RESUMO
High-pressure and temperature extraction (HPTE) can effectively recover bioactive compounds from olive pomace (OP). HPTE extract obtained by extracting OP with ethanol and water (50:50 v/v) at 180 °C for 90 min demonstrated a pronounced ability to preserve intracellular calcium homeostasis, shielding neurons from the harmful effects induced by N-methyl-d-aspartate (NMDA) receptor (NMDAR) overactivation, such as aberrant calpain activation. In this study, the extraction temperature was changed from 37 to 180 °C, and the extracts were evaluated for their antioxidant potency and ability to preserve crucial intracellular Ca2+-homeostasis necessary for neuronal survival. Additionally, to verify the temperature-induced activity of the extract, further extractions on the exhausted olive pomace were conducted, aiming to identify variations in the quality and quantity of extracted phenolic molecules through HPLC analysis. The results revealed a significant increase in bioactive compounds as a function of temperature variation, reaching 6.31 ± 0.09 mgCAE/mL extract for the extraction performed at 180 °C. Subsequent extraction of the exhausted residues yielded extracts that remained active in preventing calcium-induced cell death. Moreover, despite increased antiradical power, extracts re-treated at 180 °C did not display cell protection activity. Our results indicate that the molecules able to maintain physiological Ca2+-homeostasis in murine cortical neurons in conditions of cytotoxic stimulation of NMDAR are wholly recovered from olive pomace only following extraction performed at 180 °C.
Assuntos
Olea , Animais , Camundongos , Cálcio , Temperatura , Neurônios , Receptores de N-Metil-D-Aspartato , Extratos Vegetais/farmacologiaRESUMO
BACKGROUND: The most recent modulator combination, elexacaftor/tezacaftor/ivacaftor (Trikafta®), has been shown to improve clinical outcomes in most patients with cystic fibrosis (PwCF). Unfortunately, the clinical benefits are sometimes variable; thus, improving our knowledge of the possible causes of this variability can help reduce it. METHODS: Circulating mononuclear cells (CMCs) and plasma were collected from 16 PwCF (including those on Trikafta® therapy) and 4 non-CF subjects. Cystic fibrosis transmembrane conductance regulator (CFTR) activity and matrix metalloprotease 9 (MMP9) expression were monitored before and after therapy, together with some clinical parameters. The relationship between MMP9 expression and the modulation of the extracellular-regulated 1/2 (ERK1/2) and nuclear factor-kB (NF-kB) pathways was also analyzed. RESULTS: MMP9, markedly expressed in the CMCs and plasma of all the patients included in the study, was downregulated in the clinically responsive PwCF. In the non-responder, the MMP9 levels remained high. The modulation of MMP9 following treatment with Trikafta® may be controlled by the NF-kB pathway. CONCLUSIONS: These data strongly suggest that MMP9 downregulation is a potential biomarker of therapy efficacy and that it could be useful in understanding the molecular events underlying the variable clinical responses of patients to Trikafta®. This knowledge could be helpful for future studies of personalized medicine and thereby ensure improvements in individual responses to therapies.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Humanos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Metaloproteinase 9 da Matriz/genética , NF-kappa BRESUMO
The ability of oxytocin (OT) to interact with the dopaminergic system through facilitatory D2-OT receptor (OTR) receptor-receptor interaction in the limbic system is increasingly considered to play roles in social or emotional behavior, and suggested to serve as a potential therapeutic target. Although roles of astrocytes in the modulatory effects of OT and dopamine in the central nervous system are well recognized, the possibility of D2-OTR receptor-receptor interaction in astrocytes has been neglected. In purified astrocyte processes from adult rat striatum, we assessed OTR and dopamine D2 receptor expression by confocal analysis. The effects of activation of these receptors were evaluated in the processes through a neurochemical study of glutamate release evoked by 4-aminopyridine; D2-OTR heteromerization was assessed by co-immunoprecipitation and proximity ligation assay (PLA). The structure of the possible D2-OTR heterodimer was estimated by a bioinformatic approach. We found that both D2 and OTR were expressed on the same astrocyte processes and controlled the release of glutamate, showing a facilitatory receptor-receptor interaction in the D2-OTR heteromers. Biochemical and biophysical evidence confirmed D2-OTR heterodimers on striatal astrocytes. The residues in the transmembrane domains four and five of both receptors are predicted to be mainly involved in the heteromerization. In conclusion, roles for astrocytic D2-OTR in the control of glutamatergic synapse functioning through modulation of astrocytic glutamate release should be taken into consideration when considering interactions between oxytocinergic and dopaminergic systems in striatum.
Assuntos
Astrócitos , Corpo Estriado , Receptores de Dopamina D2 , Receptores de Ocitocina , Animais , Ratos , Astrócitos/metabolismo , Corpo Estriado/metabolismo , Dopamina/metabolismo , Ácido Glutâmico/metabolismo , Ocitocina/metabolismo , Receptores de Ocitocina/química , Receptores de Ocitocina/metabolismo , Receptores de Dopamina D2/química , Receptores de Dopamina D2/metabolismoRESUMO
BACKGROUND: Roles of astrocytes in the modulatory effects of oxytocin (OT) in central nervous system are increasingly considered. Nevertheless, OT effects on gliotransmitter release have been neglected. METHODS: In purified astrocyte processes from adult rat striatum, we assessed OT receptor (OTR) and adenosine A2A receptor expression by confocal analysis. The effects of receptors activation on glutamate release from the processes were evaluated; A2A-OTR heteromerization was assessed by co-immunoprecipitation and PLA. Structure of the possible heterodimer of A2A and OT receptors was estimated by a bioinformatic approach. RESULTS: Both A2A and OT receptors were expressed on the same astrocyte processes. Evidence for A2A-OTR receptor-receptor interaction was obtained by measuring the release of glutamate: OT inhibited the evoked glutamate release, while activation of A2A receptors, per se ineffective, abolished the OT effect. Biochemical and biophysical evidence for A2A-OTR heterodimers on striatal astrocytes was also obtained. The residues in the transmembrane domains 4 and 5 of both receptors are predicted to be mainly involved in the heteromerization. CONCLUSIONS: When considering effects of OT in striatum, modulation of glutamate release from the astrocyte processes and of glutamatergic synapse functioning, and the interaction with A2A receptors on the astrocyte processes should be taken into consideration.
Assuntos
Astrócitos/metabolismo , Ácido Glutâmico/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptores de Ocitocina/metabolismo , Animais , Corpo Estriado/metabolismo , Masculino , Neostriado/metabolismo , Ocitocina/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The aim of this study was the identification of specific proteomic profiles, related to a restored cystic fibrosis transmembrane conductance regulator (CFTR) activity in cystic fibrosis (CF) leukocytes before and after ex vivo treatment with the potentiator VX770. We used leukocytes, isolated from CF patients carrying residual function mutations and eligible for Ivacaftor therapy, and performed CFTR activity together with proteomic analyses through micro-LC-MS. Bioinformatic analyses of the results obtained revealed the downregulation of proteins belonging to the leukocyte transendothelial migration and regulation of actin cytoskeleton pathways when CFTR activity was rescued by VX770 treatment. In particular, we focused our attention on matrix metalloproteinase 9 (MMP9), because the high expression of this protease potentially contributes to parenchyma lung destruction and dysfunction in CF. Thus, the downregulation of MMP9 could represent one of the possible positive effects of VX770 in decreasing the disease progression, and a potential biomarker for the prediction of the efficacy of therapies targeting the defect of Cl- transport in CF.
Assuntos
Aminofenóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Metaloproteinase 9 da Matriz/genética , Quinolonas/farmacologia , Citoesqueleto de Actina/genética , Adulto , Biomarcadores/sangue , Movimento Celular/efeitos dos fármacos , Fibrose Cística/sangue , Fibrose Cística/genética , Fibrose Cística/patologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Proteoma/genéticaRESUMO
We have recently demonstrated that bioactive molecules, extracted by high pressure and temperature from olive pomace, counteract calcium-induced cell damage to different cell lines. Here, our aim was to study the effect of the same extract on murine cortical neurons, since the preservation of the intracellular Ca2+-homeostasis is essential for neuronal function and survival. Accordingly, we treated neurons with different stimuli in order to evoke cytotoxic glutamatergic activation. In these conditions, the high-pressure and temperature extract from olive pomace (HPTOPE) only abolished the effects of N-methyl-d-aspartate (NMDA). Particularly, we observed that HPTOPE was able to promote the neuron rescue from NMDA-induced cell death. Moreover, we demonstrated that HPTOPE is endowed with the ability to maintain the intracellular Ca2+-homeostasis following NMDA receptor overactivation, protecting neurons from Ca2+-induced adverse effects, including aberrant calpain proteolytic activity. Moreover, we highlight the importance of the extraction conditions used that, without producing toxic molecules, allow us to obtain protecting molecules belonging to proanthocyanidin derivatives like procyanidin B2. In conclusion, we can hypothesize that HPTOPE, due to its functional and nontoxic properties on neuronal primary culture, can be utilized for future therapeutic interventions for neurodegeneration.
Assuntos
Biflavonoides/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Catequina/farmacologia , N-Metilaspartato/efeitos adversos , Neurônios/metabolismo , Olea/química , Extratos Vegetais/farmacologia , Proantocianidinas/farmacologia , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Biflavonoides/química , Catequina/química , Morte Celular/efeitos dos fármacos , Células Cultivadas , Camundongos , N-Metilaspartato/farmacologia , Neurônios/patologia , Extratos Vegetais/química , Proantocianidinas/químicaRESUMO
Our previous findings indicate that A2A and D2 receptors are co-expressed on adult rat striatal astrocytes and on the astrocyte processes, and that A2A-D2 receptorâ»receptor interaction can control the release of glutamate from the processes. Functional evidence suggests that the receptorâ»receptor interaction was based on heteromerization of native A2A and D2 receptors at the plasma membrane of striatal astrocyte processes. We here provide biochemical and biophysical evidence confirming that receptorâ»receptor interaction between A2A and D2 receptors at the astrocyte plasma membrane is based on A2A-D2 heteromerization. To our knowledge, this is the first direct demonstration of the ability of native A2A and D2 receptors to heteromerize on glial cells. As striatal astrocytes are recognized to be involved in Parkinson's pathophysiology, the findings that adenosine A2A and dopamine D2 receptors can form A2A-D2 heteromers on the astrocytes in the striatum (and that these heteromers can play roles in the control of the striatal glutamatergic transmission) may shed light on the molecular mechanisms involved in the pathogenesis of the disease.
Assuntos
Astrócitos/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptores de Dopamina D2/metabolismo , Animais , Membrana Celular/metabolismo , Corpo Estriado/metabolismo , Ácido Glutâmico/metabolismo , Multimerização Proteica , Ratos , Ratos Sprague-Dawley , Receptor A2A de Adenosina/química , Receptores de Dopamina D2/químicaRESUMO
Matrix metalloproteinase 9 (MMP9) is physiologically involved in remodeling the extracellular matrix components but its abnormal release has been observed in several human pathologies. We here report that peripheral blood mononuclear cells (PBMCs), isolated from cystic fibrosis (CF) patients homozygous for F508del-cystic fibrosis transmembrane conductance regulator (CFTR), express constitutively and release at high rate MMP9 due to the alteration in their intracellular Ca(2+) homeostasis. This spontaneous and sustained MMP9 secretion may contribute to the accumulation of this protease in fluids of CF patients. Conversely, in PBMCs isolated from healthy donors, expression and secretion of MMP9 are undetectable but can be evoked, after 12 h of culture, by paracrine stimulation which also promotes an increase in [Ca(2+)]i. We also demonstrate that in both CF and control PBMCs the Ca(2+)-dependent MMP9 secretion is mediated by the concomitant activation of calpain and protein kinase Cα (PKCα), and that MMP9 expression involves extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) phosphorylation. Our results are supported by the fact that either the inhibition of Ca(2+) entry or chelation of [Ca(2+)]i as well as the inhibition of single components of the signaling pathway or the restoration of CFTR activity all promote the reduction of MMP9 secretion.
Assuntos
Calpaína/sangue , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/sangue , Leucócitos Mononucleares/metabolismo , Metaloproteinase 9 da Matriz/sangue , Proteína Quinase C-alfa/sangue , Adolescente , Adulto , Idoso , Cálcio/metabolismo , Ativação Enzimática , Células Epiteliais/citologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Deleção de Genes , Regulação Enzimológica da Expressão Gênica , Homeostase , Homozigoto , Humanos , Pessoa de Meia-Idade , Fosforilação , Valor Preditivo dos Testes , Adulto JovemRESUMO
Here we demonstrate that the presence of the L-domain in calpastatins induces biphasic interaction with calpain. Competition experiments revealed that the L-domain is involved in positioning the first inhibitory unit in close and correct proximity to the calpain active site cleft, both in the closed and in the open conformation. At high concentrations of calpastatin, the multiple EF-hand structures in domains IV and VI of calpain can bind calpastatin, maintaining the active site accessible to substrate. Based on these observations, we hypothesize that two distinct calpain-calpastatin complexes may occur in which calpain can be either fully inhibited (I) or fully active (II). In complex II the accessible calpain active site can be occupied by an additional calpastatin molecule, now a cleavable substrate. The consequent proteolysis promotes the accumulation of calpastatin free inhibitory units which are able of improving the capacity of the cell to inhibit calpain. This process operates under conditions of prolonged [Ca(2+)] alteration, as seen for instance in Familial Amyotrophic Lateral Sclerosis (FALS) in which calpastatin levels are increased. Our findings show that the L-domain of calpastatin plays a crucial role in determining the formation of complexes with calpain in which calpain can be either inhibited or still active. Moreover, the presence of multiple inhibitory domains in native full-length calpastatin molecules provides a reservoir of potential inhibitory units to be used to counteract aberrant calpain activity.
RESUMO
Glioblastoma (GBM) is the most malignant brain tumor, characterized by cell heterogeneity comprising stem cells (GSCs) responsible for aggressiveness. The calpain/calpastatin (calp/cast) proteolytic system is involved in critical physiological processes and cancer progression. In this work we showed the expression profile of hcast 3-25 (a Type III calpastatin variant devoid of inhibitory units) and the members of the system in several patient-derived GSCs exploring the relationship between hcast 3-25 and activation/activity of calpains. Each GSC shows a peculiar calp/cast mRNA and protein expression pattern, and hcast 3-25 is the least expressed. Differentiation promotes upregulation of all the calp/cast system components except hcast 3-25 mRNA, which increased or decreased depending on individual GSC culture. Transfection of hcast 3-25-V5 into two selected GSCs indicated that hcast 3-25 effectively associates with calpains, supporting the digestion of selected calpain targets. Hcast 3-25 possibly affects the stem state promoting a differentiated, less aggressive phenotype.
RESUMO
We are here showing that peripheral mononuclear blood cells (PBMC) from cystic fibrosis (CF) patients contain almost undetectable amounts of mature 170 kDa CF-transmembrane conductance regulator (CFTR) and a highly represented 100 kDa form. This CFTR protein, resembling the form produced by calpain digestion and present, although in lower amounts, also in normal PBMC, is localized in cytoplasmic internal vesicles. These observations are thus revealing that the calpain-mediated proteolysis is largely increased in cells from CF patients. To characterize the process leading to the accumulation of such split CFTR, FRT cells expressing the F508del-CFTR mutated channel protein and human leukaemic T cell line (JA3), expressing wild type CFTR were used. In in vitro experiments, the sensitivity of the mutated channel to the protease is identical to that of the wild type, whereas in Ca(2+)-loaded cells F508del-CFTR is more susceptible to digestion. Inhibition of intracellular calpain activity prevents CFTR degradation and leads to a 10-fold increase in the level of F508del-CFTR at the plasma membrane, further indicating the involvement of calpain activity in the maintenance of very low levels of mature channel form. The higher sensitivity to calpain of the mutated 170 kDa CFTR results from a reduced affinity for HSP90 causing a lower degree of protection from calpain digestion. The recovery of HSP90 binding capacity in F508del-CFTR, following digestion, explains the large accumulation of the 100 kDa CFTR form in circulating PBMC from CF patients.
Assuntos
Calpaína/metabolismo , Membrana Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Immunoblotting , Imunoprecipitação , Mutação/genética , Transporte Proteico , Ratos , Ratos Endogâmicos F344 , Deleção de SequênciaRESUMO
We are here reporting that in peripheral blood mononuclear cells (PBMC) of patients homozygous for F508del-CFTR the calpain-calpastatin system undergoes a profound alteration. In fact, calpain basal activity, almost undetectable in control PBMC, becomes measurable at a significant extent in cells from cystic fibrosis (CF) patients, also due to a 40-60% decrease in both calpastatin protein and inhibitory activity. Constitutive protease activation in CF patients' cells induces a large accumulation of the mutated cystic fibrosis transmembrane conductance regulator (CFTR) in the 100kD+70kD split forms as well as a degradation of proteins associated to the CFTR complex. Specifically, the scaffolding protein Na(+)/H(+) exchanger 3 regulatory factor-1 (NHERF-1) is converted in two distinct fragments showing masses of 35kD and 20kD, being however the latter form the most represented one, thereby indicating that in CF-PBMC the CFTR complex undergoes a large disorganization. In conclusion, our observations are providing new information on the role of calpain in the regulation of plasma membrane ion conductance and provide additional evidence on the transition of this protease activity from a physiological to a pathological function.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Calpaína/metabolismo , Fibrose Cística/metabolismo , Leucócitos Mononucleares/metabolismo , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Adolescente , Adulto , Proteínas de Ligação ao Cálcio/química , Calpaína/antagonistas & inibidores , Estudos de Casos e Controles , Criança , Fibrose Cística/sangue , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Proteínas do Citoesqueleto/metabolismo , Ativação Enzimática , Ensaios Enzimáticos , Feminino , Humanos , Leucócitos Mononucleares/enzimologia , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Isoformas de Proteínas/metabolismo , Transporte Proteico , Proteólise , Adulto JovemRESUMO
In mammalian cells, the content of polyamines is tightly regulated. Polyamines, including spermine, spermidine and putrescine, are involved in many cellular processes. Spermine oxidase specifically oxidizes spermine, and its deregulated activity has been reported to be linked to brain pathologies involving neuron damage. Spermine is a neuromodulator of a number of ionotropic glutamate receptors and types of ion channels. In this respect, the Dach-SMOX mouse model overexpressing spermine oxidase in the neocortex neurons was revealed to be a model of chronic oxidative stress, excitotoxicity and neuronal damage. Reactive astrocytosis, chronic oxidative and excitotoxic stress, neuron loss and the susceptibility to seizure in the Dach-SMOX are discussed here. This genetic model would help researchers understand the linkage between polyamine dysregulation and neurodegeneration and unveil the roles of polyamines in the crosstalk between astrocytes and neurons in neuroprotection or neurodegeneration.
RESUMO
Persistent dysregulation in Ca(2+) homeostasis is a pervasive pathogenic mechanism in most neurodegenerative diseases, and accordingly, calpain activation has been implicated in neuronal cells dysfunction and death. In this study we examined the intracellular functional state of the calpain-calpastatin system in -G93A(+) SOD1 transgenic mice to establish if and how uncontrolled activation of calpain can be prevented in vivo during the course of prolonged [Ca(2+)](i) elevation. The presented data indicate that 1) calpain activation is more extensive in motor cortex, in lumbar, and sacral spinal cord segments compared with the lower or almost undetectable activation of the protease in other brain areas, 2) direct measurements of the variations of Ca(2+) levels established that the degree of the protease activation is correlated to the extent of elevation of [Ca(2+)](i), 3) intracellular activation of calpain is always associated with diffusion of calpastatin from perinuclear aggregated forms into the cytosol and the formation of a calpain-calpastatin complex, and 4) a conservative fragmentation of calpastatin is accompanied by its increased expression and inhibitory capacity in conditions of prolonged increase in [Ca(2+)](i). Thus, calpastatin diffusion and formation of the calpain-calpastatin complex together with an increased synthesis of the inhibitor protein represent a cellular defense response to conditions of prolonged dysregulation in intracellular Ca(2+) homeostasis. Altogether these findings provide a new understanding of the in vivo molecular mechanisms governing calpain activation that can be extended to many neurodegenerative diseases, potentially useful for the development of new therapeutic approaches.
Assuntos
Encéfalo/citologia , Encéfalo/enzimologia , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Calpaína/metabolismo , Homeostase , Animais , Proteínas de Ligação ao Cálcio/genética , Calpaína/genética , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica , Humanos , Espaço Intracelular/enzimologia , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Camundongos Transgênicos , Músculos/citologia , Músculos/enzimologia , Neurônios/citologia , Neurônios/enzimologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Medula Espinal/citologia , Medula Espinal/enzimologia , Especificidade por Substrato , Extratos de TecidosRESUMO
The level of the mature native 170 kDa form of CFTR (cystic fibrosis transmembrane conductance regulator) at the plasma membrane is under the control of a selective proteolysis catalysed by calpain. The product of this limited digestion, consisting of discrete fragments still associated by strong interactions, is removed from the plasma membrane and internalized in vesicles and subject to an additional degradation. This process can be monitored by visualizing the accumulation of a 100 kDa fragment in a proliferating human leukaemic T-cell line and in human circulating lymphocytes. In reconstructed systems, and in intact cells, the conversion of native CFTR into the 100 kDa fragment linearly correlated with calpain activation and was prevented by addition of synthetic calpain inhibitors. A reduction in Ca2+ influx, by blocking the NMDA (N-methyl-D-aspartate) receptor Ca2+ channel, inhibited the conversion of the native 170 kDa fragment into the 100 kDa fragment, whereas an endosome acidification blocker promoted accumulation of the digested 100 kDa CFTR form. An important role in calpain-mediated turnover of CFTR is exerted by HSP90 (heat-shock protein 90), which, via association with the protein channel, modulates the degradative effect of calpain through a selective protection. Taken together these results indicate that CFTR turnover is initiated by calpain activation, which is induced by an increased Ca2+ influx and, following internalization of the cleaved channel protein, and completed by the lysosomal proteases. These findings provide new insights into the molecular mechanisms responsible for the defective functions of ion channels in human pathologies.
Assuntos
Calpaína/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Animais , Calpaína/genética , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Transporte Proteico , RatosRESUMO
Cystic fibrosis in characterized by pulmonary bacterial colonization and hyperinflammation. Lymphocytes, monocytes/macrophages, neutrophils, and dendritic cells of patients with CF express functional CFTR and are directly affected by altered CFTR expression/function, impairing their ability to resolve infections and inflammation. However, the mechanism behind and the contribution of leukocytes in the pathogenesis of CF are still poorly characterized. The recent clinical introduction of specific CFTR modulators added an important tool not only for the clinical management of the disease but also to the investigation of the pathophysiological mechanisms related to CFTR dysfunction and dysregulated immunity. These drugs treat the basic defect in cystic fibrosis (CF) by increasing CFTR function with improvement of lung function and quality of life, and may improve clinical outcomes also by correcting the dysregulated immune function that characterizes CF. Measure of CFTR function, protein expression profiling and several omics methods were used to identify molecular changes in freshly isolated leukocytes of CF patients, highlighting two roles of leukocytes in CF: one more generally related to the mechanism(s) causing immune dysregulation in CF and unresolved inflammation, and another more applicative role, which identifies in myeloid cells, an important tool predictive of the therapeutic response of CF patients. In this review we will summarize available data on CFTR expression and function in leukocyte populations and will discuss potential clinical applications based on available data.
Assuntos
Fibrose Cística/patologia , Leucócitos/patologia , Animais , Ensaios Clínicos como Assunto , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Modelos BiológicosRESUMO
BACKGROUND: In the brain, polyamines are mainly synthesized in neurons, but preferentially accumulated in astrocytes, and are proposed to be involved in neurodegenerative/neuroinflammatory disorders and neuron injury. A transgenic mouse overexpressing spermine oxidase (SMOX, which specifically oxidizes spermine) in the neocortex neurons (Dach-SMOX mouse) was proved to be a model of increased susceptibility to excitotoxic injury. METHODS: To investigate possible alterations in synapse functioning in Dach-SMOX mouse, both cerebrocortical nerve terminals (synaptosomes) and astrocytic processes (gliosomes) were analysed by assessing polyamine levels, ezrin and vimentin content, glutamate AMPA receptor activation, calcium influx, and catalase activity. RESULTS: The main findings are as follows: (i) the presence of functional calcium-permeable AMPA receptors in synaptosomes from both control and Dach-SMOX mice, and in gliosomes from Dach-SMOX mice only; (ii) reduced content of spermine in gliosomes from Dach-SMOX mice; and (iii) down-regulation and up-regulation of catalase activity in synaptosomes and gliosomes, respectively, from Dach-SMOX mice. CONCLUSIONS: Chronic activation of SMOX in neurons leads to major changes in the astrocyte processes including reduced spermine levels, increased calcium influx through calcium-permeable AMPA receptors, and stimulation of catalase activity. Astrocytosis and the astrocyte process alterations, depending on chronic activation of polyamine catabolism, result in synapse dysregulation and neuronal suffering.
Assuntos
Gliose/metabolismo , Gliose/patologia , Poliaminas/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Cálcio/metabolismo , Catalase/metabolismo , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Terminações Nervosas/efeitos dos fármacos , Terminações Nervosas/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Receptores de AMPA/metabolismo , Espermina/análogos & derivados , Espermina/metabolismo , Espermina/farmacologia , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Vimentina/metabolismo , Poliamina OxidaseRESUMO
BACKGROUND: To establish the physiological role of calpain, it is necessary to define how the protease can escape from the effect of its natural inhibitor calpastatin, since both proteins co-localize into the cell cytosol. METHODS: To answer this question, we have overexpressed four fluorescent calpastatin constructs, differing in the composition of their XL- and L-domains, and the intracellular trafficking of this protein inhibitor has been followed by single cell fluorescence imaging. RESULTS AND CONCLUSIONS: By the use of these calpastatin forms differing in the type of exon-derived sequences contained in the XL- and L-domains, we have demonstrated that the sequence coded by exon 6, containing multiple phosphorylation sites, is directly involved in determining the cell localization of calpastatin. In fact, exposure to cAMP promotes the recruitment into aggregates of those calpastatin forms containing the exon 6 sequence. These protein movements are directly related to the level of cytosolic inhibitory capacity and thereby to the extent of intracellular calpain activation. GENERAL SIGNIFICANCE: The recruitment of calpastatin into aggregates allows the translocation and activation of the protease to the membranes; on the contrary, the presence of large amounts of calpastatin in the cytosol prevents both processes, protecting the cell from undesired proteolysis.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Calpaína/metabolismo , Éxons , Sequência de Bases , Linhagem Celular , AMP Cíclico/metabolismo , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Humanos , Microscopia de Fluorescência , Frações Subcelulares/enzimologiaRESUMO
In resting human neuronal cells, nitric oxide synthase (nNOS) is present in its native 160 kDa form in a quiescent state predominantly co-localized on the plasma membrane, via its PDZ (Psd-95/Discs-large/Zona Occludens) domain, with NMDA receptor (NMDA-R) and in tight association with heat shock protein 90 (HSP90). Following exposure of the cells to Ca(2+)-ionophore or to NMDA, nNOS undergoes proteolytic removal of the PDZ domain being converted into a fully active 130 kDa form. The newly generated nNO synthase form dissociates from NMDA-R and extensively diffuses into the cytosol in direct correlation with NO production. Intracellular redistribution and activation of nNOS are completely prevented in cells preloaded with calpain inhibitor-1, indicating that these processes are triggered by a concomitant activation of calpain. The role of calpain has been confirmed by immunoprecipitation experiments revealing that also mu-calpain is specifically recruited into the NMDA-R-nNOS-HSP90 complex following calcium loading. Thus, the formation of clusters containing HSP90, mu-calpain, nNOS and NMDA-R represents the limiting step for the operation of the mechanism that links an efficient synthesis of NO to a local increase in Ca(2+) influx.
Assuntos
Sinalização do Cálcio/fisiologia , Calpaína/metabolismo , Neurônios/enzimologia , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico/biossíntese , Receptores de N-Metil-D-Aspartato/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Calpaína/antagonistas & inibidores , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Citosol/metabolismo , Difusão , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Proteínas de Choque Térmico HSP90/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Ionóforos/farmacologia , Neuroblastoma , Neurônios/efeitos dos fármacos , Óxido Nítrico Sintase Tipo I/efeitos dos fármacos , Peptídeo Hidrolases/metabolismo , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/fisiologiaRESUMO
The production of recombinant calpastatin in E. coli has become an efficient tool to obtain discrete amounts of a specific calpastatin species that can be present concomitantly with other calpastatin fragments/forms in the same tissue or cell type in a given condition. Indeed, at present, it is still difficult to distinguish the various calpastatin species for several reasons among which: calpastatins differ only at the N-terminus, can undergo calpain-dependent cleavage generating discrete fragments, and show anomalous electrophoretic mobility. Another benefit of using recombinant calpastatin is that, as the wild-type forms, it is heat resistant and thus can be efficiently isolated taking advantage of a simple quick purification step. Finally, the lack of posttranslational modifications makes recombinant calpastatin species particularly suitable for studying in vitro the biochemical features of specific parts of the inhibitor that following controlled posttranslational modifications change their functional interaction with calpain. In this chapter, we describe, starting from the mRNA sequence, how to produce rat calpastatin Type I in E. coli. We use routinely the same method, with minor modifications, for the production of other calpastatin species deriving from different tissues or organisms and calpastatin constructs having only specific domains. The possibility to obtain large amounts of a single calpain inhibitor form is a great advantage for studying the calpain/calpastatin system in vitro.