|Institution:||University of New South Wales|
|Department:||Biotechnology & Biomolecular Sciences|
|Keywords:||Cholesterol; DHCR24; Desmosterol; Cholesterol homeostasis; Bloch pathway; Oxysterol; Progesterone; SREBP-2|
|Full text PDF:||http://handle.unsw.edu.au/1959.4/53433|
Cholesterol is necessary for mammalian life, as an essential component in cell membranes, foetal development, and a precursor for steroid hormones. Hence, cholesterol levels must be tightly regulated. Previous research has focused on an early step in cholesterol synthesis: 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), target of the cholesterol-lowering statin drugs. However, less is known about other steps in the pathway. We investigated 3β-hydroxysterol Δ24-reductase (DHCR24); involved in the last step of cholesterol synthesis, and implicated in inflammation, oxidative stress and hepatitis C virus infection. There is a paucity of fundamental information on the structure of DHCR24 and how it interacts with cellular membranes, as well as how this critical enzyme is regulated. We found that DHCR24 is an integral endoplasmic reticulum (ER) membrane protein, with multiple atypical membrane associated regions. We present biochemical evidence that the majority of the enzyme is associated with the ER membrane, contrary to published membrane topology models and in silico predictions. This has important consequences for the many functions attributed to DHCR24. In particular, those that suggest DHCR24 alters its localisation within the cell should be reassessed in light of this new information. We show that DHCR24 is regulated at multiple levels, with potent effects on cholesterol synthesis. Our findings demonstrate feedback regulation at the transcriptional and post‑translational level. Transcriptional regulation occurs through sterol regulatory element binding protein 2 (SREBP-2), mediated by dual sterol regulatory elements (SREs) within the DHCR24 promoter, which work cooperatively to regulate expression. DHCR24 activity is potently inhibited at the post‑translational level by endogenous side-chain oxysterols, in particular 24(S),25‑epoxycholesterol (24,25EC), which was independent of DHCR24 protein levels. This observation was extended to another structurally similar byproduct of the cholesterol synthesis pathway, progesterone. Another mode of regulation is through signalling. We found that phosphorylation affects DHCR24 activity, at a known phosphorylation site, T110. We found that protein kinase C (PKC) also ablated DHCR24 activity, through an unknown phosphorylation site. These findings provide fundamental new insights into DHCR24 and its regulation, indicating it may be an important regulatory step in cholesterol synthesis and maintaining cellular cholesterol homeostasis.