A DNMT3A mutation common in AML exhibits dominant-negative effects in murine ES 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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

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

Download Full-text

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.

Download Full-text

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

Journal of Molecular Neuroscience ◽

10.1007/s12031-016-0868-7 ◽

2016 ◽

Vol 61 (2) ◽

pp. 215-220 ◽

Cited By ~ 19

Author(s):

Frederik Tibert Larsen ◽

Nina Jensen ◽

Jacob Kwasi Autzen ◽

Iben Boutrup Kongsfelt ◽

Lene Pedersen

Keyword(s):

Mammalian Cells ◽

Dominant Negative ◽

Transport Function ◽

Wild Type ◽

Negative Effects ◽

Brain Calcification

Download Full-text

Phenotypic behavior of caveolin-3 R26Q, a mutant associated with hyperCKemia, distal myopathy, and rippling muscle disease

AJP Cell Physiology ◽

10.1152/ajpcell.00166.2003 ◽

2003 ◽

Vol 285 (5) ◽

pp. C1150-C1160 ◽

Cited By ~ 28

Author(s):

Federica Sotgia ◽

Scott E. Woodman ◽

Gloria Bonuccelli ◽

Franco Capozza ◽

Carlo Minetti ◽

...

Keyword(s):

Golgi Complex ◽

Cell Function ◽

Cultured Cells ◽

Dominant Negative ◽

Muscle Disease ◽

Mutant Protein ◽

Dominant Negative Effect ◽

Distal Myopathy ◽

Wild Type ◽

Disease Phenotypes

Four different phenotypes have been associated with CAV3 mutations: limb girdle muscular dystrophy-1C (LGMD-1C), rippling muscle disease (RMD), and distal myopathy (DM), as well as idiopathic and familial hyperCKemia (HCK). Detailed molecular characterization of two caveolin-3 mutations (P104L and ΔTFT), associated with LGMD-1C, shows them to impart a dominant-negative effect on wild-type caveolin-3, rendering it dysfunctional through sequestration in the Golgi complex. Interestingly, substitution of glutamine for arginine at amino acid position 26 (R26Q) of caveolin-3 is associated not only with RMD but also with DM and HCK. However, the phenotypic behavior of the caveolin-3 R26Q mutation has never been evaluated in cultured cells. Thus we characterized the cellular and molecular properties of the R26Q mutant protein to better understand how this mutation can manifest as such distinct disease phenotypes. Here, we show that the caveolin-3 R26Q mutant is mostly retained at the level of the Golgi complex. The caveolin-3 R26Q mutant formed oligomers of a much larger size than wild-type caveolin-3 and was excluded from caveolae-enriched membranes. However, caveolin-3 R26Q did not behave in a dominant-negative fashion when coexpressed with wild-type caveolin-3. Thus the R26Q mutation behaves differently from other caveolin-3 mutations (P104L and ΔTFT) that have been previously characterized. These data provide a possible explanation for the scope of the various disease phenotypes associated with the caveolin-3 R26Q mutation. We propose a haploinsufficiency model in which reduced levels of wild-type caveolin-3, although not rendered dysfunctional due to the caveolin-3 R26Q mutant protein, are insufficient for normal muscle cell function.

Download Full-text

The DFNA15 Deafness Mutation Affects POU4F3 Protein Stability, Localization, and Transcriptional Activity

Molecular and Cellular Biology ◽

10.1128/mcb.23.22.7957-7964.2003 ◽

2003 ◽

Vol 23 (22) ◽

pp. 7957-7964 ◽

Cited By ~ 39

Author(s):

Sigal Weiss ◽

Irit Gottfried ◽

Itay Mayrose ◽

Suvarna L. Khare ◽

Mengqing Xiang ◽

...

Keyword(s):

Hair Cell ◽

Transcriptional Activity ◽

Autosomal Dominant ◽

Dominant Negative ◽

Mutant Protein ◽

Wild Type ◽

Nuclear Entry ◽

Nuclear Localization Signals ◽

Series Of Experiments ◽

Human Mutation

ABSTRACT A mutation in the POU4F3 gene (BRN-3.1, BRN3C) is responsible for DFNA15 (MIM 602459), autosomal-dominant nonsyndromic hearing loss. POU4F3 is a member of the POU family of transcription factors and is essential for inner-ear hair cell maintenance. To test the potential effects of the human POU4F3 mutation, we performed a series of experiments in cell culture to mimic the human mutation. Mutant POU4F3 loses most of its transcriptional activity and most of its ability to bind to DNA and does not function in a dominant-negative manner. Moreover, whereas wild-type POU4F3 is found exclusively in the nucleus, our studies demonstrate that the mutant protein is localized both to the nucleus and the cytoplasm. Two nuclear localization signals were identified; both are essential for proper nuclear entry of POU4F3 protein. We found that the mutant protein half-life is longer than that of the wild type. We propose that the combination of defects caused by the mutation on the function of the POU4F3 transcription factor eventually leads to hair cell morbidity in affected family H members.

Download Full-text

CHCHD2 harboring Parkinson’s disease-linked T61I mutation precipitates inside mitochondria and induces precipitation of wild-type CHCHD2

Human Molecular Genetics ◽

10.1093/hmg/ddaa028 ◽

2020 ◽

Vol 29 (7) ◽

pp. 1096-1106

Author(s):

Tom Cornelissen ◽

Marco Spinazzi ◽

Shaun Martin ◽

Dorien Imberechts ◽

Peter Vangheluwe ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Coiled Coil ◽

Dominant Negative ◽

Mutant Protein ◽

Dominant Negative Effect ◽

Cysteine Residues ◽

Intermembrane Space ◽

Mitochondrial Targeting ◽

Wild Type

Abstract The T61I mutation in coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2), a protein residing in the mitochondrial intermembrane space (IMS), causes an autosomal dominant form of Parkinson’s disease (PD), but the underlying pathogenic mechanisms are not well understood. Here, we compared the subcellular localization and solubility of wild-type (WT) and T61I mutant CHCHD2 in human cells. We found that mitochondrial targeting of both WT and T61I CHCHD2 depended on the four cysteine residues in the C-terminal coiled-coil-helix-coiled-coil-helix (CHCH) domain but not on the N-terminal predicted mitochondrial targeting sequence. The T61I mutation did not interfere with mitochondrial targeting of the mutant protein but induced its precipitation in the IMS. Moreover, T61I CHCHD2 induced increased mitochondrial production of reactive oxygen species and apoptosis, which was prevented by treatment with anti-oxidants. Retention of T61I CHCHD2 in the cytosol through mutation of the cysteine residues in the CHCH domain prevented its precipitation as well as its apoptosis-inducing effect. Importantly, T61I CHCHD2 potently impaired the solubility of WT CHCHD2. In conclusion, our data show that the T61I mutation renders mutant CHCHD2 insoluble inside mitochondria, suggesting loss of function of the mutant protein. In addition, T61I CHCHD2 exerts a dominant-negative effect on the solubility of WT CHCHD2, explaining the dominant inheritance of this form of PD.

Download Full-text

Mutants of FtsZ Targeting the Protofilament Interface: Effects on Cell Division and GTPase Activity

Journal of Bacteriology ◽

10.1128/jb.187.8.2727-2736.2005 ◽

2005 ◽

Vol 187 (8) ◽

pp. 2727-2736 ◽

Cited By ~ 72

Author(s):

Sambra D. Redick ◽

Jesse Stricker ◽

Gina Briscoe ◽

Harold P. Erickson

Keyword(s):

Cell Division ◽

Structural Model ◽

Dominant Negative ◽

Site Directed Mutagenesis ◽

Bottom Surface ◽

Gtpase Activity ◽

Bacterial Cell Division ◽

Wild Type ◽

Negative Effects ◽

A Subunit

ABSTRACT The bacterial cell division protein FtsZ assembles into straight protofilaments, one subunit thick, in which subunits appear to be connected by identical bonds or interfaces. These bonds involve the top surface of one subunit making extensive contact with the bottom surface of the subunit above it. We have investigated this interface by site-directed mutagenesis. We found nine bottom and eight top mutants that were unable to function for cell division. We had expected that some of the mutants might poison cell division substoichiometrically, but this was not found for any mutant. Eight of the bottom mutants exhibited dominant negative effects (reduced colony size) and four completely blocked colony formation, but this required expression of the mutant protein at four to five times the wild-type FtsZ level. Remarkably, the top mutants were even weaker, most showing no effect at the highest expression level. This suggests a directional assembly or treadmilling, where subunit addition is primarily to the bottom end of the protofilament. Selected pairs of top and bottom mutants showed no GTPase activity up to 10 to 20 μM, in contrast to the high GTPase activity of wild-type FtsZ above 1 μM. Overall, these results suggest that in order for a subunit to bind a protofilament at the 1 μM Kd for elongation, it must have functional interfaces at both the top and bottom. This is inconsistent with the present model of the protofilament, as a simple stack of subunits one on top of the other, and may require a new structural model.

Download Full-text