The regulation and membrane topology of DHCR24, a key enzyme in cholesterol synthesis by Eser J. Zerenturk A thesis submitted in fulfilment of the requirements for the Degree of Doctorate of Philosophy (Biochemistry and Molecular Genetics) School of Biotechnology and Biomolecular Sciences The University of New South Wales Submitted: December 2013 Revised: April 2014 THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Zerenturk First name: Eser Other name/s: Jane Abbreviation for degree as given in the University calendar: PhD School: Biotechnology and Biomolecular Sciences Faculty: Science Title: The regulation and membrane topology of DHCR24, a key enzyme in cholesterol synthesis Abstract 350 words maximum: 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. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). 16/05/2014 …………………………………………………………… ……………………………………..……………… ……….……………………...…….… Signature Witness Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: ORIGINALITY STATEMENT ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed …………………………………………… Date …………………………………………… i COPYRIGHT STATEMENT ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signed …………………………………………… Date …………………………………………… AUTHENTICITY STATEMENT ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed …………………………………………… Date …………………………………………… ii ABSTRACT Cholesterol is necessary for mammalian life, as an essential component in cell membranes, fetal 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 infection infection. There is a paucity of fundamental information on the structure 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. Prediction programs and previous studies have shown conflicting results regarding which regions of DHCR24 are associated with the membrane, although there was general agreement that this was limited to only the N-terminal portion. Here, we present biochemical evidence that in fact the majority of the enzyme is associated with the ER membrane. 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 dual sterol regulatory elements (SREs) within the DHCR24 promoter, which work cooperatively to regulate expression. DHCR24 is potently inhibited at the post-translational level by endogenous side-chain oxysterols, in particular 24(S),25-epoxycholesterol, and the steroid hormone, 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 indicate DHCR24 may be an important regulatory step in cholesterol synthesis and maintaining cellular cholesterol homeostasis. iii iv LIST OF PUBLICATIONS Zerenturk E.J.*, Kristiana I.*, Gill S., Brown A.J. (2012) The endogenous regulator 24(S),25-epoxycholesterol inhibits cholesterol synthesis at DHCR24 (Seladin-1). Biochim Biophys Acta 1821: 1269-1277. Zerenturk E.J.*, Sharpe L.J.*, Brown A.J. (2012) Sterols regulate 3β-hydroxysterol Δ24-reductase (DHCR24) via dual sterol regulatory elements: cooperative induction of key enzymes in lipid synthesis by Sterol Regulatory Element Binding Proteins. Biochim Biophys Acta 1821: 1350-1360. Zerenturk E.J., Sharpe L.J.,