RESUMO
Aldo-keto reductases (AKRs) comprise a superfamily of proteins involved in the reduction and oxidation of biogenic and xenobiotic carbonyls. In humans, at least 15 AKR superfamily members have been identified so far. One of these is a newly identified gene locus, AKR1B15, which clusters on chromosome 7 with the other human AKR1B subfamily members (i.e. AKR1B1 and AKR1B10). We show that alternative splicing of the AKR1B15 gene transcript gives rise to two protein isoforms with different N termini: AKR1B15.1 is a 316-amino acid protein with 91% amino acid identity to AKR1B10; AKR1B15.2 has a prolonged N terminus and consists of 344 amino acid residues. The two gene products differ in their expression level, subcellular localization, and activity. In contrast with other AKR enzymes, which are mostly cytosolic, AKR1B15.1 co-localizes with the mitochondria. Kinetic studies show that AKR1B15.1 is predominantly a reductive enzyme that catalyzes the reduction of androgens and estrogens with high positional selectivity (17ß-hydroxysteroid dehydrogenase activity) as well as 3-keto-acyl-CoA conjugates and exhibits strong cofactor selectivity toward NADP(H). In accordance with its substrate spectrum, the enzyme is expressed at the highest levels in steroid-sensitive tissues, namely placenta, testis, and adipose tissue. Placental and adipose expression could be reproduced in the BeWo and SGBS cell lines, respectively. In contrast, AKR1B15.2 localizes to the cytosol and displays no enzymatic activity with the substrates tested. Collectively, these results demonstrate the existence of a novel catalytically active AKR, which is associated with mitochondria and expressed mainly in steroid-sensitive tissues.
Assuntos
Acil Coenzima A/metabolismo , Processamento Alternativo/genética , Mitocôndrias/enzimologia , Oxirredutases Atuantes sobre Doadores de Grupos Aldeído ou Oxo/metabolismo , Esteroides/metabolismo , Acil Coenzima A/genética , Tecido Adiposo/metabolismo , Sequência de Aminoácidos , Regulação Enzimológica da Expressão Gênica , Humanos , Cinética , Mitocôndrias/metabolismo , Oxirredução , Oxirredutases Atuantes sobre Doadores de Grupos Aldeído ou Oxo/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Recent research suggests that in addition to their role as soluble electron carriers, pyridine nucleotides [NAD(P)(H)] also regulate ion transport mechanisms. This mode of regulation seems to have been conserved through evolution. Several bacterial ion-transporting proteins or their auxiliary subunits possess nucleotide-binding domains. In eukaryotes, the Kv1 and Kv4 channels interact with pyridine nucleotide-binding ß-subunits that belong to the aldo-keto reductase superfamily. Binding of NADP(+) to Kvß removes N-type inactivation of Kv currents, whereas NADPH stabilizes channel inactivation. Pyridine nucleotides also regulate Slo channels by interacting with their cytosolic regulator of potassium conductance domains that show high sequence homology to the bacterial TrkA family of K(+) transporters. These nucleotides also have been shown to modify the activity of the plasma membrane K(ATP) channels, the cystic fibrosis transmembrane conductance regulator, the transient receptor potential M2 channel, and the intracellular ryanodine receptor calcium release channels. In addition, pyridine nucleotides also modulate the voltage-gated sodium channel by supporting the activity of its ancillary subunit-the glycerol-3-phosphate dehydrogenase-like protein. Moreover, the NADP(+) metabolite, NAADP(+), regulates intracellular calcium homeostasis via the 2-pore channel, ryanodine receptor, or transient receptor potential M2 channels. Regulation of ion channels by pyridine nucleotides may be required for integrating cell ion transport to energetics and for sensing oxygen levels or metabolite availability. This mechanism also may be an important component of hypoxic pulmonary vasoconstriction, memory, and circadian rhythms, and disruption of this regulatory axis may be linked to dysregulation of calcium homeostasis and cardiac arrhythmias.
Assuntos
Cátions/metabolismo , Canais Iônicos/fisiologia , Transporte de Íons/fisiologia , NADP/fisiologia , NAD/fisiologia , Animais , Sítios de Ligação , Sinalização do Cálcio/fisiologia , Proteínas de Transporte/fisiologia , ADP-Ribose Cíclica/fisiologia , Células Eucarióticas/metabolismo , Homeostase/fisiologia , Humanos , Ativação do Canal Iônico/fisiologia , Canais Iônicos/química , Mamíferos/metabolismo , NADP/análogos & derivados , Fosforilação , Potássio/metabolismo , Células Procarióticas/metabolismo , Sódio/metabolismoRESUMO
Oxidation of unsaturated lipids generates reactive aldehydes that accumulate in tissues during inflammation, ischemia, or aging. These aldehydes form covalent adducts with histidine-containing dipeptides such as carnosine and anserine, which are present in high concentration in skeletal muscle, heart, and brain. The metabolic pathways involved in the detoxification and elimination of these conjugates are, however, poorly defined, and their significance in regulating oxidative stress is unclear. Here we report that conjugates of carnosine with aldehydes such as acrolein are produced during normal metabolism and excreted in the urine of mice and adult human non-smokers as carnosine-propanols. Our studies show that the reduction of carnosine-propanals is catalyzed by the enzyme aldose reductase (AR). Carnosine-propanals were converted to carnosine-propanols in the lysates of heart, skeletal muscle, and brain tissue from wild-type (WT) but not AR-null mice. In comparison with WT mice, the urinary excretion of carnosine-propanols was decreased in AR-null mice. Carnosine-propanals formed covalent adducts with nucleophilic amino acids leading to the generation of carnosinylated proteins. Deletion of AR increased the abundance of proteins bound to carnosine in skeletal muscle, brain, and heart of aged mice and promoted the accumulation of carnosinylated proteins in hearts subjected to global ischemia ex vivo. Perfusion with carnosine promoted post-ischemic functional recovery in WT but not in AR-null mouse hearts. Collectively, these findings reveal a previously unknown metabolic pathway for the removal of carnosine-propanal conjugates and suggest a new role of AR as a critical regulator of protein carnosinylation and carnosine-mediated tissue protection.
Assuntos
Acroleína/metabolismo , Aldeído Redutase/metabolismo , Carnosina/metabolismo , Acetilcisteína/análise , Animais , Antioxidantes/metabolismo , Humanos , Inflamação , Peroxidação de Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Traumatismo por Reperfusão , Distribuição TecidualRESUMO
OBJECTIVE: Atherosclerotic lesions are associated with the accumulation of reactive aldehydes derived from oxidized lipids. Although inhibition of aldehyde metabolism has been shown to exacerbate atherosclerosis and enhance the accumulation of aldehyde-modified proteins in atherosclerotic plaques, no therapeutic interventions have been devised to prevent aldehyde accumulation in atherosclerotic lesions. APPROACH AND RESULTS: We examined the efficacy of carnosine, a naturally occurring ß-alanyl-histidine dipeptide, in preventing aldehyde toxicity and atherogenesis in apolipoprotein E-null mice. In vitro, carnosine reacted rapidly with lipid peroxidation-derived unsaturated aldehydes. Gas chromatography mass-spectrometry analysis showed that carnosine inhibits the formation of free aldehydes 4-hydroxynonenal and malonaldialdehyde in Cu(2+)-oxidized low-density lipoprotein. Preloading bone marrow-derived macrophages with cell-permeable carnosine analogs reduced 4-hydroxynonenal-induced apoptosis. Oral supplementation with octyl-D-carnosine decreased atherosclerotic lesion formation in aortic valves of apolipoprotein E-null mice and attenuated the accumulation of protein-acrolein, protein-4-hydroxyhexenal, and protein-4-hydroxynonenal adducts in atherosclerotic lesions, whereas urinary excretion of aldehydes as carnosine conjugates was increased. CONCLUSIONS: The results of this study suggest that carnosine inhibits atherogenesis by facilitating aldehyde removal from atherosclerotic lesions. Endogenous levels of carnosine may be important determinants of atherosclerotic lesion formation, and treatment with carnosine or related peptides could be a useful therapy for the prevention or the treatment of atherosclerosis.
Assuntos
Aterosclerose/prevenção & controle , Carnosina/farmacologia , Suplementos Nutricionais , Peroxidação de Lipídeos/fisiologia , Lipoproteínas LDL/metabolismo , Aldeídos/metabolismo , Animais , Apolipoproteínas E/deficiência , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , Distribuição Aleatória , Valores de ReferênciaRESUMO
Lipid peroxidation products, such as 4-hydroxy-trans-2-nonenal (HNE), cause endothelial activation, and they increase the adhesion of the endothelium to circulating leukocytes. Nevertheless, the mechanisms underlying these effects remain unclear. We observed that in HNE-treated human umbilical vein endothelial cells, some of the protein-HNE adducts colocalize with the endoplasmic reticulum (ER) and that HNE forms covalent adducts with several ER chaperones that assist in protein folding. We also found that at concentrations that did not induce apoptosis or necrosis, HNE activated the unfolded protein response, leading to an increase in XBP-1 splicing, phosphorylation of protein kinase-like ER kinase and eukaryotic translation initiation factor 2α, and the induction of ATF3 and ATF4. This increase in eukaryotic translation initiation factor 2α phosphorylation was prevented by transfection with protein kinase-like ER kinase siRNA. Treatment with HNE increased the expression of the ER chaperones, GRP78 and HERP. Exposure to HNE led to a depletion of reduced glutathione and an increase in the production of reactive oxygen species (ROS); however, glutathione depletion and ROS production by tert-butyl-hydroperoxide did not trigger the unfolded protein response. Pretreatment with a chemical chaperone, phenylbutyric acid, or adenoviral transfection with ATF6 attenuated HNE-induced monocyte adhesion and IL-8 induction. Moreover, phenylbutyric acid and taurine-conjugated ursodeoxycholic acid attenuated HNE-induced leukocyte rolling and their firm adhesion to the endothelium in rat cremaster muscle. These data suggest that endothelial activation by HNE is mediated in part by ER stress, induced by mechanisms independent of ROS production or glutathione depletion. The induction of ER stress may be a significant cause of vascular inflammation induced by products of oxidized lipids.
Assuntos
Aldeídos/metabolismo , Aldeídos/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Peroxidação de Lipídeos , Sequência de Aminoácidos , Animais , Chaperona BiP do Retículo Endoplasmático , Endotélio/citologia , Endotélio/efeitos dos fármacos , Endotélio/metabolismo , Glutationa/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Dados de Sequência Molecular , Transporte Proteico/efeitos dos fármacos , Proteínas/química , Proteínas/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
Voltage-gated potassium (Kv) channels are tetrameric assemblies of transmembrane Kv proteins with cytosolic N- and C-termini. The N-terminal domain of Kv1 proteins binds to ß-subunits, but the role of the C-terminus is less clear. Therefore, we studied the role of the C-terminus in regulating Kv1.5 channel and its interactions with Kvß-subunits. When expressed in COS-7 cells, deletion of the C-terminal domain of Kv1.5 did not affect channel gating or kinetics. Coexpression of Kv1.5 with Kvß3 increased current inactivation, whereas Kvß2 caused a hyperpolarizing shift in the voltage dependence of current activation. Inclusion of NADPH in the patch pipette solution accelerated the inactivation of Kv1.5-Kvß3 currents. In contrast, NADP(+) decreased the rate and the extent of Kvß3-induced inactivation and reversed the hyperpolarizing shift in the voltage dependence of activation induced by Kvß2. Currents generated by Kv1.5ΔC+Kvß3 or Kv1.5ΔC+Kvß2 complexes did not respond to changes in intracellular pyridine nucleotide concentration, indicating that the C-terminus is required for pyridine nucleotide-dependent interactions between Kvß and Kv1.5. A glutathione-S-transferase (GST) fusion protein containing the C-terminal peptide of Kv1.5 did not bind to apoKvß2, but displayed higher affinity for Kvß2:NADPH than Kvß2:NADP(+). The GST fusion protein also precipitated Kvß proteins from mouse brain lysates. Pull-down experiments, structural analysis and electrophysiological data indicated that a specific region of the C-terminus (Arg543-Val583) is required for Kvß binding. These results suggest that the C-terminal domain of Kv1.5 interacts with ß-subunits and that this interaction is essential for the differential regulation of Kv currents by oxidized and reduced nucleotides.
Assuntos
Ativação do Canal Iônico/fisiologia , Canal de Potássio Kv1.5/fisiologia , Subunidades Proteicas/fisiologia , Piridinas/metabolismo , Animais , Células COS , Chlorocebus aethiops , Deleção de Genes , Rim/citologia , Rim/metabolismo , Canal de Potássio Kv1.5/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Técnicas de Patch-ClampRESUMO
The generation of oxidized phospholipids in lipoproteins has been linked to vascular inflammation in atherosclerotic lesions. Products of phospholipid oxidation increase endothelial activation; however, their effects on macrophages are poorly understood, and it is unclear whether these effects are regulated by the biochemical pathways that metabolize oxidized phospholipids. We found that incubation of 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) with THP-1-derived macrophages upregulated the expression of cytokine genes, including granulocyte/macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF)-α, monocyte chemotactic protein 1 (MCP-1), interleukin (IL)-1ß, IL-6, and IL-8. In these cells, reagent POVPC was either hydrolyzed to lyso-phosphatidylcholine (lyso-PC) or reduced to 1-palmitoyl-2-(5-hydroxy-valeroyl)-sn-glycero-3-phosphocholine (PHVPC). Treatment with the phospholipase A(2) (PLA(2)) inhibitor, pefabloc, decreased POVPC hydrolysis and increased PHVPC accumulation. Pefabloc also increased the induction of cytokine genes in POVPC-treated cells. In contrast, PHVPC accumulation and cytokine production were decreased upon treatment with the aldose reductase (AR) inhibitor, tolrestat. In comparison with POVPC, lyso-PC led to 2- to 3-fold greater and PHVPC 10- to 100-fold greater induction of cytokine genes. POVPC-induced cytokine gene induction was prevented in bone-marrow derived macrophages from AR-null mice. These results indicate that although hydrolysis is the major pathway of metabolism, reduction further increases the proinflammatory responses to POVPC. Thus, vascular inflammation in atherosclerotic lesions is likely to be regulated by metabolism of phospholipid aldehydes in macrophages.
Assuntos
Inflamação/metabolismo , Éteres Fosfolipídicos/metabolismo , Éteres Fosfolipídicos/farmacologia , Aldeído Redutase/metabolismo , Animais , Linhagem Celular , Citocinas/genética , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/metabolismo , Camundongos , Oxirredução , Regulação para Cima/efeitos dos fármacosRESUMO
Aldose reductase (AR) is a multifunctional enzyme that catalyzes the reduction of glucose and lipid peroxidation-derived aldehydes. During myocardial ischemia, the activity of AR is increased due to the oxidation of its cysteine residues to sulfenic acids. It is not known, however, whether the activated, sulfenic form of the protein (AR-SOH) is converted back to its reduced, unactivated state (AR-SH). We report here that in perfused mouse hearts activation of AR during 15 min of global ischemia is completely reversed by 30 min of reperfusion. During reperfusion, AR-SOH was converted to a mixed disulfide (AR-SSG). Deactivation of AR and the appearance of AR-SSG during reperfusion were delayed in hearts of mice lacking glutathione S-transferase P (GSTP). In vitro, GSTP accelerated glutathiolation and inactivation of AR-SOH. Reduction of AR-SSG to AR-SH was facilitated by glutaredoxin (GRX). Ischemic activation of AR was increased in GRX-null hearts but was attenuated in the hearts of cardiospecific GRX transgenic mice. Incubation of AR-SSG with GRX led to the regeneration of the reduced form of the enzyme. In ischemic cardiospecific AR transgenic hearts, AR was co-immunoprecipitated with GSTP, whereas in reperfused hearts, the association of AR with GRX was increased. These findings suggest that upon reperfusion of the ischemic heart AR-SOH is converted to AR-SSG via GSTP-assisted glutathiolation. AR-SSG is then reduced by GRX to AR-SH. Sequential catalysis by GSTP and GRX may be a general redox switching mechanism that regulates the reduction of protein sulfenic acids to cysteines.
Assuntos
Glutarredoxinas/metabolismo , Glutationa Transferase/metabolismo , Isquemia Miocárdica/metabolismo , Compostos de Sulfidrila/metabolismo , Aldeído Redutase/metabolismo , Animais , Cisteína/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Isquemia Miocárdica/enzimologia , Oxirredução , Ácidos Sulfênicos/metabolismoRESUMO
RATIONALE: Atherosclerotic lesion formation is associated with the accumulation of oxidized lipids. Products of lipid oxidation, particularly aldehydes, stimulate cytokine production and enhance monocyte adhesion; however, their contribution to atherosclerotic lesion formation remains unclear. OBJECTIVE: To test the hypothesis that inhibition of aldehyde removal by aldose reductase (AR), which metabolizes both free and phospholipid aldehydes, exacerbates atherosclerotic lesion formation. METHODS AND RESULTS: In atherosclerotic lesions of apolipoprotein (apo)E-null mice, AR protein was located in macrophage-rich regions and its abundance increased with lesion progression. Treatment of apoE-null mice with AR inhibitors sorbinil or tolrestat increased early lesion formation but did not affect the formation of advanced lesions. Early lesions of AR(-/-)/apoE(-/-) mice maintained on high-fat diet were significantly larger when compared with age-matched AR(+/+)/apoE(-/-) mice. The increase in lesion area attributable to deletion of the AR gene was seen in both male and female mice. Pharmacological inhibition or genetic ablation of AR also increased the lesion formation in male mice made diabetic by streptozotocin treatment. Lesions in AR(-/-)/apoE(-/-) mice exhibited increased collagen and macrophage content and a decrease in smooth muscle cells. AR(-/-)/apoE(-/-) mice displayed a greater accumulation of the AR substrate 4-hydroxy trans-2-nonenal (HNE) in the plasma and protein-HNE adducts in arterial lesions than AR(+/+)/apoE(-/-) mice. CONCLUSIONS: These observations indicate that AR is upregulated in atherosclerotic lesions and it protects against early stages of atherogenesis by removing toxic aldehydes generated in oxidized lipids.
Assuntos
Aldeído Redutase/metabolismo , Aldeídos/metabolismo , Apolipoproteínas E , Aterosclerose/enzimologia , Fosfolipídeos/metabolismo , Aldeído Redutase/genética , Animais , Aterosclerose/genética , Aterosclerose/prevenção & controle , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/genética , Gorduras na Dieta/efeitos adversos , Gorduras na Dieta/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Imidazolidinas/farmacologia , Macrófagos/enzimologia , Masculino , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso/enzimologia , Naftalenos/farmacologia , Oxirredução/efeitos dos fármacos , Fosfolipídeos/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Aldehydes such as acrolein are ubiquitous pollutants present in automobile exhaust, cigarette, wood, and coal smoke. Such aldehydes are also constituents of several food substances and are present in drinking water, irrigation canals, and effluents from manufacturing plants. Oral intake represents the most significant source of exposure to acrolein and related aldehydes. To study the effects of short-term oral exposure to acrolein on lipoprotein levels and metabolism, adult mice were gavage-fed 0.1 to 5 mg acrolein/kg bwt and changes in plasma lipoproteins were assessed. Changes in hepatic gene expression related to lipid metabolism and cytokines were examined by qRT-PCR analysis. Acrolein feeding did not affect body weight, blood urea nitrogen, plasma creatinine, electrolytes, cytokines or liver enzymes, but increased plasma cholesterol and triglycerides. Similar results were obtained with apoE-null mice. Plasma lipoproteins from acrolein-fed mice showed altered electrophoretic mobility on agarose gels. Chromatographic analysis revealed elevated VLDL cholesterol, phospholipids, and triglycerides levels with little change in LDL or HDL. NMR analysis indicated shifts from small to large VLDL and from large to medium-small LDL with no change in the size of HDL particles. Increased plasma VLDL was associated with a significant decrease in post-heparin plasma hepatic lipase activity and a decrease in hepatic expression of hepatic lipase. These observations suggest that oral exposure to acrolein could induce or exacerbate systemic dyslipidemia and thereby contribute to cardiovascular disease risk.
Assuntos
Acroleína/toxicidade , Dislipidemias/induzido quimicamente , Lipoproteínas/sangue , Acroleína/administração & dosagem , Acroleína/farmacologia , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Relação Dose-Resposta a Droga , Vias de Administração de Medicamentos , Esquema de Medicação , Regulação da Expressão Gênica , Lipase/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Acrolein is a ubiquitous component of environmental pollutants such as automobile exhaust, cigarette, wood, and coal smoke. It is also a natural constituent of several foods and is generated endogenously during inflammation or oxidation of unsaturated lipids. Because increased inflammation and episodic exposure to acrolein-rich pollutants such as traffic emissions or cigarette smoke have been linked to acute myocardial infarction, we examined the effects of acrolein on matrix metalloproteinases (MMPs), which destabilize atherosclerotic plaques. Our studies show that exposure to acrolein resulted in the secretion of MMP-9 from differentiated THP-1 macrophages. Acrolein-treatment of macrophages also led to an increase in reactive oxygen species (ROS), free intracellular calcium ([Ca2+](i)), and xanthine oxidase (XO) activity. ROS production was prevented by allopurinol, but not by rotenone or apocynin and by buffering changes in [Ca2+](I) with BAPTA-AM. The increase in MMP production was abolished by pre-treatment with the antioxidants Tiron and N-acetyl cysteine (NAC) or with the xanthine oxidase inhibitors allopurinol or oxypurinol. Finally, MMP activity was significantly stimulated in aortic sections from apoE-null mice containing advanced atherosclerotic lesions after exposure to acrolein ex vivo. These observations suggest that acrolein exposure results in MMP secretion from macrophages via a mechanism that involves an increase in [Ca2+](I), leading to xanthine oxidase activation and an increase in ROS production. ROS-dependent activation of MMPs by acrolein could destabilize atherosclerotic lesions during brief episodes of inflammation or pollutant exposure.
Assuntos
Acroleína/toxicidade , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Metaloproteinases da Matriz/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerose/enzimologia , Aterosclerose/genética , Cálcio/metabolismo , Linhagem Celular , Poluentes Ambientais/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Knockout , Xantina Oxidase/metabolismoRESUMO
The beta-subunits of voltage-gated potassium (Kv) channels are members of the aldo-keto reductase (AKR) superfamily. These proteins regulate inactivation and membrane localization of Kv1 and Kv4 channels. The Kvbeta proteins bind to pyridine nucleotides with high affinity; however, their catalytic properties remain unclear. Here we report that recombinant rat Kvbeta2 catalyzes the reduction of a wide range of aldehydes and ketones. The rate of catalysis was slower (0.06-0.2 min(-1)) than those of most other AKRs but displayed the expected hyperbolic dependence on substrate concentration, with no evidence of allosteric cooperativity. Catalysis was prevented by site-directed substitution of Tyr-90 with phenylalanine, indicating that the acid-base catalytic residue, identified in other AKRs, has a conserved function in Kvbeta2. The protein catalyzed the reduction of a broad range of carbonyls, including aromatic carbonyls, electrophilic aldehydes and prostaglandins, phospholipids, and sugar aldehydes. Little or no activity was detected with carbonyl steroids. Initial velocity profiles were consistent with an ordered bi-bi rapid equilibrium mechanism in which NADPH binding precedes carbonyl binding. Significant primary kinetic isotope effects (2.0-3.1) were observed under single- and multiple-turnover conditions, indicating that the bond-breaking chemical step is rate-limiting. Structure-activity relationships with a series of para-substituted benzaldehydes indicated that the electronic interactions predominate during substrate binding and that no significant charge develops during the transition state. These data strengthen the view that Kvbeta proteins are catalytically active AKRs that impart redox sensitivity to Kv channels.
Assuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Aldeído Redutase , Aldeídos/metabolismo , Aldo-Ceto Redutases , Animais , Sítios de Ligação , Catálise , Deutério , Cromatografia Gasosa-Espectrometria de Massas , Concentração de Íons de Hidrogênio , Cetonas/metabolismo , Cinética , Mutagênese Sítio-Dirigida , Mutação , NADP/metabolismo , Fosfolipídeos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Prostaglandinas/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Especificidade por SubstratoRESUMO
The aldo-keto reductase (AKR) superfamily comprises enzymes that catalyze redox transformations involved in biosynthesis, intermediary metabolism, and detoxification. Substrates of AKRs include glucose, steroids, glycosylation end-products, lipid peroxidation products, and environmental pollutants. These proteins adopt a (beta/alpha)(8) barrel structural motif interrupted by a number of extraneous loops and helixes that vary between proteins and bring structural identity to individual families. The human AKR family differs from the rodent families. Due to their broad substrate specificity, AKRs play an important role in the phase II detoxification of a large number of pharmaceuticals, drugs, and xenobiotics.
Assuntos
Oxirredutases do Álcool/química , Oxirredutases do Álcool/fisiologia , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo , Oxirredutases do Álcool/genética , Aldeído Redutase , Aldo-Ceto Redutases , Animais , Humanos , Inativação Metabólica/genética , Inativação Metabólica/fisiologia , Estrutura Secundária de Proteína/efeitos dos fármacos , Estrutura Secundária de Proteína/genética , Estrutura Secundária de Proteína/fisiologiaRESUMO
Scaffold attachment factor B1 (SAFB1) is a multifunctional protein that can bind both DNA and RNA and is involved in RNA processing and stress response. In addition, SAFB1 contains a transcriptional repression domain and can bind certain hormone receptors and repress their activity. To assess the role of SAFB1 in vivo, we generated SAFB1 mutant mice through targeted deletion in embryonic stem cells. While viable homozygous mutant (SAFB1-/-) mice were obtained, genotypic distribution indicated that homozygous deficiency resulted in both prenatal and neonatal lethality. Mice lacking SAFB1 exhibited dwarfism, as a result of in utero growth retardation, and had low serum insulin-like growth factor 1 (IGF1) levels. In agreement with the previous characterization of SAFB1 as a corepressor for hormone receptors, we found that SAFB1-/- mice displayed dramatic defects in the development and function of the reproductive system. Male SAFB1 null mice were infertile, apparently because of low circulating levels of testosterone. SAFB1-/- testes were small and showed progressive degeneration of the germinal epithelium, increased apoptosis of germ cells, and Leydig cell hyperplasia. SAFB-/- female mice were subfertile and showed progressive infertility, in part because of defects in oviductal transport and reduced numbers of follicles. Immortalized SAFB1-/- mouse embryonic fibroblasts showed cell-intrinsic defects including increased transcriptional estrogen receptor alpha activity and enhanced responsiveness to IGF1. Together, these in vivo findings establish a critical role for SAFB1 in development, growth regulation, and reproduction.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Nanismo/genética , Desenvolvimento Embrionário/genética , Genes Letais , Proteínas de Ligação a RNA/fisiologia , Reprodução/genética , Animais , Apoptose , Linhagem Celular Transformada , Proteínas de Ligação a DNA/genética , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/fisiologia , Feminino , Retardo do Crescimento Fetal/genética , Fibroblastos/metabolismo , Hormônios Esteroides Gonadais/sangue , Infertilidade Feminina/genética , Infertilidade Masculina/genética , Fator de Crescimento Insulin-Like I/análise , Fator de Crescimento Insulin-Like I/metabolismo , Células Intersticiais do Testículo/patologia , Masculino , Camundongos , Camundongos Knockout , Gravidez , Proteínas de Ligação a RNA/genética , Transcrição GênicaRESUMO
Phospholipid oxidation generates several bioactive aldehydes that remain esterified to the glycerol backbone ('core' aldehydes). These aldehydes induce endothelial cells to produce monocyte chemotactic factors and enhance monocyte-endothelium adhesion. They also serve as ligands of scavenger receptors for the uptake of oxidized lipoproteins or apoptotic cells. The biochemical pathways involved in phospholipid aldehyde metabolism, however, remain largely unknown. In the present study, we have examined the efficacy of the three mammalian AKR (aldo-keto reductase) families in catalysing the reduction of phospholipid aldehydes. The model phospholipid aldehyde POVPC [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine] was efficiently reduced by members of the AKR1, but not by the AKR6 or the ARK7 family. In the AKR1 family, POVPC reductase activity was limited to AKR1A and B. No significant activity was observed with AKR1C enzymes. Among the active proteins, human AR (aldose reductase) (AKR1B1) showed the highest catalytic activity. The catalytic efficiency of human small intestinal AR (AKR1B10) was comparable with the murine AKR1B proteins 1B3 and 1B8. Among the murine proteins AKR1A4 and AKR1B7 showed appreciably lower catalytic activity as compared with 1B3 and 1B8. The human AKRs, 1B1 and 1B10, and the murine proteins, 1B3 and 1B8, also reduced C-7 and C-9 sn-2 aldehydes as well as POVPE [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphoethanolamine]. AKR1A4, B1, B7 and B8 catalysed the reduction of aldehydes generated in oxidized C(16:0-20:4) phosphatidylcholine with acyl, plasmenyl or alkyl linkage at the sn-1 position or C(16:0-20:4) phosphatidylglycerol or phosphatidic acid. AKR1B1 displayed the highest activity with phosphatidic acids; AKR1A4 was more efficient with long-chain aldehydes such as 5-hydroxy-8-oxo-6-octenoyl derivatives, whereas AKR1B8 preferred phosphatidylglycerol. These results suggest that proteins of the AKR1A and B families are efficient phospholipid aldehyde reductases, with non-overlapping substrate specificity, and may be involved in tissue-specific metabolism of endogenous or dietary phospholipid aldehydes.
Assuntos
Aldeído Redutase/metabolismo , Aldeídos/metabolismo , Isoenzimas/metabolismo , Fosfolipídeos/metabolismo , Aldeído Redutase/classificação , Aldeído Redutase/genética , Aldeídos/química , Animais , Humanos , Isoenzimas/genética , Camundongos , Dados de Sequência Molecular , Estrutura Molecular , Oxirredução , Fosfolipídeos/química , Especificidade por SubstratoRESUMO
Aldo-keto reductases (AKRs) are distributed in three families and multiple subfamilies in mammals. The mouse Akr1b3 gene is clearly orthologous to human AKR1B1, both coding for aldose reductase, and their gene products show similar tissue distribution, regulation by osmotic stress and kinetic properties. In contrast, no unambiguous orthologs of human AKR1B10 and AKR1B15.1 have been identified in rodents. Although two more AKRs, AKR1B7 and AKR1B8, have been identified and characterized in mouse, none of them seems to exhibit properties similar to the human AKRs. Recently, a novel mouse AKR gene, Akr1b16, was annotated and the respective gene product, AKR1B16 (sharing 83% and 80% amino acid sequence identity with AKR1B10 and AKR1B15.1, respectively), was expressed as insoluble and inactive protein in a bacterial expression system. Here we describe the expression and purification of a soluble and enzymatically active AKR1B16 from E. coli using three chaperone systems. A structural model of AKR1B16 allowed the estimation of its active-site pocket volume, which was much wider (402 Å3) than those of AKR1B10 (279 Å3) and AKR1B15.1 (60 Å3). AKR1B16 reduced aliphatic and aromatic carbonyl compounds, using NADPH as a cofactor, with moderate or low activity (highest kcat values around 5 min-1). The best substrate for the enzyme was pyridine-3-aldehyde. AKR1B16 showed poor inhibition with classical AKR inhibitors, tolrestat being the most potent. Kinetics and inhibition properties resemble those of rat AKR1B17 but differ from those of the human enzymes. In addition, AKR1B16 catalyzed the oxidation of 17ß-hydroxysteroids in a NADP+-dependent manner. These results, together with a phylogenetic analysis, suggest that mouse AKR1B16 is an ortholog of rat AKR1B17, but not of human AKR1B10 or AKR1B15.1. These human enzymes have no counterpart in the murine species, which is evidenced by forming a separate cluster in the phylogenetic tree and by their unique activity with retinaldehyde.
Assuntos
Oxirredutases Atuantes sobre Doadores de Grupos Aldeído ou Oxo/metabolismo , Aldo-Ceto Redutases/química , Aldo-Ceto Redutases/metabolismo , Sequência de Aminoácidos , Animais , Biocatálise , Células HeLa , Humanos , Cinética , Camundongos , NADP/química , NADP/metabolismo , Oxirredutases Atuantes sobre Doadores de Grupos Aldeído ou Oxo/classificação , Oxirredutases Atuantes sobre Doadores de Grupos Aldeído ou Oxo/genética , Filogenia , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Retinaldeído/química , Retinaldeído/metabolismo , Alinhamento de Sequência , Espectrofotometria , Esteroides/química , Esteroides/metabolismo , Especificidade por SubstratoRESUMO
RNA interference (RNAi) is a posttranscriptional silencing mechanism triggered by double-stranded RNA that was recently shown to function in mammalian cells. Expression of cancer-associated genes was knocked down by expressing short hairpin RNAs (shRNAs) in cancer cells. By virtue of its excellent target specificity, RNAi may be used as a new therapeutic modality for cancer. The success of this approach will largely depend on efficient delivery of shRNAs to tumor cells. Tumor-selective replication competent viruses are especially suited to efficiently deliver anticancer genes to tumors. In addition, their intrinsic capacity to kill cancer cells makes these viruses promising anticancer agents per se. In this study, conditionally replicating adenoviruses were constructed encoding shRNAs targeted against firefly luciferase. These replicating viruses were shown to specifically silence the expression of the target gene in human cancer cells down to 30% relative to control virus. This finding offers the promise of using RNAi in the context of cancer gene therapy with oncolytic viruses.
Assuntos
Adenoviridae/genética , Inativação Gênica , Terapia Genética/métodos , Neoplasias/genética , Neoplasias/terapia , RNA Interferente Pequeno/genética , Adenoviridae/fisiologia , Linhagem Celular Tumoral , Humanos , Luciferases/antagonistas & inibidores , Luciferases/biossíntese , Luciferases/genética , Interferência de RNA , Replicação ViralRESUMO
Human aldo-keto reductase 1B15 (AKR1B15) is a newly discovered enzyme which shares 92% amino acid sequence identity with AKR1B10. While AKR1B10 is a well characterized enzyme with high retinaldehyde reductase activity, involved in the development of several cancer types, the enzymatic activity and physiological role of AKR1B15 are still poorly known. Here, the purified recombinant enzyme has been subjected to substrate specificity characterization, kinetic analysis and inhibitor screening, combined with structural modeling. AKR1B15 is active towards a variety of carbonyl substrates, including retinoids, with lower kcat and Km values than AKR1B10. In contrast to AKR1B10, which strongly prefers all-trans-retinaldehyde, AKR1B15 exhibits superior catalytic efficiency with 9-cis-retinaldehyde, the best substrate found for this enzyme. With ketone and dicarbonyl substrates, AKR1B15 also shows higher catalytic activity than AKR1B10. Several typical AKR inhibitors do not significantly affect AKR1B15 activity. Amino acid substitutions clustered in loops A and C result in a smaller, more hydrophobic and more rigid active site in AKR1B15 compared with the AKR1B10 pocket, consistent with distinct substrate specificity and narrower inhibitor selectivity for AKR1B15.
Assuntos
Oxirredutases Atuantes sobre Doadores de Grupos Aldeído ou Oxo/metabolismo , Retinaldeído/metabolismo , Aldeído Redutase/antagonistas & inibidores , Aldeído Redutase/genética , Aldeído Redutase/metabolismo , Aldo-Ceto Redutases , Sequência de Aminoácidos , Substituição de Aminoácidos , Domínio Catalítico/genética , Diterpenos , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Modelos Moleculares , Oxirredutases Atuantes sobre Doadores de Grupos Aldeído ou Oxo/antagonistas & inibidores , Oxirredutases Atuantes sobre Doadores de Grupos Aldeído ou Oxo/genética , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína , Especificidade por SubstratoRESUMO
The aldo-keto reductase superfamily contains 173 proteins which are present in all phyla. Examination of the human and mouse genomes has identified that in some instances a single AKR gene can give rise to alternatively spliced mRNA variants which in some cases can give rise to more than one protein isoform. This is currently well documented in the AKR6A subfamily which contains the ß-subunits of the voltage-gated potassium ion channels. With the emergence of second generation sequencing it is likely that the occurrence of transcript variants and protein isoforms from a single AKR gene may become common place. To deal with this issue we recommend that the Ensembl data-base nomenclature be used to annotate the transcript variants from a single AKR gene. However, since multiple transcript variants could give rise to either the same or multiple protein isoforms from the same AKR gene we also propose to expand the nomenclature of the AKR protein superfamily, so that when a protein isoform is shown to be expressed and is functional it would be assigned the standard AKR name followed by a "period or full-stop" and a number for that unique isoform. Numbers will be assigned chronologically and linked to the respective transcripts annotated in Ensembl e.g. AKR6A5.1 (Kvß2.1) (AKR6A5-001, -006 and -201), followed by AKR6A5.2 (Kvß2.2) (AKR6A5-002,-202). This nomenclature is expandable and it enables multiple protein isoforms to be assigned to their respective transcripts when they arise from the same AKR gene or for a single protein isoform to be assigned to multiple transcripts when the transcripts encode the same AKR protein.
Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Aldeído Redutase , Aldo-Ceto Redutases , Processamento Alternativo , Animais , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Isoformas de Proteínas , RNA Mensageiro/genética , Regiões não TraduzidasRESUMO
Aldehydes are generated by oxidized lipids and carbohydrates at increased levels under conditions of metabolic imbalance and oxidative stress during atherosclerosis, myocardial and cerebral ischemia, diabetes, neurodegenerative diseases and trauma. In most tissues, aldehydes are detoxified by oxidoreductases that catalyze the oxidation or the reduction of aldehydes or enzymatic and nonenzymatic conjugation with low molecular weight thiols and amines, such as glutathione and histidine dipeptides. Histidine dipeptides are present in micromolar to millimolar range in the tissues of vertebrates, where they are involved in a variety of physiological functions such as pH buffering, metal chelation, oxidant and aldehyde scavenging. Histidine dipeptides such as carnosine form Michael adducts with lipid-derived unsaturated aldehydes, and react with carbohydrate-derived oxo- and hydroxy-aldehydes forming products of unknown structure. Although these peptides react with electrophilic molecules at lower rate than glutathione, they can protect glutathione from modification by oxidant and they may be important for aldehyde quenching in glutathione-depleted cells or extracellular space where glutathione is scarce. Consistent with in vitro findings, treatment with carnosine has been shown to diminish ischemic injury, improve glucose control, ameliorate the development of complications in animal models of diabetes and obesity, promote wound healing and decrease atherosclerosis. The protective effects of carnosine have been linked to its anti-oxidant properties, its ability to promote glycolysis, detoxify reactive aldehydes and enhance histamine levels. Thus, treatment with carnosine and related histidine dipeptides may be a promising strategy for the prevention and treatment of diseases associated with high carbonyl load.