TY - JOUR
T1 - Proteomics-Based Transporter Identification by the PICK Method
T2 - Involvement of TM7SF3 and LHFPL6 in Proton-Coupled Organic Cation Antiport at the Blood–Brain Barrier
AU - Kurosawa, Toshiki
AU - Tega, Yuma
AU - Uchida, Yasuo
AU - Higuchi, Kei
AU - Tabata, Hidetsugu
AU - Sumiyoshi, Takaaki
AU - Kubo, Yoshiyuki
AU - Terasaki, Tetsuya
AU - Deguchi, Yoshiharu
N1 - Publisher Copyright:
© 2022 by the authors.
PY - 2022/8
Y1 - 2022/8
N2 - A proton-coupled organic cation (H+/OC) antiporter working at the blood–brain barrier (BBB) in humans and rodents is thought to be a promising candidate for the efficient delivery of cationic drugs to the brain. Therefore, it is important to identify the molecular entity that exhibits this activity. Here, for this purpose, we established the Proteomics-based Identification of transporter by Crosslinking substrate in Keyhole (PICK) method, which combines photo-affinity labeling with comprehensive proteomics analysis using SWATH-MS. Using preselected criteria, the PICK method generated sixteen candidate proteins. From these, knockdown screening in hCMEC/D3 cells, an in vitro BBB model, identified two proteins, TM7SF3 and LHFPL6, as candidates for the H+/OC antiporter. We synthesized a novel H+/OC antiporter substrate for functional analysis of TM7SF3 and LHFPL6 in hCMEC/D3 cells and HEK293 cells. The results suggested that both TM7SF3 and LHFPL6 are components of the H+/OC antiporter.
AB - A proton-coupled organic cation (H+/OC) antiporter working at the blood–brain barrier (BBB) in humans and rodents is thought to be a promising candidate for the efficient delivery of cationic drugs to the brain. Therefore, it is important to identify the molecular entity that exhibits this activity. Here, for this purpose, we established the Proteomics-based Identification of transporter by Crosslinking substrate in Keyhole (PICK) method, which combines photo-affinity labeling with comprehensive proteomics analysis using SWATH-MS. Using preselected criteria, the PICK method generated sixteen candidate proteins. From these, knockdown screening in hCMEC/D3 cells, an in vitro BBB model, identified two proteins, TM7SF3 and LHFPL6, as candidates for the H+/OC antiporter. We synthesized a novel H+/OC antiporter substrate for functional analysis of TM7SF3 and LHFPL6 in hCMEC/D3 cells and HEK293 cells. The results suggested that both TM7SF3 and LHFPL6 are components of the H+/OC antiporter.
KW - SWATH-MS (sequential window acquisition of all theoretical-mass spectra)
KW - blood–brain barrier
KW - photo-affinity labeling
KW - proteomics
KW - proton-coupled organic cation antiporter
UR - http://www.scopus.com/inward/record.url?scp=85137380922&partnerID=8YFLogxK
U2 - 10.3390/pharmaceutics14081683
DO - 10.3390/pharmaceutics14081683
M3 - 学術論文
C2 - 36015309
AN - SCOPUS:85137380922
SN - 1999-4923
VL - 14
JO - Pharmaceutics
JF - Pharmaceutics
IS - 8
M1 - 1683
ER -