Molecular and cellular aspects of the SREBP pathway

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dc.contributor.advisor McCarthy, Tommie V. en Barriscale, Katherine A. 2015-01-19T10:21:41Z 2014 2014
dc.identifier.citation Barriscale, K. A. 2014. Molecular and cellular aspects of the SREBP pathway. PhD Thesis, University College Cork. en
dc.identifier.endpage 292
dc.description.abstract The SREBP (sterol response element binding proteins) transcription factors are central to regulating de novo biosynthesis of cholesterol and fatty acids. The SREBPs are regulated by retention or escape from the ER to the Golgi where they are proteolytically cleaved into active forms. The SREBP cleavage activating protein (SCAP) and the INSIG proteins are essential in this regulatory process. The aim of this thesis is to further characterise the molecular and cellular aspects surrounding regulation of SREBP processing. SREBP and SCAP are known to interact via their carboxy-terminal regulatory domains (CTDs) but this interaction is poorly characterised. Significant steps were achieved in this thesis towards specific mapping of the interaction site. These included cloning and over expression and partial purification of tagged SREBP1 and SREBP2 CTDs and probing of a SCAP peptide array with the CTDs. Results from the SREBP2 probing were difficult to interpret due to insolubility issues with the protein, however, probing with SREBP1 revealed five potential binding sites which were detected reproducibly. Further research is necessary to overcome SREBP2 insolubility issues and to confirm the identified SREBP1 interaction site(s) on SCAP. INSIG1 has a central role in regulating SREBP processing and in regulating stability of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), a rate limiting enzyme in cholesterol biosynthesis. There are two protein isoforms of human INSIG1 produced through the use of two in-frame alternative start sites. Bioinformatic analysis indicated that the presence of two in-frame start sites within the 5-prime region of INSIG1 mRNA is highly conserved and that production of two isoforms of INSIG1is likely a conserved event. Functional differences between these two isoforms were explored. No difference in either the regulation of SREBP processing or HMGCR degradation between the INSIG1 isoforms was observed and the functional significance of the two isoforms is as yet unclear. The final part of this thesis focused on enhancing the cytotoxicity of statins by targeted inhibition of SREBP processing by oxysterols. Statins have significant potential as anti-cancer agents as they inhibit the activity of HMGCR leading to a deficiency in mevalonate which is essential for cell survival. The levels of HMGCR fluctuate widely due to cholesterol feedback of SREBP processing. The relationship between sterol feedback and statin mediated cell death was investigated in depth in HeLa cells. Down regulation of SREBP processing by sterols significantly enhanced the efficacy of statin mediated cell death. Investigation of sterol feedback in additional cancer cell lines showed that sterol feedback was absent in cell lines A- 498, DU-145, MCF-7 and MeWo but was present in cell lines HT-29, HepG2 and KYSE-70. In the latter inhibition of SREBP processing using oxysterols significantly enhanced statin cytotoxicity. The results indicate that this approach is valid to enhance statin cytotoxicity in cancer cells, but may be limited by deregulation of SREBP processing and off target effects of statins, which were observed for some of the cancer cell lines screened. en
dc.format.mimetype application/pdf en
dc.language.iso en en
dc.publisher University College Cork en
dc.rights © 2014, Katherine A. Barriscale en
dc.rights.uri en
dc.subject Statins en
dc.subject Cholesterol biosynthesis en
dc.subject SREBP pathway en
dc.subject Cancer biology en
dc.title Molecular and cellular aspects of the SREBP pathway en
dc.type Doctoral thesis en
dc.type.qualificationlevel Doctoral Degree (Structured) en
dc.type.qualificationname PhD Scholars Programme in Cancer Biology en
dc.internal.availability Full text not available en Restricted to everyone for three years en 2020-01-01T10:21:41Z
dc.description.version Accepted Version
dc.contributor.funder Health Research Board en
dc.description.status Not peer reviewed en Biochemistry en
dc.check.reason This thesis is due for publication or the author is actively seeking to publish this material en
dc.check.opt-out Not applicable en
dc.thesis.opt-out false
dc.check.entireThesis Entire Thesis Restricted
dc.check.embargoformat Both hard copy thesis and e-thesis en
dc.internal.conferring Summer Conferring 2014

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© 2014, Katherine A. Barriscale Except where otherwise noted, this item's license is described as © 2014, Katherine A. Barriscale
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