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

2003 ◽  
Vol 23 (22) ◽  
pp. 7957-7964 ◽  
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.

scholarly journals Impaired Peroxisome Proliferator-Activated Receptor γ Function through Mutation of a Conserved Salt Bridge (R425C) in Familial Partial Lipodystrophy

2007 ◽  
Vol 21 (5) ◽  
pp. 1049-1065 ◽  
Author(s):  
Ellen H. Jeninga ◽  
Olivier van Beekum ◽  
Aalt D. J. van Dijk ◽  
Nicole Hamers ◽  
Brenda I. Hendriks-Stegeman ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Transcriptional Activity ◽  
Salt Bridge ◽  
Dominant Negative ◽  
Heterozygous Mutation ◽  
Peroxisome Proliferator ◽  
Wild Type ◽  
Peroxisome Proliferator Activated Receptor ◽  
Partial Lipodystrophy ◽  
Familial Partial Lipodystrophy

Abstract The nuclear receptor peroxisome proliferator-activated receptor (PPAR) γ plays a key role in the regulation of glucose and lipid metabolism in adipocytes by regulating their differentiation, maintenance, and function. A heterozygous mutation in the PPARG gene, which changes an arginine residue at position 425 into a cysteine (R425C), has been reported in a patient with familial partial lipodystrophy subtype 3 (FPLD3). The strong conservation of arginine 425 among nuclear receptors that heterodimerize with retinoic acid X receptor prompted us to investigate the functional consequences of the R425C mutation on PPARγ function. Here we show that this mutant displayed strongly reduced transcriptional activity compared with wild-type PPARγ, irrespective of cell type, promoter context, or ligand, whereas transrepression of nuclear factor-κB activity remained largely intact. Our data indicate that the reduced transcriptional activity of PPARγ R425C is not caused by impaired corepressor release, but due to reduced dimerization with retinoic acid X receptor α in combination with reduced ligand binding and subsequent coactivator binding. As a consequence of these molecular defects, the R425C mutant was less effective in inducing adipocyte differentiation. PPARγ R425C did not inhibit its wild-type counterpart in a dominant-negative manner, suggesting a haploinsufficiency mechanism in at least some FPLD3 patients. Using molecular dynamics simulations, substitution of R425 with cysteine is predicted to cause the formation of an alternative salt bridge. This structural change provides a likely explanation of how mutation of a single conserved residue in a patient with FPLD3 can disrupt the function of the adipogenic transcription factor PPARγ on multiple levels.

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

2003 ◽  
Vol 285 (5) ◽  
pp. C1150-C1160 ◽  
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.

scholarly journals A Missense POU4F3 Variant Associated with Autosomal Dominant Midfrequency Hearing Loss Alters Subnuclear Localization and Transcriptional Capabilities

2021 ◽  
Vol 2021 ◽  
pp. 1-6
Author(s):  
Dan Bai ◽  
Xudong Zhang ◽  
Yu Li ◽  
Jing Ni ◽  
Kai Lan
Keyword(s):  
Hearing Loss ◽  
Transcriptional Activity ◽  
Autosomal Dominant ◽  
Luciferase Activity ◽  
Cytoplasmic Localization ◽  
Cellular Activity ◽  
Wild Type ◽  
Structural Alterations ◽  
Functional Changes ◽  
Subnuclear Localization

Background. The pathogenic variant, POU class 4 transcription factor 3 (POU4F3), is reported to cause autosomal dominant nonsyndromic hearing loss (ADNSHL). Previously, we have examined a four-generation midfrequency sensorineural hearing loss (MFSNHL) family (no. 6126) and established POU4F3 c.602T>C (p.Leu201Pro) as a potential disease-causing variant. Objectives. We explored the structural and functional alterations that the c.602T>C (p.Leu201Pro) variant enforces on the POU4F3 protein. Methods. We utilized wild-type (WT) and mutant (MUT) POU4F3 c.602T>C plasmid incorporation into HeLa cells to assess functional changes, by immunofluorescence and luciferase assays. To predict protein structural alterations in the MUT versus WT POU4F3, we also generated 3D structures to compare both types of POU4F3 proteins. Results. The WT POU4F3 is ubiquitously present in the nucleus, whereas the MUT form of POU4F3 exhibits a more restricted nuclear presence. This finding is different from other publications, which report a cytoplasmic localization of the MUT POU4F3. We also demonstrated that, as opposed to WT POU4F3, the MUT POU4F3 had 40% reduced luciferase activity. Conclusions. The reduced nuclear presence, combined with reduced transcriptional activity, suggests that the POU4F3 c.602T>C variant alters cellular activity and may contribute to the pathogenicity of POU4F3-related hearing loss. It, also, provides more evidence of the pathophysiological characteristics of MFSNHL.

A Dominant-Negative GFI1B Mutation in Gray Platelet Syndrome

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. LBA-3-LBA-3
Author(s):  
Bert A. Van der Reijden ◽  
Davide Monteferrario ◽  
Nikhita Bolar ◽  
Anna Marneth ◽  
Konnie Hebeda ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Autosomal Dominant ◽  
Dominant Negative ◽  
Bleeding Complications ◽  
Wild Type ◽  
Platelet Factor ◽  
Dominant Type ◽  
Platelet Surface ◽  
Gray Platelet Syndrome

Abstract Gray platelet syndrome (GPS) is a hereditary, usually autosomal recessive bleeding disorder caused by defective production of α-granules in platelets. GPS patients show reduced numbers of platelets that are larger and have a typical gray appearance under light microscopy, caused by the lack of α-granules. We describe a large family with an autosomal dominant type of GPS characterized by mild to severe bleeding complications. In addition to large gray platelets, other GPS-associated phenomena like myelofibrosis, thrombocytopenia, and low platelet factor 4 expression were observed in affected individuals. Histopathological examination of a BM biopsy from a patient showed a cellular marrow with increased numbers of megakaryocytes that were pleomorphic in size and shape. Megakaryocytes clustered along BM sinuses and showed dysmorphic stretched features. To determine the disease causing mutation we performed linkage analysis and identified a candidate locus on chromosome 9q34 with a LOD score of 3.9. We considered GFI1B (Growth Factor Independence 1B), located within this region, an excellent candidate gene because of its function as a transcriptional repressor in megakaryocyte development. Sequence analysis identified a nonsense mutation in GFI1B exon 6 (c.859C>T, p.Gln287*) that completely co-segregated with the GPS disease in this family. The mutated transcript predicts a 44 amino acid C-terminally truncated protein, GFI1BTr. The truncation is located within zinc finger 5 of GFI1B, deleting all of its four amino acids that directly interact with DNA. Luciferase gene reporter assays showed that GFI1BTr was unable to repress gene expression. Importantly, GFI1BTr inhibited gene repression mediated by wild type GFI1B, indicating that the mutant interferes with wild type GFI1B in a dominant-negative manner. To validate that GFI1BTr adversely affects normal GFI1B, we expressed the mutant in mouse bone marrow cells followed by induction of megakaryocytic differentiation. Compared to control cells, GFI1BTr-positive megakaryocytes showed dysplastic features including hypolobulation of the nuclei, irregular contours and multiple separate nuclei, that were very similar to those observed in patient cells. This indicates that GFI1BTr causes megakaryocytic abnormalities and that it functions in a dominant-negative manner. GFI1B silencing inhibits the development of human megakaryocyte colonies in vitro. We observed that megakaryocyte colony forming cells were significantly more frequent in patient bone marrow compared to controls. In addition, patient-derived megakaryocyte colonies were significantly larger compared to controls. Immunophenotypic analyses of peripheral blood showed no differences in myeloid and erythroid lineages and the platelet markers GP3B, ITGA2B and ITGB3 among affected an non-affected individuals. However, within the ITGA2B/CD41-positive platelet population, 5 of 6 affected members showed a marked decrease in the platelet surface membrane glycoprotein 1b-α (GP1BA/CD42b), compared to unaffected members. In addition, a strong expression of CD34, which is usually confined to immature hematopoietic progenitors, was detected on platelets from all studied affected individuals. Immunostaining of a BM biopsy from a patient showed the presence of ITGB3/CD61 positive megakaryocytes that intensely expressed CD34. Electron microscopy analysis showed megakaryocytes with few, small, irregularly shaped and centrally located α-granules characterized by an extensive peripheral cytoplasm with irregular proplatelets, largely devoid of cell organelles. To test whether these abnormalities were cell intrinsic, we stimulated CD34+ cells from two patients to differentiate along the megakaryocytic lineage in vitro. Megakaryocytic cells showed dysplastic features reminiscent of those observed in the bone marrow aspirates. In addition, increased CD34 and decreased GP1BA/CD42b expression were observed on megakaryocytes, indicating that GFI1BTr-induced abnormalities are intrinsic to the cell. In summary, we have identified GFI1B as a causative gene in autosomal dominant GPS. GFI1BTr acts in a dominant-negative manner over wild type GFI1B and affects the development of megakaryocytes and platelets, demonstrating a pivotal role of GFI1B in governing normal megakaryopoiesis and platelet production. Disclosures: No relevant conflicts of interest to declare.

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

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.

scholarly journals A Novel, C-Terminal Dominant Negative Mutation of the GR Causes Familial Glucocorticoid Resistance through Abnormal Interactions with p160 Steroid Receptor Coactivators

2002 ◽  
Vol 87 (6) ◽  
pp. 2658-2667 ◽  
Author(s):  
Alessandra Vottero ◽  
Tomoshige Kino ◽  
Herve Combe ◽  
Pierre Lecomte ◽  
George P. Chrousos
Keyword(s):  
Ligand Binding ◽  
Transcriptional Activity ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Glucocorticoid Resistance ◽  
Base Substitution ◽  
Wild Type ◽  
Nuclear Speckles ◽  
Dominant Negative Mutation ◽  
Type Receptor

Primary cortisol resistance is a rare, inherited or sporadic form of generalized end-organ insensitivity to glucocorticoids. Here, we report a kindred in which affected members had a heterozygous T to G base substitution at nucleotide 2373 of exon 9α of the GR gene, causing substitution of Ile by Met at position 747. This mutation was located close to helix 12, at the C terminus of the ligand-binding domain, which has a pivotal role in the formation of activation function (AF)-2, a subdomain that interacts with p160 coactivators. The affinity of the mutant GR for dexamethasone was decreased by about 2-fold, and its transcriptional activity on the glucocorticoid-responsive mouse mammary tumor virus promoter was compromised by 20- to 30-fold. In addition, the mutant GR functioned as a dominant negative inhibitor of wild-type receptor-induced transactivation. The mutant GR through its intact AF-1 domain bound to a p160 coactivator, but failed to do so through its AF-2 domain. Overexpression of a p160 coactivator restored the transcriptional activity and reversed the negative transdominant activity of the mutant GR. Interestingly, green fluorescent protein (GFP)-fused GRαI747M had a slight delay in its translocation from the cytoplasm into the nucleus and formed coarser nuclear speckles than GFP-fused wild-type GRα. Similarly, a GFP-fused p160 coactivator had a distinctly different distribution in the nucleus in the presence of mutant vs. wild-type receptor, presenting also as coarser speckling. We conclude that the mutation at amino acid 747 of the GR causes familial, autosomal dominant glucocorticoid resistance by decreasing ligand binding affinity and transcriptional activity, and by exerting a negative transdominant effect on the wild-type receptor. The mutant receptor has an ineffective AF-2 domain, which leads to an abnormal interaction with p160 coactivators and a distinct nuclear distribution of both.

scholarly journals The cellular fate of mutant rhodopsin: quality control, degradation and aggresome formation

2002 ◽  
Vol 115 (14) ◽  
pp. 2907-2918 ◽  
Author(s):  
Richard S. Saliba ◽  
Peter M. G. Munro ◽  
Philip J. Luthert ◽  
Michael E. Cheetham
Keyword(s):  
Quality Control ◽  
Retinitis Pigmentosa ◽  
Autosomal Dominant ◽  
Cultured Cells ◽  
State Level ◽  
Detailed Examination ◽  
Mutant Protein ◽  
Wild Type ◽  
Autosomal Dominant Retinitis Pigmentosa ◽  
Aggresome Formation

Mutations in the photopigment rhodopsin are the major cause of autosomal dominant retinitis pigmentosa. The majority of mutations in rhodopsin lead to misfolding of the protein. Through the detailed examination of P23H and K296E mutant opsin processing in COS-7 cells, we have shown that the mutant protein does not accumulate in the Golgi, as previously thought, instead it forms aggregates that have many of the characteristic features of an aggresome. The aggregates form close to the centrosome and lead to the dispersal of the Golgi apparatus. Furthermore, these aggregates are ubiquitinated, recruit cellular chaperones and disrupt the intermediate filament network. Mutant opsin expression can disrupt the processing of normal opsin, as co-transfection revealed that the wild-type protein is recruited to mutant opsin aggregates. The degradation of mutant opsin is dependent on the proteasome machinery. Unlike the situation with ΔF508-CFTR, proteasome inhibition does not lead to a marked increase in aggresome formation but increases the retention of the protein within the ER, suggesting that the proteasome is required for the efficient retrotranslocation of the mutant protein. Inhibition of N-linked glycosylation with tunicamycin leads to the selective retention of the mutant protein within the ER and increases the steady state level of mutant opsin. Glycosylation, however, has no influence on the biogenesis and targeting of wild-type opsin in cultured cells. This demonstrates that N-linked glycosylation is required for ER-associated degradation of the mutant protein but is not essential for the quality control of opsin folding. The addition of 9-cis-retinal to the media increased the amount of P23H, but not K296E, that was soluble and reached the plasma membrane. These data show that rhodopsin autosomal dominant retinitis pigmentosa is similar to many other neurodegenerative diseases in which the formation of intracellular protein aggregates is central to disease pathogenesis, and they suggest a mechanism for disease dominance.

scholarly journals A DNMT3A mutation common in AML exhibits dominant-negative effects in murine ES cells

Blood ◽  
2013 ◽  
Vol 122 (25) ◽  
pp. 4086-4089 ◽  
Author(s):  
Soo Jin Kim ◽  
Hongbo Zhao ◽  
Swanand Hardikar ◽  
Anup Kumar Singh ◽  
Margaret A. Goodell ◽  
...  
Keyword(s):  
Es Cells ◽  
Dominant Negative ◽  
Mutant Protein ◽  
Wild Type ◽  
Negative Effects ◽  
Key Points

Key Points Mouse Dnmt3a R878H (human R882H) mutant protein inhibits wild-type Dnmt3a/Dnmt3b in murine ES cells, suggesting dominant-negative effects.

scholarly journals Haploinsufficiency of MSX1: a Mechanism for Selective Tooth Agenesis

1998 ◽  
Vol 18 (10) ◽  
pp. 6044-6051 ◽  
Author(s):  
Gezhi Hu ◽  
Heleni Vastardis ◽  
Andrew J. Bendall ◽  
Zhaoqing Wang ◽  
Malcolm Logan ◽  
...  
Keyword(s):  
Missense Mutation ◽  
Transcriptional Repression ◽  
Autosomal Dominant ◽  
Ectopic Expression ◽  
Dominant Negative ◽  
Tooth Agenesis ◽  
Wild Type ◽  
Proline Substitution ◽  
Functional Analyses

ABSTRACT Previously, we found that the cause of autosomal dominant selective tooth agenesis in one family is a missense mutation resulting in an arginine-to-proline substitution in the homeodomain of MSX1. To determine whether the tooth agenesis phenotype may result from haploinsufficiency or a dominant-negative mechanism, we have performed biochemical and functional analyses of the mutant protein Msx1(R31P). We show that Msx1(R31P) has perturbed structure and reduced thermostability compared with wild-type Msx1. As a consequence, the biochemical activities of Msx1(R31P) are severely impaired, since it exhibits little or no ability to interact with DNA or other protein factors or to function in transcriptional repression. We also show that Msx1(R31P) is inactive in vivo, since it does not display the activities of wild-type Msx1 in assays of ectopic expression in the limb. Furthermore, Msx1(R31P) does not antagonize the activity of wild-type Msx1 in any of these assays. Because Msx1(R31P) appears to be inactive and does not affect the action of wild-type Msx1, we propose that the phenotype of affected individuals with selective tooth agenesis is due to haploinsufficiency.

scholarly journals Mapping of a Domain Required for Protein-Protein Interactions and Inhibitory Activity of a Helicobacter pylori Dominant-Negative VacA Mutant Protein

2006 ◽  
Vol 74 (4) ◽  
pp. 2093-2101 ◽  
Author(s):  
Victor J. Torres ◽  
Mark S. McClain ◽  
Timothy L. Cover
Keyword(s):  
Amino Acids ◽  
Helicobacter Pylori ◽  
Protein Interactions ◽  
Inhibitory Activity ◽  
Mammalian Cells ◽  
Mutational Analysis ◽  
Dominant Negative ◽  
Mutant Protein ◽  
Wild Type ◽  
Protein Protein Interactions

ABSTRACT The Helicobacter pylori VacA toxin is an 88-kDa secreted protein that causes multiple alterations in mammalian cells and is considered an important virulence factor in the pathogenesis of peptic ulcer disease and gastric cancer. We have shown previously that a VacA mutant protein lacking amino acids 6 to 27 (Δ6-27p88 VacA) is able to inhibit many activities of wild-type VacA in a dominant-negative manner. Analysis of a panel of C-terminally truncated Δ6-27p88 VacA proteins indicated that a fragment containing amino acids 1 to 478 (Δ6-27p48) exhibited a dominant-negative phenotype similar to that of the full-length Δ6-27p88 VacA protein. In contrast, a shorter VacA fragment lacking amino acids 6 to 27 (Δ6-27p33) did not exhibit detectable inhibitory activity. The Δ6-27p48 protein physically interacted with wild-type p88 VacA, whereas the Δ6-27p33 protein did not. Mutational analysis indicated that amino acids 351 to 360 are required for VacA protein-protein interactions and for dominant-negative inhibitory activity. The C-terminal portion (p55 domain) of wild-type p88 VacA could complement either Δ6-27p33 or Δ(6-27/351-360)p48, reconstituting dominant-negative inhibitory activity. Collectively, our data provide strong evidence that the inhibitory properties of dominant-negative VacA mutant proteins are dependent on interactions between the mutant VacA proteins and wild-type VacA, and they allow mapping of a domain involved in the formation of oligomeric VacA complexes.

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