Solution Structure of an Intramembrane Aspartyl Protease via Small Angle Neutron Scattering

DOE PAGES

Naing, Swe-Htet; Oliver, Ryan C.; Weiss, Kevin L.; ...

2018-02-06

Intramembrane aspartyl proteases (IAPs) comprise one of four families of integral membrane proteases that hydrolyze substrates within the hydrophobic lipid bilayer. IAPs include signal peptide peptidase, which processes remnant signal peptides from nascent polypeptides in the endoplasmic reticulum, and presenilin, the catalytic component of the γ-secretase complex that processes Notch and amyloid precursor protein. Despite their broad biomedical reach, basic structure-function relationships of IAPs remain active areas of research. Characterization of membrane-bound proteins is notoriously challenging due to their inherently hydrophobic character. For IAPs, oligomerization state in solution is one outstanding question, with previous proposals for monomer, dimer, tetramer, andmore » octamer. Here we used small angle neutron scattering (SANS) to characterize n-dodecyl-β-D-maltopyranoside (DDM) detergent solutions containing and absent a microbial IAP ortholog. A unique feature of SANS is the ability to modulate the solvent composition to mask all but the enzyme of interest. The signal from the IAP was enhanced by deuteration and, uniquely, scattering from DDM and buffers were matched by the use of both tail-deuterated DDM and D 2O. The radius of gyration calculated for IAP and the corresponding ab initio consensus model are consistent with a monomer. The model is slightly smaller than the crystallographic IAP monomer, suggesting a more compact protein in solution compared with the crystal lattice. In conclusion, our study provides direct insight into the oligomeric state of purified IAP in surfactant solution, and demonstrates the utility of fully contrast-matching the detergent in SANS to characterize other intramembrane proteases and their membrane-bound substrates.« less

Amyloid precursor protein and Presenilin1 interact with the adaptor GRB2 and modulate ERK 1,2 signaling.

PubMed

Nizzari, Mario; Venezia, Valentina; Repetto, Emanuela; Caorsi, Valentina; Magrassi, Raffaella; Gagliani, Maria Cristina; Carlo, Pia; Florio, Tullio; Schettini, Gennaro; Tacchetti, Carlo; Russo, Tommaso; Diaspro, Alberto; Russo, Claudio

2007-05-04

The amyloid precursor protein (APP) and the presenilins 1 and 2 are genetically linked to the development of familial Alzheimer disease. APP is a single-pass transmembrane protein and precursor of fibrillar and toxic amyloid-beta peptides, which are considered responsible for Alzheimer disease neurodegeneration. Presenilins are multipass membrane proteins, involved in the enzymatic cleavage of APP and other signaling receptors and transducers. The role of APP and presenilins in Alzheimer disease development seems to be related to the formation of amyloid-beta peptides; however, their physiological function, reciprocal interaction, and molecular mechanisms leading to neurodegeneration are unclear. APP and presenilins are also involved in multiple interactions with intracellular proteins, the significance of which is under investigation. Among the different APP-interacting proteins, we focused our interest on the GRB2 adaptor protein, which connects cell surface receptors to intracellular signaling pathways. In this study we provide evidence by co-immunoprecipitation experiments, confocal and electron microscopy, and by fluorescence resonance energy transfer experiments that both APP and presenilin1 interact with GRB2 in vesicular structures at the centrosome of the cell. The final target for these interactions is ERK1,2, which is activated in mitotic centrosomes in a PS1- and APP-dependent manner. These data suggest that both APP and presenilin1 can be part of a common signaling pathway that regulates ERK1,2 and the cell cycle.

Presenilin 1 mutations influence processing and trafficking of the ApoE receptor apoER2.

PubMed

Wang, Wei; Moerman-Herzog, Andrea M; Slaton, Arthur; Barger, Steven W

2017-01-01

Presenilin (PS)-1 is an intramembrane protease serving as the catalytic component of γ-secretase. Mutations in the PS1 gene are the most common cause of familial Alzheimer's disease (FAD). The low-density lipoprotein (LDL)-receptor family member apoER2 is a γ-secretase substrate that has been associated with AD in several ways, including acting as a receptor for apolipoprotein E (ApoE). ApoER2 is processed by γ-secretase into a C-terminal fragment (γ-CTF) that appears to regulate gene expression. FAD PS1 mutations were tested for effects on apoER2. PS1 mutation R278I showed impaired γ-secretase activity for apoER2 in the basal state or after exposure to Reelin. PS1 M146V mutation permitted accumulation of apoER2 CTFs after Reelin treatment, whereas no difference was seen between wild-type (WT) and M146V in the basal state. PS1 L282V mutation, combined with the γ-secretase inhibitor N-(N-[3,5-Difluorophenacetyl]-L-alanyl)-S-phenylglycine t-butyl ester, greatly reduced the cell-surface levels of apoER2 without affecting total apoER2 levels, suggesting a defect in receptor trafficking. These findings indicate that impaired processing or localization of apoER2 may contribute to the pathogenic effects of FAD mutations in PS1. Published by Elsevier Inc.

PRESENILIN-NULL CELLS HAVE ALTERED TWO-PORE CALCIUM CHANNEL EXPRESSION AND LYSOSOMAL CALCIUM; IMPLICATIONS FOR LYSOSOMAL FUNCTION

PubMed Central

Kayala, Kara M Neely; Dickinson, George D; Minassian, Anet; Walls, Ken C; Green, Kim N; LaFerla, Frank M

2012-01-01

Presenilins are necessary for calcium homeostasis and also for efficient proteolysis through the autophagy/lysosome system. Presenilin regulates both endoplasmic reticulum calcium stores and autophagic proteolysis in a γ-secretase independent fashion. The endo-lysosome system can also act as a calcium store, with calcium efflux channels being recently identified as two-pore channels 1 and 2. Here we investigated lysosomal calcium content and the channels that mediate calcium release from these acidic stores in presenilin knockout cells. We report that presenilin loss leads to a lower total lysosomal calcium store despite the buildup of lysosomes found in these cells. Additionally, we find alterations in two-pore calcium channel protein expression, with loss of presenilin preventing the formation of a high molecular weight species of TPC1 and TPC2. Finally, we find that treatments that disturb lysosomal calcium release lead to a reduction in autophagy function yet lysosomal inhibitors do not alter two-pore calcium channel expression. These data indicate that alterations in lysosomal calcium in the absence of presenilins might be leading to disruptions in autophagy. PMID:23103503

Activation of Phospholipase C Increases Intramembrane Electric Fields in N1E-115 Neuroblastoma Cells

PubMed Central

Xu, Chang; Loew, Leslie M.

2003-01-01

We imaged the intramembrane potential (a combination of transmembrane, surface, and dipole potential) on N1E-115 neuroblastoma cells with a voltage-sensitive dye. After activation of the B2 bradykinin receptor, the electric field sensed by the dye increased by an amount equivalent to a depolarization of 83 mV. The increase in intramembrane potential was blocked by the phospholipase C (PLC) inhibitors U-73122 and neomycin, and was invariably accompanied by a transient rise of [Ca2+]i. A depolarized inner surface potential, as the membrane loses negative charges via phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, and an increase in the dipole potential, as PIP2 is hydrolyzed to 1,2-diacylglycerol (DAG), can each account for a small portion of the change in intramembrane potential. The primary contribution to the measured change in intramembrane potential may arise from an increased dipole potential, as DAG molecules are generated from hydrolysis of other phospholipids. We found bradykinin produced an inhibition of a M-type voltage-dependent K+ current (IK(M)). This inhibition was also blocked by the PLC inhibitors and had similar kinetics as the bradykinin-induced modulation of intramembrane potential. Our results suggest that the change in the local intramembrane potential induced by bradykinin may play a role in mediating the IK(M) inhibition. PMID:12770917

MBTPS2 mutations cause defective regulated intramembrane proteolysis in X-linked osteogenesis imperfecta

PubMed Central

Lindert, Uschi; Cabral, Wayne A.; Ausavarat, Surasawadee; Tongkobpetch, Siraprapa; Ludin, Katja; Barnes, Aileen M.; Yeetong, Patra; Weis, Maryann; Krabichler, Birgit; Srichomthong, Chalurmpon; Makareeva, Elena N.; Janecke, Andreas R.; Leikin, Sergey; Röthlisberger, Benno; Rohrbach, Marianne; Kennerknecht, Ingo; Eyre, David R.; Suphapeetiporn, Kanya; Giunta, Cecilia; Marini, Joan C.; Shotelersuk, Vorasuk

2016-01-01

Osteogenesis imperfecta (OI) is a collagen-related bone dysplasia. We identified an X-linked recessive form of OI caused by defects in MBTPS2, which encodes site-2 metalloprotease (S2P). MBTPS2 missense mutations in two independent kindreds with moderate/severe OI cause substitutions at highly conserved S2P residues. Mutant S2P has normal stability, but impaired functioning in regulated intramembrane proteolysis (RIP) of OASIS, ATF6 and SREBP transcription factors, consistent with decreased proband secretion of type I collagen. Further, hydroxylation of the collagen lysine residue (K87) critical for crosslinking is reduced in proband bone tissue, consistent with decreased lysyl hydroxylase 1 in proband osteoblasts. Reduced collagen crosslinks presumptively undermine bone strength. Also, proband osteoblasts have broadly defective differentiation. These mutations provide evidence that RIP plays a fundamental role in normal bone development. PMID:27380894

Taking a position on intramembrane proteolysis.

PubMed

Lemieux, M Joanne

2018-03-30

Decades of work have contributed to our in-depth mechanistic understanding of soluble proteases, but much less is known about the catalytic mechanism of intramembrane proteolysis due to inherent difficulties in both preparing and analyzing integral membrane enzymes and transmembrane substrates. New work from Naing et al. tackles this challenge by examining the catalytic parameters of an aspartyl intramembrane protease homologous to the enzyme that cleaves amyloid precursor protein, finding that both chemistry and register contribute to specificity in substrate cleavage. © 2018 Joanne Lemieux.

Molecular dynamics simulation study reveals potential substrate entry path into γ-secretase/presenilin-1.

PubMed

Kong, Ren; Chang, Shan; Xia, Weiming; Wong, Stephen T C

2015-08-01

Presenilin 1 (PS1) is the catalytic unit of γ-secretase which cleaves more than one hundred substrates. Among them, amyloid precursor protein (APP) and Notch are notable for their pivotal role in the pathogenesis of Alzheimer's disease (AD) and certain types of cancer. The hydrolysis process occurring inside the hydrophobic lipid bilayer remains unclear. With the aim to understand the mechanism of intramembrane proteolysis by γ-secretase, we constructed a homology model of human PS1 and performed molecular dynamics simulation in explicit membrane phospholipids with different components. During the simulation, TM9 was found to exhibit a high level of flexibility that involved in "gate-open" movement of TM2 and TM6, and thus partially exposed the catalytic residues. The highly conserved PALP motif acts as an anchor to mediate the conformation changes of TM6 induced by TM9. Moreover, direct interactions were observed between 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and the active site of γ-secretase, indicating that the lipid molecules have the potential to modulate γ-secretase by contacting with the catalytic residues, i.e., ASP 257 and ASP 385 of PS1. The intermediate states indicate a potential substrate penetration pathway through the interface of TM2 and TM6, which may be induced by changes of TM9. To our knowledge, this is the first molecular simulation study that reveals dynamic behavior of the human PS1 structure in the lipid bilayer and provides insight into the substrate entry path for subsequent intramembrane hydrolysis, which is critical information required for new strategy development of γ-secretase modulators to alleviate devastating AD. Copyright © 2015 Elsevier Inc. All rights reserved.

An Evolutionarily Conserved Role of Presenilin in Neuronal Protection in the Aging Drosophila Brain.

PubMed

Kang, Jongkyun; Shin, Sarah; Perrimon, Norbert; Shen, Jie

2017-07-01

Mutations in the Presenilin genes are the major genetic cause of Alzheimer's disease. Presenilin and Nicastrin are essential components of γ-secretase, a multi-subunit protease that cleaves Type I transmembrane proteins. Genetic studies in mice previously demonstrated that conditional inactivation of Presenilin or Nicastrin in excitatory neurons of the postnatal forebrain results in memory deficits, synaptic impairment, and age-dependent neurodegeneration. The roles of Drosophila Presenilin ( Psn ) and Nicastrin ( Nct ) in the adult fly brain, however, are unknown. To knockdown (KD) Psn or Nct selectively in neurons of the adult brain, we generated multiple shRNA lines. Using a ubiquitous driver, these shRNA lines resulted in 80-90% reduction of mRNA and pupal lethality-a phenotype that is shared with Psn and Nct mutants carrying nonsense mutations. Furthermore, expression of these shRNAs in the wing disc caused notching wing phenotypes, which are also shared with Psn and Nct mutants. Similar to Nct , neuron-specific Psn KD using two independent shRNA lines led to early mortality and rough eye phenotypes, which were rescued by a fly Psn transgene. Interestingly, conditional KD (cKD) of Psn or Nct in adult neurons using the elav-Gal4 and tubulin-Gal80 ts system caused shortened lifespan, climbing defects, increases in apoptosis, and age-dependent neurodegeneration. Together, these findings demonstrate that, similar to their mammalian counterparts, Drosophila Psn and Nct are required for neuronal survival during aging and normal lifespan, highlighting an evolutionarily conserved role of Presenilin in neuronal protection in the aging brain. Copyright © 2017 by the Genetics Society of America.

Evidence that the C-terminus of Human Presenilin 1 is Located in the Extra-cytoplasmic Space

PubMed Central

James Turner, R.

2005-01-01

The polytopic membrane protein presenilin 1 (PS1) is a component of the γ-secretase complex that is responsible for the intramembranous cleavage of a number of type I transmembrane proteins including the β-amyloid precursor protein (APP). Mutations of PS1, apparently leading to aberrant processing of APP, have been genetically linked to early-onset familial Alzheimer's disease. PS1 contains ten hydrophobic regions (HRs) sufficiently long to be α-helical membrane spanning segments. Most topology models for PS1 place its C-terminal ∼40 amino acids, which include the 10th HR, in the cytosolic space. However, several recent observations suggest that HR 10 may be integrated into the membrane and involved in the interaction between PS1 and APP. We have applied three independent methodologies to investigate the location of HR 10 and the extreme C-terminus of PS1. The results from these methods indicate that HR 10 spans the membrane and that the C-terminal amino acids of PS1 lie in the extra-cytoplasmic space. PMID:15843437

Cleavage of amyloid precursor protein by an archaeal presenilin homologue PSH

PubMed Central

Dang, Shangyu; Wu, Shenjie; Wang, Jiawei; Li, Hongbo; Huang, Min; He, Wei; Li, Yue-Ming; Wong, Catherine C. L.; Shi, Yigong

2015-01-01

Aberrant cleavage of amyloid precursor protein (APP) by γ-secretase contributes to the development of Alzheimer’s disease. More than 200 disease-derived mutations have been identified in presenilin (the catalytic subunit of γ-secretase), making modulation of γ-secretase activity a potentially attractive therapeutic opportunity. Unfortunately, the technical challenges in dealing with intact γ-secretase have hindered discovery of modulators and demand a convenient substitute approach. Here we report that, similar to γ-secretase, the archaeal presenilin homolog PSH faithfully processes the substrate APP C99 into Aβ42, Aβ40, and Aβ38. The molar ratio of the cleavage products Aβ42 over Aβ40 by PSH is nearly identical to that by γ-secretase. The proteolytic activity of PSH is specifically suppressed by presenilin-specific inhibitors. Known modulators of γ-secretase also modulate PSH similarly in terms of the Aβ42/Aβ40 ratio. Structural analysis reveals association of a known γ-secretase inhibitor with PSH between its two catalytic aspartate residues. These findings identify PSH as a surrogate protease for the screening of agents that may regulate the protease activity and the cleavage preference of γ-secretase. PMID:25733893

Sequential and γ-secretase-dependent processing of the betacellulin precursor generates a palmitoylated intracellular-domain fragment that inhibits cell growth

PubMed Central

Stoeck, Alexander; Shang, Li; Dempsey, Peter J.

2010-01-01

Betacellulin (BTC) belongs to the family of epidermal growth factor (EGF)-like growth factors that are expressed as transmembrane precursors and undergo proteolytic ectodomain shedding to release soluble mature ligands. BTC is a dual-specificity ligand for ErbB1 and ErbB4 receptors, and can activate unique signal-transduction pathways that are beneficial for the function, survival and regeneration of pancreatic β-cells. We have previously shown that BTC precursor (proBTC) is cleaved by ADAM10 to generate soluble ligand and a stable, transmembrane remnant (BTC-CTF). In this study, we analyzed the fate of the BTC-CTF in greater detail. We demonstrated that proBTC is cleaved by ADAM10 to produce BTC-CTF, which then undergoes intramembrane processing by presenilin-1- and/or presenilin-2-dependent γ-secretase to generate an intracellular-domain fragment (BTC-ICD). We found that the proBTC cytoplasmic domain is palmitoylated and that palmitoylation is not required for ADAM10-dependent cleavage but is necessary for the stability and γ-secretase-dependent processing of BTC-CTF to generate BTC-ICD. Additionally, palmitoylation is required for nuclear-membrane localization of BTC-ICD, as demonstrated by the redistribution of non-palmitoylated BTC-ICD mutant to the nucleoplasm. Importantly, a novel receptor-independent role for BTC-ICD signaling is suggested by the ability of BTC-ICD to inhibit cell growth in vitro. PMID:20530572

p53-dependent control of cell death by nicastrin: lack of requirement for presenilin-dependent gamma-secretase complex.

PubMed

Pardossi-Piquard, Raphaëlle; Dunys, Julie; Giaime, Emilie; Guillot-Sestier, Marie-Victoire; St George-Hyslop, Peter; Checler, Frédéric; Alves da Costa, Cristine

2009-04-01

Nicastrin (NCT) is a component of the presenilin (PS)-dependent gamma-secretase complexes that liberate amyloid beta-peptides from the beta-Amyloid Precursor Protein. Several lines of evidence indicate that the members of these complexes could also contribute to the control of cell death. Here we show that over-expression of NCT increases the viability of human embryonic kidney (HEK293) cells and decreases staurosporine (STS)- and thapsigargin (TPS)-induced caspase-3 activation in various cell lines from human and neuronal origins by Akt-dependent pathway. NCT lowers p53 expression, transcriptional activity and promoter transactivation and reduces p53 phosphorylation. NCT-associated protection against STS-stimulated cell death was completely abolished by p53 deficiency. Conversely, the depletion of NCT drastically enhances STS-induced caspase-3 activation and p53 pathway and favored p53 nuclear translocation. We examined whether NCT protective function depends on PS-dependent gamma-secretase activity. First, a 29-amino acid deletion known to reduce NCT-dependent amyloid beta-peptide production did not affect NCT-associated protective phenotype. Second, NCT still reduces STS-induced caspase-3 activation in fibroblasts lacking PS1 and PS2. Third, the gamma-secretase inhibitor DFK167 did not affect NCT-mediated reduction of p53 activity. Altogether, our study indicates that NCT controls cell death via phosphoinositide 3-kinase/Akt and p53-dependent pathways and that this function remains independent of the activity and molecular integrity of the gamma-secretase complexes.

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

PubMed

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

2015-12-01

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

Presenilin-1 affects trafficking and processing of βAPP and is targeted in a complex with nicastrin to the plasma membrane

PubMed Central

Kaether, Christoph; Lammich, Sven; Edbauer, Dieter; Ertl, Michaela; Rietdorf, Jens; Capell, Anja; Steiner, Harald; Haass, Christian

2002-01-01

Amyloid β-peptide (Aβ) is generated by the consecutive cleavages of β- and γ-secretase. The intramembraneous γ-secretase cleavage critically depends on the activity of presenilins (PS1 and PS2). Although there is evidence that PSs are aspartyl proteases with γ-secretase activity, it remains controversial whether their subcellular localization overlaps with the cellular sites of Aβ production. We now demonstrate that biologically active GFP-tagged PS1 as well as endogenous PS1 are targeted to the plasma membrane (PM) of living cells. On the way to the PM, PS1 binds to nicastrin (Nct), an essential component of the γ-secretase complex. This complex is targeted through the secretory pathway where PS1-bound Nct becomes endoglycosidase H resistant. Moreover, surface-biotinylated Nct can be coimmunoprecipitated with PS1 antibodies, demonstrating that this complex is located to cellular sites with γ-secretase activity. Inactivating PS1 or PS2 function by mutagenesis of one of the critical aspartate residues or by γ-secretase inhibitors results in delayed reinternalization of the β-amyloid precursor protein and its accumulation at the cell surface. Our data suggest that PS is targeted as a biologically active complex with Nct through the secretory pathway to the cell surface and suggest a dual function of PS in γ-secretase processing and in trafficking. PMID:12147673

Genotyping of presenilin-1 polymorphism in amyotrophic lateral sclerosis.

PubMed

Panas, M; Karadima, G; Kalfakis, N; Psarrou, O; Floroskoufi, P; Kladi, A; Petersen, M B; Vassilopoulos, D

2000-12-01

The mechanisms underlying motor neuron degeneration in amyotrophic lateral sclerosis are not fully understood. Recent studies suggest that apoptosis is involved in the abnormal neural death that occurs in this devastating disease. Presenilin-1, a transmembrane protein, seems to be implicated in apoptosis. To determine whether presenilin-1 intron 8 polymorphism has an influence in the course of amyotrophic lateral sclerosis, we examined this polymorphism genotypes in a large group of patients (n = 72) with amyotrophic lateral sclerosis and in a random sample of 213 healthy individuals. The results showed a significant difference in genotype (P < 0.04) and allele (P < 0.03) distribution between patients controls. These results suggest a possible intervention of presenilin-1 in the pathogenesis of amyotrophic lateral sclerosis.

Endochondral vs. intramembranous demineralized bone matrices as implants for osseous defects.

PubMed

Nidoli, M C; Nielsen, F F; Melsen, B

1999-05-01

This study focuses on the difference in regenerative capacity between endochondral and intramembranous demineralized bone matrices (DBMs) when implanted into bony defects. It also focuses on the possible influence of the type of skeletal recipient site (orthotopic or heterotopic). Of 34 Wistar rats, 10 served as a source of DBM, and 24 were divided into two groups of 12 animals. In group A identical defects were produced in the parietal bones, whereas in group B the defects were produced in each radius. The right defects were implanted with endochondral DBM and the left defects were implanted with intramembranous DBM. Descriptive and/or histomorphometric analyses were performed by means of light and polarized microscopy, and radiography (group B). Right and left data were compared to disclose differences in bone-healing capacity. The quantitative results demonstrated that endochondral DBM displays a greater regenerative capacity than intramembranous DBM when implanted heterotopically. The different clinical performances of endochondral and intramembranous bone grafts might be explained on the basis of the mechanical rather than the osteoinductive principle. The qualitative results suggest that the type of bone deposition induced by the DBMs is not related to the type of implanted DBM. Recipient site characteristics and/or environmental factors seem decisive in the occurrence of either types of ossification.

Alzheimer's Disease Phenotypes and Genotypes Associated with Mutations in Presenilin 2

ERIC Educational Resources Information Center

Jayadev, Suman; Leverenz, James B.; Steinbart, Ellen; Stahl, Justin; Klunk, William; Yu, Cheng-En; Bird, Thomas D.

2010-01-01

Mutations in presenilin 2 are rare causes of early onset familial Alzheimer's disease. Eighteen presenilin 2 mutations have been reported, although not all have been confirmed pathogenic. Much remains to be learned about the range of phenotypes associated with these mutations. We have analysed our unique collection of 146 affected cases in 11…

Calorie restriction ameliorates neurodegenerative phenotypes in forebrain-specific presenilin-1 and presenilin-2 double knockout mice.

PubMed

Wu, Pu; Shen, Qian; Dong, Suzhen; Xu, Zhiliang; Tsien, Joe Z; Hu, Yinghe

2008-10-01

Conditional double knockout of presenilin-1 and presenilin-2 (cDKO) in forebrain of mice led to brain atrophy, tau hyperphosphorylation, synaptic dysfunction and cognitive deficit. These brain changes recapitulated most of the neurodegenerative phenotypes of Alzheimer's disease (AD). In this report, we have investigated the effects of 4-month calorie restriction (CR) regimen on different phenotypes in cDKO mice. We found that CR improved novel object recognition and contextual fear conditioning memory in the cDKO mice. Histological and biochemical analysis showed that CR attenuated ventricle enlargement, caspase-3 activation and astrogliosis. In addition, the induction of tau hyperphosphorylation in the cDKO mice was reduced by CR, possibly through reduction of p25 accumulation and aberrant CDK5 activation. Finally, DNA microarray analysis demonstrated that CR could increase the expression of neurogenesis related genes and decrease the expression of inflammation related genes in the hippocampus of cDKO mice. The possible molecular mechanisms of the CR effects on alleviating AD pathogenesis have been discussed.

Rhomboid intramembrane protease RHBDL4 triggers ER-export and non-canonical secretion of membrane-anchored TGFα

PubMed Central

Wunderle, Lina; Knopf, Julia D.; Kühnle, Nathalie; Morlé, Aymeric; Hehn, Beate; Adrain, Colin; Strisovsky, Kvido; Freeman, Matthew; Lemberg, Marius K.

2016-01-01

Rhomboid intramembrane proteases are the enzymes that release active epidermal growth factor receptor (EGFR) ligands in Drosophila and C. elegans, but little is known about their functions in mammals. Here we show that the mammalian rhomboid protease RHBDL4 (also known as Rhbdd1) promotes trafficking of several membrane proteins, including the EGFR ligand TGFα, from the endoplasmic reticulum (ER) to the Golgi apparatus, thereby triggering their secretion by extracellular microvesicles. Our data also demonstrate that RHBDL4-dependent trafficking control is regulated by G-protein coupled receptors, suggesting a role for this rhomboid protease in pathological conditions, including EGFR signaling. We propose that RHBDL4 reorganizes trafficking events within the early secretory pathway in response to GPCR signaling. Our work identifies RHBDL4 as a rheostat that tunes secretion dynamics and abundance of specific membrane protein cargoes. PMID:27264103

Carbon-11-Pittsburgh compound B positron emission tomography imaging of amyloid deposition in presenilin 1 mutation carriers.

PubMed

Knight, William D; Okello, Aren A; Ryan, Natalie S; Turkheimer, Federico E; Rodríguez Martinez de Llano, Sofia; Edison, Paul; Douglas, Jane; Fox, Nick C; Brooks, David J; Rossor, Martin N

2011-01-01

(11)Carbon-Pittsburgh compound B positron emission tomography studies have suggested early and prominent amyloid deposition in the striatum in presenilin 1 mutation carriers. This cross-sectional study examines the (11)Carbon-Pittsburgh compound B positron emission tomography imaging profiles of presymptomatic and mildly affected (mini-mental state examination ≥ 20) carriers of seven presenilin 1 mutations, comparing them with groups of controls and symptomatic sporadic Alzheimer's disease cases. Parametric ratio images representing (11)Carbon-Pittsburgh compound B retention from 60 to 90 min were created using the pons as a reference region and nine regions of interest were studied. We confirmed that increased amyloid load may be detected in presymptomatic presenilin 1 mutation carriers with (11)Carbon-Pittsburgh compound B positron emission tomography and that the pattern of retention is heterogeneous. Comparison of presenilin 1 and sporadic Alzheimer's disease groups revealed significantly greater thalamic retention in the presenilin 1 group and significantly greater frontotemporal retention in the sporadic Alzheimer's disease group. A few individuals with presenilin 1 mutations showed increased cerebellar (11)Carbon-Pittsburgh compound B retention suggesting that this region may not be as suitable a reference region in familial Alzheimer's disease.

Familial Alzheimer disease-linked mutations specifically disrupt Ca2+ leak function of presenilin 1.

PubMed

Nelson, Omar; Tu, Huiping; Lei, Tianhua; Bentahir, Mostafa; de Strooper, Bart; Bezprozvanny, Ilya

2007-05-01

Mutations in presenilins are responsible for approximately 40% of all early-onset familial Alzheimer disease (FAD) cases in which a genetic cause has been identified. In addition, a number of mutations in presenilin-1 (PS1) have been suggested to be associated with the occurrence of frontal temporal dementia (FTD). Presenilins are highly conserved transmembrane proteins that support cleavage of the amyloid precursor protein by gamma-secretase. Recently, we discovered that presenilins also function as passive ER Ca(2+) leak channels. Here we used planar lipid bilayer reconstitution assays and Ca(2+) imaging experiments with presenilin-null mouse embryonic fibroblasts to analyze ER Ca(2+) leak function of 6 FAD-linked PS1 mutants and 3 known FTD-associated PS1 mutants. We discovered that L166P, A246E, E273A, G384A, and P436Q FAD mutations in PS1 abolished ER Ca(2+) leak function of PS1. In contrast, A79V FAD mutation or FTD-associated mutations (L113P, G183V, and Rins352) did not appear to affect ER Ca(2+) leak function of PS1 in our experiments. We validated our findings in Ca(2+) imaging experiments with primary fibroblasts obtained from an FAD patient possessing mutant PS1-A246E. Our results indicate that many FAD mutations in presenilins are loss-of-function mutations affecting ER Ca(2+) leak activity. In contrast, none of the FTD-associated mutations affected ER Ca(2+) leak function of PS1, indicating that the observed effects are disease specific. Our observations are consistent with the potential role of disturbed Ca(2+) homeostasis in Alzheimer disease pathogenesis.

Presenilin 1 Maintains Lysosomal Ca(2+) Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification.

PubMed

Lee, Ju-Hyun; McBrayer, Mary Kate; Wolfe, Devin M; Haslett, Luke J; Kumar, Asok; Sato, Yutaka; Lie, Pearl P Y; Mohan, Panaiyur; Coffey, Erin E; Kompella, Uday; Mitchell, Claire H; Lloyd-Evans, Emyr; Nixon, Ralph A

2015-09-01

Presenilin 1 (PS1) deletion or Alzheimer's disease (AD)-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit, causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in Presenilin 1 knockout (PS1KO) cells induces abnormal Ca(2+) efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca(2+). In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca(2+) homeostasis, but correcting lysosomal Ca(2+) deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss-of-function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca(2+) homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Regulation of 1, 4, 5-triphosphate receptor channel gating dynamics by mutant presenilin in Alzheimer's disease cells

NASA Astrophysics Data System (ADS)

Wei, Fang; Li, Xiang; Cai, Meichun; Liu, Yanping; Jung, Peter; Shuai, Jianwei

2017-06-01

In neurons of patients with Alzheimer's disease, the intracellular Ca2+ concentration is increased by its release from the endoplasmic reticulum via the inositol 1, 4, 5-triphosphate receptor (IP3R). In this paper, we discuss the IP3R gating dynamics in familial Alzheimer's disease (FAD) cells induced with presenilin mutation PS1. By fitting the parameters of an IP3R channel model to experimental data of the open probability, the mean open time and the mean closed time of IP3R channels, in control cells and FAD mutant cells, we suggest that the interaction of presenilin mutation PS1 with IP3R channels leads the decrease in the unbinding rates of IP3 and the activating Ca2+ from IP3Rs. As a result, the increased affinities of IP3 and activating Ca2+ for IP3R channels induce the increase in the Ca2+ signal in FAD mutant cells. Specifically, the PS1 mutation decreases the IP3 dissociation rate of IP3R channels significantly in FAD mutant cells. Our results suggest possible novel targets for FAD therapeutic intervention.

A Patient with Posterior Cortical Atrophy Possesses a Novel Mutation in the Presenilin 1 Gene

PubMed Central

Sitek, Emilia J.; Narożańska, Ewa; Pepłońska, Beata; Filipek, Sławomir; Barczak, Anna; Styczyńska, Maria; Mlynarczyk, Krzysztof; Brockhuis, Bogna; Portelius, Erik; Religa, Dorota; Barcikowska, Maria

2013-01-01

Posterior cortical atrophy is a dementia syndrome with symptoms of cortical visual dysfunction, associated with amyloid plaques and neurofibrillary tangles predominantly affecting visual association cortex. Most patients diagnosed with posterior cortical atrophy will finally develop a typical Alzheimer's disease. However, there are a variety of neuropathological processes, which could lead towards a clinical presentation of posterior cortical atrophy. Mutations in the presenilin 1 gene, affecting the function of γ-secretase, are the most common genetic cause of familial, early-onset Alzheimer's disease. Here we present a patient with a clinical diagnosis of posterior cortical atrophy who harbors a novel Presenilin 1 mutation (I211M). In silico analysis predicts that the mutation could influence the interaction between presenilin 1 and presenilin1 enhancer-2 protein, a protein partner within the γ-secretase complex. These findings along with published literature support the inclusion of posterior cortical atrophy on the Alzheimer's disease spectrum. PMID:23593396

Antioxidant proteins TSA and PAG interact synergistically with Presenilin to modulate Notch signaling in Drosophila.

PubMed

Wangler, Michael F; Reiter, Lawrence T; Zimm, Georgianna; Trimble-Morgan, Jennifer; Wu, Jane; Bier, Ethan

2011-07-01

Alzheimer's disease (AD) pathogenesis is characterized by senile plaques in the brain and evidence of oxidative damage. Oxidative stress may precede plaque formation in AD; however, the link between oxidative damage and plaque formation remains unknown. Presenilins are transmembrane proteins in which mutations lead to accelerated plaque formation and early-onset familial Alzheimer's disease. Presenilins physically interact with two antioxidant enzymes thiol-specific antioxidant (TSA) and proliferation-associated gene (PAG) of the peroxiredoxin family. The functional consequences of these interactions are unclear. In the current study we expressed a presenilin transgene in Drosophila wing and sensory organ precursors of the fly. This caused phenotypes typical of Notch signaling loss-of-function mutations. We found that while expression of TSA or PAG alone produced no phenotype, co-expression of TSA and PAG with presenilin led to an enhanced Notch loss-of-function phenotype. This phenotype was more severe and more penetrant than that caused by the expression of Psn alone. In order to determine whether these phenotypes were indeed affecting Notch signaling, this experiment was performed in a genetic background carrying an activated Notch (Abruptex) allele. The phenotypes were almost completely rescued by this activated Notch allele. These results link peroxiredoxins with the in vivo function of Presenilin, which ultimately connects two key pathogenetic mechanisms in AD, namely, antioxidant activity and plaque formation, and raises the possibility of a role for peroxiredoxin family members in Alzheimer's pathogenesis.

Probing catalytic rate enhancement during intramembrane proteolysis.

PubMed

Arutyunova, Elena; Smithers, Cameron C; Corradi, Valentina; Espiritu, Adam C; Young, Howard S; Tieleman, D Peter; Lemieux, M Joanne

2016-09-01

Rhomboids are ubiquitous intramembrane serine proteases involved in various signaling pathways. While the high-resolution structures of the Escherichia coli rhomboid GlpG with various inhibitors revealed an active site comprised of a serine-histidine dyad and an extensive oxyanion hole, the molecular details of rhomboid catalysis were unclear because substrates are unknown for most of the family members. Here we used the only known physiological pair of AarA rhomboid with its psTatA substrate to decipher the contribution of catalytically important residues to the reaction rate enhancement. An MD-refined homology model of AarA was used to identify residues important for catalysis. We demonstrated that the AarA active site geometry is strict and intolerant to alterations. We probed the roles of H83 and N87 oxyanion hole residues and determined that substitution of H83 either abolished AarA activity or reduced the transition state stabilization energy (ΔΔG‡) by 3.1 kcal/mol; substitution of N87 decreased ΔΔG‡ by 1.6-3.9 kcal/mol. Substitution M154, a residue conserved in most rhomboids that stabilizes the catalytic general base, to tyrosine, provided insight into the mechanism of nucleophile generation for the catalytic dyad. This study provides a quantitative evaluation of the role of several residues important for hydrolytic efficiency and oxyanion stabilization during intramembrane proteolysis.

Intramembranous ossification of scleral ossicles in Chelydra serpentina.

PubMed

Franz-Odendaal, Tamara A

2006-01-01

Scleral ossicles are present in many reptiles, including turtles and birds. In both groups the sclerotic ring situated in the eye is composed of a number of imbricating scleral ossicles or plates. Despite this gross morphological similarity, Andrews (1996. An endochondral rather than a dermal origin for scleral ossicles in Cryptodiran turtles. J. Herpetol. 30, 257-260) reported that the scleral ossicles of turtles develop endochondrally unlike those in birds, which develop intramembranously after a complex epithelial-mesenchymal inductive event. This study re-explores one of the species examined by Andrews in order to determine the mode of ossification of scleral ossicles in turtles. A growth series of Chelydra serpentina embryos, including the stages examined by Andrews, were examined by staining separately for cartilage and bone. Results clearly contradict Andrews (1996) and show that the scleral ossicles of Chelydra serpentina develop similarly to those in birds. That is, they develop intramembranously without a cartilage precursor and are likely induced by transient scleral papillae. The sequence of scleral papillae development is broadly similar, but the papillae themselves are not as distinct as those seen in chicken embryos. This study has important consequences for understanding the homology of scleral ossicles among tetrapods.

Caspase-6 Activation in Familial Alzheimer Disease Brains Carrying Amyloid Precursor Protein, Presenilin I or Presenilin II Mutations

PubMed Central

Albrecht, Steffen; Bogdanovic, Nenad; Ghetti, Bernardino; Winblad, Bengt; LeBlanc, Andréa C.

2010-01-01

We previously demonstrated the activation of Caspase-6 in the hippocampus and cortex in cases of mild, moderate, severe and very severe Alzheimer disease (AD). To determine whether Caspase-6 is also activated in familial AD, we performed an immunohistochemical analysis of active Caspase-6 and Tau cleaved by Caspase-6 in temporal cortex and hippocampal tissue sections from cases of familial AD. The cases included 5 carrying the amyloid precursor protein K670N, M671L Swedish mutation, 1 carrying the amyloid precursor protein E693G Arctic mutation, 2 each carrying the Presenilin I M146V, F105L, A431E, V261F, Y115C mutations, and 1 with the Presenilin II N141I mutation. Active Caspase-6 immunoreactivity was found in all cases. Caspase-6 immunoreactivity was observed in neuritic plaques or cotton wool plaques in some cases, neuropil threads and neurofibrillary tangles. These results indicate that Caspase-6 is activated in familial forms of AD, as previously observed in sporadic forms. Since sporadic and familial AD cases have similar pathological features, these results support a fundamental role of Caspase-6 in the pathophysiology of both familial and sporadic AD. PMID:19915487

Different Hippocampus Functional Connectivity Patterns in Healthy Young Adults with Mutations of APP/Presenilin-1/2 and APOEε4.

PubMed

Zheng, Li Juan; Su, Yun Yan; Wang, Yun Fei; Schoepf, U Joseph; Varga-Szemes, Akos; Pannell, Jonathan; Liang, Xue; Zheng, Gang; Lu, Guang Ming; Yang, Gui Fen; Zhang, Long Jiang

2018-04-01

This study aims to explore the hippocampus-based functional connectivity patterns in young, healthy APP and/or presenilin-1/2 mutation carriers and APOE ε4 subjects. Seventy-eight healthy young adults (33 male, mean age 24.0 ± 2.2 years; 18 APP and/or presenilin1/2 mutation carriers [APP/presenilin-1/2 group], 30 APOE ε4 subjects [APOE ε4 group], and 30 subjects without the above-mentioned genes [control group]) underwent resting-state functional MR imaging and neuropsychological assessments. Bilateral hippocampus functional connectivity patterns were compared among three groups. The brain regions with statistical differences were then extracted, and correlation analyses were performed between Z values of the brain regions and neuropsychological results. Compared with control group, both APOE ε4 group and APP/presenilin-1/2 group showed increased functional connectivity in medial prefrontal cortex and precuneus for the seeds of bilateral hippocampi. The APOE ε4 group displayed increased functional connectivity from bilateral hippocampi to the left middle temporal gyrus compared with the control group. Moreover, compared with the APP/presenilin-1/2 group, the APOE ε4 group also had markedly increased functional connectivity in right hippocampus-left middle temporal gyrus. The Z values of right hippocampus-left middle temporal gyrus correlated with various neuropsychological results across all the subjects, as well as in APOE ε4 group. Young healthy adults carrying APOE ε4 and APP/presenilin-1/2 displayed different hippocampus functional connectivity patterns, which may underlie the discrepant mechanisms of gene-modulated cognitive dysfunction in Alzheimer's disease.

Generation of amyloid-β is reduced by the interaction of calreticulin with amyloid precursor protein, presenilin and nicastrin.

PubMed

Stemmer, Nina; Strekalova, Elena; Djogo, Nevena; Plöger, Frank; Loers, Gabriele; Lutz, David; Buck, Friedrich; Michalak, Marek; Schachner, Melitta; Kleene, Ralf

2013-01-01

Dysregulation of the proteolytic processing of amyloid precursor protein by γ-secretase and the ensuing generation of amyloid-β is associated with the pathogenesis of Alzheimer's disease. Thus, the identification of amyloid precursor protein binding proteins involved in regulating processing of amyloid precursor protein by the γ-secretase complex is essential for understanding the mechanisms underlying the molecular pathology of the disease. We identified calreticulin as novel amyloid precursor protein interaction partner that binds to the γ-secretase cleavage site within amyloid precursor protein and showed that this Ca(2+)- and N-glycan-independent interaction is mediated by amino acids 330-344 in the C-terminal C-domain of calreticulin. Co-immunoprecipitation confirmed that calreticulin is not only associated with amyloid precursor protein but also with the γ-secretase complex members presenilin and nicastrin. Calreticulin was detected at the cell surface by surface biotinylation of cells overexpressing amyloid precursor protein and was co-localized by immunostaining with amyloid precursor protein and presenilin at the cell surface of hippocampal neurons. The P-domain of calreticulin located between the N-terminal N-domain and the C-domain interacts with presenilin, the catalytic subunit of the γ-secretase complex. The P- and C-domains also interact with nicastrin, another functionally important subunit of this complex. Transfection of amyloid precursor protein overexpressing cells with full-length calreticulin leads to a decrease in amyloid-β42 levels in culture supernatants, while transfection with the P-domain increases amyloid-β40 levels. Similarly, application of the recombinant P- or C-domains and of a synthetic calreticulin peptide comprising amino acid 330-344 to amyloid precursor protein overexpressing cells result in elevated amyloid-β40 and amyloid-β42 levels, respectively. These findings indicate that the interaction of calreticulin with

Type IV pilins regulate their own expression via direct intramembrane interactions with the sensor kinase PilS.

PubMed

Kilmury, Sara L N; Burrows, Lori L

2016-05-24

Type IV pili are important virulence factors for many pathogens, including Pseudomonas aeruginosa Transcription of the major pilin gene-pilA-is controlled by the PilS-PilR two-component system in response to unknown signals. The absence of a periplasmic sensing domain suggested that PilS may sense an intramembrane signal, possibly PilA. We suggest that direct interactions between PilA and PilS in the inner membrane reduce pilA transcription when PilA levels are high. Overexpression in trans of PilA proteins with diverse and/or truncated C termini decreased native pilA transcription, suggesting that the highly conserved N terminus of PilA was the regulatory signal. Point mutations in PilA or PilS that disrupted their interaction prevented autoregulation of pilA transcription. A subset of PilA point mutants retained the ability to interact with PilS but could no longer decrease pilA transcription, suggesting that interaction between the pilin and sensor kinase is necessary but not sufficient for pilA autoregulation. Furthermore, PilS's phosphatase motif was required for the autoregulation of pilA transcription, suggesting that under conditions where PilA is abundant, the PilA-PilS interaction promotes PilR dephosphorylation and thus down-regulation of further pilA transcription. These data reveal a clever bacterial inventory control strategy in which the major subunit of an important P. aeruginosa virulence factor controls its own expression.

Transient chondrogenic phase in the intramembranous pathway during normal skeletal development.

PubMed

Nah, H D; Pacifici, M; Gerstenfeld, L C; Adams, S L; Kirsch, T

2000-03-01

Calvarial and facial bones form by intramembranous ossification, in which bone cells arise directly from mesenchyme without an intermediate cartilage anlage. However, a number of studies have reported the emergence of chondrocytes from in vitro calvarial cell or organ cultures and the expression of type II collagen, a cartilage-characteristic marker, in developing calvarial bones. Based on these findings we hypothesized that a covert chondrogenic phase may be an integral part of the normal intramembranous pathway. To test this hypothesis, we analyzed the temporal and spatial expression patterns of cartilage characteristic genes in normal membranous bones from chick embryos at various developmental stages (days 12, 15 and 19). Northern and RNAse protection analyses revealed that embryonic frontal bones expressed not only the type I collagen gene but also a subset of cartilage characteristic genes, types IIA and XI collagen and aggrecan, thus resembling a phenotype of prechondrogenic-condensing mesenchyme. The expression of cartilage-characteristic genes decreased with the progression of bone maturation. Immunohistochemical analyses of developing embryonic chick heads indicated that type II collagen and aggrecan were produced by alkaline phosphatase activity positive cells engaged in early stages of osteogenic differentiation, such as cells in preosteogenic-condensing mesenchyme, the cambium layer of periosteum, the advancing osteogenic front, and osteoid bone. Type IIB and X collagen messenger RNAs (mRNA), markers for mature chondrocytes, were also detected at low levels in calvarial bone but not until late embryonic stages (day 19), indicating that some calvarial cells may undergo overt chondrogenesis. On the basis of our findings, we propose that the normal intramembranous pathway in chicks includes a previously unrecognized transient chondrogenic phase similar to prechondrogenic mesenchyme, and that the cells in this phase retain chondrogenic potential that can

Action potential propagation: ion current or intramembrane electric field?

PubMed

Martí, Albert; Pérez, Juan J; Madrenas, Jordi

2018-01-01

The established action potential propagation mechanisms do not satisfactorily explain propagation on myelinated axons given the current knowledge of biological channels and membranes. The flow across ion channels presents two possible effects: the electric potential variations across the lipid bilayers (action potential) and the propagation of an electric field through the membrane inner part. The proposed mechanism is based on intra-membrane electric field propagation, this propagation can explain the action potential saltatory propagation and its constant delay independent of distance between Ranvier nodes in myelinated axons.

Early growth response 1 (EGR-1) is a transcriptional regulator of mitochondrial carrier homolog 1 (MTCH 1)/presenilin 1-associated protein (PSAP).

PubMed

Nelo-Bazán, María Alejandra; Latorre, Pedro; Bolado-Carrancio, Alfonso; Pérez-Campo, Flor M; Echenique-Robba, Pablo; Rodríguez-Rey, José Carlos; Carrodeguas, José Alberto

2016-03-01

Attempts to elucidate the cellular function of MTCH1 (mitochondrial carrier homolog 1) have not yet rendered a clear insight into the function of this outer mitochondrial membrane protein. Classical biochemical and cell biology approaches have not produced the expected outcome. In vitro experiments have indicated a likely role in the regulation of cell death by apoptosis, and its reported interaction with presenilin 1 suggests a role in the cellular pathways in which this membrane protease participates, nevertheless in vivo data are missing. In an attempt to identify cellular pathways in which this protein might participate, we have studied its promoter looking for transcriptional regulators. We have identified several putative binding sites for EGR-1 (Early growth response 1; a protein involved in growth, proliferation and differentiation), in the proximal region of the MTCH1 promoter. Chromatin immunoprecipitation showed an enrichment of these sequences in genomic DNA bound to EGR-1 and transient overexpression of EGR-1 in cultured HEK293T cells induces an increase of endogenous MTCH1 levels. We also show that MTCH1 levels increase in response to treatment of cells with doxorubicin, an apoptosis inducer through DNA damage. The endogenous levels of MTCH1 decrease when EGR-1 levels are lowered by RNA interference. Our results indicate that EGR-1 is a transcriptional regulator of MTCH1 and give some clues about the cellular processes in which MTCH1 might participate. Copyright © 2015 Elsevier B.V. All rights reserved.

Membrane-bound transcription factors: regulated release by RIP or RUP.

PubMed

Hoppe, T; Rape, M; Jentsch, S

2001-06-01

Regulated nuclear transport of transcription factors from cytoplasmic pools is a major route by which eukaryotes control gene expression. Exquisite examples are transcription factors that are kept in a dormant state in the cytosol by membrane anchors; such proteins are released from membranes by proteolytic cleavage, which enables these transcription factors to enter the nucleus. Cleavage can be mediated either by regulated intramembrane proteolysis (RIP) catalysed by specific membrane-bound proteases or by regulated ubiquitin/proteasome-dependent processing (RUP). In both cases processing can be controlled by cues that originate at or in the vicinity of the membrane.

Attenuation of neurodegenerative phenotypes in Alzheimer-like presenilin 1/presenilin 2 conditional double knockout mice by EUK1001, a promising derivative of xanomeline.

PubMed

Wang, Dong; Yang, Liguo; Su, Jingjing; Niu, Yan; Lei, Xiaoping; Xiong, Juan; Cao, Xiaohua; Hu, Yinghe; Mei, Bing; Hu, Jin-Feng

2011-07-01

The M1/M4-preferring muscarinic agonist xanomeline was found to have some benefit in the treatment of the memory impairment of Alzheimer's disease (AD), but side effects precluded further development. EUK1001, a fluorinated derivative of xanomeline, because of greater affinity for M1 muscarinic receptors, is likely to have a significantly better side effect profile than xanomeline. We have now studied the effects of 3-month chronic administration of EUK1001 and xanomeline (0.5mg/kg/day) in AD-like presenilin 1/presenilin 2 conditional double knockout (PS cDKO) mice. Only EUK1001 was found to significantly ameliorate the deficit in recognition memory. Histological analysis demonstrated partial attenuation of the brain atrophy in EUK1001-treated PS cDKO mice and minimal effect in the xanomeline-treated mice. Both compounds effectively suppressed the elevation of brain tau phosphorylation in the PS cDKO mice, but neither inhibited the increased inflammatory responses. These results indicate that EUK1001 showed superiority to xanomeline with regard to attenuation of several AD-like neurodegenerative phenotypes in PS cDKO mice. These results suggest further investigation of the development of EUK1001 for the treatment of AD is indicated. Copyright © 2011 Elsevier Inc. All rights reserved.

Protein Aggregates and Novel Presenilin Gene Variants in Idiopathic Dilated Cardiomyopathy

PubMed Central

Gianni, Davide; Li, Airong; Tesco, Giuseppina; McKay, Kenneth M.; Moore, John; Raygor, Kunal; Rota, Marcello; Gwathmey, Judith K; Dec, G William; Aretz, Thomas; Leri, Annarosa; Semigran, Marc J; Anversa, Piero; Macgillivray, Thomas E; Tanzi, Rudolph E.; Monte, Federica del

2010-01-01

Background Heart failure (HF) is a debilitating condition resulting in severe disability and death. In a subset of cases, clustered as Idiopathic Dilated Cardiomyopathy (iDCM), the origin of HF is unknown. In the brain of patients with dementia, proteinaceous aggregates and abnormal oligomeric assemblies of β-amyloid impair cell function and lead to cell death. Methods and Results We have similarly characterized fibrillar and oligomeric assemblies in the hearts of iDCM patients pointing to abnormal protein aggregation as a determinant of iDCM. We also showed that oligomers alter myocyte Ca2+ homeostasis. Additionally, we have identified two new sequence variants in the presenilin-1 (PSEN1) gene promoter leading to reduced gene and protein expression. We also show that presenilin-1 co-immunoprecipitates with SERCA2a. Conclusions Based on these findings we propose that two mechanisms may link protein aggregation and cardiac function: oligomer-induced changes on Ca2+ handling and a direct effect of PSEN1 sequence variants on EC-coupling protein function. PMID:20194882

Pen-2 is dispensable for endoproteolysis of presenilin 1, and nicastrin-Aph subcomplex is important for both γ-secretase assembly and substrate recruitment

PubMed Central

Mao, Guozhang; Cui, Mei-Zhen; Li, Tong; Jin, Yipeng; Xu, Xuemin

2012-01-01

γ-secretase is a protease complex with at least four components: presenilin, nicastrin (NCT), anterior pharynx-defective 1 (Aph-1), and presenilin enhancer 2 (Pen-2). In this study, using knockout cell lines and small interfering RNA technology, our data demonstrated that the disappeared presenilin 1 C-terminal fragment (PS1C) caused by knockdown of pen-2 or knockout of NCT or Aph-1 was recovered by the addition of proteasome inhibitors, indicating that Pen-2, as well as NCT and Aph-1α , is dispensable for presenilin endoproteolysis. Our data also demonstrate that the formation of the nicastrin-Aph-1 subcomplex plays not only an important role in γ-secretase complex assembly but also in recruiting substrate C-terminal fragment of amyloid precursor protein generated by β-cleavage (CTFβ). Ablating any one component resulted in the instability of other components of the γ-secretase complex, and the presence of all three of the other components is required for full maturation of NCT. PMID:22973949

Presenilin expression during induced differentiation of the human neuroblastoma SH-SY5Y cell line.

PubMed

Flood, Fiona; Sundström, Erik; Samuelsson, Eva-Britt; Wiehager, Birgitta; Seiger, Ake; Johnston, Janet A; Cowburn, Richard F

2004-06-01

Human neuroblastoma SH-SY5Y cells stably transfected with both wild-type and exon-9 deleted (deltaE9) presenilin constructs were used to study the role of the presenilin proteins during differentiation. Cells transfected with either wild-type or deltaE9 PS1, of which the latter abolishes normal endoproteolytic cleavage of the protein, showed no obvious differences in their ability to differentiate to a neuronal-like phenotype upon treatment with retinoic acid (RA). A defined pattern of PS1 expression was observed during differentiation with both RA and the phorbol ester TPA. Full-length PS1 was shown to increase dramatically within 5-24 h of RA treatment. TPA gave an earlier and longer lasting increase in full-length PS1 levels. The intracellular distribution pattern of PS1 was markedly altered following RA treatment. Within 24h PS1 was highly up-regulated throughout the cell body around the nucleus. Between 2 and 4 weeks PS1 staining appeared punctate and also localised to the nucleus. Increases in PS1 expression upon treatment with RA and TPA were blocked by treatment with cycloheximide, indicating a role of de-novo protein synthesis in this effect. PS2 expression remained unchanged during differentiation. Levels of full-length PS1 were also seen to increase during neurogenesis and neuronal differentiation in the forebrain of first trimester human foetuses between 6.5 and 11 weeks. These combined observations support the idea that PS1 is involved in neuronal differentiation by a mechanism likely independent of endoproteolysis of the protein.

An Internal Signal Sequence Directs Intramembrane Proteolysis of a Cellular Immunoglobulin Domain Protein*S⃞

PubMed Central

Robakis, Thalia; Bak, Beata; Lin, Shu-huei; Bernard, Daniel J.; Scheiffele, Peter

2008-01-01

Precursor proteolysis is a crucial mechanism for regulating protein structure and function. Signal peptidase (SP) is an enzyme with a well defined role in cleaving N-terminal signal sequences but no demonstrated function in the proteolysis of cellular precursor proteins. We provide evidence that SP mediates intraprotein cleavage of IgSF1, a large cellular Ig domain protein that is processed into two separate Ig domain proteins. In addition, our results suggest the involvement of signal peptide peptidase (SPP), an intramembrane protease, which acts on substrates that have been previously cleaved by SP. We show that IgSF1 is processed through sequential proteolysis by SP and SPP. Cleavage is directed by an internal signal sequence and generates two separate Ig domain proteins from a polytopic precursor. Our findings suggest that SP and SPP function are not restricted to N-terminal signal sequence cleavage but also contribute to the processing of cellular transmembrane proteins. PMID:18981173

Specificity of Intramembrane Protein–Lipid Interactions

PubMed Central

Contreras, Francesc-Xabier; Ernst, Andreas Max; Wieland, Felix; Brügger, Britta

2011-01-01

Our concept of biological membranes has markedly changed, from the fluid mosaic model to the current model that lipids and proteins have the ability to separate into microdomains, differing in their protein and lipid compositions. Since the breakthrough in crystallizing membrane proteins, the most powerful method to define lipid-binding sites on proteins has been X-ray and electron crystallography. More recently, chemical biology approaches have been developed to analyze protein–lipid interactions. Such methods have the advantage of providing highly specific cellular probes. With the advent of novel tools to study functions of individual lipid species in membranes together with structural analysis and simulations at the atomistic resolution, a growing number of specific protein–lipid complexes are defined and their functions explored. In the present article, we discuss the various modes of intramembrane protein–lipid interactions in cellular membranes, including examples for both annular and nonannular bound lipids. Furthermore, we will discuss possible functional roles of such specific protein–lipid interactions as well as roles of lipids as chaperones in protein folding and transport. PMID:21536707

γ-Secretase Heterogeneity in the Aph1 Subunit: Relevance for Alzheimer’s Disease

PubMed Central

Serneels, Lutgarde; Van Biervliet, Jérôme; Craessaerts, Katleen; Dejaegere, Tim; Horré, Katrien; Van Houtvin, Tine; Esselmann, Hermann; Paul, Sabine; Schäfer, Martin K.; Berezovska, Oksana; Hyman, Bradley T.; Sprangers, Ben; Sciot, Raf; Moons, Lieve; Jucker, Mathias; Yang, Zhixiang; May, Patrick C.; Karran, Eric; Wiltfang, Jens; D’Hooge, Rudi; De Strooper, Bart

2009-01-01

The γ-secretase complex plays a role in Alzheimer’s disease (AD) and cancer progression. The development of clinical useful inhibitors, however, is complicated by the role of the γ-secretase complex in regulated intramembrane proteolysis of Notch and other essential proteins. Different γ-secretase complexes containing different Presenilin or Aph1 protein subunits are present in various tissues. Here we show that these complexes have heterogeneous biochemical and physiological properties. Specific inactivation of the Aph1B γ-secretase in a murine Alzheimer’s disease model led to improvements of Alzheimer’s disease-relevant phenotypic features without any Notch-related side effects. The Aph1B complex contributes to total γ-secretase activity in the human brain, thus specific targeting of Aph1B-containing γ-secretase complexes may be helpful in generating less toxic therapies for Alzheimer’s disease. PMID:19299585

Inhibition of γ-Secretase Leads to an Increase in Presenilin-1.

PubMed

Sogorb-Esteve, Aitana; García-Ayllón, María-Salud; Llansola, Marta; Felipo, Vicente; Blennow, Kaj; Sáez-Valero, Javier

2018-06-01

γ-Secretase inhibitors (GSIs) are potential therapeutic agents for Alzheimer's disease (AD); however, trials have proven disappointing. We addressed the possibility that γ-secretase inhibition can provoke a rebound effect, elevating the levels of the catalytic γ-secretase subunit, presenilin-1 (PS1). Acute treatment of SH-SY5Y cells with the GSI LY-374973 (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, DAPT) augments PS1, in parallel with increases in other γ-secretase subunits nicastrin, presenilin enhancer 2, and anterior pharynx-defective 1, yet with no increase in messenger RNA expression. Over-expression of the C-terminal fragment (CTF) of APP, C99, also triggered an increase in PS1. Similar increases in PS1 were evident in primary neurons treated repeatedly (4 days) with DAPT or with the GSI BMS-708163 (avagacestat). Likewise, rats examined after 21 days administered with avagacestat (40 mg/kg/day) had more brain PS1. Sustained γ-secretase inhibition did not exert a long-term effect on PS1 activity, evident through the decrease in CTFs of APP and ApoER2. Prolonged avagacestat treatment of rats produced a subtle impairment in anxiety-like behavior. The rebound increase in PS1 in response to GSIs must be taken into consideration for future drug development.

Enzymatic characteristics of I213T mutant presenilin-1/gamma-secretase in cell models and knock-in mouse brains: familial Alzheimer disease-linked mutation impairs gamma-site cleavage of amyloid precursor protein C-terminal fragment beta.

PubMed

Shimojo, Masafumi; Sahara, Naruhiko; Mizoroki, Tatsuya; Funamoto, Satoru; Morishima-Kawashima, Maho; Kudo, Takashi; Takeda, Masatoshi; Ihara, Yasuo; Ichinose, Hiroshi; Takashima, Akihiko

2008-06-13

Presenilin (PS)/gamma-secretase-mediated intramembranous proteolysis of amyloid precursor protein produces amyloid beta (Abeta) peptides in which Abeta species of different lengths are generated through multiple cleavages at the gamma-, zeta-, and epsilon-sites. An increased Abeta42/Abeta40 ratio is a common characteristic of most cases of familial Alzheimer disease (FAD)-linked PS mutations. However, the molecular mechanisms underlying amyloid precursor protein proteolysis leading to increased Abeta42/Abeta40 ratios still remain unclear. Here, we report our findings on the enzymatic analysis of gamma-secretase derived from I213T mutant PS1-expressing PS1/PS2-deficient (PS(-/-)) cells and from the brains of I213T mutant PS1 knock-in mice. Kinetics analyses revealed that the FAD mutation reduced de novo Abeta generation, suggesting that mutation impairs the total catalytic rate of gamma-secretase. Analysis of each Abeta species revealed that the FAD mutation specifically reduced Abeta40 levels more drastically than Abeta42 levels, leading to an increased Abeta42/Abeta40 ratio. By contrast, the FAD mutation increased the generation of longer Abeta species such as Abeta43, Abeta45, and >Abeta46. These results were confirmed by analyses of gamma-secretase derived from I213T knock-in mouse brains, in which the reduction of de novo Abeta generation was mutant allele dose-dependent. Our findings clearly indicate that the mechanism underlying the increased Abeta42/Abeta40 ratio observed in cases of FAD mutations is related to the differential inhibition of gamma-site cleavage reactions, in which the reaction producing Abeta40 is subject to more inhibition than that producing Abeta42. Our results also provide novel insight into how enhancing the generation of longer Abetas may contribute to Alzheimer disease onset.

Impaired JIP3-dependent axonal lysosome transport promotes amyloid plaque pathology

PubMed Central

Gowrishankar, Swetha; Wu, Yumei

2017-01-01

Lysosomes robustly accumulate within axonal swellings at Alzheimer’s disease (AD) amyloid plaques. However, the underlying mechanisms and disease relevance of such lysosome accumulations are not well understood. Motivated by these problems, we identified JNK-interacting protein 3 (JIP3) as an important regulator of axonal lysosome transport and maturation. JIP3 knockout mouse neuron primary cultures accumulate lysosomes within focal axonal swellings that resemble the dystrophic axons at amyloid plaques. These swellings contain high levels of amyloid precursor protein processing enzymes (BACE1 and presenilin 2) and are accompanied by elevated Aβ peptide levels. The in vivo importance of the JIP3-dependent regulation of axonal lysosomes was revealed by the worsening of the amyloid plaque pathology arising from JIP3 haploinsufficiency in a mouse model of AD. These results establish the critical role of JIP3-dependent axonal lysosome transport in regulating amyloidogenic amyloid precursor protein processing and support a model wherein Aβ production is amplified by plaque-induced axonal lysosome transport defects. PMID:28784610

Neurodegeneration in Alzheimer disease: role of amyloid precursor protein and presenilin 1 intracellular signaling.

PubMed

Nizzari, Mario; Thellung, Stefano; Corsaro, Alessandro; Villa, Valentina; Pagano, Aldo; Porcile, Carola; Russo, Claudio; Florio, Tullio

2012-01-01

Alzheimer disease (AD) is a heterogeneous neurodegenerative disorder characterized by (1) progressive loss of synapses and neurons, (2) intracellular neurofibrillary tangles, composed of hyperphosphorylated Tau protein, and (3) amyloid plaques. Genetically, AD is linked to mutations in few proteins amyloid precursor protein (APP) and presenilin 1 and 2 (PS1 and PS2). The molecular mechanisms underlying neurodegeneration in AD as well as the physiological function of APP are not yet known. A recent theory has proposed that APP and PS1 modulate intracellular signals to induce cell-cycle abnormalities responsible for neuronal death and possibly amyloid deposition. This hypothesis is supported by the presence of a complex network of proteins, clearly involved in the regulation of signal transduction mechanisms that interact with both APP and PS1. In this review we discuss the significance of novel finding related to cell-signaling events modulated by APP and PS1 in the development of neurodegeneration.

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

PubMed

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

2016-01-01

γ-Secretase is a multisubunit membrane protein complex containing presenilin (PS1) as a catalytic subunit. Familial Alzheimer disease (FAD) mutations within PS1 were analyzed in yeast cells artificially expressing membrane-bound substrate, amyloid precursor protein, or Notch fused to Gal4 transcriptional activator. The FAD mutations, L166P and G384A (Leu-166 to Pro and Gly-384 to Ala substitution, respectively), were loss-of-function in yeast. We identified five amino acid substitutions that suppress the FAD mutations. The cleavage of amyloid precursor protein or Notch was recovered by the secondary mutations. We also found that secondary mutations alone activated the γ-secretase activity. FAD mutants with suppressor mutations, L432M or S438P within TMD9 together with a missense mutation in the second or sixth loops, regained γ-secretase activity when introduced into presenilin null mouse fibroblasts. Notably, the cells with suppressor mutants produced a decreased amount of Aβ42, which is responsible for Alzheimer disease. These results indicate that the yeast system is useful to screen for mutations and chemicals that modulate γ-secretase activity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Regulation of DREAM Expression by Group I mGluR

PubMed Central

Lee, Jinu; Kim, Insook; Oh, So Ra; Ko, Suk Jin; Lim, Mi Kyung; Kim, Dong Goo

2011-01-01

DREAM (downstream regulatory element antagonistic modulator) is a calcium-binding protein that regulates dynorphin expression, promotes potassium channel surface expression, and enhances presenilin processing in an expression level-dependent manner. However, no molecular mechanism has yet explained how protein levels of DREAM are regulated. Here we identified group I mGluR (mGluR1/5) as a positive regulator of DREAM protein expression. Overexpression of mGluR1/5 increased the cellular level of DREAM. Up-regulation of DREAM resulted in increased DREAM protein in both the nucleus and cytoplasm, where the protein acts as a transcriptional repressor and a modulator of its interacting proteins, respectively. DHPG (3,5-dihydroxyphenylglycine), a group I mGluR agonist, also up-regulated DREAM expression in cortical neurons. These results suggest that group I mGluR is the first identified receptor that may regulate DREAM activity in neurons. PMID:21660149

A novel presenilin 1 mutation (L174 M) in a large Cuban family with early onset Alzheimer disease.

PubMed

Bertoli Avella, A M; Marcheco Teruel, B; Llibre Rodriguez, J J; Gomez Viera, N; Borrajero Martinez, I; Severijnen, E A; Joosse, M; van Duijn, C M; Heredero Baute, L; Heutink, P

2002-10-01

We studied a Cuban family with presenile dementia (autosomal dominant) consisting of 281 members within six generations, the proband descended from a Spanish founder. Mean age at onset was 59 years of age. Memory impairment was the main symptom in all patients, additionally, ischemic episodes were described in 4 (n = 18) patients. Neuropathological examination of brain material (1 patient) revealed neuronal loss, amyloid plaques, and neurofibrillary tangles. Thirty DNA samples were genotyped (regions on chromosome 1, 3, 10, 12, 14, 17, 19, 20, and 21). A maximum Lod score of 3.79 at theta = 0 was obtained for marker D14S43, located in a 9-cM interval in which all patients shared the same haplotype. Sequencing of the PSEN1 gene revealed a heterozygous base substitution, C520A (exon 6), which is predicted to cause an amino acid change from leucine to methionine in the TMIII of the presenilin 1 protein. The mutation was found to co-segregate with the disease phenotype and the associated disease haplotype. The C --> A change was not observed in 80 control chromosomes from the Cuban population. Leucine at position 174 is highly conserved among species and is identical in presenilin 1 and presenilin 2 proteins. We propose the L174 M mutation might lead to an abnormal N-terminal and probably C-terminal fragments and malfunction of the protein complex. In conclusion, we found a novel PSEN1 mutation in a large family with clinical and pathological diagnosis of early onset familial Alzheimer disease, which may be relevant for other Hispanic populations.

Bacillus subtilis Intramembrane Protease RasP Activity in Escherichia coli and In Vitro.

PubMed

Parrell, Daniel; Zhang, Yang; Olenic, Sandra; Kroos, Lee

2017-10-01

RasP is a predicted intramembrane metalloprotease of Bacillus subtilis that has been proposed to cleave the stress response anti-sigma factors RsiW and RsiV, the cell division protein FtsL, and remnant signal peptides within their transmembrane segments. To provide evidence for direct effects of RasP on putative substrates, we developed a heterologous coexpression system. Since expression of catalytically inactive RasP E21A inhibited expression of other membrane proteins in Escherichia coli , we added extra transmembrane segments to RasP E21A, which allowed accumulation of most other membrane proteins. A corresponding active version of RasP appeared to promiscuously cleave coexpressed membrane proteins, except those with a large periplasmic domain. However, stable cleavage products were not observed, even in clpP mutant E. coli Fusions of transmembrane segment-containing parts of FtsL and RsiW to E. coli maltose-binding protein (MBP) also resulted in proteins that appeared to be RasP substrates upon coexpression in E. coli , including FtsL with a full-length C-terminal domain (suggesting that prior cleavage by a site 1 protease is unnecessary) and RsiW designed to mimic the PrsW site 1 cleavage product (suggesting that further trimming by extracytoplasmic protease is unnecessary). Purified RasP cleaved His 6 -MBP-RsiW(73-118) in vitro within the RsiW transmembrane segment based on mass spectrometry analysis, demonstrating that RasP is an intramembrane protease. Surprisingly, purified RasP failed to cleave His 6 -MBP-FtsL(23-117). We propose that the lack of α-helix-breaking residues in the FtsL transmembrane segment creates a requirement for the membrane environment and/or an additional protein(s) in order for RasP to cleave FtsL. IMPORTANCE Intramembrane proteases govern important signaling pathways in nearly all organisms. In bacteria, they function in stress responses, cell division, pathogenesis, and other processes. Their membrane-associated substrates are

Thermal Transients Excite Neurons through Universal Intramembrane Mechanoelectrical Effects

NASA Astrophysics Data System (ADS)

Plaksin, Michael; Shapira, Einat; Kimmel, Eitan; Shoham, Shy

2018-01-01

Modern advances in neurotechnology rely on effectively harnessing physical tools and insights towards remote neural control, thereby creating major new scientific and therapeutic opportunities. Specifically, rapid temperature pulses were shown to increase membrane capacitance, causing capacitive currents that explain neural excitation, but the underlying biophysics is not well understood. Here, we show that an intramembrane thermal-mechanical effect wherein the phospholipid bilayer undergoes axial narrowing and lateral expansion accurately predicts a potentially universal thermal capacitance increase rate of ˜0.3 % /°C . This capacitance increase and concurrent changes in the surface charge related fields lead to predictable exciting ionic displacement currents. The new MechanoElectrical Thermal Activation theory's predictions provide an excellent agreement with multiple experimental results and indirect estimates of latent biophysical quantities. Our results further highlight the role of electro-mechanics in neural excitation; they may also help illuminate subthreshold and novel physical cellular effects, and could potentially lead to advanced new methods for neural control.

A Presenilin-1 Mutation Identified in Familial Alzheimer Disease with Cotton Wool Plaques Causes a Nearly Complete Loss of γ-Secretase Activity*

PubMed Central

Heilig, Elizabeth A.; Xia, Weiming; Shen, Jie; Kelleher, Raymond J.

2010-01-01

Mutations in presenilin-1 and presenilin-2 (PS1 and PS2) are the most common cause of familial Alzheimer disease. PS1 and PS2 are the presumptive catalytic components of the multisubunit γ-secretase complex, which proteolyzes a number of type I transmembrane proteins, including the amyloid precursor protein (APP) and Notch. APP processing by γ-secretase produces β-amyloid peptides (Aβ40 and Aβ42) that accumulate in the Alzheimer disease brain. Here we identify a pathogenic L435F mutation in PS1 in two affected siblings with early-onset familial Alzheimer disease characterized by deposition of cerebral cotton wool plaques. The L435F mutation resides in a conserved C-terminal PAL sequence implicated in active site conformation and catalytic activity. The impact of PS1 mutations in and around the PAL motif on γ-secretase activity was assessed by expression of mutant PS1 in mouse embryo fibroblasts lacking endogenous PS1 and PS2. Surprisingly, the L435F mutation caused a nearly complete loss of γ-secretase activity, including >90% reductions in the generation of Aβ40, Aβ42, and the APP and Notch intracellular domains. Two nonpathogenic PS1 mutations, P433L and L435R, caused essentially complete loss of γ-secretase activity, whereas two previously identified pathogenic PS1 mutations, P436Q and P436S, caused partial loss of function with substantial reductions in production of Aβ40, Aβ42, and the APP and Notch intracellular domains. These results argue against overproduction of Aβ42 as an essential property of presenilin proteins bearing pathogenic mutations. Rather, our findings provide support for the hypothesis that pathogenic mutations cause a general loss of presenilin function. PMID:20460383

Presenilin-1 familial Alzheimer’s disease mutation alters hippocampal neurogenesis and memory function in CCL2 null mice

PubMed Central

Kiyota, Tomomi; Morrison, Christine M; Tu, Guihua; Dyavarshetty, Bhagyalaxmi; Weir, Robert A; Zhang, Gang; Xiong, Huangui; Gendelman, Howard E

2015-01-01

Aberrations in hippocampal neurogenesis are associated with learning and memory, synaptic plasticity and neurodegeneration in Alzheimer’s disease (AD). However, the linkage between them, β-amyloidosis and neuroinflammation is not well understood. To this end, we generated a mouse overexpressing familial AD (FAD) mutant human presenilin-1 (PS1) crossed with a knockout (KO) of the CC-chemokine ligand 2 (CCL2) gene. The PS1/CCL2KO mice developed robust age-dependent deficits in hippocampal neurogenesis associated with impairments in learning and memory, synaptic plasticity and long-term potentiation. Neurogliogenesis gene profiling supported β-amyloid independent pathways for FAD-associated deficits in hippocampal neurogenesis. We conclude that these PS1/CCL2KO mice are suitable for studies linking host genetics, immunity and hippocampal function. PMID:26112421

Association between presenilin-1 polymorphism and maternal meiosis II errors in Down syndrome.

PubMed

Petersen, M B; Karadima, G; Samaritaki, M; Avramopoulos, D; Vassilopoulos, D; Mikkelsen, M

2000-08-28

Several lines of evidence suggest a shared genetic susceptibility to Down syndrome (DS) and Alzheimer disease (AD). Rare forms of autosomal-dominant AD are caused by mutations in the APP and presenilin genes (PS-1 and PS-2). The presenilin proteins have been localized to the nuclear membrane, kinetochores, and centrosomes, suggesting a function in chromosome segregation. A genetic association between a polymorphism in intron 8 of the PS-1 gene and AD has been described in some series, and an increased risk of AD has been reported in mothers of DS probands. We therefore studied 168 probands with free trisomy 21 of known parental and meiotic origin and their parents from a population-based material, by analyzing the intron 8 polymorphism in the PS-1 gene. An increased frequency of allele 1 in mothers with a meiosis II error (70.8%) was found compared with mothers with a meiosis I error (52.7%, P < 0.01), with an excess of the 11 genotype in the meiosis II mothers. The frequency of allele 1 in mothers carrying apolipoprotein E (APOE) epsilon4 allele (68.0%) was higher than in mothers without epsilon4 (52.2%, P < 0.01). We hypothesize that the PS-1 intronic polymorphism might be involved in chromosomal nondisjunction through an influence on the expression level of PS-1 or due to linkage disequilibrium with biologically relevant polymorphisms in or outside the PS-1 gene. Copyright 2000 Wiley-Liss, Inc.

Regulation of raft-dependent endocytosis

PubMed Central

Lajoie, P; Nabi, IR

2007-01-01

Abstract Raft-dependent endocytosis is in large part defined as the cholesterol-sensitive, clathrin-independent internalization of ligands and receptors from the plasma membrane. It encompasses the endocytosis of caveo-lae, smooth plasmalemmal vesicles that form a subdomain of cholesterol and sphingolipid-rich lipid rafts and that are enriched for caveolin-1. While sharing common mechanisms, like cholesterol sensitivity, raft endocytic routes show differential regulation by various cellular components including caveolin-1, dynamin-2 and regulators of the actin cytoskeleton. Dynamin-dependent raft pathways, mediated by caveolae and morphologically equivalent non-caveolin vesicular intermediates, are referred to as caveolae/raft-dependent endocytosis. In contrast, dynamin-independent raft pathways are mediated by non-caveolar intermediates. Raft-dependent endocytosis is regulated by tyrosine kinase inhibitors and, through the regulation of the internalization of various ligands, receptors and effectors, is also a determinant of cellular signaling. In this review, we characterize and discuss the regulation of raft-dependent endocytic pathways and the role of key regulators such as caveolin-1. PMID:17760830

Ibuprofen partially attenuates neurodegenerative symptoms in presenilin conditional double-knockout mice.

PubMed

Dong, Z; Yan, L; Huang, G; Zhang, L; Mei, B; Meng, B

2014-06-13

Ibuprofen is a widely used nonsteroidal anti-inflammatory drug that reportedly reduces the risk of Alzheimer's disease (AD) development. The anti-inflammatory effect of ibuprofen occurred via inhibition of cyclooxygenases and anti-amyloidogenesis through modulation of γ-secretase. Presenilin 1 and 2 conditional double-knockout (cDKO) mice exhibited age-dependent memory impairment and forebrain degeneration without elevation of amyloid β deposition. Therefore, cDKO mice can be an ideal animal model on which to independently test the effects of ibuprofen anti-inflammatory properties on the prevention of AD. Three- and six-month-old cDKO mice were fed diet containing 375 ppm ibuprofen for six months. After multiple, well-validated behavioral tests, treatment with ibuprofen improved cognition-related behavioral performance, and drug efficacy was correlated with the timing of administration. Ibuprofen was more effective on six-month-old than on three-month-old cDKO mice. Biochemical analysis demonstrated that the effects of ibuprofen on glial fibrillary acidic protein and CD68 expression levels were uneven in different brain regions of cDKO mice and that age also influenced such effects. Tau hyperphosphorylation and the cleavage of caspase-3 decreased after ibuprofen treatment, and this effect was more significant in the older than the younger group of mice, which was consistent with the results of behavioral tests. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Pseudomonas aeruginosa Regulated Intramembrane Proteolysis (RIP): Protease MucP can Overcome Mutations in the AlgO Periplasmic Protease to Restore Alginate Production in Nonmucoid Revertants.

PubMed

Delgado, Camila; Florez, Laura; Lollett, Ivonne; Lopez, Christine; Kangeyan, Shiva; Kumari, Hansi; Stylianou, Marios; Smiddy, Robert J; Schneper, Lisa; Sautter, Robert T; Szatmari, George; Mathee, Kalai

2018-05-21

The progression of cystic fibrosis (CF) from an acute to a chronic disease is often associated with the conversion of the opportunistic pathogen Pseudomonas aeruginosa from a nonmucoid form to a mucoid form in the lung. This conversion involves the overproduction of the exopolysaccharide alginate, whose production is under control of the AlgT/U sigma factor. This factor is regulated posttranslationally by an extremely unstable process and has been commonly attributed to mutations in the algT/U gene. By exploiting this unstable phenotype, we isolated 34 spontaneous nonmucoid variants arising from the mucoid strain PDO300, a PAO1 derivative containing the mucA22 allele commonly found in mucoid CF isolates. Complementation analysis using a minimal tiling path cosmid library revealed that most of these mutants mapped to two protease-encoding genes, algO also known as prc or PA3257 , and mucP. Interestingly, our algO mutations were complemented by both mucP and algO , leading us to delete, clone and overexpress mucP , algO , mucE and mucD in both wild-type PAO1 and in PDO300 backgrounds to better understand the regulation of this complex regulatory mechanism. Our findings suggest the regulatory proteases follow two pathways for regulated intramembrane proteolysis (RIP), where both the AlgO/MucP pathway and MucE/AlgW pathway are required in the wild type strain, but where the AlgO/MucP pathway can bypass the MucE/AlgW pathway in mucoid strains with membrane-associated forms of MucA with shortened C-termini, such as the MucA22 variant. This work gives us a better understanding of how alginate production is regulated in the clinically important mucoid variants of Pseudomonas aeruginosa. IMPORTANCE: Infection by the opportunistic pathogen Pseudomonas aeruginosa is the leading cause of morbidity and mortality seen in cystic fibrosis (CF) patients. Poor patient prognosis correlates with the genotypic and phenotypic change of the bacteria from a typical nonmucoid to a mucoid

The Large Hydrophilic Loop of Presenilin 1 Is Important for Regulating γ-Secretase Complex Assembly and Dictating the Amyloid β Peptide (Aβ) Profile without Affecting Notch Processing*

PubMed Central

Wanngren, Johanna; Frånberg, Jenny; Svensson, Annelie I.; Laudon, Hanna; Olsson, Fredrik; Winblad, Bengt; Liu, Frank; Näslund, Jan; Lundkvist, Johan; Karlström, Helena

2010-01-01

γ-Secretase is an enzyme complex that mediates both Notch signaling and β-amyloid precursor protein (APP) processing, resulting in the generation of Notch intracellular domain, APP intracellular domain, and the amyloid β peptide (Aβ), the latter playing a central role in Alzheimer disease (AD). By a hitherto undefined mechanism, the activity of γ-secretase gives rise to Aβ peptides of different lengths, where Aβ42 is considered to play a particular role in AD. In this study we have examined the role of the large hydrophilic loop (amino acids 320–374, encoded by exon 10) of presenilin 1 (PS1), the catalytic subunit of γ-secretase, for γ-secretase complex formation and activity on Notch and APP processing. Deletion of exon 10 resulted in impaired PS1 endoproteolysis, γ-secretase complex formation, and had a differential effect on Aβ-peptide production. Although the production of Aβ38, Aβ39, and Aβ40 was severely impaired, the effect on Aβ42 was affected to a lesser extent, implying that the production of the AD-related Aβ42 peptide is separate from the production of the Aβ38, Aβ39, and Aβ40 peptides. Interestingly, formation of the intracellular domains of both APP and Notch was intact, implying a differential cleavage activity between the ϵ/S3 and γ sites. The most C-terminal amino acids of the hydrophilic loop were important for regulating APP processing. In summary, the large hydrophilic loop of PS1 appears to differentially regulate the relative production of different Aβ peptides without affecting Notch processing, two parameters of significance when considering γ-secretase as a target for pharmaceutical intervention in AD. PMID:20106965

Environment enrichment rescues the neurodegenerative phenotypes in presenilins-deficient mice.

PubMed

Dong, Suzhen; Li, Chunxia; Wu, Pu; Tsien, Joe Z; Hu, Yinghe

2007-07-01

Presenilin (PS) 1 and 2 conditional double knockout (cDKO) mice show progressive memory dysfunction and forebrain degeneration. Gene expression profiling results revealed a strong activation of immunity and inflammation responses in the brains of 10-month-old cDKO mice. As environmental enrichment (EE) has been shown to be able to improve memory and induce neurogenesis of the brain, we assessed the effects of EE on the memory performance and the neurodegeneration in cDKO mice. We found that EE effectively enhanced memory and partially rescued the forebrain atrophy of the cDKO mice. Our results suggest that immunity and inflammation could play important roles in the neurodegeneration of cDKO mice. Furthermore, the beneficial effects of EE may be associated with the inhibition of the expression of immunity and inflammation-related genes in the brain.

Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by Alzheimer-related PS1 mutations.

PubMed

Lee, Ju-Hyun; Yu, W Haung; Kumar, Asok; Lee, Sooyeon; Mohan, Panaiyur S; Peterhoff, Corrinne M; Wolfe, Devin M; Martinez-Vicente, Marta; Massey, Ashish C; Sovak, Guy; Uchiyama, Yasuo; Westaway, David; Cuervo, Ana Maria; Nixon, Ralph A

2010-06-25

Macroautophagy is a lysosomal degradative pathway essential for neuron survival. Here, we show that macroautophagy requires the Alzheimer's disease (AD)-related protein presenilin-1 (PS1). In PS1 null blastocysts, neurons from mice hypomorphic for PS1 or conditionally depleted of PS1, substrate proteolysis and autophagosome clearance during macroautophagy are prevented as a result of a selective impairment of autolysosome acidification and cathepsin activation. These deficits are caused by failed PS1-dependent targeting of the v-ATPase V0a1 subunit to lysosomes. N-glycosylation of the V0a1 subunit, essential for its efficient ER-to-lysosome delivery, requires the selective binding of PS1 holoprotein to the unglycosylated subunit and the Sec61alpha/oligosaccharyltransferase complex. PS1 mutations causing early-onset AD produce a similar lysosomal/autophagy phenotype in fibroblasts from AD patients. PS1 is therefore essential for v-ATPase targeting to lysosomes, lysosome acidification, and proteolysis during autophagy. Defective lysosomal proteolysis represents a basis for pathogenic protein accumulations and neuronal cell death in AD and suggests previously unidentified therapeutic targets.

Presenilins and γ-Secretase: Structure, Function, and Role in Alzheimer Disease

PubMed Central

De Strooper, Bart; Iwatsubo, Takeshi; Wolfe, Michael S.

2012-01-01

Presenilins were first discovered as sites of missense mutations responsible for early-onset Alzheimer disease (AD). The encoded multipass membrane proteins were subsequently found to be the catalytic components of γ-secretases, membrane-embedded aspartyl protease complexes responsible for generating the carboxyl terminus of the amyloid β-protein (Aβ) from the amyloid protein precursor (APP). The protease complex also cleaves a variety of other type I integral membrane proteins, most notably the Notch receptor, signaling from which is involved in many cell differentiation events. Although γ-secretase is a top target for developing disease-modifying AD therapeutics, interference with Notch signaling should be avoided. Compounds that alter Aβ production by γ-secretase without affecting Notch proteolysis and signaling have been identified and are currently at various stages in the drug development pipeline. PMID:22315713

Reversible Unfolding of Rhomboid Intramembrane Proteases.

PubMed

Panigrahi, Rashmi; Arutyunova, Elena; Panwar, Pankaj; Gimpl, Katharina; Keller, Sandro; Lemieux, M Joanne

2016-03-29

Denaturant-induced unfolding of helical membrane proteins provides insights into their mechanism of folding and domain organization, which take place in the chemically heterogeneous, anisotropic environment of a lipid membrane. Rhomboid proteases are intramembrane proteases that play key roles in various diseases. Crystal structures have revealed a compact helical bundle with a buried active site, which requires conformational changes for the cleavage of transmembrane substrates. A dimeric form of the rhomboid protease has been shown to be important for activity. In this study, we examine the mechanism of refolding for two distinct rhomboids to gain insight into their secondary structure-activity relationships. Although helicity is largely abolished in the unfolded states of both proteins, unfolding is completely reversible for HiGlpG but only partially reversible for PsAarA. Refolding of both proteins results in reassociation of the dimer, with a 90% regain of catalytic activity for HiGlpG but only a 70% regain for PsAarA. For both proteins, a broad, gradual transition from the native, folded state to the denatured, partly unfolded state was revealed with the aid of circular dichroism spectroscopy as a function of denaturant concentration, thus arguing against a classical two-state model as found for many globular soluble proteins. Thermal denaturation has irreversible destabilizing effects on both proteins, yet reveals important functional details regarding substrate accessibility to the buried active site. This concerted biophysical and functional analysis demonstrates that HiGlpG, with a simple six-transmembrane-segment organization, is more robust than PsAarA, which has seven predicted transmembrane segments, thus rendering HiGlpG amenable to in vitro studies of membrane-protein folding. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Relative hyperperfusion by SPECT in a family with a presenilin 1 (T245P) mutation.

PubMed

Edwards-Lee, Terri; Wen, Johnny; Chung, Julia A; Vasinrapee, Panukorn; Mishkin, Frederick S

2008-01-01

Clinical characteristics of autosomal dominant Alzheimer's disease often differ clinically from sporadic disease with the onset of seizures, spasticity and myoclonus early in the disease course. Similarly imaging characteristics may also differ. We report the findings of relative hyperperfusion by Tc-99m HMPAO SPECT in the medial orbitofrontal cortex and anterior temporal lobe in four affected family members carrying a presenilin 1 mutation. SPECT of the four individuals was compared to an age-matched normal database. We speculate that the findings of relative medial orbitofrontal and anterior temporal lobe hyperperfusion may be a marker of early onset Alzheimer's disease in this family.

Emotion Regulation in Alcohol Dependence.

PubMed

Petit, Géraldine; Luminet, Olivier; Maurage, François; Tecco, Juan; Lechantre, Stéphane; Ferauge, Marc; Gross, James J; de Timary, Philippe

2015-12-01

The main aim of this study was to investigate, in alcohol-dependent (AD) patients, the use of the 5 emotion regulation strategies specified in Gross's (1998, Rev Gen Psychol, 2, 271) process model of emotion regulation with the use of a semi-structured interview allowing a detailed and high-quality assessment of emotion regulation strategies. A secondary aim was to examine the possible influence of protracted abstinence and detoxification on emotion dysregulation. Finally, the association between the level of craving and the types of regulation strategies was investigated. Forty-four treatment-seeking AD patients with varying time spent in rehabilitation, and 26 healthy controls were interviewed using a version of the Emotion Regulation Interview (Werner et al., 2011, J Psychopathol Behav Assess, 33, 346) adapted to alcohol dependence. Compared to controls, AD patients reported significantly greater use of response modulation and attentional deployment, but lesser use of cognitive change. Among patients, (1) rehabilitation duration was positively correlated with the use of cognitive change and (2) the use of response modulation was positively associated with the level of craving. These findings clarify the specific pattern of emotion dysregulation associated with alcohol dependence. They also suggest that (1) abstinence is associated with a shift toward more adaptive emotion regulation patterns and that (2) inefficient regulation strategies may lead to craving and the maintenance of alcohol use. If these findings are confirmed through longitudinal and mediation designs, they will have important clinical implications. Copyright © 2015 by the Research Society on Alcoholism.

A comparative study of self-regulation in substance dependent and non-dependent individuals.

PubMed

Bakhshani, Nour Mohammad; Hosseinbor, Mohsen

2013-08-05

Several factors influence the beginning and maintenance of substance use. The purpose of this study was to examine as well as to compare 'self-regulation' in both substance dependent and non-substance dependent individuals. In a cross-sectional study 228 (118 substance dependent and 110 with no history of using substance) participants aged 16-55 were recruited. All of the participants were asked to complete the Self-Regulation Inventory (SRI-25) and a demographic characteristics data checklist. Data was analyzed using descriptive statistics (frequency, mean and standard deviation) and the t-test. The results showed significant differences between substance dependent and non- substance dependent groups in all the scales of the self-regulation inventory including positive actions, controllability, expression of feelings and needs, assertiveness, and well-being seeking (p<0.01). Self-regulation and self-control skills in drug dependent individuals are lower than those without substance dependence individuals. It is concluded that substance use may related to a deficiency in self-control and regulation of feelings. Therefore, for prevention and treatment of substance dependence disorder, it is necessary to work out and exploit strategies that include the improvement of self-regulation.

Amyloid Precursor Protein (APP) Mediated Regulation of Ganglioside Homeostasis Linking Alzheimer's Disease Pathology with Ganglioside Metabolism

PubMed Central

Grimm, Marcus O. W.; Zinser, Eva G.; Grösgen, Sven; Hundsdörfer, Benjamin; Rothhaar, Tatjana L.; Burg, Verena K.; Kaestner, Lars; Bayer, Thomas A.; Lipp, Peter; Müller, Ulrike; Grimm, Heike S.; Hartmann, Tobias

2012-01-01

Gangliosides are important players for controlling neuronal function and are directly involved in AD pathology. They are among the most potent stimulators of Aβ production, are enriched in amyloid plaques and bind amyloid beta (Aβ). However, the molecular mechanisms linking gangliosides with AD are unknown. Here we identified the previously unknown function of the amyloid precursor protein (APP), specifically its cleavage products Aβ and the APP intracellular domain (AICD), of regulating GD3-synthase (GD3S). Since GD3S is the key enzyme converting a- to b-series gangliosides, it therefore plays a major role in controlling the levels of major brain gangliosides. This regulation occurs by two separate and additive mechanisms. The first mechanism directly targets the enzymatic activity of GD3S: Upon binding of Aβ to the ganglioside GM3, the immediate substrate of the GD3S, enzymatic turnover of GM3 by GD3S was strongly reduced. The second mechanism targets GD3S expression. APP cleavage results, in addition to Aβ release, in the release of AICD, a known candidate for gene transcriptional regulation. AICD strongly down regulated GD3S transcription and knock-in of an AICD deletion mutant of APP in vivo, or knock-down of Fe65 in neuroblastoma cells, was sufficient to abrogate normal GD3S functionality. Equally, knock-out of the presenilin genes, presenilin 1 and presenilin 2, essential for Aβ and AICD production, or of APP itself, increased GD3S activity and expression and consequently resulted in a major shift of a- to b-series gangliosides. In addition to GD3S regulation by APP processing, gangliosides in turn altered APP cleavage. GM3 decreased, whereas the ganglioside GD3, the GD3S product, increased Aβ production, resulting in a regulatory feedback cycle, directly linking ganglioside metabolism with APP processing and Aβ generation. A central aspect of this homeostatic control is the reduction of GD3S activity via an Aβ-GM3 complex and AICD

Assignment of Alzheimer's presenilin-2 (PS-2) gene to 1q42.1 by fluorescence in situ hybridization.

PubMed

Takano, T; Sahara, N; Yamanouchi, Y; Mori, H

1997-01-17

Presenilin-2 (PS-2) was suggested to be localized on 1q31-42 based on linkage analysis and cDNA cloning. The final identification of PS-2 as the causal gene for early-onset familial Alzheimer's disease in Voga-German pedigrees was concluded based on the point mutation found in the candidate cDNA isolated from this familial AD. We present evidence of its physical genome mapping of PS-2 on chromosome 1q42.1 by fluorescence in situ hybridization method.

Familial Alzheimer's disease mutations in presenilin 1 do not alter levels of the secreted amyloid-beta protein precursor generated by beta-secretase cleavage.

PubMed

Zhang, Can; Browne, Andrew; Kim, Doo Yeon; Tanzi, Rudolph E

2010-02-01

Alzheimer's disease (AD) is an insidious and progressive disease with a genetically complex and heterogenous etiology. More than 200 fully penetrant mutations in the amyloid beta-protein precursor (APP), presenilin 1 (or PSEN1), and presenilin 2 (PSEN2) have been linked to early-onset familial AD (FAD). 177 PSEN1 FAD mutations have been identified so far and account for more than approximately 80% of all FAD mutations. All PSEN1 FAD mutations can increase the Abeta42:Abeta40 ratio with seemingly different and incompletely understood mechanisms. A recent study has shown that the 286 amino acid N-terminal fragment of APP (N-APP), a proteolytic product of beta-secretase-derived secreted form of APP (sAPPbeta), could bind the death receptor, DR6, and lead to neurodegeneration. Here we asked whether PSEN1 FAD mutations lead to neurodegeneration by modulating sAPPbeta levels. All four different PSEN1 FAD mutations tested (in three mammalian cell lines) did not alter sAPPbeta levels. Therefore PS1 mutations do not appear to contribute to AD pathogenesis via altered production of sAPPbeta.

Neurotrophins regulate ApoER2 proteolysis through activation of the Trk signaling pathway.

PubMed

Larios, Jorge A; Jausoro, Ignacio; Benitez, Maria-Luisa; Bronfman, Francisca C; Marzolo, Maria-Paz

2014-09-19

ApoER2 and the neurotrophin receptors Trk and p75(NTR) are expressed in the CNS and regulate key functional aspects of neurons, including development, survival, and neuronal function. It is known that both ApoER2 and p75(NTR) are processed by metalloproteinases, followed by regulated intramembrane proteolysis. TrkA activation by nerve growth factor (NGF) increases the proteolytic processing of p75(NTR) mediated by ADAM17. Reelin induces the sheeding of ApoER2 ectodomain depending on metalloproteinase activity. However, it is not known if there is a common regulation mechanism for processing these receptors. We found that TrkA activation by NGF in PC12 cells induced ApoER2 processing, which was dependent on TrkA activation and metalloproteinases. NGF-induced ApoER2 proteolysis was independent of mitogen activated protein kinase activity and of phosphatidylinositol-3 kinase activity. In contrast, the basal proteolysis of ApoER2 increased when both kinases were pharmacologically inhibited. The ApoER2 ligand reelin regulated the proteolytic processing of its own receptor but not of p75(NTR). Finally, in primary cortical neurons, which express both ApoER2 and TrkB, we found that the proteolysis of ApoER2 was also regulated by brain-derived growth factor (BDNF). Our results highlight a novel relationship between neurotrophins and the reelin-ApoER2 system, suggesting that these two pathways might be linked to regulate brain development, neuronal survival, and some pathological conditions.

Dependence regulation in newlywed couples: A prospective examination.

PubMed

Derrick, Jaye L; Leonard, Kenneth E; Homish, Gregory G

2012-12-01

According to the Risk Regulation Model (Murray, S. L., Holmes, J. G., & Collins, N. L. (2006). Optimizing assurance: The risk regulation system in relationships. Psychological Bulletin, 132 , 641-666), people need to trust in their partner's regard before they risk interdependence. The current study prospectively examines the association between perceived regard and levels of dependence in newlywed couples over nine years of marriage. Analyses demonstrate that changes in perceived regard predict levels of dependence, changes in dependence do not predict perceived regard, and alternative explanations cannot account for these effects. Further, changes in perceived regard prospectively predict divorce, and levels of dependence mediate this association. Results are discussed in terms of the dependence regulation component of the Risk Regulation Model.

Cell-cell interactions of isolated and cultured oligodendrocytes: formation of linear occluding junctions and expression of peculiar intramembrane particles.

PubMed

Massa, P T; Szuchet, S; Mugnaini, E

1984-12-01

Oligodendrocytes were isolated from lamb brain. Freshly isolated cells and cultured cells, either 1- to 4-day-old unattached or 1- to 5-week-old attached, were examined by thin section and freeze-fracture electron microscopy. Freeze-fracture of freshly isolated oligodendrocytes showed globular and elongated intramembrane particles similar to those previously described in oligodendrocytes in situ. Enrichment of these particles was seen at sites of inter-oligodendrocyte contact. Numerous gap junctions and scattered linear tight junctional arrays were apparent. Gap junctions were connected to blebs of astrocytic plasma membrane sheared off during isolation, whereas tight junctions were facing extracellular space or blebs of oligodendrocytic plasma membrane. Thin sections of cultured, unattached oligodendrocytes showed rounded cell bodies touching one another at points without forming specialized cell junctions. Cells plated on polylysine-coated aclar dishes attached, emanated numerous, pleomorphic processes, and expressed galactocerebroside and myelin basic protein, characteristic markers for oligodendrocytes. Thin sections showed typical oligodendrocyte ultrastructure but also intermediate filaments not present in unattached cultures. Freeze-fracture showed intramembrane particles similar to but more numerous, and with a different fracture face repartition, than those seen in oligodendrocytes, freshly isolated or in situ. Gap junctions were small and rare. Apposed oligodendrocyte plasma membrane formed linear tight junctions which became more numerous with time in culture. Thus, cultured oligodendrocytes isolated from ovine brains develop and maintain features characteristic of mature oligodendrocytes in situ and can be used to explore formation and maintenance of tight junctions and possibly other classes of cell-cell interactions important in the process of myelination.

Apolipoprotein E and presenilin-1 genotypes in Huntington's disease.

PubMed

Panas, M; Avramopoulos, D; Karadima, G; Petersen, M B; Vassilopoulos, D

1999-07-01

Huntington's disease (HD) is an autosomal dominant degenerative disease of the central nervous system manifested by involuntary movements (chorea), psychiatric manifestations, and cognitive impairment with a variable age at onset. This variability is mainly attributed to genetic factors. The so-called aging genes [e.g., those for apolipoprotein E (APOE) and presenilin-1 (PS-1) have been implicated in determining the age at onset of Alzheimer's disease, a disease sharing common clinical features with HD. In 60 unrelated patients suffering from HD (mean age at onset 40.1 years, range 20-65) we determined number of CAG repeats and the distribution of the APOE alleles (epsilon2, epsilon3, epsilon4) and PS-1 alleles. The results showed that: (a) The age at onset was higher in the group of patients with the epsilon4 allele (51.6 vs. 38.0 P<0.002), (b) The correlation between the age at onset and the number of CAG repeats was strong in patients with the epsilon3/epsilon3 genotype while it was not detected in patients with epsilon3/epsilon4 genotype. (c) No correlation was found between age at onset and PS-1 alleles. In conclusion, APOE seems to be a significant factor influencing the age at onset of Huntington's disease.

Investigation of the Role of Sialomucin Complex (SCMC)/Muc4, a Unique Intramembranous HER-2/ErbB-2 Ligand, as a Suppressor of Apoptosis

DTIC Science & Technology

2005-04-01

Complex (SCMC)/ Muc4 , a Unique Intramembranous HER-2/ErbB-2 Ligand, as a Suppressor of Apoptosis PRINCIPAL INVESTIGATOR: George Theodore...1 Apr 2002 – 31 Mar 2005 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Investigation of the Role of Sialomucin Complex (SCMC)/ Muc4 , a Unique...Seminar and Journal Club course in the Department of Cell Biology and Anatomy RESEARCH ACCOMPLISHMENTS Introduction Muc4 /sialomucin complex (SMC) is

1,25-Dihydroxyvitamin D3 and 22-oxa-1,25-dihydroxyvitamin D3 in vivo nuclear receptor binding in developing bone during endochondral and intramembranous ossification.

PubMed

Stumpf, W E; Koike, N; Hayakawa, N; Tokuda, K; Nishimiya, K; Tsuchiya, Y; Hirate, J; Okazaki, A; Kumaki, K

1994-09-01

Target cells for 3H-labeled 1 alpha, 25(OH)2 vitamin D3 [1,25(OH)2D3, vitamin D] and its analog 3H-labeled 22-oxa-1 alpha, 25(OH)2 vitamin D3 (OCT) have been identified during endochondral and intramembranous ossification in developing, undecalcified, unembedded bone, using thaw-mount autoradiography. Two-day-old neonatal rats were injected with [3H]1,25(OH)2D3 or [3H]OCT; after 2 h leg, spine, and head were frozen and sectioned. In the epiphyseal-metaphyseal region specific nuclear concentrations of [3H]1,25(OH)2D3 and [3H]OCT were observed in identical cell populations, being low in cells of the articular and resting zone, intermediate in the proliferating zone, and highest in hypertrophic chondrocytes and in osteoblasts and precursor cells. In the primary spongiosa intertrabecular spaces there were a large number of cells with nuclear labeling--probably osteoblasts and precursor cells. In contrast, in the secondary spongiosa intertrabecular spaces, apparent blood-forming cells were mostly unlabeled. Osteoblasts along bone spicules and compact bone in long bones, vertebrae, and head also showed strong nuclear labeling, as did cells of the periosteum. These data suggest that 1,25(OH)2D3 and OCT regulate development, differentiation, and activities of chondrocytes and osteoblasts, including differentiation of resting chondrocytes into proliferating and hypertrophic chondrocytes that involve "chondroclastic" enlargement of lacunae and "trans-differentiation" of surviving hypertrophic chondrocytes; differentiation of stroma cells into osteoblasts; and in periosteum and other regions of intramembranous ossification differentiation of precursor cells and osteoblasts. Nuclear receptor binding and their selective and hierarchical distribution during cell differentiation appear to correspond to multiple genomic effects toward growth, regeneration and repair. The findings indicate a physiological significance and therapeutic potential of 1,25(OH)2D3 and in particular of

Epigenetic regulation of estrogen-dependent memory

PubMed Central

Fortress, Ashley M.; Frick, Karyn M.

2014-01-01

Hippocampal memory formation is highly regulated by post-translational histone modifications and DNA methylation. Accordingly, these epigenetic processes play a major role in the effects of modulatory factors, such as sex steroid hormones, on hippocampal memory. Our laboratory recently demonstrated that the ability of the potent estrogen 17β-estradiol (E2) to enhance hippocampal-dependent novel object recognition memory in ovariectomized female mice requires ERK-dependent histone H3 acetylation and DNA methylation in the dorsal hippocampus. Although these data provide valuable insight into the chromatin modifications that mediate the memory-enhancing effects of E2, epigenetic regulation of gene expression is enormously complex. Therefore, more research is needed to fully understand how E2 and other hormones employ epigenetic alterations to shape behavior. This review discusses the epigenetic alterations shown thus far to regulate hippocampal memory, briefly reviews the effects of E2 on hippocampal function, and describes in detail our work on epigenetic regulation of estrogenic memory enhancement. PMID:24878494

Epigenetic regulation of estrogen-dependent memory.

PubMed

Fortress, Ashley M; Frick, Karyn M

2014-10-01

Hippocampal memory formation is highly regulated by post-translational histone modifications and DNA methylation. Accordingly, these epigenetic processes play a major role in the effects of modulatory factors, such as sex steroid hormones, on hippocampal memory. Our laboratory recently demonstrated that the ability of the potent estrogen 17β-estradiol (E2) to enhance hippocampal-dependent novel object recognition memory in ovariectomized female mice requires ERK-dependent histone H3 acetylation and DNA methylation in the dorsal hippocampus. Although these data provide valuable insight into the chromatin modifications that mediate the memory-enhancing effects of E2, epigenetic regulation of gene expression is enormously complex. Therefore, more research is needed to fully understand how E2 and other hormones employ epigenetic alterations to shape behavior. This review discusses the epigenetic alterations shown thus far to regulate hippocampal memory, briefly reviews the effects of E2 on hippocampal function, and describes in detail our work on epigenetic regulation of estrogenic memory enhancement. Copyright © 2014 Elsevier Inc. All rights reserved.

A founder mutation in presenilin 1 causing early-onset Alzheimer disease in unrelated Caribbean Hispanic families.

PubMed

Athan, E S; Williamson, J; Ciappa, A; Santana, V; Romas, S N; Lee, J H; Rondon, H; Lantigua, R A; Medrano, M; Torres, M; Arawaka, S; Rogaeva, E; Song, Y Q; Sato, C; Kawarai, T; Fafel, K C; Boss, M A; Seltzer, W K; Stern, Y; St George-Hyslop, P; Tycko, B; Mayeux, R

2001-11-14

Genetic determinants of Alzheimer disease (AD) have not been comprehensively examined in Caribbean Hispanics, a population in the United States in whom the frequency of AD is higher compared with non-Hispanic whites. To identify variant alleles in genes related to familial early-onset AD among Caribbean Hispanics. Family-based case series conducted in 1998-2001 at an AD research center in New York, NY, and clinics in the Dominican Republic. Among 206 Caribbean Hispanic families with 2 or more living members with AD who were identified, 19 (9.2%) had at least 1 individual with onset of AD before the age of 55 years. The entire coding region of the presenilin 1 gene and exons 16 and 17 of the amyloid precursor protein gene were sequenced in probands from the 19 families and their living relatives. A G-to-C nucleotide change resulting in a glycine-alanine amino acid substitution at codon 206 (Gly206Ala) in exon 7 of presenilin 1 was observed in 23 individuals from 8 (42%) of the 19 families. A Caribbean Hispanic individual with the Gly206Ala mutation and early-onset familial disease was also found by sequencing the corresponding genes of 319 unrelated individuals in New York City. The Gly206Ala mutation was not found in public genetic databases but was reported in 5 individuals from 4 Hispanic families with AD referred for genetic testing. None of the members of these families were related to one another, yet all carriers of the Gly206Ala mutation tested shared a variant allele at 2 nearby microsatellite polymorphisms, indicating a common ancestor. No mutations were found in the amyloid precursor protein gene. The Gly206Ala mutation was found in 8 of 19 unrelated Caribbean Hispanic families with early-onset familial AD. This genetic change may be a prevalent cause of early-onset familial AD in the Caribbean Hispanic population.

An Anti-Parkinson’s Disease Drug via Targeting Adenosine A2A Receptor Enhances Amyloid-β Generation and γ-Secretase Activity

PubMed Central

Li, Xiaohang; Wang, Xin; Zhou, Yue; Yang, Wenjuan; Chen, Ming; Zhao, Jian; Pei, Gang

2016-01-01

γ-secretase mediates the intramembranous proteolysis of amyloid precursor protein (APP) and determines the generation of Aβ which is associated with Alzheimer’s disease (AD). Here we identified that an anti-Parkinson’s disease drug, Istradefylline, could enhance Aβ generation in various cell lines and primary neuronal cells of APP/PS1 mouse. Moreover, the increased generation of Aβ42 was detected in the cortex of APP/PS1 mouse after chronic treatment with Istradefylline. Istradefylline promoted the activity of γ-secretase which could lead to increased Aβ production. These effects of Istradefylline were reduced by the knockdown of A2AR but independent of A2AR-mediated G protein- or β-arrestin-dependent signal pathway. We further observed that A2AR colocalized with γ-secretase in endosomes and physically interacted with the catalytic subunit presenilin-1 (PS1). Interestingly, Istradefylline attenuated the interaction in time- and dosage-dependent manners. Moreover the knockdown of A2AR which in theory would release PS1 potentiated both Aβ generation and γ-secretase activity. Thus, our study implies that the association of A2AR could modulate γ-secretase activity. Istradefylline enhance Aβ generation and γ-secretase activity possibly via modulating the interaction between A2AR and γ-secretase, which may bring some undesired effects in the central nervous system (CNS). PMID:27835671

Intramembrane cavitation as a unifying mechanism for ultrasound-induced bioeffects.

PubMed

Krasovitski, Boris; Frenkel, Victor; Shoham, Shy; Kimmel, Eitan

2011-02-22

The purpose of this study was to develop a unified model capable of explaining the mechanisms of interaction of ultrasound and biological tissue at both the diagnostic nonthermal, noncavitational (<100 mW · cm(-2)) and therapeutic, potentially cavitational (>100 mW · cm(-2)) spatial peak temporal average intensity levels. The cellular-level model (termed "bilayer sonophore") combines the physics of bubble dynamics with cell biomechanics to determine the dynamic behavior of the two lipid bilayer membrane leaflets. The existence of such a unified model could potentially pave the way to a number of controlled ultrasound-assisted applications, including CNS modulation and blood-brain barrier permeabilization. The model predicts that the cellular membrane is intrinsically capable of absorbing mechanical energy from the ultrasound field and transforming it into expansions and contractions of the intramembrane space. It further predicts that the maximum area strain is proportional to the acoustic pressure amplitude and inversely proportional to the square root of the frequency (ε A,max ∝ P(A)(0.8f - 0.5) and is intensified by proximity to free surfaces, the presence of nearby microbubbles in free medium, and the flexibility of the surrounding tissue. Model predictions were experimentally supported using transmission electron microscopy (TEM) of multilayered live-cell goldfish epidermis exposed in vivo to continuous wave (CW) ultrasound at cavitational (1 MHz) and noncavitational (3 MHz) conditions. Our results support the hypothesis that ultrasonically induced bilayer membrane motion, which does not require preexistence of air voids in the tissue, may account for a variety of bioeffects and could elucidate mechanisms of ultrasound interaction with biological tissue that are currently not fully understood.

The role of oxygen as a regulator of stem cell fate during fracture repair in TSP2-null mice.

PubMed

Burke, Darren; Dishowitz, Michael; Sweetwyne, Mariya; Miedel, Emily; Hankenson, Kurt D; Kelly, Daniel J

2013-10-01

It is often difficult to decouple the relative importance of different factors in regulating MSC differentiation. Genetically modified mice provide model systems whereby some variables can be manipulated while others are kept constant. Fracture repair in thrombospondin-2 (TSP2)-null mice is characterized by reduced endochondral ossification and enhanced intramembranous bone formation. The proposed mechanism for this shift in MSC fate is that increased vascular density and hence oxygen availability in TSP2-null mice regulates differentiation. However, TSP2 is multifunctional and regulates other aspects of the regenerative cascade, such as MSC proliferation. The objective of this study is to use a previously developed computational model of tissue differentiation, in which substrate stiffness and oxygen tension regulate stem cell differentiation, to simulate potential mechanisms which may drive alterations in MSC fate in TSP2-null mice. Four models (increased cell proliferation, increased numbers of MSCs in the marrow decreased cellular oxygen consumption, and an initially stiffer callus) were not predictive of experimental observations in TSP2-null mice. In contrast, increasing the rate of angiogenic progression led to a prediction of greater intramembranous ossification, diminished endochondral ossification, and a reduced region of hypoxia in the fracture callus similar to that quantified experimentally by the immunohistochemical detection of pimonidazole adducts that develop with hypoxia. This study therefore provides further support for the hypothesis that oxygen availability during early fracture healing is a key regulator of MSC bipotential differentiation, and furthermore, it highlights the advantages of integrating computational models with genetically modified mouse studies for further elucidating mechanisms regulating stem cell fate. Copyright © 2013 Orthopaedic Research Society.

Conditional forebrain inactivation of nicastrin causes progressive memory impairment and age-related neurodegeneration.

PubMed

Tabuchi, Katsuhiko; Chen, Guiquan; Südhof, Thomas C; Shen, Jie

2009-06-03

Loss of presenilin function in adult mouse brains causes memory loss and age-related neurodegeneration. Since presenilin possesses gamma-secretase-dependent and -independent activities, it remains unknown which activity is required for presenilin-dependent memory formation and neuronal survival. To address this question, we generated postnatal forebrain-specific nicastrin conditional knock-out (cKO) mice, in which nicastrin, a subunit of gamma-secretase, is inactivated selectively in mature excitatory neurons of the cerebral cortex. nicastrin cKO mice display progressive impairment in learning and memory and exhibit age-dependent cortical neuronal loss, accompanied by astrocytosis, microgliosis, and hyperphosphorylation of the microtubule-associated protein Tau. The neurodegeneration observed in nicastrin cKO mice likely occurs via apoptosis, as evidenced by increased numbers of apoptotic neurons. These findings demonstrate an essential role of nicastrin in the execution of learning and memory and the maintenance of neuronal survival in the brain and suggest that presenilin functions in memory and neuronal survival via its role as a gamma-secretase subunit.

Pharmacological evidences for DFK167-sensitive presenilin-independent gamma-secretase-like activity.

PubMed

Sevalle, Jean; Ayral, Erwan; Hernandez, Jean-François; Martinez, Jean; Checler, Frédéric

2009-07-01

Amyloid-beta (Abeta) peptides production is thought to be a key event in the neurodegenerative process ultimately leading to Alzheimer's disease (AD) pathology. A bulk of studies concur to propose that the C-terminal moiety of Abeta is released from its precursor beta-amyloid precursor protein by a high molecular weight enzymatic complex referred to as gamma-secretase, that is composed of at least, nicastrin (NCT), Aph-1, Pen-2, and presenilins (PS) 1 or 2. They are thought to harbor the gamma-secretase catalytic activity. However, several lines of evidence suggest that additional gamma-secretase-like activities could potentially contribute to Abeta production. By means of a quenched fluorimetric substrate (JMV2660) mimicking the beta-amyloid precursor protein sequence targeted by gamma-secretase, we first show that as expected, this probe allows monitoring of an activity detectable in several cell systems including the neuronal cell line telencephalon specific murine neurons (TSM1). This activity is reduced by DFK167, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), and LY68458, three inhibitors known to functionally interact with PS. Interestingly, JMV2660 but not the unrelated peptide JMV2692, inhibits Abeta production in an in vitrogamma-secretase assay as expected from a putative substrate competitor. This activity is enhanced by PS1 and PS2 mutations known to be responsible for familial forms of AD and reduced by aspartyl mutations inactivating PS or in cells devoid of PS or NCT. However, we clearly establish that residual JMV2660-hydrolysing activity could be recovered in PS- and NCT-deficient fibroblasts and that this activity remained inhibited by DFK167. Overall, our study describes the presence of a proteolytic activity displaying gamma-secretase-like properties but independent of PS and still blocked by DFK167, suggesting that the PS-dependent complex could not be the unique gamma-secretase activity responsible for Abeta

Amyloid precursor protein and Presenilin 1 interaction studied by FRET in human H4 cells.

PubMed

Nizzari, Mario; Venezia, Valentina; Bianchini, Paolo; Caorsi, Valentina; Diaspro, Alberto; Repetto, Emanuela; Thellung, Stefano; Corsaro, Alessandro; Carlo, Pia; Schettini, Gennaro; Florio, Tullio; Russo, Claudio

2007-01-01

The mayor pathologic hallmarks of Alzheimer's disease (AD) are senile plaque and neurofibrillary tangles. Senile plaque are primarily made up of deposits of amyloid-beta protein, a proteolytic product derived from the amyloid precursor protein (APP). APP is a transmembrane protein detected into the endoplasmic reticulum, in the Golgi apparatus, at the cell surface, recycled by endocytosis to endosomes, whose physiological function is unclear. Presenilins (PS), are a component of gamma-secretase complex that cleave alpha-CTFs (carboxy-terminal fragment), or beta-CTFs, leaving 40 or 42 amino acids amyloid-beta peptides and 58 or 56 amino acids intracellular domains (AICD). Where the amyloid-beta peptides is generated is not clear. The study of APP-PS interaction in specific cell compartments provides a good opportunity to light upon the molecular mechanisms regulating the activity of the "gamma-secretase complex," and where beta-amyloid is generated. In our study we used a biophysical assay of protein proximity: fluorescence resonance energy transfer (FRET), that can provide information about molecular interactions when two proteins are in the close proximity (<10 nm), to examine the subcellular localization and interaction between APP and PS1 in human neuroglioma cells (H4). Confocal microscopic analysis reveals extensive colocalization in different cells' compartment, and centrosomal or microtubule organizing center (MTOC) localization of APP and PS1, but not necessarily a close molecular interaction. We used FRET to determine if APP and PS1 interact at the cell centrosome. FRET data suggest a close interaction between APP and PS1 in subcellular compartments and at the centrosome of H4 cells. Using this approach we show that APP and PS1 are closely associated in the centrosomes of the H4 cell.

The alexithymia, emotion regulation, emotion regulation difficulties, positive and negative affects, and suicidal risk in alcohol-dependent outpatients.

PubMed

Ghorbani, Fatemeh; Khosravani, Vahid; Sharifi Bastan, Farangis; Jamaati Ardakani, Razieh

2017-06-01

The aim of this study was to evaluate the potential contributing factors such as alexithymia, emotion regulation and difficulties in emotion regulation, positive/negative affects and clinical factors including severity of alcohol dependence and depression connected to high suicidality in alcohol-dependent outpatients. 205 alcohol-dependent outpatients and 100 normal controls completed the demographic questionnaire, the Persian version of the Toronto Alexithymia Scale (FTAS-20), the Difficulties in Emotion Regulation Scale (DERS), the Emotion Regulation Questionnaire (ERQ), the Positive/Negative Affect Scales, the Alcohol Use Disorders Identification Test (AUDIT), and the Beck Depression Inventory-II (BDI-II). The suicidal risk was assessed using the Scale for Suicide Ideation (SSI) and history taking. Alcohol-dependent outpatients showed higher means in alexithymia, difficulties in emotion regulation, suppression subscale, negative affect, and suicide ideation than normal controls. Logistic regression analysis revealed that negative affect, duration of alcohol use, externally-oriented thinking, and severity of alcohol dependence explained lifetime suicide attempts. Depression, impulsivity, severity of alcohol dependence, reappraisal (reversely), externally-oriented thinking, difficulties engaging in goal-directed behaviors, and negative affect significantly predicted the suicidal risk. The findings may constitute useful evidence of the relevancies of alexithymia, emotion regulation, emotion regulation difficulties, and affects to suicidality in alcoholic patients. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Timescales and bottlenecks in miRNA-dependent gene regulation.

PubMed

Hausser, Jean; Syed, Afzal Pasha; Selevsek, Nathalie; van Nimwegen, Erik; Jaskiewicz, Lukasz; Aebersold, Ruedi; Zavolan, Mihaela

2013-12-03

MiRNAs are post-transcriptional regulators that contribute to the establishment and maintenance of gene expression patterns. Although their biogenesis and decay appear to be under complex control, the implications of miRNA expression dynamics for the processes that they regulate are not well understood. We derived a mathematical model of miRNA-mediated gene regulation, inferred its parameters from experimental data sets, and found that the model describes well time-dependent changes in mRNA, protein and ribosome density levels measured upon miRNA transfection and induction. The inferred parameters indicate that the timescale of miRNA-dependent regulation is slower than initially thought. Delays in miRNA loading into Argonaute proteins and the slow decay of proteins relative to mRNAs can explain the typically small changes in protein levels observed upon miRNA transfection. For miRNAs to regulate protein expression on the timescale of a day, as miRNAs involved in cell-cycle regulation do, accelerated miRNA turnover is necessary.

Copper Regulates Cyclic AMP-Dependent Lipolysis

PubMed Central

Krishnamoorthy, Lakshmi; Cotruvo, Joseph A.; Chan, Jefferson; Kaluarachchi, Harini; Muchenditsi, Abigael; Pendyala, Venkata S.; Jia, Shang; Aron, Allegra T.; Ackerman, Cheri M.; Vander Wal, Mark N.; Guan, Timothy; Smaga, Lukas P.; Farhi, Samouil L.; New, Elizabeth J.; Lutsenko, Svetlana; Chang, Christopher J.

2016-01-01

Cell signaling relies extensively on dynamic pools of redox-inactive metal ions such as sodium, potassium, calcium, and zinc, but their redox-active transition metal counterparts such as copper and iron have been studied primarily as static enzyme cofactors. Here we report that copper is an endogenous regulator of lipolysis, the breakdown of fat, which is an essential process in maintaining the body's weight and energy stores. Utilizing a murine model of genetic copper misregulation, in combination with pharmacological alterations in copper status and imaging studies in a 3T3-L1 white adipocyte model, we demonstrate that copper regulates lipolysis at the level of the second messenger, cyclic AMP (cAMP), by altering the activity of the cAMP-degrading phosphodiesterase PDE3B. Biochemical studies of the copper-PDE3B interaction establish copper-dependent inhibition of enzyme activity and identify a key conserved cysteine residue within a PDE3-specific loop that is essential for the observed copper-dependent lipolytic phenotype. PMID:27272565

38 CFR 3.270 - Applicability of various dependency, income and estate regulations.

Code of Federal Regulations, 2012 CFR

2012-07-01

... dependency, income and estate regulations. 3.270 Section 3.270 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS ADJUDICATION Pension, Compensation, and Dependency and Indemnity Compensation Dependency, Income and Estate § 3.270 Applicability of various dependency, income and estate regulations. (a...

38 CFR 3.270 - Applicability of various dependency, income and estate regulations.

Code of Federal Regulations, 2011 CFR

2011-07-01

... dependency, income and estate regulations. 3.270 Section 3.270 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS ADJUDICATION Pension, Compensation, and Dependency and Indemnity Compensation Dependency, Income and Estate § 3.270 Applicability of various dependency, income and estate regulations. (a...

38 CFR 3.270 - Applicability of various dependency, income and estate regulations.

Code of Federal Regulations, 2010 CFR

2010-07-01

... dependency, income and estate regulations. 3.270 Section 3.270 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS ADJUDICATION Pension, Compensation, and Dependency and Indemnity Compensation Dependency, Income and Estate § 3.270 Applicability of various dependency, income and estate regulations. (a...

38 CFR 3.270 - Applicability of various dependency, income and estate regulations.

Code of Federal Regulations, 2013 CFR

2013-07-01

... dependency, income and estate regulations. 3.270 Section 3.270 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS ADJUDICATION Pension, Compensation, and Dependency and Indemnity Compensation Dependency, Income and Estate § 3.270 Applicability of various dependency, income and estate regulations. (a...

38 CFR 3.270 - Applicability of various dependency, income and estate regulations.

Code of Federal Regulations, 2014 CFR

2014-07-01

... dependency, income and estate regulations. 3.270 Section 3.270 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS ADJUDICATION Pension, Compensation, and Dependency and Indemnity Compensation Dependency, Income and Estate § 3.270 Applicability of various dependency, income and estate regulations. (a...

Ubiquitin-Dependent Degradation of Mitochondrial Proteins Regulates Energy Metabolism.

PubMed

Lavie, Julie; De Belvalet, Harmony; Sonon, Sessinou; Ion, Ana Madalina; Dumon, Elodie; Melser, Su; Lacombe, Didier; Dupuy, Jean-William; Lalou, Claude; Bénard, Giovanni

2018-06-05

The ubiquitin proteasome system (UPS) regulates many cellular functions by degrading key proteins. Notably, the role of UPS in regulating mitochondrial metabolic functions is unclear. Here, we show that ubiquitination occurs in different mitochondrial compartments, including the inner mitochondrial membrane, and that turnover of several metabolic proteins is UPS dependent. We specifically detailed mitochondrial ubiquitination and subsequent UPS-dependent degradation of succinate dehydrogenase subunit A (SDHA), which occurred when SDHA was minimally involved in mitochondrial energy metabolism. We demonstrate that SDHA ubiquitination occurs inside the organelle. In addition, we show that the specific inhibition of SDHA degradation by UPS promotes SDHA-dependent oxygen consumption and increases ATP, malate, and citrate levels. These findings suggest that the mitochondrial metabolic machinery is also regulated by the UPS. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

When rejection sensitivity matters: regulating dependence within daily interactions with family and friends.

PubMed

Overall, Nickola C; Sibley, Chris G

2009-08-01

This diary study examined situational dependence within daily interactions with family members and close friends over a 2-week period. Experiencing low personal control (high situational dependence) when interacting with family members and friends was associated with lower perceived regard and intimacy. Participants generally regulated felt dependence by derogating and withdrawing from their interaction partner (self-protective dependence regulation) and exhibiting lower levels of positive behavior, such as expressing thoughts and feelings or trying to improve the interaction (relationship-promotive dependence regulation). Furthermore, higher rejection sensitivity (but not low self-esteem) was associated with more negative evaluations of perceived regard and intimacy, greater self-protective dependence regulation, and lower relationship-promotive dependence regulation within low control interactions. These results identify dependence as a key element of rejection risk contexts and support the situation-specific nature of rejection sensitivity.

Cartilage-Specific and Cre-Dependent Nkx3.2 Overexpression In Vivo Causes Skeletal Dwarfism by Delaying Cartilage Hypertrophy.

PubMed

Jeong, Da-Un; Choi, Je-Yong; Kim, Dae-Won

2017-01-01

Nkx3.2, the vertebrate homologue of Drosophila bagpipe, has been implicated as playing a role in chondrogenic differentiation. In brief, Nkx3.2 is initially expressed in chondrocyte precursor cells and later during cartilage maturation, its expression is diminished in hypertrophic chondrocytes. In addition to Nkx3.2 expression analyses, previous studies using ex vivo chick embryo cultures and in vitro cell cultures have suggested that Nkx3.2 can suppress chondrocyte hypertrophy. However, it has never been demonstrated that Nkx3.2 functions in regulating chondrocyte hypertrophy during cartilage development in vivo. Here, we show that cartilage-specific and Cre-dependent Nkx3.2 overexpression in mice results in significant postnatal dwarfism in endochondral skeletons, while intramembranous bones remain unaltered. Further, we observed significant delays in cartilage hypertrophy in conditional transgenic ciTg-Nkx3.2 mice. Together, these findings confirm that Nkx3.2 is capable of controlling hypertrophic maturation of cartilage in vivo, and this regulation plays a significant role in endochondral ossification and longitudinal bone growth. J. Cell. Physiol. 232: 78-90, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Small systems and regulator dependence in relativistic hydrodynamics

NASA Astrophysics Data System (ADS)

Spaliński, Michał

2016-10-01

Consistent theories of hydrodynamics necessarily include nonhydrodynamic modes, which can be viewed as a regulator necessary to ensure causality. Under many circumstances the choice of regulator is not relevant, but this is not always the case. In particular, for sufficiently small systems (such as those arising in pA or pp collisions) such dependence may be inevitable. We address this issue in the context of the modern version of Müller-Israel-Stewart theory of relativistic hydrodynamics. In this case, by demanding that the nonhydrodynamic modes do not dominate, we find that regulator dependence becomes inevitable only for multiplicities d N /d Y of the order of a few. This conclusion supports earlier studies based on hydrodynamic simulations of small systems, at the same time providing a simple physical picture of how hydrodynamics can be reliable even in such seemingly extreme conditions.

Redox-dependent regulation of epidermal growth factor receptor signaling.

PubMed

Heppner, David E; van der Vliet, Albert

2016-08-01

Tyrosine phosphorylation-dependent cell signaling represents a unique feature of multicellular organisms, and is important in regulation of cell differentiation and specialized cell functions. Multicellular organisms also contain a diverse family of NADPH oxidases (NOXs) that have been closely linked with tyrosine kinase-based cell signaling and regulate tyrosine phosphorylation via reversible oxidation of cysteine residues that are highly conserved within many proteins involved in this signaling pathway. An example of redox-regulated tyrosine kinase signaling involves the epidermal growth factor receptor (EGFR), a widely studied receptor system with diverse functions in normal cell biology as well as pathologies associated with oxidative stress such as cancer. The purpose of this Graphical Redox Review is to highlight recently emerged concepts with respect to NOX-dependent regulation of this important signaling pathway. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Therapeutic impact of rHuEPO on abnormal platelet APP, BACE 1, presenilin 1, ADAM 10 and Aβ expressions in chronic kidney disease patients with cognitive dysfunction like Alzheimer's disease: A pilot study.

PubMed

G, Vinothkumar; S, Krishnakumar; Sureshkumar; G, Shivashekar; S, Sreedhar; Preethikrishnan; S, Dinesh; A, Sundaram; D, Balakrishnan; Riya; P, Venkataraman

2018-08-01

Cognitive dysfunction is reported to be a major cause of morbidity in chronic kidney disease (CKD). The senile plaques (SPs) in the brain are one of the most pathophysiological characteristics of cognitive dysfunction and its major constituent amyloid β (Aβ) released from amyloid precursor protein (APP) by β (BACE1) and γ (presenilin 1) secretases . Platelets contain more than 95% of the circulating APP and implicate as a candidate biomarker for cognitive decline. Recombinant human erythropoietin (rHuEPO) is a standard therapy for anemia in CKD and also acts as a neuroprotective agent. The aim of the study is to determine the impact of rHuEPO therapy on platelet APP processing in CKD with Cognitive Dysfunction. A total of 60 subjects comprising of 30 CKD without cognitive dysfunction and 30 CKD with cognitive dysfunction based on neuropsychological assessment. APP, BACE1, Presenilin 1, ADAM 10 (α secretase) and Aβ expressions in platelets were determined by western blotting and lipid peroxidation (LPO) in platelet rich plasma (PRP) was done by spectrophotometrically. The parameters were statistically compared with Alzheimer's disease (AD), Normocytic normochromic anemic and healthy subjects. Significantly (p < 0.05) decreased APP, ADAM 10 while increased BACE1, Presenilin 1, Aβ and LPO were observed in CKD with cognitive dysfunction like AD subjects compared to other groups. The parameters were reassessed in CKD with cognitive dysfunction subjects after rHuEPO (100 IU/ kg, weekly twice, 6 months) therapy. All the parameters were retrieved significantly (p < 0.05) along with improved neuropsychological tests scoring after rHuEPO therapy. This study demonstrated that rHuEPO is an effective neuroprotective agent in the context of CKD associated cognitive dysfunction and proved its clinical usefulness. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

APP intracellular domain derived from amyloidogenic β- and γ-secretase cleavage regulates neprilysin expression

PubMed Central

Grimm, Marcus O. W.; Mett, Janine; Stahlmann, Christoph P.; Grösgen, Sven; Haupenthal, Viola J.; Blümel, Tamara; Hundsdörfer, Benjamin; Zimmer, Valerie C.; Mylonas, Nadine T.; Tanila, Heikki; Müller, Ulrike; Grimm, Heike S.; Hartmann, Tobias

2015-01-01

Alzheimer's disease (AD) is characterized by an accumulation of Amyloid-β (Aβ), released by sequential proteolytic processing of the amyloid precursor protein (APP) by β - and γ-secretase. Aβ peptides can aggregate, leading to toxic Aβ oligomers and amyloid plaque formation. Aβ accumulation is not only dependent on de novo synthesis but also on Aβ degradation. Neprilysin (NEP) is one of the major enzymes involved in Aβ degradation. Here we investigate the molecular mechanism of NEP regulation, which is up to now controversially discussed to be affected by APP processing itself. We found that NEP expression is highly dependent on the APP intracellular domain (AICD), released by APP processing. Mouse embryonic fibroblasts devoid of APP processing, either by the lack of the catalytically active subunit of the γ-secretase complex [presenilin (PS) 1/2] or by the lack of APP and the APP-like protein 2 (APLP2), showed a decreased NEP expression, activity and protein level. Similar results were obtained by utilizing cells lacking a functional AICD domain (APPΔCT15) or expressing mutations in the genes encoding for PS1. AICD supplementation or retransfection with an AICD encoding plasmid could rescue the down-regulation of NEP further strengthening the link between AICD and transcriptional NEP regulation, in which Fe65 acts as an important adaptor protein. Especially AICD generated by the amyloidogenic pathway seems to be more involved in the regulation of NEP expression. In line, analysis of NEP gene expression in vivo in six transgenic AD mouse models (APP and APLP2 single knock-outs, APP/APLP2 double knock-out, APP-swedish, APP-swedish/PS1Δexon9, and APPΔCT15) confirmed the results obtained in cell culture. In summary, in the present study we clearly demonstrate an AICD-dependent regulation of the Aβ-degrading enzyme NEP in vitro and in vivo and elucidate the underlying mechanisms that might be beneficial to develop new therapeutic strategies for the

Wild-type presenilin 1 protects against Alzheimer disease mutation-induced amyloid pathology.

PubMed

Wang, Runsheng; Wang, Baiping; He, Wanxia; Zheng, Hui

2006-06-02

Mutations in presenilin 1 (PS1) lead to dominant inheritance of early onset familial Alzheimer disease (FAD). These mutations are known to alter the gamma-secretase cleavage of the amyloid precursor protein, resulting in increased ratio of Abeta42/Abeta40 and accelerated amyloid plaque pathology in transgenic mouse models. To investigate the factors that drive the Abeta42/Abeta40 ratio and amyloid pathogenesis and to investigate the possible interactions between wild-type and FAD mutant PS1, which are co-expressed in transgenic animals, we expressed the PS1 M146V knock-in allele either on wild-type PS1 (PS1M146V/+) or PS1 null (PS1M146V/-) background and crossed these alleles with the Tg2576 APP transgenic mice. Introduction of the PS1 M146V mutation on Tg2576 background resulted in earlier onset of plaque pathology. Surprisingly, removing the wild-type PS1 in the presence of the PS1 M146V mutation (PS1M146V/-) greatly exacerbated the amyloid burden; and this was attributed to a reduction of gamma-secretase activity rather than an increase in Abeta42. Our findings establish a protective role of the wild-type PS1 against the FAD mutation-induced amyloid pathology through a partial loss-of-function mechanism.

Mechanism of Intramembrane Cleavage of Alcadeins by γ-Secretase

PubMed Central

Piao, Yi; Kimura, Ayano; Urano, Satomi; Saito, Yuhki; Taru, Hidenori; Yamamoto, Tohru; Hata, Saori; Suzuki, Toshiharu

2013-01-01

Background Alcadein proteins (Alcs; Alcα, Alcβand Alcγ) are predominantly expressed in neurons, as is Alzheimer's β-amyloid (Aβ) precursor protein (APP). Both Alcs and APP are cleaved by primary α- or β-secretase to generate membrane-associated C-terminal fragments (CTFs). Alc CTFs are further cleaved by γ-secretase to secrete p3-Alc peptide along with the release of intracellular domain fragment (Alc ICD) from the membrane. In the case of APP, APP CTFβ is initially cleaved at the ε-site to release the intracellular domain fragment (AICD) and consequently the γ-site is determined, by which Aβ generates. The initial ε-site is thought to define the final γ-site position, which determines whether Aβ40/43 or Aβ42 is generated. However, initial intracellular ε-cleavage sites of Alc CTF to generate Alc ICD and the molecular mechanism that final γ-site position is determined remains unclear in Alcs. Methodology Using HEK293 cells expressing Alcs plus presenilin 1 (PS1, a catalytic unit of γ-secretase) and the membrane fractions of these cells, the generation of p3-Alc possessing C-terminal γ-cleavage site and Alc ICD possessing N-terminal ε-cleavage site were analysed with MALDI-TOF/MS. We determined the initial ε-site position of all Alcα, Alcβ and Alcγ, and analyzed the relationship between the initially determined ε-site position and the final γ-cleavage position. Conclusions The initial ε-site position does not always determine the final γ-cleavage position in Alcs, which differed from APP. No additional γ-cleavage sites are generated from artificial/non-physiological positions of ε-cleavage for Alcs, while the artificial ε-cleavage positions can influence in selection of physiological γ-site positions. Because alteration of γ-secretase activity is thought to be a pathogenesis of sporadic Alzheimer's disease, Alcs are useful and sensitive substrate to detect the altered cleavage of substrates by γ-secretase, which may be induced by

Presenilin E318G variant and Alzheimer's disease risk: the Cache County study.

PubMed

Hippen, Ariel A; Ebbert, Mark T W; Norton, Maria C; Tschanz, JoAnn T; Munger, Ronald G; Corcoran, Christopher D; Kauwe, John S K

2016-06-29

Alzheimer's disease is the leading cause of dementia in the elderly and the third most common cause of death in the United States. A vast number of genes regulate Alzheimer's disease, including Presenilin 1 (PSEN1). Multiple studies have attempted to locate novel variants in the PSEN1 gene that affect Alzheimer's disease status. A recent study suggested that one of these variants, PSEN1 E318G (rs17125721), significantly affects Alzheimer's disease status in a large case-control dataset, particularly in connection with the APOEε4 allele. Our study looks at the same variant in the Cache County Study on Memory and Aging, a large population-based dataset. We tested for association between E318G genotype and Alzheimer's disease status by running a series of Fisher's exact tests. We also performed logistic regression to test for an additive effect of E318G genotype on Alzheimer's disease status and for the existence of an interaction between E318G and APOEε4. In our Fisher's exact test, it appeared that APOEε4 carriers with an E318G allele have slightly higher risk for AD than those without the allele (3.3 vs. 3.8); however, the 95 % confidence intervals of those estimates overlapped completely, indicating non-significance. Our logistic regression model found a positive but non-significant main effect for E318G (p = 0.895). The interaction term between E318G and APOEε4 was also non-significant (p = 0.689). Our findings do not provide significant support for E318G as a risk factor for AD in APOEε4 carriers. Our calculations indicated that the overall sample used in the logistic regression models was adequately powered to detect the sort of effect sizes observed previously. However, the power analyses of our Fisher's exact tests indicate that our partitioned data was underpowered, particularly in regards to the low number of E318G carriers, both AD cases and controls, in the Cache county dataset. Thus, the differences in types of datasets used may help to

Metabolic-flux dependent regulation of microbial physiology.

PubMed

Litsios, Athanasios; Ortega, Álvaro D; Wit, Ernst C; Heinemann, Matthias

2018-04-01

According to the most prevalent notion, changes in cellular physiology primarily occur in response to altered environmental conditions. Yet, recent studies have shown that changes in metabolic fluxes can also trigger phenotypic changes even when environmental conditions are unchanged. This suggests that cells have mechanisms in place to assess the magnitude of metabolic fluxes, that is, the rate of metabolic reactions, and use this information to regulate their physiology. In this review, we describe recent evidence for metabolic flux-sensing and flux-dependent regulation. Furthermore, we discuss how such sensing and regulation can be mechanistically achieved and present a set of new candidates for flux-signaling metabolites. Similar to metabolic-flux sensing, we argue that cells can also sense protein translation flux. Finally, we elaborate on the advantages that flux-based regulation can confer to cells. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Subjects harboring presenilin familial Alzheimer’s disease mutations exhibit diverse white matter biochemistry alterations

PubMed Central

Roher, Alex E; Maarouf, Chera L; Malek-Ahmadi, Michael; Wilson, Jeffrey; Kokjohn, Tyler A; Daugs, Ian D; Whiteside, Charisse M; Kalback, Walter M; Macias, MiMi P; Jacobson, Sandra A; Sabbagh, Marwan N; Ghetti, Bernardino; Beach, Thomas G

2013-01-01

Alzheimer’s disease (AD) dementia impacts all facets of higher order cognitive function and is characterized by the presence of distinctive pathological lesions in the gray matter (GM). The profound alterations in GM structure and function have fostered the view that AD impacts are primarily a consequence of GM damage. However, the white matter (WM) represents about 50% of the cerebrum and this area of the brain is substantially atrophied and profoundly abnormal in both sporadic AD (SAD) and familial AD (FAD). We examined the WM biochemistry by ELISA and Western blot analyses of key proteins in 10 FAD cases harboring mutations in the presenilin genes PSEN1 and PSEN2 as well as in 4 non-demented control (NDC) individuals and 4 subjects with SAD. The molecules examined were direct substrates of PSEN1 such as Notch-1 and amyloid precursor protein (APP). In addition, apolipoproteins, axonal transport molecules, cytoskeletal and structural proteins, neurotrophic factors and synaptic proteins were examined. PSEN-FAD subjects had, on average, higher amounts of WM amyloid-beta (Aβ) peptides compared to SAD, which may play a role in the devastating dysfunction of the brain. However, the PSEN-FAD mutations we examined did not produce uniform increases in the relative proportions of Aβ42 and exhibited substantial variability in total Aβ levels. These observations suggest that neurodegeneration and dementia do not depend solely on enhanced Aβ42 levels. Our data revealed additional complexities in PSEN-FAD individuals. Some direct substrates of γ-secretase, such as Notch, N-cadherin, Erb-B4 and APP, deviated substantially from the NDC group baseline for some, but not all, mutation types. Proteins that were not direct γ-secretase substrates, but play key structural and functional roles in the WM, likewise exhibited varied concentrations in the distinct PSEN mutation backgrounds. Detailing the diverse biochemical pathology spectrum of PSEN mutations may offer valuable

Microtubule-dependent regulation of mitotic protein degradation

PubMed Central

Song, Ling; Craney, Allison; Rape, Michael

2014-01-01

Accurate cell division depends on tightly regulated ubiquitylation events catalyzed by the anaphase-promoting complex. Among its many substrates, the APC/C triggers the degradation of proteins that stabilize the mitotic spindle, and loss or accumulation of such spindle assembly factors can result in aneuploidy and cancer. Although critical for cell division, it has remained poorly understood how the timing of spindle assembly factor degradation is established during mitosis. Here, we report that active spindle assembly factors are protected from APC/C-dependent degradation by microtubules. In contrast, those molecules that are not bound to microtubules are highly susceptible to proteolysis and turned over immediately after APC/C-activation. The correct timing of spindle assembly factor degradation, as achieved by this regulatory circuit, is required for accurate spindle structure and function. We propose that the localized stabilization of APC/C-substrates provides a mechanism for the selective disposal of cell cycle regulators that have fulfilled their mitotic roles. PMID:24462202

Cdk5 Regulates Activity-Dependent Gene Expression and Dendrite Development.

PubMed

Liang, Zhuoyi; Ye, Tao; Zhou, Xiaopu; Lai, Kwok-On; Fu, Amy K Y; Ip, Nancy Y

2015-11-11

The proper growth and arborization of dendrites in response to sensory experience are essential for neural connectivity and information processing in the brain. Although neuronal activity is important for sculpting dendrite morphology, the underlying molecular mechanisms are not well understood. Here, we report that cyclin-dependent kinase 5 (Cdk5)-mediated transcriptional regulation is a key mechanism that controls activity-dependent dendrite development in cultured rat neurons. During membrane depolarization, Cdk5 accumulates in the nucleus to regulate the expression of a subset of genes, including that of the neurotrophin brain-derived neurotrophic factor, for subsequent dendritic growth. Furthermore, Cdk5 function is mediated through the phosphorylation of methyl-CpG-binding protein 2, a key transcriptional repressor that is mutated in the mental disorder Rett syndrome. These findings collectively suggest that the nuclear import of Cdk5 is crucial for activity-dependent dendrite development by regulating neuronal gene transcription during neural development. Neural activity directs dendrite development through the regulation of gene transcription. However, how molecular signals link extracellular stimuli to the transcriptional program in the nucleus remains unclear. Here, we demonstrate that neuronal activity stimulates the translocation of the kinase Cdk5 from the cytoplasmic compartment into the nucleus; furthermore, the nuclear localization of Cdk5 is required for dendrite development in cultured neurons. Genome-wide transcriptome analysis shows that Cdk5 deficiency specifically disrupts activity-dependent gene transcription of bdnf. The action of Cdk5 is mediated through the modulation of the transcriptional repressor methyl-CpG-binding protein 2. Therefore, this study elucidates the role of nuclear Cdk5 in the regulation of activity-dependent gene transcription and dendritic growth. Copyright © 2015 the authors 0270-6474/15/3515127-08$15.00/0.

Presenilin 1 mutation decreases both calcium and contractile responses in cerebral arteries.

PubMed

Toussay, Xavier; Morel, Jean-Luc; Biendon, Nathalie; Rotureau, Lolita; Legeron, François-Pierre; Boutonnet, Marie-Charlotte; Cho, Yoon H; Macrez, Nathalie

2017-10-01

Mutations or upregulation in presenilin 1 (PS1) gene are found in familial early-onset Alzheimer's disease or sporadic late-onset Alzheimer's disease, respectively. PS1 has been essentially studied in neurons and its mutation was shown to alter intracellular calcium (Ca 2+ ) signals. Here, we showed that PS1 is expressed in smooth muscle cells (SMCs) of mouse cerebral arteries, and we assessed the effects of the deletion of exon 9 of PS1 (PS1dE9) on Ca 2+ signals and contractile responses of vascular SMC. Agonist-induced contraction of cerebral vessels was significantly decreased in PS1dE9 both in vivo and ex vivo. Spontaneous activity of Ca 2+ sparks through ryanodine-sensitive channels (RyR) was unchanged, whereas the RyR-mediated Ca 2+ -release activated by caffeine was shorter in PS1dE9 SMC when compared with control. Moreover, PS1dE9 mutation decreased the caffeine-activated capacitive Ca 2+ entry, and inhibitors of SERCA pumps reversed the effects of PS1dE9 on Ca 2+ signals. PS1dE9 mutation also leads to the increased expression of SERCA3, phospholamban, and RyR3. These results show that PS1 plays a crucial role in the cerebrovascular system and the vascular reactivity is decreased through altered Ca 2+ signals in PS1dE9 mutant mice. Copyright © 2017 Elsevier Inc. All rights reserved.

The zinc ionophore clioquinol reverses autophagy arrest in chloroquine-treated ARPE-19 cells and in APP/mutant presenilin-1-transfected Chinese hamster ovary cells.

PubMed

Seo, Bo-Ra; Lee, Sook-Jeong; Cho, Kyung Sook; Yoon, Young Hee; Koh, Jae-Young

2015-12-01

Arrested autophagy may contribute to the pathogenesis of Alzheimer's disease. Because we found that chloroquine (CQ) causes arrested autophagy but clioquinol (ClioQ), a zinc ionophore, activates autophagic flux, in the present study, we examined whether ClioQ can overcome arrested autophagy induced by CQ or mutant presenilin-1 (mPS1). CQ induced vacuole formation and cell death in adult retinal pigment epithelial (ARPE-19) cells, but co-treatment with ClioQ attenuated CQ-associated toxicity in a zinc-dependent manner. Increases in lysosome dilation and blockage of autophagic flux by CQ were also markedly attenuated by ClioQ treatment. Interestingly, CQ increased lysosomal pH in amyloid precursor protein (APP)/mPS1-expressing Chinese hamster ovary 7WΔE9 (CHO-7WΔE9) cell line, and ClioQ partially re-acidified lysosomes. Furthermore, accumulation of amyloid-β (Aβ) oligomers in CHO-7WΔE9 cells was markedly attenuated by ClioQ. Moreover, intracellular accumulation of exogenously applied fluorescein isothiocyanate-conjugated Aβ(1-42) was also increased by CQ but was returned to control levels by ClioQ. These results suggest that modulation of lysosomal functions by manipulating lysosomal zinc levels may be a useful strategy for clearing intracellular Aβ oligomers. Copyright © 2015 Elsevier Inc. All rights reserved.

Cell fate determination by ubiquitin-dependent regulation of translation

PubMed Central

Werner, Achim; Iwasaki, Shintaro; McGourty, Colleen; Medina-Ruiz, Sofia; Teerikorpi, Nia; Fedrigo, Indro; Ingolia, Nicholas T.; Rape, Michael

2015-01-01

Metazoan development depends on accurate execution of differentiation programs that allow pluripotent stem cells to adopt specific fates 1. Differentiation requires changes to chromatin architecture and transcriptional networks, yet whether other regulatory events support cell fate determination is less well understood. Here, we have identified the vertebrate-specific ubiquitin ligase CUL3KBTBD8 as an essential regulator of neural crest specification. CUL3KBTBD8 monoubiquitylates NOLC1 and its paralog TCOF1, whose mutation underlies the neurocristopathy Treacher Collins Syndrome 2,3. Ubiquitylation drives formation of a TCOF1-NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodels the translational program of differentiating cells in favor of neural crest specification. We conclude that ubiquitin-dependent regulation of translation is an important feature of cell fate determination. PMID:26399832

An atomic structure of human γ-secretase

NASA Astrophysics Data System (ADS)

Bai, Xiao-Chen; Yan, Chuangye; Yang, Guanghui; Lu, Peilong; Ma, Dan; Sun, Linfeng; Zhou, Rui; Scheres, Sjors H. W.; Shi, Yigong

2015-09-01

Dysfunction of the intramembrane protease γ-secretase is thought to cause Alzheimer's disease, with most mutations derived from Alzheimer's disease mapping to the catalytic subunit presenilin 1 (PS1). Here we report an atomic structure of human γ-secretase at 3.4 Å resolution, determined by single-particle cryo-electron microscopy. Mutations derived from Alzheimer's disease affect residues at two hotspots in PS1, each located at the centre of a distinct four transmembrane segment (TM) bundle. TM2 and, to a lesser extent, TM6 exhibit considerable flexibility, yielding a plastic active site and adaptable surrounding elements. The active site of PS1 is accessible from the convex side of the TM horseshoe, suggesting considerable conformational changes in nicastrin extracellular domain after substrate recruitment. Component protein APH-1 serves as a scaffold, anchoring the lone transmembrane helix from nicastrin and supporting the flexible conformation of PS1. Ordered phospholipids stabilize the complex inside the membrane. Our structure serves as a molecular basis for mechanistic understanding of γ-secretase function.

Complex Formed between Intramembrane Metalloprotease SpoIVFB and Its Substrate, Pro-σK*

PubMed Central

Zhang, Yang; Halder, Sabyasachi; Kerr, Richard A.; Parrell, Daniel; Ruotolo, Brandon; Kroos, Lee

2016-01-01

Intramembrane metalloproteases (IMMPs) are conserved from bacteria to humans and control many important signaling pathways, but little is known about how IMMPs interact with their substrates. SpoIVFB is an IMMP that cleaves Pro-σK during Bacillus subtilis endospore formation. When catalytically inactive SpoIVFB was coexpressed with C-terminally truncated Pro-σK(1–126) (which can be cleaved by active SpoIVFB) in Escherichia coli, the substrate dramatically improved solubilization of the enzyme from membranes with mild detergents. Both the Pro(1–20) and σK(21–126) parts contributed to improving SpoIVFB solubilization from membranes, but only the σK part was needed to form a stable complex with SpoIVFB in a pulldown assay. The last 10 residues of SpoIVFB were required for improved solubilization from membranes by Pro-σK(1–126) and for normal interaction with the substrate. The inactive SpoIVFB·Pro-σK(1–126)-His6 complex was stable during affinity purification and gel filtration chromatography. Disulfide cross-linking of the purified complex indicated that it resembled the complex formed in vivo. Ion mobility-mass spectrometry analysis resulted in an observed mass consistent with a 4:2 SpoIVFB·Pro-σK(1–126)-His6 complex. Stepwise photobleaching of SpoIVFB fused to a fluorescent protein supported the notion that the enzyme is tetrameric during B. subtilis sporulation. The results provide the first evidence that an IMMP acts as a tetramer, give new insights into how SpoIVFB interacts with its substrate, and lay the foundation for further biochemical analysis of the enzyme·substrate complex and future structural studies. PMID:26953342

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

Frampton, Gabriel; Coufal, Monique; Li, Huang

The endocannabinoids anandamide (AEA) and 2-arachidonylglycerol (2-AG) have opposing effects on cholangiocarcinoma growth. Implicated in cancer, Notch signaling requires the {gamma}-secretase complex for activation. The aims of this study were to determine if the opposing effects of endocannabinoids depend on the differential activation of the Notch receptors and to demonstrate that the differential activation of these receptors are due to presenilin 1 containing- and presenilin 2 containing-{gamma}-secretase complexes. Mz-ChA-1 cells were treated with AEA or 2-AG. Notch receptor expression, activation, and nuclear translocation were determined. Specific roles for Notch 1 and 2 on cannabinoid-induced effects were determined by transient transfectionmore » of Notch 1 or 2 shRNA vectors before stimulation with AEA or 2-AG. Expression of presenilin 1 and 2 was determined after AEA or 2-AG treatment, and the involvement of presenilin 1 and 2 in the cannabinoid-induced effects was demonstrated in cell lines with low presenilin 1 or 2 expression. Antiproliferative effects of AEA required increased Notch 1 mRNA, activation, and nuclear translocation, whereas the growth-promoting effects induced by 2-AG required increased Notch 2 mRNA expression, activation, and nuclear translocation. AEA increased presenilin 1 expression and recruitment into the {gamma}-secretase complex, whereas 2-AG increased expression and recruitment of presenilin 2. The development of novel therapeutic strategies aimed at modulating the endocannabinoid system or mimicking the mode of action of AEA on Notch signaling pathways would prove beneficial for cholangiocarcinoma management.« less

TRANSPARENT TESTA GLABRA 1-Dependent Regulation of Flavonoid Biosynthesis

PubMed Central

Zhang, Bipei

2017-01-01

The flavonoid composition of various tissues throughout plant development is of biological relevance and particular interest for breeding. Arabidopsis thaliana TRANSPARENT TESTA GLABRA 1 (AtTTG1) is an essential regulator of late structural genes in flavonoid biosynthesis. Here, we provide a review of the regulation of the pathway’s core enzymes through AtTTG1-containing R2R3-MYELOBLASTOSIS-basic HELIX-LOOP-HELIX-WD40 repeat (MBW(AtTTG1)) complexes embedded in an evolutionary context. We present a comprehensive collection of A. thaliana ttg1 mutants and AtTTG1 orthologs. A plethora of MBW(AtTTG1) mechanisms in regulating the five major TTG1-dependent traits is highlighted. PMID:29261137

Transport, metabolism, and endosomal trafficking-dependent regulation of intestinal fructose absorption

PubMed Central

Patel, Chirag; Douard, Veronique; Yu, Shiyan; Gao, Nan; Ferraris, Ronaldo P.

2015-01-01

Dietary fructose that is linked to metabolic abnormalities can up-regulate its own absorption, but the underlying regulatory mechanisms are not known. We hypothesized that glucose transporter (GLUT) protein, member 5 (GLUT5) is the primary fructose transporter and that fructose absorption via GLUT5, metabolism via ketohexokinase (KHK), as well as GLUT5 trafficking to the apical membrane via the Ras-related protein-in-brain 11 (Rab11)a-dependent endosomes are each required for regulation. Introducing fructose but not lysine and glucose solutions into the lumen increased by 2- to 10-fold the heterogeneous nuclear RNA, mRNA, protein, and activity levels of GLUT5 in adult wild-type mice consuming chow. Levels of GLUT5 were >100-fold that of candidate apical fructose transporters GLUTs 7, 8, and 12 whose expression, and that of GLUT 2 and the sodium-dependent glucose transporter protein 1 (SGLT1), was not regulated by luminal fructose. GLUT5-knockout (KO) mice exhibited no facilitative fructose transport and no compensatory increases in activity and expression of SGLT1 and other GLUTs. Fructose could not up-regulate GLUT5 in GLUT5-KO, KHK-KO, and intestinal epithelial cell-specific Rab11a-KO mice. The fructose-specific metabolite glyceraldehyde did not increase GLUT5 expression. GLUT5 is the primary transporter responsible for facilitative absorption of fructose, and its regulation specifically requires fructose uptake and metabolism and normal GLUT5 trafficking to the apical membrane.—Patel, C., Douard, V., Yu, S., Gao, N., Ferraris, R. P. Transport, metabolism, and endosomal trafficking-dependent regulation of intestinal fructose absorption. PMID:26071406

Exact time-dependent solutions for a self-regulating gene.

PubMed

Ramos, A F; Innocentini, G C P; Hornos, J E M

2011-06-01

The exact time-dependent solution for the stochastic equations governing the behavior of a binary self-regulating gene is presented. Using the generating function technique to rephrase the master equations in terms of partial differential equations, we show that the model is totally integrable and the analytical solutions are the celebrated confluent Heun functions. Self-regulation plays a major role in the control of gene expression, and it is remarkable that such a microscopic model is completely integrable in terms of well-known complex functions.

Cell-fate determination by ubiquitin-dependent regulation of translation.

PubMed

Werner, Achim; Iwasaki, Shintaro; McGourty, Colleen A; Medina-Ruiz, Sofia; Teerikorpi, Nia; Fedrigo, Indro; Ingolia, Nicholas T; Rape, Michael

2015-09-24

Metazoan development depends on the accurate execution of differentiation programs that allow pluripotent stem cells to adopt specific fates. Differentiation requires changes to chromatin architecture and transcriptional networks, yet whether other regulatory events support cell-fate determination is less well understood. Here we identify the ubiquitin ligase CUL3 in complex with its vertebrate-specific substrate adaptor KBTBD8 (CUL3(KBTBD8)) as an essential regulator of human and Xenopus tropicalis neural crest specification. CUL3(KBTBD8) monoubiquitylates NOLC1 and its paralogue TCOF1, the mutation of which underlies the neurocristopathy Treacher Collins syndrome. Ubiquitylation drives formation of a TCOF1-NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodels the translational program of differentiating cells in favour of neural crest specification. We conclude that ubiquitin-dependent regulation of translation is an important feature of cell-fate determination.

Curcumin and derivatives function through protein phosphatase 2A and presenilin orthologues in Dictyostelium discoideum

PubMed Central

Cocorocchio, Marco; Baldwin, Amy J.; Stewart, Balint; Kim, Lou; Harwood, Adrian J.; Thompson, Christopher R. L.; Andrews, Paul L. R.

2018-01-01

ABSTRACT Natural compounds often have complex molecular structures and unknown molecular targets. These characteristics make them difficult to analyse using a classical pharmacological approach. Curcumin, the main curcuminoid of turmeric, is a complex molecule possessing wide-ranging biological activities, cellular mechanisms and roles in potential therapeutic treatment, including Alzheimer's disease and cancer. Here, we investigate the physiological effects and molecular targets of curcumin in Dictyostelium discoideum. We show that curcumin exerts acute effects on cell behaviour, reduces cell growth and slows multicellular development. We employed a range of structurally related compounds to show the distinct role of different structural groups in curcumin's effects on cell behaviour, growth and development, highlighting active moieties in cell function, and showing that these cellular effects are unrelated to the well-known antioxidant activity of curcumin. Molecular mechanisms underlying the effect of curcumin and one synthetic analogue (EF24) were then investigated to identify a curcumin-resistant mutant lacking the protein phosphatase 2A regulatory subunit (PsrA) and an EF24-resistant mutant lacking the presenilin 1 orthologue (PsenB). Using in silico docking analysis, we then showed that curcumin might function through direct binding to a key regulatory region of PsrA. These findings reveal novel cellular and molecular mechanisms for the function of curcumin and related compounds. PMID:29361519

Curcumin and derivatives function through protein phosphatase 2A and presenilin orthologues in Dictyostelium discoideum.

PubMed

Cocorocchio, Marco; Baldwin, Amy J; Stewart, Balint; Kim, Lou; Harwood, Adrian J; Thompson, Christopher R L; Andrews, Paul L R; Williams, Robin S B

2018-01-29

Natural compounds often have complex molecular structures and unknown molecular targets. These characteristics make them difficult to analyse using a classical pharmacological approach. Curcumin, the main curcuminoid of turmeric, is a complex molecule possessing wide-ranging biological activities, cellular mechanisms and roles in potential therapeutic treatment, including Alzheimer's disease and cancer. Here, we investigate the physiological effects and molecular targets of curcumin in Dictyostelium discoideum We show that curcumin exerts acute effects on cell behaviour, reduces cell growth and slows multicellular development. We employed a range of structurally related compounds to show the distinct role of different structural groups in curcumin's effects on cell behaviour, growth and development, highlighting active moieties in cell function, and showing that these cellular effects are unrelated to the well-known antioxidant activity of curcumin. Molecular mechanisms underlying the effect of curcumin and one synthetic analogue (EF24) were then investigated to identify a curcumin-resistant mutant lacking the protein phosphatase 2A regulatory subunit (PsrA) and an EF24-resistant mutant lacking the presenilin 1 orthologue (PsenB). Using in silico docking analysis, we then showed that curcumin might function through direct binding to a key regulatory region of PsrA. These findings reveal novel cellular and molecular mechanisms for the function of curcumin and related compounds. © 2018. Published by The Company of Biologists Ltd.

Activity-Dependent Bidirectional Regulation of GAD Expression in a Homeostatic Fashion Is Mediated by BDNF-Dependent and Independent Pathways

PubMed Central

Hanno-Iijima, Yoko; Tanaka, Masami; Iijima, Takatoshi

2015-01-01

Homeostatic synaptic plasticity, or synaptic scaling, is a mechanism that tunes neuronal transmission to compensate for prolonged, excessive changes in neuronal activity. Both excitatory and inhibitory neurons undergo homeostatic changes based on synaptic transmission strength, which could effectively contribute to a fine-tuning of circuit activity. However, gene regulation that underlies homeostatic synaptic plasticity in GABAergic (GABA, gamma aminobutyric) neurons is still poorly understood. The present study demonstrated activity-dependent dynamic scaling in which NMDA-R (N-methyl-D-aspartic acid receptor) activity regulated the expression of GABA synthetic enzymes: glutamic acid decarboxylase 65 and 67 (GAD65 and GAD67). Results revealed that activity-regulated BDNF (brain-derived neurotrophic factor) release is necessary, but not sufficient, for activity-dependent up-scaling of these GAD isoforms. Bidirectional forms of activity-dependent GAD expression require both BDNF-dependent and BDNF-independent pathways, both triggered by NMDA-R activity. Additional results indicated that these two GAD genes differ in their responsiveness to chronic changes in neuronal activity, which could be partially caused by differential dependence on BDNF. In parallel to activity-dependent bidirectional scaling in GAD expression, the present study further observed that a chronic change in neuronal activity leads to an alteration in neurotransmitter release from GABAergic neurons in a homeostatic, bidirectional fashion. Therefore, the differential expression of GAD65 and 67 during prolonged changes in neuronal activity may be implicated in some aspects of bidirectional homeostatic plasticity within mature GABAergic presynapses. PMID:26241953

Activity-Dependent Bidirectional Regulation of GAD Expression in a Homeostatic Fashion Is Mediated by BDNF-Dependent and Independent Pathways.

PubMed

Hanno-Iijima, Yoko; Tanaka, Masami; Iijima, Takatoshi

2015-01-01

Homeostatic synaptic plasticity, or synaptic scaling, is a mechanism that tunes neuronal transmission to compensate for prolonged, excessive changes in neuronal activity. Both excitatory and inhibitory neurons undergo homeostatic changes based on synaptic transmission strength, which could effectively contribute to a fine-tuning of circuit activity. However, gene regulation that underlies homeostatic synaptic plasticity in GABAergic (GABA, gamma aminobutyric) neurons is still poorly understood. The present study demonstrated activity-dependent dynamic scaling in which NMDA-R (N-methyl-D-aspartic acid receptor) activity regulated the expression of GABA synthetic enzymes: glutamic acid decarboxylase 65 and 67 (GAD65 and GAD67). Results revealed that activity-regulated BDNF (brain-derived neurotrophic factor) release is necessary, but not sufficient, for activity-dependent up-scaling of these GAD isoforms. Bidirectional forms of activity-dependent GAD expression require both BDNF-dependent and BDNF-independent pathways, both triggered by NMDA-R activity. Additional results indicated that these two GAD genes differ in their responsiveness to chronic changes in neuronal activity, which could be partially caused by differential dependence on BDNF. In parallel to activity-dependent bidirectional scaling in GAD expression, the present study further observed that a chronic change in neuronal activity leads to an alteration in neurotransmitter release from GABAergic neurons in a homeostatic, bidirectional fashion. Therefore, the differential expression of GAD65 and 67 during prolonged changes in neuronal activity may be implicated in some aspects of bidirectional homeostatic plasticity within mature GABAergic presynapses.

TRC8-dependent degradation of hepatitis C virus immature core protein regulates viral propagation and pathogenesis

PubMed Central

Aizawa, Sayaka; Okamoto, Toru; Sugiyama, Yukari; Kouwaki, Takahisa; Ito, Ayano; Suzuki, Tatsuya; Ono, Chikako; Fukuhara, Takasuke; Yamamoto, Masahiro; Okochi, Masayasu; Hiraga, Nobuhiko; Imamura, Michio; Chayama, Kazuaki; Suzuki, Ryosuke; Shoji, Ikuo; Moriishi, Kohji; Moriya, Kyoji; Koike, Kazuhiko; Matsuura, Yoshiharu

2016-01-01

Signal-peptide peptidase (SPP) is an intramembrane protease that participates in the production of the mature core protein of hepatitis C virus (HCV). Here we show that SPP inhibition reduces the production of infectious HCV particles and pathogenesis. The immature core protein produced in SPP-knockout cells or by treatment with an SPP inhibitor is quickly degraded by the ubiquitin–proteasome pathway. Oral administration of the SPP inhibitor to transgenic mice expressing HCV core protein (CoreTg) reduces the expression of core protein and ameliorates insulin resistance and liver steatosis. Moreover, the haploinsufficiency of SPP in CoreTg has similar effects. TRC8, an E3 ubiquitin ligase, is required for the degradation of the immature core protein. The expression of the HCV core protein alters endoplasmic reticulum (ER) distribution and induces ER stress in SPP/TRC8 double-knockout cells. These data suggest that HCV utilizes SPP cleavage to circumvent the induction of ER stress in host cells. PMID:27142248

Bmp2 conditional knockout in osteoblasts and endothelial cells does not impair bone formation after injury or mechanical loading in adult mice

PubMed Central

McKenzie, Jennifer A.; Buettmann, Evan G.; Gardner, Michael J.; Silva, Matthew J.

2015-01-01

Post-natal osteogenesis after mechanical trauma or stimulus occurs through either endochondral healing, intramembranous healing or lamellar bone formation. Bone morphogenetic protein 2 (BMP2) is up-regulated in each of these osteogenic processes and is expressed by a variety of cells including osteoblasts and vascular cells. It is known that genetic knockout of Bmp2 in all cells or in osteo-chondroprogenitor cells completely abrogates endochondral healing after full fracture. However, the importance of BMP2 from differentiated osteoblasts and endothelial cells is not known. Moreover, the importance of BMP2 in non-endochondral bone formation such as intramembranous healing or lamellar bone formation is not known. Using inducible and tissue-specific Cre-lox mediated targeting of Bmp2 in adult (10–24 week old) mice, we assessed the role of BMP2 expression globally, by osteoblasts, and by vascular endothelial cells in endochondral healing, intramembranous healing and lamellar bone formation. These three osteogenic processes were modeled using full femur fracture, ulnar stress fracture, and ulnar non-damaging cyclic loading, respectively. Our results confirmed the requirement of BMP2 for endochondral fracture healing, as mice in which Bmp2 was knocked out in all cells prior to fracture failed to form a callus. Targeted deletion of Bmp2 in osteoblasts (osterix-expressing) or vascular endothelial cells (vascular endothelial cadherin-expressing) did not impact fracture healing in any way. Regarding non-endochondral bone formation, we found that BMP2 is largely dispensable for intramembranous bone formation after stress fracture and also not required for lamellar bone formation induced by mechanical loading. Taken together our results indicate that osteoblasts and endothelial cells are not a critical source of BMP2 in endochondral fracture healing, and that non-endochondral bone formation in the adult mouse is not as critically dependent on BMP2. PMID:26344756

Parathyroid hormone-dependent signaling pathways regulating genes in bone cells

NASA Technical Reports Server (NTRS)

Swarthout, John T.; D'Alonzo, Richard C.; Selvamurugan, Nagarajan; Partridge, Nicola C.

2002-01-01

Parathyroid hormone (PTH) is an 84-amino-acid polypeptide hormone functioning as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. PTH and PTH-related protein (PTHrP) indirectly activate osteoclasts resulting in increased bone resorption. During this process, PTH changes the phenotype of the osteoblast from a cell involved in bone formation to one directing bone resorption. In addition to these catabolic effects, PTH has been demonstrated to be an anabolic factor in skeletal tissue and in vitro. As a result, PTH has potential medical application to the treatment of osteoporosis, since intermittent administration of PTH stimulates bone formation. Activation of osteoblasts by PTH results in expression of genes important for the degradation of the extracellular matrix, production of growth factors, and stimulation and recruitment of osteoclasts. The ability of PTH to drive changes in gene expression is dependent upon activation of transcription factors such as the activator protein-1 family, RUNX2, and cAMP response element binding protein (CREB). Much of the regulation of these processes by PTH is protein kinase A (PKA)-dependent. However, while PKA is linked to many of the changes in gene expression directed by PTH, PKA activation has been shown to inhibit mitogen-activated protein kinase (MAPK) and proliferation of osteoblasts. It is now known that stimulation of MAPK and proliferation by PTH at low concentrations is protein kinase C (PKC)-dependent in both osteoblastic and kidney cells. Furthermore, PTH has been demonstrated to regulate components of the cell cycle. However, whether this regulation requires PKC and/or extracellular signal-regulated kinases or whether PTH is able to stimulate other components of the cell cycle is unknown. It is possible that stimulation of this signaling pathway by PTH mediates a unique pattern of gene expression resulting in proliferation in osteoblastic and kidney cells; however, specific

Regulation of gamma-Secretase in Alzheimer's Disease

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

Zhou, Shuxia; Zhou, Hua; Walian, Peter

2007-02-07

The {gamma}-secretase complex is an intramembrane aspartyl protease that cleaves its substrates along their transmembrane regions. Sequential proteolytic processing of amyloid precursor protein by {beta}- and {gamma}-secretase produces amyloid {beta}-peptides, which are the major components of amyloid plaques in the brains of Alzheimer's disease patients. The {gamma}-secretase complex is therefore believed to be critical in the pathogenesis of Alzheimer's disease. Here we review the range of factors found to affect the nature and degree of {gamma}-secretase complex activity; these include {gamma}-secretase complex assembly and activation, the integral regulatory subunit CD147, transient or weak binding partners, the levels of cholesterol andmore » sphingolipids in cell membranes, and inflammatory cytokines. Integrated knowledge of the molecular mechanisms supporting the actions of these factors is expected to lead to a comprehensive understanding of the functional regulation of the {gamma}-secretase complex, and this, in turn, should facilitate the development of novel therapeutic strategies for the treatment of Alzheimer's disease.« less

Understanding leptin-dependent regulation of skeletal homeostasis

PubMed Central

Motyl, Katherine J.; Rosen, Clifford J.

2012-01-01

Despite growing evidence for adipose tissue regulation of bone mass, the role of the adipokine leptin in bone remodeling remains controversial. The majority of in vitro studies suggest leptin enhances osteoblastic proliferation and differentiation while inhibiting adipogenic differentiation from marrow stromal cells. Alternatively, some evidence demonstrates either no effect or a pro-apoptotic action of leptin on stromal cells. Similarly, in vivo work has demonstrated both positive and negative effects of leptin on bone mass. Most of the literature supports the idea that leptin suppresses bone mass by acting in the brainstem to reduce serotonin-dependent sympathetic signaling from the ventromedial hypothalamus to bone. However, other studies have found partly or entirely contrasting actions of leptin. Recently one study found a significant effect of surgery alone with intracerebroventricular administration of leptin, a technique crucial for understanding centrally-mediated leptin regulation of bone. Thus, two mainstream hypotheses for the role of leptin on bone emerge: 1) direct regulation through increased osteoblast proliferation and differentiation and 2) indirect suppression of bone formation through a hypothalamic relay. At the present time, it remains unclear whether these effects are relevant in only extreme circumstances (i.e. models with complete deficiency) or play an important homeostatic role in the regulation of peak bone acquisition and skeletal remodeling. Ultimately, determining the actions of leptin on the skeleton will be critical for understanding how the obesity epidemic may be impacting the prevalence of osteoporosis. PMID:22534195

Context-dependent interactions and the regulation of species richness in freshwater fish.

PubMed

MacDougall, Andrew S; Harvey, Eric; McCune, Jenny L; Nilsson, Karin A; Bennett, Joseph; Firn, Jennifer; Bartley, Timothy; Grace, James B; Kelly, Jocelyn; Tunney, Tyler D; McMeans, Bailey; Matsuzaki, Shin-Ichiro S; Kadoya, Taku; Esch, Ellen; Cazelles, Kevin; Lester, Nigel; McCann, Kevin S

2018-03-06

Species richness is regulated by a complex network of scale-dependent processes. This complexity can obscure the influence of limiting species interactions, making it difficult to determine if abiotic or biotic drivers are more predominant regulators of richness. Using integrative modeling of freshwater fish richness from 721 lakes along an 11 o latitudinal gradient, we find negative interactions to be a relatively minor independent predictor of species richness in lakes despite the widespread presence of predators. Instead, interaction effects, when detectable among major functional groups and 231 species pairs, were strong, often positive, but contextually dependent on environment. These results are consistent with the idea that negative interactions internally structure lake communities but do not consistently 'scale-up' to regulate richness independently of the environment. The importance of environment for interaction outcomes and its role in the regulation of species richness highlights the potential sensitivity of fish communities to the environmental changes affecting lakes globally.

Context-dependent interactions and the regulation of species richness in freshwater fish

USGS Publications Warehouse

MacDougall, Andrew S.; Harvey, Eric; McCune, Jenny L.; Nilsson, Karin A.; Bennett, Joseph; Firn, Jennifer; Bartley, Timothy; Grace, James B.; Kelly, Jocelyn; Tunney, Tyler D.; McMeans, Bailey; Matsuzaki, Shin-Ichiro S.; Kadoya, Taku; Esch, Ellen; Cazelles, Kevin; Lester, Nigel; McCann, Kevin S.

2018-01-01

Species richness is regulated by a complex network of scale-dependent processes. This complexity can obscure the influence of limiting species interactions, making it difficult to determine if abiotic or biotic drivers are more predominant regulators of richness. Using integrative modeling of freshwater fish richness from 721 lakes along an 11olatitudinal gradient, we find negative interactions to be a relatively minor independent predictor of species richness in lakes despite the widespread presence of predators. Instead, interaction effects, when detectable among major functional groups and 231 species pairs, were strong, often positive, but contextually dependent on environment. These results are consistent with the idea that negative interactions internally structure lake communities but do not consistently ‘scale-up’ to regulate richness independently of the environment. The importance of environment for interaction outcomes and its role in the regulation of species richness highlights the potential sensitivity of fish communities to the environmental changes affecting lakes globally.

Get Over It! A Multilevel Threshold Autoregressive Model for State-Dependent Affect Regulation.

PubMed

De Haan-Rietdijk, Silvia; Gottman, John M; Bergeman, Cindy S; Hamaker, Ellen L

2016-03-01

Intensive longitudinal data provide rich information, which is best captured when specialized models are used in the analysis. One of these models is the multilevel autoregressive model, which psychologists have applied successfully to study affect regulation as well as alcohol use. A limitation of this model is that the autoregressive parameter is treated as a fixed, trait-like property of a person. We argue that the autoregressive parameter may be state-dependent, for example, if the strength of affect regulation depends on the intensity of affect experienced. To allow such intra-individual variation, we propose a multilevel threshold autoregressive model. Using simulations, we show that this model can be used to detect state-dependent regulation with adequate power and Type I error. The potential of the new modeling approach is illustrated with two empirical applications that extend the basic model to address additional substantive research questions.

PERK Regulates Working Memory and Protein Synthesis-Dependent Memory Flexibility

PubMed Central

Zhu, Siying; Henninger, Keely; McGrath, Barbara C.; Cavener, Douglas R.

2016-01-01

PERK (EIF2AK3) is an ER-resident eIF2α kinase required for memory flexibility and metabotropic glutamate receptor-dependent long-term depression, processes known to be dependent on new protein synthesis. Here we investigated PERK’s role in working memory, a cognitive ability that is independent of new protein synthesis, but instead is dependent on cellular Ca2+ dynamics. We found that working memory is impaired in forebrain-specific Perk knockout and pharmacologically PERK-inhibited mice. Moreover, inhibition of PERK in wild-type mice mimics the fear extinction impairment observed in forebrain-specific Perk knockout mice. Our findings reveal a novel role of PERK in cognitive functions and suggest that PERK regulates both Ca2+ -dependent working memory and protein synthesis-dependent memory flexibility. PMID:27627766

NLR-Dependent Regulation of Inflammation in Multiple Sclerosis

PubMed Central

Gharagozloo, Marjan; Gris, Katsiaryna V.; Mahvelati, Tara; Amrani, Abdelaziz; Lukens, John R.; Gris, Denis

2018-01-01

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) associated with inappropriate activation of lymphocytes, hyperinflammatory responses, demyelination, and neuronal damage. In the past decade, a number of biological immunomodulators have been developed that suppress the peripheral immune responses and slow down the progression of the disease. However, once the inflammation of the CNS has commenced, it can cause serious permanent neuronal damage. Therefore, there is a need for developing novel therapeutic approaches that control and regulate inflammatory responses within the CNS. Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are intracellular regulators of inflammation expressed by many cell types within the CNS. They redirect multiple signaling pathways initiated by pathogens and molecules released by injured tissues. NLR family members include positive regulators of inflammation, such as NLRP3 and NLRC4 and anti-inflammatory NLRs, such as NLRX1 and NLRP12. They exert immunomodulatory effect at the level of peripheral immune responses, including antigen recognition and lymphocyte activation and differentiation. Also, NLRs regulate tissue inflammatory responses. Understanding the molecular mechanisms that are placed at the crossroad of innate and adaptive immune responses, such as NLR-dependent pathways, could lead to the discovery of new therapeutic targets. In this review, we provide a summary of the role of NLRs in the pathogenesis of MS. We also summarize how anti-inflammatory NLRs regulate the immune response within the CNS. Finally, we speculate the therapeutic potential of targeting NLRs in MS. PMID:29403486

Frequency and Clinicopathological Characteristics of Presenilin 1 Gly206Ala Mutation in Puerto Rican Hispanics with Dementia

PubMed Central

Arnold, Steven E.; Vega, Irving E.; Karlawish, Jason H.; WoIk, David A.; Nunez, Jessica; Negron, Mirna; Xie, Sharon X.; Wang, Li-San; Dubroff, Jacob G.; McCarty-Wood, Elisabeth; Trojanowski, John Q.; Van Deerlin, Vivianna

2012-01-01

The frequency and clinical and pathological characteristics associated with the Gly206Ala presenilin 1 (PSEN1) mutation in Puerto Rican and non-Puerto Rican Hispanics were evaluated at the University of Pennsylvania’s Alzheimer’s Disease Center. DNAs from all cohort subjects were genotyped for the Gly206Ala PSEN1 mutation. Carriers and non-carriers with neurodegenerative disease dementias were compared for demographic, clinical, psychometric, and biomarker variables. Nineteen (12.6%) of 151 unrelated subjects with dementia were discovered to carry the PSEN1 Gly 206Ala mutation. Microsatellite marker genotyping determined a common ancestral haplotype for all carriers. Carriers were all of Puerto Rican heritage with significantly younger age of onset, but otherwise were clinically and neuropsychologically comparable to those of non-carriers with AD. Three subjects had extensive topographic and biochemical biomarker assessments that were also typical of non-carriers with AD. Neuropathological examination in one subject revealed severe, widespread plaque and tangle pathology without other meaningful disease lesions. The PSEN1 Gly206Ala mutation is notably frequent in unrelated Puerto Rican immigrants with dementia in Philadelphia. Considered together with the increased prevalence and mortality of AD reported in Puerto Rico, these high rates may reflect hereditary risk concentrated in the island which warrants further study. PMID:23114514

Redox-dependent transcriptional regulation.

PubMed

Liu, Hongjun; Colavitti, Renata; Rovira, Ilsa I; Finkel, Toren

2005-11-11

Reactive oxygen species contribute to the pathogenesis of a number of disparate disorders including tissue inflammation, heart failure, hypertension, and atherosclerosis. In response to oxidative stress, cells activate expression of a number of genes, including those required for the detoxification of reactive molecules as well as for the repair and maintenance of cellular homeostasis. In many cases, these induced genes are regulated by transcription factors whose structure, subcellular localization, or affinity for DNA is directly or indirectly regulated by the level of oxidative stress. This review summarizes the recent progress on how cellular redox status can regulate transcription-factor activity and the implications of this regulation for cardiovascular disease.

Cargo-mediated regulation of a rapid Rab4-dependent recycling pathway.

PubMed

Yudowski, Guillermo A; Puthenveedu, Manojkumar A; Henry, Anastasia G; von Zastrow, Mark

2009-06-01

Membrane trafficking is well known to regulate receptor-mediated signaling processes, but less is known about whether signaling receptors conversely regulate the membrane trafficking machinery. We investigated this question by focusing on the beta-2 adrenergic receptor (B2AR), a G protein-coupled receptor whose cellular signaling activity is controlled by ligand-induced endocytosis followed by recycling. We used total internal reflection fluorescence microscopy (TIR-FM) and tagging with a pH-sensitive GFP variant to image discrete membrane trafficking events mediating B2AR endo- and exocytosis. Within several minutes after initiating rapid endocytosis of B2ARs by the adrenergic agonist isoproterenol, we observed bright "puffs" of locally increased surface fluorescence intensity representing discrete Rab4-dependent recycling events. These events reached a constant frequency in the continuous presence of isoproterenol, and agonist removal produced a rapid (observed within 1 min) and pronounced (approximately twofold) increase in recycling event frequency. This regulation required receptor signaling via the cAMP-dependent protein kinase (PKA) and a specific PKA consensus site located in the carboxyl-terminal cytoplasmic tail of the B2AR itself. B2AR-mediated regulation was not restricted to this membrane cargo, however, as transferrin receptors packaged in the same population of recycling vesicles were similarly affected. In contrast, net recycling measured over a longer time interval (10 to 30 min) was not detectably regulated by B2AR signaling. These results identify rapid regulation of a specific recycling pathway by a signaling receptor cargo.

Faster voltage-dependent activation of Na+ channels in growth cones versus somata of neuroblastoma N1E-115 cells.

PubMed Central

Zhang, J; Loew, L M; Davidson, R M

1996-01-01

Kinetics of voltage-gated ionic channels fundamentally reflect the response of the channels to local electric fields. In this report cell-attached patch-clamp studies reveal that the voltage-dependent activation rate of sodium channels residing in the growth cone membrane differs from that of soma sodium channels in differentiating N1E-115 neuroblastoma cells. Because other electrophysiological properties of these channels do not differ, this finding may be a reflection of the difference in intramembrane electric field in these two regions of the cell. This represents a new mechanism for channels to attain a range of activities both within and between cells. PMID:8913589

Faster voltage-dependent activation of Na+ channels in growth cones versus somata of neuroblastoma N1E-115 cells.

PubMed

Zhang, J; Loew, L M; Davidson, R M

1996-11-01

Kinetics of voltage-gated ionic channels fundamentally reflect the response of the channels to local electric fields. In this report cell-attached patch-clamp studies reveal that the voltage-dependent activation rate of sodium channels residing in the growth cone membrane differs from that of soma sodium channels in differentiating N1E-115 neuroblastoma cells. Because other electrophysiological properties of these channels do not differ, this finding may be a reflection of the difference in intramembrane electric field in these two regions of the cell. This represents a new mechanism for channels to attain a range of activities both within and between cells.

Impaired Na⁺-dependent regulation of acetylcholine-activated inward-rectifier K⁺ current modulates action potential rate dependence in patients with chronic atrial fibrillation.

PubMed

Voigt, Niels; Heijman, Jordi; Trausch, Anne; Mintert-Jancke, Elisa; Pott, Lutz; Ravens, Ursula; Dobrev, Dobromir

2013-08-01

Shortened action-potential duration (APD) and blunted APD rate adaptation are hallmarks of chronic atrial fibrillation (cAF). Basal and muscarinic (M)-receptor-activated inward-rectifier K(+) currents (IK1 and IK,ACh, respectively) contribute to regulation of human atrial APD and are subject to cAF-dependent remodeling. Intracellular Na(+) ([Na(+)]i) enhances IK,ACh in experimental models but the effect of [Na(+)]i-dependent regulation of inward-rectifier K(+) currents on APD in human atrial myocytes is currently unknown. Here, we report a [Na(+)]i-dependent inhibition of outward IK1 in atrial myocytes from sinus rhythm (SR) or cAF patients. In contrast, IK,ACh activated by carbachol, a non-selective M-receptor agonist, increased with elevation of [Na(+)]i in SR. This [Na(+)]i-dependent IK,ACh regulation was absent in cAF. Including [Na(+)]i dependence of IK1 and IK,ACh in a recent computational model of the human atrial myocyte revealed that [Na(+)]i accumulation at fast rates inhibits IK1 and blunts physiological APD rate dependence in both groups. [Na(+)]i-dependent IK,ACh augmentation at fast rates increased APD rate dependence in SR, but not in cAF. These results identify impaired Na(+)-sensitivity of IK,ACh as one potential mechanism contributing to the blunted APD rate dependence in patients with cAF. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes". Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

Unusually long-lived pause required for regulation of a Rho-dependent transcription terminator.

PubMed

Hollands, Kerry; Sevostiyanova, Anastasia; Groisman, Eduardo A

2014-05-13

Up to half of all transcription termination events in bacteria rely on the RNA-dependent helicase Rho. However, the nucleic acid sequences that promote Rho-dependent termination remain poorly characterized. Defining the molecular determinants that confer Rho-dependent termination is especially important for understanding how such terminators can be regulated in response to specific signals. Here, we identify an extraordinarily long-lived pause at the site where Rho terminates transcription in the 5'-leader region of the Mg(2+) transporter gene mgtA in Salmonella enterica. We dissect the sequence elements required for prolonged pausing in the mgtA leader and establish that the remarkable longevity of this pause is required for a riboswitch to stimulate Rho-dependent termination in the mgtA leader region in response to Mg(2+) availability. Unlike Rho-dependent terminators described previously, where termination occurs at multiple pause sites, there is a single site of transcription termination directed by Rho in the mgtA leader. Our data suggest that Rho-dependent termination events that are subject to regulation may require elements distinct from those operating at constitutive Rho-dependent terminators.

Unusually long-lived pause required for regulation of a Rho-dependent transcription terminator

PubMed Central

Hollands, Kerry; Sevostiyanova, Anastasia; Groisman, Eduardo A.

2014-01-01

Up to half of all transcription termination events in bacteria rely on the RNA-dependent helicase Rho. However, the nucleic acid sequences that promote Rho-dependent termination remain poorly characterized. Defining the molecular determinants that confer Rho-dependent termination is especially important for understanding how such terminators can be regulated in response to specific signals. Here, we identify an extraordinarily long-lived pause at the site where Rho terminates transcription in the 5′-leader region of the Mg2+ transporter gene mgtA in Salmonella enterica. We dissect the sequence elements required for prolonged pausing in the mgtA leader and establish that the remarkable longevity of this pause is required for a riboswitch to stimulate Rho-dependent termination in the mgtA leader region in response to Mg2+ availability. Unlike Rho-dependent terminators described previously, where termination occurs at multiple pause sites, there is a single site of transcription termination directed by Rho in the mgtA leader. Our data suggest that Rho-dependent termination events that are subject to regulation may require elements distinct from those operating at constitutive Rho-dependent terminators. PMID:24778260

Bicarbonate-regulated adenylyl cyclase (sAC) is a sensor that regulates pH-dependent V-ATPase recycling.

PubMed

Pastor-Soler, Nuria; Beaulieu, Valerie; Litvin, Tatiana N; Da Silva, Nicolas; Chen, Yanqiu; Brown, Dennis; Buck, Jochen; Levin, Lonny R; Breton, Sylvie

2003-12-05

Modulation of environmental pH is critical for the function of many biological systems. However, the molecular identity of the pH sensor and its interaction with downstream effector proteins remain poorly understood. Using the male reproductive tract as a model system in which luminal acidification is critical for sperm maturation and storage, we now report a novel pathway for pH regulation linking the bicarbonate activated soluble adenylyl cyclase (sAC) to the vacuolar H+ATPase (V-ATPase). Clear cells of the epididymis and vas deferens contain abundant V-ATPase in their apical pole and are responsible for acidifying the lumen. Proton secretion is regulated via active recycling of V-ATPase. Here we demonstrate that this recycling is regulated by luminal pH and bicarbonate. sAC is highly expressed in clear cells, and apical membrane accumulation of V-ATPase is triggered by a sAC-dependent rise in cAMP in response to alkaline luminal pH. As sAC is expressed in other acid/base transporting epithelia, including kidney and choroid plexus, this cAMP-dependent signal transduction pathway may be a widespread mechanism that allows cells to sense and modulate extracellular pH.

Flow-dependent regulation of endothelial nitric oxide synthase: role of protein kinases

NASA Technical Reports Server (NTRS)

Boo, Yong Chool; Jo, Hanjoong

2003-01-01

Vascular endothelial cells are directly and continuously exposed to fluid shear stress generated by blood flow. Shear stress regulates endothelial structure and function by controlling expression of mechanosensitive genes and production of vasoactive factors such as nitric oxide (NO). Though it is well known that shear stress stimulates NO production from endothelial nitric oxide synthase (eNOS), the underlying molecular mechanisms remain unclear and controversial. Shear-induced production of NO involves Ca2+/calmodulin-independent mechanisms, including phosphorylation of eNOS at several sites and its interaction with other proteins, including caveolin and heat shock protein-90. There have been conflicting results as to which protein kinases-protein kinase A, protein kinase B (Akt), other Ser/Thr protein kinases, or tyrosine kinases-are responsible for shear-dependent eNOS regulation. The functional significance of each phosphorylation site is still unclear. We have attempted to summarize the current status of understanding in shear-dependent eNOS regulation.

Regulating Surface Facets of Metallic Aerogel Electrocatalysts by Size-dependent Localized Ostwald Ripening.

PubMed

Wenchao, Duan; Zhang, Peina; Xiahou, Yujiao; Song, Yahui; Bi, Cuixia; Zhan, Jie; Du, Wei; Huang, Lihui; Möhwald, Helmuth; Xia, Haibing

2018-06-21

It is well known that the activity and stability of electrocatalysts are largely dependent on their surface facets. In this work, we have successfully regulated surface facets of three-dimensional (3D) metallic Au m-n aerogels by salt-induced assembly of citrate-stabilized gold nanoparticles (Au NPs) of two different sizes and further size-dependent localized Ostwald ripening at controlled particle-number ratios, where m and n represent the size of Au NPs, respectively. In addition, 3D Au m-n @Pd aerogels were further synthesized on the basis of Au m-n aerogels and also bear controlled surface facets due to the formation of ultrathin Pd layers on Au m-n aerogels. Taking the electrooxidation of small organic molecules (such as methanol and ethanol) by the resulting Au m-n and Au m-n @Pd aerogels as examples, it is found that surface facets of metallic aerogels with excellent performance can be regulated to realize preferential surface facets for methanol oxidation and ethanol oxidation, respectively. Moreover, they also indeed simultaneously bear high activity and excellent stability. Furthermore, their activities and stability are also highly dependent on the area ratio of active facets and inactive facets on their surfaces, respectively, and these ratios are varied via the mismatch of sizes of adjacent nanoparticles. Thus, this work not only demonstrates the realization of the regulation of the surface facets of metallic aerogels by size-dependent localized Ostwald ripening, but also will open up a new way to improve electrocatalytic performance of three-dimensional metallic aerogels by surface regulation.

Fibroblast growth factor rescues brain endothelial cells lacking presenilin 1 from apoptotic cell death following serum starvation.

PubMed

Gama Sosa, Miguel A; De Gasperi, Rita; Hof, Patrick R; Elder, Gregory A

2016-07-22

Presenilin 1 (Psen1) is important for vascular brain development and is known to influence cellular stress responses. To understand the role of Psen1 in endothelial stress responses, we investigated the effects of serum withdrawal on wild type (wt) and Psen1-/- embryonic brain endothelial cells. Serum starvation induced apoptosis in Psen1-/- cells but did not affect wt cells. PI3K/AKT signaling was reduced in serum-starved Psen1-/- cells, and this was associated with elevated levels of phospho-p38 consistent with decreased pro-survival AKT signaling in the absence of Psen1. Fibroblast growth factor (FGF1 and FGF2), but not vascular endothelial growth factor (VEGF) rescued Psen1-/- cells from serum starvation induced apoptosis. Inhibition of FGF signaling induced apoptosis in wt cells under serum withdrawal, while blocking γ-secretase activity had no effect. In the absence of serum, FGF2 immunoreactivity was distributed diffusely in cytoplasmic and nuclear vesicles of wt and Psen1-/- cells, as levels of FGF2 in nuclear and cytosolic fractions were not significantly different. Thus, sensitivity of Psen1-/- cells to serum starvation is not due to lack of FGF synthesis but likely to effects of Psen1 on FGF release onto the cell surface and impaired activation of the PI3K/AKT survival pathway.

A novel presenilin 1 mutation (Ala275Val) as cause of early-onset familial Alzheimer disease.

PubMed

Luedecke, Daniel; Becktepe, Jos S; Lehmbeck, Jan T; Finckh, Ulrich; Yamamoto, Raina; Jahn, Holger; Boelmans, Kai

2014-04-30

Mutations in the presenilin 1 (PS1) gene (PSEN1) are associated with familial Alzheimer disease (FAD). Here, we report on a 50-year-old patient presenting with progressive deterioration of his short-term memory and a family history of early-onset dementia. Diagnostic workup included a neuropsychological examination, structural magnetic resonance (MR) imaging, cerebrospinal fluid (CSF) biomarkers including total tau, phosphorylated tau, and Aβ42 levels, as well as sequencing relevant fragments of the genes PSEN1, PSEN2, and APP. Additionally, we were able to obtain archival paraffin-embedded cerebellar tissue from the patient's father for cosegregation analysis. Clinical, neuropsychological and MR imaging data were indicative of early-onset Alzheimer disease. Furthermore, CSF biomarkers showed a typical pattern for Alzheimer disease. DNA sequencing revealed a heterozygous nucleotide transition (c.824C>T) in exon 8 of PSEN1, leading to an amino acid change from alanine to valine at codon 275 (Ala275Val). The same mutation was found in an archival brain specimen of the patient's demented father, but not in a blood sample of the non-demented mother. This mutation alters a conserved residue in the large hydrophilic loop of PS1, suggesting pathogenic relevance. Cosegregegation analysis and the structural as well as the presumed functional role of the mutated and highly conserved residue suggest FAD causing characteristics of the novel PSEN1 mutation Ala275Val. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Global architecture of the F-actin cytoskeleton regulates cell shape-dependent endothelial mechanotransduction.

PubMed

Shao, Yue; Mann, Jennifer M; Chen, Weiqiang; Fu, Jianping

2014-03-01

Uniaxial stretch is an important biophysical regulator of cell morphology (or shape) and functions of vascular endothelial cells (ECs). However, it is unclear whether and how cell shape can independently regulate EC mechanotransductive properties under uniaxial stretch. Herein, utilizing a novel uniaxial cell-stretching device integrated with micropost force sensors, we reported the first experimental evidence showing cell shape-dependent EC mechanotransduction via cytoskeleton (CSK) contractile forces in response to uniaxial stretch. Combining experiments and theoretical modeling from first principles, we showed that it was the global architecture of the F-actin CSK that instructed the cell shape-dependent EC mechanotransductive process. Furthermore, a cell shape-dependent nature was relayed in EC mechanotransduction via dynamic focal adhesion (FA) assembly. Our results suggested a novel mechanotransductive process in ECs wherein the global architecture of the F-actin CSK, governed by cell shape, controls mechanotransduction via CSK contractile forces and force-dependent FA assembly under uniaxial stretch.

Evolution of Nova-Dependent Splicing Regulation in the Brain

PubMed Central

Živin, Marko; Darnell, Robert B

2007-01-01

A large number of alternative exons are spliced with tissue-specific patterns, but little is known about how such patterns have evolved. Here, we study the conservation of the neuron-specific splicing factors Nova1 and Nova2 and of the alternatively spliced exons they regulate in mouse brain. Whereas Nova RNA binding domains are 94% identical across vertebrate species, Nova-dependent splicing silencer and enhancer elements (YCAY clusters) show much greater divergence, as less than 50% of mouse YCAY clusters are conserved at orthologous positions in the zebrafish genome. To study the relation between the evolution of tissue-specific splicing and YCAY clusters, we compared the brain-specific splicing of Nova-regulated exons in zebrafish, chicken, and mouse. The presence of YCAY clusters in lower vertebrates invariably predicted conservation of brain-specific splicing across species, whereas their absence in lower vertebrates correlated with a loss of alternative splicing. We hypothesize that evolution of Nova-regulated splicing in higher vertebrates proceeds mainly through changes in cis-acting elements, that tissue-specific splicing might in some cases evolve in a single step corresponding to evolution of a YCAY cluster, and that the conservation level of YCAY clusters relates to the functions encoded by the regulated RNAs. PMID:17937501

Glutamate-dependent transcriptional regulation in bergmann glia cells: involvement of p38 MAP kinase.

PubMed

Zepeda, Rossana C; Barrera, Iliana; Castelán, Francisco; Soto-Cid, Abraham; Hernández-Kelly, Luisa C; López-Bayghen, Esther; Ortega, Arturo

2008-07-01

Glutamate (Glu) is the major excitatory neurotransmitter in the Central Nervous System (CNS). Ionotropic and metabotropic glutamate receptors (GluRs) are present in neurons and glial cells and are involved in gene expression regulation. Mitogen-activated proteins kinases (MAPK) are critical for all the membrane to nuclei signaling pathways described so far. In cerebellar Bergmann glial cells, glutamate-dependent transcriptional regulation is partially dependent on p42/44 MAPK activity. Another member of this kinase family, p38 MAPK is activated by non-mitogenic stimuli through its Thr180/Tyr182 phosphorylation and phosphorylates cytoplasmic and nuclear protein targets involved in translational and transcriptional events. Taking into consideration that the role of p38MAPK in glial cells is not well understood, we demonstrate here that glutamate increases p38 MAPK phosphorylation in a time and dose dependent manner in cultured chick cerebellar Bergmann glial cells (BGC). Moreover, p38 MAPK is involved in the glutamate-induced transcriptional activation in these cells. Ionotropic as well as metabotropic glutamate receptors participate in p38 MAPK activation. The present findings demonstrate the involvement of p38 MAPK in glutamate-dependent gene expression regulation in glial cells.

Mediator complex dependent regulation of cardiac development and disease.

PubMed

Grueter, Chad E

2013-06-01

Cardiovascular disease (CVD) is a leading cause of morbidity and mortality. The risk factors for CVD include environmental and genetic components. Human mutations in genes involved in most aspects of cardiovascular function have been identified, many of which are involved in transcriptional regulation. The Mediator complex serves as a pivotal transcriptional regulator that functions to integrate diverse cellular signals by multiple mechanisms including recruiting RNA polymerase II, chromatin modifying proteins and non-coding RNAs to promoters in a context dependent manner. This review discusses components of the Mediator complex and the contribution of the Mediator complex to normal and pathological cardiac development and function. Enhanced understanding of the role of this core transcriptional regulatory complex in the heart will help us gain further insights into CVD. Copyright © 2013. Production and hosting by Elsevier Ltd.

Negative regulation of quorum-sensing systems in Pseudomonas aeruginosa by ATP-dependent Lon protease.

PubMed

Takaya, Akiko; Tabuchi, Fumiaki; Tsuchiya, Hiroko; Isogai, Emiko; Yamamoto, Tomoko

2008-06-01

Lon protease, a member of the ATP-dependent protease family, regulates numerous cellular systems by degrading specific substrates. Here, we demonstrate that Lon is involved in the regulation of quorum-sensing (QS) signaling systems in Pseudomonas aeruginosa, an opportunistic human pathogen. The organism has two acyl-homoserine lactone (HSL)-mediated QS systems, LasR/LasI and RhlR/RhlI. Many reports have demonstrated that these two systems are regulated and interconnected by global regulators. We found that lon-disrupted cells overproduce pyocyanin, the biosynthesis of which depends on the RhlR/RhlI system, and show increased levels of a transcriptional regulator, RhlR. The QS systems are organized hierarchically: the RhlR/RhlI system is subordinate to LasR/LasI. To elucidate the mechanism by which Lon negatively regulates RhlR/RhlI, we examined the effect of lon disruption on the LasR/LasI system. We found that Lon represses the expression of LasR/LasI by degrading LasI, an HSL synthase, leading to negative regulation of the RhlR/RhlI system. RhlR/RhlI was also shown to be regulated by Lon independently of LasR/LasI via regulation of RhlI, an HSL synthase. In view of these findings, it is suggested that Lon protease is a powerful negative regulator of both HSL-mediated QS systems in P. aeruginosa.

HIF-1α regulates Cx40-dependent vasodilatation following hemorrhagic shock in rats

PubMed Central

Duan, Chenyang; Chen, Ken; Yang, Guangming; Li, Tao; Liu, Liangming

2017-01-01

HIF-1α plays an essential role in hemorrhagic shock-induced vasoconstriction. However, the underlying mechanisms remain poorly understood. Here, we studied both the role of HIF-1α in regulating vasodilatation, and the involvement of Cx40 in this process. We found that endothelium-dependent vasodilatation exhibited an overall decline after hemorrhagic shock: at the beginning of shock vasodilatation reactivity significantly decreased, followed by a slight increase from 0.5 h to 2 h after shock. After 2 h vasodilatation dropped again. Throughout this process, protein levels of HIF-1α gradually increased. In the late period of shock, vasodilatation reactivity was enhanced by oligomycin, an HIF-1α inhibitor, suggesting that HIF-1α may promote vasoconstriction. Moreover, in the late period of shock Cx40 levels gradually increased and exhibited a negative correlation with endothelium-dependent vasoconstriction reactivity. Furthermore, Cx40 AODN significantly improved vasoconstriction reactivity and could be regulated by either an HIF-1α inhibitor or an agonist. Together, these data suggest that HIF-1α may inhibit endothelium-dependent vasodilatation reactivity following hemorrhagic shock by up-regulating Cx40, especially in the late period of shock. PMID:28386353

Regulation of the Proteasome by Neuronal Activity and Calcium/Calmodulin-dependent Protein Kinase II*

PubMed Central

Djakovic, Stevan N.; Schwarz, Lindsay A.; Barylko, Barbara; DeMartino, George N.; Patrick, Gentry N.

2009-01-01

Protein degradation via the ubiquitin proteasome system has been shown to regulate changes in synaptic strength that underlie multiple forms of synaptic plasticity. It is plausible, therefore, that the ubiquitin proteasome system is itself regulated by synaptic activity. By utilizing live-cell imaging strategies we report the rapid and dynamic regulation of the proteasome in hippocampal neurons by synaptic activity. We find that the blockade of action potentials (APs) with tetrodotoxin inhibited the activity of the proteasome, whereas the up-regulation of APs with bicuculline dramatically increased the activity of the proteasome. In addition, the regulation of the proteasome is dependent upon external calcium entry in part through N-methyl-d-aspartate receptors and L-type voltage-gated calcium channels and requires the activity of calcium/calmodulin-dependent protein kinase II (CaMKII). Using in vitro and in vivo assays we find that CaMKII stimulates proteasome activity and directly phosphorylates Rpt6, a subunit of the 19 S (PA700) subcomplex of the 26 S proteasome. Our data provide a novel mechanism whereby CaMKII may regulate the proteasome in neurons to facilitate remodeling of synaptic connections through protein degradation. PMID:19638347

Regulation of the proteasome by neuronal activity and calcium/calmodulin-dependent protein kinase II.

PubMed

Djakovic, Stevan N; Schwarz, Lindsay A; Barylko, Barbara; DeMartino, George N; Patrick, Gentry N

2009-09-25

Protein degradation via the ubiquitin proteasome system has been shown to regulate changes in synaptic strength that underlie multiple forms of synaptic plasticity. It is plausible, therefore, that the ubiquitin proteasome system is itself regulated by synaptic activity. By utilizing live-cell imaging strategies we report the rapid and dynamic regulation of the proteasome in hippocampal neurons by synaptic activity. We find that the blockade of action potentials (APs) with tetrodotoxin inhibited the activity of the proteasome, whereas the up-regulation of APs with bicuculline dramatically increased the activity of the proteasome. In addition, the regulation of the proteasome is dependent upon external calcium entry in part through N-methyl-D-aspartate receptors and L-type voltage-gated calcium channels and requires the activity of calcium/calmodulin-dependent protein kinase II (CaMKII). Using in vitro and in vivo assays we find that CaMKII stimulates proteasome activity and directly phosphorylates Rpt6, a subunit of the 19 S (PA700) subcomplex of the 26 S proteasome. Our data provide a novel mechanism whereby CaMKII may regulate the proteasome in neurons to facilitate remodeling of synaptic connections through protein degradation.

Regulator dependence of fixed points in quantum Einstein gravity with R 2 truncation

NASA Astrophysics Data System (ADS)

Nagy, S.; Fazekas, B.; Peli, Z.; Sailer, K.; Steib, I.

2018-03-01

We performed a functional renormalization group analysis for the quantum Einstein gravity including a quadratic term in the curvature. The ultraviolet non-gaussian fixed point and its critical exponent for the correlation length are identified for different forms of regulators in case of dimension 3. We searched for that optimized regulator where the physical quantities show the least regulator parameter dependence. It is shown that the Litim regulator satisfies this condition. The infrared fixed point has also been investigated, it is found that the exponent is insensitive to the third coupling introduced by the R 2 term.

Density-dependent intraspecific aggression regulates survival in northern Yellowstone wolves (Canis lupus).

PubMed

Cubaynes, Sarah; MacNulty, Daniel R; Stahler, Daniel R; Quimby, Kira A; Smith, Douglas W; Coulson, Tim

2014-11-01

Understanding the population dynamics of top-predators is essential to assess their impact on ecosystems and to guide their management. Key to this understanding is identifying the mechanisms regulating vital rates. Determining the influence of density on survival is necessary to understand the extent to which human-caused mortality is compensatory or additive. In wolves (Canis lupus), empirical evidence for density-dependent survival is lacking. Dispersal is considered the principal way in which wolves adjust their numbers to prey supply or compensate for human exploitation. However, studies to date have primarily focused on exploited wolf populations, in which density-dependent mechanisms are likely weak due to artificially low wolf densities. Using 13 years of data on 280 collared wolves in Yellowstone National Park, we assessed the effect of wolf density, prey abundance and population structure, as well as winter severity, on age-specific survival in two areas (prey-rich vs. prey-poor) of the national park. We further analysed cause-specific mortality and explored the factors driving intraspecific aggression in the prey-rich northern area of the park. Overall, survival rates decreased during the study. In northern Yellowstone, density dependence regulated adult survival through an increase in intraspecific aggression, independent of prey availability. In the interior of the park, adult survival was less variable and density-independent, despite reduced prey availability. There was no effect of prey population structure in northern Yellowstone, or of winter severity in either area. Survival was similar among yearlings and adults, but lower for adults older than 6 years. Our results indicate that density-dependent intraspecific aggression is a major driver of adult wolf survival in northern Yellowstone, suggesting intrinsic density-dependent mechanisms have the potential to regulate wolf populations at high ungulate densities. When low prey availability or high

Age-dependent regulation of ERF-VII transcription factor activity in Arabidopsis thaliana.

PubMed

Giuntoli, Beatrice; Shukla, Vinay; Maggiorelli, Federica; Giorgi, Federico M; Lombardi, Lara; Perata, Pierdomenico; Licausi, Francesco

2017-10-01

The Group VII Ethylene Responsive Factors (ERFs-VII) RAP2.2 and RAP2.12 have been mainly characterized with regard to their contribution as activators of fermentation in plants. However, transcriptional changes measured in conditions that stabilize these transcription factors exceed the mere activation of this biochemical pathway, implying additional roles performed by the ERF-VIIs in other processes. We evaluated gene expression in transgenic Arabidopsis lines expressing a stabilized form of RAP2.12, or hampered in ERF-VII activity, and identified genes affected by this transcriptional regulator and its homologs, including some involved in oxidative stress response, which are not universally induced under anaerobic conditions. The contribution of the ERF-VIIs in regulating this set of genes in response to chemically induced or submergence-stimulated mitochondria malfunctioning was found to depend on the plant developmental stage. A similar age-dependent mechanism also restrained ERF-VII activity upon the core-hypoxic genes, independently of the N-end rule pathway, which is accounted for the control of the anaerobic response. To conclude, this study shed new light on a dual role of ERF-VII proteins under submergence: as positive regulators of the hypoxic response and as repressors of oxidative-stress related genes, depending on the developmental stage at which plants are challenged by stress conditions. © 2017 John Wiley & Sons Ltd.

Drosophila Fragile X Mental Retardation Protein Developmentally Regulates Activity-Dependent Axon Pruning

PubMed Central

Tessier, Charles R.; Broadie, Kendal

2014-01-01

Summary Fragile X Syndrome (FraX) is a broad-spectrum neurological disorder with symptoms ranging from hyperexcitability to mental retardation and autism. Loss of the fragile X mental retardation 1 (fmr1) gene product, the mRNA-binding translational regulator FMRP, causes structural over-elaboration of dendritic and axonal processes as well as functional alterations in synaptic plasticity at maturity. It is unclear, however, whether FraX is primarily a disease of development, a disease of plasticity or both; a distinction vital for engineering intervention strategies. To address this critical issue, we have used the Drosophila FraX model to investigate the developmental roles of Drosophila FMRP (dFMRP). dFMRP expression and regulation of chickadee/profilin coincides with a transient window of late brain development. During this time, dFMRP is positively regulated by sensory input activity, and required to limit axon growth and for efficient activity-dependent pruning of axon branches in the Mushroom Body learning/memory center. These results demonstrate that dFMRP has a primary role in activity-dependent neural circuit refinement in late brain development. PMID:18321984

Structural basis for gene regulation by a B12-dependent photoreceptor

PubMed Central

Jost, Marco; Fernández-Zapata, Jésus; Polanco, María Carmen; Ortiz-Guerrero, Juan Manuel; Chen, Percival Yang-Ting; Kang, Gyunghoon; Padmanabhan, S.; Elías-Arnanz, Montserrat; Drennan, Catherine L.

2015-01-01

Summary Photoreceptor proteins enable organisms to sense and respond to light. The newly discovered CarH-type photoreceptors use a vitamin B12 derivative, adenosylcobalamin, as the light-sensing chromophore to mediate light-dependent gene regulation. Here, we present crystal structures of Thermus thermophilus CarH in all three relevant states: in the dark, both free and bound to operator DNA, and after light exposure. These structures provide a visualization of how adenosylcobalamin mediates CarH tetramer formation in the dark, how this tetramer binds to the promoter −35 element to repress transcription, and how light exposure leads to a large-scale conformational change that activates transcription. In addition to the remarkable functional repurposing of adenosylcobalamin from an enzyme cofactor to a light sensor, we find that nature also repurposed two independent protein modules in assembling CarH. These results expand the biological role of vitamin B12 and provide fundamental insight into a new mode of light-dependent gene regulation. PMID:26416754

PRMT5 regulates IRES-dependent translation via methylation of hnRNP A1

PubMed Central

Gao, Guozhen; Dhar, Surbhi

2017-01-01

Abstract The type II arginine methyltransferase PRMT5 is responsible for the symmetric dimethylation of histone to generate the H3R8me2s and H4R3me2s marks, which correlate with the repression of transcription. However, the protein level of a number of genes (MEP50, CCND1, MYC, HIF1a, MTIF and CDKN1B) are reported to be downregulated by the loss of PRMT5, while their mRNA levels remain unchanged, which is counterintuitive for PRMT5's proposed role as a transcription repressor. We noticed that the majority of the genes regulated by PRMT5, at the posttranscriptional level, express mRNA containing an internal ribosome entry site (IRES). Using an IRES-dependent reporter system, we established that PRMT5 facilitates the translation of a subset of IRES-containing genes. The heterogeneous nuclear ribonucleoprotein, hnRNP A1, is an IRES transacting factor (ITAF) that regulates the IRES-dependent translation of Cyclin D1 and c-Myc. We showed that hnRNP A1 is methylated by PRMT5 on two residues, R218 and R225, and that this methylation facilitates the interaction of hnRNP A1 with IRES RNA to promote IRES-dependent translation. This study defines a new role for PRMT5 regulation of cellular protein levels, which goes beyond the known functions of PRMT5 as a transcription and splicing regulator. PMID:28115626

Canonical wnt signaling regulates hematopoiesis in a dosage-dependent fashion.

PubMed

Luis, Tiago C; Naber, Brigitta A E; Roozen, Paul P C; Brugman, Martijn H; de Haas, Edwin F E; Ghazvini, Mehrnaz; Fibbe, Willem E; van Dongen, Jacques J M; Fodde, Riccardo; Staal, Frank J T

2011-10-04

Canonical Wnt signaling has been implicated in the regulation of hematopoiesis. By employing a Wnt-reporter mouse, we observed that Wnt signaling is differentially activated during hematopoiesis, suggesting an important regulatory role for specific Wnt signaling levels. To investigate whether canonical Wnt signaling regulates hematopoiesis in a dosage-dependent fashion, we analyzed the effect of different mutations in the Adenomatous polyposis coli gene (Apc), a negative modulator of the canonical Wnt pathway. By combining different targeted hypomorphic alleles and a conditional deletion allele of Apc, a gradient of five different Wnt signaling levels was obtained in vivo. We here show that different, lineage-specific Wnt dosages regulate hematopoietic stem cells (HSCs), myeloid precursors, and T lymphoid precursors during hematopoiesis. Differential, lineage-specific optimal Wnt dosages provide a unifying concept that explains the differences reported among inducible gain-of-function approaches, leading to either HSC expansion or depletion of the HSC pool. Copyright © 2011 Elsevier Inc. All rights reserved.

FAD-dependent lysine-specific demethylase-1 regulates cellular energy expenditure

PubMed Central

Hino, Shinjiro; Sakamoto, Akihisa; Nagaoka, Katsuya; Anan, Kotaro; Wang, Yuqing; Mimasu, Shinya; Umehara, Takashi; Yokoyama, Shigeyuki; Kosai, Ken-ichiro; Nakao, Mitsuyoshi

2012-01-01

Environmental factors such as nutritional state may act on the epigenome that consequently contributes to the metabolic adaptation of cells and the organisms. The lysine-specific demethylase-1 (LSD1) is a unique nuclear protein that utilizes flavin adenosine dinucleotide (FAD) as a cofactor. Here we show that LSD1 epigenetically regulates energy-expenditure genes in adipocytes depending on the cellular FAD availability. We find that the loss of LSD1 function, either by short interfering RNA or by selective inhibitors in adipocytes, induces a number of regulators of energy expenditure and mitochondrial metabolism such as PPARγ coactivator-1α resulting in the activation of mitochondrial respiration. In the adipose tissues from mice on a high-fat diet, expression of LSD1-target genes is reduced, compared with that in tissues from mice on a normal diet, which can be reverted by suppressing LSD1 function. Our data suggest a novel mechanism where LSD1 regulates cellular energy balance through coupling with cellular FAD biosynthesis. PMID:22453831

FAD-dependent lysine-specific demethylase-1 regulates cellular energy expenditure.

PubMed

Hino, Shinjiro; Sakamoto, Akihisa; Nagaoka, Katsuya; Anan, Kotaro; Wang, Yuqing; Mimasu, Shinya; Umehara, Takashi; Yokoyama, Shigeyuki; Kosai, Ken-Ichiro; Nakao, Mitsuyoshi

2012-03-27

Environmental factors such as nutritional state may act on the epigenome that consequently contributes to the metabolic adaptation of cells and the organisms. The lysine-specific demethylase-1 (LSD1) is a unique nuclear protein that utilizes flavin adenosine dinucleotide (FAD) as a cofactor. Here we show that LSD1 epigenetically regulates energy-expenditure genes in adipocytes depending on the cellular FAD availability. We find that the loss of LSD1 function, either by short interfering RNA or by selective inhibitors in adipocytes, induces a number of regulators of energy expenditure and mitochondrial metabolism such as PPARγ coactivator-1α resulting in the activation of mitochondrial respiration. In the adipose tissues from mice on a high-fat diet, expression of LSD1-target genes is reduced, compared with that in tissues from mice on a normal diet, which can be reverted by suppressing LSD1 function. Our data suggest a novel mechanism where LSD1 regulates cellular energy balance through coupling with cellular FAD biosynthesis.

Phosphatidylinositol 4,5-bisphosphate regulates SNARE-dependent membrane fusion.

PubMed

James, Declan J; Khodthong, Chuenchanok; Kowalchyk, Judith A; Martin, Thomas F J

2008-07-28

Phosphatidylinositol 4,5-bisphosphate (PI 4,5-P(2)) on the plasma membrane is essential for vesicle exocytosis but its role in membrane fusion has not been determined. Here, we quantify the concentration of PI 4,5-P(2) as approximately 6 mol% in the cytoplasmic leaflet of plasma membrane microdomains at sites of docked vesicles. At this concentration of PI 4,5-P(2) soluble NSF attachment protein receptor (SNARE)-dependent liposome fusion is inhibited. Inhibition by PI 4,5-P(2) likely results from its intrinsic positive curvature-promoting properties that inhibit formation of high negative curvature membrane fusion intermediates. Mutation of juxtamembrane basic residues in the plasma membrane SNARE syntaxin-1 increase inhibition by PI 4,5-P(2), suggesting that syntaxin sequesters PI 4,5-P(2) to alleviate inhibition. To define an essential rather than inhibitory role for PI 4,5-P(2), we test a PI 4,5-P(2)-binding priming factor required for vesicle exocytosis. Ca(2+)-dependent activator protein for secretion promotes increased rates of SNARE-dependent fusion that are PI 4,5-P(2) dependent. These results indicate that PI 4,5-P(2) regulates fusion both as a fusion restraint that syntaxin-1 alleviates and as an essential cofactor that recruits protein priming factors to facilitate SNARE-dependent fusion.

Load-dependent regulation of neuromuscular system

NASA Technical Reports Server (NTRS)

Ohira, Yoshinobu; Kawano, Fuminori; Stevens, James L.; Wang, Xiao D.; Ishihara, Akihiko

2004-01-01

Roles of gravitational loading, sarcomere length, and/or tension development on the electromyogram (EMG) of soleus and afferent neurogram recorded at the L5 segmental level of spinal cord were investigated during parabolic flight of a jet airplane or hindlimb suspension in conscious rats. Both EMG and neurogram levels were increased when the gravity levels were elevated from 1-G to 2-G during the parabolic flight. They were decreased when the hindlimbs were unloaded by exposure to actual microgravity or by suspension. These phenomena were related to passive shortening of muscle fibers and/or sarcomeres. Unloading-related decrease in sarcomere length was greater at the central rather than the proximal and distal regions of fibers. These activities and tension development were not detected when the mean sarcomere length was less than 2.03 micrometers. It is suggested that load-dependent regulation of neuromuscular system is related to the tension development which is influenced by sarcomere length.

Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis

PubMed Central

Khan, Meraj A.; Philip, Lijy M.; Cheung, Guillaume; Vadakepeedika, Shawn; Grasemann, Hartmut; Sweezey, Neil; Palaniyar, Nades

2018-01-01

Neutrophils migrating from the blood (pH 7.35–7.45) into the surrounding tissues encounter changes in extracellular pH (pHe) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H+ ions reducing the intracellular pH (pHi). Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET) formation (NETosis) is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS) and neutrophil protease activity, stimulating NETosis. Here, we found that raising pHe (ranging from 6.6 to 7.8; every 0.2 units) increased pHi of both activated and resting neutrophils within 10–20 min (Seminaphtharhodafluor dual fluorescence measurements). Since Nox activity generates H+ ions, pHi is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging) during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs). In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots) and NETosis. Raising pH increased Escherichia coli lipopolysaccharide-, Pseudomonas aeruginosa (Gram-negative)-, and Staphylococcus aureus (Gram-positive)-induced NETosis. Thus, higher pHe promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pHe-mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM) increases NETosis. Each Tris molecule can bind 3H+ ions, whereas each bicarbonate HCO3− ion binds 1H+ ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar

Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis.

PubMed

Khan, Meraj A; Philip, Lijy M; Cheung, Guillaume; Vadakepeedika, Shawn; Grasemann, Hartmut; Sweezey, Neil; Palaniyar, Nades

2018-01-01

Neutrophils migrating from the blood (pH 7.35-7.45) into the surrounding tissues encounter changes in extracellular pH (pH e ) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H + ions reducing the intracellular pH (pH i ). Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET) formation (NETosis) is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS) and neutrophil protease activity, stimulating NETosis. Here, we found that raising pH e (ranging from 6.6 to 7.8; every 0.2 units) increased pH i of both activated and resting neutrophils within 10-20 min (Seminaphtharhodafluor dual fluorescence measurements). Since Nox activity generates H + ions, pH i is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging) during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs). In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots) and NETosis. Raising pH increased Escherichia coli lipopolysaccharide-, Pseudomonas aeruginosa (Gram-negative)-, and Staphylococcus aureus (Gram-positive)-induced NETosis. Thus, higher pH e promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pH e -mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM) increases NETosis. Each Tris molecule can bind 3H + ions, whereas each bicarbonate HCO3 - ion binds 1H + ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar

Circadian clock-dependent and -independent posttranscriptional regulation underlies temporal mRNA accumulation in mouse liver

PubMed Central

Wang, Jingkui; Yeung, Jake; Gobet, Cédric; Sobel, Jonathan; Lück, Sarah; Molina, Nacho; Naef, Felix

2018-01-01

The mammalian circadian clock coordinates physiology with environmental cycles through the regulation of daily oscillations of gene expression. Thousands of transcripts exhibit rhythmic accumulations across mouse tissues, as determined by the balance of their synthesis and degradation. While diurnally rhythmic transcription regulation is well studied and often thought to be the main factor generating rhythmic mRNA accumulation, the extent of rhythmic posttranscriptional regulation is debated, and the kinetic parameters (e.g., half-lives), as well as the underlying regulators (e.g., mRNA-binding proteins) are relatively unexplored. Here, we developed a quantitative model for cyclic accumulations of pre-mRNA and mRNA from total RNA-seq data, and applied it to mouse liver. This allowed us to identify that about 20% of mRNA rhythms were driven by rhythmic mRNA degradation, and another 15% of mRNAs regulated by both rhythmic transcription and mRNA degradation. The method could also estimate mRNA half-lives and processing times in intact mouse liver. We then showed that, depending on mRNA half-life, rhythmic mRNA degradation can either amplify or tune phases of mRNA rhythms. By comparing mRNA rhythms in wild-type and Bmal1−/− animals, we found that the rhythmic degradation of many transcripts did not depend on a functional BMAL1. Interestingly clock-dependent and -independent degradation rhythms peaked at distinct times of day. We further predicted mRNA-binding proteins (mRBPs) that were implicated in the posttranscriptional regulation of mRNAs, either through stabilizing or destabilizing activities. Together, our results demonstrate how posttranscriptional regulation temporally shapes rhythmic mRNA accumulation in mouse liver. PMID:29432155

CodY-Dependent Regulation of Sporulation in Clostridium difficile.

PubMed

Nawrocki, Kathryn L; Edwards, Adrianne N; Daou, Nadine; Bouillaut, Laurent; McBride, Shonna M

2016-08-01

Clostridium difficile must form a spore to survive outside the gastrointestinal tract. The factors that trigger sporulation in C. difficile remain poorly understood. Previous studies have suggested that a link exists between nutritional status and sporulation initiation in C. difficile In this study, we investigated the impact of the global nutritional regulator CodY on sporulation in C. difficile strains from the historical 012 ribotype and the current epidemic 027 ribotype. Sporulation frequencies were increased in both backgrounds, demonstrating that CodY represses sporulation in C. difficile The 027 codY mutant exhibited a greater increase in spore formation than the 012 codY mutant. To determine the role of CodY in the observed sporulation phenotypes, we examined several factors that are known to influence sporulation in C. difficile Using transcriptional reporter fusions and quantitative reverse transcription-PCR (qRT-PCR) analysis, we found that two loci associated with the initiation of sporulation, opp and sinR, are regulated by CodY. The data demonstrate that CodY is a repressor of sporulation in C. difficile and that the impact of CodY on sporulation and expression of specific genes is significantly influenced by the strain background. These results suggest that the variability of CodY-dependent regulation is an important contributor to virulence and sporulation in current epidemic isolates. This report provides further evidence that nutritional state, virulence, and sporulation are linked in C. difficile This study sought to examine the relationship between nutrition and sporulation in C. difficile by examining the global nutritional regulator CodY. CodY is a known virulence and nutritional regulator of C. difficile, but its role in sporulation was unknown. Here, we demonstrate that CodY is a negative regulator of sporulation in two different ribotypes of C. difficile We also demonstrate that CodY regulates known effectors of sporulation, Opp and Sin

CodY-Dependent Regulation of Sporulation in Clostridium difficile

PubMed Central

Nawrocki, Kathryn L.; Edwards, Adrianne N.; Daou, Nadine; Bouillaut, Laurent

2016-01-01

ABSTRACT Clostridium difficile must form a spore to survive outside the gastrointestinal tract. The factors that trigger sporulation in C. difficile remain poorly understood. Previous studies have suggested that a link exists between nutritional status and sporulation initiation in C. difficile. In this study, we investigated the impact of the global nutritional regulator CodY on sporulation in C. difficile strains from the historical 012 ribotype and the current epidemic 027 ribotype. Sporulation frequencies were increased in both backgrounds, demonstrating that CodY represses sporulation in C. difficile. The 027 codY mutant exhibited a greater increase in spore formation than the 012 codY mutant. To determine the role of CodY in the observed sporulation phenotypes, we examined several factors that are known to influence sporulation in C. difficile. Using transcriptional reporter fusions and quantitative reverse transcription-PCR (qRT-PCR) analysis, we found that two loci associated with the initiation of sporulation, opp and sinR, are regulated by CodY. The data demonstrate that CodY is a repressor of sporulation in C. difficile and that the impact of CodY on sporulation and expression of specific genes is significantly influenced by the strain background. These results suggest that the variability of CodY-dependent regulation is an important contributor to virulence and sporulation in current epidemic isolates. This report provides further evidence that nutritional state, virulence, and sporulation are linked in C. difficile. IMPORTANCE This study sought to examine the relationship between nutrition and sporulation in C. difficile by examining the global nutritional regulator CodY. CodY is a known virulence and nutritional regulator of C. difficile, but its role in sporulation was unknown. Here, we demonstrate that CodY is a negative regulator of sporulation in two different ribotypes of C. difficile. We also demonstrate that CodY regulates known effectors of

Structural basis for gene regulation by a B 12-dependent photoreceptor

DOE PAGES

Jost, Marco; Fernández-Zapata, Jésus; Polanco, María Carmen; ...

2015-09-28

Photoreceptor proteins enable organisms to sense and respond to light. The newly discovered CarH-type photoreceptors use a vitamin B 12 derivative, adenosylcobalamin, as the light-sensing chromophore to mediate light-dependent gene regulation. Here in this paper, we present crystal structures of Thermus thermophilus CarH in all three relevant states: in the dark, both free and bound to operator DNA, and after light exposure. These structures provide visualizations of how adenosylcobalamin mediates CarH tetramer formation in the dark, how this tetramer binds to the promoter -35 element to repress transcription, and how light exposure leads to a large-scale conformational change that activatesmore » transcription. In addition to the remarkable functional repurposing of adenosylcobalamin from an enzyme cofactor to a light sensor, we find that nature also repurposed two independent protein modules in assembling CarH. Finally, these results expand the biological role of vitamin B 12 and provide fundamental insight into a new mode of light-dependent gene regulation.« less

The ZO-1–associated Y-box factor ZONAB regulates epithelial cell proliferation and cell density

PubMed Central

Balda, Maria S.; Garrett, Michelle D.; Matter, Karl

2003-01-01

Epithelial tight junctions regulate paracellular permeability, restrict apical/basolateral intramembrane diffusion of lipids, and have been proposed to participate in the control of epithelial cell proliferation and differentiation. Previously, we have identified ZO-1–associated nucleic acid binding proteins (ZONAB), a Y-box transcription factor whose nuclear localization and transcriptional activity is regulated by the tight junction–associated candidate tumor suppressor ZO-1. Now, we found that reduction of ZONAB expression using an antisense approach or by RNA interference strongly reduced proliferation of MDCK cells. Transfection of wild-type or ZONAB-binding fragments of ZO-1 reduced proliferation as well as nuclear ZONAB pools, indicating that promotion of proliferation by ZONAB requires its nuclear accumulation. Overexpression of ZONAB resulted in increased cell density in mature monolayers, and depletion of ZONAB or overexpression of ZO-1 reduced cell density. ZONAB was found to associate with cell division kinase (CDK) 4, and reduction of nuclear ZONAB levels resulted in reduced nuclear CDK4. Thus, our data indicate that tight junctions can regulate epithelial cell proliferation and cell density via a ZONAB/ZO-1–based pathway. Although this regulatory process may also involve regulation of transcription by ZONAB, our data suggest that one mechanism by which ZONAB and ZO-1 influence proliferation is by regulating the nuclear accumulation of CDK4. PMID:12566432

Ral-Arf6 crosstalk regulates Ral dependent exocyst trafficking and anchorage independent growth signalling.

PubMed

Pawar, Archana; Meier, Jeremy A; Dasgupta, Anwesha; Diwanji, Neha; Deshpande, Neha; Saxena, Kritika; Buwa, Natasha; Inchanalkar, Siddhi; Schwartz, Martin Alexander; Balasubramanian, Nagaraj

2016-09-01

Integrin dependent regulation of growth factor signalling confers anchorage dependence that is deregulated in cancers. Downstream of integrins and oncogenic Ras the small GTPase Ral is a vital mediator of adhesion dependent trafficking and signalling. This study identifies a novel regulatory crosstalk between Ral and Arf6 that controls Ral function in cells. In re-adherent mouse fibroblasts (MEFs) integrin dependent activation of RalA drives Arf6 activation. Independent of adhesion constitutively active RalA and RalB could both however activate Arf6. This is further conserved in oncogenic H-Ras containing bladder cancer T24 cells, which express anchorage independent active Ral that supports Arf6 activation. Arf6 mediates active Ral-exocyst dependent delivery of raft microdomains to the plasma membrane that supports anchorage independent growth signalling. Accordingly in T24 cells the RalB-Arf6 crosstalk is seen to preferentially regulate anchorage independent Erk signalling. Active Ral we further find uses a Ral-RalBP1-ARNO-Arf6 pathway to mediate Arf6 activation. This study hence identifies Arf6, through this regulatory crosstalk, to be a key downstream mediator of Ral isoform function along adhesion dependent pathways in normal and cancer cells. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

NLRC5: a key regulator of MHC class I-dependent immune responses.

PubMed

Kobayashi, Koichi S; van den Elsen, Peter J

2012-12-01

The expression of MHC class I molecules is crucial for the initiation and regulation of adaptive immune responses against pathogens. NOD-, LRR- and CARD-containing 5 (NLRC5) was recently identified as a specific transactivator of MHC class I genes (CITA). NLRC5 and the master regulator for MHC class II genes, class II transactivator (CIITA), interact with similar MHC promoter-bound factors. Here, we provide a broad overview of the molecular mechanisms behind MHC class I transcription and the role of the class I transactivator NLRC5 in MHC class I-dependent immune responses.

Inactivation of Nitric Oxide Synthesis Exacerbates the Development of Alzheimer Disease Pathology in APPPS1 Mice (Amyloid Precursor Protein/Presenilin-1).

PubMed

Cifuentes, Diana; Poittevin, Marine; Bonnin, Philippe; Ngkelo, Anta; Kubis, Nathalie; Merkulova-Rainon, Tatyana; Lévy, Bernard I

2017-07-31

The epidemiological link between hypertension and Alzheimer disease is established. We previously reported that hypertension aggravates the Alzheimer-like pathology in APPPS1 mice (amyloid precursor protein/presenilin-1, mouse model of Alzheimer disease) with angiotensin II-induced hypertension, in relation with hypertension and nitric oxide deficiency. To provide further insights into the role of nitric oxide in the hypertension-Alzheimer disease cross-talk, we studied the effects of nitric oxide blockade in APPPS1 mice using N (ω)-nitro-l-arginine methyl ester (l-NAME) alone or in combination with hydralazine, to normalize blood pressure. Compared with normotensive APPPS1 mice, those with l-NAME-induced hypertension had greater amyloid burden ( P <0.05), increased cortical amyloid angiopathy ( P <0.01), decreased regional microvascular density ( P <0.05), and deficient long-term spatial reference memory ( P <0.001). Blood pressure normalization with hydralazine did not protect APPPS1 mice from l-NAME-induced deterioration except for cortical amyloid angiopathy, linked to hypertension-induced arterial wall remodeling. By testing the cerebrovascular response to hypercapnic breathing, we evidenced early functional impairment of cerebral vasomotor activity in APPPS1 mice. Whereas in control wild-type normotensive mice, carbon dioxide breathing resulted in 15±1.3% increase in the mean blood flow velocity ( P <0.001), paradoxical mild decrease (1.5±0.4%) was recorded in normotensive APPPS1 mice ( P <0.001). Carbon dioxide-induced decrease in mean blood flow velocity was not significantly modified in l-NAME-treated hypertensive APPPS1 mice (2.5±1.2%) and partly reversed to mild vasodilation by hydralazine (3.2±1.5%, P <0.01). These results suggest that impaired nitric oxide bioavailability exacerbates the pathophysiology of Alzheimer disease, essentially impacting amyloid load and cognitive impairment, independently of l-NAME-induced hypertension. Only cerebral

The microRNA miR-1 regulates a MEF-2 dependent retrograde signal at neuromuscular junctions

PubMed Central

Simon, David J.; Madison, Jon M.; Conery, Annie L.; Thompson-Peer, Katherine L.; Soskis, Michael; Ruvkun, Gary B.; Kaplan, Joshua M.; Kim, John K.

2008-01-01

Summary We show that miR-1, a conserved muscle specific microRNA, regulates aspects of both pre- and post-synaptic function at C. elegans neuromuscular junctions. miR-1 regulates the expression level of two nicotinic acetylcholine receptor (nAChR) subunits (UNC-29 and UNC-63), thereby altering muscle sensitivity to acetylcholine (ACh). miR-1 also regulates the muscle transcription factor MEF-2, which results in altered pre-synaptic ACh secretion, suggesting that MEF-2 activity in muscles controls a retrograde signal. The effect of the MEF-2-dependent retrograde signal on secretion is mediated by the synaptic vesicle protein RAB-3. Finally, acute activation of levamisole-sensitive nAChRs stimulates MEF-2-dependent transcriptional responses, and induces the MEF-2-dependent retrograde signal. We propose that miR-1 refines synaptic function by coupling changes in muscle activity to changes in pre-synaptic function. PMID:18510933

Profound regulation of Na/K pump activity by transient elevations of cytoplasmic calcium in murine cardiac myocytes

PubMed Central

Lu, Fang-Min; Deisl, Christine; Hilgemann, Donald W

2016-01-01

Small changes of Na/K pump activity regulate internal Ca release in cardiac myocytes via Na/Ca exchange. We now show conversely that transient elevations of cytoplasmic Ca strongly regulate cardiac Na/K pumps. When cytoplasmic Na is submaximal, Na/K pump currents decay rapidly during extracellular K application and multiple results suggest that an inactivation mechanism is involved. Brief activation of Ca influx by reverse Na/Ca exchange enhances pump currents and attenuates current decay, while repeated Ca elevations suppress pump currents. Pump current enhancement reverses over 3 min, and results are similar in myocytes lacking the regulatory protein, phospholemman. Classical signaling mechanisms, including Ca-activated protein kinases and reactive oxygen, are evidently not involved. Electrogenic signals mediated by intramembrane movement of hydrophobic ions, such as hexyltriphenylphosphonium (C6TPP), increase and decrease in parallel with pump currents. Thus, transient Ca elevation and Na/K pump inactivation cause opposing sarcolemma changes that may affect diverse membrane processes. DOI: http://dx.doi.org/10.7554/eLife.19267.001 PMID:27627745

Kinetics of electrically and chemically induced swelling in polyelectrolyte gels

NASA Astrophysics Data System (ADS)

Grimshaw, P. E.; Nussbaum, J. H.; Grodzinsky, A. J.; Yarmush, M. L.

1990-09-01

Controlled swelling and shrinking of polyelectrolyte gels is useful for regulating the transport of solutes into, out of, and through these materials. A macroscopic continuum model is presented to predict the kinetics of swelling in polyelectrolyte gel membranes induced by augmentation of electrostatic swelling forces arising from membrane fixed charge groups. The model accounts for ionic transport within the membrane, electrodiffusion phenomena, dissociation of membrane charge groups, intramembrane fluid flow, and mechanical deformation of the membrane matrix. Model predictions are compared with measurements of chemically and electrically induced swelling and shrinking in crosslinked polymethacrylic acid (PMAA) membranes. Large, reversible changes in PMAA membrane hydration were observed after changing the bath pH or by applying an electric field to modify the intramembrane ionic environment and fixed charge density. A relatively slow swelling process and more rapid shrinking for both chemical and electrical modulation of the intramembrane pH are observed. The model indicates that retardation of membrane swelling is dominated by diffusion-limited reaction of H+ ions with membrane charge groups, and that the more rapid shrinking is limited primarily by mechanical processes.

Monoamine oxidase-A physically interacts with presenilin-1(M146V) in the mouse cortex.

PubMed

Wei, Zelan; Gabriel, Geraldine G; Rui, Lewei; Cao, Xia; Pennington, Paul R; Chlan-Fourney, Jennifer; Nazarali, Adil J; Baker, Glen B; Mousseau, Darrell D

2012-01-01

The concentration of presenilin-1 (PS-1) protein at the mitochondrial-associated aspect of the endoplasmic reticulum supports the potential for a mitochondrial influence of PS-1. Given that carriers of certain Alzheimer's disease (AD)-related PS-1 variants are predisposed to clinical depression and that depression has been historically associated with the mitochondrial enzyme, monoamine oxidase-A (MAO-A), we investigated cortical MAO-A function in the AD-related PS-1(M146V) knock-in mouse. The MAO-A system was clearly altered in the PS-1(M146V) mouse as revealed by (a) a mismatch between MAO-A protein expression and MAO-A activity; (b) changes in MAO-A-mediated monoaminergic neurotransmitter metabolism; (c) changes in non-cognitive behavior following treatment with the irreversible MAO-A inhibitor clorgyline; and (d) an increase in the potency of clorgyline in these same mice. We next investigated whether PS-1(M146V) could be influencing MAO-A directly. We observed (a) an enhanced MAO-A activity in necropsied PS-1(M146V) mouse cortical extracts incubated with DAPT (a PS-1 substrate-competitor); (b) the proximity of PS-1 with MAO-A and mitochondrial markers in cortical sections and in primary cortical neurons; (c) the co-segregation and co-immunoprecipitation of PS-1 and MAO-A within the mitochondrial fraction; and (d) the co-immunoprecipitation of overexpressed PS-1(M146V) and MAO-A proteins from N2a lysates. The PS-1(ΔEx9) and PS-1(D257A) variants, known to have low substrate-binding capacity, co-immunoprecipitated weakly with MAO-A. These combined data support a physical interaction between PS-1 and MAO-A that could influence MAO-A activity and contribute to the monoaminergic disruptions common to disorders as seemingly diverse as depression and AD.

Hypocotyl Transcriptome Reveals Auxin Regulation of Growth-Promoting Genes through GA-Dependent and -Independent Pathways

PubMed Central

Castillejo, Cristina; Sartor, Ryan; Bialy, Agniezska; Sun, Tai-ping; Estelle, Mark

2012-01-01

Many processes critical to plant growth and development are regulated by the hormone auxin. Auxin responses are initiated through activation of a transcriptional response mediated by the TIR1/AFB family of F-box protein auxin receptors as well as the AUX/IAA and ARF families of transcriptional regulators. However, there is little information on how auxin regulates a specific cellular response. To begin to address this question, we have focused on auxin regulation of cell expansion in the Arabidopsis hypocotyl. We show that auxin-mediated hypocotyl elongation is dependent upon the TIR1/AFB family of auxin receptors and degradation of AUX/IAA repressors. We also use microarray studies of elongating hypocotyls to show that a number of growth-associated processes are activated by auxin including gibberellin biosynthesis, cell wall reorganization and biogenesis, and others. Our studies indicate that GA biosynthesis is required for normal response to auxin in the hypocotyl but that the overall transcriptional auxin output consists of PIF-dependent and -independent genes. We propose that auxin acts independently from and interdependently with PIF and GA pathways to regulate expression of growth-associated genes in cell expansion. PMID:22590525

The structure and function of Alzheimer's gamma secretase enzyme complex.

PubMed

Krishnaswamy, Sudarsan; Verdile, Giuseppe; Groth, David; Kanyenda, Limbikani; Martins, Ralph N

2009-01-01

The production and accumulation of the beta amyloid protein (Abeta) is a key event in the cascade of oxidative and inflammatory processes that characterizes Alzheimer's disease (AD). A multi-subunit enzyme complex, referred to as gamma (gamma) secretase, plays a pivotal role in the generation of Abeta from its parent molecule, the amyloid precursor protein (APP). Four core components (presenilin, nicastrin, aph-1, and pen-2) interact in a high-molecular-weight complex to perform intramembrane proteolysis on a number of membrane-bound proteins, including APP and Notch. Inhibitors and modulators of this enzyme have been assessed for their therapeutic benefit in AD. However, although these agents reduce Abeta levels, the majority have been shown to have severe side effects in pre-clinical animal studies, most likely due to the enzymes role in processing other proteins involved in normal cellular function. Current research is directed at understanding this enzyme and, in particular, at elucidating the roles that each of the core proteins plays in its function. In addition, a number of interacting proteins that are not components of gamma-secretase also appear to play important roles in modulating enzyme activity. This review will discuss the structural and functional complexity of the gamma-secretase enzyme and the effects of inhibiting its activity.

Somatostatin regulates brain amyloid beta peptide Abeta42 through modulation of proteolytic degradation.

PubMed

Saito, Takashi; Iwata, Nobuhisa; Tsubuki, Satoshi; Takaki, Yoshie; Takano, Jiro; Huang, Shu-Ming; Suemoto, Takahiro; Higuchi, Makoto; Saido, Takaomi C

2005-04-01

Expression of somatostatin in the brain declines during aging in various mammals including apes and humans. A prominent decrease in this neuropeptide also represents a pathological characteristic of Alzheimer disease. Using in vitro and in vivo paradigms, we show that somatostatin regulates the metabolism of amyloid beta peptide (Abeta), the primary pathogenic agent of Alzheimer disease, in the brain through modulating proteolytic degradation catalyzed by neprilysin. Among various effector candidates, only somatostatin upregulated neprilysin activity in primary cortical neurons. A genetic deficiency of somatostatin altered hippocampal neprilysin activity and localization, and increased the quantity of a hydrophobic 42-mer form of Abeta, Abeta(42), in a manner similar to presenilin gene mutations that cause familial Alzheimer disease. These results indicate that the aging-induced downregulation of somatostatin expression may be a trigger for Abeta accumulation leading to late-onset sporadic Alzheimer disease, and suggest that somatostatin receptors may be pharmacological-target candidates for prevention and treatment of Alzheimer disease.

Cytokines and growth factors which regulate bone cell function

NASA Astrophysics Data System (ADS)

Seino, Yoshiki

Everybody knows that growth factors are most important in making bone. Hormones enhance bone formation from a long distance. Growth factors promote bone formation as an autocrine or paracrine factor in nearby bone. BMP-2 through BMP-8 are in the TGF-β family. BMP makes bone by enchondral ossification. In bone, IGF-II is most abundant, second, TGF-β, and third IGF-I. TGF-β enhances bone formation mainly by intramembranous ossification in vivo. TGF-β affects both cell proliferation and differentiation, however, TGF-β mainly enhances bone formation by intramembranous ossification. Interestingly, TGF-β is increased by estrogen(E 2), androgen, vitamin D, TGF-β and FGF. IGF-I and IGF-II also enhance bone formation. At present it remains unclear why IGF-I is more active in bone formation than IGF-II, although IGF-II is more abundant in bone compared to IGF-I. However, if only type I receptor signal transduction promotes bone formation, the strong activity of IGF-I in bone formation is understandable. GH, PTH and E 2 promotes IGF-I production. Recent data suggest that hormones containing vitamin D or E 2 enhance bone formation through growth factors. Therefore, growth factors are the key to clarifying the mechanism of bone formation.

Estrogen Receptor Alpha (ESR1)-Dependent Regulation of the Mouse Oviductal Transcriptome.

PubMed

Cerny, Katheryn L; Ribeiro, Rosanne A C; Jeoung, Myoungkun; Ko, CheMyong; Bridges, Phillip J

2016-01-01

Estrogen receptor-α (ESR1) is an important transcriptional regulator in the mammalian oviduct, however ESR1-dependent regulation of the transcriptome of this organ is not well defined, especially at the genomic level. The objective of this study was therefore to investigate estradiol- and ESR1-dependent regulation of the transcriptome of the oviduct using transgenic mice, both with (ESR1KO) and without (wild-type, WT) a global deletion of ESR1. Oviducts were collected from ESR1KO and WT littermates at 23 days of age, or ESR1KO and WT mice were treated with 5 IU PMSG to stimulate follicular development and the production of ovarian estradiol, and the oviducts collected 48 h later. RNA extracted from whole oviducts was hybridized to Affymetrix Genechip Mouse Genome 430-2.0 arrays (n = 3 arrays per genotype and treatment) or reverse transcribed to cDNA for analysis of the expression of selected mRNAs by real-time PCR. Following microarray analysis, a statistical two-way ANOVA and pairwise comparison (LSD test) revealed 2428 differentially expressed transcripts (DEG's, P < 0.01). Genotype affected the expression of 2215 genes, treatment (PMSG) affected the expression of 465 genes, and genotype x treatment affected the expression of 438 genes. With the goal of determining estradiol/ESR1-regulated function, gene ontology (GO) and bioinformatic pathway analyses were performed on DEG's in the oviducts of PMSG-treated ESR1KO versus PMSG-treated WT mice. Significantly enriched GO molecular function categories included binding and catalytic activity. Significantly enriched GO cellular component categories indicated the extracellular region. Significantly enriched GO biological process categories involved a single organism, modulation of a measurable attribute and developmental processes. Bioinformatic analysis revealed ESR1-regulation of the immune response within the oviduct as the primary canonical pathway. In summary, a transcriptomal profile of estradiol- and ESR1

Development and Mechanism of γ-Secretase Modulators for Alzheimer Disease

PubMed Central

Crump, Christina J.; Johnson, Douglas S.; Li, Yue-Ming

2013-01-01

γ-Secretase is an aspartyl intramembranal protease composed of presenilin, Nicastrin, Aph1 and Pen2 with 19 transmembrane domains. γ-Secretase cleaves the amyloid precursor proteins (APP) to release Aβ peptides that likely play a causative role in the pathogenesis of Alzheimer disease (AD). In addition, γ-secretase cleaves Notch and other type I membrane proteins. γ-Secretase inhibitors (GSIs) have been developed and used for clinical studies. However, clinical trials have shown adverse effects of GSIs that are potentially linked with non-discriminatory inhibition of Notch signaling, overall APP processing and other substrate cleavages. Therefore, these findings call for the development of disease modifying agents that target γ-secretase activity to lower Aβ42 production without blocking the overall processing of γ-secretase substrates. γ-Secretase modulators (GSMs) originally derived from non-steroidal anti-inflammatory drugs (NSAIDs) display such characteristics and are the focus of this review. However, first generation GSMs have limited potential due to low potency and undesired neuropharmacokinetic properties. This generation of GSMs has been suggested to interact with the APP substrate, γ-secretase or both. To improve the potency and brain availability, second generation GSMs including NSAID-derived carboxylic acid and non-NSAID-derived heterocyclic chemotypes as well as natural product-derived GSMs have been developed. Animal studies of this generation of GSMs have shown encouraging preclinical profiles. Moreover, using potent GSM photoaffinity probes, multiple studies unambiguously have showed that both carboxylic acid and heterocyclic GSMs specifically target presenilin, the catalytic subunit of γ-secretase. In addition, two types of GSMs have distinct binding sites within the γ-secretase complex and exhibit different Aβ profiles. GSMs induce a conformational change of γ-secretase to achieve modulation. Various models are proposed and

Carbon dioxide-dependent regulation of NF-κB family members RelB and p100 gives molecular insight into CO2-dependent immune regulation.

PubMed

Keogh, Ciara E; Scholz, Carsten C; Rodriguez, Javier; Selfridge, Andrew C; von Kriegsheim, Alexander; Cummins, Eoin P

2017-07-07

CO 2 is a physiological gas normally produced in the body during aerobic respiration. Hypercapnia (elevated blood pCO 2 >≈50 mm Hg) is a feature of several lung pathologies, e.g. chronic obstructive pulmonary disease. Hypercapnia is associated with increased susceptibility to bacterial infections and suppression of inflammatory signaling. The NF-κB pathway has been implicated in these effects; however, the molecular mechanisms underpinning cellular sensitivity of the NF-κB pathway to CO 2 are not fully elucidated. Here, we identify several novel CO 2 -dependent changes in the NF-κB pathway. NF-κB family members p100 and RelB translocate to the nucleus in response to CO 2 A cohort of RelB protein-protein interactions ( e.g. with Raf-1 and IκBα) are altered by CO 2 exposure, although others are maintained ( e.g. with p100). RelB is processed by CO 2 in a manner dependent on a key C-terminal domain located in its transactivation domain. Loss of the RelB transactivation domain alters NF-κB-dependent transcriptional activity, and loss of p100 alters sensitivity of RelB to CO 2 Thus, we provide molecular insight into the CO 2 sensitivity of the NF-κB pathway and implicate altered RelB/p100-dependent signaling in the CO 2 -dependent regulation of inflammatory signaling. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Cartilage-specific RBPjκ-dependent and -independent Notch signals regulate cartilage and bone development

PubMed Central

Kohn, Anat; Dong, Yufeng; Mirando, Anthony J.; Jesse, Alana M.; Honjo, Tasuku; Zuscik, Michael J.; O’Keefe, Regis J.; Hilton, Matthew J.

2012-01-01

The Notch signaling pathway has emerged as an important regulator of endochondral bone formation. Although recent studies have examined the role of Notch in mesenchymal and chondro-osteo progenitor cell populations, there has yet to be a true examination of Notch signaling specifically within developing and committed chondrocytes, or a determination of whether cartilage and bone formation are regulated via RBPjκ-dependent or -independent Notch signaling mechanisms. To develop a complete understanding of Notch signaling during cartilage and bone development we generated and compared general Notch gain-of-function (Rosa-NICDf/+), RBPjκ-deficient (Rbpjκf/f), and RBPjκ-deficient Notch gain-of-function (Rosa-NICDf/+;Rbpjκf/f) conditional mutant mice, where activation or deletion of floxed alleles were specifically targeted to mesenchymal progenitors (Prx1Cre) or committed chondrocytes (inducible Col2CreERT2). These data demonstrate, for the first time, that Notch regulation of chondrocyte maturation is solely mediated via the RBPjκ-dependent pathway, and that the perichodrium or osteogenic lineage probably influences chondrocyte terminal maturation and turnover of the cartilage matrix. Our study further identifies the cartilage-specific RBPjκ-independent pathway as crucial for the proper regulation of chondrocyte proliferation, survival and columnar chondrocyte organization. Unexpectedly, the RBPjκ-independent Notch pathway was also identified as an important long-range cell non-autonomous regulator of perichondral bone formation and an important cartilage-derived signal required for coordinating chondrocyte and osteoblast differentiation during endochondral bone development. Finally, cartilage-specific RBPjκ-independent Notch signaling likely regulates Ihh responsiveness during cartilage and bone development. PMID:22354840

The DNA Replication Checkpoint Directly Regulates MBF-Dependent G1/S Transcription▿

PubMed Central

Dutta, Chaitali; Patel, Prasanta K.; Rosebrock, Adam; Oliva, Anna; Leatherwood, Janet; Rhind, Nicholas

2008-01-01

The DNA replication checkpoint transcriptionally upregulates genes that allow cells to adapt to and survive replication stress. Our results show that, in the fission yeast Schizosaccharomyces pombe, the replication checkpoint regulates the entire G1/S transcriptional program by directly regulating MBF, the G1/S transcription factor. Instead of initiating a checkpoint-specific transcriptional program, the replication checkpoint targets MBF to maintain the normal G1/S transcriptional program during replication stress. We propose a mechanism for this regulation, based on in vitro phosphorylation of the Cdc10 subunit of MBF by the Cds1 replication-checkpoint kinase. Replacement of two potential phosphorylation sites with phosphomimetic amino acids suffices to promote the checkpoint transcriptional program, suggesting that Cds1 phosphorylation directly regulates MBF-dependent transcription. The conservation of MBF between fission and budding yeast, and recent results implicating MBF as a target of the budding yeast replication checkpoint, suggests that checkpoint regulation of the MBF transcription factor is a conserved strategy for coping with replication stress. Furthermore, the structural and regulatory similarity between MBF and E2F, the metazoan G1/S transcription factor, suggests that this checkpoint mechanism may be broadly conserved among eukaryotes. PMID:18662996

Deficits in Emotion-Regulation Skills Predict Alcohol Use during and after Cognitive-Behavioral Therapy for Alcohol Dependence

ERIC Educational Resources Information Center

Berking, Matthias; Margraf, Matthias; Ebert, David; Wupperman, Peggilee; Hofmann, Stefan G.; Junghanns, Klaus

2011-01-01

Objective: As emotion regulation is widely considered to be a primary motive in the misuse of alcohol, our aim in the study was to investigate whether deficits in adaptive emotion-regulation skills maintain alcohol dependence (AD). Method: A prospective study investigated whether emotion-regulation skills were associated with AD and whether these…

Microarray and functional analysis of growth-phase dependent gene regulation in Bordetella bronchiseptica

USDA-ARS?s Scientific Manuscript database

Growth-phase dependent gene regulation has recently been demonstrated to occur in B. pertussis, with many transcripts, including known virulence factors, significantly decreasing during the transition from logarithmic to stationary-phase growth. Given that B. pertussis is thought to have derived fro...

Altered cell cycle-related gene expression in brain and lymphocytes from a transgenic mouse model of Alzheimer's disease [amyloid precursor protein/presenilin 1 (PS1)].

PubMed

Esteras, Noemí; Bartolomé, Fernando; Alquézar, Carolina; Antequera, Desireé; Muñoz, Úrsula; Carro, Eva; Martín-Requero, Ángeles

2012-09-01

Cumulative evidence indicates that aberrant re-expression of many cell cycle-related proteins and inappropriate neuronal cell cycle control are critical events in Alzheimer's disease (AD) pathogenesis. Evidence of cell cycle activation in post-mitotic neurons has also been observed in murine models of AD, despite the fact that most of these mice do not show massive loss of neuronal bodies. Dysfunction of the cell cycle appears to affect cells other than neurons, as peripheral cells, such as lymphocytes and fibroblasts from patients with AD, show an altered response to mitogenic stimulation. We sought to determine whether cell cycle disturbances are present simultaneously in both brain and peripheral cells from the amyloid precursor protein (APP)/presenilin 1 (PS1) mouse model of AD, in order to validate the use of peripheral cells from patients not only to study cell cycle abnormalities as a pathogenic feature of AD, but also as a means to test novel therapeutic approaches. By using cell cycle pathway-specific RT(2)Profiler™ PCR Arrays, we detected changes in a number of cell cycle-related genes in brain as well as in lymphocytes from APP/PS1 mice. Moreover, we found enhanced 5'-bromo-2'-deoxyuridine incorporation into DNA in lymphocytes from APP/PS1 mice, and increased expression of the cell proliferation marker proliferating cell nuclear antigen (PCNA), and the cyclin-dependent kinase (CDK) inhibitor Cdkn2a, as detected by immunohistochemistry in cortical neurons of the APP/PS1 mice. Taken together, the cell cycle-related changes in brain and blood cells reported here support the mitosis failure hypothesis in AD and validate the use of peripheral cells as surrogate tissue to study the molecular basis of AD pathogenesis. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

SMU.940 regulates dextran-dependent aggregation and biofilm formation in Streptococcus mutans.

PubMed

Senpuku, Hidenobu; Yonezawa, Hideo; Yoneda, Saori; Suzuki, Itaru; Nagasawa, Ryo; Narisawa, Naoki

2018-02-01

The oral bacterium Streptococcus mutans is the principal agent in the development of dental caries. Biofilm formation by S. mutans requires bacterial attachment, aggregation, and glucan formation on the tooth surface under sucrose supplementation conditions. Our previous microarray analysis of clinical strains identified 74 genes in S. mutans that were related to biofilm morphology; however, the roles of almost all of these genes in biofilm formation are poorly understood. We investigated the effects of 21 genes randomly selected from our previous study regarding S. mutans biofilm formation, regulation by the complement pathway, and responses to competence-stimulating peptide. Eight competence-stimulating peptide-dependent genes were identified, and their roles in biofilm formation and aggregation were examined by mutational analyses of the S. mutansUA159 strain. Of these eight genes, the inactivation of the putative hemolysin III family SMU.940 gene of S. mutansUA159 promoted rapid dextran-dependent aggregation and biofilm formation in tryptic soy broth without dextrose (TSB) with 0.25% glucose and slightly reduced biofilm formation in TSB with 0.25% sucrose. The SMU.940 mutant showed higher expression of GbpC and gbpC gene than wild-type. GbpC is known to be involved in the dextran-dependent aggregation of S. mutans. An SMU.940-gbpC double mutant strain was constructed in the SMU.940 mutant background. The gbpC mutation completely abolished the dextran-dependent aggregation of the SMU.940 mutant. In addition, the aggregation of the mutant was abrogated by dextranase. These findings suggest that SMU.940 controls GbpC expression, and contributes to the regulation of dextran-dependent aggregation and biofilm formation. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Deletion of exons 9 and 10 of the Presenilin 1 gene in a patient with Early-onset Alzheimer Disease generates longer amyloid seeds.

PubMed

Le Guennec, Kilan; Veugelen, Sarah; Quenez, Olivier; Szaruga, Maria; Rousseau, Stéphane; Nicolas, Gaël; Wallon, David; Fluchere, Frédérique; Frébourg, Thierry; De Strooper, Bart; Campion, Dominique; Chávez-Gutiérrez, Lucía; Rovelet-Lecrux, Anne

2017-08-01

Presenilin 1 (PSEN1) mutations are the main cause of autosomal dominant Early-onset Alzheimer Disease (EOAD). Among them, deletions of exon 9 have been reported to be associated with a phenotype of spastic paraparesis. Using exome data from a large sample of 522 EOAD cases and 584 controls to search for genomic copy-number variations (CNVs), we report here a novel partial, in-frame deletion of PSEN1, removing both exons 9 and 10. The patient presented with memory impairment associated with spastic paraparesis, both starting from the age of 56years. He presented a positive family history of EOAD. We performed functional analysis to elucidate the impact of this novel deletion on PSEN1 activity as part of the γ-secretase complex. The deletion does not affect the assembly of a mature protease complex but has an extreme impact on its global endopeptidase activity. The mutant carboxypeptidase-like activity is also strongly impaired and the deleterious mutant effect leads to an incomplete digestion of long Aβ peptides and enhances the production of Aβ43, which has been shown to be potently amyloidogenic and neurotoxic in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

Lanthanide-Dependent Regulation of Methylotrophy in Methylobacteriumaquaticum Strain 22A.

PubMed

Masuda, Sachiko; Suzuki, Yutaka; Fujitani, Yoshiko; Mitsui, Ryoji; Nakagawa, Tomoyuki; Shintani, Masaki; Tani, Akio

2018-01-01

Methylobacterium species are representative of methylotrophic bacteria. Their genomes usually encode two types of methanol dehydrogenases (MDHs): MxaF and XoxF. The former is a Ca 2+ -dependent enzyme, and the latter was recently determined to be a lanthanide-dependent enzyme that is necessary for the expression of mxaF . This finding revealed the unexpected and important roles of lanthanides in bacterial methylotrophy. In this study, we performed transcriptome sequencing (RNA-seq) analysis using M. aquaticum strain 22A grown in the presence of different lanthanides. Expression of mxaF and xoxF1 genes showed a clear inverse correlation in response to La 3+ . We observed downregulation of formaldehyde oxidation pathways, high formaldehyde dehydrogenase activity, and low accumulation of formaldehyde in the reaction with cells grown in the presence of La 3+ ; this might be due to the direct oxidation of methanol to formate by XoxF1. Lanthanides induced the transcription of AT-rich genes, the function of most of which was unknown, and genes possibly related to cellular survival, as well as other MDH homologues. These results revealed not only the metabolic response toward altered primary methanol oxidation, but also the possible targets to be investigated further in order to better understand methylotrophy in the presence of lanthanides. IMPORTANCE Lanthanides have been considered unimportant for biological processes. In methylotrophic bacteria, however, a methanol dehydrogenase (MDH) encoded by xoxF was recently found to be lanthanide dependent, while the classic-type mxaFI is calcium dependent. XoxF-type MDHs are more widespread in diverse bacterial genera, suggesting their importance for methylotrophy. Methylobacterium species, representative methylotrophic and predominating alphaproteobacteria in the phyllosphere, contain both types and regulate their expression depending on the availability of lanthanides. RNA-seq analysis showed that the regulation takes place

Density regulation in Northeast Atlantic fish populations: Density dependence is stronger in recruitment than in somatic growth.

PubMed

Zimmermann, Fabian; Ricard, Daniel; Heino, Mikko

2018-05-01

Population regulation is a central concept in ecology, yet in many cases its presence and the underlying mechanisms are difficult to demonstrate. The current paradigm maintains that marine fish populations are predominantly regulated by density-dependent recruitment. While it is known that density-dependent somatic growth can be present too, its general importance remains unknown and most practical applications neglect it. This study aimed to close this gap by for the first time quantifying and comparing density dependence in growth and recruitment over a large set of fish populations. We fitted density-dependent models to time-series data on population size, recruitment and age-specific weight from commercially exploited fish populations in the Northeast Atlantic Ocean and the Baltic Sea. Data were standardized to enable a direct comparison within and among populations, and estimated parameters were used to quantify the impact of density regulation on population biomass. Statistically significant density dependence in recruitment was detected in a large proportion of populations (70%), whereas for density dependence in somatic growth the prevalence of density dependence depended heavily on the method (26% and 69%). Despite age-dependent variability, the density dependence in recruitment was consistently stronger among age groups and between alternative approaches that use weight-at-age or weight increments to assess growth. Estimates of density-dependent reduction in biomass underlined these results: 97% of populations with statistically significant parameters for growth and recruitment showed a larger impact of density-dependent recruitment on population biomass. The results reaffirm the importance of density-dependent recruitment in marine fishes, yet they also show that density dependence in somatic growth is not uncommon. Furthermore, the results are important from an applied perspective because density dependence in somatic growth affects productivity and

The Transcription Factor ABI4 Is Required for the Ascorbic Acid–Dependent Regulation of Growth and Regulation of Jasmonate-Dependent Defense Signaling Pathways in Arabidopsis[C][W

PubMed Central

Kerchev, Pavel I.; Pellny, Till K.; Vivancos, Pedro Diaz; Kiddle, Guy; Hedden, Peter; Driscoll, Simon; Vanacker, Hélène; Verrier, Paul; Hancock, Robert D.; Foyer, Christine H.

2011-01-01

Cellular redox homeostasis is a hub for signal integration. Interactions between redox metabolism and the ABSCISIC ACID-INSENSITIVE-4 (ABI4) transcription factor were characterized in the Arabidopsis thaliana vitamin c defective1 (vtc1) and vtc2 mutants, which are defective in ascorbic acid synthesis and show a slow growth phenotype together with enhanced abscisic acid (ABA) levels relative to the wild type (Columbia-0). The 75% decrease in the leaf ascorbate pool in the vtc2 mutants was not sufficient to adversely affect GA metabolism. The transcriptome signatures of the abi4, vtc1, and vtc2 mutants showed significant overlap, with a large number of transcription factors or signaling components similarly repressed or induced. Moreover, lincomycin-dependent changes in LIGHT HARVESTING CHLOROPHYLL A/B BINDING PROTEIN 1.1 expression were comparable in these mutants, suggesting overlapping participation in chloroplast to nucleus signaling. The slow growth phenotype of vtc2 was absent in the abi4 vtc2 double mutant, as was the sugar-insensitive phenotype of the abi4 mutant. Octadecanoid derivative-responsive AP2/ERF-domain transcription factor 47 (ORA47) and AP3 (an ABI5 binding factor) transcripts were enhanced in vtc2 but repressed in abi4 vtc2, suggesting that ABI4 and ascorbate modulate growth and defense gene expression through jasmonate signaling. We conclude that low ascorbate triggers ABA- and jasmonate-dependent signaling pathways that together regulate growth through ABI4. Moreover, cellular redox homeostasis exerts a strong influence on sugar-dependent growth regulation. PMID:21926335

Amino acid-dependent signaling via S6K1 and MYC is essential for regulation of rDNA transcription

PubMed Central

Kang, Jian; Kusnadi, Eric P.; Ogden, Allison J.; Hicks, Rodney J.; Bammert, Lukas; Kutay, Ulrike; Hung, Sandy; Sanij, Elaine; Hannan, Ross D.; Hannan, Katherine M.; Pearson, Richard B.

2016-01-01

Dysregulation of RNA polymerase I (Pol I)-dependent ribosomal DNA (rDNA) transcription is a consistent feature of malignant transformation that can be targeted to treat cancer. Understanding how rDNA transcription is coupled to the availability of growth factors and nutrients will provide insight into how ribosome biogenesis is maintained in a tumour environment characterised by limiting nutrients. We demonstrate that modulation of rDNA transcription initiation, elongation and rRNA processing is an immediate, co-regulated response to altered amino acid abundance, dependent on both mTORC1 activation of S6K1 and MYC activity. Growth factors regulate rDNA transcription initiation while amino acids modulate growth factor-dependent rDNA transcription by primarily regulating S6K1-dependent rDNA transcription elongation and processing. Thus, we show for the first time amino acids regulate rRNA synthesis by a distinct, post-initiation mechanism, providing a novel model for integrated control of ribosome biogenesis that has implications for understanding how this process is dysregulated in cancer. PMID:27385002

Alzheimer disease-related presenilin-1 variants exert distinct effects on monoamine oxidase-A activity in vitro.

PubMed

Pennington, Paul R; Wei, Zelan; Rui, Lewei; Doig, Jennifer A; Graham, Brett; Kuski, Kelly; Gabriel, Geraldine G; Mousseau, Darrell D

2011-07-01

Monoamine oxidase-A (MAO-A) has been associated with both depression and Alzheimer disease (AD). Recently, carriers of AD-related presenilin-1 (PS-1) alleles have been found to be at higher risk for developing clinical depression. We chose to examine whether PS-1 could influence MAO-A function in vitro. Overexpression of selected AD-related PS-1 variants (wildtype, Y115H, ΔEx9 and M146V) in mouse hippocampal HT-22 cells affects MAO-A catalytic activity in a variant-specific manner. The ability of the PS-1 substrate-competitor DAPT to induce MAO-A activity in cells expressing either PS-1 wildtype or PS-1(M146V) suggests the potential for a direct influence of PS-1 on MAO-A function. In support of this, we were able to co-immunoprecipitate MAO-A with FLAG-tagged PS-1 wildtype and M146V proteins. This potential for a direct protein-protein interaction between PS-1 and MAO-A is not specific for HT-22 cells as we were also able to co-immunoprecipitate MAO-A with FLAG-PS-1 variants in N2a mouse neuroblastoma cells and in HEK293 human embryonic kidney cells. Finally, we demonstrate that the two PS-1 variants reported to be associated with an increased incidence of clinical depression [e.g., A431E and L235V] both induce MAO-A activity in HT-22 cells. A direct influence of PS-1 variants on MAO-A function could provide an explanation for the changes in monoaminergic tone observed in several neurodegenerative processes including AD. The ability to induce MAO-A catalytic activity with a PS-1/γ-secretase inhibitor should also be considered when designing secretase inhibitor-based therapeutics.

Down-regulation of respiration in pear fruit depends on temperature.

PubMed

Ho, Quang Tri; Hertog, Maarten L A T M; Verboven, Pieter; Ambaw, Alemayehu; Rogge, Seppe; Verlinden, Bert E; Nicolaï, Bart M

2018-04-09

The respiration rate of plant tissues decreases when the amount of available O2 is reduced. There is, however, a debate on whether the respiration rate is controlled either by diffusion limitation of oxygen or through regulatory processes at the level of the transcriptome. We used experimental and modelling approaches to demonstrate that both diffusion limitation and metabolic regulation affect the response of respiration of bulky plant organs such as fruit to reduced O2 levels in the surrounding atmosphere. Diffusion limitation greatly affects fruit respiration at high temperature, but at low temperature respiration is reduced through a regulatory process, presumably a response to a signal generated by a plant oxygen sensor. The response of respiration to O2 is time dependent and is highly sensitive, particularly at low O2 levels in the surrounding atmosphere. Down-regulation of the respiration at low temperatures may save internal O2 and relieve hypoxic conditions in the fruit.

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

PubMed

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.

CRTC1 Function During Memory Encoding Is Disrupted in Neurodegeneration.

PubMed

Parra-Damas, Arnaldo; Chen, Meng; Enriquez-Barreto, Lilian; Ortega, Laura; Acosta, Sara; Perna, Judith Camats; Fullana, M Neus; Aguilera, José; Rodríguez-Alvarez, José; Saura, Carlos A

2017-01-15

Associative memory impairment is an early clinical feature of dementia patients, but the molecular and cellular mechanisms underlying these deficits are largely unknown. In this study, we investigated the functional regulation of the cyclic adenosine monophosphate response element binding protein (CREB)-regulated transcription coactivator 1 (CRTC1) by associative learning in physiological and neurodegenerative conditions. We evaluated the activation of CRTC1 in the hippocampus of control mice and mice lacking the Alzheimer's disease-linked presenilin genes (presenilin conditional double knockout [PS cDKO]) after one-trial contextual fear conditioning by using biochemical, immunohistochemical, and gene expression analyses. PS cDKO mice display classical features of neurodegeneration occurring in Alzheimer's disease including age-dependent cortical atrophy, neuron loss, dendritic degeneration, and memory deficits. Context-associative learning, but not single context or unconditioned stimuli, induces rapid dephosphorylation (Ser151) and translocation of CRTC1 from the cytosol/dendrites to the nucleus of hippocampal neurons in the mouse brain. Accordingly, context-associative learning induces differential CRTC1-dependent transcription of c-fos and the nuclear receptor subfamily 4 (Nr4a) genes Nr4a1-3 in the hippocampus through a mechanism that involves CRTC1 recruitment to CRE promoters. Deregulation of CRTC1 dephosphorylation, nuclear translocation, and transcriptional function are associated with long-term contextual memory deficits in PS cDKO mice. Importantly, CRTC1 gene therapy in the hippocampus ameliorates context memory and transcriptional deficits and dendritic degeneration despite ongoing cortical degeneration in this neurodegeneration mouse model. These findings reveal a critical role of CRTC1 in the hippocampus during associative memory, and provide evidence that CRTC1 deregulation underlies memory deficits during neurodegeneration. Copyright © 2016

Focal adhesion kinase regulates neuronal growth, synaptic plasticity and hippocampus-dependent spatial learning and memory.

PubMed

Monje, Francisco J; Kim, Eun-Jung; Pollak, Daniela D; Cabatic, Maureen; Li, Lin; Baston, Arthur; Lubec, Gert

2012-01-01

The focal adhesion kinase (FAK) is a non-receptor tyrosine kinase abundantly expressed in the mammalian brain and highly enriched in neuronal growth cones. Inhibitory and facilitatory activities of FAK on neuronal growth have been reported and its role in neuritic outgrowth remains controversial. Unlike other tyrosine kinases, such as the neurotrophin receptors regulating neuronal growth and plasticity, the relevance of FAK for learning and memory in vivo has not been clearly defined yet. A comprehensive study aimed at determining the role of FAK in neuronal growth, neurotransmitter release and synaptic plasticity in hippocampal neurons and in hippocampus-dependent learning and memory was therefore undertaken using the mouse model. Gain- and loss-of-function experiments indicated that FAK is a critical regulator of hippocampal cell morphology. FAK mediated neurotrophin-induced neuritic outgrowth and FAK inhibition affected both miniature excitatory postsynaptic potentials and activity-dependent hippocampal long-term potentiation prompting us to explore the possible role of FAK in spatial learning and memory in vivo. Our data indicate that FAK has a growth-promoting effect, is importantly involved in the regulation of the synaptic function and mediates in vivo hippocampus-dependent spatial learning and memory. Copyright © 2011 S. Karger AG, Basel.

Caveolin-1 regulates shear stress-dependent activation of extracellular signal-regulated kinase

NASA Technical Reports Server (NTRS)

Park, H.; Go, Y. M.; Darji, R.; Choi, J. W.; Lisanti, M. P.; Maland, M. C.; Jo, H.

2000-01-01

Fluid shear stress activates a member of the mitogen-activated protein (MAP) kinase family, extracellular signal-regulated kinase (ERK), by mechanisms dependent on cholesterol in the plasma membrane in bovine aortic endothelial cells (BAEC). Caveolae are microdomains of the plasma membrane that are enriched with cholesterol, caveolin, and signaling molecules. We hypothesized that caveolin-1 regulates shear activation of ERK. Because caveolin-1 is not exposed to the outside, cells were minimally permeabilized by Triton X-100 (0.01%) to deliver a neutralizing, polyclonal caveolin-1 antibody (pCav-1) inside the cells. pCav-1 then bound to caveolin-1 and inhibited shear activation of ERK but not c-Jun NH(2)-terminal kinase. Epitope mapping studies showed that pCav-1 binds to caveolin-1 at two regions (residues 1-21 and 61-101). When the recombinant proteins containing the epitopes fused to glutathione-S-transferase (GST-Cav(1-21) or GST-Cav(61-101)) were preincubated with pCav-1, only GST-Cav(61-101) reversed the inhibitory effect of the antibody on shear activation of ERK. Other antibodies, including m2234, which binds to caveolin-1 residues 1-21, had no effect on shear activation of ERK. Caveolin-1 residues 61-101 contain the scaffolding and oligomerization domains, suggesting that binding of pCav-1 to these regions likely disrupts the clustering of caveolin-1 or its interaction with signaling molecules involved in the shear-sensitive ERK pathway. We suggest that caveolae-like domains play a critical role in the mechanosensing and/or mechanosignal transduction of the ERK pathway.

Cyclin-dependent kinase 5 regulates degranulation in human eosinophils.

PubMed

Odemuyiwa, Solomon O; Ilarraza, Ramses; Davoine, Francis; Logan, Michael R; Shayeganpour, Anooshirvan; Wu, Yingqi; Majaesic, Carina; Adamko, Darryl J; Moqbel, Redwan; Lacy, Paige

2015-04-01

Degranulation from eosinophils in response to secretagogue stimulation is a regulated process that involves exocytosis of granule proteins through specific signalling pathways. One potential pathway is dependent on cyclin-dependent kinase 5 (Cdk5) and its effector molecules, p35 and p39, which play a central role in neuronal cell exocytosis by phosphorylating Munc18, a regulator of SNARE binding. Emerging evidence suggests a role for Cdk5 in exocytosis in immune cells, although its role in eosinophils is not known. We sought to examine the expression of Cdk5 and its activators in human eosinophils, and to assess the role of Cdk5 in eosinophil degranulation. We used freshly isolated human eosinophils and analysed the expression of Cdk5, p35, p39 and Munc18c by Western blot, RT-PCR, flow cytometry and immunoprecipitation. Cdk5 kinase activity was determined following eosinophil activation. Cdk5 inhibitors were used (roscovitine, AT7519 and small interfering RNA) to determine its role in eosinophil peroxidase (EPX) secretion. Cdk5 was expressed in association with Munc18c, p35 and p39, and phosphorylated following human eosinophil activation with eotaxin/CCL11, platelet-activating factor, and secretory IgA-Sepharose. Cdk5 inhibitors (roscovitine, AT7519) reduced EPX release when cells were stimulated by PMA or secretory IgA. In assays using small interfering RNA knock-down of Cdk5 expression in human eosinophils, we observed inhibition of EPX release. Our findings suggest that in activated eosinophils, Cdk5 is phosphorylated and binds to Munc18c, resulting in Munc18c release from syntaxin-4, allowing SNARE binding and vesicle fusion, with subsequent eosinophil degranulation. Our work identifies a novel role for Cdk5 in eosinophil mediator release by agonist-induced degranulation. © 2014 John Wiley & Sons Ltd.

An apple NAC transcription factor negatively regulates cold tolerance via CBF-dependent pathway.

PubMed

An, Jian-Ping; Li, Rui; Qu, Feng-Jia; You, Chun-Xiang; Wang, Xiao-Fei; Hao, Yu-Jin

2018-02-01

Cold stress is an adverse stimulus that affects plant growth and development, and the C-repeat binding factor (CBF) cold-regulatory cascade has been regarded as a master regulator in the plant response to cold stress. Here, we showed that a NAC transcription factor modulated low-temperature tolerance. MdNAC029/MdNAP, an apple NAC gene was isolated and its role in regulating cold tolerance was investigated. MdNAC029 was responsive to low-temperature treatment, and over-expression of MdNAC029 reduced cold tolerance in apple calli and Arabidopsis. Furthermore, EMSA assays and transient expression assays demonstrated that MdNAC029 directly repressed the expression of MdCBF1 and MdCBF4 by binding to their promoters. Taken together, our data suggest that MdNAC029 functions as a negative regulator in regulating plant cold tolerance in a CBF-dependent manner, providing a deeper understanding of NAC transcription-factor-mediated cold tolerance. Copyright © 2017 Elsevier GmbH. All rights reserved.

Size-Dependent Regulation of Intracellular Trafficking of Polystyrene Nanoparticle-Based Drug-Delivery Systems.

PubMed

Wang, Ting; Wang, Lu; Li, Xiaoming; Hu, Xingjie; Han, Yuping; Luo, Yao; Wang, Zejun; Li, Qian; Aldalbahi, Ali; Wang, Lihua; Song, Shiping; Fan, Chunhai; Zhao, Yun; Wang, Maolin; Chen, Nan

2017-06-07

Nanoparticles (NPs) have shown great promise as intracellular imaging probes or nanocarriers and are increasingly being used in biomedical applications. A detailed understanding of how NPs get "in and out" of cells is important for developing new nanomaterials with improved selectivity and less cytotoxicity. Both physical and chemical characteristics have been proven to regulate the cellular uptake of NPs. However, the exocytosis process and its regulation are less explored. Herein, we investigated the size-regulated endocytosis and exocytosis of carboxylated polystyrene (PS) NPs. PS NPs with a smaller size were endocytosed mainly through the clathrin-dependent pathway, whereas PS NPs with a larger size preferred caveolae-mediated endocytosis. Furthermore, our results revealed exocytosis of larger PS NPs and tracked the dynamic process at the single-particle level. These results indicate that particle size is a key factor for the regulation of intracellular trafficking of NPs and provide new insight into the development of more effective cellular nanocarriers.

Root Cell-Specific Regulators of Phosphate-Dependent Growth1[OPEN

PubMed Central

Ding, Wona

2017-01-01

Cellular specialization in abiotic stress responses is an important regulatory feature driving plant acclimation. Our in silico approach of iterative coexpression, interaction, and enrichment analyses predicted root cell-specific regulators of phosphate starvation response networks in Arabidopsis (Arabidopsis thaliana). This included three uncharacterized genes termed Phosphate starvation-induced gene interacting Root Cell Enriched (PRCE1, PRCE2, and PRCE3). Root cell-specific enrichment of 12 candidates was confirmed in promoter-GFP lines. T-DNA insertion lines of 11 genes showed changes in phosphate status and growth responses to phosphate availability compared with the wild type. Some mutants (cbl1, cipk2, prce3, and wdd1) displayed strong biomass gain irrespective of phosphate supply, while others (cipk14, mfs1, prce1, prce2, and s6k2) were able to sustain growth under low phosphate supply better than the wild type. Notably, root or shoot phosphate accumulation did not strictly correlate with organ growth. Mutant response patterns markedly differed from those of master regulators of phosphate homeostasis, PHOSPHATE STARVATION RESPONSE1 (PHR1) and PHOSPHATE2 (PHO2), demonstrating that negative growth responses in the latter can be overcome when cell-specific regulators are targeted. RNA sequencing analysis highlighted the transcriptomic plasticity in these mutants and revealed PHR1-dependent and -independent regulatory circuits with gene coexpression profiles that were highly correlated to the quantified physiological traits. The results demonstrate how in silico prediction of cell-specific, stress-responsive genes uncovers key regulators and how their manipulation can have positive impacts on plant growth under abiotic stress. PMID:28465462

Oligomerization-Dependent Regulation of Motility and Morphogenesis by the Collagen Xviii Nc1/Endostatin Domain

PubMed Central

Kuo, Calvin J.; LaMontagne, Kenneth R.; Garcia-Cardeña, Guillermo; Ackley, Brian D.; Kalman, Daniel; Park, Susan; Christofferson, Rolf; Kamihara, Junne; Ding, Yuan-Hua; Lo, Kin-Ming; Gillies, Stephen; Folkman, Judah; Mulligan, Richard C.; Javaherian, Kashi

2001-01-01

Collagen XVIII (c18) is a triple helical endothelial/epithelial basement membrane protein whose noncollagenous (NC)1 region trimerizes a COOH-terminal endostatin (ES) domain conserved in vertebrates, Caenorhabditis elegans and Drosophila. Here, the c18 NC1 domain functioned as a motility-inducing factor regulating the extracellular matrix (ECM)-dependent morphogenesis of endothelial and other cell types. This motogenic activity required ES domain oligomerization, was dependent on rac, cdc42, and mitogen-activated protein kinase, and exhibited functional distinction from the archetypal motogenic scatter factors hepatocyte growth factor and macrophage stimulatory protein. The motility-inducing and mitogen-activated protein kinase–stimulating activities of c18 NC1 were blocked by its physiologic cleavage product ES monomer, consistent with a proteolysis-dependent negative feedback mechanism. These data indicate that the collagen XVIII NC1 region encodes a motogen strictly requiring ES domain oligomerization and suggest a previously unsuspected mechanism for ECM regulation of motility and morphogenesis. PMID:11257123

BMAL1-dependent regulation of the mTOR signaling pathway delays aging

PubMed Central

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. PMID:24481314

Novel microscopy-based screening method reveals regulators of contact-dependent intercellular transfer

PubMed Central

Michael Frei, Dominik; Hodneland, Erlend; Rios-Mondragon, Ivan; Burtey, Anne; Neumann, Beate; Bulkescher, Jutta; Schölermann, Julia; Pepperkok, Rainer; Gerdes, Hans-Hermann; Kögel, Tanja

2015-01-01

Contact-dependent intercellular transfer (codeIT) of cellular constituents can have functional consequences for recipient cells, such as enhanced survival and drug resistance. Pathogenic viruses, prions and bacteria can also utilize this mechanism to spread to adjacent cells and potentially evade immune detection. However, little is known about the molecular mechanism underlying this intercellular transfer process. Here, we present a novel microscopy-based screening method to identify regulators and cargo of codeIT. Single donor cells, carrying fluorescently labelled endocytic organelles or proteins, are co-cultured with excess acceptor cells. CodeIT is quantified by confocal microscopy and image analysis in 3D, preserving spatial information. An siRNA-based screening using this method revealed the involvement of several myosins and small GTPases as codeIT regulators. Our data indicates that cellular protrusions and tubular recycling endosomes are important for codeIT. We automated image acquisition and analysis to facilitate large-scale chemical and genetic screening efforts to identify key regulators of codeIT. PMID:26271723

Thioredoxin h regulates calcium dependent protein kinases in plasma membranes.

PubMed

Ueoka-Nakanishi, Hanayo; Sazuka, Takashi; Nakanishi, Yoichi; Maeshima, Masayoshi; Mori, Hitoshi; Hisabori, Toru

2013-07-01

Thioredoxin (Trx) is a key player in redox homeostasis in various cells, modulating the functions of target proteins by catalyzing a thiol-disulfide exchange reaction. Target proteins of cytosolic Trx-h of higher plants were studied, particularly in the plasma membrane, because plant plasma membranes include various functionally important protein molecules such as transporters and signal receptors. Plasma membrane proteins from Arabidopsis thaliana cell cultures were screened using a resin Trx-h1 mutant-immobilized, and a total of 48 candidate proteins obtained. These included two calcium-sensing proteins: a phosphoinositide-specific phospholipase 2 (AtPLC2) and a calcium-dependent protein kinase 21 (AtCPK21). A redox-dependent change in AtCPK21 kinase activity was demonstrated in vitro. Oxidation of AtCPK21 resulted in a decrease in kinase activity to 19% of that of untreated AtCPK21, but Trx-h1 effectively restored the activity to 90%. An intramolecular disulfide bond (Cys97-Cys108) that is responsible for this redox modulation was then identified. In addition, endogenous AtCPK21 was shown to be oxidized in vivo when the culture cells were treated with H2 O2 . These results suggest that redox regulation of AtCPK21 by Trx-h in response to external stimuli is important for appropriate cellular responses. The relationship between the redox regulation system and Ca(2+) signaling pathways is discussed. © 2013 The Authors. FEBS Journal published by John Wiley & Sons Ltd on behalf of FEBS.

RhoG regulates anoikis through a phosphatidylinositol 3-kinase-dependent mechanism

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

Yamaki, Nao; Negishi, Manabu; Katoh, Hironori

2007-08-01

In normal epithelial cells, cell-matrix interaction is required for cell survival and proliferation, whereas disruption of this interaction causes epithelial cells to undergo apoptosis called anoikis. Here we show that the small GTPase RhoG plays an important role in the regulation of anoikis. HeLa cells are capable of anchorage-independent cell growth and acquire resistance to anoikis. We found that RNA interference-mediated knockdown of RhoG promoted anoikis in HeLa cells. Previous studies have shown that RhoG activates Rac1 and induces several cellular functions including promotion of cell migration through its effector ELMO and the ELMO-binding protein Dock180 that function as amore » Rac-specific guanine nucleotide exchange factor. However, RhoG-induced suppression of anoikis was independent of the ELMO- and Dock180-mediated activation of Rac1. On the other hand, the regulation of anoikis by RhoG required phosphatidylinositol 3-kinase (PI3K) activity, and constitutively active RhoG bound to the PI3K regulatory subunit p85{alpha} and induced the PI3K-dependent phosphorylation of Akt. Taken together, these results suggest that RhoG protects cells from apoptosis caused by the loss of anchorage through a PI3K-dependent mechanism, independent of its activation of Rac1.« less

Ethylene regulates Apple (Malus x domestica) fruit softening through a dose x time-dependent mechanism and through differential sensitivities and dependencies of cell wall-modifying genes.

PubMed

Ireland, Hilary S; Gunaseelan, Kularajathevan; Muddumage, Ratnasiri; Tacken, Emma J; Putterill, Jo; Johnston, Jason W; Schaffer, Robert J

2014-05-01

In fleshy fruit species that have a strong requirement for ethylene to ripen, ethylene is synthesized autocatalytically, producing increasing concentrations as the fruits ripen. Apple fruit with the ACC OXIDASE 1 (ACO1) gene suppressed cannot produce ethylene autocatalytically at ripening. Using these apple lines, an ethylene sensitivity dependency model was previously proposed, with traits such as softening showing a high dependency for ethylene as well as low sensitivity. In this study, it is shown that the molecular control of fruit softening is a complex process, with different cell wall-related genes being independently regulated and exhibiting differential sensitivities to and dependencies on ethylene at the transcriptional level. This regulation is controlled through a dose × time mechanism, which results in a temporal transcriptional response that would allow for progressive cell wall disassembly and thus softening. This research builds on the sensitivity dependency model and shows that ethylene-dependent traits can progress over time to the same degree with lower levels of ethylene. This suggests that a developmental clock measuring cumulative ethylene controls the fruit ripening process.

Regulation of Arabidopsis leaf hydraulics involves light-dependent phosphorylation of aquaporins in veins.

PubMed

Prado, Karine; Boursiac, Yann; Tournaire-Roux, Colette; Monneuse, Jean-Marc; Postaire, Olivier; Da Ines, Olivier; Schäffner, Anton R; Hem, Sonia; Santoni, Véronique; Maurel, Christophe

2013-03-01

The water status of plant leaves depends on the efficiency of the water supply, from the vasculature to inner tissues. This process is under hormonal and environmental regulation and involves aquaporin water channels. In Arabidopsis thaliana, the rosette hydraulic conductivity (Kros) is higher in darkness than it is during the day. Knockout plants showed that three plasma membrane intrinsic proteins (PIPs) sharing expression in veins (PIP1;2, PIP2;1, and PIP2;6) contribute to rosette water transport, and PIP2;1 can fully account for Kros responsiveness to darkness. Directed expression of PIP2;1 in veins of a pip2;1 mutant was sufficient to restore Kros. In addition, a positive correlation, in both wild-type and PIP2;1-overexpressing plants, was found between Kros and the osmotic water permeability of protoplasts from the veins but not from the mesophyll. Thus, living cells in veins form a major hydraulic resistance in leaves. Quantitative proteomic analyses showed that light-dependent regulation of Kros is linked to diphosphorylation of PIP2;1 at Ser-280 and Ser-283. Expression in pip2;1 of phosphomimetic and phosphorylation-deficient forms of PIP2;1 demonstrated that phosphorylation at these two sites is necessary for Kros enhancement under darkness. These findings establish how regulation of a single aquaporin isoform in leaf veins critically determines leaf hydraulics.

Effect of intra-membrane C60 fullerenes on the modulus of elasticity and the mechanical resistance of gel and fluid lipid bilayers

NASA Astrophysics Data System (ADS)

Zhou, Jihan; Liang, Dehai; Contera, Sonia

2015-10-01

Penetration and partition of C60 to the lipid bilayer core are both relevant to C60 toxicity, and useful to realise C60 biomedical potential. A key aspect is the effect of C60 on bilayer mechanical properties. Here, we present an experimental study on the mechanical effect of the incorporation of C60 into the hydrophobic core of fluid and gel phase zwitterionic phosphatidylcholine (PC) lipid bilayers. We demonstrate its incorporation inside the hydrophobic lipid core and the effect on the packing of the lipids and the vesicle size using a combination of infrared (IR) spectroscopy, atomic force microscopy (AFM) and laser light scattering. Using AFM we measured the Young's modulus of elasticity (E) of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in the absence (presence) of intra-membranous C60 at 24.5 °C. E of fluid phase supported bilayers is not altered by C60, but E increases with incorporation of C60 in gel phase bilayers. The increase is higher for longer hydrocarbon chains: 1.6 times for DPPC and 2 times for DSPC. However the mechanical resistance of gel phase bilayers of curved bilayered structures decreases with the incorporation of C60. Our combined results indicate that C60 causes a decrease in gel phase lipid mobility, i.e. an increase in membrane viscosity.

The Golgi apparatus regulates cGMP-dependent protein kinase I compartmentation and proteolysis.

PubMed

Kato, Shin; Chen, Jingsi; Cornog, Katherine H; Zhang, Huili; Roberts, Jesse D

2015-06-01

cGMP-dependent protein kinase I (PKGI) is an important effector of cGMP signaling that regulates vascular smooth muscle cell (SMC) phenotype and proliferation. PKGI has been detected in the perinuclear region of cells, and recent data indicate that proprotein convertases (PCs) typically resident in the Golgi apparatus (GA) can stimulate PKGI proteolysis and generate a kinase fragment that localizes to the nucleus and regulates gene expression. However, the role of the endomembrane system in PKGI compartmentation and processing is unknown. Here, we demonstrate that PKGI colocalizes with endoplasmic reticulum (ER), ER-Golgi intermediate compartment, GA cisterna, and trans-Golgi network proteins in pulmonary artery SMC and cell lines. Moreover, PKGI localizes with furin, a trans-Golgi network-resident PC known to cleave PKGI. ER protein transport influences PKGI localization because overexpression of a constitutively inactive Sar1 transgene caused PKGI retention in the ER. Additionally, PKGI appears to reside within the GA because PKGI immunoreactivity was determined to be resistant to cytosolic proteinase K treatment in live cells. The GA appears to play a role in PKGI proteolysis because overexpression of inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate, not only tethered heterologous PKGI-β to the ER and decreased its localization to the GA, but also diminished PKGI proteolysis and nuclear translocation. Also, inhibiting intra-GA protein transport with monensin was observed to decrease PKGI cleavage. These studies detail a role for the endomembrane system in regulating PKGI compartmentation and proteolysis. Moreover, they support the investigation of mechanisms regulating PKGI-dependent nuclear cGMP signaling in the pulmonary vasculature with Golgi dysfunction. Copyright © 2015 the American Physiological Society.

MicroRNAs-Dependent Regulation of PPARs in Metabolic Diseases and Cancers

PubMed Central

Portius, Dorothea; Sobolewski, Cyril

2017-01-01

Peroxisome proliferator-activated receptors (PPARs) are a family of ligand-dependent nuclear receptors, which control the transcription of genes involved in energy homeostasis and inflammation and cell proliferation/differentiation. Alterations of PPARs' expression and/or activity are commonly associated with metabolic disorders occurring with obesity, type 2 diabetes, and fatty liver disease, as well as with inflammation and cancer. Emerging evidence now indicates that microRNAs (miRNAs), a family of small noncoding RNAs, which fine-tune gene expression, play a significant role in the pathophysiological mechanisms regulating the expression and activity of PPARs. Herein, the regulation of PPARs by miRNAs is reviewed in the context of metabolic disorders, inflammation, and cancer. The reciprocal control of miRNAs expression by PPARs, as well as the therapeutic potential of modulating PPAR expression/activity by pharmacological compounds targeting miRNA, is also discussed. PMID:28167956

Restricted growth and insulin-like growth factor-1 deficiency in mice lacking presenilin-1 in the neural crest cell lineage

PubMed Central

Nakajima, Mitsunari; Watanabe, Sono; Okuyama, Satoshi; Shen, Jie; Furukawa, Yoshiko

2012-01-01

Presenilin-1 (PS1) is a transmembrane protein that is in many cases responsible for the development of early-onset familial Alzheimer’s disease. PS1 is essential for neurogenesis, somitogenesis, angiogenesis, and cardiac morphogenesis. We report here that PS1 is also required for maturation and/or maintenance of the pituitary gland. We generated PS1-conditional knockout (PS1-cKO) mice by crossing floxed PS1 and Wnt1-cre mice, in which PS1 was lacking in the neural crest-derived cell lineage. Although the PS1-cKO mice exhibited no obvious phenotypic abnormalities for several days after birth, reduced body weight in the mutant was evident by the age of 3 to 5 weeks. Pituitary weight and serum insulin-like growth factor (IGF)-1 level were also reduced in the mutant. Histologic analysis revealed severe atrophy of the cytosol in the anterior and intermediate pituitary lobes of the mutant. Immunohistochemistry did not reveal clear differences in the expression levels of thyroid-stimulating hormone, adrenocorticotropic hormone, or prolactin in the mutant pituitary. In contrast, growth hormone expression levels were reduced in the anterior lobe of the mutant. PS1 was defective in the posterior lobe, but not the anterior or intermediate lobes, in the mutant pituitary. These findings suggest that PS1 indirectly mediates the development and/or maintenance of the anterior and intermediate lobes in the pituitary gland via actions in other regions, such as the posterior lobe. PMID:19665542

Cell cycle-dependent regulation of Greatwall kinase by protein phosphatase 1 and regulatory subunit 3B.

PubMed

Ren, Dapeng; Fisher, Laura A; Zhao, Jing; Wang, Ling; Williams, Byron C; Goldberg, Michael L; Peng, Aimin

2017-06-16

Greatwall (Gwl) kinase plays an essential role in the regulation of mitotic entry and progression. Mitotic activation of Gwl requires both cyclin-dependent kinase 1 (CDK1)-dependent phosphorylation and its autophosphorylation at an evolutionarily conserved serine residue near the carboxyl terminus (Ser-883 in Xenopus ). In this study we show that Gwl associates with protein phosphatase 1 (PP1), particularly PP1γ, which mediates the dephosphorylation of Gwl Ser-883. Consistent with the mitotic activation of Gwl, its association with PP1 is disrupted in mitotic cells and egg extracts. During mitotic exit, PP1-dependent dephosphorylation of Gwl Ser-883 occurs prior to dephosphorylation of other mitotic substrates; replacing endogenous Gwl with a phosphomimetic S883E mutant blocks mitotic exit. Moreover, we identified PP1 regulatory subunit 3B (PPP1R3B) as a targeting subunit that can direct PP1 activity toward Gwl. PPP1R3B bridges PP1 and Gwl association and promotes Gwl Ser-883 dephosphorylation. Consistent with the cell cycle-dependent association of Gwl and PP1, Gwl and PPP1R3B dissociate in M phase. Interestingly, up-regulation of PPP1R3B facilitates mitotic exit and blocks mitotic entry. Thus, our study suggests PPP1R3B as a new cell cycle regulator that functions by governing Gwl dephosphorylation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

An interactive network of elastase, secretases, and PAR-2 protein regulates CXCR1 receptor surface expression on neutrophils.

PubMed

Bakele, Martina; Lotz-Havla, Amelie S; Jakowetz, Anja; Carevic, Melanie; Marcos, Veronica; Muntau, Ania C; Gersting, Soeren W; Hartl, Dominik

2014-07-25

CXCL8 (IL-8) recruits and activates neutrophils through the G protein-coupled chemokine receptor CXCR1. We showed previously that elastase cleaves CXCR1 and thereby impairs antibacterial host defense. However, the molecular intracellular machinery involved in this process remained undefined. Here we demonstrate by using flow cytometry, confocal microscopy, subcellular fractionation, co-immunoprecipitation, and bioluminescence resonance energy transfer that combined α- and γ-secretase activities are functionally involved in elastase-mediated regulation of CXCR1 surface expression on human neutrophils, whereas matrix metalloproteases are dispensable. We further demonstrate that PAR-2 is stored in mobilizable compartments in neutrophils. Bioluminescence resonance energy transfer and co-immunoprecipitation studies showed that secretases, PAR-2, and CXCR1 colocalize and physically interact in a novel protease/secretase-chemokine receptor network. PAR-2 blocking experiments provided evidence that elastase increased intracellular presenilin-1 expression through PAR-2 signaling. When viewed in combination, these studies establish a novel functional network of elastase, secretases, and PAR-2 that regulate CXCR1 expression on neutrophils. Interfering with this network could lead to novel therapeutic approaches in neutrophilic diseases, such as cystic fibrosis or rheumatoid arthritis.

An Interactive Network of Elastase, Secretases, and PAR-2 Protein Regulates CXCR1 Receptor Surface Expression on Neutrophils*

PubMed Central

Bakele, Martina; Lotz-Havla, Amelie S.; Jakowetz, Anja; Carevic, Melanie; Marcos, Veronica; Muntau, Ania C.; Gersting, Soeren W.; Hartl, Dominik

2014-01-01

CXCL8 (IL-8) recruits and activates neutrophils through the G protein-coupled chemokine receptor CXCR1. We showed previously that elastase cleaves CXCR1 and thereby impairs antibacterial host defense. However, the molecular intracellular machinery involved in this process remained undefined. Here we demonstrate by using flow cytometry, confocal microscopy, subcellular fractionation, co-immunoprecipitation, and bioluminescence resonance energy transfer that combined α- and γ-secretase activities are functionally involved in elastase-mediated regulation of CXCR1 surface expression on human neutrophils, whereas matrix metalloproteases are dispensable. We further demonstrate that PAR-2 is stored in mobilizable compartments in neutrophils. Bioluminescence resonance energy transfer and co-immunoprecipitation studies showed that secretases, PAR-2, and CXCR1 colocalize and physically interact in a novel protease/secretase-chemokine receptor network. PAR-2 blocking experiments provided evidence that elastase increased intracellular presenilin-1 expression through PAR-2 signaling. When viewed in combination, these studies establish a novel functional network of elastase, secretases, and PAR-2 that regulate CXCR1 expression on neutrophils. Interfering with this network could lead to novel therapeutic approaches in neutrophilic diseases, such as cystic fibrosis or rheumatoid arthritis. PMID:24914212

Phenotypic Variability in Autosomal Dominant Familial Alzheimer Disease due to the S170F Mutation of Presenilin-1.

PubMed

Tiedt, Hannes O; Benjamin, Beate; Niedeggen, Michael; Lueschow, Andreas

2018-02-22

In rare cases, patients with Alzheimer disease (AD) present at an early age and with a family history suggestive of an autosomal dominant mode of inheritance. Mutations of the presenilin-1 (PSEN1) gene are the most common causes of dementia in these patients. Early-onset and particularly familial AD patients frequently present with variable non-amnestic cognitive symptoms such as visual, language or behavioural changes as well as non-cognitive, e.g. motor, symptoms. To investigate the phenotypic variability in carriers of the PSEN1 S170F mutation. We report a family with 4 patients carrying the S170F mutation of whom 2 underwent detailed clinical examinations. We discuss our current findings in the context of previously reported S170F cases. The clinical phenotype was consistent regarding initial memory impairment and early onset in the late twenties found in all S170F patients. There were frequent non-amnestic cognitive changes and, at early stages of the disease, indications of a more pronounced disturbance of visuospatial abilities as compared to face and object recognition. Non-cognitive symptoms most often included myoclonus and cerebellar ataxia. A review of the available case reports indicates some phenotypic variability associated with the S170F mutation including different constellations of symptoms such as parkinsonism and delusions. The variable clinical findings associated with the S170F mutation highlight the relevance of atypical phenotypes in the context of research and under a clinical perspective. CSF sampling and detection of Aβ species may be essential to indicate AD pathology in unclear cases presenting with cognitive and motor symptoms at a younger age. © 2018 S. Karger AG, Basel.

Lanthanide-Dependent Regulation of Methylotrophy in Methylobacterium aquaticum Strain 22A

PubMed Central

Masuda, Sachiko; Suzuki, Yutaka; Fujitani, Yoshiko; Mitsui, Ryoji; Nakagawa, Tomoyuki

2018-01-01

ABSTRACT Methylobacterium species are representative of methylotrophic bacteria. Their genomes usually encode two types of methanol dehydrogenases (MDHs): MxaF and XoxF. The former is a Ca2+-dependent enzyme, and the latter was recently determined to be a lanthanide-dependent enzyme that is necessary for the expression of mxaF. This finding revealed the unexpected and important roles of lanthanides in bacterial methylotrophy. In this study, we performed transcriptome sequencing (RNA-seq) analysis using M. aquaticum strain 22A grown in the presence of different lanthanides. Expression of mxaF and xoxF1 genes showed a clear inverse correlation in response to La3+. We observed downregulation of formaldehyde oxidation pathways, high formaldehyde dehydrogenase activity, and low accumulation of formaldehyde in the reaction with cells grown in the presence of La3+; this might be due to the direct oxidation of methanol to formate by XoxF1. Lanthanides induced the transcription of AT-rich genes, the function of most of which was unknown, and genes possibly related to cellular survival, as well as other MDH homologues. These results revealed not only the metabolic response toward altered primary methanol oxidation, but also the possible targets to be investigated further in order to better understand methylotrophy in the presence of lanthanides. IMPORTANCE Lanthanides have been considered unimportant for biological processes. In methylotrophic bacteria, however, a methanol dehydrogenase (MDH) encoded by xoxF was recently found to be lanthanide dependent, while the classic-type mxaFI is calcium dependent. XoxF-type MDHs are more widespread in diverse bacterial genera, suggesting their importance for methylotrophy. Methylobacterium species, representative methylotrophic and predominating alphaproteobacteria in the phyllosphere, contain both types and regulate their expression depending on the availability of lanthanides. RNA-seq analysis showed that the regulation takes place

Phosphorylation-dependent down-regulation of apolipoprotein A5 by insulin

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

Nowak, Maxine; Helleboid-Chapman, Audrey; Jakel, Heidelinde

2004-02-15

The apolipoprotein A5 (APOA5) gene has been shown to be important in lowering plasma triglyceride levels. Since several studies have shown that hyperinsulinemia is associated with hypertriglyceridemia, we sought to determine whether APOA5 gene is regulated by insulin. We show here that cell and mouse treatments with insulin down-regulated APOA5 expression in a dose-dependent manner. Furthermore, we determined that insulin decreases APOA5 promoter activity and subsequent deletion analyses revealed an E-box-containing fragment. We showed that Upstream Stimulatory Factors, USF1/USF2, bind to the identified E-box in the APOA5 promoter. Moreover, in cotransfection studies, USF1 stimulates APOA5 promoter activity. The treatment withmore » insulin reduces the binding of USF1/USF2 to APOA5 promoter. The inhibition of PI3K pathway with wortmannin abolished the insulin s effect on APOA5 gene transcription. Using oligoprecipitation method of USF from nuclear extracts, we demonstrated that phosphorylated USF1 failed to bind to APOA5 promoter. This indicates that the APOA5 gene transrepression by insulin involves a phosphorylation of USF through PI3K, that modulate their binding to APOA5 promoter and results in APOA5 down-regulation. The effect of exogenous hyperinsulinemia in healthy men shows a decrease of the plasma ApoAV level. These data suggest a potential mechanism involving APOA5 gene in hypertriglyceridemia associated with hyperinsulinemia.« less

Redox regulation and reaction mechanism of human cystathionine-beta-synthase: a PLP-dependent hemesensor protein.

PubMed

Banerjee, Ruma; Zou, Cheng-Gang

2005-01-01

Cystathionine beta-synthase in mammals lies at a pivotal crossroad in methionine metabolism directing flux toward cysteine synthesis and catabolism. The enzyme exhibits a modular organization and complex regulation. It catalyzes the beta-replacement of the hydroxyl group of serine with the thiolate of homocysteine and is unique in being the only known pyridoxal phosphate-dependent enzyme that also contains heme b as a cofactor. The heme functions as a sensor and modulates enzyme activity in response to redox change and to CO binding. Mutations in this enzyme are the single most common cause of hereditary hyperhomocysteinemia. Elucidation of the crystal structure of a truncated and highly active form of the human enzyme containing the heme- and pyridoxal phosphate binding domains has afforded a structural perspective on mechanistic and mutation analysis studies. The C-terminal regulatory domain containing two CBS motifs exerts intrasteric regulation and binds the allosteric activator, S-adenosylmethionine. Studies with mammalian cells in culture as well as with animal models have unraveled multiple layers of regulation of cystathionine beta-synthase in response to redox perturbations and reveal the important role of this enzyme in glutathione-dependent redox homestasis. This review discusses the recent advances in our understanding of the structure, mechanism, and regulation of cystathionine beta-synthase from the perspective of its physiological function, focusing on the clinically relevant human enzyme.

Osteogenic and chondrogenic master genes expression is dependent on the Kir2.1 potassium channel through the bone morphogenetic protein pathway.

PubMed

Pini, Jonathan; Giuliano, Serena; Matonti, Julia; Simkin, Dina; Rouleau, Matthieu; Bendahhou, Saïd

2018-05-29

Andersen's syndrome is a rare disorder affecting muscle, heart, and bone, that is associated with mutations leading to a loss of function of the inwardly rectifying K + channel Kir2.1. While the Kir2.1 function can be anticipated in excitable cells by controlling the electrical activity, its role in non-excitable cells remains to be investigated. Using Andersen's syndrome induced Pluripotent Stem cells, we investigated the cellular and molecular events during the osteoblastic and chondrogenic differentiation that are affected by the loss of the Ik1 current. We show that loss of Kir2.1 channel function impairs both osteoblastic and chondrogenic processes through the down regulation master gene expression. This down regulation is due to an impairment of the bone morphogenetic proteins signaling pathway through de-phosphorylation of the Smad proteins. Restoring Kir2.1 channel function in Andersen's syndrome cells rescued master genes expression, and restored normal osteoblasts and chondrocytes behavior. Our results show that Kir2.1-mediated activity controls endochondral and intramembranous ossification signaling pathways. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

State-Dependent Riccati Equation Regulation of Systems with State and Control Nonlinearities

NASA Technical Reports Server (NTRS)

Beeler, Scott C.; Cox, David E. (Technical Monitor)

2004-01-01

The state-dependent Riccati equations (SDRE) is the basis of a technique for suboptimal feedback control of a nonlinear quadratic regulator (NQR) problem. It is an extension of the Riccati equation used for feedback control of linear problems, with the addition of nonlinearities in the state dynamics of the system resulting in a state-dependent gain matrix as the solution of the equation. In this paper several variations on the SDRE-based method will be considered for the feedback control problem with control nonlinearities. The control nonlinearities may result in complications in the numerical implementation of the control, which the different versions of the SDRE method must try to overcome. The control methods will be applied to three test problems and their resulting performance analyzed.

MK-STYX, a Catalytically Inactive Phosphatase Regulating Mitochondrially Dependent Apoptosis ▿

PubMed Central

Niemi, Natalie M.; Lanning, Nathan J.; Klomp, Jeff A.; Tait, Stephen W.; Xu, Yong; Dykema, Karl J.; Murphy, Leon O.; Gaither, L. Alex; Xu, H. Eric; Furge, Kyle A.; Green, Douglas R.; MacKeigan, Jeffrey P.

2011-01-01

Evasion of apoptosis is a significant problem affecting an array of cancers. In order to identify novel regulators of apoptosis, we performed an RNA interference (RNAi) screen against all kinases and phosphatases in the human genome. We identified MK-STYX (STYXL1), a catalytically inactive phosphatase with homology to the mitogen-activated protein kinase (MAPK) phosphatases. Despite this homology, MK-STYX knockdown does not significantly regulate MAPK signaling in response to growth factors or apoptotic stimuli. Rather, RNAi-mediated knockdown of MK-STYX inhibits cells from undergoing apoptosis induced by cellular stressors activating mitochondrion-dependent apoptosis. This MK-STYX phenotype mimics the loss of Bax and Bak, two potent guardians of mitochondrial apoptotic potential. Similar to loss of both Bax and Bak, cells without MK-STYX expression are unable to release cytochrome c. Proapoptotic members of the BCL-2 family (Bax, Bid, and Bim) are unable to trigger cytochrome c release in MK-STYX-depleted cells, placing the apoptotic deficiency at the level of mitochondrial outer membrane permeabilization (MOMP). MK-STYX was found to localize to the mitochondria but is neither released from the mitochondria upon apoptotic stress nor proximal to the machinery currently known to control MOMP, indicating that MK-STYX regulates MOMP using a distinct mechanism. PMID:21262771

Netrin-4 regulates thalamocortical axon branching in an activity-dependent fashion.

PubMed

Hayano, Yasufumi; Sasaki, Kensuke; Ohmura, Nami; Takemoto, Makoto; Maeda, Yurie; Yamashita, Toshihide; Hata, Yoshio; Kitada, Kazuhiro; Yamamoto, Nobuhiko

2014-10-21

Axon branching is remodeled by sensory-evoked and spontaneous neuronal activity. However, the underlying molecular mechanism is largely unknown. Here, we demonstrate that the netrin family member netrin-4 (NTN4) contributes to activity-dependent thalamocortical (TC) axon branching. In the postnatal developmental stages of rodents, ntn4 expression was abundant in and around the TC recipient layers of sensory cortices. Neuronal activity dramatically altered the ntn4 expression level in the cortex in vitro and in vivo. TC axon branching was promoted by exogenous NTN4 and suppressed by depletion of the endogenous protein. Moreover, unc-5 homolog B (Unc5B), which strongly bound to NTN4, was expressed in the sensory thalamus, and knockdown of Unc5B in thalamic cells markedly reduced TC axon branching. These results suggest that NTN4 acts as a positive regulator for TC axon branching through activity-dependent expression.

Thiamin diphosphate-dependent enzymes: from enzymology to metabolic regulation, drug design and disease models.

PubMed

Bunik, Victoria I; Tylicki, Adam; Lukashev, Nikolay V

2013-12-01

Bringing a knowledge of enzymology into research in vivo and in situ is of great importance in understanding systems biology and metabolic regulation. The central metabolic significance of thiamin (vitamin B1 ) and its diphosphorylated derivative (thiamin diphosphate; ThDP), and the fundamental differences in the ThDP-dependent enzymes of metabolic networks in mammals versus plants, fungi and bacteria, or in health versus disease, suggest that these enzymes are promising targets for biotechnological and medical applications. Here, the in vivo action of known regulators of ThDP-dependent enzymes, such as synthetic structural analogs of the enzyme substrates and thiamin, is analyzed in light of the enzymological data accumulated during half a century of research. Mimicking the enzyme-specific catalytic intermediates, the phosphonate analogs of 2-oxo acids selectively inhibit particular ThDP-dependent enzymes. Because of their selectivity, use of these compounds in cellular and animal models of ThDP-dependent enzyme malfunctions improves the validity of the model and its predictive power when compared with the nonselective and enzymatically less characterized oxythiamin and pyrithiamin. In vitro studies of the interaction of thiamin analogs and their biological derivatives with potential in vivo targets are necessary to identify and attenuate the analog selectivity. For both the substrate and thiamin synthetic analogs, in vitro reactivities with potential targets are highly relevant in vivo. However, effective concentrations in vivo are often higher than in vitro studies would suggest. The significance of specific inihibition of the ThDP-dependent enzymes for the development of herbicides, antibiotics, anticancer and neuroprotective strategies is discussed. © 2013 FEBS.

RIPK3 regulates p62-LC3 complex formation via the caspase-8-dependent cleavage of p62.

PubMed

Matsuzawa, Yu; Oshima, Shigeru; Nibe, Yoichi; Kobayashi, Masanori; Maeyashiki, Chiaki; Nemoto, Yasuhiro; Nagaishi, Takashi; Okamoto, Ryuichi; Tsuchiya, Kiichiro; Nakamura, Tetsuya; Watanabe, Mamoru

2015-01-02

RIPK3 is a key molecule for necroptosis, initially characterized by necrotic cell death morphology and the activation of autophagy. Cell death and autophagic signaling are believed to tightly regulate each other. However, the associated recruitment of signaling proteins remains poorly understood. p62/sequestosome-1 is a selective autophagy substrate and a selective receptor for ubiquitinated proteins. In this study, we illustrated that both mouse and human RIPK3 mediate p62 cleavage and that RIPK3 interacts with p62, resulting in complex formation. In addition, RIPK3-dependent p62 cleavage is restricted by the inhibition of caspases, especially caspase-8. Moreover, overexpression of A20, a ubiquitin-editing enzyme and an inhibitor of caspase-8 activity, inhibits RIPK3-dependent p62 cleavage. To further investigate the potential role of RIPK3 in selective autophagy, we analyzed p62-LC3 complex formation, revealing that RIPK3 prevents the localization of LC3 and ubiquitinated proteins to the p62 complex. In addition, RIPK3-dependent p62-LC3 complex disruption is regulated by caspase inhibition. Taken together, these results demonstrated that RIPK3 interacts with p62 and regulates p62-LC3 complex formation. These findings suggested that RIPK3 serves as a negative regulator of selective autophagy and provides new insights into the mechanism by which RIPK3 regulates autophagic signaling. Copyright © 2014 Elsevier Inc. All rights reserved.

Cop9/signalosome subunits and Pcu4 regulate ribonucleotide reductase by both checkpoint-dependent and -independent mechanisms

PubMed Central

Liu, Cong; Powell, Kelly A.; Mundt, Kirsten; Wu, LeJung; Carr, Antony M.; Caspari, Thomas

2003-01-01

The signalosome is implicated in regulating cullin-dependent ubiquitin ligases. We find that two signalosome subunits, Csn1 and Csn2, are required to regulate ribonucleotide reductase (RNR) through the degradation of a small protein, Spd1, that acts to anchor the small RNR subunit in the nucleus. Spd1 destruction correlates with the nuclear export of the small RNR subunit, which, in turn, correlates with a requirement for RNR in replication and repair. Spd1 degradation is promoted by two separate CSN-dependent mechanisms. During unperturbed S phase, Spd1 degradation is independent of checkpoint proteins. In irradiated G2 cells, Spd1 degradation requires the DNA damage checkpoint. The signalosome copurifies with Pcu4 (cullin 4). Pcu4, Csn1, and Csn2 promote the degradation of Spd1, identifying a new function for the signalosome as a regulator of Pcu4-containing E3 ubiquitin ligase. PMID:12695334

Microtubule-regulating proteins and cAMP-dependent signaling in neuroblastoma differentiation.

PubMed

Muñoz-Llancao, Pablo; de Gregorio, Cristian; Las Heras, Macarena; Meinohl, Christopher; Noorman, Kevin; Boddeke, Erik; Cheng, Xiaodong; Lezoualc'h, Frank; Schmidt, Martina; Gonzalez-Billault, Christian

2017-03-01

Neurons are highly differentiated cells responsible for the conduction and transmission of information in the nervous system. The proper function of a neuron relies on the compartmentalization of their intracellular domains. Differentiated neuroblastoma cells have been extensively used to study and understand the physiology and cell biology of neuronal cells. Here, we show that differentiation of N1E-115 neuroblastoma cells is more pronounced upon exposure of a chemical analog of cyclic AMP (cAMP), db-cAMP. We next analysed the expression of key microtubule-regulating proteins in differentiated cells and the expression and activation of key cAMP players such as EPAC, PKA and AKAP79/150. Most of the microtubule-promoting factors were up regulated during differentiation of N1E-115 cells, while microtubule-destabilizing proteins were down regulated. We observed an increase in tubulin post-translational modifications related to microtubule stability. As expected, db-cAMP increased PKA- and EPAC-dependent signalling. Consistently, pharmacological modulation of EPAC activity instructed cell differentiation, number of neurites, and neurite length in N1E-115 cells. Moreover, disruption of the PKA-AKAP interaction reduced these morphometric parameters. Interestingly, PKA and EPAC act synergistically to induce neuronal differentiation in N1E-115. Altogether these results show that the changes observed in the differentiation of N1E-115 cells proceed by regulating several microtubule-stabilizing factors, and the acquisition of a neuronal phenotype is a process involving concerted although independent functions of EPAC and PKA. © 2017 Wiley Periodicals, Inc.

Human I-mfa domain proteins specifically interact with KSHV LANA and affect its regulation of Wnt signaling-dependent transcription

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

Kusano, Shuichi, E-mail: skusano@m2.kufm.kagoshima-u.ac.jp; Eizuru, Yoshito

2010-06-04

Kaposi's sarcoma-associated herpes virus (KSHV)-encoded latency-associated nuclear antigen (LANA) protein has been reported to interact with glycogen synthase kinase 3{beta} (GSK-3{beta}) and to negatively regulate its activity, leading to stimulation of GSK-3{beta}-dependent {beta}-catenin degradation. We show here that the I-mfa domain proteins, HIC (human I-mfa domain-containing protein) and I-mfa (inhibitor of MyoD family a), interacted in vivo with LANA through their C-terminal I-mfa domains. This interaction affected the intracellular localization of HIC, inhibited the LANA-dependent transactivation of a {beta}-catenin-regulated reporter construct, and decreased the level of the LANA.GSK-3{beta} complex. These data reveal for the first time that I-mfa domain proteinsmore » interact with LANA and negatively regulate LANA-mediated activation of Wnt signaling-dependent transcription by inhibiting the formation of the LANA.GSK-3{beta} complex.« less

Differential regulation of cyclin-dependent kinase inhibitors in neuroblastoma cells

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

Qiao, Lan; Department of Pharmaceutical Sciences, Jilin University, Changchun 130021; Paul, Pritha

2013-05-31

Highlights: •GRP-R signaling differentially regulated the expression of p21 and p27. •Silencing GRP/GRP-R downregulated p21, while p27 expression was upregulated. •Inhibition of GRP/GRP-R signaling enhanced PTEN expression, correlative to the increased expression of p27. •PTEN and p27 co-localized in cytoplasm and silencing PTEN decreased p27 expression. -- Abstract: Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are highly expressed in undifferentiated neuroblastoma, and they play critical roles in oncogenesis. We previously reported that GRP activates the PI3K/AKT signaling pathway to promote DNA synthesis and cell cycle progression in neuroblastoma cells. Conversely, GRP-R silencing induces cell cycle arrest. Here, we speculated thatmore » GRP/GRP-R signaling induces neuroblastoma cell proliferation via regulation of cyclin-dependent kinase (CDK) inhibitors. Surprisingly, we found that GRP/GRP-R differentially induced expressions of p21 and p27. Silencing GRP/GRP-R decreased p21, but it increased p27 expressions in neuroblastoma cells. Furthermore, we found that the intracellular localization of p21 and p27 in the nuclear and cytoplasmic compartments, respectively. In addition, we found that GRP/GRP-R silencing increased the expression and accumulation of PTEN in the cytoplasm of neuroblastoma cells where it co-localized with p27, thus suggesting that p27 promotes the function of PTEN as a tumor suppressor by stabilizing PTEN in the cytoplasm. GRP/GRP-R regulation of CDK inhibitors and tumor suppressor PTEN may be critical for tumoriogenesis of neuroblastoma.« less

Emotion Regulation and Substance Use Frequency in Women with Substance Dependence and Borderline Personality Disorder Receiving Dialectical Behavior Therapy

PubMed Central

Axelrod, Seth R.; Perepletchikova, Francheska; Holtzman, Kevin; Sinha, Rajita

2011-01-01

Background Dialectical Behavior Therapy (DBT) identifies emotion dysregulation as central to the dangerous impulsivity of borderline personality disorder (BPD) including substance use disorders, and DBT targets improved emotion regulation as a primary mechanism of change. However, improved emotion regulation with DBT and associations between such improvement and behavioral outcomes such as substance use has not been previously reported. Objective Thus, the goal of this study was to assess for improvement in emotion regulation and to examine the relationship between improvements in the emotion regulation and substance use problems following DBT treatment. Method Emotion regulation as assessed by the Difficulties in Emotion Regulation Scale, depressed mood as assessed by the Beck Depression Inventory, and their associations with substance use frequency were investigated in 27 women with substance dependence and BPD receiving 20 weeks of DBT in an academic community outpatient substance abuse treatment program. Results indicated improved emotion regulation, improved mood, and decreased substance use frequency. Further, emotion regulation improvement, but not improved mood, explained the variance of decreased substance use frequency. Conclusions This is the first study to demonstrate improved emotion regulation in BPD patients treated with DBT and to show that improved emotion regulation can account for increased behavioral control in BPD patients. Significance and Future Research Emotion regulation assessment is recommended for future studies to further clarify the etiology and maintenance of disorders associated with emotional dyregulation such as BPD and substance dependence, and to further explore emotion regulation as a potential mechanism of change for clinical interventions. PMID:21091162

SUMO regulates proteasome-dependent degradation of FLASH/Casp8AP2

PubMed Central

Vennemann, Astrid; Hofmann, Thomas G.

2013-01-01

FLASH/Casp8AP2 is a huge multifunctional protein involved in multiple cellular processes, reaching from death receptor signaling to regulation of histone gene transcription and histone mRNA processing. Previous work has shown that FLASH localizes to Cajal bodies and promyelocytic leukemia (PML) bodies. However, the function of its nuclear body association remains unclear. Here we demonstrate that murine FLASH is covalently modified by SUMO at Lys residue 1792. Interestingly, ectopic expression of SUMO results in proteasome-dependent degradation of FLASH. A point mutant of FLASH with a mutated SUMO acceptor lysine residue, FLASHK1792R, is resistant to SUMO-induced degradation. Finally, we show that arsenic trioxide, a drug known to potentiate SUMO modification and degradation of PML, triggers recruitment of FLASH to PML bodies and concomitant loss of FLASH protein. Our data suggest that SUMO targets FLASH for proteasome-dependent degradation, which is associated with recruitment of FLASH to PML bodies. PMID:23673342

Intracellular pH regulation in unstimulated Calliphora salivary glands is Na+ dependent and requires V-ATPase activity.

PubMed

Schewe, Bettina; Blenau, Wolfgang; Walz, Bernd

2012-04-15

Salivary gland cells of the blowfly Calliphora vicina have a vacuolar-type H(+)-ATPase (V-ATPase) that lies in their apical membrane and energizes the secretion of a KCl-rich primary saliva upon stimulation with serotonin (5-hydroxytryptamine). Whether and to what extent V-ATPase contributes to intracellular pH (pH(i)) regulation in unstimulated gland cells is unknown. We used the fluorescent dye BCECF to study intracellular pH(i) regulation microfluorometrically and show that: (1) under resting conditions, the application of Na(+)-free physiological saline induces an intracellular alkalinization attributable to the inhibition of the activity of a Na(+)-dependent glutamate transporter; (2) the maintenance of resting pH(i) is Na(+), Cl(-), concanamycin A and DIDS sensitive; (3) recovery from an intracellular acid load is Na(+) sensitive and requires V-ATPase activity; (4) the Na(+)/H(+) antiporter is not involved in pH(i) recovery after a NH(4)Cl prepulse; and (5) at least one Na(+)-dependent transporter and the V-ATPase maintain recovery from an intracellular acid load. Thus, under resting conditions, the V-ATPase and at least one Na(+)-dependent transporter maintain normal pH(i) values of pH 7.5. We have also detected the presence of a Na(+)-dependent glutamate transporter, which seems to act as an acid loader. Despite this not being a common pH(i)-regulating transporter, its activity affects steady-state pH(i) in C. vicina salivary gland cells.

Reciprocal and activity-dependent regulation of surface AMPA and NMDA receptors in cultured neurons

PubMed Central

Li, Guo Hua; Jackson, Michael F; Orser, Beverley A; MacDonald, John F

2010-01-01

Activation of NMDA receptors (NMDARs) can modulate excitatory synaptic transmission in the central nervous system by dynamically altering the number of synaptic AMPA receptors (AMPARs). The surface expression of NMDARs themselves is also subject to modulation in an activity-dependent manner. In addition to NMDAR-induced changes in AMPAR expression, AMPARs have also been found to regulate their own surface expression, independently of NMDARs. However, whether or not AMPARs and NMDARs might reciprocally regulate their surface expression has not previously been systematically explored. We utilized surface biotinylation assays and stimulation protocols intended to selectively stimulate various glutamate receptor subpopulations (e.g. AMPARs vs NMDARs; synaptic vs extrasynaptic). We reveal that activation of synaptic NMDARs increases the surface expression of both NMDAR and AMPAR subunits, while activation of extrasynaptic NMDAR produces the opposite effect. Surprisingly, we find that selective activation of AMPARs reduces the surface expression of not only AMPARs but also of NMDARs. These results suggest that both AMPARs and NMDARs at synaptic sites are subject to modulation by multiple signalling pathways in an activity-dependent way. PMID:21383896

Sox2 acts in a dose-dependent fashion to regulate proliferation of cortical progenitors.

PubMed

Hagey, Daniel W; Muhr, Jonas

2014-12-11

Organ formation and maintenance depends on slowly self-renewing stem cells that supply an intermediate population of rapidly dividing progenitors, but how this proliferative hierarchy is regulated is unknown. By performing genome-wide single-cell and functional analyses in the cortex, we demonstrate that reduced Sox2 expression is a key regulatory signature of the transition between stem cells and rapidly dividing progenitors. In stem cells, Sox2 is expressed at high levels, which enables its repression of proproliferative genes, of which Cyclin D1 is the most potent target. Sox2 confers this function through binding to low-affinity motifs, which facilitate the recruitment of Gro/Tle corepressors in synergy with Tcf/Lef proteins. Upon differentiation, proneural factors reduce Sox2 expression, which derepresses Cyclin D1 and promotes proliferation. Our results show how concentration-dependent Sox2 occupancy of DNA motifs of varying affinities translates into recruitment of repressive complexes, which regulate the proliferative dynamics of neural stem and progenitor cells. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells

PubMed Central

Bhattacharya, Sujoy; Ray, Ramesh M.; Johnson, Leonard R.

2014-01-01

Homeostasis of the gastrointestinal epithelium is dependent upon a balance between cell proliferation and apoptosis. Cyclin-dependent kinases (Cdks) are well known for their role in cell proliferation. Previous studies from our group have shown that polyamine-depletion of intestinal epithelial cells (IEC-6) decreases cyclin-dependent kinase 2 (Cdk2) activity, increases p53 and p21Cip1 protein levels, induces G1 arrest, and protects cells from camptothecin (CPT)-induced apoptosis. Although emerging evidence suggests that members of the Cdk family are involved in the regulation of apoptosis, their roles directing apoptosis of IEC-6 cells are not known. In this study, we report that inhibition of Cdk1, 2, and 9 (with the broad range Cdk inhibitor, AZD5438) in proliferating IEC-6 cells triggered DNA damage, activated p53 signaling, inhibited proliferation, and induced apoptosis. By contrast, inhibition of Cdk2 (with NU6140) increased p53 protein and activity, inhibited proliferation, but had no effect on apoptosis. Notably, AZD5438 sensitized, whereas, NU6140 rescued proliferating IEC-6 cells from CPT-induced apoptosis. However, in colon carcinoma (Caco2) cells with mutant p53, treatment with either AZD5438 or NU6140 blocked proliferation, albeit more robustly with AZD5438. Both Cdk inhibitors induced apoptosis in Caco2 cells in a p53-independent manner. In serum starved quiescent IEC-6 cells, both AZD5438 and NU6140 decreased TNF- /CPT-induced activation of p53 and, consequently, rescued cells from apoptosis, indicating that sustained Cdk activity is required for apoptosis of quiescent cells. Furthermore, AZD5438 partially reversed the protective effect of polyamine depletion whereas NU6140 had no effect. Together, these results demonstrate that Cdks possess opposing roles in the control of apoptosis in quiescent and proliferating cells. In addition, Cdk inhibitors uncouple proliferation from apoptosis in a p53-dependent manner. PMID:24242917

Output regulation control for switched stochastic delay systems with dissipative property under error-dependent switching

NASA Astrophysics Data System (ADS)

Li, L. L.; Jin, C. L.; Ge, X.

2018-01-01

In this paper, the output regulation problem with dissipative property for a class of switched stochastic delay systems is investigated, based on an error-dependent switching law. Under the assumption that none subsystem is solvable for the problem, a sufficient condition is derived by structuring multiple Lyapunov-Krasovskii functionals with respect to multiple supply rates, via designing error feedback regulators. The condition is also established when dissipative property reduces to passive property. Finally, two numerical examples are given to demonstrate the feasibility and efficiency of the present method.

Interferon-gamma regulates nucleoside transport systems in macrophages through signal transduction and activator of transduction factor 1 (STAT1)-dependent and -independent signalling pathways.

PubMed Central

Soler, Concepció; Felipe, Antonio; García-Manteiga, José; Serra, Maria; Guillén-Gómez, Elena; Casado, F Javier; MacLeod, Carol; Modolell, Manuel; Pastor-Anglada, Marçal; Celada, Antonio

2003-01-01

The expressions of CNT and ENT (concentrative and equilibrative nucleoside transporters) in macrophages are differentially regulated by IFN-gamma (interferon-gamma). This cytokine controls gene expression through STAT1-dependent and/or -independent pathways (where STAT1 stands for signal transduction and activator of transcription 1). In the present study, the role of STAT1 in the response of nucleoside transporters to IFN-gamma was studied using macrophages from STAT1 knockout mice. IFN-gamma triggered an inhibition of ENT1-related nucleoside transport activity through STAT1-dependent mechanisms. Such inhibition of macrophage growth and ENT1 activity by IFN-gamma is required for DNA synthesis. Interestingly, IFN-gamma led to an induction of the CNT1- and CNT2-related nucleoside transport activities independent of STAT1, thus ensuring the supply of extracellular nucleosides for the STAT1-independent RNA synthesis. IFN-gamma up-regulated CNT2 mRNA and CNT1 protein levels and down-regulated ENT1 mRNA in both wild-type and STAT1 knockout macrophages. This is consistent with a STAT1-independent, long-term-mediated, probably transcription-dependent, regulation of nucleoside transporter genes. Moreover, STAT1-dependent post-transcriptional mechanisms are implicated in the regulation of ENT1 activity. Although nitric oxide is involved in the regulation of ENT1 activity in B-cells at a post-transcriptional level, our results show that STAT1-dependent induction of nitric oxide by IFN-gamma is not implicated in the regulation of ENT1 activity in macrophages. Our results indicate that both STAT1-dependent and -independent pathways are involved in the regulation of nucleoside transporters by IFN-gamma in macrophages. PMID:12868960

p35 Regulates the CRM1-Dependent Nucleocytoplasmic Shuttling of Nuclear Hormone Receptor Coregulator-Interacting Factor 1 (NIF-1)

PubMed Central

Zhao, Xiao-Su; Fu, Wing-Yu; Chien, Winnie W. Y.; Li, Zhen; Fu, Amy K. Y.; Ip, Nancy Y.

2014-01-01

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase, which plays critical roles in a wide spectrum of neuronal functions including neuronal survival, neurite outgrowth, and synapse development and plasticity. Cdk5 activity is controlled by its specific activators: p35 or p39. While knockout studies reveal that Cdk5/p35 is critical for neuronal migration during early brain development, functions of Cdk5/p35 have been unraveled through the identification of the interacting proteins of p35, most of which are Cdk5/p35 substrates. However, it remains unclear whether p35 can regulate neuronal functions independent of Cdk5 activity. Here, we report that a nuclear protein, nuclear hormone receptor coregulator (NRC)-interacting factor 1 (NIF-1), is a new interacting partner of p35. Interestingly, p35 regulates the functions of NIF-1 independent of Cdk5 activity. NIF-1 was initially discovered as a transcriptional regulator that enhances the transcriptional activity of nuclear hormone receptors. Our results show that p35 interacts with NIF-1 and regulates its nucleocytoplasmic trafficking via the nuclear export pathway. Furthermore, we identified a nuclear export signal on p35; mutation of this site or blockade of the CRM1/exportin-dependent nuclear export pathway resulted in the nuclear accumulation of p35. Intriguingly, blocking the nuclear export of p35 attenuated the nuclear accumulation of NIF-1. These findings reveal a new p35-dependent mechanism in transcriptional regulation that involves the nucleocytoplasmic shuttling of transcription regulators. PMID:25329792

p35 regulates the CRM1-dependent nucleocytoplasmic shuttling of nuclear hormone receptor coregulator-interacting factor 1 (NIF-1).

PubMed

Zhao, Xiao-Su; Fu, Wing-Yu; Chien, Winnie W Y; Li, Zhen; Fu, Amy K Y; Ip, Nancy Y

2014-01-01

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase, which plays critical roles in a wide spectrum of neuronal functions including neuronal survival, neurite outgrowth, and synapse development and plasticity. Cdk5 activity is controlled by its specific activators: p35 or p39. While knockout studies reveal that Cdk5/p35 is critical for neuronal migration during early brain development, functions of Cdk5/p35 have been unraveled through the identification of the interacting proteins of p35, most of which are Cdk5/p35 substrates. However, it remains unclear whether p35 can regulate neuronal functions independent of Cdk5 activity. Here, we report that a nuclear protein, nuclear hormone receptor coregulator (NRC)-interacting factor 1 (NIF-1), is a new interacting partner of p35. Interestingly, p35 regulates the functions of NIF-1 independent of Cdk5 activity. NIF-1 was initially discovered as a transcriptional regulator that enhances the transcriptional activity of nuclear hormone receptors. Our results show that p35 interacts with NIF-1 and regulates its nucleocytoplasmic trafficking via the nuclear export pathway. Furthermore, we identified a nuclear export signal on p35; mutation of this site or blockade of the CRM1/exportin-dependent nuclear export pathway resulted in the nuclear accumulation of p35. Intriguingly, blocking the nuclear export of p35 attenuated the nuclear accumulation of NIF-1. These findings reveal a new p35-dependent mechanism in transcriptional regulation that involves the nucleocytoplasmic shuttling of transcription regulators.

Regulation of ATM-Dependent DNA Damage Responses in Breast Cancer by the RhoGEF Net1

DTIC Science & Technology

2013-04-01

Science 279: 509-514. 5. Jaffe AB. et al., (2010) RhoGTPases: Biochemistry and Biology. Annu. Rev. Cell Dev. Biol. 21:247-269. 6. Rossman KL, et al...exchange factor Net1 is regulated by nuclear sequestration. J. Biol. Chem. 277:17, 14581-14588. 17. Harper JW, et al., (2007) The DNA Damage Response: Ten...Research (AACR) Annual Meeting and 2013 Annual Cancer Research Biochemistry Retreat Regulation of ATM-dependent DNA damage signaling in human breast

Calmodulin Bound to the First IQ Motif Is Responsible for Calcium-dependent Regulation of Myosin 5a*

PubMed Central

Lu, Zekuan; Shen, Mei; Cao, Yang; Zhang, Hai-Man; Yao, Lin-Lin; Li, Xiang-dong

2012-01-01

Myosin 5a is as yet the best-characterized unconventional myosin motor involved in transport of organelles along actin filaments. It is well-established that myosin 5a is regulated by its tail in a Ca2+-dependent manner. The fact that the actin-activated ATPase activity of myosin 5a is stimulated by micromolar concentrations of Ca2+ and that calmodulin (CaM) binds to IQ motifs of the myosin 5a heavy chain indicates that Ca2+ regulates myosin 5a function via bound CaM. However, it is not known which IQ motif and bound CaM are responsible for the Ca2+-dependent regulation and how the head-tail interaction is affected by Ca2+. Here, we found that the CaM in the first IQ motif (IQ1) is responsible for Ca2+ regulation of myosin 5a. In addition, we demonstrate that the C-lobe fragment of CaM in IQ1 is necessary for mediating Ca2+ regulation of myosin 5a, suggesting that the C-lobe fragment of CaM in IQ1 participates in the interaction between the head and the tail. We propose that Ca2+ induces a conformational change of the C-lobe of CaM in IQ1 and prevents interaction between the head and the tail, thus activating motor function. PMID:22437832

ARH directs megalin to the endocytic recycling compartment to regulate its proteolysis and gene expression

PubMed Central

Shah, Mehul; Baterina, Oscar Y.; Taupin, Vanessa

2013-01-01

Receptors internalized by endocytosis can return to the plasma membrane (PM) directly from early endosomes (EE; fast recycling) or they can traffic from EE to the endocytic recycling compartment (ERC) and recycle from there (slow recycling). How receptors are sorted for trafficking along these two pathways remains unclear. Here we show that autosomal recessive hypercholesterolemia (ARH) is required for trafficking of megalin, a member of the LDL receptor family, from EE to the ERC by coupling it to dynein; in the absence of ARH, megalin returns directly to the PM from EE via the connecdenn2/Rab35 fast recycling pathway. Binding of ARH to the endocytic adaptor AP-2 prevents fast recycling of megalin. ARH-mediated trafficking of megalin to the ERC is necessary for γ-secretase mediated cleavage of megalin and release of a tail fragment that mediates transcriptional repression. These results identify a novel mechanism for sorting receptors for trafficking to the ERC and link ERC trafficking to regulated intramembrane proteolysis (RIP) and expression of megalin. PMID:23836931

Regulated proteolysis of Trop2 drives epithelial hyperplasia and stem cell self-renewal via β-catenin signaling.

PubMed

Stoyanova, Tanya; Goldstein, Andrew S; Cai, Houjian; Drake, Justin M; Huang, Jiaoti; Witte, Owen N

2012-10-15

The cell surface protein Trop2 is expressed on immature stem/progenitor-like cells and is overexpressed in many epithelial cancers. However the biological function of Trop2 in tissue maintenance and tumorigenesis remains unclear. In this study, we demonstrate that Trop2 is a regulator of self-renewal, proliferation, and transformation. Trop2 controls these processes through a mechanism of regulated intramembrane proteolysis that leads to cleavage of Trop2, creating two products: the extracellular domain and the intracellular domain. The intracellular domain of Trop2 is released from the membrane and accumulates in the nucleus. Heightened expression of the Trop2 intracellular domain promotes stem/progenitor self-renewal through signaling via β-catenin and is sufficient to initiate precursor lesions to prostate cancer in vivo. Importantly, we demonstrate that loss of β-catenin or Trop2 loss-of-function cleavage mutants abrogates Trop2-driven self-renewal and hyperplasia in the prostate. These findings suggest that heightened expression of Trop2 is selected for in epithelial cancers to enhance the stem-like properties of self-renewal and proliferation. Defining the mechanism of Trop2 function in self-renewal and transformation is essential to identify new therapeutic strategies to block Trop2 activation in cancer.

Regulated proteolysis of Trop2 drives epithelial hyperplasia and stem cell self-renewal via β-catenin signaling

PubMed Central

Stoyanova, Tanya; Goldstein, Andrew S.; Cai, Houjian; Drake, Justin M.; Huang, Jiaoti; Witte, Owen N.

2012-01-01

The cell surface protein Trop2 is expressed on immature stem/progenitor-like cells and is overexpressed in many epithelial cancers. However the biological function of Trop2 in tissue maintenance and tumorigenesis remains unclear. In this study, we demonstrate that Trop2 is a regulator of self-renewal, proliferation, and transformation. Trop2 controls these processes through a mechanism of regulated intramembrane proteolysis that leads to cleavage of Trop2, creating two products: the extracellular domain and the intracellular domain. The intracellular domain of Trop2 is released from the membrane and accumulates in the nucleus. Heightened expression of the Trop2 intracellular domain promotes stem/progenitor self-renewal through signaling via β-catenin and is sufficient to initiate precursor lesions to prostate cancer in vivo. Importantly, we demonstrate that loss of β-catenin or Trop2 loss-of-function cleavage mutants abrogates Trop2-driven self-renewal and hyperplasia in the prostate. These findings suggest that heightened expression of Trop2 is selected for in epithelial cancers to enhance the stem-like properties of self-renewal and proliferation. Defining the mechanism of Trop2 function in self-renewal and transformation is essential to identify new therapeutic strategies to block Trop2 activation in cancer. PMID:23070813

The Hsp90 chaperone complex regulates GDI-dependent Rab recycling.

PubMed

Chen, Christine Y; Balch, William E

2006-08-01

Rab GTPase regulated hubs provide a framework for an integrated coding system, the membrome network, that controls the dynamics of the specialized exocytic and endocytic membrane architectures found in eukaryotic cells. Herein, we report that Rab recycling in the early exocytic pathways involves the heat-shock protein (Hsp)90 chaperone system. We find that Hsp90 forms a complex with guanine nucleotide dissociation inhibitor (GDI) to direct recycling of the client substrate Rab1 required for endoplasmic reticulum (ER)-to-Golgi transport. ER-to-Golgi traffic is inhibited by the Hsp90-specific inhibitors geldanamycin (GA), 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), and radicicol. Hsp90 activity is required to form a functional GDI complex to retrieve Rab1 from the membrane. Moreover, we find that Hsp90 is essential for Rab1-dependent Golgi assembly. The observation that the highly divergent Rab GTPases Rab1 involved in ER-to-Golgi transport and Rab3A involved in synaptic vesicle fusion require Hsp90 for retrieval from membranes lead us to now propose that the Hsp90 chaperone system may function as a general regulator for Rab GTPase recycling in exocytic and endocytic trafficking pathways involved in cell signaling and proliferation.

Regulation of resveratrol production in Vitis amurensis cell cultures by calcium-dependent protein kinases.

PubMed

Aleynova, O A; Dubrovina, A S; Manyakhin, A Y; Karetin, Y A; Kiselev, K V

2015-02-01

Resveratrol is a naturally occurring plant stilbene that exhibits a wide range of valuable biological and pharmacological properties. Although the beneficial effects of trans-resveratrol to human health and plant protection against fungal pathogens are well-established, little is known about the molecular mechanisms regulating stilbene biosynthesis in plant cells. It has been recently shown that overexpression of the calcium-dependent protein kinase VaCPK20 gene considerably increased resveratrol accumulation in cell cultures of Vitis amurensis. It is possible that calcium-dependent protein kinases (CDPKs) play an important role in the regulation of resveratrol biosynthesis. In the present work, we investigated the effects of overexpression of other members of the CDPK multigene family (VaCPK9, VaCPK13, VaCPK21, and VaCPK29) on resveratrol accumulation and growth parameters of grape cell cultures. The obtained data show that overexpression of VaCPK29 increased resveratrol content 1.6-2.4-fold and fresh biomass accumulation 1.1-1.4-fold in the four independently transformed cell lines of V. amurensis compared with that in the empty vector-transformed calli. However, overexpression of the VaCPK9, VaCPK13, and VaCPK21 genes did not considerably affect resveratrol content and fresh/dry biomass accumulation in the independently transformed cell lines of V. amurensis. VaCPK29-transformed calli were capable of producing between 1.02 and 1.39 mg/l of resveratrol, while the control calli produced 0.48 to 0.79 mg/l of resveratrol. The data indicate that the VaCPK9, VaCPK13, and VaCPK21 genes are not involved in the regulation of stilbene biosynthesis in grape cells, while the VaCPK29 and VaCPK20 genes are implicated in resveratrol biosynthesis as positive regulators.

Thioredoxin-dependent Redox Regulation of Chloroplastic Phosphoglycerate Kinase from Chlamydomonas reinhardtii*

PubMed Central

Morisse, Samuel; Michelet, Laure; Bedhomme, Mariette; Marchand, Christophe H.; Calvaresi, Matteo; Trost, Paolo; Fermani, Simona; Zaffagnini, Mirko; Lemaire, Stéphane D.

2014-01-01

In photosynthetic organisms, thioredoxin-dependent redox regulation is a well established mechanism involved in the control of a large number of cellular processes, including the Calvin-Benson cycle. Indeed, 4 of 11 enzymes of this cycle are activated in the light through dithiol/disulfide interchanges controlled by chloroplastic thioredoxin. Recently, several proteomics-based approaches suggested that not only four but all enzymes of the Calvin-Benson cycle may withstand redox regulation. Here, we characterized the redox features of the Calvin-Benson enzyme phosphoglycerate kinase (PGK1) from the eukaryotic green alga Chlamydomonas reinhardtii, and we show that C. reinhardtii PGK1 (CrPGK1) activity is inhibited by the formation of a single regulatory disulfide bond with a low midpoint redox potential (−335 mV at pH 7.9). CrPGK1 oxidation was found to affect the turnover number without altering the affinity for substrates, whereas the enzyme activation appeared to be specifically controlled by f-type thioredoxin. Using a combination of site-directed mutagenesis, thiol titration, mass spectrometry analyses, and three-dimensional modeling, the regulatory disulfide bond was shown to involve the not strictly conserved Cys227 and Cys361. Based on molecular mechanics calculation, the formation of the disulfide is proposed to impose structural constraints in the C-terminal domain of the enzyme that may lower its catalytic efficiency. It is therefore concluded that CrPGK1 might constitute an additional light-modulated Calvin-Benson cycle enzyme with a low activity in the dark and a TRX-dependent activation in the light. These results are also discussed from an evolutionary point of view. PMID:25202015

CFTR/ENaC dependent regulation of membrane potential during human sperm capacitation is initiated by bicarbonate uptake through NBC.

PubMed

Puga Molina, Lis C; Pinto, Nicolas A; Torres, Nicolás I; Gonzalez-Cota, Ana L; Luque, Guillermina M; Balestrini, Paula A; Romarowski, Ana; Krapf, Dario; Santi, Celia M; Trevino, Claudia L; Darszon, Alberto; Buffone, Mariano G

2018-05-09

To fertilize an egg, sperm must reside in the female reproductive tract to undergo several maturational changes that are collectively referred to as capacitation. From a molecular point of view, the HCO3--dependent activation of the atypical soluble adenylyl cyclase (ADCY10) is one of the first events that occurs during capacitation and leads to the subsequent cAMP-dependent activation of protein kinase A (PKA). Capacitation is also accompanied by hyperpolarization of the sperm plasma membrane. We previously reported that PKA activation is necessary for CFTR (Cystic Fibrosis Transmembrane Conductance Regulator Channel) activity and for the modulation of membrane potential (Em). However, the main HCO3- transporters involved in the initial transport and the PKA-dependent Em changes are not well known nor characterized. Here, we analyzed how the activity of CFTR regulates Em during capacitation and examined its relationship with an electrogenic Na+/HCO3- cotransporter (NBC) and epithelial Na+ channels (ENaCs). We observed that inhibition of both CFTR and NBC decreased HCO3- influx, resulting in lower PKA activity, and that events downstream the cAMP-activation of PKA are essential for the regulation of Em. Addition of a permeable cAMP analog partially rescued the inhibitory effects caused by these inhibitors. HCO3-  also produced a rapid membrane hyperpolarization mediated by ENaC channels, which contribute to the regulation of Em during capacitation. Altogether, we demonstrate for the first time, that NBC cotransporters and ENaC channels are essential in the CFTR-dependent activation of the cAMP/PKA signaling pathway and Em regulation during human sperm capacitation. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of a calcium/calmodulin-dependent protein kinase homolog from maize roots showing light-regulated gravitropism

NASA Technical Reports Server (NTRS)

Lu, Y. T.; Hidaka, H.; Feldman, L. J.

1996-01-01

Roots of many species respond to gravity (gravitropism) and grow downward only if illuminated. This light-regulated root gravitropism is phytochrome-dependent, mediated by calcium, and inhibited by KN-93, a specific inhibitor of calcium/calmodulin-dependent protein kinase II (CaMK II). A cDNA encoding MCK1, a maize homolog of mammalian CaMK, has been isolated from roots of maize (Zea mays L.). The MCK1 gene is expressed in root tips, the site of perception for both light and gravity. Using the [35S]CaM gel-overlay assay we showed that calmodulin-binding activity of the MCK1 is abolished by 50 microM KN-93, but binding is not affected by 5 microM KN-93, paralleling physiological findings that light-regulated root gravitropism is inhibited by 50 microM KN-93, but not by 5 microM KN-93. KN-93 inhibits light-regulated gravitropism by interrupting transduction of the light signal, not light perception, suggesting that MCK1 may play a role in transducing light. This is the first report suggesting a physiological function for a CaMK homolog in light signal transduction.

Myosin IIb-dependent Regulation of Actin Dynamics Is Required for N-Methyl-D-aspartate Receptor Trafficking during Synaptic Plasticity.

PubMed

Bu, Yunfei; Wang, Ning; Wang, Shaoli; Sheng, Tao; Tian, Tian; Chen, Linlin; Pan, Weiwei; Zhu, Minsheng; Luo, Jianhong; Lu, Wei

2015-10-16

N-Methyl-d-aspartate receptor (NMDAR) synaptic incorporation changes the number of NMDARs at synapses and is thus critical to various NMDAR-dependent brain functions. To date, the molecules involved in NMDAR trafficking and the underlying mechanisms are poorly understood. Here, we report that myosin IIb is an essential molecule in NMDAR synaptic incorporation during PKC- or θ burst stimulation-induced synaptic plasticity. Moreover, we demonstrate that myosin light chain kinase (MLCK)-dependent actin reorganization contributes to NMDAR trafficking. The findings from additional mutual occlusion experiments demonstrate that PKC and MLCK share a common signaling pathway in NMDAR-mediated synaptic regulation. Because myosin IIb is the primary substrate of MLCK and can regulate actin dynamics during synaptic plasticity, we propose that the MLCK- and myosin IIb-dependent regulation of actin dynamics is required for NMDAR trafficking during synaptic plasticity. This study provides important insights into a mechanical framework for understanding NMDAR trafficking associated with synaptic plasticity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms

NASA Technical Reports Server (NTRS)

Liu, Z.; Xia, M.; Poovaiah, B. W.

1998-01-01

cDNA clones of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) from tobacco (TCCaMK-1 and TCCaMK-2) were isolated and characterized. The polypeptides encoded by TCCaMK-1 and TCCaMK-2 have 15 different amino acid substitutions, yet they both contain a total of 517 amino acids. Northern analysis revealed that CCaMK is expressed in a stage-specific manner during anther development. Messenger RNA was detected when tobacco bud sizes were between 0.5 cm and 1.0 cm. The appearance of mRNA coincided with meiosis and became undetectable at later stages of anther development. The reverse polymerase chain reaction (RT-PCR) amplification assay using isoform-specific primers showed that both of the CCaMK mRNAs were expressed in anther with similar expression patterns. The CCaMK protein expressed in Escherichia coli showed Ca2+-dependent autophosphorylation and Ca2+/calmodulin-dependent substrate phosphorylation. Calmodulin isoforms (PCM1 and PCM6) had differential effects on the regulation of autophosphorylation and substrate phosphorylation of tobacco CCaMK, but not lily CCaMK. The evolutionary tree of plant serine/threonine protein kinases revealed that calmodulin-dependent kinases form one subgroup that is distinctly different from Ca2+-dependent protein kinases (CDPKs) and other serine/threonine kinases in plants.

MdHY5 positively regulates cold tolerance via CBF-dependent and CBF-independent pathways in apple.

PubMed

An, Jian-Ping; Yao, Ji-Fang; Wang, Xiao-Na; You, Chun-Xiang; Wang, Xiao-Fei; Hao, Yu-Jin

2017-11-01

Cold stress is a major external stimulator that affects crop quality and productivity. The CBF cold regulatory pathway has been regarded as a master regulator in the response to cold stress. In this study, we found that the apple bZIP transcription factor, MdHY5, was responsive to cold treatment both at the transcriptional and at the post-translational levels. Moreover, overexpression of MdHY5 enhanced cold tolerance in apple calli and Arabidopsis. Subsequently, EMSA assay and transient expression assay demonstrated that MdHY5 positively regulated the transcript of MdCBF1 by binding to G-Box motif of its promoter. Furthermore, MdHY5 also regulated the expression of CBF-independent cold-regulated genes. Taken together, our data suggest that MdHY5 positively modulates plant cold tolerance through CBF-dependent and CBF-independent pathways, providing a deeper understanding of MdHY5-regulated cold tolerance in apple. Copyright © 2017 Elsevier GmbH. All rights reserved.

Nitric Oxide Is an Activity-Dependent Regulator of Target Neuron Intrinsic Excitability

PubMed Central

Steinert, Joern R.; Robinson, Susan W.; Tong, Huaxia; Haustein, Martin D.; Kopp-Scheinpflug, Cornelia; Forsythe, Ian D.

2011-01-01

Summary Activity-dependent changes in synaptic strength are well established as mediating long-term plasticity underlying learning and memory, but modulation of target neuron excitability could complement changes in synaptic strength and regulate network activity. It is thought that homeostatic mechanisms match intrinsic excitability to the incoming synaptic drive, but evidence for involvement of voltage-gated conductances is sparse. Here, we show that glutamatergic synaptic activity modulates target neuron excitability and switches the basis of action potential repolarization from Kv3 to Kv2 potassium channel dominance, thereby adjusting neuronal signaling between low and high activity states, respectively. This nitric oxide-mediated signaling dramatically increases Kv2 currents in both the auditory brain stem and hippocampus (>3-fold) transforming synaptic integration and information transmission but with only modest changes in action potential waveform. We conclude that nitric oxide is a homeostatic regulator, tuning neuronal excitability to the recent history of excitatory synaptic inputs over intervals of minutes to hours. PMID:21791288

Age–dependent regulation of synaptic connections by dopamine D2 receptors

PubMed Central

Jia, Jie–Min; Zhao, Jun; Hu, Zhonghua; Lindberg, Daniel; Li, Zheng

2013-01-01

Dopamine D2 receptors (D2R) are G protein–coupled receptors that modulate synaptic transmission and play an important role in various brain functions including affect learning and working memory. Abnormal D2R signaling has been implicated in psychiatric disorders such as schizophrenia. Here we report a new function of D2R in dendritic spine morphogenesis. Activation of D2R reduces spine number via GluN2B– and cAMP–dependent mechanisms in mice. Notably, this regulation takes place only during adolescence. During this period, D2R overactivation caused by mutations in the schizophrenia–risk–gene dysbindin leads to spine deficiency, dysconnectivity within the entorhinal–hippocampal circuit and impairment of spatial working memory. Notably, these defects can be ameliorated by D2R blockers administered during adolescence. These findings uncover a novel age–dependent function of D2R in spine development, provide evidence that D2R dysfunction during adolescence impairs neuronal circuits and working memory, and suggest that adolescent interventions of aberrant D2R activity protect against cognitive impairment. PMID:24121738

Dopamine D1 Receptors Regulate the Light Dependent Development of Retinal Synaptic Responses

PubMed Central

He, Quanhua; Xu, Hong-ping; Wang, Ping; Tian, Ning

2013-01-01

Retinal synaptic connections and function are developmentally regulated. Retinal synaptic activity plays critical roles in the development of retinal synaptic circuitry. Dopamine receptors have been thought to play important roles in the activity-dependent synaptic plasticity in central nervous system. The primary goal of this study is to determine whether dopamine D1 receptor regulates the activity-dependent development of retinal light responsiveness. Accordingly, we recorded electroretinogram from wild type mice and mice with genetic deletion of D1 dopamine receptor (D1−/− mice) raised under cyclic light conditions and constant darkness. Our results demonstrated that D1−/− mice have reduced amplitudes of all three major components of electroretinogram in adulthood. When the relative strength of the responses is considered, the D1−/− mice have selective reduction of the amplitudes of a-wave and oscillatory potentials evoked by low-intermediate intensities of lights. During postnatal development, D1−/− mice have increased amplitude of b-wave at the time of eye-opening but reduced developmental increase of the amplitude of b-wave after eye opening. Light deprivation from birth significantly reduced the amplitudes of b-wave and oscillatory potentials, increased the outer retinal light response gain and altered the light response kinetics of both a- and b-waves of wild type mice. In D1−/− mice, the effect of dark rearing on the amplitude of oscillatory potentials was diminished and dark rearing induced effects on the response gain of outer retina and the kinetics of a-wave were reversed. These results demonstrated roles of dopamine D1 receptor in the activity-dependent functional development of mouse retina. PMID:24260267

Regulation of Arabidopsis Leaf Hydraulics Involves Light-Dependent Phosphorylation of Aquaporins in Veins[C][W

PubMed Central

Prado, Karine; Boursiac, Yann; Tournaire-Roux, Colette; Monneuse, Jean-Marc; Postaire, Olivier; Da Ines, Olivier; Schäffner, Anton R.; Hem, Sonia; Santoni, Véronique; Maurel, Christophe

2013-01-01

The water status of plant leaves depends on the efficiency of the water supply, from the vasculature to inner tissues. This process is under hormonal and environmental regulation and involves aquaporin water channels. In Arabidopsis thaliana, the rosette hydraulic conductivity (Kros) is higher in darkness than it is during the day. Knockout plants showed that three plasma membrane intrinsic proteins (PIPs) sharing expression in veins (PIP1;2, PIP2;1, and PIP2;6) contribute to rosette water transport, and PIP2;1 can fully account for Kros responsiveness to darkness. Directed expression of PIP2;1 in veins of a pip2;1 mutant was sufficient to restore Kros. In addition, a positive correlation, in both wild-type and PIP2;1-overexpressing plants, was found between Kros and the osmotic water permeability of protoplasts from the veins but not from the mesophyll. Thus, living cells in veins form a major hydraulic resistance in leaves. Quantitative proteomic analyses showed that light-dependent regulation of Kros is linked to diphosphorylation of PIP2;1 at Ser-280 and Ser-283. Expression in pip2;1 of phosphomimetic and phosphorylation-deficient forms of PIP2;1 demonstrated that phosphorylation at these two sites is necessary for Kros enhancement under darkness. These findings establish how regulation of a single aquaporin isoform in leaf veins critically determines leaf hydraulics. PMID:23532070

A Unique cis-Encoded Small Noncoding RNA Is Regulating Legionella pneumophila Hfq Expression in a Life Cycle-Dependent Manner.

PubMed

Oliva, Giulia; Sahr, Tobias; Rolando, Monica; Knoth, Maike; Buchrieser, Carmen

2017-01-10

Legionella pneumophila is an environmental bacterium that parasitizes protozoa, but it may also infect humans, thereby causing a severe pneumonia called Legionnaires' disease. To cycle between the environment and a eukaryotic host, L. pneumophila is regulating the expression of virulence factors in a life cycle-dependent manner: replicating bacteria do not express virulence factors, whereas transmissive bacteria are highly motile and infective. Here we show that Hfq is an important regulator in this network. Hfq is highly expressed in transmissive bacteria but is expressed at very low levels in replicating bacteria. A L. pneumophila hfq deletion mutant exhibits reduced abilities to infect and multiply in Acanthamoeba castellanii at environmental temperatures. The life cycle-dependent regulation of Hfq expression depends on a unique cis-encoded small RNA named Anti-hfq that is transcribed antisense of the hfq transcript and overlaps its 5' untranslated region. The Anti-hfq sRNA is highly expressed only in replicating L. pneumophila where it regulates hfq expression through binding to the complementary regions of the hfq transcripts. This results in reduced Hfq protein levels in exponentially growing cells. Both the small noncoding RNA (sRNA) and hfq mRNA are bound and stabilized by the Hfq protein, likely leading to the cleavage of the RNA duplex by the endoribonuclease RNase III. In contrast, after the switch to transmissive bacteria, the sRNA is not expressed, allowing now an efficient expression of the hfq gene and consequently Hfq. Our results place Hfq and its newly identified sRNA anti-hfq in the center of the regulatory network governing L. pneumophila differentiation from nonvirulent to virulent bacteria. The abilities of L. pneumophila to replicate intracellularly and to cause disease depend on its capacity to adapt to different extra- and intracellular environmental conditions. Therefore, a timely and fine-tuned expression of virulence factors and

Cellular and molecular mechanisms regulating vascular tone. Part 1: basic mechanisms controlling cytosolic Ca2+ concentration and the Ca2+-dependent regulation of vascular tone.

PubMed

Akata, Takashi

2007-01-01

General anesthetics cause hemodynamic instability and alter blood flow to various organs. There is mounting evidence that most general anesthetics, at clinical concentrations, influence a wide variety of cellular and molecular mechanisms regulating the contractile state of vascular smooth muscle cells (i.e., vascular tone). In addition, in current anesthetic practice, various types of vasoactive agents are often used to control vascular reactivity and to sustain tissue blood flow in high-risk surgical patients with impaired vital organ function and/or hemodynamic instability. Understanding the physiological mechanisms involved in the regulation of vascular tone thus would be beneficial for anesthesiologists. This review, in two parts, provides an overview of current knowledge about the cellular and molecular mechanisms regulating vascular tone-i.e., targets for general anesthetics, as well as for vasoactive drugs that are used in intraoperative circulatory management. This first part of the two-part review focuses on basic mechanisms regulating cytosolic Ca2+ concentration and the Ca2+-dependent regulation of vascular tone.

Phosphorylation-Dependent Regulation of Ryanodine Receptors

PubMed Central

Marx, Steven O.; Reiken, Steven; Hisamatsu, Yuji; Gaburjakova, Marta; Gaburjakova, Jana; Yang, Yi-Ming; Rosemblit, Nora; Marks, Andrew R.

2001-01-01

Ryanodine receptors (RyRs), intracellular calcium release channels required for cardiac and skeletal muscle contraction, are macromolecular complexes that include kinases and phosphatases. Phosphorylation/dephosphorylation plays a key role in regulating the function of many ion channels, including RyRs. However, the mechanism by which kinases and phosphatases are targeted to ion channels is not well understood. We have identified a novel mechanism involved in the formation of ion channel macromolecular complexes: kinase and phosphatase targeting proteins binding to ion channels via leucine/isoleucine zipper (LZ) motifs. Activation of kinases and phosphatases bound to RyR2 via LZs regulates phosphorylation of the channel, and disruption of kinase binding via LZ motifs prevents phosphorylation of RyR2. Elucidation of this new role for LZs in ion channel macromolecular complexes now permits: (a) rapid mapping of kinase and phosphatase targeting protein binding sites on ion channels; (b) predicting which kinases and phosphatases are likely to regulate a given ion channel; (c) rapid identification of novel kinase and phosphatase targeting proteins; and (d) tools for dissecting the role of kinases and phosphatases as modulators of ion channel function. PMID:11352932

Light-regulated root gravitropism: a role for, and characterization of, a calcium/calmodulin-dependent protein kinase homolog

NASA Technical Reports Server (NTRS)

Lu, Y. T.; Feldman, L. J.

1997-01-01

Roots of many species grow downward (orthogravitropism) only when illuminated. Previous work suggests that this is a calcium-regulated response and that both calmodulin and calcium/calmodulin-dependent kinases participate in transducing gravity and light stimuli. A genomic sequence has been obtained for a calcium/calmodulin-dependent kinase homolog (MCK1) expressed in root caps, the site of perception for both light and gravity. This homolog consists of 7265 base pairs and contains 11 exons and 10 introns. Since MCK1 is expressed constitutively in both light and dark, it is unlikely that the light directly affects MCK1 expression, though the activity of the protein may be affected by light. In cultivars showing light-regulated gravitropism, we hypothesize that MCK1, or a homolog, functions in establishing the auxin asymmetry necessary for orthogravitropism.

Genetic Feedback Regulation of Frontal Cortical Neuronal Ensembles Through Activity-Dependent Arc Expression and Dopaminergic Input.

PubMed

Mastwal, Surjeet; Cao, Vania; Wang, Kuan Hong

2016-01-01

Mental functions involve coordinated activities of specific neuronal ensembles that are embedded in complex brain circuits. Aberrant neuronal ensemble dynamics is thought to form the neurobiological basis of mental disorders. A major challenge in mental health research is to identify these cellular ensembles and determine what molecular mechanisms constrain their emergence and consolidation during development and learning. Here, we provide a perspective based on recent studies that use activity-dependent gene Arc/Arg3.1 as a cellular marker to identify neuronal ensembles and a molecular probe to modulate circuit functions. These studies have demonstrated that the transcription of Arc is activated in selective groups of frontal cortical neurons in response to specific behavioral tasks. Arc expression regulates the persistent firing of individual neurons and predicts the consolidation of neuronal ensembles during repeated learning. Therefore, the Arc pathway represents a prototypical example of activity-dependent genetic feedback regulation of neuronal ensembles. The activation of this pathway in the frontal cortex starts during early postnatal development and requires dopaminergic (DA) input. Conversely, genetic disruption of Arc leads to a hypoactive mesofrontal dopamine circuit and its related cognitive deficit. This mutual interaction suggests an auto-regulatory mechanism to amplify the impact of neuromodulators and activity-regulated genes during postnatal development. Such a mechanism may contribute to the association of mutations in dopamine and Arc pathways with neurodevelopmental psychiatric disorders. As the mesofrontal dopamine circuit shows extensive activity-dependent developmental plasticity, activity-guided modulation of DA projections or Arc ensembles during development may help to repair circuit deficits related to neuropsychiatric disorders.

Targeted Deletion of a Plasmodium Site-2 Protease Impairs Life Cycle Progression in the Mammalian Host

PubMed Central

Goulielmaki, Evi; Chalari, Anna; Withers-Martinez, Chrislaine; Siden-Kiamos, Inga; Matuschewski, Kai

2017-01-01

Site-2 proteases (S2P) belong to the M50 family of metalloproteases, which typically perform essential roles by mediating activation of membrane–bound transcription factors through regulated intramembrane proteolysis (RIP). Protease-dependent liberation of dormant transcription factors triggers diverse cellular responses, such as sterol regulation, Notch signalling and the unfolded protein response. Plasmodium parasites rely on regulated proteolysis for controlling essential pathways throughout the life cycle. In this study we examine the Plasmodium-encoded S2P in a murine malaria model and show that it is expressed in all stages of Plasmodium development. Localisation studies by endogenous gene tagging revealed that in all invasive stages the protein is in close proximity to the nucleus. Ablation of PbS2P by reverse genetics leads to reduced growth rates during liver and blood infection and, hence, virulence attenuation. Strikingly, absence of PbS2P was compatible with parasite life cycle progression in the mosquito and mammalian hosts under physiological conditions, suggesting redundant or dispensable roles in vivo. PMID:28107409

Targeted Deletion of a Plasmodium Site-2 Protease Impairs Life Cycle Progression in the Mammalian Host.

PubMed

Koussis, Konstantinos; Goulielmaki, Evi; Chalari, Anna; Withers-Martinez, Chrislaine; Siden-Kiamos, Inga; Matuschewski, Kai; Loukeris, Thanasis G

2017-01-01

Site-2 proteases (S2P) belong to the M50 family of metalloproteases, which typically perform essential roles by mediating activation of membrane-bound transcription factors through regulated intramembrane proteolysis (RIP). Protease-dependent liberation of dormant transcription factors triggers diverse cellular responses, such as sterol regulation, Notch signalling and the unfolded protein response. Plasmodium parasites rely on regulated proteolysis for controlling essential pathways throughout the life cycle. In this study we examine the Plasmodium-encoded S2P in a murine malaria model and show that it is expressed in all stages of Plasmodium development. Localisation studies by endogenous gene tagging revealed that in all invasive stages the protein is in close proximity to the nucleus. Ablation of PbS2P by reverse genetics leads to reduced growth rates during liver and blood infection and, hence, virulence attenuation. Strikingly, absence of PbS2P was compatible with parasite life cycle progression in the mosquito and mammalian hosts under physiological conditions, suggesting redundant or dispensable roles in vivo.

Bombyx mori cyclin-dependent kinase inhibitor is involved in regulation of the silkworm cell cycle.

PubMed

Tang, X-F; Zhou, X-L; Zhang, Q; Chen, P; Lu, C; Pan, M-H

2018-06-01

Cyclin-dependent kinase inhibitors (CKIs) are negative regulators of the cell cycle. They can bind to cyclin-dependent kinase (CDK)-cyclin complexes and inhibit CDK activities. We identified a single homologous gene of the CDK interacting protein/kinase inhibitory protein (Cip/Kip) family, BmCKI, in the silkworm, Bombyx mori. The gene transcribes two splice variants: a 654-bp-long BmCKI-L (the longer splice variant) encoding a protein with 217 amino acids and a 579-bp-long BmCKI-S (the shorter splice variant) encoding a protein with 192 amino acids. BmCKI-L and BmCKI-S contain the Cip/Kip family conserved cyclin-binding domain and the CDK-binding domain. They are localized in the nucleus and have an unconventional bipartite nuclear localization signal at amino acid residues 181-210. Overexpression of BmCKI-L or BmCKI-S affected cell cycle progression; the cell cycle was arrested in the first gap phase of cell cycle (G1). RNA interference of BmCKI-L or BmCKI-S led to cells accumulating in the second gap phase and the mitotic phase of cell cycle (G2/M). Both BmCKI-L and BmCKI-S are involved in cell cycle regulation and probably have similar effects. The transgenic silkworm with BmCKI-L overexpression (BmCKI-L-OE), exhibited embryonic lethal, larva developmental retardation and lethal phenotypes. These results suggest that BmCKI-L might regulate the growth and development of silkworm. These findings clarify the function of CKIs and increase our understanding of cell cycle regulation in the silkworm. © 2018 The Royal Entomological Society.

Calcium/calmodulin-dependent protein kinase II activity regulates the proliferative potential of growth plate chondrocytes.

PubMed

Li, Yuwei; Ahrens, Molly J; Wu, Amy; Liu, Jennifer; Dudley, Andrew T

2011-01-01

For tissues that develop throughout embryogenesis and into postnatal life, the generation of differentiated cells to promote tissue growth is at odds with the requirement to maintain the stem cell/progenitor cell population to preserve future growth potential. In the growth plate cartilage, this balance is achieved in part by establishing a proliferative phase that amplifies the number of progenitor cells prior to terminal differentiation into hypertrophic chondrocytes. Here, we show that endogenous calcium/calmodulin-dependent protein kinase II (CamkII, also known as Camk2) activity is upregulated prior to hypertrophy and that loss of CamkII function substantially blocks the transition from proliferation to hypertrophy. Wnt signaling and Pthrp-induced phosphatase activity negatively regulate CamkII activity. Release of this repression results in activation of multiple effector pathways, including Runx2- and β-catenin-dependent pathways. We present an integrated model for the regulation of proliferation potential by CamkII activity that has important implications for studies of growth control and adult progenitor/stem cell populations.

Emotion self-regulation and empathy depend upon longer stimulus exposure.

PubMed

Ikezawa, Satoru; Corbera, Silvia; Wexler, Bruce E

2014-10-01

Observation of others in pain induces positive elevation (pain effect) in late event-related potentials (ERP). This effect is associated with top-down attention regulating processes. It has previously been shown that stimulus exposure duration can affect top-down attentional modulation of response to threat-related stimuli. We investigated the effect of exposure duration on ERP response to others in pain. Two late ERP components, P3 and late positive potentials (LPP), from 18 healthy people were measured while they viewed pictures of hands in painful or neutral situations for either 200 or 500 ms, during two task conditions (pain judgment and counting hands). P3 and LPP pain effects during the pain judgment condition were significantly greater with 500 ms than 200 ms stimulus presentation. Ours is the first study to suggest that engagement of empathy-related self-regulatory processes reflected in late potentials requires longer exposure to the pain-related stimulus. Although this is important information about the relationship between early sensory and subsequent brain processing, and about engagement of self-regulatory processes, the neural basis of this time-dependence remains unclear. It might be important to investigate the relationship between stimulus duration and empathic response in clinical populations where issues of self-regulation, empathic response and speed of information processing exist. © The Author (2013). Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

Cell Cycle-Dependent Rho GTPase Activity Dynamically Regulates Cancer Cell Motility and Invasion In Vivo

PubMed Central

Kagawa, Yoshinori; Matsumoto, Shinji; Kamioka, Yuji; Mimori, Koshi; Naito, Yoko; Ishii, Taeko; Okuzaki, Daisuke; Nishida, Naohiro; Maeda, Sakae; Naito, Atsushi; Kikuta, Junichi; Nishikawa, Keizo; Nishimura, Junichi; Haraguchi, Naotsugu; Takemasa, Ichiro; Mizushima, Tsunekazu; Ikeda, Masataka; Yamamoto, Hirofumi; Sekimoto, Mitsugu; Ishii, Hideshi; Doki, Yuichiro; Matsuda, Michiyuki; Kikuchi, Akira; Mori, Masaki; Ishii, Masaru

2013-01-01

The mechanism behind the spatiotemporal control of cancer cell dynamics and its possible association with cell proliferation has not been well established. By exploiting the intravital imaging technique, we found that cancer cell motility and invasive properties were closely associated with the cell cycle. In vivo inoculation of human colon cancer cells bearing fluorescence ubiquitination-based cell cycle indicator (Fucci) demonstrated an unexpected phenomenon: S/G2/M cells were more motile and invasive than G1 cells. Microarray analyses showed that Arhgap11a, an uncharacterized Rho GTPase-activating protein (RhoGAP), was expressed in a cell-cycle-dependent fashion. Expression of ARHGAP11A in cancer cells suppressed RhoA-dependent mechanisms, such as stress fiber formation and focal adhesion, which made the cells more prone to migrate. We also demonstrated that RhoA suppression by ARHGAP11A induced augmentation of relative Rac1 activity, leading to an increase in the invasive properties. RNAi-based inhibition of Arhgap11a reduced the invasion and in vivo expansion of cancers. Additionally, analysis of human specimens showed the significant up-regulation of Arhgap11a in colon cancers, which was correlated with clinical invasion status. The present study suggests that ARHGAP11A, a cell cycle-dependent RhoGAP, is a critical regulator of cancer cell mobility and is thus a promising therapeutic target in invasive cancers. PMID:24386239

Cell cycle-dependent Rho GTPase activity dynamically regulates cancer cell motility and invasion in vivo.

PubMed

Kagawa, Yoshinori; Matsumoto, Shinji; Kamioka, Yuji; Mimori, Koshi; Naito, Yoko; Ishii, Taeko; Okuzaki, Daisuke; Nishida, Naohiro; Maeda, Sakae; Naito, Atsushi; Kikuta, Junichi; Nishikawa, Keizo; Nishimura, Junichi; Haraguchi, Naotsugu; Takemasa, Ichiro; Mizushima, Tsunekazu; Ikeda, Masataka; Yamamoto, Hirofumi; Sekimoto, Mitsugu; Ishii, Hideshi; Doki, Yuichiro; Matsuda, Michiyuki; Kikuchi, Akira; Mori, Masaki; Ishii, Masaru

2013-01-01

The mechanism behind the spatiotemporal control of cancer cell dynamics and its possible association with cell proliferation has not been well established. By exploiting the intravital imaging technique, we found that cancer cell motility and invasive properties were closely associated with the cell cycle. In vivo inoculation of human colon cancer cells bearing fluorescence ubiquitination-based cell cycle indicator (Fucci) demonstrated an unexpected phenomenon: S/G2/M cells were more motile and invasive than G1 cells. Microarray analyses showed that Arhgap11a, an uncharacterized Rho GTPase-activating protein (RhoGAP), was expressed in a cell-cycle-dependent fashion. Expression of ARHGAP11A in cancer cells suppressed RhoA-dependent mechanisms, such as stress fiber formation and focal adhesion, which made the cells more prone to migrate. We also demonstrated that RhoA suppression by ARHGAP11A induced augmentation of relative Rac1 activity, leading to an increase in the invasive properties. RNAi-based inhibition of Arhgap11a reduced the invasion and in vivo expansion of cancers. Additionally, analysis of human specimens showed the significant up-regulation of Arhgap11a in colon cancers, which was correlated with clinical invasion status. The present study suggests that ARHGAP11A, a cell cycle-dependent RhoGAP, is a critical regulator of cancer cell mobility and is thus a promising therapeutic target in invasive cancers.

The Developmental Intestinal Regulator ELT-2 Controls p38-Dependent Immune Responses in Adult C. elegans

PubMed Central

Block, Dena H. S.; Twumasi-Boateng, Kwame; Kang, Hae Sung; Carlisle, Jolie A.; Hanganu, Alexandru; Lai, Ty Yu-Jen; Shapira, Michael

2015-01-01

GATA transcription factors play critical roles in cellular differentiation and development. However, their roles in mature tissues are less understood. In C. elegans larvae, the transcription factor ELT-2 regulates terminal differentiation of the intestine. It is also expressed in the adult intestine, where it was suggested to maintain intestinal structure and function, and where it was additionally shown to contribute to infection resistance. To study the function of elt-2 in adults we characterized elt-2-dependent gene expression following its knock-down specifically in adults. Microarray analysis identified two ELT-2-regulated gene subsets: one, enriched for hydrolytic enzymes, pointed at regulation of constitutive digestive functions as a dominant role of adult elt-2; the second was enriched for immune genes that are induced in response to Pseudomonas aeruginosa infection. Focusing on the latter, we used genetic analyses coupled to survival assays and quantitative RT-PCR to interrogate the mechanism(s) through which elt-2 contributes to immunity. We show that elt-2 controls p38-dependent gene induction, cooperating with two p38-activated transcription factors, ATF-7 and SKN-1. This demonstrates a mechanism through which the constitutively nuclear elt-2 can impact induced responses, and play a dominant role in C. elegans immunity. PMID:26016853

LKB1 Regulates Mitochondria-Dependent Presynaptic Calcium Clearance and Neurotransmitter Release Properties at Excitatory Synapses along Cortical Axons.

PubMed

Kwon, Seok-Kyu; Sando, Richard; Lewis, Tommy L; Hirabayashi, Yusuke; Maximov, Anton; Polleux, Franck

2016-07-01

Individual synapses vary significantly in their neurotransmitter release properties, which underlie complex information processing in neural circuits. Presynaptic Ca2+ homeostasis plays a critical role in specifying neurotransmitter release properties, but the mechanisms regulating synapse-specific Ca2+ homeostasis in the mammalian brain are still poorly understood. Using electrophysiology and genetically encoded Ca2+ sensors targeted to the mitochondrial matrix or to presynaptic boutons of cortical pyramidal neurons, we demonstrate that the presence or absence of mitochondria at presynaptic boutons dictates neurotransmitter release properties through Mitochondrial Calcium Uniporter (MCU)-dependent Ca2+ clearance. We demonstrate that the serine/threonine kinase LKB1 regulates MCU expression, mitochondria-dependent Ca2+ clearance, and thereby, presynaptic release properties. Re-establishment of MCU-dependent mitochondrial Ca2+ uptake at glutamatergic synapses rescues the altered neurotransmitter release properties characterizing LKB1-null cortical axons. Our results provide novel insights into the cellular and molecular mechanisms whereby mitochondria control neurotransmitter release properties in a bouton-specific way through presynaptic Ca2+ clearance.

An asymmetrically localized Staufen2-dependent RNA complex regulates maintenance of mammalian neural stem cells.

PubMed

Vessey, John P; Amadei, Gianluca; Burns, Sarah E; Kiebler, Michael A; Kaplan, David R; Miller, Freda D

2012-10-05

The cellular mechanisms that regulate self-renewal versus differentiation of mammalian somatic tissue stem cells are still largely unknown. Here, we asked whether an RNA complex regulates this process in mammalian neural stem cells. We show that the RNA-binding protein Staufen2 (Stau2) is apically localized in radial glial precursors of the embryonic cortex, where it forms a complex with other RNA granule proteins including Pumilio2 (Pum2) and DDX1, and the mRNAs for β-actin and mammalian prospero, prox1. Perturbation of this complex by functional knockdown of Stau2, Pum2, or DDX1 causes premature differentiation of radial glial precursors into neurons and mislocalization and misexpression of prox1 mRNA. Thus, a Stau2- and Pum2-dependent RNA complex directly regulates localization and, potentially, expression of target mRNAs like prox1 in mammalian neural stem cells, and in so doing regulates the balance of stem cell maintenance versus differentiation. Copyright © 2012 Elsevier Inc. All rights reserved.

Stochastic seasonality and nonlinear density-dependent factors regulate population size in an African rodent

USGS Publications Warehouse

Leirs, H.; Stenseth, N.C.; Nichols, J.D.; Hines, J.E.; Verhagen, R.; Verheyen, W.

1997-01-01

Ecology has long been troubled by the controversy over how populations are regulated. Some ecologists focus on the role of environmental effects, whereas others argue that density-dependent feedback mechanisms are central. The relative importance of both processes is still hotly debated, but clear examples of both processes acting in the same population are rare. Keyfactor analysis (regression of population changes on possible causal factors) and time-series analysis are often used to investigate the presence of density dependence, but such approaches may be biased and provide no information on actual demographic rates. Here we report on both density-dependent and density-independent effects in a murid rodent pest species, the multimammate rat Mastomys natalensis (Smith, 1834), using statistical capture-recapture models. Both effects occur simultaneously, but we also demonstrate that they do not affect all demographic rates in the same way. We have incorporated the obtained estimates of demographic rates in a population dynamics model and show that the observed dynamics are affected by stabilizing nonlinear density-dependent components coupled with strong deterministic and stochastic seasonal components.

Light-dependent gene regulation by a coenzyme B12-based photoreceptor

PubMed Central

Ortiz-Guerrero, Juan Manuel; Polanco, María Carmen; Murillo, Francisco J.; Padmanabhan, S.; Elías-Arnanz, Montserrat

2011-01-01

Cobalamin (B12) typically functions as an enzyme cofactor but can also regulate gene expression via RNA-based riboswitches. B12-directed gene regulatory mechanisms via protein factors have, however, remained elusive. Recently, we reported down-regulation of a light-inducible promoter in the bacterium Myxococcus xanthus by two paralogous transcriptional repressors, of which one, CarH, but not the other, CarA, absolutely requires B12 for activity even though both have a canonical B12-binding motif. Unanswered were what underlies this striking difference, what is the specific cobalamin used, and how it acts. Here, we show that coenzyme B12 (5′-deoxyadenosylcobalamin, AdoB12), specifically dictates CarH function in the dark and on exposure to light. In the dark, AdoB12-binding to the autonomous domain containing the B12-binding motif foments repressor oligomerization, enhances operator binding, and blocks transcription. Light, at various wavelengths at which AdoB12 absorbs, dismantles active repressor oligomers by photolysing the bound AdoB12 and weakens repressor–operator binding to allow transcription. By contrast, AdoB12 alters neither CarA oligomerization nor operator binding, thus accounting for its B12-independent activity. Our findings unveil a functional facet of AdoB12 whereby it serves as the chromophore of a unique photoreceptor protein class acting in light-dependent gene regulation. The prevalence of similar proteins of unknown function in microbial genomes suggests that this distinct B12-based molecular mechanism for photoregulation may be widespread in bacteria. PMID:21502508

Acid-induced off-response of PKD2L1 channel in Xenopus oocytes and its regulation by Ca2+

PubMed Central

Hussein, Shaimaa; Zheng, Wang; Dyte, Chris; Wang, Qian; Yang, JungWoo; Zhang, Fan; Tang, Jingfeng; Cao, Ying; Chen, Xing-Zhen

2015-01-01

Polycystic kidney disease (PKD) protein 2 Like 1 (PKD2L1), also called transient receptor potential polycystin-3 (TRPP3), regulates Ca2+-dependent hedgehog signalling in primary cilia, intestinal development and sour tasting but with an unclear mechanism. PKD2L1 is a Ca2+-permeable cation channel that is activated by extracellular Ca2+ (on-response) in Xenopus oocytes. PKD2L1 co-expressed with PKD protein 1 Like 3 (PKD1L3) exhibits extracellular acid-induced activation (off-response, i.e., activation following acid removal) but whether PKD1L3 participates in acid sensing remains unclear. Here we used the two-microelectrode voltage-clamp, site directed mutagenesis, Western blotting, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence, and showed that PKD2L1 expressed in oocytes exhibits sustained off-response currents in the absence of PKD1L3. PKD1L3 co-expression augmented the PKD2L1 plasma membrane localization but did not alter the observed properties of the off-response. PKD2L1 off-response was inhibited by an increase in intracellular Ca2+. We also identified two intra-membrane residues aspartic acid 349 (D349) and glutamic acid 356 (E356) in the third transmembrane domain that are critical for PKD2L1 channel function. Our study suggests that PKD2L1 may itself sense acids and defines off-response properties in the absence of PKD1L3. PMID:26502994

Manduca Contactin Regulates Amyloid Precursor Protein-Dependent Neuronal Migration

PubMed Central

Ramaker, Jenna M.; Swanson, Tracy L.

2016-01-01

Amyloid precursor protein (APP) was originally identified as the source of β-amyloid peptides that accumulate in Alzheimer's disease (AD), but it also has been implicated in the control of multiple aspects of neuronal motility. APP belongs to an evolutionarily conserved family of transmembrane proteins that can interact with a variety of adapter and signaling molecules. Recently, we showed that both APP and its insect ortholog [APPL (APP-Like)] directly bind the heterotrimeric G-protein Goα, supporting the model that APP can function as an unconventional Goα-coupled receptor. We also adapted a well characterized assay of neuronal migration in the hawkmoth, Manduca sexta, to show that APPL–Goα signaling restricts ectopic growth within the developing nervous system, analogous to the role postulated for APP family proteins in controlling migration within the mammalian cortex. Using this assay, we have now identified Manduca Contactin (MsContactin) as an endogenous ligand for APPL, consistent with previous work showing that Contactins interact with APP family proteins in other systems. Using antisense-based knockdown protocols and fusion proteins targeting both proteins, we have shown that MsContactin is selectively expressed by glial cells that ensheath the migratory neurons (expressing APPL), and that MsContactin–APPL interactions normally prevent inappropriate migration and outgrowth. These results provide new evidence that Contactins can function as authentic ligands for APP family proteins that regulate APP-dependent responses in the developing nervous system. They also support the model that misregulated Contactin–APP interactions might provoke aberrant activation of Goα and its effectors, thereby contributing to the neurodegenerative sequelae that typify AD. SIGNIFICANCE STATEMENT Members of the amyloid precursor protein (APP) family participate in many aspects of neuronal development, but the ligands that normally activate APP signaling have remained

Distinct phosphodiesterase 5A-containing compartments allow selective regulation of cGMP-dependent signalling in human arterial smooth muscle cells.

PubMed

Wilson, Lindsay S; Guo, Manhong; Umana, M Bibiana; Maurice, Donald H

2017-08-01

Cyclic GMP (cGMP) translates and integrates much of the information encoded by nitric oxide (NO · ) and several natriuretic peptides, including the atrial natriuretic peptide (ANP). Previously, we reported that integration of a cGMP-specific cyclic nucleotide phosphodiesterase, namely phosphodiesterase 5A (PDE5A), into a protein kinase G (PKG)- and inositol-1,4,5-trisphosphate receptor (IP 3 R)-containing endoplasmic reticulum (ER) signalosome allows localized control of PDE5A activity and of PKG-dependent inhibition of IP 3 -mediated release of ER Ca 2+ in human platelets. Herein, we report that PDE5A integrates into an analogous signalosome in human arterial smooth muscle cells (HASMC), wherein it regulates muscarinic agonist-dependent Ca 2+ release and is activated selectively by PKG-dependent phosphorylation. In addition, we report that PDE5A also regulates HASMC functions via events independent of PKG, but rather through actions coordinated by competitive cGMP-mediated inhibition of cAMP hydrolysis by the so-called cGMP-inhibited cAMP PDE, namely phosphodiesterase 3A (PDE3A). Indeed, we show that ANP increases both cGMP and cAMP levels in HASMC and promotes phosphorylation of vasodilator-stimulated phospho-protein (VASP) at each the PKG and PKA phospho-acceptor sites. Since selective inhibition of PDE5 decreased DNA synthesis and chemotaxis of HASMC, and that PDE3A knockdown obviated these effects, our findings are consistent with a role for a PDE5A-PDE3A-PKA axis in their regulation. Our findings provide insight into the existence of distinct "pools" of PDE5A in HASMC and support the idea that these discrete compartments regulate distinct cGMP-dependent events. As a corollary, we suggest that it may be possible to target these distinct PDE5A-regulated pools and in so-doing differentially impact selected cGMP-regulated functions in these cells. Copyright © 2017. Published by Elsevier Inc.

Genome-wide analysis of a Wnt1-regulated transcriptional network implicates neurodegenerative pathways.

PubMed

Wexler, Eric M; Rosen, Ezra; Lu, Daning; Osborn, Gregory E; Martin, Elizabeth; Raybould, Helen; Geschwind, Daniel H

2011-10-04

Wnt proteins are critical to mammalian brain development and function. The canonical Wnt signaling pathway involves the stabilization and nuclear translocation of β-catenin; however, Wnt also signals through alternative, noncanonical pathways. To gain a systems-level, genome-wide view of Wnt signaling, we analyzed Wnt1-stimulated changes in gene expression by transcriptional microarray analysis in cultured human neural progenitor (hNP) cells at multiple time points over a 72-hour time course. We observed a widespread oscillatory-like pattern of changes in gene expression, involving components of both the canonical and the noncanonical Wnt signaling pathways. A higher-order, systems-level analysis that combined independent component analysis, waveform analysis, and mutual information-based network construction revealed effects on pathways related to cell death and neurodegenerative disease. Wnt effectors were tightly clustered with presenilin1 (PSEN1) and granulin (GRN), which cause dominantly inherited forms of Alzheimer's disease and frontotemporal dementia (FTD), respectively. We further explored a potential link between Wnt1 and GRN and found that Wnt1 decreased GRN expression by hNPs. Conversely, GRN knockdown increased WNT1 expression, demonstrating that Wnt and GRN reciprocally regulate each other. Finally, we provided in vivo validation of the in vitro findings by analyzing gene expression data from individuals with FTD. These unbiased and genome-wide analyses provide evidence for a connection between Wnt signaling and the transcriptional regulation of neurodegenerative disease genes.

State-Dependent Differences in Emotion Regulation Between Unmedicated Bipolar Disorder and Major Depressive Disorder.

PubMed

Rive, Maria M; Mocking, Roel J T; Koeter, Maarten W J; van Wingen, Guido; de Wit, Stella J; van den Heuvel, Odile A; Veltman, Dick J; Ruhé, Henricus G; Schene, Aart H

2015-07-01

Major depressive disorder (MDD) and bipolar disorder (BD) are difficult to distinguish clinically during the depressed or remitted states. Both mood disorders are characterized by emotion regulation disturbances; however, little is known about emotion regulation differences between MDD and BD. Better insight into these differences would be helpful for differentiation based on disorder-specific underlying pathophysiological mechanisms. Previous studies comparing these disorders often allowed medication use, limiting generalizability and validity. Moreover, patients with MDD and BD were mostly compared during the depressed, but not the remitted, state, while state might potentially modulate differences between MDD and BD. To investigate positive and negative emotion regulation in medication-free patients with MDD and BD in 2 mood states: depressed or remitted. A cross-sectional study conducted from May 2009 to August 2013 comparing behavioral and functional magnetic resonance imaging emotion regulation data of 42 patients with MDD, 35 with BD, and 36 healthy control (HC) participants free of psychotropic medication recruited from several psychiatric institutions across the Netherlands. A voluntary emotion regulation functional magnetic resonance imaging task using positive and negative pictures. Behavioral and functional magnetic resonance imaging blood oxygen level-dependent responses during emotion regulation. In the remitted state, only patients with BD showed impaired emotion regulation (t = 3.39; P < .001; Cohen d = 0.70), irrespective of emotion type and associated with increased dorsolateral prefrontal cortex activity compared with those with MDD and healthy control participants (P = .008). In the depressed state, patients with MDD and BD differed with regard to happy vs sad emotion regulation (t = 4.19; P < .001; Cohen d = 1.66) associated with differences in rostral anterior cingulate activity (P < .001). Patients with MDD

The NH2 terminus regulates voltage-dependent gating of CALHM ion channels.

PubMed

Tanis, Jessica E; Ma, Zhongming; Foskett, J Kevin

2017-08-01

Calcium homeostasis modulator protein-1 (CALHM1) and its Caenorhabditis elegans (ce) homolog, CLHM-1, belong to a new family of physiologically important ion channels that are regulated by voltage and extracellular Ca 2+ (Ca 2+ o ) but lack a canonical voltage-sensing domain. Consequently, the intrinsic voltage-dependent gating mechanisms for CALHM channels are unknown. Here, we performed voltage-clamp experiments on ceCLHM-1 chimeric, deletion, insertion, and point mutants to assess the role of the NH 2 terminus (NT) in CALHM channel gating. Analyses of chimeric channels in which the ceCLHM-1 and human (h)CALHM1 NH 2 termini were interchanged showed that the hCALHM1 NT destabilized channel-closed states, whereas the ceCLHM-1 NT had a stabilizing effect. In the absence of Ca 2+ o , deletion of up to eight amino acids from the ceCLHM-1 NT caused a hyperpolarizing shift in the conductance-voltage relationship with little effect on voltage-dependent slope. However, deletion of nine or more amino acids decreased voltage dependence and induced a residual conductance at hyperpolarized voltages. Insertion of amino acids into the NH 2 -terminal helix also decreased voltage dependence but did not prevent channel closure. Mutation of ceCLHM-1 valine 9 and glutamine 13 altered half-maximal activation and voltage dependence, respectively, in 0 Ca 2+ In 2 mM Ca 2+ o , ceCLHM-1 NH 2 -terminal deletion and point mutant channels closed completely at hyperpolarized voltages with apparent affinity for Ca 2+ o indistinguishable from wild-type ceCLHM-1, although the ceCLHM-1 valine 9 mutant exhibited an altered conductance-voltage relationship and kinetics. We conclude that the NT plays critical roles modulating voltage dependence and stabilizing the closed states of CALHM channels. Copyright © 2017 the American Physiological Society.

Insulin-induced Gene Protein (INSIG)-dependent Sterol Regulation of Hmg2 Endoplasmic Reticulum-associated Degradation (ERAD) in Yeast*

PubMed Central

Theesfeld, Chandra L.; Hampton, Randolph Y.

2013-01-01

Insulin-induced gene proteins (INSIGs) function in control of cellular cholesterol. Mammalian INSIGs exert control by directly interacting with proteins containing sterol-sensing domains (SSDs) when sterol levels are elevated. Mammalian 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (HMGR) undergoes sterol-dependent, endoplasmic-reticulum (ER)-associated degradation (ERAD) that is mediated by INSIG interaction with the HMGR SSD. The yeast HMGR isozyme Hmg2 also undergoes feedback-regulated ERAD in response to the early pathway-derived isoprene gernanylgeranyl pyrophosphate (GGPP). Hmg2 has an SSD, and its degradation is controlled by the INSIG homologue Nsg1. However, yeast Nsg1 promotes Hmg2 stabilization by inhibiting GGPP-stimulated ERAD. We have proposed that the seemingly disparate INSIG functions can be unified by viewing INSIGs as sterol-dependent chaperones of SSD clients. Accordingly, we tested the role of sterols in the Nsg1 regulation of Hmg2. We found that both Nsg1-mediated stabilization of Hmg2 and the Nsg1-Hmg2 interaction required the early sterol lanosterol. Lowering lanosterol in the cell allowed GGPP-stimulated Hmg2 ERAD. Thus, Hmg2-regulated degradation is controlled by a two-signal logic; GGPP promotes degradation, and lanosterol inhibits degradation. These data reveal that the sterol dependence of INSIG-client interaction has been preserved for over 1 billion years. We propose that the INSIGs are a class of sterol-dependent chaperones that bind to SSD clients, thus harnessing ER quality control in the homeostasis of sterols. PMID:23306196

Effectiveness of Gross Model-Based Emotion Regulation Strategies Training on Anger Reduction in Drug-Dependent Individuals and its Sustainability in Follow-up

PubMed Central

Massah, Omid; Sohrabi, Faramarz; A’azami, Yousef; Doostian, Younes; Farhoudian, Ali; Daneshmand, Reza

2016-01-01

Background Emotion plays an important role in adapting to life changes and stressful events. Difficulty regulating emotions is one of the problems drug abusers often face, and teaching these individuals to express and manage their emotions can be effective on improving their difficult circumstances. Objectives The present study aimed to determine the effectiveness of the Gross model-based emotion regulation strategies training on anger reduction in drug-dependent individuals. Patients and Methods The present study had a quasi-experimental design wherein pretest-posttest evaluations were applied using a control group. The population under study included addicts attending Marivan’s methadone maintenance therapy centers in 2012 - 2013. Convenience sampling was used to select 30 substance-dependent individuals undergoing maintenance treatment who were then randomly assigned to the experiment and control groups. The experiment group received its training in eight two-hour sessions. Data were analyzed using analysis of co-variance and paired t-test. Results There was significant reduction in anger symptoms of drug-dependent individuals after gross model based emotion regulation training (ERT) (P < 0.001). Moreover, the effectiveness of the training on anger was persistent in the follow-up period. Conclusions Symptoms of anger in drug-dependent individuals of this study were reduced by gross model-based emotion regulation strategies training. Based on the results of this study, we may conclude that the gross model based emotion regulation strategies training can be applied alongside other therapies to treat drug abusers undergoing rehabilitation. PMID:27162759

Effectiveness of Gross Model-Based Emotion Regulation Strategies Training on Anger Reduction in Drug-Dependent Individuals and its Sustainability in Follow-up.

PubMed

Massah, Omid; Sohrabi, Faramarz; A'azami, Yousef; Doostian, Younes; Farhoudian, Ali; Daneshmand, Reza

2016-03-01

Emotion plays an important role in adapting to life changes and stressful events. Difficulty regulating emotions is one of the problems drug abusers often face, and teaching these individuals to express and manage their emotions can be effective on improving their difficult circumstances. The present study aimed to determine the effectiveness of the Gross model-based emotion regulation strategies training on anger reduction in drug-dependent individuals. The present study had a quasi-experimental design wherein pretest-posttest evaluations were applied using a control group. The population under study included addicts attending Marivan's methadone maintenance therapy centers in 2012 - 2013. Convenience sampling was used to select 30 substance-dependent individuals undergoing maintenance treatment who were then randomly assigned to the experiment and control groups. The experiment group received its training in eight two-hour sessions. Data were analyzed using analysis of co-variance and paired t-test. There was significant reduction in anger symptoms of drug-dependent individuals after gross model based emotion regulation training (ERT) (P < 0.001). Moreover, the effectiveness of the training on anger was persistent in the follow-up period. Symptoms of anger in drug-dependent individuals of this study were reduced by gross model-based emotion regulation strategies training. Based on the results of this study, we may conclude that the gross model based emotion regulation strategies training can be applied alongside other therapies to treat drug abusers undergoing rehabilitation.

Regulation of the Drosophila Hypoxia-Inducible Factor α Sima by CRM1-Dependent Nuclear Export ▿

PubMed Central

Romero, Nuria M.; Irisarri, Maximiliano; Roth, Peggy; Cauerhff, Ana; Samakovlis, Christos; Wappner, Pablo

2008-01-01

Hypoxia-inducible factor α (HIF-α) proteins are regulated by oxygen levels through several different mechanisms that include protein stability, transcriptional coactivator recruitment, and subcellular localization. It was previously reported that these transcription factors are mainly nuclear in hypoxia and cytoplasmic in normoxia, but so far the molecular basis of this regulation is unclear. We show here that the Drosophila melanogaster HIF-α protein Sima shuttles continuously between the nucleus and the cytoplasm. We identified the relevant nuclear localization signal and two functional nuclear export signals (NESs). These NESs are in the Sima basic helix-loop-helix (bHLH) domain and promote CRM1-dependent nuclear export. Site-directed mutagenesis of either NES provoked Sima nuclear retention and increased transcriptional activity, suggesting that nuclear export contributes to Sima regulation. The identified NESs are conserved and probably functional in the bHLH domains of several bHLH-PAS proteins. We propose that rapid nuclear export of Sima regulates the duration of cellular responses to hypoxia. PMID:18332128

Chloroquine uptake, altered partitioning and the basis of drug resistance: evidence for chloride-dependent ionic regulation.

PubMed

Martiney, J A; Ferrer, A S; Cerami, A; Dzekunov, S; Roepe, P

1999-01-01

The biochemical mechanism of chloroquine resistance in Plasmodium falciparum remains unknown. We postulated that chloroquine-resistant strains could alter ion fluxes that then indirectly control drug accumulation within the parasite by affecting pH and/or membrane potential ('altered partitioning mechanism'). Two principal intracellular pH-regulating systems in many cell types are the amiloride-sensitive Na+/H+ exchanger (NHE), and the sodium-independent, stilbene-sensitive Cl-/HCO3- antiporter (AE). We report that under physiological conditions (balanced CO2 and HCO3-) chloroquine uptake and susceptibility are not altered by amiloride analogues. We also do not detect a significant difference in NHE activity between chloroquine-sensitive and chloroquine-resistant strains via single cell photometry methods. AE activity is dependent on the intracellular and extracellular concentrations of Cl- and HCO3- ions. Chloroquine-resistant strains differentially respond to experimental modifications in chloride-dependent homeostasis, including growth, cytoplasmic pH and pH regulation. Chloroquine susceptibility is altered by stilbene DIDS only on chloroquine-resistant strains. Our results suggest that a Cl(-)-dependent system (perhaps AE) has a significant effect on the uptake of chloroquine by the infected erythrocyte, and that alterations of this biophysical parameter may be part of the mechanism of chloroquine resistance in P. falciparum.

Characterization of the Drosophila Methoprene -tolerant gene product. Juvenile hormone binding and ligand-dependent gene regulation.

PubMed

Miura, Ken; Oda, Masahito; Makita, Sumiko; Chinzei, Yasuo

2005-03-01

Juvenile hormones (JHs) of insects are sesquiterpenoids that regulate a great diversity of processes in development and reproduction. As yet the molecular modes of action of JH are poorly understood. The Methoprene-tolerant (Met) gene of Drosophila melanogaster has been found to be responsible for resistance to a JH analogue (JHA) insecticide, methoprene. Previous studies on Met have implicated its involvement in JH signaling, although direct evidence is lacking. We have now examined the product of Met (MET) in terms of its binding to JH and ligand-dependent gene regulation. In vitro synthesized MET directly bound to JH III with high affinity (Kd = 5.3 +/- 1.5 nm, mean +/- SD), consistent with the physiological JH concentration. In transient transfection assays using Drosophila S2 cells the yeast GAL4-DNA binding domain fused to MET exerted JH- or JHA-dependent activation of a reporter gene. Activation of the reporter gene was highly JH- or JHA-specific with the order of effectiveness: JH III > JH II > JH I > methoprene; compounds which are only structurally related to JH or JHA did not induce any activation. Localization of MET in the S2 cells was nuclear irrespective of the presence or absence of JH. These results suggest that MET may function as a JH-dependent transcription factor.

Regulation of Aedes aegypti Population Dynamics in Field Systems: Quantifying Direct and Delayed Density Dependence

PubMed Central

Walsh, Rachael K.; Aguilar, Cristobal L.; Facchinelli, Luca; Valerio, Laura; Ramsey, Janine M.; Scott, Thomas W.; Lloyd, Alun L.; Gould, Fred

2013-01-01

Transgenic strains of Aedes aegypti have been engineered to help control transmission of dengue virus. Although resources have been invested in developing the strains, we lack data on the ecology of mosquitoes that could impact the success of this approach. Although studies of intra-specific competition have been conducted using Ae. aegypti larvae, none of these studies examine mixed age cohorts at densities that occur in the field, with natural nutrient levels. Experiments were conducted in Mexico to determine the impact of direct and delayed density dependence on Ae. aegypti populations. Natural water, food, and larval densities were used to estimate the impacts of density dependence on larval survival, development, and adult body size. Direct and delayed density-dependent factors had a significant impact on larval survival, larval development, and adult body size. These results indicate that control methods attempting to reduce mosquito populations may be counteracted by density-dependent population regulation. PMID:23669230

Coordinated Expression of Cyclin-dependent Kinase-4 and its Regulators in Human Oral Tumors

PubMed Central

POI, MING J.; KNOBLOCH, THOMAS J.; SEARS, MARTA T.; UHRIG, LANA K.; WARNER, BLAKE M.; WEGHORST, CHRISTOPHER M.; LI, JUNAN

2014-01-01

Background/Aim While aberrant expression of cyclin-dependent kinase-4 (CDK4) has been found in squamous cell carcinoma of the head and neck (SCCHN), the associations between CDK4 and its regulators, namely, cyclin D1, cyclin E, gankyrin, SEI1, and BMI1 in gene expression remain to be explored. Herein we investigated the mRNA profiles of these oncogenes and their interrelations in different oral lesion tissues. Materials and Methods Thirty SCCHN specimens and patient-matched high at-risk mucosa (HARM) and 16 healthy control specimens were subjected to quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses. Results The mRNA levels of CDK4, cyclin D1, gankyrin, SEI1, BMI1 were significantly elevated in both HARM and SCCHN (in comparison with control specimens), and statistically significant correlations were found among these markers in gene expression. Conclusion Up-regulation of CDK4 and its regulators takes place in oral cancer progression in a coordinate manner, and HARM and SCCHN share a similar molecular signature within the CDK4-pRB pathway. PMID:24982332

Interaction with epsin 1 regulates the constitutive clathrin-dependent internalization of ErbB3.

PubMed

Szymanska, Monika; Fosdahl, Anne Marthe; Raiborg, Camilla; Dietrich, Markus; Liestøl, Knut; Stang, Espen; Bertelsen, Vibeke

2016-06-01

In contrast to other members of the EGF receptor family, ErbB3 is constitutively internalized in a clathrin-dependent manner. Previous studies have shown that ErbB3 does not interact with the coated pit localized adaptor complex 2 (AP-2), and that ErbB3 lacks two AP-2 interacting internalization signals identified in the EGF receptor. Several other clathrin-associated sorting proteins which may recruit cargo into coated pits have, however, been identified, and the study was performed to identify adaptors needed for constitutive internalization of ErbB3. A high-throughput siRNA screen was used to identify adaptor proteins needed for internalization of ErbB3. Upon knock-down of candidate proteins internalization of ErbB3 was identified using an antibody-based internalization assay combined with automatic fluorescence microscopy. Among 29 candidates only knock-down of epsin 1 turned out to inhibit ErbB3. Epsin 1 has ubiquitin interacting motifs (UIMs) and we show that ErbB3 interacts with an epsin 1 deletion mutant containing these UIMs. In support of an ErbB3-epsin 1 UIM dependent interaction, we show that ErbB3 is constitutively ubiquitinated, but that both ubiquitination and the ErbB3-epsin 1 interaction increase upon ligand binding. Altogether the results are consistent with a model whereby both constitutive and ligand-induced internalization of ErbB3 are regulated through interaction with epsin 1. Internalization is an important regulator of growth factor receptor mediated signaling and the current study identify mechanisms regulating plasma membrane turnover of ErbB3. Copyright © 2016 Elsevier B.V. All rights reserved.

CREB at the Crossroads of Activity-Dependent Regulation of Nervous System Development and Function.

PubMed

Belgacem, Yesser H; Borodinsky, Laura N

2017-01-01

The central nervous system is a highly plastic network of cells that constantly adjusts its functions to environmental stimuli throughout life. Transcription-dependent mechanisms modify neuronal properties to respond to external stimuli regulating numerous developmental functions, such as cell survival and differentiation, and physiological functions such as learning, memory, and circadian rhythmicity. The discovery and cloning of the cyclic adenosine monophosphate (cAMP) responsive element binding protein (CREB) constituted a big step toward deciphering the molecular mechanisms underlying neuronal plasticity. CREB was first discovered in learning and memory studies as a crucial mediator of activity-dependent changes in target gene expression that in turn impose long-lasting modifications of the structure and function of neurons. In this chapter, we review the molecular and signaling mechanisms of neural activity-dependent recruitment of CREB and its cofactors. We discuss the crosstalk between signaling pathways that imprints diverse spatiotemporal patterns of CREB activation allowing for the integration of a wide variety of stimuli.

Translation Stress Positively Regulates MscL-Dependent Excretion of Cytoplasmic Proteins

PubMed Central

Morra, Rosa; Del Carratore, Francesco; Muhamadali, Howbeer; Horga, Luminita Gabriela; Halliwell, Samantha

2018-01-01

ABSTRACT The apparent mislocalization or excretion of cytoplasmic proteins is a commonly observed phenomenon in both bacteria and eukaryotes. However, reports on the mechanistic basis and the cellular function of this so-called “nonclassical protein secretion” are limited. Here we report that protein overexpression in recombinant cells and antibiotic-induced translation stress in wild-type Escherichia coli cells both lead to excretion of cytoplasmic protein (ECP). Condition-specific metabolomic and proteomic analyses, combined with genetic knockouts, indicate a role for both the large mechanosensitive channel (MscL) and the alternative ribosome rescue factor A (ArfA) in ECP. Collectively, the findings indicate that MscL-dependent protein excretion is positively regulated in response to both osmotic stress and arfA-mediated translational stress. PMID:29382730

MicroRNA-dependent regulation of transcription in non-small cell lung cancer.

PubMed

Molina-Pinelo, Sonia; Gutiérrez, Gabriel; Pastor, Maria Dolores; Hergueta, Marta; Moreno-Bueno, Gema; García-Carbonero, Rocío; Nogal, Ana; Suárez, Rocío; Salinas, Ana; Pozo-Rodríguez, Francisco; Lopez-Rios, Fernando; Agulló-Ortuño, Maria Teresa; Ferrer, Irene; Perpiñá, Asunción; Palacios, José; Carnero, Amancio; Paz-Ares, Luis

2014-01-01

Squamous cell lung cancer (SCC) and adenocarcinoma are the most common histological subtypes of non-small cell lung cancer (NSCLC), and have been traditionally managed in the clinic as a single entity. Increasing evidence, however, illustrates the biological diversity of these two histological subgroups of lung cancer, and supports the need to improve our understanding of the molecular basis beyond the different phenotypes if we aim to develop more specific and individualized targeted therapy. The purpose of this study was to identify microRNA (miRNA)-dependent transcriptional regulation differences between SCC and adenocarcinoma histological lung cancer subtypes. In this work, paired miRNA (667 miRNAs by TaqMan Low Density Arrays (TLDA)) and mRNA profiling (Whole Genome 44 K array G112A, Agilent) was performed in tumor samples of 44 NSCLC patients. Nine miRNAs and 56 mRNAs were found to be differentially expressed in SCC versus adenocarcinoma samples. Eleven of these 56 mRNA were predicted as targets of the miRNAs identified to be differently expressed in these two histological conditions. Of them, 6 miRNAs (miR-149, miR-205, miR-375, miR-378, miR-422a and miR-708) and 9 target genes (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B, MUC1) were validated by quantitative PCR in an independent cohort of 41 lung cancer patients. Furthermore, the inverse correlation between mRNAs and microRNAs expression was also validated. These results suggest miRNA-dependent transcriptional regulation differences play an important role in determining key hallmarks of NSCLC, and may provide new biomarkers for personalized treatment strategies.

Heart rate variability predicts alcohol craving in alcohol dependent outpatients: further evidence for HRV as a psychophysiological marker of self-regulation.

PubMed

Quintana, Daniel S; Guastella, Adam J; McGregor, Iain S; Hickie, Ian B; Kemp, Andrew H

2013-09-01

Past research has highlighted an important role of the autonomic nervous system in alcohol dependence and capacity for self-regulation. While previous studies have examined alcohol dependent inpatients, it remains unclear whether resting-state HRV, a potential psychophysiological marker of ones capacity for self-regulation, is related to craving in patients who currently consume alcohol. Thus, the aim of the present study was to determine whether HRV predicts alcohol craving in dependent individuals in the community. Resting-state HRV and alcohol craving, as indexed by the obsessive compulsive drinking scale, were assessed in 26 alcohol dependent outpatients. Results supported hypotheses indicating that HRV accounts for an additional 12.1% of the variance in craving after controlling for age, anxiety and levels of alcohol consumption. Here we show for the first time that resting-state HRV predicts craving in alcohol dependent outpatients. Results provide important new evidence for a role of the autonomic nervous system in the maintenance of dependence disorders. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Sulfide-responsive transcriptional repressor SqrR functions as a master regulator of sulfide-dependent photosynthesis.

PubMed

Shimizu, Takayuki; Shen, Jiangchuan; Fang, Mingxu; Zhang, Yixiang; Hori, Koichi; Trinidad, Jonathan C; Bauer, Carl E; Giedroc, David P; Masuda, Shinji

2017-02-28

Sulfide was used as an electron donor early in the evolution of photosynthesis, with many extant photosynthetic bacteria still capable of using sulfur compounds such as hydrogen sulfide (H 2 S) as a photosynthetic electron donor. Although enzymes involved in H 2 S oxidation have been characterized, mechanisms of regulation of sulfide-dependent photosynthesis have not been elucidated. In this study, we have identified a sulfide-responsive transcriptional repressor, SqrR, that functions as a master regulator of sulfide-dependent gene expression in the purple photosynthetic bacterium Rhodobacter capsulatus SqrR has three cysteine residues, two of which, C41 and C107, are conserved in SqrR homologs from other bacteria. Analysis with liquid chromatography coupled with an electrospray-interface tandem-mass spectrometer reveals that SqrR forms an intramolecular tetrasulfide bond between C41 and C107 when incubated with the sulfur donor glutathione persulfide. SqrR is oxidized in sulfide-stressed cells, and tetrasulfide-cross-linked SqrR binds more weakly to a target promoter relative to unmodified SqrR. C41S and C107S R. capsulatus SqrRs lack the ability to respond to sulfide, and constitutively repress target gene expression in cells. These results establish that SqrR is a sensor of H 2 S-derived reactive sulfur species that maintain sulfide homeostasis in this photosynthetic bacterium and reveal the mechanism of sulfide-dependent transcriptional derepression of genes involved in sulfide metabolism.

PKA- and PKC-dependent regulation of angiopoietin 2 mRNA in human granulosa lutein cells.

PubMed

Witt, P S; Pietrowski, D; Keck, C

2004-02-01

New blood vessels develop from preexisting vessels in response to growth factors or hypoxic conditions. Recent studies have shown that angiopoietin 2 (ANGPT-2) plays an important role in the modulation of angiogenesis and vasculogenesis in humans and mice. The signaling pathways that lead to the regulation of ANGPT-2 are largely unclear. Here, we report that protein kinase C and protein kinase A activators (ADMB, 8-Cl-cAMP) increased the mRNA levels of ANGPT-2 in human Granulosa cells, whereas PKC and PKA Inhibitors (Rp-cAMP, GO 6983) decreased markedly the level of ANGPT-2 mRNA. Due to varying specificity of the modulators for certain protein kinases subunits, we conclude that the conventional PKCs, but not PKC alpha and beta1, the atypical PKCs and the PKA I, are involved in the regulation of ANGPT-2. These findings may help to explain the role of both PKA and PKC dependent signaling cascades in the regulation of ANGPT-2 mRNA.

TALE activators regulate gene expression in a position- and strand-dependent manner in mammalian cells.

PubMed

Uhde-Stone, Claudia; Cheung, Edna; Lu, Biao

2014-01-24

Transcription activator-like effectors (TALEs) are a class of transcription factors that are readily programmable to regulate gene expression. Despite their growing popularity, little is known about binding site parameters that influence TALE-mediated gene activation in mammalian cells. We demonstrate that TALE activators modulate gene expression in mammalian cells in a position- and strand-dependent manner. To study the effects of binding site location, we engineered TALEs customized to recognize specific DNA sequences located in either the promoter or the transcribed region of reporter genes. We found that TALE activators robustly activated reporter genes when their binding sites were located within the promoter region. In contrast, TALE activators inhibited the expression of reporter genes when their binding sites were located on the sense strand of the transcribed region. Notably, this repression was independent of the effector domain utilized, suggesting a simple blockage mechanism. We conclude that TALE activators in mammalian cells regulate genes in a position- and strand-dependent manner that is substantially different from gene activation by native TALEs in plants. These findings have implications for optimizing the design of custom TALEs for genetic manipulation in mammalian cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Temperature-dependent endogenous oxygen concentration regulates microsomal oleate desaturase in developing sunflower seeds.

PubMed

Rolletschek, Hardy; Borisjuk, Ljudmilla; Sánchez-García, Alicia; Gotor, Cecilia; Romero, Luis C; Martínez-Rivas, José M; Mancha, Manuel

2007-01-01

Oleoyl-phosphatidylcholine desaturase (FAD2) is a key enzyme involved in fatty acid desaturation in oilseeds, which is affected by environmental temperature. The results of this study show that FAD2 is regulated in vivo via temperature-dependent endogenous oxygen concentrations in developing sunflower (Helianthus annuus L.) seeds. By combining in vivo oxygen profiling, in situ hybridization of FAD2 genes, an assay of energy status, fatty acid analysis, and an in vitro FAD2 enzyme activity assay, it is shown that: (i) the oil-storing embryo is characterized by a very low oxygen level that is developmentally regulated. Oxygen supply is mainly limited by the thin seed coat. (ii) Elevations of external oxygen supply raised the energy status of seed and produced a dramatic increase of the FAD2 enzyme activity as well as the linoleic acid content. (iii) A clear negative correlation exists between temperature and internal oxygen concentration. The changes occurred almost instantly and the effect was fully reversible. The results indicate that the internal oxygen level acts as a key regulator for the activity of the FAD2 enzyme. It is concluded that a major mechanism by which temperature modifies the unsaturation degree of the sunflower oil is through its effect on dissolved oxygen levels in the developing seed.

Acute Fasting Regulates Retrograde Synaptic Enhancement through a 4E-BP-Dependent Mechanism.

PubMed

Kauwe, Grant; Tsurudome, Kazuya; Penney, Jay; Mori, Megumi; Gray, Lindsay; Calderon, Mario R; Elazouzzi, Fatima; Chicoine, Nicole; Sonenberg, Nahum; Haghighi, A Pejmun

2016-12-21

While beneficial effects of fasting on organismal function and health are well appreciated, we know little about the molecular details of how fasting influences synaptic function and plasticity. Our genetic and electrophysiological experiments demonstrate that acute fasting blocks retrograde synaptic enhancement that is normally triggered as a result of reduction in postsynaptic receptor function at the Drosophila larval neuromuscular junction (NMJ). This negative regulation critically depends on transcriptional enhancement of eukaryotic initiation factor 4E binding protein (4E-BP) under the control of the transcription factor Forkhead box O (Foxo). Furthermore, our findings indicate that postsynaptic 4E-BP exerts a constitutive negative input, which is counteracted by a positive regulatory input from the Target of Rapamycin (TOR). This combinatorial retrograde signaling plays a key role in regulating synaptic strength. Our results provide a mechanistic insight into how cellular stress and nutritional scarcity could acutely influence synaptic homeostasis and functional stability in neural circuits. Copyright © 2016 Elsevier Inc. All rights reserved.

NAD(+)- dependent deacetylase SIRT3 regulates mitochondrial protein synthesis by deacetylation of the ribosomal protein MRPL10

USDA-ARS?s Scientific Manuscript database

A member of the sirtuin family of NAD (+)-dependent deacetylases, SIRT3, is located in mammalian mitochondria and is important for regulation of mitochondrial metabolism, cell survival, and longevity. In this study, MRPL10 (mitochondrial ribosomal protein L10) was identified as the major acetylated ...

Glucocorticoid- and Protein Kinase A–Dependent Transcriptome Regulation in Airway Smooth Muscle

PubMed Central

Misior, Anna M.; Deshpande, Deepak A.; Loza, Matthew J.; Pascual, Rodolfo M.; Hipp, Jason D.; Penn, Raymond B.

2009-01-01

Glucocorticoids (GCs) and protein kinase A (PKA)–activating agents (β-adrenergic receptor agonists) are mainstream asthma therapies based on their ability to prevent or reverse excessive airway smooth muscle (ASM) constriction. Their abilities to regulate another important feature of asthma—excessive ASM growth—are poorly understood. Recent studies have suggested that GCs render agents of inflammation such as IL-1β and TNF-α mitogenic to ASM, via suppression of (antimitogenic) induced cyclooxygenase-2–dependent PKA activity. To further explore the mechanistic basis of these observations, we assessed the effects of epidermal growth factor and IL-1β stimulation, and the modulatory effects of GC treatment and PKA inhibition, on the ASM transcriptome by microarray analysis. Results demonstrate that ASM stimulated with IL-1β, in a manner that is often cooperative with stimulation with epidermal growth factor, exhibit a profound capacity to function as immunomodulatory cells. Moreover, results implicate an important role for induced autocrine/paracrine factors (many whose regulation was minimally affected by GCs or PKA inhibition) as regulators of both airway inflammation and ASM growth. Induction of numerous chemokines, in conjunction with regulation of proteases and agents of extracellular matrix remodeling, is suggested as an important mechanism promoting upregulated G protein–coupled receptor signaling capable of stimulating ASM growth. Additional functional assays suggest that intracellular PKA plays a critical role in suppressing the promitogenic effects of induced autocrine factors in ASM. Finally, identification and comparison of GC- and PKA-sensitive genes in ASM provide insight into the complementary effects of β-agonist/GC combination therapies, and suggest specific genes as important targets for guiding the development of new generations of GCs and adjunct asthma therapies. PMID:19059887

Arrestin-dependent angiotensin AT1 receptor signaling regulates Akt and mTor-mediated protein synthesis.

PubMed

Kendall, Ryan T; Lee, Mi-Hye; Pleasant, Dorea L; Robinson, Katherine; Kuppuswamy, Dhandapani; McDermott, Paul J; Luttrell, Louis M

2014-09-19

Control of protein synthesis is critical to both cell growth and proliferation. The mammalian target of rapamycin (mTOR) integrates upstream growth, proliferation, and survival signals, including those transmitted via ERK1/2 and Akt, to regulate the rate of protein translation. The angiotensin AT1 receptor has been shown to activate both ERK1/2 and Akt in arrestin-based signalsomes. Here, we examine the role of arrestin-dependent regulation of ERK1/2 and Akt in the stimulation of mTOR-dependent protein translation by the AT1 receptor using HEK293 and primary vascular smooth muscle cell models. Nascent protein synthesis stimulated by both the canonical AT1 receptor agonist angiotensin II (AngII), and the arrestin pathway-selective agonist [Sar(1)-Ile(4)-Ile(8)]AngII (SII), is blocked by shRNA silencing of βarrestin1/2 or pharmacological inhibition of Akt, ERK1/2, or mTORC1. In HEK293 cells, SII activates a discrete arrestin-bound pool of Akt and promotes Akt-dependent phosphorylation of mTOR and its downstream effector p70/p85 ribosomal S6 kinase (p70/85S6K). In parallel, SII-activated ERK1/2 helps promote mTOR and p70/85S6K phosphorylation, and is required for phosphorylation of the known ERK1/2 substrate p90 ribosomal S6 kinase (p90RSK). Thus, arrestins coordinate AT1 receptor regulation of ERK1/2 and Akt activity and stimulate protein translation via both Akt-mTOR-p70/85S6K and ERK1/2-p90RSK pathways. These results suggest that in vivo, arrestin pathway-selective AT1 receptor agonists may promote cell growth or hypertrophy through arrestin-mediated mechanisms despite their antagonism of G protein signaling. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Chk2 and REGγ-dependent DBC1 regulation in DNA damage induced apoptosis

PubMed Central

Magni, Martina; Ruscica, Vincenzo; Buscemi, Giacomo; Kim, Ja-Eun; Nachimuthu, Benjamin Tamilselvan; Fontanella, Enrico; Delia, Domenico; Zannini, Laura

2014-01-01

Human DBC1 (Deleted in Breast Cancer 1; KIAA1967; CCAR2) is a protein implicated in the regulation of apoptosis, transcription and histone modifications. Upon DNA damage, DBC1 is phosphorylated by ATM/ATR on Thr454 and this modification increases its inhibitory interaction with SIRT1, leading to p53 acetylation and p53-dependent apoptosis. Here, we report that the inhibition of SIRT1 by DBC1 in the DNA damage response (DDR) also depends on Chk2, the transducer kinase that is activated by ATM upon DNA lesions and contributes to the spreading of DNA damage signal. Indeed we found that inactivation of Chk2 reduces DBC1-SIRT1 binding, thus preventing p53 acetylation and DBC1-induced apoptosis. These events are mediated by Chk2 phosphorylation of the 11S proteasome activator REGγ on Ser247, which increases REGγ-DBC1 interaction and SIRT1 inhibition. Overall our results clarify the mechanisms underlying the DBC1-dependent SIRT1 inhibition and link, for the first time, Chk2 and REGγ to the ATM-DBC1-SIRT1 axis. PMID:25361978

Vitamin B12–dependent taurine synthesis regulates growth and bone mass

PubMed Central

Roman-Garcia, Pablo; Quiros-Gonzalez, Isabel; Mottram, Lynda; Lieben, Liesbet; Sharan, Kunal; Wangwiwatsin, Arporn; Tubio, Jose; Lewis, Kirsty; Wilkinson, Debbie; Santhanam, Balaji; Sarper, Nazan; Clare, Simon; Vassiliou, George S.; Velagapudi, Vidya R.; Dougan, Gordon; Yadav, Vijay K.

2014-01-01

Both maternal and offspring-derived factors contribute to lifelong growth and bone mass accrual, although the specific role of maternal deficiencies in the growth and bone mass of offspring is poorly understood. In the present study, we have shown that vitamin B12 (B12) deficiency in a murine genetic model results in severe postweaning growth retardation and osteoporosis, and the severity and time of onset of this phenotype in the offspring depends on the maternal genotype. Using integrated physiological and metabolomic analysis, we determined that B12 deficiency in the offspring decreases liver taurine production and associates with abrogation of a growth hormone/insulin-like growth factor 1 (GH/IGF1) axis. Taurine increased GH-dependent IGF1 synthesis in the liver, which subsequently enhanced osteoblast function, and in B12-deficient offspring, oral administration of taurine rescued their growth retardation and osteoporosis phenotypes. These results identify B12 as an essential vitamin that positively regulates postweaning growth and bone formation through taurine synthesis and suggests potential therapies to increase bone mass. PMID:24911144

Local Adaptation of Sun-Exposure-Dependent Gene Expression Regulation in Human Skin.

PubMed

Kita, Ryosuke; Fraser, Hunter B

2016-10-01

Sun-exposure is a key environmental variable in the study of human evolution. Several skin-pigmentation genes serve as classical examples of positive selection, suggesting that sun-exposure has significantly shaped worldwide genomic variation. Here we investigate the interaction between genetic variation and sun-exposure, and how this impacts gene expression regulation. Using RNA-Seq data from 607 human skin samples, we identified thousands of transcripts that are differentially expressed between sun-exposed skin and non-sun-exposed skin. We then tested whether genetic variants may influence each individual's gene expression response to sun-exposure. Our analysis revealed 10 sun-exposure-dependent gene expression quantitative trait loci (se-eQTLs), including genes involved in skin pigmentation (SLC45A2) and epidermal differentiation (RASSF9). The allele frequencies of the RASSF9 se-eQTL across diverse populations correlate with the magnitude of solar radiation experienced by these populations, suggesting local adaptation to varying levels of sunlight. These results provide the first examples of sun-exposure-dependent regulatory variation and suggest that this variation has contributed to recent human adaptation.

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

PubMed

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.

Fibronectin Matrix Polymerization Regulates Smooth Muscle Cell Phenotype through a Rac1 Dependent Mechanism

PubMed Central

Shi, Feng; Long, Xiaochun; Hendershot, Allison; Miano, Joseph M.; Sottile, Jane

2014-01-01

Smooth muscle cells are maintained in a differentiated state in the vessel wall, but can be modulated to a synthetic phenotype following injury. Smooth muscle phenotypic modulation is thought to play an important role in the pathology of vascular occlusive diseases. Phenotypically modulated smooth muscle cells exhibit increased proliferative and migratory properties that accompany the downregulation of smooth muscle cell marker proteins. Extracellular matrix proteins, including fibronectin, can regulate the smooth muscle phenotype when used as adhesive substrates. However, cells produce and organize a 3-dimensional fibrillar extracellular matrix, which can affect cell behavior in distinct ways from the protomeric 2-dimensional matrix proteins that are used as adhesive substrates. We previously showed that the deposition/polymerization of fibronectin into the extracellular matrix can regulate the deposition and organization of other extracellular matrix molecules in vitro. Further, our published data show that the presence of a fibronectin polymerization inhibitor results in increased expression of smooth muscle cell differentiation proteins and inhibits vascular remodeling in vivo. In this manuscript, we used an in vitro cell culture system to determine the mechanism by which fibronectin polymerization affects smooth muscle phenotypic modulation. Our data show that fibronectin polymerization decreases the mRNA levels of multiple smooth muscle differentiation genes, and downregulates the levels of smooth muscle α-actin and calponin proteins by a Rac1-dependent mechanism. The expression of smooth muscle genes is transcriptionally regulated by fibronectin polymerization, as evidenced by the increased activity of luciferase reporter constructs in the presence of a fibronectin polymerization inhibitor. Fibronectin polymerization also promotes smooth muscle cell growth, and decreases the levels of actin stress fibers. These data define a Rac1-dependent pathway wherein

Epigenetic regulation of opioid-induced hyperalgesia, dependence, and tolerance in mice.

PubMed

Liang, De-Yong; Li, XiangQi; Clark, J David

2013-01-01

Repeated administration of opioids such as morphine induces persistent behavioral changes including opioid-induced hyperalgesia (OIH), tolerance, and physical dependence. In the current work we explored how the balance of histone acetyltransferase (HAT) versus histone deacetylase (HDAC) might regulate these morphine-induced changes. Nociceptive thresholds, analgesia, and physical dependence were assessed during and for a period of several weeks after morphine exposure. To probe the roles of histone acetylation, the HAT inhibitor curcumin or a selective HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) was administered daily to groups of animals. Histone acetylation in spinal cord was assessed by Western blot and immunohistochemistry. Concurrent administration of curcumin with morphine for 4 days significantly reduced development of opioid-induced mechanical allodynia, thermal hyperalgesia, tolerance, and physical dependence. Conversely, the HDAC inhibitor SAHA enhanced these responses. Interestingly, SAHA treatment after the termination of opioid administration sustained these behavioral changes for at least 4 weeks. Histone H3 acetylation in the dorsal horn of the spinal cord was increased after chronic morphine treatment, but H4 acetylation was unchanged. Moreover, we observed a decrease in HDAC activity in the spinal cords of morphine-treated mice while overall HAT activity was unchanged, suggesting a shift toward a state of enhanced histone acetylation. The current study indicates that epigenetic mechanisms play a crucial role in opioid-induced long-lasting neuroplasticity. These results provide new sight into understanding the mechanisms of opioid-induced neuroplasticity and suggest new strategies to limit opioid abuse potential and increase the value of these drugs as analgesics. Copyright © 2013 American Pain Society. All rights reserved.

Quantitative Phosphoproteomics Reveals SLP-76 Dependent Regulation of PAG and Src Family Kinases in T Cells

PubMed Central

Cao, Lulu; Ding, Yiyuan; Hung, Norris; Yu, Kebing; Ritz, Anna; Raphael, Benjamin J.; Salomon, Arthur R.

2012-01-01

The SH2-domain-containing leukocyte protein of 76 kDa (SLP-76) plays a critical scaffolding role in T cell receptor (TCR) signaling. As an adaptor protein that contains multiple protein-binding domains, SLP-76 interacts with many signaling molecules and links proximal receptor stimulation to downstream effectors. The function of SLP-76 in TCR signaling has been widely studied using the Jurkat human leukaemic T cell line through protein disruption or site-directed mutagenesis. However, a wide-scale characterization of SLP-76-dependant phosphorylation events is still lacking. Quantitative profiling of over a hundred tyrosine phosphorylation sites revealed new modes of regulation of phosphorylation of PAG, PI3K, and WASP while reconfirming previously established regulation of Itk, PLCγ, and Erk phosphorylation by SLP-76. The absence of SLP-76 also perturbed the phosphorylation of Src family kinases (SFKs) Lck and Fyn, and subsequently a large number of SFK-regulated signaling molecules. Altogether our data suggests unique modes of regulation of positive and negative feedback pathways in T cells by SLP-76, reconfirming its central role in the pathway. PMID:23071622

Quantitative phosphoproteomics reveals SLP-76 dependent regulation of PAG and Src family kinases in T cells.

PubMed

Cao, Lulu; Ding, Yiyuan; Hung, Norris; Yu, Kebing; Ritz, Anna; Raphael, Benjamin J; Salomon, Arthur R

2012-01-01

The SH2-domain-containing leukocyte protein of 76 kDa (SLP-76) plays a critical scaffolding role in T cell receptor (TCR) signaling. As an adaptor protein that contains multiple protein-binding domains, SLP-76 interacts with many signaling molecules and links proximal receptor stimulation to downstream effectors. The function of SLP-76 in TCR signaling has been widely studied using the Jurkat human leukaemic T cell line through protein disruption or site-directed mutagenesis. However, a wide-scale characterization of SLP-76-dependant phosphorylation events is still lacking. Quantitative profiling of over a hundred tyrosine phosphorylation sites revealed new modes of regulation of phosphorylation of PAG, PI3K, and WASP while reconfirming previously established regulation of Itk, PLCγ, and Erk phosphorylation by SLP-76. The absence of SLP-76 also perturbed the phosphorylation of Src family kinases (SFKs) Lck and Fyn, and subsequently a large number of SFK-regulated signaling molecules. Altogether our data suggests unique modes of regulation of positive and negative feedback pathways in T cells by SLP-76, reconfirming its central role in the pathway.

Blue Light–Dependent Interaction between Cryptochrome2 and CIB1 Regulates Transcription and Leaf Senescence in Soybean[W

PubMed Central

Meng, Yingying; Li, Hongyu; Wang, Qin; Liu, Bin; Lin, Chentao

2013-01-01

Cryptochromes are blue light receptors that regulate light responses in plants, including various crops. The molecular mechanism of plant cryptochromes has been extensively investigated in Arabidopsis thaliana, but it has not been reported in any crop species. Here, we report a study of the mechanism of soybean (Glycine max) cryptochrome2 (CRY2a). We found that CRY2a regulates leaf senescence, which is a life history trait regulated by light and photoperiods via previously unknown mechanisms. We show that CRY2a undergoes blue light–dependent interaction with the soybean basic helix-loop-helix transcription activator CIB1 (for cryptochrome-interacting bHLH1) that specifically interacts with the E-box (CANNTG) DNA sequences. Analyses of transgenic soybean plants expressing an elevated or reduced level of the CRY2a or CIB1 demonstrate that CIB1 promotes leaf senescence, whereas CRY2a suppresses leaf senescence. Results of the gene expression and molecular interaction analyses support the hypothesis that CIB1 activates transcription of senescence-associated genes, such as WRKY DNA BINDING PROTEIN53b (WRKY53b), and leaf senescence. CIB1 interacts with the E-box–containing promoter sequences of the WRKY53b chromatin, whereas photoexcited CRY2a interacts with CIB1 to inhibit its DNA binding activity. These findings argue that CIB-dependent transcriptional regulation is an evolutionarily conserved CRY-signaling mechanism in plants, and this mechanism is opted in evolution to mediate light regulation of different aspects of plant development in different plant species. PMID:24272488

Regulation of lipogenesis by cyclin-dependent kinase 8–mediated control of SREBP-1

PubMed Central

Zhao, Xiaoping; Feng, Daorong; Wang, Qun; Abdulla, Arian; Xie, Xiao-Jun; Zhou, Jie; Sun, Yan; Yang, Ellen S.; Liu, Lu-Ping; Vaitheesvaran, Bhavapriya; Bridges, Lauren; Kurland, Irwin J.; Strich, Randy; Ni, Jian-Quan; Wang, Chenguang; Ericsson, Johan; Pessin, Jeffrey E.; Ji, Jun-Yuan; Yang, Fajun

2012-01-01

Altered lipid metabolism underlies several major human diseases, including obesity and type 2 diabetes. However, lipid metabolism pathophysiology remains poorly understood at the molecular level. Insulin is the primary stimulator of hepatic lipogenesis through activation of the SREBP-1c transcription factor. Here we identified cyclin-dependent kinase 8 (CDK8) and its regulatory partner cyclin C (CycC) as negative regulators of the lipogenic pathway in Drosophila, mammalian hepatocytes, and mouse liver. The inhibitory effect of CDK8 and CycC on de novo lipogenesis was mediated through CDK8 phosphorylation of nuclear SREBP-1c at a conserved threonine residue. Phosphorylation by CDK8 enhanced SREBP-1c ubiquitination and protein degradation. Importantly, consistent with the physiologic regulation of lipid biosynthesis, CDK8 and CycC proteins were rapidly downregulated by feeding and insulin, resulting in decreased SREBP-1c phosphorylation. Moreover, overexpression of CycC efficiently suppressed insulin and feeding–induced lipogenic gene expression. Taken together, these results demonstrate that CDK8 and CycC function as evolutionarily conserved components of the insulin signaling pathway in regulating lipid homeostasis. PMID:22684109

MicroRNA-Dependent Regulation of Transcription in Non-Small Cell Lung Cancer

PubMed Central

Molina-Pinelo, Sonia; Gutiérrez, Gabriel; Pastor, Maria Dolores; Hergueta, Marta; Moreno-Bueno, Gema; García-Carbonero, Rocío; Nogal, Ana; Suárez, Rocío; Salinas, Ana; Pozo-Rodríguez, Francisco; Lopez-Rios, Fernando; Agulló-Ortuño, Maria Teresa; Ferrer, Irene; Perpiñá, Asunción; Palacios, José; Carnero, Amancio; Paz-Ares, Luis

2014-01-01

Squamous cell lung cancer (SCC) and adenocarcinoma are the most common histological subtypes of non-small cell lung cancer (NSCLC), and have been traditionally managed in the clinic as a single entity. Increasing evidence, however, illustrates the biological diversity of these two histological subgroups of lung cancer, and supports the need to improve our understanding of the molecular basis beyond the different phenotypes if we aim to develop more specific and individualized targeted therapy. The purpose of this study was to identify microRNA (miRNA)-dependent transcriptional regulation differences between SCC and adenocarcinoma histological lung cancer subtypes. In this work, paired miRNA (667 miRNAs by TaqMan Low Density Arrays (TLDA)) and mRNA profiling (Whole Genome 44 K array G112A, Agilent) was performed in tumor samples of 44 NSCLC patients. Nine miRNAs and 56 mRNAs were found to be differentially expressed in SCC versus adenocarcinoma samples. Eleven of these 56 mRNA were predicted as targets of the miRNAs identified to be differently expressed in these two histological conditions. Of them, 6 miRNAs (miR-149, miR-205, miR-375, miR-378, miR-422a and miR-708) and 9 target genes (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B, MUC1) were validated by quantitative PCR in an independent cohort of 41 lung cancer patients. Furthermore, the inverse correlation between mRNAs and microRNAs expression was also validated. These results suggest miRNA-dependent transcriptional regulation differences play an important role in determining key hallmarks of NSCLC, and may provide new biomarkers for personalized treatment strategies. PMID:24625834

PRMT5-Dependent Methylation of the TIP60 Coactivator RUVBL1 Is a Key Regulator of Homologous Recombination.

PubMed

Clarke, Thomas L; Sanchez-Bailon, Maria Pilar; Chiang, Kelly; Reynolds, John J; Herrero-Ruiz, Joaquin; Bandeiras, Tiago M; Matias, Pedro M; Maslen, Sarah L; Skehel, J Mark; Stewart, Grant S; Davies, Clare C

2017-03-02

Protein post-translation modification plays an important role in regulating DNA repair; however, the role of arginine methylation in this process is poorly understood. Here we identify the arginine methyltransferase PRMT5 as a key regulator of homologous recombination (HR)-mediated double-strand break (DSB) repair, which is mediated through its ability to methylate RUVBL1, a cofactor of the TIP60 complex. We show that PRMT5 targets RUVBL1 for methylation at position R205, which facilitates TIP60-dependent mobilization of 53BP1 from DNA breaks, promoting HR. Mechanistically, we demonstrate that PRMT5-directed methylation of RUVBL1 is critically required for the acetyltransferase activity of TIP60, promoting histone H4K16 acetylation, which facilities 53BP1 displacement from DSBs. Interestingly, RUVBL1 methylation did not affect the ability of TIP60 to facilitate ATM activation. Taken together, our findings reveal the importance of PRMT5-mediated arginine methylation during DSB repair pathway choice through its ability to regulate acetylation-dependent control of 53BP1 localization. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

COPPER RESPONSE REGULATOR1–Dependent and –Independent Responses of the Chlamydomonas reinhardtii Transcriptome to Dark Anoxia[W

PubMed Central

Hemschemeier, Anja; Casero, David; Liu, Bensheng; Benning, Christoph; Pellegrini, Matteo; Happe, Thomas; Merchant, Sabeeha S.

2013-01-01

Anaerobiosis is a stress condition for aerobic organisms and requires extensive acclimation responses. We used RNA-Seq for a whole-genome view of the acclimation of Chlamydomonas reinhardtii to anoxic conditions imposed simultaneously with transfer to the dark. Nearly 1.4 × 103 genes were affected by hypoxia. Comparing transcript profiles from early (hypoxic) with those from late (anoxic) time points indicated that cells activate oxidative energy generation pathways before employing fermentation. Probable substrates include amino acids and fatty acids (FAs). Lipid profiling of the C. reinhardtii cells revealed that they degraded FAs but also accumulated triacylglycerols (TAGs). In contrast with N-deprived cells, the TAGs in hypoxic cells were enriched in desaturated FAs, suggesting a distinct pathway for TAG accumulation. To distinguish transcriptional responses dependent on COPPER RESPONSE REGULATOR1 (CRR1), which is also involved in hypoxic gene regulation, we compared the transcriptomes of crr1 mutants and complemented strains. In crr1 mutants, ∼40 genes were aberrantly regulated, reaffirming the importance of CRR1 for the hypoxic response, but indicating also the contribution of additional signaling strategies to account for the remaining differentially regulated transcripts. Based on transcript patterns and previous results, we conclude that nitric oxide–dependent signaling cascades operate in anoxic C. reinhardtii cells. PMID:24014546

Keap1 redox-dependent regulation of doxorubicin-induced oxidative stress response in cardiac myoblasts

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

Nordgren, Kendra K.S., E-mail: knordgre@d.umn.edu; Wallace, Kendall B., E-mail: kwallace@d.umn.edu

Doxorubicin (DOX) is a widely prescribed treatment for a broad scope of cancers, but clinical utility is limited by the cumulative, dose-dependent cardiomyopathy that occurs with repeated administration. DOX-induced cardiotoxicity is associated with the production of reactive oxygen species (ROS) and oxidation of lipids, DNA and proteins. A major cellular defense mechanism against such oxidative stress is activation of the Keap1/Nrf2-antioxidant response element (ARE) signaling pathway, which transcriptionally regulates expression of antioxidant genes such as Nqo1 and Gstp1. In the present study, we address the hypothesis that an initial event associated with DOX-induced oxidative stress is activation of the Keap1/Nrf2-dependentmore » expression of antioxidant genes and that this is regulated through drug-induced changes in redox status of the Keap1 protein. Incubation of H9c2 rat cardiac myoblasts with DOX resulted in a time- and dose-dependent decrease in non-protein sulfhydryl groups. Associated with this was a near 2-fold increase in Nrf2 protein content and enhanced transcription of several of the Nrf2-regulated down-stream genes, including Gstp1, Ugt1a1, and Nqo1; the expression of Nfe2l2 (Nrf2) itself was unaltered. Furthermore, both the redox status and the total amount of Keap1 protein were significantly decreased by DOX, with the loss of Keap1 being due to both inhibited gene expression and increased autophagic, but not proteasomal, degradation. These findings identify the Keap1/Nrf2 pathway as a potentially important initial response to acute DOX-induced oxidative injury, with the primary regulatory events being the oxidation and autophagic degradation of the redox sensor Keap1 protein. - Highlights: • DOX caused a ∼2-fold increase in Nrf2 protein content. • DOX enhanced transcription of several Nrf2-regulated down-stream genes. • Redox status and total amount of Keap1 protein were significantly decreased by DOX. • Loss of Keap1 protein was

Phosphorylation-dependent Regulation of Connecdenn/DENND1 Guanine Nucleotide Exchange Factors*

PubMed Central

Kulasekaran, Gopinath; Nossova, Nadya; Marat, Andrea L.; Lund, Ingrid; Cremer, Christopher; Ioannou, Maria S.; McPherson, Peter S.

2015-01-01

Connecdenn 1/2 are DENN (differentially expressed in normal and neoplastic cells) domain-bearing proteins that function as GEFs (guanine nucleotide exchange factors) for the small GTPase Rab35. Disruption of connecdenn/Rab35 function leads to defects in the recycling of multiple cargo proteins from endosomes with altered cell function, yet the regulation of connecdenn GEF activity is unexplored. We now demonstrate that connecdenn 1/2 are autoinhibited such that the purified, full-length proteins have significantly less Rab35 binding and GEF activity than the isolated DENN domain. Both proteins are phosphorylated with prominent phosphorylation sites between residues 500 and 600 of connecdenn 1. A large scale proteomics screen revealed that connecdenn 1 is phosphorylated at residues Ser-536 and Ser-538 in an Akt-dependent manner in response to insulin stimulation of adipocytes. Interestingly, we find that an Akt inhibitor reduces connecdenn 1 interaction with Rab35 after insulin treatment of adipocytes. Remarkably, a peptide flanking Ser-536/Ser-538 binds the DENN domain of connecdenn 1, whereas a phosphomimetic peptide does not. Moreover, connecdenn 1 interacts with 14-3-3 proteins, and this interaction is also disrupted by Akt inhibition and by mutation of Ser-536/Ser-538. We propose that Akt phosphorylation of connecdenn 1 downstream of insulin activation regulates connecdenn 1 function through an intramolecular interaction. PMID:26055712

Regulation of NT-PGC-1alpha subcellular localization and function by protein kinase A-dependent modulation of nuclear export by CRM1.

PubMed

Chang, Ji Suk; Huypens, Peter; Zhang, Yubin; Black, Chelsea; Kralli, Anastasia; Gettys, Thomas W

2010-06-04

Peroxisome proliferator-activated receptor gamma co-activator-1alpha (PGC-1alpha) plays a central role in the regulation of cellular energy metabolism and metabolic adaptation to environmental and nutritional stimuli. We recently described a novel, biologically active splice variant of PGC-1alpha (NT-PGC-1alpha, amino acids 1-270) that retains the ability to interact with and transactivate nuclear hormone receptors through its N-terminal transactivation domain. Whereas PGC-1alpha is an unstable nuclear protein sensitive to ubiquitin-mediated targeting to the proteasome, NT-PGC-1alpha is relatively stable and predominantly cytoplasmic, suggesting that its ability to interact with and activate nuclear receptors and transcription factors is dependent upon regulated access to the nucleus. We provide evidence that NT-PGC-1alpha interacts with the nuclear exportin, CRM1, through a specific leucine-rich domain (nuclear export sequence) that regulates its export to the cytoplasm. The nuclear export of NT-PGC-1alpha is inhibited by protein kinase A-dependent phosphorylation of Ser-194, Ser-241, and Thr-256 on NT-PGC-1alpha, which effectively increases its nuclear concentration. Using site-directed mutagenesis to prevent or mimic phosphorylation at these sites, we show that the transcriptional activity of NT-PGC-1alpha is regulated in part through regulation of its subcellular localization. These findings suggest that the function of NT-PGC-1alpha as a transcriptional co-activator is regulated by protein kinase A-dependent inhibition of CRM1-mediated export from the nucleus.

Genetic regulation by NLA and microRNA827 for maintaining nitrate-dependent phosphate homeostasis in arabidopsis.

PubMed

Kant, Surya; Peng, Mingsheng; Rothstein, Steven J

2011-03-01

Plants need abundant nitrogen and phosphorus for higher yield. Improving plant genetics for higher nitrogen and phosphorus use efficiency would save potentially billions of dollars annually on fertilizers and reduce global environmental pollution. This will require knowledge of molecular regulators for maintaining homeostasis of these nutrients in plants. Previously, we reported that the NITROGEN LIMITATION ADAPTATION (NLA) gene is involved in adaptive responses to low-nitrogen conditions in Arabidopsis, where nla mutant plants display abrupt early senescence. To understand the molecular mechanisms underlying NLA function, two suppressors of the nla mutation were isolated that recover the nla mutant phenotype to wild type. Map-based cloning identified these suppressors as the phosphate (Pi) transport-related genes PHF1 and PHT1.1. In addition, NLA expression is shown to be regulated by the low-Pi induced microRNA miR827. Pi analysis revealed that the early senescence in nla mutant plants was due to Pi toxicity. These plants accumulated over five times the normal Pi content in shoots specifically under low nitrate and high Pi but not under high nitrate conditions. Also the Pi overaccumulator pho2 mutant shows Pi toxicity in a nitrate-dependent manner similar to the nla mutant. Further, the nitrate and Pi levels are shown to have an antagonistic crosstalk as displayed by their differential effects on flowering time. The results demonstrate that NLA and miR827 have pivotal roles in regulating Pi homeostasis in plants in a nitrate-dependent fashion.

Position-dependent and neuron-specific splicing regulation by the CELF family RNA-binding protein UNC-75 in Caenorhabditis elegans

PubMed Central

Kuroyanagi, Hidehito; Watanabe, Yohei; Suzuki, Yutaka; Hagiwara, Masatoshi

2013-01-01

A large fraction of protein-coding genes in metazoans undergo alternative pre-mRNA splicing in tissue- or cell-type-specific manners. Recent genome-wide approaches have identified many putative-binding sites for some of tissue-specific trans-acting splicing regulators. However, the mechanisms of splicing regulation in vivo remain largely unknown. To elucidate the modes of splicing regulation by the neuron-specific CELF family RNA-binding protein UNC-75 in Caenorhabditis elegans, we performed deep sequencing of poly(A)+ RNAs from the unc-75(+)- and unc-75-mutant worms and identified more than 20 cassette and mutually exclusive exons repressed or activated by UNC-75. Motif searches revealed that (G/U)UGUUGUG stretches are enriched in the upstream and downstream introns of the UNC-75-repressed and -activated exons, respectively. Recombinant UNC-75 protein specifically binds to RNA fragments carrying the (G/U)UGUUGUG stretches in vitro. Bi-chromatic fluorescence alternative splicing reporters revealed that the UNC-75-target exons are regulated in tissue-specific and (G/U)UGUUGUG element-dependent manners in vivo. The unc-75 mutation affected the splicing reporter expression specifically in the nervous system. These results indicate that UNC-75 regulates alternative splicing of its target exons in neuron-specific and position-dependent manners through the (G/U)UGUUGUG elements in C. elegans. This study thus reveals the repertoire of target events for the CELF family in the living organism. PMID:23416545

Calcium-mediated signaling and calmodulin-dependent kinase regulate hepatocyte-inducible nitric oxide synthase expression.

PubMed

Zhang, Baochun; Crankshaw, Will; Nesemeier, Ryan; Patel, Jay; Nweze, Ikenna; Lakshmanan, Jaganathan; Harbrecht, Brian G

2015-02-01

Induced nitric oxide synthase (iNOS) is induced in hepatocytes by shock and inflammatory stimuli. Excessive NO from iNOS mediates shock-induced hepatic injury and death, so understanding the regulation of iNOS will help elucidate the pathophysiology of septic shock. In vitro, cytokines induce iNOS expression through activation of signaling pathways including mitogen-activated protein kinases and nuclear factor κB. Cytokines also induce calcium (Ca(2+)) mobilization and activate calcium-mediated intracellular signaling pathways, typically through activation of calmodulin-dependent kinases (CaMK). Calcium regulates NO production in macrophages but the role of calcium and calcium-mediated signaling in hepatocyte iNOS expression has not been defined. Primary rat hepatocytes were isolated, cultured, and induced to produce NO with proinflammatory cytokines. Calcium mobilization and Ca(2+)-mediated signaling were altered with ionophore, Ca(2+) channel blockers, and inhibitors of CaMK. The Ca(2+) ionophore A23187 suppressed cytokine-stimulated NO production, whereas Ethylene glycol tetraacetic acid and nifedipine increased NO production, iNOS messenger RNA, and iNOS protein expression. Inhibition of CaMK with KN93 and CBD increased NO production but the calcineurin inhibitor FK 506 decreased iNOS expression. These data demonstrate that calcium-mediated signaling regulates hepatocyte iNOS expression and does so through a mechanism independent of calcineurin. Changes in intracellular calcium levels may regulate iNOS expression during hepatic inflammation induced by proinflammatory cytokines. Copyright © 2015 Elsevier Inc. All rights reserved.

Clonorchis sinensis excretory-secretory products regulate migration and invasion in cholangiocarcinoma cells via extracellular signal-regulated kinase 1/2/nuclear factor-κB-dependent matrix metalloproteinase-9 expression.

PubMed

Pak, Jhang Ho; Shin, Jimin; Song, In-Sung; Shim, Sungbo; Jang, Sung-Wuk

2017-01-01

Matrix metalloproteinase-9 plays an important role in the invasion and metastasis of various types of cancer cells. We have previously reported that excretory-secretory products from Clonorchis sinensis increases matrix metalloproteinase-9 expression. However, the regulatory mechanisms through which matrix metalloproteinase-9 expression affects cholangiocarcinoma development remain unclear. In the current study, we examined the potential role of excretory-secretory products in regulating the migration and invasion of various cholangiocarcinoma cell lines. We demonstrated that excretory-secretory products significantly induced matrix metalloproteinase-9 expression and activity in a concentration-dependent manner. Reporter gene and chromatin immunoprecipitation assays showed that excretory-secretory products induced matrix metalloproteinase-9 expression by enhancing the activity of nuclear factor-kappa B. Moreover, excretory-secretory products induced the degradation and phosphorylation of IκBα and stimulated nuclear factor-kappa B p65 nuclear translocation, which was regulated by extracellular signal-regulated kinase 1/2. Taken together, our findings indicated that the excretory-secretory product-dependent enhancement of matrix metalloproteinase-9 activity and subsequent induction of IκBα and nuclear factor-kappa B activities may contribute to the progression of cholangiocarcinoma. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

Thought Suppression, Impaired Regulation of Urges, and Addiction-Stroop Predict Affect-Modulated Cue-Reactivity among Alcohol Dependent Adults

PubMed Central

Garland, Eric L.; Carter, Kristin; Ropes, Katie; Howard, Matthew O.

2011-01-01

Abstinent alcohol dependent individuals commonly employ thought suppression to cope with stress and intrusive cognitions about alcohol. This strategy may inadvertently bias attention toward alcohol-related stimuli while depleting neurocognitive resources needed to regulate urges, manifested as decreased heart rate variability (HRV) responsivity to alcohol cues. The present study tested the hypothesis that trait and state thought suppression, impaired regulation of urges, and alcohol attentional bias as measured by the Addiction-Stroop would have significant effects on the HRV responsivity of 58 adults in residential treatment for alcohol dependence (mean age = 39.6 ± 9.4, 81% female) who participated in an affect-modulated cue-reactivity protocol. Regression analyses controlling for age, level of pre-treatment alcohol consumption, and baseline HRV indicated that higher levels of trait thought suppression, impaired regulation of alcohol urges, and attentional fixation on alcohol cues were associated with lower HRV responsivity during stress-primed alcohol cue-exposure. Moreover, there was a significant state X trait suppression interaction on HRV cue-responsivity, such that alcohol dependent persons reporting high levels of state and trait suppression exhibited less HRV during cue-exposure than persons reporting low levels of state and trait suppression. Results suggest that chronic thought suppression taxes regulatory resources reflected in reduced HRV responsivity, an effect that is particularly evident when high trait suppressors engage in intensive suppression of drinking-related thoughts under conditions of stress. Treatment approaches that offer effective alternatives to the maladaptive strategy of suppressing alcohol urges may be crucial for relapse prevention. PMID:21967855

Thought suppression, impaired regulation of urges, and Addiction-Stroop predict affect-modulated cue-reactivity among alcohol dependent adults.

PubMed

Garland, Eric L; Carter, Kristin; Ropes, Katie; Howard, Matthew O

2012-01-01

Abstinent alcohol dependent individuals commonly employ thought suppression to cope with stress and intrusive cognitions about alcohol. This strategy may inadvertently bias attention towards alcohol-related stimuli while depleting neurocognitive resources needed to regulate urges, manifested as decreased heart rate variability (HRV) responsivity to alcohol cues. The present study tested the hypothesis that trait and state thought suppression, impaired regulation of urges, and alcohol attentional bias as measured by the Addiction-Stroop would have significant effects on the HRV responsivity of 58 adults in residential treatment for alcohol dependence (mean age=39.6 ± 9.4, 81% female) who participated in an affect-modulated cue-reactivity protocol. Regression analyses controlling for age, level of pre-treatment alcohol consumption, and baseline HRV indicated that higher levels of trait thought suppression, impaired regulation of alcohol urges, and attentional fixation on alcohol cues were associated with lower HRV responsivity during stress-primed alcohol cue-exposure. Moreover, there was a significant state × trait suppression interaction on HRV cue-responsivity, such that alcohol dependent persons reporting high levels of state and trait suppression exhibited less HRV during cue-exposure than persons reporting low levels of state and trait suppression. Results suggest that chronic thought suppression taxes regulatory resources reflected in reduced HRV responsivity, an effect that is particularly evident when high trait suppressors engage in intensive suppression of drinking-related thoughts under conditions of stress. Treatment approaches that offer effective alternatives to the maladaptive strategy of suppressing alcohol urges may be crucial for relapse prevention. Copyright © 2011 Elsevier B.V. All rights reserved.

Cold-induced ultrastructural changes in bull and boar sperm plasma membranes.

PubMed

De Leeuw, F E; Chen, H C; Colenbrander, B; Verkleij, A J

1990-04-01

The effect of low temperatures on the ultrastructure of the plasma membrane of bull and boar spermatozoa was investigated. Cold-induced changes in the organization of sperm plasma membrane components were demonstrated by the use of fast-freezing combined with freeze-fracture electron microscopy. This preparation technique ensures fixation without artifacts. At 38 degrees C bull and boar spermatozoa exhibited a random distribution of intramembranous particles over the plasma membrane of both head and tail. Exposure to 0 degree C resulted in redistribution of the intramembranous particles: on the head and principal piece of bull spermatozoa and on the principal piece of boar spermatozoa, particle-free areas were observed, whereas on the boar sperm head, particle aggregates were present. The original particle distribution was restored upon rewarming of bull and boar spermatozoa to 38 degrees C, as well as after freezing and thawing of bull spermatozoa. Dilution of bull and boar semen into Tris-dilution buffer and Beltsville Thaw Solution-dilution buffer, respectively, could not prevent cold-induced redistribution of intramembranous particles. The observed particle reorganization upon cooling was interpreted as the result of lateral phase separation in the plasma membrane. Species-dependent differences in cold-induced ultrastructural changes were considered to be determined by lipid composition and asymmetry of the plasma membrane, and might be related to differences in cold resistance between species.

Negative regulation of multifunctional Ca2+/calmodulin-dependent protein kinases: physiological and pharmacological significance of protein phosphatases

PubMed Central

Ishida, A; Sueyoshi, N; Shigeri, Y; Kameshita, I

2008-01-01

Multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) play pivotal roles in intracellular Ca2+ signaling pathways. There is growing evidence that CaMKs are involved in the pathogenic mechanisms underlying various human diseases. In this review, we begin by briefly summarizing our knowledge of the involvement of CaMKs in the pathogenesis of various diseases suggested to be caused by the dysfunction/dysregulation or aberrant expression of CaMKs. It is widely known that the activities of CaMKs are strictly regulated by protein phosphorylation/dephosphorylation of specific phosphorylation sites. Since phosphorylation status is balanced by protein kinases and protein phosphatases, the mechanism of dephosphorylation/deactivation of CaMKs, corresponding to their ‘switching off', is extremely important, as is the mechanism of phosphorylation/activation corresponding to their ‘switching on'. Therefore, we focus on the regulation of multifunctional CaMKs by protein phosphatases. We summarize the current understanding of negative regulation of CaMKs by protein phosphatases. We also discuss the biochemical properties and physiological significance of a protein phosphatase that we designated as Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP), and those of its homologue CaMKP-N. Pharmacological applications of CaMKP inhibitors are also discussed. These compounds may be useful not only for exploring the physiological functions of CaMKP/CaMKP-N, but also as novel chemotherapies for various diseases. PMID:18454172

JMJD6 and U2AF65 co-regulate alternative splicing in both JMJD6 enzymatic activity dependent and independent manner

PubMed Central

Yi, Jia; Shen, Hai-Feng; Qiu, Jin-Song; Huang, Ming-Feng; Zhang, Wen-Juan; Ding, Jian-Cheng; Zhu, Xiao-Yan; Zhou, Yu

2017-01-01

Abstract JMJD6, a jumonji C (Jmj C) domain-containing protein demethylase and hydroxylase, has been implicated in an array of biological processes. It has been shown that JMJD6 interacts with and hydroxylates multiple serine/arginine-rich (SR) proteins and SR related proteins, including U2AF65, all of which are known to function in alternative splicing regulation. However, whether JMJD6 is widely involved in alternative splicing and the molecular mechanism underlying JMJD6-regulated alternative splicing have remained incompletely understood. Here, by using RASL-Seq, we investigated the functional impact of RNA-dependent interaction between JMJD6 and U2AF65, revealing that JMJD6 and U2AF65 co-regulated a large number of alternative splicing events. We further demonstrated the JMJD6 function in alternative splicing in jmjd6 knockout mice. Mechanistically, we showed that the enzymatic activity of JMJD6 was required for a subset of JMJD6-regulated splicing, and JMJD6-mediated lysine hydroxylation of U2AF65 could account for, at least partially, their co-regulated alternative splicing events, suggesting both JMJD6 enzymatic activity-dependent and independent control of alternative splicing. These findings reveal an intimate link between JMJD6 and U2AF65 in alternative splicing regulation, which has important implications in development and disease processes. PMID:27899633

Studies of protein aggregation in A53T α-synuclein transgenic, Tg2576 transgenic, and P246L presenilin-1 knock-in cross bred mice.

PubMed

Emmer, Kristel L; Covy, Jason P; Giasson, Benoit I

2012-01-24

Synucleinopathies are a group of neurodegenerative disorders, including Parkinson disease, associated with neuronal amyloid inclusions comprised of the presynaptic protein α-synuclein (α-syn); however the biological events that initiate and lead to the formation of these inclusions are still poorly understood. There is mounting evidence that intracellular α-syn aggregation may proceed via a seeding mechanism and could spread between neurons through a prion-like mechanism that may involve other amyloidogenic proteins. Several lines of evidence suggest that Aβ peptides and/or extracellular Aβ deposits may directly or indirectly promote intracellular α-syn aggregation. To assess the effects of Aβ peptides and extracellular Aβ deposits on α-syn aggregate formation, transgenic mice (line M83) expressing A53T human α-syn that are sensitive to developing α-syn pathological inclusions were cross bred to Tg2576 transgenic mice that generated elevated levels of Aβ peptides and develop abundant Aβ plaques. In addition these mice were bred to mice with the P264L presenilin-1 knock-in mutation that further promotes Aβ plaque formation. These mice demonstrated the expected formation of Aβ plaques; however despite the accumulation of hyperphosphorylated α-syn dystrophic neurites within or surrounding Aβ plaques, no additional α-syn pathologies were observed. These studies show that Aβ amyloid deposits can cause the local aggregation of α-syn, but these did not lead to more extensive α-syn pathology. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Hippo signaling regulates Microprocessor and links cell density-dependent miRNA biogenesis to cancer

PubMed Central

Mori, Masaki; Triboulet, Robinson; Mohseni, Morvarid; Schlegelmilch, Karin; Shrestha, Kriti; Camargo, Fernando D.; Gregory, Richard I.

2014-01-01

SUMMARY Global downregulation of microRNAs (miRNAs) is commonly observed in human cancers and can have a causative role in tumorigenesis. The mechanisms responsible for this phenomenon remain poorly understood. Here we show that YAP, the downstream target of the tumor-suppressive Hippo signaling pathway regulates miRNA biogenesis in a cell density-dependent manner. At low cell density, nuclear YAP binds and sequesters p72 (DDX17), a regulatory component of the miRNA processing machinery. At high cell density, Hippo-mediated cytoplasmic retention of YAP facilitates p72 association with Microprocessor and binding to a specific sequence motif in pri-miRNAs. Inactivation of the Hippo pathway or expression of constitutively active YAP causes widespread miRNA suppression in cells and tumors and a corresponding post-transcriptional induction of MYC expression. Thus, the Hippo pathway links contact-inhibition regulation to miRNA biogenesis and may be responsible for the widespread miRNA repression observed in cancer. PMID:24581491

Borrelia oxidative stress response regulator, BosR: A distinctive Zn-dependent transcriptional activator

PubMed Central

Boylan, Julie A.; Posey, James E.; Gherardini, Frank C.

2003-01-01

The ability of a pathogen to cause infection depends on successful colonization of the host, which, in turn, requires adaptation to various challenges presented by that host. For example, host immune cells use a variety of mechanisms to control infection by bacterial pathogens, including the production of bactericidal reactive oxygen species. Prokaryotic and eukaryotic cells have developed ways of protecting themselves against this oxidative damage; for instance, Borrelia burgdorferi alters the expression of oxidative-stress-related proteins, such as a Dps/Dpr homolog NapA (BB0690), in response to increasing levels of oxygen and reactive oxygen species. These stress-related genes appear to be regulated by a putative metal-dependent DNA-binding protein (BB0647) that has 50.7% similarity to the peroxide-specific stress response repressor of Bacillus subtilis, PerR. We overexpressed and purified this protein from Escherichia coli and designated it Borrelia oxidative stress regulator, BosR. BosR bound to a 50-nt region 180 bp upstream of the napA transcriptional start site and required DTT and Zn2+ for optimal binding. Unlike the Bacillus subtilis PerR repressor, BosR did not require Fe2+ and Mn2+ for binding, and oxidizing agents, such as t-butyl peroxide, enhanced, not eliminated, BosR binding to the napA promoter region. Surprisingly, transcriptional fusion analysis indicated that BosR exerted a positive regulatory effect on napA that is inducible with t-butyl peroxide. On the basis of these data, we propose that, despite the similarity to PerR, BosR functions primarily as a transcriptional activator, not a repressor of oxidative stress response, in B. burgdorferi. PMID:12975527

Regulation of extrafloral nectar secretion by jasmonates in lima bean is light dependent

PubMed Central

Radhika, Venkatesan; Kost, Christian; Mithöfer, Axel; Boland, Wilhelm

2010-01-01

To maximize fitness, plants need to perceive changes in their light environment and adjust their physiological responses accordingly. Whether and how such changes also affect the regulation of their defense responses against herbivores remains largely unclear. We addressed this issue by studying the secretion of extrafloral nectar (EFN) in lima bean (Phaseolus lunatus), which is known to be activated by the phytohormone jasmonic acid (JA) and functions as an indirect defense mechanism against herbivores. We found that the plant’s EFN secretion in response to JA was light dependent: In the dark, JA reduced EFN secretion, whereas under light conditions, JA induced EFN secretion relative to controls. This modulation was affected by the light’s spectral composition [i.e., ratio of red to far-red (R:FR) radiation], but not light intensity. These findings demonstrate a unique differential effect of JA on EFN secretion depending on the ambient light conditions. Interestingly, treatment with the isoleucine–JA conjugate (JA–Ile) enhanced EFN secretion under light conditions yet did not reduce EFN secretion in the dark. Moreover, inhibition of Ile biosynthesis in light-exposed plants significantly decreased the EFN secretion rate. This reduction could be recovered by additional application of JA–Ile, suggesting that JA–Ile is the active compound required to up-regulate EFN secretion. Finally, experiments with mechanically damaged plants revealed that light was required for the formation of JA–Ile, but not of JA. These results demonstrate that in lima bean, the light environment modulates the plant’s response to jasmonates as well as JA–Ile biosynthesis, which controls the subsequent EFN secretion. PMID:20855624

Palmitoylation-dependent CDKL5-PSD-95 interaction regulates synaptic targeting of CDKL5 and dendritic spine development.

PubMed

Zhu, Yong-Chuan; Li, Dan; Wang, Lu; Lu, Bin; Zheng, Jing; Zhao, Shi-Lin; Zeng, Rong; Xiong, Zhi-Qi

2013-05-28

The X-linked gene cyclin-dependent kinase-like 5 (CDKL5) is mutated in severe neurodevelopmental disorders, including some forms of atypical Rett syndrome, but the function and regulation of CDKL5 protein in neurons remain to be elucidated. Here, we show that CDKL5 binds to the scaffolding protein postsynaptic density (PSD)-95, and that this binding promotes the targeting of CDKL5 to excitatory synapses. Interestingly, this binding is not constitutive, but governed by palmitate cycling on PSD-95. Furthermore, pathogenic mutations that truncate the C-terminal tail of CDKL5 diminish its binding to PSD-95 and synaptic accumulation. Importantly, down-regulation of CDKL5 by RNA interference (RNAi) or interference with the CDKL5-PSD-95 interaction inhibits dendritic spine formation and growth. These results demonstrate a critical role of the palmitoylation-dependent CDKL5-PSD-95 interaction in localizing CDKL5 to synapses for normal spine development and suggest that disruption of this interaction by pathogenic mutations may be implicated in the pathogenesis of CDKL5-related disorders.

The Iron-Dependent Regulator Fur Controls Pheromone Signaling Systems and Luminescence in the Squid Symbiont Vibrio fischeri ES114

PubMed Central

Septer, Alecia N.; Lyell, Noreen L.

2013-01-01

Bacteria often use pheromones to coordinate group behaviors in specific environments. While high cell density is required for pheromones to achieve stimulatory levels, environmental cues can also influence pheromone accumulation and signaling. For the squid symbiont Vibrio fischeri ES114, bioluminescence requires pheromone-mediated regulation, and this signaling is induced in the host to a greater extent than in culture, even at an equivalent cell density. Our goal is to better understand this environment-specific control over pheromone signaling and bioluminescence. Previous work with V. fischeri MJ1 showed that iron limitation induces luminescence, and we recently found that ES114 encounters a low-iron environment in its host. Here we show that ES114 induces luminescence at lower cell density and achieves brighter luminescence in low-iron media. This iron-dependent effect on luminescence required ferric uptake regulator (Fur), which we propose influences two pheromone signaling master regulators, LitR and LuxR. Genetic and bioinformatic analyses suggested that under low-iron conditions, Fur-mediated repression of litR is relieved, enabling more LitR to perform its established role as an activator of luxR. Interestingly, Fur may similarly control the LitR homolog SmcR of Vibrio vulnificus. These results reveal an intriguing regulatory link between low-iron conditions, which are often encountered in host tissues, and pheromone-dependent master regulators. PMID:23315731

Genetic Regulation by NLA and MicroRNA827 for Maintaining Nitrate-Dependent Phosphate Homeostasis in Arabidopsis

PubMed Central

Kant, Surya; Peng, Mingsheng; Rothstein, Steven J.

2011-01-01

Plants need abundant nitrogen and phosphorus for higher yield. Improving plant genetics for higher nitrogen and phosphorus use efficiency would save potentially billions of dollars annually on fertilizers and reduce global environmental pollution. This will require knowledge of molecular regulators for maintaining homeostasis of these nutrients in plants. Previously, we reported that the NITROGEN LIMITATION ADAPTATION (NLA) gene is involved in adaptive responses to low-nitrogen conditions in Arabidopsis, where nla mutant plants display abrupt early senescence. To understand the molecular mechanisms underlying NLA function, two suppressors of the nla mutation were isolated that recover the nla mutant phenotype to wild type. Map-based cloning identified these suppressors as the phosphate (Pi) transport-related genes PHF1 and PHT1.1. In addition, NLA expression is shown to be regulated by the low-Pi induced microRNA miR827. Pi analysis revealed that the early senescence in nla mutant plants was due to Pi toxicity. These plants accumulated over five times the normal Pi content in shoots specifically under low nitrate and high Pi but not under high nitrate conditions. Also the Pi overaccumulator pho2 mutant shows Pi toxicity in a nitrate-dependent manner similar to the nla mutant. Further, the nitrate and Pi levels are shown to have an antagonistic crosstalk as displayed by their differential effects on flowering time. The results demonstrate that NLA and miR827 have pivotal roles in regulating Pi homeostasis in plants in a nitrate-dependent fashion. PMID:21455488

Dual regulation of Ca2+-dependent glutamate release from astrocytes: vesicular glutamate transporters and cytosolic glutamate levels.

PubMed

Ni, Yingchun; Parpura, Vladimir

2009-09-01

Vesicular glutamate transporters (VGLUTs) are responsible for vesicular glutamate storage and exocytotic glutamate release in neurons and astrocytes. Here, we selectively and efficiently overexpressed individual VGLUT proteins (VGLUT1, 2, or 3) in solitary astrocytes and studied their effects on mechanical stimulation-induced Ca2+-dependent glutamate release. Neither VGLUT1 nor VGLUT2 overexpression changed the amount of glutamate release, whereas overexpression of VGLUT3 significantly enhanced Ca2+-dependent glutamate release from astrocytes. None of the VGLUT overexpression affected mechanically induced intracellular Ca2+ increase. Inhibition of glutamine synthetase activity by L-methionine sulfoximine in astrocytes, which leads to increased cytosolic glutamate concentration, greatly increased their mechanically induced Ca2+-dependent glutamate release, without affecting intracellular Ca2+ dynamics. Taken together, these data indicate that both VGLUT3 and the cytosolic concentration of glutamate are key limiting factors in regulating the Ca2+-dependent release of glutamate from astrocytes.

Light-regulated leaf expansion in two Populus species: dependence on developmentally controlled ion transport.

PubMed

Stiles, Kari A; Van Volkenburgh, Elizabeth

2002-07-01

Leaf growth responses to light have been compared in two species of Populus, P. deltoides and P. trichocarpa. These species differ markedly in morphology, anatomy, and dependence on light during leaf expansion. Light stimulates the growth rate and acidification of cell walls in P. trichocarpa but not in P. deltoides, whereas leaves of P. deltoides maintain growth in the dark. Light-induced growth is promoted in P. deltoides when cells are provided 50-100 mM KCl. In both species, light initially depolarizes, then hyperpolarizes mesophyll plasma membranes. However, in the dark, the resting E(m) of mesophyll cells in P. deltoides, but not in P. trichocarpa, is relatively insensitive to decade changes in external [K+]. Results suggest that light-stimulated leaf growth depends on developmentally regulated cellular mechanisms controlling ion fluxes across the plasma membrane. These developmental differences underlie species-level differences in growth and physiological responses to the photoenvironment.

Density-dependent regulation of growth of BSC-1 cells in cell culture: growth inhibitors formed by the cells.

PubMed Central

Holley, R W; Armour, R; Baldwin, J H

1978-01-01

Inhibitors formed by a monkey epithelial cell line, BSC-1, play an important role in limiting growth at high cell densities. At least three inhibitors are formed: lactic acid, ammonia, and an unidentified inhibitor that may be an unstable protein. The unidentified inhibitor is destroyed by shaking the conditioned medium, by bubbling gas through the medium, or by heating or storing the medium in the absence of cells. The concentrations of lactic acid and ammonia that accumulate in conditioned medium inhibit growth when added to fresh medium. These results, together with earlier studies, indicate that density-dependent regulation of growth of BSC-1 cells results from the combined effects of (a) inhibitors formed by the cells, (b) decreased availability of receptor sites for serum growth factors as the cells become crowded, and (c) limiting concentrations of low molecular weight nutrients in the medium. In contrast, density-dependent regulation of growth in 3T3 mouse embryo fibroblasts results almost entirely from inactivation of serum factors. PMID:273914

Time-dependent upregulation of electron transport with concomitant induction of regulated excitation dissipation in Haslea diatoms.

PubMed

Perkins, R; Williamson, C; Lavaud, J; Mouget, J-L; Campbell, D A

2018-04-16

Photoacclimation by strains of Haslea "blue" diatom species H. ostrearia and H. silbo sp. nov. ined. was investigated with rapid light curves and induction-recovery curves using fast repetition rate fluorescence. Cultures were grown to exponential phase under 50 µmol m -2  s -1 photosynthetic available radiation (PAR) and then exposed to non-sequential rapid light curves where, once electron transport rate (ETR) had reached saturation, light intensity was decreased and then further increased prior to returning to near growth light intensity. The non-sequential rapid light curve revealed that ETR was not proportional to the instantaneously applied light intensity, due to rapid photoacclimation. Changes in the effective absorption cross sections for open PSII reaction centres (σ PSII ') or reaction centre connectivity (ρ) did not account for the observed increases in ETR under extended high light. σ PSII ' in fact decreased as a function of a time-dependent induction of regulated excitation dissipation Y(NPQ), once cells were at or above a PAR coinciding with saturation of ETR. Instead, the observed increases in ETR under extended high light were explained by an increase in the rate of PSII reopening, i.e. Q A - oxidation. This acceleration of electron transport was strictly light dependent and relaxed within seconds after a return to low light or darkness. The time-dependent nature of ETR upregulation and regulated NPQ induction was verified using induction-recovery curves. Our findings show a time-dependent induction of excitation dissipation, in parallel with very rapid photoacclimation of electron transport, which combine to make ETR independent of short-term changes in PAR. This supports a selective advantage for these diatoms when exposed to fluctuating light in their environment.

Oxygen-dependent regulation of c-di-GMP synthesis by SadC controls alginate production in Pseudomonas aeruginosa.

PubMed

Schmidt, Annika; Hammerbacher, Anna Silke; Bastian, Mike; Nieken, Karen Jule; Klockgether, Jens; Merighi, Massimo; Lapouge, Karine; Poschgan, Claudia; Kölle, Julia; Acharya, K Ravi; Ulrich, Martina; Tümmler, Burkhard; Unden, Gottfried; Kaever, Volkhard; Lory, Stephen; Haas, Dieter; Schwarz, Sandra; Döring, Gerd

2016-10-01

Pseudomonas aeruginosa produces increased levels of alginate in response to oxygen-deprived conditions. The regulatory pathway(s) that links oxygen limitation to increased synthesis of alginate has remained elusive. In the present study, using immunofluorescence microscopy, we show that anaerobiosis-induced alginate production by planktonic PAO1 requires the diguanylate cyclase (DGC) SadC, previously identified as a regulator of surface-associated lifestyles. Furthermore, we found that the gene products of PA4330 and PA4331, located in a predicted operon with sadC, have a major impact on alginate production: deletion of PA4330 (odaA, for oxygen-dependent alginate synthesis activator) caused an alginate production defect under anaerobic conditions, whereas a PA4331 (odaI, for oxygen-dependent alginate synthesis inhibitor) deletion mutant produced alginate also in the presence of oxygen, which would normally inhibit alginate synthesis. Based on their sequence, OdaA and OdaI have predicted hydratase and dioxygenase reductase activities, respectively. Enzymatic assays using purified protein showed that unlike OdaA, which did not significantly affect DGC activity of SadC, OdaI inhibited c-di-GMP production by SadC. Our data indicate that SadC, OdaA and OdaI are components of a novel response pathway of P. aeruginosa that regulates alginate synthesis in an oxygen-dependent manner. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Suppression of Mediator is regulated by Cdk8-dependent Grr1 turnover of the Med3 coactivator.

PubMed

Gonzalez, Deyarina; Hamidi, Nurul; Del Sol, Ricardo; Benschop, Joris J; Nancy, Thomas; Li, Chao; Francis, Lewis; Tzouros, Manuel; Krijgsveld, Jeroen; Holstege, Frank C P; Conlan, R Steven

2014-02-18

Mediator, an evolutionary conserved large multisubunit protein complex with a central role in regulating RNA polymerase II-transcribed genes, serves as a molecular switchboard at the interface between DNA binding transcription factors and the general transcription machinery. Mediator subunits include the Cdk8 module, which has both positive and negative effects on activator-dependent transcription through the activity of the cyclin-dependent kinase Cdk8, and the tail module, which is required for positive and negative regulation of transcription, correct preinitiation complex formation in basal and activated transcription, and Mediator recruitment. Currently, the molecular mechanisms governing Mediator function remain largely undefined. Here we demonstrate an autoregulatory mechanism used by Mediator to repress transcription through the activity of distinct components of different modules. We show that the function of the tail module component Med3, which is required for transcription activation, is suppressed by the kinase activity of the Cdk8 module. Med3 interacts with, and is phosphorylated by, Cdk8; site-specific phosphorylation triggers interaction with and degradation by the Grr1 ubiquitin ligase, thereby preventing transcription activation. This active repression mechanism involving Grr1-dependent ubiquitination of Med3 offers a rationale for the substoichiometric levels of the tail module that are found in purified Mediator and the corresponding increase in tail components seen in cdk8 mutants.

The tetraspanin CD63 regulates ESCRT-independent and dependent endosomal sorting during melanogenesis

PubMed Central

van Niel, Guillaume; Charrin, Stéphanie; Simoes, Sabrina; Romao, Maryse; Rochin, Leila; Saftig, Paul; Marks, Michael S.; Rubinstein, Eric; Raposo, Graça

2011-01-01

Summary Cargo sorting to intraluminal vesicles (ILVs) of multivesicular endosomes is required for numerous physiological processes including lysosome-related organelle (LRO) biogenesis. PMEL – a component of melanocyte LROs (melanosomes) – is sorted to ILVs in an ESCRT-independent manner, where it is proteolytically processed and assembled into functional amyloid fibrils during melanosome maturation. Here we show that the tetraspanin CD63 directly participates in ESCRT-independent sorting of the PMEL luminal domain, but not of traditional ESCRT-dependent cargoes, to ILVs. Inactivating CD63 in cell culture or in mice impairs amyloidogenesis and downstream melanosome morphogenesis. Whereas CD63 is required for normal PMEL luminal domain sorting, the disposal of the remaining PMEL transmembrane fragment requires functional ESCRTs but not CD63. In the absence of CD63, the PMEL luminal domain follows this fragment and is targeted for ESCRT-dependent degradation. Our data thus reveal a tight interplay regulated by CD63 between two distinct endosomal ILV sorting processes for a single cargo during LRO biogenesis. PMID:21962903

Presynaptic Regulation of Leptin in a Defined Lateral Hypothalamus-Ventral Tegmental Area Neurocircuitry Depends on Energy State.

PubMed

Liu, Jing-Jing; Bello, Nicholas T; Pang, Zhiping P

2017-12-06

Synaptic transmission controls brain activity and behaviors, including food intake. Leptin, an adipocyte-derived hormone, acts on neurons located in the lateral hypothalamic area (LHA) to maintain energy homeostasis and regulate food intake behavior. The specific synaptic mechanisms, cell types, and neural projections mediating this effect remain unclear. In male mice, using pathway-specific retrograde tracing, whole-cell patch-clamp recordings and post hoc cell type identification, we found that leptin reduces excitatory synaptic strength onto both melanin-concentrating hormone- and orexin-expressing neurons projecting from the LHA to the ventral tegmental area (VTA), which may affect dopamine signaling and motivation for feeding. A presynaptic mechanism mediated by distinct intracellular signaling mechanisms may account for this regulation by leptin. The regulatory effects of leptin depend on intact leptin receptor signaling. Interestingly, the synaptic regulatory function of leptin in the LHA-to-VTA neuronal pathway is highly sensitive to energy states: both energy deficiency (acute fasting) and excessive energy storage (high-fat diet-induced obesity) blunt the effect of leptin. These data revealed that leptin may regulate synaptic transmission in the LHA-to-VTA neurocircuitry in an inverted "U-shape" fashion dependent on plasma glucose levels and related to metabolic states. SIGNIFICANCE STATEMENT The lateral hypothalamic area (LHA) to ventral tegmental area (VTA) projection is an important neural pathway involved in balancing whole-body energy states and reward. We found that the excitatory synaptic inputs to both orexin- and melanin-concentrating hormone expressing LHA neurons projecting to the VTA were suppressed by leptin, a peptide hormone derived from adipocytes that signals peripheral energy status to the brain. Interestingly, energy states seem to affect how leptin regulates synaptic transmission since both the depletion of energy induced by acute food

Damage-dependent regulation of MUS81-EME1 by Fanconi anemia complementation group A protein

PubMed Central

Benitez, Anaid; Yuan, Fenghua; Nakajima, Satoshi; Wei, Leizhen; Qian, Liangyue; Myers, Richard; Hu, Jennifer J.; Lan, Li; Zhang, Yanbin

2014-01-01

MUS81-EME1 is a DNA endonuclease involved in replication-coupled repair of DNA interstrand cross-links (ICLs). A prevalent hypothetical role of MUS81-EME1 in ICL repair is to unhook the damage by incising the leading strand at the 3′ side of an ICL lesion. In this study, we report that purified MUS81-EME1 incises DNA at the 5′ side of a psoralen ICL residing in fork structures. Intriguingly, ICL repair protein, Fanconi anemia complementation group A protein (FANCA), greatly enhances MUS81-EME1-mediated ICL incision. On the contrary, FANCA exhibits a two-phase incision regulation when DNA is undamaged or the damage affects only one DNA strand. Studies using truncated FANCA proteins indicate that both the N- and C-moieties of the protein are required for the incision regulation. Using laser-induced psoralen ICL formation in cells, we find that FANCA interacts with and recruits MUS81 to ICL lesions. This report clarifies the incision specificity of MUS81-EME1 on ICL damage and establishes that FANCA regulates the incision activity of MUS81-EME1 in a damage-dependent manner. PMID:24170812

Amino acid transporter SLC7A11/xCT at the crossroads of regulating redox homeostasis and nutrient dependency of cancer.

PubMed

Koppula, Pranavi; Zhang, Yilei; Zhuang, Li; Gan, Boyi

2018-04-25

Cancer cells often upregulate nutrient transporters to fulfill their increased biosynthetic and bioenergetic needs, and to maintain redox homeostasis. One nutrient transporter frequently overexpressed in human cancers is the cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11; also known as xCT). SLC7A11 promotes cystine uptake and glutathione biosynthesis, resulting in protection from oxidative stress and ferroptotic cell death. Recent studies have unexpectedly revealed that SLC7A11 also plays critical roles in glutamine metabolism and regulates the glucose and glutamine dependency of cancer cells. This review discusses the roles of SLC7A11 in regulating the antioxidant response and nutrient dependency of cancer cells, explores our current understanding of SLC7A11 regulation in cancer metabolism, and highlights key open questions for future studies in this emerging research area. A deeper understanding of SLC7A11 in cancer metabolism may identify new therapeutic opportunities to target this important amino acid transporter for cancer treatment.

Radiation-induced cyclooxygenase 2 up-regulation is dependent on redox status in prostate cancer cells.

PubMed

Li, Lingyun; Steinauer, Kirsten K; Dirks, Amie J; Husbeck, Bryan; Gibbs, Iris; Knox, Susan J

2003-12-01

Cyclooxygenase 2 (COX2) is the inducible isozyme of COX, a key enzyme in arachidonate metabolism and the conversion of arachidonic acid (AA) to prostaglandins (PGs) and other eicosanoids. Previous studies have demonstrated that the COX2 protein is up-regulated in prostate cancer cells after irradiation and that this results in elevated levels of PGE(2). In the present study, we further investigated whether radiation-induced COX2 up-regulation is dependent on the redox status of cells from the prostate cancer cell line PC-3. l-Buthionine sulfoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), and the antioxidants alpha-lipoic acid and N-acetyl-l-cysteine (NAC) were used to modulate the cellular redox status. BSO decreased the cellular GSH level and increased cellular reactive oxygen species (ROS) in PC-3 cells, whereas alpha-lipoic acid and NAC increased the GSH level and decreased cellular ROS. Both radiation and the oxidant H(2)O(2) had similar effects on COX2 up-regulation and PGE(2) production in PC-3 cells, suggesting that radiation-induced COX2 up-regulation is secondary to the production of ROS. The relative increases in COX2 expression and PGE(2) production induced by radiation and H(2)O(2) were even greater when PC-3 cells were pretreated with BSO. When the cells were pretreated with alpha-lipoic acid or NAC for 24 h, both radiation- and H(2)O(2)-induced COX2 up-regulation and PGE(2) production were markedly inhibited. These results demonstrate that radiation-induced COX2 up-regulation in prostate cancer cells is modulated by the cellular redox status. Radiation-induced increases in ROS levels contribute to the adaptive response of PC-3 cells, resulting in elevated levels of COX2.

JMJD6 and U2AF65 co-regulate alternative splicing in both JMJD6 enzymatic activity dependent and independent manner.

PubMed

Yi, Jia; Shen, Hai-Feng; Qiu, Jin-Song; Huang, Ming-Feng; Zhang, Wen-Juan; Ding, Jian-Cheng; Zhu, Xiao-Yan; Zhou, Yu; Fu, Xiang-Dong; Liu, Wen

2017-04-07

JMJD6, a jumonji C (Jmj C) domain-containing protein demethylase and hydroxylase, has been implicated in an array of biological processes. It has been shown that JMJD6 interacts with and hydroxylates multiple serine/arginine-rich (SR) proteins and SR related proteins, including U2AF65, all of which are known to function in alternative splicing regulation. However, whether JMJD6 is widely involved in alternative splicing and the molecular mechanism underlying JMJD6-regulated alternative splicing have remained incompletely understood. Here, by using RASL-Seq, we investigated the functional impact of RNA-dependent interaction between JMJD6 and U2AF65, revealing that JMJD6 and U2AF65 co-regulated a large number of alternative splicing events. We further demonstrated the JMJD6 function in alternative splicing in jmjd6 knockout mice. Mechanistically, we showed that the enzymatic activity of JMJD6 was required for a subset of JMJD6-regulated splicing, and JMJD6-mediated lysine hydroxylation of U2AF65 could account for, at least partially, their co-regulated alternative splicing events, suggesting both JMJD6 enzymatic activity-dependent and independent control of alternative splicing. These findings reveal an intimate link between JMJD6 and U2AF65 in alternative splicing regulation, which has important implications in development and disease processes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Cyclic AMP Regulates Bacterial Persistence through Repression of the Oxidative Stress Response and SOS-Dependent DNA Repair in Uropathogenic Escherichia coli.

PubMed

Molina-Quiroz, Roberto C; Silva-Valenzuela, Cecilia; Brewster, Jennifer; Castro-Nallar, Eduardo; Levy, Stuart B; Camilli, Andrew

2018-01-09

Bacterial persistence is a transient, nonheritable physiological state that provides tolerance to bactericidal antibiotics. The stringent response, toxin-antitoxin modules, and stochastic processes, among other mechanisms, play roles in this phenomenon. How persistence is regulated is relatively ill defined. Here we show that cyclic AMP, a global regulator of carbon catabolism and other core processes, is a negative regulator of bacterial persistence in uropathogenic Escherichia coli , as measured by survival after exposure to a β-lactam antibiotic. This phenotype is regulated by a set of genes leading to an oxidative stress response and SOS-dependent DNA repair. Thus, persister cells tolerant to cell wall-acting antibiotics must cope with oxidative stress and DNA damage and these processes are regulated by cyclic AMP in uropathogenic E. coli IMPORTANCE Bacterial persister cells are important in relapsing infections in patients treated with antibiotics and also in the emergence of antibiotic resistance. Our results show that in uropathogenic E. coli , the second messenger cyclic AMP negatively regulates persister cell formation, since in its absence much more persister cells form that are tolerant to β-lactams antibiotics. We reveal the mechanism to be decreased levels of reactive oxygen species, specifically hydroxyl radicals, and SOS-dependent DNA repair. Our findings suggest that the oxidative stress response and DNA repair are relevant pathways to target in the design of persister-specific antibiotic compounds. Copyright © 2018 Molina-Quiroz et al.

3-Phosphoinositide-dependent PDK1 negatively regulates transforming growth factor-beta-induced signaling in a kinase-dependent manner through physical interaction with Smad proteins.

PubMed

Seong, Hyun-A; Jung, Haiyoung; Kim, Kyong-Tai; Ha, Hyunjung

2007-04-20

We have reported previously that PDK1 physically interacts with STRAP, a transforming growth factor-beta (TGF-beta) receptor-interacting protein, and enhances STRAP-induced inhibition of TGF-beta signaling. In this study we show that PDK1 coimmunoprecipitates with Smad proteins, including Smad2, Smad3, Smad4, and Smad7, and that this association is mediated by the pleckstrin homology domain of PDK1. The association between PDK1 and Smad proteins is increased by insulin treatment but decreased by TGF-beta treatment. Analysis of the interacting proteins shows that Smad proteins enhance PDK1 kinase activity by removing 14-3-3, a negative regulator of PDK1, from the PDK1-14-3-3 complex. Knockdown of endogenous Smad proteins, including Smad3 and Smad7, by transfection with small interfering RNA produced the opposite trend and decreased PDK1 activity, protein kinase B/Akt phosphorylation, and Bad phosphorylation. Moreover, coexpression of Smad proteins and wild-type PDK1 inhibits TGF-beta-induced transcription, as well as TGF-beta-mediated biological functions, such as apoptosis and cell growth arrest. Inhibition was dose-dependent on PDK1, but no inhibition was observed in the presence of an inactive kinase-dead PDK1 mutant. In addition, confocal microscopy showed that wild-type PDK1 prevents translocation of Smad3 and Smad4 from the cytoplasm to the nucleus, as well as the redistribution of Smad7 from the nucleus to the cytoplasm in response to TGF-beta. Taken together, our results suggest that PDK1 negatively regulates TGF-beta-mediated signaling in a PDK1 kinase-dependent manner via a direct physical interaction with Smad proteins and that Smad proteins can act as potential positive regulators of PDK1.

Fragile X Mental Retardation Protein Regulates Activity-Dependent Membrane Trafficking and Trans-Synaptic Signaling Mediating Synaptic Remodeling

PubMed Central

Sears, James C.; Broadie, Kendal

2018-01-01

Fragile X syndrome (FXS) is the leading monogenic cause of autism and intellectual disability. The disease arises through loss of fragile X mental retardation protein (FMRP), which normally exhibits peak expression levels in early-use critical periods, and is required for activity-dependent synaptic remodeling during this transient developmental window. FMRP canonically binds mRNA to repress protein translation, with targets that regulate cytoskeleton dynamics, membrane trafficking, and trans-synaptic signaling. We focus here on recent advances emerging in these three areas from the Drosophila disease model. In the well-characterized central brain mushroom body (MB) olfactory learning/memory circuit, FMRP is required for activity-dependent synaptic remodeling of projection neurons innervating the MB calyx, with function tightly restricted to an early-use critical period. FMRP loss is phenocopied by conditional removal of FMRP only during this critical period, and rescued by FMRP conditional expression only during this critical period. Consistent with FXS hyperexcitation, FMRP loss defects are phenocopied by heightened sensory experience and targeted optogenetic hyperexcitation during this critical period. FMRP binds mRNA encoding Drosophila ESCRTIII core component Shrub (human CHMP4 homolog) to restrict Shrub translation in an activity-dependent mechanism only during this same critical period. Shrub mediates endosomal membrane trafficking, and perturbing Shrub expression is known to interfere with neuronal process pruning. Consistently, FMRP loss and Shrub overexpression targeted to projection neurons similarly causes endosomal membrane trafficking defects within synaptic boutons, and genetic reduction of Shrub strikingly rescues Drosophila FXS model defects. In parallel work on the well-characterized giant fiber (GF) circuit, FMRP limits iontophoretic dye loading into central interneurons, demonstrating an FMRP role controlling core neuronal properties through the

Novel Phosphorylation and Ubiquitination Sites Regulate Reactive Oxygen Species-dependent Degradation of Anti-apoptotic c-FLIP Protein*

PubMed Central

Wilkie-Grantham, Rachel P.; Matsuzawa, Shu-Ichi; Reed, John C.

2013-01-01

The cytosolic protein c-FLIP (cellular Fas-associated death domain-like interleukin 1β-converting enzyme inhibitory protein) is an inhibitor of death receptor-mediated apoptosis that is up-regulated in a variety of cancers, contributing to apoptosis resistance. Several compounds found to restore sensitivity of cancer cells to TRAIL, a TNF family death ligand with promising therapeutic potential, act by targeting c-FLIP ubiquitination and degradation by the proteasome. The generation of reactive oxygen species (ROS) has been implicated in c-FLIP protein degradation. However, the mechanism by which ROS post-transcriptionally regulate c-FLIP protein levels is not well understood. We show here that treatment of prostate cancer PPC-1 cells with the superoxide generators menadione, paraquat, or buthionine sulfoximine down-regulates c-FLIP long (c-FLIPL) protein levels, which is prevented by the proteasome inhibitor MG132. Furthermore, pretreatment of PPC-1 cells with a ROS scavenger prevented ubiquitination and loss of c-FLIPL protein induced by menadione or paraquat. We identified lysine 167 as a novel ubiquitination site of c-FLIPL important for ROS-dependent degradation. We also identified threonine 166 as a novel phosphorylation site and demonstrate that Thr-166 phosphorylation is required for ROS-induced Lys-167 ubiquitination. The mutation of either Thr-166 or Lys-167 was sufficient to stabilize c-FLIP protein levels in PPC-1, HEK293T, and HeLa cancer cells treated with menadione or paraquat. Accordingly, expression of c-FLIP T166A or K167R mutants protected cells from ROS-mediated sensitization to TRAIL-induced cell death. Our findings reveal novel ROS-dependent post-translational modifications of the c-FLIP protein that regulate its stability, thus impacting sensitivity of cancer cells to TRAIL. PMID:23519470

Novel phosphorylation and ubiquitination sites regulate reactive oxygen species-dependent degradation of anti-apoptotic c-FLIP protein.

PubMed

Wilkie-Grantham, Rachel P; Matsuzawa, Shu-Ichi; Reed, John C

2013-05-03

The cytosolic protein c-FLIP (cellular Fas-associated death domain-like interleukin 1β-converting enzyme inhibitory protein) is an inhibitor of death receptor-mediated apoptosis that is up-regulated in a variety of cancers, contributing to apoptosis resistance. Several compounds found to restore sensitivity of cancer cells to TRAIL, a TNF family death ligand with promising therapeutic potential, act by targeting c-FLIP ubiquitination and degradation by the proteasome. The generation of reactive oxygen species (ROS) has been implicated in c-FLIP protein degradation. However, the mechanism by which ROS post-transcriptionally regulate c-FLIP protein levels is not well understood. We show here that treatment of prostate cancer PPC-1 cells with the superoxide generators menadione, paraquat, or buthionine sulfoximine down-regulates c-FLIP long (c-FLIP(L)) protein levels, which is prevented by the proteasome inhibitor MG132. Furthermore, pretreatment of PPC-1 cells with a ROS scavenger prevented ubiquitination and loss of c-FLIP(L) protein induced by menadione or paraquat. We identified lysine 167 as a novel ubiquitination site of c-FLIP(L) important for ROS-dependent degradation. We also identified threonine 166 as a novel phosphorylation site and demonstrate that Thr-166 phosphorylation is required for ROS-induced Lys-167 ubiquitination. The mutation of either Thr-166 or Lys-167 was sufficient to stabilize c-FLIP protein levels in PPC-1, HEK293T, and HeLa cancer cells treated with menadione or paraquat. Accordingly, expression of c-FLIP T166A or K167R mutants protected cells from ROS-mediated sensitization to TRAIL-induced cell death. Our findings reveal novel ROS-dependent post-translational modifications of the c-FLIP protein that regulate its stability, thus impacting sensitivity of cancer cells to TRAIL.

CYCLIN-DEPENDENT KINASE8 differentially regulates plant immunity to fungal pathogens through kinase-dependent and -independent functions in Arabidopsis.

PubMed

Zhu, Yingfang; Schluttenhoffer, Craig M; Wang, Pengcheng; Fu, Fuyou; Thimmapuram, Jyothi; Zhu, Jian-Kang; Lee, Sang Yeol; Yun, Dae-Jin; Mengiste, Tesfaye

2014-10-01

CYCLIN-DEPENDENT KINASE8 (CDK8) is a widely studied component of eukaryotic Mediator complexes. However, the biological and molecular functions of plant CDK8 are not well understood. Here, we provide evidence for regulatory functions of Arabidopsis thaliana CDK8 in defense and demonstrate its functional and molecular interactions with other Mediator and non-Mediator subunits. The cdk8 mutant exhibits enhanced resistance to Botrytis cinerea but susceptibility to Alternaria brassicicola. The contributions of CDK8 to the transcriptional activation of defensin gene PDF1.2 and its interaction with MEDIATOR COMPLEX SUBUNIT25 (MED25) implicate CDK8 in jasmonate-mediated defense. Moreover, CDK8 associates with the promoter of AGMATINE COUMAROYLTRANSFERASE to promote its transcription and regulate the biosynthesis of the defense-active secondary metabolites hydroxycinnamic acid amides. CDK8 also interacts with the transcription factor WAX INDUCER1, implying its additional role in cuticle development. In addition, overlapping functions of CDK8 with MED12 and MED13 and interactions between CDK8 and C-type cyclins suggest the conserved configuration of the plant Mediator kinase module. In summary, while CDK8's positive transcriptional regulation of target genes and its phosphorylation activities underpin its defense functions, the impaired defense responses in the mutant are masked by its altered cuticle, resulting in specific resistance to B. cinerea. © 2014 American Society of Plant Biologists. All rights reserved.

Proinflammatory TLR signaling is regulated by a TRAF2-dependent proteolysis mechanism in macrophages

PubMed Central

Jin, Jin; Xiao, Yichuan; Hu, Hongbo; Zou, Qiang; Li, Yanchuan; Gao, Yanpan; Ge, Wei; Cheng, Xuhong; Sun, Shao-Cong

2014-01-01

Signal transduction from toll-like receptors (TLRs) is important for innate immunity against infections, but deregulated TLR signaling contributes to inflammatory disorders. Here we show that myeloid cell-specific ablation of TRAF2 greatly promotes TLR-stimulated proinflammatory cytokine expression in macrophages and exacerbates colitis in an animal model of inflammatory bowel disease. TRAF2 deficiency does not enhance upstream signaling events, but it causes accumulation of two transcription factors, c-Rel and IRF5, known to mediate proinflammatory cytokine induction. Interestingly, TRAF2 controls the fate of c-Rel and IRF5 via a proteasome-dependent mechanism that also requires TRAF3 and the E3 ubiquitin ligase cIAP. We further show that TRAF2 also regulates inflammatory cytokine production in tumor-associated macrophages and facilitates tumor growth. These findings demonstrate an unexpected anti-inflammatory function of TRAF2 and suggest a proteasome-dependent mechanism that limits the proinflammatory TLR signaling. PMID:25565375

HGG-22. TARGETING NEURONAL ACTIVITY-REGULATED NEUROLIGIN-3 DEPENDENCY FOR HIGH-GRADE GLIOMA THERAPY

PubMed Central

Venkatesh, Humsa S; Tam, Lydia T; Woo, Pamelyn J; Monje, Michelle

2017-01-01

Abstract Neuronal activity promotes high-grade glioma (HGG) growth. An important mechanism mediating this neural regulation of brain cancer is activity-dependent cleavage and secretion of the synaptic molecule and glioma mitogen neuroligin-3 (Nlgn3), but the therapeutic potential of targeting Nlgn3 in glioma remains to be defined. Here, we demonstrate a striking dependence of HGG growth on microenvironmental Nlgn3 and determine a targetable mechanism of secretion. Patient-derived orthotopic xenografts of pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG) fail to grow in Nlgn3 knockout mice. Using genetic mouse models, we illustrate that Nlgn3 is cleaved from both neurons and oligodendrocyte precursor cells via the ADAM10 sheddase. Administration of an ADAM10 inhibitor robustly blocks pGBM and DIPG xenograft growth via modulation of the tumor microenvironment. This work defines the therapeutic potential of and a promising strategy for targeting Nlgn3 secretion in the glioma microenvironment, which could prove transformative for treatment of HGG.

pH regulation in barnacle muscle fibers: dependence on extracellular sodium and bicarbonate.

PubMed

Boron, W F; McCormick, W C; Roos, A

1981-01-01

Intracellular pH (pHi) regulation was studied in barnacle muscle fibers with pH-sensitive microelectrodes. The cells were acid loaded, and the subsequent recovery of pHi was monitored. The rate of recovery was reduced by one-third when external Na+ ([Na+]o) was replaced by Li+, but recovery was completely abolished when Na+ was replaced by choline or N-methyl-D-glucamine. In other experiments, varying amounts of Na+ were replaced by choline, and the acid extrusion rate, derived from the recovery rate of pHi, was calculated at a single value of pHi, 6.80. The dependence of the acid extrusion rate on [Na+]o could be described by Michaelis-Menten kinetics; at pHo (extracellular) = 8.0 and [HCO3-]o (extracellular) = 10 mM, the apparent Km and Vmax were 59 mM and 1.3 mmol x l(-1) x min-1. When [HCO3-]o was reduced to 2.5 mM at the same pHo, Km did not change significantly, but Vmax was substantially reduced. On the other hand, when pHo was reduced to 7.4 at constant [HCO3-]o, Vmax changed only slightly, but Km increased substantially. In similar experiments, we examined the dependence of the acid extrusion rate on [HCO3-]o. At pHo = 8.0 and [Na+]o = 440 mM, the apparent Km and Vmax were 4.1 mM and 2.1 mmol x 1-1 x min-1. When pHo was reduced to 7.4, Vmax was not altered, but Km substantially increased. The kinetic data are discussed in terms of the role of pHo, [Na+]o, and [HCO3-]o in the pHi-regulating system.

Double-Stranded RNA-Dependent Protein Kinase Regulates the Motility of Breast Cancer Cells

PubMed Central

Xu, Mei; Chen, Gang; Wang, Siying; Liao, Mingjun; Frank, Jacqueline A.; Bower, Kimberly A.; Zhang, Zhuo; Shi, Xianglin; Luo, Jia

2012-01-01

Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is an interferon-induced protein kinase that plays a central role in the anti-viral process. Due to its pro-apoptotic and anti-proliferative action, there is an increased interest in PKR modulation as an anti-tumor strategy. PKR is overexpressed in breast cancer cells; however, the role of PKR in breast cancer cells is unclear. The expression/activity of PKR appears inversely related to the aggressiveness of breast cancer cells. The current study investigated the role of PKR in the motility/migration of breast cancer cells. The activation of PKR by a synthesized dsRNA (PIC) significantly decreased the motility of several breast cancer cell lines (BT474, MDA-MB231 and SKBR3). PIC inhibited cell migration and blocked cell membrane ruffling without affecting cell viability. PIC also induced the reorganization of the actin cytoskeleton and impaired the formation of lamellipodia. These effects of PIC were reversed by the pretreatment of a selective PKR inhibitor. PIC also activated p38 mitogen-activated protein kinase (MAPK) and its downstream MAPK-activated protein kinase 2 (MK2). PIC-induced activation of p38 MAPK and MK2 was attenuated by the PKR inhibitor and the PKR siRNA, but a selective p38 MAPK inhibitor (SB203580) or other MAPK inhibitors did not affect PKR activity, indicating that PKR is upstream of p38 MAPK/MK2. Cofilin is an actin severing protein and regulates membrane ruffling, lamellipodia formation and cell migration. PIC inhibited cofilin activity by enhancing its phosphorylation at Ser3. PIC activated LIM kinase 1 (LIMK1), an upstream kinase of cofilin in a p38 MAPK-dependent manner. We concluded that the activation of PKR suppressed cell motility by regulating the p38 MAPK/MK2/LIMK/cofilin pathway. PMID:23112838

Protein kinase C -dependent regulation of synaptosomal glutamate uptake under conditions of hypergravity

NASA Astrophysics Data System (ADS)

Borisova, Tatiana; Krisanova, Natalia; Borisov, Arseniy; Sivko, Roman

Glutamate is not only the main excitatory neurotransmitter in the mammalian CNS, but also a potent neurotoxin. Excessive concentration of ambient glutamate over activates glutamate receptors and causes neurotoxicity. Uptake of glutamate from the extracellular space into nerve cells was mediated by sodium-dependent glutamate transporters located in the plasma membrane. It was shown that the activity of glutamate transporters in rat brain nerve terminals was decreased after hypergravity (centrifugation of rats in special containers at 10 G for 1 hour). This decrease may result from the reduction in the number of glutamate transporters expressed in the plasma membrane of nerve terminals after hypergravity that was regulated by protein kinase C. The possibility of the involvement of protein kinase C in the regulation of the activity of glutamate transporters was assessed under conditions of hypergravity. The effect of protein kinase C inhibitor GF 109 203X on synaptosomal L-[14C]glutamate uptake was analysed. It was shown that the inhibitor decreased L-[14C]glutamate uptake by 15 % in control but did not influence it after hypergravity. In control, the initial velocity of L-[14C]glutamate uptake in the presence of the inhibitor decreased from 2.5 ± 0.2 nmol x min-1 x mg-1 of proteins to 2.17 ± 0.1 nmol x min-1 x mg-1 of proteins, whereas after hypergravity this value lowered from 2.05 ± 0.1 nmol x min-1 x mg-1 of proteins to 2.04 ± 0.1 nmol x min-1 x mg-1 of proteins. Thus, protein kinase C -dependent alteration in the cell surface expression of glutamate transporters may be one of the causes of a decrease in the activity of glutamate transporters after hypergravity.

Caffeic acid attenuates rat liver reperfusion injury through sirtuin 3-dependent regulation of mitochondrial respiratory chain.

PubMed

Mu, Hong-Na; Li, Quan; Pan, Chun-Shui; Liu, Yu-Ying; Yan, Li; Hu, Bai-He; Sun, Kai; Chang, Xin; Zhao, Xin-Rong; Fan, Jing-Yu; Han, Jing-Yan

2015-08-01

Sirtuin 3 (Sirt3) plays critical roles in regulating mitochondrial oxidative metabolism. However, whether Sirt3 is involved in liver ischemia and reperfusion (I/R) injury remains elusive. Caffeic acid (CA) is a natural antioxidant derived from Salvia miltiorrhiza. Whether CA protects against liver I/R injury through regulating Sirt3 and the mitochondrial respiratory chain (MRC) is unclear. This study investigated the effect of CA on liver I/R injury, microcirculatory disturbance, and potential mechanisms, particularly focusing on Sirt3-dependent MRC. Liver I/R of male Sprague-Dawley rats was established by occlusion of portal area vessels for 30 min followed by 120 min of reperfusion. CA (15 mg/kg/h) was continuously infused via the femoral vein starting 30 min before ischemia. After I/R, Sirt3 expression, and MRC activity decreased, acetylation of NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 and succinate dehydrogenase complex, subunit A, flavoprotein variant provoked, and the liver microcirculatory disturbance and injury were observed. Treatment with CA attenuated liver injury, inhibited Sirt3 down-expression, and up-regulated MRC activity. CA attenuated rat liver microcirculatory disturbance and oxidative injury through regulation of Sirt3 and the mitochondrial respiratory chain. Copyright © 2015 Elsevier Inc. All rights reserved.

Ca2+-Dependent Regulations and Signaling in Skeletal Muscle: From Electro-Mechanical Coupling to Adaptation

PubMed Central

Gehlert, Sebastian; Bloch, Wilhelm; Suhr, Frank

2015-01-01

Calcium (Ca2+) plays a pivotal role in almost all cellular processes and ensures the functionality of an organism. In skeletal muscle fibers, Ca2+ is critically involved in the innervation of skeletal muscle fibers that results in the exertion of an action potential along the muscle fiber membrane, the prerequisite for skeletal muscle contraction. Furthermore and among others, Ca2+ regulates also intracellular processes, such as myosin-actin cross bridging, protein synthesis, protein degradation and fiber type shifting by the control of Ca2+-sensitive proteases and transcription factors, as well as mitochondrial adaptations, plasticity and respiration. These data highlight the overwhelming significance of Ca2+ ions for the integrity of skeletal muscle tissue. In this review, we address the major functions of Ca2+ ions in adult muscle but also highlight recent findings of critical Ca2+-dependent mechanisms essential for skeletal muscle-regulation and maintenance. PMID:25569087

Regulator-dependent mechanisms of C3b processing by factor I allow differentiation of immune responses.

PubMed

Xue, Xiaoguang; Wu, Jin; Ricklin, Daniel; Forneris, Federico; Di Crescenzio, Patrizia; Schmidt, Christoph Q; Granneman, Joke; Sharp, Thomas H; Lambris, John D; Gros, Piet

2017-08-01

The complement system labels microbes and host debris for clearance. Degradation of surface-bound C3b is pivotal to direct immune responses and protect host cells. How the serine protease factor I (FI), assisted by regulators, cleaves either two or three distant peptide bonds in the CUB domain of C3b remains unclear. We present a crystal structure of C3b in complex with FI and regulator factor H (FH; domains 1-4 with 19-20). FI binds C3b-FH between FH domains 2 and 3 and a reoriented C3b C-terminal domain and docks onto the first scissile bond, while stabilizing its catalytic domain for proteolytic activity. One cleavage in C3b does not affect its overall structure, whereas two cleavages unfold CUB and dislodge the thioester-containing domain (TED), affecting binding of regulators and thereby determining the number of cleavages. These data explain how FI generates late-stage opsonins iC3b or C3dg in a context-dependent manner, to react to foreign, danger or healthy self signals.

Listeria monocytogenes differential transcriptome analysis reveals temperature-dependent Agr regulation and suggests overlaps with other regulons.

PubMed

Garmyn, Dominique; Augagneur, Yoann; Gal, Laurent; Vivant, Anne-Laure; Piveteau, Pascal

2012-01-01

Listeria monocytogenes is a ubiquitous, opportunistic pathogenic organism. Environmental adaptation requires constant regulation of gene expression. Among transcriptional regulators, AgrA is part of an auto-induction system. Temperature is an environmental cue critical for in vivo adaptation. In order to investigate how temperature may affect AgrA-dependent transcription, we compared the transcriptomes of the parental strain L. monocytogenes EGD-e and its ΔagrA mutant at the saprophytic temperature of 25°C and in vivo temperature of 37°C. Variations of transcriptome were higher at 37°C than at 25°C. Results suggested that AgrA may be involved in the regulation of nitrogen transport, amino acids, purine and pyrimidine biosynthetic pathways and phage-related functions. Deregulations resulted in a growth advantage at 37°C, but affected salt tolerance. Finally, our results suggest overlaps with PrfA, σB, σH and CodY regulons. These overlaps may suggest that through AgrA, Listeria monocytogenes integrates information on its biotic environment.

Listeria monocytogenes Differential Transcriptome Analysis Reveals Temperature-Dependent Agr Regulation and Suggests Overlaps with Other Regulons

PubMed Central

Garmyn, Dominique; Augagneur, Yoann; Gal, Laurent; Vivant, Anne-Laure; Piveteau, Pascal

2012-01-01

Listeria monocytogenes is a ubiquitous, opportunistic pathogenic organism. Environmental adaptation requires constant regulation of gene expression. Among transcriptional regulators, AgrA is part of an auto-induction system. Temperature is an environmental cue critical for in vivo adaptation. In order to investigate how temperature may affect AgrA-dependent transcription, we compared the transcriptomes of the parental strain L. monocytogenes EGD-e and its ΔagrA mutant at the saprophytic temperature of 25°C and in vivo temperature of 37°C. Variations of transcriptome were higher at 37°C than at 25°C. Results suggested that AgrA may be involved in the regulation of nitrogen transport, amino acids, purine and pyrimidine biosynthetic pathways and phage-related functions. Deregulations resulted in a growth advantage at 37°C, but affected salt tolerance. Finally, our results suggest overlaps with PrfA, σB, σH and CodY regulons. These overlaps may suggest that through AgrA, Listeria monocytogenes integrates information on its biotic environment. PMID:23024744

Ca2+-Binding Protein 1 Regulates Hippocampal-dependent Memory and Synaptic Plasticity.

PubMed

Yang, Tian; Britt, Jeremiah K; Cintrón-Pérez, Coral J; Vázquez-Rosa, Edwin; Tobin, Kevin V; Stalker, Grant; Hardie, Jason; Taugher, Rebecca J; Wemmie, John; Pieper, Andrew A; Lee, Amy

2018-06-01

Ca 2+ -binding protein 1 (CaBP1) is a Ca 2+ -sensing protein similar to calmodulin that potently regulates voltage-gated Ca 2+ channels. Unlike calmodulin, however, CaBP1 is mainly expressed in neuronal cell-types and enriched in the hippocampus, where its function is unknown. Here, we investigated the role of CaBP1 in hippocampal-dependent behaviors using mice lacking expression of CaBP1 (C-KO). By western blot, the largest CaBP1 splice variant, caldendrin, was detected in hippocampal lysates from wild-type (WT) but not C-KO mice. Compared to WT mice, C-KO mice exhibited mild deficits in spatial learning and memory in both the Barnes maze and in Morris water maze reversal learning. In contextual but not cued fear-conditioning assays, C-KO mice showed greater freezing responses than WT mice. In addition, the number of adult-born neurons in the hippocampus of C-KO mice was ∼40% of that in WT mice, as measured by bromodeoxyuridine labeling. Moreover, hippocampal long-term potentiation was significantly reduced in C-KO mice. We conclude that CaBP1 is required for cellular mechanisms underlying optimal encoding of hippocampal-dependent spatial and fear-related memories. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Protein arginine methyltransferase 1 modulates innate immune responses through regulation of peroxisome proliferator-activated receptor γ-dependent macrophage differentiation.

PubMed

Tikhanovich, Irina; Zhao, Jie; Olson, Jody; Adams, Abby; Taylor, Ryan; Bridges, Brian; Marshall, Laurie; Roberts, Benjamin; Weinman, Steven A

2017-04-28

Arginine methylation is a common posttranslational modification that has been shown to regulate both gene expression and extranuclear signaling events. We recently reported defects in protein arginine methyltransferase 1 (PRMT1) activity and arginine methylation in the livers of cirrhosis patients with a history of recurrent infections. To examine the role of PRMT1 in innate immune responses in vivo , we created a cell type-specific knock-out mouse model. We showed that myeloid-specific PRMT1 knock-out mice demonstrate higher proinflammatory cytokine production and a lower survival rate after cecal ligation and puncture. We found that this defect is because of defective peroxisome proliferator-activated receptor γ (PPARγ)-dependent M2 macrophage differentiation. PPARγ is one of the key transcription factors regulating macrophage polarization toward a more anti-inflammatory and pro-resolving phenotype. We found that PRMT1 knock-out macrophages failed to up-regulate PPARγ expression in response to IL4 treatment resulting in 4-fold lower PPARγ expression in knock-out cells than in wild-type cells. Detailed study of the mechanism revealed that PRMT1 regulates PPARγ gene expression through histone H4R3me2a methylation at the PPARγ promoter. Supplementing with PPARγ agonists rosiglitazone and GW1929 was sufficient to restore M2 differentiation in vivo and in vitro and abrogated the difference in survival between wild-type and PRMT1 knock-out mice. Taken together these data suggest that PRMT1-dependent regulation of macrophage PPARγ expression contributes to the infection susceptibility in PRMT1 knock-out mice. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Ca2+ -dependent regulation of phototransduction.

PubMed

Stephen, Ricardo; Filipek, Sławomir; Palczewski, Krzysztof; Sousa, Marcelo Carlos

2008-01-01

Photon absorption by rhodopsin triggers the phototransduction signaling pathway that culminates in degradation of cGMP, closure of cGMP-gated ion channels and hyperpolarization of the photoreceptor membrane. This process is accompanied by a decrease in free Ca(2+) concentration in the photoreceptor cytosol sensed by Ca(2+)-binding proteins that modulate phototransduction and activate the recovery phase to reestablish the photoreceptor dark potential. Guanylate cyclase-activating proteins (GCAPs) belong to the neuronal calcium sensor (NCS) family and are responsible for activating retinal guanylate cyclases (retGCs) at low Ca(2+) concentrations triggering synthesis of cGMP and recovery of the dark potential. Here we review recent structural insight into the role of the N-terminal myristoylation in GCAPs and compare it to other NCS family members. We discuss previous studies identifying regions of GCAPs important for retGC1 regulation in the context of the new structural data available for myristoylated GCAP1. In addition, we present a hypothetical model for the Ca(2+)-triggered conformational change in GCAPs and retGC1 regulation. Finally, we briefly discuss the involvement of mutant GCAP1 proteins in the etiology of retinal degeneration as well as the importance of other Ca(2+) sensors in the modulation of phototransduction.

Adrenergic Signaling Regulates Mitochondrial Ca2+ Uptake Through Pyk2-Dependent Tyrosine Phosphorylation of the Mitochondrial Ca2+ Uniporter

PubMed Central

Jhun, Bong Sook; Xu, Shangcheng; Hurst, Stephen; Raffaello, Anna; Liu, Xiaoyun; Yi, Bing; Zhang, Huiliang; Gross, Polina; Mishra, Jyotsna; Ainbinder, Alina; Kettlewell, Sarah; Smith, Godfrey L.; Dirksen, Robert T.; Wang, Wang; Rizzuto, Rosario

2014-01-01

Abstract Aims: Mitochondrial Ca2+ homeostasis is crucial for balancing cell survival and death. The recent discovery of the molecular identity of the mitochondrial Ca2+ uniporter pore (MCU) opens new possibilities for applying genetic approaches to study mitochondrial Ca2+ regulation in various cell types, including cardiac myocytes. Basal tyrosine phosphorylation of MCU was reported from mass spectroscopy of human and mouse tissues, but the signaling pathways that regulate mitochondrial Ca2+ entry through posttranslational modifications of MCU are completely unknown. Therefore, we investigated α1-adrenergic-mediated signal transduction of MCU posttranslational modification and function in cardiac cells. Results: α1-adrenoceptor (α1-AR) signaling translocated activated proline-rich tyrosine kinase 2 (Pyk2) from the cytosol to mitochondrial matrix and accelerates mitochondrial Ca2+ uptake via Pyk2-dependent MCU phosphorylation and tetrametric MCU channel pore formation. Moreover, we found that α1-AR stimulation increases reactive oxygen species production at mitochondria, mitochondrial permeability transition pore activity, and initiates apoptotic signaling via Pyk2-dependent MCU activation and mitochondrial Ca2+ overload. Innovation: Our data indicate that inhibition of α1-AR-Pyk2-MCU signaling represents a potential novel therapeutic target to limit or prevent mitochondrial Ca2+ overload, oxidative stress, mitochondrial injury, and myocardial death during pathophysiological conditions, where chronic adrenergic stimulation is present. Conclusion: The α1-AR-Pyk2-dependent tyrosine phosphorylation of the MCU regulates mitochondrial Ca2+ entry and apoptosis in cardiac cells. Antioxid. Redox Signal. 21, 863–879. PMID:24800979

WEREWOLF, a MYB-related protein in Arabidopsis, is a position-dependent regulator of epidermal cell patterning.

PubMed

Lee, M M; Schiefelbein, J

1999-11-24

The formation of the root epidermis of Arabidopsis provides a simple and elegant model for the analysis of cell patterning. A novel gene, WEREWOLF (WER), is described here that is required for position-dependent patterning of the epidermal cell types. The WER gene encodes a MYB-type protein and is preferentially expressed within cells destined to adopt the non-hair fate. Furthermore, WER is shown to regulate the position-dependent expression of the GLABRA2 homeobox gene, to interact with a bHLH protein, and to act in opposition to the CAPRICE MYB. These results suggest a simple model to explain the specification of the two root epidermal cell types, and they provide insight into the molecular mechanisms used to control cell patterning.

Pheromone-Regulated Expression of Sex Pheromone Plasmid pAD1-Encoded Aggregation Substance Depends on at Least Six Upstream Genes and a cis-Acting, Orientation-Dependent Factor

PubMed Central

Muscholl-Silberhorn, Albrecht B.

2000-01-01

Conjugative transfer of Enterococcus faecalis-specific sex pheromone plasmids relies on an adhesin, called aggregation substance, to confer a tight cell-to-cell contact between the mating partners. To analyze the dependence of pAD1-encoded aggregation substance, Asa1, on pheromone induction, a variety of upstream fragments were fused to an α-amylase reporter gene, amyL, by use of a novel promoter probe vector, pAMY-em1. For pheromone-regulated α-amylase activity, a total of at least six genes, traB, traC, traA, traE1, orfY, and orf1, are required: TraB efficiently represses asa1 (by a mechanism unrelated to its presumptive function in pheromone shutdown, since a complete shutdown is observed exclusively in the presence of traC); only traC can relieve traB-mediated repression in a pheromone-dependent manner. In addition to traB, traA is required but not sufficient for negative control. Mutational inactivation of traE1, orfY, or orf1, respectively, results in a total loss of α-amylase activity for constructs normally mediating constitutive expression. Inversion of a fragment covering traA, P0, and traE1 without disrupting any gene or control element switches off amyL or asa1 expression, indicating the involvement of a cis-acting, orientation-dependent factor (as had been shown for plasmid pCF10). Unexpectedly, pAD1 represses all pAMY-em1 derivatives in trans, while its own pheromone-dependent functions are unaffected. The discrepancy between the new data and those of former studies defining TraE1 as a trans-acting positive regulator is discussed. PMID:10850999

Cell cycle-dependent regulation of Aurora kinase B mRNA by the Microprocessor complex.

PubMed

Jung, Eunsun; Seong, Youngmo; Seo, Jae Hong; Kwon, Young-Soo; Song, Hoseok

2014-03-28

Aurora kinase B regulates the segregation of chromosomes and the spindle checkpoint during mitosis. In this study, we showed that the Microprocessor complex, which is responsible for the processing of the primary transcripts during the generation of microRNAs, destabilizes the mRNA of Aurora kinase B in human cells. The Microprocessor-mediated cleavage kept Aurora kinase B at a low level and prevented premature entrance into mitosis. The cleavage was reduced during mitosis leading to the accumulation of Aurora kinase B mRNA and protein. In addition to Aurora kinase B mRNA, the processing of other primary transcripts of miRNAs were also decreased during mitosis. We found that the cleavage was dependent on an RNA helicase, DDX5, and the association of DDX5 and DDX17 with the Microprocessor was reduced during mitosis. Thus, we propose a novel mechanism by which the Microprocessor complex regulates stability of Aurora kinase B mRNA and cell cycle progression. Copyright © 2014 Elsevier Inc. All rights reserved.

Inhibition of NFκB and Pancreatic Cancer Cell and Tumor Growth by Curcumin Is Dependent on Specificity Protein Down-regulation*

PubMed Central

Jutooru, Indira; Chadalapaka, Gayathri; Lei, Ping; Safe, Stephen

2010-01-01

Curcumin activates diverse anticancer activities that lead to inhibition of cancer cell and tumor growth, induction of apoptosis, and antiangiogenic responses. In this study, we observed that curcumin inhibits Panc28 and L3.6pL pancreatic cancer cell and tumor growth in nude mice bearing L3.6pL cells as xenografts. In addition, curcumin decreased expression of p50 and p65 proteins and NFκB-dependent transactivation and also decreased Sp1, Sp3, and Sp4 transcription factors that are overexpressed in pancreatic cancer cells. Because both Sp transcription factors and NFκB regulate several common genes such as cyclin D1, survivin, and vascular endothelial growth factor that contribute to the cancer phenotype, we also investigated interactions between Sp and NFκB transcription factors. Results of Sp1, Sp3, and Sp4 knockdown by RNA interference demonstrate that both p50 and p65 are Sp-regulated genes and that inhibition of constitutive or tumor necrosis factor-induced NFκB by curcumin is dependent on down-regulation of Sp1, Sp3, and Sp4 proteins by this compound. Curcumin also decreased mitochondrial membrane potential and induced reactive oxygen species in pancreatic cancer cells, and this pathway is required for down-regulation of Sp proteins in these cells, demonstrating that the mitochondriotoxic effects of curcumin are important for its anticancer activities. PMID:20538607

Calcium Co-regulates Oxidative Metabolism and ATP Synthase-dependent Respiration in Pancreatic Beta Cells

PubMed Central

De Marchi, Umberto; Thevenet, Jonathan; Hermant, Aurelie; Dioum, Elhadji; Wiederkehr, Andreas

2014-01-01

Mitochondrial energy metabolism is essential for glucose-induced calcium signaling and, therefore, insulin granule exocytosis in pancreatic beta cells. Calcium signals are sensed by mitochondria acting in concert with mitochondrial substrates for the full activation of the organelle. Here we have studied glucose-induced calcium signaling and energy metabolism in INS-1E insulinoma cells and human islet beta cells. In insulin secreting cells a surprisingly large fraction of total respiration under resting conditions is ATP synthase-independent. We observe that ATP synthase-dependent respiration is markedly increased after glucose stimulation. Glucose also causes a very rapid elevation of oxidative metabolism as was followed by NAD(P)H autofluorescence. However, neither the rate of the glucose-induced increase nor the new steady-state NAD(P)H levels are significantly affected by calcium. Our findings challenge the current view, which has focused mainly on calcium-sensitive dehydrogenases as the target for the activation of mitochondrial energy metabolism. We propose a model of tight calcium-dependent regulation of oxidative metabolism and ATP synthase-dependent respiration in beta cell mitochondria. Coordinated activation of matrix dehydrogenases and respiratory chain activity by calcium allows the respiratory rate to change severalfold with only small or no alterations of the NAD(P)H/NAD(P)+ ratio. PMID:24554722

Leptin Regulates Amyloid β Production Via the γ-Secretase Complex

PubMed Central

Niedowicz, Dana M.; Studzinski, Christa M.; Weidner, Adam M.; Platt, Thomas L.; Kingry, Kristen N.; Beckett, Tina L.; Bruce-Keller, Annadora J.; Keller, Jeffrey N.; Murphy, M. Paul

2013-01-01

Alzheimer’s Disease (AD) is the most common age-related neurodegenerative disease, affecting an estimated 5.3 million people in the United States. While many factors likely contribute to AD progression, it is widely accepted that AD is driven by the accumulation of β-amyloid (Aβ), a small, fibrillogenic peptide generated by the sequential proteolysis of the amyloid precursor protein by the β- and γ-secretases. Though the underlying causes of Aβ accumulation in sporadic AD are myriad, it is clear that lifestyle and overall health play a significant role. The adipocyte-derived hormone leptin has varied systemic affects, including neuropeptide release and neuroprotection. A recent study by Lieb et al (2009) showed that individuals with low plasma leptin levels are at greater risk of developing AD, through unknown mechanisms. In this report, we show that plasma leptin is a strong negative predictor of Aβ levels in the mouse brain, supporting a protective role for the hormone in AD onset. We also show that the inhibition of Aβ accumulation is due to the downregulation of transcription of the γ-secretase components. On the other hand, β-secretase expression is either unchanged (BACE1) or increased (BACE2). Finally, we show that only presenilin 1 (PS1) is negatively correlated with plasma leptin at the protein level (p<0.0001). These data are intriguing and may highlight a role for leptin in regulating the onset of amyloid pathology and AD. PMID:23274884

Leptin regulates amyloid β production via the γ-secretase complex.

PubMed

Niedowicz, Dana M; Studzinski, Christa M; Weidner, Adam M; Platt, Thomas L; Kingry, Kristen N; Beckett, Tina L; Bruce-Keller, Annadora J; Keller, Jeffrey N; Murphy, M Paul

2013-03-01

Alzheimer's disease (AD) is the most common age-related neurodegenerative disease, affecting an estimated 5.3million people in the United States. While many factors likely contribute to AD progression, it is widely accepted that AD is driven by the accumulation of β-amyloid (Aβ), a small, fibrillogenic peptide generated by the sequential proteolysis of the amyloid precursor protein by the β- and γ-secretases. Though the underlying causes of Aβ accumulation in sporadic AD are myriad, it is clear that lifestyle and overall health play a significant role. The adipocyte-derived hormone leptin has varied systemic affects, including neuropeptide release and neuroprotection. A recent study by Lieb et al. (2009) showed that individuals with low plasma leptin levels are at greater risk of developing AD, through unknown mechanisms. In this report, we show that plasma leptin is a strong negative predictor of Aβ levels in the mouse brain, supporting a protective role for the hormone in AD onset. We also show that the inhibition of Aβ accumulation is due to the downregulation of transcription of the γ-secretase components. On the other hand, β-secretase expression is either unchanged (BACE1) or increased (BACE2). Finally, we show that only presenilin 1 (PS1) is negatively correlated with plasma leptin at the protein level (p<0.0001). These data are intriguing and may highlight a role for leptin in regulating the onset of amyloid pathology and AD. Copyright © 2013 Elsevier B.V. All rights reserved.

Palmitoylation-dependent CDKL5–PSD-95 interaction regulates synaptic targeting of CDKL5 and dendritic spine development

PubMed Central

Zhu, Yong-Chuan; Li, Dan; Wang, Lu; Lu, Bin; Zheng, Jing; Zhao, Shi-Lin; Zeng, Rong; Xiong, Zhi-Qi

2013-01-01

The X-linked gene cyclin-dependent kinase-like 5 (CDKL5) is mutated in severe neurodevelopmental disorders, including some forms of atypical Rett syndrome, but the function and regulation of CDKL5 protein in neurons remain to be elucidated. Here, we show that CDKL5 binds to the scaffolding protein postsynaptic density (PSD)-95, and that this binding promotes the targeting of CDKL5 to excitatory synapses. Interestingly, this binding is not constitutive, but governed by palmitate cycling on PSD-95. Furthermore, pathogenic mutations that truncate the C-terminal tail of CDKL5 diminish its binding to PSD-95 and synaptic accumulation. Importantly, down-regulation of CDKL5 by RNA interference (RNAi) or interference with the CDKL5–PSD-95 interaction inhibits dendritic spine formation and growth. These results demonstrate a critical role of the palmitoylation-dependent CDKL5–PSD-95 interaction in localizing CDKL5 to synapses for normal spine development and suggest that disruption of this interaction by pathogenic mutations may be implicated in the pathogenesis of CDKL5-related disorders. PMID:23671101

SARS coronavirus papain-like protease induces Egr-1-dependent up-regulation of TGF-β1 via ROS/p38 MAPK/STAT3 pathway.

PubMed

Li, Shih-Wein; Wang, Ching-Ying; Jou, Yu-Jen; Yang, Tsuey-Ching; Huang, Su-Hua; Wan, Lei; Lin, Ying-Ju; Lin, Cheng-Wen

2016-05-13

SARS coronavirus (SARS-CoV) papain-like protease (PLpro) has been identified in TGF-β1 up-regulation in human promonocytes (Proteomics 2012, 12: 3193-205). This study investigates the mechanisms of SARS-CoV PLpro-induced TGF-β1 promoter activation in human lung epithelial cells and mouse models. SARS-CoV PLpro dose- and time-dependently up-regulates TGF-β1 and vimentin in A549 cells. Dual luciferase reporter assays with TGF-β1 promoter plasmids indicated that TGF-β1 promoter region between -175 to -60, the Egr-1 binding site, was responsible for TGF-β1 promoter activation induced by SARS-CoV PLpro. Subcellular localization analysis of transcription factors showed PLpro triggering nuclear translocation of Egr-1, but not NF-κB and Sp-1. Meanwhile, Egr-1 silencing by siRNA significantly reduced PLpro-induced up-regulation of TGF-β1, TSP-1 and pro-fibrotic genes. Furthermore, the inhibitors for ROS (YCG063), p38 MAPK (SB203580), and STAT3 (Stattic) revealed ROS/p38 MAPK/STAT3 pathway involving in Egr-1 dependent activation of TGF-β1 promoter induced by PLpro. In a mouse model with a direct pulmonary injection, PLpro stimulated macrophage infiltration into lung, up-regulating Egr-1, TSP-1, TGF-β1 and vimentin expression in lung tissues. The results revealed that SARS-CoV PLpro significantly triggered Egr-1 dependent activation of TGF-β1 promoter via ROS/p38 MAPK/STAT3 pathway, correlating with up-regulation of pro-fibrotic responses in vitro and in vivo.

The phosphorylation-dependent regulation of nuclear SREBP1 during mitosis links lipid metabolism and cell growth

PubMed Central

Bengoechea-Alonso, Maria Teresa; Ericsson, Johan

2016-01-01

ABSTRACT The SREBP transcription factors are major regulators of lipid metabolism. Disturbances in lipid metabolism are at the core of several health issues facing modern society, including cardiovascular disease, obesity and diabetes. In addition, the role of lipid metabolism in cancer cell growth is receiving increased attention. Transcriptionally active SREBP molecules are unstable and rapidly degraded in a phosphorylation-dependent manner by Fbw7, a ubiquitin ligase that targets several cell cycle regulatory proteins for degradation. We have previously demonstrated that active SREBP1 is stabilized during mitosis. We have now delineated the mechanisms involved in the stabilization of SREBP1 in mitotic cells. This process is initiated by the phosphorylation of a specific serine residue in nuclear SREBP1 by the mitotic kinase Cdk1. The phosphorylation of this residue creates a docking site for a separate mitotic kinase, Plk1. Plk1 interacts with nuclear SREBP1 in mitotic cells and phosphorylates a number of residues in the C-terminal domain of the protein, including a threonine residue in close proximity of the Fbw7 docking site in SREBP1. The phosphorylation of these residues by Plk1 blocks the interaction between SREBP1 and Fbw7 and attenuates the Fbw7-dependent degradation of nuclear SREBP1 during cell division. Inactivation of SREBP1 results in a mitotic defect, suggesting that SREBP1 could regulate cell division. We propose that the mitotic phosphorylation and stabilization of nuclear SREBP1 during cell division provides a link between lipid metabolism and cell proliferation. Thus, the current study provides additional support for the emerging hypothesis that SREBP-dependent lipid metabolism may be important for cell growth. PMID:27579997

Estrogen receptor alpha is cell cycle-regulated and regulates the cell cycle in a ligand-dependent fashion

PubMed Central

JavanMoghadam, Sonia; Weihua, Zhang; Hunt, Kelly K.; Keyomarsi, Khandan

2016-01-01

ABSTRACT Estrogen receptor alpha (ERα) has been implicated in several cell cycle regulatory events and is an important predictive marker of disease outcome in breast cancer patients. Here, we aimed to elucidate the mechanism through which ERα influences proliferation in breast cancer cells. Our results show that ERα protein is cell cycle-regulated in human breast cancer cells and that the presence of 17-β-estradiol (E2) in the culture medium shortened the cell cycle significantly (by 4.5 hours, P < 0.05) compared with unliganded conditions. The alterations in cell cycle duration were observed in the S and G2/M phases, whereas the G1 phase was indistinguishable under liganded and unliganded conditions. In addition, ERα knockdown in MCF-7 cells accelerated mitotic exit, whereas transfection of ERα-negative MDA-MB-231 cells with exogenous ERα significantly shortened the S and G2/M phases (by 9.1 hours, P < 0.05) compared with parental cells. Finally, treatment of MCF-7 cells with antiestrogens revealed that tamoxifen yields a slower cell cycle progression through the S and G2/M phases than fulvestrant does, presumably because of the destabilizing effect of fulvestrant on ERα protein. Together, these results show that ERα modulates breast cancer cell proliferation by regulating events during the S and G2/M phases of the cell cycle in a ligand-dependent fashion. These results provide the rationale for an effective treatment strategy that includes a cell cycle inhibitor in combination with a drug that lowers estrogen levels, such as an aromatase inhibitor, and an antiestrogen that does not result in the degradation of ERα, such as tamoxifen. PMID:27049344

Estrogen receptor alpha is cell cycle-regulated and regulates the cell cycle in a ligand-dependent fashion.

PubMed

JavanMoghadam, Sonia; Weihua, Zhang; Hunt, Kelly K; Keyomarsi, Khandan

2016-06-17

Estrogen receptor alpha (ERα) has been implicated in several cell cycle regulatory events and is an important predictive marker of disease outcome in breast cancer patients. Here, we aimed to elucidate the mechanism through which ERα influences proliferation in breast cancer cells. Our results show that ERα protein is cell cycle-regulated in human breast cancer cells and that the presence of 17-β-estradiol (E2) in the culture medium shortened the cell cycle significantly (by 4.5 hours, P < 0.05) compared with unliganded conditions. The alterations in cell cycle duration were observed in the S and G2/M phases, whereas the G1 phase was indistinguishable under liganded and unliganded conditions. In addition, ERα knockdown in MCF-7 cells accelerated mitotic exit, whereas transfection of ERα-negative MDA-MB-231 cells with exogenous ERα significantly shortened the S and G2/M phases (by 9.1 hours, P < 0.05) compared with parental cells. Finally, treatment of MCF-7 cells with antiestrogens revealed that tamoxifen yields a slower cell cycle progression through the S and G2/M phases than fulvestrant does, presumably because of the destabilizing effect of fulvestrant on ERα protein. Together, these results show that ERα modulates breast cancer cell proliferation by regulating events during the S and G2/M phases of the cell cycle in a ligand-dependent fashion. These results provide the rationale for an effective treatment strategy that includes a cell cycle inhibitor in combination with a drug that lowers estrogen levels, such as an aromatase inhibitor, and an antiestrogen that does not result in the degradation of ERα, such as tamoxifen.

A Ca2+ channel differentially regulates Clathrin-mediated and activity-dependent bulk endocytosis.

PubMed

Yao, Chi-Kuang; Liu, Yu-Tzu; Lee, I-Chi; Wang, You-Tung; Wu, Ping-Yen

2017-04-01

Clathrin-mediated endocytosis (CME) and activity-dependent bulk endocytosis (ADBE) are two predominant forms of synaptic vesicle (SV) endocytosis, elicited by moderate and strong stimuli, respectively. They are tightly coupled with exocytosis for sustained neurotransmission. However, the underlying mechanisms are ill defined. We previously reported that the Flower (Fwe) Ca2+ channel present in SVs is incorporated into the periactive zone upon SV fusion, where it triggers CME, thus coupling exocytosis to CME. Here, we show that Fwe also promotes ADBE. Intriguingly, the effects of Fwe on CME and ADBE depend on the strength of the stimulus. Upon mild stimulation, Fwe controls CME independently of Ca2+ channeling. However, upon strong stimulation, Fwe triggers a Ca2+ influx that initiates ADBE. Moreover, knockout of rodent fwe in cultured rat hippocampal neurons impairs but does not completely abolish CME, similar to the loss of Drosophila fwe at the neuromuscular junction, suggesting that Fwe plays a regulatory role in regulating CME across species. In addition, the function of Fwe in ADBE is conserved at mammalian central synapses. Hence, Fwe exerts different effects in response to different stimulus strengths to control two major modes of endocytosis.

A Ca2+ channel differentially regulates Clathrin-mediated and activity-dependent bulk endocytosis

PubMed Central

Liu, Yu-Tzu; Lee, I-Chi; Wang, You-Tung; Wu, Ping-Yen

2017-01-01

Clathrin-mediated endocytosis (CME) and activity-dependent bulk endocytosis (ADBE) are two predominant forms of synaptic vesicle (SV) endocytosis, elicited by moderate and strong stimuli, respectively. They are tightly coupled with exocytosis for sustained neurotransmission. However, the underlying mechanisms are ill defined. We previously reported that the Flower (Fwe) Ca2+ channel present in SVs is incorporated into the periactive zone upon SV fusion, where it triggers CME, thus coupling exocytosis to CME. Here, we show that Fwe also promotes ADBE. Intriguingly, the effects of Fwe on CME and ADBE depend on the strength of the stimulus. Upon mild stimulation, Fwe controls CME independently of Ca2+ channeling. However, upon strong stimulation, Fwe triggers a Ca2+ influx that initiates ADBE. Moreover, knockout of rodent fwe in cultured rat hippocampal neurons impairs but does not completely abolish CME, similar to the loss of Drosophila fwe at the neuromuscular junction, suggesting that Fwe plays a regulatory role in regulating CME across species. In addition, the function of Fwe in ADBE is conserved at mammalian central synapses. Hence, Fwe exerts different effects in response to different stimulus strengths to control two major modes of endocytosis. PMID:28414717

GLUT-5 expression in neonatal rats: crypt-villus location and age-dependent regulation.

PubMed

Jiang, L; David, E S; Espina, N; Ferraris, R P

2001-09-01

The rat fructose transporter normally appears after completion of weaning but can be precociously induced by early feeding of a high-fructose diet. In this study, the crypt-villus site, the metabolic nature of the signal, and the age dependence of induction were determined. In weaning rats fed high-glucose pellets, GLUT-5 mRNA expression was modest, localized mainly in the upper three-fourths of the villus, and there was little expression in the villus base. When fed high-fructose pellets, GLUT-5 mRNA expression was two to three times greater in all regions except the villus base. Intestinal perfusion in vivo of a nonmetabolizable fructose analog, 3-O-methylfructose, tended to increase fructose uptake rate and moderately increased GLUT-5 mRNA abundance but had no effect on glucose uptake rates and SGLT1 mRNA abundance. Gavage feeding of high-fructose, but not high-glucose, solutions enhanced fructose uptake only in pups > or =14 days, suggesting that GLUT-5 regulation is markedly age dependent. Fructose or its metabolites upregulate GLUT-5 expression in all enterocytes, except those in the crypt and villus base and in pups <14 days old.

Nutrient-dependent phosphorylation channels lipid synthesis to regulate PPARα

PubMed Central

Jensen-Urstad, Anne P. L.; Song, Haowei; Lodhi, Irfan J.; Funai, Katsuhiko; Yin, Li; Coleman, Trey; Semenkovich, Clay F.

2013-01-01

Peroxisome proliferator-activated receptor (PPAR)α is a nuclear receptor that coordinates liver metabolism during fasting. Fatty acid synthase (FAS) is an enzyme that stores excess calories as fat during feeding, but it also activates hepatic PPARα by promoting synthesis of an endogenous ligand. Here we show that the mechanism underlying this paradoxical relationship involves the differential regulation of FAS in at least two distinct subcellular pools: cytoplasmic and membrane-associated. In mouse liver and cultured hepatoma cells, the ratio of cytoplasmic to membrane FAS-specific activity was increased with fasting, indicating higher cytoplasmic FAS activity under conditions associated with PPARα activation. This effect was due to a nutrient-dependent and compartment-selective covalent modification of FAS. Cytoplasmic FAS was preferentially phosphorylated during feeding or insulin treatment at Thr-1029 and Thr-1033, which flank a dehydratase domain catalytic residue. Mutating these sites to alanines promoted PPARα target gene expression. Rapamycin-induced inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1), a mediator of the feeding/insulin signal to induce lipogenesis, reduced FAS phosphorylation, increased cytoplasmic FAS enzyme activity, and increased PPARα target gene expression. Rapamycin-mediated induction of the same gene was abrogated with FAS knockdown. These findings suggest that hepatic FAS channels lipid synthesis through specific subcellular compartments that allow differential gene expression based on nutritional status. PMID:23585690

Pro‐migratory and TGF‐β‐activating functions of αvβ6 integrin in pancreatic cancer are differentially regulated via an Eps8‐dependent GTPase switch

PubMed Central

Tod, Jo; Hanley, Christopher J; Morgan, Mark R; Rucka, Marta; Mellows, Toby; Lopez, Maria‐Antoinette; Kiely, Philip; Moutasim, Karwan A; Frampton, Steven J; Sabnis, Durgagauri; Fine, David R; Johnson, Colin; Marshall, John F; Scita, Giorgio; Jenei, Veronika

2017-01-01

Abstract The integrin αvβ6 is up‐regulated in numerous carcinomas, where expression commonly correlates with poor prognosis. αvβ6 promotes tumour invasion, partly through regulation of proteases and cell migration, and is also the principal mechanism by which epithelial cells activate TGF‐β1; this latter function complicates therapeutic targeting of αvβ6, since TGF‐β1 has both tumour‐promoting and ‐suppressive effects. It is unclear how these different αvβ6 functions are linked; both require actin cytoskeletal reorganization, and it is suggested that tractive forces generated during cell migration activate TGF‐β1 by exerting mechanical tension on the ECM‐bound latent complex. We examined the functional relationship between cell invasion and TGF‐β1 activation in pancreatic ductal adenocarcinoma (PDAC) cells, and confirmed that both processes are αvβ6‐dependent. Surprisingly, we found that cellular functions could be biased towards either motility or TGF‐β1 activation depending on the presence or absence of epidermal growth factor receptor pathway substrate 8 (Eps8), a regulator of actin remodelling, endocytosis, and GTPase activation. Similar to αvβ6, we found that Eps8 was up‐regulated in >70% of PDACs. In complex with Abi1/Sos1, Eps8 regulated αvβ6‐dependent cell migration through activation of Rac1. Down‐regulation of Eps8, Sos1 or Rac1 suppressed cell movement, while simultaneously increasing αvβ6‐dependent TGF‐β1 activation. This latter effect was modulated through increased cell tension, regulated by Rho activation. Thus, the Eps8/Abi1/Sos1 tricomplex acts as a key molecular switch altering the balance between Rac1 and Rho activation; its presence or absence in PDAC cells modulates αvβ6‐dependent functions, resulting in a pro‐migratory (Rac1‐dependent) or a pro‐TGF‐β1 activation (Rho‐dependent) functional phenotype, respectively. © 2017 The Authors. The Journal of Pathology published by John

Role of CBS and Bateman Domains in Phosphorylation-Dependent Regulation of a CLC Anion Channel.

PubMed

Yamada, Toshiki; Krzeminski, Mickael; Bozoky, Zoltan; Forman-Kay, Julie D; Strange, Kevin

2016-11-01

Eukaryotic CLC anion channels and transporters are homodimeric proteins composed of multiple α-helical membrane domains and large cytoplasmic C-termini containing two cystathionine-β-synthase domains (CBS1 and CBS2) that dimerize to form a Bateman domain. The Bateman domains of adjacent CLC subunits interact to form a Bateman domain dimer. The functions of CLC CBS and Bateman domains are poorly understood. We utilized the Caenorhabditis elegans CLC-1/2/Ka/Kb anion channel homolog CLH-3b to characterize the regulatory roles of CLC cytoplasmic domains. CLH-3b activity is reduced by phosphorylation or deletion of a 14-amino-acid activation domain (AD) located on the linker connecting CBS1 and CBS2. We demonstrate here that phosphorylation-dependent reductions in channel activity require an intact Bateman domain dimer and concomitant phosphorylation or deletion of both ADs. Regulation of a CLH-3b AD deletion mutant is reconstituted by intracellular perfusion with recombinant 14-amino-acid AD peptides. The sulfhydryl reactive reagent 2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET) alters in a phosphorylation-dependent manner the activity of channels containing single cysteine residues that are engineered into the short intracellular loop connecting membrane α-helices H and I (H-I loop), the AD, CBS1, and CBS2. In contrast, MTSET has no effect on channels in which cysteine residues are engineered into intracellular regions that are dispensable for regulation. These studies together with our previous work suggest that binding and unbinding of the AD to the Bateman domain dimer induces conformational changes that are transduced to channel membrane domains via the H-I loop. Our findings provide new, to our knowledge, insights into the roles of CLC Bateman domains and the structure-function relationships that govern the regulation of CLC protein activity by diverse ligands and signaling pathways. Copyright © 2016 Biophysical Society. Published by Elsevier

Hydroxyoctadecadienoic acids regulate apoptosis in human THP-1 cells in a PPARγ-dependent manner.

PubMed

Vangaveti, Venkat N; Shashidhar, Venkatesh M; Rush, Catherine; Malabu, Usman H; Rasalam, Roy R; Collier, Fiona; Baune, Bernhard T; Kennedy, Richard L

2014-12-01

Macrophage apoptosis, a key process in atherogenesis, is regulated by oxidation products, including hydroxyoctadecadienoic acids (HODEs). These stable oxidation products of linoleic acid (LA) are abundant in atherosclerotic plaque and activate PPARγ and GPR132. We investigated the mechanisms through which HODEs regulate apoptosis. The effect of HODEs on THP-1 monocytes and adherent THP-1 cells were compared with other C18 fatty acids, LA and α-linolenic acid (ALA). The number of cells was reduced within 24 hours following treatment with 9-HODE (p < 0.01, 30 μM) and 13 HODE (p < 0.01, 30 μM), and the equivalent cell viability was also decreased (p < 0.001). Both 9-HODE and 13-HODE (but not LA or ALA) markedly increased caspase-3/7 activity (p < 0.001) in both monocytes and adherent THP-1 cells, with 9-HODE the more potent. In addition, 9-HODE and 13-HODE both increased Annexin-V labelling of cells (p < 0.001). There was no effect of LA, ALA, or the PPARγ agonist rosiglitazone (1 μM), but the effect of HODEs was replicated with apoptosis-inducer camptothecin (10 μM). Only 9-HODE increased DNA fragmentation. The pro-apoptotic effect of HODEs was blocked by the caspase inhibitor DEVD-CHO. The PPARγ antagonist T0070907 further increased apoptosis, suggestive of the PPARγ-regulated apoptotic effects induced by 9-HODE. The use of siRNA for GPR132 showed no evidence that the effect of HODEs was mediated through this receptor. 9-HODE and 13-HODE are potent--and specific--regulators of apoptosis in THP-1 cells. Their action is PPARγ-dependent and independent of GPR132. Further studies to identify the signalling pathways through which HODEs increase apoptosis in macrophages may reveal novel therapeutic targets for atherosclerosis.

Thioredoxin and NADPH-Dependent Thioredoxin Reductase C Regulation of Tetrapyrrole Biosynthesis.

PubMed

Da, Qingen; Wang, Peng; Wang, Menglong; Sun, Ting; Jin, Honglei; Liu, Bing; Wang, Jinfa; Grimm, Bernhard; Wang, Hong-Bin

2017-10-01

In chloroplasts, thioredoxin (TRX) isoforms and NADPH-dependent thioredoxin reductase C (NTRC) act as redox regulatory factors involved in multiple plastid biogenesis and metabolic processes. To date, less is known about the functional coordination between TRXs and NTRC in chlorophyll biosynthesis. In this study, we aimed to explore the potential functions of TRX m and NTRC in the regulation of the tetrapyrrole biosynthesis (TBS) pathway. Silencing of three genes, TRX m1 , TRX m2 , and TRX m4 ( TRX ms ), led to pale-green leaves, a significantly reduced 5-aminolevulinic acid (ALA)-synthesizing capacity, and reduced accumulation of chlorophyll and its metabolic intermediates in Arabidopsis ( Arabidopsis thaliana ). The contents of ALA dehydratase, protoporphyrinogen IX oxidase, the I subunit of Mg-chelatase, Mg-protoporphyrin IX methyltransferase (CHLM), and NADPH-protochlorophyllide oxidoreductase were decreased in triple TRX m- silenced seedlings compared with the wild type, although the transcript levels of the corresponding genes were not altered significantly. Protein-protein interaction analyses revealed a physical interaction between the TRX m isoforms and CHLM. 4-Acetoamido-4-maleimidylstilbene-2,2-disulfonate labeling showed the regulatory impact of TRX ms on the CHLM redox status. Since CHLM also is regulated by NTRC (Richter et al., 2013), we assessed the concurrent functions of TRX m and NTRC in the control of CHLM. Combined deficiencies of three TRX m isoforms and NTRC led to a cumulative decrease in leaf pigmentation, TBS intermediate contents, ALA synthesis rate, and CHLM activity. We discuss the coordinated roles of TRX m and NTRC in the redox control of CHLM stability with its corollary activity in the TBS pathway. © 2017 American Society of Plant Biologists. All Rights Reserved.

Nerve-dependent factors regulating transcript levels of glycogen phosphorylase in skeletal muscle.

PubMed

Matthews, C C; Carlsen, R C; Froman, B; Tait, R; Gorin, F

1998-06-01

.40% (w/v) VIN for 1 hr reduced axonal transport and decreased MGP transcript levels by 50% within 7 days, but also reduced sensory and motor nerve conduction velocities and depressed TA contractile function. 6. Myogenin, a member of a family of regulatory factors shown to influence the transcription of many muscle genes, including MGP, was used as a molecular marker for muscle inactivity. Myogenin transcript levels were increased following denervation and after treatment with TTX or 0.40% VIN but not after treatment with 0.10% VIN. 7. The results suggest that MGP transcript levels in TA are regulated predominantly by muscle activity, rather than by the delivery of neurotrophic factors. Intrinsic myogenic factors, however, also play a role in MGP expression, since denervation did not reduce MGP transcript levels below 30% of control TA. The dominant influence of activity in the regulation of MGP contrasts with the proposed regulation of oxidative enzyme expression, which appears to depend on both activity and trophic factor influences.

Protein Phosphatase 2A (PP2A)-specific Ubiquitin Ligase MID1 Is a Sequence-dependent Regulator of Translation Efficiency Controlling 3-Phosphoinositide-dependent Protein Kinase-1 (PDPK-1)*

PubMed Central

Aranda-Orgillés, Beatriz; Rutschow, Désirée; Zeller, Raphael; Karagiannidis, Antonios I.; Köhler, Andrea; Chen, Changwei; Wilson, Timothy; Krause, Sven; Roepcke, Stefan; Lilley, David; Schneider, Rainer; Schweiger, Susann

2011-01-01

We have shown previously that the ubiquitin ligase MID1, mutations of which cause the midline malformation Opitz BBB/G syndrome (OS), serves as scaffold for a microtubule-associated protein complex that regulates protein phosphatase 2A (PP2A) activity in a ubiquitin-dependent manner. Here, we show that the MID1 protein complex associates with mRNAs via a purine-rich sequence motif called MIDAS (MID1 association sequence) and thereby increases stability and translational efficiency of these mRNAs. Strikingly, inclusion of multiple copies of the MIDAS motif into mammalian mRNAs increases production of the encoded proteins up to 20-fold. Mutated MID1, as found in OS patients, loses its influence on MIDAS-containing mRNAs, suggesting that the malformations in OS patients could be caused by failures in the regulation of cytoskeleton-bound protein translation. This is supported by the observation that the majority of mRNAs that carry MIDAS motifs is involved in developmental processes and/or energy homeostasis. Further analysis of one of the proteins encoded by a MIDAS-containing mRNA, namely PDPK-1 (3-phosphoinositide dependent protein kinase-1), which is an important regulator of mammalian target of rapamycin/PP2A signaling, showed that PDPK-1 protein synthesis is significantly reduced in cells from an OS patient compared with an age-matched control and can be rescued by functional MID1. Together, our data uncover a novel messenger ribonucleoprotein complex that regulates microtubule-associated protein translation. They suggest a novel mechanism underlying OS and point at an enormous potential of the MIDAS motif to increase the efficiency of biotechnological protein production in mammalian cells. PMID:21930711

SARS coronavirus papain-like protease induces Egr-1-dependent up-regulation of TGF-β1 via ROS/p38 MAPK/STAT3 pathway

PubMed Central

Li, Shih-Wein; Wang, Ching-Ying; Jou, Yu-Jen; Yang, Tsuey-Ching; Huang, Su-Hua; Wan, Lei; Lin, Ying-Ju; Lin, Cheng-Wen

2016-01-01

SARS coronavirus (SARS-CoV) papain-like protease (PLpro) has been identified in TGF-β1 up-regulation in human promonocytes (Proteomics 2012, 12: 3193-205). This study investigates the mechanisms of SARS-CoV PLpro-induced TGF-β1 promoter activation in human lung epithelial cells and mouse models. SARS-CoV PLpro dose- and time-dependently up-regulates TGF-β1 and vimentin in A549 cells. Dual luciferase reporter assays with TGF-β1 promoter plasmids indicated that TGF-β1 promoter region between −175 to −60, the Egr-1 binding site, was responsible for TGF-β1 promoter activation induced by SARS-CoV PLpro. Subcellular localization analysis of transcription factors showed PLpro triggering nuclear translocation of Egr-1, but not NF-κB and Sp-1. Meanwhile, Egr-1 silencing by siRNA significantly reduced PLpro-induced up-regulation of TGF-β1, TSP-1 and pro-fibrotic genes. Furthermore, the inhibitors for ROS (YCG063), p38 MAPK (SB203580), and STAT3 (Stattic) revealed ROS/p38 MAPK/STAT3 pathway involving in Egr-1 dependent activation of TGF-β1 promoter induced by PLpro. In a mouse model with a direct pulmonary injection, PLpro stimulated macrophage infiltration into lung, up-regulating Egr-1, TSP-1, TGF-β1 and vimentin expression in lung tissues. The results revealed that SARS-CoV PLpro significantly triggered Egr-1 dependent activation of TGF-β1 promoter via ROS/p38 MAPK/STAT3 pathway, correlating with up-regulation of pro-fibrotic responses in vitro and in vivo. PMID:27173006

Aberrant Subcellular Neuronal Calcium Regulation in Aging and Alzheimer’s Disease

PubMed Central

Camandola, Simonetta; Mattson, Mark P.

2010-01-01

In this mini-review/opinion article we describe evidence that multiple cellular and molecular alterations in Alzheimer’s disease (AD) pathogenesis involve perturbed cellular calcium regulation, and that alterations in synaptic calcium handling may be early and pivotal events in the disease process. With advancing age neurons encounter increased oxidative stress and impaired energy metabolism, which compromise the function of proteins that control membrane excitability and subcellular calcium dynamics. Altered proteolytic cleavage of the β-amyloid precursor protein (APP) in response to the aging process in combination with genetic and environmental factors results in the production and accumulation of neurotoxic forms of amyloid β-peptide (Aβ ). Aβ undergoes a self-aggregation process and concomitantly generates reactive oxygen species that can trigger membrane-associated oxidative stress which, in turn, impairs the functions of ion-motive ATPases and glutamate and glucose transporters thereby rendering neurons vulnerable to excitotoxicity and apoptosis. Mutations in presenilin-1 that cause early-onset AD increase Aβ production, but also result in an abnormal increase in the size of endoplasmic reticulum calcium stores. Some of the events in the neurodegenerative cascade can be counteracted in animal models by manipulations that stabilize neuronal calcium homeostasis including dietary energy restriction, agonists of glucagon-like peptide 1 receptors and drugs that activate mitochondrial potassium channels. Emerging knowledge of the actions of calcium upstream and downstream of Aβ provides opportunities to develop novel preventative and therapeutic interventions for AD. PMID:20950656

Pax2 regulates a fadd-dependent molecular switch that drives tissue fusion during eye development.

PubMed

Viringipurampeer, Ishaq A; Ferreira, Todd; DeMaria, Shannon; Yoon, Jookyung J; Shan, Xianghong; Moosajee, Mariya; Gregory-Evans, Kevin; Ngai, John; Gregory-Evans, Cheryl Y

2012-05-15

Tissue fusion is an essential morphogenetic mechanism in development, playing a fundamental role in developing neural tube, palate and the optic fissure. Disruption of genes associated with the tissue fusion can lead to congenital malformations, such as spina bifida, cleft lip/palate and ocular coloboma. For instance, the Pax2 transcription factor is required for optic fissure closure, although the mechanism of Pax2 action leading to tissue fusion remains elusive. This lack of information defining how transcription factors drive tissue morphogenesis at the cellular level is hampering new treatments options. Through loss- and gain-of-function analysis, we now establish that pax2 in combination with vax2 directly regulate the fas-associated death domain (fadd) gene. In the presence of fadd, cell proliferation is restricted in the developing eye through a caspase-dependent pathway. However, the loss of fadd results in a proliferation defect and concomitant activation of the necroptosis pathway through RIP1/RIP3 activity, leading to an abnormal open fissure. Inhibition of RIP1 with the small molecule drug necrostatin-1 rescues the pax2 eye fusion defect, thereby overcoming the underlying genetic defect. Thus, fadd has an essential physiological function in protecting the developing optic fissure neuroepithelium from RIP3-dependent necroptosis. This study demonstrates the molecular hierarchies that regulate a cellular switch between proliferation and the apoptotic and necroptotic cell death pathways, which in combination drive tissue morphogenesis. Furthermore, our data suggest that future therapeutic strategies may be based on small molecule drugs that can bypass the gene defects causing common congenital tissue fusion defects.

The Neurotrophin-Inducible Gene Vgf Regulates Hippocampal Function and Behavior Through a BDNF-Dependent Mechanism

PubMed Central

Bozdagi, Ozlem; Rich, Erin; Tronel, Sophie; Sadahiro, Masato; Patterson, Kamara; Shapiro, Matthew L.; Alberini, Cristina M.; Huntley, George W.; Salton, Stephen R. J.

2009-01-01

VGF is a neurotrophin-inducible, activity-regulated gene product that is expressed in CNS and PNS neurons, where it is processed into peptides and secreted. VGF synthesis is stimulated by BDNF, a critical regulator of hippocampal development and function, and two VGF C-terminal peptides increase synaptic activity in cultured hippocampal neurons. To assess VGF function in the hippocampus, we tested heterozygous and homozygous VGF knockout mice in two different learning tasks, assessed long-term potentiation (LTP) and depression (LTD) in hippocampal slices from VGF mutant mice, and investigated how VGF C-terminal peptides modulate synaptic plasticity. Treatment of rat hippocampal slices with the VGF-derived peptide TLQP62 resulted in transient potentiation through a mechanism that was selectively blocked by the BDNF scavenger TrkB-Fc, the Trk tyrosine kinase inhibitor K252a (100 nM), and by tPASTOP, an inhibitor of tissue plasminogen activator (tPA), an enzyme involved in pro-BDNF cleavage to BDNF, but was not blocked by the NMDA receptor antagonist APV, anti-p75NTR function-blocking antiserum, nor by prior tetanic stimulation. Although LTP was normal in slices from VGF knockout mice, LTD could not be induced, and VGF mutant mice were impaired in hippocampal-dependent spatial learning and contextual fear conditioning tasks. Our studies indicate that the VGF C-terminal peptide TLQP62 modulates hippocampal synaptic transmission through a BDNF-dependent mechanism, and that VGF deficiency in mice impacts synaptic plasticity and memory in addition to depressive behavior. PMID:18815270

Regulation of expression of Na+,K+-ATPase in androgen-dependent and androgen-independent prostate cancer

PubMed Central

Blok, L J; Chang, G T G; Steenbeek-Slotboom, M; Weerden, W M van; Swarts, H G P; Pont, J J H H M De; Steenbrugge, G J van; Brinkmann, A O

1999-01-01

The β1-subunit of Na+,K+-ATPase was isolated and identified as an androgen down-regulated gene. Expression was observed at high levels in androgen-independent as compared to androgen-dependent (responsive) human prostate cancer cell lines and xenografts when grown in the presence of androgens. Down-regulation of the β1-subunit was initiated at concentrations between 0.01 nM and 0.03 nM of the synthetic androgen R1881 after relatively long incubation times (> 24 h). Using polyclonal antibodies, the concentration of β1-subunit protein, but not of the α1-subunit protein, was markedly reduced in androgen-dependent human prostate cancer cells (LNCaP-FGC) cultured in the presence of androgens. In line with these observations it was found that the protein expression of total Na+,K+-ATPase in the membrane (measured by 3H-ouabain binding) was also markedly decreased. The main function of Na+,K+-ATPase is to maintain sodium and potassium homeostasis in animal cells. The resulting electrochemical gradient is facilitative for transport of several compounds over the cell membrane (for example cisplatin, a chemotherapeutic agent experimentally used in the treatment of hormone-refractory prostate cancer). Here we observed that a ouabain-induced decrease of Na+,K+-ATPase activity in LNCaP-FGC cells results in reduced sensitivity of these cells to cisplatin-treatment. Surprisingly, androgen-induced decrease of Na+,K+-ATPase expression, did not result in significant protection against the chemotherapeutic agent. © 1999 Cancer Research Campaign PMID:10487609

NO-dependent blood pressure regulation in RGS2-deficient mice

PubMed Central

Obst, Michael; Tank, Jens; Plehm, Ralph; Blumer, Kendall J.; Diedrich, Andrè; Jordan, Jens; Luft, Friedrich C.; Gross, Volkmar

2009-01-01

The regulator of G protein signaling (RGS) 2, a GTPase-activating protein, is activated via the nitric oxide (NO)-cGMP pathway and thereby may influence blood pressure regulation. To test that notion, we measured mean arterial blood pressure (MAP) and heart rate (HR) with telemetry in Nω-nitro-L-arginine methyl ester (L-NAME, 5 mg L-NAME/10 ml tap water)-treated RGS2-deficient (RGS2−/−) and RGS2-sufficient (RGS2+/+) mice and assessed autonomic function. Without L-NAME, RGS2−/− mice showed during day and night a similar increase of MAP compared with controls. L-NAME treatment increased MAP in both strains. nNOS is involved in this L-NAME-dependent blood pressure increase, since 7-nitroindazole increased MAP by 8 and 9 mmHg (P < 0.05) in both strains. The L-NAME-induced MAP increase of 14–15 mmHg during night was similar in both strains. However, the L-NAME-induced MAP increase during the day was smaller in RGS2−/− than in RGS2+/+ (11 ± 1 vs. 17 ± 2 mmHg; P < 0.05). Urinary norepinephrine and epinephrine excretion was higher in RGS2−/− than in RGS2+/+ mice. The MAP decrease after prazosin was more pronounced in L-NAME-RGS2−/−. HR variability parameters [root mean square of successive differences (RMSSD), low-frequency (LF) power, and high-frequency (HF) power] and baroreflex sensitivity were increased in RGS2−/−. Atropine and atropine plus metoprolol markedly reduced RMSSD, LF, and HF. Our data suggest an interaction between RGS2 and the NO-cGMP pathway. The blunted L-NAME response in RGS2−/− during the day suggests impaired NO signaling. The MAP increases during the active phase in RGS2−/− mice may be related to central sympathetic activation and increased vascular adrenergic responsiveness. PMID:16269576

The effects of emotion regulation on explicit memory depend on strategy and testing method.

PubMed

Knight, Marisa; Ponzio, Allison

2013-12-01

Although previous work has shown that emotion regulation strategies can influence memory, the mechanisms through which different strategies produce different memory outcomes are not well understood. We examined how two cognitive reappraisal strategies with similar elaboration demands but diverging effects on visual attention and emotional arousal influenced explicit memory for emotional stimuli and for the strategies used to evaluate the stimuli. At encoding, participants used reappraisal to increase and decrease the personal relevance of neutral and emotional pictures. In two experiments, recall accuracy was highest for emotional pictures featured on increase trials, intermediate for emotional pictures featured on look (respond naturally) trials, and lowest for emotional pictures featured on decrease trials. This recall pattern emerged after a short delay (15 min) and persisted over a longer delay (48 hr). Memory accuracy for the strategies used to evaluate the pictures showed a different pattern: Strategy memory was better for emotional pictures featured on decrease and increase trials than for pictures featured on look trials. Our findings show that the effects of emotion regulation on memory depend both on the particular strategy engaged and the particular aspect of memory being tested.

Fatty Acids Regulate Germline Sex Determination through ACS-4-Dependent Myristoylation.

PubMed

Tang, Hongyun; Han, Min

2017-04-20

Fat metabolism has been linked to fertility and reproductive adaptation in animals and humans, and environmental sex determination potentially plays a role in the process. To investigate the impact of fatty acids (FA) on sex determination and reproductive development, we examined and observed an impact of FA synthesis and mobilization by lipolysis in somatic tissues on oocyte fate in Caenorhabditis elegans. The subsequent genetic analysis identified ACS-4, an acyl-CoA synthetase and its FA-CoA product, as key germline factors that mediate the role of FA in promoting oocyte fate through protein myristoylation. Further tests indicated that ACS-4-dependent protein myristoylation perceives and translates the FA level into regulatory cues that modulate the activities of MPK-1/MAPK and key factors in the germline sex-determination pathway. These findings, including a similar role of ACS-4 in a male/female species, uncover a likely conserved mechanism by which FA, an environmental factor, regulates sex determination and reproductive development. Copyright © 2017 Elsevier Inc. All rights reserved.

Plant chimeric Ca2+/Calmodulin-dependent protein kinase. Role of the neural visinin-like domain in regulating autophosphorylation and calmodulin affinity

NASA Technical Reports Server (NTRS)

Sathyanarayanan, P. V.; Cremo, C. R.; Poovaiah, B. W.

2000-01-01

Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) is characterized by a serine-threonine kinase domain, an autoinhibitory domain, a calmodulin-binding domain and a neural visinin-like domain with three EF-hands. The neural visinin-like Ca(2+)-binding domain at the C-terminal end of the CaM-binding domain makes CCaMK unique among all the known calmodulin-dependent kinases. Biological functions of the plant visinin-like proteins or visinin-like domains in plant proteins are not well known. Using EF-hand deletions in the visinin-like domain, we found that the visinin-like domain regulated Ca(2+)-stimulated autophosphorylation of CCaMK. To investigate the effects of Ca(2+)-stimulated autophosphorylation on the interaction with calmodulin, the equilibrium binding constants of CCaMK were measured by fluorescence emission anisotropy using dansylated calmodulin. Binding was 8-fold tighter after Ca(2+)-stimulated autophosphorylation. This shift in affinity did not occur in CCaMK deletion mutants lacking Ca(2+)-stimulated autophosphorylation. A variable calmodulin affinity regulated by Ca(2+)-stimulated autophosphorylation mediated through the visinin-like domain is a new regulatory mechanism for CCaMK activation and calmodulin-dependent protein kinases. Our experiments demonstrate the existence of two functional molecular switches in a protein kinase regulating the kinase activity, namely a visinin-like domain acting as a Ca(2+)-triggered switch and a CaM-binding domain acting as an autophosphorylation-triggered molecular switch.

AtPP2CG1, a protein phosphatase 2C, positively regulates salt tolerance of Arabidopsis in abscisic acid-dependent manner

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

Liu, Xin, E-mail: fangfei6073@126.com; Zhu, Yanming, E-mail: ymzhu2001@neau.edu.cn; Zhai, Hong, E-mail: Zhai.h@neigaehrb.ac.cn

2012-06-15

Highlights: Black-Right-Pointing-Pointer AtPP2CG1 positively regulates salt tolerance in ABA-dependent manner. Black-Right-Pointing-Pointer AtPP2CG1 up-regulates the expression of marker genes in different pathways. Black-Right-Pointing-Pointer AtPP2CG1 expresses in the vascular system and trichomes of Arabidopsis. -- Abstract: AtPP2CG1 (Arabidopsis thaliana protein phosphatase 2C G Group 1) was predicted as an abiotic stress candidate gene by bioinformatic analysis in our previous study. The gene encodes a putative protein phosphatase 2C that belongs to Group G of PP2C. There is no report of Group G genes involved in abiotic stress so far. Real-time RT-PCR analysis showed that AtPP2CG1 expression was induced by salt, drought, andmore » abscisic acid (ABA) treatment. The expression levels of AtPP2CG1 in the ABA synthesis-deficient mutant abi2-3 were much lower than that in WT plants under salt stress suggesting that the expression of AtPP2CG1 acts in an ABA-dependent manner. Over-expression of AtPP2CG1 led to enhanced salt tolerance, whereas its loss of function caused decreased salt tolerance. These results indicate that AtPP2CG1 positively regulates salt stress in an ABA-dependent manner. Under salt treatment, AtPP2CG1 up-regulated the expression levels of stress-responsive genes, including RD29A, RD29B, DREB2A and KIN1. GUS activity was detected in roots, leaves, stems, flower, and trichomes of AtPP2CG1 promoter-GUS transgenic plants. AtPP2CG1 protein was localized in nucleus and cytoplasm via AtPP2CG1:eGFP and YFP:AtPP2CG1 fusion approaches.« less

Exercise dependence score in patients with longstanding eating disorders and controls: the importance of affect regulation and physical activity intensity.

PubMed

Bratland-Sanda, Solfrid; Martinsen, Egil W; Rosenvinge, Jan H; Rø, Oyvind; Hoffart, Asle; Sundgot-Borgen, Jorunn

2011-01-01

To examine associations among exercise dependence score, amount of physical activity and eating disorder (ED) symptoms in patients with longstanding ED and non-clinical controls. Adult female inpatients (n = 59) and 53 age-matched controls participated in this cross sectional study. Assessments included the eating disorders examination, eating disorders inventory, exercise dependence scale, reasons for exercise inventory, and MTI Actigraph accelerometer. Positive associations were found among vigorous, not moderate, physical activity, exercise dependence score and ED symptoms in patients. In the controls, ED symptoms were negatively associated with vigorous physical activity and not correlated with exercise dependence score. Exercise for negative affect regulation, not weight/appearance, and amount of vigorous physical activity were explanatory variables for exercise dependence score in both groups. The positive associations among exercise dependence score, vigorous physical activity and ED symptoms need proper attention in the treatment of longstanding ED. Copyright © 2011 John Wiley & Sons, Ltd and Eating Disorders Association.

Two Components of Voltage-Dependent Inactivation in Cav1.2 Channels Revealed by Its Gating Currents

PubMed Central

Ferreira, Gonzalo; Ríos, Eduardo; Reyes, Nicolás

2003-01-01

Voltage-dependent inactivation (VDI) was studied through its effects on the voltage sensor in Cav1.2 channels expressed in tsA 201 cells. Two kinetically distinct phases of VDI in onset and recovery suggest the presence of dual VDI processes. Upon increasing duration of conditioning depolarizations, the half-distribution potential (V1/2) of intramembranous mobile charge was negatively shifted as a sum of two exponential terms, with time constants 0.5 s and 4 s, and relative amplitudes near 50% each. This kinetics behavior was consistent with that of increment of maximal charge related to inactivation (Qn). Recovery from inactivation was also accompanied by a reduction of Qn that varied with recovery time as a sum of two exponentials. The amplitudes of corresponding exponential terms were strongly correlated in onset and recovery, indicating that channels recover rapidly from fast VDI and slowly from slow VDI. Similar to charge “immobilization,” the charge moved in the repolarization (OFF) transient became slower during onset of fast VDI. Slow VDI had, instead, hallmarks of interconversion of charge. Confirming the mechanistic duality, fast VDI virtually disappeared when Li+ carried the current. A nine-state model with parallel fast and slow inactivation pathways from the open state reproduces most of the observations. PMID:12770874

Regulation of replicative senescence by NADP+ -dependent isocitrate dehydrogenase.

PubMed

Kil, In Sup; Huh, Tae Lin; Lee, Young Sup; Lee, You Mie; Park, Jeen-Woo

2006-01-01

The free radical hypothesis of aging postulates that senescence is due to an accumulation of cellular oxidative damage, caused largely by reactive oxygen species that are produced as by-products of normal metabolic processes. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of cytosolic (IDPc) and mitochondrial NADP+ -dependent isocitrate dehydrogenase (IDPm) by supplying NADPH for antioxidant systems. In this paper, we demonstrate that modulation of IDPc or IDPm activity in IMR-90 cells regulates cellular redox status and replicative senescence. When we examined the regulatory role of IDPc and IDPm against the aging process with IMR-90 cells transfected with cDNA for IDPc or IDPm in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPc or IDPm expressed in target cells and their susceptibility to senescence, which was reflected by changes in replicative potential, cell cycle, senescence-associated beta-galactosidase activity, expression of p21 and p53, and morphology of cells. Furthermore, lipid peroxidation, oxidative DNA damage, and intracellular peroxide generation were higher and cellular redox status shifted to a prooxidant condition in the cell lines expressing the lower level of IDPc or IDPm. The results suggest that IDPc and IDPm play an important regulatory role in cellular defense against oxidative stress and in the senescence of IMR-90 cells.

Proteasome-dependent degradation of replisome components regulates faithful DNA replication.

PubMed

Roseaulin, Laura C; Noguchi, Chiaki; Noguchi, Eishi

2013-08-15

The replication machinery, or the replisome, collides with a variety of obstacles during the normal process of DNA replication. In addition to damaged template DNA, numerous chromosome regions are considered to be difficult to replicate owing to the presence of DNA secondary structures and DNA-binding proteins. Under these conditions, the replication fork stalls, generating replication stress. Stalled forks are prone to collapse, posing serious threats to genomic integrity. It is generally thought that the replication checkpoint functions to stabilize the replisome and replication fork structure upon replication stress. This is important in order to allow DNA replication to resume once the problem is solved. However, our recent studies demonstrated that some replisome components undergo proteasome-dependent degradation during DNA replication in the fission yeast Schizosaccharomyces pombe. Our investigation has revealed the involvement of the SCF(Pof3) (Skp1-Cullin/Cdc53-F-box) ubiquitin ligase in replisome regulation. We also demonstrated that forced accumulation of the replisome components leads to abnormal DNA replication upon replication stress. Here we review these findings and present additional data indicating the importance of replisome degradation for DNA replication. Our studies suggest that cells activate an alternative pathway to degrade replisome components in order to preserve genomic integrity.

The Shine-Dalgarno sequence of riboswitch-regulated single mRNAs shows ligand-dependent accessibility bursts

NASA Astrophysics Data System (ADS)

Rinaldi, Arlie J.; Lund, Paul E.; Blanco, Mario R.; Walter, Nils G.

2016-01-01

In response to intracellular signals in Gram-negative bacteria, translational riboswitches--commonly embedded in messenger RNAs (mRNAs)--regulate gene expression through inhibition of translation initiation. It is generally thought that this regulation originates from occlusion of the Shine-Dalgarno (SD) sequence upon ligand binding; however, little direct evidence exists. Here we develop Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) to investigate the ligand-dependent accessibility of the SD sequence of an mRNA hosting the 7-aminomethyl-7-deazaguanine (preQ1)-sensing riboswitch. Spike train analysis reveals that individual mRNA molecules alternate between two conformational states, distinguished by `bursts' of probe binding associated with increased SD sequence accessibility. Addition of preQ1 decreases the lifetime of the SD's high-accessibility (bursting) state and prolongs the time between bursts. In addition, ligand-jump experiments reveal imperfect riboswitching of single mRNA molecules. Such complex ligand sensing by individual mRNA molecules rationalizes the nuanced ligand response observed during bulk mRNA translation.

Propeptides are sufficient to regulate organelle-specific pH-dependent activation of furin and proprotein convertase 1/3.

PubMed

Dillon, Stephanie L; Williamson, Danielle M; Elferich, Johannes; Radler, David; Joshi, Rajendra; Thomas, Gary; Shinde, Ujwal

2012-10-12

The proprotein convertases (PCs) furin and proprotein convertase 1/3 (PC1) cleave substrates at dibasic residues along the eukaryotic secretory/endocytic pathway. PCs are evolutionarily related to bacterial subtilisin and are synthesized as zymogens. They contain N-terminal propeptides (PRO) that function as dedicated catalysts that facilitate folding and regulate activation of cognate proteases through multiple-ordered cleavages. Previous studies identified a histidine residue (His69) that functions as a pH sensor in the propeptide of furin (PRO(FUR)), which regulates furin activation at pH~6.5 within the trans-Golgi network. Although this residue is conserved in the PC1 propeptide (PRO(PC1)), PC1 nonetheless activates at pH~5.5 within the dense core secretory granules. Here, we analyze the mechanism by which PRO(FUR) regulates furin activation and examine why PRO(FUR) and PRO(PC1) differ in their pH-dependent activation. Sequence analyses establish that while both PRO(FUR) and PRO(PC1) are enriched in histidines when compared with cognate catalytic domains and prokaryotic orthologs, histidine content in PRO(FUR) is ~2-fold greater than that in PRO(PC1), which may augment its pH sensitivity. Spectroscopy and molecular dynamics establish that histidine protonation significantly unfolds PRO(FUR) when compared to PRO(PC1) to enhance autoproteolysis. We further demonstrate that PRO(FUR) and PRO(PC1) are sufficient to confer organelle sensing on folding and activation of their cognate proteases. Swapping propeptides between furin and PC1 transfers pH-dependent protease activation in a propeptide-dictated manner in vitro and in cells. Since prokaryotes lack organelles and eukaryotic PCs evolved from propeptide-dependent, not propeptide-independent prokaryotic subtilases, our results suggest that histidine enrichment may have enabled propeptides to evolve to exploit pH gradients to activate within specific organelles. Copyright © 2012 Elsevier Ltd. All rights reserved.

Proteomic analysis reveals APC-dependent post-translational modifications and identifies a novel regulator of β-catenin.

PubMed

Blundon, Malachi A; Schlesinger, Danielle R; Parthasarathy, Amritha; Smith, Samantha L; Kolev, Hannah M; Vinson, David A; Kunttas-Tatli, Ezgi; McCartney, Brooke M; Minden, Jonathan S

2016-07-15

Wnt signaling generates patterns in all embryos, from flies to humans, and controls cell fate, proliferation and metabolic homeostasis. Inappropriate Wnt pathway activation results in diseases, including colorectal cancer. The adenomatous polyposis coli (APC) tumor suppressor gene encodes a multifunctional protein that is an essential regulator of Wnt signaling and cytoskeletal organization. Although progress has been made in defining the role of APC in a normal cellular context, there are still significant gaps in our understanding of APC-dependent cellular function and dysfunction. We expanded the APC-associated protein network using a combination of genetics and a proteomic technique called two-dimensional difference gel electrophoresis (2D-DIGE). We show that loss of Drosophila Apc2 causes protein isoform changes reflecting misregulation of post-translational modifications (PTMs), which are not dependent on β-catenin transcriptional activity. Mass spectrometry revealed that proteins involved in metabolic and biosynthetic pathways, protein synthesis and degradation, and cell signaling are affected by Apc2 loss. We demonstrate that changes in phosphorylation partially account for the altered PTMs in APC mutants, suggesting that APC mutants affect other types of PTM. Finally, through this approach Aminopeptidase P was identified as a new regulator of β-catenin abundance in Drosophila embryos. This study provides new perspectives on the cellular effects of APC that might lead to a deeper understanding of its role in development. © 2016. Published by The Company of Biologists Ltd.

Connexin43high prostate cancer cells induce endothelial connexin43 up-regulation through the activation of intercellular ERK1/2-dependent signaling axis.

PubMed

Piwowarczyk, Katarzyna; Paw, Milena; Ryszawy, Damian; Rutkowska-Zapała, Magdalena; Madeja, Zbigniew; Siedlar, Maciej; Czyż, Jarosław

2017-06-01

Connexin(Cx)43 regulates the invasive potential of prostate cancer cells and participates in their extravasation. To address the role of endothelial Cx43 in this process, we analyzed Cx43 regulation in human umbilical vein endothelial cells in the proximity of Cx43 high (DU-145 and MAT-LyLu) and Cx43 low prostate cancer cells (PC-3 and AT-2). Endothelial Cx43 up-regulation was observed during the diapedesis of DU-145 and MAT-LyLu cells. This process was attenuated by transient Cx43 silencing in cancer cells and by chemical inhibition of ERK1/2-dependent signaling in endothelial cells. Cx43 expression in endothelial cells was insensitive to the inhibition of gap junctional intercellular coupling between Cx43 high prostate cancer and endothelial cells by 18α-glycyrrhetinic acid. Instead, endothelial Cx43 up-regulation was correlated with the local contraction of endothelial cells and with their activation in the proximity of Cx43 high DU-145 and MAT-LyLu cells. It was also sensitive to pro-inflammatory factors secreted by peripheral blood monocytes, such as TNFα. In contrast to Cx43 low AT-2 cells, Cx43 low PC-3 cells produced angioactive factors that locally activated the endothelial cells in the absence of endothelial Cx43 up-regulation. Collectively, these data show that Cx43 low and Cx43 high prostate cancer cells can adapt discrete, Cx43-independent and Cx43-dependent strategies of diapedesis. Our observations identify a novel strategy of prostate cancer cell diapedesis, which depends on the activation of intercellular Cx43/ERK1/2/Cx43 signaling axis at the interfaces between Cx43 high prostate cancer and endothelial cells. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

PKB/SGK-dependent GSK3-phosphorylation in the regulation of LPS-induced Ca2+ increase in mouse dendritic cells.

PubMed

Russo, Antonella; Schmid, Evi; Nurbaeva, Meerim K; Yang, Wenting; Yan, Jing; Bhandaru, Madhuri; Faggio, Caterina; Shumilina, Ekaterina; Lang, Florian

2013-08-02

The function of dendritic cells (DCs) is modified by glycogen synthase kinase GSK3 and GSK3 inhibitors have been shown to protect against inflammatory disease. Regulators of GSK3 include the phosphoinositide 3 kinase (PI3K) pathway leading to activation of protein kinase B (PKB/Akt) and serum and glucocorticoid inducible kinase (SGK) isoforms, which in turn phosphorylate and thus inhibit GSK3. The present study explored, whether PKB/SGK-dependent inhibition of GSK3 contributes to the regulation of cytosolic Ca(2+) concentration following stimulation with bacterial lipopolysaccharides (LPS). To this end DCs from mutant mice, in which PKB/SGK-dependent GSK3α,β regulation was disrupted by replacement of the serine residues in the respective SGK/PKB-phosphorylation consensus sequence by alanine (gsk3(KI)), were compared to DCs from respective wild type mice (gsk3(WT)). According to Western blotting, GSK3 phosphorylation was indeed absent in gsk3(KI) DCs. According to flow cytometry, expression of antigen-presenting molecule major histocompatibility complex II (MHCII) and costimulatory molecule CD86, was similar in unstimulated and LPS (1μg/ml, 24h)-stimulated gsk3(WT) and gsk3(KI) DCs. Moreover, production of cytokines IL-6, IL-10, IL-12 and TNFα was not significantly different in gsk3(KI) and gsk3(WT) DCs. In gsk3(WT) DCs, stimulation with LPS (1μg/ml) within 10min led to transient phosphorylation of GSK3. According to Fura2 fluorescence, LPS (1μg/ml) increased cytosolic Ca(2+) concentration, an effect significantly more pronounced in gsk3(KI) DCs than in gsk3(WT) DCs. Conversely, GSK3 inhibitor SB216763 (3-[2,4-Dichlorophenyl]-4-[1-methyl-1H-indol-3-yl]-1H-pyrrole-2,5-dione, 10μM, 30min) significantly blunted the increase of cytosolic Ca(2+) concentration following LPS exposure. In conclusion, PKB/SGK-dependent GSK3α,β activity participates in the regulation of Ca(2+) signaling in dendritic cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Aryl hydrocarbon receptor-dependent regulation of miR-196a expression controls lung fibroblast apoptosis but not proliferation

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

Hecht, Emelia; Zago, Michela; Sarill, Miles

2014-11-01

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor implicated in the regulation of apoptosis and proliferation. Although activation of the AhR by xenobiotics such as dioxin inhibits the cell cycle and control apoptosis, paradoxically, AhR expression also promotes cell proliferation and survival independent of exogenous ligands. The microRNA (miRNA) miR-196a has also emerged as a regulator of proliferation and apoptosis but a relationship between the AhR and miR-196a is not known. Therefore, we hypothesized that AhR-dependent regulation of endogenous miR-196a expression would promote cell survival and proliferation. Utilizing lung fibroblasts from AhR deficient (AhR{sup −/−}) and wild-type (AhR{supmore » +/+}) mice, we show that there is ligand-independent regulation of miRNA, including low miR-196a in AhR{sup −/−} cells. Validation by qRT-PCR revealed a significant decrease in basal expression of miR-196a in AhR{sup −/−} compared to AhR{sup +/+} cells. Exposure to AhR agonists benzo[a]pyrene (B[a]P) and FICZ as well as AhR antagonist CH-223191 decreased miR-196a expression in AhR{sup +/+} fibroblasts concomitant with decreased AhR protein levels. There was increased proliferation only in AhR{sup +/+} lung fibroblasts in response to serum, corresponding to a decrease in p27{sup KIP1} protein, a cyclin-dependent kinase inhibitor. Increasing the cellular levels of miR-196a had no effect on proliferation or expression of p27{sup KIP1} in AhR{sup −/−} fibroblasts but attenuated cigarette smoke-induced apoptosis. This study provides the first evidence that AhR expression is essential for the physiological regulation of cellular miRNA levels- including miR-196a. Future experiments designed to elucidate the functional relationship between the AhR and miR-196a may delineate additional novel ligand-independent roles for the AhR. - Highlights: • The AhR controls proliferation and apoptosis in lung cells. • The AhR regulates

Regulation of singlet oxygen-induced apoptosis by cytosolic NADP+-dependent isocitrate dehydrogenase.

PubMed

Kim, Sun Yee; Lee, Su Min; Tak, Jean Kyoung; Choi, Kyeong Sook; Kwon, Taeg Kyu; Park, Jeen-Woo

2007-08-01

Singlet oxygen is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules and it also promotes deleterious processes such as cell death. Recently, we demonstrated that the control of redox balance and the cellular defense against oxidative damage are the primary functions of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) through supplying NADPH for antioxidant systems. In this report, we demonstrate that modulation of IDPc activity in HL-60 cells regulates singlet oxygen-induced apoptosis. When we examined the protective role of IDPc against singlet oxygen-induced apoptosis with HL-60 cells transfected with the cDNA for mouse IDPc in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPc expressed in target cells and their susceptibility to apoptosis. The results suggest that IDPc plays an important protective role in apoptosis of HL-60 cells induced by singlet oxygen.

Nardilysin controls intestinal tumorigenesis through HDAC1/p53-dependent transcriptional regulation.

PubMed

Kanda, Keitaro; Sakamoto, Jiro; Matsumoto, Yoshihide; Ikuta, Kozo; Goto, Norihiro; Morita, Yusuke; Ohno, Mikiko; Nishi, Kiyoto; Eto, Koji; Kimura, Yuto; Nakanishi, Yuki; Ikegami, Kanako; Yoshikawa, Takaaki; Fukuda, Akihisa; Kawada, Kenji; Sakai, Yoshiharu; Ito, Akihiro; Yoshida, Minoru; Kimura, Takeshi; Chiba, Tsutomu; Nishi, Eiichiro; Seno, Hiroshi

2018-04-19

Colon cancer is a complex disease affected by a combination of genetic and epigenetic factors. Here we demonstrate that nardilysin (N-arginine dibasic convertase; NRDC), a metalloendopeptidase of the M16 family, regulates intestinal tumorigenesis via its nuclear functions. NRDC is highly expressed in human colorectal cancers. Deletion of the Nrdc gene in ApcMin mice crucially suppressed intestinal tumor development. In ApcMin mice, epithelial cell-specific deletion of Nrdc recapitulated the tumor suppression observed in Nrdc-null mice. Moreover, epithelial cell-specific overexpression of Nrdc significantly enhanced tumor formation in ApcMin mice. Notably, epithelial NRDC controlled cell apoptosis in a gene dosage-dependent manner. In human colon cancer cells, nuclear NRDC directly associated with HDAC1, and controlled both acetylation and stabilization of p53, with alterations of p53 target apoptotic factors. These findings demonstrate that NRDC is critically involved in intestinal tumorigenesis through its epigenetic regulatory function, and targeting NRDC may lead to a novel prevention or therapeutic strategy against colon cancer.

Unraveling the Molecular Basis of Temperature-Dependent Genetic Regulation in Penicillium marneffei

PubMed Central

Yang, Ence; Wang, Gang; Woo, Patrick C. Y.; Lau, Susanna K. P.; Chow, Wang-Ngai; Chong, Ken T. K.; Tse, Herman; Kao, Richard Y. T.; Chan, Che-Man; Che, Xiaoyan; Yuen, Kwok-Yung

2013-01-01

Penicillium marneffei is an opportunistic fungal pathogen endemic in Southeast Asia, causing lethal systemic infections in immunocompromised patients. P. marneffei grows in a mycelial form at the ambient temperature of 25°C and transitions to a yeast form at 37°C. The ability to alternate between the mycelial and yeast forms at different temperatures, namely, thermal dimorphism, has long been considered critical for the pathogenicity of P. marneffei, yet the underlying genetic mechanisms remain elusive. Here we employed high-throughput sequencing to unravel global transcriptional profiles of P. marneffei PM1 grown at 25 and 37°C. Among ∼11,000 protein-coding genes, 1,447 were overexpressed and 1,414 were underexpressed at 37°C. Counterintuitively, heat-responsive genes, predicted in P. marneffei through sequence comparison, did not tend to be overexpressed at 37°C. These results suggest that P. marneffei may take a distinct strategy of genetic regulation at the elevated temperature; the current knowledge concerning fungal heat response, based on studies of model fungal organisms, may not be applicable to P. marneffei. Our results further showed that the tandem repeat sequences (TRSs) are overrepresented in coding regions of P. marneffei genes, and TRS-containing genes tend to be overexpressed at 37°C. Furthermore, genomic sequences and expression data were integrated to characterize gene clusters, multigene families, and species-specific genes of P. marneffei. In sum, we present an integrated analysis and a comprehensive resource toward a better understanding of temperature-dependent genetic regulation in P. marneffei. PMID:23851338

Peroxisomes in Different Skeletal Cell Types during Intramembranous and Endochondral Ossification and Their Regulation during Osteoblast Differentiation by Distinct Peroxisome Proliferator-Activated Receptors

PubMed Central

Qian, Guofeng; Karnati, Srikanth; Baumgart-Vogt, Eveline

2015-01-01

Ossification defects leading to craniofacial dysmorphism or rhizomelia are typical phenotypes in patients and corresponding knockout mouse models with distinct peroxisomal disorders. Despite these obvious skeletal pathologies, to date no careful analysis exists on the distribution and function of peroxisomes in skeletal tissues and their alterations during ossification. Therefore, we analyzed the peroxisomal compartment in different cell types of mouse cartilage and bone as well as in primary cultures of calvarial osteoblasts. The peroxisome number and metabolism strongly increased in chondrocytes during endochondral ossification from the reserve to the hypertrophic zone, whereas in bone, metabolically active osteoblasts contained a higher numerical abundance of this organelle than osteocytes. The high abundance of peroxisomes in these skeletal cell types is reflected by high levels of Pex11β gene expression. During culture, calvarial pre-osteoblasts differentiated into secretory osteoblasts accompanied by peroxisome proliferation and increased levels of peroxisomal genes and proteins. Since many peroxisomal genes contain a PPAR-responsive element, we analyzed the gene expression of PPARɑ/ß/ɣ in calvarial osteoblasts and MC3T3-E1 cells, revealing higher levels for PPARß than for PPARɑ and PPARɣ. Treatment with different PPAR agonists and antagonists not only changed the peroxisomal compartment and associated gene expression, but also induced complex alterations of the gene expression patterns of the other PPAR family members. Studies in M3CT3-E1 cells showed that the PPARß agonist GW0742 activated the PPRE-mediated luciferase expression and up-regulated peroxisomal gene transcription (Pex11, Pex13, Pex14, Acox1 and Cat), whereas the PPARß antagonist GSK0660 led to repression of the PPRE and a decrease of the corresponding mRNA levels. In the same way, treatment of calvarial osteoblasts with GW0742 increased in peroxisome number and related gene expression

Role of Dlx6 in regulation of an endothelin-1-dependent, dHAND branchial arch enhancer

PubMed Central

Charité, Jeroen; McFadden, David G.; Merlo, Giorgio; Levi, Giovanni; Clouthier, David E.; Yanagisawa, Masashi; Richardson, James A.; Olson, Eric N.

2001-01-01

Neural crest cells play a key role in craniofacial development. The endothelin family of secreted polypeptides regulates development of several neural crest sublineages, including the branchial arch neural crest. The basic helix–loop–helix transcription factor dHAND is also required for craniofacial development, and in endothelin-1 (ET-1) mutant embryos, dHAND expression in the branchial arches is down-regulated, implicating it as a transcriptional effector of ET-1 action. To determine the mechanism that links ET-1 signaling to dHAND transcription, we analyzed the dHAND gene for cis-regulatory elements that control transcription in the branchial arches. We describe an evolutionarily conserved dHAND enhancer that requires ET-1 signaling for activity. This enhancer contains four homeodomain binding sites that are required for branchial arch expression. By comparing protein binding to these sites in branchial arch extracts from endothelin receptor A (EdnrA) mutant and wild-type mouse embryos, we identified Dlx6, a member of the Distal-less family of homeodomain proteins, as an ET-1-dependent binding factor. Consistent with this conclusion, Dlx6 was down-regulated in branchial arches from EdnrA mutant mice. These results suggest that Dlx6 acts as an intermediary between ET-1 signaling and dHAND transcription during craniofacial morphogenesis. PMID:11711438

Adaptive Significance of Quorum Sensing-Dependent Regulation of Rhamnolipids by Integration of Growth Rate in Burkholderia glumae: A Trade-Off between Survival and Efficiency.

PubMed

Nickzad, Arvin; Déziel, Eric

2016-01-01

Quorum sensing (QS) is a cell density-dependent mechanism which enables a population of bacteria to coordinate cooperative behaviors in response to the accumulation of self-produced autoinducer signals in their local environment. An emerging framework is that the adaptive significance of QS in the regulation of production of costly extracellular metabolites ("public goods") is to maintain the homeostasis of cooperation. We investigated this model using the phytopathogenic bacterium Burkholderia glumae, which we have previously demonstrated uses QS to regulate the production of rhamnolipids, extracellular surface-active glycolipids promoting the social behavior called "swarming motility." Using mass spectrometric quantification and chromosomal lux-based gene expression, we made the unexpected finding that when unrestricted nutrient resources are provided, production of rhamnolipids is carried out completely independently of QS regulation. This is a unique observation among known QS-controlled factors in bacteria. On the other hand, under nutrient-limited conditions, QS then becomes the main regulating mechanism, significantly enhancing the specific rhamnolipids yield. Accordingly, decreasing nutrient concentrations amplifies rhamnolipid biosynthesis gene expression, revealing a system where QS-dependent regulation is specifically triggered by the growth rate of the population, rather than by its cell density. Furthermore, a gradual increase in QS signal specific concentration upon decrease of specific growth rate suggests a reduction in quorum threshold, which reflects an increase in cellular demand for production of QS-dependent target gene product at low density populations. Integration of growth rate with QS as a decision-making mechanism for biosynthesis of costly metabolites, such as rhamnolipids, could serve to assess the demand and timing for expanding the carrying capacity of a population through spatial expansion mechanisms, such as swarming motility, thus

Ryanodine receptors regulate arterial diameter and wall [Ca2+] in cerebral arteries of rat via Ca2+-dependent K+ channels

PubMed Central

Knot, Harm J; Standen, Nicholas B; Nelson, Mark T

1998-01-01

The effects of inhibitors of ryanodine-sensitive calcium release (RyR) channels in the sarcoplasmic reticulum (SR) and Ca2+-dependent potassium (KCa) channels on the membrane potential, intracellular [Ca2+], and diameters of small pressurized (60 mmHg) cerebral arteries (100–200 μm) were studied using digital fluorescence video imaging of arterial diameter and wall [Ca2+], combined with microelectrode measurements of arterial membrane potential. Ryanodine (10 μm), an inhibitor of RyR channels, depolarized by 9 mV, increased intracellular [Ca2+] by 46 nm and constricted pressurized (to 60 mmHg) arteries with myogenic tone by 44 μm (∼22 %). Iberiotoxin (100 nm), a blocker of KCa channels, under the same conditions, depolarized the arteries by 10 mV, increased arterial wall calcium by 51 nm, and constricted by 37 μm (∼19 %). The effects of ryanodine and iberiotoxin were not additive and were blocked by inhibitors of voltage-dependent Ca2+ channels. Caffeine (10 mm), an activator of RyR channels, transiently increased arterial wall [Ca2+] by 136 ± 9 nm in control arteries and by 158 ± 12 nm in the presence of iberiotoxin. Caffeine was relatively ineffective in the presence of ryanodine, increasing [calcium] by 18 ± 5 nm. In the presence of blockers of voltage-dependent Ca2+ channels (nimodipine, diltiazem), ryanodine and inhibitors of the SR calcium ATPase (thapsigargin, cyclopiazonic acid) were without effect on arterial wall [Ca2+] and diameter. These results suggest that local Ca2+ release originating from RyR channels (Ca2+ sparks) in the SR of arterial smooth muscle regulates myogenic tone in cerebral arteries solely through activation of KCa channels, which regulate membrane potential through tonic hyperpolarization, thus limiting Ca2+ entry through L-type voltage-dependent Ca2+ channels. KCa channels therefore act as a negative feedback control element regulating arterial diameter through a reduction in global intracellular free [Ca2+]. PMID:9490841

Rice NAD+-dependent histone deacetylase OsSRT1 represses glycolysis and regulates the moonlighting function of GAPDH as a transcriptional activator of glycolytic genes.

PubMed

Zhang, Hua; Zhao, Yu; Zhou, Dao-Xiu

2017-12-01

Sirtuins, a family of proteins with homology to the yeast silent information regulator 2 (Sir2), are NAD+-dependent histone deacetylases and play crucial roles in energy sensing and regulation in yeast and animal cells. Plants are autotrophic organisms and display distinct features of carbon and energy metabolism. It remains largely unexplored whether and how plant cells sense energy/redox status to control carbon metabolic flux under various growth conditions. In this work, we show that the rice nuclear sirtuin OsSRT1 not only functions as an epigenetic regulator to repress glycolytic genes expression and glycolysis in seedlings, but also inhibits transcriptional activity of glyceraldehyde-3-phosphatedehydrogenase (GAPDH) that is enriched on glycolytic genes promoters and stimulates their expression. We show that OsSRT1 reduces GAPDH lysine acetylation and nuclear accumulation that are enhanced by oxidative stress. Mass spectrometry identified six acetylated lysines regulated by OsSRT1. OsSRT1-dependent lysine deacetylation of OsGAPDH1 represses transcriptional activity of the protein. The results indicate that OsSRT1 represses glycolysis by both regulating epigenetic modification of histone and inhibiting the moonlighting function of GAPDH as a transcriptional activator of glycolytic genes in rice. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Activity-dependent regulation of release probability at excitatory hippocampal synapses: a crucial role of FMRP in neurotransmission

PubMed Central

Wang, Xiao-Sheng; Peng, Chun-Zi; Cai, Wei-Jun; Xia, Jian; Jin, Daozhong; Dai, Yuqiao; Luo, Xue-Gang; Klyachko, Vitaly A.; Deng, Pan-Yue

2014-01-01

Transcriptional silencing of the Fmr1 gene encoding fragile X mental retardation protein (FMRP) causes Fragile X Syndrome (FXS), the most common form of inherited intellectual disability and the leading genetic cause of autism. FMRP has been suggested to play important roles in regulating neurotransmission and short-term synaptic plasticity at excitatory hippocampal and cortical synapses. However, the origins and the mechanisms of these FMRP actions remain incompletely understood, and the role of FMRP in regulating synaptic release probability and presynaptic function remains debated. Here we used variance-mean analysis and peak scaled nonstationary variance analysis to examine changes in both pre- and postsynaptic parameters during repetitive activity at excitatory CA3-CA1 hippocampal synapses in a mouse model of FXS. Our analyses revealed that loss of FMRP did not affect the basal release probability or basal synaptic transmission, but caused an abnormally elevated release probability specifically during repetitive activity. These abnormalities were not accompanied by changes in EPSC kinetics, quantal size or postsynaptic AMPA receptor conductance. Our results thus indicate that FMRP regulates neurotransmission at excitatory hippocampal synapses specifically during repetitive activity via modulation of release probability in a presynaptic manner. Our study suggests that FMRP function in regulating neurotransmitter release is an activity-dependent phenomenon that may contribute to the pathophysiology of FXS. PMID:24646437

The dependence of neuronal encoding efficiency on Hebbian plasticity and homeostatic regulation of neurotransmitter release

PubMed Central

Faghihi, Faramarz; Moustafa, Ahmed A.

2015-01-01

Synapses act as information filters by different molecular mechanisms including retrograde messenger that affect neuronal spiking activity. One of the well-known effects of retrograde messenger in presynaptic neurons is a change of the probability of neurotransmitter release. Hebbian learning describe a strengthening of a synapse between a presynaptic input onto a postsynaptic neuron when both pre- and postsynaptic neurons are coactive. In this work, a theory of homeostatic regulation of neurotransmitter release by retrograde messenger and Hebbian plasticity in neuronal encoding is presented. Encoding efficiency was measured for different synaptic conditions. In order to gain high encoding efficiency, the spiking pattern of a neuron should be dependent on the intensity of the input and show low levels of noise. In this work, we represent spiking trains as zeros and ones (corresponding to non-spike or spike in a time bin, respectively) as words with length equal to three. Then the frequency of each word (here eight words) is measured using spiking trains. These frequencies are used to measure neuronal efficiency in different conditions and for different parameter values. Results show that neurons that have synapses acting as band-pass filters show the highest efficiency to encode their input when both Hebbian mechanism and homeostatic regulation of neurotransmitter release exist in synapses. Specifically, the integration of homeostatic regulation of feedback inhibition with Hebbian mechanism and homeostatic regulation of neurotransmitter release in the synapses leads to even higher efficiency when high stimulus intensity is presented to the neurons. However, neurons with synapses acting as high-pass filters show no remarkable increase in encoding efficiency for all simulated synaptic plasticity mechanisms. This study demonstrates the importance of cooperation of Hebbian mechanism with regulation of neurotransmitter release induced by rapid diffused retrograde

[Ultrastructural organization of cytoplasmatic membrane of Anaerobacter polyendosporus studied by electron microscopic cryofractography].

PubMed

Duda, V I; Suzina, N E; Dmitriev, V V

2001-01-01

Anaerobacter polyendosporus cells do not have typical mesosomes. However, the analysis of this anaerobic multispore bacterium by electron microscopic cryofractography showed that its cytoplasmic membrane contains specific intramembrane structures in the form of flat lamellar inverted lipid membranes tenths of nanometers to several microns in size. It was found that these structures are located in the hydrophobic interior between the outer and inner leaflets of the cytoplasmic membrane and do not contain intramembrane particles that are commonly present on freeze-fracture replicas. The flat inverted lipid membranes were revealed in bacterial cells cultivated under normal growth conditions, indicating the existence of a complex-type compartmentalization in biological membranes, which manifests itself in the formation of intramembrane compartments having the appearance of vesicles and inverted lipid membranes.

Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking.

PubMed

Andersen, Martin N; Hefting, Louise L; Steffensen, Annette B; Schmitt, Nicole; Olesen, Søren-Peter; Olsen, Jesper V; Lundby, Alicia; Rasmussen, Hanne B

2015-11-15

The potassium channel Kv7.1 plays critical physiological roles in both heart and epithelial tissues. In heart, Kv7.1 and the accessory subunit KCNE1 forms the slowly activating delayed-rectifier potassium current current, which is enhanced by protein kinase A (PKA)-mediated phosphorylation. The observed current increase requires both phosphorylation of Kv7.1 and the presence of KCNE1. However, PKA also stimulates Kv7.1 currents in epithelial tissues, such as colon, where the channel does not coassemble with KCNE1. Here, we demonstrate that PKA activity significantly impacts the subcellular localization of Kv7.1 in Madin-Darby canine kidney cells. While PKA inhibition reduced the fraction of channels at the cell surface, PKA activation increased it. We show that PKA inhibition led to intracellular accumulation of Kv7.1 in late endosomes/lysosomes. By mass spectroscopy we identified eight phosphorylated residues on Kv7.1, however, none appeared to play a role in the observed response. Instead, we found that PKA acted by regulating endocytic trafficking involving the ubiquitin ligase Nedd4-2. We show that a Nedd4-2-resistant Kv7.1-mutant displayed significantly reduced intracellular accumulation upon PKA inhibition. Similar effects were observed upon siRNA knockdown of Nedd4-2. However, although Nedd4-2 is known to regulate Kv7.1 by ubiquitylation, biochemical analyses demonstrated that PKA did not influence the amount of Nedd4-2 bound to Kv7.1 or the ubiquitylation level of the channel. This suggests that PKA influences Nedd4-2-dependent Kv7.1 transport though a different molecular mechanism. In summary, we identify a novel mechanism whereby PKA can increase Kv7.1 current levels, namely by regulating Nedd4-2-dependent Kv7.1 transport. Copyright © 2015 the American Physiological Society.

Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

PubMed Central

Andersen, Martin N.; Hefting, Louise L.; Steffensen, Annette B.; Schmitt, Nicole; Olesen, Søren-Peter; Olsen, Jesper V.; Lundby, Alicia

2015-01-01

The potassium channel Kv7.1 plays critical physiological roles in both heart and epithelial tissues. In heart, Kv7.1 and the accessory subunit KCNE1 forms the slowly activating delayed-rectifier potassium current current, which is enhanced by protein kinase A (PKA)-mediated phosphorylation. The observed current increase requires both phosphorylation of Kv7.1 and the presence of KCNE1. However, PKA also stimulates Kv7.1 currents in epithelial tissues, such as colon, where the channel does not coassemble with KCNE1. Here, we demonstrate that PKA activity significantly impacts the subcellular localization of Kv7.1 in Madin-Darby canine kidney cells. While PKA inhibition reduced the fraction of channels at the cell surface, PKA activation increased it. We show that PKA inhibition led to intracellular accumulation of Kv7.1 in late endosomes/lysosomes. By mass spectroscopy we identified eight phosphorylated residues on Kv7.1, however, none appeared to play a role in the observed response. Instead, we found that PKA acted by regulating endocytic trafficking involving the ubiquitin ligase Nedd4-2. We show that a Nedd4-2-resistant Kv7.1-mutant displayed significantly reduced intracellular accumulation upon PKA inhibition. Similar effects were observed upon siRNA knockdown of Nedd4-2. However, although Nedd4-2 is known to regulate Kv7.1 by ubiquitylation, biochemical analyses demonstrated that PKA did not influence the amount of Nedd4-2 bound to Kv7.1 or the ubiquitylation level of the channel. This suggests that PKA influences Nedd4-2-dependent Kv7.1 transport though a different molecular mechanism. In summary, we identify a novel mechanism whereby PKA can increase Kv7.1 current levels, namely by regulating Nedd4-2-dependent Kv7.1 transport. PMID:26405101

Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1.

PubMed

Furihata, Takashi; Maruyama, Kyonoshin; Fujita, Yasunari; Umezawa, Taishi; Yoshida, Riichiro; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

2006-02-07

bZIP-type transcription factors AREBs/ABFs bind an abscisic acid (ABA)-responsive cis-acting element named ABRE and transactivate downstream gene expression in Arabidopsis. Because AREB1 overexpression could not induce downstream gene expression, activation of AREB1 requires ABA-dependent posttranscriptional modification. We confirmed that ABA activated 42-kDa kinase activity, which, in turn, phosphorylated Ser/Thr residues of R-X-X-S/T sites in the conserved regions of AREB1. Amino acid substitutions of R-X-X-S/T sites to Ala suppressed transactivation activity, and multiple substitution of these sites resulted in almost complete suppression of transactivation activity in transient assays. In contrast, substitution of the Ser/Thr residues to Asp resulted in high transactivation activity without exogenous ABA application. A phosphorylated, transcriptionally active form was achieved by substitution of Ser/Thr in all conserved R-X-X-S/T sites to Asp. Transgenic plants overexpressing the phosphorylated active form of AREB1 expressed many ABA-inducible genes, such as RD29B, without ABA treatment. These results indicate that the ABA-dependent multisite phosphorylation of AREB1 regulates its own activation in plants.

Synaptic Strength Is Bidirectionally Controlled by Opposing Activity-Dependent Regulation of Nedd4-1 and USP8

PubMed Central

Scudder, Samantha L.; Goo, Marisa S.; Cartier, Anna E.; Molteni, Alice; Schwarz, Lindsay A.; Wright, Rebecca

2014-01-01

The trafficking of AMPA receptors (AMPARs) to and from synapses is crucial for synaptic plasticity. Previous work has demonstrated that AMPARs undergo activity-dependent ubiquitination by the E3 ubiquitin ligase Nedd4-1, which promotes their internalization and degradation in lysosomes. Here, we define the molecular mechanisms involved in ubiquitination and deubiquitination of AMPARs. We report that Nedd4-1 is rapidly redistributed to dendritic spines in response to AMPAR activation and not in response to NMDA receptor (NMDAR) activation in cultured rat neurons. In contrast, NMDAR activation directly antagonizes Nedd4-1 function by promoting the deubiquitination of AMPARs. We show that NMDAR activation causes the rapid dephosphorylation and activation of the deubiquitinating enzyme (DUB) USP8. Surface AMPAR levels and synaptic strength are inversely regulated by Nedd4-1 and USP8. Strikingly, we show that homeostatic downscaling of synaptic strength is accompanied by an increase and decrease in Nedd4-1 and USP8 protein levels, respectively. Furthermore, we show that Nedd4-1 is required for homeostatic loss of surface AMPARs and downscaling of synaptic strength. This study provides the first mechanistic evidence for rapid and opposing activity-dependent control of a ubiquitin ligase and DUB at mammalian CNS synapses. We propose that the dynamic regulation of these opposing forces is critical in maintaining synapses and scaling them during homeostatic plasticity. PMID:25505317

Arginine methylation regulates c-Myc-dependent transcription by altering promoter recruitment of the acetyltransferase p300.

PubMed

Tikhanovich, Irina; Zhao, Jie; Bridges, Brian; Kumer, Sean; Roberts, Ben; Weinman, Steven A

2017-08-11

Protein arginine methyltransferase 1 (PRMT1) is an essential enzyme controlling about 85% of the total cellular arginine methylation in proteins. We have shown previously that PRMT1 is an important regulator of innate immune responses and that it is required for M2 macrophage differentiation. c-Myc is a transcription factor that is critical in regulating cell proliferation and also regulates the M2 transcriptional program in macrophages. Here, we sought to determine whether c-Myc in myeloid cells is regulated by PRMT1-dependent arginine methylation. We found that PRMT1 activity was necessary for c-Myc binding to the acetyltransferase p300. PRMT1 inhibition decreased p300 recruitment to c-Myc target promoters and increased histone deacetylase 1 (HDAC1) recruitment, thereby decreasing transcription at these sites. Moreover, PRMT1 inhibition blocked c-Myc-mediated induction of several of its target genes, including peroxisome proliferator-activated receptor γ ( PPARG ) and mannose receptor C-type 1 ( MRC1 ), suggesting that PRMT1 is necessary for c-Myc function in M2 macrophage differentiation. Of note, in primary human blood monocytes, p300-c-Myc binding was strongly correlated with PRMT1 expression, and in liver sections, PRMT1, c-Myc, and M2 macrophage levels were strongly correlated with each other. Both PRMT1 levels and M2 macrophage numbers were significantly lower in livers from individuals with a history of spontaneous bacterial peritonitis, known to have defective cellular immunity. In conclusion, our findings demonstrate that PRMT1 is an important regulator of c-Myc function in myeloid cells. PRMT1 loss in individuals with cirrhosis may contribute to their immune defects. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

LRP1 regulates architecture of the vascular wall by controlling PDGFRbeta-dependent phosphatidylinositol 3-kinase activation.

PubMed

Zhou, Li; Takayama, Yoshiharu; Boucher, Philippe; Tallquist, Michelle D; Herz, Joachim

2009-09-09

Low density lipoprotein receptor-related protein 1 (LRP1) protects against atherosclerosis by regulating the activation of platelet-derived growth factor receptor beta (PDGFRbeta) in vascular smooth muscle cells (SMCs). Activated PDGFRbeta undergoes tyrosine phosphorylation and subsequently interacts with various signaling molecules, including phosphatidylinositol 3-kinase (PI3K), which binds to the phosphorylated tyrosine 739/750 residues in mice, and thus regulates actin polymerization and cell movement. In this study, we found disorganized actin in the form of membrane ruffling and enhanced cell migration in LRP1-deficient (LRP1-/-) SMCs. Marfan syndrome-like phenotypes such as tortuous aortas, disrupted elastic layers and abnormally activated transforming growth factor beta (TGFbeta) signaling are present in smooth muscle-specific LRP1 knockout (smLRP1-/-) mice. To investigate the role of LRP1-regulated PI3K activation by PDGFRbeta in atherogenesis, we generated a strain of smLRP1-/- mice in which tyrosine 739/750 of the PDGFRbeta had been mutated to phenylalanines (PDGFRbeta F2/F2). Spontaneous atherosclerosis was significantly reduced in the absence of hypercholesterolemia in these mice compared to smLRP1-/- animals that express wild type PDGFR. Normal actin organization was restored and spontaneous SMC migration as well as PDGF-BB-induced chemotaxis was dramatically reduced, despite continued overactivation of TGFbeta signaling, as indicated by high levels of nuclear phospho-Smad2. Our data suggest that LRP1 regulates actin organization and cell migration by controlling PDGFRbeta-dependent activation of PI3K. TGFbeta activation alone is not sufficient for the expression of the Marfan-like vascular phenotype. Thus, regulation of PI3 Kinase by PDGFRbeta is essential for maintaining vascular integrity, and for the prevention of atherosclerosis as well as Marfan syndrome.

Characterization of the Regulation and Function of Zinc-Dependent Histone Deacetylases During Mouse Liver Regeneration

PubMed Central

Huang, Jiansheng; Barr, Emily; Rudnick, David A.

2013-01-01

The studies reported here were undertaken to define the regulation and functional importance of zinc-dependent histone deacetylase (Zn-HDAC) activity during liver regeneration using the mouse partial hepatectomy (PH) model. The results showed that hepatic HDAC activity was significantly increased in nuclear and cytoplasmic fractions following PH. Further analyses showed isoform-specific effects of PH on HDAC mRNA and protein expression, with increased expression of the class I HDACs, 1 and 8, and class II HDAC4 in regenerating liver. Hepatic expression of (class II) HDAC5 was unchanged after PH; however HDAC5 exhibited transient nuclear accumulation in regenerating liver. These changes in hepatic HDAC expression, subcellular localization, and activity coincided with diminished histone acetylation in regenerating liver. The significance of these events was investigated by determining the effects of suberoylanilide hydroxyamic acid (SAHA, a specific inhibitor of Zn-HDAC activity) on hepatic regeneration. The results showed that SAHA-treatment suppressed the effects of PH on histone deacetylation and hepatocellular BrdU incorporation. Further examination showed that SAHA blunted hepatic expression and activation of cell cycle signals downstream of induction of cyclin D1 expression in mice subjected to PH. Conclusion The data reported here demonstrate isoform-specific regulation of Zn-HDAC expression, subcellular localization, and activity in regenerating liver. These studies also indicate that HDAC activity promotes liver regeneration by regulating hepatocellular cell cycle progression at a step downstream of cyclin D1 induction. PMID:23258575

NMDA receptor- and ERK-dependent histone methylation changes in the lateral amygdala bidirectionally regulate fear memory formation.

PubMed

Gupta-Agarwal, Swati; Jarome, Timothy J; Fernandez, Jordan; Lubin, Farah D

2014-07-01

It is well established that fear memory formation requires de novo gene transcription in the amygdala. We provide evidence that epigenetic mechanisms in the form of histone lysine methylation in the lateral amygdala (LA) are regulated by NMDA receptor (NMDAR) signaling and involved in gene transcription changes necessary for fear memory consolidation. Here we found increases in histone H3 lysine 9 dimethylation (H3K9me2) levels in the LA at 1 h following auditory fear conditioning, which continued to be temporally regulated up to 25 h following behavioral training. Additionally, we demonstrate that inhibiting the H3K9me2 histone lysine methyltransferase G9a (H/KMTs-G9a) in the LA impaired fear memory, while blocking the H3K9me2 histone lysine demethylase LSD1 (H/KDM-LSD1) enhanced fear memory, suggesting that H3K9me2 in the LA can bidirectionally regulate fear memory formation. Furthermore, we show that NMDAR activity differentially regulated the recruitment of H/KMT-G9a, H/KDM-LSD1, and subsequent H3K9me2 levels at a target gene promoter. This was largely regulated by GluN2B- but not GluN2A-containing NMDARs via ERK activation. Moreover, fear memory deficits associated with NMDAR or ERK blockade were successfully rescued through pharmacologically inhibiting LSD1, suggesting that enhancements of H3K9me2 levels within the LA can rescue fear memory impairments that result from hypofunctioning NMDARs or loss of ERK signaling. Together, the present study suggests that histone lysine methylation regulation in the LA via NMDAR-ERK-dependent signaling is involved in fear memory formation. © 2014 Gupta-Agarwal et al.; Published by Cold Spring Harbor Laboratory Press.

Uroguanylin levels in intestine and plasma are regulated by nutritional status in a leptin-dependent manner.

PubMed

Folgueira, C; Sanchez-Rebordelo, E; Barja-Fernandez, S; Leis, R; Tovar, S; Casanueva, F F; Dieguez, C; Nogueiras, R; Seoane, L M

2016-03-01

Uroguanylin (UGN) is a 16 amino acid peptide produced mainly by intestinal epithelial cells. Nutrients intake increases circulating levels of prouroguanylin that is processed and converted to UGN to activate the guanylyl cyclase 2C receptor (GUCY2C). Given that the UGN-GUCY2C system has been proposed as a novel gut-brain endocrine axis regulating energy balance, the aim of the present study was to investigate the regulation of UGN protein levels in duodenum and circulating levels in lean and obese mice under different nutritional conditions and its potential interaction with leptin. Swiss, C57BL/6 wild-type and ob/ob male adult mice under different nutritional conditions were used: fed ad libitum standard diet (control); 48 h fasting (fasted); 48 h fasting followed by 24 h of feeding (refed); and fed high-fat diet (45 %) during 10 weeks. In addition, peripheral leptin administration was performed. Intestinal uroguanylin expression was studied by Western blot analysis; plasma levels were measured by ELISA. Food deprivation significantly reduced plasma UGN levels, which were correlated with the lower protein levels of UGN in duodenum. These effects were reverted after refeeding and leptin challenge. Consistently, in ob/ob mice UGN expression was decreased, whereas leptin treatment up-regulated UGN levels in duodenum in these genetically modified mice compared to WT. Diet-induced obese mice displayed increased UGN levels in intestine and plasma in comparison with lean mice. Our findings suggest that UGN levels are correlated with energy balance status and that the regulation of UGN by nutritional status is leptin-dependent.

Role of an Iron-Dependent Transcriptional Regulator in the Pathogenesis and Host Response to Infection with Streptococcus pneumoniae

PubMed Central

Gupta, Radha; Bhatty, Minny; Swiatlo, Edwin; Nanduri, Bindu

2013-01-01

Iron is a critical cofactor for many enzymes and is known to regulate gene expression in many bacterial pathogens. Streptococcus pneumoniae normally inhabits the upper respiratory mucosa but can also invade and replicate in lungs and blood. These anatomic sites vary considerably in both the quantity and form of available iron. The genome of serotype 4 pneumococcal strain TIGR4 encodes a putative iron-dependent transcriptional regulator (IDTR). A mutant deleted at idtr (Δidtr) exhibited growth kinetics similar to parent strain TIGR4 in vitro and in mouse blood for up to 48 hours following infection. However, Δidtr was significantly attenuated in a murine model of sepsis. IDTR down-regulates the expression of ten characterized and putative virulence genes in nasopharyngeal colonization and pneumonia. The host cytokine response was significantly suppressed in sepsis with Δidtr. Since an exaggerated inflammatory response is associated with a poor prognosis in sepsis, the decreased inflammatory response could explain the increased survival with Δidtr. Our results suggest that IDTR, which is dispensable for pneumococcal growth in vitro, is associated with regulation of pneumococcal virulence in specific host environments. Additionally, IDTR ultimately modulates the host cytokine response and systemic inflammation that contributes to morbidity and mortality of invasive pneumococcal disease. PMID:23437050

Regulation of Neuronal Cav3.1 Channels by Cyclin-Dependent Kinase 5 (Cdk5)

PubMed Central

González-Ramírez, Ricardo; González-Billault, Christian; Felix, Ricardo

2015-01-01

Low voltage-activated (LVA) T-type Ca2+ channels activate in response to subthreshold membrane depolarizations and therefore represent an important source of Ca2+ influx near the resting membrane potential. In neurons, these proteins significantly contribute to control relevant physiological processes including neuronal excitability, pacemaking and post-inhibitory rebound burst firing. Three subtypes of T-type channels (Cav3.1 to Cav3.3) have been identified, and using functional expression of recombinant channels diverse studies have validated the notion that T-type Ca2+ channels can be modulated by various endogenous ligands as well as by second messenger pathways. In this context, the present study reveals a previously unrecognized role for cyclin-dependent kinase 5 (Cdk5) in the regulation of native T-type channels in N1E-115 neuroblastoma cells, as well as recombinant Cav3.1channels heterologously expressed in HEK-293 cells. Cdk5 and its co-activators play critical roles in the regulation of neuronal differentiation, cortical lamination, neuronal cell migration and axon outgrowth. Our results show that overexpression of Cdk5 causes a significant increase in whole cell patch clamp currents through T-type channels in N1E-115 cells, while siRNA knockdown of Cdk5 greatly reduced these currents. Consistent with this, overexpression of Cdk5 in HEK-293 cells stably expressing Cav3.1channels upregulates macroscopic currents. Furthermore, using site-directed mutagenesis we identified a major phosphorylation site at serine 2234 within the C-terminal region of the Cav3.1subunit. These results highlight a novel role for Cdk5 in the regulation of T-type Ca2+ channels. PMID:25760945

Regulation of neuronal cav3.1 channels by cyclin-dependent kinase 5 (Cdk5).

PubMed

Calderón-Rivera, Aida; Sandoval, Alejandro; González-Ramírez, Ricardo; González-Billault, Christian; Felix, Ricardo

2015-01-01

Low voltage-activated (LVA) T-type Ca2+ channels activate in response to subthreshold membrane depolarizations and therefore represent an important source of Ca2+ influx near the resting membrane potential. In neurons, these proteins significantly contribute to control relevant physiological processes including neuronal excitability, pacemaking and post-inhibitory rebound burst firing. Three subtypes of T-type channels (Cav3.1 to Cav3.3) have been identified, and using functional expression of recombinant channels diverse studies have validated the notion that T-type Ca2+ channels can be modulated by various endogenous ligands as well as by second messenger pathways. In this context, the present study reveals a previously unrecognized role for cyclin-dependent kinase 5 (Cdk5) in the regulation of native T-type channels in N1E-115 neuroblastoma cells, as well as recombinant Cav3.1channels heterologously expressed in HEK-293 cells. Cdk5 and its co-activators play critical roles in the regulation of neuronal differentiation, cortical lamination, neuronal cell migration and axon outgrowth. Our results show that overexpression of Cdk5 causes a significant increase in whole cell patch clamp currents through T-type channels in N1E-115 cells, while siRNA knockdown of Cdk5 greatly reduced these currents. Consistent with this, overexpression of Cdk5 in HEK-293 cells stably expressing Cav3.1channels upregulates macroscopic currents. Furthermore, using site-directed mutagenesis we identified a major phosphorylation site at serine 2234 within the C-terminal region of the Cav3.1subunit. These results highlight a novel role for Cdk5 in the regulation of T-type Ca2+ channels.

The Effect of Methadone-Maintenance Therapy With and Without Interactive Treatment on Improving Emotion-Regulation Strategies and Resilience Among Opiate-Dependent Clients

PubMed Central

Hoseiny, Hadis; Jadidi, Mohsen; Habiballah Nataj, Leila; Saberi- Zafarghandi, Mohammad Bagher

2015-01-01

Background: Due to the chronic and recurrent nature of addiction, many people who quit drug addiction may slip back into the pattern of using drugs shortly after the detoxification period. Emotion-regulation strategies and resilience play an important role in preventing the recurrences of substance abuse. Objectives: This study aimed to compare the effects of methadone-maintenance therapy (MMT) and interactive therapy (a combination of MMT and cognitive-behavioral therapy) on improving emotion-regulation strategies and resilience among opiate-dependent clients. Patients and Methods: This pretest-posttest quasi-experimental study was performed on 60 patients with substance abuse admitted to Methadone Addiction Treatment Centers and Detox Centers in Sari within three months of therapy for their addiction (from October to December 2013). Then, the participants were randomly assigned to two different groups (n = 30) were examined in two groups of 30 people targeted to be available in the selected population. Participants in all three groups, before and after the intervention, filled out the questionnaires of Schutte emotional intelligence scale and Connor-Davidson resiliency questionnaire. Data were analyzed using the analysis of covariance method. Results: The results showed that an interactive therapy would be significantly more effective than the MMT on improving emotion-regulation strategies and promoting the resilience level among opiate-dependent clients. Moreover, the results showed that cognitive- behavior therapy combined with MMT may improve emotion-regulation strategies, and promote the amount of resiliency and recovery. Conclusions: The cognitive-behavior therapy combined with MMT can improve emotion-regulation strategies and resiliency and thus prevent the substance-abuse relapse. PMID:25821751

Phosphorylation-dependent cleavage regulates von Hippel Lindau proteostasis and function

PubMed Central

German, Peter; Bai, Shanshan; Liu, Xian-De; Sun, Mianen; Zhou, Lijun; Kalra, Sarathi; Zhang, Xuesong; Minelli, Rosalba; Scott, Kenneth L.; Mills, Gordon B.; Jonasch, Eric; Ding, Zhiyong

2016-01-01

Loss of von Hippel Lindau (VHL) protein function is a key driver of VHL diseases, including sporadic and inherited clear cell renal cell carcinoma. Modulation of the proteostasis of VHL, especially missense point-mutated VHL, is a promising approach to augmenting VHL levels and function. VHL proteostasis is regulated by multiple mechanisms including folding, chaperone binding, complex formation, and phosphorylation. Nevertheless, many details underlying the regulations of VHL proteostasis are unknown. VHL is expressed as two variants, VHL30 and VHL19. Furthermore, the long form variant of VHL was often detected as multiple bands by Western blotting. However, how these multiple species of VHL are generated and whether the process regulates VHL proteostasis and function are unknown. We hypothesized that the two major species are generated by VHL protein cleavage, and the cleavage regulates VHL proteostasis and subsequent function. We characterized VHL species using genetic and pharmacologic approaches and showed that VHL was first cleaved at the N-terminus by chymotrypsin C before being directed for proteasomal degradation. Casein kinase 2-mediated phosphorylation at VHL N-terminus was required for the cleavage. Furthermore, inhibition of cleavage stabilized VHL protein, thereby promoting HIF downregulation. Our study reveals a novel mechanism regulating VHL proteostasis and function, which is significant for identifying new drug targets and developing new therapeutic approaches targeting VHL deficiency in VHL diseases. PMID:26973240

Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma.

PubMed

Venkatesh, Humsa S; Tam, Lydia T; Woo, Pamelyn J; Lennon, James; Nagaraja, Surya; Gillespie, Shawn M; Ni, Jing; Duveau, Damien Y; Morris, Patrick J; Zhao, Jean J; Thomas, Craig J; Monje, Michelle

2017-09-28

High-grade gliomas (HGG) are a devastating group of cancers, and represent the leading cause of brain tumour-related death in both children and adults. Therapies aimed at mechanisms intrinsic to glioma cells have translated to only limited success; effective therapeutic strategies will need also to target elements of the tumour microenvironment that promote glioma progression. Neuronal activity promotes the growth of a range of molecularly and clinically distinct HGG types, including adult and paediatric glioblastoma (GBM), anaplastic oligodendroglioma, and diffuse intrinsic pontine glioma (DIPG). An important mechanism that mediates this neural regulation of brain cancer is activity-dependent cleavage and secretion of the synaptic adhesion molecule neuroligin-3 (NLGN3), which promotes glioma proliferation through the PI3K-mTOR pathway. However, the necessity of NLGN3 for glioma growth, the proteolytic mechanism of NLGN3 secretion, and the further molecular consequences of NLGN3 secretion in glioma cells remain unknown. Here we show that HGG growth depends on microenvironmental NLGN3, identify signalling cascades downstream of NLGN3 binding in glioma, and determine a therapeutically targetable mechanism of secretion. Patient-derived orthotopic xenografts of paediatric GBM, DIPG and adult GBM fail to grow in Nlgn3 knockout mice. NLGN3 stimulates several oncogenic pathways, such as early focal adhesion kinase activation upstream of PI3K-mTOR, and induces transcriptional changes that include upregulation of several synapse-related genes in glioma cells. NLGN3 is cleaved from both neurons and oligodendrocyte precursor cells via the ADAM10 sheddase. ADAM10 inhibitors prevent the release of NLGN3 into the tumour microenvironment and robustly block HGG xenograft growth. This work defines a promising strategy for targeting NLGN3 secretion, which could prove transformative for HGG therapy.

Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma

PubMed Central

Venkatesh, Humsa S.; Tam, Lydia T.; Woo, Pamelyn J.; Lennon, James; Nagaraja, Surya; Gillespie, Shawn M.; Ni, Jing; Duveau, Damien Y.; Morris, Patrick J.; Zhao, Jean J.; Thomas, Craig J.; Monje, Michelle

2017-01-01

Summary High-grade gliomas (HGG) are a devastating group of cancers, representing the leading cause of brain tumor-related death in both children and adults. Therapies aimed at mechanisms intrinsic to the glioma cell have translated to only limited success; effective therapeutic strategies will need to also target elements of the tumor microenvironment that promote glioma progression. We recently demonstrated that neuronal activity robustly promotes the growth of a range of molecularly and clinically distinct HGG types, including adult glioblastoma (GBM), anaplastic oligodendroglioma, pediatric GBM, and diffuse intrinsic pontine glioma (DIPG)1. An important mechanism mediating this neural regulation of brain cancer is activity-dependent cleavage and secretion of the synaptic molecule neuroligin-3 (NLGN3), which promotes glioma proliferation through the PI3K-mTOR pathway1. However, neuroligin-3 necessity to glioma growth, proteolytic mechanism of secretion and further molecular consequences in glioma remain to be clarified. Here, we demonstrate a striking dependence of HGG growth on microenvironmental neuroligin-3, elucidate signaling cascades downstream of neuroligin-3 binding in glioma and determine a therapeutically targetable mechanism of secretion. Patient-derived orthotopic xenografts of pediatric GBM, DIPG and adult GBM fail to grow in Nlgn3 knockout mice. Neuroligin-3 stimulates numerous oncogenic pathways, including early focal adhesion kinase activation upstream of PI3K-mTOR, and induces transcriptional changes including upregulation of numerous synapse-related genes in glioma cells. Neuroligin-3 is cleaved from both neurons and oligodendrocyte precursor cells via the ADAM10 sheddase. ADAM10 inhibitors prevent release of neuroligin-3 into the tumor microenvironment and robustly block HGG xenograft growth. This work defines a promising strategy for targeting neuroligin-3 secretion, which could prove transformative for HGG therapy. PMID:28959975

PACAP signaling to DREAM: a cAMP-dependent pathway that regulates cortical astrogliogenesis.

PubMed

Vallejo, Mario

2009-04-01

Astrocytes constitute a very abundant cell type in the mammalian central nervous system and play critical roles in brain function. During development, astrocytes are generated from neural progenitor cells only after these cells have generated neurons. This so called gliogenic switch is tightly regulated by intrinsic factors that inhibit the generation of astrocytes during the neurogenic period. Once neural progenitors acquire gliogenic competence, they differentiate into astrocytes in response to specific extracellular signals. Some of these signals are delivered by neurotrophic cytokines via activation of the gp130-JAK-signal transducer and activator of transcription system, whereas others depend on the activity of pituitary adenylate cyclase-activating polypeptide (PACAP) on specific PAC1 receptors that stimulate the production of cAMP. This results in the activation of the small GTPases Rap1 and Ras, and in the cAMP-dependent entry of extracellular calcium into the cell. Calcium, in turn, stimulates the transcription factor downstream regulatory element antagonist modulator (DREAM), which is bound to specific sites of the promoter of the glial fibrillary acidic protein gene, stimulating its expression during astrocyte differentiation. Lack of DREAM in vivo results in alterations in the number of neurons and astrocytes generated during development. Thus, the PACAP-cAMP-Ca(2+)-DREAM signaling cascade constitutes an important pathway to activate glial-specific gene expression during astrocyte differentiation.

Temperature-dependent regulation of rDNA condensation in Saccharomyces cerevisiae.

PubMed

Shen, Donglai; Skibbens, Robert V

2017-06-03

Chromatin condensation during mitosis produces detangled and discrete DNA entities required for high fidelity sister chromatid segregation during mitosis and positions DNA away from the cleavage furrow during cytokinesis. Regional condensation during G1 also establishes a nuclear architecture through which gene transcription is regulated but remains plastic so that cells can respond to changes in nutrient levels, temperature and signaling molecules. To date, however, the potential impact of this plasticity on mitotic chromosome condensation remains unknown. Here, we report results obtained from a new condensation assay that wildtype budding yeast cells exhibit dramatic changes in rDNA conformation in response to temperature. rDNA hypercondenses in wildtype cells maintained at 37°C, compared with cells maintained at 23°C. This hypercondensation machinery can be activated during preanaphase but readily inactivated upon exposure to lower temperatures. Extended mitotic arrest at 23°C does not result in hypercondensation, negating a kinetic-based argument in which condensation that typically proceeds slowly is accelerated when cells are placed at 37°C. Neither elevated recombination nor reduced transcription appear to promote this hypercondensation. This heretofore undetected temperature-dependent hypercondensation pathway impacts current views of chromatin structure based on conditional mutant gene analyses and significantly extends our understanding of physiologic changes in chromatin architecture in response to hypothermia.

Temperature-dependent regulation of rDNA condensation in Saccharomyces cerevisiae

PubMed Central

Shen, Donglai; Skibbens, Robert V.

2017-01-01

ABSTRACT Chromatin condensation during mitosis produces detangled and discrete DNA entities required for high fidelity sister chromatid segregation during mitosis and positions DNA away from the cleavage furrow during cytokinesis. Regional condensation during G1 also establishes a nuclear architecture through which gene transcription is regulated but remains plastic so that cells can respond to changes in nutrient levels, temperature and signaling molecules. To date, however, the potential impact of this plasticity on mitotic chromosome condensation remains unknown. Here, we report results obtained from a new condensation assay that wildtype budding yeast cells exhibit dramatic changes in rDNA conformation in response to temperature. rDNA hypercondenses in wildtype cells maintained at 37°C, compared with cells maintained at 23°C. This hypercondensation machinery can be activated during preanaphase but readily inactivated upon exposure to lower temperatures. Extended mitotic arrest at 23°C does not result in hypercondensation, negating a kinetic-based argument in which condensation that typically proceeds slowly is accelerated when cells are placed at 37°C. Neither elevated recombination nor reduced transcription appear to promote this hypercondensation. This heretofore undetected temperature-dependent hypercondensation pathway impacts current views of chromatin structure based on conditional mutant gene analyses and significantly extends our understanding of physiologic changes in chromatin architecture in response to hypothermia. PMID:28426272

Density-dependent regulation of growth of BSC-1 cells in cell culture: Control of growth by low molecular weight nutrients

PubMed Central

Holley, Robert W.; Armour, Rosemary; Baldwin, Julia H.

1978-01-01