scholarly journals Oral Glucosamine Ameliorates Aggravated Neurological Phenotype in Mucopolysaccharidosis III Type C Mouse Model Expressing Misfolded HGSNAT Variant

2021 ◽  
Author(s):  
Xuefang Pan ◽  
Mahsa Taherzadeh ◽  
Poulomee Bose ◽  
Rachel Heon-Roberts ◽  
Annie L.A. Nguyen ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Missense Variant ◽  
Dominant Negative ◽  
Synaptic Proteins ◽  
Missense Mutations ◽  
Negative Effects ◽  
Object Recognition Test ◽  
Y Maze ◽  
Neurological Phenotype

Objective: Over 55% of mucopolysaccharidosis IIIC (MPS IIIC) patients have at least one allelic missense variant responsible for misfolding of heparan sulfate acetyl-CoA:α-glucosaminide N-acetyltransferase (HGSNAT). These variants are potentially treatable with pharmacological chaperones, such as a competitive HGSNAT inhibitor, glucosamine. Since the constitutive HGSNAT knockout mice, we generated previously cannot be used to test such strategy in vivo, we generated a novel model, the HgsnatP304L strain, expressing misfolded mutant HGSNAT with human missense mutation Pro311Leu (Pro304Leu in the mouse enzyme). Results: HgsnatP304L mice present deficits in short-term (novel object recognition test) and working/spatial (Y-maze test) memory at 4 months of age, 2-4 months earlier than previously described gene-targeted Hgsnat-Geo mice, which lack HGSNAT protein. HgsnatP304L mice also show increased severity of synaptic deficits in CA1 neurons, and accelerated course of CNS pathology including neuronal storage of heparan sulfate, accumulation of misfolded proteins, increase of simple gangliosides, and neuroinflammation as compared with Hgsnat-Geo mice. Expression of misfolded human Pro311Leu HGSNAT protein in cultured hippocampal Hgsnat-Geo neurons aggravated reduction of synaptic proteins. Memory deficits and majority of pathological changes in the brain were rescued in mice receiving daily doses of oral glucosamine. Interpretation: Altogether, our data for the first time demonstrate dominant-negative effects of the misfolded HGSNAT Pro304Leu variant and show that these effects are treatable by oral administration of glucosamine, suggesting that patients, affected with missense mutations preventing normal folding of the enzyme, could benefit from chaperone therapy.

scholarly journals 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 ◽  
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.

Lack of dominant-negative effects of a truncated γc on retroviral-mediated gene correction of immunodeficient mice

Blood ◽  
2001 ◽  
Vol 97 (6) ◽  
pp. 1618-1624 ◽  
Author(s):  
Makoto Otsu ◽  
Kazuo Sugamura ◽  
Fabio Candotti
Keyword(s):  
Gene Therapy ◽  
Murine Model ◽  
Severe Combined Immunodeficiency ◽  
Dominant Negative ◽  
Interleukin 4 ◽  
Gene Correction ◽  
Negative Effects ◽  
Gamma Chain ◽  
Immunodeficient Mice

A recent clinical trial of gene therapy for X-linked severe combined immunodeficiency (XSCID) has shown that retroviral-mediated gene correction of bone marrow stem cells can lead to the development of normal immune function. These exciting results have been preceded by successful immune reconstitution in several XSCID mouse models, all carrying null mutations of the common gamma chain (γc). One question not formally addressed by these previous studies is that of possible dominant-negative effects of the endogenous mutant γc protein on the activity of the wild-type transferred gene product. The present work was therefore undertaken to study whether corrective gene transfer was applicable to an XSCID murine model with preserved expression of a truncated γc molecule (Δγc+-XSCID). Gene correction of Δγc+-XSCID mice resulted in the reconstitution of lymphoid development, and preferential repopulation of lymphoid organs by gene-corrected cells demonstrated the selective advantage of γc-expressing cells in vivo. Newly developed B cells showed normalization of lipopolysaccharide-mediated proliferation and interleukin-4 (IL-4)–induced immunoglobulin G1 isotype switching. Splenic T cells and thymocytes of treated animals proliferated normally to mitogens and responded to the addition of IL-2, IL-4, and IL-7, indicating functional reconstitution of γc-sharing receptors. Repopulated thymi showed a clear increase of CD4−/CD8− and CD8+fractions, both dramatically reduced in untreated Δγc+-XSCID mice. These improvements were associated with the restoration of Bcl-2 expression levels and enhanced cell survival. These data indicate that residual expression of the endogenous truncated γc did not lead to dominant-negative effects in this murine model and suggest that patient selection may not be strictly necessary for gene therapy of XSCID.

scholarly journals Novel Transcription Coactivator Complex Containing Activating Signal Cointegrator 1

2002 ◽  
Vol 22 (14) ◽  
pp. 5203-5211 ◽  
Author(s):  
Dong-Ju Jung ◽  
Hee-Sook Sung ◽  
Young-Wha Goo ◽  
Hyun Mi Lee ◽  
Ok Ku Park ◽  
...  
Keyword(s):  
Nuclear Receptors ◽  
Serum Response Factor ◽  
Ectopic Expression ◽  
Nuclear Factor Κb ◽  
Dominant Negative ◽  
Mutant Form ◽  
Negative Effects ◽  
Direct Binding ◽  
Serum Response

ABSTRACT Human activating signal cointegrator 1 (hASC-1) was originally isolated as a transcriptional coactivator of nuclear receptors. Here we report that ASC-1 exists as a steady-state complex associated with three polypeptides, P200, P100, and P50, in HeLa nuclei; stimulates transactivation by serum response factor (SRF), activating protein 1 (AP-1), and nuclear factor κB (NF-κB) through direct binding to SRF, c-Jun, p50, and p65; and relieves the previously described transrepression between nuclear receptors and either AP-1 or NF-κB. Interestingly, ectopic expression of Caenorhabditis elegans ASC-1 (ceASC-1), an ASC-1 homologue that binds P200 and P100, like hASC-1, while weakly interacting only with p65, in HeLa cells appears to replace endogenous hASC-1 from the hASC-1 complex and exerts potent dominant-negative effects on AP-1, NF-κB, and SRF transactivation. In addition, neutralization of endogenous P50 by single-cell microinjection of a P50 antibody inhibits AP-1 transactivation; the inhibition is relieved by coexpression of wild-type P50, but not of P50ΔKH, a mutant form that does not interact with P200. Overall, these results suggest that the endogenous hASC-1 complex appears to play an essential role in AP-1, SRF, and NF-κB transactivation and to mediate the transrepression between nuclear receptors and either AP-1 or NF-κB in vivo.

scholarly journals Heparan Sulfate-dependent RAGE oligomerization is indispensable for pathophysiological functions of RAGE

2021 ◽  
Author(s):  
Miaomiao Li ◽  
Chih Yean Ong ◽  
Christophe J Langouet-Astrie ◽  
Lisi TAN ◽  
Ashwni Verma ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Genetic Model ◽  
Point Mutations ◽  
Active Role ◽  
Dominant Negative ◽  
Unexpected Finding ◽  
Rna Seq ◽  
Dominant Negative Form

RAGE, a druggable inflammatory receptor, is known to function as an oligomer but the exact oligomerization mechanism remains poorly understood. Previously we have shown that heparan sulfate (HS) plays an active role in RAGE oligomerization. To understand the physiological significance of HS-induced RAGE oligomerization in vivo, we generated RAGE knock-in mice (RageAHA/AHA) by introducing point mutations to specifically disrupt HS-RAGE interaction. The RAGE mutant demonstrated normal ligand-binding but impaired capacity of HS-binding and oligomerization. Remarkably, RageAHA/AHA mice phenocopied Rage-/- mice in two different pathophysiological processes, namely bone remodeling and neutrophil-mediated liver injury, which demonstrates that HS-induced RAGE oligomerization is essential for RAGE signaling. Our findings suggest that it should be possible to block RAGE signaling by inhibiting HS-RAGE interaction. To test this, we generated a monoclonal antibody that targets the HS-binding site of RAGE. This antibody blocks RAGE signaling in vitro and in vivo, recapitulating the phenotype of RageAHA/AHA mice. By inhibiting HS-RAGE interaction genetically and pharmacologically, our work validated an alternative strategy to antagonize RAGE. Finally, we have performed RNA-seq analysis of neutrophils and lungs and found that while Rage-/- mice had a broad alteration of transcriptome in both tissues compared to wild-type mice, the changes of transcriptome in RageAHA/AHA mice were much more restricted. This unexpected finding suggests that by preserving the expression of RAGE protein (in a dominant-negative form), RageAHA/AHA mouse might represent a cleaner genetic model to study physiological roles of RAGE in vivo compared to Rage-/- mice.

Drosophila transcription factor AP-2 in proboscis, leg and brain central complex development

Development ◽  
2001 ◽  
Vol 128 (8) ◽  
pp. 1239-1252 ◽  
Author(s):  
I. Monge ◽  
R. Krishnamurthy ◽  
D. Sims ◽  
F. Hirth ◽  
M. Spengler ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dominant Negative ◽  
Central Complex ◽  
Missense Mutations ◽  
Leg Length ◽  
New Paradigm ◽  
Negative Effects ◽  
Wing Vein ◽  
Normal Length ◽  
Hypomorphic Alleles

We report loss- and gain-of-function analyses that identify essential roles in development for Drosophila transcription factor AP-2. A mutagenesis screen yielded 16 lethal point mutant alleles of dAP-2. Null mutants die as adults or late pupae with a reduced proboscis, severely shortened legs (~30% of normal length) lacking tarsal joints, and disruptions in the protocerebral central complex, a brain region critical for locomotion. Seven hypomorphic alleles constitute a phenotypic series yielding hemizygous adults with legs ranging from 40–95% of normal length. Hypomorphic alleles show additive effects with respect to leg length and viability; and several heteroallelic lines were established. Heteroallelic adults have moderately penetrant defects that include necrotic leg joints and ectopic growths (sometimes supernumerary antennae) invading medial eye territory. Several dAP-2 alleles with DNA binding domain missense mutations are null in hemizygotes but have dominant negative effects when paired with hypomorphic alleles. In wild-type leg primordia, dAP-2 is restricted to presumptive joints. Ectopic dAP-2 in leg discs can inhibit but not enhance leg elongation indicating that functions of dAP-2 in leg outgrowth are region restricted. In wing discs, ectopic dAP-2 cell autonomously transforms presumptive wing vein epithelium to ectopic sensory bristles, consistent with an instructive role in sensory organ development. These findings reveal multiple functions for dAP-2 during morphogenesis of feeding and locomotor appendages and their neural circuitry, and provide a new paradigm for understanding AP-2 family transcription factors.

scholarly journals Identification of a druggable protein-protein interaction site between mutant p53 and its stabilizing chaperone DNAJA1

2020 ◽  
pp. jbc.RA120.014749
Author(s):  
Xin Tong ◽  
Dandan Xu ◽  
Rama K. Mishra ◽  
Ryan D Jones ◽  
Leyu Sun ◽  
...  
Keyword(s):  
Small Molecule ◽  
Tp53 Gene ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Dominant Negative Effect ◽  
Mutant P53 ◽  
Missense Mutations ◽  
Oncogenic Function

The TP53 gene is the most frequently mutated gene in human cancers, and the majority of TP53 mutations are missense mutations. As a result, these mutant p53 (mutp53) either directly lose wild-type p53 (wtp53) tumor suppressor function or exhibit a dominant negative effect over wtp53. In addition, some mutp53 have acquired new oncogenic function (gain of function). Therefore, targeting mutp53 for its degradation, may serve as a promising strategy for cancer prevention and therapy. Based on our previous finding that farnesylated DNAJA1 is a crucial chaperone in maintaining mutp53 stabilization, and by using an in silico approach, we built 3-D homology models of human DNAJA1 and mutp53R175H proteins, identified the interacting pocket in the DNAJA1-mutp53R175H complex, and found one critical druggable small molecule binding  site in the DNAJA1 glycine/phenylalanine rich region. We confirmed that the interacting pocket in the DNAJA1-mutp53R175H complex was crucial for stabilizing mutp53R175H using a site-directed mutagenesis approach. We further screened a drug-like library to identify a promising small molecule hit (GY1-22) against the interacting pocket in DNAJA1-mutp53R175H complex. The GY1-22 compound displayed an effective activity against DNAJA1-mutp53R175H complex. Treatment with GY1-22 significantly reduced mutp53 protein levels, enhanced Waf1p21 expression, suppressed cyclin D1 expression, and inhibited mutp53-driven pancreatic cancer growth both in vitro and in vivo. Together, our results indicate that the interacting pocket in the DNAJA1-mutp53R175H complex is critical for mutp53’s stability and oncogenic function, and DNAJA1 is a robust therapeutic target for developing the efficient small molecule inhibitors against oncogenic mutp53.

scholarly journals SRP54 mutations induce Congenital Neutropenia via dominant-negative effects on XBP1 splicing

Blood ◽  
2020 ◽  
Author(s):  
Christoph Schürch ◽  
Thorsten Schaefer ◽  
Joëlle Seraina Müller ◽  
Pauline Hanns ◽  
Marlon Arnone ◽  
...  
Keyword(s):  
De Novo ◽  
Dominant Negative ◽  
Severe Neutropenia ◽  
In Vivo Model ◽  
Factor X ◽  
Negative Effects ◽  
Congenital Neutropenia ◽  
Developmental Defects ◽  
Xbp1 Mrna

Heterozygous de novo missense variants of SRP54 were recently identified in patients with congenital neutropenia (CN), displaying symptoms overlapping with Shwachman-Diamond-Syndrome (SDS).1 Here, we investigate srp54 KO zebrafish as the first in vivo model of SRP54 deficiency. srp54-/- zebrafish are embryonically lethal and display, next to severe neutropenia, multi-systemic developmental defects. In contrast, srp54+/- zebrafish are viable, fertile and only show mild neutropenia. Interestingly, injection of human SRP54 mRNAs carrying mutations observed in patients (T115A, T117Δ and G226E) aggravated neutropenia and induced pancreatic defects in srp54+/- fish, mimicking the corresponding human clinical phenotypes. These data suggest that the variable phenotypes observed in patients may be due to mutation-specific dominant negative effects on the functionality of the residual wildtype SRP54 protein. Consistently, overexpression of mutated SRP54 also induced neutropenia in wildtype fish and impaired granulocytic maturation of human promyelocytic HL-60 cells as well as of healthy cord-blood derived CD34+ HSPCs. Mechanistically, srp54 mutant fish and human cells show impaired unconventional splicing of the transcription factor X-box binding protein 1 (Xbp1). Vice-versa, xbp1 morphants recapitulate phenotypes observed in srp54 deficiency and, importantly, injection of spliced, but not unspliced xbp1 mRNA rescues neutropenia in srp54+/- zebrafish. Together, these data indicate that SRP54 is critical for the development of various tissues, with neutrophils reacting most sensitively to SRP54 loss. The heterogenic phenotypes observed in patients, ranging from mild CN to SDS-like disease, may be due to different dominant negative effects of mutated SRP54 proteins on downstream XBP1 splicing, which represents a potential therapeutic target.

Ectopic expression of a Drosophila InsP3R channel mutant has dominant-negative effects in vivo

Cell Calcium ◽  
2006 ◽  
Vol 39 (2) ◽  
pp. 187-196 ◽  
Author(s):  
Sonal Srikanth ◽  
Santanu Banerjee ◽  
Gaiti Hasan
Keyword(s):  
Ectopic Expression ◽  
Dominant Negative ◽  
Negative Effects

scholarly journals OP0314 DOCK8 MUTATIONS IN COVID-19 AND MIS-C CYTOKINE STORM SYNDROME

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 192.1-192
Author(s):  
R. Cron ◽  
M. Zhang ◽  
D. Absher ◽  
J. Bridges ◽  
A. Schnell ◽  
...  
Keyword(s):  
Nk Cell ◽  
Foamy Virus ◽  
Dominant Negative ◽  
Target Cells ◽  
Missense Mutations ◽  
Cytokine Storm ◽  
Negative Effects ◽  
Research Support ◽  
Rare Mutations ◽  
Lytic Function

Background:We recently identified DOCK8 as a novel gene associated with cytokine storm syndrome (CSS)1. Heterozygous missense mutations in DOCK8 diminish NK cell lytic function and contribute to increased pro-inflammatory cytokine production (CSS). CSS is a potential complication of COVID-19 with severe consequences2. Children are at risk of a SARS-CoV-2 post-infectious CSS, multisystem inflammatory syndrome in children (MIS-C)3. Host genetic factors associated with COVID-19 CSS and MIS-C CSS are unknown.Objectives:The goals are to identify and functionally study rare mutations in DOCK8 in patients with SARS-CoV-2 COVID-19 and MIS-C.Methods:To date, 16 adult patients enrolled in a COVID-19 CSS clinical trial at UAB had whole genome sequencing. Four (25%) had rare heterozygous DOCK8 mutations (3 missense, 1 intronic). A COVID-19 CSS adult patient in Seattle also had a DOCK8 missense mutation. In addition, DOCK8 missense mutations were identified in five children (UAB & Northwell) hospitalized with MIS-C. DOCK8 mutations, or wild-type (WT) sequence controls, were introduced into human NK-92 cells by FOAMY virus transduction. WT and mutant DOCK8-expressing NK-92 cells were incubated with K562 target cells and compared for cytolysis and degranulation (CD107a).Results:One COVID-19 patient DOCK8 mutation (Gly523Arg) reduced NK cell degranulation by 30% and cytolysis by 23% (n=3) (Figure 1). Similar studies of 3 MIS-C patients with DOCK8 missense mutations (Arg899Trp, Ala2Thr, Pro687Leu) revealed up to 31% reduced NK cell degranulation and 48% reduction in cytolysis by 3 distinct mutations (n=3). Two-way ANOVA analysis revealed statistically significant (p<0.05) differences in NK cell degranulation and lysis for four unique DOCK8 mutations.Conclusion:Heterozygous DOCK8 missense mutations may contribute to severe COVID-19 and MIS-C CSS by partial dominant-negative effects yielding decreased NK cell cytolysis.References:[1]Schulert GS, Cron RQ. The genetics of macrophage activation syndrome. Genes Immun 2020:21:169-181.[2] Cron RQ, Chatham WW. The rheumatologist’s role in COVID-19. J Rheumatol 2020:47:639-642.[3]Reiff D, Mannion ML, Samuy N, Scalici P, Cron RQ. Distinguishing active pediatric COVID-19 from MIS-C. Pediatr Rheumatol Online J, in press.Disclosure of Interests:Randy Cron Consultant of: SOBI, Novartis, Pfizer, Sironax, Grant/research support from: SOBI, Mingce Zhang: None declared, Remy Cron: None declared, Devin Absher: None declared, John Bridges: None declared, Amanda Schnell: None declared, Pavan Bhatraju: None declared, Anshul Vagrecha: None declared, Shannon Lozinsky: None declared, Suchitra Acharya: None declared, Carolyn Levy: None declared, Winn Chatham Grant/research support from: SOBI.

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