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1.
Int J Mol Sci ; 22(18)2021 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-34576210

RESUMO

G protein-coupled receptors (GPCRs) are targets of extracellular stimuli and hence occupy a key position in drug discovery. By specific and not yet fully elucidated coupling profiles with α subunits of distinct G protein families, they regulate cellular responses. The histamine H2 and H4 receptors (H2R and H4R) are prominent members of Gs- and Gi-coupled GPCRs. Nevertheless, promiscuous G protein and selective Gi signaling have been reported for the H2R and H4R, respectively, the molecular mechanism of which remained unclear. Using a combination of cellular experimental assays and Gaussian accelerated molecular dynamics (GaMD) simulations, we investigated the coupling profiles of the H2R and H4R to engineered mini-G proteins (mG). We obtained coupling profiles of the mGs, mGsi, or mGsq proteins to the H2R and H4R from the mini-G protein recruitment assays using HEK293T cells. Compared to H2R-mGs expressing cells, histamine responses were weaker (pEC50, Emax) for H2R-mGsi and -mGsq. By contrast, the H4R selectively bound to mGsi. Similarly, in all-atom GaMD simulations, we observed a preferential binding of H2R to mGs and H4R to mGsi revealed by the structural flexibility and free energy landscapes of the complexes. Although the mG α5 helices were consistently located within the HR binding cavity, alternative binding orientations were detected in the complexes. Due to the specific residue interactions, all mG α5 helices of the H2R complexes adopted the Gs-like orientation toward the receptor transmembrane (TM) 6 domain, whereas in H4R complexes, only mGsi was in the Gi-like orientation toward TM2, which was in agreement with Gs- and Gi-coupled GPCRs structures resolved by X-ray/cryo-EM. These cellular and molecular insights support (patho)physiological profiles of the histamine receptors, especially the hitherto little studied H2R function in the brain, as well as of the pharmacological potential of H4R selective drugs.

2.
J Cell Biochem ; 2021 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-34516024

RESUMO

Variants of concern of the SARS-CoV-2 virus with an asparagine-to-tyrosine substitution at position 501 (N501Y) in the receptor-binding domain (RBD) show enhanced infectivity compared to wild-type, resulting in an altered pandemic situation in affected areas. These SARS-Cov-2 variants comprise the two Alpha variants (B.1.1.7, United Kingdom and B.1.1.7 with the additional E484K mutation), the Beta variant (B.1.351, South Africa), and the Gamma variant (P.1, Brazil). Understanding the binding modalities between these viral variants and the host cell receptor ACE2 allows to depict changes, but also common motifs of virus-host cell interaction. The trimeric spike protein expressed at the viral surface contains the RBD that forms the molecular interface with ACE2. All the above-mentioned variants carry between one and three amino acid exchanges within the interface-forming region of the RBD, thereby altering the binding interface with ACE2. Using molecular dynamics (MD) simulations and decomposition of intermolecular contacts between the RBD and ACE2, we identified phenylalanine 486, glutamine 498, threonine 500, and tyrosine 505 as important interface-forming residues across viral variants. However, especially the N501Y exchange increased contact formation for this residue and also induced some local conformational changes. Comparing here, the in silico generated B.1.1.7 RBD-ACE2 complex with the now available experimentally solved structure reveals very similar behavior during MD simulation. We demonstrate, how computational methods can help to identify differences in conformation as well as contact formation for newly emerging viral variants. Altogether, we provide extensive data on all N501Y expressing SARS-CoV-2 variants of concern with respect to their interaction with ACE2 and how this induces reshaping of the RBD-ACE2 interface.

4.
Nitric Oxide ; 115: 34-43, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34329739

RESUMO

Ferroptosis is a type of iron-dependent necrotic cell death, which is typically triggered by the depletion of intracellular glutathione (GSH), which is associated with increased lipid peroxidation. Nitric oxide (NO) is a highly reactive gaseous radical mediator with anti-oxidation properties that terminates lipid peroxidation reactions. In the current study, we report the anti-ferroptotic action of NOC18, an NO donor that spontaneously releases NO, in cells under various ferroptotic conditions in vitro. Our results indicate that, when mouse hepatoma Hepa 1-6 cells are incubated with NOC18, cell death induced by various ferroptotic stimuli such as cysteine (Cys) starvation, the inhibition of glutathione peroxidase 4 (GPX4) and treatment with tertiary-butyl hydroperoxide (TBHP) is significantly reduced. Treatment with NOC18 failed to improve the decrease in the levels of Cys or GSH and the accumulation of ferrous iron upon ferroptotic stimuli. The fluorescent intensity of C11-BODIPY581/591, a probe that is used to detect lipid peroxidation products, was increased somewhat by treatment with NOC18 under conditions of Cys starvation, and the accumulation of lipid peroxidation end-products, as evidenced by the levels of 4-hydroxynonenal, were effectively suppressed. The pre-incubation of TBHP with NOC7, a short-lived NO donor completely eliminated its ability to trigger ferroptosis. These collective results indicate that NO exerts a cytoprotective action against various ferroptotic stimuli by aborting the lipid peroxidation chain reaction.

5.
Cell Death Dis ; 12(7): 698, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257282

RESUMO

Sorafenib, a protein kinase inhibitor approved for the treatment of hepatocellular carcinoma and advanced renal cell carcinoma, has been repeatedly reported to induce ferroptosis by possibly involving inhibition of the cystine/glutamate antiporter, known as system xc-. Using a combination of well-defined genetically engineered tumor cell lines and canonical small molecule ferroptosis inhibitors, we now provide unequivocal evidence that sorafenib does not induce ferroptosis in a series of tumor cell lines unlike the cognate system xc- inhibitors sulfasalazine and erastin. We further show that only a subset of tumor cells dies by ferroptosis upon sulfasalazine and erastin treatment, implying that certain cell lines appear to be resistant to system xc- inhibition, while others undergo ferroptosis-independent cell death. From these findings, we conclude that sorafenib does not qualify as a bona fide ferroptosis inducer and that ferroptosis induced by system xc- inhibitors can only be achieved in a fraction of tumor cell lines despite robust expression of SLC7A11, the substrate-specific subunit of system xc-.


Assuntos
Antineoplásicos/farmacologia , Ferroptose/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Sorafenibe/farmacologia , Sistema y+ de Transporte de Aminoácidos/antagonistas & inibidores , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Células HEK293 , Humanos , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Piperazinas/farmacologia , Sulfassalazina/farmacologia
6.
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
7.
Redox Biol ; 45: 102056, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34229160

RESUMO

Ferroptosis is primarily triggered by a failure of the glutathione (GSH)-glutathione peroxidase 4 (GPX4) reductive system and associated overwhelming lipid peroxidation, in which enzymes regulating polyunsaturated fatty acid (PUFA) metabolism, and in particular acyl-CoA synthetase long chain family member 4 (ACSL4), are central. Here, we found that exogenous oxygen radicals generated by photodynamic therapy (PDT) can directly peroxidize PUFAs and initiate lipid autoxidation, coinciding with cellular GSH depletion. Different from canonical ferroptosis induced by RSL3 or erastin, PDT-initiated lipid peroxidation and ferroptotis-like cell death is independent of lipoxygenase (ALOXs) and ACSL4. Especially, this form of cell death modality can be triggered in malignant cells insensitive to or acquired resistance to canonical ferroptosis inducers. We also observed a distinct iron metabolism pathway in this PDT-triggered cell death modality, in which cytosolic labile iron is decreased probably due to its relocation to mitochondria. Inhibition of the mitochondrial Ca2+ and Fe2+ uniporter (MCU) effectively prevented PDT-triggered lipid peroxidation and subsequent cell death. Therefore, we tentatively term this distinct ferroptosis-like cell death as liperoptosis. Moreover, using the clinically approved photosensitizer Verteporfin, PDT inhibited tumor growth through inducing prevailing ferroptosis-like cell death in a mouse xenograft model. With its site-specific advantages, these findings highlight the value of using PDT to trigger lipid peroxidation and ferroptosis-like cell death in vivo, and will benefit exploring the exact molecular mechanism of immunological effects of PDT in cancer treatment.


Assuntos
Ferroptose , Fotoquimioterapia , Animais , Morte Celular , Peroxidação de Lipídeos , Camundongos , Fosfolipídeo Hidroperóxido Glutationa Peroxidase
8.
Biomedicines ; 9(5)2021 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-34066729

RESUMO

The B.1.1.7 variant of the SARS-CoV-2 virus shows enhanced infectiousness over the wild type virus, leading to increasing patient numbers in affected areas. Amino acid exchanges within the SARS-CoV-2 spike protein variant of B.1.1.7 affect inter-monomeric contact sites within the trimer (A570D and D614G) as well as the ACE2-receptor interface region (N501Y), which comprises the receptor-binding domain (RBD) of the spike protein. However, the molecular consequences of mutations within B.1.1.7 on spike protein dynamics and stability or ACE2 binding are largely unknown. Here, molecular dynamics simulations comparing SARS-CoV-2 wild type with the B.1.1.7 variant revealed inter-trimeric contact rearrangements, altering the structural flexibility within the spike protein trimer. Furthermore, we found increased flexibility in direct spatial proximity of the fusion peptide due to salt bridge rearrangements induced by the D614G mutation in B.1.1.7. This study also implies a reduced binding affinity for B.1.1.7 with ACE2, as the N501Y mutation restructures the RBD-ACE2 interface, significantly decreasing the linear interaction energy between the RBD and ACE2. Our results demonstrate how mutations found within B.1.1.7 enlarge the flexibility around the fusion peptide and change the RBD-ACE2 interface. We anticipate our findings to be starting points for in depth biochemical and cell biological analyses of B.1.1.7.

9.
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
10.
Trends Endocrinol Metab ; 32(7): 463-473, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33985897

RESUMO

Lipid peroxidation (LPO) is the molecular mechanism involved in oxidative damage of cellular membranes and the hallmark of a nonapoptotic form of cell death, known as ferroptosis. This iron-dependent cell death is an emerging strategy in cancer treatment and one of the central cell death mechanisms accounting for early cell loss and organ dysfunction in both neurodegenerative disease and ischemia-reperfusion injury. Although the biological roles of LPO products have attracted considerable attention, not only for their pathological mechanisms but also for their potential clinical application as biomarkers, the existence of a common lethal lipid death signal generated during ferroptosis remains poorly explored. A better understanding of the LPO process, however, may unleash unprecedented opportunities for therapeutic intervention of as-yet incurable diseases.

11.
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
12.
Proc Natl Acad Sci U S A ; 118(7)2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33579817

RESUMO

The mitochondrial thioredoxin/peroxiredoxin system encompasses NADPH, thioredoxin reductase 2 (TrxR2), thioredoxin 2, and peroxiredoxins 3 and 5 (Prx3 and Prx5) and is crucial to regulate cell redox homeostasis via the efficient catabolism of peroxides (TrxR2 and Trxrd2 refer to the mitochondrial thioredoxin reductase protein and gene, respectively). Here, we report that endothelial TrxR2 controls both the steady-state concentration of peroxynitrite, the product of the reaction of superoxide radical and nitric oxide, and the integrity of the vascular system. Mice with endothelial deletion of the Trxrd2 gene develop increased vascular stiffness and hypertrophy of the vascular wall. Furthermore, they suffer from renal abnormalities, including thickening of the Bowman's capsule, glomerulosclerosis, and functional alterations. Mechanistically, we show that loss of Trxrd2 results in enhanced peroxynitrite steady-state levels in both vascular endothelial cells and vessels by using a highly sensitive redox probe, fluorescein-boronate. High steady-state peroxynitrite levels were further found to coincide with elevated protein tyrosine nitration in renal tissue and a substantial change of the redox state of Prx3 toward the oxidized protein, even though glutaredoxin 2 (Grx2) expression increased in parallel. Additional studies using a mitochondria-specific fluorescence probe (MitoPY1) in vessels revealed that enhanced peroxynitrite levels are indeed generated in mitochondria. Treatment with Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin [Mn(III)TMPyP], a peroxynitrite-decomposition catalyst, blunted intravascular formation of peroxynitrite. Our data provide compelling evidence for a yet-unrecognized role of TrxR2 in balancing the nitric oxide/peroxynitrite ratio in endothelial cells in vivo and thus establish a link between enhanced mitochondrial peroxynitrite and disruption of vascular integrity.


Assuntos
Endotélio Vascular/metabolismo , Ácido Peroxinitroso/metabolismo , Tiorredoxina Redutase 2/metabolismo , Animais , Rim/irrigação sanguínea , Rim/metabolismo , Camundongos , Mitocôndrias/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo , Tiorredoxina Redutase 2/genética , Remodelação Vascular
13.
Nat Rev Mol Cell Biol ; 22(4): 266-282, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33495651

RESUMO

The research field of ferroptosis has seen exponential growth over the past few years, since the term was coined in 2012. This unique modality of cell death, driven by iron-dependent phospholipid peroxidation, is regulated by multiple cellular metabolic pathways, including redox homeostasis, iron handling, mitochondrial activity and metabolism of amino acids, lipids and sugars, in addition to various signalling pathways relevant to disease. Numerous organ injuries and degenerative pathologies are driven by ferroptosis. Intriguingly, therapy-resistant cancer cells, particularly those in the mesenchymal state and prone to metastasis, are exquisitely vulnerable to ferroptosis. As such, pharmacological modulation of ferroptosis, via both its induction and its inhibition, holds great potential for the treatment of drug-resistant cancers, ischaemic organ injuries and other degenerative diseases linked to extensive lipid peroxidation. In this Review, we provide a critical analysis of the current molecular mechanisms and regulatory networks of ferroptosis, the potential physiological functions of ferroptosis in tumour suppression and immune surveillance, and its pathological roles, together with a potential for therapeutic targeting. Importantly, as in all rapidly evolving research areas, challenges exist due to misconceptions and inappropriate experimental methods. This Review also aims to address these issues and to provide practical guidelines for enhancing reproducibility and reliability in studies of ferroptosis. Finally, we discuss important concepts and pressing questions that should be the focus of future ferroptosis research.


Assuntos
Ferroptose/genética , Neoplasias/genética , Animais , Redes Reguladoras de Genes/genética , Humanos , Peroxidação de Lipídeos , Oxirredução , Reprodutibilidade dos Testes
14.
Trends Mol Med ; 27(2): 113-122, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32958404

RESUMO

Attaining control over life and death decisions facilitates the identification of new therapeutic strategies for diseases affected by early cell loss or resistance to cell death. In this context, ferroptosis, a prevailing form of non-apoptotic cell death marked by the iron-dependent oxidative destruction of lipid bilayers and metabolic aberrations, has attracted overwhelming interest among basic researchers and clinicians due to its relevance for a number of degenerative diseases, such as neurodegeneration, ischemia/reperfusion injury (IRI), and organ failure, as well as therapy-resistant tumors. As the ferroptotic death pathway offers various druggable nodes, it is anticipated that the preclinical and clinical development of ferroptosis modulators will unleash unprecedented opportunities for the treatment of as-yet-incurable diseases.

15.
Biol Chem ; 402(3): 271-287, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33055310

RESUMO

Maintenance of cellular redox control is pivotal for normal cellular functions and cell fate decisions including cell death. Among the key cellular redox systems in mammals, the glutathione peroxidase (GPX) family of proteins is the largest conferring multifaceted functions and affecting virtually all cellular processes. The endoplasmic reticulum (ER)-resident GPXs, designated as GPX7 and GPX8, are the most recently added members of this family of enzymes. Recent studies have provided exciting insights how both enzymes support critical processes of the ER including oxidative protein folding, maintenance of ER redox control by eliminating H2O2, and preventing palmitic acid-induced lipotoxicity. Consequently, numerous pathological conditions, such as neurodegeneration, cancer and metabolic diseases have been linked with altered GPX7 and GPX8 expression. Studies in mice have demonstrated that loss of GPX7 leads to increased differentiation of preadipocytes, increased tumorigenesis and shortened lifespan. By contrast, GPX8 deficiency in mice results in enhanced caspase-4/11 activation and increased endotoxic shock in colitis model. With the increasing recognition that both types of enzymes are dysregulated in various tumor entities in man, we deem a review of the emerging roles played by GPX7 and GPX8 in health and disease development timely and appropriate.

16.
Biochim Biophys Acta Mol Cell Res ; 1868(3): 118928, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33340545

RESUMO

Ferroptosis is a necrotic form of cell death caused by inactivation of the glutathione system and uncontrolled iron-mediated lipid peroxidation. Increasing evidence implicates ferroptosis in a wide range of diseases from neurotrauma to cancer, highlighting the importance of identifying an executioner system that can be exploited for clinical applications. In this study, using pharmacological and genetic models of ferroptosis, we observed that lysosomal membrane permeabilization and cytoplasmic leakage of cathepsin B unleashes structural and functional changes in mitochondria and promotes a not previously reported cleavage of histone H3. Inhibition of cathepsin-B robustly rescued cellular membrane integrity and chromatin degradation. We show that these protective effects are independent of glutathione peroxidase-4 and are mediated by preventing lysosomal membrane damage. This was further confirmed when cathepsin B knockout primary fibroblasts remained unaffected in response to various ferroptosis inducers. Our work identifies new and yet-unrecognized aspects of ferroptosis and identifies cathepsin B as a mediator of ferroptotic cell death.


Assuntos
Catepsina B/genética , Catepsina B/metabolismo , Mitocôndrias/metabolismo , Neurônios/citologia , Animais , Linhagem Celular , Ferroptose , Técnicas de Silenciamento de Genes , Histonas/metabolismo , Peroxidação de Lipídeos , Lisossomos/metabolismo , Potencial da Membrana Mitocondrial , Camundongos , Células NIH 3T3 , Neurônios/metabolismo
17.
Signal Transduct Target Ther ; 5(1): 261, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33149129

Assuntos
Ferro , Melanoma , Ferroptose , Humanos
18.
Cell Res ; 30(12): 1061-1062, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33149249

Assuntos
Ferroptose , Éter , Lipídeos
19.
Cell Metab ; 32(6): 920-937, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33217331

RESUMO

Acute or chronic cellular stress resulting from aberrant metabolic and biochemical processes may trigger a pervasive non-apoptotic form of cell death, generally known as ferroptosis. Ferroptosis is unique among the different cell death modalities, as it has been mostly linked to pathophysiological conditions and because several metabolic pathways, such as (seleno)thiol metabolism, fatty acid metabolism, iron handling, mevalonate pathway, and mitochondrial respiration, directly impinge on the cells' sensitivity toward lipid peroxidation and ferroptosis. Additionally, key cellular redox systems, such as selenium-dependent glutathione peroxidase 4 and the NAD(P)H/ferroptosis suppressor protein-1/ubiquinone axis, are at play that constantly surveil and neutralize oxidative damage to cellular membranes. Since this form of cell death emerges to be the root cause of a number of diseases and since it offers various pharmacologically tractable nodes for therapeutic intervention, there has been overwhelming interest in the last few years aiming for a better molecular understanding of the ferroptotic death process.

20.
Int J Mol Sci ; 21(18)2020 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-32932742

RESUMO

The histamine H2 receptor (H2R) plays an important role in the regulation of gastric acid secretion. Therefore, it is a main drug target for the treatment of gastroesophageal reflux or peptic ulcer disease. However, there is as of yet no 3D-structural information available hampering a mechanistic understanding of H2R. Therefore, we created a model of the histamine-H2R-Gs complex based on the structure of the ternary complex of the ß2-adrenoceptor and investigated the conformational stability of this active GPCR conformation. Since the physiologically relevant motions with respect to ligand binding and conformational changes of GPCRs can only partly be assessed on the timescale of conventional MD (cMD) simulations, we also applied metadynamics and Gaussian accelerated molecular dynamics (GaMD) simulations. A multiple walker metadynamics simulation in combination with cMD was applied for the determination of the histamine binding mode. The preferential binding pose detected is in good agreement with previous data from site directed mutagenesis and provides a basis for rational ligand design. Inspection of the H2R-Gs interface reveals a network of polar interactions that may contribute to H2R coupling selectivity. The cMD and GaMD simulations demonstrate that the active conformation is retained on a µs-timescale in the ternary histamine-H2R-Gs complex and in a truncated complex that contains only Gs helix α5 instead of the entire G protein. In contrast, histamine alone is unable to stabilize the active conformation, which is in line with previous studies of other GPCRs.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores Histamínicos H2/metabolismo , Sequência de Aminoácidos , Histamina/metabolismo , Humanos , Ligantes , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica
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