2-hydroxyoleic acid

Abstract

2-hydroxyoleic acid (OHOA, Minerval®) is an example of a substance used for membrane lipid therapy, where the cellular membranes rather than specific proteins constitute the therapeutical target. OHOA is thought to mediate its anti-tumor effect by affecting the biophysical properties of membranes, which leads to altered recruitment and activation of amphitropic proteins, altered cellular signaling, and eventual cell death. Little is known about the initial signaling events upon treatment with OHOA, and whether the altered membrane properties would have any impact on the dynamic intracellular transport system. In the present study we demonstrate that treatment with OHOA led to a rapid release of intracellular calcium and activation of multiple signaling pathways in HeLa cells, including the PI3K-AKT1-MTOR pathway and several MAP kinases, in a process independent of the EGFR. By lipidomics we confirmed that OHOA was incorporated into several lipid classes. Concomitantly, OHOA potently increased retrograde transport of the plant toxin ricin from endosomes to the Golgi and further to the endoplasmic reticulum. The OHOA-stimulated ricin transport seemed to require several amphitropic proteins, including Src, phospholipase C, protein kinase C, and also Ca2+/calmodulin. Interestingly, OHOA induced a slight increase in endosomal localization of the retromer component VPS35. Thus, our data show that addition of a lipid known to alter membrane properties not only affects signaling, but also intracellular transport.

OHOA stimulates transport of ricin to the Golgi and the ER

To verify that sorting into the retrograde pathway was stimulated by OHOA, we specifically measured the arrival of ricin to the TGN by the sulfation assay. This assay takes advantage of the trans-Golgi-specific sulfation process, in which tyrosyl protein sulfotransferases couple sulfate groups to proteins displaying a sulfation site []. By addition of recombinant ricin molecules containing a sulfation site, the arrival of toxin molecules to the Golgi can be detected. Indeed, treatment with increasing concentrations of OHOA potentiated the ricin sulfation (Figure ). The total amount of ricin associated with the cells was unchanged. To address the specificity of the stimulatory effect of OHOA, unmodified OA was used in parallel. In agreement with the toxicity data, OA had a very limited effect on ricin sulfation (Figure ).

OHOA stimulates transport of ricin to the Golgi and the ER

Along the same lines, and as previously shown for cholera toxin [], we established a SNAP-tag assay for measuring the arrival of cargo molecules to the ER. The SNAP-tag enzyme was maintained selectively in the ER lumen by fusion to an ER-signal peptide and the KDEL-retrieval sequence (). In line with the data for Golgi-transport, ricin transport to the ER was stimulated by OHOA in a concentration-dependent manner (Figure ).