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1.
Nat Commun ; 12(1): 5243, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34475406

RESUMO

Peroxisome, a special cytoplasmic organelle, possesses one or more kinds of oxidases for hydrogen peroxide (H2O2) production and catalase for H2O2 degradation, which serves as an intracellular H2O2 regulator to degrade toxic peroxides to water. Inspired by this biochemical pathway, we demonstrate the reactive oxygen species (ROS) induced tumor therapy by integrating lactate oxidase (LOx) and catalase (CAT) into Fe3O4 nanoparticle/indocyanine green (ICG) co-loaded hybrid nanogels (designated as FIGs-LC). Based on the O2 redistribution and H2O2 activation by cascading LOx and CAT catalytic metabolic regulation, hydroxyl radical (·OH) and singlet oxygen (1O2) production can be modulated for glutathione (GSH)-activated chemodynamic therapy (CDT) and NIR-triggered photodynamic therapy (PDT), by manipulating the ratio of LOx and CAT to catalyze endogenous lactate to produce H2O2 and further cascade decomposing H2O2 into O2. The regulation reactions of FIGs-LC significantly elevate the intracellular ROS level and cause fatal damage to cancer cells inducing the effective inhibition of tumor growth. Such enzyme complex loaded hybrid nanogel present potential for biomedical ROS regulation, especially for the tumors with different redox state, size, and subcutaneous depth.


Assuntos
Antineoplásicos/farmacologia , Nanogéis/química , Peroxissomos/enzimologia , Fotoquimioterapia/métodos , Animais , Antineoplásicos/química , Catalase/química , Catalase/metabolismo , Catálise , Linhagem Celular Tumoral , Óxido Ferroso-Férrico/química , Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Verde de Indocianina/química , Camundongos , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Nanopartículas/química , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Oxigênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Microambiente Tumoral/efeitos dos fármacos
2.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360635

RESUMO

Salivary gland cancers are rare but aggressive tumors that have poor prognosis and lack effective cure. Of those, parotid tumors constitute the majority. Functioning as metabolic machinery contributing to cellular redox balance, peroxisomes have emerged as crucial players in tumorigenesis. Studies on murine and human cells have examined the role of peroxisomes in carcinogenesis with conflicting results. These studies either examined the consequences of altered peroxisomal proliferators or compared their expression in healthy and neoplastic tissues. None, however, examined such differences exclusively in human parotid tissue or extended comparison to peroxisomal proteins and their associated gene expressions. Therefore, we examined differences in peroxisomal dynamics in parotid tumors of different morphologies. Using immunofluorescence and quantitative PCR, we compared the expression levels of key peroxisomal enzymes and proliferators in healthy and neoplastic parotid tissue samples. Three parotid tumor subtypes were examined: pleomorphic adenoma, mucoepidermoid carcinoma and acinic cell carcinoma. We observed higher expression of peroxisomal matrix proteins in neoplastic samples with exceptional down regulation of certain enzymes; however, the degree of expression varied between tumor subtypes. Our findings confirm previous experimental results on other organ tissues and suggest peroxisomes as possible therapeutic targets or markers in all or certain subtypes of parotid neoplasms.


Assuntos
Adenoma Pleomorfo/enzimologia , Carcinoma de Células Acinares/enzimologia , Carcinoma Mucoepidermoide/enzimologia , Neoplasias Parotídeas/enzimologia , Peroxissomos/enzimologia , Adenoma Pleomorfo/patologia , Carcinoma de Células Acinares/patologia , Carcinoma Mucoepidermoide/patologia , Estudos de Casos e Controles , Humanos , Proteínas de Neoplasias/metabolismo , Glândula Parótida/patologia , Neoplasias Parotídeas/patologia , Receptores Ativados por Proliferador de Peroxissomo/metabolismo
3.
Am J Physiol Heart Circ Physiol ; 320(5): H1813-H1821, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33666503

RESUMO

Although peroxisomes have been extensively studied in other cell types, their presence and function have gone virtually unexamined in cardiac myocytes. Here, in neonatal rat ventricular myocytes (NRVM) we showed that several known peroxisomal proteins co-localize to punctate structures with a morphology typical of peroxisomes. Surprisingly, we found that the peroxisomal protein, fatty acyl-CoA reductase 1 (FAR1), was upregulated by pharmacological and pathophysiological ER stress induced by tunicamycin (TM) and simulated ischemia-reperfusion (sI/R), respectively. Moreover, FAR1 induction in NRVM was mediated by the ER stress sensor, activating transcription factor 6 (ATF6). Functionally, FAR1 knockdown reduced myocyte death during oxidative stress induced by either sI/R or hydrogen peroxide (H2O2). Thus, Far1 is an ER stress-inducible gene, which encodes a protein that localizes to peroxisomes of cardiac myocytes, where it reduces myocyte viability during oxidative stress. Since FAR1 is critical for plasmalogen synthesis, these results imply that plasmalogens may exert maladaptive effects on the viability of myocytes exposed to oxidative stress.NEW & NOTEWORTHY The peroxisomal enzyme, FAR1, was shown to be an ER stress- and ATF6-inducible protein that localizes to peroxisomes in cardiac myocytes. FAR1 decreases myocyte viability during oxidative stress.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Aldeído Oxirredutases/biossíntese , Estresse do Retículo Endoplasmático , Traumatismo por Reperfusão Miocárdica/enzimologia , Miócitos Cardíacos/enzimologia , Peroxissomos/enzimologia , Fator 6 Ativador da Transcrição/genética , Aldeído Oxirredutases/genética , Animais , Animais Recém-Nascidos , Hipóxia Celular , Sobrevivência Celular , Células Cultivadas , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Indução Enzimática , Peróxido de Hidrogênio/toxicidade , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Estresse Oxidativo , Peroxissomos/efeitos dos fármacos , Peroxissomos/metabolismo , Ratos , Tunicamicina/toxicidade
4.
Mol Biotechnol ; 63(6): 544-555, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33786739

RESUMO

Candida tropicalis can metabolize alkanes or fatty acids to produce long-chain dicarboxylic acids (DCAs). Fatty acid transporters located on the cell or peroxisome membrane may play an important role in this process. Using amino acid sequence homologous alignment, two putative proteins, CtFat1p and CtPxa1p, located on the cell and peroxisome membrane were found, respectively. Moreover, single- and double-knockout homologous recombination technology was used to study ctfat1p and ctpxa1p gene effects on DCA synthesis. In comparison to the wild-type strain, long-chain DCA yield decreased by 65.14%, 88.38% and 56.19% after single and double-copy knockout of ctfat1p genes and double-copy knockout of ctpxa1p genes, respectively, indicating that the knockout of ctfat1p and ctpxa1p genes had a significant effect on the conversion of oils and fats into long-chain DCAs by C. tropicalis. However, the yield of long-chain DCAs increased by 21.90% after single-knockout of the ctpxa1p gene, indicating that the single-knockout of the ctpxa1p gene may reduce fatty acid transport to peroxisome for further oxidation. Moreover, to improve the intracellular transport rate of fatty acids, ctfat1p copy number increased, increasing DCA yield by 30.10%. These results may provide useful information for enhancing the production of long-chain DCAs by C. tropicalis.


Assuntos
Alcanos/química , Candida tropicalis/química , Ácidos Graxos/química , Engenharia de Proteínas , Alcanos/metabolismo , Sequência de Aminoácidos/genética , Candida tropicalis/enzimologia , Candida tropicalis/metabolismo , Proteínas de Transporte de Ácido Graxo/metabolismo , Ácidos Graxos/metabolismo , Fermentação , Redes e Vias Metabólicas/genética , Oxirredução , Peroxissomos/enzimologia , Peroxissomos/genética , Engenharia de Proteínas/métodos , Alinhamento de Sequência
5.
PLoS One ; 15(12): e0242445, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33301490

RESUMO

Acyl-CoA dehydrogenase 10 (Acad10)-deficient mice develop impaired glucose tolerance, peripheral insulin resistance, and abnormal weight gain. In addition, they exhibit biochemical features of deficiencies of fatty acid oxidation, such as accumulation of metabolites consistent with abnormal mitochondrial energy metabolism and fasting induced rhabdomyolysis. ACAD10 has significant expression in mouse brain, unlike other acyl-CoA dehydrogenases (ACADs) involved in fatty acid oxidation. The presence of ACAD10 in human tissues was determined using immunohistochemical staining. To characterize the effect of ACAD10 deficiency on the brain, micro-MRI and neurobehavioral evaluations were performed. Acad10-deficient mouse behavior was examined using open field testing and DigiGait analysis for changes in general activity as well as indices of gait, respectively. ACAD10 protein was shown to colocalize to mitochondria and peroxisomes in lung, muscle, kidney, and pancreas human tissue. Acad10-deficient mice demonstrated subtle behavioral abnormalities, which included reduced activity and increased time in the arena perimeter in the open field test. Mutant animals exhibited brake and propulsion metrics similar to those of control animals, which indicates normal balance, stability of gait, and the absence of significant motor impairment. The lack of evidence for motor impairment combined with avoidance of the center of an open field arena and reduced vertical and horizontal exploration are consistent with a phenotype characterized by elevated anxiety. These results implicate ACAD10 function in normal mouse behavior, which suggests a novel role for ACAD10 in brain metabolism.


Assuntos
Acil-CoA Desidrogenase/genética , Ansiedade/genética , Encéfalo/enzimologia , Metabolismo Energético/genética , Mitocôndrias/enzimologia , Acil-CoA Desidrogenase/deficiência , Acil-CoA Desidrogenase/metabolismo , Animais , Ansiedade/enzimologia , Ansiedade/fisiopatologia , Comportamento Animal , Encéfalo/diagnóstico por imagem , Carnitina/análogos & derivados , Carnitina/metabolismo , Marcha/fisiologia , Humanos , Rim/enzimologia , Fígado/enzimologia , Pulmão/enzimologia , Imageamento por Ressonância Magnética , Aprendizagem em Labirinto , Camundongos , Camundongos Knockout , Músculo Esquelético/enzimologia , Pâncreas/enzimologia , Peroxissomos/enzimologia
6.
Plant J ; 104(6): 1472-1490, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33031578

RESUMO

Benzoic acid-derived compounds, such as polyprenylated benzophenones and xanthones, attract the interest of scientists due to challenging chemical structures and diverse biological activities. The genus Hypericum is of high medicinal value, as exemplified by H. perforatum. It is rich in benzophenone and xanthone derivatives, the biosynthesis of which requires the catalytic activity of benzoate-coenzyme A (benzoate-CoA) ligase (BZL), which activates benzoic acid to benzoyl-CoA. Despite remarkable research so far done on benzoic acid biosynthesis in planta, all previous structural studies of BZL genes and proteins are exclusively related to benzoate-degrading microorganisms. Here, a transcript for a plant acyl-activating enzyme (AAE) was cloned from xanthone-producing Hypericum calycinum cell cultures using transcriptomic resources. An increase in the HcAAE1 transcript level preceded xanthone accumulation after elicitor treatment, as previously observed with other pathway-related genes. Subcellular localization of reporter fusions revealed the dual localization of HcAAE1 to cytosol and peroxisomes owing to a type 2 peroxisomal targeting signal. This result suggests the generation of benzoyl-CoA in Hypericum by the CoA-dependent non-ß-oxidative route. A luciferase-based substrate specificity assay and the kinetic characterization indicated that HcAAE1 exhibits promiscuous substrate preference, with benzoic acid being the sole aromatic substrate accepted. Unlike 4-coumarate-CoA ligase and cinnamate-CoA ligase enzymes, HcAAE1 did not accept 4-coumaric and cinnamic acids, respectively. The substrate preference was corroborated by in silico modeling, which indicated valid docking of both benzoic acid and its adenosine monophosphate intermediate in the HcAAE1/BZL active site cavity.


Assuntos
Acil Coenzima A/metabolismo , Coenzima A Ligases/metabolismo , Hypericum/metabolismo , Proteínas de Plantas/metabolismo , Xantonas/metabolismo , Clonagem Molecular , Coenzima A Ligases/genética , Citosol/enzimologia , Hypericum/enzimologia , Redes e Vias Metabólicas , Simulação de Acoplamento Molecular , Peroxissomos/enzimologia , Filogenia , Proteínas de Plantas/genética
7.
Biol Pharm Bull ; 43(9): 1382-1392, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32879213

RESUMO

The effects of different dietary fats on hepatic fatty acid oxidation were compared in male ICR mice and Sprague-Dawley rats. Animals were fed diets containing 100 g/kg of either palm oil (saturated fat), safflower oil (rich in linoleic acid), an oil of evening primrose origin (γ-linolenic acid, GLA oil), perilla oil (α-linolenic acid) or fish oil (eicosapentaenoic and doxosahexaenoic acids) for 21 d. GLA, perilla and fish oils, compared with palm and safflower oils, increased the activity of fatty acid oxidation enzymes in both mice and rats, with some exceptions. In mice, GLA and fish oils greatly increased the peroxisomal palmitoyl-CoA oxidation rate, and the activity of acyl-CoA oxidase and enoyl-CoA hydratase to the same degree. The effects were much smaller with perilla oil. In rats, enhancing effects were more notable with fish oil than with GLA and perilla oils, excluding the activity of enoyl-CoA hydratase, and were comparable between GLA and perilla oils. In mice, strong enhancing effects of GLA oil, which were greater than with perilla oil and comparable to those of fish oil, were confirmed on mRNA levels of peroxisomal but not mitochondrial fatty acid oxidation enzymes. In rats, the effects of GLA and perilla oils on mRNA levels of peroxisomal and mitochondrial enzymes were indistinguishable, and lower than those observed with fish oil. Therefore, considerable diversity in the response to dietary polyunsaturated fats, especially the oil rich in γ-linolenic acid and fish oil, of hepatic fatty acid oxidation pathway exists between mice and rats.


Assuntos
Gorduras na Dieta/administração & dosagem , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Ácido gama-Linolênico/administração & dosagem , Acil-CoA Oxidase/metabolismo , Ração Animal , Animais , Enoil-CoA Hidratase/metabolismo , Óleos de Peixe/administração & dosagem , Óleos de Peixe/química , Fígado/citologia , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Oxirredução/efeitos dos fármacos , Peroxissomos/efeitos dos fármacos , Peroxissomos/enzimologia , Óleos Vegetais/administração & dosagem , Óleos Vegetais/química , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie
8.
Sci Rep ; 10(1): 10846, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32616740

RESUMO

In plants, the shikimate pathway generally occurs in plastids and leads to the biosynthesis of aromatic amino acids. Chorismate synthase (CS) catalyses the last step of the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate, but the role of CS in the metabolism of higher plants has not been reported. In this study, we found that PhCS, which is encoded by a single-copy gene in petunia (Petunia hybrida), contains N-terminal plastidic transit peptides and peroxisomal targeting signals. Green fluorescent protein (GFP) fusion protein assays revealed that PhCS was localized in chloroplasts and, unexpectedly, in peroxisomes. Petunia plants with reduced PhCS activity were generated through virus-induced gene silencing and further characterized. PhCS silencing resulted in reduced CS activity, severe growth retardation, abnormal flower and leaf development and reduced levels of folate and pigments, including chlorophylls, carotenoids and anthocyanins. A widely targeted metabolomics analysis showed that most primary and secondary metabolites were significantly changed in pTRV2-PhCS-treated corollas. Overall, the results revealed a clear connection between primary and specialized metabolism related to the shikimate pathway in petunia.


Assuntos
Antocianinas/metabolismo , Cloroplastos/enzimologia , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Peroxissomos/enzimologia , Petunia/crescimento & desenvolvimento , Fósforo-Oxigênio Liases/metabolismo , Flores/metabolismo , Petunia/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
9.
Planta ; 251(5): 98, 2020 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-32306103

RESUMO

MAIN CONCLUSION: This work reveals information about new peroxisomal targeting signals type 1 and identifies trehalose-6-phosphate phosphatase I as multitargeted and is implicated in plant development, reproduction, and stress response. A putative, non-canonical peroxisomal targeting signal type 1 (PTS1) Pro-Arg-Met > was identified in the extreme C-terminus of trehalose-6-phosphate phosphatase (TPP)I. TPP catalyzes the final step of trehalose synthesis, and the enzyme was previously characterized to be nuclear only (Krasensky et al. in Antioxid Redox Signal 21(9):1289-1304, 2014). Here we show that the TPPI C-terminal decapeptide ending with Pro-Arg-Met > or Pro-Lys-Met > can indeed function as a PTS1. Upon transient expression in two plant expression systems, the free C- or N-terminal end led to the full-length TPPI targeting to peroxisomes and plastids, respectively. The nucleus and nucleolus targeting of the full-length TPPI was observed in both cases. The homozygous T-DNA insertion line of TPPI showed a pleiotropic phenotype including smaller leaves, shorter roots, delayed flowering, hypersensitivity to salt, and a sucrose dependent seedling development. Our results identify novel PTS1s, and TPPI as a protein multi-targeted to peroxisomes, plastids, nucleus, and nucleolus. Altogether our findings implicate an essential role for TPPI in development, reproduction, and cell signaling.


Assuntos
Arabidopsis/enzimologia , Flores/enzimologia , Sinais de Orientação para Peroxissomos , Monoéster Fosfórico Hidrolases/metabolismo , Transdução de Sinais , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Biologia Computacional , Flores/genética , Flores/crescimento & desenvolvimento , Flores/fisiologia , Peroxissomos/enzimologia , Monoéster Fosfórico Hidrolases/genética , Filogenia , Plastídeos/metabolismo , Reprodução
10.
Biochim Biophys Acta Mol Basis Dis ; 1866(5): 165720, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32057943

RESUMO

Carnitine plays an essential role in mitochondrial fatty acid ß-oxidation as a part of a cycle that transfers long-chain fatty acids across the mitochondrial membrane and involves two carnitine palmitoyltransferases (CPT1 and CPT2). Two distinct carnitine acyltransferases, carnitine octanoyltransferase (COT) and carnitine acetyltransferase (CAT), are peroxisomal enzymes, which indicates that carnitine is not only important for mitochondrial, but also for peroxisomal metabolism. It has been demonstrated that after peroxisomal metabolism, specific intermediates can be exported as acylcarnitines for subsequent and final mitochondrial metabolism. There is also evidence that peroxisomes are able to degrade fatty acids that are typically handled by mitochondria possibly after transport as acylcarnitines. Here we review the biochemistry and physiological functions of metabolite exchange between peroxisomes and mitochondria with a special focus on acylcarnitines.


Assuntos
Carnitina Aciltransferases/metabolismo , Carnitina/análogos & derivados , Ácidos Graxos/metabolismo , Mitocôndrias/enzimologia , Peroxissomos/enzimologia , Carnitina/metabolismo
11.
J Exp Bot ; 71(3): 823-836, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31641750

RESUMO

Recent work revealed that PGD2, an Arabidopsis 6-phosphogluconate dehydrogenase (6-PGD) catalysing the third step of the oxidative pentose-phosphate pathway (OPPP) in peroxisomes, is essential during fertilization. Earlier studies on the second step, catalysed by PGL3, a dually targeted Arabidopsis 6-phosphogluconolactonase (6-PGL), reported the importance of OPPP reactions in plastids but their irrelevance in peroxisomes. Assuming redundancy of 6-PGL activity in peroxisomes, we examined the sequences of other higher plant enzymes. In tomato, there exist two 6-PGL isoforms with the strong PTS1 motif SKL. However, their analysis revealed problems regarding peroxisomal targeting: reporter-PGL detection in peroxisomes required construct modification, which was also applied to the Arabidopsis isoforms. The relative contribution of PGL3 versus PGL5 during fertilization was assessed by mutant crosses. Reduced transmission ratios were found for pgl3-1 (T-DNA-eliminated PTS1) and also for knock-out allele pgl5-2. The prominent role of PGL3 showed as compromised growth of pgl3-1 seedlings on sucrose and higher activity of mutant PGL3-1 versus PGL5 using purified recombinant proteins. Evidence for PTS1-independent uptake was found for PGL3-1 and other Arabidopsis PGL isoforms, indicating that peroxisome import may be supported by a piggybacking mechanism. Thus, multiple redundancy at the level of the second OPPP step in peroxisomes explains the occurrence of pgl3-1 mutant plants.


Assuntos
Arabidopsis/enzimologia , Hidrolases de Éster Carboxílico/metabolismo , Peroxissomos/enzimologia , Arabidopsis/genética , Hidrolases de Éster Carboxílico/genética , Isoenzimas/metabolismo , Lycopersicon esculentum/enzimologia
12.
Adv Exp Med Biol ; 1299: 55-70, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33417207

RESUMO

Peroxisomes play a central role in metabolism as exemplified by the fact that many genetic disorders in humans have been identified through the years in which there is an impairment in one or more of these peroxisomal functions, in most cases associated with severe clinical signs and symptoms. One of the key functions of peroxisomes is the ß-oxidation of fatty acids which differs from the oxidation of fatty acids in mitochondria in many respects which includes the different substrate specificities of the two organelles. Whereas mitochondria are the main site of oxidation of medium-and long-chain fatty acids, peroxisomes catalyse the ß-oxidation of a distinct set of fatty acids, including very-long-chain fatty acids, pristanic acid and the bile acid intermediates di- and trihydroxycholestanoic acid. Peroxisomes require the functional alliance with multiple subcellular organelles to fulfil their role in metabolism. Indeed, peroxisomes require the functional interaction with lysosomes, lipid droplets and the endoplasmic reticulum, since these organelles provide the substrates oxidized in peroxisomes. On the other hand, since peroxisomes lack a citric acid cycle as well as respiratory chain, oxidation of the end-products of peroxisomal fatty acid oxidation notably acetyl-CoA, and different medium-chain acyl-CoAs, to CO2 and H2O can only occur in mitochondria. The same is true for the reoxidation of NADH back to NAD+. There is increasing evidence that these interactions between organelles are mediated by tethering proteins which bring organelles together in order to allow effective exchange of metabolites. It is the purpose of this review to describe the current state of knowledge about the role of peroxisomes in fatty acid oxidation, the transport of metabolites across the peroxisomal membrane, its functional interaction with other subcellular organelles and the disorders of peroxisomal fatty acid ß-oxidation identified so far in humans.


Assuntos
Ácidos Graxos/metabolismo , Transtornos Peroxissômicos/metabolismo , Peroxissomos/metabolismo , Humanos , Metabolismo dos Lipídeos , Oxirredução , Transtornos Peroxissômicos/enzimologia , Peroxissomos/enzimologia
13.
Adv Exp Med Biol ; 1299: 161-167, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33417214

RESUMO

This paper reports that the human peroxisomal 3-ketoacyl-CoA thiolase expression shows three transcripts: Tr1 (1705 bp), Tr2 (1375 bp) and Tr3 (1782 bp). Their highest expression is observed in the human liver and at a lesser extent in hepatic-derived HepG2 cells. The intestine and blood and endothelial cells show lower expression. The lowest expression is found in adipocytes. The transcript Tr3 appears to be the most abundant. So far, no data have been published regarding the regulation of the human peroxisomal thiolase. After cloning a fragment of the 5' region involved in the regulation of the human thiolase gene, the effects of different treatments have been studied on the thiolase expression in the hepatoma HepG2 human cell line. Biocomputing analysis indicates that (i) a GRE (glucocorticoid response element) is located at -650 bp upstream of the transcription initiation site; (ii) a C/EBPα (CCAAT/enhancer-binding protein) binding site is located at - 1000 bp upstream of the transcription initiation site - and (iii) there is no putative PPRE (peroxisome proliferator-activated receptor response element). In the human HepG2 cells, thiolase expression is upregulated by glucose and downregulated by insulin and sterols, while dexamethasone and fatty acids have no effect. The ciprofibrate, a peroxisome proliferator, leads only to a weak stimulation of the mRNA expression as compared to thiolase B expression in the rat liver.


Assuntos
Acetil-CoA C-Acetiltransferase/metabolismo , Peroxissomos/enzimologia , Animais , Glucose/farmacologia , Humanos , Insulina/farmacologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Especificidade de Órgãos , Esteróis/farmacologia , Distribuição Tecidual
14.
Int J Mol Sci ; 20(24)2019 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-31817290

RESUMO

There are three human enzymes with HMG-CoA lyase activity that are able to synthesize ketone bodies in different subcellular compartments. The mitochondrial HMG-CoA lyase was the first to be described, and catalyzes the cleavage of 3-hydroxy-3-methylglutaryl CoA to acetoacetate and acetyl-CoA, the common final step in ketogenesis and leucine catabolism. This protein is mainly expressed in the liver and its function is metabolic, since it produces ketone bodies as energetic fuels when glucose levels are low. Another isoform is encoded by the same gene for the mitochondrial HMG-CoA lyase (HMGCL), but it is located in peroxisomes. The last HMG-CoA lyase to be described is encoded by a different gene, HMGCLL1, and is located in the cytosolic side of the endoplasmic reticulum membrane. Some activity assays and tissue distribution of this enzyme have shown the brain and lung as key tissues for studying its function. Although the roles of the peroxisomal and cytosolic HMG-CoA lyases remain unknown, recent studies highlight the role of ketone bodies in metabolic remodeling, homeostasis, and signaling, providing new insights into the molecular and cellular function of these enzymes.


Assuntos
Citosol/enzimologia , Mitocôndrias/enzimologia , Oxo-Ácido-Liases/metabolismo , Peroxissomos/enzimologia , Metabolismo Energético , Evolução Molecular , Humanos , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/metabolismo , Corpos Cetônicos/metabolismo , Fígado/enzimologia , Oxo-Ácido-Liases/classificação , Oxo-Ácido-Liases/genética
15.
Sci Rep ; 9(1): 10502, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324846

RESUMO

The peroxisomal ABC transporter, Comatose (CTS), a full length transporter from Arabidopsis has intrinsic acyl-CoA thioesterase (ACOT) activity, important for physiological function. We used molecular modelling, mutagenesis and biochemical analysis to identify amino acid residues important for ACOT activity. D863, Q864 and T867 lie within transmembrane helix 9. These residues are orientated such that they might plausibly contribute to a catalytic triad similar to type II Hotdog fold thioesterases. When expressed in Saccharomyces cerevisiae, mutation of these residues to alanine resulted in defective of ß-oxidation. All CTS mutants were expressed and targeted to peroxisomes and retained substrate-stimulated ATPase activity. When expressed in insect cell membranes, Q864A and S810N had similar ATPase activity to wild type but greatly reduced ACOT activity, whereas the Walker A mutant K487A had greatly reduced ATPase and no ATP-dependent ACOT activity. In wild type CTS, ATPase but not ACOT was stimulated by non-cleavable C14 ether-CoA. ACOT activity was stimulated by ATP but not by non-hydrolysable AMPPNP. Thus, ACOT activity depends on functional ATPase activity but not vice versa, and these two activities can be separated by mutagenesis. Whether D863, Q864 and T867 have a catalytic role or play a more indirect role in NBD-TMD communication is discussed.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Ácido Graxo Sintases/metabolismo , Tioléster Hidrolases/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Animais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Domínio Catalítico , Linhagem Celular , Ácido Graxo Sintases/genética , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Ácido Oleico/metabolismo , Oxirredução , Peroxissomos/enzimologia , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Spodoptera , Relação Estrutura-Atividade , Tioléster Hidrolases/genética
16.
Free Radic Biol Med ; 141: 279-290, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31238127

RESUMO

Catalases are among the main scavengers of reactive oxygen species (ROS) present in the peroxisome, thereby preventing oxidative cellular and tissular damage. In human, multiple diseases are associated with malfunction of these organelles, which causes accumulation of ROS species and consequently the inefficient detoxification of cells. Despite intense research, much remains to be clarified about the precise molecular role of catalase in cellular homeostasis. Yeast peroxisomes and their peroxisomal catalases have been used as eukaryotic models for oxidative metabolism, ROS generation and detoxification, and associated pathologies. In order to provide reliable models for oxidative metabolism research, we have determined the high-resolution crystal structures of peroxisomal catalase from two important biotechnology and basic biology yeast models, Pichia pastoris and Kluyveromyces lactis. We have performed an extensive functional, biochemical and stability characterization of both enzymes in order to establish their differential activity profiles. Furthermore, we have analyzed the role of the peroxisomal catalase under study in the survival of yeast to oxidative burst challenges combining methanol, water peroxide, and sodium chloride. Interestingly, whereas catalase activity was induced 200-fold upon challenging the methylotrophic P. pastoris cells with methanol, the increase in catalase activity in the non-methylotrophic K. lactis was only moderate. The inhibitory effect of sodium azide and ß-mercaptoethanol over both catalases was analyzed, establishing IC50 values for both compounds that are consistent with an elevated resistance of both enzymes toward these inhibitors. Structural comparison of these two novel catalase structures allows us to rationalize the differential susceptibility to inhibitors and oxidative bursts. The inherent worth and validity of the P. pastoris and K. lactis yeast models for oxidative damage will be strengthened by the availability of reliable structural-functional information on these enzymes, which are central to our understanding of peroxisomal response toward oxidative stress.


Assuntos
Catalase/metabolismo , Sequestradores de Radicais Livres/metabolismo , Estresse Oxidativo/genética , Catalase/química , Catalase/genética , Eucariotos/enzimologia , Eucariotos/genética , Humanos , Kluyveromyces/enzimologia , Oxirredução , Peroxissomos/enzimologia , Peroxissomos/metabolismo , Pichia/enzimologia , Espécies Reativas de Oxigênio/metabolismo
17.
Plant Physiol ; 180(2): 783-792, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30886114

RESUMO

The oxygenation of ribulose 1,5-bisphosphate by Rubisco is the first step in photorespiration and reduces the efficiency of photosynthesis in C3 plants. Our recent data indicate that mutants in photorespiration have increased rates of photosynthetic cyclic electron flow around photosystem I. We investigated mutant lines lacking peroxisomal hydroxypyruvate reductase to determine if there are connections between 2-phosphoglycolate accumulation and cyclic electron flow in Arabidopsis (Arabidopsis thaliana). We found that 2-phosphoglycolate is a competitive inhibitor of triose phosphate isomerase, an enzyme in the Calvin-Benson cycle that converts glyceraldehyde 3-phosphate to dihydroxyacetone phosphate. This block in metabolism could be overcome if glyceraldehyde 3-phosphate is exported to the cytosol, where cytosolic triose phosphate isomerase could convert it to dihydroxyacetone phosphate. We found evidence that carbon is reimported as glucose-6-phosphate, forming a cytosolic bypass around the block of stromal triose phosphate isomerase. However, this also stimulates a glucose-6-phosphate shunt, which consumes ATP, which can be compensated by higher rates of cyclic electron flow.


Assuntos
Citosol/metabolismo , Glucose-6-Fosfato/metabolismo , Hidroxipiruvato Redutase/metabolismo , Peroxissomos/enzimologia , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Carbono/metabolismo , Dióxido de Carbono/metabolismo , Carotenoides/metabolismo , Clorofila/metabolismo , Fosfato de Di-Hidroxiacetona/metabolismo , Transporte de Elétrons , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Gliceraldeído 3-Fosfato/metabolismo , Glicolatos , Cinética , Modelos Biológicos , Mutação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Triose-Fosfato Isomerase/metabolismo
18.
Arch Pharm Res ; 42(5): 393-406, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30739266

RESUMO

Peroxisomes and their (patho-)physiological importance in heath and disease have attracted increasing interest during last few decades. Together with mitochondria, peroxisomes comprise key metabolic platforms for oxidation of various fatty acids and redox regulation. In addition, peroxisomes contribute to bile acid, cholesterol, and plasmalogen biosynthesis. The importance of functional peroxisomes for cellular metabolism is demonstrated by the marked brain and systemic organ abnormalities occuring in peroxisome biogenesis disorders and peroxisomal enzyme deficiencies. Current evidences indicate that peroxisomal function is declined with aging, with peroxisomal dysfunction being linked to early onset of multiple age-related diseases including neurodegenerative diseases. Herein, we review recent progress toward understanding the physiological roles and pathological implications of peroxisomal dysfunctions, focusing on neurodegenerative disease.


Assuntos
Encéfalo/patologia , Doenças Neurodegenerativas/etiologia , Transtornos Peroxissômicos/etiologia , Peroxissomos/patologia , Envelhecimento/fisiologia , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Modelos Animais de Doenças , Humanos , Metabolismo dos Lipídeos/fisiologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Oxirredução , Transtornos Peroxissômicos/metabolismo , Transtornos Peroxissômicos/patologia , Peroxissomos/enzimologia , Peroxissomos/metabolismo , Espécies Reativas de Oxigênio/metabolismo
19.
Mol Cell Biochem ; 456(1-2): 53-62, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30604065

RESUMO

The retinal pathology in peroxisomal disorders suggests that peroxisomes are important to maintain retinal homeostasis and function. These ubiquitous cell organelles are mainly involved in lipid metabolism, which comprises α- and ß-oxidation and ether lipid synthesis. Although peroxisomes were extensively studied in liver, their role in the retina still remains to be elucidated. As a first step in gaining more insight into the role of peroxisomes in retinal physiology, we performed immunohistochemical stainings, immunoblotting and enzyme activity measurements to reveal the distribution of peroxisomes and peroxisomal lipid metabolizing enzymes in the murine retina. Whereas peroxisomes were detected in every retinal layer, we found a clear differential distribution of the peroxisomal lipid metabolizing enzymes in the neural retina compared to the retinal pigment epithelium. In particular, the ABC transporters that transfer lipid substrates into the organelle as well as several enzymes of the ß-oxidation pathway were enriched either in the neural retina or in the retinal pigment epithelium. In conclusion, our results strongly indicate that peroxisome function varies between different regions in the murine retina.


Assuntos
Proteínas do Olho/metabolismo , Metabolismo dos Lipídeos/fisiologia , Peroxissomos/enzimologia , Retina/enzimologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Camundongos
20.
FASEB J ; 33(3): 4355-4364, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30540494

RESUMO

Peroxisomes are essential organelles for the specialized oxidation of a wide variety of fatty acids, but they are also able to degrade fatty acids that are typically handled by mitochondria. Using a combination of pharmacological inhibition and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 genome editing technology to simultaneously manipulate peroxisomal and mitochondrial fatty acid ß-oxidation (FAO) in HEK-293 cells, we identified essential players in the metabolic crosstalk between these organelles. Depletion of carnitine palmitoyltransferase (CPT)2 activity through pharmacological inhibition or knockout (KO) uncovered a significant residual peroxisomal oxidation of lauric and palmitic acid, leading to the production of peroxisomal acylcarnitine intermediates. Generation and analysis of additional single- and double-KO cell lines revealed that the D-bifunctional protein (HSD17B4) and the peroxisomal ABC transporter ABCD3 are essential in peroxisomal oxidation of lauric and palmitic acid. Our results indicate that peroxisomes not only accept acyl-CoAs but can also oxidize acylcarnitines in a similar biochemical pathway. By using an Hsd17b4 KO mouse model, we demonstrated that peroxisomes contribute to the plasma acylcarnitine profile after acute inhibition of CPT2, proving in vivo relevance of this pathway. We summarize that peroxisomal FAO is important when mitochondrial FAO is defective or overloaded.-Violante, S., Achetib, N., van Roermund, C. W. T., Hagen, J., Dodatko, T., Vaz, F. M., Waterham, H. R., Chen, H., Baes, M., Yu, C., Argmann, C. A., Houten, S. M. Peroxisomes can oxidize medium- and long-chain fatty acids through a pathway involving ABCD3 and HSD17B4.


Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Ácidos Graxos/metabolismo , Proteína Multifuncional do Peroxissomo-2/fisiologia , Peroxissomos/enzimologia , Transportadores de Cassetes de Ligação de ATP/deficiência , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Sistemas CRISPR-Cas , Carnitina/análogos & derivados , Carnitina/metabolismo , Carnitina O-Palmitoiltransferase/antagonistas & inibidores , Carnitina O-Palmitoiltransferase/deficiência , Carnitina O-Palmitoiltransferase/fisiologia , Células HEK293 , Humanos , Ácidos Láuricos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/enzimologia , Oxirredução , Ácido Palmítico/metabolismo , Enzima Bifuncional do Peroxissomo/deficiência , Proteína Multifuncional do Peroxissomo-2/deficiência , Proteína Multifuncional do Peroxissomo-2/genética , Proteínas Recombinantes/metabolismo
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