RESUMO
Compensatory angiogenesis is an important adaptation for recovery from critical ischemia. We recently identified 20-hydroxyeicosatetraenoic acid (20-HETE) as a novel contributor of ischemia-induced angiogenesis. However, the precise mechanisms by which ischemia promotes 20-HETE increases that drive angiogenesis are unknown. This study aims to address the hypothesis that inflammatory neutrophil-derived myeloperoxidase (MPO) and hypochlorous acid (HOCl) critically contribute to 20-HETE increases leading to ischemic angiogenesis. Using Liquid Chromatography-Mass Spectrometry/Mass Spectrometry, Laser Doppler Perfusion Imaging, and Microvascular Density analysis, we found that neutrophil depletion and MPO knockout mitigate angiogenesis and 20-HETE production in the gracilis muscles of mice subjected to hindlimb ischemia. Furthermore, we found MPO and HOCl to be elevated in these tissues postischemia as assessed by immunofluorescence microscopy and in vivo live imaging of HOCl. Next, we demonstrated that the additions of either HOCl or an enzymatic system for generating HOCl to endothelial cells increase the expression of CYP4A11 and its product, 20-HETE. Finally, pharmacological interference of hypoxia inducible factor (HIF) signaling results in ablation of HOCl-induced CYP4A11 transcript and significant reductions in CYP4A11 protein. Collectively, we conclude that neutrophil-derived MPO and its product HOCl activate HIF-1α and CYP4A11 leading to increased 20-HETE production that drives postischemic compensatory angiogenesis. SIGNIFICANCE STATEMENT: Traditionally, neutrophil derived MPO and HOCl are exclusively associated in the innate immunity as potent bactericidal/virucidal factors. The present study establishes a novel paradigm by proposing a unique function for MPO/HOCl as signaling agents that drive critical physiological angiogenesis by activating the CYP4A11-20-HETE signaling axis via a HIF-1α-dependent mechanism. The findings from this study potentially identify novel therapeutic targets for the treatment of ischemia and other diseases associated with abnormal angiogenesis.
Assuntos
Ácido Hipocloroso , Peroxidase , Animais , Células Endoteliais/metabolismo , Ácidos Hidroxieicosatetraenoicos , Ácido Hipocloroso/metabolismo , Ácido Hipocloroso/farmacologia , Isquemia/metabolismo , Camundongos , Neovascularização Patológica/metabolismo , Neutrófilos/metabolismo , Peroxidase/metabolismoRESUMO
Mammalian transglutaminase 2 exhibits poor long-term stability in solution. Reconstituting lyophilized transglutaminase 2 in solutions containing dithiothreitol and EDTA alone and together with glycerol stabilizes the activity of this enzyme for several weeks.
Assuntos
Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases , Animais , Ditiotreitol , Ácido Edético , Glicerol , Cobaias , Fígado , MamíferosRESUMO
Cancer cells require lipids to fulfill energetic, proliferative, and signaling requirements. Even though these cells can take up exogenous fatty acids, the majority exhibit a dependency on de novo fatty acid synthesis. Fatty acid synthase (FASN) is the rate-limiting enzyme in this process. Expression and activity of FASN is elevated in multiple cancers, where it correlates with disease progression and poor prognosis. These observations have sparked interest in developing methods of detecting FASN expression in vivo. One promising approach is the imaging of radiolabeled molecular probes targeting FASN by positron emission tomography (PET). However, although [11C]acetate uptake by prostate cancer cells correlates with FASN expression, no FASN-specific PET probes currently exist. Our aim was to synthesize and evaluate a series of small molecule triazolones based on GSK2194069, an FASN inhibitor with IC50 = 7.7 ± 4.1 nM, for PET imaging of FASN expression. These triazolones were labeled with carbon-11 in good yield and excellent radiochemical purity, and binding to FASN-positive LNCaP cells was significantly higher than FASN-negative PC3 cells. Despite these promising characteristics, however, these molecules exhibited poor in vivo pharmacokinetics and were predominantly retained in lymph nodes and the hepatobiliary system. Future studies will seek to identify structural modifications that improve tumor targeting while maintaining the excretion profile of these first-generation 11C-methyltriazolones.
Assuntos
Ácido Graxo SintasesRESUMO
Neurodegenerative disorders represent the culmination of numerous insults including oxidative stress. The long etiology of most of these disorders suggests that lessening the effects of one or more of the insults could significantly delay disease onset. Antioxidants have been tested as possible therapeutics for neurodegenerative disorders, but with little success. Here we report that serotonin acts as a scavenger of hypochlorous acid (HOCl) in the brain. Serotonin was shown to prevent the oxidation of 2-thio-5-nitrobenzoate by HOCl in a biphasic manner. The first phase was a partial scavenging that occurred at concentrations of serotonin that exceeded those of HOCl. (1)H-NMR studies indicated that HOCl chlorinates both the aryl and akyl nitrogen atoms of serotonin. Thus, the oxidation of 2-thio-5-nitrobenzoate that occurred during the first phase of scavenging is likely due to the formation of serotonergic chloramines. A second phase of scavenging occurred at concentrations of HOCl that exceeded those of serotonin. Under these conditions, the chlorinated serotonin polymerized and formed inert aggregates. Serotonin was further shown to prevent the loss of cells and cellular α-ketoglutarate dehydrogenase complex activity caused by HOCl. Extracellular concentrations of serotonin in the brain can be elevated with selective serotonin reuptake inhibitors and suggests that such compounds could be used to increase the cerebral antioxidant capacity. Acute administration of selective serotonin reuptake inhibitors to mice treated with endotoxin partially mitigated sickness behavior and protein chlorination in the brain. These observations suggest that serotonin may act to suppress chlorinative stress in the brain.
Assuntos
Encéfalo/metabolismo , Sequestradores de Radicais Livres/metabolismo , Ácido Hipocloroso/metabolismo , Estresse Oxidativo , Serotonina/metabolismo , Animais , Sequestradores de Radicais Livres/farmacologia , Halogenação , Humanos , Complexo Cetoglutarato Desidrogenase/metabolismo , Masculino , Camundongos , Serotonina/farmacologia , Células THP-1RESUMO
Two 4-fluoro-L-glutamine diastereoisomers [(2S,4R)-4-FGln, (2S,4S)-4-FGln] were previously developed for positron emission tomography. Label uptake into two tumor cell types was greater with [18F](2S,4R)-4-FGln than with [18F](2S,4S)-4-FGln. In the present work we investigated the enzymology of two diastereoisomers of 4-FGln, two diastereoisomers of 4-fluoroglutamate (4-FGlu) (potential metabolites of the 4-FGln diastereoisomers) and another fluoro-derivative of L-glutamine [(2S,4S)-4-(3-fluoropropyl)glutamine (FP-Gln)]. The two 4-FGlu diastereoisomers were found to be moderate-to-good substrates relative to L-glutamate of glutamate dehydrogenase, aspartate aminotransferase and alanine aminotransferase. Additionally, alanine aminotransferase was shown to catalyze an unusual γ-elimination reaction with both 4-FGlu diastereoisomers. Both 4-FGlu diastereoisomers were shown to be poor substrates, but strong inhibitors of glutamine synthetase. Both 4-FGln diastereoisomers were shown to be poor substrates compared to L-glutamine of glutamine transaminase L and α-aminoadipate aminotransferase. However, (2S,4R)-4-FGln was found to be a poor substrate of glutamine transaminase K, whereas (2S,4S)-4-FGln was shown to be an excellent substrate. By contrast, FP-Gln was found to be a poor substrate of all enzymes examined. Evidently, substitution of H in position 4 by F in L-glutamine/L-glutamate has moderate-to-profound effects on enzyme-catalyzed reactions. The present results: 1) show that 4-FGln and 4-FGlu diastereoisomers may be useful for studying active site topology of glutamate- and glutamine-utilizing enzymes; 2) provide a framework for understanding possible metabolic transformations in tumors of 18F-labeled (2S,4R)-4-FGln, (2S,4S)-4-FGln, (2S,4R)-4-FGlu or (2S,4S)-4-FGlu; and 3) show that [18F]FP-Gln is likely to be much less metabolically active in vivo than are the [18F]4-FGln diastereoisomers.
RESUMO
The following article addresses some seemingly paradoxical observations concerning cerebral glutamine synthetase in ischemia-reperfusion injury. In the brain, this enzyme is predominantly found in astrocytes and catalyzes part of the glutamine-glutamate cycle. Glutamine synthetase is also thought to be especially sensitive to inactivation by the oxygen- and nitrogen-centered radicals generated during strokes. Despite this apparent sensitivity, glutamine synthetase specific activity is elevated in the affected tissues during reperfusion. Given the central role of the glutamine-glutamate cycle in the brain, we sought to resolve these conflicting observations with the view of providing an alternative perspective for therapeutic intervention in stroke.
Assuntos
Glutamato-Amônia Ligase/metabolismo , Acidente Vascular Cerebral/enzimologia , Animais , Astrócitos/metabolismo , Modelos Animais de Doenças , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Humanos , Traumatismo por Reperfusão/enzimologiaRESUMO
The following report describes a simple and optimized assay for the detection of iron in solution based on the binding of this metal by ferrozine. This assay accurately measures between 1 and 200 µM sample iron concentrations within 2½ hours.
Assuntos
Técnicas de Química Analítica/métodos , Ferrozina/química , Ferro/análise , Ferro/química , Absorção Fisico-Química , Limite de DetecçãoRESUMO
At high concentrations, the glutamine synthetase inhibitor L-methionine-S,R-sulfoximine (MSO) is a convulsant, especially in dogs. Nevertheless, sub-convulsive doses of MSO are neuroprotective in rodent models of hyperammonemia, acute liver disease, and amyotrophic lateral sclerosis and suggest MSO may be clinically useful. Previous work has also shown that much lower doses of MSO are required to produce convulsions in dogs than in primates. Evidence from the mid-20th century suggests that humans are also less sensitive. In the present work, the inhibition of recombinant human glutamine synthetase by MSO is shown to be biphasic-an initial reversible competitive inhibition (K i 1.19 mM) is followed by rapid irreversible inactivation. This K i value for the human enzyme accounts, in part, for relative insensitivity of primates to MSO and suggests that this inhibitor could be used to safely inhibit glutamine synthetase activity in humans.
Assuntos
Convulsivantes/farmacologia , Inibidores Enzimáticos/farmacologia , Glutamato-Amônia Ligase/antagonistas & inibidores , Metionina Sulfoximina/farmacologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Doenças do Sistema Nervoso/tratamento farmacológico , Trifosfato de Adenosina/metabolismo , Esclerose Lateral Amiotrófica/tratamento farmacológico , Animais , Proteínas de Bactérias/antagonistas & inibidores , Cães/metabolismo , Humanos , Cinética , Doenças do Sistema Nervoso/enzimologia , Proteínas de Plantas/antagonistas & inibidores , Ligação Proteica , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Ovinos/metabolismo , Especificidade da EspécieRESUMO
Solutions of hypochlorous acid (HOCl) decay over time. This decay indicates the necessity for methods and reagents for the routine measurement of this oxidant. 2-Nitro-5-thiobenzoate is commonly used to measure HOCl concentrations. This article describes a method for the preparation of 2-nitro-5-thiobenzoate that is stable for at least 3 months. This method relies on the partial rather than full reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) and the resulting equilibrium between the substrate and the product.
Assuntos
Ácido Ditionitrobenzoico/química , Ácido Hipocloroso/análise , Oxidantes/química , Oxirredução , SoluçõesRESUMO
Transglutaminases catalyze the formation of γ-glutamylamines utilizing glutamyl residues and amine-bearing compounds such as lysyl residues and polyamines. These γ-glutamylamines can be released from proteins by proteases in an intact form. The free γ-glutamylamines can be catabolized to 5-oxo-L-proline and the free amine by γ-glutamylamine cyclotransferase. Free γ-glutamylamines, however, accumulate in the CSF and affected areas of Huntington Disease brain. This observation suggests transglutaminase-derived γ-glutamylamines may play a more significant role in neurodegeneration than previously thought. The following monograph reviews the metabolism of γ-glutamylamines and examines the possibility that these species contribute to neurodegeneration.
Assuntos
Dipeptídeos/metabolismo , Doenças Neurodegenerativas/enzimologia , Processamento de Proteína Pós-Traducional , Aminas/metabolismo , Animais , Glutamina/metabolismo , Humanos , Lisina/metabolismo , Doenças Neurodegenerativas/metabolismo , Transglutaminases/fisiologia , gama-Glutamilciclotransferase/fisiologiaRESUMO
Dietary methionine restriction (MR) increases longevity by improving health. In experimental models, MR is accompanied by decreased cystathionine ß-synthase activity and increased cystathionine γ-lyase activity. These enzymes are parts of the transsulfuration pathway which produces cysteine and 2-oxobutanoate. Thus, the decrease in cystathionine ß-synthase activity is likely to account for the loss of tissue cysteine observed in MR animals. Despite this decrease in cysteine levels, these tissues exhibit increased H2S production which is thought to be generated by ß-elimination of the thiol moiety of cysteine, as catalyzed by cystathionine ß-synthase or cystathionine γ-lyase. Another possibility for this H2S production is the cystathionine γ-lyase-catalyzed ß-elimination of cysteine persulfide from cystine, which upon reduction yields H2S and cysteine. Here, we demonstrate that MR increases cystathionine γ-lyase production and activities in the liver and kidneys, and that cystine is a superior substrate for cystathionine γ-lyase catalyzed ß-elimination as compared to cysteine. Moreover, cystine and cystathionine exhibit comparable Kcat/Km values (6000 M-1 s-1) as substrates for cystathionine γ-lyase-catalyzed ß-elimination. By contrast, cysteine inhibits cystathionine γ-lyase in a non-competitive manner (Ki ~ 0.5 mM), which limits its ability to function as a substrate for ß-elimination by this enzyme. Cysteine inhibits the enzyme by reacting with its pyridoxal 5'-phosphate cofactor to form a thiazolidine and in so doing prevents further catalysis. These enzymological observations are consistent with the notion that during MR cystathionine γ-lyase is repurposed to catabolize cystine and thereby form cysteine persulfide, which upon reduction produces cysteine.
RESUMO
The validation of prostate specific membrane antigen (PSMA) as a molecular target in metastatic castration-resistant prostate cancer has stimulated the development of multiple classes of theranostic ligands that specifically target PSMA. Theranostic ligands are used to image disease or selectively deliver cytotoxic radioactivity to cells expressing PSMA according to the radioisotope conjugated to the ligand. PSMA theranostics is a rapidly advancing field that is now integrating into clinical management of prostate cancer patients. In this review we summarize published research describing the biological role(s) and activity of PSMA, highlight the most clinically advanced PSMA targeting molecules and biomacromolecules, and identify next generation PSMA ligands that aim to further improve treatment efficacy. The goal of this review is to provide a comprehensive assessment of the current state-of-play and a roadmap to achieving further advances in PSMA theranostics.
RESUMO
PURPOSE: Fibroblast activation protein-α (FAPα) is uniquely expressed in activated fibroblasts, including cancer-associated fibroblasts that populate tumor stroma and contribute to proliferation and immunosuppression. Radiolabeled FAPα inhibitors enable imaging of multiple human cancers, but time-dependent clearance from tumors currently limits their utility as FAPα-targeted radiotherapeutics. We sought to increase the area under the curve (AUC) by constructing a trifunctional ligand that binds FAPα with high affinity and also binds albumin and theranostic radiometals. PROCEDURES: RPS-309 comprised a FAPα-targeting moiety, an albumin-binding group, and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Inhibition of recombinant human FAPα (rhFAPα) was determined by colorimetric assay. Affinity for human serum albumin (HSA) was determined by high-performance affinity chromatography. The tissue distribution of [68Ga]Ga-RPS-309 in SW872 tumor xenograft-bearing mice was imaged by microPET/CT and quantified by biodistribution studies performed from 30 min to 3 h post injection (p.i.). The biodistribution of [177Lu]Lu-RPS-309 was determined at 4, 24, and 96 h p.i. RESULTS: RPS-309 inhibits rhFAPα with IC50 = 7.3 ± 1.4 nM. [68Ga]Ga-RPS-309 is taken up specifically by FAPα-expressing cells and binds HSA with Kd = 4.6 ± 0.1 µM. Uptake of the radiolabeled ligand in tumors was evident from 30 min p.i. (> 5 %ID/g) and was significantly reduced by co-injection of RPS-309. Specific skeletal uptake was also observed. Activity in tumors was constant through 4 h p.i., but cleared significantly by 24 h. The AUC in this period was 127 (%ID/g) × h. CONCLUSIONS: RPS-309 is a high-affinity FAPα inhibitor with prolonged plasma residence. Introduction of the albumin-binding group did not compromise FAPα binding. Although initial tumor uptake was high and FAPα-specific, RPS-309 also progressively cleared from tumors. Nevertheless, RPS-309 incorporates multiple sites in which structural diversity can be introduced, and therefore serves as a platform for future structure-activity relationship studies.
Assuntos
Antineoplásicos , Fibroblastos Associados a Câncer/metabolismo , Endopeptidases/metabolismo , Proteínas de Membrana/metabolismo , Medicina de Precisão/métodos , Compostos Radiofarmacêuticos , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Feminino , Humanos , Ligantes , Camundongos , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Transglutaminases (TGs) are Ca2+-dependent enzymes that catalyze a variety of modifications of glutaminyl (Q) residues. In the brain, these modifications include the covalent attachment of a number of amine-bearing compounds, including lysyl (K) residues and polyamines, which serve to either regulate enzyme activity or attach the TG substrates to biological matrices. Aberrant TG activity is thought to contribute to Alzheimer disease, Parkinson disease, Huntington disease, and supranuclear palsy. Strategies designed to interfere with TG activity have some benefit in animal models of Huntington and Parkinson diseases. The following review summarizes the involvement of TGs in neurodegenerative diseases and discusses the possible use of selective inhibitors as therapeutic agents in these diseases.
Assuntos
Degeneração Neural/enzimologia , Transglutaminases/metabolismo , Animais , Encéfalo/enzimologia , Humanos , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologiaRESUMO
In this study the effects of lovastatin on DU145 prostate cancer cells treated with phenylbutyrate (PB) was investigated in order to determine the NMR-detectable metabolic changes resulting from the cooperative activity of these two agents. DU145 cells were perfused with PB in the presence or absence of 10 microM of the HMG-CoA reductase inhibitor lovastatin, and the results monitored by 31P and diffusion-weighted 1H NMR spectroscopy. Lovastatin had additive effects on the PB-induced NMR-visible total choline in 1H spectra, and glycerophosphocholine in 31P spectra but no significant effect on NMR-visible lipid. Moreover, lovastatin had no effect on the ability of PB to either promote the formation of oil red O-detectable lipid droplets or arrest the cell cycle. The most remarkable observations from these studies were that lovastatin enhanced the increase in glycerophosphocholine while reversing late markers of apoptosis and the loss of NTP caused by PB. These results identify a branch point separating the neutral lipid production and the apoptotic cell death caused by the actions of differentiating agents.
Assuntos
Apoptose/fisiologia , Linhagem Celular Tumoral , Glicerofosfatos/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases , Lovastatina , Fenilbutiratos , Fosforilcolina/metabolismo , Animais , Caspase 3/metabolismo , Ciclo Celular , Linhagem Celular Tumoral/diagnóstico por imagem , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Ativação Enzimática , Glicerofosfatos/química , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Corpos de Inclusão/química , Corpos de Inclusão/metabolismo , Lipídeos/química , Lovastatina/metabolismo , Lovastatina/farmacologia , Masculino , Ressonância Magnética Nuclear Biomolecular , Fenilbutiratos/metabolismo , Fenilbutiratos/farmacologia , Fosforilcolina/química , Neoplasias da Próstata , RadiografiaRESUMO
Transglutaminases (TGases) catalyze several reactions with protein substrates, including formation of gamma-glutamyl-epsilon-lysine cross-links and gamma-glutamylpolyamine residues. The resulting gamma-glutamylamines are excised intact during proteolysis. TGase activity is altered in several diseases, highlighting the importance of in situ enzymatic determinations. Previous work showed that TGase activity (as measured by an in vitro assay) and free gamma-glutamyl-epsilon-lysine levels are elevated in Huntington disease (HD) and that gamma-glutamyl-epsilon-lysine is increased in HD CSF. Although free gamma-glutamyl-epsilon-lysine was used in these studies as an index of in situ TGase activity, gamma-glutamylpolyamines may also be diagnostic. We have devised methods for the simultaneous determination of four gamma-glutamylamines in CSF: gamma-glutamyl-epsilon-lysine, gamma-glutamylspermidine, gamma-glutamylputrescine, and bis-gamma-glutamylputrescine and showed that all are present in normal human CSF at concentrations of approximately 150, 670, 40, and 240 nM, respectively. The high gamma-glutamylspermidine/gamma-glutamylputrescine and gamma-glutamylspermidine/bis-gamma-glutamylputrescine ratios presumably reflect in part the large spermidine to putrescine mole ratio in human brain. We also showed that all four gamma-glutamylamines are elevated in HD CSF. Our findings support the hypotheses that (i) gamma-glutamylpolyamines are reflective of TGase activity in human brain, (ii) polyamination is an important post-translational modification of brain proteins, and (iii) TGase-catalyzed modification of proteins is increased in HD brain.
Assuntos
Poliaminas Biogênicas/metabolismo , Encéfalo/enzimologia , Glutamina/metabolismo , Doença de Huntington/líquido cefalorraquidiano , Doença de Huntington/enzimologia , Transglutaminases/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Aminação , Poliaminas Biogênicas/sangue , Biomarcadores/análise , Biomarcadores/líquido cefalorraquidiano , Encéfalo/fisiopatologia , Feminino , Humanos , Lisina/metabolismo , Masculino , Pessoa de Meia-Idade , Neuroquímica/métodos , Processamento de Proteína Pós-Traducional , Putrescina/análogos & derivados , Putrescina/metabolismo , Espermidina/metabolismo , Regulação para Cima/fisiologiaRESUMO
Cystamine is commonly used as a transglutaminase inhibitor. This disulphide undergoes reduction in vivo to the aminothiol compound, cysteamine. Thus, the mechanism by which cystamine inhibits transglutaminase activity in vivo could be due to either cystamine or cysteamine, which depends on the local redox environment. Cystamine inactivates transglutaminases by promoting the oxidation of two vicinal cysteine residues on the enzyme to an allosteric disulphide, whereas cysteamine acts as a competitive inhibitor for transamidation reactions catalyzed by this enzyme. The latter mechanism is likely to result in the formation of a unique biomarker, N-(γ-glutamyl)cysteamine that could serve to indicate how cyst(e)amine acts to inhibit transglutaminases inside cells and the body.
Assuntos
Cistamina/farmacologia , Cisteamina/análogos & derivados , Cisteamina/farmacologia , Inibidores Enzimáticos/farmacologia , Transglutaminases/antagonistas & inibidores , Biomarcadores/metabolismo , Cistamina/farmacocinética , Cisteamina/farmacocinética , Cisteína/metabolismo , Humanos , Oxirredução , Transglutaminases/metabolismoRESUMO
Many enzymes make "mistakes". Consequently, repair enzymes have evolved to correct these mistakes. For example, lactate dehydrogenase (LDH) and mitochondrial malate dehydrogenase (mMDH) slowly catalyze the reduction of 2-oxoglutarate (2-OG) to the oncometabolite l-2-hydroxyglutarate (l-2-HG). l-2-HG dehydrogenase corrects this error by converting l-2-HG to 2-OG. LDH also catalyzes the reduction of the oxo group of 2-oxoglutaramate (2-OGM; transamination product of l-glutamine). We show here that human glutamine synthetase (GS) catalyzes the amidation of the terminal carboxyl of both the l- and d- isomers of 2-HG. The reaction of 2-OGM with LDH and the reaction of l-2-HG with GS generate l-2-hydroxyglutaramate (l-2-HGM). We also show that l-2-HGM is a substrate of human ω-amidase. The product (l-2-HG) can then be converted to 2-OG by l-2-HG dehydrogenase. Previous work showed that 2-oxosuccinamate (2-OSM; transamination product of l-asparagine) is an excellent substrate of LDH. Finally, we also show that human ω-amidase converts the product of this reaction (i.e., l-2-hydroxysuccinamate; l-2-HSM) to l-malate. Thus, ω-amidase may act together with hydroxyglutarate dehydrogenases to repair certain "mistakes" of GS and LDH. The present findings suggest that non-productive pathways for nitrogen metabolism occur in mammalian tissues in vivo. Perturbations of these pathways may contribute to symptoms associated with hydroxyglutaric acidurias and to tumor progression. Finally, methods for the synthesis of l-2-HGM and l-2-HSM are described that should be useful in determining the roles of ω-amidase/4- and 5-C compounds in photorespiration in plants.
RESUMO
Glutamate is present in the brain at an average concentration-typically 10-12 mM-far in excess of those of other amino acids. In glutamate-containing vesicles in the brain, the concentration of glutamate may even exceed 100 mM. Yet because glutamate is a major excitatory neurotransmitter, the concentration of this amino acid in the cerebral extracellular fluid must be kept low-typically µM. The remarkable gradient of glutamate in the different cerebral compartments: vesicles > cytosol/mitochondria > extracellular fluid attests to the extraordinary effectiveness of glutamate transporters and the strict control of enzymes of glutamate catabolism and synthesis in well-defined cellular and subcellular compartments in the brain. A major route for glutamate and ammonia removal is via the glutamine synthetase (glutamate ammonia ligase) reaction. Glutamate is also removed by conversion to the inhibitory neurotransmitter γ-aminobutyrate (GABA) via the action of glutamate decarboxylase. On the other hand, cerebral glutamate levels are maintained by the action of glutaminase and by various α-ketoglutarate-linked aminotransferases (especially aspartate aminotransferase and the mitochondrial and cytosolic forms of the branched-chain aminotransferases). Although the glutamate dehydrogenase reaction is freely reversible, owing to rapid removal of ammonia as glutamine amide, the direction of the glutamate dehydrogenase reaction in the brain in vivo is mainly toward glutamate catabolism rather than toward the net synthesis of glutamate, even under hyperammonemia conditions. During hyperammonemia, there is a large increase in cerebral glutamine content, but only small changes in the levels of glutamate and α-ketoglutarate. Thus, the channeling of glutamate toward glutamine during hyperammonemia results in the net synthesis of 5-carbon units. This increase in 5-carbon units is accomplished in part by the ammonia-induced stimulation of the anaplerotic enzyme pyruvate carboxylase. Here, we suggest that glutamate may constitute a buffer or bulwark against changes in cerebral amine and ammonia nitrogen. Although the glutamate transporters are briefly discussed, the major emphasis of the present review is on the enzymology contributing to the maintenance of glutamate levels under normal and hyperammonemic conditions. Emphasis will also be placed on the central role of glutamate in the glutamine-glutamate and glutamine-GABA neurotransmitter cycles between neurons and astrocytes. Finally, we provide a brief and selective discussion of neuropathology associated with altered cerebral glutamate levels.
Assuntos
Encéfalo/metabolismo , Ácido Glutâmico/metabolismo , Nitrogênio/metabolismo , Barreira Hematoencefálica/metabolismo , Glutaminase/metabolismo , Humanos , Hiperamonemia , Transaminases/metabolismoRESUMO
Inflammation is a common feature of Parkinson Disease and other neurodegenerative disorders. Hypochlorous acid (HOCl) is a reactive oxygen species formed by neutrophils and other myeloperoxidase-containing cells during inflammation. HOCl chlorinates the amine and catechol moieties of dopamine to produce chlorinated derivatives collectively termed chlorodopamine. Here, we report that chlorodopamine is toxic to dopaminergic neurons both in vivo and in vitro Intrastriatal administration of 90 nmol chlorodopamine to mice resulted in loss of dopaminergic neurons from the substantia nigra and decreased ambulation-results that were comparable to those produced by the same dose of the parkinsonian poison, 1-methyl-4-phenylpyridinium (MPP+). Chlorodopamine was also more toxic to differentiated SH SY5Y cells than HOCl. The basis of this selective toxicity is likely mediated by chlorodopamine uptake through the dopamine transporter, as expression of this transporter in COS-7 cells conferred sensitivity to chlorodopamine toxicity. Pharmacological blockade of the dopamine transporter also mitigated the deleterious effects of chlorodopamine in vivo The cellular actions of chlorodopamine included inactivation of the α-ketoglutarate dehydrogenase complex, as well as inhibition of mitochondrial respiration. The latter effect is consistent with inhibition of cytochrome c oxidase. Illumination at 670 nm, which stimulates cytochrome c oxidase, reversed the effects of chlorodopamine. The observed changes in mitochondrial biochemistry were also accompanied by the swelling of these organelles. Overall, our findings suggest that chlorination of dopamine by HOCl generates toxins that selectively kill dopaminergic neurons in the substantia nigra in a manner comparable to MPP+.