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Here are few details about the ongoing research that is being conducted in this lab.

Background

Cardiovascular disease is the number one killer in the United States, and the accumulation of cholesterol is a significant cause.  High-density lipoprotein (HDL) has been the focus of intensive study because it can remove cholesterol from arteries, yet details of how this molecule is synthesized remain sketchy.  Additionally, there are human diseases in which HDL synthesis appears to be disrupted by defective genes that code for proteins involved in lipid transport.  Examples of such diseases are Tangier disease, in which the defective gene codes for a cholesterol transporter, the ABC1 transporter protein, and abetalipoproteinemia (ABL), caused by a mutation in a protein called microsomal triglyceride transfer protein (MTP).  In both of these diseases, HDL synthesis is effected.  Without a clear understanding of how HDL biosynthesis normally occurs, it is difficult to understand the extent to which defects in proteins like the ABC1 transporter protein and MTP disrupt its synthesis.  The focus of this project is to elucidate details of lipoprotein biosynthesis.  Does it use a novel pathway?  Are there accessory proteins involved in the lipidation process?  What portion of the apolipoprotein sequence codes for lipidation?

Lipoproteins 

Lipoproteins, protein-lipid complexes that have a polar exterior and a non-polar interior, have been found in many vertebrate and insect species, and their basic structure and function appear to be conserved.  They facilitate the intercellular transport of hydrophobic lipids through aqueous media.  Their synthesis requires an unusual process referred to as lipidation, whereby lipids are added to the protein component of the lipoprotein.  Lipidation is thought to occur during or immediately following translation of these proteins, but how this process occurs is unknown.  Of particular interest is the extent to which the protein sequence of the apoproteins drives lipidation and the level of involvement of other proteins in this process.

Project Goals

The goal of the on going research projects in this lab, is to study lipidation by expressing the apolipoproteins from the tobacco hornworm, Manduca sexta, in two different expression systems. The first uses budding yeast, Sacchormyces cerevisiae, and second uses Drosophila Schneider 2 cells to begin to examine the apolipoprotein sequence for regions that are crucial to lipoprotein biosynthesis. 

The Yeast Expression System

Previous research has revealed that the unicellular organism, S. cerevisiae, is capable of expressing, lipidating, and secreting M. sexta apolipoproteins.  The yeast cells produce an intact lipoprotein, a particle required for intercellular transport of lipids.  This is a major finding—the first demonstration of any apolipoprotein being expressed in a unicellular organism.  It is remarkable that the yeast cells are able to lipidate this protein, and produce a particle that is so similar to M. sexta nascent lipophorin.  The fact that yeast can produce this particle indicates that the expression, lipidation, and secretion of lipoproteins occurs via universal pathways--if any lipidation accessory or “helper” proteins are required, they are present in yeast.  These accessory proteins may not be the same proteins that are used for the secretion of non-lipidated proteins, and one of the projects in this lab focuses on their identification.

The S2 Cell System

It has been demonstrated that lipophorin production occurs in the absence of the full apoLp-I sequence.  This finding indicates that the lipidation code resides within the first 45% of the precursor protein sequence.  Deletion analysis has revealed that removal of any portion of the apoLp-II sequence prevents expression of the apolipoprotein.  Therefore, it is possible to conclude that all of the information required to make a lipoprotein is included in the apoLp-II sequence.  An excellent expression system, the Drosophila S2 expression system, has been developed, and can be used for the examination of the apoLp-II sequence.  A closer examination of the apolipoprotein sequence through deletion analysis and alanine-scanning mutagenesis should provide insights into the code for lipidation included in the amino acid sequence of apoLp-II.

 

This page was last updated on 05/29/03 .

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