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1.
Nat Commun ; 12(1): 4402, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285231

RESUMO

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.


Assuntos
Injúria Renal Aguda/patologia , Ferroptose/fisiologia , Túbulos Renais/patologia , Traumatismo por Reperfusão/patologia , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/etiologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Cisplatino/administração & dosagem , Cisplatino/toxicidade , Modelos Animais de Doenças , Células Epiteliais , Feminino , Ferroptose/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HT29 , Transplante de Coração/efeitos adversos , Humanos , Imidazóis/química , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Indóis/química , Indóis/farmacologia , Indóis/uso terapêutico , Masculino , Camundongos , Camundongos Transgênicos , Microssomos Hepáticos , Proteínas Mitocondriais/metabolismo , Células NIH 3T3 , Necrose/tratamento farmacológico , Necrose/etiologia , Necrose/patologia , Oxirredutases/genética , Oxirredutases/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/antagonistas & inibidores , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Cultura Primária de Células , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/etiologia
2.
Nat Cell Biol ; 23(6): 652-663, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34083785

RESUMO

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.


Assuntos
Processamento Alternativo , Fatores de Transcrição Forkhead/metabolismo , Ensaios de Triagem em Larga Escala , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteômica , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Proteínas tau/metabolismo , Sistemas CRISPR-Cas , Éxons , Fatores de Transcrição Forkhead/genética , Células HEK293 , Humanos , Isoformas de Proteínas , Proteoma , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Análise de Célula Única , Proteínas tau/genética
3.
Nat Commun ; 12(1): 2244, 2021 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-33854057

RESUMO

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.


Assuntos
Coenzima A Ligases/metabolismo , Ferroptose , Ácidos Linolênicos/metabolismo , Neoplasias de Mama Triplo Negativas/enzimologia , Neoplasias de Mama Triplo Negativas/fisiopatologia , Animais , Morte Celular , Coenzima A Ligases/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo
4.
Nature ; 575(7784): 693-698, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31634899

RESUMO

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.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Ferroptose/genética , Glutationa/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Peroxidação de Lipídeos/genética , Camundongos , Proteínas Mitocondriais/genética , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo
5.
Cell ; 172(3): 409-422.e21, 2018 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-29290465

RESUMO

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.


Assuntos
Apoptose , Glutationa Peroxidase/metabolismo , Convulsões/metabolismo , Selênio/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Feminino , Glutationa Peroxidase/genética , Células HEK293 , Humanos , Peróxido de Hidrogênio/toxicidade , Interneurônios/metabolismo , Peroxidação de Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Convulsões/etiologia
6.
BMC Genomics ; 18(1): 905, 2017 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-29178829

RESUMO

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.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Apoptose , Sistemas CRISPR-Cas , Sequenciamento de Nucleotídeos em Larga Escala , Software , Animais , Linhagem Celular , Camundongos , Mutação
7.
IUBMB Life ; 69(6): 423-434, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28276141

RESUMO

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.


Assuntos
Antioxidantes/farmacologia , Glutationa Peroxidase/antagonistas & inibidores , Distúrbios do Metabolismo do Ferro/metabolismo , Ferro/metabolismo , Distrofias Neuroaxonais/metabolismo , Insuficiência Renal/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Cicloexilaminas/farmacologia , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Humanos , Quelantes de Ferro/uso terapêutico , Distúrbios do Metabolismo do Ferro/tratamento farmacológico , Distúrbios do Metabolismo do Ferro/genética , Distúrbios do Metabolismo do Ferro/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Necrose/genética , Necrose/metabolismo , Necrose/patologia , Distrofias Neuroaxonais/tratamento farmacológico , Distrofias Neuroaxonais/genética , Distrofias Neuroaxonais/patologia , Fenilenodiaminas/farmacologia , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Quinoxalinas/farmacologia , Insuficiência Renal/tratamento farmacológico , Insuficiência Renal/genética , Insuficiência Renal/patologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/prevenção & controle , Compostos de Espiro/farmacologia
8.
Nat Chem Biol ; 13(1): 81-90, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27842066

RESUMO

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.


Assuntos
Ácido Araquidônico/metabolismo , Ácidos Graxos Insaturados/metabolismo , Fosfolipídeos/metabolismo , Animais , Ácido Araquidônico/antagonistas & inibidores , Morte Celular/efeitos dos fármacos , Linhagem Celular , Coenzima A Ligases/antagonistas & inibidores , Coenzima A Ligases/deficiência , Coenzima A Ligases/metabolismo , Ácidos Graxos Insaturados/antagonistas & inibidores , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
9.
Nat Chem Biol ; 13(1): 91-98, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27842070

RESUMO

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.


Assuntos
Apoptose , Coenzima A Ligases/metabolismo , Glutationa Peroxidase/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Coenzima A Ligases/antagonistas & inibidores , Coenzima A Ligases/deficiência , Feminino , Glutationa Peroxidase/deficiência , Humanos , Hipoglicemiantes/farmacologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Knockout , Necrose , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Tiazolidinedionas/farmacologia
10.
J Biol Chem ; 290(23): 14668-78, 2015 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-25922076

RESUMO

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.


Assuntos
Substituição de Aminoácidos , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Infertilidade Masculina/genética , Espermatogênese , Animais , Domínio Catalítico , Células Cultivadas , Perda do Embrião/genética , Perda do Embrião/metabolismo , Perda do Embrião/patologia , Feminino , Heterozigoto , Homozigoto , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Camundongos , Camundongos Transgênicos , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Selenocisteína/genética , Serina/genética , Espermatozoides/metabolismo , Espermatozoides/patologia , Espermatozoides/ultraestrutura
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