A CRISPR screen identifies a pathway required for paraquat-induced cell death

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

Mitochondrial electron transport chain is necessary for NLRP3 inflammasome activation

The NLRP3 inflammasome is linked to sterile and pathogen-dependent inflammation, and its dysregulation underlies many chronic diseases. Mitochondria have been implicated as regulators of the NLRP3 inflammasome through several mechanisms including generation of mitochondrial reactive oxygen species (ROS). Here, we report that mitochondrial electron transport chain (ETC) complex I, II, III and V inhibitors all prevent NLRP3 inflammasome activation. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI1) or Ciona intestinalis alternative oxidase, which can complement the functional loss of mitochondrial complex I or III, respectively, without generation of ROS, rescued NLRP3 inflammasome activation in the absence of endogenous mitochondrial complex I or complex III function. Metabolomics revealed phosphocreatine (PCr), which can sustain ATP levels, as a common metabolite that is diminished by mitochondrial ETC inhibitors. PCr depletion decreased ATP levels and NLRP3 inflammasome activation. Thus, the mitochondrial ETC sustains NLRP3 inflammasome activation through PCr-dependent generation of ATP, but via a ROS-independent mechanism.publishedVersionPeer reviewe

Mitochondrial integrated stress response controls lung epithelial cell fate

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.Alveolar epithelial type 1 (AT1) cells are necessary to transfer oxygen and carbon dioxide between the blood and air. Alveolar epithelial type 2 (AT2) cells serve as a partially committed stem cell population, producing AT1 cells during postnatal alveolar development and repair after influenza A and SARS-CoV-2 pneumonia1,2,3,4,5,6. Little is known about the metabolic regulation of the fate of lung epithelial cells. Here we report that deleting the mitochondrial electron transport chain complex I subunit Ndufs2 in lung epithelial cells during mouse gestation led to death during postnatal alveolar development. Affected mice displayed hypertrophic cells with AT2 and AT1 cell features, known as transitional cells. Mammalian mitochondrial complex I, comprising 45 subunits, regenerates NAD+ and pumps protons. Conditional expression of yeast NADH dehydrogenase (NDI1) protein that regenerates NAD+ without proton pumping7,8 was sufficient to correct abnormal alveolar development and avert lethality. Single-cell RNA sequencing revealed enrichment of integrated stress response (ISR) genes in transitional cells. Administering an ISR inhibitor9,10 or NAD+ precursor reduced ISR gene signatures in epithelial cells and partially rescued lethality in the absence of mitochondrial complex I function. Notably, lung epithelial-specific loss of mitochondrial electron transport chain complex II subunit Sdhd, which maintains NAD+ regeneration, did not trigger high ISR activation or lethality. These findings highlight an unanticipated requirement for mitochondrial complex I-dependent NAD+ regeneration in directing cell fate during postnatal alveolar development by preventing pathological ISR induction.This work was supported by the National Institutes for Health (NIH): R35CA197532 to N.S.C.; P01HL071643 and P01AG049665 to N.S.C. and G.R.S.B.; K08HL143138 to S.H.; R01HL134800 to C.J.G.; T32 HL076139 to C.R.R.; and K08HL146943 to P.A.R. S.H. was supported by the American Heart Association Career Development Award (19CDA34630070), the Parker B. Francis Fellowship and the Doris Duke Charitable Foundation/Walder Foundation and Feinberg School of Medicine COVID-19 Fund to Retain Clinician Scientists. G.R.S.B. was supported by the Veterans Administration grant CX001777. M.L. and Y.S. were supported by the National Research Foundation of Korea (2022R1A2C2093050), KREONET (Korea Research Environment Open NETwork), which is managed and operated by KISTI (Korea Institute of Science and Technology Information) and the Dongguk University Research Fund. R.P.C. and M.M.H. were supported by the Northwestern University Pulmonary and Critical Care Cugell fellowship.Peer reviewe

The Two Faces of Reactive Oxygen Species in Cancer

Reactive oxygen species (ROS), now appreciated for their cellular signaling capabilities, have a dual role in cancer. On the one hand, ROS can promote protumorigenic signaling, facilitating cancer cell proliferation, survival, and adaptation to hypoxia. On the other hand, ROS can promote antitumorigenic signaling and trigger oxidative stress–induced cancer cell death. To hyperactivate the cell signaling pathways necessary for cellular transformation and tumorigenesis, cancer cells increase their rate of ROS production compared with normal cells. Concomitantly, in order to maintain ROS homeostasis and evade cell death, cancer cells increase their antioxidant capacity. Compared with normal cells, this altered redox environment of cancer cells may increase their susceptibility to ROS-manipulation therapies. In this review, we discuss the two faces of ROS in cancer, the potential mechanisms underlying ROS signaling, and the opposing cancer therapeutic approaches to targeting ROS. </jats:p

The Mitochondrial Respiratory Chain Is Required for Organismal Adaptation to Hypoxia

SummaryHypoxia-inducible factors (HIFs) are crucial for cellular and organismal adaptation to hypoxia. The mitochondrial respiratory chain is the largest consumer of oxygen in most mammalian cells; however, it is unknown whether the respiratory chain is necessary for in vivo activation of HIFs and organismal adaptation to hypoxia. HIF-1 activation in the epidermis has been shown to be a key regulator of the organismal response to hypoxic conditions, including renal production of erythropoietin (Epo). Therefore, we conditionally deleted expression of TFAM in mouse epidermal keratinocytes. TFAM is required for maintenance of the mitochondrial genome, and TFAM-null cells are respiratory deficient. TFAM loss in epidermal keratinocytes reduced epidermal levels of HIF-1α protein and diminished the hypoxic induction of HIF-dependent transcription in epidermis. Furthermore, epidermal TFAM deficiency impaired hypoxic induction of renal Epo expression. Our results demonstrate that the mitochondrial respiratory chain is essential for in vivo HIF activation and organismal adaptation to hypoxia

Wwox Binding to the Murine Brca1-BRCT Domain Regulates Timing of Brip1 and CtIP Phospho-Protein Interactions with This Domain at DNA Double-Strand Breaks, and Repair Pathway Choice

Wwox-deficient human cells show elevated homologous recombination, leading to resistance to killing by double-strand break-inducing agents. Human Wwox binds to the Brca1 981-PPLF-984 Wwox-binding motif, likely blocking the pChk2 phosphorylation site at Brca1-S988. This phosphorylation site is conserved across mammalian species; the PPLF motif is conserved in primates but not in rodents. We now show that murine Wwox does not bind Brca1 near the conserved mouse Brca1 phospho-S971 site, leaving it open for Chk2 phosphorylation and Brca1 activation. Instead, murine Wwox binds to Brca1 through its BRCT domain, where pAbraxas, pBrip1, and pCtIP, of the A, B, and C binding complexes, interact to regulate double-strand break repair pathway response. In Wwox-deficient mouse cells, the Brca1-BRCT domain is thus accessible for immediate binding of these phospho-proteins. We confirm elevated homologous recombination in Wwox-silenced murine cells, as in human cells. Wwox-deficient murine cells showed increased ionizing radiation-induced Abraxas, Brca1, and CtIP foci and long resected single-strand DNA, early after ionizing radiation. Wwox deletion increased the basal level of Brca1-CtIP interaction and the expression level of the MRN-CtIP protein complex, key players in end-resection, and facilitated Brca1 release from foci. Inhibition of phospho-Chk2 phosphorylation of Brca1-S971 delays the end-resection; the delay of premature end-resection by combining Chk2 inhibition with ionizing radiation or carboplatin treatment restored ionizing radiation and platinum sensitivity in Wwox-deficient murine cells, as in human cells, supporting the use of murine in vitro and in vivo models in preclinical cancer treatment research.</jats:p

Wwox Binding to the Murine Brca1-BRCT Domain Regulates Timing of Brip1 and CtIP Phospho-Protein Interactions with This Domain at DNA Double-Strand Breaks, and Repair Pathway Choice

Wwox-deficient human cells show elevated homologous recombination, leading to resistance to killing by double-strand break-inducing agents. Human Wwox binds to the Brca1 981-PPLF-984 Wwox-binding motif, likely blocking the pChk2 phosphorylation site at Brca1-S988. This phosphorylation site is conserved across mammalian species; the PPLF motif is conserved in primates but not in rodents. We now show that murine Wwox does not bind Brca1 near the conserved mouse Brca1 phospho-S971 site, leaving it open for Chk2 phosphorylation and Brca1 activation. Instead, murine Wwox binds to Brca1 through its BRCT domain, where pAbraxas, pBrip1, and pCtIP, of the A, B, and C binding complexes, interact to regulate double-strand break repair pathway response. In Wwox-deficient mouse cells, the Brca1-BRCT domain is thus accessible for immediate binding of these phospho-proteins. We confirm elevated homologous recombination in Wwox-silenced murine cells, as in human cells. Wwox-deficient murine cells showed increased ionizing radiation-induced Abraxas, Brca1, and CtIP foci and long resected single-strand DNA, early after ionizing radiation. Wwox deletion increased the basal level of Brca1-CtIP interaction and the expression level of the MRN-CtIP protein complex, key players in end-resection, and facilitated Brca1 release from foci. Inhibition of phospho-Chk2 phosphorylation of Brca1-S971 delays the end-resection; the delay of premature end-resection by combining Chk2 inhibition with ionizing radiation or carboplatin treatment restored ionizing radiation and platinum sensitivity in Wwox-deficient murine cells, as in human cells, supporting the use of murine in vitro and in vivo models in preclinical cancer treatment research