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 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.

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.

Both the paired domain and homeodomain are required for in vivo function of Drosophila Paired

Development ◽  
1996 ◽  
Vol 122 (9) ◽  
pp. 2709-2718 ◽  
Author(s):  
P. Miskiewicz ◽  
D. Morrissey ◽  
Y. Lan ◽  
L. Raj ◽  
S. Kessler ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Ectopic Expression ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Paired Domain ◽  
Mutant Proteins ◽  
Segment Polarity ◽  
Negative Effect

Drosophila paired, a homolog of mammalian Pax-3, is key to the coordinated regulation of segment-polarity genes during embryogenesis. The paired gene and its homologs are unusual in encoding proteins with two DNA-binding domains, a paired domain and a homeodomain. We are using an in vivo assay to dissect the functions of the domains of this type of molecule. In particular, we are interested in determining whether one or both DNA-binding activities are required for individual in vivo functions of Paired. We constructed point mutants in each domain designed to disrupt DNA binding and tested the mutants with ectopic expression assays in Drosophila embryos. Mutations in either domain abolished the normal regulation of the target genes engrailed, hedgehog, gooseberry and even-skipped, suggesting that these in vivo functions of Paired require DNA binding through both domains rather than either domain alone. However, when the two mutant proteins were placed in the same embryo, Paired function was restored, indicating that the two DNA-binding activities need not be present in the same molecule. Quantitation of this effect shows that the paired domain mutant has a dominant-negative effect consistent with the observations that Paired protein can bind DNA as a dimer.

Regulation of Spemann organizer formation by the intracellular kinase Xgsk-3

Development ◽  
1995 ◽  
Vol 121 (3) ◽  
pp. 755-765 ◽  
Author(s):  
S.B. Pierce ◽  
D. Kimelman
Keyword(s):  
Ectopic Expression ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Axis Formation ◽  
Serine Threonine Kinase ◽  
Spemann Organizer ◽  
Isolation And Characterization ◽  
Secondary Axis ◽  
Dorsal Axis ◽  
Negative Effect

Dorsal axis formation in the Xenopus embryo can be induced by the ectopic expression of several Wnt family members. In Drosophila, the protein encoded by the Wnt family gene, wingless, signals through a pathway that antagonizes the effects of the serine/threonine kinase zeste-white 3/shaggy. We describe the isolation and characterization of a Xenopus homolog of zeste-white 3/shaggy, Xgsk-3. A kinase-dead mutant of Xgsk-3, Xgsk-3K-->R, has a dominant negative effect and mimics the ability of Wnt to induce a secondary axis by induction of an ectopic Spemann organizer. Xgsk-3K-->R, like Wnt, induces dorsal axis formation when expressed in the deep vegetal cells, which do not contribute to the axis. These results indicate that the dorsal fate is actively repressed by Xgsk-3, which must be inactivated for dorsal axis formation to occur. Furthermore, our work suggests that the effects of Xgsk-3K-->R are mediated by an additional intercellular signal.

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 Mammalian end binding proteins control persistent microtubule growth

2009 ◽  
Vol 184 (5) ◽  
pp. 691-706 ◽  
Author(s):  
Yulia Komarova ◽  
Christian O. De Groot ◽  
Ilya Grigoriev ◽  
Susana Montenegro Gouveia ◽  
E. Laura Munteanu ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Microtubule Dynamics ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Dimerization Domain ◽  
Negative Effect ◽  
Microtubule Growth ◽  
Tracking Behavior ◽  
In Cells

End binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners.

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 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.

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