Targeted Proteomics-Based Quantitative Protein Atlas of Pannexin and Connexin Subtypes in Mouse and Human Tissues and Cancer Cell Lines.

The pannexin (Px) and connexin (Cx) families form multimeric hemichannels that mediate cellular transport of a wide variety of signaling and other molecules and exhibit pathophysiological and pharmacological functions. Twenty-four Px and Cx subtypes have been identified in humans and 23 in mice. The purpose of this study is to establish a quantitative protein atlas of Px and Cx subtypes in mouse and human tissues and cancer cell lines by means of quantitative targeted absolute proteomics, using an internal standard protein in which stable-isotope-labeled target peptides selected according to in silico criteria are concatenated together with internal reference peptides for the determination of the protein amount. This quantification system enabled us to cover 20 of 24 subtypes (83%) in humans, and 21 of 23 subtypes (91%) in mice. In mice, Px1, Cx32, and Cx43 were most abundantly expressed in the small intestine, liver and pancreas, and brain capillary, brain, and heart, respectively. Human blood-brain barrier endothelial cells (human cerebral microvessel endothelial cells) highly expressed Px1 and Cx43. Among human cancer cells, Panc-1 selectively expressed Px1, and Caco-2 cells abundantly expressed Cx32, while MCF-7 and AsPC-1 did not express any subtypes of hemichannels tested. These results suggest that Px1, Cx32, and Cx43 appear to play predominant roles in normal tissues and some cancer cells.

Selective Protein Expression Changes of Leukocyte-Migration-Associated Cluster of Differentiation Antigens at the Blood-Brain Barrier in a Lipopolysaccharide-Induced Systemic Inflammation Mouse Model without Alteration of Transporters, Receptors or Tight Junction-Related Protein.

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.

Cluster of Differentiation 46 Is the Major Receptor in Human Blood-Brain Barrier Endothelial Cells for Uptake of Exosomes Derived from Brain-Metastatic Melanoma Cells (SK-Mel-28).

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).

Identification of Blood-Brain Barrier-Permeable Proteins Derived from a Peripheral Organ: In Vivo and in Vitro Evidence of Blood-to-Brain Transport of Creatine Kinase.

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.

Liver Zonation Index of Drug Transporter and Metabolizing Enzyme Protein Expressions in Mouse Liver Acinus.

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.

Identification of blood biomarkers in glioblastoma by SWATH mass spectrometry and quantitative targeted absolute proteomics.

Molecular biomarkers in blood are needed to aid the early diagnosis and clinical assessment of glioblastoma (GBM). Here, in order to identify biomarker candidates in plasma of GBM patients, we performed quantitative comparisons of the plasma proteomes of GBM patients (n = 14) and healthy controls (n = 15) using SWATH mass spectrometry analysis. The results were validated by means of quantitative targeted absolute proteomics analysis. As a result, we identified eight biomarker candidates for GBM (leucine-rich alpha-2-glycoprotein (LRG1), complement component C9 (C9), C-reactive protein (CRP), alpha-1-antichymotrypsin (SERPINA3), apolipoprotein B-100 (APOB), gelsolin (GSN), Ig alpha-1 chain C region (IGHA1), and apolipoprotein A-IV (APOA4)). Among them, LRG1, C9, CRP, GSN, IGHA1, and APOA4 gave values of the area under the receiver operating characteristics curve of greater than 0.80. To investigate the relationships between the biomarker candidates and GBM biology, we examined correlations between plasma concentrations of biomarker candidates and clinical presentation (tumor size, progression-free survival time, or overall survival time) in GBM patients. The plasma concentrations of LRG1, CRP, and C9 showed significant positive correlations with tumor size (R2 = 0.534, 0.495, and 0.452, respectively).

ATP-Binding Cassette Transporter A Subfamily 8 Is a Sinusoidal Efflux Transporter for Cholesterol and Taurocholate in Mouse and Human Liver.

The ATP-binding cassette (ABC) transporter A subfamily 8 (ABCA8) belongs to the ABCA6-like transporters subgroup, which is distinct from the ABCA1-like subgroup in the ABCA family. The expression and function of the short-size human ABCA8 lacking one of the two ATP-binding domains for ATP hydrolysis, which are regularly present in the other ABCA transporters, have been reported. However, the functional differences between the short-size human ABCA8 and full-size human ABCA8, which has the two ATP-binding domains, remain unknown. The purpose of the present study was to clarify the tissue expression profiles of ABCA6-like and ABCA1-like subgroup transporters and the functional characteristics of ABCA8 in mouse and human. The tissue distribution of mouse ABCA (mABCA) transporter protein and the changes in mABCA8 protein expression levels in a mouse model of obstructive cholestasis were elucidated by means of quantitative targeted absolute proteomics (QTAP). The transport characteristics were clarified in a HEK293 cell line overexpressing full-size ABCA8 protein. QTAP and immunohistochemical analyses revealed that mABCA transporters exhibited the distinct protein expression patterns in the tissues, and mABCA8b, its mouse orthologue, was abundant in the liver and predominantly distributed in sinusoidal membranes of the hepatocytes. Further, protein expression of mABCA8b was decreased in the mouse cholestasis liver. Changes of mABCA8b expression level in cholestasis were similar to those of mABCA1, a sinusoidal cholesterol efflux transporter. Uptake and efflux assays showed that ABCA8 mediates efflux of [3H]cholesterol and [3H]taurocholate, while it showed no significant efflux activity for [3H]estrone sulfate, [3H]digoxin, [3H]vinblastine, [3H]para-aminohippuric acid, [3H]oleic acid, [14C]nicotine, or [3H]methotrexate. [3H]Cholesterol efflux was increased by extracellularly applied taurocholate. These results suggest that mABCA8b/ABCA8 functions as a sinusoidal efflux transporter for at least cholesterol and taurocholate in mouse and human liver.

Quantification of Transporter and Receptor Proteins in Dog Brain Capillaries and Choroid Plexus: Relevance for the Distribution in Brain and CSF of Selected BCRP and P-gp Substrates.

Transporters at the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) play a pivotal role as gatekeepers for efflux or uptake of endogenous and exogenous molecules. The protein expression of a number of them has already been determined in the brains of rodents, nonhuman primates, and humans using quantitative targeted absolute proteomics (QTAP). The dog is an important animal model for drug discovery and development, especially for safety evaluations. The purpose of the present study was to clarify the relevance of the transporter protein expression for drug distribution in the dog brain and CSF. We used QTAP to examine the protein expression of 17 selected transporters and receptors at the dog BBB and BCSFB. For the first time, we directly linked the expression of two efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), to regional brain and CSF distribution using specific substrates. Two cocktails, each containing one P-gp substrate (quinidine or apafant) and one BCRP substrate (dantrolene or daidzein) were infused intravenously prior to collection of the brain. Transporter expression varied only slightly between the capillaries of different brain regions and did not result in region-specific distribution of the investigated substrates. There were, however, distinct differences between brain capillaries and choroid plexus. Largest differences were observed for BCRP and P-gp: both were highly expressed in brain capillaries, but no BCRP and only low amounts of P-gp were detected in the choroid plexus. Kp,uu,brain and Kp,uu,CSF of both P-gp substrates were indicative of drug efflux. Also, Kp,uu,brain for the BCRP substrates was low. In contrast, Kp,uu,CSF for both BCRP substrates was close to unity, resulting in Kp,uu,CSF/Kp,uu,brain ratios of 7 and 8, respectively. We conclude that the drug transporter expression profiles differ between the BBB and BCSFB in dogs, that there are species differences in the expression profiles, and that CSF is not a suitable surrogate for unbound brain concentrations of BCRP substrates in dogs.

LC-MS/MS Based Quantitation of ABC and SLC Transporter Proteins in Plasma Membranes of Cultured Primary Human Retinal Pigment Epithelium Cells and Immortalized ARPE19 Cell Line.

The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier between neural retina and choroid. The RPE has several important vision supporting functions, such as transport mechanisms that may also modify pharmacokinetics in the posterior eye segment. Expression of plasma membrane transporters in the RPE cells has not been quantitated. The aim of this study was to characterize and compare transporter protein expression in the ARPE19 cell line and hfRPE (human fetal RPE) cells by using quantitative targeted absolute proteomics (QTAP). Among 41 studied transporters, 16 proteins were expressed in hfRPE and 13 in ARPE19 cells. MRP1, MRP5, GLUT1, 4F2hc, TAUT, CAT1, LAT1, and MATE1 proteins were detected in both cell lines within 4-fold differences. MPR7, OAT2 and RFC1 were detected in the hfRPE cells, but their expression levels were below the limit of quantification in ARPE19 cells. PCFT was detected in both studied cell lines, but the expression was over 4-fold higher in hfRPE cells. MCT1, MCT4, MRP4, and Na+/K+ ATPase were upregulated in the ARPE19 cell line showing over 4-fold differences in the quantitative expression values. Expression levels of 25 transporters were below the limit of quantification in both cell models. In conclusion, we present the first systematic and quantitative study on transporter protein expression in the plasma membranes of ARPE19 and hfRPE cells. Overall, transporter expression in the ARPE19 and hfRPE cells correlated well and the absolute expression levels were similar, but not identical. The presented quantitative expression levels could be a useful basis for further studies on drug permeation in the outer blood-retinal barrier.

Global and Targeted Proteomics of Prostate Cancer Cell Secretome: Combination of 2-Dimensional Image-Converted Analysis of Liquid Chromatography and Mass Spectrometry and In Silico Selection Selected Reaction Monitoring Analysis.

Prostate-specific antigen is currently the only protein biomarker routinely used as a diagnostic tool for early detection and treatment monitoring of prostate cancer. However, it remains questionable whether prostate-specific antigen-based screening can sensitively and selectively identify the presence and progression status of primary and metastatic prostate cancers. Hence, the purpose of this study was to identify potential biomarker candidates in the secretome of primary and metastatic prostate cancer cells by using a combination of global and targeted proteomics. Quantitative comparisons among secretome proteins derived from androgen-responsive primary cancer cells (P-22Rv1), androgen-irresponsive bone metastatic cancer cells (M-PC-3), and noncancerous prostate cells (N-PNT2) were performed using 2-dimensional image-converted analysis of liquid chromatography and mass spectrometry followed by in silico selection selected reaction monitoring analysis. Mediator of RNA polymerase II transcription subunit 13-like, insulin-like growth factor-binding protein 2, and hepatocyte growth factor were identified as highly secreted proteins from P-22Rv1 cells compared with N-PNT2 cells. Prostate-associated microseminoprotein, proactivator polypeptide, collagen-α-1 (VI) chain, and neuropilin-1 were identified as predominantly secreted proteins in M-PC-3 cells compared with N-PNT2 cells. These proteins in biological fluids are considered to be candidate biomarkers of primary and/or metastatic prostate cancer.

Roles of calmodulin and calmodulin-binding proteins in synaptic vesicle recycling during regulated exocytosis at submicromolar Ca2+ concentrations.

Calcium ion is required at various concentrations for vesicular recycling in the presynaptic terminal. Although calmodulin (CaM) is the most abundant Ca2+-binding protein and has a submicromolar affinity for Ca2+, it is not the Ca2+ sensor for vesicular fusion because this process requires Ca2+ concentrations above 1 microM. Several lines of evidence, however, suggest that CaM mediates the regulation of vesicular recycling by submicromolar Ca2+ via novel protein-protein interactions. In this review, we discuss recent findings on how CaM regulates synaptic vesicle recycling by controlling the SNARE mechanism, which is the molecular machinery that mediates exocytosis.

Drebrin attenuates the interaction between actin and myosin-V.

Drebrin-A is an actin-binding protein localized in the dendritic spines of mature neurons, and has been suggested to affect spine morphology [K. Hayashi, T. Shirao, Change in the shape of dendritic spines caused by overexpression of drebrin in cultured cortical neurons, J. Neurosci. 19 (1999) 3918-3925]. However, no biochemical analysis of drebrin-A has yet been reported. In this study, we purified drebrin-A using a bacterial expression system, and characterized it in vitro. Drebrin-A bound to actin filaments with a stoichiometry of one drebrin molecule to 5-6 actin molecules. Furthermore, drebrin-A decreased the Mg-ATPase activity of myosin V. In vitro motility assay revealed that the attachment of F-actin to glass surface coated with myosin-V was decreased by drebrin-A, but once F-actin attached to the surface, the sliding speed of F-actin was unaffected by the presence of drebrin A. These findings suggest that drebrin-A may affect spine dynamics, vesicle transport, and other myosin-V-driven motility in neurons through attenuating the interaction between actin and myosin-V.

Myosin-Va regulates exocytosis through the submicromolar Ca2+-dependent binding of syntaxin-1A.

Myosin-Va is an actin-based processive motor that conveys intracellular cargoes. Synaptic vesicles are one of the most important cargoes for myosin-Va, but the role of mammalian myosin-Va in secretion is less clear than for its yeast homologue, Myo2p. In the current studies, we show that myosin-Va on synaptic vesicles interacts with syntaxin-1A, a t-SNARE involved in exocytosis, at or above 0.3 microM Ca2+. Interference with formation of the syntaxin-1A-myosin-Va complex reduces the exocytotic frequency in chromaffin cells. Surprisingly, the syntaxin-1A-binding site was not in the tail of myosin-Va but rather in the neck, a region that contains calmodulin-binding IQ-motifs. Furthermore, we found that syntaxin-1A binding by myosin-Va in the presence of Ca2+ depends on the release of calmodulin from the myosin-Va neck, allowing syntaxin-1A to occupy the vacant IQ-motif. Using an anti-myosin-Va neck antibody, which blocks this binding, we demonstrated that the step most important for the antibody's inhibitory activity is the late sustained phase, which is involved in supplying readily releasable vesicles. Our results demonstrate that the interaction between myosin-Va and syntaxin-1A is involved in exocytosis and suggest that the myosin-Va neck contributes not only to the large step size but also to the regulation of exocytosis by Ca2+.

Role of Ser50 phosphorylation in SCG10 regulation of microtubule depolymerization.

Members of the stathmin-like protein family depolymerize microtubules (MTs), probably due to the ability of each stathmin monomer to bind two tubulin heterodimers in a complex (T(2)S complex). SCG10, a member of this family, is localized in the growth cone of neurons. It has four identified sites of serine phosphorylation (S50, S63, S73, and S97). Of these, S50 and S97 are phosphorylated by cAMP-dependent protein kinase, an enzyme involved in growth cone guidance. When the equivalent sites in stathmins are phosphorylated, they lose their ability to depolymerize MTs. We investigated the specific role of the two cAMP-dependent protein kinase (PKA) phosphorylation sites in SCG10. A mutant of SCG10 phosphorylated only on S50 retained the ability to depolymerize MTs, but SCG10 phosphorylated on S97 or on both S50 and S97 lost MT-depolymerizing activity. Surface plasmon resonance studies revealed that the phosphorylation of SCG10 at these sites reduced the tubulin heterodimer binding, mainly due to a reduced rate of association. In particular, compared to the two other phosphorylated forms, SCG10 phosphorylated at S50 had a significantly smaller dissociation constant for the binding of the first tubulin heterodimer and larger association and dissociation rate constants for the binding of the second heterodimer. This indicates that the phosphorylation of S50 compensates for the effect of phosphorylation at other sites by modulating T2S complex formation. Furthermore, these results suggest that S50-P maintains MT-depolymerizing activity, which indicates that the biological functions of phosphorylation at S50 and S97 are different.

NY-ESO-1 expression and its serum immunoreactivity in esophageal cancer.

PURPOSE: NY-ESO-1, a member of the cancer/testis antigen (CTA) family, elicits humoral and cellular immune responses in patients with advanced cancer. Unresectable or metastatic esophageal carcinoma patients do not benefit from the present multimodality treatment regimens in terms of survival. The objectives of this study were to analyze the antibody response to NY-ESO-1 antigen in patients with esophageal cancer and to determine the potential of NY-ESO-1 for use in tumor-specific immunotherapy. METHODS: Serum from 69 patients with esophageal cancer was investigated for antibody production against NY-ESO-1 by Western blot analysis. Also analyzed by immunohistochemistry were 56 tissue samples from these patients for NY-ESO-1 protein expression. RESULTS: NY-ESO-1 protein expression was found in 18 of 56 (32%) esophageal carcinomas. Serum immunoreactivity specific for NY-ESO-1 was found in 9 patients (13%) of whom 8 were in the advanced stage (stages III and IV). There was no relationship between clinicopathologic features and serum immunoreactivity for NY-ESO-1. NY-ESO-1 protein expression was detected in three of five antibody-positive patients whose tissue was available for analysis. Survival analysis showed no significant difference between antibody-positive and antibody-negative patient groups. CONCLUSIONS: A humoral immune response to NY-ESO-1 antigen was established in patients with advanced esophageal cancer. NY-ESO-1 is a good candidate for vaccine-based immunotherapy for advanced esophageal carcinoma.