scholarly journals A mutated human homologue to yeast Upf1 protein has a dominant-negative effect on the decay of nonsensecontaining mRNAs in mammalian cells

1998 ◽  
Vol 95 (17) ◽  
pp. 10009-10014 ◽  
Author(s):  
X. Sun ◽  
H. A. Perlick ◽  
H. C. Dietz ◽  
L. E. Maquat
Keyword(s):  
Mammalian Cells ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Human Homologue ◽  
Negative Effect

scholarly journals Crystal structure of MICU2 and comparison with MICU1 reveal insights into the uniporter gating mechanism

2019 ◽  
Vol 116 (9) ◽  
pp. 3546-3555 ◽  
Author(s):  
Kimberli J. Kamer ◽  
Wei Jiang ◽  
Virendar K. Kaushik ◽  
Vamsi K. Mootha ◽  
Zenon Grabarek
Keyword(s):  
Binding Sites ◽  
Mammalian Cells ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Functional Coupling ◽  
Gating Mechanism ◽  
Ef Hand ◽  
Negative Effect ◽  
Oligomeric Complex

The mitochondrial uniporter is a Ca2+-channel complex resident within the organelle’s inner membrane. In mammalian cells the uniporter’s activity is regulated by Ca2+ due to concerted action of MICU1 and MICU2, two paralogous, but functionally distinct, EF-hand Ca2+-binding proteins. Here we present the X-ray structure of the apo form of Mus musculus MICU2 at 2.5-Å resolution. The core structure of MICU2 is very similar to that of MICU1. It consists of two lobes, each containing one canonical Ca2+-binding EF-hand (EF1, EF4) and one structural EF-hand (EF2, EF3). Two molecules of MICU2 form a symmetrical dimer stabilized by highly conserved hydrophobic contacts between exposed residues of EF1 of one monomer and EF3 of another. Similar interactions stabilize MICU1 dimers, allowing exchange between homo- and heterodimers. The tight EF1–EF3 interface likely accounts for the structural and functional coupling between the Ca2+-binding sites in MICU1, MICU2, and their complex that leads to the previously reported Ca2+-binding cooperativity and dominant negative effect of mutation of the Ca2+-binding sites in either protein. The N- and C-terminal segments of the two proteins are distinctly different. In MICU2 the C-terminal helix is significantly longer than in MICU1, and it adopts a more rigid structure. MICU2’s C-terminal helix is dispensable in vitro for its interaction with MICU1 but required for MICU2’s function in cells. We propose that in the MICU1–MICU2 oligomeric complex the C-terminal helices of both proteins form a central semiautonomous assembly which contributes to the gating mechanism of the uniporter.

scholarly journals FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells

2008 ◽  
Vol 181 (3) ◽  
pp. 497-510 ◽  
Author(s):  
Taichi Hara ◽  
Akito Takamura ◽  
Chieko Kishi ◽  
Shun-ichiro Iemura ◽  
Tohru Natsume ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Cellular Functions ◽  
Autophagosome Formation ◽  
Interacting Protein ◽  
Autophagy Induction ◽  
Negative Effect ◽  
And Migration ◽  
Starvation Conditions

Autophagy is a membrane-mediated intracellular degradation system. The serine/threonine kinase Atg1 plays an essential role in autophagosome formation. However, the role of the mammalian Atg1 homologues UNC-51–like kinase (ULK) 1 and 2 are not yet well understood. We found that murine ULK1 and 2 localized to autophagic isolation membrane under starvation conditions. Kinase-dead alleles of ULK1 and 2 exerted a dominant-negative effect on autophagosome formation, suggesting that ULK kinase activity is important for autophagy. We next screened for ULK binding proteins and identified the focal adhesion kinase family interacting protein of 200 kD (FIP200), which regulates diverse cellular functions such as cell size, proliferation, and migration. We found that FIP200 was redistributed from the cytoplasm to the isolation membrane under starvation conditions. In FIP200-deficient cells, autophagy induction by various treatments was abolished, and both stability and phosphorylation of ULK1 were impaired. These results suggest that FIP200 is a novel mammalian autophagy factor that functions together with ULKs.

scholarly journals Entamoeba histolyticaExpressing a Dominant Negative N-Truncated Light Subunit of Its Gal-Lectin Are Less Virulent

2002 ◽  
Vol 13 (12) ◽  
pp. 4256-4265 ◽  
Author(s):  
Uriel Katz ◽  
Serge Ankri ◽  
Tamara Stolarsky ◽  
Yael Nuchamowitz ◽  
David Mirelman
Keyword(s):  
Antisense Rna ◽  
Mammalian Cells ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Impaired Ability ◽  
Reducing Conditions ◽  
Negative Effect ◽  
Terminal Amino ◽  
Heterodimeric Complex

The 260-kDa heterodimeric Gal/GalNAc-specific Lectin (Gal-lectin) of Entamoeba histolytica dissociates under reducing conditions into a heavy (hgl, 170 kDa) and a light subunit (lgl, 35 kDa). We have previously shown that inhibition of expression of the 35-kDa subunit by antisense RNA causes a decrease in virulence. To further understand the role of the light subunit of the Gal-lectin in pathogenesis, amoebae were transfected with plasmids encoding intact, mutated, and truncated forms of the light subunit lgl1 gene. A transfectant in which the 55 N-terminal amino acids of the lgl were removed, overproduced an N-truncated lgl protein (32 kDa), which replaced most of the native 35-kDa lgl in the formation of the Gal-lectin heterodimeric complex and exerted a dominant negative effect. Amoebae transfected with this construct showed a significant decrease in their ability to adhere to and kill mammalian cells as well as in their capacity to form rosettes with and to phagocytose erythrocytes. In addition, immunofluorescence confocal microscopy of this transfectant with anti–Gal-lectin antibodies showed an impaired ability to cap. These results indicate that the light subunit has a role in enabling the clustering of Gal-lectin complexes and that its N-truncation affects this function, which is required for virulence.

Cloning and characterization of dominant negative splice variants of the human histamine H4 receptor

2008 ◽  
Vol 414 (1) ◽  
pp. 121-131 ◽  
Author(s):  
Richard M. van Rijn ◽  
André van Marle ◽  
Paul L. Chazot ◽  
Ellen Langemeijer ◽  
Yongjun Qin ◽  
...  
Keyword(s):  
Mast Cells ◽  
Mammalian Cells ◽  
Splice Variants ◽  
Inflammatory Diseases ◽  
Dominant Negative ◽  
Full Length ◽  
Dominant Negative Effect ◽  
Histamine H4 Receptor ◽  
Negative Effect ◽  
H4 Receptor

The H4R (histamine H4 receptor) is the latest identified member of the histamine receptor subfamily of GPCRs (G-protein-coupled receptors) with potential functional implications in inflammatory diseases and cancer. The H4R is primarily expressed in eosinophils and mast cells and has the highest homology with the H3R. The occurrence of at least twenty different hH3R (human H3R) isoforms led us to investigate the possible existence of H4R splice variants. In the present paper, we report on the cloning of the first two alternatively spliced H4R isoforms from CD34+ cord blood-cell-derived eosinophils and mast cells. These H4R splice variants are localized predominantly intracellularly when expressed recombinantly in mammalian cells. We failed to detect any ligand binding, H4R–ligand induced signalling or constitutive activity for these H4R splice variants. However, when co-expressed with full-length H4R [H4R(390) (H4R isoform of 390 amino acids)], the H4R splice variants have a dominant negative effect on the surface expression of H4R(390). We detected H4R(390)–H4R splice varianthetero-oligomers by employing both biochemical (immunoprecipitation and cell-surface labelling) and biophysical [time-resolved FRET (fluorescence resonance energy transfer)] techniques. mRNAs encoding the H4R splice variants were detected in various cell types and expressed at similar levels to the full-length H4R(390) mRNA in, for example, pre-monocytes. We conclude that the H4R splice variants described here have a dominant negative effect on H4R(390) functionality, as they are able to retain H4R(390) intracellularly and inactivate a population of H4R(390), presumably via hetero-oligomerization.

Functional analysis of mutations in the kinase domain of the TGF-β receptor ALK1 reveals different mechanisms for induction of hereditary hemorrhagic telangiectasia

Blood ◽  
2006 ◽  
Vol 107 (5) ◽  
pp. 1951-1954 ◽  
Author(s):  
Yi Gu ◽  
Peng Jin ◽  
Long Zhang ◽  
Xingang Zhao ◽  
Xia Gao ◽  
...  
Keyword(s):  
Hereditary Hemorrhagic Telangiectasia ◽  
Mammalian Cells ◽  
Kinase Domain ◽  
Dominant Negative ◽  
Receptor Activity ◽  
Dominant Negative Effect ◽  
Loss Of Function ◽  
Negative Effect ◽  
Single Allele ◽  
Β Receptor

Genetic studies in mouse and zebrafish have established the importance of activin receptor-like kinase 1 (ALK1) in formation and remodeling of blood vessels. Single-allele mutations in the ALK1 gene have been linked to the human type 2 hereditary hemorrhagic telangiectasia (HHT2). However, how these ALK1 mutations contribute to this disorder remains unclear. To explore the mechanism underlying effect of the HHT-related ALK1 mutations on receptor activity, we generated 11 such mutants and investigated their signaling activities using reporter assay in mammalian cells and examined their effect on zebrafish embryogenesis. Here we show that some of the HHT2-related mutations generate a dominant-negative effect whereas the others give rise to a null phenotype via loss of protein expression or receptor activity. These data indicate that loss-of-function mutations in a single allele of the ALK1 locus are sufficient to contribute to defects in maintaining endothelial integrity.

scholarly journals Defining the Roles of β-Catenin and Plakoglobin in LEF/T-Cell Factor-Dependent Transcription Using β-Catenin/Plakoglobin-Null F9 Cells

2007 ◽  
Vol 28 (2) ◽  
pp. 825-835 ◽  
Author(s):  
Masayuki Shimizu ◽  
Yoshitaka Fukunaga ◽  
Junichi Ikenouchi ◽  
Akira Nagafuchi
Keyword(s):  
T Cell ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Dominant Negative ◽  
Nuclear Accumulation ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
F9 Cells ◽  
T Cell Factor ◽  
Negative Effect

ABSTRACT β-Catenin functions as a transcriptional regulator in Wnt signaling. Its function is regulated by a specific destruction system. Plakoglobin is a close homologue of β-catenin in mammalian cells and is regulated in a similar fashion. When β-catenin or plakoglobin is exogenously expressed in cells, endogenous β-catenin is stabilized, which complicates estimation of the transcriptional activities of exogenously expressed proteins. To facilitate the design of experiments aimed at investigating the transcriptional activities of β-catenin and plakoglobin, we utilized F9 cells in which we knocked out endogenous β-catenin and/or plakoglobin by gene deletion and exogenously expressed wild-type and mutant β-catenin and/or plakoglobin. We show that C-terminally deleted β-catenin, but not plakoglobin, has a strong dominant-negative effect on transcription without altering the nuclear accumulation of β-catenin. Moreover, we show that Wnt-3a activation of LEF/T-cell factor (TCF)-dependent transcription depends on β-catenin but not on plakoglobin. Using chimeras of β-catenin and plakoglobin, we demonstrate that plakoglobin has the potential to function in transcriptional regulation but is not responsible for Wnt-3a signaling in F9 cells. Our data show that preferential nuclear accumulation of β-catenin is not necessarily linked to its transcriptional activity. We also clearly demonstrate that plakoglobin is insufficient for LEF/TCF-dependent transcriptional activation by Wnt-3a in F9 cells.

scholarly journals A Dominant Negative Effect of eth-1r, a Mutant Allele of the Neurospora crassa S-Adenosylmethionine Synthetase-Encoding Gene Conferring Resistance to the Methionine Toxic Analogue Ethionine

Genetics ◽  
1996 ◽  
Vol 144 (4) ◽  
pp. 1455-1462
Author(s):  
José L Barra ◽  
Mario R Mautino ◽  
Alberto L Rosa
Keyword(s):  
Neurospora Crassa ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Resistant Phenotype ◽  
Negative Effect ◽  
Encoding Gene ◽  
Adenosylmethionine Synthetase ◽  
Adomet Synthetase ◽  
Identical Gene

eth-1r a thermosensitive allele of the Neurospora crassa S-adenosylmethionine (AdoMet) synthetase gene that confers ethionine resistance, has been cloned and sequenced. Replacement of an aspartic amino acid residue (D48 → N48), perfectly conserved in prokaryotic, fungal and higher eukaryotic AdoMet synthetases, was found responsible for both thermosensitivity and ethionine resistance conferred by eth-1r. Gene fusion constructs, designed to overexpress eth-1r in vivo, render transformant cells resistant to ethionine. Dominance of ethionine resistance was further demonstrated in eth-1  +/eth-1r partial diploids carrying identical gene doses of both alleles. Heterozygous eth-1  +/eth-1r cells have, at the same time, both the thermotolerance conferred by eth-1  + and the ethionine-resistant phenotype conferred by eth-1r. AdoMet levels and AdoMet synthetase activities were dramatically decreased in heterozygous eth-1  +/eth-1r cells. We propose that this negative effect exerted by eth-1r results from the in vivo formation of heteromeric eth-1  +/eth-1r AdoMet synthetase molecules.

scholarly journals Restoration of Silencing in Saccharomyces cerevisiae by Tethering of a Novel Sir2-Interacting Protein, Esc8

Genetics ◽  
2002 ◽  
Vol 162 (2) ◽  
pp. 633-645 ◽  
Author(s):  
Guido Cuperus ◽  
David Shore
Keyword(s):  
Protein A ◽  
Dominant Negative ◽  
Class I ◽  
Dominant Negative Effect ◽  
Rna Pol Ii ◽  
Interacting Protein ◽  
Nucleosome Remodeling ◽  
Negative Effect ◽  
Close Homolog

Abstract We previously described two classes of SIR2 mutations specifically defective in either telomeric/HM silencing (class I) or rDNA silencing (class II) in S. cerevisiae. Here we report the identification of genes whose protein products, when either overexpressed or directly tethered to the locus in question, can establish silencing in SIR2 class I mutants. Elevated dosage of SCS2, previously implicated as a regulator of both inositol biosynthesis and telomeric silencing, suppressed the dominant-negative effect of a SIR2-143 mutation. In a genetic screen for proteins that restore silencing when tethered to a telomere, we isolated ESC2 and an uncharacterized gene, (YOL017w), which we call ESC8. Both Esc2p and Esc8p interact with Sir2p in two-hybrid assays, and the Esc8p-Sir2 interaction is detected in vitro. Interestingly, Esc8p has a single close homolog in yeast, the ISW1-complex factor Ioc3p, and has also been copurified with Isw1p, raising the possibility that Esc8p is a component of an Isw1p-containing nucleosome remodeling complex. Whereas esc2 and esc8 deletion mutants alone have only marginal silencing defects, cells lacking Isw1p show a strong silencing defect at HMR but not at telomeres. Finally, we show that Esc8p interacts with the Gal11 protein, a component of the RNA pol II mediator complex.

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