Novel Role for Hedgehog Acyltransferase in the Uptake of Palmitoyl‐CoA into the Endoplasmic Reticulum

Abstract

Post‐translational attachment of fatty acids to proteins plays a fundamental role in signaling pathways in normal and cancer cells. Most fatty acylated proteins are localized within cells, but a subset are secreted, including Hedgehog and Wnt proteins. Hedgehog proteins, of which Sonic Hedgehog (Shh) is the best studied, are modified by attachment of the 16‐carbon fatty acid palmitate during passage through the secretory pathway. Palmitoylation of Shh is mediated by Hedgehog Acyltransferase (Hhat) and is essential for short and long range Shh signaling in development and tumorigenesis. Hhat is localized to the endoplasmic reticulum (ER) and is a member of the membrane bound‐O‐acyltransferase (MBOAT) family of enzymes. The palmitoylation reaction occurs in the lumen of the ER, and thus palmitoyl‐CoA must be present in the ER lumen for Hhat to serve as a substrate. However, how palmitoyl‐CoA, which is not permeable across membranes, enters the ER lumen remains unknown. In mitochondria, a carnitine palmitoyltransferase (CPT1/2) system shuttles palmitoyl‐CoA across both the inner and outer mitochondrial membranes. We were interested in determining if a similar system exists in the ER or if the CPT1/2 system is shared at ER‐mitochondrial contact sites. Using cell‐based radiolabeling experiments, we showed that Etomoxir, a CPT1 inhibitor, strongly reduced palmitoylation of Shh but had no effect on palmitoylation of the Src family kinase Fyn, which is palmitoylated by cytoplasmic palmitoyl‐CoA. Etomoxir treatment had no effect on the ability of purified Hhat to palmitoylate Shh in vitro. We then developed in vitro assays to monitor palmitoyl‐CoA uptake into the lumen of microsomal membranes and to determine if Hhat plays a role in promoting this process. Using either fluorescent (NBD) or radiolabeled (125I) analogs of palmitoyl‐CoA, we observed a 3‐fold increase in uptake of palmitoyl‐CoA into the lumen of microsomal membranes prepared from cells overexpressing Hhat compared to membranes from cells transfected with empty vector. Increased palmitoyl‐CoA uptake was dependent on Hhat catalytic activity, as it was inhibited by a small molecule Hhat inhibitor and was not observed with H379A, an inactive Hhat mutant. Palmitoyl‐CoA uptake was also reduced after treatment of membranes with Etomoxir, and addition of the CPT1 substrate carnitine counteracted this inhibition. Microsomal membranes from Hhat −/− cells exhibited 2‐fold lower palmitoyl‐CoA uptake, compared to membranes from wild type cells, which supports the hypothesis that endogenous Hhat enhances uptake of this fatty acyl CoA. Taken together, this data suggests that a CPT1/2‐like system exists in the ER, or as part of an ER‐mitochondrial contact site. This system, along with Hhat, promotes uptake of palmitoyl CoA into the ER lumen to enable Hhat to catalyze fatty acylation of Shh. Understanding the mechanism by which fatty acids enter the ER lumen and are attached to proteins involved in cell signaling pathways can potentially reveal novel therapeutic targets in cancers characterized by aberrant signaling pathways.‐‐‐‐‐Support or Funding InformationNIH GM116860This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.