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Novel calcineurin A (PPP3CA) variant associated with epilepsy, constitutive enzyme activation and downregulation of protein expression.

ABSTRACT: PPP3CA encodes calmodulin-binding catalytic subunit of calcineurin, a ubiquitously expressed calcium/calmodulin-regulated protein phosphatase. Recently de novo PPP3CA variants were reported as a cause of disease in 12 subjects presenting with epileptic encephalopathy and dysmorphic features. We describe a boy with similar phenotype and severe early onset epileptic encephalopathy in whom a novel de novo c.1324C>T (p.(Gln442Ter)) PPP3CA variant was found by whole exome sequencing. Western blot experiments in patient's cells (EBV transformed lymphocytes and neuronal cells derived through reprogramming) indicate that despite normal mRNA abundance the protein expression level is strongly reduced both for the mutated and wild-type protein. By in vitro studies with recombinant protein expressed in E. coli we show that c.1324C>T (p.(Gln442Ter)) results in constitutive activation of the enzyme. Our results confirm the role of PPP3CA defects in pathogenesis of a distinct neurodevelopmental disorder including severe epilepsy and dysmorphism and provide further functional clues regarding the pathogenic mechanism.

SUBMITTER: Rydzanicz M

PROVIDER: S-EPMC6303256 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Novel calcineurin A (PPP3CA) variant associated with epilepsy, constitutive enzyme activation and downregulation of protein expression.

Rydzanicz Małgorzata M Wachowska Małgorzata M Cook Erik C EC Lisowski Paweł P Kuźniewska Bożena B Szymańska Krystyna K Diecke Sebastian S Prigione Alessandro A Szczałuba Krzysztof K Szybińska Aleksandra A Koppolu Agnieszka A Murcia Pienkowski Victor V Kosińska Joanna J Wiweger Małgorzata M Kostrzewa Grażyna G Brzozowska Małgorzata M Domańska-Pakieła Dorota D Jurkiewicz Elżbieta E Stawiński Piotr P Gromadka Agnieszka A Zielenkiewicz Piotr P Demkow Urszula U Dziembowska Magdalena M Kuźnicki Jacek J Creamer Trevor P TP Płoski Rafał R

European journal of human genetics : EJHG 20180925 1

PPP3CA encodes calmodulin-binding catalytic subunit of calcineurin, a ubiquitously expressed calcium/calmodulin-regulated protein phosphatase. Recently de novo PPP3CA variants were reported as a cause of disease in 12 subjects presenting with epileptic encephalopathy and dysmorphic features. We describe a boy with similar phenotype and severe early onset epileptic encephalopathy in whom a novel de novo c.1324C>T (p.(Gln442Ter)) PPP3CA variant was found by whole exome sequencing. Western blot exp ...[more]

PMID: 30254215

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Project description:BackgroundMicrobial transglutaminase (mTG) is a robust enzyme catalyzing the formation of an isopeptide bond between glutamine and lysine residues. It has found use in food applications, pharmaceuticals, textiles, and biomedicine. Overexpression of soluble and active mTG in E. coli has been limited due to improper protein folding and requirement for proteolytic cleavage of the pro-domain. Furthermore, to integrate mTG more fully industrially and academically, thermostable and solvent-stable variants may be imperative.ResultsA novel expression system constitutively producing active mTG was designed. Wild-type (WT) mTG and a S2P variant had similar expression levels, comparable to previous studies. Kinetic constants were determined by a glutamate dehydrogenase-coupled assay, and the S2P variant showed an increased affinity and a doubled enzyme efficiency towards Z-Gln-Gly. The melting temperature (Tm) of the WT was determined by intrinsic fluorescence measurements to be 55.8 ± 0.1 °C and of the S2P variant to be 56.3 ± 0.4 °C and 45.5 ± 0.1 °C, showing a moderately different thermostability profile. Stability in water miscible organic solvents was determined for both the WT and S2P variant. Of the solvents tested, incubation of mTG in isopropanol for 24 h at 4 °C showed the strongest stabilizing effect with mTG retaining 61 and 72% activity for WT and S2P respectively in 70% isopropanol. Both enzymes also showed an increased initial activity in the presence of organic solvents with the highest activity increase in 40% DMSO. Nevertheless, both enzymes were inactivated in 70% of all organic solvents tested.ConclusionsA constitutive expression system of active mTG in E. coli without downstream proteolytic cleavage processing was used for overexpression and characterization. High throughput techniques for testing thermostability and kinetics were useful in streamlining analysis and could be used in the future for quickly identifying beneficial mutants. Hitherto untested thermostability and stability of mTG in organic solvents was evaluated, which can pave the way for use of the enzyme in novel applications and processes.

Dataset Information

Novel calcineurin A (PPP3CA) variant associated with epilepsy, constitutive enzyme activation and downregulation of protein expression.

Publications

Novel calcineurin A (PPP3CA) variant associated with epilepsy, constitutive enzyme activation and downregulation of protein expression.

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