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1.
Chem Commun (Camb) ; 57(71): 8961-8964, 2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34486587

RESUMO

Optical properties of anisotropic gold nanorod arrays inside anodic aluminium oxide substrates enhance the longitudinal absorption intensities and the hyperthermia cancer cell killing at 42.1 °C under photothermal laser exposures at 671 nm.


Assuntos
Antineoplásicos/farmacologia , Nanotubos/química , Terapia Fototérmica/métodos , Óxido de Alumínio/química , Óxido de Alumínio/farmacologia , Óxido de Alumínio/efeitos da radiação , Antineoplásicos/química , Antineoplásicos/efeitos da radiação , Morte Celular/fisiologia , Ouro/química , Ouro/farmacologia , Ouro/efeitos da radiação , Células HeLa , Humanos , Nanotubos/efeitos da radiação
2.
Int J Mol Sci ; 22(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445796

RESUMO

Obesity and metabolic syndrome are associated with cognitive decline and dementia. Palmitic acid (PA) is increased in the cerebrospinal fluid of obese patients with cognitive impairment. This study was therefore designed to examine fatty acid (FA) lipotoxicity in BV2 microglia cells. We found that PA induced time- and dose-dependent decrease in cell viability and increase in cell death without affecting the cell cycle profile and that PA lipotoxicity did not depend on cell surface free fatty acid receptors but rather on FA uptake. Treatment with sulfosuccinimidyl oleate (SSO), an irreversible inhibitor of fatty acid translocase CD36, significantly inhibited FA uptake in BSA- and PA-treated cells and blocked PA-induced decrease in cell viability. Inhibition of ER stress or treatment with N-acetylcysteine was not able to rescue PA lipotoxicity. Our study also showed that unsaturated fatty acids (UFAs), such as linoleic acid (LA), oleic acid (OA), α-linolenic acid (ALA), and docosahexaenoic acid (DHA), were not lipotoxic but instead protected microglia against PA-induced decrease in cell viability. Co-treatment of PA with LA, OA, and DHA significantly inhibited FA uptake in PA-treated cells. All UFAs tested induced the incorporation of FAs into and the amount of neutral lipids, while PA did not significantly affect the amount of neutral lipids compared with BSA control.


Assuntos
Ácidos Graxos Insaturados/metabolismo , Microglia/metabolismo , Ácido Palmítico/metabolismo , Animais , Morte Celular/fisiologia , Sobrevivência Celular/fisiologia , Ácidos Graxos não Esterificados/metabolismo , Lipídeos/química , Camundongos
3.
Int J Mol Sci ; 22(14)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34298888

RESUMO

We recently demonstrated that chemical proteasome inhibition induced inner retinal degeneration, supporting the pivotal roles of the ubiquitin-proteasome system in retinal structural integrity maintenance. In this study, using beclin1-heterozygous (Becn1-Het) mice with autophagic dysfunction, we tested our hypothesis that autophagy could be a compensatory retinal protective mechanism for proteasomal impairment. Despite the reduced number of autophagosome, the ocular tissue morphology and intraocular pressure were normal. Surprisingly, Becn1-Het mice experienced the same extent of retinal degeneration as was observed in wild-type mice, following an intravitreal injection of a chemical proteasome inhibitor. Similarly, these mice equally responded to other chemical insults, including endoplasmic reticulum stress inducer, N-methyl-D-aspartate, and lipopolysaccharide. Interestingly, in cultured neuroblastoma cells, we found that the mammalian target of rapamycin-independent autophagy activators, lithium chloride and rilmenidine, rescued these cells against proteasome inhibition-induced death. These results suggest that Becn1-mediated autophagy is not an effective intrinsic protective mechanism for retinal damage induced by insults, including impaired proteasomal activity; furthermore, autophagic activation beyond normal levels is required to alleviate the cytotoxic effect of proteasomal inhibition. Further studies are underway to delineate the precise roles of different forms of autophagy, and investigate the effects of their activation in rescuing retinal neurons under various pathological conditions.


Assuntos
Autofagia/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Retina/metabolismo , Degeneração Retiniana/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/fisiologia , Humanos , Camundongos , Retina/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
4.
Plant Sci ; 310: 110982, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34315598

RESUMO

The spotted leaf lesion mimic trait simulates cell death in a plant responding to pathogen infection. Some spotted leaf genes are recessive, while others are dominant. A small number of plants with a lesion mimic phenotype appeared in a segregating population obtained by crossing two normal green wheat strains, XN509 and N07216. Here, we clarified the genetic model and its breeding value. Phenotyping of the consecutive progeny populations over six cropping seasons showed that the spotted leaf lesion mimic phenotype was controlled by a dominant gene designated TaSpl1, which was inhibited by two other dominant genes, designated TaSpl1-I1 and TaSpl1-I2. Using bulked segregant analysis RNA-seq (BSR-Seq) and newly developed KASP-PCR markers, the TaSpl1 and TaSpl1-I1 loci in N07216 were mapped to the end of chromosomes 3DS and 3BS, respectively. Plants with the spotted phenotype showed lower levels of stripe rust and powdery mildew than those with the normal green phenotype. Compared with normal leaves, the differentially expressed genes in spotted leaves were significantly enriched in plant-pathogen interaction and endocytosis pathways. There were no differences in the yield parameters of the F5 and F6 sister lines, N13039S with TaSpl1 and N13039 N without TaSpl1. These results provide a greater understanding of spotted leaf phenotyping and the breeding value of the lesion mimic allele in developing disease-resistance varieties.


Assuntos
Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Triticum/metabolismo , Triticum/microbiologia , Morte Celular/genética , Morte Celular/fisiologia , Resistência à Doença/fisiologia , Endocitose/fisiologia
5.
Cells ; 10(7)2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201847

RESUMO

Cell death mechanisms are crucial to maintain an appropriate environment for the functionality of healthy cells. However, during viral infections, dysregulation of these processes can be present and can participate in the pathogenetic mechanisms of the disease. In this review, we describe some features of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and some immunopathogenic mechanisms characterizing the present coronavirus disease (COVID-19). Lymphopenia and monocytopenia are important contributors to COVID-19 immunopathogenesis. The fine mechanisms underlying these phenomena are still unknown, and several hypotheses have been raised, some of which assign a role to cell death as far as the reduction of specific types of immune cells is concerned. Thus, we discuss three major pathways such as apoptosis, necroptosis, and pyroptosis, and suggest that all of them likely occur simultaneously in COVID-19 patients. We describe that SARS-CoV-2 can have both a direct and an indirect role in inducing cell death. Indeed, on the one hand, cell death can be caused by the virus entry into cells, on the other, the excessive concentration of cytokines and chemokines, a process that is known as a COVID-19-related cytokine storm, exerts deleterious effects on circulating immune cells. However, the overall knowledge of these mechanisms is still scarce and further studies are needed to delineate new therapeutic strategies.


Assuntos
COVID-19/patologia , Morte Celular/fisiologia , SARS-CoV-2/patogenicidade , Apoptose/fisiologia , COVID-19/imunologia , COVID-19/virologia , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/patologia , Citocinas/metabolismo , Humanos , Necroptose/fisiologia , Internalização do Vírus
6.
Biomolecules ; 11(6)2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207338

RESUMO

White matter (WM) injury and survival after intracerebral hemorrhage (ICH) has received insufficient attention. WM disruption surrounding the hematoma has been documented in animal models with histology, but rarely in human ICH with noninvasive means, like magnetic resonance imaging (MRI). A few human MRI studies have investigated changes in long WM tracts after ICH remote from the hematoma, like the corticospinal tract, but have not attempted to obtain an unbiased quantification of WM changes within and around the hematoma over time. This study attempts such quantification from 3 to 30 days post ictus. Thirteen patients with mild to moderate ICH underwent diffusion tensor imaging (DTI) MRI at 3, 14, and 30 days. Fractional anisotropy (FA) maps were used to calculate the volume of tissue with FA > 0.5, both within the hematoma (lesion) and in the perilesional tissue. At day 3, the percentages of both lesional and perilesional tissue with an FA > 0.5 were significantly less than contralateral, unaffected, anatomically identical tissue. This perilesional contralateral difference persisted at day 14, but there was no significant difference at day 30. The loss of perilesional tissue with FA > 0.5 increased with increasing hematoma size at day 3 and day 14. All patients had some tissue within the lesion with FA > 0.5 at all time points. This did not decrease with duration after ictus, suggesting the persistence of white matter within the hematoma/lesion. These results outline an approach to quantify WM injury, both within and surrounding the hematoma, after mild to moderate ICH using DTI MRI. This may be important for monitoring treatment strategies, such as hematoma evacuation, and assessing efficacy noninvasively.


Assuntos
Hemorragia Cerebral/diagnóstico por imagem , Substância Branca/diagnóstico por imagem , Substância Branca/lesões , Adulto , Idoso , Idoso de 80 Anos ou mais , Anisotropia , Morte Celular/fisiologia , Hemorragia Cerebral/metabolismo , Hemorragia Cerebral/patologia , Imagem de Tensor de Difusão/métodos , Feminino , Hematoma/diagnóstico por imagem , Hematoma/metabolismo , Hematoma/patologia , Humanos , Imageamento por Ressonância Magnética/métodos , Masculino , Pessoa de Meia-Idade , Substância Branca/patologia
7.
Artigo em Inglês | MEDLINE | ID: mdl-34266626

RESUMO

Nucleotide excision repair (NER) is the main pathway to repair bulky DNA damages including pyrimidine dimers, and the genetic dysregulation of NER associated proteins is well known to cause diseases such as cancer and neurological disorder. Other than the genetic defects, 'external factors' such as oxidative stress and environmental chemicals also affect NER. In this study, we examined the impact of extracellular pH on NER. We prepared the culture media, whose pH values are 8.4 (normal condition), 7.6, 6.6 and 6.2 under atmospheric CO2 conditions. Human keratinocytes, HaCaT, slightly died after 48 h incubation in DMEM at pH 8.4, 7.6 and 6.6, while in pH 6.2 condition, marked cell death was induced. UV-induced pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) and cyclobutane pyrimidine dimers (CPDs), were effectively repaired at 60 min and 24 h, respectively, which were remarkably inhibited at pH 6.6 and 6.2. The associated repair molecule, TFIIH, was accumulated to the damaged sites 5 min after UVC irradiation in all pH conditions, but the release was delayed as the pH got lower. Furthermore, accumulation of XPG at 5 min was delayed at pH 6.2 and 6.6, and the release at 60 min was completely suppressed. At the low pH, the DNA synthesis at the gaps created by incision of oligonucleotides containing pyrimidine dimers was significantly delayed. In this study, we found that the low extracellular pH inhibited NER pathway. This might partially contribute to carcinogenesis in inflamed tissues, which exhibit acidic pH.


Assuntos
Reparo do DNA/genética , Morte Celular/genética , Morte Celular/fisiologia , Células Cultivadas , Dano ao DNA/genética , Dano ao DNA/fisiologia , Replicação do DNA/genética , Replicação do DNA/fisiologia , Fibroblastos/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Queratinócitos/efeitos dos fármacos , Queratinócitos/fisiologia , Dímeros de Pirimidina/genética , Raios Ultravioleta/efeitos adversos
8.
Molecules ; 26(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208178

RESUMO

The heat shock protein (HSP) 70 is considered the main hallmark in preclinical studies to stain the peri-infarct region defined area penumbra in preclinical models of brain ischemia. This protein is also considered as a potential disease modifier, which may improve the outcome of ischemic damage. In fact, the molecule HSP70 acts as a chaperonine being able to impact at several level the homeostasis of neurons. Despite being used routinely to stain area penumbra in light microscopy, the subcellular placement of this protein within area penumbra neurons, to our knowledge, remains undefined. This is key mostly when considering studies aimed at deciphering the functional role of this protein as a determinant of neuronal survival. The general subcellular placement of HSP70 was grossly reported in studies using confocal microscopy, although no direct visualization of this molecule at electron microscopy was carried out. The present study aims to provide a direct evidence of HSP70 within various subcellular compartments. In detail, by using ultrastructural morphometry to quantify HSP70 stoichiometrically detected by immuno-gold within specific organelles we could compare the compartmentalization of the molecule within area penumbra compared with control brain areas. The study indicates that two cell compartments in control conditions own a high density of HSP70, cytosolic vacuoles and mitochondria. In these organelles, HSP70 is present in amount exceeding several-fold the presence in the cytosol. Remarkably, within area penumbra a loss of such a specific polarization is documented. This leads to the depletion of HSP70 from mitochondria and mostly cell vacuoles. Such an effect is expected to lead to significant variations in the ability of HSP70 to exert its physiological roles. The present findings, beyond defining the neuronal compartmentalization of HSP70 within area penumbra may lead to a better comprehension of its beneficial/detrimental role in promoting neuronal survival.


Assuntos
Isquemia Encefálica/metabolismo , Citosol/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Mitocôndrias/metabolismo , Neurônios/metabolismo , Vacúolos/metabolismo , Animais , Isquemia Encefálica/patologia , Morte Celular/fisiologia , Citosol/patologia , Modelos Animais de Doenças , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia Eletrônica de Varredura , Mitocôndrias/patologia , Neurônios/patologia , Vacúolos/patologia
9.
Int J Mol Sci ; 22(10)2021 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067535

RESUMO

We recently discovered an anti-ferroptotic mechanism inherent to M1 macrophages whereby high levels of NO● suppressed ferroptosis via inhibition of hydroperoxy-eicosatetraenoyl-phosphatidylethanolamine (HpETE-PE) production by 15-lipoxygenase (15LOX) complexed with PE-binding protein 1 (PEBP1). However, the mechanism of NO● interference with 15LOX/PEBP1 activity remained unclear. Here, we use a biochemical model of recombinant 15LOX-2 complexed with PEBP1, LC-MS redox lipidomics, and structure-based modeling and simulations to uncover the mechanism through which NO● suppresses ETE-PE oxidation. Our study reveals that O2 and NO● use the same entry pores and channels connecting to 15LOX-2 catalytic site, resulting in a competition for the catalytic site. We identified residues that direct O2 and NO● to the catalytic site, as well as those stabilizing the esterified ETE-PE phospholipid tail. The functional significance of these residues is supported by in silico saturation mutagenesis. We detected nitrosylated PE species in a biochemical system consisting of 15LOX-2/PEBP1 and NO● donor and in RAW264.7 M2 macrophages treated with ferroptosis-inducer RSL3 in the presence of NO●, in further support of the ability of NO● to diffuse to, and react at, the 15LOX-2 catalytic site. The results provide first insights into the molecular mechanism of repression of the ferroptotic Hp-ETE-PE production by NO●.


Assuntos
Ferroptose/fisiologia , Óxido Nítrico/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Araquidonato 15-Lipoxigenase/metabolismo , Morte Celular/fisiologia , Humanos , Lipidômica , Macrófagos/metabolismo , Simulação de Dinâmica Molecular , Oxirredução , Fosfatidiletanolaminas , Fosfolipídeos/metabolismo
10.
Methods Mol Biol ; 2276: 215-225, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060044

RESUMO

Mitochondria play a key role in various modes of cell death. Analysis of mitochondrial dysfunction and the release of proteins from the intermembrane space of mitochondria represent essential tools in cell death investigation. Here we describe how to evaluate release of intermembrane space proteins during apoptosis, alterations in the mitochondrial membrane potential, and oxygen consumption in apoptotic cells.


Assuntos
Membranas Intracelulares/patologia , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/patologia , Morte Celular/fisiologia , Células Cultivadas , Citocromos c/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Mitocôndrias/metabolismo , Consumo de Oxigênio/fisiologia
11.
FASEB J ; 35(7): e21748, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34152016

RESUMO

Although adipose-derived human mesenchymal stem cell (hADSC) transplantation has recently emerged as a promising therapeutic modality for Parkinson's disease (PD), its underlying mechanism of action has not been fully elucidated. This study evaluated the therapeutic effects of stereotaxic injection of hADSCs in the striatum of the 6-OHDA-induced mouse model. Furthermore, an in vitro PD model was constructed using tissue-organized brain slices. The therapeutic effect was also evaluated using a co-culture of the hADSCs and 6-OHDA-treated brain slice. The analysis of hADSC exocrine proteins using RNA-sequencing, human protein cytokine arrays, and label-free quantitative proteomics identified key extracellular factors in the hADSC secretion environment. The degeneration and apoptosis of the dopaminergic neurons were measured in the PD samples in vivo and in vitro, and the beneficial effects were evaluated using quantitative reverse transcription-polymerase chain reaction, western blotting, Fluoro-Jade C, TUNEL assay, and immunofluorescence analysis. This study found that hADSCs protected the dopaminergic neurons in the in vivo and vitro models. We identified Pentraxin 3 (PTX3) as a key extracellular factor in the hADSC secretion environment. Moreover, we found that human recombinant PTX3 (rhPTX3) treatment could rescue the pathophysiological behavior of the PD mice in vivo, prevent dopaminergic neuronal death, and increase neuronal terminals in the ventral tegmental area + substantia nigra pars compacta and striatum in the PD brain slices in vitro. Furthermore, testing of the pro-apoptotic markers in the PD mouse brain following rhPTX3 treatment revealed that rhPTX3 can prevent apoptosis and degeneration of the dopaminergic neurons. This study discovered that PTX3, a hADSC-secreted protein, potentially protected the dopaminergic neurons against apoptosis and degeneration during PD progression and improved motor performance in PD mice, indicating the possible mechanism of action of hADSC replacement therapy for PD. Thus, our study discovered potential translational implications for the development of PTX3-based therapeutics for PD.


Assuntos
Tecido Adiposo/metabolismo , Apoptose/fisiologia , Proteína C-Reativa/metabolismo , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Doença de Parkinson/metabolismo , Componente Amiloide P Sérico/metabolismo , Animais , Morte Celular/fisiologia , Células Cultivadas , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL
12.
FASEB J ; 35(7): e21706, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34160104

RESUMO

Acute kidney injury (AKI) is a devastating condition with high morbidity and mortality rates. The pathological features of AKI are tubular injury, infiltration of inflammatory cells, and impaired vascular integrity. Pyruvate kinase is the final rate-limiting enzyme in the glycolysis pathway. We previously showed that pyruvate kinase M2 (PKM2) plays an important role in regulating the glycolytic reprogramming of fibroblasts in renal interstitial fibrosis. The present study aimed to determine the role of PKM2 in fibroblast activation during the pathogenesis of AKI. We found increased numbers of S100A4 positive cells expressing PKM2 in renal tissues from mice with AKI induced via folic acid or ischemia/reperfusion (I/R). The loss of PKM2 in fibroblasts impaired fibroblast proliferation and promoted tubular epithelial cell death including apoptosis, necroptosis, and ferroptosis. Mechanistically, fibroblasts produced less hepatocyte growth factor (HGF) in response to a loss of PKM2. Moreover, in two AKI mouse models, fibroblast-specific deletion of PKM2 blocked HGF signal activation and aggravated AKI after it was induced in mice via ischemia or folic acid. Fibroblast proliferation mediated by PKM2 elicits pro-survival signals that repress tubular cell death and may help to prevent AKI progression. Fibroblast activation mediated by PKM2 in AKI suggests that targeting PKM2 expression could be a novel strategy for treating AKI.


Assuntos
Injúria Renal Aguda/metabolismo , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Piruvato Quinase/metabolismo , Animais , Apoptose/fisiologia , Morte Celular/fisiologia , Linhagem Celular , Modelos Animais de Doenças , Fibrose/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Necroptose/fisiologia , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/fisiologia
13.
Nat Rev Endocrinol ; 17(8): 497-510, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34135504

RESUMO

The death of endocrine cells is involved in type 1 diabetes mellitus, autoimmunity, adrenopause and hypogonadotropism. Insights from research on basic cell death have revealed that most pathophysiologically important cell death is necrotic in nature, whereas regular metabolism is maintained by apoptosis programmes. Necrosis is defined as cell death by plasma membrane rupture, which allows the release of damage-associated molecular patterns that trigger an immune response referred to as necroinflammation. Regulated necrosis comes in different forms, such as necroptosis, pyroptosis and ferroptosis. In this Perspective, with a focus on the endocrine environment, we introduce these cell death pathways and discuss the specific consequences of regulated necrosis. Given that clinical trials of necrostatins for the treatment of autoimmune conditions have already been initiated, we highlight the therapeutic potential of such novel therapeutic approaches that, in our opinion, should be tested in endocrine disorders in the future.


Assuntos
Doenças do Sistema Endócrino/etiologia , Necrose/fisiopatologia , Animais , Apoptose/fisiologia , Morte Celular/fisiologia , Doenças do Sistema Endócrino/patologia , Doenças do Sistema Endócrino/fisiopatologia , Doenças do Sistema Endócrino/terapia , Humanos , Transdução de Sinais/fisiologia , Terapias em Estudo/métodos , Terapias em Estudo/tendências
15.
Biochim Biophys Acta Mol Cell Res ; 1868(7): 119037, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33839168

RESUMO

Interleukin-6 (IL-6) enhanced TNF-α and TRAIL/Apo2L induced cell death in various human cancer cells derived from malignant glioma, melanoma, breast cancer and leukemia, although the effect was not detected with IL-6 alone. The effects of IL-6 using SKBR3 cells were associated with the generation of apoptotic cells as analyzed by fluorescence microscopy and flow cytometry. IL-6 activated p53 and upregulated TRAIL death receptors (DR-4 and DR-5) and stimulated the TNF-α and TRAIL dependent extrinsic apoptotic pathway without activation of the p53 mediated intrinsic apoptotic pathway. TNF-α and TRAIL induced cleavage of caspase-8 and caspase-3 was more enhanced by IL-6, although these caspases were not cleaved by IL-6 alone. The dead cell generation elicited by the combination with IL-6 was blocked by anti-human TRAIL R2/TNFRSF10B Fc chimera antibody which can neutralize the DR-5 mediated death signal. These findings indicate that IL-6 could contribute to the enhancement of TNF-α or TRAIL induced apoptosis through p53 dependent upregulation of DR-4 and DR-5. The data suggest that a favorable therapeutic interaction could occur between TNF-α or TRAIL and IL-6, and provide an experimental basis for rational clinical treatments in various cancers.


Assuntos
Interleucina-6/metabolismo , Neoplasias/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 3/metabolismo , Caspase 8/metabolismo , Caspases/metabolismo , Morte Celular/fisiologia , Linhagem Celular Tumoral/metabolismo , Humanos , Interleucina-6/fisiologia , Neoplasias/fisiopatologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Morte Celular/metabolismo , Receptores de Morte Celular/fisiologia , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
16.
Nat Commun ; 12(1): 2211, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33850121

RESUMO

Phosphorylation of the MLKL pseudokinase by the RIPK3 kinase leads to MLKL oligomerization, translocation to, and permeabilization of, the plasma membrane to induce necroptotic cell death. The precise choreography of MLKL activation remains incompletely understood. Here, we report Monobodies, synthetic binding proteins, that bind the pseudokinase domain of MLKL within human cells and their crystal structures in complex with the human MLKL pseudokinase domain. While Monobody-32 constitutively binds the MLKL hinge region, Monobody-27 binds MLKL via an epitope that overlaps the RIPK3 binding site and is only exposed after phosphorylated MLKL disengages from RIPK3 following necroptotic stimulation. The crystal structures identified two distinct conformations of the MLKL pseudokinase domain, supporting the idea that a conformational transition accompanies MLKL disengagement from RIPK3. These studies provide further evidence that MLKL undergoes a large conformational change upon activation, and identify MLKL disengagement from RIPK3 as a key regulatory step in the necroptosis pathway.


Assuntos
Morte Celular/fisiologia , Necroptose/fisiologia , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Sítios de Ligação , Membrana Celular , Cristalografia por Raios X , Células HT29 , Humanos , Camundongos , Conformação Molecular , Simulação de Dinâmica Molecular , Mutação , Fosforilação , Conformação Proteica , Proteínas Quinases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteínas Recombinantes , Alinhamento de Sequência , Células U937
19.
PLoS Pathog ; 17(3): e1009432, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33760879

RESUMO

Neuronal damage is a major consequence of bacterial meningitis, but little is known about mechanisms of bacterial interaction with neurons leading to neuronal cell death. Streptococcus pneumoniae (pneumococcus) is a leading cause of bacterial meningitis and many survivors develop neurological sequelae after the acute infection has resolved, possibly due to neuronal damage. Here, we studied mechanisms for pneumococcal interactions with neurons. Using human primary neurons, pull-down experiments and mass spectrometry, we show that pneumococci interact with the cytoskeleton protein ß-actin through the pilus-1 adhesin RrgA and the cytotoxin pneumolysin (Ply), thereby promoting adhesion and invasion of neurons, and neuronal death. Using our bacteremia-derived meningitis mouse model, we observe that RrgA- and Ply-expressing pneumococci co-localize with neuronal ß-actin. Using purified proteins, we show that Ply, through its cholesterol-binding domain 4, interacts with the neuronal plasma membrane, thereby increasing the exposure on the outer surface of ß-actin filaments, leading to more ß-actin binding sites available for RrgA binding, and thus enhanced pneumococcal interactions with neurons. Pneumococcal infection promotes neuronal death possibly due to increased intracellular Ca2+ levels depending on presence of Ply, as well as on actin cytoskeleton disassembly. STED super-resolution microscopy showed disruption of ß-actin filaments in neurons infected with pneumococci expressing RrgA and Ply. Finally, neuronal death caused by pneumococcal infection could be inhibited using antibodies against ß-actin. The generated data potentially helps explaining mechanisms for why pneumococci frequently cause neurological sequelae.


Assuntos
Actinas/metabolismo , Proteínas de Fímbrias/metabolismo , Meningite Pneumocócica/patologia , Neurônios/patologia , Estreptolisinas/metabolismo , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Morte Celular/fisiologia , Humanos , Meningite Pneumocócica/metabolismo , Camundongos , Neurônios/metabolismo
20.
Arthritis Rheumatol ; 73(9): 1683-1693, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33750029

RESUMO

OBJECTIVE: Eosinophils are tissue-dwelling immune cells. Accumulating evidence indicates that a type of cell death termed ETosis is an important cell fate involved in the pathophysiology of inflammatory diseases. Although the critical role of eosinophils in eosinophilic granulomatosis with polyangiitis (EGPA; formerly Churg-Strauss syndrome) is well established, the presence of eosinophil ETosis (EETosis) is poorly understood. We undertook this study to better understand the characteristics of EETosis. METHODS: In vitro studies using blood-derived eosinophils were conducted to characterize EETosis. The occurrence of EETosis in tissues from patients with EGPA was studied by immunostaining and electron microscopy. Serum concentrations of eosinophil-derived proteins in healthy controls, patients with asthma, and EGPA patients with active disease or with disease in remission (n = 15 per group) were examined. RESULTS: EETosis was reliant on reactive oxygen species and peptidylarginine deiminase type 4-dependent histone citrullination, resulting in the cytolytic release of net-like eosinophil extracellular traps, free galectin-10, and membrane-bound intact granules. The signature of EETosis, including loss of cytoplasmic galectin-10 and deposition of granules, was observed in eosinophils infiltrating various tissues from EGPA patients. Serum eosinophil granule proteins and galectin-10 levels were increased in EGPA and positively correlated with disease activity as assessed by the Birmingham Vasculitis Activity Score (r = 0.8531, P < 0.0001 for galectin-10). When normalized to blood eosinophil counts, this correlation remained for galectin-10 (r = 0.7168, P < 0.0001) but not for granule proteins. Galectin-10 levels in active EGPA positively correlated with serum interleukin-5 levels. CONCLUSION: Eosinophils infiltrating diseased tissues in EGPA undergo EETosis. Considering the exclusive expression and large pool of cytoplasmic galectin-10 in eosinophils, elevated serum galectin-10 levels in patients with EGPA might reflect the systemic occurrence of cytolytic EETosis.


Assuntos
Morte Celular/fisiologia , Eosinófilos/metabolismo , Galectinas/sangue , Granulomatose com Poliangiite/metabolismo , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Espécies Reativas de Oxigênio/metabolismo
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