scholarly journals Mutation-specific non-canonical pathway of PTEN as a distinct therapeutic target for glioblastoma

2021 ◽  
Vol 12 (4) ◽  
Author(s):  
Seung Won Choi ◽  
Yeri Lee ◽  
Kayoung Shin ◽  
Harim Koo ◽  
Donggeon Kim ◽  
...  
Keyword(s):  
Functional Properties ◽  
Therapeutic Target ◽  
Medical Center ◽  
Dominant Negative ◽  
The Cancer Genome Atlas ◽  
Dominant Negative Effect ◽  
Cell Periphery ◽  
Missense Mutations ◽  
Phosphatase Domain ◽  
Mutant Proteins

AbstractPTEN is one of the most frequently altered tumor suppressor genes in malignant tumors. The dominant-negative effect of PTEN alteration suggests that the aberrant function of PTEN mutation might be more disastrous than deletion, the most frequent genomic event in glioblastoma (GBM). This study aimed to understand the functional properties of various PTEN missense mutations and to investigate their clinical relevance. The genomic landscape of PTEN alteration was analyzed using the Samsung Medical Center GBM cohort and validated via The Cancer Genome Atlas dataset. Several hotspot mutations were identified, and their subcellular distributions and phenotypes were evaluated. We established a library of cancer cell lines that overexpress these mutant proteins using the U87MG and patient-derived cell models lacking functional PTEN. PTEN mutations were categorized into two major subsets: missense mutations in the phosphatase domain and truncal mutations in the C2 domain. We determined the subcellular compartmentalization of four mutant proteins (H93Y, C124S, R130Q, and R173C) from the former group and found that they had distinct localizations; those associated with invasive phenotypes (‘edge mutations’) localized to the cell periphery, while the R173C mutant localized to the nucleus. Invasive phenotypes derived from edge substitutions were unaffected by an anti-PI3K/Akt agent but were disrupted by microtubule inhibitors. PTEN mutations exhibit distinct functional properties regarding their subcellular localization. Further, some missense mutations (‘edge mutations’) in the phosphatase domain caused enhanced invasiveness associated with dysfunctional cytoskeletal assembly, thus suggesting it to be a potent therapeutic target.

Preclinical proof-of-concept study: antisense-mediated knockdown of CALM as a therapeutic strategy for calmodulinopathy

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
Y Yamamoto ◽  
T Makiyama ◽  
Y Wuriyanghai ◽  
H Kohjitani ◽  
J Gao ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Dominant Negative ◽  
Locked Nucleic Acid ◽  
Polymorphic Ventricular Tachycardia ◽  
Dominant Negative Effect ◽  
Homologous Sequence ◽  
Funding Source ◽  
Missense Mutations ◽  
Private Company ◽  
Ips Cell

Abstract Background Calmodulin (CaM) is a ubiquitous Ca2+ sensor molecule encoded by three distinct calmodulin genes, CALM1–3, and has an important role for cardiac ion channel function. Recently, heterozygous missense mutations in CALM genes were reported to cause a new category of life-threatening genetic arrhythmias such as long-QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT), which is called as “calmodulinopathy”. The patients with calmodulinopathy show poor prognosis and there is no effective treatment for them. Purpose Considering the dominant-negative effect of mutant calmodulin proteins produced by heterozygous missense mutations in CALMs, we aimed to prove the concept of antisense-based therapy to treat calmodulinopathy using human iPS cell-derived cardiomyocyte (hiPSC-CM) model. Methods We designed multiple locked nucleic acid (LNA) gapmer-antisense oligonucleotides (ASOs) targeting CALM2 and analyzed the silencing efficiency and toxicity in cultured cells to select the most potent ASO. Using CMs differentiated from hiPSCs which were generated form a 12-year-old boy with LQTS carrying a heterozygous CALM2-N98S mutation, CALM2 expression and action potentials (APs) were analyzed to evaluate the efficacy of ASOs. Results We identified several ASOs which reduced CALM2 expression without affecting cell viability in human cultured cells (HepG2) (ASO 50 nM, n=2; Figure 1A). Considering further experiments in vivo mouse model, we investigated the CALM2 silencing activity in mouse cultured cells (3T3-L1) without transfection (free-uptake) (ASO 1 μM, n=2; †ASOs have homologous sequence between human and mouse; Figure B). After free-uptake CALM2 silencing analysis in 3T3-L1 cells, we identified that ASO #2 has the most potent CALM2 silencing activity and low cytotoxicity (Figure 1B). ASO #2 effectively reduced CALM2 expression even in hiPSC-CMs (ASO(−): n=3, lipofection: n=4, free-uptake: n=3; P<0.05; Figure 1C). In action potential recordings, we demonstrated that ASO #2 ameliorated prolonged AP durations (APD90) in N98S-hiPSC-CMs at 0.5 Hz pacing (ASO(−): 666±123 ms (n=7), lipofection: 329±21 ms (n=8), free-uptake: 388±34 ms (n=12); P<0.05; Figure 1D). Conclusion Our results using patient-derived hiPSC-CM model suggest that ASO-based therapy might be a promising strategy for the treatment of calmodulinopathy. Figure 1 Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Nissan Chemical Corporation

Both the paired domain and homeodomain are required for in vivo function of Drosophila Paired

Development ◽  
1996 ◽  
Vol 122 (9) ◽  
pp. 2709-2718 ◽  
Author(s):  
P. Miskiewicz ◽  
D. Morrissey ◽  
Y. Lan ◽  
L. Raj ◽  
S. Kessler ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Ectopic Expression ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Paired Domain ◽  
Mutant Proteins ◽  
Segment Polarity ◽  
Negative Effect

Drosophila paired, a homolog of mammalian Pax-3, is key to the coordinated regulation of segment-polarity genes during embryogenesis. The paired gene and its homologs are unusual in encoding proteins with two DNA-binding domains, a paired domain and a homeodomain. We are using an in vivo assay to dissect the functions of the domains of this type of molecule. In particular, we are interested in determining whether one or both DNA-binding activities are required for individual in vivo functions of Paired. We constructed point mutants in each domain designed to disrupt DNA binding and tested the mutants with ectopic expression assays in Drosophila embryos. Mutations in either domain abolished the normal regulation of the target genes engrailed, hedgehog, gooseberry and even-skipped, suggesting that these in vivo functions of Paired require DNA binding through both domains rather than either domain alone. However, when the two mutant proteins were placed in the same embryo, Paired function was restored, indicating that the two DNA-binding activities need not be present in the same molecule. Quantitation of this effect shows that the paired domain mutant has a dominant-negative effect consistent with the observations that Paired protein can bind DNA as a dimer.

Functional analysis of the BMP9 response of ALK1 mutants from HHT2 patients: a diagnostic tool for novel ACVRL1 mutations

Blood ◽  
2010 ◽  
Vol 116 (9) ◽  
pp. 1604-1612 ◽  
Author(s):  
Nicolas Ricard ◽  
Marie Bidart ◽  
Christine Mallet ◽  
Gaetan Lesca ◽  
Sophie Giraud ◽  
...  
Keyword(s):  
Diagnostic Tool ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Novel Mutations ◽  
Mutant Proteins ◽  
Genes Encoding ◽  
Negative Effect ◽  
Bone Morphogenetic Protein 9

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant genetically inheritable vascular dysplasia caused by mutations in genes encoding receptors of the transforming growth factor-β (TGF-β) family: ENG, encoding endoglin (HHT1), and ACVRL1, encoding activin receptor-like kinase-1 (ALK1; HHT2). Our recent discovery of bone morphogenetic protein 9 (BMP9) as the specific ligand for ALK1 allowed us to reevaluate the functional significance of ACVRL1 mutations. We generated 19 ALK1 mutants reproducing HHT2 mutations (4 were novel mutations) found throughout the protein. We show that all ALK1 mutant proteins were expressed by transfected cells; most of them were present at the cell surface and retained their ability to bind BMP9 (except for the extracellular mutants). However, most were defective in BMP9 signaling. None of the ALK1 mutants had a dominant negative effect on wild-type ALK1 activity. These data demonstrate that mutations of ACVRL1 fit with a functional haploinsufficiency model affecting BMP9 signaling. Our study also identified 4 ACVRL1 mutations (D179A, R386C, R454W, and A482V) that did not alter the BMP9 responses that are polymorphisms and 2 novel mutations that are pathogenic (L381P and I485F). This demonstrates that the analysis of BMP9 responses can be used as a diagnostic tool by geneticists confronted with novel or conflicting ACVRL1 mutations.

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 De novo Mutations in NALCN Cause a Syndrome of Congenital Contractures of the Limbs and Face with Hypotonia, and Developmental Delay

2015 ◽  
Author(s):  
Jessica X Chong ◽  
Margaret J McMillin ◽  
Kathryn M Shively ◽  
Anita E Beck ◽  
Colby T Marvin ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Developmental Delay ◽  
De Novo ◽  
Dominant Negative ◽  
Protein Domain ◽  
Dominant Negative Effect ◽  
Global Developmental Delay ◽  
Missense Mutations ◽  
Severe Intellectual Disability ◽  
Dominant Condition

Freeman-Sheldon syndrome, or distal arthrogryposis type 2A (DA2A), is an autosomal dominant condition caused by mutations in MYH3 and characterized by multiple congenital contractures of the face and limbs and normal cognitive development. We identified a subset of five simplex cases putatively diagnosed with “DA2A with severe neurological abnormalities” in which the proband had Congenital Contractures of the LImbs and FAce, Hypotonia, and global Developmental Delay often resulting in early death, a unique condition that we now refer to as CLIFAHDD syndrome. Exome sequencing identified missense mutations in sodium leak channel, nonselective (NALCN) in four families with CLIFAHDD syndrome. Using molecular inversion probes to screen NALCN in a cohort of 202 DA cases as well as concurrent exome sequencing of six other DA cases revealed NALCN mutations in ten additional families with “atypical” forms of DA. All fourteen mutations were missense variants predicted to alter amino acid residues in or near the S5 and S6 pore-forming segments of NALCN, highlighting the functional importance of these segments. In vitro functional studies demonstrated that mutant NALCN nearly abolished the expression of wildtype NALCN, suggesting that mutations that cause CLIFAHDD syndrome have a dominant negative effect. In contrast, homozygosity for mutations in other regions of NALCN has been reported in three families with an autosomal recessive condition characterized mainly by hypotonia and severe intellectual disability. Accordingly, mutations in NALCN can cause either a recessive or dominant condition with varied though overlapping phenotypic features perhaps depending on the type of mutation and affected protein domain(s).

scholarly journals KDM6A mutations promote acute cytoplasmic DNA release, DNA damage response and mitosis defects

2021 ◽  
Author(s):  
Julian Koch ◽  
Alexander Lang ◽  
Patcharawalai Whongsiri ◽  
Wolfgang A. Schulz ◽  
Michele Janine Hoffmann ◽  
...  
Keyword(s):  
Dna Damage ◽  
Nuclear Dna ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Dominant Negative Effect ◽  
Future Research ◽  
Missense Mutations ◽  
Cancer Genes ◽  
Intrinsically Disordered ◽  
Demethylase Activity

Abstract Background KDM6A, encoding a histone demethylase, is one of the top ten mutated epigenetic cancer genes. The effect of mutations on its structure and function are however poorly characterized. Methods Database search identified nonsense and missense mutations in the N-terminal TPR motifs and the C-terminal, catalytic JmjC domain, but also in the intrinsically disordered region connecting both well-structured domains. KDM6A variants with cancer-derived mutations were generated using site directed mutagenesis and fused to eGFP, which served as an all-in-one affinity and fluorescence tag to study demethylase activity by an ELISA based assay in vitro, complex assembly by Co-immunoprecipitation and localization by microscopy in cellulo. Results Independent of the mutation and demethylase activity, all KDM6A variants were detectable in the nucleus. KDM6A truncations displayed changes in complex assemblies: affecting (1) known interactions with the COMPASS complex component RBBP5 and (2) KDM6A-DNA associated assemblies with the nucleolar protein Nucleophosmin. Furthermore, we observed a severe cellular phenotype characterized by multiple acute effects on nuclear integrity, namely, release of nuclear DNA into the cytoplasm, increased level of DNA damage indicators RAD51 and p-γH2A.X, and hence mitosis defects. Conclusion These observations reveal novel effects of pathogenic variants pointing at new specific functions of KDM6A as well as at a dominant negative effect of KDM6A truncation variants. The underlying mechanisms and affected pathways have to be investigated in future research to understand how tumor cells cope with and benefit from KDM6A truncations.

scholarly journals Four novel mutations in the ALPL gene in Chinese patients with odonto, childhood, and adult hypophosphatasia

2018 ◽  
Vol 38 (4) ◽  
Author(s):  
Lijun Xu ◽  
Qianqian Pang ◽  
Yan Jiang ◽  
Ou Wang ◽  
Mei Li ◽  
...  
Keyword(s):  
Serum Alkaline Phosphatase ◽  
Gene Mutations ◽  
Dominant Negative ◽  
Chinese Patients ◽  
Dominant Negative Effect ◽  
Healthy Controls ◽  
Missense Mutations ◽  
Genetic Characteristics ◽  
Chinese Families ◽  
Negative Effect

Hypophosphatasia (HPP) is a rare inherited disorder characterized by defective bone and/or dental mineralization, and decreased serum alkaline phosphatase (ALP) activity. ALPL, the only gene related with HPP, encodes tissue non-specific ALP (TNSALP). Few studies were carried out in ALPL gene mutations in the Chinese population with HPP. The purpose of the present study is to elucidate the clinical and genetic characteristics of HPP in five unrelated Chinese families and two sporadic patients. Ten clinically diagnosed HPP patients from five unrelated Chinese families and two sporadic patients and fifty healthy controls were genetically investigated. All 12 exons and exon–intron boundaries of the ALPL gene were amplified by PCR and directly sequenced. The laboratory and radiological investigations were conducted simultaneously in these HPP ten patients. A 3D model of the TNSALP was used to predict the dominant negative effect of identified missense mutations. Three odonto, three childhood, and four adult types of HPP were clinically diagnosed. Ten mutations were identified in five unrelated Chinese families and two sporadic patients, including eight missense mutations and two frameshift mutations. Of which, four were novel: one frameshift mutation (p.R138Pfsx45); three missense mutations (p.C201R, p.V459A, p.C497S). No identical mutations and any other new ALPL mutations were found in unrelated 50 healthy controls. Our study demonstrated that the ALPL gene mutations are responsible for HPP in these Chinese families. These findings will be useful for clinicians to improve understanding of this heritable bone disorder.

scholarly journals Characterization of ANGPT2 mutations associated with primary lymphedema

2020 ◽  
Vol 12 (560) ◽  
pp. eaax8013 ◽  
Author(s):  
Veli-Matti Leppänen ◽  
Pascal Brouillard ◽  
Emilia A. Korhonen ◽  
Tuomas Sipilä ◽  
Sawan Kumar Jha ◽  
...  
Keyword(s):  
De Novo ◽  
Vascular System ◽  
Lymphatic Vessels ◽  
Mouse Skin ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Missense Mutations ◽  
Activation Mechanisms ◽  
Primary Lymphedema ◽  
Negative Effect

Primary lymphedema is caused by developmental and functional defects of the lymphatic vascular system that result in accumulation of protein-rich fluid in tissues, resulting in edema. The 28 currently known genes causing primary lymphedema can explain <30% of cases. Angiopoietin 1 (ANGPT1) and ANGPT2 function via the TIE1-TIE2 (tyrosine kinase with immunoglobulin-like and epidermal growth factor–like domains 1 and 2) receptor complex and α5β1 integrin to form an endothelial cell signaling pathway that is critical for blood and lymphatic vessel formation and remodeling during embryonic development, as well as for homeostasis of the mature vasculature. By screening a cohort of 543 individuals affected by primary lymphedema, we identified one heterozygous de novo ANGPT2 whole-gene deletion and four heterozygous ANGPT2 missense mutations. Functional analyses revealed three missense mutations that resulted in decreased ANGPT2 secretion and inhibited the secretion of wild-type (WT)–ANGPT2, suggesting that they have a dominant-negative effect on ANGPT2 signaling. WT-ANGPT2 and soluble mutants T299M and N304K activated TIE1 and TIE2 in an autocrine assay in human lymphatic endothelial cells. Molecular modeling and biophysical studies showed that amino-terminally truncated ANGPT subunits formed asymmetrical homodimers that bound TIE2 in a 2:1 ratio. The T299M mutant, located in the dimerization interphase, showed reduced integrin α5 binding, and its expression in mouse skin promoted hyperplasia and dilation of cutaneous lymphatic vessels. These results demonstrate that primary lymphedema can be associated with ANGPT2 mutations and provide insights into TIE1 and TIE2 activation mechanisms.

scholarly journals A dominant-negative effect drives selection of TP53 missense mutations in myeloid malignancies

Science ◽  
2019 ◽  
Vol 365 (6453) ◽  
pp. 599-604 ◽  
Author(s):  
Steffen Boettcher ◽  
Peter G. Miller ◽  
Rohan Sharma ◽  
Marie McConkey ◽  
Matthew Leventhal ◽  
...  
Keyword(s):  
Dna Binding ◽  
Leukemia Cell ◽  
Dominant Negative ◽  
Single Amino Acid ◽  
Dominant Negative Effect ◽  
Human Leukemia ◽  
Missense Mutations ◽  
Myeloid Malignancies ◽  
Missense Variants ◽  
Negative Effect

TP53, which encodes the tumor suppressor p53, is the most frequently mutated gene in human cancer. The selective pressures shaping its mutational spectrum, dominated by missense mutations, are enigmatic, and neomorphic gain-of-function (GOF) activities have been implicated. We used CRISPR-Cas9 to generate isogenic human leukemia cell lines of the most common TP53 missense mutations. Functional, DNA-binding, and transcriptional analyses revealed loss of function but no GOF effects. Comprehensive mutational scanning of p53 single–amino acid variants demonstrated that missense variants in the DNA-binding domain exert a dominant-negative effect (DNE). In mice, the DNE of p53 missense variants confers a selective advantage to hematopoietic cells on DNA damage. Analysis of clinical outcomes in patients with acute myeloid leukemia showed no evidence of GOF for TP53 missense mutations. Thus, a DNE is the primary unit of selection for TP53 missense mutations in myeloid malignancies.

Sign in / Sign up

Export Citation Format

Share Document