26 research outputs found

    Quantitative Targeted Absolute Proteomics of Transporters and Pharmacoproteomics-Based Reconstruction of P‑Glycoprotein Function in Mouse Small Intestine

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    The purpose of this study was to investigate whether a pharmacokinetic model integrating in vitro mdr1a efflux activity (which we previously reported) with in vitro/in vivo differences in protein expression level can reconstruct intestinal mdr1a function. In situ intestinal permeability–surface area product ratio between wild-type and mdr1<i>a</i>/1b (−/−) mice is one of the parameters used to describe intestinal mdr1a function. The reconstructed ratios of six mdr1a substrates (dexamethasone, digoxin, loperamide, quinidine, verapamil, vinblastine) and one nonsubstrate (diazepam) were consistent with the observed values reported by Adachi et al. within 2.1-fold difference. Thus, intestinal mdr1a function can be reconstructed by our pharmacoproteomic modeling approach. Furthermore, we evaluated regional differences in protein expression levels of mouse intestinal transporters. Sixteen (mdr1a, mrp4, bcrp, abcg5, abcg8, glut1, 4f2hc, sglt1, lat2, pept1, mct1, slc22a18, ostβ, villin1, Na<sup>+</sup>/K<sup>+</sup>-ATPase, γ-gtp) out of 46 target molecules were detected by employing our established quantitative targeted absolute proteomics technique. The protein expression amounts of mdr1a and bcrp increased progressively from duodenum to ileum. Sglt1, lat2, and 4f2hc were highly expressed in jejunum and ileum. Mct1 and ostβ were highly expressed in ileum. The quantitative expression profiles established here should be helpful to understand and predict intestinal transporter functions

    Combining Genomics To Identify the Pathways of Post-Transcriptional Nongenotoxic Signaling and Energy Homeostasis in Livers of Rats Treated with the Pregnane X Receptor Agonist, Pregnenolone Carbonitrile

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    Transcriptomic, proteomic, phosphoproteomic, and metabolomic analyses were combined to determine the role of pregnane X receptor (PXR) in nongenotoxic signaling and energy homeostasis in liver after rats were repeatedly orally dosed with the PXR agonist pregnenolone carbonitrile (PCN) for 7 days. Analyses of mRNAs and proteins in the supernatant, membrane, and cytosolic fractions of enlarged liver homogenates showed diverse expression profiles. Gene set enrichment analysis showed that the synchronous increase in mRNAs and proteins involved in chemical carcinogenesis and the response to drug was possibly mediated by the PXR pathway and proteasome core complex assembly was possibly mediated by the Nrf2 pathway. In addition, levels of proteins in the endoplasmic reticulum lumen and involved in the acute-phase response showed specific increase with no change in mRNA level, and those composed of the mitochondrial inner membrane showed specific decrease. The analysis of phosphorylated peptides of poly­(A) RNA binding proteins showed a decrease in phosphorylation, possibly by casein kinase 2, which may be related to the regulation of protein expression. Proteins involved in insulin signaling pathways showed an increase in phosphorylation, possibly by protein kinase A, and those involved in apoptosis showed a decrease. Metabolomic analysis suggested the activation of the pentose phosphate and anaerobic glycolysis pathways and the increase of amino acid and fatty acid levels, as occurs in the Warburg effect. In conclusion, the results of combined analyses suggest that PXR’s effects are due to transcriptional and post-transcriptional regulation with alteration of nongenotoxic signaling pathways and energy homeostasis

    High Expression of UGT1A1/1A6 in Monkey Small Intestine: Comparison of Protein Expression Levels of Cytochromes P450, UDP-Glucuronosyltransferases, and Transporters in Small Intestine of Cynomolgus Monkey and Human

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    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

    Quantitative Targeted Absolute Proteomics-Based Large-Scale Quantification of Proline-Hydroxylated α‑Fibrinogen in Plasma for Pancreatic Cancer Diagnosis

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    Pancreatic cancer is a devastating disease and early diagnosis and treatment are essential to improve the prognosis. We previously showed that α-fibrinogen containing hydroxylated proline residues at positions 530 and 565 is increased in plasma of pancreatic cancer patients. However, no antibody specific for hydroxylated proline-530 is available. Therefore, the purposes of this study were to develop a quantification method specific for both proline-hydroxylated α-fibrinogens by selected/multiple reaction monitoring (SRM/MRM), and to validate these modifications as pancreatic cancer markers. The target peptide for hydroxylated proline-530 contained methionine, and since variable partial oxidation of this residue would affect the quantification, hydrogen peroxide treatment was carried out to ensure complete oxidation. Quantification values of modified and unmodified α-fibrinogen were well correlated with those obtained by immunoblotting. Concentrations of modified and unmodified α-fibrinogen were quantified in 70 pancreatic cancer patients and 27 healthy controls. Percent hydroxylation of α-fibrinogen and concentration of hydroxylated α-fibrinogen were significantly greater in the plasma of patients. Furthermore, among 8 carbohydrate antigen 19-9 (CA19-9)-negative patients in stages I/II, 6 were positive for proline-hydroxylated α-fibrinogen. These results indicate that plasma concentration of proline-hydroxylated α-fibrinogen measured by SRM/MRM analysis may be a good pancreatic cancer marker, especially in CA19-9-negative patients

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

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    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 [<sup>3</sup>H]­cholesterol and [<sup>3</sup>H]­taurocholate, while it showed no significant efflux activity for [<sup>3</sup>H]­estrone sulfate, [<sup>3</sup>H]­digoxin, [<sup>3</sup>H]­vinblastine, [<sup>3</sup>H]<i>para</i>-aminohippuric acid, [<sup>3</sup>H]­oleic acid, [<sup>14</sup>C]­nicotine, or [<sup>3</sup>H]­methotrexate. [<sup>3</sup>H]­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

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

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    <div><p>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 (R<sup>2</sup> = 0.534, 0.495, and 0.452, respectively).</p></div

    Identification of blood biomarkers in glioblastoma by SWATH mass spectrometry and quantitative targeted absolute proteomics - Fig 3

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    <p><b>Kaplan–Meier curve of progression-free survival time (PFS) (A) and overall survival time (OS) (B) in patients with glioblastoma (GBM) showed prognostic significance of gelsolin (GSN).</b> GBM patients were classified into two categories on the basis of GSN level: low (0–472 fmol/μL plasma) and high (> 472 fmol/μL plasma). Mean GSN plasma level in GBM patients was selected as the cut-off point. (A) PFS interval was determined as the interval between the date of initial operation and the date of patient’s recurrence or determined endpoint (for those no recurrent on August 1, 2015). (B) OS interval was determined as the interval between the date of the initial operation and date of patient’s death or determined end point (for those alive on August 1, 2015).</p
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