Regulation of acid sphingomyelinase by lysosomal free cholesterol

Acid sphingomyelinase (aSMase) is a lysosomal enzyme that converts sphingomyelin to phosphorylcholine and ceramide, a potent lipid second messenger. aSMase activity partially controls sphingomyelin concentrations, which is necessary for the maintenance of a proper cholesterol to sphingomyelin ratio in intracellular membranes and is a key enzyme in the coordinate regulation of cholesterol and sphingomyelin metabolism. Accumulation of lysosomal free cholesterol either due to a defect in the Niemann-Pick type C1 or C2 proteins, or treatment with compounds that block normal cholesterol egress from lysosomes, reduces aSMase activity (<15% of normal) while stimulation of lysosomal cholesterol egress by removal of lipoproteins from the culture media restores aSMase activity to normal levels. The studies in this dissertation were designed to elucidate the mechanism responsible for the lysosomal cholesterol-mediated regulation of aSMase.;Initial studies demonstrated that a CHO cell line mutant (CT-60), which accumulates lysosomal free cholesterol due to a defect in the NP-C1 protein, has approximately 5--10% of the aSMase activity of its parental cell line (25-RA) or wild type (CHO-K1) cells. A similar reduction in aSMase activity could be induced in CHO-K1 cells through incubation with low density lipoproteins (LDL) and progesterone, which induced the accumulation of lysosomal free cholesterol. Western blot analysis demonstrated that both the CT60 and LDL/progesterone treated CHO-K1 cells possessed near normal levels of aSMase protein, suggesting that the cholesterol-induced inhibition is a post-translational event, perhaps involving cofactor mediated modulation of enzymatic activity or alterations in aSMase protein trafficking and maturation.;Niemann-Pick type C fibroblasts also displayed normal aSMase protein but negligible levels of aSMase activity. Further analysis using a polyclonal antibody directed against aSMase demonstrated that lysosomal cholesterol accumulation is coupled with a redistribution of aSMase to the trans-Golgi network. Filipin staining revealed that the removal of lipoproteins from the culture media decreased the late endosomal/lysosomal free cholesterol concentration and restored aSMase activity to normal levels. This restoration of aSMase activity corresponded to a redistribution of aSMase to late endosomes/lysosomas, as determined by co-localization with lysosomal associated membrane protein II. These studies suggest a novel mechanism by which an increase in intracellular cholesterol concentration arrests the normal trafficking and/or processing of aSMase thereby eliminating a critical event required for the generation of active lysosomal aSMase.;To investigate the posttranslational processing and elucidate the mechanism by which lysosomal cholesterol concentration regulates aSMase activity, a CHO cell line was produced that stably expressed an aSMase construct under the control of a constitutive promoter, CMV, and tagged with the FLAG (DYKDDDDK) epitope at the carboxy-terminal end. Time course experiments revealed that induction of lysosomal free cholesterol accumulation reduced aSMase activity in a biphasic time-dependent manner. The rapid down-regulation of aSMase activity was not accounted for by a decrease in aSMase mass. However, changes in cellular aSMase mass were apparent following prolonged incubation in cholesterol loading media. In these studies aSMase activity was regulated by dual mechanisms including an apparent posttranslational mechanism that resulted in the rapid reduction in activity without a corresponding change in mass and a second mechanism that involved a reduction in aSMase protein levels.