RESUMEN
To understand the functional role of the peroxisomal membrane channel Pxmp2, mice with a targeted disruption of the Pxmp2 gene were generated. These mice were viable, grew and bred normally. However, Pxmp2(-/-) female mice were unable to nurse their pups. Lactating mammary gland epithelium displayed secretory lipid droplets and milk proteins, but the size of the ductal system was greatly reduced. Examination of mammary gland development revealed that retarded mammary ductal outgrowth was due to reduced proliferation of epithelial cells during puberty. Transplantation experiments established the Pxmp2(-/-) mammary stroma as a tissue responsible for suppression of epithelial growth. Morphological and biochemical examination confirmed the presence of peroxisomes in the mammary fat pad adipocytes, and functional Pxmp2 was detected in the stroma of wild-type mammary glands. Deletion of Pxmp2 led to an elevation in the expression of peroxisomal proteins in the mammary fat pad but not in liver or kidney of transgenic mice. Lipidomics of Pxmp2(-/-)mammary fat pad showed a decrease in the content of myristic acid (C14), a principal substrate for protein myristoylation and a potential peroxisomal ß-oxidation product. Analysis of complex lipids revealed a reduced concentration of a variety of diacylglycerols and phospholipids containing mostly polyunsaturated fatty acids that may be caused by activation of lipid peroxidation. However, an antioxidant-containing diet did not stimulate mammary epithelial proliferation in Pxmp2(-/-) mice. The results point to disturbances of lipid metabolism in the mammary fat pad that in turn may result in abnormal epithelial growth. The work reveals impaired mammary gland development as a new category of peroxisomal disorders.
Asunto(s)
Metabolismo de los Lípidos , Glándulas Mamarias Animales/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Peroxisomas/metabolismo , Tejido Adiposo/metabolismo , Animales , Ácidos y Sales Biliares/química , Células Epiteliales/citología , Ácidos Grasos/química , Femenino , Homeostasis , Lactancia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , Poliaminas/química , Fracciones Subcelulares , Factores de TiempoRESUMEN
Bile acids play multiple roles in the physiology of vertebrates; they facilitate lipid absorption, serve as signaling molecules to control carbohydrate and lipid metabolism, and provide a disposal route for cholesterol. Unexpectedly, the α-methylacyl-CoA racemase (Amacr) deficient mice, which are unable to complete the peroxisomal cleavage of C27-precursors to the mature C24-bile acids, are physiologically asymptomatic when maintained on a standard laboratory diet. The aim of this study was to uncover the underlying adaptive mechanism with special reference to cholesterol and bile acid metabolism that allows these mice to have a normal life span. Intestinal cholesterol absorption in Amacr-/- mice is decreased resulting in a 2-fold increase in daily cholesterol excretion. Also fecal excretion of bile acids (mainly C27-sterols) is enhanced 3-fold. However, the body cholesterol pool remains unchanged, although Amacr-deficiency accelerates hepatic sterol synthesis 5-fold. Changes in lipoprotein profiles are mainly due to decreased phospholipid transfer protein activity. Thus Amacr-deficient mice provide a unique example of metabolic regulation, which allows them to have a normal lifespan in spite of the disruption of a major metabolic pathway. This metabolic adjustment can be mainly explained by setting cholesterol and bile acid metabolism to a new balanced level in the Amacr-deficient mouse.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Colesterol/metabolismo , Metabolismo de los Lípidos/fisiología , Hígado/metabolismo , Racemasas y Epimerasas/metabolismo , Animales , Ácidos y Sales Biliares/genética , Colesterol/genética , Longevidad/fisiología , Ratones , Ratones Noqueados , Racemasas y Epimerasas/genéticaRESUMEN
The mitochondrial beta-oxidation system is one of the central metabolic pathways of energy metabolism in mammals. Enzyme defects in this pathway cause fatty acid oxidation disorders. To elucidate the role of 2,4-dienoyl-CoA reductase (DECR) as an auxiliary enzyme in the mitochondrial beta-oxidation of unsaturated fatty acids, we created a DECR-deficient mouse line. In Decr(-/-) mice, the mitochondrial beta-oxidation of unsaturated fatty acids with double bonds is expected to halt at the level of trans-2, cis/trans-4-dienoyl-CoA intermediates. In line with this expectation, fasted Decr(-/-) mice displayed increased serum acylcarnitines, especially decadienoylcarnitine, a product of the incomplete oxidation of linoleic acid (C(18:2)), urinary excretion of unsaturated dicarboxylic acids, and hepatic steatosis, wherein unsaturated fatty acids accumulate in liver triacylglycerols. Metabolically challenged Decr(-/-) mice turned on ketogenesis, but unexpectedly developed hypoglycemia. Induced expression of peroxisomal beta-oxidation and microsomal omega-oxidation enzymes reflect the increased lipid load, whereas reduced mRNA levels of PGC-1alpha and CREB, as well as enzymes in the gluconeogenetic pathway, can contribute to stress-induced hypoglycemia. Furthermore, the thermogenic response was perturbed, as demonstrated by intolerance to acute cold exposure. This study highlights the necessity of DECR and the breakdown of unsaturated fatty acids in the transition of intermediary metabolism from the fed to the fasted state.
Asunto(s)
Hipoglucemia/fisiopatología , Cuerpos Cetónicos/biosíntesis , Mitocondrias/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/deficiencia , Estrés Fisiológico , Animales , Ácidos Grasos Insaturados/metabolismo , Femenino , Glucosa/metabolismo , Hipoglucemia/enzimología , Hipoglucemia/genética , Hipoglucemia/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/metabolismo , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Triglicéridos/metabolismoRESUMEN
Coumarin derivatives such as warfarin represent the therapy of choice for the long-term treatment and prevention of thromboembolic events. Coumarins target blood coagulation by inhibiting the vitamin K epoxide reductase multiprotein complex (VKOR). This complex recycles vitamin K 2,3-epoxide to vitamin K hydroquinone, a cofactor that is essential for the post-translational gamma-carboxylation of several blood coagulation factors. Despite extensive efforts, the components of the VKOR complex have not been identified. The complex has been proposed to be involved in two heritable human diseases: combined deficiency of vitamin-K-dependent clotting factors type 2 (VKCFD2; Online Mendelian Inheritance in Man (OMIM) 607473), and resistance to coumarin-type anticoagulant drugs (warfarin resistance, WR; OMIM 122700). Here we identify, by using linkage information from three species, the gene vitamin K epoxide reductase complex subunit 1 (VKORC1), which encodes a small transmembrane protein of the endoplasmic reticulum. VKORC1 contains missense mutations in both human disorders and in a warfarin-resistant rat strain. Overexpression of wild-type VKORC1, but not VKORC1 carrying the VKCFD2 mutation, leads to a marked increase in VKOR activity, which is sensitive to warfarin inhibition.
Asunto(s)
Trastornos de las Proteínas de Coagulación/genética , Resistencia a Medicamentos/genética , Oxigenasas de Función Mixta/genética , Mutación Missense/genética , Warfarina/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Línea Celular , Mapeo Cromosómico , Trastornos de las Proteínas de Coagulación/enzimología , Análisis Mutacional de ADN , Humanos , Ratones , Oxigenasas de Función Mixta/antagonistas & inhibidores , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Vitamina K Epóxido ReductasasRESUMEN
Cathepsins B and L are lysosomal cysteine proteases which have been implicated in a variety of pathological processes such as cancer, tumor angiogenesis, and neurodegeneration. However, only a few protein substrates have thus far been described and the mechanisms by which cathepsins B and L regulate cell proliferation, invasion, and apoptosis are poorly understood. Combined deficiency of both cathepsins results in early-onset neurodegeneration in mice reminiscent of neuronal ceroid lipofuscinoses in humans. Therefore, we intended to quantify accumulated proteins in brain lysosomes of double deficient mice. A combination of subcellular fractionation and LC-MS/MS using isobaric tagging for relative and absolute quantitation (iTRAQ) allowed us to simultaneously assess wildtype and cathepsin B(-/-)L(-/-) cerebral lysosomes. Altogether, 19 different proteins were significantly increased in cathepsin B(-/-)L(-/-) lysosomes. Most elevated proteins had previously been localized to neuronal biosynthetic, recycling/endocytic or lysosomal compartments. A more than 10-fold increase was observed for Rab14, the Delta/Notch-like epidermal growth factor-related receptor (DNER), calcyon, and carboxypeptidase E. Intriguingly, immunohistochemistry demonstrated that Rab14 and DNER specifically stain swollen axons in double deficient brains. Since dense accumulations of expanded axons are the earliest phenotypic and pathognomonic feature of cathepsin B(-/-)L(-/-) brains, our data suggest a role for cathepsins B and L in recycling processes during axon outgrowth and synapse formation in the developing postnatal central nervous system.
Asunto(s)
Química Encefálica/genética , Catepsina B/deficiencia , Catepsina B/genética , Catepsinas/deficiencia , Catepsinas/genética , Cisteína Endopeptidasas/deficiencia , Cisteína Endopeptidasas/genética , Lisosomas/enzimología , Lisosomas/genética , Proteoma , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Axones/enzimología , Química Encefálica/fisiología , Catepsina B/fisiología , Catepsina L , Catepsinas/fisiología , Cisteína Endopeptidasas/fisiología , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Proteoma/genética , Sinapsis/enzimologíaRESUMEN
Alpha-methylacyl-CoA racemases are essential enzymes for branched-chain fatty acid metabolism. Their reaction mechanism and the structural basis of their wide substrate specificity are poorly understood. High-resolution crystal structures of Mycobacterium tuberculosis alpha-methylacyl-CoA racemase (MCR) complexed with substrate molecules show the active site geometry required for catalysis of the interconversion of (2S) and (2R)-methylacyl-CoA. The thioester oxygen atom and the 2-methyl group are in a cis-conformation with respect to each other. The thioester oxygen atom fits into an oxyanion hole and the 2-methyl group points into a hydrophobic pocket. The active site geometry agrees with a 1,1-proton transfer mechanism in which the acid/base-pair residues are His126 and Asp156. The structures of the complexes indicate that the acyl chains of the S-substrate and the R-substrate bind in an S-pocket and an R-pocket, respectively. A unique feature of MCR is a large number of methionine residues in the acyl binding region, located between the S-pocket and the R-pocket. It appears that the (S) to (R) interconversion of the 2-methylacyl chiral center is coupled to a movement of the acyl group over this hydrophobic, methionine-rich surface, when moving from its S-pocket to its R-pocket, whereas the 2-methyl moiety and the CoA group remain fixed in their respective pockets.
Asunto(s)
Acilcoenzima A/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Metionina/química , Protones , Racemasas y Epimerasas/química , Racemasas y Epimerasas/metabolismo , Acilcoenzima A/química , Sitios de Unión , Catálisis , Dominio Catalítico , Modelos Biológicos , Modelos Moleculares , Conformación Molecular , Mycobacterium tuberculosis/enzimologíaRESUMEN
Lipids are essential for normal and malignant cells during growth and differentiation. The turnover is strictly regulated because an uncontrolled uptake and accumulation is cytotoxic and can lead to lipoapoptosis: lipoptosis. The human monoclonal antibody SAM-6 binds to a cell surface receptor on malignant cells and to oxidized low-density lipoprotein (LDL). SAM-6 induces an excess of intracellular lipids, by overfeeding malignant cells with oxidized LDL, via a receptor-mediated endocytosis. The treated cells overaccumulate depots of cholesteryl esters and triglycerides. This lipid overaccumulation is tumor specific; nonmalignant cells neither bind the antibody nor harvest lipids after incubation. Because for both forms of apoptosis, the death domain dependent ("extrinsic") and independent ("intrinsic"), the activation of proteases is crucial, we also investigated this pathway in more detail. It was found that shortly after internalization of antibody/oxidized LDL/receptor complex and formation of lipid depots, cytochrome c is released by mitochondria. Followed by this, initiator caspase-8 and caspase-9 and effector caspase-3 and caspase-6 are activated. The mechanism of mitochondrial trigger (e.g., by free fatty acids) is under investigation. However, the present data indicate that the SAM-6 antibody induces an intrinsic-like form of apoptosis by overfeeding malignant cells with lipoproteins.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Apoptosis/efectos de los fármacos , Inmunoglobulina M/farmacología , Lipoproteínas LDL/metabolismo , Neoplasias/patología , Animales , Caspasas/metabolismo , Citocromos c/metabolismo , Fragmentación del ADN/efectos de los fármacos , Endocitosis/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Lípidos/análisis , Ratones , Ratones Desnudos , Unión ProteicaRESUMEN
In the active site of the bacterial α-methylacyl-CoA racemase of Mycobacterium tuberculosis (MCR), the chirality of the 2-methyl branched C2-atom is interconverted between (S) and (R) isomers. Protein crystallographic data and quantum mechanics/molecular mechanics (QM/MM) computational approaches show that this interconversion is achieved via a planar enolate intermediate. The crystal structure, at 1.4 Å, visualizes the mode of binding of a reaction intermediate analogue, 2-methylacetoacetyl-CoA, in a well-defined planar enolate form. The computational studies confirm that in the conversion from (S) to (R), first a proton is abstracted by Nδ1 (His126), and subsequently the planar enolate form is reprotonated by Oδ2 (Asp156). The calculations also show that the negatively charged thioester oxygen of the enolate intermediate is stabilized by an oxyanion hole formed by N (Asp127), as well as by the side chain atoms of the catalytic residues, Asp156 and His126, both being protonated simultaneously, at the intermediate stage of the catalytic cycle. The computational analysis also reveals that the conversion of the (S)- to (R)- chirality is achieved by a movement of 1.7 Å of the chiral C2-carbon, with smaller shifts (approximately 1 Å) of the carbon atom of the 2-methyl group, the C3-atom of the fatty acid tail, and the C1-carbon and O1-oxygen atoms of the thioester moiety.
Asunto(s)
Simulación de Dinámica Molecular , Teoría Cuántica , Racemasas y Epimerasas/química , Dominio Catalítico , Cristalografía por Rayos X , Enlace de Hidrógeno , Mycobacterium tuberculosis/enzimología , Racemasas y Epimerasas/metabolismoRESUMEN
Alpha-methylacyl-CoA racemase (Amacr) catalyzes the racemization of alpha-methyl-branched CoA esters. Sequence comparisons have shown that this enzyme is a member of the family III CoA transferases. The mammalian Amacr is involved in bile acid synthesis and branched-chain fatty acid degradation. In human, mutated variants of Amacr have been shown to be associated with disease states. Amino acid sequence alignment of Amacrs and its homologues from various species revealed 26 conserved protic residues, assumed to be potential candidates as catalytic residues. Amacr from Mycobacterium tuberculosis (MCR) was taken as a representative of the racemases. To determine their importance for efficient catalysis, each of these 26 protic residues of MCR was mutated into an alanine, respectively, and the mutated variants were overexpressed in Escherichia coli. It was found that four variants (R91A, H126A, D156A, and E241A) were properly folded but had much decreased catalytic efficiency. Apparently, Arg91, His126, Asp156, and Glu241 are important catalytic residues of MCR. The importance of these residues for catalysis can be rationalized by the 1.8 A resolution crystal structure of MCR, which shows that the catalytic site is at the interface between the large and small domain of two different subunits of the dimeric enzyme. This crystal structure is the first structure of a complete enzyme of the bile acid synthesis pathway. It shows that MCR has unique structural features, not seen in the structures of the sequence related formyl-CoA transferases, suggesting that the family III CoA transferases can be subdivided in at least two classes, being racemases and CoA transferases.
Asunto(s)
Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Racemasas y Epimerasas/química , Racemasas y Epimerasas/genética , Alanina/química , Secuencia de Aminoácidos , Animales , Ácidos y Sales Biliares/metabolismo , Sitios de Unión , Catálisis , Dicroismo Circular , Clonación Molecular , Cristalografía por Rayos X , Dimerización , Escherichia coli/metabolismo , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Ratas , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Rayos UltravioletaRESUMEN
According to current views, the second peroxisomal beta-oxidation pathway is responsible for the degradation of the side chain of bile acid intermediates. Peroxisomal multifunctional enzyme type 2 [peroxisomal multifunctional 2-enoyl-CoA hydratase/(R)-3-hydroxyacyl-CoA dehydrogenase; MFE-2] catalyses the second (hydration) and third (dehydrogenation) reactions of the pathway. Deficiency of MFE-2 leads to accumulation of very-long-chain fatty acids, 2-methyl-branched fatty acids and C(27) bile acid intermediates in plasma, but bile acid synthesis is not blocked completely. In this study we describe an alternative pathway, which allows MFE-2 deficiency to be overcome. The alternative pathway consists of alpha-methylacyl-CoA racemase and peroxisomal multifunctional enzyme type 1 [peroxisomal multifunctional 2-enoyl-CoA hydratase/(S)-3-hydroxyacyl-CoA dehydrogenase; MFE-1]. (24E)-3alpha,7alpha,12alpha-Trihydroxy-5beta-cholest-24-enoyl-CoA, the presumed physiological isomer, is hydrated by MFE-1 with the formation of (24S,25S)-3alpha,7alpha,12alpha,24-tetrahydroxy-5beta-cholestanoyl-CoA [(24S,25S)-24-OH-THCA-CoA], which after conversion by a alpha-methylacyl-CoA racemase into the (24S,25R) isomer can again be dehydrogenated by MFE-1 to 24-keto-3alpha,7alpha,12alpha-trihydroxycholestanoyl-CoA, a physiological intermediate in cholic acid synthesis. The discovery of the alternative pathway of cholesterol side-chain oxidation will improve diagnosis of peroxisomal deficiencies by identification of serum 24-OH-THCA-CoA diastereomer profiles.
Asunto(s)
Ácidos y Sales Biliares/biosíntesis , Colesterol/análogos & derivados , Racemasas y Epimerasas/fisiología , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , 3-Hidroxiacil-CoA Deshidrogenasas/fisiología , Acilcoenzima A/metabolismo , Animales , Colesterol/metabolismo , Cromatografía Líquida de Alta Presión , Enoil-CoA Hidratasa/metabolismo , Enoil-CoA Hidratasa/fisiología , Isomerasas/fisiología , Modelos Químicos , Complejos Multienzimáticos/metabolismo , Complejos Multienzimáticos/fisiología , Enzima Bifuncional Peroxisomal , Racemasas y Epimerasas/metabolismo , Ratas , EstereoisomerismoRESUMEN
The peroxisome biogenesis disorders (PBD) are a group of autosomal-recessive diseases with complex developmental and metabolic phenotypes, including the Zellweger spectrum and rhizomelic chondrodysplasia punctata. The diseases are caused by defects in peroxisomal matrix protein import and are characterized by the loss of multiple peroxisomal metabolic functions. In humans, 12 complementation groups have been identified, with complementation group 1 accounting for more than two thirds of all PBD patients. Mutations in the PEX1 gene encoding a member of the AAA protein family of ATPases are responsible for the defects in this group, and a variety of PEX1 mutant alleles have been described. We characterized the PEX1 gene mutations and associated haplotypes in a group of thoroughly documented Zellweger spectrum patients in complementation group 1 who represent the broad range of phenotypic variation. We compared the type of mutation with the age of survival, clinical manifestations, and biochemical alterations and found a close relationship between genotype and age of survival. Missense mutations cause a milder form of disease, whereas insertions, deletions, and nonsense mutations are associated with severe clinical phenotypes. Thus, knowing the PEX1 gene mutation is helpful in predicting the course of disease in individual cases.
Asunto(s)
Proteínas de la Membrana/genética , Índice de Severidad de la Enfermedad , Síndrome de Zellweger/genética , ATPasas Asociadas con Actividades Celulares Diversas , Adolescente , Adulto , Niño , Preescolar , Ácidos Grasos/sangre , Prueba de Complementación Genética , Genotipo , Haplotipos , Humanos , Fenotipo , Polimorfismo Conformacional Retorcido-SimpleRESUMEN
alpha-Methylacyl-CoA racemase is a key enzyme in the metabolism of 2-methyl-branched fatty acids and, in mammals, in the conversion of cholesterol to bile acids. The enzyme from Mycobacterium tuberculosis has been purified to homogeneity and crystallized by the hanging-drop vapour-diffusion method. The crystals of the unliganded racemase belong to space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 122.0, c = 256.4 A. Data sets were collected at 100 K. The crystals diffract to 2.8 A using synchrotron radiation.
Asunto(s)
Mycobacterium tuberculosis/enzimología , Racemasas y Epimerasas/química , Secuencia de Aminoácidos , Animales , Cristalización , Cristalografía por Rayos X , Humanos , Datos de Secuencia Molecular , Racemasas y Epimerasas/metabolismo , Ratas , Homología de Secuencia de Aminoácido , SincrotronesRESUMEN
alpha-Methylacyl-CoA racemase (Amacr) deficiency in humans leads to sensory motor neuronal and liver abnormalities. The disorder is recessively inherited and caused by mutations in the AMACR gene, which encodes Amacr, an enzyme presumed to be essential for bile acid synthesis and to participate in the degradation of methyl-branched fatty acids. To generate a model to study the pathophysiology in Amacr deficiency we inactivated the mouse Amacr gene. As per human Amacr deficiency, the Amacr(-/-) mice showed accumulation (44-fold) of C27 bile acid precursors and decreased (over 50%) primary (C24) bile acids in bile, serum and liver, however the Amacr(-/-) mice were clinically symptomless. Real-time quantitative PCR analysis showed that, among other responses, the level of mRNA for peroxisomal multifunctional enzyme type 1 (pMFE-1) was increased 3-fold in Amacr(-/-) mice. This enzyme can be placed, together with CYP3A11 and CYP46A1, to make an Amacr-independent pathway for the generation of C24 bile acids. Exposure of Amacr(-/-) mice to a diet supplemented with phytol, a source for branched-chain fatty acids, triggered the development of a disease state with liver manifestations, redefining the physiological significance of Amacr. Amacr is indispensable for the detoxification of dietary methyl-branched lipids and, although it contributes normally to bile acid synthesis from cholesterol, the putative pMFE-1-mediated cholesterol degradation can provide for generation of bile acids, allowing survival without Amacr. Based upon our mouse model, we propose elimination of phytol from the diet of patients suffering from Amacr deficiency.
Asunto(s)
Ácidos y Sales Biliares/biosíntesis , Enfermedades Carenciales/etiología , Lípidos/farmacología , Racemasas y Epimerasas/deficiencia , Animales , Hidrocarburo de Aril Hidroxilasas/genética , Hidrocarburo de Aril Hidroxilasas/metabolismo , Ácidos y Sales Biliares/metabolismo , Peso Corporal/genética , Colesterol/sangre , Colesterol/metabolismo , Colesterol 24-Hidroxilasa , Clofibrato/farmacología , Citocromo P-450 CYP3A , Enfermedades Carenciales/tratamiento farmacológico , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Grasas de la Dieta/farmacología , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Hipolipemiantes/farmacología , Riñón/enzimología , Lípidos/sangre , Lípidos/química , Hígado/enzimología , Hígado/patología , Masculino , Proteínas de la Membrana , Ratones , Ratones Mutantes , Oxidorreductasas N-Desmetilantes/genética , Oxidorreductasas N-Desmetilantes/metabolismo , Fitol/farmacología , Racemasas y Epimerasas/genética , Racemasas y Epimerasas/metabolismo , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismo , Vitamina K/metabolismoRESUMEN
Alpha-methylacyl-CoA racemase (AMACR) is a peroxisomal and mitochondrial enzyme involved in the beta-oxidation of branched fatty acids, shown to be elevated in prostate cancer by several recent studies. Sequence variants of AMACR have been linked to prostate cancer risk. Although mRNA transcript, protein, and sequence variants of AMACR have been studied in the context of prostate cancer, AMACR enzymatic activity has not been addressed. Here we present evidence that AMACR activity is consistently elevated in prostate cancer tissue specimens. This activity can be immunodepleted from prostate cancer tissue extracts. Furthermore, mock needle biopsy cores containing foci of prostate cancer exhibited increased AMACR enzymatic activity, correlating with both protein levels and histopathology. Taken together, our studies suggest that AMACR activity is increased in prostate cancer relative to benign epithelia and suggests that monitoring AMACR activity levels in prostate needle biopsies may have clinical applications.
Asunto(s)
Neoplasias de la Próstata/enzimología , Racemasas y Epimerasas/metabolismo , Biomarcadores de Tumor , Biopsia con Aguja , Humanos , Immunoblotting , Inmunohistoquímica , Masculino , Microdisección , Neoplasias de la Próstata/patología , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Skin fibroblast cultures from patients with inherited lysosomal enzymopathies, alpha-N-acetylgalactosaminidase (alpha-NAGA) and alpha-galactosidase A deficiencies (Schindler and Fabry disease, respectively), and from normal controls were used to study in situ degradation of blood group A and B glycosphingolipids. Glycosphingolipids A-6-2 (GalNAc (alpha 1-->3)[Fuc alpha 1-->2]Gal(beta1-->4)GlcNAc(beta 1-->3)Gal(beta 1--> 4)Glc (beta 1-->1')Cer, IV(2)-alpha-fucosyl-IV(3)-alpha-N-acetylgalactosaminylneolactotetraosylceramide), B-6-2 (Gal(alpha 1-->3)[Fuc alpha 1--> 2] Gal (beta 1-->4)GlcNAc(beta 1-->3)Gal(beta 1-->4)Glc(beta 1-->1')Cer, IV(2)- alpha-fucosyl-IV(3)-alpha-galactosylneolactotetraosylceramide), and globoside (GalNAc(beta 1-->3)Gal(alpha 1-->4)Gal(beta 1-->4)Glc(beta 1-->1') Cer, globotetraosylceramide) were tritium labeled in their ceramide moiety and used as natural substrates. The degradation rate of glycolipid A-6-2 was very low in fibroblasts of all the alpha-NAGA-deficient patients (less than 7% of controls), despite very heterogeneous clinical pictures, ruling out different residual enzyme activities as an explanation for the clinical heterogeneity. Strongly elevated urinary excretion of blood group A glycolipids was detected in one patient with blood group A, secretor status (five times higher than upper limit of controls), in support of the notion that blood group A-active glycolipids may contribute as storage compounds in blood group A patients. When glycolipid B-6-2 was fed to alpha-galactosidase A-deficient cells, the degradation rate was surprisingly high (50% of controls), while that of globotriaosylceramide was reduced to less than 15% of control average, presumably reflecting differences in the lysosomal enzymology of polar glycolipids versus less-polar ones. Relatively high-degree degradation of substrates with alpha-D-Galactosyl moieties hints at a possible contribution of other enzymes.