scholarly journals Novel Functional Properties of Missense Mutations in the Glycine Receptor β Subunit in Startle Disease

2021 ◽  
Vol 14 ◽  
Author(s):  
Inken Piro ◽  
Anna-Lena Eckes ◽  
Vikram Babu Kasaragod ◽  
Claudia Sommer ◽  
Robert J. Harvey ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Glycine Receptor ◽  
Scaffolding Protein ◽  
Binding Motif ◽  
Missense Mutations ◽  
Β Subunit ◽  
Wild Type ◽  
Gain Of Function ◽  
Aromatic Residues ◽  
Startle Disease

Startle disease is a rare disorder associated with mutations in GLRA1 and GLRB, encoding glycine receptor (GlyR) α1 and β subunits, which enable fast synaptic inhibitory transmission in the spinal cord and brainstem. The GlyR β subunit is important for synaptic localization via interactions with gephyrin and contributes to agonist binding and ion channel conductance. Here, we have studied three GLRB missense mutations, Y252S, S321F, and A455P, identified in startle disease patients. For Y252S in M1 a disrupted stacking interaction with surrounding aromatic residues in M3 and M4 is suggested which is accompanied by an increased EC50 value. By contrast, S321F in M3 might stabilize stacking interactions with aromatic residues in M1 and M4. No significant differences in glycine potency or efficacy were observed for S321F. The A455P variant was not predicted to impact on subunit folding but surprisingly displayed increased maximal currents which were not accompanied by enhanced surface expression, suggesting that A455P is a gain-of-function mutation. All three GlyR β variants are trafficked effectively with the α1 subunit through intracellular compartments and inserted into the cellular membrane. In vivo, the GlyR β subunit is transported together with α1 and the scaffolding protein gephyrin to synaptic sites. The interaction of these proteins was studied using eGFP-gephyrin, forming cytosolic aggregates in non-neuronal cells. eGFP-gephyrin and β subunit co-expression resulted in the recruitment of both wild-type and mutant GlyR β subunits to gephyrin aggregates. However, a significantly lower number of GlyR β aggregates was observed for Y252S, while for mutants S321F and A455P, the area and the perimeter of GlyR β subunit aggregates was increased in comparison to wild-type β. Transfection of hippocampal neurons confirmed differences in GlyR-gephyrin clustering with Y252S and A455P, leading to a significant reduction in GlyR β-positive synapses. Although none of the mutations studied is directly located within the gephyrin-binding motif in the GlyR β M3-M4 loop, we suggest that structural changes within the GlyR β subunit result in differences in GlyR β-gephyrin interactions. Hence, we conclude that loss- or gain-of-function, or alterations in synaptic GlyR clustering may underlie disease pathology in startle disease patients carrying GLRB mutations.

scholarly journals Novel missense mutations in the glycine receptor β subunit gene (GLRB) in startle disease

2013 ◽  
Vol 52 ◽  
pp. 137-149 ◽  
Author(s):  
Victoria M. James ◽  
Anna Bode ◽  
Seo-Kyung Chung ◽  
Jennifer L. Gill ◽  
Maartje Nielsen ◽  
...  
Keyword(s):  
Glycine Receptor ◽  
Missense Mutations ◽  
Β Subunit ◽  
Subunit Gene ◽  
Startle Disease

scholarly journals Transient neuromotor phenotype in transgenic spastic mice expressing low levels of glycine receptor β-subunit: an animal model of startle disease

2000 ◽  
Vol 12 (1) ◽  
pp. 27-32 ◽  
Author(s):  
Lore Becker ◽  
Bettina Hartenstein ◽  
Johannes Schenkel ◽  
Jochen Kuhse ◽  
Heinrich Betz ◽  
...  
Keyword(s):  
Animal Model ◽  
Glycine Receptor ◽  
Β Subunit ◽  
Startle Disease ◽  
Low Levels

scholarly journals Identification of a gephyrin binding motif on the glycine receptor β subunit

Neuron ◽  
1995 ◽  
Vol 15 (3) ◽  
pp. 563-572 ◽  
Author(s):  
Guido Meyer ◽  
Joachim Kirsch ◽  
Heinrich Betz ◽  
Dieter Langosch
Keyword(s):  
Glycine Receptor ◽  
Binding Motif ◽  
Β Subunit

scholarly journals Gain-of-function cardiomyopathic mutations in RBM20 rewire splicing regulation and re-distribute ribonucleoprotein aggregates within processing bodies

2021 ◽  
Author(s):  
Aidan M Fenix ◽  
Yuichiro Miyaoka ◽  
Alessandro Bertero ◽  
Steven Blue ◽  
Matthew J Spindler ◽  
...  
Keyword(s):  
Rna Binding ◽  
Acute Stress ◽  
Rna Binding Proteins ◽  
Super Resolution ◽  
Circular Rna ◽  
Binding Motif ◽  
Missense Mutations ◽  
Gain Of Function ◽  
Processing Bodies ◽  
Heart Pathology

RNA binding motif protein 20 (RBM20) is a key regulator of alternative splicing in the heart, and its mutation leads to malignant dilated cardiomyopathy (DCM). To understand the mechanism of RBM20-associated DCM, we engineered isogenic human induced pluripotent stem cells (iPSCs) with heterozygous or homozygous DCM-associated missense mutations in RBM20 (R636S) as well as RBM20 knockout (KO) iPSCs. iPSC-derived engineered heart tissues made from these cell lines recapitulated contractile dysfunction of RBM20-associated DCM and revealed greater dysfunction with missense mutations than KO. Analysis of RBM20 RNA binding by eCLIP revealed a gain-of-function preference of mutant RBM20 for 3′ UTR sequences that are shared with amyotrophic lateral sclerosis (ALS) and processing-body associated RNA binding proteins (FUS, DDX6). Deep RNA sequencing revealed that the RBM20 R636S mutant has unique gene, splicing, polyadenylation and circular RNA defects that differ from RBM20 KO, impacting distinct cardiac signaling pathways. Splicing defects specific to KO or R636S mutations were supported by data from R636S gene-edited pig hearts and eCLIP. Super-resolution microscopy verified that mutant RBM20 maintains limited nuclear localization potential; rather, the mutant protein associates with cytoplasmic processing bodies (DDX6) under basal conditions, and with stress granules (G3BP1) following acute stress. Taken together, our results highlight a novel pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways that are likely to mediate differential contractile phenotypes and stress-associated heart pathology.

Significance of distinct specifically enriched missense TP53 mutations in prostate cancer.

2020 ◽  
Vol 38 (6_suppl) ◽  
pp. 377-377
Author(s):  
Irina Vasilevskaya ◽  
Jennifer McCann ◽  
Christopher McNair ◽  
Neermala Poudel Neupane ◽  
Peter Gallagher ◽  
...  
Keyword(s):  
Gene Expression ◽  
Prostate Cancer ◽  
Missense Mutations ◽  
Wild Type ◽  
Gain Of Function ◽  
Expression Arrays ◽  
Mutational Status ◽  
Underlying Mechanisms

377 Background: The most common TP53 alterations are missense mutations occurring in the DNA binding domain. The majority of missense p53 mutants (mut-p53) demonstrate oncogenic gain-of-function (GOF) abilities, irrespective of wild-type p53 presence, and thus contribute to a more aggressive disease. In prostate cancer (PCa), characterized by comparatively low overall mutational burden, TP53 is frequently mutated in both primary and advanced disease. Despite significant progress made in the field, detailed mechanisms of GOF in PCa remain undefined due to differing features of p53 mutants. Methods: Analysis of available datasets was performed to assess TP53 mutational status in PCa patient samples and its correlation with the clinical outcome. Using hormone therapy sensitive and CRPC cells, a panel of cell lines was generated to model the two most frequently occurring mutations in the presence or absence of wild-type TP53, as occurs clinically. CHIP-seq, gene expression arrays, and in vitro and in vivo biological assays were performed to interrogate the significance of mut-p53 in PCa. Results: In PCa, missense mutations are significantly associated with decreased progression-free and overall survival. In PCa patient samples these mutations most commonly occur at the R273 residue, demonstrating specific enrichment when compared to other cancers, with R273C alteration being the most frequent. Using our cell panel, CHIP-seq data revealed an expansion of the p53 cistrome upon expression of R273C and R273H mutants in a manner distinct from p53 stabilization in the presence of wt-p53. Moreover, analysis of the TP53 missense mutant-sensitive transcriptomes demonstrated differential gene expression between these mutants, related to the expression of wild-type TP53 in those cells. Finally, R273C and R273H p53 mutants elicited context dependent effects on canonical p53 functions, thereby modulating distinct downstream biological outcomes. Conclusions: These data expand our knowledge of the underlying mechanisms by which distinct gain-of-function p53 mutants affect prostate cancer, and can lead to identification of novel therapeutic targets to improve clinical outcomes in PCa patients harboring these mutations.

Functional plasticity of putative TP53 gain of function mutations in human gastrointestinal tract adenocarcinomas.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e15530-e15530
Author(s):  
David R. Braxton ◽  
Joanne Xiu ◽  
Wafik S. El-Deiry ◽  
Sourat Darabi ◽  
Wolfgang Michael Korn ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Tp53 Gene ◽  
Immune Checkpoint Blockade ◽  
Missense Mutations ◽  
Cell Cycle Regulators ◽  
Wild Type ◽  
Functional Plasticity ◽  
Gain Of Function ◽  
Multifunctional Protein ◽  
Gi Cancers

e15530 Background: The TP53 gene product is a multifunctional protein and prototypical tumor suppressor. Certain missense mutations occurring in hotspots in the TP53 gene may confer gain of function (GOF) oncogenic properties. A recent mouse model (Kadosh et. al., Nature, 2020) demonstrated that secreted factors in the gut microbiome permit GOF p53 to be oncogenic in lower GI tract cancers (LT). By contrast, the GOF p53 mutants in the upper tract cancers (UT) retained tumor suppressor functions. We aimed to provide evidence of the plasticity of TP53 GOF mutants in human GI cancers. We hypothesized that UT should have a lower rate of TP53 GOF than the LT, and UT cancers with putative GOF TP53 mutations should display a higher degree of co-occurring oncogenic alterations such as dysregulation of CCND1, MYCS, or WNT. We also explored outcome differences for TP53 GOF variants. Methods: Tumors of UT (stomach, esophagus, small intestine) and LT (colorectum, anus) were tested at Caris Life Sciences (Phoenix, AZ) by NGS (NextSeq, 592 or NovoSeq, whole exome). MSI/MMRP status was determined by IHC and NGS. Real-world overall survival (OS) was obtained from payor claims data and Kaplan-Meier estimates were calculated. P values adjusted for multiple correction (q) of < 0.05 was considered significant. Six p53 variants were classified as GOF (p.R175, p.G245, p.R248, p.R249, p.R273, and p.R282). Results: Of the 5311 UT and 14810 LT tumors sequenced, 4799 harbored a TP53 GOF variant. UT had TP53 GOF in 22.9% of cases compared to 27.0% of LT (q < 0.05). 67 genes were enriched for amplification (q < 0.05) in the UT TP53 GOF cancers compared to the LT TP53 GOF, including cell cycle regulators( CCND1, CCNE1, CDK6, CDK12), and oncogenes ( KRAS, ERBB2, EGFR). LT TP53 GOF cancers were enriched for eight gene amplifications, including CDX2, FLT3, and SRC. Differences in SNV/InDel patterns were not remarkable. OS analysis revealed TP53 GOF in UT had shorter OS when compared to TP53 wild-type (WT) (Median OS: 418 vs 515 days; HR: 0.864; 95%CI [0.772-0.966]; p = 0.011), but not when MSI/MMRP status was considered separately. LT TP53 GOF did not show OS difference in aggregate, however, MSI-High TP53 GOF cancers showed a shorter OS (Median OS: 762 vs 1479 days; HR: 1.522; [1.068 - 2.171]; p = 0.019). Sub-analysis of LT MSI-High cancers treated by immune checkpoint blockade showed a non-significant trend toward shorter OS of TP53 GOF versus TP53 wild type cancers (HR: 2.55; [0.90-7.19], p: 0.067), but no such trend was seen in UT (HR: 1.434; [0.304-6.758], p:0.646). Conclusions: Our study is the first to compare putative TP53 GOF variants across UT and LT GI adenocarcinoma. TP53 GOF variants are associated with decreased OS in both UT cancers and MSI-High LT cancers. Our findings support the functional plasticity of TP53 GOF variants in human GI cancers. Therefore, the purported effects of secreted microbiome factors on TP53 GOF variants cannot be ruled out.

Platelet Activation and Aggregation Induced by Recombinant von Willebrand Factors Reproducing Four Type 2B von Willebrand Disease Missense Mutations

1998 ◽  
Vol 79 (01) ◽  
pp. 211-216 ◽  
Author(s):  
Lysiane Hilbert ◽  
Claudine Mazurier ◽  
Christophe de Romeuf
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Missense Mutations ◽  
Inhibit Platelet Aggregation ◽  
Wild Type ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryType 2B of von Willebrand disease (vWD) refers to qualitative variants with increased affinity of von Willebrand factor (vWF) for platelet glycoprotein Ib (GPIb). All the mutations responsible for type 2B vWD have been located in the A1 domain of vWF. In this study, various recombinant von Willebrand factors (rvWF) reproducing four type 2B vWD missense mutations were compared to wild-type rvWF (WT-rvWF) for their spontaneous binding to platelets and their capacity to induce platelet activation and aggregation. Our data show that the multimeric pattern of each mutated rvWF is similar to that of WT-rvWF but the extent of spontaneous binding and the capacity to induce platelet activation and aggregation are more important for the R543Q and V553M mutations than for the L697V and A698V mutations. Both the binding of mutated rvWFs to platelets and platelet aggregation induced by type 2B rvWFs are inhibited by monoclonal anti-GPIb and anti-vWF antibodies, inhibitors of vWF binding to platelets in the presence of ristocetin, as well as by aurin tricarboxylic acid. On the other hand, EDTA and a monoclonal antibody directed against GPIIb/IIIa only inhibit platelet aggregation. Furthermore, the incubation of type 2B rvWFs with platelets, under stirring conditions, results in the decrease in high molecular weight vWF multimers in solution, the extent of which appears correlated with that of plasma vWF from type 2B vWD patients harboring the corresponding missense mutation. This study supports that the binding of different mutated type 2B vWFs onto platelet GPIb induces various degrees of platelet activation and aggregation and thus suggests that the phenotypic heterogeneity of type 2B vWD may be related to the nature and/or location of the causative point mutation.

The Effect of Mianserin on Lifespan of Caenorhabditis elegans is Abolished by Glucose

2021 ◽  
Vol 13 ◽  
Author(s):  
Abdullah Almotayri ◽  
Jency Thomas ◽  
Mihiri Munasinghe ◽  
Markandeya Jois
Keyword(s):  
Caenorhabditis Elegans ◽  
Scaffolding Protein ◽  
Lifespan Extension ◽  
Wild Type ◽  
E Coli ◽  
C Elegans ◽  
Risk Of Death ◽  
Increased Risk ◽  
Antidepressant Use ◽  
Extension Effect

Background: The antidepressant mianserin has been shown to extend the lifespan of Caenorhabditis elegans (C. elegans), a well-established model organism used in aging research. The extension of lifespan in C. elegans was shown to be dependent on increased expression of the scaffolding protein (ANK3/unc-44). In contrast, antidepressant use in humans is associated with an increased risk of death. The C. elegans in the laboratory are fed Escherichia coli (E. coli), a diet high in protein and low in carbohydrate, whereas a typical human diet is high in carbohydrates. We hypothesized that dietary carbohydrates might mitigate the lifespan-extension effect of mianserin. Objective: To investigate the effect of glucose added to the diet of C. elegans on the lifespan-extension effect of mianserin. Methods: Wild-type Bristol N2 and ANK3/unc-44 inactivating mutants were cultured on agar plates containing nematode growth medium and fed E. coli. Treatment groups included (C) control, (M50) 50 μM mianserin, (G) 73 mM glucose, and (M50G) 50 μM mianserin and 73 mM glucose. Lifespan was determined by monitoring the worms until they died. Statistical analysis was performed using the Kaplan-Meier version of the log-rank test. Results: Mianserin treatment resulted in a 12% increase in lifespan (P<0.05) of wild-type Bristol N2 worms but reduced lifespan by 6% in ANK3/unc-44 mutants, consistent with previous research. The addition of glucose to the diet reduced the lifespan of both strains of worms and abolished the lifespan-extension by mianserin. Conclusion: The addition of glucose to the diet of C. elegans abolishes the lifespan-extension effects of mianserin.

scholarly journals The Mitochondrial Genome Integrity Gene, MGI1, of Kluyveromyces lactis Encodes the β-Subunit of F1-ATPase

Genetics ◽  
1996 ◽  
Vol 144 (4) ◽  
pp. 1445-1454 ◽  
Author(s):  
Xin Jie Chen ◽  
G Desmond Clark-Walker
Keyword(s):  
Mitochondrial Genome ◽  
Kluyveromyces Lactis ◽  
Three Dimensional ◽  
Genome Integrity ◽  
Dimensional Structure ◽  
Deletion Mutants ◽  
Β Subunit ◽  
Gain Of Function ◽  
F1 Atpase ◽  
Γ Subunit

In a previous report, we found that mutations at the mitochondrial genome integrity locus, MGI1, can convert Kluyveromyces lactis into a petite-positive yeast. In this report, we describe the isolation of the MGI1 gene and show that it encodes the β-subunit of the mitochondrial F1-ATPase. The site of mutation in four independently isolated mgi1 alleles is at Arg435, which has changed to Gly in three cases and Ile in the fourth isolate. Disruption of MGI1 does not lead to the production of mitochondrial genome deletion mutants, indicating that an assembled F1 complex is needed for the “gain-of-function” phenotype found in mgi1 point mutants. The location of Arg435 in the β-subunit, as deduced from the three-dimensional structure of the bovine F1-ATPase, together with mutational sites in the previously identified mgi2 and mgi5 alleles, suggests that interaction of the β- and α- (MGI2) subunits with the γ-subunit (MGI5) is likely to be affected by the mutations.

scholarly journals Structural and Thermodynamic Analysis of the Resistance Development to Pimodivir (VX-787), the Clinical Inhibitor of Cap Binding to PB2 Subunit of Influenza A Polymerase

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 1007
Author(s):  
Jiří Gregor ◽  
Kateřina Radilová ◽  
Jiří Brynda ◽  
Jindřich Fanfrlík ◽  
Jan Konvalinka ◽  
...  
Keyword(s):  
Thermodynamic Analysis ◽  
Influenza A ◽  
Polymerase Activity ◽  
Second Phase ◽  
Missense Mutations ◽  
Wild Type ◽  
Resistance Development ◽  
Third Phase ◽  
Major Mutation ◽  
Control And Prevention

Influenza A virus (IAV) encodes a polymerase composed of three subunits: PA, with endonuclease activity, PB1 with polymerase activity and PB2 with host RNA five-prime cap binding site. Their cooperation and stepwise activation include a process called cap-snatching, which is a crucial step in the IAV life cycle. Reproduction of IAV can be blocked by disrupting the interaction between the PB2 domain and the five-prime cap. An inhibitor of this interaction called pimodivir (VX-787) recently entered the third phase of clinical trial; however, several mutations in PB2 that cause resistance to pimodivir were observed. First major mutation, F404Y, causing resistance was identified during preclinical testing, next the mutation M431I was identified in patients during the second phase of clinical trials. The mutation H357N was identified during testing of IAV strains at Centers for Disease Control and Prevention. We set out to provide a structural and thermodynamic analysis of the interactions between cap-binding domain of PB2 wild-type and PB2 variants bearing these mutations and pimodivir. Here we present four crystal structures of PB2-WT, PB2-F404Y, PB2-M431I and PB2-H357N in complex with pimodivir. We have thermodynamically analysed all PB2 variants and proposed the effect of these mutations on thermodynamic parameters of these interactions and pimodivir resistance development. These data will contribute to understanding the effect of these missense mutations to the resistance development and help to design next generation inhibitors.

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