A Heterozygous Mutation in the Third Transmembrane Domain Causes a Dominant-Negative Effect on Signalling Capability of the MC4R

ABSTRACTPlatelets are anucleate and mostly ribosome-free cells within the bloodstream, derived from megakaryocytes within bone marrow and crucial for cessation of bleeding at sites of injury. Inherited thrombocytopenias are a group of disorders characterized by alow platelet count and are frequently associated with excessive bleeding. SLFN14 is one of the most recently discovered genes linked to inherited thrombocytopenia where several heterozygous missense mutations in SLFN14 were identified to cause defective megakaryocyte maturation and platelet dysfunction. Yet, SLFN14 was recently described as a ribosome-associated protein resulting in rRNA and ribosome-bound mRNA degradation in rabbit reticulocytes. To unveil the cellular function of SLFN14 and the link between SLFN14 and thrombocytopenia, we examined SLFN14 (WT/mutants) in in vitro models. Here, we show that all SLFN14 variants co-localize with ribosomes and mediate rRNA endonucleolytic degradation and ribosome clearance. Compare dto SLFN14 WT, expression of mutants is dramatically reduced as a result of post-translational degradation due to partial misfolding of the protein. Moreover, all SLFN14 variants tend to form oligomers. These findings could explain the dominant negative effect of heterozygous mutation on SLFN14 expression in patients’ platelets. Overall we suggest that SLFN14 could be involved in ribosome degradation during platelet formation and maturation.

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Physical interaction of junctophilin and the CaV1.1 C terminus is crucial for skeletal muscle contraction

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1716649115 ◽

2018 ◽

Vol 115 (17) ◽

pp. 4507-4512 ◽

Cited By ~ 16

Author(s):

Tsutomu Nakada ◽

Toshihide Kashihara ◽

Masatoshi Komatsu ◽

Katsuhiko Kojima ◽

Toshikazu Takeshita ◽

...

Keyword(s):

Skeletal Muscle ◽

Transmembrane Domain ◽

Ryanodine Receptors ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Physical Interaction ◽

Membrane Expression ◽

C Terminus ◽

Negative Effect

Close physical association of CaV1.1 L-type calcium channels (LTCCs) at the sarcolemmal junctional membrane (JM) with ryanodine receptors (RyRs) of the sarcoplasmic reticulum (SR) is crucial for excitation–contraction coupling (ECC) in skeletal muscle. However, the molecular mechanism underlying the JM targeting of LTCCs is unexplored. Junctophilin 1 (JP1) and JP2 stabilize the JM by bridging the sarcolemmal and SR membranes. Here, we examined the roles of JPs in localization and function of LTCCs. Knockdown of JP1 or JP2 in cultured myotubes inhibited LTCC clustering at the JM and suppressed evoked Ca2+ transients without disrupting JM structure. Coimmunoprecipitation and GST pull-down assays demonstrated that JPs physically interacted with 12-aa residues in the proximal C terminus of the CaV1.1. A JP1 mutant lacking the C terminus including the transmembrane domain (JP1ΔCT) interacted with the sarcolemmal/T-tubule membrane but not the SR membrane. Expression of this mutant in adult mouse muscles in vivo exerted a dominant-negative effect on endogenous JPs, impairing LTCC–RyR coupling at triads without disrupting JM morphology, and substantially reducing Ca2+ transients without affecting SR Ca2+ content. Moreover, the contractile force of the JP1ΔCT-expressed muscle was dramatically reduced compared with the control. Taken together, JPs recruit LTCCs to the JM through physical interaction and ensure robust ECC at triads in skeletal muscle.

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The Impact of Different DNMT3A Mutations on Outcome in Younger Adults with Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v124.21.67.67 ◽

2014 ◽

Vol 124 (21) ◽

pp. 67-67

Author(s):

Rosemary E Gale ◽

Katarina Lamb ◽

Christopher Allen ◽

Dima El-Sharkawi ◽

Cassandra Stowe ◽

...

Keyword(s):

Conflicts Of Interest ◽

Multivariable Analysis ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Heterozygous Mutation ◽

Prognostic Impact ◽

Missense Mutations ◽

Limited Information ◽

Negative Effect ◽

The Impact

Abstract DNMT3A mutations (DNMT3AMUT) are recurrent in AML. They predominate in patients with intermediate-risk (IR) cytogenetics and are often co-incident with FLT3ITD and NPM1MUT. Their prognostic impact is unclear. Most reports suggest they are associated with a worse outcome, but a large study including 1060 younger adult IR patients found that DNMT3AMUT had no significant impact on survival endpoints. Variable results have also been reported for different FLT3/NPM1 subgroups. Missense mutations at R882 in exon 23 occur in ≈65% of patients, but other missense and truncation mutations occur throughout the gene, mainly in exons 13-23. There is limited information on the prognostic impact of the different mutations, although they may have differing functional consequences. We therefore screened exons 13-23 in DNA samples from 914 younger adult AML patients (median age 43 years) with IR cytogenetics treated on UK MRC trials and evaluated outcome according to type of DNMT3Amutation. Overall, 278 mutations were detected in 272 (30%) patients; 175 (64%) had R882 missense mutations, 59 (22%) other missense mutations, 35 (13%) truncations or in-frame deletions; 3 (1%) had 2 mutations of differing types. Median R882 mutant level in 172 mutated cases was 47% (range 15-85%), consistent with a heterozygous mutation in most cells. Patients with DNMT3AMUT were significantly older than those with DNMT3A wild-type (DNMT3AWT) (P<.0001), more likely to be female (P=.004), have a higher presenting WBC (P<.0001), and a normal rather than abnormal karyotype (P<.0001). The presence of DNMT3AMUT positively correlated with FLT3ITD (P=.0003) and NPM1MUT (P<.0001) and negatively correlated with CEBPAMUT (P<.0001). Patients with R882 mutations had significantly higher WBC (P=.005) and correlation with NPM1MUT (P=.01) than non-R882 mutated patients; non-R882 missense mutated patients had higher WBC (P=.05) and non-significant higher co-incidence with FLT3ITD than those with truncations. Presence of DNMT3AMUT was associated with a poorer prognosis, but this difference was only seen if the results were analyzed separately according to NPM1 genotype, where DNMT3AMUT was associated with higher cumulative incidence of relapse (CIR) than DNMT3AWT in cases with NPM1MUT (49% vs 40%, P=.01) and NPM1WT (61% vs 58%, P=.5) genotype. Similarly, DNMT3AMUT patients had worse overall survival (OS) than DNMT3AWT patients with NPM1MUT (38% vs 50%, P=.008) and NPM1WT (15% vs 25%, P=.09) genotype. This statistical anomaly is an example of Simpson’s paradox. It results from the strong co-incidence of DNMT3A and NPM1 mutations with opposing prognostic associations that mask the effect seen separately when the groups are combined. Although the differences were smaller for NPM1WT cases, tests for heterogeneity showed that the impact of a DNMT3A mutation did not differ between NPM1MUT and NPM1WT for either CIR or OS, nor between the 4 genotypes defined by the combination of NPM1 and FLT3ITD genotypes. In multivariable analysis including age, WBC, NPM1 and FLT3ITD, DNMT3AMUT was a significant adverse risk factor for CIR (HR=1.27, CI=1.01-1.61; P=.04), and showed a trend for being adverse for OS (HR=1.19, CI=.98-1.45; P=.08). When outcome was considered according to the type of mutation (R882, other missense or truncations), for the NPM1MUT genotype cases CIR was highest in R882 and other missense cases (51%, 50%) and truncation cases were similar to DNMT3AWT (35%, 40%). For NPM1WT, CIR was highest in R882 cases (76%), similar in other missense and DNMT3AWT cases (55%, 58%) and lowest in truncation cases (40%). Consistent with this data, for NPM1MUT genotype, OS was lowest in R882 and other missense cases (35%, 38%), better in DNMT3AWT (50%) and highest in truncation cases (57%). For NPM1WT, OS was lowest in R882 cases (11%), and similar in DNMT3AWT, other missense and truncation cases (25%, 21%, 18% respectively). These data suggest that screening cannot be limited to the hotspot R882 mutations and that cases with missense mutations should be treated as poor risk, including those patients currently considered as favorable risk such as NPM1MUTFLT3WT. Conversely, truncation mutations have a different functional impact from missense mutations, more likely to result in haploinsufficiency than a dominant-negative effect, and these cases should be considered as equivalent to DNMT3AWT for prognostication and selection of therapy in 1st remission. Disclosures No relevant conflicts of interest to declare.

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A Dominant Negative Effect of eth-1r, a Mutant Allele of the Neurospora crassa S-Adenosylmethionine Synthetase-Encoding Gene Conferring Resistance to the Methionine Toxic Analogue Ethionine

Genetics ◽

10.1093/genetics/144.4.1455 ◽

1996 ◽

Vol 144 (4) ◽

pp. 1455-1462

Author(s):

José L Barra ◽

Mario R Mautino ◽

Alberto L Rosa

Keyword(s):

Neurospora Crassa ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Resistant Phenotype ◽

Negative Effect ◽

Encoding Gene ◽

Adenosylmethionine Synthetase ◽

Adomet Synthetase ◽

Identical Gene

eth-1r a thermosensitive allele of the Neurospora crassa S-adenosylmethionine (AdoMet) synthetase gene that confers ethionine resistance, has been cloned and sequenced. Replacement of an aspartic amino acid residue (D48 → N48), perfectly conserved in prokaryotic, fungal and higher eukaryotic AdoMet synthetases, was found responsible for both thermosensitivity and ethionine resistance conferred by eth-1r. Gene fusion constructs, designed to overexpress eth-1r in vivo, render transformant cells resistant to ethionine. Dominance of ethionine resistance was further demonstrated in eth-1 +/eth-1r partial diploids carrying identical gene doses of both alleles. Heterozygous eth-1 +/eth-1r cells have, at the same time, both the thermotolerance conferred by eth-1 + and the ethionine-resistant phenotype conferred by eth-1r. AdoMet levels and AdoMet synthetase activities were dramatically decreased in heterozygous eth-1 +/eth-1r cells. We propose that this negative effect exerted by eth-1r results from the in vivo formation of heteromeric eth-1 +/eth-1r AdoMet synthetase molecules.

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Evidence for a dominant‐negative effect of a missense mutation in the SERPING1 gene responsible for hereditary angioedema type I

The Journal of Dermatology ◽

10.1111/1346-8138.15930 ◽

2021 ◽

Author(s):

Shuichiro Yasuno ◽

Osamu Ansai ◽

Ryota Hayashi ◽

Sawako Nakamura ◽

Yutaka Shimomura

Keyword(s):

Missense Mutation ◽

Hereditary Angioedema ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Type I ◽

Negative Effect ◽

Serping1 Gene

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Restoration of Silencing in Saccharomyces cerevisiae by Tethering of a Novel Sir2-Interacting Protein, Esc8

Genetics ◽

10.1093/genetics/162.2.633 ◽

2002 ◽

Vol 162 (2) ◽

pp. 633-645 ◽

Cited By ~ 2

Author(s):

Guido Cuperus ◽

David Shore

Keyword(s):

Protein A ◽

Dominant Negative ◽

Class I ◽

Dominant Negative Effect ◽

Rna Pol Ii ◽

Interacting Protein ◽

Nucleosome Remodeling ◽

Negative Effect ◽

Close Homolog

Abstract We previously described two classes of SIR2 mutations specifically defective in either telomeric/HM silencing (class I) or rDNA silencing (class II) in S. cerevisiae. Here we report the identification of genes whose protein products, when either overexpressed or directly tethered to the locus in question, can establish silencing in SIR2 class I mutants. Elevated dosage of SCS2, previously implicated as a regulator of both inositol biosynthesis and telomeric silencing, suppressed the dominant-negative effect of a SIR2-143 mutation. In a genetic screen for proteins that restore silencing when tethered to a telomere, we isolated ESC2 and an uncharacterized gene, (YOL017w), which we call ESC8. Both Esc2p and Esc8p interact with Sir2p in two-hybrid assays, and the Esc8p-Sir2 interaction is detected in vitro. Interestingly, Esc8p has a single close homolog in yeast, the ISW1-complex factor Ioc3p, and has also been copurified with Isw1p, raising the possibility that Esc8p is a component of an Isw1p-containing nucleosome remodeling complex. Whereas esc2 and esc8 deletion mutants alone have only marginal silencing defects, cells lacking Isw1p show a strong silencing defect at HMR but not at telomeres. Finally, we show that Esc8p interacts with the Gal11 protein, a component of the RNA pol II mediator complex.

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Dominant Negative Mutants Implicate STAT5 in Myeloid Cell Proliferation and Neutrophil Differentiation

Blood ◽

10.1182/blood.v93.12.4154 ◽

1999 ◽

Vol 93 (12) ◽

pp. 4154-4166 ◽

Cited By ~ 80

Author(s):

Robert L. Ilaria ◽

Robert G. Hawley ◽

Richard A. Van Etten

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Cytokine Signaling ◽

Transactivation Domain ◽

Negative Effects ◽

Murine Bone Marrow ◽

Negative Effect

Abstract STAT5 is a member of the signal transducers and activation of transcription (STAT) family of latent transcription factors activated in a variety of cytokine signaling pathways. We introduced alanine substitution mutations in highly conserved regions of murine STAT5A and studied the mutants for dimerization, DNA binding, transactivation, and dominant negative effects on erythropoietin-induced STAT5-dependent transcriptional activation. The mutations included two near the amino-terminus (W255KR→AAA and R290QQ→AAA), two in the DNA-binding domain (E437E→AA and V466VV→AAA), and a carboxy-terminal truncation of STAT5A (STAT5A/▵53C) analogous to a naturally occurring isoform of rat STAT5B. All of the STAT mutant proteins were tyrosine phosphorylated by JAK2 and heterodimerized with STAT5B except for the WKR mutant, suggesting an important role for this region in STAT5 for stabilizing dimerization. The WKR, EE, and VVV mutants had no detectable DNA-binding activity, and the WKR and VVV mutants, but not EE, were defective in transcriptional induction. The VVV mutant had a moderate dominant negative effect on erythropoietin-induced STAT5 transcriptional activation, which was likely due to the formation of heterodimers that are defective in DNA binding. Interestingly, the WKR mutant had a potent dominant negative effect, comparable to the transactivation domain deletion mutant, ▵53C. Stable expression of either the WKR or ▵53C STAT5 mutants in the murine myeloid cytokine-dependent cell line 32D inhibited both interleukin-3–dependent proliferation and granulocyte colony-stimulating factor (G-CSF)–dependent differentiation, without induction of apoptosis. Expression of these mutants in primary murine bone marrow inhibited G-CSF–dependent granulocyte colony formation in vitro. These results demonstrate that mutations in distinct regions of STAT5 exert dominant negative effects on cytokine signaling, likely through different mechanisms, and suggest a role for STAT5 in proliferation and differentiation of myeloid cells.

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Insight into the AcrAB-TolC Complex Assembly Process Learned from Competition Studies

Antibiotics ◽

10.3390/antibiotics10070830 ◽

2021 ◽

Vol 10 (7) ◽

pp. 830

Author(s):

Prasangi Rajapaksha ◽

Isoiza Ojo ◽

Ling Yang ◽

Ankit Pandeya ◽

Thilini Abeywansha ◽

...

Keyword(s):

Efflux Pump ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Assembly Process ◽

Multidrug Efflux ◽

E Coli ◽

Multidrug Efflux Pumps ◽

Negative Effect ◽

Dynamic Assembly ◽

Pump Assembly

The RND family efflux pump AcrAB-TolC in E. coli and its homologs in other Gram-negative bacteria are major players in conferring multidrug resistance to the cells. While the structure of the pump complex has been elucidated with ever-increasing resolution through crystallography and Cryo-EM efforts, the dynamic assembly process remains poorly understood. Here, we tested the effect of overexpressing functionally defective pump components in wild type E. coli cells to probe the pump assembly process. Incorporation of a defective component is expected to reduce the efflux efficiency of the complex, leading to the so called “dominant negative” effect. Being one of the most intensively studied bacterial multidrug efflux pumps, many AcrA and AcrB mutations have been reported that disrupt efflux through different mechanisms. We examined five groups of AcrB and AcrA mutants, defective in different aspects of assembly and substrate efflux. We found that none of them demonstrated the expected dominant negative effect, even when expressed at concentrations many folds higher than their genomic counterpart. The assembly of the AcrAB-TolC complex appears to have a proof-read mechanism that effectively eliminated the formation of futile pump complex.

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The Diagnostic Journey of a Patient with Prader–Willi-Like Syndrome and a Unique Homozygous SNURF-SNRPN Variant; Bio-Molecular Analysis and Review of the Literature

Genes ◽

10.3390/genes12060875 ◽

2021 ◽

Vol 12 (6) ◽

pp. 875

Author(s):

Karlijn Pellikaan ◽

Geeske M. van Woerden ◽

Lotte Kleinendorst ◽

Anna G. W. Rosenberg ◽

Bernhard Horsthemke ◽

...

Keyword(s):

Intellectual Disability ◽

Single Nucleotide Polymorphism Array ◽

Genetic Defect ◽

Missense Variant ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Pituitary Hormone ◽

Negative Effect

Prader–Willi syndrome (PWS) is a rare genetic condition characterized by hypotonia, intellectual disability, and hypothalamic dysfunction, causing pituitary hormone deficiencies and hyperphagia, ultimately leading to obesity. PWS is most often caused by the loss of expression of a cluster of genes on chromosome 15q11.2-13. Patients with Prader–Willi-like syndrome (PWLS) display features of the PWS phenotype without a classical PWS genetic defect. We describe a 46-year-old patient with PWLS, including hypotonia, intellectual disability, hyperphagia, and pituitary hormone deficiencies. Routine genetic tests for PWS were normal, but a homozygous missense variant NM_003097.3(SNRPN):c.193C>T, p.(Arg65Trp) was identified. Single nucleotide polymorphism array showed several large regions of homozygosity, caused by high-grade consanguinity between the parents. Our functional analysis, the ‘Pipeline for Rapid in silico, in vivo, in vitro Screening of Mutations’ (PRiSM) screen, showed that overexpression of SNRPN-p.Arg65Trp had a dominant negative effect, strongly suggesting pathogenicity. However, it could not be confirmed that the variant was responsible for the phenotype of the patient. In conclusion, we present a unique homozygous missense variant in SNURF-SNRPN in a patient with PWLS. We describe the diagnostic trajectory of this patient and the possible contributors to her phenotype in light of the current literature on the genotype–phenotype relationship in PWS.

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