The aim of this project is to study the cellular regulation of the polyisoprenoid pathway as it proceeds through HMG-CoA to mevalonic acid, thence to farnesyl pyrophosphate and from thence to squalene and to sterols. In addition to the well-established regulation of this pathway at the HMG-CoA reductase step, there is another regulatory point beyond squalene which appears subject to feedback control by sterols. We have discovered a possible link between these two control points which is related to the activity of squalene oxidocyclase (SOC), and which may explain the mechanism by which LDL represses HMG-CoA reductase activity. This relationship was elucidated with the aid of U-18666A, a SOC inhibitor. When the activity of SOC is impaired, squaleneepoxide (SO) accumulates and is converted to squalene 2,3:22,23 diepoxide (SDO). SDO is converted to a polar sterol via a cyclization reaction catalyzed by SOC. The polar steroid is an inhibitor of HMG-CoA reductase and shuts down the entire sterol synthetic pathway. When SOIC is completely inhibited by U-18666A, then the conversion of SDO to polar steroids is prevented and HMG-CoA reductase activity is relieved of inhibition. The effect of LDL in repressing HMG-CoA reductase in cells in culture is prevented by U-18666A without any effect on LDL degradation. We plan to devote a major part of our effort to examine the following points: (1) To identify the product of SDO which is responsible for inhibition of HMG-CoA reductase; (2) To test whether the inhibitory product of SDO acts in a fashion similar to well known oxysterol inhibitor; (3) To study further the nature of the repression of reductase activity exerted by LDL and its release by U-18666A; (4) To understand the physiological factors which modulate the activity of SOC and to compare these in different tissues and cell lines and to relate these to HMG-CoA reductase activity. Our work on HMG-CoA reductase will continue to be directed to examining the role of the essential SH group at the catalytic site as part of a study of the mechanism of catalysis by this enzyme. In addition, we will examine the synthesis and degradation of the whole enzyme in various phases of cell development with immunoprecipitation techniques.
