26 research outputs found
Quantitative Targeted Absolute Proteomics of Transporters and Pharmacoproteomics-Based Reconstruction of P‑Glycoprotein Function in Mouse Small Intestine
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
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 polyÂ(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
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
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
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]Âcholesterol and [<sup>3</sup>H]Âtaurocholate, while it showed no
significant efflux activity for [<sup>3</sup>H]Âestrone sulfate, [<sup>3</sup>H]Âdigoxin, [<sup>3</sup>H]Âvinblastine, [<sup>3</sup>H]<i>para</i>-aminohippuric acid, [<sup>3</sup>H]Âoleic acid, [<sup>14</sup>C]Ânicotine, or [<sup>3</sup>H]Âmethotrexate. [<sup>3</sup>H]ÂCholesterol 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
<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
Summary of the differentially expressed proteins in plasma validated by QTAP analysis.
<p>Summary of the differentially expressed proteins in plasma validated by QTAP analysis.</p
Identification of blood biomarkers in glioblastoma by SWATH mass spectrometry and quantitative targeted absolute proteomics - Fig 3
<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