RESUMEN
The stepwise addition of monosaccharides to N-glycans attached to client proteins to generate a repertoire of mature proteins involves a concerted action of many glycosidases and glycosyltransferases. Here, we report that Golgi α-mannosidase II (GMII), a pivotal enzyme catalyzing the first step in the conversion of hybrid- to complex-type N-glycans, is activated by Zn2+ supplied by the early secretory compartment-resident ZNT5-ZNT6 heterodimers (ZNT5-6) and ZNT7 homodimers (ZNT7). Loss of ZNT5-6 and ZNT7 function results in marked accumulation of hybrid-type and complex/hybrid glycans with concomitant reduction of complex- and high-mannose-type glycans. In cells lacking the ZNT5-6 and ZNT7 functions, the GMII activity is substantially decreased. In contrast, the activity of its homolog, lysosomal mannosidase (LAMAN), is not decreased. Moreover, we show that the growth of pancreatic cancer MIA PaCa-2 cells lacking ZNT5-6 and ZNT7 is significantly decreased in a nude mouse xenograft model. Our results indicate the integral roles of ZNT5-6 and ZNT7 in N-glycosylation and highlight their potential as novel target proteins for cancer therapy.
Asunto(s)
Proteínas de Transporte de Catión , Aparato de Golgi , Zinc , Humanos , Glicosilación , Proteínas de Transporte de Catión/metabolismo , Proteínas de Transporte de Catión/genética , Animales , Zinc/metabolismo , Ratones , Aparato de Golgi/metabolismo , Manosidasas/metabolismo , Manosidasas/genética , Polisacáridos/metabolismo , Línea Celular Tumoral , Ratones Desnudos , Transportador 8 de ZincRESUMEN
The transcorneal route is the main entry route for drugs to the intraocular parts, after topical administration. The outer surface, the corneal epithelium (CE), forms the rate-limiting barrier for drug permeability. Information about the role and protein expression of drug and amino acid transporter proteins in the CE is sparse and lacking. The aim of our study was to characterize transporter protein expression in rabbit and porcine CE to better understand potential drug and nutrient absorption after topical administration. Proteins, mainly Abc and Slc transporters, were characterized with quantitative targeted absolute proteomics and global untargeted proteomics methods. In the rabbit CE, 24 of 48 proteins were detected in the targeted approach, and 21 of these were quantified. In the porcine CE, 26 of 58 proteins were detected in the targeted approach, and 20 of these were quantified. Among these, 15 proteins were quantified in both animals: 4f2hc (Slc3a2), Aqp0, Asct1 (Slc1a4), Asct2 (Slc1a5), Glut1 (Slc2a1), Hmit (Slc2a13), Insr, Lat1 (Slc7a5), Mct1 (Slc16a1), Mct2 (Slc16a7), Mct4 (Slc16a3), Mrp 4 (Abcc4), Na+/K+-ATPase, Oatp3a1 (Slco3a1), and Snat2 (Slc38a2). Overall, the global proteomics results supported the targeted proteomics results. Organic anion transporting polypeptide Oatp3a1 was detected and quantified for the first time in both rabbit (1.4 ± 0.4 fmol/cm2) and porcine (11.1 ± 5.3 fmol/cm2) CE. High expression levels were observed for L-type amino acid transporter, Lat1, which was quantified with newly selected extracellular domain peptides in rabbit (48.9 ± 11.8 fmol/cm2) and porcine (37.6 ± 11.5 fmol/cm2) CE. The knowledge of transporter protein expression in ocular barriers is a key factor in the successful design of new ocular drugs, pharmacokinetic modeling, understanding ocular diseases, and the translation to human.
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Epitelio Corneal , Proteómica , Animales , Conejos , Porcinos , Epitelio Corneal/metabolismo , Proteómica/métodos , Transporte Biológico , Proteínas de Transporte de Membrana/metabolismo , Administración OftálmicaRESUMEN
Glycosylphosphatidylinositol (GPI)-anchored proteins play crucial roles in various enzyme activities, cell signaling and adhesion, and immune responses. While the molecular mechanism underlying GPI-anchored protein biosynthesis has been well studied, the role of zinc transport in this process has not yet been elucidated. Zn transporter (ZNT) proteins mobilize cytosolic zinc to the extracellular space and to intracellular compartments. Here, we report that the early secretory pathway ZNTs (ZNT5-ZNT6 heterodimers [ZNT5-6] and ZNT7-ZNT7 homodimers [ZNT7]), which supply zinc to the lumen of the early secretory pathway compartments are essential for GPI-anchored protein expression on the cell surface. We show, using overexpression and gene disruption/re-expression strategies in cultured human cells, that loss of ZNT5-6 and ZNT7 zinc transport functions results in significant reduction in GPI-anchored protein levels similar to that in mutant cells lacking phosphatidylinositol glycan anchor biosynthesis (PIG) genes. Furthermore, medaka fish with disrupted Znt5 and Znt7 genes show touch-insensitive phenotypes similar to zebrafish Pig mutants. These findings provide a previously unappreciated insight into the regulation of GPI-anchored protein expression and protein quality control in the early secretory pathway.
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Proteínas de Transporte de Catión , Proteínas Ligadas a GPI , Zinc , Animales , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Pollos/metabolismo , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Glicosilfosfatidilinositoles/genética , Proteínas de la Membrana/metabolismo , Pez Cebra/metabolismo , Zinc/metabolismoRESUMEN
Blood-brain barrier (BBB) dysfunction is a fundamental cause of multiple sclerosis and identifying the molecules that are responsible is an urgent matter. Protein expression was comprehensively quantified at the BBB of experimental autoimmune encephalomyelitis (EAE) mice, a model of multiple sclerosis, using the SWATH method. Concerning tight junction molecules, the level of expression of Claudin-5, which, in a previous immunohistochemical analysis, was confirmed to be down-regulated by EAE, remained unchanged, but the expression of Claudin-11 and Occludin was decreased by 0.69- and 0.62-fold, respectively, in brain capillaries isolated from EAE mice. A number of other cell-cell junctional molecules including ESAM, CADM1, CADM2, CADM3, CADM4, and HEPACAM were also down-regulated. The levels of expression of intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1), which directly mediate the infiltration of lymphocytes across the BBB, were increased in EAE mice by 3.3- and 2.6-fold, respectively. The expression of CXADR, which possibly facilitates the adhesion of migrating cells, was also increased by 3.5-fold. Interestingly, various members of the Annexin A (ANXA) family were also up-regulated in brain capillaries that were isolated from EAE mice. In a pathway associated with cell infiltration and tight junction disruption, a series of molecules that are involved in ANXA2 signaling (ANXA2, PTP1B, Ahnak, S100A11, CD44, Kindlin2, Integrin α5, Fibronectin, Fibrinogen) were up-regulated. ANXA2 is selectively and abundantly expressed in endothelial cells in the brain. The daily administration of an ANXA2 inhibitor (LCKLSL peptide) significantly suppressed the development of EAE in mice. In summary, the activation of ANXA2 signaling at the BBB appear to play an important role in the pathogenesis of EAE.
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Anexina A2 , Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Animales , Anexina A2/metabolismo , Barrera Hematoencefálica/metabolismo , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/patología , Células Endoteliales/metabolismo , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/metabolismoRESUMEN
The purpose of this study was to elucidate the absolute abundance of transporters, enzymes, receptors, and tight junction and marker proteins at human blood-arachnoid barrier (BAB) and compare with those of dogs and pigs. Protein expression levels in plasma membrane fractions of brain leptomeninges were determined by quantitative targeted absolute proteomics. To realistically compare the absolute abundance of target molecules at the BAB among humans, dogs, and pigs, the unit was converted from fmol/µg-protein to pmol/cm2 -leptomeninges. Of a total of 70 proteins, 52 were detected. OAT1, OAT3, GLUT1, 4F2hc, EAAT1, EAAT2, MCT8, SMVT, CTL2, GFAP, Claudin-5, Na+ /K+ -ATPase, COMT, GSTP1, and CES1 were abundantly expressed at the human BAB (>1 pmol/cm2 ). The protein expression levels were within a 3-fold difference for 16 out of 33 proteins between humans and dogs and for 13 out of 28 proteins between humans and pigs. Both human-dog and human-pig differences in protein expression levels were within 3-fold for OAT1, OAT3, 4F2hc, xCT, OCT2, MDR1, BCRP, PEPT2, SYP, and MCT1. In contrast, OCT3, MCT4, and OATP1A2 were detected in humans but not in dogs or pigs. MRP3 was detected in dogs and pigs but not in humans. The absolute level of GLUT1 in humans was nearly the same as that in dogs but was 6.14-fold greater in pigs. No significant differences in the levels were observed between male and female dogs for nearly all molecules. These results should be helpful in understanding the physiological roles of BAB and cerebrospinal fluid pharmacokinetics in humans and their differences from dogs and pigs.
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Barrera Hematoencefálica , Uniones Estrechas , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Animales , Aracnoides/metabolismo , Biomarcadores/metabolismo , Barrera Hematoencefálica/metabolismo , Perros , Femenino , Transportador de Glucosa de Tipo 1/metabolismo , Humanos , Masculino , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Porcinos , Uniones Estrechas/metabolismoRESUMEN
One of the major reasons why central nervous system (CNS)-drug development has been challenging in the past, is the barriers that prevent substances entering from the blood circulation into the brain. These barriers include the blood-brain barrier (BBB), blood-spinal cord barrier (BSCB), blood-cerebrospinal fluid barrier (BCSFB), and blood-arachnoid barrier (BAB), and they differ from each other in their transporter protein expression and function as well as among the species. The quantitative expression profiles of the transporters in the CNS-barriers have been recently revealed, and in this review, it is described how they affect the pharmacokinetics of compounds and how these expression differences can be taken into account in the prediction of brain drug disposition in humans, an approach called pharmacoproteomics. In recent years, also structural biology and computational resources have progressed remarkably, enabling a detailed understanding of the dynamic processes of transporters. Molecular dynamics simulations (MDS) are currently used commonly to reveal the conformational changes of the transporters and to find the interactions between the substrates and the protein during the binding, translocation in the transporter cavity, and release of the substrate on the other side of the membrane. The computational advancements have also aided in the rational design of transporter-utilizing compounds, including prodrugs that can be actively transported without losing potency towards the pharmacological target. In this review, the state-of-art of these approaches will be also discussed to give insights into the transporter-mediated drug delivery to the CNS.
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Barrera Hematoencefálica , Encéfalo , Sistemas de Liberación de Medicamentos , Proteínas de Transporte de Membrana , Transporte Biológico , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Humanos , Proteínas de Transporte de Membrana/metabolismo , Proteómica , Médula Espinal/metabolismoRESUMEN
PURPOSE: The purpose of the present study was to quantitatively determine the expression of transporters, receptors and tight junction molecules at the blood-arachnoid barrier (BAB) and blood-spinal cord barrier (BSCB) in cervical, thoracic and lumbar spines from dogs. METHODS: The expression levels of 31 transporters, 3 receptors, 1 tight junction protein, and 3 marker proteins in leptomeninges and capillaries isolated from spines (3 male and 2 female dogs) were determined by quantitative Targeted Absolute Proteomics (qTAP). The units were converted from fmol/µg protein to pmol/cm (absolute abundance at the BAB and the BSCB in a 1 cm section of spine). RESULTS: The expression of MDR1 and BCRP were greater at the BSCB compared to the BAB (especially in the cervical cord), and the expressions at the lumbar BSCB were lower than that for the cervical BSCB. Among the organic anionic and cationic drug transporters, OAT1, OAT3, MRP1, OCT2 and MATE1/2 were detected only in the BAB, and not at the BSCB). The expression of these transporters was higher in the order: lumbar > thoracic > cervical BAB. The expressions of GLUT1, 4F2hc, EAAT1, 2, PEPT2, CTL1, and MCT1 at the BSCB of the cervical cord were higher than the corresponding values for the cervical BAB, and these values decreased in going down the spinal cord. CONCLUSION: These results provide a better understanding of the molecular mechanisms underlying the concentration gradients of drugs and endogenous substances in the cerebrospinal fluid and parenchyma of the spinal cord.
Asunto(s)
Barrera Hematoencefálica , Uniones Estrechas , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Animales , Aracnoides/metabolismo , Barrera Hematoencefálica/metabolismo , Perros , Femenino , Masculino , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Médula Espinal/metabolismo , Uniones Estrechas/metabolismoRESUMEN
From the viewpoint of drug discovery, it is an important issue to elucidate the drug permeability at the human central nervous system (CNS) barriers and the molecular mechanisms in the cells forming CNS barriers especially during CNS diseases. I introduced quantitative proteomics techniques into the blood-brain barrier (BBB) study, then quantitatively investigated the transport system at the human BBB and clarified the quantitative differences in protein expression levels and functions of transporters and receptors between animals and humans, or in vitro and in vivo. Based on the difference in the absolute expression level of transporters between in vitro and in vivo, I demonstrated that the drug efflux activity of P-glycoprotein (P-gp) at in vivo BBB can be accurately reconstructed from the in vitro system, not only in mouse models but also monkeys similar to humans and pathological conditions. Furthermore, I discovered Claudin-11 as another tight junction molecule expressed at the CNS barriers, and clarified that it contributes to the disruption of the CNS barriers in multiple sclerosis. Furthermore, it was also elucidated that the P-gp dysfunction causes excessive brain entry of glucocorticoid which causes a nerve damage in cerebral infarct, and it can be suppressed by targeting Abl/Src kinases. These suggest that targeting the tight junctions and transporters, which are important molecules at the CNS barriers, would potentially lead to the treatment of CNS diseases. In this review, I would like to introduce a new CNS barrier study opened by quantitative proteomics research.
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Barrera Hematoencefálica/metabolismo , Proteómica , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Infarto Cerebral/metabolismo , Claudinas/metabolismo , Descubrimiento de Drogas , Humanos , Esclerosis Múltiple/metabolismo , Estrés Oxidativo , Uniones Estrechas/metabolismoRESUMEN
The purpose of the present study was to identify a plasma protein biomarker able to predict pre-eclampsia (PE). Comprehensive quantitative proteomics using mass spectrometry with sequential window acquisition of all theoretical fragment ion spectra (SWATH-MS) was applied to plasma samples of 7 PE and 14 healthy pregnant women (for PE subjects, plasma samples were taken before onset of PE), and 11 proteins were selected as candidates potentially able to differentiate the two groups. Plasmas collected at gestational weeks 14-24 from 36 PE and 120 healthy pregnant women (for PE subjects, plasma samples were taken before onset of PE) were used to conduct selected reaction monitoring quantification analysis, optimize protein combinations and conduct internal validation, which consisted of 30 iterations of 10-fold cross-validation using multivariate logistic regression and receiver operating characteristic (ROC) analysis. The combination of afamin, fibronectin, and sex-hormone-binding globulin was selected as the best candidate. The 3-protein combination predictive model (predictive equation and cut-off value) generated using the internal validation subjects was successfully validated in another group of validation subjects (36 PE and 54 healthy (for PE subjects, plasma samples were taken before onset of PE)) and showed good predictive performance, with the area under the curve (AUC) 0.835 and odds ratio 13.43. In conclusion, we newly identified a 3-protein combination biomarker and established a predictive equation and cut-off value that can predict the onset of PE based on analysis of plasma samples collected during gestational weeks 14-24.
Asunto(s)
Proteínas Portadoras/sangre , Fibronectinas/sangre , Glicoproteínas/sangre , Preeclampsia/sangre , Globulina de Unión a Hormona Sexual/análisis , Adulto , Biomarcadores/sangre , Femenino , Humanos , Embarazo , Albúmina Sérica Humana , Adulto JovenRESUMEN
We have previously shown that gelsolin (GSN) levels are significantly lower in the blood of patients with glioblastoma (GBM) than in healthy controls. Here, we analyzed the function of GSN in GBM and examined its clinical significance. Furthermore, microRNAs involved in GSN expression were also identified. The expression of GSN was determined using western blot analysis and found to be significantly lower in GBM samples than normal ones. Gelsolin was mainly localized in normal astrocytes, shown using immunohistochemistry and immunofluorescence. Higher expression of GSN was correlated with more prolonged progression-free survival and overall survival. Gelsolin knockdown using siRNA and shRNA markedly accelerated cell proliferation and invasion in GBM in vitro and in vivo. The inactive form of glycogen synthase kinase-3ß was dephosphorylated by GSN knockdown. In GBM tissues, the expression of GSN and microRNA (miR)-654-5p and miR-450b-5p showed an inverse correlation. The miR-654-5p and miR-450b-5p inhibitors enhanced GSN expression, resulting in reduced proliferation and invasion. In conclusion, GSN, which inhibits cell proliferation and invasion, is suppressed by miR-654-5p and miR-450b-5p in GBM, suggesting that these miRNAs can be targets for treating GBM.
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Gelsolina/genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , MicroARNs/genética , Animales , Apoptosis/genética , Biomarcadores de Tumor , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Supervivencia Celular/genética , Modelos Animales de Enfermedad , Femenino , Gelsolina/metabolismo , Técnicas de Inactivación de Genes , Glioblastoma/metabolismo , Glioblastoma/mortalidad , Glioblastoma/patología , Humanos , Ratones , Clasificación del Tumor , Fenotipo , Pronóstico , Interferencia de ARNRESUMEN
The physiologic and pharmacologic roles of the blood-arachnoid barrier (BAB) remain unclear. Therefore, the purpose of the present study was to comprehensively evaluate and compare the absolute protein expression levels of transporters in the leptomeninges and plexus per cerebrum, and to determine the localizations of transporters at the cerebrospinal fluid (CSF)-facing and blood (dura)-facing plasma membranes of the BAB in pig. Using multidrug resistance protein 1 (MDR1) and organic anion transporter (OAT) 1 as blood (dura)-facing and CSF-facing plasma membrane marker proteins, respectively, we established that breast cancer resistance protein (BCRP), multidrug resistance-associated protein (MRP) 4, organic anion-transporting polypeptide (OATP) 2B1, multidrug and toxin extrusion protein 1 (MATE1), and glucose transporter 1 (GLUT1) are localized at the blood-facing plasma membrane, and OAT3, peptide transporter (PEPT) 2, MRP3, organic cation transporter (OCT) 2, xCT, monocarboxylate transporter (MCT) 1, MCT4, and MCT8 are localized at the CSF-facing plasma membrane of the BAB. The absolute protein expression levels of OAT1, OAT3, MDR1, BCRP, PEPT2, xCT, MATE1, OCT2, and 4f2hc in the whole BAB surrounding the entire cerebrum were much larger than those in the total of the choroid plexuses forming the blood-cerebrospinal fluid barrier (BCSFB). Although MRP4, OATP2B1, MCT8, GLUT1, and MCT1 were also statistically significantly more abundant in the BAB than in the choroid plexuses per porcine cerebrum, these transporters were nevertheless almost equally distributed between the two barriers. In contrast, OATP1A2, MRP1, OATP3A1, and OCTN2 were specifically expressed in the choroid plexus. These results should be helpful in understanding the relative overall importance of transport at the BAB compared with that at the BCSFB, as well as the rank order of transport capacities among different transporters at the BAB, and the directions of transport mediated by individual transporters. SIGNIFICANCE STATEMENT: We found that BCRP, MRP4, OATP2B1, MATE1, and GLUT1 localize at the blood-facing plasma membrane of the blood-arachnoid barrier (BAB), while OAT3, PEPT2, MRP3, OCT2, xCT, MCT1, MCT4, and MCT8 localize at the CSF-facing plasma membrane. 4F2hc is expressed in both membranes. For OAT1, OAT3, MDR1, BCRP, PEPT2, xCT, MATE1, OCT2, and 4f2hc, the absolute protein expression levels in the whole BAB surrounding the entire cerebrum are much greater than the total amounts in the choroid plexuses.
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Aracnoides/metabolismo , Barrera Hematoencefálica/metabolismo , Membrana Celular/metabolismo , Líquido Cefalorraquídeo/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Animales , Transporte Biológico/fisiología , Plexo Coroideo/metabolismo , PorcinosRESUMEN
This work was designed to clarify the absolute abundances of transporters and receptors at different cerebral regions of the blood-brain barriers (BBB) and blood-spinal cord barrier (BSCB) in humans and rats, using physiologically relevant units (pmol/g tissue and fmol/cm2); 39 and 29 proteins including tight-junction proteins and markers were quantified in human and rat capillary samples, respectively. Protein expression levels of almost all proteins were identical within a 2-fold range between BBB and BSCB in rats, while many proteins showed >2-fold smaller expression levels in BSCB than BBB in humans. Protein expression levels of transporters and receptors in humans were remarkably smaller than those in rats in both BBB and BSCB in units of pmol/g tissue and fmol/cm2. Protein expression levels (fmol/cm2) of MDR1 and BCRP at the BBB in humans were 9.88-fold and 5.23-fold smaller than those in rats, respectively. GLUT1 expression (pmol/g tissue) at cortical BBB in a human was 2.49- and 3.76-fold greater than that at white matter BBB and BSCB, respectively. INSR and LRP1 proteins were detected at cortical BBB, but not at white matter BBB or BSCB in humans. These findings throw light on regional differences and species differences in pharmacokinetics and physiological functions in the central nervous system.
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Barrera Hematoencefálica/metabolismo , Médula Espinal/metabolismo , Anciano , Animales , Transportador de Glucosa de Tipo 1/metabolismo , Humanos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , Ratas , Receptor de Insulina/metabolismoRESUMEN
Decreased levels of docosahexaenoic acid (DHA), an endogenous neuroprotective compound, in the brain are associated with the development of Alzheimer's disease (AD). We previously showed that DHA is a substrate of fatty acid transport protein 1 (FATP1/SLC27A1), and FATP1 is localized at the abluminal membrane of brain capillary endothelial cells. We hypothesized that amyloid ß (Aß) decreases FATP1-mediated cellular efflux (i.e. supply to the brain) of DHA at the blood-brain barrier (BBB). Here, we tested this hypothesis using a human cerebral microvascular endothelial cell line, human cerebral microvessel endothelial cells (hCMEC/D3), as a BBB model. The efflux of DHA-d5 by hCMEC/D3 cells increased time-dependently up to 3 min. Knock-down of FATP1 with specific siRNA indicated that FATP1-mediated efflux accounts for 47.0% of this DHA-d5 efflux. In hCMEC/D3 cells treated with Aß25-35 (10 µM/24 h), which we employed as an in vitro model of the BBB in AD, FATP1 protein expression in the plasma membrane was decreased by 96.0%, which was greater than the decrease in the whole-cell lysate, and the DHA-d5 efflux was decreased by 68.3%. Of this 68.3% decrease, 45.1% (47.0 × 0.96) is accounted for by the decrease in FATP1-mediated efflux and the remaining 23.2% is presumably mediated by other mechanism(s). Thus, we have established for the first time that FATP1 is a major contributor to DHA efflux from human brain capillary endothelial cells, and its efflux activity at the abluminal membrane of the cells is blocked by Aß. This may explain the decreased DHA level in the brain of AD patients. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.
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Péptidos beta-Amiloides/metabolismo , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Ácidos Docosahexaenoicos/metabolismo , Proteínas de Transporte de Ácidos Grasos/metabolismo , Línea Celular , Regulación hacia Abajo , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , HumanosRESUMEN
Certain proteins, such as inflammatory cytokines, that are released from injured or diseased organs are transported from the circulating blood through the blood-brain barrier (BBB) into the brain and contribute to the pathogenesis of related central nervous system dysfunctions. However, little is known about the protein transport mechanisms involved in the central nervous system dysfunctions. The aims of the present study were to identify BBB-permeable protein(s) derived from liver and to clarify their transport characteristics at the BBB. After administration of biotin-labeled liver cytosolic protein fraction to mice in vivo, we identified 9 biotin-labeled proteins in the brain. Among them, we focused here on creatine kinase (CK). In vitro uptake studies with human brain microvessel endothelial cells (hCMEC/D3 cells) showed preferential uptake of muscle-type CK (CK-MM) compared with brain-type CK (CK-BB) at the BBB. Integration plot analysis revealed that CK-MM readily penetrated into brain parenchyma from the circulating blood across the BBB. The uptake of CK-MM by hCMEC/D3 cells was decreased at 4 °C and in the presence of clathrin- and caveolin-dependent endocytosis inhibitors. These results indicate that entry of CK into the brain is mediated by a transport system(s) at the BBB.
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Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Creatina Quinasa/metabolismo , Animales , Transporte Biológico/fisiología , Línea Celular , Cerebro/metabolismo , Humanos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteómica , ConejosRESUMEN
Brain metastasis is a frequent complication of cancer and may be mediated, at least in part, by the internalization of cancer-cell-derived exosomes into brain capillary endothelial cells. Clarifying the mechanism(s) of this internalization is of interest because it could help us to develop ways to block brain metastasis, as well as affording a potential new route for drug delivery into the brain. Therefore, the purpose of the present study was to address this issue by identifying the receptors involved in the internalization of exosomes derived from a brain-metastatic cancer cell line (SK-Mel-28) into human blood-brain barrier endothelial cells (hCMEC/D3 cells). The combination of sulfo-SBED-based cross-linking and comprehensive proteomics yielded 20 proteins as exosome receptor candidates in hCMEC/D3 cells. The uptake of PKH67-labeled exosomes by hCMEC/D3 cells measured at 37 °C was significantly reduced by 95.6% at 4 °C and by 15.3% in the presence of 1 mM RGD peptide, an integrin ligand. Therefore, we focused on the identified RGD receptors, integrin α5 and integrin αV, and CD46, which is reported to act as an adenovirus receptor, together with integrin αV. A mixture of neutralizing antibodies against integrin α5 and integrin αV significantly decreased the exosome uptake by 11.8%, while application of CD46 siRNA reduced it by 39.0%. Immunohistochemical analysis confirmed the presence of CD46 in human brain capillary endothelial cells. These results suggest that CD46 is a major receptor for the uptake of SK-Mel-28-derived exosomes by human blood-brain barrier endothelial cells (hCMEC/D3 cells).
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Encéfalo/metabolismo , Exosomas/metabolismo , Proteómica/métodos , Transporte Biológico/fisiología , Barrera Hematoencefálica/metabolismo , Línea Celular Tumoral , Células Endoteliales/metabolismo , Humanos , Melanoma/metabolismo , Proteína Cofactora de Membrana/metabolismo , ARN Interferente Pequeño , Receptores Virales/metabolismoRESUMEN
Leukocyte migration across the blood-brain barrier (BBB) is an important step in the progression of brain dysfunction in systemic inflammation. The purpose of this study was to identify the key regulatory molecule(s) at the BBB among the cluster of differentiation (CD) antigens involved in leucocyte migration in lipopolysaccharide (LPS)-induced systemic inflammation based on their absolute protein expressions. Here, we identified the absolute expression levels of 17 CD antigens in isolated brain capillaries (Bcap) of LPS-administered mice. Among them, the expression levels of CD54 and CD106 were dramatically increased in LPS-administered mice compared to the control by 6.21- and 3.67-fold, respectively. In peripheral blood mononuclear cells, the expression levels of CD11a/CD18, the counter-receptor for CD54, were similar to those of CD54 in Bcap of LPS-administered mice. On the other hand, the expression level of CD49d, part of CD29/CD49d complex, which is the counter-receptor for CD106, was under the limit of quantification. It is thus likely that CD54 at the BBB is predominantly involved in promoting leukocyte migration across the BBB in systemic inflammation. The expression levels of CD9, CD49c and CD97, which are thought to be involved in cell-to-cell interaction, were decreased by 40-60% in Bcap of LPS-administered mice. In contrast, the expression levels of 9 transporters, 2 receptors, and 1 tight junction-related protein in Bcap of LPS-administered mice were essentially unchanged compared to the control. These results suggest that enhancement of leucocyte migration in systemic inflammation involves dynamic changes of CD antigens without alterations of other major functional molecules.
Asunto(s)
Antígenos CD/inmunología , Barrera Hematoencefálica/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Movimiento Celular , Femenino , Leucocitos Mononucleares/fisiología , Lipopolisacáridos/farmacología , Masculino , Proteínas de Transporte de Membrana/inmunología , Ratones Endogámicos C57BLRESUMEN
The present study aimed to establish a humanized mouse model with which to explore OATP1A2-mediated transcellular transport of drug substrates across the blood-brain barrier (BBB) and to evaluate the usefulness of the humanized mice in preclinical studies. Sulpiride, amisulpride, sultopride, and triptans were used as probes to discriminate OATP1A2 and Oatp1a4. We generated a mouse line humanized for OATP1A2 by introducing the coding region downstream of the Oatp1a4 promoter using the CRISPR/Cas9 technique. In the mice generated, OATP1A2 mRNA in the brain was increased corresponding to disappearance of Oatp1a4. OATP1A2 was localized on both the luminal and abluminal sides of the BBB. Unfortunately, study in vivo employing sulpiride, sumatriptan, and zolmitriptan as probes did not indicate any difference in their brain-to-plasma ratio between the control and humanized mice. Quantitative targeted absolute proteomic analysis of the BBB fraction from the humanized mice revealed that almost all analyzed transporters and membrane proteins were expressed at similar levels to those in control mice. The quantitative levels of OATP1A2 differed depending on the peptide quantified, which suggests that incomplete translation or posttranslational modification may occur. The blood-to-brain transport of zolmitriptan, determined by brain perfusion in situ, was 1.6-fold higher in the humanized mice than in the controls, whereas that of sulpiride was not significantly changed. To our knowledge, we established a mouse line humanized for a BBB uptake transporter for the first time. Unfortunately, because of limited impact, there is still room for improvement of the model system.
Asunto(s)
Transporte Biológico/fisiología , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Transportadores de Anión Orgánico/metabolismo , Preparaciones Farmacéuticas/metabolismo , Animales , Línea Celular , Células HEK293 , Humanos , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Oxazolidinonas/metabolismo , Proteómica/métodos , Triptaminas/metabolismoRESUMEN
The purpose of the present study was to clarify the molecular basis of zonated drug distributions in mouse liver based on the protein expression levels of transporters and metabolizing enzymes in periportal (PP) and pericentral (PC) vein regions of mouse hepatic lobules. The distributions of sulforhodamine 101 (SR-101), a substrate of organic anion transporting polypeptides (Oatps), and ribavirin, a substrate of equilibrative nucleoside transporter 1 (Ent1), were elucidated in frozen liver sections of mice, to which each compound had been intravenously administered. Regions strongly positive for SR-101 (SR-101+) and regions weakly positive or negative for SR-101 (SR-101-) were separated by laser microdissection. The zonated distribution of protein expression was quantified in terms of the liver zonation index. Quantitative targeted absolute proteomics revealed the selective expression of glutamine synthetase in the SR-101+ region, indicating predominant distribution of SR-101 in hepatocytes of the PC vein region. The protein levels of Oatp1a1, Oatp1b2, organic cation transporter 1 (Oct1), and cytochrome P450 (P450) 2e1 were greater in the PC vein regions, whereas the level of organic anion transporter 2 (Oat2) was greater in the PP vein regions. Mouse Oatp1a1 mediated SR-101 transport. On the other hand, there were no statistically significant differences in expression of Ent1, Na+-taurocholate cotransporting polypeptide, several canalicular transporters, P450 enzymes, and UDP-glucuronosyltransferases between the PP and PC vein regions. This is consistent with the almost uniform distribution of ribavirin in the liver. In conclusion, sinusoidal membrane transporters such as Oatp1a1, Oatp1b2, Oct1, and Oat2 appear to be determinants of the zonated distribution of drugs in the liver.
Asunto(s)
Transporte Biológico/fisiología , Hígado/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Transportador 1 de Catión Orgánico/metabolismo , Preparaciones Farmacéuticas/metabolismo , Animales , Sistema Enzimático del Citocromo P-450/metabolismo , Hepatocitos/metabolismo , Masculino , Ratones , Ribavirina/metabolismoRESUMEN
Although arachnoid mater epithelial cells form the blood-arachnoid barrier (BAB), acting as a blood-CSF interface, it has been generally considered that the BAB is impermeable to water-soluble substances and plays a largely passive role. Here, we aimed to clarify the function of transporters at the BAB in regulating CSF clearance of water-soluble organic anion drugs based on quantitative targeted absolute proteomics (QTAP) and in vivo analyses. Protein expression levels of 61 molecules, including 19 ATP-binding-cassette (ABC) transporters and 32 solute-carrier (SLC) transporters, were measured in plasma membrane fraction of rat leptomeninges using QTAP. Thirty-three proteins were detected; others were under the quantification limits. Expression levels of multidrug resistance protein 1 (Mdr1a/P-gp/Abcb1a) and breast cancer resistance protein (Bcrp/Abcg2) were 16.6 and 3.27 fmol/µg protein (51.9- and 9.82-fold greater than in choroid plexus, respectively). Among those organic anion transporters detected only at leptomeninges, not choroid plexus, organic anion transporter 1 (oat1/Slc22a6) showed the greatest expression (2.73 fmol/µg protein). On the other hand, the protein expression level of oat3 at leptomeninges was 6.65 fmol/µg protein, and the difference from choroid plexus was within two-fold. To investigate oat1's role, we injected para-aminohippuric acid (PAH) with or without oat1 inhibitors into cisterna magna (to minimize the contribution of choroid plexus function) of rats. A bulk flow marker, FITC-inulin, was not taken up from CSF up to 15 min, whereas uptake clearance of PAH was 26.5 µL/min. PAH uptake was completely blocked by 3 mM cephalothin (inhibits both oat1 and oat3), while 17% of PAH uptake was inhibited by 0.2 mM cephalothin (selectively inhibits oat3). These results indicate that oat1 and oat3 at the BAB provide a distinct clearance pathway of organic anion drugs from CSF independently of choroid plexus.
Asunto(s)
Aniones/farmacocinética , Aracnoides/metabolismo , Barrera Hematoencefálica/metabolismo , Proteína 1 de Transporte de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Animales , Aniones/administración & dosificación , Aniones/líquido cefalorraquídeo , Aracnoides/irrigación sanguínea , Barrera Hematoencefálica/efectos de los fármacos , Cefalotina/farmacología , Líquido Cefalorraquídeo/química , Plexo Coroideo/irrigación sanguínea , Plexo Coroideo/metabolismo , Colorantes Fluorescentes/administración & dosificación , Colorantes Fluorescentes/farmacocinética , Inyecciones Intraventriculares , Masculino , Tasa de Depuración Metabólica , Proteína 1 de Transporte de Anión Orgánico/antagonistas & inhibidores , Transportadores de Anión Orgánico Sodio-Independiente/antagonistas & inhibidores , Proteómica/métodos , Ratas , Ratas Wistar , Rodamina 123/administración & dosificación , Rodamina 123/líquido cefalorraquídeo , Rodamina 123/farmacocinéticaRESUMEN
Cynomolgus monkeys have been widely used for the prediction of drug absorption in humans. The purpose of this study was to clarify the regional protein expression levels of cytochromes P450 (CYPs), UDP-glucuronosyltransferases (UGTs), and transporters in small intestine of cynomolgus monkey using liquid chromatography-tandem mass spectrometry, and to compare them with the corresponding levels in human. UGT1A1 in jejunum and ileum were >4.57- and >3.11-fold and UGT1A6 in jejunum and ileum were >16.1- and >8.57-fold, respectively, more highly expressed in monkey than in human. Also, jejunal expression of monkey CYP3A8 (homologue of human CYP3A4) was >3.34-fold higher than that of human CYP3A4. Among apical drug efflux transporters, BCRP showed the most abundant expression in monkey and human, and the expression levels of BCRP in monkey and human were >1.74- and >1.25-fold greater than those of P-gp and >2.76- and >4.50-fold greater than those of MRP2, respectively. These findings should be helpful to understand species differences of the functions of CYPs, UGTs, and transporters between monkey and human. The UGT1A1/1A6 data would be especially important because it is difficult to identify isoforms responsible for species differences of intestinal glucuronidation by means of functional studies due to overlapping substrate specificity.