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1.
ACS Appl Mater Interfaces ; 13(33): 38959-38968, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34379404

RESUMO

Chemotherapy continues to be the most commonly applied strategy for cancer. Despite the impressive clinical success obtained with several drugs, increasing numbers of (multi)drug-resistant tumors are reported. To overcome this shortcoming, novel drug candidates and delivery systems are urgently needed. Herein, a therapeutic copper polypyridine complex encapsulated in natural nanocarrier apoferritin is reported. The generated nanoparticles showed higher cytotoxicity toward various (drug-resistant) cancer cell lines than noncancerous cells. The study of the mechanism revealed that the compound triggers cell autophagy-dependent apoptosis. Promisingly, upon injection of the nanodrug conjugate into the bloodstream of a mouse model bearing a multidrug-resistant colon tumor, a strong tumor growth inhibition effect was observed. To date, this is the first study describing the encapsulation of a copper complex in apoferritin that acts by autophagy-dependent apoptosis.


Assuntos
Antineoplásicos/química , Apoferritinas/química , Neoplasias do Colo/tratamento farmacológico , Complexos de Coordenação/química , Cobre/química , Nanocápsulas/química , Animais , Antineoplásicos/farmacologia , Apoferritinas/metabolismo , Morte Celular Autofágica/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Complexos de Coordenação/farmacologia , Composição de Medicamentos , Liberação Controlada de Fármacos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Camundongos Endogâmicos BALB C , Neoplasias Experimentais
2.
Phys Chem Chem Phys ; 23(32): 17158-17165, 2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34318824

RESUMO

Due to its unique structure, recent years have witnessed the use of apo-ferritin to accumulate various non-natural metal ions as a scaffold for nanomaterial synthesis. However, the transport mechanism of metal ions into the cavity of apo-ferritin is still unclear, limiting the rational design and controllable preparation of nanomaterials. Here, we conducted all-atom classical molecular dynamics (MD) simulations combined with Markov state models (MSMs) to explore the transportation behavior of Au(iii) ions. We exhibited the complete transportation paths of Au(iii) from solution into the apo-ferritin cage at the atomic level. We also revealed that the transportation of Au(iii) ions is accompanied by coupled protein structural changes. It is shown that the 3-fold axis channel serves as the only entrance with the longest residence time of Au(iii) ions. Besides, there are eight binding clusters and five 3-fold structural metastable states, which are important during Au(iii) transportation. The conformational changes of His118, Asp127, and Glu130, acting as doors, were observed to highly correlate with the Au(iii) ion's position. The MSM analysis and Potential Mean Force (PMF) calculation suggest a remarkable energy barrier near Glu130, making it the rate-limiting step of the whole process. The dominant transportation pathway is from cluster 3 in the 3-fold channel to the inner cavity to cluster 5 on the inner surface, and then to cluster 6. These findings provide inspiration and theoretical guidance for the further rational design and preparation of new nanomaterials using apo-ferritin.


Assuntos
Apoferritinas/metabolismo , Ouro/metabolismo , Cadeias de Markov , Simulação de Dinâmica Molecular/estatística & dados numéricos , Animais , Apoferritinas/química , Sítios de Ligação , Ouro/química , Cavalos , Ligação de Hidrogênio , Ligação Proteica , Conformação Proteica , Eletricidade Estática
3.
Biomolecules ; 11(3)2021 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-33670982

RESUMO

BACKGROUND: Currently, no blood biomarkers exist that can diagnose unstable angina (UA) patients. Nourin is an early inflammatory mediator rapidly released within 5 min by reversible ischemic myocardium, and if ischemia persists, it is also released by necrosis. Nourin is elevated in acute coronary syndrome (ACS) patients but not in symptomatic noncardiac and healthy subjects. Recently, circulating microRNAs (miRNAs) have been established as markers of disease, including cardiac injury and inflammation. OBJECTIVES: To profile and validate the potential diagnostic value of Nourin-dependent miR-137 (marker of cell damage) and miR-106b-5p (marker of inflammation) as early biomarkers in suspected UA patients and to investigate the association of their target and regulating genes. METHODS: Using Nourin amino acid sequence, an integrated bioinformatics analysis was conducted. Analysis indicated that Nourin is a direct target for miR-137 and miR-106b-5p in myocardial ischemic injury. Two linked molecular networks of lncRNA/miRNAs/mRNAs were also retrieved, including CTB89H12.4/miR-137/FTHL-17 and CTB89H12.4/miR-106b-5p/ANAPC11. Gene expression profiling was assessed in serum samples collected at presentation to an emergency department (ED) from: (1) UA patients (n = 30) (confirmed by invasive coronary angiography with stenosis greater than 50% and troponin level below the clinical decision limit); (2) patients with acute ST elevation myocardial infarction (STEMI) (n = 16) (confirmed by persistent ST-segment changes and elevated troponin level); and 3) healthy subjects (n = 16). RESULTS: Gene expression profiles showed that miR-137 and miR-106b-5p were significantly upregulated by 1382-fold and 192-fold in UA compared to healthy, and by 2.5-fold and 4.6-fold in STEMI compared to UA, respectively. Healthy subjects showed minimal expression profile. Receiver operator characteristics (ROC) analysis revealed that the two miRNAs were sensitive and specific biomarkers for assessment of UA and STEMI patients. Additionally, Spearman's correlation analysis revealed a significant association of miRNAs with the associated mRNA targets and the regulating lncRNA. CONCLUSIONS: Nourin-dependent gene expression of miR-137 and miR-106b-5p are novel blood-based biomarkers that can diagnose UA and STEMI patients at presentation and stratify severity of myocardial ischemia, with higher expression in STEMI compared to UA. Early diagnosis of suspected UA patients using the novel Nourin biomarkers is key for initiating guideline-based therapy that improves patients' health outcomes.


Assuntos
Angina Instável/diagnóstico , Angina Instável/genética , Diagnóstico Precoce , MicroRNAs/metabolismo , Síndrome Coronariana Aguda/genética , Apoferritinas/genética , Apoferritinas/metabolismo , Estudos de Casos e Controles , Feminino , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Curva ROC
4.
Int J Mol Med ; 47(4)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33649797

RESUMO

Parkinson's disease (PD) is a neurodegenerative disease characterized by the selective loss of dopaminergic neurons in the substantia nigra (SN). In a previous study, the authors demonstrated that ferritin heavy chain 1 (FTH1) inhibited ferroptosis in a model of 6­hydroxydopamine (6­OHDA)­induced PD. However, whether and how microRNAs (miRNAs/miRs) modulate FTH1 in PD ferroptosis is not yet well understood. In the present study, in vivo and in vitro models of PD induced by 6­OHDA were established. The results in vivo and in vitro revealed that the levels of the ferroptosis marker protein, glutathione peroxidase 4 (GPX4), and the PD marker protein, tyrosine hydroxylase (TH), were decreased in the model group, associated with a decreased FTH1 expression and the upregulation of miR­335. In both the in vivo and in vitro models, miR­335 mimic led to a lower FTH1 expression, exacerbated ferroptosis and an enhanced PD pathology. The luciferase 3'­untranslated region reporter results identified FTH1 as the direct target of miR­335. The silencing of FTH1 in 6­OHDA­stimulated cells enhanced the effects of miR­335 on ferroptosis and promoted PD pathology. Mechanistically, miR­335 enhanced ferroptosis through the degradation of FTH1 to increase iron release, lipid peroxidation and reactive oxygen species (ROS) accumulation, and to decrease mitochondrial membrane potential (MMP). On the whole, the findings of the present study reveal that miR­335 promotes ferroptosis by targeting FTH1 in in vitro and in vivo models of PD, providing a potential therapeutic target for the treatment of PD.


Assuntos
Apoferritinas/metabolismo , Ferroptose/genética , MicroRNAs/genética , Doença de Parkinson/patologia , Animais , Modelos Animais de Doenças , Ferro/metabolismo , Peroxidação de Lipídeos/fisiologia , Masculino , Potencial da Membrana Mitocondrial/fisiologia , Oxidopamina/toxicidade , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/análise , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Tirosina 3-Mono-Oxigenase/análise
5.
Biochem Biophys Res Commun ; 553: 114-118, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33765555

RESUMO

Major depressive disorder (MDD) is a debilitating mental illness that can cause significant emotional disturbances and severe socioeconomic burdens. Rodent and nonhuman primate-based depression models have been studied, such as brain-derived neurotrophic factor (BDNF) and monoamine acid disorder hypotheses, as well as peripheral microbiota disturbances causing MDD; however, the pathogenesis is still largely unknown. This study aims to explore the relationship between ferritin and MDD. First, alterations in ferritin, including ferritin light chain (FTL) and ferritin heavy chain (FTH), in MDD patient plasma compared with healthy control (HC) plasma were detected using ELISA. Then, serum ferritin expression in cLPS-depressed mice was measured by ELISA. The existence of FTH in the hippocampus was validated by immunofluorescence, and the change in FTH levels in the hippocampus of mice injected with cLPS was detected by western blotting. FTL levels in MDD patients were decreased compared with those in HCs. In cLPS-depressed mice, serum ferritin was not different from that in the control group, while the expression of FTH in the hippocampus was significantly reduced in depressed mice. Our findings demonstrate the alteration of ferritin expression in MDD and provide new insight into the pathogenesis of MDD.


Assuntos
Apoferritinas/sangue , Apoferritinas/metabolismo , Transtorno Depressivo Maior/sangue , Transtorno Depressivo Maior/metabolismo , Hipocampo/metabolismo , Animais , Apoferritinas/deficiência , Apoferritinas/genética , Biomarcadores/sangue , Biomarcadores/metabolismo , Estudos de Casos e Controles , Transtorno Depressivo Maior/genética , Transtorno Depressivo Maior/patologia , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Humanos , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Adulto Jovem
6.
Cell Mol Life Sci ; 78(7): 3355-3367, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33439270

RESUMO

Neuroferritinopathy is a rare autosomal dominant inherited movement disorder caused by alteration of the L-ferritin gene that results in the production of a ferritin molecule that is unable to properly manage iron, leading to the presence of free redox-active iron in the cytosol. This form of iron has detrimental effects on cells, particularly severe for neuronal cells, which are highly sensitive to oxidative stress. Although very rare, the disorder is notable for two reasons. First, neuroferritinopathy displays features also found in a larger group of disorders named Neurodegeneration with Brain Iron Accumulation (NBIA), such as iron deposition in the basal ganglia and extrapyramidal symptoms; thus, the elucidation of its pathogenic mechanism may contribute to clarifying the incompletely understood aspects of NBIA. Second, neuroferritinopathy shows the characteristic signs of an accelerated process of aging; thus, it can be considered an interesting model to study the progress of aging. Here, we will review the clinical and neurological features of neuroferritinopathy and summarize biochemical studies and data from cellular and animal models to propose a pathogenic mechanism of the disorder.


Assuntos
Apoferritinas/metabolismo , Distúrbios do Metabolismo do Ferro/patologia , Ferro/metabolismo , Distrofias Neuroaxonais/patologia , Animais , Humanos , Distúrbios do Metabolismo do Ferro/metabolismo , Distrofias Neuroaxonais/metabolismo
7.
Ann Neurol ; 89(3): 498-510, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33244761

RESUMO

OBJECTIVE: Multiple sclerosis (MS) is a heterogeneous inflammatory demyelinating disease. Iron distribution is altered in MS patients' brains, suggesting iron liberation within active lesions amplifies demyelination and neurodegeneration. Whether the amount and distribution of iron are similar or different among different MS immunopatterns is currently unknown. METHODS: We used synchrotron X-ray fluorescence imaging, histology, and immunohistochemistry to compare the iron quantity and distribution between immunopattern II and III early active MS lesions. We analyzed archival autopsy and biopsy tissue from 21 MS patients. RESULTS: Immunopattern II early active lesions contain 64% more iron (95% confidence interval [CI] = 17-127%, p = 0.004) than immunopattern III lesions, and 30% more iron than the surrounding periplaque white matter (95% CI = 3-64%, p = 0.03). Iron in immunopattern III lesions is 28% lower than in the periplaque white matter (95% CI = -40 to -14%, p < 0.001). When normalizing the iron content of early active lesions to that of surrounding periplaque white matter, the ratio is significantly higher in immunopattern II (p < 0.001). Microfocused X-ray fluorescence imaging shows that iron in immunopattern II lesions localizes to macrophages, whereas macrophages in immunopattern III lesions contain little iron. INTERPRETATION: Iron distribution and content are heterogeneous in early active MS lesions. Iron accumulates in macrophages in immunopattern II, but not immunopattern III lesions. This heterogeneity in the two most common MS immunopatterns may be explained by different macrophage polarization, origin, or different demyelination mechanisms, and paves the way for developing new or using existing iron-sensitive magnetic resonance imaging techniques to differentiate among immunopatterns in the general nonbiopsied MS patient population. ANN NEUROL 2021;89:498-510.


Assuntos
Encéfalo/metabolismo , Ferro/metabolismo , Esclerose Múltipla/metabolismo , Adolescente , Adulto , Idoso , Apoferritinas/metabolismo , Apoptose , Encéfalo/imunologia , Encéfalo/patologia , Criança , Proteínas do Sistema Complemento/metabolismo , Feminino , Compostos Férricos/metabolismo , Compostos Ferrosos/metabolismo , Humanos , Imunoglobulinas/metabolismo , Imuno-Histoquímica , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Proteínas da Mielina/metabolismo , Glicoproteína Associada a Mielina/metabolismo , Oligodendroglia/metabolismo , Imagem Óptica , Espectrometria por Raios X , Síncrotrons , Adulto Jovem
8.
Artigo em Inglês | MEDLINE | ID: mdl-33383192

RESUMO

Ferritin H can participate in the regulation of teleostean immunity. ORF sequences of RCC/WCC/WR-ferritin H were 609 bp, while WR-ferritin H gene possessed chimeric fragments or offspring-specific mutations. In order to elucidate regulation of immune-related signal transduction, three fibroblast-like cell lines derived from caudal fin of red crucian carp (RCC), white crucian carp (WCC) and their hybrid offspring (WR) were characterized and designated as RCCFCs, WCCFCs and WRFCs. A sharp increase of ferritin H mRNA was observed in RCCFCs, WCCFCs and WRFCs following lipopolysaccharide (LPS) challenge. Overexpression of RCC/WCC/WR-ferritin H can decrease MyD88-IRAK4 signal and antagonize NF-κB, TNFα promoter activity in RCCFCs, WCCFCs and WRFCs, respectively. These results indicated that ferritin H in hybrid offspring harbors highly-conserved domains with a close sequence similarity to those of its parents, playing a regulatory role in inflammatory signals.


Assuntos
Apoferritinas/metabolismo , Carpas/genética , Inflamação/metabolismo , Lipopolissacarídeos/toxicidade , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Apoferritinas/genética , Células Cultivadas , Clonagem Molecular , Regulação para Baixo , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Conformação Proteica , Regulação para Cima
9.
Sci Rep ; 10(1): 20666, 2020 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-33244127

RESUMO

The role of abnormal brain iron metabolism in neurodegenerative diseases is still insufficiently understood. Here, we investigate the molecular basis of the neurodegenerative disease hereditary ferritinopathy (HF), in which dysregulation of brain iron homeostasis is the primary cause of neurodegeneration. We mutagenized ferritin's three-fold pores (3FPs), i.e. the main entry route for iron, to investigate ferritin's iron management when iron must traverse the protein shell through the disrupted four-fold pores (4FPs) generated by mutations in the ferritin light chain (FtL) gene in HF. We assessed the structure and properties of ferritins using cryo-electron microscopy and a range of functional analyses in vitro. Loss of 3FP function did not alter ferritin structure but led to a decrease in protein solubility and iron storage. Abnormal 4FPs acted as alternate routes for iron entry and exit in the absence of functional 3FPs, further reducing ferritin iron-storage capacity. Importantly, even a small number of MtFtL subunits significantly compromises ferritin solubility and function, providing a rationale for the presence of ferritin aggregates in cell types expressing different levels of FtLs in patients with HF. These findings led us to discuss whether modifying pores could be used as a pharmacological target in HF.


Assuntos
Apoferritinas/metabolismo , Ferro/metabolismo , Polímeros/metabolismo , Apoferritinas/genética , Encéfalo/metabolismo , Microscopia Crioeletrônica/métodos , Homeostase/genética , Homeostase/fisiologia , Humanos , Distúrbios do Metabolismo do Ferro/genética , Distúrbios do Metabolismo do Ferro/metabolismo , Mutação/genética , Distrofias Neuroaxonais/genética , Distrofias Neuroaxonais/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo
10.
Int J Mol Sci ; 21(17)2020 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-32878313

RESUMO

Various pathological processes in humans are associated with biogenic iron accumulation and the mineralization of iron oxide nanoparticles, especially magnetite. Ferritin has been proposed as a precursor to pathological magnetite mineralization. This study quantifies spectroscopically the release of ferrous ions from native ferritin and magnetoferritin as a model system for pathological ferritin in the presence of potent natural reducing agents (vitamins C and B2) over time. Ferrous cations are required for the transformation of ferrihydrite (physiological) into a magnetite (pathological) mineral core and are considered toxic at elevated levels. The study shows a significant difference in the reduction and iron release from native ferritin compared to magnetoferritin for both vitamins. The amount of reduced iron formed from a magnetoferritin mineral core is two to five times higher than from native ferritin. Surprisingly, increasing the concentration of the reducing agent affects only iron release from native ferritin. Magnetoferritin cores with different loading factors seem to be insensitive to different concentrations of vitamins. An alternative hypothesis of human tissue magnetite mineralization and the process of iron-induced pathology is proposed. The results could contribute to evidence of the molecular mechanisms of various iron-related pathologies, including neurodegeneration.


Assuntos
Apoferritinas/metabolismo , Ácido Ascórbico/farmacologia , Ferritinas/metabolismo , Ferro/metabolismo , Óxidos/metabolismo , Riboflavina/farmacologia , Apoferritinas/efeitos dos fármacos , Ferritinas/efeitos dos fármacos , Humanos , Complexo Vitamínico B/farmacologia , Vitaminas/farmacologia
11.
Biochim Biophys Acta Gen Subj ; 1864(11): 129700, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32798636

RESUMO

BACKGROUND: The mechanism of iron oxidation and core formation in homopolymeric H-type ferritins has been extensively studied in-vitro, so has the reductive mobilization of iron from the inorganic iron(III) core. However, neither process is well-understood in-vivo despite recent scientific advances. SCOPE OF REVIEW: Here, we provide a summary of our current understanding of iron mineralization and iron core dissolution in homopolymeric H-type ferritins and highlight areas of interest and further studies that could answer some of the outstanding questions of iron metabolism. MAJOR CONCLUSIONS: The overall iron oxidation mechanism in homopolymeric H-type ferritins from vertebrates (i.e. human H and frog M ferritins) is similar, despite nuances in the individual oxidation steps due to differences in the iron ligand environments inside the three fold channels, and at the dinuclear ferroxidase centers. Ferrous cations enter the protein shell through hydrophilic channels, followed by their rapid oxidization at di­iron centers. Hydrogen peroxide produced during iron oxidation can react with additional iron(II) at ferroxidase centers, or at separate sites, or possibly on the surface of the mineral core. In-vitro ferritin iron mobilization can be achieved using a variety of reducing agents, but in-vivo iron retrieval may occur through a variety of processes, including proteolytic degradation, auxiliary iron mobilization mechanisms involving physiological reducing agents, and/or oxidoreductases. GENERAL SIGNIFICANCE: This review provides important insights into the mechanisms of iron oxidation and mobilization in homopolymeric H-type ferritins, and different strategies in maintaining iron homeostasis.


Assuntos
Apoferritinas/metabolismo , Ferro/metabolismo , Animais , Apoferritinas/química , Transporte Biológico , Ceruloplasmina/química , Ceruloplasmina/metabolismo , Ferritinas/química , Ferritinas/metabolismo , Humanos , Modelos Moleculares , Oxirredução , Proteólise
12.
J Exp Clin Cancer Res ; 39(1): 137, 2020 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-32677981

RESUMO

BACKGROUND: Hypoxia, a fundamental characteristic of glioma, is considered to promote tumor malignancy by inducing process of epithelial mesenchymal transition (EMT). Ferritin Light Chain (FTL) is one of the iron metabolism regulators and is overexpressed in glioma. However, relationship between hypoxia and FTL expression and its role in regulating EMT remains unclear. METHODS: Immunohistochemistry (IHC), western blot and public datasets were used to evaluate FTL level in glioma. Wound healing, transwell assays, CCK8, annexin V staining assay were used to measure migration, invasion, proliferation and apoptosis of glioma cells in vitro. Interaction between HIF1A and FTL was assessed by luciferase reporter and Chromatin immunoprecipitation (ChIP) assays. Subcutaneous xenograft model was established to investigate in vivo growth. RESULTS: FTL expression was enriched in high grade glioma (HGG) and its expression significantly associated with IDH1/2 wildtype and unfavorable prognosis of glioma patients. FTL expression positively correlated with HIF1A in glioma tissues and obviously increased in U87 and U251 cells under hypoxia in a time-dependent manner. Mechanistically, HIF-1α regulates FTL expression by directly binding to HRE-3 in FTL promoter region. Furthermore, we found that knockdown FTL dramatically repressed EMT and reduced migration and invasion of glioma by regulating AKT/GSK3ß/ ß-catenin signaling both in vitro and in vivo. Moreover, our study found downregulation FTL decreased the survival rate and increased the apoptosis of glioma cells treated with temozolomide (TMZ). FTL expression segregated glioma patients who were treated with TMZ or with high MGMT promoter methylation into survival groups in TCGA dataset. Patients with methylated MGMT who had high FTL expression presented similar prognosis with patients with unmethylated MGMT. CONCLUSION: Our study strongly suggested that hypoxia-inducible FTL was a regulator of EMT and acted not only as a prognostic marker but also a novel biomarker of response to TMZ in glioma.


Assuntos
Apoferritinas/metabolismo , Biomarcadores Tumorais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Glioma/tratamento farmacológico , Hipóxia/fisiopatologia , Temozolomida/farmacologia , Animais , Antineoplásicos Alquilantes/farmacologia , Apoferritinas/genética , Apoptose , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Sci Rep ; 10(1): 11693, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32678124

RESUMO

Current immunohistochemical methods of studying microglia in the post-mortem human brain do not capture the heterogeneity of microglial function in response to damage and disease. We therefore investigated the expression of eight myeloid cell proteins associated with changes in function alongside Iba1. To study the myeloid cells we used immunohistochemistry on post-mortem human middle temporal gyrus sections from neurologically normal individuals. First we investigated co-labelling between the classical 'activation' marker, HLA-DR and each of the other markers of interest. Significant co-labelling between HLA-DR with CD206, CD32, CD163, or L-Ferritin was observed, although complete overlap of expression of HLA-DR with aforementioned markers was not observed. A qualitative assessment also demonstrated that perivascular macrophages expressed higher levels of the markers of interest we investigated than microglia, suggesting perivascular macrophages show a more phagocytic and antigen presentation state in the human brain. To determine whether the markers of interest were expressed in different functional states, the immunoreactivity for each marker was qualitatively assessed on microglial morphologies. Degenerating marker, L-Ferritin, was specific for dystrophic microglia. We demonstrate that microglial heterogeneity can be investigated in immunohistochemically stain post-mortem human tissue by integrating the single-cell abundance of proteins and cell morphology to infer function.


Assuntos
Imunofluorescência/métodos , Microglia/metabolismo , Células Mieloides/metabolismo , Lobo Temporal/metabolismo , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Apoferritinas/metabolismo , Autopsia , Biomarcadores/metabolismo , Feminino , Antígenos HLA-DR/metabolismo , Humanos , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Receptores de Superfície Celular/metabolismo , Receptores de IgG/metabolismo , Receptores Imunológicos/metabolismo
14.
Sci Rep ; 10(1): 12232, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32699419

RESUMO

Ferritin is an iron-binding molecule, which comprises 24 subunits, heavy (FeH) and light (FeL) subunits, suggested to have a pathogenic role by the 'hyperferritinemic syndrome'. In this work, we tested (1) FeH and FeL in bone marrow (BM) and sera in patients with macrophage activation syndrome (MAS); (2) pro-inflammatory effects of ferritin, FeL, and FeH on macrophages; (3) ability of FeH-stimulated macrophages to stimulate the proliferation of peripheral blood mononuclear cells (PBMCs); (4) production of mature IL-1ß and IL-12p70 in extracellular compartments of FeH-stimulated macrophages. Immunofluorescence analysis and liquid chromatography mass spectrometry (LC-MS/MS) based proteomics were performed to identify FeL and FeH in BM and sera, respectively, in the same patients. Macrophages were stimulated with ferritin, FeH, and FeL to assess pro-inflammatory effects by RT-PCR and western blot. The proliferation of co-cultured PBMCs with FeH-stimulated macrophages was tested. Immunofluorescence showed an increased FeH expression in BMs, whereas LC-MS/MS identified that FeL was mainly represented in sera. FeH induced a significant increase of gene expressions of IL-1ß, IL-6, IL-12, and TNF-α, more marked with FeH, which also stimulated NLRP3. FeH-stimulated macrophages enhanced the proliferation of PBMCs. The ELISA assays showed that mature form of IL-1ß and IL-12p70 were increased, in extracellular compartments of FeH-stimulated macrophages. Our results showed FeH in BM biopsies of MAS patients, whereas, LC-MS/MS identified FeL in the sera. FeH showed pro-inflammatory effects on macrophages, stimulated NLRP3, and increased PBMCs proliferation.


Assuntos
Apoferritinas/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Células Cultivadas , Expressão Gênica/fisiologia , Humanos , Interleucina-12/metabolismo , Interleucina-1beta/metabolismo , Leucócitos Mononucleares/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
15.
PLoS One ; 15(7): e0235904, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32663208

RESUMO

Pancreatic ductal adenocarcinoma is one of the most aggressive types of cancer. Certain proteins in the tumor microenvironment have attracted considerable attention owing to their association with tumor invasion and metastasis. Here, we used proteomics to identify proteins associated with lymph-node metastasis, which is one of the prognostic factors. We selected lymph node metastasis-positive and -negative patients (n = 5 each) who underwent pancreatectomy between 2005 and 2015 and subjected to comprehensive proteomic profiling of tumor stroma. A total of 490 proteins were detected by mass spectrometry. Software analysis revealed that nine of these proteins were differentially expressed between the two patient groups. We focused on hemopexin and ferritin light chain based on immunohistochemistry results. We assessed the clinicopathological data of 163 patients and found that hemopexin expression was associated with UICC N2 (p = 0.0399), lymph node ratio (p = 0.0252), venous invasion (p = 0.0096), and lymphatic invasion (p = 0.0232). Notably, in vitro assays showed that hemopexin promotes invasion of the pancreatic cancer cells. Our findings suggest that hemopexin is a lymph node metastasis-associated protein that could potentially serve as a useful therapeutic target or biomarker of pancreatic ductal adenocarcinoma.


Assuntos
Carcinoma Ductal Pancreático/patologia , Hemopexina/metabolismo , Neoplasias Pancreáticas/patologia , Idoso , Apoferritinas/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Imuno-Histoquímica , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Neoplasias Pancreáticas/metabolismo , Prognóstico , Proteoma/análise , Proteômica/métodos , Espectrometria de Massas em Tandem
16.
Biochemistry ; 59(29): 2707-2717, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32608971

RESUMO

Ferritinophagy is a ferritin autophagic degradation process mediated by the selective nuclear receptor coactivator-4 (NCOA4). NCOA4 binds to ferritin and delivers it to nascent autophagosomes, which then merge with the lysosomes for ferritin degradation and iron release. Earlier studies have demonstrated a specific association of NCOA4 with ferritin H-subunits, but not L-subunits. However, neither the thermodynamics of this interaction nor the effect of NCOA4 on iron oxidation, iron mineral core formation, or iron mobilization in ferritin has been explored. Using isothermal titration calorimetry, light absorption spectroscopy, and a soluble fragment (residues 383-522) of human NCOA4 expressed in Escherichia coli, we show that the NCOA4 fragment specifically binds H-rich ferritins with a binding stoichiometry of ∼8 NCOA4 molecules per ferritin shell, and Kd values of ∼0.4 and ∼2 µM for homopolymer H-chain ferritin and heteropolymer H-rich ferritin, respectively. The binding reaction was both enthalpically and entropically favored. Whereas the iron oxidation kinetics were not affected by the presence of NCOA4, iron mobilization from ferritin by two different reducing agents (FMN/NADH and sodium dithionite) showed a strong inhibitory effect that was dependent on the concentration of NCOA4 present in solution. Our results suggest that the binding of NCOA4 to ferritin may interfere in the electron transfer pathway through the ferritin shell and may have important biological implications on cellular iron homeostasis.


Assuntos
Apoferritinas/metabolismo , Ferritinas/metabolismo , Coativadores de Receptor Nuclear/metabolismo , Oxirredutases/metabolismo , Apoferritinas/química , Sítios de Ligação , Ferritinas/química , Humanos , Cinética , Coativadores de Receptor Nuclear/química , Oxirredutases/química , Ligação Proteica , Mapas de Interação de Proteínas , Termodinâmica
17.
Nutrients ; 12(5)2020 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-32397086

RESUMO

Despite the crucial role of the liver as the central regulator of iron homeostasis, no studies have directly tested the modulation of liver gene and protein expression patterns during iron deficiency instauration and recovery with fermented milks. Fermented goat milk consumption improves the key proteins of intestinal iron metabolism during iron deficiency recovery, enhancing the digestive and metabolic utilization of iron. The aim of this study was to assess the influence of fermented goat or cow milk consumption on liver iron homeostasis during iron-deficiency anemia recovery with normal or iron-overload diets. Analysis included iron status biomarkers, gene and protein expression in hepatocytes. In general, fermented goat milk consumption either with normal or high iron content up-regulated liver DMT1, FPN1 and FTL1 gene expression and DMT1 and FPN1 protein expression. However, HAMP mRNA expression was lower in all groups of animals fed fermented goat milk. Additionally, hepcidin protein expression decreased in control and anemic animals fed fermented goat milk with normal iron content. In conclusion, fermented goat milk potentiates the up-regulation of key genes coding for proteins involved in iron metabolism, such as DMT1, and FPN1, FTL1 and down-regulation of HAMP, playing a key role in enhanced iron repletion during anemia recovery, inducing a physiological adaptation of the liver key genes and proteins coordinated with the fluctuation of the cellular iron levels, favoring whole-body iron homeostasis.


Assuntos
Anemia Ferropriva/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Ingestão de Alimentos/fisiologia , Fermentação , Expressão Gênica , Hepcidinas/genética , Hepcidinas/metabolismo , Homeostase/genética , Ferro/metabolismo , Fígado/metabolismo , Leite , Animais , Apoferritinas/genética , Apoferritinas/metabolismo , Apoferritinas/fisiologia , Proteínas de Transporte de Cátions/fisiologia , Bovinos , Cabras , Hepcidinas/fisiologia , Humanos , Mucosa Intestinal/metabolismo , Ratos Wistar
18.
J Inorg Biochem ; 206: 111016, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32142941

RESUMO

Ferritin is a globular hollow protein that acts as the major iron storage protein across living organisms. The 8 nm-diameter internal cavity of ferritin has been used as a nanoreactor for the synthesis of various metallic nanoparticles different to iron oxides. For this purpose, ferritin is incubated in solution with metallic ions that enter the cavity through its natural channels. Then, these ions are subjected to a reduction step to obtain highly monodisperse metallic nanoparticles, with enhanced stability and biocompatibility provided by the ferritin structure. Potential biomedical applications of ferritin-nanoparticle complex will require the use of human ferritin to provide a safer and low-risk alternative for the delivery of metallic nanoparticles into the body. However, most of the reported protocols for metallic nanoparticles synthesis uses horse spleen ferritin as nanocontainer. Previous studies have acknowledged technical difficulties with recombinant human ferritin during the synthesis of metallic nanoparticles, like protein precipitation, which is translated into low recovery yields. In this study, we tested a novel photochemical reduction method for silver nanoparticle synthesis in human recombinant ferritin and compared it with the traditional chemical reduction method. The results show that photoreduction of silver ions inside ferritin cavity provides a universal method for silver nanoparticle synthesis in both recombinant human ferritin homopolymers (Light and Heavy ferritin). Additionally, we report important parameters that account for the efficiency of the method, such as ferritin recovery yield (~60%) and ferritin­silver nanoparticle yield (34% for H-ferritin and 17% for L-ferritin).


Assuntos
Apoferritinas/química , Nanopartículas Metálicas/química , Fotoquímica , Proteínas Recombinantes/química , Prata/química , Apoferritinas/metabolismo , Humanos , Proteínas Recombinantes/metabolismo
19.
Chemistry ; 26(26): 5770-5773, 2020 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-32027764

RESUMO

X-ray structures of homopolymeric human L-ferritin and horse spleen ferritin were solved by freezing protein crystals at different time intervals after exposure to a ferric salt and revealed the growth of an octa-nuclear iron cluster on the inner surface of the protein cage with a key role played by some glutamate residues. An atomic resolution view of how the cluster formation develops starting from a (µ3 -oxo)tris[(µ2 -glutamato-κO:κO')](glutamato-κO)(diaquo)triiron(III) seed is provided. The results support the idea that iron biomineralization in ferritin is a process initiating at the level of the protein surface, capable of contributing coordination bonds and electrostatic guidance.


Assuntos
Apoferritinas/química , Ferritinas/química , Ferro/química , Animais , Apoferritinas/metabolismo , Fenômenos Biológicos , Cavalos , Humanos
20.
Sci Rep ; 10(1): 2725, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-32066785

RESUMO

Superparamagnetic iron oxide nanoparticles (SPIONs) have been investigated for wide variety of applications. Their unique properties render them highly applicable as MRI contrast agents, in magnetic hyperthermia or targeted drug delivery. SPIONs surface properties affect a whole array of parameters such as: solubility, toxicity, stability, biodistribution etc. Therefore, progress in the field of SPIONs surface functionalization is crucial for further development of therapeutic or diagnostic agents. In this study, SPIONs were synthesized by thermal decomposition of iron (III) acetylacetonate Fe(acac)3 and functionalized with dihexadecyl phosphate (DHP) via phase transfer. Bioactivity of the SPION-DHP was assessed on SW1353 and TCam-2 cancer derived cell lines. The following test were conducted: cytotoxicity and proliferation assay, reactive oxygen species (ROS) assay, SPIONs uptake (via Iron Staining and ICP-MS), expression analysis of the following genes: alkaline phosphatase (ALPL); ferritin light chain (FTL); serine/threonine protein phosphatase 2A (PP2A); protein tyrosine phosphatase non-receptor type 11 (PTPN11); transferrin receptor 1 (TFRC) via RT-qPCR. SPION-DHP nanoparticles were successfully obtained and did not reveal significant cytotoxicity in the range of tested concentrations. ROS generation was elevated, however not correlated with the concentrations. Gene expression profile was slightly altered only in SW1353 cells.


Assuntos
Condrócitos/efeitos dos fármacos , Compostos Férricos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Nanopartículas de Magnetita/química , Organofosfatos/química , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Apoferritinas/genética , Apoferritinas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Compostos Férricos/química , Humanos , Hidroxibutiratos/química , Pentanonas/química , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Succímero/química
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