INFORMATION FOR PROSPECTIVE STUDENTS AND POSTDOCS

Projects in my laboratory are directed at understanding how cholesterol synthesis is modulated at the protein level through regulatory proteins (i.e., kinases, accessory protein factors), and how botanicals and over-the-counter pharmaceuticals (policosanol, garlic, green and black tea) inhibit cholesterol synthesis.  Techniques typically involve the cloning of cDNAs to various cholesterolgenic enzymes and expression  in both E. coli and in hepatoma cells.  Results are confirmed with whole animal studies and through site-directed mutagenesis of relevant proteins.  

Supernatant protein factor (SPF) is a recently cloned member of a family of cytosolic lipid-binding proteins.  SPF increases cholesterol synthesis by two-fold when expressed in hepatoma cells, suggesting that SPF may play a role in the regulation of cholesterol synthesis.  SPF has long been known to stimulate squalene monooxygenase activity in microsomal preparations; our recent studies reveal that SPF also stimulates HMG-CoA reductase activity, both in cell culture and in vitro.  The combined activation of these two cholesterolgenic enzymes appears to be by a common mechanism that has yet to be clearly defined.  SPF, in turn, is activated by phosphorylation by protein kinases A and C, suggesting a mechanism by which both hormonal and intracellular signaling pathways can rapidly modulate cholesterol synthesis through SPF. 

What projects might a new student undertake?

Determine how SPF activity is regulated in cell culture: 

We have shown that SPF is phosphorylated in freshly isolated liver preparations, and the purified enzyme can be phosphorylated in vitro with specific protein kinases.   Phosphorylation activates SPF by two-fold, suggesting that phosphorylation regulates its activity, and perhaps also cholesterol synthesis.  SPF activity in hepatoma cells also appears to be dependent upon phosphorylation, but the factors that regulate this phosphorylation are not known.  Studies are needed to determine the role of phosphorylation in the regulation of SPF in cultured hepatoma cells by using specific inhibitors and activators of protein kinases and protein phosphatases, and to determine if sterol levels, sterol precursors, and vitamin E congeners regulate SPF activity.

Determine how SPF expression and activity is regulated in vivo:  

SPF activity is decreased in the livers of rats fed a high-fat diet, concomitant with a corresponding decrease in squalene monooxygenase activity.  It is not known if this down-regulation is mediated at the transcriptional or translational level, or through enhanced enzyme degradation.  Studies are needed to determine the mechanism of this decrease by measuring SPF mRNA and protein in the livers of rats fed either a high-cholesterol diet or a normal diet supplemented with cholestyramine to reduce dietary cholesterol.  The effect of these diets on SPF activity and its phosphorylation state is also unknown and needs to be determined.  Future studies would be directed at obtaining an 'SPF-knockout' mouse, to assess the role of this protein in vivo. 

Identify the structural elements of SPF that govern its function:  

SPF contains several structural elements in common with related lipid-binding proteins.  These structural motifs include a CRAL/TRIO domain thought to be involved in lipid binding, a lipid-exchange loop that is proposed to interact with the membrane, and an N-terminal “coiled-coil” segment and carboxyl-terminal extension that may be involved in Golgi and nucleotide (GTP/GDP) binding.  Studies to explore the function of these elements in SPF will involve segmental deletions and site-directed mutagenesis experiments, with the modified proteins being expressed and purified to determine 1) the binding affinity with the proposed ligands squalene and phosphatidylinositol; 2) the ability to stimulate squalene monooxygenase and HMG-CoA reductase in microsomes; and 3) the ability to increase cholesterol synthesis in hepatoma cells.

Determine the subcellular location of SPF and whether its location changes in response to potential ligands and factors that alter its activity: 

SPF is regarded as a cytosolic lipid-transfer protein that transiently associates with intracellular membranes, including the endoplasmic reticulum, where cholesterol synthesis takes place.  Our studies reveal that Golgi are necessary for the SPF-mediated stimulation of cholesterol synthesis, suggesting a transient association with Golgi.  SPF contains two domains that are potential Golgi-association signals; studies using structurally modified proteins and immunofluorescent co-localization with confocal microscopy will determine if these domains are involved in targeting SPF to Golgi or other intracellular locations.  The effect of phosphorylation/dephosphorylation on the subcellular location of SPF is also of interest, as is the effect of addition of proposed SPF ligands (tocopherols, tocotrienols, and tocopherylquinone).  

Garlic, tea, and policosanol (a by-product of sugar cane)  have been shown to reduce blood cholesterol levels, although the mechanistic basis for this effect has not been well described.  A significant component of this effect with these botanicals appears to be a reduction in cholesterol synthesis.  Knowledge of how these widely used botanicals affect cholesterol synthesis is essential to understanding the benefits and potential adverse effects of their increased use, particularly in combination with each other and with prescription pharmaceuticals that inhibit cholesterol synthesis (statin drugs).  The proposed studies will use state-of-the-art analytical techniques to evaluate the ability of these botanicals to inhibit cholesterol synthesis both in vitro and in vivo, to compare their relative effectiveness, and to identify the specific enzymes inhibited and the active components of each extract.

Inhibition of cholesterol synthesis is likely to be a significant component of the overall ability of garlic and tea to lower blood cholesterol levels.  Present evidence suggests that garlic acts in the downstream, or committed pathway, whereas tea may act in the upstream isoprenoid pathway (see Preliminary Studies).  The proposed studies will rigorously evaluate cholesterol synthesis in the presence of these botanicals to identify the active components of each and the affected enzymes, and thereby establish a solid, mechanistic understanding of how garlic and tea reduce cholesterol synthesis in hepatic cells.

What projects might a new student undertake?

Determine which cholesterolgenic enzymes are inhibited by garlic, tea, and policosanols:

These studies will be carried out with cultured hepatoma cells in which cholesterol synthesis can be readily followed, and various radiolabeled substrates can be added to determine which enzymes are affected by these botanicals.  Sterol precursors and intermediates that accumulate in the presence of these botanicals or their components will be fractionated by thin-layer chromatography and identified by mass spectrometry.  Results obtained in vitro will need to be confirmed by whole-animal studies. 

Determine which components of garlic, tea, and policosanol are responsible for the inhibition by these substances:

Most of the chemical constituents of these compounds are known and can be purchased from commercial suppliers.  These compounds will be tested for their ability to inhibit cholesterol synthesis in hepatoma cells, in isolated enzyme preparations, and in whole animals.  The site(s) of inhibition will be identified and, if appropriate, components will be combined to yield an inhibitory pattern that most closely reflects that seen with the extracts.

Identify the cysteines in squalene monooxygenase that are covalently modified by tellurium, selenium, and garlic compounds:

We have shown that tellurium, selenium, and garlic compounds bind to cysteine sulfhydryls on squalene monooxygenase (a downstream enzyme in cholesterol synthesis), and that binding to one or more of these cysteines inhibits the enzyme.  Identification of the cysteines that are modified by these compounds would provide information on the active site of squalene monooxygenase and the possible catalytic role of these cysteines.  These structure-function studies would include chemical labeling followed by mass spectrometric analysis of peptides, protein sequencing, and site-directed mutagenesis of the six cysteines present in squalene monooxygenase, with expression in bacterial cells and characterization in vitro of the mutated enzyme.  These studies may also provide further information on how garlic inhibits cholesterol synthesis. 

Squalene monooxygenase catalyzes the second step in the downstream, or committed pathway for cholesterol biosynthesis.  Squalene monooxygenase requires a second enzyme to provide the electrons necessary for catalysis, and for many years it has been assumed that cytochrome P450 reductase (CPR) is this requisite electron donor protein.  Our recent studies with mice engineered to lack CPR in the liver have revealed the presence of a second enzyme that can support squalene monooxygenase activity.  Proposed studies will identify and characterize this unknown reductase.  Because CPR is believed to serve as the redox partner for several other enzymes involved in cholesterol synthesis, studies using these hepatic CPR-null mice will allow us to determine the role of CPR in other steps in the cholesterolgenic pathway.  

What projects might a new student undertake?

Purify the reductase from CPR-null mouse liver:

Standard chromatographic techniques including affinity chromatography, as well as innovative experiments using cross-linking reagents and two-hybrid assays will be used to purify the unknown reductase from CPR-null mouse liver.  The ability of the purified reductase to support squalene monooxygenase will be determined with purified enzyme, and the kinetic parameters of the reaction will be characterized.

Clone and express a cDNA to the reductase and confirm its ability to support squalene monooxygenase:

Peptides will be isolated from the purified reductase and used to design primers for the amplification of a reductase cDNA from a liver library.  The cloned cDNA will be expressed in E. coli and the protein purified from these cells by affinity chromatography, after which the ability of the cloned reductase to support squalene monooxygenase will be determined.  The role of supernatant protein factor (SPF) in this reaction will also be determined.

Evaluate the role of CPR in other steps in the cholesterolgenic pathway:

CPR has been reported to serve as the redox partner for several other enzymes in the cholesterolgenic pathway, including lanosterol demethylase and 7-dehydrocholesterol reductase.  CPR may also serve as an alternate electron donor to sterol 4-methyl oxidase and sterol D5-desaturase.  The role of CPR in these reactions will be determined by adding ¹⁴C-acetate to hepatocytes from CPR-null mice and determining which sterol precursors and intermediates accumulate.  The activity of the individual enzymes will also be determined in subcellular preparations from these mice.  These studies should define the role of this important reductase in cholesterol synthesis.