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1.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445509

RESUMO

Ischemia-like conditions reflect almost the entire spectrum of events that occur during cerebral ischemia, including the induction of oxidative stress, Ca2+ overload, glutamate excitotoxicity, and activation of necrosis and apoptosis in brain cells. Mechanisms for the protective effects of the antioxidant enzyme peroxiredoxin-6 (Prx-6) on hippocampal cells during oxygen-glucose deprivation/reoxygenation (OGD/R) were investigated. Using the methods of fluorescence microscopy, inhibitory analysis, vitality tests and PCR, it was shown that 24-h incubation of mixed hippocampal cell cultures with Prx-6 does not affect the generation of a reversible phase of a OGD-induced rise in Ca2+ ions in cytosol ([Ca2+]i), but inhibits a global increase in [Ca2+]i in astrocytes completely and in neurons by 70%. In addition, after 40 min of OGD, cell necrosis is suppressed, especially in the astrocyte population. This effect is associated with the complex action of Prx-6 on neuroglial networks. As an antioxidant, Prx-6 has a more pronounced and astrocyte-directed effect, compared to the exogenous antioxidant vitamin E (Vit E). Prx-6 inhibits ROS production in mitochondria by increasing the antioxidant capacity of cells and altering the expression of genes encoding redox status proteins. Due to the close bond between [Ca2+]i and intracellular ROS, this effect of Prx-6 is one of its protective mechanisms. Moreover, Prx-6 effectively suppresses not only necrosis, but also apoptosis during OGD and reoxygenation. Incubation with Prx-6 leads to activation of the basic expression of genes encoding protective kinases-PI3K, CaMKII, PKC, anti-apoptotic proteins-Stat3 and Bcl-2, while inhibiting the expression of signaling kinases and factors involved in apoptosis activation-Ikk, Src, NF-κb, Caspase-3, p53, Fas, etc. This effect on the basic expression of the genome leads to the cell preconditions, which is expressed in the inhibition of caspase-3 during OGD/reoxygenation. A significant effect of Prx-6 is directed on suppression of the level of pro-inflammatory cytokine IL-1ß and factor TNFα, as well as genes encoding NMDA- and kainate receptor subunits, which was established for the first time for this antioxidant enzyme. The protective effect of Prx-6 is due to its antioxidant properties, since mutant Prx-6 (mutPrx-6, Prx6-C47S) leads to polar opposite effects, contributing to oxidative stress, activation of apoptosis and cell death through receptor action on TLR4.


Assuntos
Astrócitos/citologia , Hipocampo/citologia , Peroxirredoxina VI/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Cálcio/metabolismo , Células Cultivadas , Citosol/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Interleucina-1beta/metabolismo , Microscopia de Fluorescência , Peroxirredoxina VI/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
2.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070501

RESUMO

Our goal was to analyze postmortem tissues of an adult patient with late-onset thymidine kinase 2 (TK2) deficiency who died of respiratory failure. Compared with control tissues, we found a low mtDNA content in the patient's skeletal muscle, liver, kidney, small intestine, and particularly in the diaphragm, whereas heart and brain tissue showed normal mtDNA levels. mtDNA deletions were present in skeletal muscle and diaphragm. All tissues showed a low content of OXPHOS subunits, and this was especially evident in diaphragm, which also exhibited an abnormal protein profile, expression of non-muscular ß-actin and loss of GAPDH and α-actin. MALDI-TOF/TOF mass spectrometry analysis demonstrated the loss of the enzyme fructose-bisphosphate aldolase, and enrichment for serum albumin in the patient's diaphragm tissue. The TK2-deficient patient's diaphragm showed a more profound loss of OXPHOS proteins, with lower levels of catalase, peroxiredoxin 6, cytosolic superoxide dismutase, p62 and the catalytic subunits of proteasome than diaphragms of ventilated controls. Strong overexpression of TK1 was observed in all tissues of the patient with diaphragm showing the highest levels. TK2 deficiency induces a more profound dysfunction of the diaphragm than of other tissues, which manifests as loss of OXPHOS and glycolytic proteins, sarcomeric components, antioxidants and overactivation of the TK1 salvage pathway that is not attributed to mechanical ventilation.


Assuntos
DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Diafragma/metabolismo , Mitocôndrias/metabolismo , Insuficiência Respiratória/metabolismo , Timidina Quinase/deficiência , Timidina Quinase/genética , Actinas/metabolismo , Adulto , Autopsia , Encéfalo/metabolismo , Catalase/metabolismo , Diafragma/enzimologia , Feminino , Frutose-Bifosfato Aldolase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Humanos , Intestino Delgado/metabolismo , Rim/metabolismo , Fígado/metabolismo , Espectrometria de Massas , Mitocôndrias/enzimologia , Mitocôndrias/genética , Músculo Esquelético/metabolismo , Fosforilação Oxidativa , Peroxirredoxina VI/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteoma/genética , Proteoma/metabolismo , Insuficiência Respiratória/genética , Insuficiência Respiratória/mortalidade , Superóxido Dismutase/metabolismo , Timidina Quinase/metabolismo , Regulação para Cima
3.
Life Sci ; 278: 119529, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33894270

RESUMO

Glomerular podocyte damage is considered to be one of the main mechanisms leading to Diabetic nephropathy (DN). However, the relevant mechanism of podocyte injury is not yet clear. This study aimed to investigate the effect of peroxiredoxin 6 (Prdx6) on the pathogenesis of podocyte injury induced by high glucose (HG). The mouse glomerular podocyte MPC5 was stimulated with 30 nM glucose, and the Prdx6 overexpression vector or specificity protein 1 (Sp1) overexpression vector was transfected into MPC5 cells before the high glucose stimulation. As results, HG treatment significantly reduced the expression of Prdx6 and Sp1 in MPC5 cells. Prdx6 overexpression increased cell viability, while inhibited podocyte death, inflammation and podocyte destruction in HG-induced MPC5 cells. Prdx6 overexpression inhibited HG-induced ROS and MDA production, while restored SOD and GSH activity in MPC5 cells. Prdx6 overexpression also eliminated ferroptosis caused by HG, which was reflected in the suppression of iron accumulation and the increase in SLC7A11 and GPX4 expression. The improvement effect of Prdx6 on HG-induced podocyte damage could be eliminated by erastin. Moreover, Sp1 could bind to the three Sp1 response elements in the Prdx6 promoter, thereby directly regulating the transcriptional activation of Prdx6 in podocytes. Silencing Sp1 could eliminate the effect of Prdx6 on HG-induced podocyte damage. Further, Prdx6 overexpression attenuated renal injuries in streptozotocin-induced DN mice. Sp1-mediated upregulation of Prdx6 expression prevents podocyte injury in diabetic nephropathy via mitigation of oxidative stress and ferroptosis, which may provide new insights for the study of the mechanism of DN.


Assuntos
Nefropatias Diabéticas/metabolismo , Ferroptose , Estresse Oxidativo , Peroxirredoxina VI/metabolismo , Podócitos/metabolismo , Fator de Transcrição Sp1/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Piperazinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Ativação Transcricional
4.
Arch Biochem Biophys ; 702: 108830, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33727039

RESUMO

Peroxiredoxin 6 (Prdx6) is a bifunctional enzyme with multi-substrate peroxidase and phospholipase activities that is involved in cell redox homeostasis and regulates intracellular processes. Previously, recombinant Prdx6 was shown to exert a radioprotective effect during whole-body exposure to a lethal dose of X-ray radiation. Moreover, a mutant form Prdx6-C47S, which lacks peroxidase activity, also had a radioprotective effect, and this indicates that the mechanism of radioprotection is unknown. The present study was aimed to test the hypothesis that the radioprotective effect of Prdx6 and Prdx6-C47S may be mediated through the TLR4/NF-κB signaling pathway. It was demonstrated that exogenously applied Prdx6 protected 3T3 fibroblast cells against LD50 X-ray radiation in vitro. Pretreatment with Prdx6 increased cell survival, stimulated proliferation, normalized the level of reactive oxygen species in culture, and suppressed apoptosis and necrosis. Wild-type Prdx6 and, to a lesser degree, the Prdx6-C47S mutant proteins promoted a significant increase in NF-κB activation in irradiated cells, which likely contributes to the antiapoptotic effect. Pretreatment with TLR4 inhibitors, especially those directed to the extracellular part of the receptor, significantly reduced the radioprotective effect, and this supports the role of TLR4 signaling in the protective effects of Prdx6. Therefore, the radioprotective effect of Prdx6 was related not only to its antioxidant properties, but also to its ability to trigger cellular defense mechanisms through interaction with the TLR4 receptor and subsequent activation of the NF-κB pathway. Recombinant Prdx6 may be useful for the development of a new class of safe radioprotective compounds that have a combination of antioxidant and immunomodulatory properties.


Assuntos
NF-kappa B/metabolismo , Peroxirredoxina VI/farmacologia , Protetores contra Radiação/farmacologia , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Células 3T3 , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Camundongos , Modelos Moleculares , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Peroxirredoxina VI/química , Peroxirredoxina VI/metabolismo , Conformação Proteica , Protetores contra Radiação/química , Protetores contra Radiação/metabolismo , Transdução de Sinais/efeitos da radiação , Receptor 4 Toll-Like/química
5.
Int J Immunopathol Pharmacol ; 35: 20587384211005645, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33779346

RESUMO

Protective effects of peroxiredoxin 6 (PRDX6) in RIN-m5F ß-cells and of thymulin in mice with alloxan-induced diabetes were recently reported. The present work was aimed at studying the efficiency of thymulin and PRDX6 in a type 1 diabetes mellitus model induced by streptozotocin in mice. Effects of prolonged treatment with PRDX6 or thymic peptide thymulin on diabetes development were evaluated. We assessed the effects of the drugs on the physiological status of diabetic mice by measuring blood glucose, body weight, and cell counts in several organs, as well as effects of thymulin and PRDX6 on the immune status of diabetic mice measuring concentrations of pro-inflammatory cytokines in blood plasma (TNF-α, interleukin-5 and 17, and interferon-γ), activity of NF-κB and JNK pathways, and Hsp90α expression in immune cells. Both thymulin and PRDX6 reduced the physiological impairments in diabetic mice at various levels. Thymulin and PRDX6 provide beneficial effects in the model of diabetes via very different mechanisms. Taken together, the results of our study indicated that the thymic peptide and the antioxidant enzyme have anti-inflammatory functions. As increasing evidences show diabetes mellitus as a distinct comorbidity leading to acute respiratory distress syndrome and increased mortality in patients with COVID-19 having cytokine storm, thymulin, and PRDX6 might serve as a supporting anti-inflammatory treatment in the therapy of COVID 19 in diabetic patients.


Assuntos
COVID-19 , MAP Quinase Quinase 4/metabolismo , NF-kappa B/metabolismo , Peroxirredoxina VI , Transdução de Sinais , Fator Tímico Circulante , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Antioxidantes/metabolismo , Antioxidantes/farmacologia , COVID-19/tratamento farmacológico , COVID-19/imunologia , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/imunologia , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/terapia , Descoberta de Drogas , Interferon gama/sangue , Interleucinas/sangue , Camundongos , Peroxirredoxina VI/metabolismo , Peroxirredoxina VI/farmacologia , SARS-CoV-2 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Fator Tímico Circulante/metabolismo , Fator Tímico Circulante/farmacologia , Fator de Necrose Tumoral alfa/sangue
6.
Cryobiology ; 100: 158-163, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33561454

RESUMO

Peroxiredoxin 6 (PRDX6) is one antioxidant enzyme which could control the levels of reactive oxygen species and to avoid oxidative damage of sperm. In this study, we aimed to investigate the position change of PRDX6 in human sperm under oxidative stress during cryopreservation. Semen samples were obtained from 98 healthy donors and 27 asthenozoospermic donors. The plasma membrane protein and cytoplasmic protein of sperm samples were extracted and analyzed after cryopreservation. Western blot and immunofluorescence were used to measure the expressions of PRDX6. Liquid chromatography mass spectrometric (LC-MS/MS) analysis was performed to confirm the component of sperm membrane complex. Western blot showed that the detection rate of PRDX6 in plasma membranes with low sperm motility (≤20%) was significantly higher than that with high sperm motility (≥40%). Western blot and Immunofluorescence revealed that cryopreservation and thawing induced the position change of the PRDX6 from cytoplasm to sperm membrane. LC-MS/MS analysis showed that PRDX6, ADP/ATP translocase 4 (ANT4) and glyceraldehyde-3-phosphte dehydrogenase (GAPDHS) were present in the components of membrane complex after cryopreservation. The present study indicated that the presence of PRDX6 in sperm plasma membrane was related to sperm motility. GAPDHS and ANT4 may be involved the position change of the PRDX6 from cytoplasm to sperm membrane under oxidative stress during cryopreservation.


Assuntos
Peroxirredoxina VI , Preservação do Sêmen , Membrana Celular/metabolismo , Cromatografia Líquida , Criopreservação/métodos , Humanos , Masculino , Estresse Oxidativo , Peroxirredoxina VI/metabolismo , Motilidade Espermática , Espermatozoides/metabolismo , Espectrometria de Massas em Tandem
7.
Int J Mol Sci ; 22(4)2021 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-33567524

RESUMO

Alzheimer's disease is a chronic neurodegenerative disorder and represents the main cause of dementia globally. Currently, the world is suffering from the coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a virus that uses angiotensin-converting enzyme 2 (ACE2) as a receptor to enter the host cells. In COVID-19, neurological manifestations have been reported to occur. The present study demonstrates that the protein expression level of ACE2 is upregulated in the brain of patients with Alzheimer's disease. The increased ACE2 expression is not age-dependent, suggesting the direct relationship between Alzheimer's disease and ACE2 expression. Oxidative stress has been implicated in the pathogenesis of Alzheimer's disease, and brains with the disease examined in this study also exhibited higher carbonylated proteins, as well as an increased thiol oxidation state of peroxiredoxin 6 (Prx6). A moderate positive correlation was found between the increased ACE2 protein expression and oxidative stress in brains with Alzheimer's disease. In summary, the present study reveals the relationships between Alzheimer's disease and ACE2, the receptor for SARS-CoV-2. These results suggest the importance of carefully monitoring patients with both Alzheimer's disease and COVID-19 in order to identify higher viral loads in the brain and long-term adverse neurological consequences.


Assuntos
Doença de Alzheimer/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/epidemiologia , COVID-19/metabolismo , Hipocampo/metabolismo , Pandemias , Receptores Virais/metabolismo , SARS-CoV-2/metabolismo , Regulação para Cima , Doença de Alzheimer/complicações , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Autopsia , COVID-19/complicações , COVID-19/virologia , Hipocampo/patologia , Humanos , Oxirredução , Estresse Oxidativo , Peroxirredoxina VI/metabolismo , Placa Amiloide/metabolismo , Carbonilação Proteica , Índice de Gravidade de Doença , Internalização do Vírus
8.
Dev Cell ; 56(1): 111-124.e6, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33238149

RESUMO

To date, the effects of specific modification types and sites on protein lifetime have not been systematically illustrated. Here, we describe a proteomic method, DeltaSILAC, to quantitatively assess the impact of site-specific phosphorylation on the turnover of thousands of proteins in live cells. Based on the accurate and reproducible mass spectrometry-based method, a pulse labeling approach using stable isotope-labeled amino acids in cells (pSILAC), phosphoproteomics, and a unique peptide-level matching strategy, our DeltaSILAC profiling revealed a global, unexpected delaying effect of many phosphosites on protein turnover. We further found that phosphorylated sites accelerating protein turnover are functionally selected for cell fitness, enriched in Cyclin-dependent kinase substrates, and evolutionarily conserved, whereas the glutamic acids surrounding phosphosites significantly delay protein turnover. Our method represents a generalizable approach and provides a rich resource for prioritizing the effects of phosphorylation sites on protein lifetime in the context of cell signaling and disease biology.


Assuntos
Marcação por Isótopo/métodos , Espectrometria de Massas/métodos , Fosfoproteínas/metabolismo , Proteólise , Proteoma/metabolismo , Proteômica/métodos , Sequência de Aminoácidos , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Glutamatos/metabolismo , Humanos , Peptídeos/metabolismo , Peroxirredoxina VI/química , Peroxirredoxina VI/metabolismo , Fosfoproteínas/química , Fosforilação , Proteoma/genética , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , Transdução de Sinais/genética
9.
Redox Biol ; 37: 101737, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33035814

RESUMO

Peroxiredoxin 6 (PRDX6) has been associated with tumor progression and cancer metastasis. Its acting on phospholipid hydroperoxides and its phospholipase-A2 activity are unique among the peroxiredoxin family and add complexity to its action mechanisms. As a first step towards the study of PRDX6 involvement in cancer, we have constructed a human hepatocarcinoma HepG2PRDX6-/- cell line using the CRISPR/Cas9 technique and have characterized the cellular response to lack of PRDX6. Applying quantitative global and redox proteomics, flow cytometry, in vivo extracellular flow analysis, Western blot and electron microscopy, we have detected diminished respiratory capacity, downregulation of mitochondrial proteins and altered mitochondrial morphology. Autophagic vesicles were abundant while the unfolded protein response (UPR), HIF1A and NRF2 transcription factors were not activated, despite increased levels of p62/SQSTM1 and reactive oxygen species (ROS). Insulin receptor (INSR), 3-phosphoinositide-dependent protein kinase 1 (PDPK1), uptake of glucose and hexokinase-2 (HK2) decreased markedly while nucleotide biosynthesis, lipogenesis and synthesis of long chain polyunsaturated fatty acids (LC-PUFA) increased. 254 Cys-peptides belonging to 202 proteins underwent significant redox changes. PRDX6 knockout had an antiproliferative effect due to cell cycle arrest at G2/M transition, without signs of apoptosis. Loss of PLA2 may affect the levels of specific lipids altering lipid signaling pathways, while loss of peroxidase activity could induce redox changes at critical sensitive cysteine residues in key proteins. Oxidation of specific cysteines in Proliferating Cell Nuclear Antigen (PCNA) could interfere with entry into mitosis. The GSH/Glutaredoxin system was downregulated likely contributing to these redox changes. Altogether the data demonstrate that loss of PRDX6 slows down cell division and alters metabolism and mitochondrial function, so that cell survival depends on glycolysis to lactate for ATP production and on AMPK-independent autophagy to obtain building blocks for biosynthesis. PRDX6 is an important link in the chain of elements connecting redox homeostasis and proliferation.


Assuntos
Mitocôndrias , Peroxirredoxina VI , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Oxirredução , Peroxirredoxina VI/metabolismo , Espécies Reativas de Oxigênio/metabolismo
10.
Sci Rep ; 10(1): 17416, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33060708

RESUMO

Peroxiredoxin 6 (Prdx6) is a ubiquitously expressed antioxidant non-selenium glutathione peroxidase that is known to play a major role in various physiological and pathological processes. It belongs to the family of peroxidases (referred to as Peroxiredoxins, Prdx's) that work independently of any prosthetic groups or co-factors, and instead utilize a peroxidatic thiol residue for peroxide reduction. Mammalian Prdx's are classified according to the number of Cys implicated in their catalytic activity by the formation of either inter-molecular (typical 2-Cys, Prdx1-4) or intra-molecular (atypical 2-Cys, Prdx5) disulfide bond, or non-covalent interactions (1-Cys, Prdx6). The typical and atypical 2-Prdx's have been identified to show decamer/dimer and monomer/dimer transition, respectively, upon oxidation of their peroxidatic cysteine. However, the alterations in the oligomeric status of Prdx6 as a function of peroxidatic thiol's redox state are still ambiguous. While the crystal structure of recombinant human Prdx6 is resolved as a dimer, the solution structures are reported to have both monomers and dimers. In the present study, we have employed several spectroscopic and electrophoretic probes to discern the impact of change in the redox status of peroxidatic cysteine on conformation and oligomeric status of Prdx6. Our study indicates Prdx6's peroxidase activity to be a redox-based conformation driven process which essentially involves monomer-dimer transition.


Assuntos
Peroxirredoxina VI/metabolismo , Catálise , Cisteína/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Peróxido de Hidrogênio/metabolismo , Oxirredução , Peroxirredoxina VI/química , Estrutura Quaternária de Proteína , Análise Espectral/métodos
11.
Dokl Biol Sci ; 493(1): 124-127, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32894426

RESUMO

The role of two heat shock proteins, Hsp70 and Hsp90α, on stress response in mice with severe diabetes mellitus induced by a high dose of alloxan (500 mg/kg body weight), as well as in RIN-m5F ß cells cultured in the presence of cytokines (IL-1 and TNF-α) was studied. Our results showed that severe type 1 diabetes mellitus (T1D) caused a higher expression of Hsp90α, but not Hsp70. Moreover, injections of the peroxiredoxin 6 antioxidant enzyme (PRDX6) did not affect the expression of these chaperones. Conversely, pro-inflammatory cytokines added to ß-cells caused a significant increase in the expression of Hsp90α and, substantially, Hsp70. Moreover, cells cultivated in the presence of PRDX6 were more susceptible to the cytokine effect. Thus, in the course of severe alloxan-induced T1D, no protective role of the heat shock proteins, was revealed, and their expression level was not increased by PRDX6. At the same time the protective potential of these proteins was shown in vitro with the use of RIN-m5F ß cells. Thus, the system of heat shock proteins was unable to prevent the devastating effects of severe T1D accompanied by high animal mortality.


Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Animais , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/patologia , Células Secretoras de Insulina/metabolismo , Camundongos Endogâmicos BALB C , Peroxirredoxina VI/metabolismo , Ratos , Baço/metabolismo , Baço/patologia , Estresse Fisiológico
12.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 28(4): 1157-1161, 2020 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-32798391

RESUMO

OBJECTIVE: To study the expression of Peroxiredoxin-6 (Prdx6) gene in elderly patients with acute myeloid leukemia (AML) and its clinical significance. METHODS: The expression level of Prdx6 in bone marrow cells of 33 cases of AML, 8 cases of CML and 11 cases of other blood diseases was detected by PCR. The correlation of the abnormal expression of Prdx6 with patient age and blood routine parameters was further analyzed. RESULTS: the expression level of Prdx6 in elderly patients with AML (≥60 years) was significantly lower than that in younger patients (<60 years) (P<0.05); the expression level of Prdx6 in low WBC (≤1×109/L) group was lower than that in medium WBC (1-10×109/L) group or high WBC (>10×109/L) group (P<0.05); the proportion of WBC count (≤1×109/L) in elderly patients with AML reached 38.5%, which was significantly higher than that in younger patients (5%) (P<0.05); the overall survival (OS) rate of elderly patients was lower than that of younger patients (P<0.05). CONCLUSION: The expression of Prdx6 in elderly patients with AML is low, moreover, it relates with low value of WBC in peripheral blood, suggesting that it may be one of poor prognostic factors for the elderly patients with AML.


Assuntos
Leucemia Mieloide Aguda , Peroxirredoxina VI , Idoso , Células da Medula Óssea , Humanos , Contagem de Leucócitos , Peroxirredoxina VI/genética , Peroxirredoxina VI/metabolismo , Prognóstico
13.
Cells ; 9(8)2020 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-32784474

RESUMO

Many disorders of aging, including blinding-diseases, are associated with deficiency of brain and muscle arnt-like protein 1 (Bmal1) and, thereby, dysregulation of antioxidant-defense pathway. However, knowledge is limited regarding the role of Bmal1 regulation of antioxidant-pathway in the eye lens/lens epithelial cells (LECs) at the molecular level. We found that, in aging human (h)LECs, a progressive decline of nuclear factor erythroid 2-related factor 2 (Nrf2)/ARE (antioxidant response element)-mediated antioxidant genes was connected to Bmal1-deficiency, leading to accumulation of reactive oxygen species (ROS) and cell-death. Bmal1-depletion disrupted Nrf2 and expression of its target antioxidant genes, like Peroxiredoxin 6 (Prdx6). DNA binding and transcription assays showed that Bmal1 controlled expression by direct binding to E-Box in Prdx6 promoter to regulate its transcription. Mutation at E-Box or ARE reduced promoter activity, while disruption of both sites diminished the activity, suggesting that both sites were required for peak Prdx6-transcription. As in aging hLECs, ROS accumulation was increased in Bmal1-deficient cells and the cells were vulnerable to death. Intriguingly, Bmal1/Nrf2/Prdx6 and PhaseII antioxidants showed rhythmic expression in mouse lenses in vivo and were reciprocally linked to ROS levels. We propose that Bmal1 is pivotal for regulating oxidative responses. Findings also reveal a circadian control of antioxidant-pathway, which is important in combating lens/LECs damage induced by aging or oxidative stress.


Assuntos
Fatores de Transcrição ARNTL/metabolismo , Envelhecimento/metabolismo , Ritmo Circadiano , Células Epiteliais/metabolismo , Olho/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Peroxirredoxina VI/metabolismo , Animais , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/patologia , Olho/citologia , Olho/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo
14.
Biomed Res Int ; 2020: 7103053, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32051828

RESUMO

Mesenchymal stem cells (MSCs) can be differentiated in vitro to form insulin-producing cells (IPCs). However, the proportion of induced cells is modest. Extracts from injured pancreata of rodents promoted this differentiation, and three upregulated proteins were identified in these extracts. The aim of this study was to evaluate the potential benefits of adding these proteins to the differentiation medium alone or in combination. Our results indicate that the proportion of IPCs among the protein(s)-supplemented samples was significantly higher than that in the samples with no added proteins. The yield from samples supplemented with PRDX6 alone was 4-fold higher than that from samples without added protein. These findings were also supported by the results of fluorophotometry. Gene expression profiles revealed higher levels among protein-supplemented samples. Significantly higher levels of GGT, SST, Glut-2, and MafB expression were noted among PRDX6-treated samples. There was a stepwise increase in the release of insulin and c-peptide, as a function of increasing glucose concentrations, indicating that the differentiated cells were glucose sensitive and insulin responsive. PRDX6 exerts its beneficial effects as a result of its biological antioxidant properties. Considering its ease of use as a single protein, PRDX6 is now routinely used in our differentiation protocols.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Insulina/biossíntese , Células-Tronco Mesenquimais/metabolismo , Peroxirredoxina VI/metabolismo , Peroxirredoxina VI/farmacologia , Peptídeo C/metabolismo , Glucose/metabolismo , Transportador de Glucose Tipo 2/metabolismo , Humanos , Fator de Transcrição MafB/metabolismo , Peroxirredoxina VI/genética , Somatostatina/metabolismo , Transcriptoma , gama-Glutamiltransferase/metabolismo
15.
Redox Biol ; 28: 101379, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31759247

RESUMO

Cystathionine γ lyase (CSE) is the major source of hydrogen sulfide-derived species (H2Sn) in endothelial cells and plays an important role in protecting against atherosclerosis. Here we investigated the molecular mechanisms underlying the regulation of CSE expression in endothelial cells by fluid shear stress/flow. Fluid shear stress decreased CSE expression in human and murine endothelial cells and was negatively correlated with the transcription factor Krüppel-like factor (KLF) 2. CSE was identified as a direct target of the KLF2-regulated microRNA, miR-27b and high expression of CSE in native human plaque-derived endothelial cells, was also inversely correlated with KLF2 and miR-27b levels. One consequence of decreased CSE expression was the loss of Prx6 sulfhydration (on Cys47), which resulted in Prx6 hyperoxidation, decamerization and inhibition, as well as a concomitant increase in endothelial cell reactive oxygen species and lipid membrane peroxidation. H2Sn supplementation in vitro was able to reverse the redox state of Prx6. Statin therapy, which is known to activate KLF2, also decreased CSE expression but increased CSE activity by preventing its phosphorylation on Ser377. As a result, the sulfhydration of Prx6 was partially restored in samples from plaque containing arteries from statin-treated donors. Taken together, the regulation of CSE expression by shear stress/disturbed flow is dependent on KLF2 and miR-27b. Moreover, in murine and human arteries CSE acts to maintain endothelial redox balance at least partly by targeting Prx6 to prevent its decamerization and inhibition of its peroxidase activity.


Assuntos
Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Peroxidação de Lipídeos , Placa Aterosclerótica/metabolismo , Animais , Células Endoteliais , Regulação da Expressão Gênica , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Sulfeto de Hidrogênio/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Camundongos , MicroRNAs/genética , Oxirredução , Peroxirredoxina VI/metabolismo , Estresse Mecânico
16.
Cells ; 8(10)2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31569690

RESUMO

Sulforaphane (SFN), an activator of transcription factor Nrf2 (NFE2-related factor), modulates antioxidant defense by Nrf2-mediated regulation of antioxidant genes like Peroxiredoxin 6 (Prdx6) and affects cellular homeostasis. We previously observed that dose levels of SFN are crucial in determining life or death of lens epithelial cells (LECs). Herein, we demonstrated that higher doses of SFN (>6 µM) activated death signaling by overstimulation of Nrf2/ARE (antioxidant response element)-mediated Kruppel-like factor (Klf9) repression of Prdx6 expression, which increased reactive oxygen species (ROS) load and cell death. Mechanistically, Klf9 bound to its repressive Klf9 binding elements (RKBE; 5-CA/GCCC-3) in the Prdx6 promoter, and repressed Prdx6 transcription. Under the condition of higher dose of SFN, excessive Nrf2 abundance caused death signaling by enforcing Klf9 activation through ARE (5-RTGAYnnnGC-3) in Klf9 promoter that suppress antioxidant genes such as Prdx6 via a Klf9-dependent fashion. Klf9-depletion showed that Klf9 independently caused ROS reduction and subsequent cell survival, demonstrating that Klf9 upregulation caused cell death. Our work revealed the molecular mechanism of dose-dependent altered activity of SFN in LECs, and demonstrated that SFN activity was linked to levels of Nrf2/Klf9/Prdx6 axis. We proposed that in the development of therapeutic interventions for aging/oxidative disorders, combinations of Klf9-ShRNA and Nrf2 inducers may prove to be a promising strategy.


Assuntos
Apoptose/efeitos dos fármacos , Células Epiteliais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Isotiocianatos/farmacologia , Fatores de Transcrição Kruppel-Like/metabolismo , Cristalino/patologia , Peroxirredoxina VI/metabolismo , Anticarcinógenos/farmacologia , Antioxidantes/metabolismo , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/genética , Cristalino/efeitos dos fármacos , Cristalino/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Peroxirredoxina VI/genética , Regiões Promotoras Genéticas , Espécies Reativas de Oxigênio/metabolismo
17.
Mol Hum Reprod ; 25(12): 787-796, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31651026

RESUMO

Peroxiredoxins (PRDXs) are antioxidant enzymes proven to control the levels of reactive oxygen species (ROS) and to avoid oxidative damage in the spermatozoon. Previously, we have shown that low amounts of PRDXs are associated with male infertility and that PRDX6 is the primary antioxidant defense in human spermatozoa, maintaining survival and DNA integrity (Gong et al., 2012, Fernandez and O'Flaherty, 2018). Oxidative stress can trigger different pathway cascades in the spermatozoa, including truncated apoptosis. It has been reported that the phosphorylation status of phosphoinositide 3-kinase (PI3K) and its target AKT (protein kinase B) prevent the spermatozoon from entering the truncated apoptotic cascade. Here, we aim to study the regulation of the PI3K/AKT pathway by PRDX6 and assess its role in maintaining sperm viability. Human semen samples were obtained over 1 year from 20 healthy non-smoking volunteers aged 22-30 years. Sperm viability, lipid peroxidation and apoptosis-like changes were determined by flow cytometry while phosphorylation of PI3K and AKT substrates were assessed by immunoblotting using anti-phospho-PI3K and anti-phospho-AKT substrates antibodies. We found that the addition of arachidonic acid and lysophosphatidic acid, products of PRDX6 calcium-independent phospholipase A2 (Ca2+-iPLA2), prevented loss of sperm viability and maintained the phosphorylation of PI3K. Antioxidant compounds such as D-penicillamine partially prevented the oxidative damage on spermatozoa that led to a reduction of their viability. Thus, other pathways can also participate in sperm survival and be regulated by PRDXs. In conclusion, PRDX6 contributes to the regulation of ROS production and the PI3K/AKT pathway for the maintenance of sperm survival.


Assuntos
Estresse Oxidativo/fisiologia , Peroxirredoxina VI/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Motilidade Espermática/fisiologia , Espermatozoides/metabolismo , Adulto , Antioxidantes/farmacologia , Apoptose/fisiologia , Ácido Araquidônico/farmacologia , Sobrevivência Celular/fisiologia , Humanos , Infertilidade Masculina/fisiopatologia , Lisofosfolipídeos/farmacologia , Masculino , Penicilamina/farmacologia , Peroxirredoxina VI/antagonistas & inibidores , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Adulto Jovem
18.
BMC Vet Res ; 15(1): 304, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438945

RESUMO

BACKGROUND: Brucellosis is a worldwide zoonotic infectious disease that is transmitted in various ways and causes great harm to humans and animals. The brucellosis pathogen is Brucella, which mainly resides in macrophage cells and survives and replicates in host cells. However, the mechanisms underlying Brucella survival in macrophage cells have not been thoroughly elucidated to date. Peroxiredoxin 6 (Prdx6) is a bifunctional protein that shows not only GSH peroxidase activity but also phospholipase A2 activity and plays important roles in combating oxidative damage and regulating apoptosis. RESULTS: Recombinant mouse (Mus musculus) Prdx6 (MmPrdx6) was expressed and purified, and monoclonal antibodies against MmPrdx6 were prepared. Using the Brucella suis S2 strain to infect RAW264.7 murine macrophages, the level of intracellular Prdx6 expression first decreased and later increased following infection. Overexpressing Prdx6 in macrophages resulted in an increase in B. suis S2 strain levels in RAW264.7 cells, while knocking down Prdx6 reduced the S2 levels in cells. CONCLUSIONS: Host Prdx6 can increase the intracellular survival of B. suis S2 strain and plays a role in Brucella infection.


Assuntos
Brucella suis/fisiologia , Brucelose/microbiologia , Peroxirredoxina VI/metabolismo , Animais , Feminino , Regulação da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Células RAW 264.7
19.
J Physiol Sci ; 69(5): 779-790, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31286450

RESUMO

Intermittent hyperbaric oxygen exposure (IE-HBO) can protect the body against oxygen toxicity, but the underlying mechanisms are not very clear. Peroxiredoxin 6 (Prdx6) is a special endogenous antioxidative protein. We explored if the protective effects of IE-HBO are related to Prdx6. Mice were exposed to 280 kPa O2 for 60 min, followed by 30-min exposure to 20% O2/N2 mixture with equal pressure, repeated for six cycles. The Prdx6 protein level and non-selenium glutathione peroxidase (NSGPx) activity in the brain and lungs were then measured and the injury degree of lung and the oxidation level of brain and lung were evaluated. On this basis, the relationship between Prdx6 and IE-HBO's protection was explored. Generally, both IE-HBO and continuous exposure to HBO (CE-HBO) could increase the protein and mRNA levels of Prdx6, and such increases were more significant 24 h after cessation of exposure; moreover, the Prdx6 level of IE-HBO was higher than that of CE-HBO in both brain and lung, also more significantly 24 h after cessation of exposure. In addition, IE-HBO exposure could more effectively potentiate the activity of NSGPx and increase GSH content in brain and lung tissues. At the same time, it could reduce oxidation products in these tissues. IE-HBO could also provide protection for the lungs against injuries resulting from prolonged HBO exposure. These data showed that IE-HBO can potentiate the production and the activity of Prdx6 and consequently mitigate oxidative damages in brain and lungs. The influences of IE-HBO on Prdx6 may form an important basis for its protection against oxygen toxicity.


Assuntos
Oxigênio/efeitos adversos , Oxigênio/metabolismo , Peroxirredoxina VI/metabolismo , Animais , Antioxidantes/metabolismo , Encéfalo/metabolismo , Glutationa Peroxidase/metabolismo , Oxigenação Hiperbárica/métodos , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/fisiologia , RNA Mensageiro/metabolismo
20.
Acta Pharmacol Sin ; 40(10): 1334-1342, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31036877

RESUMO

Ferroptosis is a newly characterized iron-dependent form of nonapoptotic regulated cell death triggered by lipid reactive oxygen species (LOOH). The dysregulation of ferroptosis is highly related to cancer, and the induction of ferroptosis is also proposed as a potential strategy for cancer therapy. Although several key regulators have been identified that are involved in ferroptosis, the molecular mechanism underlying this process remains largely unknown. Here, we report that Peroxiredoxin-6 (PRDX6) is a bona fide negative regulator of ferroptotic cell death. The knockdown of intracellular PRDX6 significantly enhances LOOH and ferroptotic cell death triggered by ferroptosis inducers (Erastin and RSL-3), which is correlated with the transcriptional activation of heme oxygenase-1. Moreover, overexpression of heme oxygenase-1 enhances both Erastin- and RSL-3-triggered LOOH, suggesting that heme oxygenase-1 mediates PRDX6 silencing-enhanced ferroptosis. More importantly, the application of a specific PRDX6 phospholipase A2 (iPLA2) inhibitor, MJ-33, synergistically enhances the ferroptosis induced by Erastin, suggesting that PRDX6 removes LOOH through its iPLA2 activity. Thus, our findings reveal an essential role of PRDX6 in protecting cells against ferroptosis and provide a potential target to improve the antitumor activity of ferroptosis-based chemotherapy.


Assuntos
Ferroptose/efeitos dos fármacos , Peroxirredoxina VI/metabolismo , Células A549 , Carbolinas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células HEK293 , Humanos , Lipídeos/biossíntese , Piperazinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo
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