AbstractsBiology & Animal Science

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