Post-translational regulation of organic anion transporting polypeptide 1B3 transport function by lysine acetylation and lysine deacetylase 6 inhibition.

Organic anion transporting polypeptide (OATP) 1B3 plays a clinically significant role in hepatic drug disposition. Lysine acetylation, a key post-translational modification, has not been investigated for OATP1B3. This study determined the lysine acetylation status of OATP1B3 by proteomics and assessed the impact of inhibition of lysine deacetylase (KDAC) 6, a major cytosolic KDAC, on OATP1B3 acetylation and transport function. Proteomics revealed 7 acetylation sites, including 5 with additional ubiquitin-like modifications, and 4 phosphorylation sites (T10, S293, S295, S683). In human embryonic kidney 293 (HEK293)-Myc-FLAG-OATP1B3 cells, preincubation with the selective KDAC6 inhibitor tubacin (TBC) (5 μM, 24 hours), markedly reduced OATP1B3-mediated transport of [3H]cholecystokinin-8 (CCK-8), a specific substrate, and [3H]estradiol-17β-D-glucuronide to 0.15 ± 0.03-fold and 0.19 ± 0.01-fold of the control, respectively, without affecting OATP1B3 mRNA, protein levels, or membrane localization determined by real-time reverse transcription polymerase chain reaction, immunoblotting, and confocal microscopy. TBC treatment increased K664 acetylation to 2.12 ± 1.03-fold of the control (P < .05). Consistently, the acetylation-mimetic K664Q variant exhibited reduced transport compared with the acetylation-null K664R variant (P < .05). Treatment with a second KDAC6 selective inhibitor, WT-161 (3 μM, 5 hours), similarly reduced OATP1B3-mediated [3H]CCK-8 transport. In cultured primary human hepatocytes, TBC treatment for 4, 8, and 24 hours decreased [3H]CCK-8 transport to 0.34 ± 0.02-fold, 0.27 ± 0.03-fold, and 0.37 ± 0.03-fold of the control, respectively (all P < .05). The study reveals a novel post-translational modification of OATP1B3 by lysine acetylation and demonstrates impaired transporter function following KDAC6 inhibition, likely involving increased acetylation at K664, thereby providing new insight into OATP1B3-mediated drug-drug interactions driven by KDAC6 activity. SIGNIFICANCE STATEMENT: This study identifies lysine acetylation as a novel post-translational modification of organic anion transporting polypeptide (OATP)1B3 and demonstrates that altered lysine acetylation following inhibition of lysine deacetylase 6 reduces OATP1B3 transport function. These findings provide a mechanistic basis for altered hepatic drug disposition and highlight a new pathway through which drug-drug interactions involving OATP1B3 may occur.

Duke Scholars

Author Erik James Soderblom Cell Biology