scholarly journals Regulated Disruption of Inositol 1,4,5-Trisphosphate Signaling in Caenorhabditis elegans Reveals New Functions in Feeding and Embryogenesis

2002 ◽  
Vol 13 (4) ◽  
pp. 1329-1337 ◽  
Author(s):  
Denise S. Walker ◽  
Nicholas J.D. Gower ◽  
Sung Ly ◽  
Gemma L. Bradley ◽  
Howard A. Baylis
Keyword(s):  
Binding Domain ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Animal Cells ◽  
Specific Expression ◽  
C Elegans ◽  
Wide Range ◽  
Inositol 1,4,5 Trisphosphate ◽  
Negative Effect ◽  
Pharyngeal Pumping

Inositol 1,4,5-trisphosphate (IP3) is an important second messenger in animal cells and is central to a wide range of cellular responses. The major intracellular activity of IP3 is to regulate release of Ca2+ from intracellular stores through IP3 receptors (IP3Rs). We describe a system for the transient disruption of IP3 signaling in the model organismCaenorhabditis elegans. The IP3 binding domain of the C. elegans IP3R, ITR-1, was expressed from heat shock-induced promoters in live animals. This results in a dominant-negative effect caused by the overexpressed IP3 binding domain acting as an IP3“sponge.” Disruption of IP3 signaling resulted in disrupted defecation, a phenotype predicted by previous genetic studies. This approach also identified two new IP3-mediated processes. First, the up-regulation of pharyngeal pumping in response to food is dependent on IP3 signaling. RNA-mediated interference studies and analysis of itr-1mutants show that this process is also IP3R dependent. Second, the tissue-specific expression of the dominant-negative construct enabled us to circumvent the sterility associated with loss of IP3 signaling through the IP3R and thus determine that IP3-mediated signaling is required for multiple steps in embryogenesis, including cytokinesis and gastrulation.

scholarly journals P0072GENERATION OF A C. ELEGANS MODEL TO STUDY THE INTERALLELIC INTERACTIONS OF PODOCIN

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Regina Légrádi ◽  
Magdolna Tália Keszthelyi ◽  
Tímea Köles ◽  
Kálmán Tory
Keyword(s):  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Chromosomal Integration ◽  
Rescue Effect ◽  
C Elegans ◽  
Filtration Barrier ◽  
Mutant Strains ◽  
Negative Effect ◽  
Touch Sensation ◽  
Fluorescent Tag

Abstract Background and Aims NPHS2 is the most frequently mutated gene in steroid-resistant nephrotic syndrome. NPHS2 encodes podocin, a key component of the glomerular filtration barrier. Podocin is a 42 kDa integral membrane protein, which accumulates in lipid raft microdomains at the podocyte slit diaphragm and is known to form oligomers. The c.686G>A, p.R229Q is the most common non-silent variant of NPHS2. We formerly described that R229Q is pathogenic only when trans-associated to specific 3’ missense mutations on the other parental allele. These C-terminal podocin mutants exert a dominant negative effect on R229Q podocin and retain it in intracellular compartments. As such, R229Q is the first variant in human genetics with a mutation-dependent pathogenicity. Based on FRET analysis and structural modeling, we formerly showed that the dominant negative effect is mediated by an altered oligomerization. However, the pathogenicity of several [mutation];[R229Q] associations is still in question and the identification of other rare NPHS2 variants with a mutation-dependent pathogenicity require an in vivo model. We therefore aimed to generate a Caenorhabditis elegans model, deficient for the homologous gene of NPHS2 (mec-2) and coexpressing differently (GFP- and mCherry-) tagged human podocin pairs. MEC-2 shares 45% identity and 83% similarity over 275aa (72%) of podocin (383aa). Expressed in six neurons, it is responsible for gentle-touch mechanosensation. The mec-2 mutants are insensitive to gentle touch in the center part of the body. Method Vectors encoding C-terminal GFP- or mCherry-tagged MEC-2 or human podocin with C. elegans codon optimization under mec-2 promoter and a selection marker (unc-119) were generated. Double (mec-2 and unc-119) mutant strains were established. Mutant strains were transformed by microparticle bombardment. The gentle-touch mechanosensation was examined by cat’s whiskers in a blinded experiment. Results The expression pattern of GFP and mCherry under mec-2 promoter corresponded to the six neurons responsible for gentle-touch sensation, indicating the proper functioning of the promoter. Strains with extrachromosomal MEC-2 or podocin coding vectors were successfully established (GFP-tagged MEC-2: n= 55 strains, GFP-tagged podocin: n= 81, mCherry-tagged podocin: n= 18). However, we found no rescue of the gentle touch sensation in any of them in blinded experiments. We hypothesized that either the fluorescent tag or the lack of chromosomal integration prevents the rescue effect of MEC-2. We therefore aimed to achieve self-cleaving of MEC-2 and the fluorescent tag, and inserted a T2A self-cleaving peptide-encoding sequence between them. To achieve chromosomal integration, we are implementing the MosSCI (Mos1-mediated Single Copy Insertion) technique. Conclusion Once the rescue with MEC-2 is achieved, the rescue effect of wild type and next different human podocin variant(s) will be aimed to analyze. The generation of the first animal model to study human interallelic interactions is challenging.

scholarly journals Yeast formin Bni1p has multiple localization regions that function in polarized growth and spindle orientation

2012 ◽  
Vol 23 (3) ◽  
pp. 412-422 ◽  
Author(s):  
Wenyu Liu ◽  
Felipe H. Santiago-Tirado ◽  
Anthony Bretscher
Keyword(s):  
Coiled Coil ◽  
Binding Domain ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Spindle Orientation ◽  
Bud Neck ◽  
Negative Effect ◽  
The Right ◽  
Actin Cables ◽  
And Function

Formins are conserved proteins that assemble unbranched actin filaments in a regulated, localized manner. Budding yeast's two formins, Bni1p and Bnr1p, assemble actin cables necessary for polarized cell growth and organelle segregation. Here we define four regions in Bni1p that contribute to its localization to the bud and at the bud neck. The first (residues 1–333) requires dimerization for its localization and encompasses the Rho-binding domain. The second (residues 334–821) covers the Diaphanous inhibitory–dimerization–coiled coil domains, and the third is the Spa2p-binding domain. The fourth region encompasses the formin homology 1–formin homology 2–COOH region of the protein. These four regions can each localize to the bud cortex and bud neck at the right stage of the cell cycle independent of both F-actin and endogenous Bni1p. The first three regions contribute cumulatively to the proper localization of Bni1p, as revealed by the effects of progressive loss of these regions on the actin cytoskeleton and fidelity of spindle orientation. The fourth region contributes to the localization of Bni1p in tiny budded cells. Expression of mislocalized Bni1p constructs has a dominant-negative effect on both growth and nuclear segregation due to mislocalized actin assembly. These results define an unexpected complexity in the mechanism of formin localization and function.

Dominant Negative Form of PAX5 Is Generated by Multimerization of Its DNA Binding Domain

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 25-25
Author(s):  
Norihiko Kawamata ◽  
Mario Pennella ◽  
Jennifer Woo ◽  
Arnold Berk ◽  
H. Phillip Koeffler
Keyword(s):  
Dna Binding ◽  
Fusion Protein ◽  
Binding Affinity ◽  
Alpha Helix ◽  
Binding Domain ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Dna Binding Domain ◽  
Negative Effect ◽  
Dna Binding Affinity

Abstract Abstract 25 We have previously cloned a number of fusion genes involving PAX5 in acute lymphoblastic leukemia (ALL) (Kawamata N. et al. PNAS, 2008). All of these fusion products exerted a dominant negative effect over the wild-type PAX5. One of these fusion PAX5 proteins, PAX5-C20orf112, was generated by the fusion between the DNA binding domain of PAX5 (PAX5DB) and the C-terminal end of C20orf112. To find the mechanism of the dominant negative effect of the PAX5-C20 fusion, we performed Fluorescence Recovery After Photobleaching (FRAP) assay using PAX5-C20 and PAX5wt constructs connected with Yellow Fluorescence Proteins (YFP). Results showed extremely strong DNA binding affinity of PAX5-C20 compared to PAX5wt. FRAP experiments using deletion mutants of PAX5-C20 showed that both the DNA binding domain and C-terminal alpha-helix region of C20 were indispensable for this strong binding to DNA. Fluorescence Resonance Energy Transfer (FRET) assay, Bi-molecule Fluorescence Complementation (BiFC) assay, and co-immunoprecipitation assay showed that C-terminal end of C20 containing an alpha-helix region encodes a homo-multimerization domain. To confirm that homo-multimerization of PAX5DB increases DNA binding affinity, PAX5DB was fused to the inducible dimerization motif of FKBP (PAX5DB-FK). PAX5DB-FK increased its DNA binding affinity with addition of FKBP ligand inducing homo-dimerization. We also fused PAX5DB to homo-dimerization of MAX (bHLH domain), or tetramerization domain of TP53. FRAP assays showed that homo-dimerization increased its DNA binding activity, and homo-tetramerization further increased its DNA binding and its dominant negative effect over PAX5wt. PAX5-ETV6, also a common fusion protein in ALL, exerts a dominant negative effect over PAX5wt. The ETV6 region of this fusion protein has a multimerization (SAM) domain and the PAX5DB-ETV6SAM mutant protein also showed a dominant negative effect and strong binding to DNA. Importantly, in further studies, co-expression of PAX5-C20 and the YFP-C20-alpha-helix-region diminished the strong DNA binding and the dominant negative activity of the fusion protein. Our data show that multimerization of the DNA binding domain of PAX5 induces strong DNA binding activity, leading to its dominant negative effect over the wild type transcription factor. We believe this represents a new paradigm explaining how a number of fusion genes containing a DB motif from one protein and a multimerization motif from the other partner, can behave in a dominant negative fashion. These observations suggest that peptides/ small molecules inhibiting the multimerization of these oncogenic fusion transcription factors can be promising reagents for treating cancers. Disclosures: No relevant conflicts of interest to declare.

scholarly journals p53 Isoforms and Their Implications in Cancer

Cancers ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 288 ◽  
Author(s):  
Maximilian Vieler ◽  
Suparna Sanyal
Keyword(s):  
Dna Binding ◽  
Tp53 Gene ◽  
Binding Domain ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Evolutionary Significance ◽  
Dna Binding Domain ◽  
Negative Effect ◽  
P53 Isoforms ◽  
P53 Inactivation

In this review we focus on the major isoforms of the tumor-suppressor protein p53, dysfunction of which often leads to cancer. Mutations of the TP53 gene, particularly in the DNA binding domain, have been regarded as the main cause for p53 inactivation. However, recent reports demonstrating abundance of p53 isoforms, especially the N-terminally truncated ones, in the cancerous tissues suggest their involvement in carcinogenesis. These isoforms are ∆40p53, ∆133p53, and ∆160p53 (the names indicate their respective N-terminal truncation). Due to the lack of structural and functional characterizations the modes of action of the p53 isoforms are still unclear. Owing to the deletions in the functional domains, these isoforms can either be defective in DNA binding or more susceptive to altered ‘responsive elements’ than p53. Furthermore, they may exert a ‘dominant negative effect’ or induce more aggressive cancer by the ‘gain of function’. One possible mechanism of p53 inactivation can be through tetramerization with the ∆133p53 and ∆160p53 isoforms—both lacking part of the DNA binding domain. A recent report and unpublished data from our laboratory also suggest that these isoforms may inactivate p53 by fast aggregation—possibly due to ectopic overexpression. We further discuss the evolutionary significance of the p53 isoforms.

scholarly journals The recessive phenotype displayed by a dominant negative microphthalmia-associated transcription factor mutant is a result of impaired nucleation potential.

1996 ◽  
Vol 16 (3) ◽  
pp. 1203-1211 ◽  
Author(s):  
K Takebayashi ◽  
K Chida ◽  
I Tsukamoto ◽  
E Morii ◽  
H Munakata ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Nuclear Localization ◽  
T Antigen ◽  
Binding Domain ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Negative Effect

In the DNA binding domain of microphthalmia-associated transcription factor (MITF), four mutations are reported: mi, Mi wh, mi ew, and mi or. MITFs encoded by the mi, Mi wh, mi ew, and Mi or mutant alleles (mi-MITF, Mi wh-MITF, Mi ew-MITF, and Mi or-MITF, respectively) interfered with the DNA binding of wild-type MITF, TFE3, and another basic helix-loop-helix leucine zipper protein in vitro. Polyclonal antibody against MITF was produced and used for investigating the subcellular localization of mutant MITFs. Immunocytochemistry and immunoblotting revealed that more than 99% of wild-type MITF and Mi wh-MITF located in nuclei of transfected NIH 3T3 and 293T cells. In contrast, mi-MITF predominantly located in the cytoplasm of cells transfected with the corresponding plasmid. When the immunoglobulin G (IgG)-conjugated peptides representing a part of the DNA binding domain containing mi and Mi wh mutations were microinjected into the cytoplasm of NRK49F cells, wild-type peptide and Mi wh-type peptide-IgG conjugate localized in nuclei but mi-type peptide-IgG conjugate was detectable only in the cytoplasm. It was also demonstrated that the nuclear translocation potential of Mi or-MITF was normal but that Mi ew-MITF was impaired as well as mi-MITF. In cotransfection assay, a strong dominant negative effect of Mi wh-MITF against wild-type MITF-dependent transactivation system on tyrosinase promoter was observed, but mi-MITF had a small effect. However, by the conjugation of simian virus 40 large-T-antigen-derived nuclear localization signal to mi-MITF, the dominant negative effect was enhanced. Furthermore, we demonstrated that the interaction between wild-type MITF and mi-MITF occurred in the cytoplasm and that mi-MITF had an inhibitory effect on nuclear localization potential of wild-type MITF.

scholarly journals Truncation of TRIM5 in the Feliformia Explains the Absence of Retroviral Restriction in Cells of the Domestic Cat

2009 ◽  
Vol 83 (16) ◽  
pp. 8270-8275 ◽  
Author(s):  
William A. McEwan ◽  
Torsten Schaller ◽  
Laura M. Ylinen ◽  
Margaret J. Hosie ◽  
Greg J. Towers ◽  
...  
Keyword(s):  
Mammalian Species ◽  
Ectopic Expression ◽  
Binding Domain ◽  
Dominant Negative ◽  
Domestic Cat ◽  
Dominant Negative Effect ◽  
Negative Effect ◽  
In Cells

ABSTRACT TRIM5α mediates a potent retroviral restriction phenotype in diverse mammalian species. Here, we identify a TRIM5 transcript in cat cells with a truncated B30.2 capsid binding domain and ablated restrictive function which, remarkably, is conserved across the Feliformia. Cat TRIM5 displayed no restriction activity, but ectopic expression conferred a dominant negative effect against human TRIM5α. Our findings explain the absence of retroviral restriction in cat cells and suggest that disruption of the TRIM5 locus has arisen independently at least twice in the Carnivora, with implications concerning the evolution of the host and pathogen in this taxon.

scholarly journals The Actin-Binding Domain of Slac2-a/Melanophilin Is Required for Melanosome Distribution in Melanocytes

2003 ◽  
Vol 23 (15) ◽  
pp. 5245-5255 ◽  
Author(s):  
Taruho S. Kuroda ◽  
Hiroyoshi Ariga ◽  
Mitsunori Fukuda
Keyword(s):  
Amino Acids ◽  
Binding Domain ◽  
Dominant Negative ◽  
Actin Binding ◽  
Dominant Negative Effect ◽  
C Terminus ◽  
Defective Mutant ◽  
Actin Binding Domain ◽  
Negative Effect ◽  
Myosin Va

ABSTRACT Melanosomes containing melanin pigments are transported from the cell body of melanocytes to the tips of their dendrites by a combination of microtubule- and actin-dependent machinery. Three proteins, Rab27A, myosin Va, and Slac2-a/melanophilin (a linker protein between Rab27A and myosin Va), are known to be essential for proper actin-based melanosome transport in melanocytes. Although Slac2-a directly interacts with Rab27A and myosin Va via its N-terminal region (amino acids 1 to 146) and the middle region (amino acids 241 to 405), respectively, the functional importance of the putative actin-binding domain of the Slac2-a C terminus (amino acids 401 to 590) in melanosome transport has never been elucidated. In this study we showed that formation of a tripartite protein complex between Rab27A, Slac2-a, and myosin Va alone is insufficient for peripheral distribution of melanosomes in melanocytes and that the C-terminal actin-binding domain of Slac2-a is also required for proper melanosome transport. When a Slac2-a deletion mutant (ΔABD) or point mutant (KA) that lacks actin-binding ability was expressed in melanocytes, the Slac2-a mutants induced melanosome accumulation in the perinuclear region, possibly by a dominant negative effect, the same as the Rab27A-binding-defective mutant of Slac2-a or the myosin Va-binding-defective mutant. Our findings indicate that Slac2-a organizes actin-based melanosome transport in cooperation with Rab27A, myosin Va, and actin.

scholarly journals Genotype-Phenotype Comparison in POGZ-Related Neurodevelopmental Disorders by Using Clinical Scoring

Genes ◽  
2022 ◽  
Vol 13 (1) ◽  
pp. 154
Author(s):  
Dóra Nagy ◽  
Sarah Verheyen ◽  
Kristen M. Wigby ◽  
Artem Borovikov ◽  
Artem Sharkov ◽  
...  
Keyword(s):  
Neurodevelopmental Disorders ◽  
Large Scale ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Published Data ◽  
Rich Domain ◽  
Wide Range ◽  
Phenotype Comparison ◽  
Negative Effect ◽  
Clinical Scoring

POGZ-related disorders (also known as White-Sutton syndrome) encompass a wide range of neurocognitive abnormalities and other accompanying anomalies. Disease severity varies widely among POGZ patients and studies investigating genotype-phenotype association are scarce. Therefore, our aim was to collect data on previously unreported POGZ patients and perform a large-scale phenotype-genotype comparison from published data. Overall, 117 POGZ patients′ genotype and phenotype data were included in the analysis, including 12 novel patients. A severity scoring system was developed for the comparison. Mild and severe phenotypes were compared with the types and location of the variants and the predicted presence or absence of nonsense-mediated RNA decay (NMD). Missense variants were more often associated with mild phenotypes (p = 0.0421) and truncating variants predicted to escape NMD presented with more severe phenotypes (p < 0.0001). Within this group, variants in the prolin-rich region of the POGZ protein were associated with the most severe phenotypes (p = 0.0004). Our study suggests that gain-of-function or dominant negative effect through escaping NMD and the location of the variants in the prolin-rich domain of the protein may play an important role in the severity of manifestations of POGZ–associated neurodevelopmental disorders.

scholarly journals NKCC1: Newly Found as a Human Disease-Causing Ion Transporter

Function ◽  
2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Rainelli Koumangoye ◽  
Lisa Bastarache ◽  
Eric Delpire
Keyword(s):  
De Novo ◽  
Stop Codon ◽  
Neurodevelopmental Disorder ◽  
Premature Stop Codon ◽  
Dominant Negative ◽  
De Novo Mutation ◽  
Dominant Negative Effect ◽  
Salt Wasting ◽  
Wide Range ◽  
Negative Effect

Abstract Among the electroneutral Na+-dependent chloride transporters, NKCC1 had until now evaded identification as a protein causing human diseases. The closely related SLC12A transporters, NKCC2 and NCC have been identified some 25 years ago as responsible for Bartter and Gitelman syndromes: two renal-dependent salt wasting disorders. Absence of disease was most surprising since the NKCC1 knockout mouse was shown in 1999 to be viable, albeit with a wide range of deleterious phenotypes. Here we summarize the work of the past 5 years that introduced us to clinical cases involving NKCC1. The most striking cases are of 3 children with inherited mutations, who have complete absence of NKCC1 expression. These cases establish that lack of NKCC1 causes deafness; CFTR-like secretory defects with mucus accumulation in lung and intestine; severe xerostomia, hypotonia, dysmorphic facial features, and severe neurodevelopmental disorder. Another intriguing case is of a patient with a dominant deleterious SLC12A2 allele. This de novo mutation introduced a premature stop codon leading to a truncated protein. This mutant transporter seems to exert dominant-negative effect on wild-type transporter only in epithelial cells. The patient who suffers from lung, bladder, intestine, pancreas, and multiple endocrine abnormalities has, however, normal hearing and cognition. Finally, new reports substantiate the haploinsufficiency prediction of the SLC12A2 gene. Cases with single allele mutations in SLC12A2 have been linked to hearing loss and neurodevelopmental disorders.

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