Year of Publication

2004

Document Type

Dissertation

College

Graduate School

Department

Toxicology

First Advisor

Todd D. Porter

Abstract

Supernatant protein factor (SPF) is a 46-kDa cytosolic protein that stimulates squalene monooxygenase, which catalyses the second committed step in cholesterol biosynthesis. The mechanism by which SPF stimulates this enzyme is not understood and the goal of these studies was to see if SPF affected cholesterol synthesis in cultured cells. Rat supernatant protein factor-like protein (SPF2) shares 77% sequence identity with human SPF. In my studies SPF2 also stimulated squalene monooxygenase in vitro and incubation of SPF2 with protein kinase A (PKA) and C increased its activity by about 2-fold, as shown earlier with SPF. GTP and GDP prevented the stimulation of squalene monooxygenase by SPF2, suggesting that binding of these nucleotides inhibits SPF2. This inhibition could be prevented by the addition of -tocopherol, although -tocopherol alone had no effect on SPF2 activity in vitro. Expression of human SPF in hepatoma cells, which lack expression of endogenous SPF, increased cholesterol synthesis by 2-fold and addition of dibuytrylcAMP, a PKA activator, to these cells yielded an additional 62% increase whereas addition of a PKA inhibitor completely blocked the ability of SPF to stimulate cholesterol synthesis. To further confirm a role for phosphorylation in the regulation of SPF, substitution of alanine for serine-289 (a putative PKA recognition site) reduced the PKA-mediated activation of SPF in vitro by 50%, as measured with microsomal squalene monooxygenase and completely blocked the ability of SPF to stimulate cholesterol synthesis in hepatoma cells. In further structure-function studies, deletion of the carboxy-terminal Golgi-dynamics domain greatly increased the ability of SPF to stimulate squalene monooxygenase in microsomes, but, paradoxically prevented SPF from stimulating cholesterol synthesis in cell culture. Addition of brefeldin A, which disrupts Golgi formation, also abolished the ability of SPF to stimulate cholesterol synthesis, supporting a role for the Golgi in SPF function. Since squalene monooxygenase is not thought to be rate-limiting with regard to cholesterol synthesis, the possibility that SPF might stimulate other enzymes in the cholesterol biosynthetic pathway was investigated. The substitution of 14Cmevalonate for 14C-acetate completely blocked an SPF-induced 1.5-fold increase in squalene synthesis, suggesting that SPF stimulated mevalonate synthesis at HMGCoA reductase. 2,3-Oxidosqualene synthesis from 14C-mevalonate remained elevated (1.3-fold) with mevalonate demonstrating that SPF also stimulated squalene monooxygenase in hepatoma cells. SPF did not increase HMG-CoA reductase or squalene monooxygenase enzyme levels in cells, indicating that SPF directly activated these enzymes. Indeed, addition of purified recombinant SPF to rat liver microsomes stimulated HMG-CoA reductase by about 1.5-fold. These results reveal that SPF directly stimulates HMG-CoA reductase, the rate-limiting step of the cholesterol biosynthetic pathway, as well as squalene monooxygenase, and, coupled with the ability of PKA-mediated phosphorylation to regulate SPF activity, suggest a new means by which cholesterol synthesis can be rapidly modulated in response to hormonal and environmental signals.

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