Studies of new factors involved in cholesterol homeostasis

The regulatory pool of cholesterol is located in the endoplasmic reticulum (ER) and is key to how mammalian cells sense and respond to changes in cellular cholesterol levels. The extent of cholesterol esterification by the ER-resident protein, acyl-coenzyme A:cholesterol acyl-transferase (ACAT), has become the standard method for monitoring cholesterol transport to the ER and is assumed to reflect the regulatory pool of ER cholesterol. The oxysterol, 25-hydroxycholesterol (25HC), is thought to trigger intracellular cholesterol transport to the ER. Chapter 3 of this thesis addressed whether or not cholesterol esterification necessarily reflects cholesterol movement to the cholesterol homeostatic machinery in the ER as determined by SREBP processing. Three agents that inhibited the ability of 25HC to induce cholesterol esterification (progesterone, nigericin, and monensin) did not have a corresponding effect on 25HC suppression of SREBP processing. Moreover, ACAT inhibition did not alter the sensitivity of SREBP processing to 25HC. These findings indicate that cholesterol esterification by the ER-resident protein ACAT is dissociable from cholesterol transport to the cholesterol homeostatic machinery in the ER.In order to readily analyse and quantify the processing of SREBP-2, the master regulator of cholesterol homeostasis, the second part of this study (Chapter 4) established a stable cell-line (13A/PS) that expresses the fusion protein PLAP-BP2 (secreted form of placental alkaline phosphatase joined to the luminal loop of SREBP-2 on the NH2- terminal side of Site-1). Processing of PLAP-BP2 mimics the cleavage of SREBP. The PLAP-BP2 cleavage assay was shown to be a valuable method for analysing and quantifying SREBP-2 processing. Compared to transiently transfected cells, the stable 13A/PS cells were more responsive to changes of sterol status in regards to PLAP secretion. Considering the reliability and consistency of the experiments, the 13A/PS cell-line is therefo