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. 2017 Dec;13(12):1274-1279.
doi: 10.1038/nchembio.2499. Epub 2017 Oct 23.

A CRISPR Screen Identifies a Pathway Required for Paraquat-Induced Cell Death

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Free PMC article

A CRISPR Screen Identifies a Pathway Required for Paraquat-Induced Cell Death

Colleen R Reczek et al. Nat Chem Biol. .
Free PMC article

Abstract

Paraquat, a herbicide linked to Parkinson's disease, generates reactive oxygen species (ROS), which causes cell death. Because the source of paraquat-induced ROS production remains unknown, we conducted a CRISPR-based positive-selection screen to identify metabolic genes essential for paraquat-induced cell death. Our screen uncovered three genes, POR (cytochrome P450 oxidoreductase), ATP7A (copper transporter), and SLC45A4 (sucrose transporter), required for paraquat-induced cell death. Furthermore, our results revealed POR as the source of paraquat-induced ROS production. Thus, our study highlights the use of functional genomic screens for uncovering redox biology.

Conflict of interest statement

COMPETING FINANCIAL CONTRIBUTIONS

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. POR, ATP7A, and SLC45A4 are essential for PQ-induced cell death
(a) Structure of paraquat (PQ; 1). (b) The gene scores for the top 25 genes following treatment with 110 μM PQ. POR is the best scoring gene, followed by ATP7A and SLC45A4. (c) Two clonal POR-null Jurkat cell lines (POR_KO1 and POR_KO2) and a non-targeting control cell line were treated with varying concentrations of PQ for 7 days and the percentage of cellular viability was assessed. (d) Live cell viability was determined following treatment with varying concentrations of PQ for 3 days in two POR-null Jurkat clones reconstituted with either empty vector (EV) or POR cDNA. (e) Clonal ATP7A-null Jurkat cells (ATP7A_KO) and non-targeting control Jurkat cells were treated with the indicated concentrations of PQ for 7 days and live cell viability was assessed. (f) Clonal SLC45A4-null Jurkat cells (SLC45A4_KO) and non-targeting control Jurkat cells were treated with the indicated concentrations of PQ for 7 days and live cell viability was assessed. For cf, error bars represent SEM (n=4 independent experiments). *, p < 0.05, and **, p < 0.01, compared to control cells.
Figure 2
Figure 2. POR is necessary for PQ-induced cytosolic ROS generation
(a) Non-targeting (NT) control, POR-null, ATP7A-null, and SLC45A4-null Jurkat cells were treated with 150 μM PQ for 48 hours and intracellular ROS levels were determined as the mean fluorescence intensity (MFI) of CM–DCF. (b) Intracellular ROS levels were measured in empty vector (EV)- or human POR cDNA-reconstituted POR-null Jurkat cells (clone POR_KO2) treated with 50 μM PQ for 48 hours. (c) Extracellular H2O2 levels were measured by Amplex red in non-targeting (NT) control, POR-null, ATP7A-null, and SLC45A4-null Jurkat cells following 150 μM PQ for 48 hours. (d) The levels of extracellular H2O2 following treatment with 50 μM PQ for 48 hours were measured in the POR_KO2 clone reconstituted with the empty vector (EV) or human POR cDNA. (e) Amplex red was used to measure intracellular H2O2 production in saponin-permeabilized non-targeting (NT) control Jurkat cells as well as POR-null, ATP7A-null, and SLC45A4-null Jurkat cells treated with 150 μM PQ for 1 hour. (f) Intracellular H2O2 levels were measured in saponin-permeabilized POR-null Jurkat cells (clone POR_KO2) reconstituted with the empty vector (EV) or human POR cDNA following treatment with 150 μM PQ for 1 hour. For all panels, error bars represent SEM (n=4 independent experiments). *, p < 0.05, and **, p < 0.01, compared to control cells. Data were background-corrected to the mean value in untreated cells.
Figure 3
Figure 3. Mitochondrial complex I is not necessary for PQ-induced cell death
(a) Non-targeting control, clonal POR-null (POR_KO2), and clonal NDUFA6-null (NDUFA6_KO) Jurkat cells were treated with varying concentrations of PQ for 7 days and the percentage of cellular viability was assessed. For statistical analysis, the POR_KO2 cells were compared. (b) Live cell viability was determined following treatment with varying concentrations of mitochondrially targeted paraquat (MitoPQ) for 7 days in non-targeting control Jurkat cells as well as clonal POR-null, ATP7A-null, SLC45A4-null, and NDUFA6-null Jurkat cells. (c) Non-targeting (NT) control, POR-null, ATP7A-null, and SLC45A4-null Jurkat cells were treated with 150 μM PQ for 24 hours and PQ uptake into the cells was analyzed by high performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS). Results are normalized to non-targeting control Jurkat cells. (d) SOD activity (U/mL) was measured in non-targeting (NT) control Jurkat cells as well as clonal POR-null, ATP7A-null, and SLC45A4-null Jurkat cells. (e) Structure of 2,3-dimethoxy-1,4-naphthoquinone (DMNQ; 4). (f) Live cell viability was determined following treatment with varying concentrations of the redox cycler and pro–oxidant DMNQ for 7 days in non-targeting control, POR-null, ATP7A-null, and SLC45A4-null Jurkat cells. For ad and f, error bars represent SEM (n=4 independent experiments for a,b,d,f; n=3 independent experiments for c). **, p < 0.01 compared to control cells.
Figure 4
Figure 4. PQ negative-selection CRISPR-based screen
(a) The gene scores as differentially required for the top 25 genes following treatment with 25 μM PQ. The copper transporter gene SLC31A1 and the copper-dependent antioxidant gene SOD1 were among the top genes identified. (b) Wild-type Jurkat cells were treated with varying concentrations of the copper-chelating agent 2,3,2-tetraamine (Tet; 5) in the presence or absence of 25 μM PQ for 7 days and the percentage of cellular viability was assessed. Error bars represent SEM (n=4 independent experiments). **, p < 0.01 compared to the 0 μM Tet condition within each PQ treatment group.

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