Molecular and cellular aspects of the SREBP pathway

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.

Step-count accuracy of three motion sensors for older and frail medical inpatients

Objectives: To measure the step-count accuracy of an ankle-worn accelerometer, a thigh-worn accelerometer and one pedometer in older and frail inpatients. Design: Cross-sectional design study. Setting: Research room within a hospital. Participants: Convenience sample of inpatients aged ≥65 years, able to walk 20 metres unassisted, with or without a walking-aid. Intervention: Patients completed a 40-minute programme of predetermined tasks while wearing the three motion sensors simultaneously. Video-recording of the procedure provided the criterion measurement of step-count. Main Outcome Measures: Mean percentage (%) errors were calculated for all tasks, slow versus fast walkers, independent versus walking-aid-users, and over shorter versus longer distances. The Intra-class Correlation was calculated and accuracy was visually displayed by Bland-Altman plots. Results: Thirty-two patients (78.1 ±7.8 years) completed the study. Fifteen were female and 17 used walking-aids. Their median speed was 0.46 m/sec (interquartile range, IQR 0.36-0.66). The ankle-worn accelerometer overestimated steps (median 1% error, IQR -3 to 13). The other motion sensors underestimated steps (40% error (IQR -51 to -35) and 38% (IQR -93 to -27), respectively). The ankle-worn accelerometer proved more accurate over longer distances (3% error, IQR 0 to 9), than shorter distances (10%, IQR -23 to 9). Conclusions: The ankle-worn accelerometer gave the most accurate step-count measurement and was most accurate over longer distances. Neither of the other motion sensors had acceptable margins of error.