11 research outputs found
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
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<sup>+</sup>/K<sup>+</sup> 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
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
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. <i>K</i><sub>p,uu,brain</sub> and <i>K</i><sub>p,uu,CSF</sub> of both P-gp substrates were indicative
of drug efflux. Also, <i>K</i><sub>p,uu,brain</sub> for
the BCRP substrates was low. In contrast, <i>K</i><sub>p,uu,CSF</sub> for both BCRP substrates was close to unity, resulting in <i>K</i><sub>p,uu,CSF</sub>/<i>K</i><sub>p,uu,brain</sub> 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
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
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
Box plot showing the plasma levels of each differentially expressed protein in GBM plasma (n = 14) compared with healthy plasma (n = 15).
<p>Each dot represents the protein level of an individual sample. In box plots, the band inside the box represents the median. The bottom and top of the box represent the first and third quartiles. The whiskers reflect the minimum and maximum values that fall within 1.5 times the interquartile range. Any data not included between the whiskers is an outlier. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Cont, Healthy controls; GBM, glioblastoma.</p
Total number of proteins and peptides in spectral library.
<p>Total number of proteins and peptides in spectral library.</p