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Results for “Dominant-negative mutations”

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PubMed1991

Dominant-negative mutants as antiviral agents: simultaneous infection with the cold-adapted live-virus vaccine for influenza A protects ferrets from disease produced by wild-type influenza A.

The attenuated cold-adapted strain of influenza A virus that is a candidate live-virus vaccine suppressed clinical disease in ferrets when given simultaneously with a virulent epidemic strain of influenza A virus. The cold-adapted virus effectively prevented disease, even when the epidemic strain was of a different subtype than the attenuated virus. In this case, ferrets given a mixed inoculum produced antibody to both subtypes in the absence of clinical disease, indicating that both viruses are replicating in the respiratory tract. These findings suggest the possibility of the development of a novel class of antivirals for influenza, namely a live virus that is a dominant-negative attenuated mutant that interferes with the replication of epidemic strains of virus.

Animals↗
PubMed1992

The v-rel oncogene: insights into the mechanism of transcriptional activation, repression, and transformation.

The v-rel oncogene product from the avian reticuloendotheliosis virus strain T corresponds to a member of the Rel-related family of enhancer-binding proteins that includes both the mammalian 50- and 65-kDa subunits of the NF-kappa B transcription factor complex. However, in contrast to NF-kappa B, v-Rel has been shown to function as a dominant-negative repressor of kappa B-dependent transcription in many mature cell types. We now demonstrate that a highly conserved motif within the Rel homology domain of v-Rel containing a consensus protein kinase A phosphorylation site is required for DNA binding, transcriptional repression, and cellular transformation mediated by this oncoprotein. However, replacement of the serine phosphate acceptor within the protein kinase A site with an alanine did not alter any of these functions of v-Rel, suggesting that phosphorylation at this site is not central to the regulation of this oncogene product. Rather, the inactive mutations appear to identify a functional domain within v-Rel required for these various biological activities. It is notable that these same mutations do not impair the ability of v-Rel to heterodimerize with the 50-kDa subunit of NF-kappa B, suggesting that v-Rel-mediated transcriptional repression likely involves direct nuclear blockade of the kappa B enhancer rather than indirect alterations in the composition of preformed cytoplasmic NF-kappa B complexes. Paradoxically, when introduced into undifferentiated F9 cells, v-Rel functions as a kappa B-specific transcriptional activator rather than as a dominant-negative repressor. These stimulatory effects of v-Rel require both the conserved protein kinase A phosphorylation site and additional unique C-terminal sequences not needed for v-Rel-mediated repression in mature cells. Retinoic acid-induced differentiation of these F9 cells restores the repressor function of v-Rel. These opposing biological actions of v-Rel occurring in cells at distinct stages of differentiation may have important implications for the mechanism of v-Rel-mediated transformation occurring in avian splenocytes.

Amino Acid Sequence↗
PubMed1991

Changes in hsp70 alter thermotolerance and heat-shock regulation in Drosophila.

To test the role of the heat shock protein hsp70 in induced thermotolerance and in the regulation of the heat-shock response, we established cell lines with altered expression of the Hsp70 gene. Underexpressing cells were created by transformation with antisense Hsp70 genes, and overexpressing cells by transformation with extra copies of the wild-type gene. Expression at normal temperatures was achieved by placing Hsp70 coding sequences under the control of the metallothionein promoter. Cells that expressed mutant hsp70s were created by transforming cells with deletion and frameshift mutations. The results indicate that hsp70 plays a major role in both thermotolerance and regulation. Surprisingly, they also indicate that these functions can be separated. Overexpression affected thermotolerance more than regulation; underexpression affected regulation more than thermotolerance. A carboxyl-terminal deletion of Hsp70 had a severe dominant-negative effect on thermotolerance but only a minor effect on regulation; an amino-terminal deletion strongly affected regulation but not thermotolerance. A model that explains these observations is presented.

Animals↗
PubMed1990

Negative control of the helix-loop-helix family of myogenic regulators in the NFB mutant.

We have characterized a nondifferentiating mouse muscle cell line, NFB, that represses the activity of the helix-loop-helix (HLH) family of myogenic regulators, yet expresses sarcomeric actins. The NFB MyoD gene is silent, but can be activated upon transfection of a long terminal region-controlled chicken MyoD cDNA, resulting in myogenesis. When NFB cells are fused with H9c2 rat muscle cells in heterokaryons, the level of rat MyoD transcripts declines. Thus, the stoichiometry of MyoD and the putative repressor controls myogenesis. Although NFB cells express myogenin and Myf-5 transcripts, the activity of these regulators is also repressed:myogenesis is not induced in 10T1/2 fibroblasts and is repressed in L6 muscle cells upon fusion with NFB cells. We conclude that the myogenic HLH regulators are not required for sarcomeric actin gene activation and that myogenesis is subject to dominant-negative control.

Actins↗
PubMed1995

Signal-induced degradation of I kappa B alpha requires site-specific ubiquitination.

The inhibitor protein I kappa B alpha controls the nuclear import of the transcription factor NF-kappa B. The inhibitory activity of I kappa B alpha is regulated from the cytoplasmic compartment by signal-induced proteolysis. Previous studies have shown that signal-dependent phosphorylation of serine residues 32 and 36 targets I kappa B alpha to the ubiquitin-proteasome pathway. Here we provide evidence that lysine residues 21 and 22 serve as the primary sites for signal-induced ubiquitination of I kappa B alpha. Conservative Lys-->Arg substitutions at both Lys-21 and Lys-22 produce dominant-negative mutants of I kappa B alpha in vivo. These constitutive inhibitors are appropriately phosphorylated but fail to release NF-kappa B in response to multiple inducers, including viral proteins, cytokines, and agents that mimic antigenic stimulation through the T-cell receptor. Moreover, these Lys-->Arg mutations prevent signal-dependent degradation of I kappa B alpha in vivo and ubiquitin conjugation in vitro. We conclude that site-specific ubiquitination of phosphorylated I kappa B alpha at Lys-21 and/or Lys-22 is an obligatory step in the activation of NF-kappa B.

Arginine↗
PubMed1989

A domain containing leucine-zipper-like motifs mediate novel in vivo interactions between the thyroid hormone and retinoic acid receptors.

The thyroid hormones and retinoic acid are potent modulators of differentiation, development, and gene expression. The transcriptional activities of these ligands are mediated by closely related nuclear receptors which bind and activate identical hormone responsive DNA elements. We noticed that a region within the ligand binding or E domain is well conserved between receptors for these hormones. This region contains hydrophobic heptad repeats that are structurally similar to the leucine-zipper dimerization domain. To study the function of this conserved domain, we examined the transcriptional responses of thyroid hormone receptor/c-erbA deletion mutants which lacked the heptad repeats. We previously reported that the chick c-erbA-alpha possesses hormone-independent (constitutive) activity in cells which express endogenous rat thyroid hormone receptor. We now demonstrate that this activity is abolished upon deletion of the conserved heptad repeats. This suggests that the heptad repeats mediate in vivo interactions between chick c-erbA and rat thyroid hormone receptors. To further test this hypothesis deletion mutants of chick c-erbA were constructed which contained all eight heptad repeats but which lacked the zinc-finger DNA binding domain. Although these mutants are transcriptionally inactive, they act in a dominant-negative fashion to block trans-activation by both the chick c-erbA-alpha and the endogenous thyroid hormone and retinoic acid receptors. We suggest that the heptad repeats mediate the formation of inactive mutant/wild-type hetero-dimers. Dimer formation suggests a mechanism to account for the dominant-negative phenotypes displayed by nonhormone binding variants of c-erbA, the proto-oncoprotein v-erbA and patients with the generalized thyroid hormone resistance syndrome.

Amino Acid Sequence↗
PubMed1992

Ras mediates translation initiation factor 4E-induced malignant transformation.

Translation initiation factor eIF-4E binds to the eukaryotic mRNA 5' cap structure (m7 GpppN, where N is any nucleotide). eIF-4E is a limiting factor in translation and plays a key role in regulation of translation. We have shown previously that overexpression of eIF-4E in rodent fibroblasts results in tumorigenic transformation. eIF-4E also exhibits mitogenic activity when microinjected into serum-starved NIH-3T3 cells. To understand the mechanisms by which eIF-4E exerts its mitogenic property, we examined the involvement of the Ras signaling pathway in this activity. Here, we report that Ras is activated in eIF-4E-overexpressing cells, as the proportion of GTP-bound Ras is increased. Overexpression of the negative effector of cellular Ras, GTPase activating protein, causes reversion of the transformed phenotype. Furthermore, we show that neutralizing antibodies to Ras, or a dominant-negative mutant of Ras, inhibit the mitogenic activity of eIF-4E. We conclude that eIF-4E exerts its mitogenic and oncogenic activities by the activation of Ras.

3T3 Cells↗
PubMed1992

Role of estrogen receptor variants in the development of hormone resistance in breast cancer.

Recent evidence suggests that the progression to hormone resistance in some breast tumors is due to mutations in the estrogen receptor (ER). Various types of ER variants have been found in breast cancer biopsies and breast cancer cell lines. The ER variants include dominant-positive receptors that are transcriptionally active in the absence of estrogen, and dominant-negative receptors that are themselves transcriptionally inactive but prevent the action of the normal receptor. The mechanisms by which these variants cause loss of hormonal control is becoming clear. ER variants may be prognostic factors for breast cancer. By modifying the action of ER variants, it should be possible to develop new strategies for treatment of malignant breast disease.

Animals↗
PubMed1991

Characterization of bovine papillomavirus E1 region deletion mutants associated with neoplastic transformation in a murine cell line.

Spontaneous focus formation in the contact-inhibited C127 cell line, cl.2, harbouring multiple copies of a bovine papillomavirus type 1 deletion mutant, was associated with the evolution of further viral genomic deletions in addition to an amplification of the viral genome copy number. Three simple frameshift deletions of 308, 605 and 1291 bp, associated with separate transformation events, were mapped within the E1 open reading frame, implying a common mechanism of spontaneous transformation in this cell line. Furthermore, each transformed cell line also retained multiple copies of the intact E1 gene, suggesting that these novel deletion mutants might function by a dominant-negative mechanism to disrupt the normal control of viral DNA replication or viral transcription. These mutants had the potential to encode truncated E1 polypeptides with a common N-terminal region encoded by the 5' end of E1, i.e. overlapping the previously described E1 modulator gene. A possible role for these mutants in diverting a lethal type of virus-cell interaction is discussed.

Animals↗
PubMed1991

The type I human T-cell leukemia virus (HTLV-I) Rex trans-activator binds directly to the HTLV-I Rex and the type 1 human immunodeficiency virus Rev RNA response elements.

The Rex protein of the type I human T-cell leukemia virus (HTLV-I) is essential for the replication of this pathogenic retrovirus and, surprisingly, can also replace the function of the structurally distinct Rev protein of the type 1 human immunodeficiency virus (HIV-1). Rex action requires a 255-nucleotide viral RNA stem-loop structure termed the Rex RNA response element (RexRE) located in the 3' retroviral long terminal repeat. Rex function leads to the induced cytoplasmic expression of the incompletely spliced family of viral mRNAs that uniquely encode the HTLV-I structural and enzymatic proteins (Gag, Pol, and Env). Our studies now demonstrate that Rex acts by binding directly to the RexRE in a sequence-specific manner. These effects of Rex require the presence of a 10-nucleotide subregion of the RexRE that is essential for Rex function in vivo. Dominant-negative mutants of Rex also bind to the RexRE with high affinity, while a recessive-negative Rex mutant altered within its arginine-rich, positively charged domain fails to engage the RexRE. Analogously, both the wild-type and dominant-negative Rex proteins specifically bind to the structurally distinct HIV-1 Rev response element, a finding that likely underlies the respective stimulatory and inhibitory effects of these HTLV-I proteins in the heterologous HIV-1 system. However, consistent with their lack of amino acid homology, the binding sites for Rex and Rev within the HIV-1 Rev response element are distinct.

Binding Sites↗
PubMed1992

Serum-, TPA-, and Ras-induced expression from Ap-1/Ets-driven promoters requires Raf-1 kinase.

Raf-1 serine-threonine protein kinase has the hallmarks of a critical switch that connects growth factor receptor activation at the cell membrane with transcriptional events in the nucleus. We show by use of Raf-1 dominant-negative mutants that Raf-1 is required for serum-, TPA-, and Ras-induced expression from the oncogene-responsive element in the polyomavirus enhancer. The minimal region of Raf-1 that displays this dominant-negative phenotype (Raf-C4) contains a cysteine finger motif. Raf-C4 appears to function by titrating out a Raf-1-activating factor that is induced by Ras following serum or TPA treatment of NIH-3T3 cells. In addition, we show that Raf-1 and Ras cooperate in trans-activation through the oncogene-responsive element and that the cysteine-rich region is necessary for this effect.

3T3 Cells↗
PubMed1991

Requirement for acidic amino acid residues immediately N-terminal to the conserved domain of Saccharomyces cerevisiae TFIID.

TFIID binds to TATA boxes and initiates the assembly of general transcription factors and pol II on promoters. TFIID proteins from various species consist of a highly conserved carboxy terminal domain and very divergent amino terminal domains. We investigated the function of the non-conserved amino terminal domain (residues 1-60) of Saccharomyces cerevisiae TFIID (YIID, 240 residues) by testing the ability of a series of YIID amino terminal deletion mutants to complement a YIID deficient yeast strain. Mutants with deletions up to amino acid 48 restored the YIID deficient yeast strain to an apparently wild type phenotype. However, deletion up to position 57 or 60 produced yeast strains which formed extremely small colonies. Moreover, overexpression of YIID delta 2-57 or YIID delta 3-60 protein in the presence of wild type YIID resulted in a dominant-negative inhibition of growth. No difference between the basal transcriptional activity of wild type YIID and these amino terminal deletion mutants was observed in vitro. However, transcriptional activation in vivo of promoter-lacZ fusions showed that the YIID delta 2-57 deletion affects the ability of certain promoters (CUP1 and an HSP UAS-CYC1 promoter hybrid promoter) to respond to upstream factor stimulation. At least one inducible promoter, PHO5, was not affected by this deletion. The defect produced by YIID delta 2-57 was due to the deletion of several acidic residues present between residues 48 and 57. The results show that the conserved carboxy terminal domain of YIID is sufficient for cell viability. However, an acidic region just amino terminal to the conserved domain is required for normal growth and transcription control in most yeast strains.

Amino Acid Sequence↗
PubMed1995

Delayed assembly of desmosomes in keratinocytes with disrupted classic-cadherin-mediated cell adhesion by a dominant negative mutant.

We examined whether classic cadherins play a role in the formation of desmosomes using a mouse keratinocyte, PAMcN390 delta cell, which shows disrupted classic-cadherin-mediated cell adhesion by introduction of a dominant-negative mutant of N-cadherin. The expression of the mutant did not alter that of endogenous E-cadherin or desmoplakin. In control cells with functional classic cadherins, we observed redistribution of desmoplakin to cell-cell borders with insertions of keratin filaments at the contact sites as soon as 2 h after calcium elevation, after an earlier event of E-cadherin translocation to the cell-cell contact sites. In contrast, in the PAMcN390 delta cells, which showed retarded translocation of E-cadherin, the redistribution of desmoplakin and the rearrangement of keratin filaments were delayed as late as 24 h after the calcium elevation. The acquisition of Nonidet P-40 insolubility of desmoplakins also was found to be delayed in the PAMcN390 delta cells. These findings indicate that the disruption of classic cadherin affected the organization of desmosomes upon calcium elevation and suggest that the proper function of classic cadherins is a prerequisite for desmosome assembly in keratinocytes.

Animals↗
PubMed1992

Insulin/IGF-1 hybrid receptors: implications for the dominant-negative phenotype in syndromes of insulin resistance.

Classical insulin and IGF-1 receptors are alpha 2 beta 2 heterotetrameric complexes synthesized from two identical alpha beta half-receptor precursors. Recent data strongly suggests, however, that nonidentical alpha beta half-receptor precursors can assemble to generate hybrid holoreceptor species both in vivo and in vitro. This review focuses primarily on two types of hybrid receptors. The first type is an insulin/IGF-1 hybrid receptor generated by the association of an alpha beta insulin half-receptor with an alpha beta IGF-1 half-receptor. The second type is one formed from a wildtype (kinase-active) insulin or IGF-1 alpha beta half-receptor and a mutant (kinase-inactive) insulin alpha beta half-receptor. Although the functional properties of insulin/IGF-1 hybrid receptors have not yet been completely defined, wildtype/mutant hybrid receptors are essentially substrate kinase inactive. These data indicate that the mutant alpha beta half-receptor exerts a transdominant inhibition upon the wildtype alpha beta half-receptor within the alpha 2 beta 2 holoreceptor complex. This defect in substrate kinase activity may contribute to the molecular defect underlying some syndromes of severe insulin resistance and diabetes. Heterozygous individuals expressing both wildtype and mutant tyrosine kinase-defective insulin receptor precursors demonstrate varying degrees of insulin resistance and diabetes. In addition, cell lines which express both endogenous wildtype and transfected kinase-defective insulin receptors display markedly decreased insulin and IGF-1 sensitivity and responsiveness. Formation of hybrid receptors which results in premature termination of insulin signal transduction may be one mechanism underlying the observation that kinase-inactive receptors inhibit the function of native receptors.

Animals↗
PubMed2025

Diagnostic Implications and Correlates of Plasma Adenosine Deaminase 2 Activity and ADA2 Variants.

OBJECTIVE: Deficiency of adenosine deaminase 2 (DADA2) is a monogenic autoinflammatory disease manifested as polyarteritis nodosa, stroke, and bone marrow failure. Leveraging an international cohort of 200 DADA2 cases, we aimed to characterize the diagnostic utility of a plasma ADA2 enzyme activity assay and understand the implications of residual ADA2 activity. METHODS: Data were collected from individuals who underwent ADA2 testing from 2018 to 2025. Plasma ADA2 activity was determined using an established spectrophotometric assay. ADA2 variants were analyzed in transfected cells by enzyme assay and western blotting. RESULTS: We determined that plasma ADA2 activity is 99.0% and 96.0% sensitive and 99.7% and 98.8% specific in distinguishing genetically confirmed DADA2 cases from controls and carriers, respectively. Eighteen individuals with DADA2 (9%) possessed detectable ADA2 activity, including several cases with levels seen in carriers. Residual ADA2 activity was associated with the vasculitis/inflammatory phenotype but not with disease severity. Genotype analysis revealed that 14 of 18 cases with residual plasma activity possessed at least one hypomorphic missense variant with greater than 20% residual ADA2 function when overexpressed in 293T cells, often occurring in trans with a more deleterious variant. In vitro analysis revealed that missense ADA2 variants exert variable dominant-negative effects by forming large intracellular protein aggregates via disulfide bond formation at a cysteine residue (Cys408). CONCLUSION: We confirmed the utility of plasma ADA2 activity as a diagnostic assay and showed that the inflammatory phenotype of DADA2 occurred in cases with residual activity. In vitro findings illustrate potential interactions of ADA2 variants to synergistically disrupt protein function.

Humans↗
PubMed2025

DNA-binding affinity and specificity determine the phenotypic diversity in BCL11B-related disorders.

BCL11B is a Cys2-His2 zinc-finger (C2H2-ZnF) domain-containing, DNA-binding, transcription factor with established roles in the development of various organs and tissues, primarily the immune and nervous systems. BCL11B germline variants have been associated with a variety of developmental syndromes. However, genotype-phenotype correlations along with pathophysiologic mechanisms of selected variants mostly remain elusive. To dissect these, we performed genotype-phenotype correlations of 92 affected individuals harboring a pathogenic or likely pathogenic BCL11B variant, followed by immune phenotyping, analysis of chromatin immunoprecipitation DNA-sequencing data, dual-luciferase reporter assays, and molecular modeling. These integrative analyses enabled us to define three clinical subtypes of BCL11B-related disorders. It is likely that gene-disruptive BCL11B variants and missense variants affecting zinc-binding cysteine and histidine residues cause mild to moderate neurodevelopmental delay with increased propensity for behavioral and dental anomalies, allergies and asthma, and reduced type 2 innate lymphoid cells. Missense variants within C2H2-ZnF DNA-contacting α helices cause highly variable clinical presentations ranging from multisystem anomalies with demise in the first years of life to late-onset, hyperkinetic movement disorder with poor fine motor skills. Those not in direct DNA contact cause a milder phenotype through reduced, target-specific transcriptional activity. However, missense variants affecting C2H2-ZnFs, DNA binding, and "specificity residues" impair BCL11B transcriptional activity in a target-specific, dominant-negative manner along with aberrant regulation of alternative DNA targets, resulting in more severe and unpredictable clinical outcomes. Taken together, we suggest that the phenotypic severity and variability is largely dependent on the DNA-binding affinity and specificity of altered BCL11B proteins.

Adolescent↗
PubMed1990

fos-jun conspiracy: implications for the cell.

Two nuclear oncoproteins, fos and jun (AP-1), cooperate in forming a very stable heterodimeric complex that binds to the AP-1 site on DNA with high affinity. The 'leucine zipper' domain of both fos and jun is necessary for the formation of this heterodimer. Mutations of single residues within the leucine zipper domain have no effect on protein complex formation. However, results from mutagenesis of the first leucine of the heptad repeat in either fos or jun basic regions and alteration of the spacing between the basic and leucine zipper domains indicate that the basic region of fos plays a crucial role in determining the DNA binding affinity of the transcriptional complex. Mutations of the basic amino acids in fos protein prevent binding to the tumour promoter response element (TRE) in the presence of wild-type jun protein. Thus fos protein appears to be dominant in jun-fos binding to DNA, even though fos alone cannot bind to TRE. Mutants in the basic region of fos and jun can be exploited as dominant-negative mutants to ablate the normal fos cellular function.

Amino Acid Sequence↗
PubMed1992

Dominant-negative mutants of a yeast G-protein beta subunit identify two functional regions involved in pheromone signalling.

The STE4 gene, which encodes the beta subunit of the mating response G-protein in the yeast Saccharomyces cerevisiae, was subjected to a saturation mutagenesis using 'doped' oligodeoxynucleotides. We employed a genetic screen to select dominant-negative STE4 mutants, which when overexpressed from the GAL1 promoter, interfered with the signalling function of the wild type protein. The identified inhibitory amino acid alterations define two small regions that are crucially involved in transmitting the mating signal from G beta to downstream components of the signalling pathway. These results underline the positive signalling role of yeast G beta and assign for the first time the positive signalling function of a G-protein beta subunit to specific structural features.

Amino Acid Sequence↗