Regulation of OATP1B3 Transport Function via Protein Acetylation
Creators: name: Powell, John
name: Kayesh, Ruhul
name: Xu, Chao
name: Soderblom, Erik
name: Waitt, Greg
name: Ho, Tricia
name: Yue, Wei
Publisher: name: Zenodo
Year: 2023
Subjects: subject: Liver Diseases
subject: Drug Interactions
subject: drug transporter

description: *This is a poster (T1230-05-32) for PharmSci 360-AAPS Annual Meeting Orlando, 2023, Oct. 22-25. This work is not peer-reviewed. Authors have no competing interests.

Purpose: The organic anion transporting polypeptide (OATP) 1B3 is a liver-specific membrane transport protein that transports many drugs and endogenous compounds. Altered transport function of OATP1B3 has significant clinical relevance in causing drug-drug interactions (DDIs). Limited information exists regarding the mechanisms regulating transport function of OATP1B3. The post-translational modification (PTM) of lysine acetylation – a common and crucial mechanism through which function of many proteins are regulated – has not been investigated for OATP1B3. The purpose of the current study is to determine the lysine-acetylation status of OATP1B3 and to determine the effects of altered lysine acetylation on OATP1B3-mediated transport.

Methods: FLAG-tagged OATP1B1 proteins were purified by FLAG immunoprecipitation from OATP1B3-overexpressing human embryonic kidney (HEK) 293 stable cell line HEK293-FLAG-OATP1B3, isolated from an SDS-PAGE gel using standardized in-gel trypsin digestion and subjected to qualitative or quantitative PTM characterization by nanoscale liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) on a Fusion Lumos Orbitrap. The effects of treatment with tubacin (TBC) (5 µM, 24 h), a specific lysine deacetylase 6 (KDAC6) inhibitor, on OATP1B3-mediated transport and on OATP1B3 protein levels were determined in HEK293-FLAG-OATP1B3 cells using [3H]-estradiol 17 β-D-glucuronide (E217βG) (1 µM, 2 min) as the substrate and by immunoblot, respectively. Effects of TBC treatment (5 µM, 24 h) on the PTM status of OATP1B3 was determined by quantitative PTM characterization, where relative peptide abundance was compared between TBC and vehicle control treatments based on area-under-the-curve measurements. Mutagenesis was conducted with substitution of arginine (R) for lysine (K) on a mammalian expression vector encoding myc-tagged wild-type (WT)-OATP1B3 to mimic acetylation-null OATP1B3 at this site. Transport function of the variant- and WT-OATP1B3 was compared after transient transfection in HEK293 cells using [3H]CCK-8 (1 µM, 3 min) as the substrate. Plasma membrane localization of variant-and WT-OATP1B3 was determined by immunofluorescent staining followed by confocal microscopy.

Results: TBC treatment significantly decreased OATP1B3-mediated transport to 0.20 ± 0.008 fold of CTL (N=3 in triplicate, p<0.05 by linear mixed effect model). TBC treatment did not significantly affect protein levels of OATP1B3. Proteomics studies showed that OATP1B3 is a lysine-acetylated protein. Treatment with TBC significantly increased OATP1B3 acetylation at K664 to 2.1 ± 1.0-fold of vehicle control treatment (p<0.05 by two-tailed heteroscedastic t-test in quadruplicate). OATP1B3-mediated transport by K664R-OATP1B3 was significantly increased by 1.42 ± 0.10 fold of WT-OATP1B3 control (p<0.01 by linear mixed effect model, N=3 in triplicate). Confocal microscopy showed primary plasma membrane localization of both K664R- and WT-OATP1B3.

Conclusion: We report the novel findings that OATP1B3 is post-translationally modified via lysine-acetylation and that transport function of OATP1B3 is regulated by the acetylation status of residue K664. Inhibition of KDAC6 activity significantly reduces OATP1B3 transport function presumably via increased lysine acetylation at K664.  The current study elucidates a novel regulatory mechanism of OATP1B3 function through altered lysine acetylation and KDAC6 inhibition.

 Supported by NIH R01 GM146956-01 (WY)