NTRK Point Mutations and Their Functional Consequences

Abstract Glutamine synthetase (GS) in the liver is expressed in a small perivenous, highly specialized hepatocyte population and is essential for the maintenance of low, non-toxic ammonia levels in the organism. However, GS activity can be impaired by tyrosine nitration of the enzyme in response to oxidative/nitrosative stress in a pH-sensitive way. The underlying molecular mechanism as investigated by combined molecular simulations and in vitro experiments indicates that tyrosine nitration can lead to a fully reversible and pH-sensitive regulation of protein function. This approach was also used to understand the functional consequences of several recently described point mutations of human GS with clinical relevance and to suggest an approach to restore impaired GS activity.

Download Full-text

Concerted action of two novel tRNA mtDNA point mutations in chronic progressive external ophthalmoplegia

Bioscience Reports ◽

10.1042/bsr20080004 ◽

2008 ◽

Vol 28 (2) ◽

pp. 89-96 ◽

Cited By ~ 6

Author(s):

Cornelia Kornblum ◽

Gábor Zsurka ◽

Rudolf J. Wiesner ◽

Rolf Schröder ◽

Wolfram S. Kunz

Keyword(s):

Skeletal Muscle ◽

Northern Blot ◽

Phenotypic Expression ◽

Point Mutations ◽

Skeletal Muscle Fibre ◽

Progressive External Ophthalmoplegia ◽

Chronic Progressive External Ophthalmoplegia ◽

Mtdna Mutations ◽

Mtdna Sequence ◽

Functional Consequences

CPEO (chronic progressive external ophthalmoplegia) is a common mitochondrial disease phenotype in adults which is due to mtDNA (mitochondrial DNA) point mutations in a subset of patients. Attributing pathogenicity to novel tRNA mtDNA mutations still poses a challenge, particularly when several mtDNA sequence variants are present. In the present study we report a CPEO patient for whom sequencing of the mitochondrial genome revealed three novel tRNA mtDNA mutations: G5835A, del4315A, T1658C in tRNATyr, tRNAIle and tRNAVal genes. In skeletal muscle, the tRNAVal and tRNAIle mutations were homoplasmic, whereas the tRNATyr mutation was heteroplasmic. To address the pathogenic relevance, we performed two types of functional tests: (i) single skeletal muscle fibre analysis comparing G5835A mutation loads and biochemical phenotypes of corresponding fibres, and (ii) Northern-blot analyses of mitochondrial tRNATyr, tRNAIle and tRNAVal. We demonstrated that both the G5835A tRNATyr and del4315A tRNAIle mutation have serious functional consequences. Single-fibre analyses displayed a high threshold of the tRNATyr mutation load for biochemical phenotypic expression at the single-cell level, indicating a rather mild pathogenic effect. In contrast, skeletal muscle tissue showed a severe decrease in respiratory-chain activities, a reduced overall COX (cytochrome c oxidase) staining intensity and abundant COX-negative fibres. Northern-blot analyses showed a dramatic reduction of tRNATyr and tRNAIle levels in muscle, with impaired charging of tRNAIle, whereas tRNAVal levels were only slightly decreased, with amino-acylation unaffected. Our findings suggest that the heteroplasmic tRNATyr and homoplasmic tRNAIle mutation act together, resulting in a concerted effect on the biochemical and histological phenotype. Thus homoplasmic mutations may influence the functional consequences of pathogenic heteroplasmic mtDNA mutations.

Download Full-text

The role of thyroid hormone receptor DNA binding in negative thyroid hormone-mediated gene transcription

Journal of Molecular Endocrinology ◽

10.1677/jme-08-0023 ◽

2008 ◽

Vol 41 (1) ◽

pp. 25-34 ◽

Cited By ~ 6

Author(s):

Anne Wulf ◽

Marianne G Wetzel ◽

Maxim Kebenko ◽

Meike Kröger ◽

Angelika Harneit ◽

...

Keyword(s):

Thyroid Hormone ◽

Dna Binding ◽

Gene Transcription ◽

Hormone Receptor ◽

Target Genes ◽

Thyroid Hormone Receptor ◽

Point Mutations ◽

Chimeric Protein ◽

Functional Consequences ◽

Glycerol 3 Phosphate Dehydrogenase

Thyroid hormone 3,3′,5-tri-iodothyronine (T3) regulates gene expression in a positive and negative manner. Here, we analyzed the regulation of a positively (mitochondrial glycerol-3-phosphate dehydrogenase) and negatively T3-regulated target gene (TSHα). Thyroid hormone receptor (TR) activates mGPDH but not TSH promoter fragments in a mammalian one-hybrid assay. Furthermore, we investigated functional consequences of targeting TR to DNA independent of its own DNA-binding domain (DBD). Using a chimeric fusion protein of the DBD of yeast transcription factor Gal4 with TR, we demonstrated a positive regulation of gene transcription in response to T3. T3-mediated activation of this chimeric protein is further increased after an introduction of point mutations within the DBD of TR. Moreover, we investigated the capacity of TR to negatively regulate gene transcription on a DNA-tethered cofactor platform. A direct binding of TR to DNA via its own DBD is dispensable in this assay. We investigated functional consequences of point mutations affecting different domains of TR. Our data indicate that the DBD of TR plays a key role in direct DNA binding on positively but not on negatively T3-regulated target genes. Nevertheless, the DBD is involved in mediating negative gene regulation independent of its capacity to bind DNA.

Download Full-text

Functional Consequences of Reduction in NMDA Receptor Glycine Affinity in Mice Carrying Targeted Point Mutations in the Glycine Binding Site

Journal of Neuroscience ◽

10.1523/jneurosci.20-11-04037.2000 ◽

2000 ◽

Vol 20 (11) ◽

pp. 4037-4049 ◽

Cited By ~ 69

Author(s):

James N. C. Kew ◽

Anja Koester ◽

Jean-Luc Moreau ◽

Francois Jenck ◽

Abdel-Mouttalib Ouagazzal ◽

...

Keyword(s):

Nmda Receptor ◽

Binding Site ◽

Point Mutations ◽

Functional Consequences

Download Full-text

Multiplexed functional genomic analysis of 5’ untranslated region mutations across the spectrum of prostate cancer

Nature Communications ◽

10.1038/s41467-021-24445-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yiting Lim ◽

Sonali Arora ◽

Samantha L. Schuster ◽

Lukas Corey ◽

Matthew Fitzgibbon ◽

...

Keyword(s):

Gene Expression ◽

Prostate Cancer ◽

Point Mutations ◽

Genomic Analysis ◽

Mrna Translation ◽

Specific Gene ◽

Functional Genomic Analysis ◽

A Genome ◽

Functional Consequences ◽

Wide Scale

AbstractThe functional consequences of genetic variants within 5’ untranslated regions (UTRs) on a genome-wide scale are poorly understood in disease. Here we develop a high-throughput multi-layer functional genomics method called PLUMAGE (Pooled full-length UTR Multiplex Assay on Gene Expression) to quantify the molecular consequences of somatic 5’ UTR mutations in human prostate cancer. We show that 5’ UTR mutations can control transcript levels and mRNA translation rates through the creation of DNA binding elements or RNA-based cis-regulatory motifs. We discover that point mutations can simultaneously impact transcript and translation levels of the same gene. We provide evidence that functional 5’ UTR mutations in the MAP kinase signaling pathway can upregulate pathway-specific gene expression and are associated with clinical outcomes. Our study reveals the diverse mechanisms by which the mutational landscape of 5’ UTRs can co-opt gene expression and demonstrates that single nucleotide alterations within 5’ UTRs are functional in cancer.

Download Full-text

Functional Consequences and Exonuclease Kinetic Parameters of Point Mutations in Bacteriophage T4DNA Polymerase†

Biochemistry ◽

10.1021/bi961552q ◽

1996 ◽

Vol 35 (51) ◽

pp. 16621-16629 ◽

Cited By ~ 29

Author(s):

A. K. M. Abdus Sattar ◽

Tsung-Chung Lin ◽

Christopher Jones ◽

William H. Konigsberg

Keyword(s):

Kinetic Parameters ◽

Point Mutations ◽

Functional Consequences

Download Full-text

Functional consequences of stably expressing a mutant calsequestrin (CASQ2D307H) in the CASQ2 null background

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00578.2011 ◽

2012 ◽

Vol 302 (1) ◽

pp. H253-H261 ◽

Cited By ~ 10

Author(s):

Anuradha Kalyanasundaram ◽

Serge Viatchenko-Karpinski ◽

Andriy E. Belevych ◽

Veronique A. Lacombe ◽

Hyun Seok Hwang ◽

...

Keyword(s):

Point Mutations ◽

Buffering Capacity ◽

Polymorphic Ventricular Tachycardia ◽

Mutant Protein ◽

Null Mouse ◽

Cardiac Sarcoplasmic Reticulum ◽

Functional Consequences ◽

Delayed Afterdepolarizations ◽

The Stability

The role of calsequestrin (CASQ2) in cardiac sarcoplasmic reticulum (SR) calcium (Ca2+) transport has gained significant attention since point mutations in CASQ2 were reported to cause ventricular arrhythmia. In the present study, we have critically evaluated the functional consequences of expressing the CASQ2D307H mutant protein in the CASQ2 null mouse. We recently reported that the mutant CASQ2D307H protein can be stably expressed in CASQ2 null hearts, and it targets appropriately to the junctional SR (Kalyanasundaram A, Bal NC, Franzini-Armstrong C, Knollmann BC, Periasamy M. J Biol Chem 285: 3076–3083, 2010). In this study, we found that introduction of CASQ2D307H protein in the CASQ2 null background partially restored triadin 1 levels, which were decreased in the CASQ2 null mice. Despite twofold expression (relative to wild-type CASQ2), the mutant protein failed to increase SR Ca2+ load. We also found that the Ca2+ transient decays slower in the CASQ2 null and CASQ2D307H cells. CASQ2D307H myocytes, when rhythmically paced and challenged with isoproterenol, exhibit spontaneous Ca2+ waves similar to CASQ2 null myocytes; however, the stability of Ca2+ cycling was increased in the CASQ2D307H myocytes. In the presence of isoproterenol, Ca2+-transient amplitude in CASQ2D307H myocytes was significantly decreased, possibly indicating an inherent defect in Ca2+ buffering capacity and release from the mutant CASQ2 at high Ca2+ concentrations. We also observed polymorphic ventricular tachycardia in the CASQ2D307H mice, although lesser than in the CASQ2 null mice. These data suggest that CASQ2D307H point mutation may affect Ca2+ buffering capacity and Ca2+ release. We propose that poor interaction between CASQ2D307H and triadin 1 could affect ryanodine receptor 2 stability, thereby increasing susceptibility to delayed afterdepolarizations and triggered arrhythmic activity.

Download Full-text

Autosomal Recessive Cutis Laxa 1C Mutations Disrupt the Structure and Interactions of Latent TGFβ Binding Protein-4

Frontiers in Genetics ◽

10.3389/fgene.2021.706662 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yasmene F. Alanazi ◽

Michael P. Lockhart-Cairns ◽

Stuart A. Cain ◽

Thomas A. Jowitt ◽

Anthony S. Weiss ◽

...

Keyword(s):

Autosomal Recessive ◽

Binding Protein ◽

Heparan Sulphate ◽

Point Mutations ◽

Elastic Fibre ◽

Terminal Region ◽

Cutis Laxa ◽

Connective Tissues ◽

Fibrillin 1 ◽

Functional Consequences

Latent TGFβ binding protein-4 (LTBP4) is a multi-domain glycoprotein, essential for regulating the extracellular bioavailability of TGFβ and assembly of elastic fibre proteins, fibrillin-1 and tropoelastin. LTBP4 mutations are linked to autosomal recessive cutis laxa type 1C (ARCL1C), a rare congenital disease characterised by high mortality and severely disrupted connective tissues. Despite the importance of LTBP4, the structure and molecular consequences of disease mutations are unknown. Therefore, we analysed the structural and functional consequences of three ARCL1C causing point mutations which effect highly conserved cysteine residues. Our structural and biophysical data show that the LTBP4 N- and C-terminal regions are monomeric in solution and adopt extended conformations with the mutations resulting in subtle changes to their conformation. Similar to LTBP1, the N-terminal region is relatively inflexible, whereas the C-terminal region is flexible. Interaction studies show that one C-terminal mutation slightly decreases binding to fibrillin-1. We also found that the LTBP4 C-terminal region directly interacts with tropoelastin which is perturbed by both C-terminal ARCL1C mutations, whereas an N-terminal mutation increased binding to fibulin-4 but did not affect the interaction with heparan sulphate. Our results suggest that LTBP4 mutations contribute to ARCL1C by disrupting the structure and interactions of LTBP4 which are essential for elastogenesis in a range of mammalian connective tissues.

Download Full-text

Extensive Ca2+ leak through K4750Q cardiac ryanodine receptors caused by cytosolic and luminal Ca2+ hypersensitivity

The Journal of General Physiology ◽

10.1085/jgp.201611624 ◽

2017 ◽

Vol 149 (2) ◽

pp. 199-218 ◽

Cited By ~ 23

Author(s):

Akira Uehara ◽

Takashi Murayama ◽

Midori Yasukochi ◽

Michael Fill ◽

Minoru Horie ◽

...

Keyword(s):

Single Channel ◽

Single Point ◽

Ryanodine Receptors ◽

Point Mutations ◽

Hek293 Cells ◽

Polymorphic Ventricular Tachycardia ◽

Single Point Mutation ◽

Control Input ◽

Life Threatening ◽

Functional Consequences

Various ryanodine receptor 2 (RyR2) point mutations cause catecholamine-induced polymorphic ventricular tachycardia (CPVT), a life-threatening arrhythmia evoked by diastolic intracellular Ca2+ release dysfunction. These mutations occur in essential regions of RyR2 that regulate Ca2+ release. The molecular dysfunction caused by CPVT-associated RyR2 mutations as well as the functional consequences remain unresolved. Here, we study the most severe CPVT-associated RyR2 mutation (K4750Q) known to date. We define the molecular and cellular dysfunction generated by this mutation and detail how it alters RyR2 function, using Ca2+ imaging, ryanodine binding, and single-channel recordings. HEK293 cells and cardiac HL-1 cells expressing RyR2-K4750Q show greatly enhanced spontaneous Ca2+ oscillations. An endoplasmic reticulum–targeted Ca2+ sensor, R-CEPIA1er, revealed that RyR2-K4750Q mediates excessive diastolic Ca2+ leak, which dramatically reduces luminal [Ca2+]. We further show that the K4750Q mutation causes three RyR2 defects: hypersensitization to activation by cytosolic Ca2+, loss of cytosolic Ca2+/Mg2+-mediated inactivation, and hypersensitization to luminal Ca2+ activation. These defects combine to kinetically stabilize RyR2-K4750Q openings, thus explaining the extensive diastolic Ca2+ leak from the sarcoplasmic reticulum, frequent Ca2+ waves, and severe CPVT phenotype. As the multiple concurrent defects are induced by a single point mutation, the K4750 residue likely resides at a critical structural point at which cytosolic and luminal RyR2 control input converge.

Download Full-text

Packpred: Predicting the functional effect of missense mutations

10.1101/2020.12.30.424909 ◽

2021 ◽

Author(s):

Kuan Pern Tan ◽

Tejashree Rajaram Kanitkar ◽

Kwoh Chee Keong ◽

M.S. Madhusudhan

Keyword(s):

Amino Acid ◽

Single Point ◽

Point Mutations ◽

Substitution Matrix ◽

Saturation Mutagenesis ◽

Data Sets ◽

Missense Mutations ◽

Wild Type ◽

Data Set ◽

Functional Consequences

1.AbstractPredicting the functional consequences of single point mutations has relevance to protein function annotation and to clinical analysis/diagnosis. We developed and tested Packpred that makes use of a multi-body clique statistical potential in combination with a depth dependent amino acid substitution matrix (FADHM) and positional Shannon Entropy to predict the functional consequences of point mutations in proteins. Parameters were trained over a saturation mutagenesis data set of T4-lysozyme (1966 mutations). The method was tested over another saturation mutagenesis data set (CcdB; 1534 mutations) and the Missense3D data set (4099 mutations). The performance of Packpred was compared against those of six other contemporary methods. With MCC values of 0.42, 0.47 and 0.36 on the training and testing data sets respectively, Packpred outperforms all method in all data sets, with the exception of marginally underperforming to FADHM in the CcdB data set. On analyzing the results, we could build meta servers that chose best performing methods of wild type amino acids and for wild type-mutant amino acid pairs. This lead to an increase of MCC value of 0.40 and 0.51 for the two meta predictors respectively on the Missense3D data set. We conjecture that it is possible to improve accuracy with better meta predictors as among the 7 methods compared, at the least one method or another is able to correctly predict ∼99% of the data.

Download Full-text