Non-synonymous substitution of evolutionarily conserved residue in Tau class glutathione transferases alters structural and catalytic features

ABSTRACT Protein kinase D2 belongs to a family of evolutionarily conserved enzymes regulating several biological processes. In a forward genetic screen for zebrafish cardiovascular mutants, we identified a mutation in the prkd2 gene. Homozygous mutant embryos develop as wild type up to 36 h post-fertilization and initiate blood flow, but fail to maintain it, resulting in a complete outflow tract stenosis. We identified a mutation in the prkd2 gene that results in a T757A substitution at a conserved residue in the kinase domain activation loop (T714A in human PRKD2) that disrupts catalytic activity and drives this phenotype. Homozygous mutants survive without circulation for several days, allowing us to study the extreme phenotype of no intracardiac flow, in the background of a functional heart. We show dysregulation of atrioventricular and outflow tract markers in the mutants and higher sensitivity to the Calcineurin inhibitor, Cyclosporin A. Finally we identify TBX5 as a potential regulator of PRKD2. Our results implicate PRKD2 catalytic activity in outflow tract development in zebrafish. This article has an associated First Person interview with the first author of the paper.

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A novel missense mutation in the γ-glutamylcysteine synthetase catalytic subunit gene causes both decreased enzymatic activity and glutathione production

Blood ◽

10.1182/blood-2002-11-3622 ◽

2003 ◽

Vol 102 (2) ◽

pp. 725-730 ◽

Cited By ~ 41

Author(s):

David Hamilton ◽

Jian Hui Wu ◽

Moulay Alaoui-Jamali ◽

Gerald Batist

Keyword(s):

Amino Acid ◽

Enzymatic Activity ◽

Missense Mutation ◽

Binding Studies ◽

Rate Limiting Step ◽

Number Of Patients ◽

Evolutionarily Conserved ◽

Glutamylcysteine Synthetase ◽

Rate Limiting ◽

Conserved Residue

Abstractγ-Glutamylcysteine synthetase (γ-GCS) catalyzes the first and rate-limiting step in glutathione (GSH) biosynthesis: the adenosine triphosphate (ATP)–dependent ligation of glutamate and cysteine. γ-GCS consists of a catalytic (γ-GCSH) and modifier (γ-GCSL) subunit. Hereditary deficiency of γ-GCS has been reported in a small number of patients and is associated with low erythrocyte levels of γ-GCS and GSH leading to hemolytic anemia. Here we report a novel γ-GCSH mutation, isolated from the cDNA of 2 related patients diagnosed with γ-GCS deficiency. Each was found to be homozygous for a C>T missense mutation at nucleotide 379, encoding for a predicted Arg127Cys amino acid change. Computerized structure modeling identified that the mutated amino acid lies within a cleft on the protein surface of γ-GCSH, and the border of this cleft was shown to contain Cys249, an evolutionarily conserved residue that has been proven to lie near the binding site of γ-GCSH. Transfection studies showed that the mutation is associated with decreased GSH production, and binding studies using purified recombinant protein showed that the mutant protein has markedly decreased enzymatic activity compared to wild type.

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Predicting human RNA quadruplex helicases through comparative sequence approaches and helicase mRNA interactome analyses

Biochemistry and Cell Biology ◽

10.1139/bcb-2020-0590 ◽

2021 ◽

Author(s):

Steven J. Dupas ◽

Daniel Gussakovsky ◽

Alvan Wai ◽

Mira J.F. Brown ◽

Georg Hausner ◽

...

Keyword(s):

In Silico Analysis ◽

Synonymous Substitution ◽

Next Generation Sequencing Data ◽

Sequencing Data ◽

Rna Helicases ◽

Level Of Evidence ◽

Disease States ◽

Specific Subset ◽

Evolutionarily Conserved ◽

Nucleic Acid Structures

RNA quadruplexes are non-canonical nucleic acid structures involved in several human disease states and are regulated by a specific subset of RNA helicases. Given the difficulty in identifying RNA quadruplex helicases due to the multifunctionality of these enzymes, we sought to provide a comprehensive in silico analysis of features found in validated RNA quadruplex helicases to predict novel human RNA quadruplex helicases. Using the 64 human RNA helicases, we correlated their amino acid compositions with subsets of RNA quadruplex helicases categorized by varying level of evidence of RNA quadruplex interaction. Utilizing phylogenetic and synonymous/non-synonymous substitution analyses, we identified an evolutionarily conserved pattern involving predicted intrinsic disorder and a previously identified motif. We analyzed available next generation sequencing data to determine which RNA helicases were directly interacting with predicted RNA quadruplex regions intracellularly and elucidated a relationship with miRNA binding sites adjacent to RNA quadruplexes. Finally, we employed a phylogenetic analysis of all 64 human RNA helicases to establish how RNA quadruplex detection and unwinding activity may be conserved among helicase subfamilies. This work furthers understanding of commonalities between RNA quadruplex helicases and provides support for the future validation of several human RNA helicases.

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Apoptosis and development

Essays in Biochemistry ◽

10.1042/bse0390011 ◽

2003 ◽

Vol 39 ◽

pp. 11-24 ◽

Cited By ~ 6

Author(s):

Justin V McCarthy

Keyword(s):

Drosophila Melanogaster ◽

Mammalian Cells ◽

Cell Number ◽

Model Organisms ◽

Biological Processes ◽

Nematode Caenorhabditis Elegans ◽

Apoptotic Pathway ◽

Genetic Pathways ◽

Evolutionarily Conserved ◽

Multicellular Organisms

Apoptosis is an evolutionarily conserved process used by multicellular organisms to developmentally regulate cell number or to eliminate cells that are potentially detrimental to the organism. The large diversity of regulators of apoptosis in mammalian cells and their numerous interactions complicate the analysis of their individual functions, particularly in development. The remarkable conservation of apoptotic mechanisms across species has allowed the genetic pathways of apoptosis determined in lower species, such as the nematode Caenorhabditis elegans and the fruitfly Drosophila melanogaster, to act as models for understanding the biology of apoptosis in mammalian cells. Though many components of the apoptotic pathway are conserved between species, the use of additional model organisms has revealed several important differences and supports the use of model organisms in deciphering complex biological processes such as apoptosis.

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