Year of Publication

2009

Degree Name

Doctor of Philosophy (PhD)

Document Type

Dissertation

College

Pharmacy

Department

Pharmaceutical Sciences

First Advisor

Dr. Todd D. Porter

Abstract

SPF (Supernatant Protein Factor) is a cytosolic protein that stimulates at least two enzymes in the cholesterol biosynthetic pathway: squalene monooxygenase and HMGCoA reductase. The mechanism of action has not been established but may be related to lipid transfer between intracellular membranes.

There are three human genes for SPF: SEC14L2 (SPF1), SEC14L3 (SPF2) and SEC14L4 (SPF3). The present study differentiates these closely related genes by evaluating their tissue-specific and relative expression levels. SPF1 mRNA was found to be most abundant in liver, mammary gland and stomach. SPF2 showed negligible expression in all tissues tested; SPF3 expression pattern was similar to that of SPF1, but at 10-50-fold lower levels than SPF1.

A cDNA to SPF3 was cloned and, upon transfection into rat hepatoma cells, was shown to increase cholesterol synthesis by approximately 50%, similar to that obtained with SPF1. However, in contrast to SPF1, SPF3 did not stimulate squalene monooxygenase activity in microsomal preparations, suggesting that it acts primarily through activation of HMG-CoA reductase.

SPF possesses a lipid binding domain (Sec14) and a Golgi dynamics domain (GOLD). SPF resides in the cytosol and requires phosphorylation and the presence of Golgi in order to stimulate cholesterol synthesis. To determine if SPF associates with specific subcellular structures, cellular immunofluorescence studies were carried out. A phosphorylationdefective mutant, a protein lacking the GOLD domain, and the effect of protein kinase A-mediated phosphorylation of endogenous SPF were examined. No change in the subcellular location of SPF could be detected with either the phosphorylation mutant or the native SPF after protein kinase A activation. However, removal of the GOLD domain resulted in a protein that co-localized with large vesicular structures around nucleus.

Studies with rat hepatoma cells showed that the expression of the two rat SPF genes is upregulated in response to serum deprivation, and is potentiated by removal of glucose. Lipid/cholesterol availability was demonstrated to be at least one of the serum components that affected SPF transcript levels. The oxysterol receptor LXR was shown not to be involved in SPF gene regulation, implicating SREBP and/or PPARα as the principal regulators of SPF gene transcription.

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