Generation and characterization of human U-2 OS cell lines with the CRISPR/Cas9-edited protoporphyrinogen oxidase IX gene

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F68378050%3A_____%2F22%3A00565700" target="_blank" >RIV/68378050:_____/22:00565700 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/86652036:_____/22:00565700 RIV/67985823:_____/22:00565700 RIV/00216208:11110/22:10456190 RIV/00216208:11310/22:10456190

Výsledek na webu

<a href="https://www.nature.com/articles/s41598-022-21147-x" target="_blank" >https://www.nature.com/articles/s41598-022-21147-x</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1038/s41598-022-21147-x" target="_blank" >10.1038/s41598-022-21147-x</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Generation and characterization of human U-2 OS cell lines with the CRISPR/Cas9-edited protoporphyrinogen oxidase IX gene

Popis výsledku v původním jazyce

In humans, disruptions in the heme biosynthetic pathway are associated with various types of porphyrias, including variegate porphyria that results from the decreased activity of protoporphyrinogen oxidase IX (PPO, E.C.1.3.3.4), the enzyme catalyzing the penultimate step of the heme biosynthesis. Here we report the generation and characterization of human cell lines, in which PPO was inactivated using the CRISPR/Cas9 system. The PPO knock-out (PPO-KO) cell lines are viable with the normal proliferation rate and show massive accumulation of protoporphyrinogen IX, the PPO substrate. Observed low heme levels trigger a decrease in the amount of functional heme containing respiratory complexes III and IV and overall reduced oxygen consumption rates. Untargeted proteomics further revealed dysregulation of 22 cellular proteins, including strong upregulation of 5-aminolevulinic acid synthase, the major regulatory protein of the heme biosynthesis, as well as additional ten targets with unknown association to heme metabolism. Importantly, knock-in of PPO into PPO-KO cells rescued their wild-type phenotype, confirming the specificity of our model. Overall, our model system exploiting a non-erythroid human U-2 OS cell line reveals physiological consequences of the PPO ablation at the cellular level and can serve as a tool to study various aspects of dysregulated heme metabolism associated with variegate porphyria.

Název v anglickém jazyce

Generation and characterization of human U-2 OS cell lines with the CRISPR/Cas9-edited protoporphyrinogen oxidase IX gene

Popis výsledku anglicky

In humans, disruptions in the heme biosynthetic pathway are associated with various types of porphyrias, including variegate porphyria that results from the decreased activity of protoporphyrinogen oxidase IX (PPO, E.C.1.3.3.4), the enzyme catalyzing the penultimate step of the heme biosynthesis. Here we report the generation and characterization of human cell lines, in which PPO was inactivated using the CRISPR/Cas9 system. The PPO knock-out (PPO-KO) cell lines are viable with the normal proliferation rate and show massive accumulation of protoporphyrinogen IX, the PPO substrate. Observed low heme levels trigger a decrease in the amount of functional heme containing respiratory complexes III and IV and overall reduced oxygen consumption rates. Untargeted proteomics further revealed dysregulation of 22 cellular proteins, including strong upregulation of 5-aminolevulinic acid synthase, the major regulatory protein of the heme biosynthesis, as well as additional ten targets with unknown association to heme metabolism. Importantly, knock-in of PPO into PPO-KO cells rescued their wild-type phenotype, confirming the specificity of our model. Overall, our model system exploiting a non-erythroid human U-2 OS cell line reveals physiological consequences of the PPO ablation at the cellular level and can serve as a tool to study various aspects of dysregulated heme metabolism associated with variegate porphyria.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10601 - Cell biology

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2022

Kód důvěrnosti údajů

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

Název periodika

Scientific Reports

ISSN

2045-2322

e-ISSN

2045-2322

Svazek periodika

12

Číslo periodika v rámci svazku

1

Stát vydavatele periodika

DE - Spolková republika Německo

Počet stran výsledku

17

Strana od-do

17081

Kód UT WoS článku

000867364300008

EID výsledku v databázi Scopus

2-s2.0-85139764065