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Cholesterol-Dependent Degradation and Unsaturated Fatty Acid-Dependent Stabilisation of Squalene Monooxygenase in the Control of Cholesterol Synthesis

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Cholesterol-Dependent Degradation and Unsaturated Fatty Acid-Dependent Stabilisation of Squalene Monooxygenase in the Control of Cholesterol Synthesis Cholesterol-dependent degradation and unsaturated fatty acid-dependent stabilisation of squalene monooxygenase in the control of cholesterol synthesis Julian Stevenson A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy School of Biotechnology and Biomolecular Sciences Faculty of Science Supervisor: Professor Andrew Brown March 2014 II PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Stevenson First name: Julian Other name/s: Abbreviation for degree as given in the University calendar: PhD School: Biotechnology and Biomolecular Sciences Faculty: Science Title: Cholesterol-dependent degradation and unsaturated fatty acid-dependent stabilisation of squalene monooxygenase in the control of cholesterol synthesis Abstract 350 words maximum: (PLEASE TYPE) Exquisite control of cholesterol synthesis is crucial for maintaining homeostasis of this vital lipid. Squalene monooxygenase (SM) catalyses the first oxygenation step in cholesterol synthesis, acting on squalene before cyclisation into the basic steroid structure. Using model cell systems, we found that cholesterol caused the accumulation of the substrate squalene, suggesting that SM may serve as a flux-controlling enzyme beyond 3- hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR, considered as rate-limiting). Cholesterol accelerated the proteasomal degradation of SM, which required the N-terminal domain (N100), partially conserved in vertebrates, but not lower organisms. Unlike HMGR, SM degradation is not mediated by Insig, 24,25-dihydrolanosterol or side-chain oxysterols, but rather by cholesterol itself. Importantly, SM’s N-terminal domain conferred cholesterol-regulated turnover on heterologous fusion proteins. Furthermore, proteasomal inhibition almost totally eliminated squalene accumulation, highlighting the importance of this degradation mechanism for the control of SM and cholesterol synthesis after mevalonate production. On the contrary, treatment with unsaturated fatty acids such as oleate, but not saturated fatty acids, increased protein levels of SM or N100, as well as reversing cholesterol-dependent squalene accumulation. Notably, the stabilisation occurred through reduced ubiquitination by the E3 ubiquitin ligase, MARCH6. Maximum stabilisation required activation of fatty acids, but not triglyceride or phosphatidylcholine synthesis. Stabilisation of a cholesterol biosynthetic enzyme by unsaturated fatty acids may help maintain a constant cholesterol/phospholipid ratio. In addition, we optimised and compared highly efficient ligation-independent cloning techniques, identifying important parametres for project design using either polymerase incomplete primer extension (PIPE) cloning, sequence and ligation-independent cloning (SLIC), or overlap extension cloning (OEC), including the need to avoid PCR artefacts such as primer-dimers and vector plasmid background. Experiments made use of a common reporter vector and a set of modular primers to clone DNA fragments of increasing size. Overall, PIPE achieved cloning efficiencies of ~95% with few manipulations, whereas SLIC provided a much higher number of transformants, but required additional steps. Our data suggest that for small inserts (<1.5 kb), OEC is a good option, requiring only two new primers, but performs poorly for larger inserts. We believe that these ligation-independent cloning approaches constitute an essential part of the researcher's molecular-tool kit 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). ………………………………………………… ……………………………………..……………… ……….……… 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: THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OR BE THE FIRST PAGE INSERTED III IV 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 ................................................................. V VI 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 ……………………………………………........................... VII VIII Abstract Exquisite control of cholesterol synthesis is crucial for maintaining homeostasis of this vital lipid. Squalene monooxygenase (SM) catalyses the first oxygenation step in cholesterol synthesis, acting on squalene before cyclisation into the basic steroid structure. Using model cell systems, we found that cholesterol caused the accumulation of the substrate squalene, suggesting that SM may serve as a flux-controlling enzyme beyond 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR, considered as rate-limiting). Cholesterol accelerated the proteasomal degradation of SM, which required the N-terminal domain (N100), partially conserved in vertebrates, but not lower organisms. Unlike HMGCR, SM degradation is not mediated by Insig, 24,25-dihydrolanosterol or side-chain oxysterols, but rather by cholesterol itself. Importantly, SM’s N-terminal domain conferred cholesterol-regulated turnover on heterologous fusion proteins. Furthermore, proteasomal inhibition almost totally eliminated squalene accumulation, highlighting the importance of this degradation mechanism for the control of SM and cholesterol synthesis after mevalonate production. On the contrary, treatment with unsaturated fatty acids such as oleate, but not saturated fatty acids, increased protein levels of SM or N100- GFP, as well as reversing cholesterol-dependent squalene accumulation. Notably, the stabilisation occurred through reduced ubiquitination by the E3 ubiquitin ligase, MARCH6. Maximum stabilisation required activation of fatty acids, but not triglyceride or phosphatidylcholine synthesis. Stabilisation of a cholesterol biosynthetic enzyme by unsaturated fatty acids may help maintain a constant cholesterol/phospholipid ratio. In addition, we optimised and compared highly efficient ligation-independent cloning techniques, identifying important parametres for project design using either polymerase incomplete primer extension (PIPE) cloning, sequence and ligation-independent cloning (SLIC), or overlap extension cloning (OEC), including the need to avoid PCR artefacts such as primer-dimers and vector plasmid background. Experiments made use of a common reporter vector and a set of modular primers to clone DNA fragments of increasing size. Overall, PIPE achieved cloning efficiencies of ~95% with few manipulations, whereas SLIC provided a much higher number of transformants, but required additional steps. Our data suggest that for small
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