scholarly journals Epitope-Tagged P0Glycoprotein Causes Charcot-Marie-Tooth–Like Neuropathy in Transgenic Mice

2000 ◽  
Vol 151 (5) ◽  
pp. 1035-1046 ◽  
Author(s):  
Stefano C. Previtali ◽  
Angelo Quattrini ◽  
Marina Fasolini ◽  
Maria Carla Panzeri ◽  
Antonello Villa ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Point Mutations ◽  
Dominant Negative ◽  
Epitope Tag ◽  
Charcot Marie Tooth ◽  
Demyelinating Neuropathies ◽  
Protein Zero ◽  
Homophilic Adhesion

In peripheral nerve myelin, the intraperiod line results from compaction of the extracellular space due to homophilic adhesion between extracellular domains (ECD) of the protein zero (P0) glycoprotein. Point mutations in this region of P0 cause human hereditary demyelinating neuropathies such as Charcot-Marie-Tooth. We describe transgenic mice expressing a full-length P0 modified in the ECD with a myc epitope tag. The presence of the myc sequence caused a dysmyelinating peripheral neuropathy similar to two distinct subtypes of Charcot-Marie-Tooth, with hypomyelination, altered intraperiod lines, and tomacula (thickened myelin). The tagged protein was incorporated into myelin and was associated with the morphological abnormalities. In vivo and in vitro experiments showed that P0myc retained partial adhesive function, and suggested that the transgene inhibits P0-mediated adhesion in a dominant-negative fashion. These mice suggest new mechanisms underlying both the pathogenesis of P0 ECD mutants and the normal interactions of P0 in the myelin sheath.

scholarly journals In vivo expression of mammalian BiP ATPase mutants causes disruption of the endoplasmic reticulum.

1995 ◽  
Vol 6 (3) ◽  
pp. 283-296 ◽  
Author(s):  
L M Hendershot ◽  
J Y Wei ◽  
J R Gaut ◽  
B Lawson ◽  
P J Freiden ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Binding ◽  
Point Mutations ◽  
Binding Domain ◽  
Dominant Negative ◽  
Atp Binding ◽  
Heavy Chains ◽  
In Vivo Expression

BiP possesses ATP binding/hydrolysis activities that are thought to be essential for its ability to chaperone protein folding and assembly in the endoplasmic reticulum (ER). We have produced a series of point mutations in a hamster BiP clone that inhibit ATPase activity and have generated a species-specific anti-BiP antibody to monitor the effects of mutant hamster BiP expression in COS monkey cells. The enzymatic inactivation of BiP did not interfere with its ability to bind to Ig heavy chains in vivo but did inhibit ATP-mediated release of heavy chains in vitro. Immunofluorescence staining and electron microscopy revealed vesiculation of the ER membranes in COS cells expressing BiP ATPase mutants. ER disruption was not observed when a "44K" fragment of BiP that did not include the protein binding domain was similarly mutated but was observed when the protein binding region of BiP was expressed without an ATP binding domain. This suggests that BiP binding to target proteins as an inactive chaperone is responsible for the ER disruption. This is the first report on the in vivo expression of mammalian BiP mutants and is demonstration that in vitro-identified ATPase mutants behave as dominant negative mutants when expressed 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.

Interaction of interleukin-6 and the BMP pathway in pulmonary smooth muscle

2007 ◽  
Vol 292 (6) ◽  
pp. L1473-L1479 ◽  
Author(s):  
Moira Hagen ◽  
Karen Fagan ◽  
Wolfgang Steudel ◽  
Michelle Carr ◽  
Kirk Lane ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Transgenic Mice ◽  
Bmp Signaling ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Second Hit ◽  
Protein Receptor ◽  
Bmp Pathway

The majority of familial pulmonary arterial hypertension (PAH) cases are caused by mutations in the type 2 bone morphogenetic protein receptor (BMPR2). However, less than one-half of BMPR2 mutation carriers develop PAH, suggesting that the most important function of BMPR2 mutation is to cause susceptibility to a “second hit.” There is substantial evidence from the literature implicating dysregulated inflammation, in particular the cytokine IL-6, in the development of PAH. We thus hypothesized that the BMP pathway regulates IL-6 in pulmonary tissues and conversely that IL-6 regulates the BMP pathway. We tested this in vivo using transgenic mice expressing an inducible dominant negative BMPR2 in smooth muscle, using mice injected with an IL-6-expressing virus, and in vitro using small interfering RNA (siRNA) to BMPR2 in human pulmonary artery smooth muscle cells (PA SMC). Consistent with our hypothesis, we found upregulation of IL-6 in both the transgenic mice and in cultured PA SMC with siRNA to BMPR2; this could be abolished with p38MAPK inhibitors. We also found that IL-6 in vivo caused a twofold increase in expression of the BMP signaling target Id1 and caused increased BMP activity in a luciferase-reporter assay in PA SMC. Thus we have shown both in vitro and in vivo a complete negative feedback loop between IL-6 and BMP, suggesting that an important consequence of BMPR2 mutations may be poor regulation of cytokines and thus vulnerability to an inflammatory second hit.

scholarly journals Regulation of SREBP1c Gene Expression in Skeletal Muscle: Role of Retinoid X Receptor/Liver X Receptor and Forkhead-O1 Transcription Factor

Endocrinology ◽  
2008 ◽  
Vol 149 (5) ◽  
pp. 2293-2305 ◽  
Author(s):  
Yasutomi Kamei ◽  
Shinji Miura ◽  
Takayoshi Suganami ◽  
Fumiko Akaike ◽  
Sayaka Kanai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Transcription Factor ◽  
Transgenic Mice ◽  
Regulatory Element ◽  
Liver X Receptor ◽  
Retinoid X Receptor ◽  
Dominant Negative

Sterol regulatory element binding protein 1c (SREBP1c) is a master regulator of lipogenic gene expression in liver and adipose tissue, where its expression is regulated by a heterodimer of nuclear receptor-type transcription factors retinoid X receptor-α (RXRα) and liver X receptor-α (LXRα). Despite the potential importance of SREBP1c in skeletal muscle, little is known about the regulation of SREBP1c in that setting. Here we report that gene expression of RXRγ is markedly decreased by fasting and is restored by refeeding in mouse skeletal muscle, in parallel with changes in gene expression of SREBP1c. RXRγ or RXRα, together with LXRα, activate the SREBP1c promoter in vitro. Moreover, transgenic mice overexpressing RXRγ specifically in skeletal muscle showed increased gene expression of SREBP1c with increased triglyceride content in their skeletal muscles. In contrast, transgenic mice overexpressing the dominant-negative form of RXRγ showed decreased SREBP1c gene expression. The expression of Forkhead-O1 transcription factor (FOXO1), which can suppress the function of multiple nuclear receptors, is negatively correlated to that of SREBP1c in skeletal muscle during nutritional change. Moreover, transgenic mice overexpressing FOXO1 specifically in skeletal muscle exhibited decreased gene expression of both RXRγ and SREBP1c. In addition, FOXO1 suppressed RXRα/LXRα-mediated SREBP1c promoter activity in vitro. These findings provide in vivo and in vitro evidence that RXR/LXR up-regulates SREBP1c gene expression and that FOXO1 antagonizes this effect of RXR/LXR in skeletal muscle.

scholarly journals Uncoupling the hydrolysis of lipid-linked oligosaccharide from the oligosaccharyl transfer reaction by point mutations in yeast oligosaccharyltransferase

2020 ◽  
Vol 295 (47) ◽  
pp. 16072-16085
Author(s):  
Takahiro Yamasaki ◽  
Daisuke Kohda
Keyword(s):  
Opposite Effect ◽  
Transfer Reaction ◽  
Point Mutations ◽  
Yeast Cells ◽  
Purification Method ◽  
Epitope Tag ◽  
Hydrolysis Of ◽  
Generation Activity

Oligosaccharyltransferase (OST) is responsible for the first step in the N-linked glycosylation, transferring an oligosaccharide chain onto asparagine residues to create glycoproteins. In the absence of an acceptor asparagine, OST hydrolyzes the oligosaccharide donor, releasing free N-glycans (FNGs) into the lumen of the endoplasmic reticulum (ER). Here, we established a purification method for mutated OSTs using a high-affinity epitope tag attached to the catalytic subunit Stt3, from yeast cells co-expressing the WT OST to support growth. The purified OST protein with mutations is useful for wide-ranging biochemical experiments. We assessed the effects of mutations in the Stt3 subunit on the two enzymatic activities in vitro, as well as their effects on the N-glycan attachment and FNG content levels in yeast cells. We found that mutations in the first DXD motif increased the FNG generation activity relative to the oligosaccharyl transfer activity, both in vitro and in vivo, whereas mutations in the DK motif had the opposite effect; the decoupling of the two activities may facilitate future deconvolution of the reaction mechanism. The isolation of the mutated OSTs also enabled us to identify different enzymatic properties in OST complexes containing either the Ost3 or Ost6 subunit and to find a 15-residue peptide as a better-quality substrate than shorter peptides. This toolbox of mutants, substrates, and methods will be useful for investigations of the molecular basis and physiological roles of the OST enzymes in yeast and other organisms.

scholarly journals Compensatory mechanisms in resistant Anopheles gambiae AcerKis and KdrKis neurons modulate insecticide-based mosquito control

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Stéphane Perrier ◽  
Eléonore Moreau ◽  
Caroline Deshayes ◽  
Marine El-Adouzi ◽  
Delphine Goven ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Anopheles Gambiae ◽  
Calcium Concentration ◽  
Mosquito Control ◽  
Point Mutations ◽  
Resting Membrane Potential ◽  
Compensatory Mechanisms ◽  
Pyrethroid Insecticides

AbstractIn the malaria vector Anopheles gambiae, two point mutations in the acetylcholinesterase (ace-1R) and the sodium channel (kdrR) genes confer resistance to organophosphate/carbamate and pyrethroid insecticides, respectively. The mechanisms of compensation that recover the functional alterations associated with these mutations and their role in the modulation of insecticide efficacy are unknown. Using multidisciplinary approaches adapted to neurons isolated from resistant Anopheles gambiae AcerKis and KdrKis strains together with larval bioassays, we demonstrate that nAChRs, and the intracellular calcium concentration represent the key components of an adaptation strategy ensuring neuronal functions maintenance. In AcerKis neurons, the increased effect of acetylcholine related to the reduced acetylcholinesterase activity is compensated by expressing higher density of nAChRs permeable to calcium. In KdrKis neurons, changes in the biophysical properties of the L1014F mutant sodium channel, leading to enhance overlap between activation and inactivation relationships, diminish the resting membrane potential and reduce the fraction of calcium channels available involved in acetylcholine release. Together with the lower intracellular basal calcium concentration observed, these factors increase nAChRs sensitivity to maintain the effect of low concentration of acetylcholine. These results explain the opposite effects of the insecticide clothianidin observed in AcerKis and KdrKis neurons in vitro and in vivo.

scholarly journals Characterization of LDD-2633 as a Novel RET Kinase Inhibitor with Anti-Tumor Effects in Thyroid Cancer

2021 ◽  
Vol 14 (1) ◽  
pp. 38
Author(s):  
Hyo Jeong Lee ◽  
Pyeonghwa Jeong ◽  
Yeongyu Moon ◽  
Jungil Choi ◽  
Jeong Doo Heo ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Thyroid Carcinoma ◽  
Kinase Inhibitor ◽  
Point Mutations ◽  
Carcinoma Cells ◽  
Medullary Thyroid ◽  
Potent Inhibition ◽  
Kinase Inhibitory Activity

Rearranged during transfection (RET), a receptor tyrosine kinase, is activated by glial cell line-derived neurotrophic factor family ligands. Chromosomal rearrangement or point mutations in RET are observed in patients with papillary thyroid and medullary thyroid carcinomas. Oncogenic alteration of RET results in constitutive activation of RET activity. Therefore, inhibiting RET activity has become a target in thyroid cancer therapy. Here, the anti-tumor activity of a novel RET inhibitor was characterized in medullary thyroid carcinoma cells. The indirubin derivative LDD-2633 was tested for RET kinase inhibitory activity. In vitro, LDD-2633 showed potent inhibition of RET kinase activity, with an IC50 of 4.42 nM. The growth of TT thyroid carcinoma cells harboring an RET mutation was suppressed by LDD-2633 treatment via the proliferation suppression and the induction of apoptosis. The effects of LDD-2633 on the RET signaling pathway were examined; LDD-2633 inhibited the phosphorylation of the RET protein and the downstream molecules Shc and ERK1/2. Oral administration of 20 or 40 mg/kg of LDD-2633 induced dose-dependent suppression of TT cell xenograft tumor growth. The in vivo and in vitro experimental results supported the potential use of LDD-2633 as an anticancer drug for thyroid cancers.

scholarly journals Inhibition of RANKL-Induced Osteoclastogenesis by Novel Mutant RANKL

2021 ◽  
Vol 22 (1) ◽  
pp. 434
Author(s):  
Yuria Jang ◽  
Hong Moon Sohn ◽  
Young Jong Ko ◽  
Hoon Hyun ◽  
Wonbong Lim
Keyword(s):  
Nuclear Translocation ◽  
Critical Role ◽  
Osteoclast Differentiation ◽  
Point Mutations ◽  
Tartrate Resistant Acid Phosphatase ◽  
Mice Model ◽  
Gsk 3Β ◽  
Rank Signaling

Background: Recently, it was reported that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL and was shown to compete with RANK to bind RANKL and suppress canonical RANK signaling during osteoclast differentiation. The critical role of the protein triad RANK–RANKL in osteoclastogenesis has made their binding an important target for the development of drugs against osteoporosis. In this study, point-mutations were introduced in the RANKL protein based on the crystal structure of the RANKL complex and its counterpart receptor RANK, and we investigated whether LGR4 signaling in the absence of the RANK signal could lead to the inhibition of osteoclastogenesis.; Methods: The effects of point-mutated RANKL (mRANKL-MT) on osteoclastogenesis were assessed by tartrate-resistant acid phosphatase (TRAP), resorption pit formation, quantitative real-time polymerase chain reaction (qPCR), western blot, NFATc1 nuclear translocation, micro-CT and histomorphological assay in wild type RANKL (mRANKL-WT)-induced in vitro and in vivo experimental mice model. Results: As a proof of concept, treatment with the mutant RANKL led to the stimulation of GSK-3β phosphorylation, as well as the inhibition of NFATc1 translocation, mRNA expression of TRAP and OSCAR, TRAP activity, and bone resorption, in RANKL-induced mouse models; and Conclusions: The results of our study demonstrate that the mutant RANKL can be used as a therapeutic agent for osteoporosis by inhibiting RANKL-induced osteoclastogenesis via comparative inhibition of RANKL. Moreover, the mutant RANKL was found to lack the toxic side effects of most osteoporosis treatments.

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.

scholarly journals Molecular Basis of In Vivo Resistance to Sulfadoxine-Pyrimethamine in African Adult Patients Infected withPlasmodium falciparum Malaria Parasites

1998 ◽  
Vol 42 (7) ◽  
pp. 1811-1814 ◽  
Author(s):  
Leonardo K. Basco ◽  
Rachida Tahar ◽  
Pascal Ringwald
Keyword(s):  
Dihydrofolate Reductase ◽  
Point Mutations ◽  
Gene Mutations ◽  
Adult Patients ◽  
Wild Type ◽  
Dihydropteroate Synthase ◽  
Reductase Gene ◽  
Parasite Populations

ABSTRACT In vitro sulfadoxine and pyrimethamine resistance has been associated with point mutations in the dihydropteroate synthase and dihydrofolate reductase domains, respectively, but the in vivo relevance of these point mutations has not been well established. To analyze the correlation between genotype and phenotype, 10 Cameroonian adult patients were treated with sulfadoxine-pyrimethamine and followed up for 28 days. After losses to follow-up (n = 1) or elimination of DNA samples due to mixed parasite populations with pyrimethamine-sensitive and pyrimethamine-resistant profiles (n = 3), parasite genomic DNA from day 0 blood samples of six patients were analyzed by DNA sequencing. Three patients who were cured had isolates characterized by a wild-type or mutant dihydrofolate reductase gene (with one or two mutations) and a wild-type dihydropteroate synthase gene. Three other patients who failed to respond to sulfadoxine-pyrimethamine treatment carried isolates with triple dihydrofolate reductase gene mutations and either a wild-type or a mutant dihydropteroate synthase gene. Three dihydrofolate reductase gene codons (51, 59, and 108) may be reliable genetic markers that can accurately predict the clinical outcome of sulfadoxine-pyrimethamine treatment in Africa.

Sign in / Sign up

Export Citation Format

Share Document