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1.
Life Sci ; 233: 116631, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31278945

RESUMO

AIMS: Prior to reperfusion, Calpains remain inactive due to the acidic pH and elevated ionic strength in the ischemic myocardium; but Calpain is activated during myocardial reperfusion. The underlying mechanism of Calpain activation in the ischemia-reperfusion (I/R) is yet to be determined. Therefore, the present study aims to investigate the mechanism of Calpain in I/R-induced mice. MAIN METHODS: In order to detect the function of Calpain and the NLRP3/ASC/Caspase-1 axis in cardiomyocyte pyroptosis, endoplasmic reticulum (ER) stress and myocardial function, the cardiomyocytes were treated with hypoxia-reoxygenation (H/R), and NLRP3 were silenced, Calpain was overexpressed and Caspase-1 inhibitors were used to determine cardiomyocyte pyroptosis. The results obtained from the cell experiments were then verified with an animal experiment in I/R mice. KEY FINDINGS: There was an overexpression in Calpain, ASC, NLRP3, GRP78 and C/EBP homologous protein (CHOP) in cardiomyocytes following H/R. A significant increase was witnessed in lactic acid dehydrogenase (LDH) activity, cardiomyocyte pyroptosis rate, Calpain activity, reactive oxygen species (ROS) concentration, as well as activation of ER stress in cardiomyocytes after H/R. However, opposing results were observed in H/R cardiomyocytes that received siRNA Calpain, siRNA NLRP3 or Caspase-1 inhibitor treatment. Overall, the results obtained from the animal experiment were consistent with the results from the cell experiment. SIGNIFICANCE: The silencing of Calpain suppresses the activation of the NLRP3/ASC/Caspase-1 axis, thus inhibiting ER stress in mice and improving myocardial dysfunction induced by I/R, providing a novel therapeutic pathway for I/R.


Assuntos
Sistema y+ de Transporte de Aminoácidos/antagonistas & inibidores , Calpaína/antagonistas & inibidores , Caspase 1/química , Estresse do Retículo Endoplasmático , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Animais , Calpaína/genética , Calpaína/metabolismo , Caspase 1/genética , Caspase 1/metabolismo , Células Cultivadas , Inflamassomos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão Miocárdica/etiologia , Traumatismo por Reperfusão Miocárdica/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , RNA Interferente Pequeno/genética
2.
J Food Sci ; 84(5): 1054-1059, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31042817

RESUMO

This study was designed to determine the effects of µ/m-calpain on the degradation of cytoskeletal proteins in pectoralis major. Four chickens were slaughtered and the breasts were removed and stored for 12 hr at 4 °C. Each sample was divided into three groups and respectively immersed in control reagent, calpain inhibitor, and caspase inhibitor at 4 °C. The samples were used to evaluate troponin-T and desmin degradation, calpain activity, and myofibril ultrastructure at 12 hr, day 1, day 3, and day 7. Casein zymography revealed that µ-calpain could not be detected in all samples after 12 hr postmortem. The calpain inhibitor inhibited µ/m-calpain activity and reduced troponin-T and desmin degradation during 7 day postmortem. The caspase inhibitor inhibited µ/m-calpain activity and, troponin-T and desmin degradation before day 3 postmortem. The findings suggest that, µ/m-calpain had an effect on cytoskeletal protein degradation after 12 hr postmortem.


Assuntos
Calpaína , Carne/análise , Proteínas Musculares , Animais , Calpaína/antagonistas & inibidores , Calpaína/metabolismo , Galinhas , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Músculo Esquelético/química , Músculo Esquelético/metabolismo
3.
Neurochem Res ; 44(7): 1636-1652, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31006091

RESUMO

HIV-1 gp120, an important subunit of the envelope spikes that decorate the surface of virions, is known to play a vital role in neuronal injury during HIV-1-associated neurocognitive disorder (HAND), although the pathological mechanism is not fully understood. Our previous studies have suggested that the V3 loop of HIV-1 gp120 (HIV-1 gp120 V3 loop) can induce neuronal apoptosis in the hippocampus, resulting in impairment in spatial learning and memory in Sprague-Dawley (SD) rats. In this study, we demonstrated that autophagy was significantly increased in rat primary hippocampal neurons in response to treatment of HIV-1 gp120 V3 loop. Importantly, HIV-1 gp120 V3 loop-induced autophagy played a dual role in the cell survival and death. An increase in autophagy for a short period inhibited apoptosis of neurons, while persistent autophagy over an extended period of time played a detrimental role by augmenting the apoptotic cascade in rat primary hippocampal neurons. In addition, we found that the HIV-1 gp120 V3 loop induced autophagy via AMPK/mTOR-dependent and calpain/mTOR-independent pathways, and the ERK/mTOR pathway plays a partial role. These findings provide evidence that HIV-1-induced autophagy plays a dual role in the survival and apoptosis of the primary rat hippocampal neurons and persistent autophagy may contribute to the pathogenesis of HAND, and autophagy modulation may represent a potential therapeutic strategy for reducing neuronal damage in HAND.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína gp120 do Envelope de HIV/farmacologia , HIV-1/química , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/fisiologia , Adenina/análogos & derivados , Adenina/farmacologia , Sequência de Aminoácidos , Animais , Apoptose/fisiologia , Autofagia/fisiologia , Calpaína/antagonistas & inibidores , Calpaína/fisiologia , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Flavonoides/farmacologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/toxicidade , Hipocampo/patologia , Masculino , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/toxicidade , Inibidores de Proteínas Quinases/farmacologia , Ratos Sprague-Dawley
4.
Biomed Res Int ; 2019: 4741252, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30895192

RESUMO

Proteolytic machineries execute vital cellular functions and their disturbances are implicated in diverse medical conditions, including neurodegenerative diseases. Interestingly, calpains, a class of Ca2+-dependent regulatory proteases, can modulate the degradational system of autophagy by cleaving proteins involved in this pathway. Moreover, both machineries are common players in many molecular pathomechanisms and have been targeted individually or together, as a therapeutic strategy in experimental setups. In this review, we briefly introduce calpains and autophagy, with their roles in health and disease, and focus on their direct pathologically relevant interplay in neurodegeneration and beyond. The modulation of calpain activity may comprise a promising treatment approach to attenuate the deregulation of these two essential mechanisms.


Assuntos
Autofagia , Calpaína/antagonistas & inibidores , Doenças Neurodegenerativas/patologia , Animais , Calpaína/química , Calpaína/metabolismo , Glicoproteínas/farmacologia , Glicoproteínas/uso terapêutico , Humanos , Modelos Biológicos , Doenças Neurodegenerativas/tratamento farmacológico
5.
Food Chem ; 288: 187-192, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-30902280

RESUMO

In this study, the activity, expression and localization of nitric oxide synthase (NOS) was investigated in beef semimembranosus muscle (SM) during postmortem aging. Five beef SM muscles were vacuum-packaged and aged for 1, 3, 7 and 14 d at 4 °C. Results showed that SM muscle retained NOS activity during the 14 d storage period. Compared to 1 d of storage, NOS activity and neuronal NOS (nNOS) content decreased significantly at 3 and 7 d (P < 0.05). Calpain played a major role in degrading nNOS during beef postmortem aging. In addition, immunofluorescence studies suggested that nNOS in beef SM muscle was mainly distributed in the sarcolemma. The S-nitrosothiol (SNO) content in SM muscle increased significantly at 1, 3 and 7 d post-slaughter. This manuscript is the first study to demonstrate the activity and expression of NOS in beef during postmortem aging.


Assuntos
Envelhecimento , Músculo Esquelético/enzimologia , Óxido Nítrico Sintase/metabolismo , Mudanças Depois da Morte , Animais , Calpaína/antagonistas & inibidores , Calpaína/metabolismo , Caspases/metabolismo , Bovinos , Proteólise
6.
Food Chem ; 275: 77-84, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30724261

RESUMO

The aim of this study was to investigate the dual effect of the nitric oxide donor NOR-3 and calpastatin on µ-calpain activity, autolysis, and proteolytic ability. µ-Calpain and calpastatin were purified and allocated to the following five treatments: µ-calpain, µ-calpain + calpastatin, µ-calpain + NOR-3, µ-calpain + calpastatin + NOR-3, and µ-calpain + NOR-3 + calpastatin. µ-Calpain autolysis and the activity against purified myofibrils was initiated by addition of calcium. Results showed that NOR-3 could induce µ-calpain S-nitrosylation and effectively block the activity via the inhibition of µ-calpain autolysis. Calpastatin inhibited µ-calpain activity in a dose-dependent manner. The combined treatment of NOR-3 and calpastatin exerted a further inhibitory effect on µ-calpain activity, autolysis and proteolysis which was affected by the addition order of NOR-3 and calpastatin. Our data suggest that S-nitrosylation may play a regulatory role in mediating µ-calpain activity in the presence of calpastatin.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Calpaína/antagonistas & inibidores , Óxido Nítrico/metabolismo , Animais , Autólise/metabolismo , Proteínas de Ligação ao Cálcio/farmacologia , Calpaína/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Relação Dose-Resposta a Droga , Miofibrilas/metabolismo , Óxido Nítrico/farmacologia , Nitrocompostos/farmacologia , Proteólise , Suínos
7.
Neurosci Lett ; 701: 106-111, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-30807795

RESUMO

Oxidative damage in neurons including glutamate excitotoxicity has been linked to increasing numbers of neuropathological conditions. Under these conditions, cells trigger several different cellular responses such as autophagy, apoptosis, necrosis and senescence. However, the connection between these responses is not well understood. In this study, we found that the 60-kDa BECN1 was specifically degraded to a 40-kDa fragment in hippocampal HT22 cells treated with 5 mM glutamate. Increased BECN1 cleavage was specifically associated with a decrease in cell viability under oxidative stress. Interestingly, this BECN1 cleavage was specifically inhibited by a calpain inhibitor ALLN but was not affected by other protease inhibitors. Also, the BECN1 cleavage was not detected in calpain-4-deficient cell lines. Furthermore, calpain cleaved BECN1 at a specific site between the coiled-coil domain and Bcl2 homology 3 domain, which is associated with the anti-apoptotic protein Bcl-2. Moreover, some cellular senescence markers, including ß-galactosidase, p21, p27Kip1, p53 and p16INK4A, increased proportionally to those of BECN1 cleaved fragments. These results suggest that calpain-mediated BECN1 cleavage under oxidative conditions is specifically associated with cell death induced by cellular senescence.


Assuntos
Proteína Beclina-1/metabolismo , Calpaína/metabolismo , Hipocampo/metabolismo , Estresse Oxidativo/fisiologia , Animais , Apoptose/fisiologia , Calpaína/antagonistas & inibidores , Calpaína/deficiência , Caspases/metabolismo , Linhagem Celular , Sobrevivência Celular/fisiologia , Senescência Celular/fisiologia , Ácido Glutâmico/toxicidade , Células HeLa , Hipocampo/patologia , Humanos , Leupeptinas/farmacologia , Camundongos , Células NIH 3T3 , Neurônios/metabolismo , Neurônios/patologia , Espécies Reativas de Oxigênio/metabolismo
8.
Methods Mol Biol ; 1915: 187-194, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30617804

RESUMO

We here describe the purification of calpastatin from human erythrocytes. When calpastatin is purified from tissues, it is necessary to measure its inhibitory activity against calpain in the presence of Ca2+ to specifically identify the protein. Thus, the purification steps necessary to obtain the inhibitor protein were originally designed to obtain calpain from the same tissue. For this reason, in addition to calpastatin purification, we also include a method for purifying human erythrocyte calpain and globin. We routinely use these two components for assaying calpastatin inhibition.


Assuntos
Proteínas de Ligação ao Cálcio/isolamento & purificação , Calpaína/química , Eritrócitos/química , Biologia Molecular/métodos , Animais , Cálcio/química , Proteínas de Ligação ao Cálcio/química , Calpaína/antagonistas & inibidores , Calpaína/metabolismo , Humanos
9.
Methods Mol Biol ; 1915: 209-218, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30617806

RESUMO

The calpain activity in cells can be experimentally manipulated in vitro by calpain inhibitors, and various types of calpain inhibitors such as peptide aldehydes and α-mercapto-acrylic acid derivatives are widely used as a valuable tool to elucidate the physiological and pathological roles of calpain. Here I describe the experimental procedures with calpain inhibitors, with human neutrophils being primarily used in this experiment. It should be noted that potent calpain inhibitors not only inhibit the calpain activity but also stimulate cell functions via direct activation of human formyl peptide receptors and/or other G protein-coupled receptors depending on the inhibitors used.


Assuntos
Calpaína/química , Inibidores de Cisteína Proteinase/farmacologia , Glicoproteínas/química , Biologia Molecular/métodos , Aldeídos/química , Aldeídos/farmacologia , Apoptose/efeitos dos fármacos , Calpaína/antagonistas & inibidores , Inibidores de Cisteína Proteinase/química , Glicoproteínas/farmacologia , Humanos , Neutrófilos/efeitos dos fármacos , Compostos de Sulfidrila/química , Compostos de Sulfidrila/farmacologia
10.
Methods Mol Biol ; 1915: 219-232, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30617807

RESUMO

Calpain is an intracellular Ca2+-dependent non-lysosomal cysteine protease expressed ubiquitously in mammals. In endothelial cells, dysregulation of calpain has been shown to be involved in a wide variety of pathological conditions such as angiogenesis, vascular inflammation, and diabetes. Cell- or tissue-targeted in vivo delivery of small interfering RNA (siRNA) is a powerful research tool in the analysis of protein function and has been proposed as an attractive therapeutic modality that is applicable against a large number of human diseases including cancer. In this chapter we describe a method to knockdown calpain 1 in mouse pulmonary vascular endothelium using delivery of siRNA/cationic liposome complex. This technique results in a greater than 80% reduction in calpain 1 protein levels 48 h after a single i.v. injection of calpain 1 siRNA (0.5 mg siRNA/kg)/cationic liposome complex. We also describe confocal imaging to verify the loss of calpain 1 expression in pulmonary microvessel endothelial cells and application of this technique in the mouse model of ventilator-induced lung injury.


Assuntos
Calpaína/genética , Células Endoteliais/química , Técnicas de Silenciamento de Genes/métodos , RNA Interferente Pequeno/genética , Animais , Calpaína/antagonistas & inibidores , Células Endoteliais/metabolismo , Endotélio Vascular/química , Endotélio Vascular/metabolismo , Humanos , Lipossomos/química , Pulmão/química , Pulmão/metabolismo , Camundongos , RNA Interferente Pequeno/química
11.
Methods Mol Biol ; 1915: 249-259, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30617809

RESUMO

Calpains are a family of calcium-dependent intracellular cysteine proteases that regulate important physiological processes by substrate cleavage. Despite the fact that the role of calpains in cell migration and other processes has been extensively studied in vitro, the same does not apply to cell migration and morphogenetic events during embryogenesis, in vivo. Herein, we describe the use of three different methods to selectively block calpain activity in vivo in order to investigate the impact on Xenopus gastrulation and neurulation, namely, a calpain inhibitor, a dominant negative, and a morpholino antisense oligonucleotide (MO). We also provide methods to determine the effectiveness of the calpain inhibition and effect on cell fate specification and morphogenetic movements, during embryogenesis in vivo.


Assuntos
Calpaína/genética , Desenvolvimento Embrionário/genética , Glicoproteínas/administração & dosagem , Biologia Molecular/métodos , Animais , Cálcio/química , Calpaína/antagonistas & inibidores , Movimento Celular/genética , Xenopus laevis/embriologia , Xenopus laevis/genética
12.
J Cell Physiol ; 234(2): 1001-1007, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30145792

RESUMO

Dysregulation of neuronal Ca2+ and oxidative stress plays an important role in the activation of cysteine proteases including calpains and caspases that contribute to neuronal death. In neurodegenerative diseases, traumatic brain injury, stroke, and neuropathic pain calpain activities are markedly increased. Melatonin is a beneficial supplement in the treatment of central nervous system (CNS) disorders. Melatonin is a potent antioxidant and works as a free-radical scavenger to regulate a large number of molecular pathways, including oxidative stress, inflammation, apoptosis, and cell death under different pathological conditions. However, limited studies have evaluated the inhibitory effect of melatonin on calpains. This review summarizes the current knowledge related to the effects of melatonin on calpains in some of the common CNS disorders.


Assuntos
Calpaína/antagonistas & inibidores , Doenças do Sistema Nervoso Central/tratamento farmacológico , Sistema Nervoso Central/efeitos dos fármacos , Inibidores de Cisteína Proteinase/uso terapêutico , Melatonina/uso terapêutico , Animais , Calpaína/metabolismo , Sistema Nervoso Central/enzimologia , Sistema Nervoso Central/patologia , Sistema Nervoso Central/fisiopatologia , Doenças do Sistema Nervoso Central/enzimologia , Doenças do Sistema Nervoso Central/patologia , Doenças do Sistema Nervoso Central/fisiopatologia , Humanos , Transdução de Sinais
13.
Mol Neurobiol ; 56(6): 4414-4427, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30327977

RESUMO

Spinal muscular atrophy (SMA), a leading genetic cause of infant death, is caused by the loss of survival motor neuron 1 (SMN1) gene. SMA is characterized by the degeneration and loss of spinal cord motoneurons (MNs), muscular atrophy, and weakness. SMN2 is the centromeric duplication of the SMN gene, whose numbers of copies determine the intracellular levels of SMN protein and define the disease onset and severity. It has been demonstrated that elevating SMN levels can be an important strategy in treating SMA and can be achieved by several mechanisms, including promotion of protein stability. SMN protein is a direct target of the calcium-dependent protease calpain and induces its proteolytic cleavage in muscle cells. In this study, we examined the involvement of calpain in SMN regulation on MNs. In vitro experiments showed that calpain activation induces SMN cleavage in CD1 and SMA mouse spinal cord MNs. Additionally, calpain 1 knockdown or inhibition increased SMN level and prevent neurite degeneration in these cells. We examined the effects of calpain inhibition on the phenotype of two severe SMA mouse models. Treatment with the calpain inhibitor, calpeptin, significantly improved the lifespan and motor function of these mice. Our observations show that calpain regulates SMN level in MNs and calpeptin administration improves SMA phenotype demonstrating the potential utility of calpain inhibitors in SMA therapy.


Assuntos
Calpaína/antagonistas & inibidores , Neurônios Motores/patologia , Atrofia Muscular Espinal/patologia , Medula Espinal/patologia , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Animais , Calpaína/metabolismo , Células Cultivadas , Dipeptídeos/administração & dosagem , Dipeptídeos/farmacologia , Técnicas de Silenciamento de Genes , Glicoproteínas/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/complicações , Atrofia Muscular Espinal/fisiopatologia , Mutação/genética , Degeneração Neural/complicações , Degeneração Neural/patologia , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Fenótipo , Potássio/farmacologia
14.
J Pharm Biomed Anal ; 164: 365-372, 2019 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-30439664

RESUMO

Vitis vinifera grape is a highly cultivated crop and solid wastes generated by the wine industry are largely under exploited. Plentiful studies have intended analyzing the polyphenolic content of grape seeds but characterization of non phenolic compounds is rather scarce. The present study aimed at the selective extraction of lipid, phenolic and aqueous phases from grape seed powder (GSP) in order to establish their intimate composition, as well as their antioxidant and chelating properties underlying partly their biological effects. Major non phenolic compounds identified in the lipid phase were glyceryl-monostearate and 2-monostearin whereas fructofuranose and sucrose were the most abundant in the aqueous phase. Among the most abundant compounds detected in the various phases, the polyphenol quercetin exhibited the best affinity and free binding energy towards the active site of the calcium-dependent protease calpain. Polyphenols likely constitute the bioactive part of GSP that should be exploited as safe modulators of intracellular signaling which is likely at the basis of their health beneficial effects. Nevertheless other compounds as lipids or sugars should be valorized along with polyphenols to improve their bioavailability into highly protected organs as brain or eye.


Assuntos
Antioxidantes/farmacologia , Calpaína/antagonistas & inibidores , Extrato de Sementes de Uva/farmacologia , Vitis/química , Antioxidantes/análise , Antioxidantes/química , Calpaína/química , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Glicerídeos/análise , Glicerídeos/química , Glicerídeos/farmacologia , Extrato de Sementes de Uva/análise , Extrato de Sementes de Uva/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estresse Oxidativo , Polifenóis/análise , Polifenóis/química , Polifenóis/farmacologia , Pós , Sementes/química , Sacarose/análise , Sacarose/química , Sacarose/farmacologia
15.
Neuroscience ; 397: 159-171, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30496824

RESUMO

The main component of Alzheimer's disease (AD) is the amyloid-beta peptide (Aß), the brain of these patients is characterized by deposits in the parenchyma and cerebral blood vessels known as cerebral amyloid angiopathy (CAA). On the other hand, the platelets are the major source of the Aß peptide in circulation and once secreted can activate the platelets and endothelial cells producing the secretion of several inflammatory mediators that finally end up unchaining the CAA and later AD. In the present study we demonstrate that cAMP/PKA pathway plays key roles in the regulation of calpain activation and secretion of Aß in human platelets. We confirmed that inhibition of platelet functionality occurred when platelets were incubated with forskolin (molecule that rapidly increased cAMP levels). In this sense we found that platelets pre-incubated with forskolin (20 µM) present a complete inhibition of calpain activity and this effect is reversed using an inhibitor of protein kinase A. Consequentially, when platelets were inhibited by forskolin a reduction in the processing of the APP with the consequent decrease in the Aß peptide secretion was observed. Therefore our study provides novel insight in relation to the mechanism of processing and release of the Aß peptide from human platelets.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Plaquetas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Adulto , Doença de Alzheimer/sangue , Precursor de Proteína beta-Amiloide/metabolismo , Plaquetas/efeitos dos fármacos , Calpaína/antagonistas & inibidores , Calpaína/metabolismo , Fármacos Cardiovasculares/farmacologia , Células Cultivadas , Colforsina/farmacologia , Simulação por Computador , Humanos , Modelos Moleculares , Selectina-P/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/fisiologia , Transdução de Sinais/efeitos dos fármacos , Adulto Jovem
16.
Sci Rep ; 8(1): 18083, 2018 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-30591714

RESUMO

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are neurodegenerative disorders of the aging population characterized by the accumulation of α-synuclein (α-syn). The mechanisms triggering α-syn toxicity are not completely understood, however, c-terminus truncation of α-syn by proteases such as calpain may have a role. Therefore, inhibition of calpain may be of value. The main objective of this study was to evaluate the effects of systemically administered novel low molecular weight calpain inhibitors on α-syn pathology in a transgenic mouse model. For this purpose, non-tg and α-syn tg mice received the calpain inhibitors - Gabadur, Neurodur or a vehicle, twice a day for 30 days. Immunocytochemical analysis showed a 60% reduction in α-syn deposition using Gabadur and a 40% reduction using Neurodur with a concomitant reduction in c-terminus α-syn and improvements in neurodegeneration. Western blot analysis showed a 77% decrease in α-spectrin breakdown products (SBDPs) SBDPs with Gabadur and 63% reduction using Neurodur. There was a 65% reduction in the active calpain form with Gabadur and a 45% reduction with Neurodur. Moreover, treatment with calpain inhibitors improved activity performance of the α-syn tg mice. Taken together, this study suggests that calpain inhibition might be considered in the treatment of synucleinopathies.


Assuntos
Calpaína/antagonistas & inibidores , Glicoproteínas/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Modelos Animais de Doenças , Imuno-Histoquímica , Doença por Corpos de Lewy/tratamento farmacológico , Doença por Corpos de Lewy/etiologia , Camundongos , Camundongos Transgênicos , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/etiologia , alfa-Sinucleína/química
17.
Physiol Rep ; 6(21): e13833, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30393967

RESUMO

Chronic rotator cuff (RC) tears are characterized by retraction, fat accumulation, and atrophy of the affected muscle. These features pose an intractable problem for surgical repair and subsequent recovery, and their prevention may be easier than reversal. Using an established ovine model, we tested the hypothesis that inhibition of the protease calpain mitigates m. infraspinatus atrophy by preservation of the myofibers' structural anchors in the sarcolemma (the costameres). Already 2 weeks of distal tendon release led to a reduction in muscle volume (-11.6 ± 9.1 cm3 , P = 0.038) and a 8.3% slow-to-fast shift of the fiber area (P = 0.046), which were both entirely abolished by chronic local administration of the calpain inhibitor calpeptin alone, and in combination with sildenafil. Calpain inhibition blunted the retraction of the muscle-tendon unit by 0.8-1.0 cm (P = 0.020) compared with the control group, and prevented cleavage of the costameric protein talin. Calpain 1 and 2 protein levels increased in the medicated groups after 4 weeks, counteracting the efficacy of calpeptin. Hence atrophic changes emerged after 4 weeks despite ongoing treatment. These findings suggest that the early muscular adaptations in the specific case of RC tear in the ovine model are indistinguishable from the atrophy and slow-to-fast fiber transformation observed with conventional unloading and can be prevented for 2 weeks. Concluding, calpain is a potential target to extend the temporal window for reconstruction of the ruptured RC tendon before recovery turns impossible.


Assuntos
Calpaína/antagonistas & inibidores , Inibidores de Cisteína Proteinase/uso terapêutico , Dipeptídeos/uso terapêutico , Atrofia Muscular/tratamento farmacológico , Lesões do Manguito Rotador/tratamento farmacológico , Animais , Calpaína/metabolismo , Feminino , Atrofia Muscular/etiologia , Lesões do Manguito Rotador/complicações , Sarcolema/metabolismo , Ovinos
18.
Artigo em Inglês | MEDLINE | ID: mdl-30319995

RESUMO

Oxygen or nitrogen oxidative species and chemical stress induce the programmed cell death (PCD) of Entamoeba histolytica trophozoites. PCD caused by the aminoglycoside G418 is reduced by incubation with the cysteine protease inhibitor E-64; however, no typical caspases or metacaspases have been detected in this parasite. Calpain, a cysteine protease activated by calcium, has been suggested to be part of a specific PCD pathway in this parasite because the specific calpain inhibitor Z-Leu-Leu-Leu-al diminishes the PCD of trophozoites. Here, we predicted the hypothetical 3D structure of a calpain-like protein of E. histolytica and produced specific antibodies against it. We detected the protein in the cytoplasm and near the nucleus. Its expression gradually increased during incubation with G418, with the highest level after 9 h of treatment. In addition, a specific calpain-like siRNA sequence reduced the cell death rate by 65%. All these results support the hypothesis that the calpain-like protein is one of the proteases involved in the execution phase of PCD in E. histolytica. The hypothetical interactome of the calpain-like protein suggests that it may activate or regulate other proteins that probably participate in PCD, including those with EF-hand domains or other calcium-binding sites.


Assuntos
Apoptose , Calpaína/metabolismo , Entamoeba histolytica/fisiologia , Calpaína/antagonistas & inibidores , Biologia Computacional , Entamoeba histolytica/efeitos dos fármacos , Ativadores de Enzimas/metabolismo , Gentamicinas/metabolismo , Mapas de Interação de Proteínas
19.
Medicina (Kaunas) ; 54(3)2018 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-30344270

RESUMO

Background and Objective: Although triptolide was effective for prostate cancer (PCa), the mechanism is still unclear. Androgen receptor (AR) plays a large role in the development and progression of PCa, even after castration. The present study aimed at investigating the effects of triptolide on AR protein stability and the possible mechanism. Materials and Methods: By blocking protein synthesis with cycloheximide (CHX), the effect of triptolide on AR protein stability was investigated with western blot assay. The potential role of calpains in triptolide reduced AR protein stability was investigated with calpain inhibitor and Ca2+ chelator. Results: Triptolide down-regulated AR protein level when protein synthesis was blocked by CHX, demonstrating the decrease of AR protein stability. The AR protein level was restored when the cells were co-treated with triptolide and calpain inhibitor or Ca2+ chelator, indicating the important role of calpains. Conclusions: The results indicate that triptolide can activate calpain via promoting intracellular Ca2+ accumulation, and thus decrease the stability of AR protein, subsequently resulting in the breakdown of the AR protein in LNCaP cells. This work provides an experimental basis and evidence to elucidate the anti-PCa mechanisms of triptolide.


Assuntos
Antineoplásicos Alquilantes/farmacologia , Diterpenos/farmacologia , Fenantrenos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Estabilidade Proteica/efeitos dos fármacos , Receptores Androgênicos/efeitos dos fármacos , Western Blotting , Calpaína/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cicloeximida , Compostos de Epóxi/farmacologia , Humanos , Masculino
20.
J Biol Chem ; 293(46): 17716-17730, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30254072

RESUMO

Calpains are intracellular, calcium-activated cysteine proteases. Calpain-3 is abundant in skeletal muscle, where its mutation-induced loss of function causes limb-girdle muscular dystrophy type 2A. Unlike the small subunit-containing calpain-1 and -2, the calpain-3 isoform homodimerizes through pairing of its C-terminal penta-EF-hand domain. It also has two unique insertion sequences (ISs) not found in the other calpains: IS1 within calpain-3's protease core and IS2 just prior to the penta-EF-hand domain. Production of either native or recombinant full-length calpain-3 to characterize the function of these ISs is challenging. Therefore, here we used recombinant rat calpain-2 as a stable surrogate and inserted IS1 into its equivalent position in the protease core. As it does in calpain-3, IS1 occupied the catalytic cleft and restricted the enzyme's access to substrate and inhibitors. Following activation by Ca2+, IS1 was rapidly cleaved by intramolecular autolysis, permitting the enzyme to freely accept substrate and inhibitors. The surrogate remained functional until extensive intermolecular autoproteolysis inactivated the enzyme, as is typical of calpain-2. Although the small-molecule inhibitors E-64 and leupeptin limited intermolecular autolysis of the surrogate, they did not block the initial intramolecular cleavage of IS1, establishing its role as a propeptide. Surprisingly, the large-molecule calpain inhibitor, calpastatin, completely blocked enzyme activity, even with IS1 intact. We suggest that calpastatin is large enough to oust IS1 from the catalytic cleft and take its place. We propose an explanation for why calpastatin can inhibit calpain-2 bearing the IS1 insertion but cannot inhibit WT calpain-3.


Assuntos
Calpaína/metabolismo , Elementos de DNA Transponíveis , Isoenzimas/metabolismo , Proteínas Musculares/metabolismo , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/química , Calpaína/antagonistas & inibidores , Calpaína/genética , Calpaína/isolamento & purificação , Inibidores de Cisteína Proteinase/química , Isoenzimas/genética , Leucina/análogos & derivados , Leucina/química , Leupeptinas/química , Proteínas Musculares/genética , Conformação Proteica , Proteólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
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