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1.
Phys Rev Lett ; 125(12): 128102, 2020 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-33016724

RESUMO

Neurodegenerative diseases, such as Alzheimer's or Parkinson's disease, show characteristic degradation of structural brain networks. This degradation eventually leads to changes in the network dynamics and degradation of cognitive functions. Here, we model the progression in terms of coupled physical processes: The accumulation of toxic proteins, given by a nonlinear reaction-diffusion transport process, yields an evolving brain connectome characterized by weighted edges on which a neuronal-mass model evolves. The progression of the brain functions can be tested by simulating the resting-state activity on the evolving brain network. We show that while the evolution of edge weights plays a minor role in the overall progression of the disease, dynamic biomarkers predict a transition over a period of 10 years associated with strong cognitive decline.


Assuntos
Demência/patologia , Modelos Neurológicos , Doenças Neurodegenerativas/patologia , Animais , Relógios Biológicos , Encéfalo/patologia , Encéfalo/fisiopatologia , Morte Celular/fisiologia , Disfunção Cognitiva/patologia , Disfunção Cognitiva/fisiopatologia , Conectoma/métodos , Demência/fisiopatologia , Humanos , Camundongos , Doenças Neurodegenerativas/fisiopatologia , Neurônios/patologia
2.
PLoS Comput Biol ; 16(8): e1007961, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32810174

RESUMO

Tumour spheroids are widely used as an in vitro assay for characterising the dynamics and response to treatment of different cancer cell lines. Their popularity is largely due to the reproducible manner in which spheroids grow: the diffusion of nutrients and oxygen from the surrounding culture medium, and their consumption by tumour cells, causes proliferation to be localised at the spheroid boundary. As the spheroid grows, cells at the spheroid centre may become hypoxic and die, forming a necrotic core. The pressure created by the localisation of tumour cell proliferation and death generates an cellular flow of tumour cells from the spheroid rim towards its core. Experiments by Dorie et al. showed that this flow causes inert microspheres to infiltrate into tumour spheroids via advection from the spheroid surface, by adding microbeads to the surface of tumour spheroids and observing the distribution over time. We use an off-lattice hybrid agent-based model to re-assess these experiments and establish the extent to which the spatio-temporal data generated by microspheres can be used to infer kinetic parameters associated with the tumour spheroids that they infiltrate. Variation in these parameters, such as the rate of tumour cell proliferation or sensitivity to hypoxia, can produce spheroids with similar bulk growth dynamics but differing internal compositions (the proportion of the tumour which is proliferating, hypoxic/quiescent and necrotic/nutrient-deficient). We use this model to show that the types of experiment conducted by Dorie et al. could be used to infer spheroid composition and parameters associated with tumour cell lines such as their sensitivity to hypoxia or average rate of proliferation, and note that these observations cannot be conducted within previous continuum models of microbead infiltration into tumour spheroids as they rely on resolving the trajectories of individual microbeads.


Assuntos
Modelos Biológicos , Esferoides Celulares , Células Tumorais Cultivadas , Animais , Fenômenos Biomecânicos , Morte Celular/fisiologia , Hipóxia Celular/fisiologia , Proliferação de Células/fisiologia , Biologia Computacional , Humanos , Esferoides Celulares/citologia , Esferoides Celulares/fisiologia , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/fisiologia
3.
Life Sci ; 258: 118227, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32781074

RESUMO

AIM: eEF1A2 is highly expressed in postmitotic cells and has been reported to interact with the antioxidant enzyme peroxiredoxin 1 (PRDX1). PRDX1 is involved in motor neuron differentiation. Here, we studied the relationship between eEF1A2 and PRDX1 during dopaminergic neuron differentiation, and examined their possible association in an oxidative stress model of Parkinson's disease (PD). MAIN METHODS: Expression of eEF1A2 and PRDX1 in SH-SY5Y cells at various durations of retinoic acid (RA) induction was detected using qRT-PCR, Western blotting and immunofluorescence. Neurons of 10-day differentiation were treated with the PRDX1 inhibitor H7, MPP+ and H7 plus MPP+. The cell viability, the amounts of apoptotic nuclei, DHE signals, and the expression of p53, p-Akt and p-mTOR were determined. The colocalization of eEF1A2 and PRDX1 was visualized using confocal microscopy. KEY FINDINGS: eEF1A2 gradually increased after RA-induced differentiation of SH-SY5Y cells, while PRDX1 protein gradually decreased. MPP+ treatment increased eEF1A2 in both undifferentiated and differentiated neurons; however, PRDX1 appeared to elevate only in mature neurons. The inhibition of the PRDX1 activity with H7 promoted MPP+-induced cell death, as evidenced by decreased cell viability, increased apoptotic nuclei, increased the DHE signal, and increased p53. However, H7 induced the activation of the prosurvival Akt and mTOR in MPP+-treated cells. Besides, a colocalization of eEF1A2 and PRDX1 was evidenced in MPP+-treated neurons. This colocalization was possibly prevented by inhibiting the PRDX1 activity, resulting in aggravated neuronal death. SIGNIFICANCE: Our results suggest that the possible association between eEF1A2 and PRDX1 may be a promising target for modifying neuronal death in PD.


Assuntos
1-Metil-4-fenilpiridínio/toxicidade , Diferenciação Celular/fisiologia , Fator 1 de Elongação de Peptídeos/metabolismo , Peroxirredoxinas/antagonistas & inibidores , Peroxirredoxinas/metabolismo , Antioxidantes/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Herbicidas/toxicidade , Humanos , Fator 1 de Elongação de Peptídeos/análise , Peroxirredoxinas/análise
4.
PLoS Pathog ; 16(7): e1008473, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32649716

RESUMO

Herpesviruses are known to encode a number of inhibitors of host cell death, including RIP Homotypic Interaction Motif (RHIM)-containing proteins. Varicella zoster virus (VZV) is a member of the alphaherpesvirus subfamily and is responsible for causing chickenpox and shingles. We have identified a novel viral RHIM in the VZV capsid triplex protein, open reading frame (ORF) 20, that acts as a host cell death inhibitor. Like the human cellular RHIMs in RIPK1 and RIPK3 that stabilise the necrosome in TNF-induced necroptosis, and the viral RHIM in M45 from murine cytomegalovirus that inhibits cell death, the ORF20 RHIM is capable of forming fibrillar functional amyloid complexes. Notably, the ORF20 RHIM forms hybrid amyloid complexes with human ZBP1, a cytoplasmic sensor of viral nucleic acid. Although VZV can inhibit TNF-induced necroptosis, the ORF20 RHIM does not appear to be responsible for this inhibition. In contrast, the ZBP1 pathway is identified as important for VZV infection. Mutation of the ORF20 RHIM renders the virus incapable of efficient spread in ZBP1-expressing HT-29 cells, an effect which can be reversed by the inhibition of caspases. Therefore we conclude that the VZV ORF20 RHIM is important for preventing ZBP1-driven apoptosis during VZV infection, and propose that it mediates this effect by sequestering ZBP1 into decoy amyloid assemblies.


Assuntos
Morte Celular/fisiologia , Herpesvirus Humano 3/metabolismo , Proteínas de Ligação a RNA/metabolismo , Infecção pelo Vírus da Varicela-Zoster/metabolismo , Proteínas Virais/metabolismo , Animais , Humanos , Camundongos
5.
PLoS Pathog ; 16(7): e1008654, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32673372

RESUMO

Prion protein (PrP) mutations are linked to genetic prion diseases, a class of phenotypically heterogeneous neurodegenerative disorders with invariably fatal outcome. How mutant PrP triggers neurodegeneration is not known. Synaptic dysfunction precedes neuronal loss but it is not clear whether, and through which mechanisms, disruption of synaptic activity ultimately leads to neuronal death. Here we show that mutant PrP impairs the secretory trafficking of AMPA receptors (AMPARs). Specifically, intracellular retention of the GluA2 subunit results in synaptic exposure of GluA2-lacking, calcium-permeable AMPARs, leading to increased calcium permeability and enhanced sensitivity to excitotoxic cell death. Mutant PrPs linked to different genetic prion diseases affect AMPAR trafficking and function in different ways. Our findings identify AMPARs as pathogenic targets in genetic prion diseases, and support the involvement of excitotoxicity in neurodegeneration. They also suggest a mechanistic explanation for how different mutant PrPs may cause distinct disease phenotypes.


Assuntos
Cálcio/metabolismo , Neurônios/metabolismo , Proteínas PrPSc/metabolismo , Receptores de AMPA/metabolismo , Sinapses/metabolismo , Animais , Morte Celular/fisiologia , Camundongos , Permeabilidade , Transporte Proteico/fisiologia
6.
Nat Commun ; 11(1): 3484, 2020 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-32661253

RESUMO

Sustainable developments of nanotechnology necessitate the exploration of structure-activity relationships (SARs) at nano-bio interfaces. While ferroptosis may contribute in the developments of some severe diseases (e.g., Parkinson's disease, stroke and tumors), the cellular pathways and nano-SARs are rarely explored in diseases elicited by nano-sized ferroptosis inducers. Here we find that WS2 and MoS2 nanosheets induce an iron-dependent cell death, ferroptosis in epithelial (BEAS-2B) and macrophage (THP-1) cells, evidenced by the suppression of glutathione peroxidase 4 (GPX4), oxygen radical generation and lipid peroxidation. Notably, nano-SAR analysis of 20 transition metal dichalcogenides (TMDs) disclosures the decisive role of surface vacancy in ferroptosis. We therefore develop methanol and sulfide passivation as safe design approaches for TMD nanosheets. These findings are validated in animal lungs by oropharyngeal aspiration of TMD nanosheets. Overall, our study highlights the key cellular events as well as nano-SARs in TMD-induced ferroptosis, which may facilitate the safe design of nanoproducts.


Assuntos
Morte Celular/fisiologia , Endocitose/fisiologia , Ferroptose/fisiologia , Animais , Biomarcadores/metabolismo , Western Blotting , Morte Celular/genética , Linhagem Celular , Sobrevivência Celular/fisiologia , Feminino , Humanos , Inflamação/metabolismo , Camundongos Endogâmicos C57BL , Microscopia Confocal , Nanotecnologia , Relação Estrutura-Atividade , Células THP-1
7.
Proc Natl Acad Sci U S A ; 117(31): 18661-18669, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32675242

RESUMO

Huntington's disease (HD) is a progressive incurable neurodegenerative disorder characterized by motor and neuropsychiatric symptoms. It is caused by expansion of a cytosine-adenine-guanine triplet in the N-terminal domain of exon 1 in the huntingtin (HTT) gene that codes for an expanded polyglutamine stretch in the protein product which becomes aggregation prone. The mutant Htt (mHtt) aggregates are associated with components of the ubiquitin-proteasome system, suggesting that mHtt is marked for proteasomal degradation and that, for reasons still debated, are not properly degraded. We used a novel HD rat model, proteomic analysis, and long-term live neuronal imaging to characterize the effects of ubiquitination on aggregation of mHtt and subsequent cellular responses. We identified two lysine residues, 6 and 9, in the first exon of mHtt that are specifically ubiquitinated in striatal and cortical brain tissues of mHtt-transgenic animals. Expression of mHtt exon 1 lacking these ubiquitination sites in cortical neurons and cultured cells was found to slow aggregate appearance rates and reduce their size but at the same time increase the number of much smaller and less visible ones. Importantly, expression of this form of mHtt was associated with elevated death rates. Proteomic analysis indicated that cellular reactions to mHtt expression were weaker in cells expressing the lysineless protein, possibly implying a reduced capacity to cope with the proteotoxic stress. Taken together, the findings suggest a novel role for ubiquitination-attenuation of the pathogenic effect of mHtt.


Assuntos
Proteína Huntingtina , Doença de Huntington , Ubiquitinação/fisiologia , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Morte Celular/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Lisina/química , Lisina/metabolismo , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma , Agregação Patológica de Proteínas/metabolismo , Ratos , Ratos Transgênicos
8.
Mol Cell ; 78(6): 1045-1054, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32516599

RESUMO

Cell death, or, more specifically, cell suicide, is a process of fundamental importance to human health. Throughout our lives, over a million cells are produced every second. When organismal growth has stopped, to balance cell division, a similar number of cells must be removed. This is achieved by activation of molecular mechanisms that have evolved so that cells can destroy themselves. The first clues regarding the nature of one of these mechanisms came from studying genes associated with cancer, in particular the gene for BCL-2. Subsequent studies revealed that mutations or other defects that inhibit cell death allow cells to accumulate, prevent removal of cells with damaged DNA, and increase the resistance of malignant cells to chemotherapy. Knowledge of this mechanism has allowed development of drugs that kill cancer cells by directly activating the cell death machinery and by synergizing with conventional chemotherapy as well as targeted agents to achieve improved outcomes for cancer patients.


Assuntos
Morte Celular/fisiologia , Neoplasias/genética , Neoplasias/terapia , Apoptose/genética , Morte Celular/genética , Dano ao DNA/genética , Humanos , Neoplasias/fisiopatologia
10.
Chem Biol Interact ; 324: 109090, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32283070

RESUMO

Epidermal growth factor receptor (EGFR) is conventionally known to play a crucial role in hepatocyte proliferation, liver regeneration and is also associated with hepatocellular carcinogenesis. In addition to these proliferative roles, EGFR has also implicated in apoptotic cell death signaling in various hepatic cells, mitochondrial dysfunction and acute liver necrosis in a clinically relevant murine model of acetaminophen overdose, warranting further comprehensive exploration of this paradoxical role of EGFR in hepatotoxicity. Apart from ligand dependent activation, EGFR can also be activated in ligand-independent manner, which is mainly associated to liver injury. Recent evidence has also emerged demonstrating important role of EGFR in lipid and fatty acid metabolism in quiescent and regenerating liver. Based on these findings, EGFR has also been shown to play an important role in steatosis in clinically relevant murine NAFLD models via regulating master transcription factors governing fatty acid synthesis and lipolysis. Moreover, several lines of evidences indicate that EGFR is also involved in hepatocellular injury, oxidative stress, inflammation, direct stellate cell activation and fibrosis in chronic liver injury models, including repeated CCl4 exposure, high-fat diet and fast-food diet models. In addition to briefly summarizing role of EGFR in liver regeneration, this review comprehensively discusses all these non-conventional emerging roles of EGFR. Considering evidences of multi-facet role of EGFR at various levels in these pathophysiological process, EGFR can be a promising therapeutic target for various liver diseases, including acute liver failure and NAFLD, requiring further exploration. These roles of EGFR are relevant for alcoholic liver diseases (ALD) as well, thus providing a valid rationale for future investigations exploring a role of EGFR in ALD.


Assuntos
Receptores ErbB/fisiologia , Metabolismo dos Lipídeos/fisiologia , Hepatopatias/fisiopatologia , Animais , Morte Celular/fisiologia , Células Estreladas do Fígado/fisiologia , Humanos , Regeneração Hepática/fisiologia
11.
Nat Commun ; 11(1): 1775, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286299

RESUMO

The increased incidence of inflammatory bowel disease (IBD) has become a global phenomenon that could be related to adoption of a Western life-style. Westernization of dietary habits is partly characterized by enrichment with the ω-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA), which entails risk for developing IBD. Glutathione peroxidase 4 (GPX4) protects against lipid peroxidation (LPO) and cell death termed ferroptosis. We report that small intestinal epithelial cells (IECs) in Crohn's disease (CD) exhibit impaired GPX4 activity and signs of LPO. PUFAs and specifically AA trigger a cytokine response of IECs which is restricted by GPX4. While GPX4 does not control AA metabolism, cytokine production is governed by similar mechanisms as ferroptosis. A PUFA-enriched Western diet triggers focal granuloma-like neutrophilic enteritis in mice that lack one allele of Gpx4 in IECs. Our study identifies dietary PUFAs as a trigger of GPX4-restricted mucosal inflammation phenocopying aspects of human CD.


Assuntos
Doença de Crohn/metabolismo , Gorduras na Dieta/efeitos adversos , Enterite/metabolismo , Ácidos Graxos Insaturados/metabolismo , Inflamação/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Adulto , Animais , Morte Celular/genética , Morte Celular/fisiologia , Doença de Crohn/genética , Enterite/etiologia , Enterite/genética , Ácidos Graxos Insaturados/genética , Feminino , Glutationa Peroxidase/metabolismo , Humanos , Inflamação/genética , Peroxidação de Lipídeos/genética , Peroxidação de Lipídeos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética
12.
Artigo em Inglês | MEDLINE | ID: mdl-32272354

RESUMO

Calcium is an essential element for plants' survival and ability to deal with environmental stresses. However, it can cause cell death due to cellular disequilibrium. Serpentine plants are sensitive to high concentrations of Ca2+, which induces lethal symptoms, especially under environmental stress. In this study, the direct effects of Ca2+ on cell death were investigated in cell cultures of Alyssum inflatum, a serpentine plant native to Western Iran, and results were compared to a non-serpentinitic congeneric species A. saxatile. The results were also compared to the effects of Mg2+ treatments in both species, as another determinative factor in serpentinite soil is high Mg2+ content. Plasma membrane permeability, reactive oxygen species (ROS), and malondialdehyde (MDA) production were measured as physiological cell injury indices. In A. inflatum higher levels of ROS and MDA were observed in Ca2+-treated cells (5 mM or more), while in A. saxatile they were measured in Mg2+-treated cells (5 mM or more). In serpentine species, results indicated that cell death by Ca2+ was more intensive than the cell death by Mg2+, which were observed with less intensity in non-serpentine plants. Microscopic studies showed that cell death occurred via apoptosis-like programmed cell death (AL-PCD). Therefore, Ca2+ sensitivity and AL-PCD as mechanistic reasons for their non-serpentine intolerance would be a crucial consideration in cellular researches concerning serpentine plants, which could be employed in green technologies such as phytoremediation.


Assuntos
Brassicaceae , Cálcio , Morte Celular , Técnicas de Cultura , Magnésio , Brassicaceae/metabolismo , Cálcio/metabolismo , Morte Celular/fisiologia , Irã (Geográfico) , Magnésio/metabolismo , Solo/química
13.
Anticancer Res ; 40(3): 1387-1394, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32132035

RESUMO

BACKGROUND/AIM: Cancer cells are frequently exposed to microenvironmental stresses, including amino acid deprivation and hypoxia, which are often targeted for cancer therapy. Here, we examined the effect of hypoxia in cysteine-deprived breast cancer cells and the mechanism to counteract the hypoxia effect. MATERIALS AND METHODS: Cell death was determined by annexin V-FITC and propidium iodide staining. Expression of mRNAs and proteins was determined by reverse transcription polymerase chain reaction and western blot analysis, respectively. RESULTS: Cysteine deprivation or sulfasalazine, a potent inhibitor of cysteine/glutamate transporter, induced cell death by activating transcription factor 4 (ATF4) up-regulation. Hypoxia significantly suppressed cell death and ATF4 up-regulation induced by cysteine deprived conditions. In addition, tumor necrosis factor-related apoptosis-inducing ligand reversed the effect of hypoxia on cysteine deprived conditions. CONCLUSION: Prevention of hypoxia may be a means for augmenting the effect of amino acid deprivation as a strategy for cancer therapy.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Neoplasias da Mama/metabolismo , Cisteína/deficiência , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Morte Celular/fisiologia , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Cisteína/antagonistas & inibidores , Cisteína/metabolismo , Feminino , Humanos , Sulfassalazina/farmacologia , Transfecção , Regulação para Cima
14.
PLoS One ; 15(3): e0229017, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32130239

RESUMO

We have earlier reported that cell-free chromatin (cfCh) particles that are released from dying cells, or those that circulate blood, can readily enter into healthy cells, illegitimately integrate into their genomes and induce dsDNA breaks, apoptosis and intense activation of inflammatory cytokines. We hypothesized that sepsis is caused by cfCh released from dying host cells following microbial infection leading to bystander host cell apoptosis and inflammation which are perpetuated in a vicious cycle with release of more cfCh from dying host cells. To test this hypothesis we used three cfCh inactivating agents namely 1) anti-histone antibody complexed nanoparticles which inactivate cfCh by binding to histones; 2) DNase I which inactivates cfCh by degrading its DNA component, and 3) a novel pro-oxidant combination of Resveratrol and Copper which, like DNase I, inactivates cfCh by degrading its DNA component. Female C57 BL/6 mice, 6-8 weeks old, were administered a single i.p. injection of LPS at a dose of 10 mg/Kg or 20 mg/Kg with or without concurrent treatment with the above cfCh inactivating agents. Administration of cfCh inactivating agents concurrently with LPS resulted in prevention of following pathological parameters: 1) release of cfCh in extra-cellular spaces of brain, lung and heart and in circulation; 2) release of inflammatory cytokines in circulation; 3) activation of DNA damage, apoptosis and inflammation in cells of thymus, spleen and in PBMCs; 4) DNA damage, apoptosis and inflammation in cells of lung, liver, heart, brain, kidney and small intestine; 5) liver and kidney dysfunction and elevation of serum lactate; 6) coagulopathy, fibrinolysis and thrombocytopenia; 7) lethality. We conclude that cfCh that are released from dying host cells in response to bacterial endotoxin represents a global instigator of sepsis. cfCh inactivation may provide a novel approach to management of sepsis in humans.


Assuntos
Morte Celular , Ácidos Nucleicos Livres/metabolismo , Cromatina/metabolismo , Endotoxinas , Sepse/metabolismo , Sepse/patologia , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Cromatina/patologia , Cromatina/fisiologia , Cobre/administração & dosagem , Citocinas/metabolismo , Dano ao DNA/efeitos dos fármacos , Desoxirribonuclease I/metabolismo , Desoxirribonuclease I/uso terapêutico , Feminino , Histonas/metabolismo , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/uso terapêutico , Resveratrol/administração & dosagem , Sepse/induzido quimicamente , Sepse/prevenção & controle
15.
J Neurosci ; 40(16): 3142-3151, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32152200

RESUMO

Prohibitin (PHB) is a critical protein involved in many cellular activities. In brain, PHB resides in mitochondria, where it forms a large protein complex with PHB2 in the inner TFmembrane, which serves as a scaffolding platform for proteins involved in mitochondrial structural and functional integrity. PHB overexpression at moderate levels provides neuroprotection in experimental brain injury models. In addition, PHB expression is involved in ischemic preconditioning, as its expression is enhanced in preconditioning paradigms. However, the mechanisms of PHB functional regulation are still unknown. Observations that nitric oxide (NO) plays a key role in ischemia preconditioning compelled us to postulate that the neuroprotective effect of PHB could be regulated by NO. Here, we test this hypothesis in a neuronal model of ischemia-reperfusion injury and show that NO and PHB are mutually required for neuronal resilience against oxygen and glucose deprivation stress. Further, we demonstrate that NO post-translationally modifies PHB through protein S-nitrosylation and regulates PHB neuroprotective function, in a nitric oxide synthase-dependent manner. These results uncover the mechanisms of a previously unrecognized form of molecular regulation of PHB that underlies its neuroprotective function.SIGNIFICANCE STATEMENT Prohibitin (PHB) is a critical mitochondrial protein that exerts a potent neuroprotective effect when mildly upregulated in mice. However, how the neuroprotective function of PHB is regulated is still unknown. Here, we demonstrate a novel regulatory mechanism for PHB that involves nitric oxide (NO) and shows that PHB and NO interact directly, resulting in protein S-nitrosylation on residue Cys69 of PHB. We further show that nitrosylation of PHB may be essential for its ability to preserve neuronal viability under hypoxic stress. Thus, our study reveals a previously unknown mechanism of functional regulation of PHB that has potential therapeutic implications for neurologic disorders.


Assuntos
Neurônios/metabolismo , Neuroproteção/fisiologia , Óxido Nítrico/metabolismo , Traumatismo por Reperfusão/metabolismo , Proteínas Repressoras/metabolismo , Animais , Morte Celular/fisiologia , Células Cultivadas , GMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Camundongos , NG-Nitroarginina Metil Éster/farmacologia , Neurônios/efeitos dos fármacos , Neuroproteção/efeitos dos fármacos , Óxido Nítrico Sintase/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
16.
J Med Chem ; 63(8): 3976-3995, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32078308

RESUMO

ERK1 and ERK5 are proposed to have pivotal roles in several types of cancer. Under some circumstance, ERK5 may provide a common bypass route, which rescues proliferation upon abrogation of ERK1 signaling. Thus, we accurately classified the tumor types from The Cancer Genome Atlas (TCGA) based on the expression levels of ERK1 and ERK5. We proposed a novel therapeutic strategy to overcome the above-mentioned compensatory mechanism in specific tumor types by co-targeting both ERK1 and ERK5. On the basis of the idea of overcoming ERK5 compensation mechanism, 22ac (ADTL-EI1712) as the first selective dual-target inhibitor of ERK1 and ERK5 was discovered to have potent antitumor effects in vitro and in vivo. Interestingly, this compound was found to induce regulated cell death accompanied by autophagy in MKN-74 cells. Taken together, our results warrant the potential of this dual-target inhibitor as a new candidate drug that conquers compensatory mechanism in certain tumor types.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Descoberta de Drogas/métodos , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Sequência de Aminoácidos , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Feminino , Células HL-60 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Simulação de Acoplamento Molecular/métodos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
17.
Cancer Sci ; 111(3): 807-816, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31908105

RESUMO

Activation-induced cell death (AICD) mediated by the Fas/Fas ligand (FasL) system plays a key role in regulating immune response. Although normal natural killer (NK) cells use this system for their homeostasis, malignant NK cells seem to disrupt the process. Extranodal NK/T-cell lymphoma, nasal type (ENKL) is a rare but fatal disease, for which novel therapeutic targets need to be identified. We confirmed that ENKL-derived NK cell lines NK-YS and Hank1, and primary lymphoma cells expressed procaspase-8/FADD-like interleukin-1ß-converting enzyme (FLICE) modulator and cellular FLICE-inhibitory protein (c-FLIP), along with Fas and FasL. Compared with Fas-sensitive Jurkat cells, NK-YS and Hank1 showed resistance to Fas-mediated apoptosis in spite of the same expression levels of c-FLIP and the death-inducing signaling complex (DISC) formation. Unexpectedly, the long isoform of c-FLIP (c-FLIPL ) was coimmunoprecipitated with Fas predominantly in both ENKL-derived NK cell lines after Fas ligation. Indeed, c-FLIPL was more sufficiently recruited to the DISC in both ENKL-derived NK cell lines than in Jurkat cells after Fas ligation. Knockdown of c-FLIPL per se enhanced autonomous cell death and restored the sensitivity to Fas in both NK-YS and Hank1 cells. Although ENKL cells are primed for AICD, they constitutively express and efficiently utilize c-FLIPL , which prevents their Fas-mediated apoptosis. Our results show that c-FLIPL could be a promising therapeutic target against ENKL.


Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Células Matadoras Naturais/metabolismo , Linfoma/metabolismo , Receptor fas/metabolismo , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 8/metabolismo , Caspases/metabolismo , Morte Celular/fisiologia , Proteína Ligante Fas , Proteína de Domínio de Morte Associada a Fas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Jurkat , Isoformas de Proteínas/metabolismo , Transdução de Sinais/fisiologia
18.
PLoS One ; 15(1): e0227950, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31978129

RESUMO

Trypan blue has long been the gold standard for staining dead cell to determine cell viability. The dye is excluded from membrane-intact live cells, but can enter and concentrate in membrane-compromised dead cells, rendering the cells dark blue. Over the years, there has been an understanding that trypan blue is inaccurate for cell viability under 80% without scientific support. We previously showed that trypan blue can alter the morphology of dead cells to a diffuse shape, which can lead to over-estimation of viability. Here, we investigate the origin of the dim and diffuse objects after trypan blue staining. Utilizing image and video acquisition, we show real-time transformation of cells into diffuse objects when stained with trypan blue. The same phenomenon was not observed when staining cells with propidium iodide. We also demonstrate the co-localization of trypan blue and propidium iodide, confirming these diffuse objects as cells that contain nuclei. The videos clearly show immediate cell rupturing after trypan blue contact. The formation of these diffuse objects was monitored and counted over time as cells die outside of the incubator. We hypothesize and demonstrate that rapid water influx may have caused the cells to rupture and disappear. Since some dead cells disappear after trypan blue staining, the total can be under-counted, leading to over-estimation of cell viability. This inaccuracy could affect the outcomes of cellular therapies, which require accurate measurements of immune cells that will be infused back into patients.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Corantes/farmacologia , Células Epiteliais/efeitos dos fármacos , Azul Tripano/farmacologia , Morte Celular/fisiologia , Rastreamento de Células/métodos , Células Epiteliais/ultraestrutura , Humanos , Iodetos/farmacologia , Células Jurkat , Imagem Óptica , Coloração e Rotulagem/métodos
19.
FASEB J ; 34(1): 1665-1678, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914676

RESUMO

Bacterial infectious diseases can lead to death or to serious illnesses. These outcomes are partly the consequence of pore-forming toxins, which are secreted by the pathogenic bacteria (eg, pneumolysin of Streptococcus pneumoniae). Pneumolysin binds to cholesterol within the plasma membrane of host cells and assembles to form trans-membrane pores, which can lead to Ca2+ influx and cell death. Membrane repair mechanisms exist that limit the extent of damage. Immune cells which are essential to fight bacterial infections critically rely on survival mechanisms after detrimental pneumolysin attacks. This study investigated the susceptibility of different immune cell types to pneumolysin. As a model system, we used the lymphoid T-cell line Jurkat, and myeloid cell lines U937 and THP-1. We show that Jurkat T cells are highly susceptible to pneumolysin attack. In contrast, myeloid THP-1 and U937 cells are less susceptible to pneumolysin. In line with these findings, human primary T cells are shown to be more susceptible to pneumolysin attack than monocytes. Differences in susceptibility to pneumolysin are due to (I) preferential binding of pneumolysin to Jurkat T cells and (II) cell type specific plasma membrane repair capacity. Myeloid cell survival is mostly dependent on Ca2+ induced expelling of damaged plasma membrane areas as microvesicles. Thus, in myeloid cells, first-line defense cells in bacterial infections, a potent cellular repair machinery ensures cell survival after pneumolysin attack. In lymphoid cells, which are important at later stages of infections, less efficient repair mechanisms and enhanced toxin binding renders the cells more sensitive to pneumolysin.


Assuntos
Toxinas Bacterianas/metabolismo , Estruturas da Membrana Celular/metabolismo , Estruturas da Membrana Celular/patologia , Membrana Celular/metabolismo , Membrana Celular/patologia , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/patologia , Cálcio/metabolismo , Morte Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Humanos , Células Jurkat , Monócitos/metabolismo , Monócitos/patologia , Células Mieloides/metabolismo , Células Mieloides/patologia , Streptococcus pneumoniae/patogenicidade , Células THP-1 , Células U937
20.
Artigo em Inglês | MEDLINE | ID: mdl-31918353

RESUMO

Infection with Cowpea severe mosaic virus (CPSMV) represents one of the main limitations for cowpea (Vigna unguiculata L. Walp.) productivity due to the severity of the disease symptoms, frequency of incidence, and difficulties in dissemination control. This study aimed to identify the proteins and metabolic pathways associated with the susceptibility and resistance of cowpea plants to CPSMV. Therefore, we treated the seeds of a naturally susceptible cowpea genotype (CE-31) with the mutagenic agent ethyl methane sulfonate (EMS) and compared the secondary leaf proteomic profile of the mutagenized resistant plants inoculated with CPSMV (MCPI plant group) to those of the naturally susceptible cowpea genotype CE-31 inoculated (CPI) and noninoculated (CPU) with CPSMV. MCPI responded to CPSMV by accumulating proteins involved in the oxidative burst, increasing H2O2 generation, promoting leaf cell death (LCD), increasing the synthesis of defense proteins, and decreasing host factors important for the establishment of CPSMV infection. In contrast, CPI accumulated several host factors that favor CPSMV infection and did not accumulate H2O2 or present LCD, which allowed CPSMV replication and systemic dissemination. Based on these results, we propose that the differential abundance of defense proteins and proteins involved in the oxidative burst, LCD, and the decrease in cowpea protein factors required for CPSMV replication are associated with the resistance trait acquired by the MCPI plant group.


Assuntos
Comovirus/fisiologia , Resistência à Doença , Peróxido de Hidrogênio/metabolismo , Mutagênese , Folhas de Planta/virologia , Vigna/metabolismo , Vigna/virologia , Morte Celular/genética , Morte Celular/fisiologia , Resistência à Doença/genética , Resistência à Doença/fisiologia , Metanossulfonato de Etila/química , Metanossulfonato de Etila/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/fisiologia , Ontologia Genética , Homeostase/efeitos dos fármacos , Homeostase/genética , Homeostase/fisiologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Mutagênicos/química , Mutagênicos/farmacologia , Oxirredução/efeitos dos fármacos , Fotossíntese/efeitos dos fármacos , Fotossíntese/genética , Doenças das Plantas/genética , Doenças das Plantas/virologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Folhas de Planta/fisiologia , Proteínas de Plantas/efeitos dos fármacos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Mapas de Interação de Proteínas , Proteoma/efeitos dos fármacos , Proteoma/genética , Proteoma/metabolismo , Proteoma/fisiologia , Vigna/genética , Vigna/fisiologia , Replicação Viral
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