RESUMEN
Most of over a thousand mitochondrial proteins are encoded by nuclear genes and must be imported from the cytosol. Little is known about the cytosolic events regulating mitochondrial protein import, partly due to the lack of appropriate tools for its assessment in living cells. We engineered an inducible biosensor for monitoring the main presequence-mediated import pathway with a quantitative, luminescence-based readout. This tool was used to explore the regulation of mitochondrial import by the PINK1 kinase-driven Parkin ubiquitin ligase, which is dysfunctional in autosomal recessive Parkinson's disease. We show that mitochondrial import was stimulated by Parkin, but not by disease-causing Parkin variants. This effect was dependent on Parkin activation by PINK1 and accompanied by an increase in the abundance of K11 ubiquitin chains on mitochondria and by ubiquitylation of subunits of the translocase of outer mitochondrial membrane. Mitochondrial import efficiency was abnormally low in cells from patients with PINK1- and PARK2-linked Parkinson's disease and was restored by phosphomimetic ubiquitin in cells with residual Parkin activity. Altogether, these findings uncover a role of ubiquitylation in mitochondrial import regulation and suggest that loss of this regulatory loop may underlie the pathophysiology of Parkinson's disease, providing novel opportunities for therapeutic intervention.
Asunto(s)
Proteínas Mitocondriales/metabolismo , Proteínas Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Técnicas Biosensibles , Células HEK293 , Humanos , Transporte de ProteínasRESUMEN
The inactivation of alkaline phosphatase (AP) from bovine intestinal mucosa caused by lowering the p2H from 10.4 to 5.4 or by increasing the temperature from 25 degrees C to 70 degrees C were not followed by significant FTIR changes, indicating that the native conformation of AP was preserved under these conditions. Further decrease of p2H from 5.4 to 3.4 leaded to small infrared spectral changes of AP in the amide I' and amide II regions that were similar to the infrared spectral changes of AP induced by raising the temperature from 70 degrees C to 80 degrees C. The increase of temperature from 70 degrees C to 80 degrees C promoted the formation of intermolecular beta-sheets at the expense of some alpha-helix structures as evidenced by the appearance of the 1684 cm-1 and 1620 cm-1 component bands and the disappearance of the 1651-1657 cm-1 component band. This conformational change was followed by a sharp increase of the 2H/H exchange rate. CD spectra confirmed the FTIR results and were very sensitive to the variation of alpha-helix content while FTIR spectra were more receptive to the changes of beta-sheet structures.
Asunto(s)
Fosfatasa Alcalina/química , Mucosa Intestinal/enzimología , Animales , Bovinos , Dicroismo Circular , Estabilidad de Enzimas , Calor , Concentración de Iones de Hidrógeno , Conformación Proteica , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
The neurohormone melatonin is the central switch of the circadian rhythm and presumably exerts its activities through a series of receptors among which MT1 and MT2 have been widely studied. The third binding site of melatonin, MT3, has been recently characterized as a melatonin-sensitive form of the quinone reductase 2 (QR2, EC 1.6.99.2). In the present work, we showed that the binding of melatonin at MT3/QR2 was better described with 2-[125I]-iodomethoxy-carbonylamino-N-acetyltryptamine (2-[125I]-I-MCA-NAT) and, most importantly, that it was measurable at 20 degrees while it has been initially described and thoroughly studied using 2-[125I]-iodomelatonin at 4 degrees. Under these novel conditions, binding to MT3 could be traced without cross-reactivity with MT1 and MT2 receptors and, moreover, under conditions similar to those used to measure MT3/QR2 catalytic activity. The pharmacology established here on hamster kidney samples using the reference compounds remained essentially as already described using other experimental conditions. A new series of compounds with nanomolar affinity for the MT3 binding site and a high MT3 selectivity versus MT1 and MT2 is reported. In addition, we further document the MT3/QR2 binding site by demonstrating that it was widely distributed among mammals, although inter-species and inter-tissues differences exist. The present report details new experimental conditions for the pharmacological study of melatonin-sensitive QR2 isoforms, and suggests that, in addition to an already demonstrated inter-species difference, inter-tissues differences in QR2 sensitivity to melatonin may exist in primates and, therefore, represent an original and interesting route of investigation on the effect of melatonin on MT3/QR2.
Asunto(s)
Receptores de Superficie Celular/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Unión Competitiva , Células Cultivadas , Cricetinae , Humanos , Radioisótopos de Yodo , Cinética , Melatonina/análogos & derivados , Melatonina/farmacología , Metalotioneína 3 , Ratones , Conejos , Ensayo de Unión Radioligante , Ratas , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores de Melatonina , Especificidad de la Especie , Temperatura , Distribución Tisular , Triptaminas/farmacologíaAsunto(s)
Compartimento Celular , Glicosilfosfatidilinositoles/metabolismo , Proteínas de la Membrana/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Transporte Biológico , Enfermedad , Glicoproteínas/biosíntesis , Glicosilfosfatidilinositoles/deficiencia , Aparato de Golgi/metabolismo , Humanos , Lipoproteínas/biosíntesis , Glicoproteínas de MembranaRESUMEN
BACKGROUND AND PURPOSE: For many years, it was suspected that sheep expressed only one melatonin receptor (closely resembling MT(1) from other mammal species). Here we report the cloning of another melatonin receptor, MT(2), from sheep. EXPERIMENTAL APPROACH: Using a thermo-resistant reverse transcriptase and polymerase chain reaction primer set homologous to the bovine MT(2) mRNA sequence, we have cloned and characterized MT(2) receptors from sheep retina. KEY RESULTS: The ovine MT(2) receptor presents 96%, 72% and 67% identity with cattle, human and rat respectively. This MT(2) receptor stably expressed in CHO-K1 cells showed high-affinity 2[(125)I]-iodomelatonin binding (K(D)= 0.04 nM). The rank order of inhibition of 2[(125)I]-iodomelatonin binding by melatonin, 4-phenyl-2-propionamidotetralin and luzindole was similar to that exhibited by MT(2) receptors of other species (melatonin > 4-phenyl-2-propionamidotetralin > luzindole). However, its pharmacological profile was closer to that of rat, rather than human MT(2) receptors. Functionally, the ovine MT(2) receptors were coupled to G(i) proteins leading to inhibition of adenylyl cyclase, as the other melatonin receptors. In sheep brain, MT(2) mRNA was expressed in pars tuberalis, choroid plexus and retina, and moderately in mammillary bodies. Real-time polymerase chain reaction showed that in sheep pars tuberalis, premammillary hypothalamus and mammillary bodies, the temporal pattern of expression of MT(1) and MT(2) mRNA was not parallel in the three tissues. CONCLUSION AND IMPLICATIONS: Co-expression of MT(1) and MT(2) receptors in all analysed sheep brain tissues suggests that MT(2) receptors may participate in melatonin regulation of seasonal anovulatory activity in ewes by modulating MT(1) receptor action.
Asunto(s)
Receptor de Melatonina MT2/genética , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Células CHO , Bovinos , Clonación Molecular , Cricetinae , Cricetulus , Femenino , Proteínas de Unión al GTP/metabolismo , Humanos , Datos de Secuencia Molecular , Especificidad de Órganos , ARN Mensajero/metabolismo , Ensayo de Unión Radioligante , Ratas , Receptor de Melatonina MT1/antagonistas & inhibidores , Receptor de Melatonina MT1/metabolismo , Receptor de Melatonina MT2/antagonistas & inhibidores , Receptor de Melatonina MT2/metabolismo , Proteínas Recombinantes/metabolismo , Retina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Ovinos , Tetrahidronaftalenos/farmacología , Triptaminas/farmacologíaRESUMEN
Zhang et al. (Research Articles, 11 November 2005, p. 996) reported that obestatin, a peptide derived from the ghrelin precursor, activated the orphan G protein-coupled receptor GPR39. However, we found that I125-obestatin does not bind GPR39 and observed no effects of obestatin on GPR39-transfected cells in various functional assays (cyclic adenosine monophosphate production, calcium mobilization, and GPR39 internalization). Our results indicate that obestatin is not the cognate ligand for GPR39.
Asunto(s)
Hormonas Peptídicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Calcio/metabolismo , Membrana Celular/metabolismo , Colforsina/farmacología , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Ghrelina , Humanos , Ligandos , Datos de Secuencia Molecular , Hormonas Peptídicas/genética , Hormonas Peptídicas/farmacología , Hipófisis/citología , Hipófisis/metabolismo , Unión Proteica , Receptores Acoplados a Proteínas G/genética , TransfecciónRESUMEN
Since 15-deoxy-delta(12,14)-prostaglandin J(2) (15dPGJ(2)) has been identified as an endogenous ligand of PPARgamma thus inducing adipogenesis, it has been reported to play active parts in numerous cellular regulatory mechanisms. As 15dPGJ(2) has been shown to covalently bind several peptides and proteins, we investigated whether it also covalently binds PPARgamma. We first observed that after incubation of 15dPGJ(2) with recombinant PPARgamma, the quantity of free 15dPGJ(2) measured was always lower than the initial amount. We then measured the ability of the labeled agonist rosiglitazone to displace the complex PPARgamma(2)/15dPGJ(2) obtained after pre-incubation. We observed that the binding of rosiglitazone was dependent on the initial concentration of 15dPGJ(2). Finally using MALDI-TOF mass spectrometry analysis, after trypsinolysis of an incubate of the PPARgamma(2) ligand binding domain (GST-LBD) with 15dPGJ2, we found a fragment (m/z = 1314.699) corresponding to the addition of 15dPGJ(2) (m/z = 316.203) to the GST-LBD peptide (m/z = 998.481). All these observations demonstrate the existence of a covalent binding of 15dPGJ(2) to PPARgamma, which opens up new perspectives to study the molecular basis for selective activities of PPARs.
Asunto(s)
Adipocitos/metabolismo , PPAR gamma/metabolismo , Prostaglandina D2/análogos & derivados , Adipocitos/citología , Hipoglucemiantes/farmacología , Ligandos , PPAR gamma/química , Prostaglandina D2/química , Prostaglandina D2/metabolismo , Unión Proteica , Rosiglitazona , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tiazolidinedionas/farmacología , Factores de TiempoRESUMEN
Glycosylphosphatidylinositol (glycosyl-PtdIns)-anchored proteins are proposed to be clustered in membrane microdomains enriched in cholesterol and glycosphingolipids (GlySphs). We have prepared biomimetic membranes in order to study the possible phenomena of surface aggregation of these membrane components. Phosphatidylcholine liposomes were treated by octylglucoside to insert a glycosyl-PtdIns-protein, alkaline phosphatase (ALP), some cholesterol, and a GlySph, the lactocerebroside. The association of these compounds was shown by centrifugation on a density gradient. The presence of ALP on the surface of the vesicles was shown by the action of a phospholipase, and the presence of the lactocerebroside was shown by the use of a galactose-specific tetravalent lectin. Our data show that total alkaline phosphatase and half to total lactocerebroside were ectoplasmically inserted in the vesicles membrane. In addition, we observed that the presence of small amounts of ALP in the liposomes led to significant changes in membrane stability with regard to detergent, as shown by the changes in the solubilization process monitored by turbidimetry. Furthermore, we have built an original method to study the cohesion of the vesicles membrane, in which some magnesium ions were trapped in the luminal space of the liposomes during several days. The ALP is magnesium-dependent for its catalytic activity and was inhibited after incubation of ALP-containing liposomes in a magnesium-free buffer. The ALP activity was restored by the addition of detergent to the liposomes, due to the release of the luminal magnesium ions. Surface aggregation phenomena will now be investigated by atomic force microscopy.
Asunto(s)
Fosfatasa Alcalina/química , Fosfatasa Alcalina/metabolismo , Colesterol/química , Glicoesfingolípidos/química , Glicosilfosfatidilinositoles/química , Fosfatidilcolinas/química , Animales , Bovinos , Centrifugación por Gradiente de Densidad , Glucósidos , Glicosilfosfatidilinositoles/metabolismo , Intestinos/enzimología , Riñón/enzimología , Cinética , Liposomas/químicaRESUMEN
A variant alkaline phosphatase (ALP), with heat-sensitivity characteristics similar to that of the bone type, was found in the serum of a patient suffering from lung cancer. In disc polyacrylamide gel electrophoretic studies most of this enzyme had migrated to the region corresponding to liver ALP, with the remainder affecting bone ALP. Like kidney ALP, this ALP was markedly inhibited by 0.5 mmol/l L-cysteine. The K(m) of this ALP for p-nitrophenylphosphate was 0.39 mmol/l, similar to that of kidney ALP. The sugar moiety of this enzyme bore greater resemblance to that of kidney ALP than liver or bone ALP. However, immunoprecipitation of this particular ALP was strong with a monoclonal antibody against liver ALP and moderate with an antibody against bone ALP.
Asunto(s)
Fosfatasa Alcalina/análisis , Carcinoma de Células Pequeñas/enzimología , Isoenzimas/análisis , Neoplasias Pulmonares/enzimología , Anciano , Fosfatasa Alcalina/química , Fosfatasa Alcalina/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Carcinoma de Células Pequeñas/patología , Humanos , Inmunohistoquímica , Isoenzimas/química , Isoenzimas/inmunología , Neoplasias Pulmonares/patología , Masculino , Ratones , Pruebas de PrecipitinaRESUMEN
To investigate the possible role(s) of glycans in human tissue non-specific alkaline phosphatase (TNAP) activity, the iso-enzymes were purified and treated with various exo- and endo-glycosidases. Catalytic activity, oligomerization, conformation and immunoreactivity of the modified TNAPs were evaluated. All TNAPs proved to be N-glycosylated, and only the liver isoform (LAP) is not O-glycosylated. Usually, the kidney (KAP) and bone (BAP) isoenzymes are similar and cannot be clearly discriminated. Differences between the immunoreactivity of KAP/BAP and LAP with a BAP antibody were mainly attributed to the N-glycosylated moieties of the TNAPs. In addition, elimination of O-glycosylations moderately affects the TNAP reactivity. Interestingly, N-glycosylation is absolutely essential for TNAP activity, but not for that of the placental or intestinal enzymes. According to the deduced amino acid sequence of TNAP cDNA, Asn-213 is a possible N-glycosylation site, and our present findings suggest that this sugar chain plays a key role in enzyme regulation. With regard to the oligomeric state of alkaline phosphatase (AP) isoforms, the dimer/tetramer equilibrium is dependent on the deglycosylation of glycosyl-phosphatidylinositol(GPI)-free APs, but not GPI-linked APs. This equilibrium does not affect the AP conformation as observed with CD. With regard to TNAPs, no data were available on the gene expression or nature of the 5'-non-translated leader exon of human KAP, as opposed to BAP and LAP genes. cDNA sequencing revealed that cortex/medulla KAP is genetically related to BAP, and medulla KAP to LAP.
Asunto(s)
Fosfatasa Alcalina/química , Fosfatasa Alcalina/inmunología , Fosfatasa Alcalina/genética , Reacciones Antígeno-Anticuerpo/efectos de los fármacos , Secuencia de Bases , Huesos/enzimología , ADN Complementario/análisis , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Glicosilación , Humanos , Riñón/enzimología , Hígado/enzimología , Datos de Secuencia Molecular , Peso Molecular , Ligamento Periodontal/enzimología , Reacción en Cadena de la Polimerasa , Conformación Proteica , ARN Mensajero/análisis , Especificidad por SustratoRESUMEN
The regulation of the circadian rhythm is relayed from the central nervous system to the periphery by melatonin, a hormone synthesized at night in the pineal gland. Besides two melatonin G-coupled receptors, mt(1) and MT(2), the existence of a novel putative melatonin receptor, MT(3), was hypothesized from the observation of a binding site in both central and peripheral hamster tissues with an original binding profile and a very rapid kinetics of ligand exchange compared with mt(1) and MT(2). In this report, we present the purification of MT(3) from Syrian hamster kidney and its identification as the hamster homologue of the human quinone reductase 2 (QR(2), EC ). Our purification strategy included the use of an affinity chromatography step which was crucial in purifying MT(3) to homogeneity. The protein was sequenced by tandem mass spectrometry and shown to align with 95% identity with human QR(2). After transfection of CHO-K1 cells with the human QR(2) gene, not only did the QR(2) enzymatic activity appear, but also the melatonin-binding sites with MT(3) characteristics, both being below the limit of detection in the native cells. We further confronted inhibition data from MT(3) binding and QR(2) enzymatic activity obtained from samples of Syrian hamster kidney or QR(2)-overexpressing Chinese hamster ovary cells, and observed an overall good correlation of the data. In summary, our results provide the identification of the melatonin-binding site MT(3) as the quinone reductase QR(2) and open perspectives as to the function of this enzyme, known so far mainly for its detoxifying properties.