RESUMEN
Stearoyl-CoA Desaturase-2 (SCD2) is a member of the Stearoyl-CoA Desaturase (SCD) family of enzymes that catalyze the rate-limiting step in monounsaturated fatty acid (MUFA) synthesis. The MUFAs palmitoleoyl-CoA (16:1n7) and oleoyl-CoA (18:1n9) are the major products of SCD2. Palmitoleoyl-CoA and oleoyl-CoA have various roles, from being a source of energy to signaling molecules. Under normal feeding conditions, SCD2 is ubiquitously expressed and is the predominant SCD isoform in the brain. However, obesogenic diets highly induce SCD2 in adipose tissue, lung, and kidney. Here we provide a comprehensive review of SCD2 in mouse development, metabolism, and various diseases, such as obesity, chronic kidney disease, Alzheimer's disease, multiple sclerosis, and Parkinson's disease. In addition, we show that bone mineral density is decreased in SCD2KO mice under high-fat feeding conditions and that SCD2 is not required for preadipocyte differentiation or the expression of PPARγ in vivo despite being required in vitro.
Asunto(s)
Adipocitos/enzimología , Diferenciación Celular , Ácidos Grasos Monoinsaturados/metabolismo , Enfermedades Neurodegenerativas/enzimología , Obesidad/enzimología , Insuficiencia Renal Crónica/enzimología , Estearoil-CoA Desaturasa/metabolismo , Acilcoenzima A/biosíntesis , Acilcoenzima A/genética , Animales , Dieta Alta en Grasa/efectos adversos , Ratones , Ratones Noqueados , Enfermedades Neurodegenerativas/genética , Obesidad/inducido químicamente , Obesidad/genética , Obesidad/metabolismo , Palmitoil Coenzima A/biosíntesis , Palmitoil Coenzima A/genética , Insuficiencia Renal Crónica/genética , Estearoil-CoA Desaturasa/genéticaRESUMEN
The mechanisms regulating oxidative phosphorylation during exercise remain poorly defined; however, key mitochondrial proteins, including carnitine palmitoyltransferase-I (CPT-I) and adenine nucleotide translocase, have redox-sensitive sites. Interestingly, muscle contraction has recently been shown to increase mitochondrial membrane potential and reactive oxygen species (ROS) production; therefore, we aimed to determine if mitochondrial-derived ROS influences bioenergetic responses to exercise. Specifically, we examined the influence of acute exercise on mitochondrial bioenergetics in WT (wild type) and transgenic mice (MCAT, mitochondrial-targeted catalase transgenic) possessing attenuated mitochondrial ROS. We found that ablating mitochondrial ROS did not alter palmitoyl-CoA (P-CoA) respiratory kinetics or influence the exercise-mediated reductions in malonyl CoA sensitivity, suggesting that mitochondrial ROS does not regulate CPT-I. In contrast, while mitochondrial protein content, maximal coupled respiration, and ADP (adenosine diphosphate) sensitivity in resting muscle were unchanged in the absence of mitochondrial ROS, exercise increased the apparent ADP Km (decreased ADP sensitivity) â¼30% only in WT mice. Moreover, while the presence of P-CoA decreased ADP sensitivity, it did not influence the basic response to exercise, as the apparent ADP Km was increased only in the presence of mitochondrial ROS. This basic pattern was also mirrored in the ability of ADP to suppress mitochondrial H2O2 emission rates, as exercise decreased the suppression of H2O2 only in WT mice. Altogether, these data demonstrate that while exercise-induced mitochondrial-derived ROS does not influence CPT-I substrate sensitivity, it inhibits ADP sensitivity independent of P-CoA. These data implicate mitochondrial redox signaling as a regulator of oxidative phosphorylation.
Asunto(s)
Adenosina Difosfato/metabolismo , Carnitina O-Palmitoiltransferasa/metabolismo , Peróxido de Hidrógeno/metabolismo , Mitocondrias Musculares/metabolismo , Condicionamiento Físico Animal , Adenosina Difosfato/genética , Animales , Carnitina O-Palmitoiltransferasa/genética , Ratones , Ratones Transgénicos , Mitocondrias Musculares/genética , Palmitoil Coenzima A/genética , Palmitoil Coenzima A/metabolismo , Especificidad por SustratoRESUMEN
Autophagy is a membrane trafficking pathway that sequesters proteins and organelles into autophagosomes. The selectivity of this pathway is determined by autophagy receptors, such as the Pichia pastoris autophagy-related protein 30 (Atg30), which controls the selective autophagy of peroxisomes (pexophagy) through the assembly of a receptor protein complex (RPC). However, how the pexophagic RPC is regulated for efficient formation of the phagophore, an isolation membrane that sequesters the peroxisome from the cytosol, is unknown. Here we describe a new, conserved acyl-CoA-binding protein, Atg37, that is an integral peroxisomal membrane protein required specifically for pexophagy at the stage of phagophore formation. Atg30 recruits Atg37 to the pexophagic RPC, where Atg37 regulates the recruitment of the scaffold protein, Atg11. Palmitoyl-CoA competes with Atg30 for Atg37 binding. The human orthologue of Atg37, acyl-CoA-binding domain containing protein 5 (ACBD5), is also peroxisomal and is required specifically for pexophagy. We suggest that Atg37/ACBD5 is a new component and positive regulator of the pexophagic RPC.
Asunto(s)
Autofagia , Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , Palmitoil Coenzima A/metabolismo , Peroxisomas/metabolismo , Fagosomas/fisiología , Pichia/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Fúngicas/genética , Células HeLa , Humanos , Proteína Huntingtina , Procesamiento de Imagen Asistido por Computador , Inmunoprecipitación , Proteínas de la Membrana/genética , Microscopía Fluorescente , Mutación/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Palmitoil Coenzima A/genética , Peroxisomas/genética , Pichia/genética , Pichia/crecimiento & desarrollo , Proteína Sequestosoma-1RESUMEN
Herein, we demonstrate that calcium-independent phospholipase A(2)γ (iPLA(2)γ) is a critical mechanistic participant in the calcium-induced opening of the mitochondrial permeability transition pore (mPTP). Liver mitochondria from iPLA(2)γ(-/-) mice were markedly resistant to calcium-induced swelling in the presence or absence of phosphate in comparison with wild-type littermates. Furthermore, the iPLA(2)γ enantioselective inhibitor (R)-(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one ((R)-BEL) was markedly more potent than (S)-BEL in inhibiting mPTP opening in mitochondria from wild-type liver in comparison with hepatic mitochondria from iPLA(2)γ(-/-) mice. Intriguingly, low micromolar concentrations of long chain fatty acyl-CoAs and the non-hydrolyzable thioether analog of palmitoyl-CoA markedly accelerated Ca(2+)-induced mPTP opening in liver mitochondria from wild-type mice. The addition of l-carnitine enabled the metabolic channeling of acyl-CoA through carnitine palmitoyltransferases (CPT-1/2) and attenuated the palmitoyl-CoA-mediated amplification of calcium-induced mPTP opening. In contrast, mitochondria from iPLA(2)γ(-/-) mice were insensitive to fatty acyl-CoA-mediated augmentation of calcium-induced mPTP opening. Moreover, mitochondria from iPLA(2)γ(-/-) mouse liver were resistant to Ca(2+)/t-butyl hydroperoxide-induced mPTP opening in comparison with wild-type littermates. In support of these findings, cytochrome c release from iPLA(2)γ(-/-) mitochondria was dramatically decreased in response to calcium in the presence or absence of either t-butyl hydroperoxide or phenylarsine oxide in comparison with wild-type littermates. Collectively, these results identify iPLA(2)γ as an important mechanistic component of the mPTP, define its downstream products as potent regulators of mPTP opening, and demonstrate the integrated roles of mitochondrial bioenergetics and lipidomic flux in modulating mPTP opening promoting the activation of necrotic and necroapoptotic pathways of cell death.
Asunto(s)
Calcio/metabolismo , Fosfolipasas A2 Grupo VI/metabolismo , Mitocondrias Hepáticas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Animales , Carnitina/genética , Carnitina/metabolismo , Carnitina O-Palmitoiltransferasa/genética , Carnitina O-Palmitoiltransferasa/metabolismo , Muerte Celular , Citocromos c/genética , Citocromos c/metabolismo , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/genética , Inhibidores Enzimáticos/farmacología , Fosfolipasas A2 Grupo VI/antagonistas & inhibidores , Fosfolipasas A2 Grupo VI/genética , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Ratones , Ratones Noqueados , Mitocondrias Hepáticas/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Poro de Transición de la Permeabilidad Mitocondrial , Palmitoil Coenzima A/genética , Palmitoil Coenzima A/metabolismo , ConejosRESUMEN
DHHC proteins catalyze the reversible S-acylation of proteins at cysteine residues, a modification important for regulating protein localization, stability, and activity. However, little is known about the kinetic mechanism of DHHC proteins. A high-performance liquid chromatography (HPLC), fluorescent peptide-based assay for protein S-acylation activity was developed to characterize mammalian DHHC2 and DHHC3. Time courses and substrate saturation curves allowed the determination of V(max) and K(m) values for both the peptide N-myristoylated-GCG and palmitoyl-coenzyme A. DHHC proteins acylate themselves upon incubation with palmitoyl-CoA, which is hypothesized to reflect a transient acyl enzyme transfer intermediate. Single turnover assays with DHHC2 and DHHC3 demonstrated that a radiolabeled acyl group on the enzyme transferred to the protein substrate, consistent with a two-step ping-pong mechanism. Enzyme autoacylation and acyltransfer to substrate displayed the same acyl-CoA specificities, further supporting a two-step mechanism. Interestingly, DHHC2 efficiently transferred acyl chains 14 carbons and longer, whereas DHHC3 activity was greatly reduced by acyl-CoAs with chain lengths longer than 16 carbons. The rate and extent of autoacylation of DHHC3, as well as the rate of acyl chain transfer to protein substrate, were reduced with stearoyl-CoA when compared with palmitoyl-CoA. This is the first observation of lipid substrate specificity among DHHC proteins and may account for the differential S-acylation of proteins observed in cells.
Asunto(s)
Acilcoenzima A/química , Aciltransferasas/química , Lipoilación/fisiología , Palmitoil Coenzima A/química , Proteínas Supresoras de Tumor/química , Acilcoenzima A/genética , Acilcoenzima A/metabolismo , Acilación/fisiología , Aciltransferasas/genética , Aciltransferasas/metabolismo , Cinética , Palmitoil Coenzima A/genética , Palmitoil Coenzima A/metabolismo , Especificidad por Sustrato/fisiología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Reports suggest that excessive ceramide accumulation in mitochondria is required to initiate the intrinsic apoptotic pathway and subsequent cell death, but how ceramide accumulates is unclear. Here we report that liver mitochondria exhibit ceramide formation from sphingosine and palmitoyl-CoA and from sphingosine and palmitate. Importantly, this activity was markedly decreased in liver from neutral ceramidase (NCDase)-deficient mice. Moreover, the levels of ceramide were dissimilar in liver mitochondria of WT and NCDase KO mice. These results suggest that NCDase is a key participant of ceramide formation in liver mitochondria. We also report that highly purified liver mitochondria have ceramidase, reverse ceramidase, and thioesterase activities. Increased accessibility of palmitoyl-CoA to the mitochondrial matrix with the pore-forming peptide zervamicin IIB resulted in 2-fold increases in palmitoyl-CoA hydrolysis by thioesterase. This increased hydrolysis was accompanied by an increase in ceramide formation, demonstrating that both outer membrane and matrix localized thioesterases can regulate ceramide formation. Also, ceramide formation might occur both in the outer mitochondrial membrane and in the mitochondrial matrix, suggesting the existence of distinct ceramide pools. Taken together, these results suggest that the reverse activity of NCDase contributes to sphingolipid homeostasis in this organelle in vivo.
Asunto(s)
Ceramidas/metabolismo , Metabolismo de los Lípidos/fisiología , Mitocondrias Hepáticas/enzimología , Proteínas Mitocondriales/metabolismo , Ceramidasa Neutra/metabolismo , Palmitoil Coenzima A/metabolismo , Esfingosina/metabolismo , Animales , Ceramidas/genética , Hidrólisis/efectos de los fármacos , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Mitocondrias Hepáticas/genética , Proteínas Mitocondriales/genética , Ceramidasa Neutra/genética , Palmitoil Coenzima A/genética , Palmitoil-CoA Hidrolasa , Peptaiboles/farmacología , Ratas , Ratas Sprague-Dawley , Esfingosina/genéticaRESUMEN
DHHC protein acyltransferases (PATs) catalyze the palmitoylation of eukaryotic proteins through an enzymatic mechanism that remains largely unexplored. In this study we have combined genetic and biochemical approaches to examine the molecular mechanism of palmitate transfer of the yeast Ras PAT, which is composed of Erf2 and Erf4. The palmitoylation reaction consists of two steps; they are autopalmitoylation of the enzyme to create a palmitoyl-Erf2 intermediate followed by the transfer of the palmitoyl moiety to the Ras substrate. Palmitoyl-CoA serves as the palmitate donor. To elucidate the kinetic properties of the Erf2·Erf4 PAT, we have developed a coupled enzyme assay that monitors the turnover of the palmitoyl-enzyme species indirectly by measuring the rate of CoASH release. Mutational analysis indicates that the DHHC motif constitutes the catalytic core of the enzyme required for autopalmitoylation and palmitoyl transfer to the Ras2 substrate. In the absence of Ras2, the palmitoyl-Erf2·Erf4 complex undergoes a cycle of hydrolysis and re-palmitoylation, implying that in the presence of palmitoyl-CoA, the complex is autopalmitoylated and competent to transfer palmitate to a protein substrate.
Asunto(s)
Aciltransferasas/metabolismo , Lipoilación/fisiología , Proteínas de la Membrana/metabolismo , Mutación Missense , Palmitoil Coenzima A/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Aciltransferasas/genética , Secuencias de Aminoácidos , Proteínas de la Membrana/genética , Palmitoil Coenzima A/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Serine palmitoyltransferase (SPT) catalyses the first step in the de novo biosynthesis of sphingolipids (SLs). It uses a decarboxylative Claisen-like condensation reaction to couple L-serine with palmitoyl-CoA to generate a long-chain base product, 3-ketodihydrosphingosine. SLs are produced by mammals, plants, yeast, and some bacteria, and we have exploited the complete genome sequence of Sphingomonas wittichii to begin a complete analysis of bacterial sphingolipid biosynthesis. Here, we describe the enzymatic characterization of the SPT from this organism and present its high-resolution x-ray structure. Moreover, we identified an open reading frame with high sequence homology to acyl carrier proteins (ACPs) that are common to fatty acid biosynthetic pathways. This small protein was co-expressed with the SPT and we isolated and characterised the apo- and holo-forms of the ACP. Our studies suggest a link between fatty acid and sphingolipid metabolism.
Asunto(s)
Proteína Transportadora de Acilo/metabolismo , Proteínas Bacterianas/metabolismo , Serina C-Palmitoiltransferasa/metabolismo , Sphingomonas/metabolismo , Proteína Transportadora de Acilo/genética , Proteínas Bacterianas/genética , Genoma Bacteriano/fisiología , Sistemas de Lectura Abierta/fisiología , Palmitoil Coenzima A/genética , Palmitoil Coenzima A/metabolismo , Serina C-Palmitoiltransferasa/genética , Sphingomonas/genética , Esfingosina/análogos & derivados , Esfingosina/biosíntesis , Esfingosina/genéticaRESUMEN
AIMS/HYPOTHESIS: Insulin resistance in skeletal muscle is a hallmark of type 2 diabetes. Therefore, we sought to identify and validate genes involved in the development of insulin resistance in skeletal muscle. MATERIALS: Differentially regulated genes in skeletal muscle of male obese insulin-resistant, and lean insulin-sensitive Zucker diabetic fatty (ZDF) rats were determined using Affymetrix microarrays. Based on these data, various aspects of glucose disposal, insulin signalling and fatty acid composition were analysed in a muscle cell line overexpressing stearoyl-CoA desaturase 1 (SCD1). RESULTS: Gene expression profiling in insulin-resistant skeletal muscle revealed the most pronounced changes in gene expression for genes involved in lipid metabolism. Among these, Scd1 showed increased expression in insulin-resistant animals, correlating with increased amounts of palmitoleoyl-CoA. This was further investigated in a muscle cell line that overexpressed SCD1 and accumulated lipids, revealing impairments of glucose uptake and of different steps of the insulin signalling cascade. We also observed differential effects of high-glucose and fatty acid treatment on glucose uptake and long-chain fatty acyl-CoA profiles, and in particular an accumulation of palmitoleoyl-CoA in cells overexpressing SCD1. CONCLUSIONS/INTERPRETATION: Insulin-resistant skeletal muscle of ZDF rats is characterised by a specific gene expression profile with increased levels of Scd1. An insulin-resistant phenotype similar to that obtained by treatment with palmitate and high glucose can be induced in vitro by overexpression of SCD1 in muscle cells. This supports the hypothesis that elevated SCD1 expression is a possible cause of insulin resistance and type 2 diabetes.