RESUMO
Oxidative stress, an imbalance between pro-oxidant and antioxidant status, favouring the pro-oxidant state is a result of increased production of reactive oxygen species (ROS) or inadequate antioxidant protection. ROS are produced through several mechanisms in cells including during mitochondrial oxidative phosphorylation. Increased mitochondrial-derived ROS are associated with mitochondrial dysfunction, an early event in age-related diseases such as Alzheimer's diseases (ADs) and in metabolic disorders including diabetes. AD post-mortem investigations of affected brain regions have shown the accumulation of oxidative damage to macromolecules, and oxidative stress has been considered an important contributor to disease pathology. An increase in oxidative stress, which leads to increased levels of superoxide, hydrogen peroxide and other ROS in a potentially vicious cycle is both causative and a consequence of mitochondrial dysfunction. Mitochondrial dysfunction may be ameliorated by molecules with antioxidant capacities that accumulate in mitochondria such as carotenoids. However, the role of carotenoids in mitigating mitochondrial dysfunction is not fully understood. A better understanding of the role of antioxidants in mitochondrial function is a promising lead towards the development of novel and effective treatment strategies for age-related diseases. This review evaluates and summarises some of the latest developments and insights into the effects of carotenoids on mitochondrial dysfunction with a focus on the antioxidant properties of carotenoids. The mitochondria-protective role of carotenoids may be key in therapeutic strategies and targeting the mitochondria ROS is emerging in drug development for age-related diseases.
Assuntos
Antioxidantes , Doenças Mitocondriais , Humanos , Antioxidantes/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Carotenoides/metabolismo , Carotenoides/farmacologia , Estresse Oxidativo , Doenças Mitocondriais/metabolismoRESUMO
Alzheimer's disease (AD) is a debilitating neurodegenerative disease that causes a progressive decline in memory, language and problem solving. For decades mechanism-based therapies have primarily focused on amyloid ß (Aß) processing and pathways that govern neurofibrillary tangle generation. With the potential exception to Aducanumab, a monotherapy to target Aß, clinical trials in these areas have been challenging and have failed to demonstrate efficacy. Currently, the prescribed therapies for AD are those that target the cholinesterase and glutamatergic systems that can moderately reduce cognitive decline, dependent on the individual. In the brain, over 40% of neuronal synapses are glutamatergic, where the glutamate level is tightly regulated through metabolite exchange in neuronal, astrocytic and endothelial cells. In AD brain, Aß can interrupt effective glutamate uptake by astrocytes, which evokes a cascade of events that leads to neuronal swelling, destruction of membrane integrity and ultimately cell death. Much work has focussed on the post-synaptic response with little insight into how glutamate is regulated more broadly in the brain and the influence of anaplerotic pathways that finely tune these mechanisms. The role of blood branched chain amino acids (BCAA) in regulating neurotransmitter profiles under disease conditions also warrant discussion. Here, we review the importance of the branched chain aminotransferase proteins in regulating brain glutamate and the potential consequence of dysregulated metabolism in the context of BCAA or glutamate accumulation. We explore how the reported benefits of BCAA supplementation or restriction in improving cognitive function in other neurological diseases may have potential application in AD. Given that memantine, the glutamate receptor agonist, shows clinical relevance it is now timely to research related pathways, an understanding of which could identify novel approaches to treatment of AD.
Assuntos
Doença de Alzheimer , Doenças Neurodegenerativas , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides , Astrócitos , Células Endoteliais , HumanosRESUMO
Cytosolic and mitochondrial human branched chain aminotransferase (hBCATc and hBCATm, respectively) play an integral role in brain glutamate metabolism. Regional increased levels of hBCATc in the CA1 and CA4 region of Alzheimer's disease (AD) brain together with increased levels of hBCATm in frontal and temporal cortex of AD brains, suggest a role for these proteins in glutamate excitotoxicity. Glutamate toxicity is a key pathogenic feature of several neurological disorders including epilepsy associated dementia, AD, vascular dementia (VaD) and dementia with Lewy bodies (DLB). To further understand if these increases are specific to AD, the expression profiles of hBCATc and hBCATm were examined in other forms of dementia including DLB and VaD. Similar to AD, levels of hBCATm were significantly increased in the frontal and temporal cortex of VaD cases and in frontal cortex of DLB cases compared to controls, however there were no observed differences in hBCATc between groups in these areas. Moreover, multiple forms of hBCATm were observed that were particular to the disease state relative to matched controls. Real-time PCR revealed similar expression of hBCATm mRNA in frontal and temporal cortex for all cohort comparisons, whereas hBCATc mRNA expression was significantly increased in VaD cases compared to controls. Collectively our results suggest that hBCATm protein expression is significantly increased in the brains of DLB and VaD cases, similar to those reported in AD brain. These findings indicate a more global response to altered glutamate metabolism and suggest common metabolic responses that might reflect shared neurodegenerative mechanisms across several forms of dementia.
Assuntos
Demência Vascular/enzimologia , Regulação Enzimológica da Expressão Gênica , Doença por Corpos de Lewy/enzimologia , Transaminases/biossíntese , Idoso , Idoso de 80 Anos ou mais , Encéfalo/enzimologia , Encéfalo/patologia , Estudos de Coortes , Demência Vascular/genética , Demência Vascular/patologia , Feminino , Humanos , Doença por Corpos de Lewy/genética , Doença por Corpos de Lewy/patologia , Masculino , Antígenos de Histocompatibilidade Menor/biossíntese , Proteínas da Gravidez/biossíntese , Transaminases/genéticaRESUMO
The human cytosolic branched-chain aminotransferase (hBCATc) enzyme is strategically located in glutamatergic neurons, where it is thought to provide approximately 30% of de novo nitrogen for brain glutamate synthesis. In health, glutamate plays a dominant role in facilitating learning and memory. However, in patients with Alzheimer's disease (AD), synaptic levels of glutamate become toxic, resulting in a direct increase in postsynaptic neuronal calcium, causing a cascade of events that contributes to the destruction of neuronal integrity and cell death, pathological features of AD. Our group is the first to map the hBCAT proteins to the human brain, where cell-specific compartmentation indicates key roles for these proteins in regulating glutamate homeostasis. Moreover, increased expression of hBCAT was observed in the brains of patients with AD relative to matched controls. We reflect on the importance of the redox-active CXXC motif, which confers novel roles for the hBCAT proteins, particularly with respect to substrate channeling and protein folding. This implies that, in addition to their role in glutamate metabolism, these proteins have additional functional roles that might impact redox cell signaling. This review discusses how these proteins behave as potential neuroprotectors during periods of oxidative stress. These findings are particularly important because an increase in misfolded proteins, linked to increased oxidative stress, occurs in several neurodegenerative conditions. Together, these studies give an overview of the diverse role that these proteins play in brain metabolism, in which a dysregulation of their expression may contribute to neurodegenerative conditions such as AD.
Assuntos
Encéfalo/enzimologia , Transaminases/metabolismo , HumanosRESUMO
There has been much interest in the use of cell culture models of neurones, to avoid the animal welfare and cost issues of using primary and human-induced pluripotent stem cell (hiPSC)-derived neurones respectively. The human neuroblastoma cell line, SH-SY5Y, is extensively used in laboratories as they can be readily expanded, are of low cost and can be differentiated into neuronal-like cells. However, much debate remains as to their phenotype once differentiated, and their ability to recapitulate the physiology of bona fide neurones. Here, we characterise a differentiation protocol using retinoic acid and BDNF, which results in extensive neurite outgrowth/branching within 10 days, and expression of key neuronal and synaptic markers. We propose that these differentiated SH-SY5Y cells may be a useful substitute for primary or hiPSC-derived neurones for cell biology studies, in order to reduce costs and animal usage. We further propose that this characterised differentiation timecourse could be used as an in vitro model for neuronal differentiation, for proof-of principle studies on neurogenesis, e.g. relating to neurodegenerative diseases. Finally, we demonstrate profound changes in Tau phosphorylation during differentiation of these cells, suggesting that they should not be used for neurodegeneration studies in their undifferentiated state.
RESUMO
The branched chain aminotransferase enzymes (BCAT) serve as nitrogen donors for the production of 30% of de novo glutamate synthesis in rat brain. Despite the importance of this major metabolite and excitatory neurotransmitter, the distribution of BCAT proteins in the human brain (hBCAT) remains unreported. We have studied this and report, for the first time, that the mitochondrial isoform, hBCATm is largely confined to vascular endothelial cells, whereas the cytosolic hBCATc is restricted to neurons. The majority of hBCATc-labelled neurons were either GABA-ergic or glutamatergic showing both cell body and axonal staining indicating a role for hBCATc in both glutamate production and glutamate release during excitation. Strong staining in hormone secreting cells suggests a further role for the transaminases in hormone regulation potentially similar to that proposed for insulin secretion. Expression of hBCATm in the endothelial cells of the vasculature demonstrates for the first time that glutamate could be metabolized by aminotranferases in these cells. This has important implications given that the dysregulation of glutamate metabolism, leading to glutamate excitotoxicity, is an important contributor to the pathogenesis of several neurodegenerative conditions, where the role of hBCATm in metabolizing excess glutamate may factor more prominently.
Assuntos
Encéfalo/enzimologia , Ácido Glutâmico/metabolismo , Proteínas da Gravidez/fisiologia , Transaminases/fisiologia , Idoso , Idoso de 80 Anos ou mais , Encéfalo/citologia , Encéfalo/metabolismo , Citosol/enzimologia , Citosol/metabolismo , Feminino , Humanos , Masculino , Antígenos de Histocompatibilidade Menor , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Neurônios/enzimologia , Neurônios/metabolismo , Proteínas da Gravidez/metabolismo , Transaminases/metabolismoRESUMO
The human branched-chain aminotransferase (hBCAT) isoenzymes are CXXC motif redox sensitive homodimers central to glutamate metabolism in the central nervous system. These proteins respond differently to oxidation by H(2)O(2), NO, and S-glutathionylation, suggesting that the redox potential is distinct between isoenzymes. Using various reduced to oxidized glutathione ratios (GSH:GSSG) to alter the redox environment, we demonstrate that hBCATc (cytosolic) has an overall redox potential that is 30 mV lower than hBCATm (mitochondrial). Furthermore, the CXXC motif of hBCATc was estimated to be 80 mV lower, suggesting that hBCATm is more oxidizing in nature. Western blot analysis revealed close correlations between hBCAT S-glutathionylation and the redox status of the assay environment, offering the hBCAT isoenzymes as novel biomarkers for cytosolic and mitochondrial oxidative stress.
Assuntos
Proteínas da Gravidez/química , Proteínas da Gravidez/metabolismo , Transaminases/química , Transaminases/metabolismo , Motivos de Aminoácidos , Citosol/enzimologia , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Humanos , Técnicas In Vitro , Antígenos de Histocompatibilidade Menor , Mitocôndrias/enzimologia , Oxirredução , Estresse Oxidativo , Proteínas da Gravidez/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transaminases/genéticaRESUMO
The cytosolic branched-chain aminotransferase (BCAT1) has received attention for its role in myeloid leukaemia development, where studies indicate metabolic adaptations due to BCAT1 up-regulation. BCAT1, like the mitochondria isoform (BCAT2), shares a conserved CXXC motif ~10 Å from the active site. This CXXC motif has been shown to act as a 'redox-switch' in the enzymatic regulation of the BCAT proteins, however the response to reactive oxygen species (ROS) differs between BCAT isoforms. Studies indicate that the BCAT1 CXXC motif is several orders of magnitude less sensitive to the effects of ROS compared with BCAT2. Moreover, estimation of the reduction mid-point potential of BCAT1, indicates that BCAT1 is more reductive in nature and may possess antioxidant properties. Therefore, the aim of this study was to further characterise the BCAT1 CXXC motif and evaluate its role in acute myeloid leukaemia. Our biochemical analyses show that purified wild-type (WT) BCAT1 protein could metabolise H2O2 in vitro, whereas CXXC motif mutant or WT BCAT2 could not, demonstrating for the first time a novel antioxidant role for the BCAT1 CXXC motif. Transformed U937 AML cells over-expressing WT BCAT1, showed lower levels of intracellular ROS compared with cells over-expressing the CXXC motif mutant (CXXS) or Vector Controls, indicating that the BCAT1 CXXC motif may buffer intracellular ROS, impacting on cell proliferation. U937 AML cells over-expressing WT BCAT1 displayed less cellular differentiation, as observed by a reduction of the myeloid markers; CD11b, CD14, CD68, and CD36. This finding suggests a role for the BCAT1 CXXC motif in cell development, which is an important pathological feature of myeloid leukaemia, a disease characterised by a block in myeloid differentiation. Furthermore, WT BCAT1 cells were more resistant to apoptosis compared with CXXS BCAT1 cells, an important observation given the role of ROS in apoptotic signalling and myeloid leukaemia development. Since CD36 has been shown to be Nrf2 regulated, we investigated the expression of the Nrf2 regulated gene, TrxRD1. Our data show that the expression of TrxRD1 was downregulated in transformed U937 AML cells overexpressing WT BCAT1, which taken with the reduction in CD36 implicates less Nrf2 activation. Therefore, this finding may implicate the BCAT1 CXXC motif in wider cellular redox-mediated processes. Altogether, this study provides the first evidence to suggest that the BCAT1 CXXC motif may contribute to the buffering of ROS levels inside AML cells, which may impact ROS-mediated processes in the development of myeloid leukaemia.
RESUMO
The metabolic enzyme branched-chain amino acid transaminase 1 (BCAT1) drives cell proliferation in aggressive cancers such as glioblastoma. Here, we show that BCAT1 localizes to mitotic structures and has a non-metabolic function as a mitotic regulator. Furthermore, BCAT1 is required for chromosome segregation in cancer and induced pluripotent stem cells and tumor growth in human cerebral organoid and mouse syngraft models. Applying gene knockout and rescue strategies, we show that the BCAT1 CXXC redox motif is crucial for controlling cysteine sulfenylation specifically in mitotic cells, promoting Aurora kinase B localization to centromeres, and securing accurate chromosome segregation. These findings offer an explanation for the well-established role of BCAT1 in promoting cancer cell proliferation. In summary, our data establish BCAT1 as a component of the mitotic apparatus that safeguards mitotic fidelity through a moonlighting redox functionality.
Assuntos
Aminoácidos de Cadeia Ramificada , Cisteína , Animais , Humanos , Camundongos , Aurora Quinase B , Modelos Animais de Doenças , Oxirredução , TransaminasesRESUMO
BACKGROUND: Biological characterisation of breast cancer subtypes is essential as it informs treatment regimens especially as different subtypes have distinct locoregional patterns. This is related to metabolic phenotype, where altered cellular metabolism is a fundamental adaptation of cancer cells during rapid proliferation. In this context, the metabolism of the essential branched-chain amino acids (BCAAs), catalysed by the human branched-chain aminotransferase proteins (hBCAT), offers multiple benefits for tumour growth. Upregulation of the cytosolic isoform of hBCAT (hBCATc), regulated by c-Myc, has been demonstrated to increase cell migration, tumour aggressiveness and proliferation in gliomas, ovarian and colorectal cancer but the importance of the mitochondrial isoform, hBCATm has not been fully investigated. METHODS: Using immunohistochemistry, the expression profile of metabolic proteins (hBCAT, IDH) was assessed between breast cancer subtypes, HER2 + , luminal A, luminal B and TNBC. Correlations between the percentage and the intensity of protein expression/co-expression with clinical parameters, such as hormone receptor status, tumour stage, lymph-node metastasis and survival, were determined. RESULTS: We show that hBCATc expression was found to be significantly associated with the more aggressive HER2 + and luminal B subtypes, whilst hBCATm and IDH1 associated with luminal A subtype. This was concomitant with better prognosis indicating a differential metabolic reliance between these two subtypes, in which enhanced expression of IDH1 may replenish the α-ketoglutarate pool in cells with increased hBCATm expression. CONCLUSION: The cytosolic isoform of BCAT is associated with tumours that express HER2 receptors, whereas the mitochondrial isoform is highly expressed in tumours that are ER + , indicating that the BCAT proteins are regulated through different signalling pathways, which may lead to the identification of novel targets for therapeutic applications targeting dysregulated cancer metabolism.
Assuntos
Neoplasias da Mama/patologia , Transaminases/metabolismo , Idoso , Biomarcadores Tumorais/metabolismo , Humanos , Imuno-Histoquímica , Metástase Linfática , Pessoa de Meia-Idade , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismoRESUMO
Specific proteins with reactive thiol(ate) groups are susceptible to nitric oxide (NO) modification, which can result in S-nitrosation, S-thiolation, or disulfide bond formation. In the present study the effect of NO modification on the functionality of human mitochondrial and cytosolic branched-chain aminotransferases (hBCATm and hBCATc, respectively) was investigated. Here, the NO reactive agents, S-nitrosoglutathione (GSNO), S-nitroso-N-acetyl-dl-penacillamine, and sodium nitroprusside, inactivated both isoforms in a dose-dependent manner. Furthermore, low concentrations of GSNO caused a time-dependent loss in BCAT activity (50 +/- 3% and 77 +/- 2% for hBCATc and hBCATm, respectively) correlating with the loss of four and one to two thiol groups, respectively, confirming the thiols as targets for NO modification. Analysis of GSNO-modified hBCATc by quadrupole time-of-flight mass spectrometry identified a major peak containing three NO adducts and a minor peak equivalent to two NO adducts and one glutathione (GSH) molecule, the latter confirmed by Western blot analysis. Moreover, prolonged exposure or increased levels of GSNO caused increased S-glutathionylation and partial dimerization of hBCATc, suggesting a possible shift from regulation by NO to one of adaptation during nitrosated stress. Although GSNO inactivated hBCATm, neither S-nitrosation, S-glutathionylation, nor dimerization could be detected, suggesting differential mechanisms of regulation through NO between isoforms in the mitochondria and cytosol. Reversal of GSNO-modified hBCAT using GSH alone was only partial, and complete reactivation was only possible using the glutaredoxin/GSH system (97 +/- 4% and 91 +/- 3% for hBCATc and hBCATm, respectively), implicating the importance of a full physiological redox system for activation/inactivation. To conclude, these results clearly demonstrate distinct functional/mechanistic responses to GSNO modification between BCAT isoforms and offer intriguing comparisons between the BCAT proteins and the respective cytosolic and mitochondrial hTrx and hGrx proteins.
Assuntos
Citosol/enzimologia , Mitocôndrias/enzimologia , Proteínas da Gravidez/metabolismo , S-Nitrosoglutationa/farmacologia , Transaminases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Cisteína/metabolismo , Citosol/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glutarredoxinas/metabolismo , Glutationa Redutase/metabolismo , Humanos , Antígenos de Histocompatibilidade Menor , Mitocôndrias/efeitos dos fármacos , Modelos Biológicos , Dados de Sequência Molecular , Nitroprussiato/farmacologia , Nitrosação/efeitos dos fármacos , Proteínas da Gravidez/antagonistas & inibidores , Proteínas da Gravidez/química , Fatores de Tempo , Transaminases/antagonistas & inibidores , Transaminases/químicaRESUMO
The human branched-chain aminotransferase (hBCAT) enzymes play an integral role in brain glutamate and branched-chain amino acid (BCAA) metabolism. Optimal hBCAT activity is dependent on the oxidation state of their redox reactive thiols, where post-translational modification by nitric oxide (NO) and glutathione results in reversible inhibition. Incubation of the cytosolic isoform (hBCATc) with S-nitrosating agents was found to inhibit in both a time and dose dependent manner through formation of a mixture of products including cysteine-nitric oxide (SNO) and S-glutathionylation. Mechanistic details of these redox interactions were studied using labeling with fluorescein-5-maleimide and confirmed via mass spectrometry and Western blot analysis. Though the mitochondrial isoform (hBCATm) was inhibited by nitrosating agents adduct formation could only be observed by DTNB titration as neither SNO, S-glutathionylation or disulfide bond formation could be detected. These studies revealed that the two isoforms of hBCAT, namely hBCATc and hBCATm, were differently regulated by S-nitrosation or S-glutathionylation pointing to distinct functional/mechanistic responses to GSNO modification. Detection of these adducts is essential for studies into the effect of NO on cells and the redox proteome which can offer insight into several pathological states and normal functioning of the cell.
Assuntos
Encéfalo/enzimologia , Citosol/enzimologia , Glutationa/metabolismo , Óxido Nítrico/metabolismo , Transaminases/metabolismo , Humanos , Mutação , Nitrosação , Oxirredução , Transaminases/química , Transaminases/genéticaRESUMO
Isolation and identification of protein targets for redox-active proteins is challenging. The human branched chain aminotransferase (hBCAT) proteins are redox active transaminases that can be regulated through oxidation, S-nitrosation and S-glutathionylation. This metabolic protein was shown to associate with the E1 decarboxylase component of the branched-chain α-keto acid dehydrogenase complex in a NADH-dependent manner, where mutation of the CXXC center was shown to prevent complex formation. To determine if the redox state of the CXXC motif can influence other NADH-dependent protein-protein interactions, proteins were extracted from neuronal cells treated under reduced and oxidized conditions and then isolated using targeted affinity chromatography, resolved using 2D electrophoresis. Select proteins spots were excised and identified using a quadrupole time of flight mass spectrometer (Thermo) with a precursor tolerance of 10 ppm and subsequently analyzed using Proteome Discoverer 2.1 with Swiss-Prot human DB. Mass tolerances for precursor/product were set to 10 ppm/0.6 Da and data were filtered by peptide confidence with PD2.1. It was determined that the protein profile considerably altered in both number and abundance dependent on the redox state of the cell and also on the availability of the redox active thiol groups. The biological relevance of the newly identified partners was determined using DAVID, the bioinformatics database, which indicated that proteins important to cytoskeletal function, protein transport, protein synthesis, chaperone activity, and cell signaling.
Assuntos
Cromatografia de Afinidade/métodos , NAD/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Transaminases/metabolismo , Motivos de Aminoácidos , Humanos , Oxirredução , Transaminases/isolamento & purificaçãoRESUMO
Redox regulation of proteins through oxidation and S-thiolation are important regulatory processes, acting in both a protective and adaptive role in the cell. In the current study, we investigated the sensitivity of the neuronal human cytosolic branched-chain aminotransferase (hBCATc) protein to oxidation and S-thiolation, with particular attention focused on functionality and modulation of its CXXC motif. Thiol specific reagents showed significant redox cycling between the reactive thiols and the TNB anion, and using NEM, four of the six reactive thiols are critical to the functionality of hBCATc. Site-directed mutagenesis studies supported these findings where a reduced kcat (ranging from 50-70% of hBCATc) for C335S, C338S, C335/8S, and C221S, respectively, followed by a modest effect on C242S was observed. However, only the thiols of the CXXC motif (C335 and C338) were directly involved in the reversible redox regulation of hBCATc through oxidation (with a loss of 40-45% BCAT activity on air oxidation alone). Concurrent with these findings, under air oxidation, the X-ray crystallography structure of hBCATc showed a disulphide bond between C335 and C338. Further oxidation of the other four thiols was not evident until levels of hydrogen peroxide were elevated. S-thiolation experiments of hBCATc exposed to GSH provided evidence for significant recycling between GSH and the thiols of hBCATc, which implied that under reducing conditions GSH was operating as a thiol donor with minimal S-glutathionylation. Western blot analysis of WT hBCATc and mutant proteins showed that as the ratio of GSH:GSSG decreased significant S-glutathionylation occurred (with a further loss of 20% BCAT activity), preferentially at the thiols of the CXXC motif, suggesting a shift in function toward a more protective role for GSH. Furthermore, the extent of S-glutathionylation increased in response to oxidative stress induced by hydrogen peroxide potentially through a C335 sulfenic acid intermediate. Deglutathionylation of hBCATc-SSG using the GSH/glutaredoxin system provides evidence that this protein may play an important role in cellular redox regulation. Moreover, redox associations between hBCATc and several neuronal proteins were identified using targeted proteomics. Thus, our data provides strong evidence that the reactive thiol groups, in particular the thiols of the CXXC motif, play an integral role in redox regulation and that hBCATc has redox mediated associations with several neuronal proteins involved in G-protein cell signaling, indicating a novel role for hBCATc in cellular redox control.
Assuntos
Citosol/enzimologia , Glutationa/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Transaminases/metabolismo , Humanos , Cinética , Mutação/genética , Oxirredução , Ligação Proteica , Compostos de Sulfidrila/química , Titulometria , Transaminases/química , Transaminases/genéticaRESUMO
The human branched chain aminotransferase enzymes are key regulators of glutamate metabolism in the brain and are among a growing number of redox-sensitive proteins. Studies that use thiol-specific reagents and electrospray ionization mass spectrometry demonstrate that the mitochondrial BCAT enzyme has a redox-active CXXC center, which on oxidation forms a disulfide bond (RSSR), via a cysteine sulfenic acid intermediate. Mechanistic details of this redox regulation were revealed by the use of mass spectrometry and dimedone modification. We discovered that the thiol group at position C315 of the CXXC motif acts a redox sensor, whereas the thiol group at position C318 permits reversible regulation by forming an intrasubunit disulphide bond. Because of their roles in redox regulation and catalysis, there is a growing interest in cysteine sulphenic acids. Therefore, development of chemical tags/methods to trap these transient intermediates is of immense importance.
Assuntos
Peróxido de Hidrogênio/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Biologia Molecular/métodos , Ácidos Sulfênicos/química , Transaminases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Bioensaio , Domínio Catalítico , Cicloexanonas/química , Ácido Ditionitrobenzoico , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/isolamento & purificação , Oxirredução/efeitos dos fármacos , Espectrofotometria , Compostos de Sulfidrila/análise , Transaminases/química , Transaminases/isolamento & purificaçãoRESUMO
Glutamate is the major excitatory neurotransmitter of the central nervous system, with the branched-chain amino acids (BCAAs) acting as key nitrogen donors for de novo glutamate synthesis. Despite the importance of these major metabolites, their metabolic pathway in the human brain is still not well characterised. The metabolic pathways that influence the metabolism of BCAAs have been well characterised in rat models. However, the expression of key proteins such as the branched-chain α-ketoacid dehydrogenase (BCKD) complex and glutamate dehydrogenase isozymes (GDH) in the human brain is still not well characterised. We have used specific antibodies to these proteins to analyse their distribution within the human brain and report, for the first time, that the E1α subunit of the BCKD is located in both neurons and vascular endothelial cells. We also demonstrate that GDH is localised to astrocytes, although vascular immunolabelling does occur. The labelling of GDH was most intense in astrocytes adjacent to the hippocampus, in keeping with glutamatergic neurotransmission in this region. GDH was also present in astrocyte processes abutting vascular endothelial cells. Previously, we demonstrated that the branched-chain aminotransferase (hBCAT) proteins were most abundant in vascular cells (hBCATm) and neurons (hBCATc). Present findings are further evidence that BCAAs are metabolised within both the vasculature and neurons in the human brain. We suggest that GDH, hBCAT and the BCKD proteins operate in conjunction with astrocytic glutamate transporters and glutamine synthetase to regulate the availability of glutamate. This has important implications given that the dysregulation of glutamate metabolism, leading to glutamate excitotoxicity, is an important contributor to the pathogenesis of several neurodegenerative conditions such as Alzheimer's disease.
Assuntos
Aminoácidos de Cadeia Ramificada/análise , Aminoácidos de Cadeia Ramificada/metabolismo , Química Encefálica/fisiologia , Encéfalo/metabolismo , Glutamato Desidrogenase/análise , Glutamato Desidrogenase/metabolismo , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Subunidades Proteicas/análise , Subunidades Proteicas/metabolismoRESUMO
A common feature among pre-malignant lesions is the induction of hypoxia through increased cell propagation and reduced access to blood flow. Hypoxia in breast cancer has been associated with poor patient prognosis, resistance to chemotherapy and increased metastasis. Although hypoxia has been correlated with factors associated with the latter stages of cancer progression, it is not well documented how hypoxia influences cells in the earliest stages of transformation. Using the immortalized MCF-10A breast epithelial cell line, we used hypoxic culture conditions to mimic reduced O2 levels found within early pre-malignant lesions and assessed various cellular parameters. In this non-transformed mammary cell line, O2 deprivation led to some changes not immediately associated with cancer progression, such as decreased proliferation, cell cycle arrest and increased apoptosis. In contrast, hypoxia did induce other changes more consistent with an increased metastatic potential. A rise in the CD44+CD24-/low-labeled cell sub-population along with increased colony forming capability indicated an expanded stem cell population. Hypoxia also induced cellular and molecular changes consistent with an epithelial-to-mesenchymal transition (EMT). Furthermore, these cells now exhibited increased migratory and invasive abilities. These results underscore the contribution of the hypoxic tumour microenvironment in cancer progression and dissemination.
Assuntos
Neoplasias da Mama/patologia , Mama/citologia , Transição Epitelial-Mesenquimal , Células-Tronco/citologia , Hipóxia Tumoral , Apoptose , Mama/metabolismo , Neoplasias da Mama/metabolismo , Antígeno CD24/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , Movimento Celular , Proliferação de Células , Células Epiteliais/citologia , Feminino , Humanos , Receptores de Hialuronatos/metabolismo , Células-Tronco/metabolismo , Células Tumorais CultivadasRESUMO
Alzheimer's disease (AD) is a progressive disease associated with the production and deposition of amyloid ß-peptide (Aß) aggregates and neurofibrillary tangles, which lead to synaptic and neuronal damage. Reduced autophagic flux has been widely associated with the accumulation of autophagic vacuoles (AV), which has been proposed to contribute to aggregate build-up observed in AD. As such, targeting autophagy regulation has received wide review, where an understanding as to how this mechanism can be controlled will be important to neuronal health. The mammalian target of rapamycin complex 1 (mTORC1), which was found to be hyperactive in AD brain, regulates autophagy and is considered to be mechanistically important to aberrant autophagy in AD. Hormones and nutrients such as insulin and leucine, respectively, positively regulate mTORC1 activation and are largely considered to inhibit autophagy. However, in AD brain there is a dysregulation of nutrient metabolism, linked to insulin resistance, where a role for insulin treatment to improve cognition has been proposed. Recent studies have highlighted that mitochondrial proteins such as glutamate dehydrogenase and the human branched chain aminotransferase protein, through metabolism of leucine and glutamate, differentially regulate mTORC1 and autophagy. As the levels of the hBCAT proteins are significantly increased in AD brain relative to aged-matched controls, we discuss how these metabolic pathways offer new potential therapeutic targets. In this review article, we highlight the core regulation of autophagy through mTORC1, focusing on how insulin and leucine will be important to consider in particular with respect to our understanding of nutrient load and AD pathogenesis.
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PURPOSE: The Limulus amebocytelysate (LAL) assay is widely used for the screening of lipopolysaccharide (LPS) in parenteral pharmaceuticals. However, correlation of LPS in Gram-negative bacterial infections by LAL assay has been problematic, partly due to the variable reactivity of different LPS structures. Recombinant factor C (rFC) has allowed the development of a new simple, specific and sensitive LPS detection system (PyroGene). In this work, the potential of the new assay for detecting various LPS structures has been investigated and compared with two LAL-based assays and a human monocyte activity assay. METHODOLOGY: The activity of the various LPS structures has been investigated by PyroGene and two LAL-based assays and a human monocyte activity assay. RESULTS: The rFC assay detected most LPS structures in picogram quantities and the potency of E. coli, B. cepacia, Salmonella smooth and Salmonella R345 LPS was no different when measured with PyroGene or LAL assays. However, the reactivity of K. pneumoniae, S. marcescens, B. pertussis and P. aeruginosa LPS differed significantly between these assays. Importantly, pairwise correlation analysis revealed that only the PyroGene assay produced a significant positive correlation with the release of IL-6 from a monocytic cell line. CONCLUSION: We conclude that the rFC-based assay is a good replacement for conventional LAL assays and as it correlates significantly with IL-6 produced by a human monocyte cell line it could potentially be more useful for detecting LPS in a clinical setting.