THE INFLUENCE OF S-ACYLATION ON OCT2 FUNCTION AND EXPRESSION

S-acylation of proteins refers to the attachment of fatty acids to the thiol group of cysteine via thioester bonds. S-acylation participates in key biological processes such as cell proliferation, apoptosis, and immune responses by regulating protein membrane localization, stability, and interaction networks. The organic cation transporter 2 (OCT2) is a membrane transporter mediating the renal elimination of several endobiotics, xenobiotics, and drugs. Based on our previous proteomic mass spectrometer (MS) analysis of mouse kidneys, we observed that mOct2 also exists in S-acylated form. The aim of this study was to determine the effect of S-acylation on OCT2 protein localization and function.

Detection of S-acylated OCT2 was assessed in membrane fractions from hOCT2-HEK293 cells isolated by differential centrifugation, subjected to resin-assisted capture (acyl-RAC) technique to pull down S-acylated proteins. OCT2 was detected by MS or western blotting (WB). Cysteine identified by MS as potential S-acylation sites were mutated to serine by site-directed mutagenesis. 2-Bromopalmitate (2-BP) was used as a pan-inhibitor of S-acylation reaction. OCT2 surface expression and transport activity were assessed by cell surface labeling coupled to WB and radiolabeled substrate transport assay, respectively. The effect of a high-fat diet (HFD) on mOct2 S-acylation was also assessed using acyl-RAC coupled to MS. The effects of palmitic acid preincubation on the transport functions of hOCT2 and mOct2 were assessed in vitro.

Approximately 10% of OCT2 was found S-acylated in hOCT2-HEK293. An 18 h-exposure to 2-BP at the extracellular concentration of 100 μM dramatically decreased the S-acylation of hOCT2 in hOCT2-HEK293. 2-BP also significantly reduced the uptake of MPP+ by hOCT2 and mOct2 with 50 μM MPP+, an effect that was dose-dependent. The biotin surface labeling experiment results indicated that hOCT2 expression did not appear to be influenced by 2-BP. Notably, the acyl-RAC and WB results indicate that 2-BP reduces the S-acylation of hOCT2 in vitro. In the WB analysis of total OCT2, two distinct bands appeared, with apparent molecular masses of approximately 80 kDa and 55 kDa. Notably, treatment with 2-BP resulted in a decrease in the intensity of the 80-kDa band and an increase in the 55-kDa band. MS analysis found C122 as a potential site of S-acylation. The C122S mutant was significantly less S-acylated and seemed to be less glycosylated compared to the wild type; these changes may reflect modifications in glycosylation status influenced by S-acylation. Although C122S can be expressed on the cell membrane, but no transport function. Interestingly, in the WB analysis of C122S-OCT2, only the 55-kDa appeared. A 19-week HFD obviously decreased the S-acylated proteins (mOct2 and Flotillilin-1) abundance in mice. However, palmitic acid preincubation showed no effect on hOCT2 and mOct2 transport function in vitro.

OCT2 is S-acylated in human and rodent. The S-acylation of OCT2 is crucial for its transport function but does not appear to influence its synthesis or surface expression in the cell. One site of S-acylation is cysteine in position 122. S-acylation and N-glycosylation crosstalk might contribute to control OCT2 localization and function in response to acute/chronic stress stimuli.