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-...
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
American Society for Microbiology
2020-08-01
|
| Series: | mBio |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/mBio.01704-20 |
| _version_ | 1819041438935023616 |
|---|---|
| author | Tamutenda Chidawanyika Kenneth M. K. Mark Surachai Supattapone |
| author_facet | Tamutenda Chidawanyika Kenneth M. K. Mark Surachai Supattapone |
| author_sort | Tamutenda Chidawanyika |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-21T09:25:00Z |
| format | Article |
| id | doaj.art-10b6aedc097946a084d333c63198c578 |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-12-21T09:25:00Z |
| publishDate | 2020-08-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-10b6aedc097946a084d333c63198c5782022-12-21T19:08:54ZengAmerican Society for MicrobiologymBio2150-75112020-08-0111410.1128/mBio.01704-20A Genome-Wide CRISPR/Cas9 Screen Reveals that Riboflavin Regulates Hydrogen Peroxide Entry into HAP1 CellsTamutenda Chidawanyika0Kenneth M. K. Mark1Surachai Supattapone2Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USADepartment of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USADepartment of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USAABSTRACT 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.https://journals.asm.org/doi/10.1128/mBio.01704-20riboflavinoxidative stresshydrogen peroxideaquaporinKEAP1riboflavin transporter |
| spellingShingle | Tamutenda Chidawanyika Kenneth M. K. Mark Surachai Supattapone A Genome-Wide CRISPR/Cas9 Screen Reveals that Riboflavin Regulates Hydrogen Peroxide Entry into HAP1 Cells mBio riboflavin oxidative stress hydrogen peroxide aquaporin KEAP1 riboflavin transporter |
| title | 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_short | 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 |
| topic | riboflavin oxidative stress hydrogen peroxide aquaporin KEAP1 riboflavin transporter |
| url | https://journals.asm.org/doi/10.1128/mBio.01704-20 |
| work_keys_str_mv | AT tamutendachidawanyika agenomewidecrisprcas9screenrevealsthatriboflavinregulateshydrogenperoxideentryintohap1cells AT kennethmkmark agenomewidecrisprcas9screenrevealsthatriboflavinregulateshydrogenperoxideentryintohap1cells AT surachaisupattapone agenomewidecrisprcas9screenrevealsthatriboflavinregulateshydrogenperoxideentryintohap1cells AT tamutendachidawanyika genomewidecrisprcas9screenrevealsthatriboflavinregulateshydrogenperoxideentryintohap1cells AT kennethmkmark genomewidecrisprcas9screenrevealsthatriboflavinregulateshydrogenperoxideentryintohap1cells AT surachaisupattapone genomewidecrisprcas9screenrevealsthatriboflavinregulateshydrogenperoxideentryintohap1cells |