Primary Brain Calcification Causal PiT2 Transport-Knockout Variants can Exert Dominant Negative Effects on Wild-Type PiT2 Transport Function in Mammalian Cells

The receptors for thyroid hormone (T3R), all-trans-retinoic acid (RAR), and 9-cis-retinoic acid (RXR) bind DNA response elements as homo- and heterodimers. The ligand-binding domains of these receptors contain nine conserved heptads proposed to play a role in dimerization. Mutant receptors with changes in the first or last hydrophobic amino acids in the highly conserved ninth heptad of chick T3R alpha [cT3R alpha(L365R) and cT3R(L372R)] and human RAR alpha (hRAR alpha) [hRAR(M377R) and hRAR(L384R)] reveal that this heptad is essential for certain heterodimeric interactions and for diverse functional activities. Without ligands, wild-type receptors form both homodimers and heterodimers, while these mutants form only homodimers. Surprisingly, the cognate ligand for each mutant enables heterodimer formation between cT3R(L365R) and RAR or RXR and between hRAR(M377R) and T3R or RXR. Both cT3R(L365R) and hRAR(M377R) mediate ligand-dependent transcriptional regulation. However, unlike the wild-type receptor, non-ligand-associated cT3R(L365R) does not suppress the basal activity of certain promoters containing thyroid hormone response elements, suggesting that this silencing effect of T3R is mediated by unliganded heterodimers of T3R and endogenous RXR or related factors. Heterodimerization is also necessary for the strong ligand-independent inhibition between T3R and RAR on a common response element, since the ninth-heptad mutants function as poor inhibitors. However, with a T3R-specific response element, hRAR(M377R) acts as a retinoic acid-dependent inhibitor of cT3R, indicating the importance of heterodimerization for this inhibition. Our studies also suggest that the ninth heptad is necessary for the dominant inhibition of wild-type T3Rs by mutant T3Rs, as has been found for the thyroid hormone-resistant syndrome in humans. Thus, the ninth heptad repeat is required for heterodimerization, suppression of basal promoter activity, and dominant negative effects of T3R and RAR. Lastly, the finding that cT3R(L365R) and hRAR(M377R) require ligands for heterodimer formation also raises the possibility that heterodimeric interactions are mediated by the ninth heptad without ligands but by a second region of these receptors with ligands.

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Analysis of ftsQ Mutant Alleles in Escherichia coli: Complementation, Septal Localization, and Recruitment of Downstream Cell Division Proteins

Journal of Bacteriology ◽

10.1128/jb.184.3.695-705.2002 ◽

2002 ◽

Vol 184 (3) ◽

pp. 695-705 ◽

Cited By ~ 34

Author(s):

Joseph C. Chen ◽

Michael Minev ◽

Jon Beckwith

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Random Mutagenesis ◽

Dominant Negative ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Wild Type ◽

Negative Effects ◽

Mutant Proteins ◽

Division Site

ABSTRACT FtsQ, a 276-amino-acid, bitopic membrane protein, is one of the nine proteins known to be essential for cell division in gram-negative bacterium Escherichia coli. To define residues in FtsQ critical for function, we performed random mutagenesis on the ftsQ gene and identified four alleles (ftsQ2, ftsQ6, ftsQ15, and ftsQ65) that fail to complement the ftsQ1(Ts) mutation at the restrictive temperature. Two of the mutant proteins, FtsQ6 and FtsQ15, are functional at lower temperatures but are unable to localize to the division site unless wild-type FtsQ is depleted, suggesting that they compete poorly with the wild-type protein for septal targeting. The other two mutants, FtsQ2 and FtsQ65, are nonfunctional at all temperatures tested and have dominant-negative effects when expressed in an ftsQ1(Ts) strain at the permissive temperature. FtsQ2 and FtsQ65 localize to the division site in the presence or absence of endogenous FtsQ, but they cannot recruit downstream cell division proteins, such as FtsL, to the septum. These results suggest that FtsQ2 and FtsQ65 compete efficiently for septal targeting but fail to promote the further assembly of the cell division machinery. Thus, we have separated the localization ability of FtsQ from its other functions, including recruitment of downstream cell division proteins, and are beginning to define regions of the protein responsible for these distinct capabilities.

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Evidence for the multimeric structure of ferroportin

Blood ◽

10.1182/blood-2006-06-032516 ◽

2006 ◽

Vol 109 (5) ◽

pp. 2205-2209 ◽

Cited By ~ 47

Author(s):

Ivana De Domenico ◽

Diane McVey Ward ◽

Giovanni Musci ◽

Jerry Kaplan

Keyword(s):

Iron Overload ◽

Cultured Cells ◽

Dominant Negative ◽

Mouse Bone Marrow ◽

C6 Cells ◽

Wild Type ◽

Negative Effects ◽

Dominant Form ◽

Rat Glioma ◽

Glioma C6

Abstract Ferroportin (Fpn) (IREG1, SLC40A1, MTP1) is an iron transporter, and mutations in Fpn result in a genetically dominant form of iron overload disease. Previously, we demonstrated that Fpn is a multimer and that mutations in Fpn are dominant negative. Other studies have suggested that Fpn is not a multimer and that overexpression or epitope tags might affect the localization, topology, or multimerization of Fpn. We generated wild-type Fpn with 3 different epitopes, GFP, FLAG, and c-myc, and expressed these constructs in cultured cells. Co-expression of any 2 different epitope-tagged proteins in the same cell resulted in their quantitative coimmunoprecipitation. Treatment of Fpn-GFP/Fpn-FLAG–expressing cells with crosslinking reagents resulted in the crosslinking of Fpn-GFP and Fpn-FLAG. Western analysis of rat glioma C6 cells or mouse bone marrow macrophages exposed to crosslinking reagents showed that endogenous Fpn is a dimer. These results support the hypothesis that the dominant inheritance of Fpn–iron overload disease is due to the dominant-negative effects of mutant Fpn proteins.

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Translational readthrough of GLA nonsense mutations suggests dominant-negative effects exerted by the interaction of wild-type and missense variants

RNA Biology ◽

10.1080/15476286.2019.1676115 ◽

2019 ◽

Vol 17 (2) ◽

pp. 254-263 ◽

Cited By ~ 4

Author(s):

Silvia Lombardi ◽

Mattia Ferrarese ◽

Saverio Marchi ◽

Paolo Pinton ◽

Mirko Pinotti ◽

...

Keyword(s):

Dominant Negative ◽

Wild Type ◽

Negative Effects ◽

Nonsense Mutations ◽

Missense Variants ◽

Translational Readthrough

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RSR1, a ras-like gene homologous to Krev-1 (smg21A/rap1A): role in the development of cell polarity and interactions with the Ras pathway in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.12.2.758 ◽

1992 ◽

Vol 12 (2) ◽

pp. 758-766 ◽

Cited By ~ 60

Author(s):

R Ruggieri ◽

A Bender ◽

Y Matsui ◽

S Powers ◽

Y Takai ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Polarity ◽

Copy Number ◽

Mammalian Cells ◽

Direct Interaction ◽

Dominant Negative ◽

Yeast Cells ◽

Wild Type ◽

Ras Gene ◽

Ras Pathway

The Saccharomyces cerevisiae ras-like gene RSR1 is particularly closely related to the mammalian gene Krev-1 (also known as smg21A and rap1A). RSR1 was originally isolated as a multicopy suppressor of a cdc24 mutation, which causes an inability to bud or establish cell polarity. Deletion of RSR1 itself does not affect growth but causes a randomization of bud position. We have now constructed mutant alleles of RSR1 encoding proteins with substitutions of Val for Gly at position 12 (analogous to constitutively activated Ras proteins) or Asn for Lys at position 16 (analogous to a dominant-negative Ras protein). rsr1Val-12 could not restore a normal budding pattern to an rsr1 deletion strain but could suppress a cdc24 mutation when overexpressed. rsr1Asn-16 could randomize the budding pattern of a wild-type strain even in low copy number but was not lethal even in high copy number. These and other results suggest that Rsr1p functions only in bud site selection and not in subsequent events of polarity establishment and bud formation, that this function involves a cycling between GTP-bound and GDP-bound forms of the protein, and that the suppression of cdc24 involves direct interaction between Rsr1p[GTP] and Cdc24p. Functional homology between Rsr1p and Krev-1 p21 was suggested by the observations that expression of the latter protein in yeast cells could both suppress a cdc24 mutation and randomize the budding pattern of wild-type cells. As Krev-1 overexpression can suppress ras-induced transformation of mammalian cells, we looked for effects of RSR1 on the S. cerevisiae Ras pathway. Although no suppression of the activated RAS2Val-19 allele was observed, overexpression of rsr1Val-12 suppressed the lethality of strains lacking RAS gene function, apparently through a direct activation of adenyl cyclase. This interaction of Rsr1p with the effector of Ras in S. cerevisiae suggests that Krev-1 may revert ras-induced transformation of mammalian cells by affecting the interaction of ras p21 with its effector.

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p53 Mutants Have Selective Dominant-Negative Effects on Apoptosis but Not Growth Arrest in Human Cancer Cell Lines

Molecular and Cellular Biology ◽

10.1128/mcb.20.3.770-778.2000 ◽

2000 ◽

Vol 20 (3) ◽

pp. 770-778 ◽

Cited By ~ 39

Author(s):

Oscar N. Aurelio ◽

Xiao-Tang Kong ◽

Swati Gupta ◽

Eric J. Stanbridge

Keyword(s):

Human Cancer ◽

Growth Arrest ◽

Carcinoma Cells ◽

Dominant Negative ◽

Wild Type ◽

Osteosarcoma Cells ◽

Negative Effects ◽

Human Cancer Cell Lines ◽

Lung Carcinoma Cells ◽

Apoptotic Pathways

ABSTRACT A bidirectional expression vector that allowed equal transcription of cloned wild-type and mutant p53 cDNAs from the same vector was developed. The vector was transfected into CaLu 6 lung carcinoma cells or Saos-2 osteosarcoma cells. All p53 mutants examined were recessive to wild-type p53 transactivation ofp21WAF1/CIP1 but dominant-negative for transactivation of Bax. An examination of effects on growth arrest and apoptotic pathways indicated that all mutants were recessive to wild type for growth arrest but only three of seven mutants were dominant negative for induction of apoptosis.

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Selective destabilization of polypeptides synthesized from NMD-targeted transcripts

Molecular Biology of the Cell ◽

10.1091/mbc.e21-08-0382 ◽

2021 ◽

Author(s):

Vincent Chu ◽

Qing Feng ◽

Yang Lim ◽

Sichen Shao

Keyword(s):

Mammalian Cells ◽

Premature Termination ◽

Proteasome Inhibition ◽

Premature Termination Codon ◽

Dominant Negative ◽

Termination Codon ◽

Negative Effects ◽

Nonsense Mediated Decay ◽

Mrna Surveillance ◽

Reporter Systems

The translation of mRNAs that contain a premature termination codon (PTC) generates truncated proteins that may have toxic dominant negative effects. Nonsense-mediated decay (NMD) is an mRNA surveillance pathway that degrades PTC-containing mRNAs to limit the production of truncated proteins. NMD activation requires a ribosome terminating translation at a PTC, but what happens to the polypeptides synthesized during the translation cycle needed to activate NMD is incompletely understood. Here, by establishing reporter systems that encode the same polypeptide sequence before a normal or premature termination codon, we show that termination of protein synthesis at a PTC is sufficient to selectively destabilize polypeptides in mammalian cells. Proteasome inhibition specifically rescues the levels of nascent polypeptides produced from PTC-containing mRNAs within an hour, but also disrupts mRNA homeostasis within a few hours. PTC-terminated polypeptide destabilization is also alleviated by depleting the central NMD factor UPF1 or SMG1, the kinase that phosphorylates UPF1 to activate NMD, but not by inhibiting SMG1 kinase activity. Our results suggest that polypeptide degradation is linked to PTC recognition in mammalian cells and clarify a framework to investigate these mechanisms.

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Fluorescence Resonance Energy Transfer Microscopy of the Helicobacter pylori Vacuolating Cytotoxin within Mammalian Cells

Infection and Immunity ◽

10.1128/iai.70.7.3824-3832.2002 ◽

2002 ◽

Vol 70 (7) ◽

pp. 3824-3832 ◽

Cited By ~ 21

Author(s):

David C. Willhite ◽

Dan Ye ◽

Steven R. Blanke

Keyword(s):

Helicobacter Pylori ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Dominant Negative ◽

Wild Type ◽

Vacuolating Cytotoxin ◽

Vacuolated Cells ◽

Mutant Forms

ABSTRACT The Helicobacter pylori vacuolating cytotoxin (VacA) binds and enters mammalian cells to induce cellular vacuolation. To investigate the quaternary structure of VacA within the intracellular environment where toxin cytotoxicity is elaborated, we employed fluorescence resonance energy transfer (FRET) microscopy. HeLa cells coexpressing full-length and truncated forms of VacA fused to cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) were analyzed for FRET to indicate direct associations. These studies revealed that VacA-CFP and VacA-YFP interact within vacuolated cells, supporting the belief that monomer associations at an intracellular site are important for the toxin's vacuolating activity. In addition, the two fragments of proteolytically nicked VacA, p37 and p58, interact when coexpressed within mammalian cells. Because p37 and p58 function in trans when expressed separately within mammalian cells, these data suggest that the mechanism by which these two fragments induce vacuolation requires direct association. FRET microscopy also demonstrated interactions between mutant forms of VacA, as well as wild-type VacA with mutant forms of the toxin within vacuolated cells. Finally, a dominant-negative form of the toxin directly associates with wild-type VacA in cells where vacuolation was not detectable, suggesting that the formation of complexes comprising wild-type and dominant-negative forms of toxin acts to block intracellular toxin function.

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The Naturally Occurring Variant of Estrogen Receptor (ER) ERΔE7 Suppresses Estrogen-Dependent Transcriptional Activation by Both Wild-Type ERα and ERβ

Endocrinology ◽

10.1210/en.2002-0027 ◽

2003 ◽

Vol 144 (7) ◽

pp. 2967-2976 ◽

Cited By ~ 41

Author(s):

Juana M. García Pedrero ◽

Pedro Zuazua ◽

Carlos Martínez-Campa ◽

Pedro S. Lazo ◽

Sofía Ramos

Keyword(s):

Estrogen Receptor ◽

Transcriptional Activation ◽

Mammalian Cells ◽

Activation Function ◽

Inhibitory Effect ◽

Dominant Negative ◽

Wild Type ◽

Independent Manner ◽

Naturally Occurring ◽

Estrogen Response

Abstract We have isolated and functionally characterized the exon 7-skipped variant (ERΔE7) of estrogen receptor (ER)α, which has emerged as the predominant variant expressed in multiple normal and tumoral tissues. However, to date no function has been established for this variant in mammalian cells. ERΔE7 exhibits a negligible ability to bind ligands, insensitivity to allosteric modulation by estrogen and antiestrogens, and loss of estrogen-dependent interaction with p160 coactivators such as SRC-1 and AIB1. ERΔE7 is able to form heterodimers with both ERα and ERβ in a ligand-independent manner. Transient expression experiments in HeLa cells show that increasing amounts of ERΔE7 result in a progressive inhibition of the estrogen-dependent transcriptional activation by both wild-type ERα and ERβ on estrogen response element-driven promoters. The inhibitory effect of ERΔE7 is due to the inhibition of binding of wild-type receptors to their responsive elements. Surprisingly, the activation function (AF)-1-dependent transactivation triggered by epithelial growth factor and phorbol-12-myristate-13-acetate is also abolished in ERΔE7 despite AF1 integrity, suggesting a cross-talk between AF1 and AF2 regions of the receptor. These results indicate that the naturally occurring variant ERΔE7 is a dominant negative receptor that, when expressed at high levels relative to wild-type ERs, might have profound effects on several estrogen-dependent functions.

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A Naturally Occurring hPMS2 Mutation Can Confer a Dominant Negative Mutator Phenotype

Molecular and Cellular Biology ◽

10.1128/mcb.18.3.1635 ◽

1998 ◽

Vol 18 (3) ◽

pp. 1635-1641 ◽

Cited By ~ 63

Author(s):

Nicholas C. Nicolaides ◽

Susan J. Littman ◽

Paul Modrich ◽

Kenneth W. Kinzler ◽

Bert Vogelstein

Keyword(s):

Mammalian Cells ◽

Germ Line ◽

Dominant Negative ◽

Wild Type ◽

Mutator Phenotype ◽

Mmr Genes ◽

Naturally Occurring ◽

Hereditary Nonpolyposis ◽

The Relationship ◽

Present Experimental Evidence

ABSTRACT Defects in mismatch repair (MMR) genes result in a mutator phenotype by inducing microsatellite instability (MI), a characteristic of hereditary nonpolyposis colorectal cancers (HNPCC) and a subset of sporadic colon tumors. Present models describing the mechanism by which germ line mutations in MMR genes predispose kindreds to HNPCC suggest a “two-hit” inactivation of both alleles of a particular MMR gene. Here we present experimental evidence that a nonsense mutation at codon 134 of the hPMS2 gene is sufficient to reduce MMR and induce MI in cells containing a wild-type hPMS2 allele. These results have significant implications for understanding the relationship between mutagenesis and carcinogenesis and the ability to generate mammalian cells with mutator phenotypes.

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