A Genome-Wide CRISPR/Cas9 Screen Reveals that Riboflavin Regulates Hydrogen Peroxide Entry into HAP1 Cells

ABSTRACT Extracellular hydrogen peroxide can induce oxidative stress, which can cause cell death if unresolved. However, the cellular mediators of H2O2-induced cell death are unknown. We determined that H2O2-induced cytotoxicity is an iron-dependent process in HAP1 cells and conducted a CRISPR/Cas9-...

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Autores principales:	Tamutenda Chidawanyika, Kenneth M. K. Mark, Surachai Supattapone
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	riboflavin oxidative stress hydrogen peroxide aquaporin KEAP1 riboflavin transporter Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/10b6aedc097946a084d333c63198c578
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spelling	oai:doaj.org-article:10b6aedc097946a084d333c63198c5782021-11-15T15:56:43ZA Genome-Wide CRISPR/Cas9 Screen Reveals that Riboflavin Regulates Hydrogen Peroxide Entry into HAP1 Cells10.1128/mBio.01704-202150-7511https://doaj.org/article/10b6aedc097946a084d333c63198c5782020-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01704-20https://doaj.org/toc/2150-7511ABSTRACT Extracellular hydrogen peroxide can induce oxidative stress, which can cause cell death if unresolved. However, the cellular mediators of H2O2-induced cell death are unknown. We determined that H2O2-induced cytotoxicity is an iron-dependent process in HAP1 cells and conducted a CRISPR/Cas9-based survival screen that identified four genes that mediate H2O2-induced cell death: POR (encoding cytochrome P450 oxidoreductase), RETSAT (retinol saturase), KEAP1 (Kelch-like ECH-associated protein-1), and SLC52A2 (riboflavin transporter). Among these genes, only POR also mediated methyl viologen dichloride hydrate (paraquat)-induced cell death. Because the identification of SLC52A2 as a mediator of H2O2 was both novel and unexpected, we performed additional experiments to characterize the specificity and mechanism of its effect. These experiments showed that paralogs of SLC52A2 with lower riboflavin affinities could not mediate H2O2-induced cell death and that riboflavin depletion protected HAP1 cells from H2O2 toxicity through a specific process that could not be rescued by other flavin compounds. Interestingly, riboflavin mediated cell death specifically by regulating H2O2 entry into HAP1 cells, likely through an aquaporin channel. Our study results reveal the general and specific effectors of iron-dependent H2O2-induced cell death and also show for the first time that a vitamin can regulate membrane transport. IMPORTANCE Using a genetic screen, we discovered that riboflavin controls the entry of hydrogen peroxide into a white blood cell line. To our knowledge, this is the first report of a vitamin playing a role in controlling transport of a small molecule across the cell membrane.Tamutenda ChidawanyikaKenneth M. K. MarkSurachai SupattaponeAmerican Society for Microbiologyarticleriboflavinoxidative stresshydrogen peroxideaquaporinKEAP1riboflavin transporterMicrobiologyQR1-502ENmBio, Vol 11, Iss 4 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	riboflavin oxidative stress hydrogen peroxide aquaporin KEAP1 riboflavin transporter Microbiology QR1-502
spellingShingle	riboflavin oxidative stress hydrogen peroxide aquaporin KEAP1 riboflavin transporter Microbiology QR1-502 Tamutenda Chidawanyika Kenneth M. K. Mark Surachai Supattapone A Genome-Wide CRISPR/Cas9 Screen Reveals that Riboflavin Regulates Hydrogen Peroxide Entry into HAP1 Cells
description	ABSTRACT Extracellular hydrogen peroxide can induce oxidative stress, which can cause cell death if unresolved. However, the cellular mediators of H2O2-induced cell death are unknown. We determined that H2O2-induced cytotoxicity is an iron-dependent process in HAP1 cells and conducted a CRISPR/Cas9-based survival screen that identified four genes that mediate H2O2-induced cell death: POR (encoding cytochrome P450 oxidoreductase), RETSAT (retinol saturase), KEAP1 (Kelch-like ECH-associated protein-1), and SLC52A2 (riboflavin transporter). Among these genes, only POR also mediated methyl viologen dichloride hydrate (paraquat)-induced cell death. Because the identification of SLC52A2 as a mediator of H2O2 was both novel and unexpected, we performed additional experiments to characterize the specificity and mechanism of its effect. These experiments showed that paralogs of SLC52A2 with lower riboflavin affinities could not mediate H2O2-induced cell death and that riboflavin depletion protected HAP1 cells from H2O2 toxicity through a specific process that could not be rescued by other flavin compounds. Interestingly, riboflavin mediated cell death specifically by regulating H2O2 entry into HAP1 cells, likely through an aquaporin channel. Our study results reveal the general and specific effectors of iron-dependent H2O2-induced cell death and also show for the first time that a vitamin can regulate membrane transport. IMPORTANCE Using a genetic screen, we discovered that riboflavin controls the entry of hydrogen peroxide into a white blood cell line. To our knowledge, this is the first report of a vitamin playing a role in controlling transport of a small molecule across the cell membrane.
format	article
author	Tamutenda Chidawanyika Kenneth M. K. Mark Surachai Supattapone
author_facet	Tamutenda Chidawanyika Kenneth M. K. Mark Surachai Supattapone
author_sort	Tamutenda Chidawanyika
title	A Genome-Wide CRISPR/Cas9 Screen Reveals that Riboflavin Regulates Hydrogen Peroxide Entry into HAP1 Cells
title_short	A Genome-Wide CRISPR/Cas9 Screen Reveals that Riboflavin Regulates Hydrogen Peroxide Entry into HAP1 Cells
title_full	A Genome-Wide CRISPR/Cas9 Screen Reveals that Riboflavin Regulates Hydrogen Peroxide Entry into HAP1 Cells
title_fullStr	A Genome-Wide CRISPR/Cas9 Screen Reveals that Riboflavin Regulates Hydrogen Peroxide Entry into HAP1 Cells
title_full_unstemmed	A Genome-Wide CRISPR/Cas9 Screen Reveals that Riboflavin Regulates Hydrogen Peroxide Entry into HAP1 Cells
title_sort	genome-wide crispr/cas9 screen reveals that riboflavin regulates hydrogen peroxide entry into hap1 cells
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/10b6aedc097946a084d333c63198c578
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A Genome-Wide CRISPR/Cas9 Screen Reveals that Riboflavin Regulates Hydrogen Peroxide Entry into HAP1 Cells

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