation and holoenzyme disassembly, illustrating the complexity of regulation/signaling, dynamic complex disassembly, and disease mutations in cancer and intellectual disability.

NF-κB regulates DNA double-strand break repair in conjunction with BRCA1-CtIP complexes.

Science.gov (United States)

Volcic, Meta; Karl, Sabine; Baumann, Bernd; Salles, Daniela; Daniel, Peter; Fulda, Simone; Wiesmüller, Lisa

2012-01-01

NF-κB is involved in immune responses, inflammation, oncogenesis, cell proliferation and apoptosis. Even though NF-κB can be activated by DNA damage via Ataxia telangiectasia-mutated (ATM) signalling, little was known about an involvement in DNA repair. In this work, we dissected distinct DNA double-strand break (DSB) repair mechanisms revealing a stimulatory role of NF-κB in homologous recombination (HR). This effect was independent of chromatin context, cell cycle distribution or cross-talk with p53. It was not mediated by the transcriptional NF-κB targets Bcl2, BAX or Ku70, known for their dual roles in apoptosis and DSB repair. A contribution by Bcl-xL was abrogated when caspases were inhibited. Notably, HR induction by NF-κB required the targets ATM and BRCA2. Additionally, we provide evidence that NF-κB interacts with CtIP-BRCA1 complexes and promotes BRCA1 stabilization, and thereby contributes to HR induction. Immunofluorescence analysis revealed accelerated formation of replication protein A (RPA) and Rad51 foci upon NF-κB activation indicating HR stimulation through DSB resection by the interacting CtIP-BRCA1 complex and Rad51 filament formation. Taken together, these results define multiple NF-κB-dependent mechanisms regulating HR induction, and thereby providing a novel intriguing explanation for both NF-κB-mediated resistance to chemo- and radiotherapies as well as for the sensitization by pharmaceutical intervention of NF-κB activation.

The cardiac copper chaperone proteins Sco1 and CCS are up-regulated, but Cox 1 and Cox4 are down-regulated, by copper deficiency.

Science.gov (United States)

Getz, Jean; Lin, Dingbo; Medeiros, Denis M

2011-10-01

Copper is ferried in a cell complexed to chaperone proteins, and in the heart much copper is required for cytochrome c oxidase (Cox). It is not completely understood how copper status affects the levels of these proteins. Here we determined if dietary copper deficiency could up- or down-regulate select copper chaperone proteins and Cox subunits 1 and 4 in cardiac tissue of rats. Sixteen weanling male Long-Evans rats were randomized into treatment groups, one group receiving a copper-deficient diet (CCS, Sco1, Ctr1, Cox17, Cox1, and Cox4 by SDS-PAGE and Western blotting. No changes were observed in the concentrations of CTR1 and Cox17 between copper-adequate and copper-deficient rats. CCS and Sco1 were up-regulated and Cox1 and Cox4 were both down-regulated as a result of copper deficiency. These data suggest that select chaperone proteins and may be up-regulated, and Cox1 and 4 down-regulated, by a dietary copper deficiency, whereas others appear not to be affected by copper status.

Rac1 Regulates the Activity of mTORC1 and mTORC2 and Controls Cellular Size

Science.gov (United States)

Saci, Abdelhafid; Cantley, Lewis C.; Carpenter, Christopher L.

2013-01-01

SUMMARY Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that exists in two separate complexes, mTORC1 and mTORC2, that function to control cell size and growth in response to growth factors, nutrients, and cellular energy levels. Low molecular weight GTP-binding proteins of the Rheb and Rag families are key regulators of the mTORC1 complex, but regulation of mTORC2 is poorly understood. Here, we report that Rac1, a member of the Rho family of GTPases, is a critical regulator of both mTORC1 and mTORC2 in response to growth-factor stimulation. Deletion of Rac1 in primary cells using an inducible-Cre/Lox approach inhibits basal and growth-factor activation of both mTORC1 and mTORC2. Rac1 appears to bind directly to mTOR and to mediate mTORC1 and mTORC2 localization at specific membranes. Binding of Rac1 to mTOR does not depend on the GTP-bound state of Rac1, but on the integrity of its C-terminal domain. This function of Rac1 provides a means to regulate mTORC1 and mTORC2 simultaneously. PMID:21474067

GATOR1 regulates nitrogenic cataplerotic reactions of the mitochondrial TCA cycle.

Science.gov (United States)

Chen, Jun; Sutter, Benjamin M; Shi, Lei; Tu, Benjamin P

2017-11-01

The GATOR1 (SEACIT) complex consisting of Iml1-Npr2-Npr3 inhibits target of rapamycin complex 1 (TORC1) in response to amino acid insufficiency. In glucose medium, Saccharomyces cerevisiae mutants lacking the function of this complex grow poorly in the absence of amino acid supplementation, despite showing hallmarks of increased TORC1 signaling. Such mutants sense that they are amino acid replete and thus repress metabolic activities that are important for achieving this state. We found that npr2Δ mutants have defective mitochondrial tricarboxylic acid (TCA)-cycle activity and retrograde response. Supplementation with glutamine, and especially aspartate, which are nitrogen-containing forms of TCA-cycle intermediates, rescues growth of npr2Δ mutants. These amino acids are then consumed in biosynthetic pathways that require nitrogen to support proliferative metabolism. Our findings revealed that negative regulators of TORC1, such as GATOR1 (SEACIT), regulate the cataplerotic synthesis of these amino acids from the TCA cycle, in tune with the amino acid and nitrogen status of cells.

Interaction of hyperalgesia and sensory loss in complex regional pain syndrome type I (CRPS I.

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Volker Huge

Full Text Available BACKGROUND: Sensory abnormalities are a key feature of Complex Regional Pain Syndrome (CRPS. In order to characterise these changes in patients suffering from acute or chronic CRPS I, we used Quantitative Sensory Testing (QST in comparison to an age and gender matched control group. METHODS: 61 patients presenting with CRPS I of the upper extremity and 56 healthy subjects were prospectively assessed using QST. The patients' warm and cold detection thresholds (WDT; CDT, the heat and cold pain thresholds (HPT; CPT and the occurrence of paradoxical heat sensation (PHS were observed. RESULTS: In acute CRPS I, patients showed warm and cold hyperalgesia, indicated by significant changes in HPT and CPT. WDT and CDT were significantly increased as well, indicating warm and cold hypoaesthesia. In chronic CRPS, thermal hyperalgesia declined, but CDT as well as WDT further deteriorated. Solely patients with acute CRPS displayed PHS. To a minor degree, all QST changes were also present on the contralateral limb. CONCLUSIONS: We propose three pathomechanisms of CRPS I, which follow a distinct time course: Thermal hyperalgesia, observed in acute CRPS, indicates an ongoing aseptic peripheral inflammation. Thermal hypoaesthesia, as detected in acute and chronic CRPS, signals a degeneration of A-delta and C-fibres, which further deteriorates in chronic CRPS. PHS in acute CRPS I indicates that both inflammation and degeneration are present, whilst in chronic CRPS I, the pathomechanism of degeneration dominates, signalled by the absence of PHS. The contralateral changes observed strongly suggest the involvement of the central nervous system.

Structural analysis of inter-genus complexes of V-antigen and its regulator and their stabilization by divalent metal ions.

Science.gov (United States)

Basu, Abhishek; Das, Atanu; Mondal, Abhisek; Datta, Saumen

2016-03-01

Gram-negative bacteria like Yersinia, Pseudomonas, and Aeromonas need type III secretion system (T3SS) for their pathogenicity. V-antigen and its regulator are essential for functioning of T3SS. There is significant functional conservation amongst V-antigen and its regulator belonging to the Ysc family. In this study, we have structurally characterized the inter-genus complexes of V-antigen and its regulator. ConSurf analysis demonstrates that V-antigens belonging to the Ysc family show high structural identity predominantly confined to the two long helical regions. The regulator of V-antigen shows high conservation in its first intramolecular coiled-coil domain, responsible for interaction with V-antigen. ∆LcrG(1-70) localizes within the groove formed by long helices of LcrV, as observed in PcrV-∆PcrG(13-72) interaction. Inter-genus complexes of LcrV-PcrG and PcrV-LcrG exhibited elongated conformation and 1:1 heterodimeric state like the native complex of PcrV-PcrG and LcrV-LcrG. Both native and inter-genus complexes showed rigid tertiary structure, solvent-exposed hydrophobic patches, and cooperative melting behavior with high melting temperature. LcrV-PcrG and PcrV-LcrG showed nanomolar affinity of interaction, identical to PcrV-PcrG interaction, but stronger than LcrV-LcrG interaction. Calcium (a secretion blocker of T3SS) propels all the complexes towards a highly monodisperse form. Calcium and magnesium increase the helicity of the native and inter-genus complexes, and causes helix-helix stabilization. Stabilization of helices leads to a slight increase in the melting temperature by 1.5-2.0 °C. However, calcium does not alter the affinity of interaction of V-antigen and its regulator, emphasizing the effect of divalent of cations at the structural level without any regulatory implications. Therefore, the structural conservation of these inter-genus complexes could be the basis for their functional complementation.

The Monopolin Complex Crosslinks Kinetochore Components to Regulate Chromosome-Microtubule Attachments

Energy Technology Data Exchange (ETDEWEB)

Corbett, Kevin D.; Yip, Calvin K.; Ee, Ly-Sha; Walz, Thomas; Amon, Angelika; Harrison, Stephen C. (Harvard-Med); (MIT)

2010-09-27

The monopolin complex regulates different types of kinetochore-microtubule attachments in fungi, ensuring sister chromatid co-orientation in Saccharomyces cerevisiae meiosis I and inhibiting merotelic attachment in Schizosaccharomyces pombe mitosis. In addition, the monopolin complex maintains the integrity and silencing of ribosomal DNA (rDNA) repeats in the nucleolus. We show here that the S. cerevisiae Csm1/Lrs4 monopolin subcomplex has a distinctive V-shaped structure, with two pairs of protein-protein interaction domains positioned {approx}10 nm apart. Csm1 presents a conserved hydrophobic surface patch that binds two kinetochore proteins: Dsn1, a subunit of the outer-kinetochore MIND/Mis12 complex, and Mif2/CENP-C. Csm1 point-mutations that disrupt kinetochore-subunit binding also disrupt sister chromatid co-orientation in S. cerevisiae meiosis I. We further show that the same Csm1 point-mutations affect rDNA silencing, probably by disrupting binding to the rDNA-associated protein Tof2. We propose that Csm1/Lrs4 functions as a molecular clamp, crosslinking kinetochore components to enforce sister chromatid co-orientation in S. cerevisiae meiosis I and to suppress merotelic attachment in S. pombe mitosis, and crosslinking rDNA repeats to aid rDNA silencing.

Lactate up-regulates the expression of lactate oxidation complex-related genes in left ventricular cardiac tissue of rats.

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Daniele Gabriel-Costa

Full Text Available Besides its role as a fuel source in intermediary metabolism, lactate has been considered a signaling molecule modulating lactate-sensitive genes involved in the regulation of skeletal muscle metabolism. Even though the flux of lactate is significantly high in the heart, its role on regulation of cardiac genes regulating lactate oxidation has not been clarified yet. We tested the hypothesis that lactate would increase cardiac levels of reactive oxygen species and up-regulate the expression of genes related to lactate oxidation complex.Isolated hearts from male adult Wistar rats were perfused with control, lactate or acetate (20mM added Krebs-Henseleit solution during 120 min in modified Langendorff apparatus. Reactive oxygen species (O2●-/H2O2 levels, and NADH and NADPH oxidase activities (in enriched microsomal or plasmatic membranes, respectively were evaluated by fluorimetry while SOD and catalase activities were evaluated by spectrophotometry. mRNA levels of lactate oxidation complex and energetic enzymes MCT1, MCT4, HK, LDH, PDH, CS, PGC1α and COXIV were quantified by real time RT-PCR. Mitochondrial DNA levels were also evaluated. Hemodynamic parameters were acquired during the experiment. The key findings of this work were that lactate elevated cardiac NADH oxidase activity but not NADPH activity. This response was associated with increased cardiac O2●-/H2O2 levels and up-regulation of MCT1, MCT4, LDH and PGC1α with no changes in HK, PDH, CS, COXIV mRNA levels and mitochondrial DNA levels. Lactate increased NRF-2 nuclear expression and SOD activity probably as counter-regulatory responses to increased O2●-/H2O2.Our results provide evidence for lactate-induced up-regulation of lactate oxidation complex associated with increased NADH oxidase activity and cardiac O2●-/H2O2 driving to an anti-oxidant response. These results unveil lactate as an important signaling molecule regulating components of the lactate oxidation complex in

Saccharomyces cerevisiae GTPase complex: Gtr1p-Gtr2p regulates cell-proliferation through Saccharomyces cerevisiae Ran-binding protein, Yrb2p

International Nuclear Information System (INIS)

Wang Yonggang; Nakashima, Nobutaka; Sekiguchi, Takeshi; Nishimoto, Takeharu

2005-01-01

A Gtr1p GTPase, the GDP mutant of which suppresses both temperature-sensitive mutants of Saccharomyces cerevisiae RanGEF/Prp20p and RanGAP/Rna1p, was presently found to interact with Yrb2p, the S. cerevisiae homologue of mammalian Ran-binding protein 3. Gtr1p bound the Ran-binding domain of Yrb2p. In contrast, Gtr2p, a partner of Gtr1p, did not bind Yrb2p, although it bound Gtr1p. A triple mutant: yrb2Δ gtr1Δ gtr2Δ was lethal, while a double mutant: gtr1Δ gtr2Δ survived well, indicating that Yrb2p protected cells from the killing effect of gtr1Δ gtr2Δ. Recombinant Gtr1p and Gtr2p were purified as a complex from Escherichia coli. The resulting Gtr1p-Gtr2p complex was comprised of an equal amount of Gtr1p and Gtr2p, which inhibited the Rna1p/Yrb2 dependent RanGAP activity. Thus, the Gtr1p-Gtr2p cycle was suggested to regulate the Ran cycle through Yrb2p

Phosphatase-regulated recruitment of the spindle- and kinetochore-associated (Ska complex to kinetochores

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Sushama Sivakumar

2017-11-01

Full Text Available Kinetochores move chromosomes on dynamic spindle microtubules and regulate signaling of the spindle checkpoint. The spindle- and kinetochore-associated (Ska complex, a hexamer composed of two copies of Ska1, Ska2 and Ska3, has been implicated in both roles. Phosphorylation of kinetochore components by the well-studied mitotic kinases Cdk1, Aurora B, Plk1, Mps1, and Bub1 regulate chromosome movement and checkpoint signaling. Roles for the opposing phosphatases are more poorly defined. Recently, we showed that the C terminus of Ska1 recruits protein phosphatase 1 (PP1 to kinetochores. Here we show that PP1 and protein phosphatase 2A (PP2A both promote accumulation of Ska at kinetochores. Depletion of PP1 or PP2A by siRNA reduces Ska binding at kinetochores, impairs alignment of chromosomes to the spindle midplane, and causes metaphase delay or arrest, phenotypes that are also seen after depletion of Ska. Artificial tethering of PP1 to the outer kinetochore protein Nuf2 promotes Ska recruitment to kinetochores, and it reduces but does not fully rescue chromosome alignment and metaphase arrest defects seen after Ska depletion. We propose that Ska has multiple functions in promoting mitotic progression and that kinetochore-associated phosphatases function in a positive feedback cycle to reinforce Ska complex accumulation at kinetochores.

The Biology of the Cytolethal Distending Toxins

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Teresa Frisan

2011-03-01

Full Text Available The cytolethal distending toxins (CDTs, produced by a variety of Gram-negative pathogenic bacteria, are the first bacterial genotoxins described, since they cause DNA damage in the target cells. CDT is an A-B2 toxin, where the CdtA and CdtC subunits are required to mediate the binding on the surface of the target cells, allowing internalization of the active CdtB subunit, which is functionally homologous to the mammalian deoxyribonuclease I. The nature of the surface receptor is still poorly characterized, however binding of CDT requires intact lipid rafts, and its internalization occurs via dynamin-dependent endocytosis. The toxin is retrograde transported through the Golgi complex and the endoplasmic reticulum, and subsequently translocated into the nuclear compartment, where it exerts the toxic activity. Cellular intoxication induces DNA damage and activation of the DNA damage responses, which results in arrest of the target cells in the G1 and/or G2 phases of the cell cycle and activation of DNA repair mechanisms. Cells that fail to repair the damage will senesce or undergo apoptosis. This review will focus on the well-characterized aspects of the CDT biology and discuss the questions that still remain unanswered.

GEFs: Dual regulation of Rac1 signaling.

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Marei, Hadir; Malliri, Angeliki

2017-04-03

GEFs play a critical role in regulating Rac1 signaling. They serve as signaling nodes converting upstream signals into downstream Rac1-driven cellular responses. Through associating with membrane-bound Rac1, GEFs facilitate the exchange of GDP for GTP, thereby activating Rac1. As a result, Rac1 undergoes conformational changes that mediate its interaction with downstream effectors, linking Rac1 to a multitude of physiological and pathological processes. Interestingly, there are at least 20 GEFs involved in Rac1 activation, suggesting a more complex role of GEFs in regulating Rac1 signaling apart from promoting the exchange of GDP for GTP. Indeed, accumulating evidence implicates GEFs in directing the specificity of Rac1-driven signaling cascades, although the underlying mechanisms were poorly defined. Recently, through conducting a comparative study, we highlighted the role of 2 Rac-specific GEFs, Tiam1 and P-Rex1, in dictating the biological outcome downstream of Rac1. Importantly, further proteomic analysis uncovered a GEF activity-independent function for both GEFs in modulating the Rac1 interactome, which results in the stimulation of GEF-specific signaling cascades. Here, we provide an overview of our recent findings and discuss the role of GEFs as master regulators of Rac1 signaling with a particular focus on GEF-mediated modulation of cell migration following Rac1 activation.

Antidiabetic phospholipid-nuclear receptor complex reveals the mechanism for phospholipid-driven gene regulation

Energy Technology Data Exchange (ETDEWEB)

Musille, Paul M; Pathak, Manish C; Lauer, Janelle L; Hudson, William H; Griffin, Patrick R; Ortlund, Eric A [Emory-MED; (Scripps)

2013-01-31

The human nuclear receptor liver receptor homolog-1 (LRH-1) has an important role in controlling lipid and cholesterol homeostasis and is a potential target for the treatment of diabetes and hepatic diseases. LRH-1 is known to bind phospholipids, but the role of phospholipids in controlling LRH-1 activation remains highly debated. Here we describe the structure of both apo LRH-1 and LRH-1 in complex with the antidiabetic phospholipid dilauroylphosphatidylcholine (DLPC). Together with hydrogen-deuterium exchange MS and functional data, our studies show that DLPC binding is a dynamic process that alters co-regulator selectivity. We show that the lipid-free receptor undergoes previously unrecognized structural fluctuations, allowing it to interact with widely expressed co-repressors. These observations enhance our understanding of LRH-1 regulation and highlight its importance as a new therapeutic target for controlling diabetes.

Parkin Regulates Mitosis and Genomic Stability through Cdc20/Cdh1.

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Lee, Seung Baek; Kim, Jung Jin; Nam, Hyun-Ja; Gao, Bowen; Yin, Ping; Qin, Bo; Yi, Sang-Yeop; Ham, Hyoungjun; Evans, Debra; Kim, Sun-Hyun; Zhang, Jun; Deng, Min; Liu, Tongzheng; Zhang, Haoxing; Billadeau, Daniel D; Wang, Liewei; Giaime, Emilie; Shen, Jie; Pang, Yuan-Ping; Jen, Jin; van Deursen, Jan M; Lou, Zhenkun

2015-10-01

Mutations in the E3 ubiquitin ligase Parkin have been linked to familial Parkinson's disease. Parkin has also been implicated in mitosis through mechanisms that are unclear. Here we show that Parkin interacts with anaphase promoting complex/cyclosome (APC/C) coactivators Cdc20 and Cdh1 to mediate the degradation of several key mitotic regulators independent of APC/C. We demonstrate that ordered progression through mitosis is orchestrated by two distinct E3 ligases through the shared use of Cdc20 and Cdh1. Furthermore, Parkin is phosphorylated and activated by polo-like kinase 1 (Plk1) during mitosis. Parkin deficiency results in overexpression of its substrates, mitotic defects, genomic instability, and tumorigenesis. These results suggest that the Parkin-Cdc20/Cdh1 complex is an important regulator of mitosis. Copyright © 2015 Elsevier Inc. All rights reserved.

Meier-Gorlin syndrome: Growth and secondary sexual development of a microcephalic primordial dwarfism disorder

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de Munnik, Sonja A.; Otten, Barto J.; Schoots, Jeroen; Bicknell, Louise S.; Aftimos, Salim; Al-Aama, Jumana Y.; van Bever, Yolande; Bober, Michael B.; Borm, George F.; Clayton-Smith, Jill; Deal, Cheri L.; Edrees, Alaa Y.; Feingold, Murray; Fryer, Alan; van Hagen, Johanna M.; Hennekam, Raoul C.; Jansweijer, Maaike C. E.; Johnson, Diana; Kant, Sarina G.; Opitz, John M.; Ramadevi, A. Radha; Reardon, Willie; Ross, Alison; Sarda, Pierre; Schrander-Stumpel, Constance T. R. M.; Sluiter, A. Erik; Temple, I. Karen; Terhal, Paulien A.; Toutain, Annick; Wise, Carol A.; Wright, Michael; Skidmore, David L.; Samuels, Mark E.; Hoefsloot, Lies H.; Knoers, Nine V. A. M.; Brunner, Han G.; Jackson, Andrew P.; Bongers, Ernie M. H. F.

2012-01-01

MeierGorlin syndrome (MGS) is a rare autosomal recessive disorder characterized by primordial dwarfism, microtia, and patellar aplasia/hypoplasia. Recently, mutations in the ORC1, ORC4, ORC6, CDT1, and CDC6 genes, encoding components of the pre-replication complex, have been identified. This complex

RNA-Mediated Regulation of HMGA1 Function

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Arndt G. Benecke

2015-05-01

Full Text Available The high mobility group protein A1 (HMGA1 is a master regulator of chromatin structure mediating its major gene regulatory activity by direct interactions with A/T-rich DNA sequences located in the promoter and enhancer regions of a large variety of genes. HMGA1 DNA-binding through three AT-hook motifs results in an open chromatin structure and subsequently leads to changes in gene expression. Apart from its significant expression during development, HMGA1 is over-expressed in virtually every cancer, where HMGA1 expression levels correlate with tumor malignancy. The exogenous overexpression of HMGA1 can lead to malignant cell transformation, assigning the protein a key role during cancerogenesis. Recent studies have unveiled highly specific competitive interactions of HMGA1 with cellular and viral RNAs also through an AT-hook domain of the protein, significantly impacting the HMGA1-dependent gene expression. In this review, we discuss the structure and function of HMGA1-RNA complexes during transcription and epigenomic regulation and their implications in HMGA1-related diseases.

JUNGBRUNNEN1, a Reactive Oxygen Species–Responsive NAC Transcription Factor, Regulates Longevity in Arabidopsis

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Wu, A.; Devi Allu, A.; Garapati, P.; Siddiqui, H.; Dortay, H.; Zanor, M.I.; Amparo Asensi-Fabado, M.; Munne´ -Bosch, S.; Antonio, C.; Tohge, T.; Fernie, A.R.; Kaufmann, K.; Xue, G.P.; Mueller-Roeber, B.; Balazadeh, S.

2012-01-01

The transition from juvenility through maturation to senescence is a complex process that involves the regulation of longevity. Here, we identify JUNGBRUNNEN1 (JUB1), a hydrogen peroxide (H2O2)-induced NAC transcription factor, as a central longevity regulator in Arabidopsis thaliana. JUB1

The fission yeast minichromosome maintenance (MCM)-binding protein (MCM-BP), Mcb1, regulates MCM function during prereplicative complex formation in DNA replication.

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Santosa, Venny; Martha, Sabrina; Hirose, Noriaki; Tanaka, Katsunori

2013-03-08

The minichromosome maintenance (MCM) complex is a replicative helicase, which is essential for chromosome DNA replication. In recent years, the identification of a novel MCM-binding protein (MCM-BP) in most eukaryotes has led to numerous studies investigating its function and its relationship to the MCM complex. However, the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood; in addition, the functional role of MCM-BP remains controversial and may vary between model organisms. The present study aims to elucidate the nature and biological function of the MCM-BP ortholog, Mcb1, in fission yeast. The Mcb1 protein continuously interacts with MCM proteins during the cell cycle in vivo and can interact with any individual MCM subunit in vitro. To understand the detailed characteristics of mcb1(+), two temperature-sensitive mcb1 gene mutants (mcb1(ts)) were isolated. Extensive genetic analysis showed that the mcb1(ts) mutants were suppressed by a mcm5(+) multicopy plasmid and displayed synthetic defects with many S-phase-related gene mutants. Moreover, cyclin-dependent kinase modulation by Cig2 repression or Rum1 overproduction suppressed the mcb1(ts) mutants, suggesting the involvement of Mcb1 in pre-RC formation during DNA replication. These data are consistent with the observation that Mcm7 loading onto replication origins is reduced and S-phase progression is delayed in mcb1(ts) mutants. Furthermore, the mcb1(ts) mutation led to the redistribution of MCM subunits to the cytoplasm, and this redistribution was dependent on an active nuclear export system. These results strongly suggest that Mcb1 promotes efficient pre-RC formation during DNA replication by regulating the MCM complex.

The Fission Yeast Minichromosome Maintenance (MCM)-binding Protein (MCM-BP), Mcb1, Regulates MCM Function during Prereplicative Complex Formation in DNA Replication*

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Santosa, Venny; Martha, Sabrina; Hirose, Noriaki; Tanaka, Katsunori

2013-01-01

The minichromosome maintenance (MCM) complex is a replicative helicase, which is essential for chromosome DNA replication. In recent years, the identification of a novel MCM-binding protein (MCM-BP) in most eukaryotes has led to numerous studies investigating its function and its relationship to the MCM complex. However, the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood; in addition, the functional role of MCM-BP remains controversial and may vary between model organisms. The present study aims to elucidate the nature and biological function of the MCM-BP ortholog, Mcb1, in fission yeast. The Mcb1 protein continuously interacts with MCM proteins during the cell cycle in vivo and can interact with any individual MCM subunit in vitro. To understand the detailed characteristics of mcb1+, two temperature-sensitive mcb1 gene mutants (mcb1ts) were isolated. Extensive genetic analysis showed that the mcb1ts mutants were suppressed by a mcm5+ multicopy plasmid and displayed synthetic defects with many S-phase-related gene mutants. Moreover, cyclin-dependent kinase modulation by Cig2 repression or Rum1 overproduction suppressed the mcb1ts mutants, suggesting the involvement of Mcb1 in pre-RC formation during DNA replication. These data are consistent with the observation that Mcm7 loading onto replication origins is reduced and S-phase progression is delayed in mcb1ts mutants. Furthermore, the mcb1ts mutation led to the redistribution of MCM subunits to the cytoplasm, and this redistribution was dependent on an active nuclear export system. These results strongly suggest that Mcb1 promotes efficient pre-RC formation during DNA replication by regulating the MCM complex. PMID:23322785

A bacterial cytotoxin identifies the RhoA exchange factor Net1 as a key effector in the response to DNA damage.

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Lina Guerra

Full Text Available BACKGROUND: Exposure of adherent cells to DNA damaging agents, such as the bacterial cytolethal distending toxin (CDT or ionizing radiations (IR, activates the small GTPase RhoA, which promotes the formation of actin stress fibers and delays cell death. The signalling intermediates that regulate RhoA activation and promote cell survival are unknown. PRINCIPAL FINDINGS: We demonstrate that the nuclear RhoA-specific Guanine nucleotide Exchange Factor (GEF Net1 becomes dephosphorylated at a critical inhibitory site in cells exposed to CDT or IR. Expression of a dominant negative Net1 or Net1 knock down by iRNA prevented RhoA activation, inhibited the formation of stress fibers, and enhanced cell death, indicating that Net1 activation is required for this RhoA-mediated responses to genotoxic stress. The Net1 and RhoA-dependent signals involved activation of the Mitogen-Activated Protein Kinase p38 and its downstream target MAPK-activated protein kinase 2. SIGNIFICANCE: Our data highlight the importance of Net1 in controlling RhoA and p38 MAPK mediated cell survival in cells exposed to DNA damaging agents and illustrate a molecular pathway whereby chronic exposure to a bacterial toxin may promote genomic instability.

GIT1/βPIX signaling proteins and PAK1 kinase regulate microtubule nucleation.

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Černohorská, Markéta; Sulimenko, Vadym; Hájková, Zuzana; Sulimenko, Tetyana; Sládková, Vladimíra; Vinopal, Stanislav; Dráberová, Eduarda; Dráber, Pavel

2016-06-01

Microtubule nucleation from γ-tubulin complexes, located at the centrosome, is an essential step in the formation of the microtubule cytoskeleton. However, the signaling mechanisms that regulate microtubule nucleation in interphase cells are largely unknown. In this study, we report that γ-tubulin is in complexes containing G protein-coupled receptor kinase-interacting protein 1 (GIT1), p21-activated kinase interacting exchange factor (βPIX), and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1) in various cell lines. Immunofluorescence microscopy revealed association of GIT1, βPIX and activated PAK1 with centrosomes. Microtubule regrowth experiments showed that depletion of βPIX stimulated microtubule nucleation, while depletion of GIT1 or PAK1 resulted in decreased nucleation in the interphase cells. These data were confirmed for GIT1 and βPIX by phenotypic rescue experiments, and counting of new microtubules emanating from centrosomes during the microtubule regrowth. The importance of PAK1 for microtubule nucleation was corroborated by the inhibition of its kinase activity with IPA-3 inhibitor. GIT1 with PAK1 thus represent positive regulators, and βPIX is a negative regulator of microtubule nucleation from the interphase centrosomes. The regulatory roles of GIT1, βPIX and PAK1 in microtubule nucleation correlated with recruitment of γ-tubulin to the centrosome. Furthermore, in vitro kinase assays showed that GIT1 and βPIX, but not γ-tubulin, serve as substrates for PAK1. Finally, direct interaction of γ-tubulin with the C-terminal domain of βPIX and the N-terminal domain of GIT1, which targets this protein to the centrosome, was determined by pull-down experiments. We propose that GIT1/βPIX signaling proteins with PAK1 kinase represent a novel regulatory mechanism of microtubule nucleation in interphase cells. Copyright © 2016 Elsevier B.V. All rights reserved.

A new functional role for mechanistic/mammalian target of rapamycin complex 1 (mTORC1 in the circadian regulation of L-type voltage-gated calcium channels in avian cone photoreceptors.

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Cathy Chia-Yu Huang

Full Text Available In the retina, the L-type voltage-gated calcium channels (L-VGCCs are responsible for neurotransmitter release from photoreceptors and are under circadian regulation. Both the current densities and protein expression of L-VGCCs are significantly higher at night than during the day. However, the underlying mechanisms of circadian regulation of L-VGCCs in the retina are not completely understood. In this study, we demonstrated that the mechanistic/mammalian target of rapamycin complex (mTORC signaling pathway participated in the circadian phase-dependent modulation of L-VGCCs. The activities of the mTOR cascade, from mTORC1 to its downstream targets, displayed circadian oscillations throughout the course of a day. Disruption of mTORC1 signaling dampened the L-VGCC current densities, as well as the protein expression of L-VGCCs at night. The decrease of L-VGCCs at night by mTORC1 inhibition was in part due to a reduction of L-VGCCα1 subunit translocation from the cytosol to the plasma membrane. Finally, we showed that mTORC1 was downstream of the phosphatidylionositol 3 kinase-protein kinase B (PI3K-AKT signaling pathway. Taken together, mTORC1 signaling played a role in the circadian regulation of L-VGCCs, in part through regulation of ion channel trafficking and translocation, which brings to light a new functional role for mTORC1: the modulation of ion channel activities.

Mediator complex cooperatively regulates transcription of retinoic acid target genes with Polycomb Repressive Complex 2 during neuronal differentiation.

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Fukasawa, Rikiya; Iida, Satoshi; Tsutsui, Taiki; Hirose, Yutaka; Ohkuma, Yoshiaki

2015-11-01

The Mediator complex (Mediator) plays key roles in transcription and functions as the nexus for integration of various transcriptional signals. Previously, we screened for Mediator cyclin-dependent kinase (CDK)-interacting factors and identified three proteins related to chromatin regulation. One of them, SUZ12 is required for both stability and activity of Polycomb Repressive Complex 2 (PRC2). PRC2 primarily suppresses gene expression through histone H3 lysine 27 trimethylation, resulting in stem cell maintenance and differentiation; perturbation of this process leads to oncogenesis. Recent work showed that Mediator contributes to the embryonic stem cell state through DNA loop formation, which is strongly associated with chromatin architecture; however, it remains unclear how Mediator regulates gene expression in cooperation with chromatin regulators (i.e. writers, readers and remodelers). We found that Mediator CDKs interact directly with the PRC2 subunit EZH2, as well as SUZ12. Known PRC2 target genes were deregulated by Mediator CDK knockdown during neuronal differentiation, and both Mediator and PRC2 complexes co-occupied the promoters of developmental genes regulated by retinoic acid. Our results provide a mechanistic link between Mediator and PRC2 during neuronal differentiation. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Sbg1 Is a Novel Regulator for the Localization of the β-Glucan Synthase Bgs1 in Fission Yeast.

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Reshma Davidson

Full Text Available Glucan synthases synthesize glucans, complex polysaccharides that are the major components in fungal cell walls and division septa. Studying regulation of glucan synthases is important as they are essential for fungal cell survival and thus popular targets for anti-fungal drugs. Linear 1,3-β-glucan is the main component of primary septum and is synthesized by the conserved β-glucan synthase Bgs1 in fission yeast cytokinesis. It is known that Rho1 GTPase regulates Bgs1 catalytic activity and the F-BAR protein Cdc15 plays a role in Bgs1 delivery to the plasma membrane. Here we characterize a novel protein Sbg1 that is present in a complex with Bgs1 and regulates its protein levels and localization. Sbg1 is essential for contractile-ring constriction and septum formation during cytokinesis. Sbg1 and Bgs1 physically interact and are interdependent for localization to the plasma membrane. Bgs1 is less stable and/or mis-targeted to vacuoles in sbg1 mutants. Moreover, Sbg1 plays an earlier and more important role in Bgs1 trafficking and localization than Cdc15. Together, our data reveal a new mode of regulation for the essential β-glucan synthase Bgs1 by the novel protein Sbg1.

CRISPR/Cas9-based knockouts reveal that CpRLP1 is a negative regulator of the sex pheromone PR-IP in the Closterium peracerosum-strigosum-littorale complex.

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Kanda, Naho; Ichikawa, Machiko; Ono, Ayaka; Toyoda, Atsushi; Fujiyama, Asao; Abe, Jun; Tsuchikane, Yuki; Nishiyama, Tomoaki; Sekimoto, Hiroyuki

2017-12-19

Heterothallic strains of the Closterium peracerosum-strigosum-littorale (C. psl.) complex have two sexes, mating-type plus (mt + ) and mating-type minus (mt - ). Conjugation between these two sexes is regulated by two sex pheromones, protoplast-release-inducing protein (PR-IP) and PR-IP Inducer, which are produced by mt + and mt - cells, respectively. PR-IP mediates the release of protoplasts from mt - cells during mating. In this study, we examined the mechanism of action of CpRLP1 (receptor-like protein 1), which was previously identified in a cDNA microarray analysis as one of the PR-IP-inducible genes. Using CRISPR/Cas9 technology, we generated CpRLP1 knockout mutants in mt - cells of the C. psl. complex. When the knockout mt - cells were mixed with wild-type mt + cells, conjugation was severely reduced. Many cells released protoplasts without pairing, suggesting a loss of synchronization between the two mating partners. Furthermore, the knockout mutants were hypersensitive to PR-IP. We conclude that CpRLP1 is a negative regulator of PR-IP that regulates the timing of protoplast release in conjugating C. psl. cells. As the first report of successful gene knockout in the class Charophyceae, this study provides a basis for research aimed at understanding the ancestral roles of genes that are indispensable for the development of land plants.

Lysosomal regulation of cholesterol homeostasis in tuberous sclerosis complex is mediated via NPC1 and LDL-R.

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Filippakis, Harilaos; Alesi, Nicola; Ogorek, Barbara; Nijmeh, Julie; Khabibullin, Damir; Gutierrez, Catherine; Valvezan, Alexander J; Cunningham, James; Priolo, Carmen; Henske, Elizabeth P

2017-06-13

Tuberous sclerosis complex (TSC) is a multisystem disease associated with hyperactive mTORC1. The impact of TSC1/2 deficiency on lysosome-mediated processes is not fully understood. We report here that inhibition of lysosomal function using chloroquine (CQ) upregulates cholesterol homeostasis genes in TSC2-deficient cells. This TSC2-dependent transcriptional signature is associated with increased accumulation and intracellular levels of both total cholesterol and cholesterol esters. Unexpectedly, engaging this CQ-induced cholesterol uptake pathway together with inhibition of de novo cholesterol synthesis allows survival of TSC2-deficient, but not TSC2-expressing cells. The underlying mechanism of TSC2-deficient cell survival is dependent on exogenous cholesterol uptake via LDL-R, and endosomal trafficking mediated by Vps34. Simultaneous inhibition of lysosomal and endosomal trafficking inhibits uptake of esterified cholesterol and cell growth in TSC2-deficient, but not TSC2-expressing cells, highlighting the TSC-dependent lysosome-mediated regulation of cholesterol homeostasis and pointing toward the translational potential of these pathways for the therapy of TSC.

Involvement of F-Actin in Chaperonin-Containing t-Complex 1 Beta Regulating Mouse Mesangial Cell Functions in a Glucose-Induction Cell Model

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Jin-Shuen Chen

2011-01-01

Full Text Available The aim of this study is to investigate the role of chaperonin-containing t-complex polypeptide 1 beta (CCT2 in the regulation of mouse mesangial cell (mMC contraction, proliferation, and migration with filamentous/globular-(F/G- actin ratio under high glucose induction. A low CCT2 mMC model induced by treatment of small interference RNA was established. Groups with and without low CCT2 induction examined in normal and high (H glucose conditions revealed the following major results: (1 low CCT2 or H glucose showed the ability to attenuate F/G-actin ratio; (2 groups with low F/G-actin ratio all showed less cell contraction; (3 suppression of CCT2 may reduce the proliferation and migration which were originally induced by H glucose. In conclusion, CCT2 can be used as a specific regulator for mMC contraction, proliferation, and migration affected by glucose, which mechanism may involve the alteration of F-actin, particularly for cell contraction.

TAK1 regulates skeletal muscle mass and mitochondrial function

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Hindi, Sajedah M.; Sato, Shuichi; Xiong, Guangyan; Bohnert, Kyle R.; Gibb, Andrew A.; Gallot, Yann S.; McMillan, Joseph D.; Hill, Bradford G.

2018-01-01

Skeletal muscle mass is regulated by a complex array of signaling pathways. TGF-β–activated kinase 1 (TAK1) is an important signaling protein, which regulates context-dependent activation of multiple intracellular pathways. However, the role of TAK1 in the regulation of skeletal muscle mass remains unknown. Here, we report that inducible inactivation of TAK1 causes severe muscle wasting, leading to kyphosis, in both young and adult mice.. Inactivation of TAK1 inhibits protein synthesis and induces proteolysis, potentially through upregulating the activity of the ubiquitin-proteasome system and autophagy. Phosphorylation and enzymatic activity of AMPK are increased, whereas levels of phosphorylated mTOR and p38 MAPK are diminished upon inducible inactivation of TAK1 in skeletal muscle. In addition, targeted inactivation of TAK1 leads to the accumulation of dysfunctional mitochondria and oxidative stress in skeletal muscle of adult mice. Inhibition of TAK1 does not attenuate denervation-induced muscle wasting in adult mice. Finally, TAK1 activity is highly upregulated during overload-induced skeletal muscle growth, and inactivation of TAK1 prevents myofiber hypertrophy in response to functional overload. Overall, our study demonstrates that TAK1 is a key regulator of skeletal muscle mass and oxidative metabolism. PMID:29415881

Border control: selectivity of chloroplast protein import and regulation at the TOC-complex.

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Demarsy, Emilie; Lakshmanan, Ashok M; Kessler, Felix

2014-01-01

Plants have evolved complex and sophisticated molecular mechanisms to regulate their development and adapt to their surrounding environment. Particularly the development of their specific organelles, chloroplasts and other plastid-types, is finely tuned in accordance with the metabolic needs of the cell. The normal development and functioning of plastids require import of particular subsets of nuclear encoded proteins. Most preproteins contain a cleavable sequence at their N terminal (transit peptide) serving as a signal for targeting to the organelle and recognition by the translocation machinery TOC-TIC (translocon of outer membrane complex-translocon of inner membrane complex) spanning the dual membrane envelope. The plastid proteome needs constant remodeling in response to developmental and environmental factors. Therefore selective regulation of preprotein import plays a crucial role in plant development. In this review we describe the diversity of transit peptides and TOC receptor complexes, and summarize the current knowledge and potential directions for future research concerning regulation of the different Toc isoforms.

Physical and biological data collected with CDT, fluorometer, and SeaSoar aboard the ship WECOMA as part of Global Ocean Ecosystem Dynamics (GLOBEC) in the North Pacific Ocean from May 30 to June 16 2000 (NODC Accession 0000986)

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National Oceanic and Atmospheric Administration, Department of Commerce — Physical and biological data collected with CDT, fluorometer, and SeaSoar aboard the ship WECOMA in the North Pacific Ocean from May 30 to June 16 2000. These data...

DAG1, no gene for RNA regulation?

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Brancaccio, Andrea

2012-04-10

DAG1 encodes for a precursor protein that liberates the two subunits featured by the dystroglycan (DG) adhesion complex that are involved in an increasing number of cellular functions in a wide variety of cells and tissues. Aside from the proteolytic events producing the α and β subunits, especially the former undergoes extensive "post-production" modifications taking place within the ER/Golgi where its core protein is both N- and O-decorated with sugars. These post-translational events, that are mainly orchestrated by a plethora of certified, or putative, glycosyltransferases, prelude to the excocytosis-mediated trafficking and targeting of the DG complex to the plasma membrane. Extensive genetic and biochemical evidences have been accumulated so far on α-DG glycosylation, while little is know on possible regulatory events underlying the chromatine activation, transcription or post-transcription (splicing and escape from the nucleus) of DAG1 or of its mRNA. A scenario is envisaged in which cells would use a sort of preferential, and scarcely regulated, route for DAG1 activation, that would imply fast mRNA transcription, maturation and export to the cytosol, and would prelude to the multiple time-consuming enzymatic post-translational activities needed for its glycosylation. Such a provocative view might be helpful to trigger future work aiming at disclosing the complete molecular mechanisms underlying DAG1 activation and at improving our knowledge of any pre-translational step that is involved in dystroglycan regulation. Copyright © 2012 Elsevier B.V. All rights reserved.

MCUR1 Is a Scaffold Factor for the MCU Complex Function and Promotes Mitochondrial Bioenergetics

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Dhanendra Tomar

2016-05-01

Full Text Available Mitochondrial Ca2+ Uniporter (MCU-dependent mitochondrial Ca2+ uptake is the primary mechanism for increasing matrix Ca2+ in most cell types. However, a limited understanding of the MCU complex assembly impedes the comprehension of the precise mechanisms underlying MCU activity. Here, we report that mouse cardiomyocytes and endothelial cells lacking MCU regulator 1 (MCUR1 have severely impaired [Ca2+]m uptake and IMCU current. MCUR1 binds to MCU and EMRE and function as a scaffold factor. Our protein binding analyses identified the minimal, highly conserved regions of coiled-coil domain of both MCU and MCUR1 that are necessary for heterooligomeric complex formation. Loss of MCUR1 perturbed MCU heterooligomeric complex and functions as a scaffold factor for the assembly of MCU complex. Vascular endothelial deletion of MCU and MCUR1 impaired mitochondrial bioenergetics, cell proliferation, and migration but elicited autophagy. These studies establish the existence of a MCU complex that assembles at the mitochondrial integral membrane and regulates Ca2+-dependent mitochondrial metabolism.

RIT1 controls actin dynamics via complex formation with RAC1/CDC42 and PAK1.

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Uta Meyer Zum Büschenfelde

2018-05-01

Full Text Available RIT1 belongs to the RAS family of small GTPases. Germline and somatic RIT1 mutations have been identified in Noonan syndrome (NS and cancer, respectively. By using heterologous expression systems and purified recombinant proteins, we identified the p21-activated kinase 1 (PAK1 as novel direct effector of RIT1. We found RIT1 also to directly interact with the RHO GTPases CDC42 and RAC1, both of which are crucial regulators of actin dynamics upstream of PAK1. These interactions are independent of the guanine nucleotide bound to RIT1. Disease-causing RIT1 mutations enhance protein-protein interaction between RIT1 and PAK1, CDC42 or RAC1 and uncouple complex formation from serum and growth factors. We show that the RIT1-PAK1 complex regulates cytoskeletal rearrangements as expression of wild-type RIT1 and its mutant forms resulted in dissolution of stress fibers and reduction of mature paxillin-containing focal adhesions in COS7 cells. This effect was prevented by co-expression of RIT1 with dominant-negative CDC42 or RAC1 and kinase-dead PAK1. By using a transwell migration assay, we show that RIT1 wildtype and the disease-associated variants enhance cell motility. Our work demonstrates a new function for RIT1 in controlling actin dynamics via acting in a signaling module containing PAK1 and RAC1/CDC42, and highlights defects in cell adhesion and migration as possible disease mechanism underlying NS.

RIT1 controls actin dynamics via complex formation with RAC1/CDC42 and PAK1.

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Meyer Zum Büschenfelde, Uta; Brandenstein, Laura Isabel; von Elsner, Leonie; Flato, Kristina; Holling, Tess; Zenker, Martin; Rosenberger, Georg; Kutsche, Kerstin

2018-05-01

RIT1 belongs to the RAS family of small GTPases. Germline and somatic RIT1 mutations have been identified in Noonan syndrome (NS) and cancer, respectively. By using heterologous expression systems and purified recombinant proteins, we identified the p21-activated kinase 1 (PAK1) as novel direct effector of RIT1. We found RIT1 also to directly interact with the RHO GTPases CDC42 and RAC1, both of which are crucial regulators of actin dynamics upstream of PAK1. These interactions are independent of the guanine nucleotide bound to RIT1. Disease-causing RIT1 mutations enhance protein-protein interaction between RIT1 and PAK1, CDC42 or RAC1 and uncouple complex formation from serum and growth factors. We show that the RIT1-PAK1 complex regulates cytoskeletal rearrangements as expression of wild-type RIT1 and its mutant forms resulted in dissolution of stress fibers and reduction of mature paxillin-containing focal adhesions in COS7 cells. This effect was prevented by co-expression of RIT1 with dominant-negative CDC42 or RAC1 and kinase-dead PAK1. By using a transwell migration assay, we show that RIT1 wildtype and the disease-associated variants enhance cell motility. Our work demonstrates a new function for RIT1 in controlling actin dynamics via acting in a signaling module containing PAK1 and RAC1/CDC42, and highlights defects in cell adhesion and migration as possible disease mechanism underlying NS.

JMJD1C demethylates MDC1 to regulate the RNF8 and BRCA1-mediated chromatin response to DNA breaks

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Watanabe, Sugiko; Watanabe, Kenji; Akimov, Vyacheslav

2013-01-01

Chromatin ubiquitylation flanking DNA double-strand breaks (DSBs), mediated by RNF8 and RNF168 ubiquitin ligases, orchestrates a two-branch pathway, recruiting repair factors 53BP1 or the RAP80-BRCA1 complex. We report that human demethylase JMJD1C regulates the RAP80-BRCA1 branch of this DNA...

Functional analysis of the cell cycle regulator Rca1 in Drosophila melanogaster

OpenAIRE

Zielke, Norman

2007-01-01

Tight regulation of APC/C activity is essential for cell cycle progression. An important class of negative APC/C regulators are the Rca1/Emi1 family proteins. All members of the Rca1/Emi1 family share a conserved zinc binding region (ZBR) which is essential for their inhibitory activity. The Rca1/Emi1 proteins belong to the class of F-box proteins that are known to act as substrate recognition subunits in SCF-E3-ligase complexes. Emi1 and Rca1 interact in vitro with members of the Skp family ...

A complex interplay between PGC-1 co-activators and mTORC1 regulates hematopoietic recovery following 5-fluorouracil treatment

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Sunanda Basu

2014-01-01

Full Text Available In vitro stimulation of HSCs with growth factors generally leads to their depletion. Understanding the molecular mechanisms underlying expansion of HSCs in vivo following myeloablation could lead to successful expansion of HSCs ex vivo for therapeutic purposes. Current findings show that mTORC1 is activated in HSPCs following 5-fluorouracil treatment and that mTORC1 activation is dependent on mitochondrial ETC capacity of HSPCs. Moreover, expression of PGC-1 family members, proteins that regulate mitochondrial biogenesis, in HSPCs following 5-fluorouracil treatment changes; also, these proteins play a stage specific role in hematopoietic recovery. While PRC regulates HSCs' expansion during early recovery phase, PGC-1α regulates progenitor cell proliferation and recovery of hematopoiesis during later phase. During early recovery phase, PRC expression, mitochondrial activity and mTORC1 activation are relatively higher in PGC-1α−/− HSCs compared to WT HSCs, and PGC-1α−/− HSCs show greater expansion. Administration of rapamycin, but not NAC, during early recovery phase improves WT HSC numbers but decreases PGC-1α−/− HSC numbers. The current findings demonstrate that mTOR activation can increase HSC numbers provided that the energy demand created by mTOR activation is successfully met. Thus, critical tuning between mTORC1 activation and mitochondrial ETC capacity is crucial for HSC maintenance/expansion in response to mitogenic stimulation.

Complex Interdependence Regulates Heterotypic Transcription Factor Distribution and Coordinates Cardiogenesis.

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Luna-Zurita, Luis; Stirnimann, Christian U; Glatt, Sebastian; Kaynak, Bogac L; Thomas, Sean; Baudin, Florence; Samee, Md Abul Hassan; He, Daniel; Small, Eric M; Mileikovsky, Maria; Nagy, Andras; Holloway, Alisha K; Pollard, Katherine S; Müller, Christoph W; Bruneau, Benoit G

2016-02-25

Transcription factors (TFs) are thought to function with partners to achieve specificity and precise quantitative outputs. In the developing heart, heterotypic TF interactions, such as between the T-box TF TBX5 and the homeodomain TF NKX2-5, have been proposed as a mechanism for human congenital heart defects. We report extensive and complex interdependent genomic occupancy of TBX5, NKX2-5, and the zinc finger TF GATA4 coordinately controlling cardiac gene expression, differentiation, and morphogenesis. Interdependent binding serves not only to co-regulate gene expression but also to prevent TFs from distributing to ectopic loci and activate lineage-inappropriate genes. We define preferential motif arrangements for TBX5 and NKX2-5 cooperative binding sites, supported at the atomic level by their co-crystal structure bound to DNA, revealing a direct interaction between the two factors and induced DNA bending. Complex interdependent binding mechanisms reveal tightly regulated TF genomic distribution and define a combinatorial logic for heterotypic TF regulation of differentiation. Copyright © 2016 Elsevier Inc. All rights reserved.

Genetic evidence suggests that GIS functions downstream of TCL1 to regulate trichome formation in Arabidopsis.

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Zhang, Na; Yang, Li; Luo, Sha; Wang, Xutong; Wang, Wei; Cheng, Yuxin; Tian, Hainan; Zheng, Kaijie; Cai, Ling; Wang, Shucai

2018-04-13

Trichome formation in Arabidopsis is regulated by a MBW complex formed by MYB, bHLH and WD40 transcriptional factors, which can activate GLABRA2 (GL2) and the R3 MYB transcription factor genes. GL2 promotes trichome formation, whereas R3 MYBs are able to block the formation of the MBW complex. It has been reported that the C2H2 transcription factor GIS (GLABROUS INFLORESCENCE STEMS) functions upstream of the MBW activator complex to regulate trichome formation, and that the expression of TCL1 is not regulated by the MBW complex. However, gis and the R3 MYB gene mutant tcl1 (trichomeless 1) have opposite inflorescence trichome phenotypes, but their relationship in regulating trichome formation remained unknown. By generating and characterization of the gis tcl1 double mutant, we found that trichome formation in the gis tcl1double and the tcl1 single mutants were largely indistinguishable, but the trichome formation in the 35S:TCL1/gis transgenic plant was similar to that in the gis mutant. By using quantitative RT-PCR analysis, we showed that expression level of GIS was increased in the triple mutant tcl1 try cpc, but the expression level of TCL1 was not affected in the gis mutant. On the other hand, trichome morphology in both gis tcl1 and 35S:TCL1/gis plants was similar to that in the gis mutant. In summary, our results indicate that GIS may work downstream of TCL1 to regulate trichome formation, and GIS has a dominant role in controlling trichome morphology.

PT-1 selectively activates AMPK-γ1 complexes in mouse skeletal muscle, but activates all three γ subunit complexes in cultured human cells by inhibiting the respiratory chain

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Jensen, Thomas Elbenhardt; Ross, Fiona A; Kleinert, Maximilian

2015-01-01

strategy to combat diseases such as cancer and type 2 diabetes. We report that the AMPK activator PT-1 selectively increased the activity of γ1- but not γ3-containing complexes in incubated mouse muscle. PT-1 increased the AMPK-dependent phosphorylation of the autophagy-regulating kinase ULK1 on Ser555...

Shaping the landscape: Metabolic regulation of S1P gradients

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Olivera, Ana; Allende, Maria Laura; Proia, Richard L.

2012-01-01

Sphingosine-1-phosphate (S1P) is a lipid that functions as a metabolic intermediate and a cellular signaling molecule. These roles are integrated when compartments with differing extracellular S1P concentrations are formed that serve to regulate functions within the immune and vascular systems, as well as during pathologic conditions. Gradients of S1P concentration are achieved by the organization of cells with specialized expression of S1P metabolic pathways within tissues. S1P concentration gradients underpin the ability of S1P signaling to regulate in vivo physiology. This review will discuss the mechanisms that are necessary for the formation and maintenance of S1P gradients, with the aim of understanding how a simple lipid controls complex physiology. PMID:22735358

Forkhead Box C1 Regulates Human Primary Keratinocyte Terminal Differentiation.

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Lianghua Bin

Full Text Available The epidermis serves as a critical protective barrier between the internal and external environment of the human body. Its remarkable barrier function is established through the keratinocyte (KC terminal differentiation program. The transcription factors specifically regulating terminal differentiation remain largely unknown. Using a RNA-sequencing (RNA-seq profiling approach, we found that forkhead box c 1 (FOXC1 was significantly up-regulated in human normal primary KC during the course of differentiation. This observation was validated in human normal primary KC from several different donors and human skin biopsies. Silencing FOXC1 in human normal primary KC undergoing differentiation led to significant down-regulation of late terminal differentiation genes markers including epidermal differentiation complex genes, keratinization genes, sphingolipid/ceramide metabolic process genes and epidermal specific cell-cell adhesion genes. We further demonstrated that FOXC1 works down-stream of ZNF750 and KLF4, and upstream of GRHL3. Thus, this study defines FOXC1 as a regulator specific for KC terminal differentiation and establishes its potential position in the genetic regulatory network.

The MYST family histone acetyltransferase complex regulates stress resistance and longevity through transcriptional control of DAF-16/FOXO transcription factors.

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Ikeda, Takako; Uno, Masaharu; Honjoh, Sakiko; Nishida, Eisuke

2017-08-09

The well-known link between longevity and the Sir2 histone deacetylase family suggests that histone deacetylation, a modification associated with repressed chromatin, is beneficial to longevity. However, the molecular links between histone acetylation and longevity remain unclear. Here, we report an unexpected finding that the MYST family histone acetyltransferase complex (MYS-1/TRR-1 complex) promotes rather than inhibits stress resistance and longevity in Caenorhabditis elegans Our results show that these beneficial effects are largely mediated through transcriptional up-regulation of the FOXO transcription factor DAF-16. MYS-1 and TRR-1 are recruited to the promoter regions of the daf-16 gene, where they play a role in histone acetylation, including H4K16 acetylation. Remarkably, we also find that the human MYST family Tip60/TRRAP complex promotes oxidative stress resistance by up-regulating the expression of FOXO transcription factors in human cells. Tip60 is recruited to the promoter regions of the foxo1 gene, where it increases H4K16 acetylation levels. Our results thus identify the evolutionarily conserved role of the MYST family acetyltransferase as a key epigenetic regulator of DAF-16/FOXO transcription factors. © 2017 The Authors.

Regulation of neurite morphogenesis by interaction between R7 regulator of G protein signaling complexes and G protein subunit Gα13.

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Scherer, Stephanie L; Cain, Matthew D; Kanai, Stanley M; Kaltenbronn, Kevin M; Blumer, Kendall J

2017-06-16

The R7 regulator of G protein signaling family (R7-RGS) critically regulates nervous system development and function. Mice lacking all R7-RGS subtypes exhibit diverse neurological phenotypes, and humans bearing mutations in the retinal R7-RGS isoform RGS9-1 have vision deficits. Although each R7-RGS subtype forms heterotrimeric complexes with Gβ 5 and R7-RGS-binding protein (R7BP) that regulate G protein-coupled receptor signaling by accelerating deactivation of G i/o α-subunits, several neurological phenotypes of R7-RGS knock-out mice are not readily explained by dysregulated G i/o signaling. Accordingly, we used tandem affinity purification and LC-MS/MS to search for novel proteins that interact with R7-RGS heterotrimers in the mouse brain. Among several proteins detected, we focused on Gα 13 because it had not been linked to R7-RGS complexes before. Split-luciferase complementation assays indicated that Gα 13 in its active or inactive state interacts with R7-RGS heterotrimers containing any R7-RGS isoform. LARG (leukemia-associated Rho guanine nucleotide exchange factor (GEF)), PDZ-RhoGEF, and p115RhoGEF augmented interaction between activated Gα 13 and R7-RGS heterotrimers, indicating that these effector RhoGEFs can engage Gα 13 ·R7-RGS complexes. Because Gα 13 /R7-RGS interaction required R7BP, we analyzed phenotypes of neuronal cell lines expressing RGS7 and Gβ 5 with or without R7BP. We found that neurite retraction evoked by Gα 12/13 -dependent lysophosphatidic acid receptors was augmented in R7BP-expressing cells. R7BP expression blunted neurite formation evoked by serum starvation by signaling mechanisms involving Gα 12/13 but not Gα i/o These findings provide the first evidence that R7-RGS heterotrimers interact with Gα 13 to augment signaling pathways that regulate neurite morphogenesis. This mechanism expands the diversity of functions whereby R7-RGS complexes regulate critical aspects of nervous system development and function. © 2017 by

Phosphorylation regulates SIRT1 function.

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Tsutomu Sasaki

Full Text Available BACKGROUND: SIR2 is an NAD(+-dependent deacetylase [1]-[3] implicated in the regulation of lifespan in species as diverse as yeast [4], worms [5], and flies [6]. We previously reported that the level of SIRT1, the mammalian homologue of SIR2 [7], [8], is coupled to the level of mitotic activity in cells both in vitro and in vivo[9]. Cells from long-lived mice maintained SIRT1 levels of young mice in tissues that undergo continuous cell replacement by proliferating stem cells. Changes in SIRT1 protein level were not associated with changes in mRNA level, suggesting that SIRT1 could be regulated post-transcriptionally. However, other than a recent report on sumoylation [10] and identification of SIRT1 as a nuclear phospho-protein by mass spectrometry [11], post-translational modifications of this important protein have not been reported. METHODOLOGY/PRINCIPAL FINDINGS: We identified 13 residues in SIRT1 that are phosphorylated in vivo using mass spectrometry. Dephosphorylation by phosphatases in vitro resulted in decreased NAD(+-dependent deacetylase activity. We identified cyclinB/Cdk1 as a cell cycle-dependent kinase that forms a complex with and phosphorylates SIRT1. Mutation of two residues phosphorylated by Cyclin B/Cdk1 (threonine 530 and serine 540 disturbs normal cell cycle progression and fails to rescue proliferation defects in SIRT1-deficient cells [12], [13]. CONCLUSIONS/SIGNIFICANCE: Pharmacological manipulation of SIRT1 activity is currently being tested as a means of extending lifespan in mammals. Treatment of obese mice with resveratrol, a pharmacological activator of SIRT1, modestly but significantly improved longevity and, perhaps more importantly, offered some protection against the development of type 2 diabetes mellitus and metabolic syndrome [14]-[16]. Understanding the endogenous mechanisms that regulate the level and activity of SIRT1, therefore, has obvious relevance to human health and disease. Our results identify

BRCA1-IRIS regulates cyclin D1 expression in breast cancer cells

International Nuclear Information System (INIS)

Nakuci, Enkeleda; Mahner, Sven; DiRenzo, James; ElShamy, Wael M.

2006-01-01

The regulator of cell cycle progression, cyclin D1, is up-regulated in breast cancer cells; its expression is, in part, dependent on ERα signaling. However, many ERα-negative tumors and tumor cell lines (e.g., SKBR3) also show over-expression of cyclin D1. This suggests that, in addition to ERα signaling, cyclin D1 expression is under the control of other signaling pathways; these pathways may even be over-expressed in the ERα-negative cells. We previously noticed that both ERα-positive and -negative cell lines over-express BRCA1-IRIS mRNA and protein. Furthermore, the level of over-expression of BRCA1-IRIS in ERα-negative cell lines even exceeded its over-expression level in ERα-positive cell lines. In this study, we show that: (1) BRCA1-IRIS forms complex with two of the nuclear receptor co-activators, namely, SRC1 and SRC3 (AIB1) in an ERα-independent manner. (2) BRCA1-IRIS alone, or in connection with co-activators, is recruited to the cyclin D1 promoter through its binding to c-Jun/AP1 complex; this binding activates the cyclin D1 expression. (3) Over-expression of BRCA1-IRIS in breast cells over-activates JNK/c-Jun; this leads to the induction of cyclin D1 expression and cellular proliferation. (4) BRCA1-IRIS activation of JNK/c-Jun/AP1 appears to account for this, because in cells that were depleted from BRCA1-IRIS, JNK remained inactive. However, depletion of SRC1 or SRC3 instead reduced c-Jun expression. Our data suggest that this novel signaling pathway links BRCA1-IRIS to cellular proliferation through c-Jun/AP1 nuclear pathway; finally, this culminates in the increased expression of the cyclin D1 gene

Ubiquitin regulates TORC1 in yeast Saccharomyces cerevisiae.

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Hu, Kejin; Guo, Shuguang; Yan, Gonghong; Yuan, Wenjie; Zheng, Yin; Jiang, Yu

2016-04-01

In the yeast Saccharomyces cerevisiae the TOR complex 1 (TORC1) controls many growth-related cellular processes and is essential for cell growth and proliferation. Macrolide antibiotic rapamycin, in complex with a cytosol protein named FKBP12, specifically inhibits TORC1, causing growth arrest. The FKBP12-rapamycin complex interferes with TORC1 function by binding to the FRB domain of the TOR proteins. In an attempt to understand the role of the FRB domain in TOR function, we identified a single point mutation (Tor2(W2041R) ) in the FRB domain of Tor2 that renders yeast cells rapamycin resistant and temperature sensitive. At the permissive temperature, the Tor2 mutant protein is partially defective for binding with Kog1 and TORC1 is impaired for membrane association. At the restrictive temperature, Kog1 but not the Tor2 mutant protein, is rapidly degraded. Overexpression of ubiquitin stabilizes Kog1 and suppresses the growth defect associated with the tor2 mutant at the nonpremissive temperature. We find that ubiquitin binds non-covalently to Kog1, prevents Kog1 from degradation and stabilizes TORC1. Our data reveal a unique role for ubiquitin in regulation of TORC1 and suggest that Kog1 requires association with the Tor proteins for stabilization. © 2016 John Wiley & Sons Ltd.

Regulation of mitogen-activated protein kinase pathways by the plasma membrane Na+/H+ exchanger, NHE1

DEFF Research Database (Denmark)

Pedersen, Stine Helene Falsig; Darborg, Barbara Vasek; Rentsch, Maria Louise

2006-01-01

activity is regulated by a three-tiered phosphorelay system, which is in turn regulated by a complex network of signaling events and scaffolding proteins. The ubiquitous plasma membrane Na(+)/H(+) exchanger NHE1 is activated by, and implicated in, the physiological/pathophysiological responses to many...... of the same stimuli that modulate MAPK activity. While under some conditions, NHE1 is regulated by MAPKs, a number of studies have, conversely, implicated NHE1 in the regulation of MAPK activity. Here, we discuss the current evidence indicating the involvement of NHE1 in MAPK regulation, the mechanisms...

Recent Progress on Liver Kinase B1 (LKB1: Expression, Regulation, Downstream Signaling and Cancer Suppressive Function

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Ren-You Gan

2014-09-01

Full Text Available Liver kinase B1 (LKB1, known as a serine/threonine kinase, has been identified as a critical cancer suppressor in many cancer cells. It is a master upstream kinase of 13 AMP-activated protein kinase (AMPK-related protein kinases, and possesses versatile biological functions. LKB1 gene is mutated in many cancers, and its protein can form different protein complexes with different cellular localizations in various cell types. The expression of LKB1 can be regulated through epigenetic modification, transcriptional regulation and post-translational modification. LKB1 dowcnstream pathways mainly include AMPK, microtubule affinity regulating kinase (MARK, salt-inducible kinase (SIK, sucrose non-fermenting protein-related kinase (SNRK and brain selective kinase (BRSK signalings, etc. This review, therefore, mainly discusses recent studies about the expression, regulation, downstream signaling and cancer suppressive function of LKB1, which can be helpful for better understanding of this molecular and its significance in cancers.

Environment-dependent regulation of spliceosome activity by the LSM2-8 complex in Arabidopsis.

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Carrasco-López, Cristian; Hernández-Verdeja, Tamara; Perea-Resa, Carlos; Abia, David; Catalá, Rafael; Salinas, Julio

2017-07-07

Spliceosome activity is tightly regulated to ensure adequate splicing in response to internal and external cues. It has been suggested that core components of the spliceosome, such as the snRNPs, would participate in the control of its activity. The experimental indications supporting this proposition, however, remain scarce, and the operating mechanisms poorly understood. Here, we present genetic and molecular evidence demonstrating that the LSM2-8 complex, the protein moiety of the U6 snRNP, regulates the spliceosome activity in Arabidopsis, and that this regulation is controlled by the environmental conditions. Our results show that the complex ensures the efficiency and accuracy of constitutive and alternative splicing of selected pre-mRNAs, depending on the conditions. Moreover, miss-splicing of most targeted pre-mRNAs leads to the generation of nonsense mediated decay signatures, indicating that the LSM2-8 complex also guarantees adequate levels of the corresponding functional transcripts. Interestingly, the selective role of the complex has relevant physiological implications since it is required for adequate plant adaptation to abiotic stresses. These findings unveil an unanticipated function for the LSM2-8 complex that represents a new layer of posttranscriptional regulation in response to external stimuli in eukaryotes. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Tsc1 is a Critical Regulator of Macrophage Survival and Function

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Chunmin Fang

2015-07-01

Full Text Available Background/Aims: Tuberous sclerosis complex 1 (Tsc1 has been shown to regulate M1/M2 polarization of macrophages, but the precise roles of Tsc1 in the function and stability of macrophages are not fully understood. Here we show that Tsc1 is required for regulating the survival, migration and phagocytosis of macrophages. Methods: Mice with Tsc1 homozygous deletion in myeloid cells (LysMCreTsc1flox/flox; Tsc1 KO were obtained by crossing Tsc1flox/flox mice with mice expressing Cre recombinase under the control of Lysozyme promoter (LysMCre. The apoptosis and growth of macrophages were determined by flow cytometry and Real-time PCR (RT-PCR. The phagocytosis was determined using a Vybrant™ phagocytosis assay kit. The migration of macrophages was determined using transwell migration assay. Results: Peritoneal macrophages of Tsc1 KO mice exhibited increased apoptosis and enlarged cell size. Both M1 and M2 phenotypes in Tsc1-deficient macrophages were elevated in steady-state as well as in inflammatory conditions. Tsc1-deficient macrophages demonstrated impaired migration and reduced expression of chemokine receptors including CCR2 and CCR5. Phagocytosis activity and ROS production were enhanced in Tsc1-deficient macrophages. Furthermore, pharmacological inhibition of the mammalian target of rapamycin complex 1 (mTORC1 partially reversed the aberrance of Tsc1-deficient macrophages. Conclusion: Tsc1 plays a critical role in regulating macrophage survival, function and polarization via inhibition of mTORC1 activity.

First report of Clostridium difficile NAP1/027 in a Mexican hospital.

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Adrián Camacho-Ortiz

Full Text Available Clostridium difficile NAP1/ribotype 027 is associated with severe disease and high mortality rates. Our aim was to determine the prevalence of NAP1/ribotype 027 among C. difficile isolates in a tertiary care hospital, and review the main clinical data.We included 106 stool samples from 106 patients. Samples were tested for A&B toxins and were cultured on CCFA agar. The genes tcdA, tcdB, tcdC, cdtA, and cdtB were amplified using PCR in clinical isolates. The tcdA 3'-end deletion analysis, PCR-ribotyping, and pulsed-field gel electrophoresis (PFGE were also performed. Stool samples that were positive for culture were tested by the GeneXpert C. difficile assay. Clinical data were collected.Thirty-six patients tested positive for A&B toxins; and 22 patients had positive culture for C. difficile, 14 of which tested positive for the A&B toxins and all 22 patients tested positive by the GeneXpert C. difficile assay. Risk factors included an average hospital stay of 16.1 days prior to toxin detection, average antibiotic use for 16.2 days, and a median of 3 antibiotics used. The 30-day crude mortality rate was 8.4%. Six of the 22 patients died, and 3 of those deaths were directly attributed to C. difficile infection. The majority of isolates, 90.9% (20/22, carried genes tcdB, tcdA, cdtA, and cdtB; and these strains carried the corresponding downregulator gene tcdC, with an 18-bp deletion. PFGE was performed on 17 isolates, and one main pattern was observed. Analysis of the ribotyping data showed similar results.The above findings represent the clonal spread of C. difficile in our institution, which mainly includes the NAP1/027 strain. This is the first report of C. difficile ribotype NAP1/027 in Mexico.

SOCS-1 localizes to the microtubule organizing complex-associated 20S proteasome.

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Vuong, Bao Q; Arenzana, Teresita L; Showalter, Brian M; Losman, Julie; Chen, X Peter; Mostecki, Justin; Banks, Alexander S; Limnander, Andre; Fernandez, Neil; Rothman, Paul B

2004-10-01

The regulation of cytokine signaling is critical for controlling cellular proliferation and activation during an immune response. SOCS-1 is a potent inhibitor of Jak kinase activity and of signaling initiated by several cytokines. SOCS-1 protein levels are tightly regulated, and recent data suggest that SOCS-1 may regulate the protein levels of some signaling proteins by the ubiquitin proteasome pathway; however, the cellular mechanism by which SOCS-1 directs proteins for degradation is unknown. In this report, SOCS-1 is found to colocalize and biochemically copurify with the microtubule organizing complex (MTOC) and its associated 20S proteasome. The SOCS-1 SH2 domain is required for the localization of SOCS-1 to the MTOC. Overexpression of SOCS-1 targets Jak1 in an SH2-dependent manner to a perinuclear distribution resembling the MTOC-associated 20S proteasome. Analysis of MTOCs fractionated from SOCS-1-deficient cells demonstrates that SOCS-1 may function redundantly to regulate the localization of Jak1 to the MTOC. Nocodazole inhibits the protein turnover of SOCS-1, demonstrating that the minus-end transport of SOCS-1 to the MTOC-associated 20S proteasome is required to regulate SOCS-1 protein levels. These data link SOCS-1 directly with the proteasome pathway and suggest another function for the SH2 domain of SOCS-1 in the regulation of Jak/STAT signaling.

CRTC1 Nuclear Translocation Following Learning Modulates Memory Strength via Exchange of Chromatin Remodeling Complexes on the Fgf1 Gene

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Shusaku Uchida

2017-01-01

Full Text Available Summary: Memory is formed by synapse-to-nucleus communication that leads to regulation of gene transcription, but the identity and organizational logic of signaling pathways involved in this communication remain unclear. Here we find that the transcription cofactor CRTC1 is a critical determinant of sustained gene transcription and memory strength in the hippocampus. Following associative learning, synaptically localized CRTC1 is translocated to the nucleus and regulates Fgf1b transcription in an activity-dependent manner. After both weak and strong training, the HDAC3-N-CoR corepressor complex leaves the Fgf1b promoter and a complex involving the translocated CRTC1, phosphorylated CREB, and histone acetyltransferase CBP induces transient transcription. Strong training later substitutes KAT5 for CBP, a process that is dependent on CRTC1, but not on CREB phosphorylation. This in turn leads to long-lasting Fgf1b transcription and memory enhancement. Thus, memory strength relies on activity-dependent changes in chromatin and temporal regulation of gene transcription on specific CREB/CRTC1 gene targets. : Uchida et al. link CRTC1 synapse-to-nucleus shuttling in memory. Weak and strong training induce CRTC1 nuclear transport and transient Fgf1b transcription by a complex including CRTC1, CREB, and histone acetyltransferase CBP, whereas strong training alone maintains Fgf1b transcription through CRTC1-dependent substitution of KAT5 for CBP, leading to memory enhancement. Keywords: memory enhancement, long-term potentiation, hippocampus, nuclear transport, epigenetics, FGF1, CRTC1, KAT5/Tip60, HDAC3, CREB

Lysosomal Regulation of mTORC1 by Amino Acids in Mammalian Cells

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Yao Yao

2017-07-01

Full Text Available The mechanistic target of rapamycin complex 1 (mTORC1 is a master regulator of cell growth in eukaryotic cells. The active mTORC1 promotes cellular anabolic processes including protein, pyrimidine, and lipid biosynthesis, and inhibits catabolic processes such as autophagy. Consistent with its growth-promoting functions, hyper-activation of mTORC1 signaling is one of the important pathomechanisms underlying major human health problems including diabetes, neurodegenerative disorders, and cancer. The mTORC1 receives multiple upstream signals such as an abundance of amino acids and growth factors, thus it regulates a wide range of downstream events relevant to cell growth and proliferation control. The regulation of mTORC1 by amino acids is a fast-evolving field with its detailed mechanisms currently being revealed as the precise picture emerges. In this review, we summarize recent progress with respect to biochemical and biological findings in the regulation of mTORC1 signaling on the lysosomal membrane by amino acids.

Cell-cycle regulation of non-enzymatic functions of the Drosophila methyltransferase PR-Set7.

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Zouaz, Amel; Fernando, Céline; Perez, Yannick; Sardet, Claude; Julien, Eric; Grimaud, Charlotte

2018-04-06

Tight cell-cycle regulation of the histone H4-K20 methyltransferase PR-Set7 is essential for the maintenance of genome integrity. In mammals, this mainly involves the interaction of PR-Set7 with the replication factor PCNA, which triggers the degradation of the enzyme by the CRL4CDT2 E3 ubiquitin ligase. PR-Set7 is also targeted by the SCFβ-TRCP ligase, but the role of this additional regulatory pathway remains unclear. Here, we show that Drosophila PR-Set7 undergoes a cell-cycle proteolytic regulation, independently of its interaction with PCNA. Instead, Slimb, the ortholog of β-TRCP, is specifically required for the degradation of the nuclear pool of PR-Set7 prior to S phase. Consequently, inactivation of Slimb leads to nuclear accumulation of PR-Set7, which triggers aberrant chromatin compaction and G1/S arrest. Strikingly, these phenotypes result from non-enzymatic PR-Set7 functions that prevent proper histone H4 acetylation independently of H4K20 methylation. Altogether, these results identify the Slimb-mediated PR-Set7 proteolysis as a new critical regulatory mechanism required for proper interphase chromatin organization at G1/S transition.

CRTC1 Nuclear Translocation Following Learning Modulates Memory Strength via Exchange of Chromatin Remodeling Complexes on the Fgf1 Gene.

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Uchida, Shusaku; Teubner, Brett J W; Hevi, Charles; Hara, Kumiko; Kobayashi, Ayumi; Dave, Rutu M; Shintaku, Tatsushi; Jaikhan, Pattaporn; Yamagata, Hirotaka; Suzuki, Takayoshi; Watanabe, Yoshifumi; Zakharenko, Stanislav S; Shumyatsky, Gleb P

2017-01-10

Memory is formed by synapse-to-nucleus communication that leads to regulation of gene transcription, but the identity and organizational logic of signaling pathways involved in this communication remain unclear. Here we find that the transcription cofactor CRTC1 is a critical determinant of sustained gene transcription and memory strength in the hippocampus. Following associative learning, synaptically localized CRTC1 is translocated to the nucleus and regulates Fgf1b transcription in an activity-dependent manner. After both weak and strong training, the HDAC3-N-CoR corepressor complex leaves the Fgf1b promoter and a complex involving the translocated CRTC1, phosphorylated CREB, and histone acetyltransferase CBP induces transient transcription. Strong training later substitutes KAT5 for CBP, a process that is dependent on CRTC1, but not on CREB phosphorylation. This in turn leads to long-lasting Fgf1b transcription and memory enhancement. Thus, memory strength relies on activity-dependent changes in chromatin and temporal regulation of gene transcription on specific CREB/CRTC1 gene targets. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Identification of a BET Family Bromodomain/Casein Kinase II/TAF-Containing Complex as a Regulator of Mitotic Condensin Function

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Hyun-Soo Kim

2014-03-01

Full Text Available Condensin is a central regulator of mitotic genome structure with mutants showing poorly condensed chromosomes and profound segregation defects. Here, we identify NCT, a complex comprising the Nrc1 BET-family tandem bromodomain protein (SPAC631.02, casein kinase II (CKII, and several TAFs, as a regulator of condensin function. We show that NCT and condensin bind similar genomic regions but only briefly colocalize during the periods of chromosome condensation and decondensation. This pattern of NCT binding at the core centromere, the region of maximal condensin enrichment, tracks the abundance of acetylated histone H4, as regulated by the Hat1-Mis16 acetyltransferase complex and recognized by the first Nrc1 bromodomain. Strikingly, mutants in NCT or Hat1-Mis16 restore the formation of segregation-competent chromosomes in cells containing defective condensin. These results are consistent with a model where NCT targets CKII to chromatin in a cell-cycle-directed manner in order to modulate the activity of condensin during chromosome condensation and decondensation.

Identification of a BET family bromodomain/casein kinase II/TAF-containing complex as a regulator of mitotic condensin function.

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Kim, Hyun-Soo; Mukhopadhyay, Rituparna; Rothbart, Scott B; Silva, Andrea C; Vanoosthuyse, Vincent; Radovani, Ernest; Kislinger, Thomas; Roguev, Assen; Ryan, Colm J; Xu, Jiewei; Jahari, Harlizawati; Hardwick, Kevin G; Greenblatt, Jack F; Krogan, Nevan J; Fillingham, Jeffrey S; Strahl, Brian D; Bouhassira, Eric E; Edelmann, Winfried; Keogh, Michael-Christopher

2014-03-13

Condensin is a central regulator of mitotic genome structure with mutants showing poorly condensed chromosomes and profound segregation defects. Here, we identify NCT, a complex comprising the Nrc1 BET-family tandem bromodomain protein (SPAC631.02), casein kinase II (CKII), and several TAFs, as a regulator of condensin function. We show that NCT and condensin bind similar genomic regions but only briefly colocalize during the periods of chromosome condensation and decondensation. This pattern of NCT binding at the core centromere, the region of maximal condensin enrichment, tracks the abundance of acetylated histone H4, as regulated by the Hat1-Mis16 acetyltransferase complex and recognized by the first Nrc1 bromodomain. Strikingly, mutants in NCT or Hat1-Mis16 restore the formation of segregation-competent chromosomes in cells containing defective condensin. These results are consistent with a model where NCT targets CKII to chromatin in a cell-cycle-directed manner in order to modulate the activity of condensin during chromosome condensation and decondensation. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Live-Cell Imaging Visualizes Frequent Mitotic Skipping During Senescence-Like Growth Arrest in Mammary Carcinoma Cells Exposed to Ionizing Radiation

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Suzuki, Masatoshi, E-mail: msuzuki@nagasaki-u.ac.jp [Department of Radiation Medical Sciences, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki (Japan); Yamauchi, Motohiro; Oka, Yasuyoshi; Suzuki, Keiji; Yamashita, Shunichi [Department of Radiation Medical Sciences, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki (Japan)

2012-06-01

Purpose: Senescence-like growth arrest in human solid carcinomas is now recognized as the major outcome of radiotherapy. This study was designed to analyze cell cycle during the process of senescence-like growth arrest in mammary carcinoma cells exposed to X-rays. Methods and Materials: Fluorescent ubiquitination-based cell cycle indicators were introduced into the human mammary carcinoma cell line MCF-7. Cell cycle was sequentially monitored by live-cell imaging for up to 5 days after exposure to 10 Gy of X-rays. Results: Live-cell imaging revealed that cell cycle transition from G2 to G1 phase without mitosis, so-called mitotic skipping, was observed in 17.1% and 69.8% of G1- and G2-irradiated cells, respectively. Entry to G1 phase was confirmed by the nuclear accumulation of mKO{sub 2}-hCdt1 as well as cyclin E, which was inversely correlated to the accumulation of G2-specific markers such as mAG-hGeminin and CENP-F. More than 90% of cells skipping mitosis were persistently arrested in G1 phase and showed positive staining for the senescent biochemical marker, which is senescence-associated ss-galactosidase, indicating induction of senescence-like growth arrest accompanied by mitotic skipping. While G2 irradiation with higher doses of X-rays induced mitotic skipping in approximately 80% of cells, transduction of short hairpin RNA (shRNA) for p53 significantly suppressed mitotic skipping, suggesting that ionizing radiation-induced mitotic skipping is associated with p53 function. Conclusions: The present study found the pathway of senescence-like growth arrest in G1 phase without mitotic entry following G2-irradiation.

Live-Cell Imaging Visualizes Frequent Mitotic Skipping During Senescence-Like Growth Arrest in Mammary Carcinoma Cells Exposed to Ionizing Radiation

International Nuclear Information System (INIS)

Suzuki, Masatoshi; Yamauchi, Motohiro; Oka, Yasuyoshi; Suzuki, Keiji; Yamashita, Shunichi

2012-01-01

Purpose: Senescence-like growth arrest in human solid carcinomas is now recognized as the major outcome of radiotherapy. This study was designed to analyze cell cycle during the process of senescence-like growth arrest in mammary carcinoma cells exposed to X-rays. Methods and Materials: Fluorescent ubiquitination-based cell cycle indicators were introduced into the human mammary carcinoma cell line MCF-7. Cell cycle was sequentially monitored by live-cell imaging for up to 5 days after exposure to 10 Gy of X-rays. Results: Live-cell imaging revealed that cell cycle transition from G2 to G1 phase without mitosis, so-called mitotic skipping, was observed in 17.1% and 69.8% of G1- and G2-irradiated cells, respectively. Entry to G1 phase was confirmed by the nuclear accumulation of mKO 2 -hCdt1 as well as cyclin E, which was inversely correlated to the accumulation of G2-specific markers such as mAG-hGeminin and CENP-F. More than 90% of cells skipping mitosis were persistently arrested in G1 phase and showed positive staining for the senescent biochemical marker, which is senescence-associated ß-galactosidase, indicating induction of senescence-like growth arrest accompanied by mitotic skipping. While G2 irradiation with higher doses of X-rays induced mitotic skipping in approximately 80% of cells, transduction of short hairpin RNA (shRNA) for p53 significantly suppressed mitotic skipping, suggesting that ionizing radiation-induced mitotic skipping is associated with p53 function. Conclusions: The present study found the pathway of senescence-like growth arrest in G1 phase without mitotic entry following G2-irradiation.

The Caenorhabditis elegans Elongator complex regulates neuronal alpha-tubulin acetylation.

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Jachen A Solinger

2010-01-01

Full Text Available Although acetylated alpha-tubulin is known to be a marker of stable microtubules in neurons, precise factors that regulate alpha-tubulin acetylation are, to date, largely unknown. Therefore, a genetic screen was employed in the nematode Caenorhabditis elegans that identified the Elongator complex as a possible regulator of alpha-tubulin acetylation. Detailed characterization of mutant animals revealed that the acetyltransferase activity of the Elongator is indeed required for correct acetylation of microtubules and for neuronal development. Moreover, the velocity of vesicles on microtubules was affected by mutations in Elongator. Elongator mutants also displayed defects in neurotransmitter levels. Furthermore, acetylation of alpha-tubulin was shown to act as a novel signal for the fine-tuning of microtubules dynamics by modulating alpha-tubulin turnover, which in turn affected neuronal shape. Given that mutations in the acetyltransferase subunit of the Elongator (Elp3 and in a scaffold subunit (Elp1 have previously been linked to human neurodegenerative diseases, namely Amyotrophic Lateral Sclerosis and Familial Dysautonomia respectively highlights the importance of this work and offers new insights to understand their etiology.

Regulation of the Tumor-Suppressor Function of the Class III Phosphatidylinositol 3-Kinase Complex by Ubiquitin and SUMO

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Reidick, Christina [Biochemie Intrazellulärer Transportprozesse, Ruhr-Universität Bochum, Bochum 44801 (Germany); El Magraoui, Fouzi; Meyer, Helmut E. [Biomedical Research, Human Brain Proteomics II, Leibniz-Institut für Analytische Wissenschaften-ISAS, Dortmund 44139 (Germany); Stenmark, Harald [Department of Biochemistry, Institute for Cancer Research, Oslo University Hospital, Montebello, Oslo 0310 (Norway); Platta, Harald W., E-mail: harald.platta@rub.de [Biochemie Intrazellulärer Transportprozesse, Ruhr-Universität Bochum, Bochum 44801 (Germany)

2014-12-23

The occurrence of cancer is often associated with a dysfunction in one of the three central membrane-involution processes—autophagy, endocytosis or cytokinesis. Interestingly, all three pathways are controlled by the same central signaling module: the class III phosphatidylinositol 3-kinase (PI3K-III) complex and its catalytic product, the phosphorylated lipid phosphatidylinositol 3-phosphate (PtdIns3P). The activity of the catalytic subunit of the PI3K-III complex, the lipid-kinase VPS34, requires the presence of the membrane-targeting factor VPS15 as well as the adaptor protein Beclin 1. Furthermore, a growing list of regulatory proteins associates with VPS34 via Beclin 1. These accessory factors define distinct subunit compositions and thereby guide the PI3K-III complex to its different cellular and physiological roles. Here we discuss the regulation of the PI3K-III complex components by ubiquitination and SUMOylation. Especially Beclin 1 has emerged as a highly regulated protein, which can be modified with Lys11-, Lys48- or Lys63-linked polyubiquitin chains catalyzed by distinct E3 ligases from the RING-, HECT-, RBR- or Cullin-type. We also point out other cross-links of these ligases with autophagy in order to discuss how these data might be merged into a general concept.

Regulation of the Tumor-Suppressor Function of the Class III Phosphatidylinositol 3-Kinase Complex by Ubiquitin and SUMO

International Nuclear Information System (INIS)

Reidick, Christina; El Magraoui, Fouzi; Meyer, Helmut E.; Stenmark, Harald; Platta, Harald W.

2014-01-01

The occurrence of cancer is often associated with a dysfunction in one of the three central membrane-involution processes—autophagy, endocytosis or cytokinesis. Interestingly, all three pathways are controlled by the same central signaling module: the class III phosphatidylinositol 3-kinase (PI3K-III) complex and its catalytic product, the phosphorylated lipid phosphatidylinositol 3-phosphate (PtdIns3P). The activity of the catalytic subunit of the PI3K-III complex, the lipid-kinase VPS34, requires the presence of the membrane-targeting factor VPS15 as well as the adaptor protein Beclin 1. Furthermore, a growing list of regulatory proteins associates with VPS34 via Beclin 1. These accessory factors define distinct subunit compositions and thereby guide the PI3K-III complex to its different cellular and physiological roles. Here we discuss the regulation of the PI3K-III complex components by ubiquitination and SUMOylation. Especially Beclin 1 has emerged as a highly regulated protein, which can be modified with Lys11-, Lys48- or Lys63-linked polyubiquitin chains catalyzed by distinct E3 ligases from the RING-, HECT-, RBR- or Cullin-type. We also point out other cross-links of these ligases with autophagy in order to discuss how these data might be merged into a general concept

TBLR1 regulates the expression of nuclear hormone receptor co-repressors

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Brown Stuart

2006-08-01

Full Text Available Abstract Background Transcription is regulated by a complex interaction of activators and repressors. The effectors of repression are large multimeric complexes which contain both the repressor proteins that bind to transcription factors and a number of co-repressors that actually mediate transcriptional silencing either by inhibiting the basal transcription machinery or by recruiting chromatin-modifying enzymes. Results TBLR1 [GenBank: NM024665] is a co-repressor of nuclear hormone transcription factors. A single highly conserved gene encodes a small family of protein molecules. Different isoforms are produced by differential exon utilization. Although the ORF of the predominant form contains only 1545 bp, the human gene occupies ~200 kb of genomic DNA on chromosome 3q and contains 16 exons. The genomic sequence overlaps with the putative DC42 [GenBank: NM030921] locus. The murine homologue is structurally similar and is also located on Chromosome 3. TBLR1 is closely related (79% homology at the mRNA level to TBL1X and TBL1Y, which are located on Chromosomes X and Y. The expression of TBLR1 overlaps but is distinct from that of TBL1. An alternatively spliced form of TBLR1 has been demonstrated in human material and it too has an unique pattern of expression. TBLR1 and the homologous genes interact with proteins that regulate the nuclear hormone receptor family of transcription factors. In resting cells TBLR1 is primarily cytoplasmic but after perturbation the protein translocates to the nucleus. TBLR1 co-precipitates with SMRT, a co-repressor of nuclear hormone receptors, and co-precipitates in complexes immunoprecipitated by antiserum to HDAC3. Cells engineered to over express either TBLR1 or N- and C-terminal deletion variants, have elevated levels of endogenous N-CoR. Co-transfection of TBLR1 and SMRT results in increased expression of SMRT. This co-repressor undergoes ubiquitin-mediated degradation and we suggest that the stabilization of

Mcl-1 Ubiquitination: Unique Regulation of an Essential Survival Protein

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Barbara Mojsa

2014-05-01

Full Text Available Mcl-1 is an anti-apoptotic protein of the Bcl-2 family that is essential for the survival of multiple cell lineages and that is highly amplified in human cancer. Under physiological conditions, Mcl-1 expression is tightly regulated at multiple levels, involving transcriptional, post-transcriptional and post-translational processes. Ubiquitination of Mcl-1, that targets it for proteasomal degradation, allows for rapid elimination of the protein and triggering of cell death, in response to various cellular events. In the last decade, a number of studies have elucidated different pathways controlling Mcl-1 ubiquitination and degradation. Four different E3 ubiquitin-ligases (e.g., Mule, SCFβ-TrCP, SCFFbw7 and Trim17 and one deubiquitinase (e.g., USP9X, that respectively mediate and oppose Mcl-1 ubiquitination, have been formerly identified. The interaction between Mule and Mcl-1 can be modulated by other Bcl-2 family proteins, while recognition of Mcl-1 by the other E3 ubiquitin-ligases and deubiquitinase is influenced by phosphorylation of specific residues in Mcl-1. The protein kinases and E3 ubiquitin-ligases that are involved in the regulation of Mcl-1 stability vary depending on the cellular context, highlighting the complexity and pivotal role of Mcl-1 regulation. In this review, we attempt to recapitulate progress in understanding Mcl-1 regulation by the ubiquitin-proteasome system.

Structural Insights into RNA Recognition by the Alternate-Splicing Regulator CUG-Binding Protein 1

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M Teplova; J Song; H Gaw; A Teplov; D Patel

2011-12-31

CUG-binding protein 1 (CUGBP1) regulates multiple aspects of nuclear and cytoplasmic mRNA processing, with implications for onset of myotonic dystrophy. CUGBP1 harbors three RRM domains and preferentially targets UGU-rich mRNA elements. We describe crystal structures of CUGBP1 RRM1 and tandem RRM1/2 domains bound to RNAs containing tandem UGU(U/G) elements. Both RRM1 in RRM1-RNA and RRM2 in RRM1/2-RNA complexes use similar principles to target UGU(U/G) elements, with recognition mediated by face-to-edge stacking and water-mediated hydrogen-bonding networks. The UG step adopts a left-handed Z-RNA conformation, with the syn guanine recognized through Hoogsteen edge-protein backbone hydrogen-bonding interactions. NMR studies on the RRM1/2-RNA complex establish that both RRM domains target tandem UGUU motifs in solution, whereas filter-binding assays identify a preference for recognition of GU over AU or GC steps. We discuss the implications of CUGBP1-mediated targeting and sequestration of UGU(U/G) elements on pre-mRNA alternative-splicing regulation, translational regulation, and mRNA decay.

Metformin anti-tumor effect via disruption of the MID1 translational regulator complex and AR downregulation in prostate cancer cells

International Nuclear Information System (INIS)

Demir, Ummuhan; Koehler, Andrea; Schneider, Rainer; Schweiger, Susann; Klocker, Helmut

2014-01-01

Metformin is an approved drug prescribed for diabetes. Its role as an anti-cancer agent has drawn significant attention because of its minimal side effects and low cost. However, its mechanism of anti-tumour action has not yet been fully clarified. The effect on cell growth was assessed by cell counting. Western blot was used for analysis of protein levels, Boyden chamber assays for analyses of cell migration and co-immunoprecipitation (CoIP) followed by western blot, PCR or qPCR for analysis of protein-protein and protein-mRNA interactions. Metformin showed an anti-proliferative effect on a wide range of prostate cancer cells. It disrupted the AR translational MID1 regulator complex leading to release of the associated AR mRNA and subsequently to downregulation of AR protein in AR positive cell lines. Inhibition of AR positive and negative prostate cancer cells by metformin suggests involvement of additional targets. The inhibitory effect of metformin was mimicked by disruption of the MID1-α4/PP2A protein complex by siRNA knockdown of MID1 or α4 whereas AMPK activation was not required. Findings reported herein uncover a mechanism for the anti-tumor activity of metformin in prostate cancer, which is independent of its anti-diabetic effects. These data provide a rationale for the use of metformin in the treatment of hormone naïve and castration-resistant prostate cancer and suggest AR is an important indirect target of metformin

Studies for the application of Boron neutron capture therapy (BNCT) to the treatment of differentiated thyroid cancer (CDT)

International Nuclear Information System (INIS)

Carpano, Marina; Thomasz, Lisa; Perona, Marina; Juvenal, Guillermo J.; Pisarev, Mario; Dagrosa, Maria A.; Nievas, Susana I.; Pozzi, Emiliano; Thorp, Silvia

2009-01-01

Boron neutron capture therapy (BNCT) is a high linear energy transfer (LET) radiotherapy for cancer, which it is based on the nuclear reaction that occurs when boron-10 that it is a non radioactive isotope of the natural elemental boron, is irradiated with low energy thermal neutrons to produce an alpha particle and a nucleus of lithium-7. Both particles have a range smaller than the diameter of a cell causing cell tumor death without significant damage to the surrounding normal tissues. In previous studies we have shown that BNCT can be a possibility for the treatment of undifferentiated thyroid cancer (UTC). However, more than 80 % of patients with thyroid neoplasm present differentiated carcinoma (CDT). These carcinomas are treated by surgery followed by therapy with 131 I and mostly these forms are well controlled. But in some patients recurrence of the tumor is observed. BNCT can be an alternative for these patients in who the tumor lost the capacity to concentrate iodide. The aim of these studies was to evaluate the possibility of treating differentiated thyroid cancer by BNCT. Materials and Methods: The human cell lines of follicular (WRO) and papillary carcinomas (TPC-1) were grown in RPMI and modified DMEM medium respectively. Both supplemented with 10 % of SFB. The cell line of thyroid rat, FRTL-5, used as control normal, was cultured in DMEM/F12. The uptakes of 125 I and p-borophenylalanine BPA (6.93mM) were studied. The intracellular boron concentration was measured by inductively coupled plasma optical emission spectroscopy (ICP-OES) at 2 hr post incubation. The NIH strain of male nude mice, aged 6 to 8 weeks and weighing 20 to 25 g were implanted (s.c) in the back right flank with different concentrations of tumor cells. The size of the tumors was measured with a caliper twice or three times a week and the volume was calculated according the following formulae: A 2 x B/2 (were A is the width and B is the length). To evaluate the BPA uptake, animals

Both sides of the same coin: Rac1 splicing regulating by EGF signaling.

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Fu, Xiang-Dong

2017-04-01

EGF, a well-studied mitogen for cancer cells, is revealed to induce an E3 ubiquitin ligase adaptor SPSB1, which recruits the Elongin B/C-Collin complex to trigger ubiquitylation of the negative splicing regulator hnRNP A1. This event is synergized with EGF-activated SR proteins to alter alternative splicing of a key small GTPase Rac1 to enhance cell migration, highlighting converging EGF signals on both negative and positive splicing regulators to jointly promote a key cancer pathway.

DMBT1 expression is down-regulated in breast cancer

International Nuclear Information System (INIS)

Braidotti, P; Pietra, GG; Nuciforo, PG; Mollenhauer, J; Poustka, A; Pellegrini, C; Moro, A; Bulfamante, G; Coggi, G; Bosari, S

2004-01-01

We studied the expression of DMBT1 (deleted in malignant brain tumor 1), a putative tumor suppressor gene, in normal, proliferative, and malignant breast epithelium and its possible relation to cell cycle. Sections from 17 benign lesions and 55 carcinomas were immunostained with anti DMBT1 antibody (DMBTh12) and sections from 36 samples, were double-stained also with anti MCM5, one of the 6 pre-replicative complex proteins with cell proliferation-licensing functions. DMBT1 gene expression at mRNA level was assessed by RT-PCR in frozen tissues samples from 39 patients. Normal glands and hyperplastic epithelium in benign lesions displayed a luminal polarized DMBTh12 immunoreactivity. Normal and hyperplastic epithelium adjacent to carcinomas showed a loss of polarization, with immunostaining present in basal and perinuclear cytoplasmic compartments. DMBT1 protein expression was down-regulated in the cancerous lesions compared to the normal and/or hyperplastic epithelium adjacent to carcinomas (3/55 positive carcinomas versus 33/42 positive normal/hyperplastic epithelia; p = 0.0001). In 72% of cases RT-PCR confirmed immunohistochemical results. Most of normal and hyperplastic mammary cells positive with DMBTh12 were also MCM5-positive. The redistribution and up-regulation of DMBT1 in normal and hyperplastic tissues flanking malignant tumours and its down-regulation in carcinomas suggests a potential role in breast cancer. Moreover, the concomitant expression of DMTB1 and MCM5 suggests its possible association with the cell-cycle regulation

Stimulus-dependent regulation of the phagocyte NADPH oxidase by a VAV1, Rac1, and PAK1 signaling axis

DEFF Research Database (Denmark)

Roepstorff, Kirstine; Rasmussen, Izabela Zorawska; Sawada, Makoto

2008-01-01

dominant-positive mutants enhanced, whereas dominant-negative mutants inhibited, NADPH oxidase-mediated superoxide generation following formyl-methionyl-leucylphenylalanine or phorbol 12-myristate 13-acetate stimulation. Both Rac1 and the GTP exchange factor VAV1 were required as upstream signaling......The p21-activated kinase-1 (PAK1) is best known for its role in the regulation of cytoskeletal and transcriptional signaling pathways. We show here in the microglia cell line Ra2 that PAK1 regulates NADPH oxidase (NOX-2) activity in a stimulus-specific manner. Thus, conditional expression of PAK1...... proteins in the formyl-methionyl-leucyl-phenylalanine-induced activation of endogenous PAK1. In contrast, PAK1 mutants had no effect on superoxide generation downstream of FcgammaR signaling during phagocytosis of IgG-immune complexes. We further present evidence that the effect of PAK1 on the respiratory...

FANCI Regulates Recruitment of the FA Core Complex at Sites of DNA Damage Independently of FANCD2.

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Maria Castella

2015-10-01

Full Text Available The Fanconi anemia (FA-BRCA pathway mediates repair of DNA interstrand crosslinks. The FA core complex, a multi-subunit ubiquitin ligase, participates in the detection of DNA lesions and monoubiquitinates two downstream FA proteins, FANCD2 and FANCI (or the ID complex. However, the regulation of the FA core complex itself is poorly understood. Here we show that the FA core complex proteins are recruited to sites of DNA damage and form nuclear foci in S and G2 phases of the cell cycle. ATR kinase activity, an intact FA core complex and FANCM-FAAP24 were crucial for this recruitment. Surprisingly, FANCI, but not its partner FANCD2, was needed for efficient FA core complex foci formation. Monoubiquitination or ATR-dependent phosphorylation of FANCI were not required for the FA core complex recruitment, but FANCI deubiquitination by USP1 was. Additionally, BRCA1 was required for efficient FA core complex foci formation. These findings indicate that FANCI functions upstream of FA core complex recruitment independently of FANCD2, and alter the current view of the FA-BRCA pathway.

Estrogenic Endocrine Disrupting Chemicals Influencing NRF1 Regulated Gene Networks in the Development of Complex Human Brain Diseases.

Science.gov (United States)

Preciados, Mark; Yoo, Changwon; Roy, Deodutta

2016-12-13

signaling pathways. Our findings suggest that in addition to estrogen signaling, EEDs influencing NRF1 regulated communities of genes across genomic and epigenomic multiple networks may contribute in the development of complex chronic human brain health disorders.

Estrogenic Endocrine Disrupting Chemicals Influencing NRF1 Regulated Gene Networks in the Development of Complex Human Brain Diseases

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Mark Preciados

2016-12-01

findings suggest that in addition to estrogen signaling, EEDs influencing NRF1 regulated communities of genes across genomic and epigenomic multiple networks may contribute in the development of complex chronic human brain health disorders.

BMAL1-dependent regulation of the mTOR signaling pathway delays aging.

Science.gov (United States)

Khapre, Rohini V; Kondratova, Anna A; Patel, Sonal; Dubrovsky, Yuliya; Wrobel, Michelle; Antoch, Marina P; Kondratov, Roman V

2014-01-01

The circadian clock, an internal time-keeping system, has been linked with control of aging, but molecular mechanisms of regulation are not known. BMAL1 is a transcriptional factor and core component of the circadian clock; BMAL1 deficiency is associated with premature aging and reduced lifespan. Here we report that activity of mammalian Target of Rapamycin Complex 1 (mTORC1) is increased upon BMAL1 deficiency both in vivo and in cell culture. Increased mTOR signaling is associated with accelerated aging; in accordance with that, treatment with the mTORC1 inhibitor rapamycin increased lifespan of Bmal1-/- mice by 50%. Our data suggest that BMAL1 is a negative regulator of mTORC1 signaling. We propose that the circadian clock controls the activity of the mTOR pathway through BMAL1-dependent mechanisms and this regulation is important for control of aging and metabolism.

The Jasmonate-ZIM-domain proteins interact with the WD-Repeat/bHLH/MYB complexes to regulate Jasmonate-mediated anthocyanin accumulation and trichome initiation in Arabidopsis thaliana.

Science.gov (United States)

Qi, Tiancong; Song, Susheng; Ren, Qingcuo; Wu, Dewei; Huang, Huang; Chen, Yan; Fan, Meng; Peng, Wen; Ren, Chunmei; Xie, Daoxin

2011-05-01

Jasmonates (JAs) mediate plant responses to insect attack, wounding, pathogen infection, stress, and UV damage and regulate plant fertility, anthocyanin accumulation, trichome formation, and many other plant developmental processes. Arabidopsis thaliana Jasmonate ZIM-domain (JAZ) proteins, substrates of the CORONATINE INSENSITIVE1 (COI1)-based SCF(COI1) complex, negatively regulate these plant responses. Little is known about the molecular mechanism for JA regulation of anthocyanin accumulation and trichome initiation. In this study, we revealed that JAZ proteins interact with bHLH (Transparent Testa8, Glabra3 [GL3], and Enhancer of Glabra3 [EGL3]) and R2R3 MYB transcription factors (MYB75 and Glabra1), essential components of WD-repeat/bHLH/MYB transcriptional complexes, to repress JA-regulated anthocyanin accumulation and trichome initiation. Genetic and physiological evidence showed that JA regulates WD-repeat/bHLH/MYB complex-mediated anthocyanin accumulation and trichome initiation in a COI1-dependent manner. Overexpression of the MYB transcription factor MYB75 and bHLH factors (GL3 and EGL3) restored anthocyanin accumulation and trichome initiation in the coi1 mutant, respectively. We speculate that the JA-induced degradation of JAZ proteins abolishes the interactions of JAZ proteins with bHLH and MYB factors, allowing the transcriptional function of WD-repeat/bHLH/MYB complexes, which subsequently activate respective downstream signal cascades to modulate anthocyanin accumulation and trichome initiation.

PRC1 Prevents Replication Stress during Chondrogenic Transit Amplification

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Frank Spaapen

2017-12-01

Full Text Available Transit amplification (TA, a state of combined, rapid proliferative expansion and differentiation of stem cell-descendants, remains poorly defined at the molecular level. The Polycomb Repressive Complex 1 (PRC1 protein BMI1 has been localized to TA compartments, yet its exact role in TA is unclear. PRC1 proteins control gene expression, cell proliferation and DNA-damage repair. Coordination of such DNA-templated activities during TA is predicted to be crucial to support DNA replication and differentiation-associated transcriptional programming. We here examined whether chondrogenesis provides a relevant biological context for synchronized coordination of these chromatin-based tasks by BMI1. Taking advantage of a prominently featuring TA-phase during chondrogenesis in vitro and in vivo, we here report that TA is completely dependent on intact PRC1 function. BMI1-depleted chondrogenic progenitors rapidly accumulate double strand DNA breaks during DNA replication, present massive non-H3K27me3-directed transcriptional deregulation and fail to undergo chondrogenic TA. Genome-wide accumulation of Topoisomerase 2α and Geminin suggests a model in which PRC1 synchronizes replication and transcription during rapid chondrogenic progenitor expansion. Our combined data reveals for the first time a vital cell-autonomous role for PRC1 during chondrogenesis. We provide evidence that chondrocyte hyper-replication and hypertrophy represent a unique example of programmed senescence in vivo. These findings provide new perspectives on PRC1 function in development and disease.

Differential regulation of the cellular response to DNA double-strand breaks in G1

DEFF Research Database (Denmark)

Barlow, Jacqueline H; Lisby, Michael; Rothstein, Rodney

2008-01-01

-induced breaks are recognized by Rfa1 only after the cell enters S phase. This difference is dependent on the DNA end-binding Yku70/Yku80 complex. Cell-cycle regulation is also observed in the DNA damage checkpoint response. Specifically, the 9-1-1 complex is required in G1 cells to recruit the Ddc2 checkpoint...... protein to damaged DNA, while, upon entry into S phase, the cyclin-dependent kinase Cdc28 and the 9-1-1 complex both serve to recruit Ddc2 to foci. Together, these results demonstrate that the DNA repair machinery distinguishes between different types of damage in G1, which translates into different modes...

Nuclear adaptor Ldb1 regulates a transcriptional program essential for the maintenance of hematopoietic stem cells.

Science.gov (United States)

Li, LiQi; Jothi, Raja; Cui, Kairong; Lee, Jan Y; Cohen, Tsadok; Gorivodsky, Marat; Tzchori, Itai; Zhao, Yangu; Hayes, Sandra M; Bresnick, Emery H; Zhao, Keji; Westphal, Heiner; Love, Paul E

2011-02-01

The nuclear adaptor Ldb1 functions as a core component of multiprotein transcription complexes that regulate differentiation in diverse cell types. In the hematopoietic lineage, Ldb1 forms a complex with the non-DNA-binding adaptor Lmo2 and the transcription factors E2A, Scl and GATA-1 (or GATA-2). Here we demonstrate a critical and continuous requirement for Ldb1 in the maintenance of both fetal and adult mouse hematopoietic stem cells (HSCs). Deletion of Ldb1 in hematopoietic progenitors resulted in the downregulation of many transcripts required for HSC maintenance. Genome-wide profiling by chromatin immunoprecipitation followed by sequencing (ChIP-Seq) identified Ldb1 complex-binding sites at highly conserved regions in the promoters of genes involved in HSC maintenance. Our results identify a central role for Ldb1 in regulating the transcriptional program responsible for the maintenance of HSCs.

Ambition, Regulation and Reality. Complex use of land and water resources in Luwu, South Sulawesi, Indonesia

NARCIS (Netherlands)

Roth, D.

2003-01-01

In this book I present three case studies of the complex regulation of use of land and water resources in Luwu. Attention to the role of legalcomplexity -the existence of different sources and definitions of normative-legal regulation in

Identification of chromatin-associated regulators of MSL complex targeting in Drosophila dosage compensation.

Directory of Open Access Journals (Sweden)

Erica Larschan

Full Text Available Sex chromosome dosage compensation in Drosophila provides a model for understanding how chromatin organization can modulate coordinate gene regulation. Male Drosophila increase the transcript levels of genes on the single male X approximately two-fold to equal the gene expression in females, which have two X-chromosomes. Dosage compensation is mediated by the Male-Specific Lethal (MSL histone acetyltransferase complex. Five core components of the MSL complex were identified by genetic screens for genes that are specifically required for male viability and are dispensable for females. However, because dosage compensation must interface with the general transcriptional machinery, it is likely that identifying additional regulators that are not strictly male-specific will be key to understanding the process at a mechanistic level. Such regulators would not have been recovered from previous male-specific lethal screening strategies. Therefore, we have performed a cell culture-based, genome-wide RNAi screen to search for factors required for MSL targeting or function. Here we focus on the discovery of proteins that function to promote MSL complex recruitment to "chromatin entry sites," which are proposed to be the initial sites of MSL targeting. We find that components of the NSL (Non-specific lethal complex, and a previously unstudied zinc-finger protein, facilitate MSL targeting and display a striking enrichment at MSL entry sites. Identification of these factors provides new insight into how MSL complex establishes the specialized hyperactive chromatin required for dosage compensation in Drosophila.

Spatio-temporal regulation of Hsp90-ligand complex leads to immune activation.

Directory of Open Access Journals (Sweden)

Yasuaki eTamura

2016-05-01

Full Text Available Hsp90 is the most abundant cytosolic HSP and is known to act as a molecular chaperone. We found that an Hsp90-cancer antigen peptide complex was efficiently cross-presented by human monocyte-derived dendritic cells and induced peptide-specific cytotoxic T lymphocytes. Furthermore, we observed that the internalized Hsp90-peptide complex was strictly sorted to the Rab5+, EEA1+ static early endosome and the Hsp90-chaperoned peptide was processed and bound to MHC class I molecules through a endosome-recycling pathway. We also found that extracellular Hsp90 complexed with CpG-A or self-DNA stimulates production of a large amount of IFN-α from pDCs via static early endosome targeting. Thus, extracellular Hsp90 can target the antigen or nucleic acid to a static early endosome by spatio-temporal regulation. Moreover, we showed that Hsp90 associates with and delivers TLR7/9 from the ER to early endosomes for ligand recognition. Hsp90 inhibitor, geldanamycin derivative inhibited the Hsp90 association with TLR7/9, resulting in inhibition IFN-α production, leading to improvement of SLE symptoms. Interstingly, we observed that serum Hsp90 is clearly increased in patients with active SLE compared with that in patients with inactive disease. Serum Hsp90 detected in SLE patients binds to self-DNA and/or anti-DNA Ab, thus leading to stimulation of pDCs to produce IFN-α. Thus, Hsp90 plays a crucial role in the pathogenesis of SLE and that an Hsp90 inhibitor will therefore provide a new therapeutic approach to SLE and other nucleic acid-related autoimmune diseases. We will discuss how spatio-temporal regulation of Hsp90-ligand complexes within antigen-presenting cells affects the innate immunity and adaptive immunity.

Ambition, Regulation and Reality. Complex use of land and water resources in Luwu, South Sulawesi, Indonesia

NARCIS (Netherlands)

Roth, D.

2003-01-01

In this book I present three case studies of the complex regulation of use of land and water resources in Luwu. Attention to the role of legalcomplexity -the existence of different sources and definitions of normative-legal regulation in

Rictor forms a complex with Cullin-1 to promote SGK1 ubiquitination and destruction

Science.gov (United States)

Gao, Daming; Wan, Lixin; Inuzuka, Hiroyuki; Berg, Anders H.; Tseng, Alan; Zhai, Bo; Shaik, Shavali; Bennett, Eric; Tron, Adriana E.; Gasser, Jessica A.; Lau, Alan; Gygi, Steven; Harper, J. Wade; DeCaprio, James A.; Toker, Alex; Wei, Wenyi

2010-01-01

Summary The Rictor/mTOR complex (also known as mTORC2) plays a critical role in cellular homeostasis by phosphorylating AGC kinases such as Akt and SGK at their hydrophobic motifs to activate downstream signaling. However, the regulation of mTORC2 and whether it has additional function(s), remains largely unknown. Here we report that Rictor associates with Cullin-1 to form a functional E3 ubiquitin ligase. Rictor, but not Raptor or mTOR alone promotes SGK1 ubiquitination. Loss of Rictor/Cullin-1-mediated ubiquitination leads to increased SGK1 protein levels as detected in Rictor null cells. Moreover, as part of a feedback mechanism, phosphorylation of Rictor at T1135 by multiple AGC kinases disrupts the interaction between Rictor and Cullin-1 to impair SGK1 ubiquitination. These findings indicate that the Rictor/Cullin-1 E3 ligase activity is regulated by a specific signal relay cascade and that misregulation of this mechanism may contribute to the frequent overexpression of SGK1 in various human cancers. PMID:20832730

Sestrin2 inhibits mTORC1 through modulation of GATOR complexes

Energy Technology Data Exchange (ETDEWEB)

Kim, Jeong Sig; Ro, Seung-Hyun; Kim, Myungjin; Park, Hwan-Woo; Semple, Ian A.; Park, Haeli; Cho, Uhn-Soo; Wang, Wei; Guan, Kun-Liang; Karin, Michael; Lee, Jun Hee (Michigan); (UCSD)

2015-03-30

Sestrins are stress-inducible metabolic regulators that suppress a wide range of age- and obesity-associated pathologies, many of which are due to mTORC1 overactivation. Upon various stresses, the Sestrins inhibit mTORC1 activity through an indirect mechanism that is still unclear. GATORs are recently identified protein complexes that regulate the activity of RagB, a small GTPase essential for mTORC1 activation. GATOR1 is a GTPase activating protein (GAP) for RagB whereas GATOR2 functions as an inhibitor of GATOR1. However, how the GATORs are physiologically regulated is unknown. Here we show that Sestrin2 binds to GATOR2, and liberates GATOR1 from GATOR2-mediated inhibition. Released GATOR1 subsequently binds to and inactivates RagB, ultimately resulting in mTORC1 suppression. Consistent with this biochemical mechanism, genetic ablation of GATOR1 nullifies the mTORC1-inhibiting effect of Sestrin2 in both cell culture and Drosophila models. Collectively, we elucidate a new signaling cascade composed of Sestrin2-GATOR2-GATOR1-RagB that mediates stress-dependent suppression of mTORC1 activity.

Fijación de radioyodo en huesos maxilares simulando metástasis en pacientes con Carcinoma Diferenciado de Tiroides (CDT): False- Positive images in patients with Differentiated Thyroid Carcinoma. (DTC)

OpenAIRE

Degrossi, O. J.; Gutiérrez, S.; Fadel, A.; Degrossi, E. B.; Valdivieso, M. C.; Balbuena, R. L.; Alak, M. del C.; de Cabrejas, M.

2008-01-01

En estudios centellográficos con 131I (CCT) para seguimiento, postablación o tratamiento de pacientes portadores de carcinoma diferenciado de tiroides (CDT) se observan frecuentemente en tiempos precoces áreas de captación del radiotrazador en macizo facial. Estas áreas corresponden, generalmente, a glándulas salivares y extremo anterior de mucosa nasal y se mantienen durante las primeras 48 horas y no son observadas generalmente a las 72 horas. Pero con menor frecuencia se presentan otras qu...

Regulation of mariner transposition: the peculiar case of Mos1.

Directory of Open Access Journals (Sweden)

Jérôme Jaillet

Full Text Available BACKGROUND: Mariner elements represent the most successful family of autonomous DNA transposons, being present in various plant and animal genomes, including humans. The introduction and co-evolution of mariners within host genomes imply a strict regulation of the transposon activity. Biochemical data accumulated during the past decade have led to a convergent picture of the transposition cycle of mariner elements, suggesting that mariner transposition does not rely on host-specific factors. This model does not account for differences of transposition efficiency in human cells between mariners. We thus wondered whether apparent similarities in transposition cycle could hide differences in the intrinsic parameters that control mariner transposition. PRINCIPAL FINDINGS: We find that Mos1 transposase concentrations in excess to the Mos1 ends prevent the paired-end complex assembly. However, we observe that Mos1 transposition is not impaired by transposase high concentration, dismissing the idea that transposase over production plays an obligatory role in the down-regulation of mariner transposition. Our main finding is that the paired-end complex is formed in a cooperative way, regardless of the transposase concentration. We also show that an element framed by two identical ITRs (Inverted Terminal Repeats is more efficient in driving transposition than an element framed by two different ITRs (i.e. the natural Mos1 copy, the latter being more sensitive to transposase concentration variations. Finally, we show that the current Mos1 ITRs correspond to the ancestral ones. CONCLUSIONS: We provide new insights on intrinsic properties supporting the self-regulation of the Mos1 element. These properties (transposase specific activity, aggregation, ITR sequences, transposase concentration/transposon copy number ratio... could have played a role in the dynamics of host-genomes invasion by Mos1, accounting (at least in part for the current low copy number of

Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

KAUST Repository

Ková cs, Krisztiá n A.; Steinmann, Myriam; Halfon, Olivier; Magistretti, Pierre J.; Cardinaux, Jean René

2015-01-01

CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

KAUST Repository

Kovács, Krisztián A.

2015-11-01

CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

Sequential multisite phospho-regulation of KNL1-BUB3 interfaces at mitotic kinetochores

NARCIS (Netherlands)

Vleugel, Mathijs; Omerzu, Manja; Groenewold, Vincent; Hadders, Michael A; Lens, Susanne M A; Kops, Geert J P L

2015-01-01

Regulated recruitment of the kinase-adaptor complex BUB1/BUB3 to kinetochores is crucial for correcting faulty chromosome-spindle attachments and for spindle assembly checkpoint (SAC) signaling. BUB1/BUB3 localizes to kinetochores by binding phosphorylated MELT motifs (MELpT) in the kinetochore

Matrix metalloproteinase-1 facilitates MSC migration via cleavage of IGF-2/IGFBP2 complex.

Science.gov (United States)

Guan, Shou P; Lam, Alan T L; Newman, Jennifer P; Chua, Kevin L M; Kok, Catherine Y L; Chong, Siao T; Chua, Melvin L K; Lam, Paula Y P

2018-01-01

The specific mechanism underlying the tumor tropism of human mesenchymal stem cells (MSCs) for cancer is not well defined. We previously showed that the migration potential of MSCs correlated with the expression and protease activity of matrix metalloproteinase (MMP)-1. Furthermore, highly tumor-tropic MSCs expressed higher levels of MMP-1 and insulin-like growth factor (IGF)-2 than poorly migrating MSCs. In this study, we examined the functional roles of IGF-2 and MMP-1 in mediating the tumor tropism of MSCs. Exogenous addition of either recombinant IGF-2 or MMP-1 could stimulate MSC migration. The correlation between IGF-2, MMP-1 expression, and MSC migration suggests that MMP-1 may play a role in regulating MSC migration via the IGF-2 signaling cascade. High concentrations of IGF binding proteins (IGFBPs) can inhibit IGF-stimulated functions by blocking its binding to its receptors and proteolysis of IGFBP is an important mechanism for the regulation of IGF signaling. We thus hypothesized that MMP-1 acts as an IGFBP2 proteinase, resulting in the cleavage of IGF-2/IGFBP2 complex and extracellular release of free IGF-2. Indeed, our results showed that conditioned media from highly migrating MSCs, which expressed high levels of MMP-1, cleaved the IGF-2/IGFBP2 complex. Taken together, these results showed that the MMP-1 secreted by highly tumor-tropic MSCs cleaved IGF-2/IGFBP2 complex. Free IGF-2 released from the complex may facilitate MSC migration toward tumor.

HTLV-1 Tax protein recruitment into IKKε and TBK1 kinase complexes enhances IFN-I expression.

Science.gov (United States)

Diani, Erica; Avesani, Francesca; Bergamo, Elisa; Cremonese, Giorgia; Bertazzoni, Umberto; Romanelli, Maria Grazia

2015-02-01

The Tax protein expressed by human T-cell leukemia virus type 1 (HTLV-1) plays a pivotal role in the deregulation of cellular pathways involved in the immune response, inflammation, cell survival, and cancer. Many of these effects derive from Tax multiple interactions with host factors, including the subunits of the IKK-complex that are required for NF-κB activation. IKKɛ and TBK1 are two IKK-related kinases that allow the phosphorylation of interferon regulatory factors that trigger IFN type I gene expression. We observed that IKKɛ and TBK1 recruit Tax into cellular immunocomplexes. We also found that TRAF3, which regulates cell receptor signaling effectors, forms complexes with Tax. Transactivation analyses revealed that expression of Tax, in presence of IKKɛ and TBK1, enhances IFN-β promoter activity, whereas the activation of NF-κB promoter is not modified. We propose that Tax may be recruited into the TBK1/IKKɛ complexes as a scaffolding-adaptor protein that enhances IFN-I gene expression. Copyright © 2014 Elsevier Inc. All rights reserved.

Convergence of the mammalian target of rapamycin complex 1- and glycogen synthase kinase 3-β-signaling pathways regulates the innate inflammatory response.

Science.gov (United States)

Wang, Huizhi; Brown, Jonathan; Gu, Zhen; Garcia, Carlos A; Liang, Ruqiang; Alard, Pascale; Beurel, Eléonore; Jope, Richard S; Greenway, Terrance; Martin, Michael

2011-05-01

The PI3K pathway and its regulation of mammalian target of rapamycin complex 1 (mTORC1) and glycogen synthase kinase 3 (GSK3) play pivotal roles in controlling inflammation. In this article, we show that mTORC1 and GSK3-β converge and that the capacity of mTORC1 to affect the inflammatory response is due to the inactivation of GSK3-β. Inhibition of mTORC1 attenuated GSK3 phosphorylation and increased its kinase activity. Immunoprecipitation and in vitro kinase assays demonstrated that GSK3-β associated with a downstream target of mTORC1, p85S6K, and phosphorylated GSK3-β. Inhibition of S6K1 abrogated the phosphorylation of GSK3-β while increasing and decreasing the levels of IL-12 and IL-10, respectively, in LPS-stimulated monocytes. In contrast, the direct inhibition of GSK3 attenuated the capacity of S6K1 inhibition to influence the levels of IL-10 and IL-12 produced by LPS-stimulated cells. At the transcriptional level, mTORC1 inhibition reduced the DNA binding of CREB and this effect was reversed by GSK3 inhibition. As a result, mTORC1 inhibition increased the levels of NF-κB p65 associated with CREB-binding protein. Inhibition of NF-κB p65 attenuated rapamycin's ability to influence the levels of pro- or anti-inflammatory cytokine production in monocytes stimulated with LPS. These studies identify the molecular mechanism by which mTORC1 affects GSK3 and show that mTORC1 inhibition regulates pro- and anti-inflammatory cytokine production via its capacity to inactivate GSK3.

Rheb may complex with RASSF1A to coordinate Hippo and TOR signaling.

Science.gov (United States)

Nelson, Nicholas; Clark, Geoffrey J

2016-06-07

The TOR pathway is a vital component of cellular homeostasis that controls the synthesis of proteins, nucleic acids and lipids. Its core is the TOR kinase. Activation of the TOR pathway suppresses autophagy, which plays a vital but complex role in tumorigenesis. The TOR pathway is regulated by activation of the Ras-related protein Rheb, which can bind mTOR. The Hippo pathway is a major growth control module that regulates cell growth, differentiation and apoptosis. Its core consists of an MST/LATS kinase cascade that can be activated by the RASSF1A tumor suppressor. The TOR and Hippo pathways may be coordinately regulated to promote cellular homeostasis. However, the links between the pathways remain only partially understood. We now demonstrate that in addition to mTOR regulation, Rheb also impacts the Hippo pathway by forming a complex with RASSF1A. Using stable clones of two human lung tumor cell lines (NCI-H1792 and NCI-H1299) with shRNA-mediated silencing or ectopic overexpression of RASSF1A, we show that activated Rheb stimulates the Hippo pathway, but is suppressed in its ability to stimulate the TOR pathway. Moreover, by selectively labeling autophagic vacuoles we show that RASSF1A inhibits the ability of Rheb to suppress autophagy and enhance cell growth. Thus, we identify a new connection that impacts coordination of Hippo and TOR signaling. As RASSF1A expression is frequently lost in human tumors, the RASSF1A status of a tumor may impact not just its Hippo pathway status, but also its TOR pathway status.

Crystallization and preliminary crystallographic analysis of the transcriptional regulator RfaH from Escherichia coli and its complex with ops DNA

International Nuclear Information System (INIS)

Vassylyeva, Marina N.; Svetlov, Vladimir; Klyuyev, Sergiy; Devedjiev, Yancho D.; Artsimovitch, Irina; Vassylyev, Dmitry G.

2006-01-01

The E. coli transcriptional regulator RfaH was cloned, expressed, purified and crystallized and the complex of RfaH with its target DNA oligonucleotide was cocrystallized. Complete diffraction data sets were collected for the apo protein and its nucleic acid complex at 2.4 and at 1.6 Å resolution, respectively. The bacterial transcriptional factor and virulence regulator RfaH binds to rapidly moving transcription elongation complexes through specific interactions with the exposed segment of the non-template DNA strand. To elucidate this unusual mechanism of recruitment, determination of the three-dimensional structure of RfaH and its complex with DNA was initiated. To this end, the Escherichia coli rfaH gene was cloned and expressed. The purified protein was crystallized by the sitting-drop vapor-diffusion technique. The space group was P6 1 22 or P6 5 22, with unit-cell parameters a = b = 45.46, c = 599.93 Å. A complex of RfaH and a nine-nucleotide oligodeoxyribonucleotide was crystallized by the same technique, but under different crystallization conditions, yielding crystals that belonged to space group P1 (unit-cell parameters a = 36.79, b = 44.01, c = 62.37 Å, α = 80.62, β = 75.37, γ = 75.41°). Complete diffraction data sets were collected for RfaH and its complex with DNA at 2.4 and 1.6 Å resolution, respectively. Crystals of selenomethionine-labeled proteins in both crystal forms were obtained by cross-microseeding using the native microcrystals. The structure determination of RfaH and its complex with DNA is in progress

Prereplicative complexes assembled in vitro support origin-dependent and independent DNA replication

Science.gov (United States)

On, Kin Fan; Beuron, Fabienne; Frith, David; Snijders, Ambrosius P; Morris, Edward P; Diffley, John F X

2014-01-01

Eukaryotic DNA replication initiates from multiple replication origins. To ensure each origin fires just once per cell cycle, initiation is divided into two biochemically discrete steps: the Mcm2-7 helicase is first loaded into prereplicative complexes (pre-RCs) as an inactive double hexamer by the origin recognition complex (ORC), Cdt1 and Cdc6; the helicase is then activated by a set of “firing factors.” Here, we show that plasmids containing pre-RCs assembled with purified proteins support complete and semi-conservative replication in extracts from budding yeast cells overexpressing firing factors. Replication requires cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK). DDK phosphorylation of Mcm2-7 does not by itself promote separation of the double hexamer, but is required for the recruitment of firing factors and replisome components in the extract. Plasmid replication does not require a functional replication origin; however, in the presence of competitor DNA and limiting ORC concentrations, replication becomes origin-dependent in this system. These experiments indicate that Mcm2-7 double hexamers can be precursors of replication and provide insight into the nature of eukaryotic DNA replication origins. PMID:24566989

Meier-Gorlin syndrome: Growth and secondary sexual development of a microcephalic primordial dwarfism disorder

NARCIS (Netherlands)

de Munnik, S.A.; Otten, B.J.; Schoots, J.; Bicknell, L.S.; Aftimos, S.; Al-Aama, J.Y.; van Bever, Y.; Bober, M.B.; Borm, G.F.; Clayton-Smith, J.; Deal, C.L.; Edrees, A.Y.; Feingold, M.; Fryer, A.; van Hagen, J.M.; Hennekam, R.C.M.; Jansweijer, M.C.E.; Johnson, D.; Kant, S.G.; Opitz, J.M.; Ramadevi, A.R.; Reardon, W.; Ross, A.; Sarda, P.; Schrander-Stumpel, C.T.R.M.; Sluiter, A.E.; Temple, I.K.; Terhal, P.A.; Toutain, A.; Wise, C.A.; Wright, M.; Skidmore, D.L.; Samuels, M.E.; Hoefsloot, L.H.; Knoers, N.V.A.M.; Brunner, H.G.; Jackson, A.P.; Bongers, M.H.F.

2012-01-01

Meier-Gorlin syndrome (MGS) is a rare autosomal recessive disorder characterized by primordial dwarfism, microtia, and patellar aplasia/hypoplasia. Recently, mutations in the ORC1, ORC4, ORC6, CDT1, and CDC6 genes, encoding components of the pre-replication complex, have been identified. This

The protein phosphatase-1/inhibitor-2 complex differentially regulates GSK3 dephosphorylation and increases sarcoplasmic/endoplasmic reticulum calcium ATPase 2 levels

International Nuclear Information System (INIS)

King, Taj D.; Gandy, Johanna C.; Bijur, Gautam N.

2006-01-01

The ubiquitously expressed protein glycogen synthase kinase-3 (GSK3) is constitutively active, however its activity is markedly diminished following phosphorylation of Ser21 of GSK3α and Ser9 of GSK3β. Although several kinases are known to phosphorylate Ser21/9 of GSK3, for example Akt, relatively much less is known about the mechanisms that cause the dephosphorylation of GSK3 at Ser21/9. In the present study KCl-induced plasma membrane depolarization of SH-SY5Y cells, which increases intracellular calcium concentrations caused a transient decrease in the phosphorylation of Akt at Thr308 and Ser473, and GSK3 at Ser21/9. Overexpression of the selective protein phosphatase-1 inhibitor protein, inhibitor-2, increased basal GSK3 phosphorylation at Ser21/9 and significantly blocked the KCl-induced dephosphorylation of GSK3β, but not GSK3α. The phosphorylation of Akt was not affected by the overexpression of inhibitor-2. GSK3 activity is known to affect sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) levels. Overexpression of inhibitor-2 or treatment of cells with the GSK3 inhibitors lithium and SB216763 increased the levels of SERCA2. These results indicate that the protein phosphatase-1/inhibitor-2 complex differentially regulates GSK3 dephosphorylation induced by KCl and that GSK3 activity regulates SERCA2 levels

JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships

Science.gov (United States)

Zeke, András; Misheva, Mariya

2016-01-01

SUMMARY The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states. PMID:27466283

Complex formation of EphB1/Nck/Caskin1 leads to tyrosine phosphorylation and structural changes of the Caskin1 SH3 domain

Directory of Open Access Journals (Sweden)

Pesti Szabolcs

2012-11-01

Full Text Available Abstract Background Scaffold proteins have an important role in the regulation of signal propagation. These proteins do not possess any enzymatic activity but can contribute to the formation of multiprotein complexes. Although scaffold proteins are present in all cell types, the nervous system contains them in the largest amount. Caskin proteins are typically present in neuronal cells, particularly, in the synapses. However, the signaling mechanisms by which Caskin proteins are regulated are largely unknown. Results Here we demonstrate that EphB1 receptor tyrosine kinase can recruit Caskin1 through the adaptor protein Nck. Upon activation of the receptor kinase, the SH2 domain of Nck binds to one of its tyrosine residues, while Nck SH3 domains interact with the proline-rich domain of Caskin1. Complex formation of the receptor, adaptor and scaffold proteins results in the tyrosine phosphorylation of Caskin1 on its SH3 domain. The phosphorylation sites were identified by mass-spectrometry as tyrosines 296 and 336. To reveal the structural consequence of this phosphorylation, CD spectroscopy was performed. This measurement suggests that upon tyrosine phosphorylation the structure of the Caskin1 SH3 domain changes significantly. Conclusion Taken together, we propose that the scaffold protein Caskin1 can form a complex with the EphB1 tyrosine kinase via the Nck protein as a linker. Complex formation results in tyrosine phosphorylation of the Caskin1 SH3 domain. Although we were not able to identify any physiological partner of the SH3 domain so far, we could demonstrate that phosphorylation on conserved tyrosine residues results in marked changes in the structure of the SH3 domain.

Ubiquitination regulates MHC class II-peptide complex retention and degradation in dendritic cells

OpenAIRE

Walseng, Even; Furuta, Kazuyuki; Bosch, Berta; Weih, Karis A.; Matsuki, Yohei; Bakke, Oddmund; Ishido, Satoshi; Roche, Paul A.

2010-01-01

The expression and turnover of MHC class II-peptide complexes (pMHC-II) on the surface of dendritic cells (DCs) is essential for their ability to activate CD4 T cells efficiently. The half-life of surface pMHC-II is significantly greater in activated (mature) DCs than in resting (immature) DCs, but the molecular mechanism leading to this difference remains unknown. We now show that ubiquitination of pMHC-II by the E3 ubiquitin ligase membrane-associated RING-CH 1 (March-I) regulates surface e...

Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury

Science.gov (United States)

Greune, Lilo; Jarosch, Kevin-André; Steil, Daniel; Zhang, Wenlan; He, Xiaohua; Lloubes, Roland; Fruth, Angelika; Kim, Kwang Sik; Schmidt, M. Alexander; Dobrindt, Ulrich; Mellmann, Alexander; Karch, Helge

2017-01-01

Outer membrane vesicles (OMVs) are important tools in bacterial virulence but their role in the pathogenesis of infections caused by enterohemorrhagic Escherichia coli (EHEC) O157, the leading cause of life-threatening hemolytic uremic syndrome, is poorly understood. Using proteomics, electron and confocal laser scanning microscopy, immunoblotting, and bioassays, we investigated OMVs secreted by EHEC O157 clinical isolates for virulence factors cargoes, interactions with pathogenetically relevant human cells, and mechanisms of cell injury. We demonstrate that O157 OMVs carry a cocktail of key virulence factors of EHEC O157 including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, and flagellin. The toxins are internalized by cells via dynamin-dependent endocytosis of OMVs and differentially separate from vesicles during intracellular trafficking. Stx2a and CdtV-B, the DNase-like CdtV subunit, separate from OMVs in early endosomes. Stx2a is trafficked, in association with its receptor globotriaosylceramide within detergent-resistant membranes, to the Golgi complex and the endoplasmic reticulum from where the catalytic Stx2a A1 fragment is translocated to the cytosol. CdtV-B is, after its retrograde transport to the endoplasmic reticulum, translocated to the nucleus to reach DNA. CdtV-A and CdtV-C subunits remain OMV-associated and are sorted with OMVs to lysosomes. EHEC hemolysin separates from OMVs in lysosomes and targets mitochondria. The OMV-delivered CdtV-B causes cellular DNA damage, which activates DNA damage responses leading to G2 cell cycle arrest. The arrested cells ultimately die of apoptosis induced by Stx2a and CdtV via caspase-9 activation. By demonstrating that naturally secreted EHEC O157 OMVs carry and deliver into cells a cocktail of biologically active virulence factors, thereby causing cell death, and by performing first comprehensive analysis of intracellular trafficking of OMVs and OMV-delivered virulence factors

Herp regulates Hrd1-mediated ubiquitylation in a ubiquitin-like domain-dependent manner

DEFF Research Database (Denmark)

Kny, Melanie; Standera, Sybille; Hartmann-Petersen, Rasmus

2011-01-01

in ER-associated protein degradation (ERAD) and interacts directly with the ubiquitin ligase Hrd1, which is found in high molecular mass complexes of the ER membrane. Here we present the first evidence that Herp regulates Hrd1-mediated ubiquitylation in a ubiquitin-like (UBL) domain-dependent manner. We...

Dock/Nck facilitates PTP61F/PTP1B regulation of insulin signalling.

Science.gov (United States)

Wu, Chia-Lun; Buszard, Bree; Teng, Chun-Hung; Chen, Wei-Lin; Warr, Coral G; Tiganis, Tony; Meng, Tzu-Ching

2011-10-01

PTP1B (protein tyrosine phosphatase 1B) is a negative regulator of IR (insulin receptor) activation and glucose homoeostasis, but the precise molecular mechanisms governing PTP1B substrate selectivity and the regulation of insulin signalling remain unclear. In the present study we have taken advantage of Drosophila as a model organism to establish the role of the SH3 (Src homology 3)/SH2 adaptor protein Dock (Dreadlocks) and its mammalian counterpart Nck in IR regulation by PTPs. We demonstrate that the PTP1B orthologue PTP61F dephosphorylates the Drosophila IR in S2 cells in vitro and attenuates IR-induced eye overgrowth in vivo. Our studies indicate that Dock forms a stable complex with PTP61F and that Dock/PTP61F associate with the IR in response to insulin. We report that Dock is required for effective IR dephosphorylation and inactivation by PTP61F in vitro and in vivo. Furthermore, we demonstrate that Nck interacts with PTP1B and that the Nck/PTP1B complex inducibly associates with the IR for the attenuation of IR activation in mammalian cells. Our studies reveal for the first time that the adaptor protein Dock/Nck attenuates insulin signalling by recruiting PTP61F/PTP1B to its substrate, the IR.

Interactions within the yeast t-SNARE Sso1p that control SNARE complex assembly.

Science.gov (United States)

Munson, M; Chen, X; Cocina, A E; Schultz, S M; Hughson, F M

2000-10-01

In the eukaryotic secretory and endocytic pathways, transport vesicles shuttle cargo among intracellular organelles and to and from the plasma membrane. Cargo delivery entails fusion of the transport vesicle with its target, a process thought to be mediated by membrane bridging SNARE protein complexes. Temporal and spatial control of intracellular trafficking depends in part on regulating the assembly of these complexes. In vitro, SNARE assembly is inhibited by the closed conformation adopted by the syntaxin family of SNAREs. To visualize this closed conformation directly, the X-ray crystal structure of a yeast syntaxin, Sso1p, has been determined and refined to 2.1 A resolution. Mutants designed to destabilize the closed conformation exhibit accelerated rates of SNARE assembly. Our results provide insight into the mechanism of SNARE assembly and its intramolecular and intermolecular regulation.

An MHC-I cytoplasmic domain/HIV-1 Nef fusion protein binds directly to the mu subunit of the AP-1 endosomal coat complex.

Directory of Open Access Journals (Sweden)

Rajendra Kumar Singh

2009-12-01

Full Text Available The down-regulation of the major histocompatibility complex class I (MHC-I from the surface of infected cells by the Nef proteins of primate immunodeficiency viruses likely contributes to pathogenesis by providing evasion of cell-mediated immunity. HIV-1 Nef-induced down-regulation involves endosomal trafficking and a cooperative interaction between the cytoplasmic domain (CD of MHC-I, Nef, and the clathrin adaptor protein complex-1 (AP-1. The CD of MHC-I contains a key tyrosine within the sequence YSQA that is required for down-regulation by Nef, but this sequence does not conform to the canonical AP-binding tyrosine-based motif Yxxphi, which mediates binding to the medium (micro subunits of AP complexes. We previously proposed that Nef allows the MHC-I CD to bind the mu subunit of AP-1 (micro1 as if it contained a Yxxphimotif.Here, we show that a direct interaction between the MHC-I CD/Nef and micro1 plays a primary role in the down-regulation of MHC-I: GST pulldown assays using recombinant proteins indicated that most of the MHC-I CD and Nef residues that are required for the down-regulation in human cells contribute to direct interactions with a truncated version of micro1. Specifically, the tyrosine residue of the YSQA sequence in the MHC-I CD as well as Nef residues E62-65 and P78 each contributed to the interaction between MHC-I CD/Nef and micro1 in vitro, whereas Nef M20 had little to no role. Conversely, residues F172/D174 and V392/L395 of the binding pocket on micro1 for Yxxphi motifs were required for a robust interaction.These data indicate that the MHC-I cytoplasmic domain, Nef, and the C-terminal two thirds of the mu subunit of AP-1 are sufficient to constitute a biologically relevant interaction. The data also reveal an unexpected role for a hydrophobic pocket in micro1 for interaction with MHC-I CD/Nef.

Overexpression of the PAP1 transcription factor reveals a complex regulation of flavonoid and phenylpropanoid metabolism in Nicotiana tabacum plants attacked by Spodoptera litura.

Science.gov (United States)

Mitsunami, Tomoko; Nishihara, Masahiro; Galis, Ivan; Alamgir, Kabir Md; Hojo, Yuko; Fujita, Kohei; Sasaki, Nobuhiro; Nemoto, Keichiro; Sawasaki, Tatsuya; Arimura, Gen-ichiro

2014-01-01

Anthocyanin pigments and associated flavonoids have demonstrated antioxidant properties and benefits for human health. Consequently, current plant bioengineers have focused on how to modify flavonoid metabolism in plants. Most of that research, however, does not consider the role of natural biotic stresses (e.g., herbivore attack). To understand the influence of herbivore attack on the metabolic engineering of flavonoids, we examined tobacco plants overexpressing the Arabidopsis PAP1 gene (encoding an MYB transcription factor), which accumulated anthocyanin pigments and other flavonoids/phenylpropanoids. In comparison to wild-type and control plants, transgenic plants exhibited greater resistance to Spodoptera litura. Moreover, herbivory suppressed the PAP1-induced increase of transcripts of flavonoid/phenylpropanoid biosynthetic genes (e.g., F3H) and the subsequent accumulation of these genes' metabolites, despite the unaltered PAP1 mRNA levels after herbivory. The instances of down-regulation were independent of the signaling pathways mediated by defense-related jasmonates but were relevant to the levels of PAP1-induced and herbivory-suppressed transcription factors, An1a and An1b. Although initially F3H transcripts were suppressed by herbivory, after the S. litura feeding was interrupted, F3H transcripts increased. We hypothesize that in transgenic plants responding to herbivory, there is a complex mechanism regulating enriched flavonoid/phenylpropanoid compounds, via biotic stress signals.

Overexpression of the PAP1 transcription factor reveals a complex regulation of flavonoid and phenylpropanoid metabolism in Nicotiana tabacum plants attacked by Spodoptera litura.

Directory of Open Access Journals (Sweden)

Tomoko Mitsunami

Full Text Available Anthocyanin pigments and associated flavonoids have demonstrated antioxidant properties and benefits for human health. Consequently, current plant bioengineers have focused on how to modify flavonoid metabolism in plants. Most of that research, however, does not consider the role of natural biotic stresses (e.g., herbivore attack. To understand the influence of herbivore attack on the metabolic engineering of flavonoids, we examined tobacco plants overexpressing the Arabidopsis PAP1 gene (encoding an MYB transcription factor, which accumulated anthocyanin pigments and other flavonoids/phenylpropanoids. In comparison to wild-type and control plants, transgenic plants exhibited greater resistance to Spodoptera litura. Moreover, herbivory suppressed the PAP1-induced increase of transcripts of flavonoid/phenylpropanoid biosynthetic genes (e.g., F3H and the subsequent accumulation of these genes' metabolites, despite the unaltered PAP1 mRNA levels after herbivory. The instances of down-regulation were independent of the signaling pathways mediated by defense-related jasmonates but were relevant to the levels of PAP1-induced and herbivory-suppressed transcription factors, An1a and An1b. Although initially F3H transcripts were suppressed by herbivory, after the S. litura feeding was interrupted, F3H transcripts increased. We hypothesize that in transgenic plants responding to herbivory, there is a complex mechanism regulating enriched flavonoid/phenylpropanoid compounds, via biotic stress signals.

Overexpression of the PAP1 Transcription Factor Reveals a Complex Regulation of Flavonoid and Phenylpropanoid Metabolism in Nicotiana tabacum Plants Attacked by Spodoptera litura

Science.gov (United States)

Mitsunami, Tomoko; Nishihara, Masahiro; Galis, Ivan; Alamgir, Kabir Md; Hojo, Yuko; Fujita, Kohei; Sasaki, Nobuhiro; Nemoto, Keichiro; Sawasaki, Tatsuya; Arimura, Gen-ichiro

2014-01-01

Anthocyanin pigments and associated flavonoids have demonstrated antioxidant properties and benefits for human health. Consequently, current plant bioengineers have focused on how to modify flavonoid metabolism in plants. Most of that research, however, does not consider the role of natural biotic stresses (e.g., herbivore attack). To understand the influence of herbivore attack on the metabolic engineering of flavonoids, we examined tobacco plants overexpressing the Arabidopsis PAP1 gene (encoding an MYB transcription factor), which accumulated anthocyanin pigments and other flavonoids/phenylpropanoids. In comparison to wild-type and control plants, transgenic plants exhibited greater resistance to Spodoptera litura. Moreover, herbivory suppressed the PAP1-induced increase of transcripts of flavonoid/phenylpropanoid biosynthetic genes (e.g., F3H) and the subsequent accumulation of these genes' metabolites, despite the unaltered PAP1 mRNA levels after herbivory. The instances of down-regulation were independent of the signaling pathways mediated by defense-related jasmonates but were relevant to the levels of PAP1-induced and herbivory-suppressed transcription factors, An1a and An1b. Although initially F3H transcripts were suppressed by herbivory, after the S. litura feeding was interrupted, F3H transcripts increased. We hypothesize that in transgenic plants responding to herbivory, there is a complex mechanism regulating enriched flavonoid/phenylpropanoid compounds, via biotic stress signals. PMID:25268129

Rac1 augments Wnt signaling by stimulating β-catenin–lymphoid enhancer factor-1 complex assembly independent of β-catenin nuclear import

Science.gov (United States)

Jamieson, Cara; Lui, Christina; Brocardo, Mariana G.; Martino-Echarri, Estefania; Henderson, Beric R.

2015-01-01

ABSTRACT β-Catenin transduces the Wnt signaling pathway and its nuclear accumulation leads to gene transactivation and cancer. Rac1 GTPase is known to stimulate β-catenin-dependent transcription of Wnt target genes and we confirmed this activity. Here we tested the recent hypothesis that Rac1 augments Wnt signaling by enhancing β-catenin nuclear import; however, we found that silencing/inhibition or up-regulation of Rac1 had no influence on nuclear accumulation of β-catenin. To better define the role of Rac1, we employed proximity ligation assays (PLA) and discovered that a significant pool of Rac1–β-catenin protein complexes redistribute from the plasma membrane to the nucleus upon Wnt or Rac1 activation. More importantly, active Rac1 was shown to stimulate the formation of nuclear β-catenin–lymphoid enhancer factor 1 (LEF-1) complexes. This regulation required Rac1-dependent phosphorylation of β-catenin at specific serines, which when mutated (S191A and S605A) reduced β-catenin binding to LEF-1 by up to 50%, as revealed by PLA and immunoprecipitation experiments. We propose that Rac1-mediated phosphorylation of β-catenin stimulates Wnt-dependent gene transactivation by enhancing β-catenin–LEF-1 complex assembly, providing new insight into the mechanism of cross-talk between Rac1 and canonical Wnt/β-catenin signaling. PMID:26403202

The Set1/COMPASS histone H3 methyltransferase helps regulate mitosis with the CDK1 and NIMA mitotic kinases in Aspergillus nidulans.

Science.gov (United States)

Govindaraghavan, Meera; Anglin, Sarah Lea; Osmani, Aysha H; Osmani, Stephen A

2014-08-01

Mitosis is promoted and regulated by reversible protein phosphorylation catalyzed by the essential NIMA and CDK1 kinases in the model filamentous fungus Aspergillus nidulans. Protein methylation mediated by the Set1/COMPASS methyltransferase complex has also been shown to regulate mitosis in budding yeast with the Aurora mitotic kinase. We uncover a genetic interaction between An-swd1, which encodes a subunit of the Set1 protein methyltransferase complex, with NIMA as partial inactivation of nimA is poorly tolerated in the absence of swd1. This genetic interaction is additionally seen without the Set1 methyltransferase catalytic subunit. Importantly partial inactivation of NIMT, a mitotic activator of the CDK1 kinase, also causes lethality in the absence of Set1 function, revealing a functional relationship between the Set1 complex and two pivotal mitotic kinases. The main target for Set1-mediated methylation is histone H3K4. Mutational analysis of histone H3 revealed that modifying the H3K4 target residue of Set1 methyltransferase activity phenocopied the lethality seen when either NIMA or CDK1 are partially functional. We probed the mechanistic basis of these genetic interactions and find that the Set1 complex performs functions with CDK1 for initiating mitosis and with NIMA during progression through mitosis. The studies uncover a joint requirement for the Set1 methyltransferase complex with the CDK1 and NIMA kinases for successful mitosis. The findings extend the roles of the Set1 complex to include the initiation of mitosis with CDK1 and mitotic progression with NIMA in addition to its previously identified interactions with Aurora and type 1 phosphatase in budding yeast. Copyright © 2014 by the Genetics Society of America.

Negative feedback regulation of Homer 1a on norepinephrine-dependent cardiac hypertrophy

Energy Technology Data Exchange (ETDEWEB)

Chiarello, Carmelina; Bortoloso, Elena; Carpi, Andrea; Furlan, Sandra; Volpe, Pompeo, E-mail: pompeo.volpe@unipd.it

2013-07-15

Homers are scaffolding proteins that modulate diverse cell functions being able to assemble signalling complexes. In this study, the presence, sub-cellular distribution and function of Homer 1 was investigated. Homer 1a and Homer 1b/c are constitutively expressed in cardiac muscle of both mouse and rat and in HL-1 cells, a cardiac cell line. As judged by confocal immunofluorescence microscopy, Homer 1a displays sarcomeric and peri-nuclear localization. In cardiomyocytes and cultured HL-1 cells, the hypertrophic agonist norepinephrine (NE) induces α{sub 1}-adrenergic specific Homer 1a over-expression, with a two-to-three-fold increase within 1 h, and no up-regulation of Homer 1b/c, as judged by Western blot and qPCR. In HL-1 cells, plasmid-driven over-expression of Homer 1a partially antagonizes activation of ERK phosphorylation and ANF up-regulation, two well-established, early markers of hypertrophy. At the morphometric level, NE-induced increase of cell size is likewise and partially counteracted by exogenous Homer 1a. Under the same experimental conditions, Homer 1b/c does not have any effect on ANF up-regulation nor on cell hypertrophy. Thus, Homer 1a up-regulation is associated to early stages of cardiac hypertrophy and appears to play a negative feedback regulation on molecular transducers of hypertrophy. -- Highlights: • Homer 1a is constitutively expressed in cardiac tissue. • In HL-1 cells, norepinephrine activates signaling pathways leading to hypertrophy. • Homer 1a up-regulation is an early event of norepinephrine-induced hypertrophy. • Homer 1a plays a negative feedback regulation modulating pathological hypertrophy. • Over-expression of Homer 1a per se does not induce hypertrophy.

Negative feedback regulation of Homer 1a on norepinephrine-dependent cardiac hypertrophy

International Nuclear Information System (INIS)

Chiarello, Carmelina; Bortoloso, Elena; Carpi, Andrea; Furlan, Sandra; Volpe, Pompeo

2013-01-01

Homers are scaffolding proteins that modulate diverse cell functions being able to assemble signalling complexes. In this study, the presence, sub-cellular distribution and function of Homer 1 was investigated. Homer 1a and Homer 1b/c are constitutively expressed in cardiac muscle of both mouse and rat and in HL-1 cells, a cardiac cell line. As judged by confocal immunofluorescence microscopy, Homer 1a displays sarcomeric and peri-nuclear localization. In cardiomyocytes and cultured HL-1 cells, the hypertrophic agonist norepinephrine (NE) induces α 1 -adrenergic specific Homer 1a over-expression, with a two-to-three-fold increase within 1 h, and no up-regulation of Homer 1b/c, as judged by Western blot and qPCR. In HL-1 cells, plasmid-driven over-expression of Homer 1a partially antagonizes activation of ERK phosphorylation and ANF up-regulation, two well-established, early markers of hypertrophy. At the morphometric level, NE-induced increase of cell size is likewise and partially counteracted by exogenous Homer 1a. Under the same experimental conditions, Homer 1b/c does not have any effect on ANF up-regulation nor on cell hypertrophy. Thus, Homer 1a up-regulation is associated to early stages of cardiac hypertrophy and appears to play a negative feedback regulation on molecular transducers of hypertrophy. -- Highlights: • Homer 1a is constitutively expressed in cardiac tissue. • In HL-1 cells, norepinephrine activates signaling pathways leading to hypertrophy. • Homer 1a up-regulation is an early event of norepinephrine-induced hypertrophy. • Homer 1a plays a negative feedback regulation modulating pathological hypertrophy. • Over-expression of Homer 1a per se does not induce hypertrophy

G1/S-regulated E2F-containing protein complexes bind to the mouse thymidine kinase gene promoter

DEFF Research Database (Denmark)

Dou, Q P; Zhao, S; Levin, A H

1994-01-01

report that MT2 includes an E2F-like binding site (GTTCGCGGGCAAA), as shown by the following evidence. (i) MT2 bound specifically to an affinity-purified fusion human E2F protein. (ii) Both MT2 and an authentic E2F site (TTTCGCGCGCTTT) bound specifically to similar or identical nuclear protein complexes...... complexes were also investigated. Studies using specific antibodies revealed that p107, a retinoblastoma-like protein, was present in both E2F-G0/G1 and E2F.S, whereas cyclin E.cyclin A.cdk2 were only present in E2F.S complex(es). These data suggest that removal of the p107-containing E2F.G0/G1 complex...

Labor Inhibits Placental Mechanistic Target of Rapamycin Complex 1 Signaling

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

2014-01-01

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

Preprotein import into chloroplasts via the Toc and Tic complexes is regulated by redox signals in Pisum sativum.

Science.gov (United States)

Stengel, Anna; Benz, J Philipp; Buchanan, Bob B; Soll, Jürgen; Bölter, Bettina

2009-11-01

The import of nuclear-encoded preproteins is necessary to maintain chloroplast function. The recognition and transfer of most precursor proteins across the chloroplast envelopes are facilitated by two membrane-inserted protein complexes, the translocons of the chloroplast outer and inner envelope (Toc and Tic complexes, respectively). Several signals have been invoked to regulate the import of preproteins. In our study, we were interested in redox-based import regulation mediated by two signals: regulation based on thiols and on the metabolic NADP+/NADPH ratio. We sought to identify the proteins participating in the regulation of these transport pathways and to characterize the preprotein subgroups whose import is redox-dependent. Our results provide evidence that the formation and reduction of disulfide bridges in the Toc receptors and Toc translocation channel have a strong influence on import yield of all tested preproteins that depend on the Toc complex for translocation. Furthermore, the metabolic NADP+/NADPH ratio influences not only the composition of the Tic complex, but also the import efficiency of most, but not all, preproteins tested. Thus, several Tic subcomplexes appear to participate in the translocation of different preprotein subgroups, and the redox-active components of these complexes likely play a role in regulating transport.

Axon-Axon Interactions Regulate Topographic Optic Tract Sorting via CYFIP2-Dependent WAVE Complex Function.

Science.gov (United States)

Cioni, Jean-Michel; Wong, Hovy Ho-Wai; Bressan, Dario; Kodama, Lay; Harris, William A; Holt, Christine E

2018-03-07

The axons of retinal ganglion cells (RGCs) are topographically sorted before they arrive at the optic tectum. This pre-target sorting, typical of axon tracts throughout the brain, is poorly understood. Here, we show that cytoplasmic FMR1-interacting proteins (CYFIPs) fulfill non-redundant functions in RGCs, with CYFIP1 mediating axon growth and CYFIP2 specifically involved in axon sorting. We find that CYFIP2 mediates homotypic and heterotypic contact-triggered fasciculation and repulsion responses between dorsal and ventral axons. CYFIP2 associates with transporting ribonucleoprotein particles in axons and regulates translation. Axon-axon contact stimulates CYFIP2 to move into growth cones where it joins the actin nucleating WAVE regulatory complex (WRC) in the periphery and regulates actin remodeling and filopodial dynamics. CYFIP2's function in axon sorting is mediated by its binding to the WRC but not its translational regulation. Together, these findings uncover CYFIP2 as a key regulatory link between axon-axon interactions, filopodial dynamics, and optic tract sorting. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

GTSE1 is a microtubule plus-end tracking protein that regulates EB1-dependent cell migration.

Directory of Open Access Journals (Sweden)

Massimilano Scolz

Full Text Available The regulation of cell migration is a highly complex process that is often compromised when cancer cells become metastatic. The microtubule cytoskeleton is necessary for cell migration, but how microtubules and microtubule-associated proteins regulate multiple pathways promoting cell migration remains unclear. Microtubule plus-end binding proteins (+TIPs are emerging as important players in many cellular functions, including cell migration. Here we identify a +TIP, GTSE1, that promotes cell migration. GTSE1 accumulates at growing microtubule plus ends through interaction with the EB1+TIP. The EB1-dependent +TIP activity of GTSE1 is required for cell migration, as well as for microtubule-dependent disassembly of focal adhesions. GTSE1 protein levels determine the migratory capacity of both nontransformed and breast cancer cell lines. In breast cancers, increased GTSE1 expression correlates with invasive potential, tumor stage, and time to distant metastasis, suggesting that misregulation of GTSE1 expression could be associated with increased invasive potential.

Characterizing complexity in socio-technical systems: a case study of a SAMU Medical Regulation Center.

Science.gov (United States)

Righi, Angela Weber; Wachs, Priscila; Saurin, Tarcísio Abreu

2012-01-01

Complexity theory has been adopted by a number of studies as a benchmark to investigate the performance of socio-technical systems, especially those that are characterized by relevant cognitive work. However, there is little guidance on how to assess, systematically, the extent to which a system is complex. The main objective of this study is to carry out a systematic analysis of a SAMU (Mobile Emergency Medical Service) Medical Regulation Center in Brazil, based on the core characteristics of complex systems presented by previous studies. The assessment was based on direct observations and nine interviews: three of them with regulator of emergencies medical doctor, three with radio operators and three with telephone attendants. The results indicated that, to a great extent, the core characteristics of complexity are magnified) due to basic shortcomings in the design of the work system. Thus, some recommendations are put forward with a view to reducing unnecessary complexity that hinders the performance of the socio-technical system.

4EGI-1 represses cap-dependent translation and regulates genome-wide translation in malignant pleural mesothelioma.

Science.gov (United States)

De, Arpita; Jacobson, Blake A; Peterson, Mark S; Jay-Dixon, Joe; Kratzke, Marian G; Sadiq, Ahad A; Patel, Manish R; Kratzke, Robert A

2018-04-01

Deregulation of cap-dependent translation has been implicated in the malignant transformation of numerous human tissues. 4EGI-1, a novel small-molecule inhibitor of cap-dependent translation, disrupts formation of the eukaryotic initiation factor 4F (eIF4F) complex. The effects of 4EGI-1-mediated inhibition of translation initiation in malignant pleural mesothelioma (MPM) were examined. 4EGI-1 preferentially inhibited cell viability and induced apoptosis in MPM cells compared to normal mesothelial (LP9) cells. This effect was associated with hypophosphorylation of 4E-binding protein 1 (4E-BP1) and decreased protein levels of the cancer-related genes, c-myc and osteopontin. 4EGI-1 showed enhanced cytotoxicity in combination with pemetrexed or gemcitabine. Translatome-wide polysome microarray analysis revealed a large cohort of genes that were translationally regulated upon treatment with 4EGI-1. The 4EGI-1-regulated translatome was negatively correlated to a previously published translatome regulated by eIF4E overexpression in human mammary epithelial cells, which is in agreement with the notion that 4EGI-1 inhibits the eIF4F complex. These data indicate that inhibition of the eIF4F complex by 4EGI-1 or similar translation inhibitors could be a strategy for treating mesothelioma. Genome wide translational profiling identified a large cohort of promising target genes that should be further evaluated for their potential significance in the treatment of MPM.

Regulation of ribonucleotide reductase by Spd1 involves multiple mechanisms

DEFF Research Database (Denmark)

Nestoras, Konstantinos; Mohammed, Asma Hadi; Schreurs, Ann-Sofie

2010-01-01

The correct levels of deoxyribonucleotide triphosphates and their relative abundance are important to maintain genomic integrity. Ribonucleotide reductase (RNR) regulation is complex and multifaceted. RNR is regulated allosterically by two nucleotide-binding sites, by transcriptional control, and...

Capital regulation: Less really can be more when incentives are socially aligned. Comments on Richard J. Herring "The Evolving Complexity of Capital Regulation". "The Interplay of Financial Regulations, Resilience, and Growth", Federal Reserve Bank of Philadelphia June 16-17, 2016

OpenAIRE

Hughes, Joseph P.

2017-01-01

Capital regulation has become increasingly complex as the largest financial institutions arbitrage differences in requirements across financial products to increase expected return for any given amount of regulatory capital, as financial regulators amend regulations to reduce arbitrage opportunities, and as financial institutions innovate to escape revised regulations - a regulatory dialectic. This increasing complexity makes monitoring bank risk-taking by markets and regulators more difficul...

Rac1 Regulates Endometrial Secretory Function to Control Placental Development

Science.gov (United States)

Davila, Juanmahel; Laws, Mary J.; Kannan, Athilakshmi; Li, Quanxi; Taylor, Robert N.; Bagchi, Milan K.; Bagchi, Indrani C.

2015-01-01

During placenta development, a succession of complex molecular and cellular interactions between the maternal endometrium and the developing embryo ensures reproductive success. The precise mechanisms regulating this maternal-fetal crosstalk remain unknown. Our study revealed that the expression of Rac1, a member of the Rho family of GTPases, is markedly elevated in mouse decidua on days 7 and 8 of gestation. To investigate its function in the uterus, we created mice bearing a conditional deletion of the Rac1 gene in uterine stromal cells. Ablation of Rac1 did not affect the formation of the decidua but led to fetal loss in mid gestation accompanied by extensive hemorrhage. To gain insights into the molecular pathways affected by the loss of Rac1, we performed gene expression profiling which revealed that Rac1 signaling regulates the expression of Rab27b, another GTPase that plays a key role in targeting vesicular trafficking. Consequently, the Rac1-null decidual cells failed to secrete vascular endothelial growth factor A, which is a critical regulator of decidual angiogenesis, and insulin-like growth factor binding protein 4, which regulates the bioavailability of insulin-like growth factors that promote proliferation and differentiation of trophoblast cell lineages in the ectoplacental cone. The lack of secretion of these key factors by Rac1-null decidua gave rise to impaired angiogenesis and dysregulated proliferation of trophoblast cells, which in turn results in overexpansion of the trophoblast giant cell lineage and disorganized placenta development. Further experiments revealed that RAC1, the human ortholog of Rac1, regulates the secretory activity of human endometrial stromal cells during decidualization, supporting the concept that this signaling G protein plays a central and conserved role in controlling endometrial secretory function. This study provides unique insights into the molecular mechanisms regulating endometrial secretions that mediate stromal

Rac1 Regulates Endometrial Secretory Function to Control Placental Development.

Directory of Open Access Journals (Sweden)

Juanmahel Davila

2015-08-01

Full Text Available During placenta development, a succession of complex molecular and cellular interactions between the maternal endometrium and the developing embryo ensures reproductive success. The precise mechanisms regulating this maternal-fetal crosstalk remain unknown. Our study revealed that the expression of Rac1, a member of the Rho family of GTPases, is markedly elevated in mouse decidua on days 7 and 8 of gestation. To investigate its function in the uterus, we created mice bearing a conditional deletion of the Rac1 gene in uterine stromal cells. Ablation of Rac1 did not affect the formation of the decidua but led to fetal loss in mid gestation accompanied by extensive hemorrhage. To gain insights into the molecular pathways affected by the loss of Rac1, we performed gene expression profiling which revealed that Rac1 signaling regulates the expression of Rab27b, another GTPase that plays a key role in targeting vesicular trafficking. Consequently, the Rac1-null decidual cells failed to secrete vascular endothelial growth factor A, which is a critical regulator of decidual angiogenesis, and insulin-like growth factor binding protein 4, which regulates the bioavailability of insulin-like growth factors that promote proliferation and differentiation of trophoblast cell lineages in the ectoplacental cone. The lack of secretion of these key factors by Rac1-null decidua gave rise to impaired angiogenesis and dysregulated proliferation of trophoblast cells, which in turn results in overexpansion of the trophoblast giant cell lineage and disorganized placenta development. Further experiments revealed that RAC1, the human ortholog of Rac1, regulates the secretory activity of human endometrial stromal cells during decidualization, supporting the concept that this signaling G protein plays a central and conserved role in controlling endometrial secretory function. This study provides unique insights into the molecular mechanisms regulating endometrial secretions

The Jasmonate-ZIM-Domain Proteins Interact with the WD-Repeat/bHLH/MYB Complexes to Regulate Jasmonate-Mediated Anthocyanin Accumulation and Trichome Initiation in Arabidopsis thaliana[C][W

Science.gov (United States)

Qi, Tiancong; Song, Susheng; Ren, Qingcuo; Wu, Dewei; Huang, Huang; Chen, Yan; Fan, Meng; Peng, Wen; Ren, Chunmei; Xie, Daoxin

2011-01-01

Jasmonates (JAs) mediate plant responses to insect attack, wounding, pathogen infection, stress, and UV damage and regulate plant fertility, anthocyanin accumulation, trichome formation, and many other plant developmental processes. Arabidopsis thaliana Jasmonate ZIM-domain (JAZ) proteins, substrates of the CORONATINE INSENSITIVE1 (COI1)–based SCFCOI1 complex, negatively regulate these plant responses. Little is known about the molecular mechanism for JA regulation of anthocyanin accumulation and trichome initiation. In this study, we revealed that JAZ proteins interact with bHLH (Transparent Testa8, Glabra3 [GL3], and Enhancer of Glabra3 [EGL3]) and R2R3 MYB transcription factors (MYB75 and Glabra1), essential components of WD-repeat/bHLH/MYB transcriptional complexes, to repress JA-regulated anthocyanin accumulation and trichome initiation. Genetic and physiological evidence showed that JA regulates WD-repeat/bHLH/MYB complex-mediated anthocyanin accumulation and trichome initiation in a COI1-dependent manner. Overexpression of the MYB transcription factor MYB75 and bHLH factors (GL3 and EGL3) restored anthocyanin accumulation and trichome initiation in the coi1 mutant, respectively. We speculate that the JA-induced degradation of JAZ proteins abolishes the interactions of JAZ proteins with bHLH and MYB factors, allowing the transcriptional function of WD-repeat/bHLH/MYB complexes, which subsequently activate respective downstream signal cascades to modulate anthocyanin accumulation and trichome initiation. PMID:21551388

Cadmium and cadmium-tolerant soil bacteria in cacao crops from northeastern Colombia.

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Bravo, D; Pardo-Díaz, S; Benavides-Erazo, J; Rengifo-Estrada, G; Braissant, O; Leon-Moreno, C

2018-05-01

This research aims to assess total-cadmium soil content and microbiological aspects to understand the dynamics of culturable cadmium-tolerant bacteria (CdtB) in cacao soils from northeastern Colombia. An integration of inverted dish plating, Cd determination and a microcalorimetry assay (IMC) was carried out. A farm in Boyacá showed the highest level of total soil Cd (3·74 mg kg -1 ) followed by farms in Santander and Arauca (2·76 and 1·16 mg kg -1 , respectively). Coefficient of determination between total soil Cd and CFU of CdtB was high (R 2  = 0·83) for the farm in Boyacá. Moreover, a pool of 129 CdtB was isolated, and phylogeny of 21 CdtB was discussed. Among CdtB strains isolated, Enterobacter sp. CdDB41 showed major Cd immobilization capacity (Q max of 2·21 and 2·32 J at 6 and 24 mg l -1 of CdCl 2 ), with an immobilization rate of 0·220 mg kg -1  h -1 . Among CdtB strains isolated, Enterobacter sp. CdDB41 showed major Cd immobilization capacity (Q max of 2·21 and 2·32 J at 6 and 24 mg l -1 of CdCl 2 ), with an immobilization rate of 0·220 mg kg -1  h -1 . Nothing is known about soil CdtB in cacao. Our data showed that CdtB such as Enterobacter sp. has high immobilization capacity. Furthermore, the otavite found in situ might be mineralized due to the bacterial metabolic activity of CdtB. © 2018 The Society for Applied Microbiology.

Clock drawing test in screening for Alzheimer's dementia and mild cognitive impairment in clinical practice.

Science.gov (United States)

Vyhnálek, Martin; Rubínová, Eva; Marková, Hana; Nikolai, Tomáš; Laczó, Jan; Andel, Ross; Hort, Jakub

2017-09-01

The clock drawing test (CDT) is a commonly used brief cognitive measure. We evaluated diagnostic accuracy of subjective ratings of CDT by physicians (with/without specialty in cognitive neurology) and neuropsychologists in discriminating amnestic mild cognitive impairment (aMCI), Alzheimer's dementia (AD) and cognitively healthy older adults. We further compared the diagnostic accuracy of subjective categorical ratings with complex scoring of CDT. Three cognitive neurologists, three neuropsychologists and six neurology residents without experience in cognitive neurology blinded to the diagnosis rated 187 CDTs (50 mild AD, 49 aMCI and 88 cognitively healthy older adults) using a "yes" (abnormal) versus "suspected" versus "no" (normal) classification. The rating suspected was combined with yes or no to obtain two sets of sensitivity estimates. We also used a 17-point CDT rating system. When using the categorical rating, neuropsychologists had highest sensitivity (89%) in differentiating patients with mild AD (yes/suspected versus no), followed by neurologic residents (80%) and cognitive neurologists (79%). When differentiating patients with aMCI (yes/suspected versus no), the sensitivity was 84% for neuropsychologists, 64% for cognitive neurologists and 62% for residents. The sensitivity using the complex scoring system was 92% in patients with mild AD and 69% in patients with aMCI. A categorical rating of CDT shows high sensitivity for mild AD even in non-experienced raters. Neuropsychologists outperformed physicians in differentiating patients with aMCI from cognitively healthy older adults (specificity), which was counterbalanced by the lower specificity of their ratings. The diagnostic accuracy was not substantially improved by using complex scoring system. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Biochemical reconstitution and phylogenetic comparison of human SET1 family core complexes involved in histone methylation.

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Shinsky, Stephen A; Monteith, Kelsey E; Viggiano, Susan; Cosgrove, Michael S

2015-03-06

Mixed lineage leukemia protein-1 (MLL1) is a member of the SET1 family of histone H3 lysine 4 (H3K4) methyltransferases that are required for metazoan development. MLL1 is the best characterized human SET1 family member, which includes MLL1-4 and SETd1A/B. MLL1 assembles with WDR5, RBBP5, ASH2L, DPY-30 (WRAD) to form the MLL1 core complex, which is required for H3K4 dimethylation and transcriptional activation. Because all SET1 family proteins interact with WRAD in vivo, it is hypothesized they are regulated by similar mechanisms. However, recent evidence suggests differences among family members that may reflect unique regulatory inputs in the cell. Missing is an understanding of the intrinsic enzymatic activities of different SET1 family complexes under standard conditions. In this investigation, we reconstituted each human SET1 family core complex and compared subunit assembly and enzymatic activities. We found that in the absence of WRAD, all but one SET domain catalyzes at least weak H3K4 monomethylation. In the presence of WRAD, all SET1 family members showed stimulated monomethyltransferase activity but differed in their di- and trimethylation activities. We found that these differences are correlated with evolutionary lineage, suggesting these enzyme complexes have evolved to accomplish unique tasks within metazoan genomes. To understand the structural basis for these differences, we employed a "phylogenetic scanning mutagenesis" assay and identified a cluster of amino acid substitutions that confer a WRAD-dependent gain-of-function dimethylation activity on complexes assembled with the MLL3 or Drosophila trithorax proteins. These results form the basis for understanding how WRAD differentially regulates SET1 family complexes in vivo. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A heteromeric molecular complex regulates the migration of lung alveolar epithelial cells during wound healing.

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Ghosh, Manik C; Makena, Patrudu S; Kennedy, Joseph; Teng, Bin; Luellen, Charlean; Sinclair, Scott E; Waters, Christopher M

2017-05-19

Alveolar type II epithelial cells (ATII) are instrumental in early wound healing in response to lung injury, restoring epithelial integrity through spreading and migration. We previously reported in separate studies that focal adhesion kinase-1 (FAK) and the chemokine receptor CXCR4 promote epithelial repair mechanisms. However, potential interactions between these two pathways were not previously considered. In the present study, we found that wounding of rat ATII cells promoted increased association between FAK and CXCR4. In addition, protein phosphatase-5 (PP5) increased its association with this heteromeric complex, while apoptosis signal regulating kinase-1 (ASK1) dissociated from the complex. Cell migration following wounding was decreased when PP5 expression was decreased using shRNA, but migration was increased in ATII cells isolated from ASK1 knockout mice. Interactions between FAK and CXCR4 were increased upon depletion of ASK1 using shRNA in MLE-12 cells, but unaffected when PP5 was depleted. Furthermore, we found that wounded rat ATII cells exhibited decreased ASK1 phosphorylation at Serine-966, decreased serine phosphorylation of FAK, and decreased association of phosphorylated ASK1 with FAK. These changes in phosphorylation were dependent upon expression of PP5. These results demonstrate a unique molecular complex comprising CXCR4, FAK, ASK1, and PP5 in ATII cells during wound healing.

Regulation of P2Y1 receptor traffic by sorting Nexin 1 is retromer independent.

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Nisar, Shaista; Kelly, Eamonn; Cullen, Pete J; Mundell, Stuart J

2010-04-01

The activity and traffic of G-protein coupled receptors (GPCRs) is tightly controlled. Recent work from our laboratory has shown that P2Y(1) and P2Y(12) responsiveness is rapidly and reversibly modulated in human platelets and that the underlying mechanism requires receptor trafficking as an essential part of this process. However, little is known about the molecular mechanisms underlying P2Y receptor traffic. Sorting nexin 1 (SNX1) has been shown to regulate the endosomal sorting of cell surface receptors either to lysosomes where they are downregulated or back to the cell surface. These functions may in part be due to interactions of SNX1 with the mammalian retromer complex. In this study, we investigated the role of SNX1 in P2Y receptor trafficking. We show that P2Y(1) receptors recycle via a slow recycling pathway that is regulated by SNX1, whereas P2Y(12) receptors return to the cell surface via a rapid route that is SNX1 independent. SNX1 inhibition caused a dramatic increase in the rate of P2Y(1) receptor recycling, whereas inhibition of Vps26 and Vps35 known to be present in retromer had no effect, indicating that SNX1 regulation of P2Y(1) receptor recycling is retromer independent. In addition, inhibition of SNX4, 6 and 17 proteins did not affect P2Y(1) receptor recycling. SNX1 has also been implicated in GPCR degradation; however, we provide evidence that P2Y receptor degradation is SNX1 independent. These data describe a novel function of SNX1 in the regulation of P2Y(1) receptor recycling and suggest that SNX1 plays multiple roles in endocytic trafficking of GPCRs.

MCT-1 protein interacts with the cap complex and modulates messenger RNA translational profiles

DEFF Research Database (Denmark)

Reinert, Line; Shi, B; Nandi, S

2006-01-01

MCT-1 is an oncogene that was initially identified in a human T cell lymphoma and has been shown to induce cell proliferation as well as activate survival-related pathways. MCT-1 contains the PUA domain, a recently described RNA-binding domain that is found in several tRNA and rRNA modification...... enzymes. Here, we established that MCT-1 protein interacts with the cap complex through its PUA domain and recruits the density-regulated protein (DENR/DRP), containing the SUI1 translation initiation domain. Through the use of microarray analysis on polysome-associated mRNAs, we showed that up......-regulation of MCT-1 was able to modulate the translation profiles of BCL2L2, TFDP1, MRE11A, cyclin D1, and E2F1 mRNAs, despite equivalent levels of mRNAs in the cytoplasm. Our data establish a role for MCT-1 in translational regulation, and support a linkage between translational control and oncogenesis....

Polycomb Group Protein PHF1 Regulates p53-dependent Cell Growth Arrest and Apoptosis*

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Yang, Yang; Wang, Chenji; Zhang, Pingzhao; Gao, Kun; Wang, Dejie; Yu, Hongxiu; Zhang, Ting; Jiang, Sirui; Hexige, Saiyin; Hong, Zehui; Yasui, Akira; Liu, Jun O.; Huang, Haojie; Yu, Long

2013-01-01

Polycomb group protein PHF1 is well known as a component of a novel EED-EZH2·Polycomb repressive complex 2 complex and plays important roles in H3K27 methylation and Hox gene silencing. PHF1 is also involved in the response to DNA double-strand breaks in human cells, promotes nonhomologous end-joining processes through interaction with Ku70/Ku80. Here, we identified another function of PHF1 as a potential p53 pathway activator in a pathway screen using luminescence reporter assay. Subsequent studies showed PHF1 directly interacts with p53 proteins both in vivo and in vitro and co-localized in nucleus. PHF1 binds to the C-terminal regulatory domain of p53. Overexpression of PHF1 elevated p53 protein level and prolonged its turnover. Knockdown of PHF1 reduced p53 protein level and its target gene expression both in normal state and DNA damage response. Mechanically, PHF1 protects p53 proteins from MDM2-mediated ubiquitination and degradation. Furthermore, we showed that PHF1 regulates cell growth arrest and etoposide-induced apoptosis in a p53-dependent manner. Finally, PHF1 expression was significantly down-regulated in human breast cancer samples. Taken together, we establish PHF1 as a novel positive regulator of the p53 pathway. These data shed light on the potential roles of PHF1 in tumorigenesis and/or tumor progression. PMID:23150668

Regulation of lipid droplet dynamics in Saccharomyces cerevisiae depends on the Rab7-like Ypt7p, HOPS complex and V1-ATPase

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Isabelle Bouchez

2015-07-01

Full Text Available It has now been clearly shown that lipid droplets (LDs play a dynamic role in the cell. This was reinforced by LD proteomics which suggest that a significant number of trafficking proteins are associated with this organelle. Using microscopy, we showed that LDs partly co-localize with the vacuole in S. cerevisiae. Immunoblot experiments confirmed the association of the vacuolar Rab GTPase Rab7-like Ypt7p with LDs. We observed an increase in fatty acid content and LD number in ypt7Δ mutant and also changes in LD morphology and intra LD fusions, revealing a direct role for Ypt7p in LD dynamics. Using co-immunoprecipitation, we isolated potential Ypt7p partners including, Vma13p, the H subunit of the V1 part of the vacuolar (H+ ATPase (V-ATPase. Deletion of the VMA13 gene, as well as deletion of three other subunits of the V1 part of the V-ATPase, also increased the cell fatty acid content and LD number. Mutants of the Homotypic fusion and vacuole protein sorting (HOPS complex showed similar phenotypes. Here, we demonstrated that LD dynamics and membrane trafficking between the vacuole and LDs are regulated by the Rab7-like Ypt7p and are impaired when the HOPS complex and the V1 domain of the V-ATPase are defective.

Mof-associated complexes have overlapping and unique roles in regulating pluripotency in embryonic stem cells and during differentiation

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Ravens, Sarina; Fournier, Marjorie; Ye, Tao; Stierle, Matthieu; Dembele, Doulaye; Chavant, Virginie; Tora, Làszlò

2014-01-01

The histone acetyltransferase (HAT) Mof is essential for mouse embryonic stem cell (mESC) pluripotency and early development. Mof is the enzymatic subunit of two different HAT complexes, MSL and NSL. The individual contribution of MSL and NSL to transcription regulation in mESCs is not well understood. Our genome-wide analysis show that i) MSL and NSL bind to specific and common sets of expressed genes, ii) NSL binds exclusively at promoters, iii) while MSL binds in gene bodies. Nsl1 regulates proliferation and cellular homeostasis of mESCs. MSL is the main HAT acetylating H4K16 in mESCs, is enriched at many mESC-specific and bivalent genes. MSL is important to keep a subset of bivalent genes silent in mESCs, while developmental genes require MSL for expression during differentiation. Thus, NSL and MSL HAT complexes differentially regulate specific sets of expressed genes in mESCs and during differentiation. DOI: http://dx.doi.org/10.7554/eLife.02104.001 PMID:24898753

Convergence of the Mammalian Target of Rapamycin Complex 1- and Glycogen Synthase Kinase 3-β–Signaling Pathways Regulates the Innate Inflammatory Response

Science.gov (United States)

Wang, Huizhi; Brown, Jonathan; Gu, Zhen; Garcia, Carlos A.; Liang, Ruqiang; Alard, Pascale; Beurel, Eléonore; Jope, Richard S.; Greenway, Terrance; Martin, Michael

2011-01-01

The PI3K pathway and its regulation of mammalian target of rapamycin complex 1 (mTORC1) and glycogen synthase kinase 3 (GSK3) play pivotal roles in controlling inflammation. In this article, we show that mTORC1 and GSK3-β converge and that the capacity of mTORC1 to affect the inflammatory response is due to the inactivation of GSK3-β. Inhibition of mTORC1 attenuated GSK3 phosphorylation and increased its kinase activity. Immunoprecipitation and in vitro kinase assays demonstrated that GSK3-β associated with a downstream target of mTORC1, p85S6K, and phosphorylated GSK3-β. Inhibition of S6K1 abrogated the phosphorylation of GSK3-β while increasing and decreasing the levels of IL-12 and IL-10, respectively, in LPS-stimulated monocytes. In contrast, the direct inhibition of GSK3 attenuated the capacity of S6K1 inhibition to influence the levels of IL-10 and IL-12 produced by LPS-stimulated cells. At the transcriptional level, mTORC1 inhibition reduced the DNA binding of CREB and this effect was reversed by GSK3 inhibition. As a result, mTORC1 inhibition increased the levels of NF-κB p65 associated with CREB-binding protein. Inhibition of NF-κB p65 attenuated rapamycin’s ability to influence the levels of pro- or anti-inflammatory cytokine production in monocytes stimulated with LPS. These studies identify the molecular mechanism by which mTORC1 affects GSK3 and show that mTORC1 inhibition regulates pro- and anti-inflammatory cytokine production via its capacity to inactivate GSK3. PMID:21422248

HEXIM1 controls satellite cell expansion after injury to regulate skeletal muscle regeneration

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Hong, Peng; Chen, Kang; Huang, Bihui; Liu, Min; Cui, Miao; Rozenberg, Inna; Chaqour, Brahim; Pan, Xiaoyue; Barton, Elisabeth R.; Jiang, Xian-Cheng; Siddiqui, M.A.Q.

2012-01-01

The native capacity of adult skeletal muscles to regenerate is vital to the recovery from physical injuries and dystrophic diseases. Currently, the development of therapeutic interventions has been hindered by the complex regulatory network underlying the process of muscle regeneration. Using a mouse model of skeletal muscle regeneration after injury, we identified hexamethylene bisacetamide inducible 1 (HEXIM1, also referred to as CLP-1), the inhibitory component of the positive transcription elongation factor b (P-TEFb) complex, as a pivotal regulator of skeletal muscle regeneration. Hexim1-haplodeficient muscles exhibited greater mass and preserved function compared with those of WT muscles after injury, as a result of enhanced expansion of satellite cells. Transplanted Hexim1-haplodeficient satellite cells expanded and improved muscle regeneration more effectively than WT satellite cells. Conversely, HEXIM1 overexpression restrained satellite cell proliferation and impeded muscle regeneration. Mechanistically, dissociation of HEXIM1 from P-TEFb and subsequent activation of P-TEFb are required for satellite cell proliferation and the prevention of early myogenic differentiation. These findings suggest a crucial role for the HEXIM1/P-TEFb pathway in the regulation of satellite cell–mediated muscle regeneration and identify HEXIM1 as a potential therapeutic target for degenerative muscular diseases. PMID:23023707

Scanning mutagenesis of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex

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Nagib eAhsan

2012-07-01

Full Text Available The mitochondrial pyruvate dehydrogenase complex is regulated by reversible seryl-phosphorylation of the E1α subunit by a dedicated, intrinsic kinase. The phospho-complex is reactivated when dephosphorylated by an intrinsic PP2C-type protein phosphatase. Both the position of the phosphorylated Ser-residue and the sequences of the flanking amino acids are highly conserved. We have used the synthetic peptide-based kinase client assay plus recombinant pyruvate dehydrogenase E1α and E1α-kinase to perform scanning mutagenesis of the residues flanking the site of phosphorylation. Consistent with the results from phylogenetic analysis of the flanking sequences, the direct peptide-based kinase assays tolerated very few changes. Even conservative changes such as Leu, Ile, or Val for Met, or Glu for Asp, gave very marked reductions in phosphorylation. Overall the results indicate that regulation of the mitochondrial pyruvate dehydrogenase complex by reversible phosphorylation is an extreme example of multiple, interdependent instances of co-evolution.

Origin Licensing Requires ATP Binding and Hydrolysis by the MCM Replicative Helicase

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Coster, Gideon; Frigola, Jordi; Beuron, Fabienne; Morris, Edward P.; Diffley, John F.X.

2014-01-01

Summary Loading of the six related Minichromosome Maintenance (MCM) proteins as head-to-head double hexamers during DNA replication origin licensing is crucial for ensuring once-per-cell-cycle DNA replication in eukaryotic cells. Assembly of these prereplicative complexes (pre-RCs) requires the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ORC, Cdc6, and MCM are members of the AAA+ family of ATPases, and pre-RC assembly requires ATP hydrolysis. Here we show that ORC and Cdc6 mutants defective in ATP hydrolysis are competent for origin licensing. However, ATP hydrolysis by Cdc6 is required to release nonproductive licensing intermediates. We show that ATP binding stabilizes the wild-type MCM hexamer. Moreover, by analyzing MCM containing mutant subunits, we show that ATP binding and hydrolysis by MCM are required for Cdt1 release and double hexamer formation. This work alters our view of how ATP is used by licensing factors to assemble pre-RCs. PMID:25087873

Brd4 and HEXIM1: Multiple Roles in P-TEFb Regulation and Cancer

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Ruichuan Chen

2014-01-01

Full Text Available Bromodomain-containing protein 4 (Brd4 and hexamethylene bisacetamide (HMBA inducible protein 1 (HEXIM1 are two opposing regulators of the positive transcription elongation factor b (P-TEFb, which is the master modulator of RNA polymerase II during transcriptional elongation. While Brd4 recruits P-TEFb to promoter-proximal chromatins to activate transcription, HEXIM1 sequesters P-TEFb into an inactive complex containing the 7SK small nuclear RNA. Besides regulating P-TEFb’s transcriptional activity, recent evidence demonstrates that both Brd4 and HEXIM1 also play novel roles in cell cycle progression and tumorigenesis. Here we will discuss the current knowledge on Brd4 and HEXIM1 and their implication as novel therapeutic options against cancer.

Five Conditions Commonly Used to Down-regulate Tor Complex 1 Generate Different Physiological Situations Exhibiting Distinct Requirements and Outcomes*

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Tate, Jennifer J.; Cooper, Terrance G.

2013-01-01

Five different physiological conditions have been used interchangeably to establish the sequence of molecular events needed to achieve nitrogen-responsive down-regulation of TorC1 and its subsequent regulation of downstream reporters: nitrogen starvation, methionine sulfoximine (Msx) addition, nitrogen limitation, rapamycin addition, and leucine starvation. Therefore, we tested a specific underlying assumption upon which the interpretation of data generated by these five experimental perturbations is premised. It is that they generate physiologically equivalent outcomes with respect to TorC1, i.e. its down-regulation as reflected by TorC1 reporter responses. We tested this assumption by performing head-to-head comparisons of the requirements for each condition to achieve a common outcome for a downstream proxy of TorC1 inactivation, nuclear Gln3 localization. We demonstrate that the five conditions for down-regulating TorC1 do not elicit physiologically equivalent outcomes. Four of the methods exhibit hierarchical Sit4 and PP2A phosphatase requirements to elicit nuclear Gln3-Myc13 localization. Rapamycin treatment required Sit4 and PP2A. Nitrogen limitation and short-term nitrogen starvation required only Sit4. G1 arrest-correlated, long-term nitrogen starvation and Msx treatment required neither PP2A nor Sit4. Starving cells of leucine or treating them with leucyl-tRNA synthetase inhibitors did not elicit nuclear Gln3-Myc13 localization. These data indicate that the five commonly used nitrogen-related conditions of down-regulating TorC1 are not physiologically equivalent and minimally involve partially differing regulatory mechanisms. Further, identical requirements for Msx treatment and long-term nitrogen starvation raise the possibility that their effects are achieved through a common regulatory pathway with glutamine, a glutamate or glutamine metabolite level as the sensed metabolic signal. PMID:23935103

“Scanning mutagenesis” of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex

Science.gov (United States)

The mitochondrial pyruvate dehydrogenase complex is regulated by reversible seryl-phosphorylation of the E1alpha subunit by a dedicated, intrinsic kinase. The phospho-complex is reactivated when dephosphorylated by an intrinsic PP2C-type protein phosphatase. Both the position of the phosphorylated...

Hypothalamic roles of mTOR complex I: integration of nutrient and hormone signals to regulate energy homeostasis.

Science.gov (United States)

Hu, Fang; Xu, Yong; Liu, Feng

2016-06-01

Mammalian or mechanistic target of rapamycin (mTOR) senses nutrient, energy, and hormone signals to regulate metabolism and energy homeostasis. mTOR activity in the hypothalamus, which is associated with changes in energy status, plays a critical role in the regulation of food intake and body weight. mTOR integrates signals from a variety of "energy balancing" hormones such as leptin, insulin, and ghrelin, although its action varies in response to these distinct hormonal stimuli as well as across different neuronal populations. In this review, we summarize and highlight recent findings regarding the functional roles of mTOR complex 1 (mTORC1) in the hypothalamus specifically in its regulation of body weight, energy expenditure, and glucose/lipid homeostasis. Understanding the role and underlying mechanisms behind mTOR-related signaling in the brain will undoubtedly pave new avenues for future therapeutics and interventions that can combat obesity, insulin resistance, and diabetes. Copyright © 2016 the American Physiological Society.

The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation

OpenAIRE

Malik, Sohail; Roeder, Robert G.

2010-01-01

The Mediator is an evolutionarily conserved, multiprotein complex that is a key regulator of protein-coding genes. In metazoan cells, multiple pathways that are responsible for homeostasis, cell growth and differentiation converge on the Mediator through transcriptional activators and repressors that target one or more of the almost 30 subunits of this complex. Besides interacting directly with RNA polymerase II, Mediator has multiple functions and can interact with and coordinate the action ...

Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex.

Science.gov (United States)

Hiraga, Shin-Ichiro; Alvino, Gina M; Chang, Fujung; Lian, Hui-Yong; Sridhar, Akila; Kubota, Takashi; Brewer, Bonita J; Weinreich, Michael; Raghuraman, M K; Donaldson, Anne D

2014-02-15

Initiation of eukaryotic DNA replication requires phosphorylation of the MCM complex by Dbf4-dependent kinase (DDK), composed of Cdc7 kinase and its activator, Dbf4. We report here that budding yeast Rif1 (Rap1-interacting factor 1) controls DNA replication genome-wide and describe how Rif1 opposes DDK function by directing Protein Phosphatase 1 (PP1)-mediated dephosphorylation of the MCM complex. Deleting RIF1 partially compensates for the limited DDK activity in a cdc7-1 mutant strain by allowing increased, premature phosphorylation of Mcm4. PP1 interaction motifs within the Rif1 N-terminal domain are critical for its repressive effect on replication. We confirm that Rif1 interacts with PP1 and that PP1 prevents premature Mcm4 phosphorylation. Remarkably, our results suggest that replication repression by Rif1 is itself also DDK-regulated through phosphorylation near the PP1-interacting motifs. Based on our findings, we propose that Rif1 is a novel PP1 substrate targeting subunit that counteracts DDK-mediated phosphorylation during replication. Fission yeast and mammalian Rif1 proteins have also been implicated in regulating DNA replication. Since PP1 interaction sites are evolutionarily conserved within the Rif1 sequence, it is likely that replication control by Rif1 through PP1 is a conserved mechanism.

MicroRNA, SND1, and alterations in translational regulation in colon carcinogenesis

International Nuclear Information System (INIS)

Tsuchiya, Naoto; Nakagama, Hitoshi

2010-01-01

Post-transcriptional regulation of gene expression by microRNA (miRNA) has recently attracted major interest in relation to its involvement in cancer development. miRNA is a member of small non-coding RNA, consists of 22-24 nucleotides and regulates expression of target mRNA species in a post-transcriptional manner by being incorporated with RNA-induced silencing complex (RISC). Staphylococcal nuclease homology domain containing 1 (SND1), a component of RISC, is frequently up-regulated in human colon cancers and also chemically induced colon cancers in animals. We here showed that SDN1 is involved in miRNA-mediated gene suppression and overexpression of SND1 in colon cancer cells causes down-regulation of APC without altering APC mRNA levels. As for the miRNA expression profile in human colon cancer, miR-34a was among the list of down-regulated miRNA. Expression of miR-34a is tightly regulated by p53, and ectopic expression of miR-34a in colon cancer cells causes remarkable reduction of cell proliferation and induces senescence-like phenotypes. MiR-34a also participates in the positive feedback loop of the p53 tumor suppressor network. This circuitry mechanism for p53 activation is of interest in understanding the tumor suppressive function of miR-34a in colon carcinogenesis. miRNA should also be considered as novel anti-cancer agents in tumor suppressive therapeutic applications.

Leucine-induced activation of translational initiation is partly regulated by the branched-chain α-keto acid dehydrogenase complex in C2C12 cells

International Nuclear Information System (INIS)

Nakai, Naoya; Shimomura, Yoshiharu; Tamura, Tomohiro; Tamura, Noriko; Hamada, Koichiro; Kawano, Fuminori; Ohira, Yoshinobu

2006-01-01

Branched-chain amino acid leucine has been shown to activate the translational regulators through the mammalian target of rapamycin. However, the leucine's effects are self-limiting because leucine promotes its own disposal by an oxidative pathway. The irreversible and rate-limiting step in the leucine oxidation pathway is catalyzed by the branched-chain α-keto acid dehydrogenase (BCKDH) complex. The complex contains E1 (α2β2), E2, and E3 subunits, and its activity is abolished by phosphorylation of the E1α subunit by BCKDH kinase. The relationship between the activity of BCKDH complex and leucine-mediated activation of the protein translation was investigated using the technique of RNA interference. The activity of BCKDH complex in C2C12 cell was modulated by transfection of small interfering RNA (siRNA) for BCKDH E2 subunit or BCKDH kinase. Transfection of siRNAs decreased the mRNA expression and protein amount of corresponding gene. Suppression of either E2 subunit or kinase produced opposite effects on the cell proliferation and the activation of translational regulators by leucine. Suppression of BCKDH kinase for 48 h resulted in decreasing cell proliferation. In contrast, E2 suppression led to increased amount of total cellular protein. The phosphorylation of p70 S6 kinase by leucine was increased in E2-siRNA transfected C2C12 cells, whereas the leucine's effect was diminished in kinase-siRNA transfected cells. These results suggest that the activation of the translational regulators by leucine was partly regulated by the activity of BCKDH complex

Different roles of the small GTPases Rac1, Cdc42, and RhoG in CALEB/NGC-induced dendritic tree complexity.

Science.gov (United States)

Schulz, Jana; Franke, Kristin; Frick, Manfred; Schumacher, Stefan

2016-10-01

Rho GTPases play prominent roles in the regulation of cytoskeletal reorganization. Many aspects have been elaborated concerning the individual functions of Rho GTPases in distinct signaling pathways leading to cytoskeletal rearrangements. However, major questions have yet to be answered regarding the integration and the signaling hierarchy of different Rho GTPases in regulating the cytoskeleton in fundamental physiological events like neuronal process differentiation. Here, we investigate the roles of the small GTPases Rac1, Cdc42, and RhoG in defining dendritic tree complexity stimulated by the transmembrane epidermal growth factor family member CALEB/NGC. Combining gain-of-function and loss-of-function analysis in primary hippocampal neurons, we find that Rac1 is essential for CALEB/NGC-mediated dendritic branching. Cdc42 reduces the complexity of dendritic trees. Interestingly, we identify the palmitoylated isoform of Cdc42 to adversely affect dendritic outgrowth and dendritic branching, whereas the prenylated Cdc42 isoform does not. In contrast to Rac1, CALEB/NGC and Cdc42 are not directly interconnected in regulating dendritic tree complexity. Unlike Rac1, the Rac1-related GTPase RhoG reduces the complexity of dendritic trees by acting upstream of CALEB/NGC. Mechanistically, CALEB/NGC activates Rac1, and RhoG reduces the amount of CALEB/NGC that is located at the right site for Rac1 activation at the cell membrane. Thus, Rac1, Cdc42, and RhoG perform very specific and non-redundant functions at different levels of hierarchy in regulating dendritic tree complexity induced by CALEB/NGC. Rho GTPases play a prominent role in dendritic branching. CALEB/NGC is a transmembrane member of the epidermal growth factor (EGF) family that mediates dendritic branching, dependent on Rac1. CALEB/NGC stimulates Rac1 activity. RhoG inhibits CALEB/NGC-mediated dendritic branching by decreasing the amount of CALEB/NGC at the plasma membrane. Palmitoylated, but not prenylated form

NECAPs are negative regulators of the AP2 clathrin adaptor complex.

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Beacham, Gwendolyn M; Partlow, Edward A; Lange, Jeffrey J; Hollopeter, Gunther

2018-01-18

Eukaryotic cells internalize transmembrane receptors via clathrin-mediated endocytosis, but it remains unclear how the machinery underpinning this process is regulated. We recently discovered that membrane-associated muniscin proteins such as FCHo and SGIP initiate endocytosis by converting the AP2 clathrin adaptor complex to an open, active conformation that is then phosphorylated (Hollopeter et al., 2014). Here we report that loss of ncap-1 , the sole C. elegans gene encoding an adaptiN Ear-binding Coat-Associated Protein (NECAP), bypasses the requirement for FCHO-1. Biochemical analyses reveal AP2 accumulates in an open, phosphorylated state in ncap-1 mutant worms, suggesting NECAPs promote the closed, inactive conformation of AP2. Consistent with this model, NECAPs preferentially bind open and phosphorylated forms of AP2 in vitro and localize with constitutively open AP2 mutants in vivo. NECAPs do not associate with phosphorylation-defective AP2 mutants, implying that phosphorylation precedes NECAP recruitment. We propose NECAPs function late in endocytosis to inactivate AP2. © 2018, Beacham et al.

Global regulator SATB1 recruits beta-catenin and regulates T(H2 differentiation in Wnt-dependent manner.

Directory of Open Access Journals (Sweden)

Dimple Notani

2010-01-01

Full Text Available In vertebrates, the conserved Wnt signalling cascade promotes the stabilization and nuclear accumulation of beta-catenin, which then associates with the lymphoid enhancer factor/T cell factor proteins (LEF/TCFs to activate target genes. Wnt/beta -catenin signalling is essential for T cell development and differentiation. Here we show that special AT-rich binding protein 1 (SATB1, the T lineage-enriched chromatin organizer and global regulator, interacts with beta-catenin and recruits it to SATB1's genomic binding sites. Gene expression profiling revealed that the genes repressed by SATB1 are upregulated upon Wnt signalling. Competition between SATB1 and TCF affects the transcription of TCF-regulated genes upon beta-catenin signalling. GATA-3 is a T helper type 2 (T(H2 specific transcription factor that regulates production of T(H2 cytokines and functions as T(H2 lineage determinant. SATB1 positively regulated GATA-3 and siRNA-mediated knockdown of SATB1 downregulated GATA-3 expression in differentiating human CD4(+ T cells, suggesting that SATB1 influences T(H2 lineage commitment by reprogramming gene expression. In the presence of Dickkopf 1 (Dkk1, an inhibitor of Wnt signalling, GATA-3 is downregulated and the expression of signature T(H2 cytokines such as IL-4, IL-10, and IL-13 is reduced, indicating that Wnt signalling is essential for T(H2 differentiation. Knockdown of beta-catenin also produced similar results, confirming the role of Wnt/beta-catenin signalling in T(H2 differentiation. Furthermore, chromatin immunoprecipitation analysis revealed that SATB1 recruits beta-catenin and p300 acetyltransferase on GATA-3 promoter in differentiating T(H2 cells in a Wnt-dependent manner. SATB1 coordinates T(H2 lineage commitment by reprogramming gene expression. The SATB1:beta-catenin complex activates a number of SATB1 regulated genes, and hence this study has potential to find novel Wnt responsive genes. These results demonstrate that SATB1

Structure of the Human Atg13-Atg101 HORMA Heterodimer: an Interaction Hub within the ULK1 Complex.

Science.gov (United States)

Qi, Shiqian; Kim, Do Jin; Stjepanovic, Goran; Hurley, James H

2015-10-06

The ULK1 complex, consisting of the ULK1 protein kinase itself, FIP200, Atg13, and Atg101, controls the initiation of autophagy in animals. We determined the structure of the complex of the human Atg13 HORMA (Hop1, Rev7, Mad2) domain in complex with the full-length HORMA domain-only protein Atg101. The two HORMA domains assemble with an architecture conserved in the Mad2 conformational heterodimer and the S. pombe Atg13-Atg101 HORMA complex. The WF finger motif that is essential for function in human Atg101 is sequestered in a hydrophobic pocket, suggesting that the exposure of this motif is regulated. Benzamidine molecules from the crystallization solution mark two hydrophobic pockets that are conserved in, and unique to, animals, and are suggestive of sites that could interact with other proteins. These features suggest that the activity of the animal Atg13-Atg101 subcomplex is regulated and that it is an interaction hub for multiple partners. Copyright © 2015 Elsevier Ltd. All rights reserved.

A Rhodium(III) Complex as an Inhibitor of Neural Precursor Cell Expressed, Developmentally Down-Regulated 8-Activating Enzyme with in Vivo Activity against Inflammatory Bowel Disease.

Science.gov (United States)

Zhong, Hai-Jing; Wang, Wanhe; Kang, Tian-Shu; Yan, Hui; Yang, Yali; Xu, Lipeng; Wang, Yuqiang; Ma, Dik-Lung; Leung, Chung-Hang

2017-01-12

We report herein the identification of the rhodium(III) complex [Rh(phq) 2 (MOPIP)] + (1) as a potent and selective ATP-competitive neural precursor cell expressed, developmentally down-regulated 8 (NEDD8)-activating enzyme (NAE) inhibitor. Structure-activity relationship analysis indicated that the overall organometallic design of complex 1 was important for anti-inflammatory activity. Complex 1 showed promising anti-inflammatory activity in vivo for the potential treatment of inflammatory bowel disease.

The interaction of mammalian Class C Vps with nSec-1/Munc18-a and syntaxin 1A regulates pre-synaptic release

International Nuclear Information System (INIS)

Kim, Bong Yoon; Sahara, Yoshinori; Yamamoto, Akitsugu; Kominami, Eiki; Kohsaka, Shinichi; Akazawa, Chihiro

2006-01-01

Membrane docking and fusion in neurons is a highly regulated process requiring the participation of a large number of SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors) and SNARE-interacting proteins. We found that mammalian Class C Vps protein complex associated specifically with nSec-1/Munc18-a, and syntaxin 1A both in vivo and in vitro. In contrast, VAMP2 and SNAP-25, other neuronal core complex proteins, did not interact. When co-transfected with the human growth hormone (hGH) reporter gene, mammalian Class C Vps proteins enhanced Ca 2+ -dependent exocytosis, which was abolished by the Ca 2+ -channel blocker nifedipine. In hippocampal primary cultures, the lentivirus-mediated overexpression of hVps18 increased asynchronous spontaneous synaptic release without changing mEPSCs. These results indicate that mammalian Class C Vps proteins are involved in the regulation of membrane docking and fusion through an interaction with neuronal specific SNARE molecules, nSec-1/Munc18-a and syntaxin 1A

Rapamycin up-regulates triglycerides in hepatocytes by down-regulating Prox1.

Science.gov (United States)

Kwon, Sora; Jeon, Ji-Sook; Kim, Su Bin; Hong, Young-Kwon; Ahn, Curie; Sung, Jung-Suk; Choi, Inho

2016-02-27

Although the prolonged use of rapamycin may cause unwanted side effects such as hyperlipidemia, the underlying mechanism remains unknown. Prox1 is a transcription factor responsible for the development of several tissues including lymphatics and liver. There is growing evidences that Prox1 participates in metabolism in addition to embryogenesis. However, whether Prox1 is directly related to lipid metabolism is currently unknown. HepG2 human hepatoma cells were treated with rapamycin and total lipids were analyzed by thin layer chromatography. The effect of rapamycin on the expression of Prox1 was determined by western blotting. To investigate the role of Prox1 in triglycerides regulation, siRNA and overexpression system were employed. Rapamycin was injected into mice for 2 weeks and total lipids and proteins in liver were measured by thin layer chromatography and western blot analysis, respectively. Rapamycin up-regulated the amount of triglyceride and down-regulated the expression of Prox1 in HepG2 cells by reducing protein half-life but did not affect its transcript. The loss-of-function of Prox1 was coincident with the increase of triglycerides in HepG2 cells treated with rapamycin. The up-regulation of triglycerides by rapamycin in HepG2 cells reverted to normal levels by the compensation of Prox1 using the overexpression system. Rapamycin also down-regulated Prox1 expression but increased triglycerides in mouse liver. This study suggests that rapamycin can increase the amount of triglycerides by down-regulating Prox1 expression in hepatocytes, which means that the mammalian target of rapamycin (mTOR) signaling is important for the regulation of triglycerides by maintaining Prox1 expression.

The subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3: dynamics and interdependence.

Science.gov (United States)

Uzunova, Sonya Dimitrova; Zarkov, Alexander Stefanov; Ivanova, Anna Marianova; Stoynov, Stoyno Stefanov; Nedelcheva-Veleva, Marina Nedelcheva

2014-01-01

The S-phase checkpoint aims to prevent cells from generation of extensive single-stranded DNA that predisposes to genome instability. The S. cerevisiae complex Tof1/Csm3/Mrc1 acts to restrain the replicative MCM helicase when DNA synthesis is prohibited. Keeping the replication machinery intact allows restart of the replication fork when the block is relieved. Although the subunits of the Tof1/Csm3/Mrc1 complex are well studied, the impact of every single subunit on the triple complex formation and function needs to be established. This work studies the cellular localization and the chromatin binding of GFP-tagged subunits when the complex is intact and when a subunit is missing. We demonstrate that the complex is formed in cell nucleus, not the cytoplasm, as Tof1, Csm3 and Mrc1 enter the nucleus independently from one another. Via in situ chromatin binding assay we show that a Tof1-Csm3 dimer formation and chromatin binding is required to ensure the attachment of Mrc1 to chromatin. Our study indicates that the translocation into the nucleus is not the process to regulate the timing of chromatin association of Mrc1. We also studied the nuclear behavior of Mrc1 subunit in the process of adaptation to the presence hydroxyurea. Our results indicate that after prolonged HU incubation, cells bypass the S-phase checkpoint and proceed throughout the cell cycle. This process is accompanied by Mrc1 chromatin detachment and Rad53 dephosphorylation. In S. cerevisiae the subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3 independently enter the cell nucleus, where a Tof1-Csm3 dimer is formed to ensure the chromatin binding of Mrc1 and favor DNA replication and S-phase checkpoint fork arrest. In the process of adaptation to the presence of hydroxyurea Mrc1 is detached from chromatin and Rad53 checkpoint activity is diminished in order to allow S-phase checkpoint escape and completion of the cell cycle.

Inhibitor of CDK interacting with cyclin A1 (INCA1) regulates proliferation and is repressed by oncogenic signaling

DEFF Research Database (Denmark)

Baumer, Nicole; Tickenbrock, Lara; Tschanter, Petra

2011-01-01

The cell cycle is driven by the kinase activity of cyclin/CDK complexes which is negatively regulated by CDK inhibitor proteins. Recently, we identified INCA1 as interaction partner and substrate of cyclin A1 in complex with CDK2. On a functional level, we identified a novel cyclin binding site...... in the INCA1 protein. INCA1 inhibited CDK2 activity and cell proliferation. The inihibitory effects depended on the cyclin-interacting domain. Mitogenic and oncogenic signals suppressed INCA1 expression, while it was induced by cell cycle arrest. We established a deletional mouse model that showed increased...... CDK2 activity in spleen with altered spleen architecture in Inca1-/- mice. Inca1-/- embryonic fibroblasts showed an increase in the fraction of S-phase cells. Furthermore, blasts from ALL and AML patients expressed significantly reduced INCA1 levels highlighting its relevance for growth control...

Tah1 helix-swap dimerization prevents mixed Hsp90 co-chaperone complexes

International Nuclear Information System (INIS)

Morgan, Rhodri M. L.; Pal, Mohinder; Roe, S. Mark; Pearl, Laurence H.; Prodromou, Chrisostomos

2015-01-01

A helix swap involving the fifth helix between two adjacently bound Tah1 molecules restores the normal binding environment of the conserved MEEVD peptide of Hsp90. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes with Hsp90 and Tah1. Specific co-chaperone adaptors facilitate the recruitment of client proteins to the Hsp90 system. Tah1 binds the C-terminal conserved MEEVD motif of Hsp90, thus linking an eclectic set of client proteins to the R2TP complex for their assembly and regulation by Hsp90. Rather than the normal complement of seven α-helices seen in other tetratricopeptide repeat (TPR) domains, Tah1 unusually consists of the first five only. Consequently, the methionine of the MEEVD peptide remains exposed to solvent when bound by Tah1. In solution Tah1 appears to be predominantly monomeric, and recent structures have failed to explain how Tah1 appears to prevent the formation of mixed TPR domain-containing complexes such as Cpr6–(Hsp90) 2 –Tah1. To understand this further, the crystal structure of Tah1 in complex with the MEEVD peptide of Hsp90 was determined, which shows a helix swap involving the fifth α-helix between two adjacently bound Tah1 molecules. Dimerization of Tah1 restores the normal binding environment of the bound Hsp90 methionine residue by reconstituting a TPR binding site similar to that in seven-helix-containing TPR domain proteins. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes

Tah1 helix-swap dimerization prevents mixed Hsp90 co-chaperone complexes

Energy Technology Data Exchange (ETDEWEB)

Morgan, Rhodri M. L.; Pal, Mohinder; Roe, S. Mark; Pearl, Laurence H., E-mail: laurence.pearl@sussex.ac.uk; Prodromou, Chrisostomos, E-mail: laurence.pearl@sussex.ac.uk [University of Sussex, Falmer, Brighton BN1 9RQ (United Kingdom)

2015-05-01

A helix swap involving the fifth helix between two adjacently bound Tah1 molecules restores the normal binding environment of the conserved MEEVD peptide of Hsp90. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes with Hsp90 and Tah1. Specific co-chaperone adaptors facilitate the recruitment of client proteins to the Hsp90 system. Tah1 binds the C-terminal conserved MEEVD motif of Hsp90, thus linking an eclectic set of client proteins to the R2TP complex for their assembly and regulation by Hsp90. Rather than the normal complement of seven α-helices seen in other tetratricopeptide repeat (TPR) domains, Tah1 unusually consists of the first five only. Consequently, the methionine of the MEEVD peptide remains exposed to solvent when bound by Tah1. In solution Tah1 appears to be predominantly monomeric, and recent structures have failed to explain how Tah1 appears to prevent the formation of mixed TPR domain-containing complexes such as Cpr6–(Hsp90){sub 2}–Tah1. To understand this further, the crystal structure of Tah1 in complex with the MEEVD peptide of Hsp90 was determined, which shows a helix swap involving the fifth α-helix between two adjacently bound Tah1 molecules. Dimerization of Tah1 restores the normal binding environment of the bound Hsp90 methionine residue by reconstituting a TPR binding site similar to that in seven-helix-containing TPR domain proteins. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes.

Mms1 is an assistant for regulating G-quadruplex DNA structures.

Science.gov (United States)

Schwindt, Eike; Paeschke, Katrin

2017-11-02

The preservation of genome stability is fundamental for every cell. Genomic integrity is constantly challenged. Among those challenges are also non-canonical nucleic acid structures. In recent years, scientists became aware of the impact of G-quadruplex (G4) structures on genome stability. It has been shown that folded G4-DNA structures cause changes in the cell, such as transcriptional up/down-regulation, replication stalling, or enhanced genome instability. Multiple helicases have been identified to regulate G4 structures and by this preserve genome stability. Interestingly, although these helicases are mostly ubiquitous expressed, they show specificity for G4 regulation in certain cellular processes (e.g., DNA replication). To this date, it is not clear how this process and target specificity of helicases are achieved. Recently, Mms1, an ubiquitin ligase complex protein, was identified as a novel G4-DNA-binding protein that supports genome stability by aiding Pif1 helicase binding to these regions. In this perspective review, we discuss the question if G4-DNA interacting proteins are fundamental for helicase function and specificity at G4-DNA structures.

COMMD1 is linked to the WASH complex and regulates endosomal trafficking of the copper transporter ATP7A

NARCIS (Netherlands)

Phillips-Krawczak, Christine A.; Singla, Amika; Starokadomskyy, Petro; Deng, Zhihui; Osborne, Douglas G.; Li, Haiying; Dick, Christopher J.; Gomez, Timothy S.; Koenecke, Megan; Zhang, Jin-San; Dai, Haiming; Sifuentes-Dominguez, Luis F.; Geng, Linda N.; Kaufmann, Scott H.; Hein, Marco Y.; Wallis, Mathew; McGaughran, Julie; Gecz, Jozef; De Sluis, Bart van; Billadeau, Daniel D.; Burstein, Ezra

2015-01-01

COMMD1 deficiency results in defective copper homeostasis, but the mechanism for this has remained elusive. Here we report that COMMD1 is directly linked to early endosomes through its interaction with a protein complex containing CCDC22, CCDC93, and C16orf62. This COMMD/CCDC22/CCDC93 (CCC) complex

Mechanisms and pathways of growth failure in primordial dwarfism.

Science.gov (United States)

Klingseisen, Anna; Jackson, Andrew P

2011-10-01

The greatest difference between species is size; however, the developmental mechanisms determining organism growth remain poorly understood. Primordial dwarfism is a group of human single-gene disorders with extreme global growth failure (which includes Seckel syndrome, microcephalic osteodysplastic primordial dwarfism I [MOPD] types I and II, and Meier-Gorlin syndrome). Ten genes have now been identified for microcephalic primordial dwarfism, encoding proteins involved in fundamental cellular processes including genome replication (ORC1 [origin recognition complex 1], ORC4, ORC6, CDT1, and CDC6), DNA damage response (ATR [ataxia-telangiectasia and Rad3-related]), mRNA splicing (U4atac), and centrosome function (CEP152, PCNT, and CPAP). Here, we review the cellular and developmental mechanisms underlying the pathogenesis of these conditions and address whether further study of these genes could provide novel insight into the physiological regulation of organism growth.

Lasp-1 regulates podosome function.

Directory of Open Access Journals (Sweden)

Miriam Stölting

Full Text Available Eukaryotic cells form a variety of adhesive structures to connect with their environment and to regulate cell motility. In contrast to classical focal adhesions, podosomes, highly dynamic structures of different cell types, are actively engaged in matrix remodelling and degradation. Podosomes are composed of an actin-rich core region surrounded by a ring-like structure containing signalling molecules, motor proteins as well as cytoskeleton-associated proteins. Lasp-1 is a ubiquitously expressed, actin-binding protein that is known to regulate cytoskeleton architecture and cell migration. This multidomain protein is predominantely present at focal adhesions, however, a second pool of Lasp-1 molecules is also found at lamellipodia and vesicle-like microdomains in the cytosol.In this report, we show that Lasp-1 is a novel component and regulator of podosomes. Immunofluorescence studies reveal a localization of Lasp-1 in the podosome ring structure, where it colocalizes with zyxin and vinculin. Life cell imaging experiments demonstrate that Lasp-1 is recruited in early steps of podosome assembly. A siRNA-mediated Lasp-1 knockdown in human macrophages affects podosome dynamics as well as their matrix degradation capacity. In summary, our data indicate that Lasp-1 is a novel component of podosomes and is involved in the regulation of podosomal function.

α-1 Antitrypsin regulates human neutrophil chemotaxis induced by soluble immune complexes and IL-8.

LENUS (Irish Health Repository)

Bergin, David A

2010-12-01

Hereditary deficiency of the protein α-1 antitrypsin (AAT) causes a chronic lung disease in humans that is characterized by excessive mobilization of neutrophils into the lung. However, the reason for the increased neutrophil burden has not been fully elucidated. In this study we have demonstrated using human neutrophils that serum AAT coordinates both CXCR1- and soluble immune complex (sIC) receptor-mediated chemotaxis by divergent pathways. We demonstrated that glycosylated AAT can bind to IL-8 (a ligand for CXCR1) and that AAT-IL-8 complex formation prevented IL-8 interaction with CXCR1. Second, AAT modulated neutrophil chemotaxis in response to sIC by controlling membrane expression of the glycosylphosphatidylinositol-anchored (GPI-anchored) Fc receptor FcγRIIIb. This process was mediated through inhibition of ADAM-17 enzymatic activity. Neutrophils isolated from clinically stable AAT-deficient patients were characterized by low membrane expression of FcγRIIIb and increased chemotaxis in response to IL-8 and sIC. Treatment of AAT-deficient individuals with AAT augmentation therapy resulted in increased AAT binding to IL-8, increased AAT binding to the neutrophil membrane, decreased FcγRIIIb release from the neutrophil membrane, and normalization of chemotaxis. These results provide new insight into the mechanism underlying the effect of AAT augmentation therapy in the pulmonary disease associated with AAT deficiency.

The SET1 Complex Selects Actively Transcribed Target Genes via Multivalent Interaction with CpG Island Chromatin

Directory of Open Access Journals (Sweden)

David A. Brown

2017-09-01

Full Text Available Chromatin modifications and the promoter-associated epigenome are important for the regulation of gene expression. However, the mechanisms by which chromatin-modifying complexes are targeted to the appropriate gene promoters in vertebrates and how they influence gene expression have remained poorly defined. Here, using a combination of live-cell imaging and functional genomics, we discover that the vertebrate SET1 complex is targeted to actively transcribed gene promoters through CFP1, which engages in a form of multivalent chromatin reading that involves recognition of non-methylated DNA and histone H3 lysine 4 trimethylation (H3K4me3. CFP1 defines SET1 complex occupancy on chromatin, and its multivalent interactions are required for the SET1 complex to place H3K4me3. In the absence of CFP1, gene expression is perturbed, suggesting that normal targeting and function of the SET1 complex are central to creating an appropriately functioning vertebrate promoter-associated epigenome.

CTC1-STN1 coordinates G- and C-strand synthesis to regulate telomere length.

Science.gov (United States)

Gu, Peili; Jia, Shuting; Takasugi, Taylor; Smith, Eric; Nandakumar, Jayakrishnan; Hendrickson, Eric; Chang, Sandy

2018-05-17

Coats plus (CP) is a rare autosomal recessive disorder caused by mutations in CTC1, a component of the CST (CTC1, STN1, and TEN1) complex important for telomere length maintenance. The molecular basis of how CP mutations impact upon telomere length remains unclear. The CP CTC1 L1142H mutation has been previously shown to disrupt telomere maintenance. In this study, we used CRISPR/Cas9 to engineer this mutation into both alleles of HCT116 and RPE cells to demonstrate that CTC1:STN1 interaction is required to repress telomerase activity. CTC1 L1142H interacts poorly with STN1, leading to telomerase-mediated telomere elongation. Impaired interaction between CTC1 L1142H :STN1 and DNA Pol-α results in increased telomerase recruitment to telomeres and further telomere elongation, revealing that C:S binding to DNA Pol-α is required to fully repress telomerase activity. CP CTC1 mutants that fail to interact with DNA Pol-α resulted in loss of C-strand maintenance and catastrophic telomere shortening. Our findings place the CST complex as an important regulator of both G-strand extensions by telomerase and C-strand synthesis by DNA Pol-α. © 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

The AP2 clathrin adaptor protein complex regulates the abundance of GLR-1 glutamate receptors in the ventral nerve cord of Caenorhabditis elegans.

Science.gov (United States)

Garafalo, Steven D; Luth, Eric S; Moss, Benjamin J; Monteiro, Michael I; Malkin, Emily; Juo, Peter

2015-05-15

Regulation of glutamate receptor (GluR) abundance at synapses by clathrin-mediated endocytosis can control synaptic strength and plasticity. We take advantage of viable, null mutations in subunits of the clathrin adaptor protein 2 (AP2) complex in Caenorhabditis elegans to characterize the in vivo role of AP2 in GluR trafficking. In contrast to our predictions for an endocytic adaptor, we found that levels of the GluR GLR-1 are decreased at synapses in the ventral nerve cord (VNC) of animals with mutations in the AP2 subunits APM-2/μ2, APA-2/α, or APS-2/σ2. Rescue experiments indicate that APM-2/μ2 functions in glr-1-expressing interneurons and the mature nervous system to promote GLR-1 levels in the VNC. Genetic analyses suggest that APM-2/μ2 acts upstream of GLR-1 endocytosis in the VNC. Consistent with this, GLR-1 accumulates in cell bodies of apm-2 mutants. However, GLR-1 does not appear to accumulate at the plasma membrane of the cell body as expected, but instead accumulates in intracellular compartments including Syntaxin-13- and RAB-14-labeled endosomes. This study reveals a novel role for the AP2 clathrin adaptor in promoting the abundance of GluRs at synapses in vivo, and implicates AP2 in the regulation of GluR trafficking at an early step in the secretory pathway. © 2015 Garafalo et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate

Energy Technology Data Exchange (ETDEWEB)

Vorhagen, Susanne; Niessen, Carien M., E-mail: carien.niessen@uni-koeln.de

2014-11-01

Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Loss of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium.

Detection of seven virulence and toxin genes of Campylobacter jejuni isolates from Danish turkeys by PCR and cytolethal distending toxin production of the isolates

DEFF Research Database (Denmark)

Bang, Dang Duong; Borck, Birgitte; Nielsen, Eva Møller

2004-01-01

A total of 117 Campylobacter jejuni isolates from Danish turkeys were tested for the presence of seven virulence and toxin genes by PCR. One hundred seventeen (100%) isolates were positive for flaA, cadF, and ceuE gene primers. One hundred three (88%) isolates were positive for cdt gene cluster PCR.......7%) in Colon 205 assays, and 109 (93.2%) in chicken embryo cell assays. The CDT titers were determined in Vero cell assays. Of 117 isolates, 50 (42.7%) produced a CDT titer of 1:100, 29 (24.8%) of 1:50, and 27 (23%) of 1:5 to 1:10; 8 (6.8%) produced a CDT titer at undiluted supernatants and 3 (2.6%) produced...

Nucleoporin Nup98 associates with Trx/MLL and NSL histone-modifying complexes and regulates Hox gene expression.

Science.gov (United States)

Pascual-Garcia, Pau; Jeong, Jieun; Capelson, Maya

2014-10-23

The nuclear pore complex is a transport channel embedded in the nuclear envelope and made up of 30 different components termed nucleoporins (Nups). In addition to their classical role in transport, a subset of Nups has a conserved role in the regulation of transcription via direct binding to chromatin. The molecular details of this function remain obscure, and it is unknown how metazoan Nups are recruited to their chromatin locations or what transcription steps they regulate. Here, we demonstrate genome-wide and physical association between Nup98 and histone-modifying complexes MBD-R2/NSL [corrected] and Trx/MLL. Importantly, we identify a requirement for MBD-R2 in recruitment of Nup98 to many of its genomic target sites. Consistent with its interaction with the Trx/MLL complex, Nup98 is shown to be necessary for Hox gene expression in developing fly tissues. These findings introduce roles of Nup98 in epigenetic regulation that may underlie the basis of oncogenicity of Nup98 fusions in leukemia.

Stat1-independent regulation of gene expression in response to IFN-γ

Science.gov (United States)

Ramana, Chilakamarti V.; Gil, M. Pilar; Han, Yulong; Ransohoff, Richard M.; Schreiber, Robert D.; Stark, George R.

2001-01-01

Although Stat1 is essential for cells to respond fully to IFN-γ, there is substantial evidence that, in the absence of Stat1, IFN-γ can still regulate the expression of some genes, induce an antiviral state and affect cell growth. We have now identified many genes that are regulated by IFN-γ in serum-starved Stat1-null mouse fibroblasts. The proteins induced by IFN-γ in Stat1-null cells can account for the substantial biological responses that remain. Some genes are induced in both wild-type and Stat1-null cells and thus are truly Stat1-independent. Others are subject to more complex regulation in response to IFN-γ, repressed by Stat1 in wild-type cells and activated in Stat1-null cells. Many genes induced by IFN-γ in Stat1-null fibroblasts also are induced by platelet-derived growth factor in wild-type cells and thus are likely to be involved in cell proliferation. In mouse cells expressing the docking site mutant Y440F of human IFN-γ receptor subunit 1, the mouse Stat1 is not phosphorylated in response to human IFN-γ, but c-myc and c-jun are still induced, showing that the Stat1 docking site is not required for Stat1-independent signaling. PMID:11390994

Ethylene Control of Fruit Ripening: Revisiting the Complex Network of Transcriptional Regulation1

Science.gov (United States)

Chervin, Christian; Bouzayen, Mondher

2015-01-01

The plant hormone ethylene plays a key role in climacteric fruit ripening. Studies on components of ethylene signaling have revealed a linear transduction pathway leading to the activation of ethylene response factors. However, the means by which ethylene selects the ripening-related genes and interacts with other signaling pathways to regulate the ripening process are still to be elucidated. Using tomato (Solanum lycopersicum) as a reference species, the present review aims to revisit the mechanisms by which ethylene regulates fruit ripening by taking advantage of new tools available to perform in silico studies at the genome-wide scale, leading to a global view on the expression pattern of ethylene biosynthesis and response genes throughout ripening. Overall, it provides new insights on the transcriptional network by which this hormone coordinates the ripening process and emphasizes the interplay between ethylene and ripening-associated developmental factors and the link between epigenetic regulation and ethylene during fruit ripening. PMID:26511917

Human T-lymphotropic virus type 1 Tax protein complexes with P-TEFb and competes for Brd4 and 7SK snRNP/HEXIM1 binding.

Science.gov (United States)

Cho, Won-Kyung; Jang, Moon Kyoo; Huang, Keven; Pise-Masison, Cynthia A; Brady, John N

2010-12-01

Positive transcription elongation factor b (P-TEFb) plays an important role in stimulating RNA polymerase II elongation for viral and cellular gene expression. P-TEFb is found in cells in either an active, low-molecular-weight (LMW) form or an inactive, high-molecular-weight (HMW) form. We report here that human T-lymphotropic virus type 1 (HTLV-1) Tax interacts with the cyclin T1 subunit of P-TEFb, forming a distinct Tax/P-TEFb LMW complex. We demonstrate that Tax can play a role in regulating the amount of HMW complex present in the cell by decreasing the binding of 7SK snRNP/HEXIM1 to P-TEFb. This is seen both in vitro using purified Tax protein and in vivo in cells transduced with Tax expression constructs. Further, we find that a peptide of cyclin T1 spanning the Tax binding domain inhibits the ability of Tax to disrupt HMW P-TEFb complexes. These results suggest that the direct interaction of Tax with cyclin T1 can dissociate P-TEFb from the P-TEFb/7SK snRNP/HEXIM1 complex for activation of the viral long terminal repeat (LTR). We also show that Tax competes with Brd4 for P-TEFb binding. Chromatin immunoprecipitation (ChIP) assays demonstrated that Brd4 and P-TEFb are associated with the basal HTLV-1 LTR, while Tax and P-TEFb are associated with the activated template. Furthermore, the knockdown of Brd4 by small interfering RNA (siRNA) activates the HTLV-1 LTR promoter, which results in an increase in viral expression and production. Our studies have identified Tax as a regulator of P-TEFb that is capable of affecting the balance between its association with the large inactive complex and the small active complex.