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1.
Cell ; 172(3): 409-422.e21, 2018 01 25.
Article in English | MEDLINE | ID: mdl-29290465

ABSTRACT

Selenoproteins are rare proteins among all kingdoms of life containing the 21st amino acid, selenocysteine. Selenocysteine resembles cysteine, differing only by the substitution of selenium for sulfur. Yet the actual advantage of selenolate- versus thiolate-based catalysis has remained enigmatic, as most of the known selenoproteins also exist as cysteine-containing homologs. Here, we demonstrate that selenolate-based catalysis of the essential mammalian selenoprotein GPX4 is unexpectedly dispensable for normal embryogenesis. Yet the survival of a specific type of interneurons emerges to exclusively depend on selenocysteine-containing GPX4, thereby preventing fatal epileptic seizures. Mechanistically, selenocysteine utilization by GPX4 confers exquisite resistance to irreversible overoxidation as cells expressing a cysteine variant are highly sensitive toward peroxide-induced ferroptosis. Remarkably, concomitant deletion of all selenoproteins in Gpx4cys/cys cells revealed that selenoproteins are dispensable for cell viability provided partial GPX4 activity is retained. Conclusively, 200 years after its discovery, a specific and indispensable role for selenium is provided.


Subject(s)
Apoptosis , Glutathione Peroxidase/metabolism , Seizures/metabolism , Selenium/metabolism , Animals , Cell Survival , Cells, Cultured , Female , Glutathione Peroxidase/genetics , HEK293 Cells , Humans , Hydrogen Peroxide/toxicity , Interneurons/metabolism , Lipid Peroxidation , Male , Mice , Mice, Inbred C57BL , Phospholipid Hydroperoxide Glutathione Peroxidase , Seizures/etiology
2.
Nature ; 626(7998): 401-410, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38297129

ABSTRACT

Ferroptosis is a form of cell death that has received considerable attention not only as a means to eradicate defined tumour entities but also because it provides unforeseen insights into the metabolic adaptation that tumours exploit to counteract phospholipid oxidation1,2. Here, we identify proferroptotic activity of 7-dehydrocholesterol reductase (DHCR7) and an unexpected prosurvival function of its substrate, 7-dehydrocholesterol (7-DHC). Although previous studies suggested that high concentrations of 7-DHC are cytotoxic to developing neurons by favouring lipid peroxidation3, we now show that 7-DHC accumulation confers a robust prosurvival function in cancer cells. Because of its far superior reactivity towards peroxyl radicals, 7-DHC effectively shields (phospho)lipids from autoxidation and subsequent fragmentation. We provide validation in neuroblastoma and Burkitt's lymphoma xenografts where we demonstrate that the accumulation of 7-DHC is capable of inducing a shift towards a ferroptosis-resistant state in these tumours ultimately resulting in a more aggressive phenotype. Conclusively, our findings provide compelling evidence of a yet-unrecognized antiferroptotic activity of 7-DHC as a cell-intrinsic mechanism that could be exploited by cancer cells to escape ferroptosis.


Subject(s)
Burkitt Lymphoma , Dehydrocholesterols , Ferroptosis , Neuroblastoma , Animals , Humans , Burkitt Lymphoma/metabolism , Burkitt Lymphoma/pathology , Cell Survival , Dehydrocholesterols/metabolism , Lipid Peroxidation , Neoplasm Transplantation , Neuroblastoma/metabolism , Neuroblastoma/pathology , Oxidation-Reduction , Phenotype , Reproducibility of Results
3.
Nature ; 608(7924): 778-783, 2022 08.
Article in English | MEDLINE | ID: mdl-35922516

ABSTRACT

Ferroptosis, a non-apoptotic form of cell death marked by iron-dependent lipid peroxidation1, has a key role in organ injury, degenerative disease and vulnerability of therapy-resistant cancers2. Although substantial progress has been made in understanding the molecular processes relevant to ferroptosis, additional cell-extrinsic and cell-intrinsic processes that determine cell sensitivity toward ferroptosis remain unknown. Here we show that the fully reduced forms of vitamin K-a group of naphthoquinones that includes menaquinone and phylloquinone3-confer a strong anti-ferroptotic function, in addition to the conventional function linked to blood clotting by acting as a cofactor for γ-glutamyl carboxylase. Ferroptosis suppressor protein 1 (FSP1), a NAD(P)H-ubiquinone reductase and the second mainstay of ferroptosis control after glutathione peroxidase-44,5, was found to efficiently reduce vitamin K to its hydroquinone, a potent radical-trapping antioxidant and inhibitor of (phospho)lipid peroxidation. The FSP1-mediated reduction of vitamin K was also responsible for the antidotal effect of vitamin K against warfarin poisoning. It follows that FSP1 is the enzyme mediating warfarin-resistant vitamin K reduction in the canonical vitamin K cycle6. The FSP1-dependent non-canonical vitamin K cycle can act to protect cells against detrimental lipid peroxidation and ferroptosis.


Subject(s)
Ferroptosis , Vitamin K , Antidotes/pharmacology , Antioxidants/metabolism , Antioxidants/pharmacology , Carbon-Carbon Ligases/metabolism , Coenzymes/metabolism , Ferroptosis/drug effects , Hydroquinones/metabolism , Hydroquinones/pharmacology , Lipid Peroxidation/drug effects , Oxidation-Reduction , S100 Calcium-Binding Protein A4/metabolism , Vitamin K/metabolism , Vitamin K/pharmacology , Warfarin/adverse effects
4.
Nature ; 575(7784): 693-698, 2019 11.
Article in English | MEDLINE | ID: mdl-31634899

ABSTRACT

Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids1,2. To date, ferroptosis has been thought to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4)3,4 and radical-trapping antioxidants5,6. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis7 is crucial to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints8 and phospholipid composition9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene11, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q10, CoQ10): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ10 using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1-CoQ10-NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Ferroptosis/genetics , Glutathione/metabolism , Mitochondrial Proteins/metabolism , Animals , Apoptosis Regulatory Proteins/genetics , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Gene Knockout Techniques , Humans , Lipid Peroxidation/genetics , Mice , Mitochondrial Proteins/genetics , Ubiquinone/analogs & derivatives , Ubiquinone/metabolism
5.
Nat Chem Biol ; 13(1): 91-98, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27842070

ABSTRACT

Ferroptosis is a form of regulated necrotic cell death controlled by glutathione peroxidase 4 (GPX4). At present, mechanisms that could predict sensitivity and/or resistance and that may be exploited to modulate ferroptosis are needed. We applied two independent approaches-a genome-wide CRISPR-based genetic screen and microarray analysis of ferroptosis-resistant cell lines-to uncover acyl-CoA synthetase long-chain family member 4 (ACSL4) as an essential component for ferroptosis execution. Specifically, Gpx4-Acsl4 double-knockout cells showed marked resistance to ferroptosis. Mechanistically, ACSL4 enriched cellular membranes with long polyunsaturated ω6 fatty acids. Moreover, ACSL4 was preferentially expressed in a panel of basal-like breast cancer cell lines and predicted their sensitivity to ferroptosis. Pharmacological targeting of ACSL4 with thiazolidinediones, a class of antidiabetic compound, ameliorated tissue demise in a mouse model of ferroptosis, suggesting that ACSL4 inhibition is a viable therapeutic approach to preventing ferroptosis-related diseases.


Subject(s)
Apoptosis , Coenzyme A Ligases/metabolism , Glutathione Peroxidase/metabolism , Mammary Neoplasms, Experimental/metabolism , Animals , Cell Death/drug effects , Cell Line , Cell Survival/drug effects , Coenzyme A Ligases/antagonists & inhibitors , Coenzyme A Ligases/deficiency , Female , Glutathione Peroxidase/deficiency , Humans , Hypoglycemic Agents/pharmacology , Mammary Neoplasms, Experimental/drug therapy , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Knockout , Necrosis , Phospholipid Hydroperoxide Glutathione Peroxidase , Thiazolidinediones/pharmacology
6.
Nat Chem Biol ; 13(1): 81-90, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27842066

ABSTRACT

Enigmatic lipid peroxidation products have been claimed as the proximate executioners of ferroptosis-a specialized death program triggered by insufficiency of glutathione peroxidase 4 (GPX4). Using quantitative redox lipidomics, reverse genetics, bioinformatics and systems biology, we discovered that ferroptosis involves a highly organized oxygenation center, wherein oxidation in endoplasmic-reticulum-associated compartments occurs on only one class of phospholipids (phosphatidylethanolamines (PEs)) and is specific toward two fatty acyls-arachidonoyl (AA) and adrenoyl (AdA). Suppression of AA or AdA esterification into PE by genetic or pharmacological inhibition of acyl-CoA synthase 4 (ACSL4) acts as a specific antiferroptotic rescue pathway. Lipoxygenase (LOX) generates doubly and triply-oxygenated (15-hydroperoxy)-diacylated PE species, which act as death signals, and tocopherols and tocotrienols (vitamin E) suppress LOX and protect against ferroptosis, suggesting a homeostatic physiological role for vitamin E. This oxidative PE death pathway may also represent a target for drug discovery.


Subject(s)
Arachidonic Acid/metabolism , Fatty Acids, Unsaturated/metabolism , Phospholipids/metabolism , Animals , Arachidonic Acid/antagonists & inhibitors , Cell Death/drug effects , Cell Line , Coenzyme A Ligases/antagonists & inhibitors , Coenzyme A Ligases/deficiency , Coenzyme A Ligases/metabolism , Fatty Acids, Unsaturated/antagonists & inhibitors , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout
7.
BMC Genomics ; 18(1): 905, 2017 Nov 25.
Article in English | MEDLINE | ID: mdl-29178829

ABSTRACT

BACKGROUND: As CRISPR/Cas9 mediated screens with pooled guide libraries in somatic cells become increasingly established, an unmet need for rapid and accurate companion informatics tools has emerged. We have developed a lightweight and efficient software to easily manipulate large raw next generation sequencing datasets derived from such screens into informative relational context with graphical support. The advantages of the software entitled ENCoRE (Easy NGS-to-Gene CRISPR REsults) include a simple graphical workflow, platform independence, local and fast multithreaded processing, data pre-processing and gene mapping with custom library import. RESULTS: We demonstrate the capabilities of ENCoRE to interrogate results from a pooled CRISPR cellular viability screen following Tumor Necrosis Factor-alpha challenge. The results not only identified stereotypical players in extrinsic apoptotic signaling but two as yet uncharacterized members of the extrinsic apoptotic cascade, Smg7 and Ces2a. We further validated and characterized cell lines containing mutations in these genes against a panel of cell death stimuli and involvement in p53 signaling. CONCLUSIONS: In summary, this software enables bench scientists with sensitive data or without access to informatic cores to rapidly interpret results from large scale experiments resulting from pooled CRISPR/Cas9 library screens.


Subject(s)
Apoptosis Regulatory Proteins/genetics , Apoptosis , CRISPR-Cas Systems , High-Throughput Nucleotide Sequencing , Software , Animals , Cell Line , Mice , Mutation
8.
IUBMB Life ; 69(6): 423-434, 2017 06.
Article in English | MEDLINE | ID: mdl-28276141

ABSTRACT

Ferroptosis is a recently described form of regulated necrotic cell death, which appears to contribute to a number of diseases, such as tissue ischemia/reperfusion injury, acute renal failure, and neurodegeneration. A hallmark of ferroptosis is iron-dependent lipid peroxidation, which can be inhibited by the key ferroptosis regulator glutathione peroxidase 4(Gpx4), radical trapping antioxidants and ferroptosis-specific inhibitors, such as ferrostatins and liproxstatins, as well as iron chelation. Although great strides have been made towards a better understanding of the proximate signals of distinctive lipid peroxides in ferroptosis, still little is known about the mechanistic implication of iron in the ferroptotic process. Hence, this review aims at summarizing recent advances in our understanding to what is known about enzymatic and nonenzymatic routes of lipid peroxidation, the involvement of iron in this process and the identification of novel players in ferroptotic cell death. Additionally, we review early works carried out long time before the term "ferroptosis" was actually introduced but which were instrumental in a better understanding of the role of ferroptosis in physiological and pathophysiological contexts. © 2017 IUBMB Life, 69(6):423-434, 2017.


Subject(s)
Antioxidants/pharmacology , Glutathione Peroxidase/antagonists & inhibitors , Iron Metabolism Disorders/metabolism , Iron/metabolism , Neuroaxonal Dystrophies/metabolism , Renal Insufficiency/metabolism , Reperfusion Injury/metabolism , Animals , Cell Death/drug effects , Cell Death/genetics , Cyclohexylamines/pharmacology , Glutathione Peroxidase/genetics , Glutathione Peroxidase/metabolism , Humans , Iron Chelating Agents/therapeutic use , Iron Metabolism Disorders/drug therapy , Iron Metabolism Disorders/genetics , Iron Metabolism Disorders/pathology , Lipid Peroxidation/drug effects , Necrosis/genetics , Necrosis/metabolism , Necrosis/pathology , Neuroaxonal Dystrophies/drug therapy , Neuroaxonal Dystrophies/genetics , Neuroaxonal Dystrophies/pathology , Phenylenediamines/pharmacology , Phospholipid Hydroperoxide Glutathione Peroxidase , Quinoxalines/pharmacology , Renal Insufficiency/drug therapy , Renal Insufficiency/genetics , Renal Insufficiency/pathology , Reperfusion Injury/genetics , Reperfusion Injury/pathology , Reperfusion Injury/prevention & control , Spiro Compounds/pharmacology
9.
J Biol Chem ; 290(23): 14668-78, 2015 Jun 05.
Article in English | MEDLINE | ID: mdl-25922076

ABSTRACT

The selenoenzyme Gpx4 is essential for early embryogenesis and cell viability for its unique function to prevent phospholipid oxidation. Recently, the cytosolic form of Gpx4 was identified as an upstream regulator of a novel form of non-apoptotic cell death, called ferroptosis, whereas the mitochondrial isoform of Gpx4 was previously shown to be crucial for male fertility. Here, we generated and analyzed mice with a targeted mutation of the active site selenocysteine of Gpx4 (Gpx4_U46S). Mice homozygous for Gpx4_U46S died at the same embryonic stage (E7.5) as Gpx4(-/-) embryos as expected. Surprisingly, male mice heterozygous for Gpx4_U46S presented subfertility. Subfertility was manifested in a reduced number of litters from heterozygous breeding and an impairment of spermatozoa to fertilize oocytes in vitro. Morphologically, sperm isolated from heterozygous Gpx4_U46S mice revealed many structural abnormalities particularly in the spermatozoa midpiece due to improper oxidation and polymerization of sperm capsular proteins and malformation of the mitochondrial capsule surrounding and stabilizing sperm mitochondria. These findings are reminiscent of sperm isolated from selenium-deprived rodents or from mice specifically lacking mitochondrial Gpx4. Due to a strongly facilitated incorporation of Ser in the polypeptide chain as compared with selenocysteine at the UGA codon, expression of the catalytically inactive Gpx4_U46S was found to be strongly increased. Because the stability of the mitochondrial capsule of mature spermatozoa depends on the moonlighting function of Gpx4 both as an enzyme oxidizing capsular protein thiols and as a structural protein, tightly controlled expression of functional Gpx4 emerges as a key for full male fertility.


Subject(s)
Amino Acid Substitution , Glutathione Peroxidase/genetics , Glutathione Peroxidase/metabolism , Infertility, Male/genetics , Spermatogenesis , Animals , Catalytic Domain , Cells, Cultured , Embryo Loss/genetics , Embryo Loss/metabolism , Embryo Loss/pathology , Female , Heterozygote , Homozygote , Infertility, Male/metabolism , Infertility, Male/pathology , Male , Mice , Mice, Transgenic , Phospholipid Hydroperoxide Glutathione Peroxidase , Selenocysteine/genetics , Serine/genetics , Spermatozoa/metabolism , Spermatozoa/pathology , Spermatozoa/ultrastructure
10.
Nat Struct Mol Biol ; 30(11): 1806-1815, 2023 Nov.
Article in English | MEDLINE | ID: mdl-37957306

ABSTRACT

Ferroptosis, marked by iron-dependent lipid peroxidation, may present an Achilles heel for the treatment of cancers. Ferroptosis suppressor protein-1 (FSP1), as the second ferroptosis mainstay, efficiently prevents lipid peroxidation via NAD(P)H-dependent reduction of quinones. Because its molecular mechanisms have remained obscure, we studied numerous FSP1 mutations present in cancer or identified by untargeted random mutagenesis. This mutational analysis elucidates the FAD/NAD(P)H-binding site and proton-transfer function of FSP1, which emerged to be evolutionarily conserved among different NADH quinone reductases. Using random mutagenesis screens, we uncover the mechanism of action of next-generation FSP1 inhibitors. Our studies identify the binding pocket of the first FSP1 inhibitor, iFSP1, and introduce the first species-independent FSP1 inhibitor, targeting the NAD(P)H-binding pocket. Conclusively, our study provides new insights into the molecular functions of FSP1 and enables the rational design of FSP1 inhibitors targeting cancer cells.


Subject(s)
Ferroptosis , Ferroptosis/genetics , NAD , Mutation , Mutagenesis , Binding Sites , Protons
11.
J Extracell Vesicles ; 11(7): e12242, 2022 07.
Article in English | MEDLINE | ID: mdl-35790086

ABSTRACT

Eukaryotic cells, including cancer cells, secrete highly heterogeneous populations of extracellular vesicles (EVs). EVs could have different subcellular origin, composition and functional properties, but tools to distinguish between EV subtypes are scarce. Here, we tagged CD63- or CD9-positive EVs secreted by triple negative breast cancer cells with Nanoluciferase enzyme, to set-up a miniaturized method to quantify secretion of these two EV subtypes directly in the supernatant of cells. We performed a cell-based high-content screening to identify clinically-approved drugs able to affect EV secretion. One of the identified hits is Homosalate, an anti-inflammatory drug found in sunscreens which robustly increased EVs' release. Comparing EVs induced by Homosalate with those induced by Bafilomycin A1, we demonstrate that: (1) the two drugs act on EVs generated in distinct subcellular compartments, and (2) EVs released by Homosalate-, but not by Bafilomycin A1-treated cells enhance resistance to anchorage loss in another recipient epithelial tumour cell line. In conclusion, we identified a new drug modifying EV release and demonstrated that under influence of different drugs, triple negative breast cancer cells release EV subpopulations from different subcellular origins harbouring distinct functional properties.


Subject(s)
Extracellular Vesicles , Triple Negative Breast Neoplasms , Dietary Supplements , Humans , Salicylates , Triple Negative Breast Neoplasms/drug therapy
12.
Nat Commun ; 13(1): 1303, 2022 03 14.
Article in English | MEDLINE | ID: mdl-35288557

ABSTRACT

Extravasation of monocytes into tissue and to the site of injury is a fundamental immunological process, which requires rapid responses via post translational modifications (PTM) of proteins. Protein arginine methyltransferase 7 (PRMT7) is an epigenetic factor that has the capacity to mono-methylate histones on arginine residues. Here we show that in chronic obstructive pulmonary disease (COPD) patients, PRMT7 expression is elevated in the lung tissue and localized to the macrophages. In mouse models of COPD, lung fibrosis and skin injury, reduced expression of PRMT7 associates with decreased recruitment of monocytes to the site of injury and hence less severe symptoms. Mechanistically, activation of NF-κB/RelA in monocytes induces PRMT7 transcription and consequential mono-methylation of histones at the regulatory elements of RAP1A, which leads to increased transcription of this gene that is responsible for adhesion and migration of monocytes. Persistent monocyte-derived macrophage accumulation leads to ALOX5 over-expression and accumulation of its metabolite LTB4, which triggers expression of ACSL4 a ferroptosis promoting gene in lung epithelial cells. Conclusively, inhibition of arginine mono-methylation might offer targeted intervention in monocyte-driven inflammatory conditions that lead to extensive tissue damage if left untreated.


Subject(s)
Protein-Arginine N-Methyltransferases , Pulmonary Disease, Chronic Obstructive , Animals , Arginine/metabolism , Histones/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Mice , Monocytes/metabolism , Protein-Arginine N-Methyltransferases/metabolism , Pulmonary Disease, Chronic Obstructive/genetics
13.
Nat Commun ; 12(1): 2244, 2021 04 14.
Article in English | MEDLINE | ID: mdl-33854057

ABSTRACT

Ferroptosis is associated with lipid hydroperoxides generated by the oxidation of polyunsaturated acyl chains. Lipid hydroperoxides are reduced by glutathione peroxidase 4 (GPX4) and GPX4 inhibitors induce ferroptosis. However, the therapeutic potential of triggering ferroptosis in cancer cells with polyunsaturated fatty acids is unknown. Here, we identify conjugated linoleates including α-eleostearic acid (αESA) as ferroptosis inducers. αESA does not alter GPX4 activity but is incorporated into cellular lipids and promotes lipid peroxidation and cell death in diverse cancer cell types. αESA-triggered death is mediated by acyl-CoA synthetase long-chain isoform 1, which promotes αESA incorporation into neutral lipids including triacylglycerols. Interfering with triacylglycerol biosynthesis suppresses ferroptosis triggered by αESA but not by GPX4 inhibition. Oral administration of tung oil, naturally rich in αESA, to mice limits tumor growth and metastasis with transcriptional changes consistent with ferroptosis. Overall, these findings illuminate a potential approach to ferroptosis, complementary to GPX4 inhibition.


Subject(s)
Coenzyme A Ligases/metabolism , Ferroptosis , Linolenic Acids/metabolism , Triple Negative Breast Neoplasms/enzymology , Triple Negative Breast Neoplasms/physiopathology , Animals , Cell Death , Coenzyme A Ligases/genetics , Humans , Mice , Mice, Inbred NOD , Phospholipid Hydroperoxide Glutathione Peroxidase/genetics , Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/metabolism
14.
Nat Cell Biol ; 23(6): 652-663, 2021 06.
Article in English | MEDLINE | ID: mdl-34083785

ABSTRACT

Expression of exon-specific isoforms from alternatively spliced mRNA is a fundamental mechanism that substantially expands the proteome of a cell. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression longitudinally at the protein level. Here, we therefore developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms of endogenous genes by scarlessly excising reporter proteins from the nascent polypeptide chain through highly efficient, intein-mediated protein splicing. We applied EXSISERS to quantify the inclusion of the disease-associated exon 10 in microtubule-associated protein tau (MAPT) in patient-derived induced pluripotent stem cells and screened Cas13-based RNA-targeting effectors for isoform specificity. We also coupled cell survival to the inclusion of exon 18b of FOXP1, which is involved in maintaining pluripotency of embryonic stem cells, and confirmed that MBNL1 is a dominant factor for exon 18b exclusion. EXSISERS enables non-disruptive and multimodal monitoring of exon-specific isoform expression with high sensitivity and cellular resolution, and empowers high-throughput screening of exon-specific therapeutic interventions.


Subject(s)
Alternative Splicing , Forkhead Transcription Factors/metabolism , High-Throughput Screening Assays , Induced Pluripotent Stem Cells/metabolism , Proteomics , RNA Stability , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Repressor Proteins/metabolism , tau Proteins/metabolism , CRISPR-Cas Systems , Exons , Forkhead Transcription Factors/genetics , HEK293 Cells , Humans , Protein Isoforms , Proteome , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , Repressor Proteins/genetics , Single-Cell Analysis , tau Proteins/genetics
15.
Nat Commun ; 12(1): 4402, 2021 07 20.
Article in English | MEDLINE | ID: mdl-34285231

ABSTRACT

Acute kidney injury (AKI) is morphologically characterized by a synchronized plasma membrane rupture of cells in a specific section of a nephron, referred to as acute tubular necrosis (ATN). Whereas the involvement of necroptosis is well characterized, genetic evidence supporting the contribution of ferroptosis is lacking. Here, we demonstrate that the loss of ferroptosis suppressor protein 1 (Fsp1) or the targeted manipulation of the active center of the selenoprotein glutathione peroxidase 4 (Gpx4cys/-) sensitize kidneys to tubular ferroptosis, resulting in a unique morphological pattern of tubular necrosis. Given the unmet medical need to clinically inhibit AKI, we generated a combined small molecule inhibitor (Nec-1f) that simultaneously targets receptor interacting protein kinase 1 (RIPK1) and ferroptosis in cell lines, in freshly isolated primary kidney tubules and in mouse models of cardiac transplantation and of AKI and improved survival in models of ischemia-reperfusion injury. Based on genetic and pharmacological evidence, we conclude that GPX4 dysfunction hypersensitizes mice to ATN during AKI. Additionally, we introduce Nec-1f, a solid inhibitor of RIPK1 and weak inhibitor of ferroptosis.


Subject(s)
Acute Kidney Injury/pathology , Ferroptosis/physiology , Kidney Tubules/pathology , Reperfusion Injury/pathology , Acute Kidney Injury/drug therapy , Acute Kidney Injury/etiology , Animals , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Cisplatin/administration & dosage , Cisplatin/toxicity , Disease Models, Animal , Epithelial Cells , Female , Ferroptosis/drug effects , Gene Knockdown Techniques , HT29 Cells , Heart Transplantation/adverse effects , Humans , Imidazoles/chemistry , Imidazoles/pharmacology , Imidazoles/therapeutic use , Indoles/chemistry , Indoles/pharmacology , Indoles/therapeutic use , Male , Mice , Mice, Transgenic , Microsomes, Liver , Mitochondrial Proteins/metabolism , NIH 3T3 Cells , Necrosis/drug therapy , Necrosis/etiology , Necrosis/pathology , Oxidoreductases/genetics , Oxidoreductases/metabolism , Phospholipid Hydroperoxide Glutathione Peroxidase/antagonists & inhibitors , Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism , Primary Cell Culture , Receptor-Interacting Protein Serine-Threonine Kinases/antagonists & inhibitors , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Reperfusion Injury/drug therapy , Reperfusion Injury/etiology
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