CRISPR-Cas9 induced mutations along de novo purine synthesis in HeLa cells result in accumulation of individual enzyme substrates and affect purinosome formation

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00064165%3A_____%2F16%3A10329213" target="_blank" >RIV/00064165:_____/16:10329213 - isvavai.cz</a>

Alternative codes found

RIV/00216208:11110/16:10329213

Result on the web

<a href="http://dx.doi.org/10.1016/j.ymgme.2016.08.004" target="_blank" >http://dx.doi.org/10.1016/j.ymgme.2016.08.004</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.ymgme.2016.08.004" target="_blank" >10.1016/j.ymgme.2016.08.004</a>

Result language

angličtina

Original language name

CRISPR-Cas9 induced mutations along de novo purine synthesis in HeLa cells result in accumulation of individual enzyme substrates and affect purinosome formation

Original language description

Purines are essential molecules for nucleic acid synthesis and are the most common carriers of chemical energy in all living organisms. The cellular pool of purines is maintained by the balance between their de novo synthesis (DNPS), recycling and degradation. DNPS includes ten reactions catalysed by six enzymes. To date, two genetically determined disorders of DNPS enzymes have been described, and the existence of other defects manifested by neurological symptoms and the accumulation of DNPS intermediates in bodily fluids is highly presumable. We prepared specific recombinant DNPS enzymes and used them for the biochemical preparation of their commercially unavailable substrates. These compounds were used as standards for the development and validation of quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). To simulate manifestations of known and putative defects of DNPS we prepared CRISPR-Cas9 genome-edited HeLa cells deficient for the individual steps of DNPS (CR-cells), assessed the substrates accumulation in cell lysates and growth media and tested how the mutations affect assembly of the purinosome, the multi-enzyme complex of DNPS enzymes. In all model cell lines with the exception of one, an accumulation of the substrate(s) for the knocked out enzyme was identified. The ability to form the purinosome was reduced. We conclude that LC-MS/MS analysis of the dephosphorylated substrates of DNPS enzymes in bodily fluids is applicable in the selective screening of the known and putative DNPS disorders. This approach should be considered in affected individuals with neurological and neuromuscular manifestations of unknown aetiology. Prepared in vitro human model systems can serve in various studies that aim to provide a better characterization and understanding of physiology and pathology of DNPS, to study the role of each DNPS protein in the purinosome formation and represent an interesting way for the screening of potential therapeutic agents.

Czech name

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Czech description

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Type

J_x - Unclassified - Peer-reviewed scientific article (Jimp, Jsc and Jost)

CEP classification

EB - Genetics and molecular biology

OECD FORD branch

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Project

Result was created during the realization of more than one project. More information in the Projects tab.

Continuities

O - Projekt operacniho programu

Publication year

2016

Confidentiality

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Name of the periodical

Molecular Genetics and Metabolism

ISSN

1096-7192

e-ISSN

—

Volume of the periodical

119

Issue of the periodical within the volume

3

Country of publishing house

US - UNITED STATES

Number of pages

8

Pages from-to

270-277

UT code for WoS article

000386987100009

EID of the result in the Scopus database

2-s2.0-84992665514