Signaling regulates activity of DHCR24, the final enzyme in cholesterol synthesis

Abstract

The role of signaling in regulating cholesterol homeostasis is gradually becoming more widely recognized. Here, we explored how kinases and phosphorylation sites regulate the activity of the enzyme involved in the final step of cholesterol synthesis (3β‐hydroxysterol Δ24‐reductase; DHCR24). Many factors are known to regulate DHCR24 transcriptionally, but little is known about its post‐translational regulation. We developed a system to specifically test human ectopic DHCR24 activity in a model cell‐line (Chinese hamster ovary‐7) using siRNA targeted only to hamster DHCR24, thus ensuring that all activity could be attributed to the human enzyme. We determined the effect of known phosphorylation sites and found that mutating certain residues (T110, Y299, and Y507) inhibited DHCR24 activity. In addition, inhibitors of protein kinase C ablated DHCR24 activity, although not through a known phosphorylation site. Our data indicate a novel mechanism whereby DHCR24 activity is regulated by signaling. Moreover, we propose that post‐translational modifications such as phosphorylation are a valuable resource for mapping the topology of membrane‐associated proteins such as DHCR24. The Brown lab is funded by grants from the National Health and Medical Research Council.

The terminal enzymes of cholesterol synthesis, DHCR24 and DHCR7, interact physically and functionally

“…Lipids were derivatized in BSTFA at 60°C for 1 h and then analyzed using a Thermo Trace gas chromatograph coupled with a Thermo DSQIII mass spectrometer and Thermo Triplus autosampler (Thermo Fisher Scientifi c), modifi ed from a previous protocol ( 22 ). Samples (1.5 l) were injected via a heated (300°C) splitless inlet into a Trace TR-50MS GC column (60 m × 0.25 mm, 0.25 m fi lm thickness) (Thermo Fisher Scientifi c).…”

“…After 24 h, cells were refreshed with medium overnight before treating/labeling/harvesting. Hamster-specifi c siRNA targeting DHCR24 [target sequence: GAGAGCCACGTGTGAAGCA; hamster versus human mRNA knockdown characterized previously ( 22 )] and DHCR7 (target sequence: GAAGCCAGGAGTAAAG-AAC) were designed by Sigma-Aldrich.…”

Cholesterol-mediated Degradation of 7-Dehydrocholesterol Reductase Switches the Balance from Cholesterol to Vitamin D Synthesis

“…In contrast, DHCR24 protein appears relatively stable (17). Because DHCR7 and DHCR24 are regulated similarly at the transcriptional level via sterol-regulatory element-binding protein (SREBP) (18), it is possible that these two alternative terminal enzymes differ at the post-translational level, providing differential regulation of the two pathways.…”

“…Lysates were processed in ethanol, with 10 M butylated hydroxytoluene and 200 M EDTA. Lipids were extracted, derivatized, and analyzed in selective ion monitoring mode using a Thermo Trace gas chromatograph coupled with a Thermo DSQII mass spectrometer and Thermo Triplus autosampler (Thermo Fisher Scientific), and data were processed with Thermo Xcalibur Software (Version 2.1.0.1140) as described previously (17). Sterols monitored are detailed in Data Presentation and Statistical Analysis-Data are presented as the mean Ϯ S.E.…”

Navigating the Shallows and Rapids of Cholesterol Synthesis Downstream of HMGCR

“…The oxysterol, 24(S),25-epoxycholesterol (24,25EC), produced in a shunt of the cholesterol biosynthesis pathway, also inhibits DHCR24 enzyme activity without affecting its protein expression (21). Recently, signalling via phosphorylation was implicated in the post-translational regulation of DHCR24 (22). Mutating DHCR24 phosphorylation sites (T110, Y299, and Y507) resulted in decreased DHCR24 activity.…”