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

Tamutenda Chidawanyika,Surachai Supattapone,Kenneth M K Mark,Tamara L Doering

doi:10.1128/mbio.01704-20

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.

Highlights

Extracellular hydrogen peroxide can induce oxidative stress, which can cause cell death if unresolved
To identify genes that mediate cell death induced by H2O2, a positive-selection screen was conducted in CRISPR/Cas9 knockout (KO) HAP1 cell libraries using H2O2 to induce oxidative stress and cell death (Fig. 1A)
SLC52A2 encodes solute carrier family 52 member 2, a riboflavin transporter localized to the plasma membrane [24, 25]

Summary

Introduction

Extracellular hydrogen peroxide can induce oxidative stress, which can cause cell death if unresolved. We determined that H2O2-induced cytotoxicity is an irondependent 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 ECHassociated protein-1), and SLC52A2 (riboflavin transporter). 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. Different cell types, including leukocytes, platelets, epithelial cells, and endothelial cells, are programmed to generate and release high levels of ROS, H2O2, in response to bacterial infection, tissue injury, and chronic inflammation [4, 11,12,13,14] While this response occurs as part of a mechanism to alleviate the stress by killing invading bacteria or attracting leukocytes to the site of infection or injury, the exposure of cells. Despite extensive knowledge about the executors of apoptosis or necrosis as a result of high extracellular H2O2 concentrations [16, 17], the genes that mediate H2O2-induced cell death in these environments are unknown

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Conclusion