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RESEARCH Overview

Function of Exchangeable Apolipoproteins
Dyslipidemia is a major epidemic in North America that leads to heart disease, obesity and the metabolic syndrome. Whereas development of dyslipidemic disease is a multi-factorial process, it is evident that aberrations in lipid metabolism represent a significant risk factor. It is widely accepted that exchangeable apolipoproteins function in regulation of plasma lipid levels, yet the molecular basis for this role is not fully understood. Increased knowledge of the properties of exchangeable apolipoproteins will be useful in the development of therapeutic strategies to influence plasma lipid levels and, thereby, reduce the risk of dyslipidemic disease. Furthermore, by understanding the molecular basis of apolipoprotein function in lipid transport and metabolism, it should be possible to design strategies to enhance or interfere with biological processes dependent upon apolipoprotein function. Knowledge gained has direct relevance to the treatment of disorders of lipid metabolism that affect children and adults.

Ryan Research Program Summary
The laboratory has three main research projects that are focused on a family of plasma proteins that function to regulate lipid transport and metabolism. In project 1 the goal is to understand how lipid transport and metabolism are regulated by molecular interactions between lipoproteins and cell surface receptors. The interaction of apolipoprotein (apo) E with members of the low-density lipoprotein receptor (LDLR) family is under investigation. Polymorphism at amino acid positions 112 or 158 in apoE give rise to three distinct isoforms. One isoform in particular, apoE4 (Arg at 112 and 158), has generated considerable interest since the discovery that it is the major genetic risk factor for development of late onset Alzheimer’s Disease (AD). Despite this correlation, the molecular mechanism underlying apoE4’s association with AD remains unclear. A tertiary structural feature distinguishing apoE4 from apoE2 and apoE3, termed domain interaction, is postulated to affect the conformation and orientation of its two independently folded structural domains (Figure 1).

Figure 1. Ribbon diagram depicting isoform specific differences in domain interaction. The NT domain 4-helix bundle is from the X-ray crystal structure (Wilson et al., 1991).  The CT domain and hinge segment have been modeled here for illustration. Adapted from Zhong and Weisgraber (2009).

It is postulated that this feature may influences apoE4 interaction with the amyloid ß peptide, sensitivity to proteolysis or its lipid accrual and receptor binding activities. In other studies the ability of apoE to modulate the signaling morphogen ”Wnt” is under investigation.  ApoE binding to the Wnt co-receptor LDLR-related protein 5/6 (LRP 5/6) is under investigation using culture cells and surface plasmon resonance spectroscopy.

In project 2 we are exploring the mechanism whereby the low abundance apolipoprotein, apoA-V, modulates plasma triacylglycerol levels. Structure-function and site directed mutagenesis experiments have been used to show that apoA-V interacts with heparan sulfate proteglycans (HSPG).  Other experiments indicate apoA-V function intracellularly to modulate triglyceride trafficking.  Current research efforts are designed to test the hypothesis that apoA-V expression drives cytosolic lipid droplet assembly at the expense of lipoprotein secretion (Figure 2).

Figure 2. Model depicting the effect of apoA-V on the fate of hepatic TG. In the absence of apoA-V (Upper Panel) TG accretion forms a lens between leaflets of the ER membrane. Expansion of this lens by continued accrual of TG leads to budding of a nascent lipid droplet from the cytoplasmic leaflet (left) or, alternatively, budding from the lumenal leaflet (right) to create a lumenal lipid droplet for utilization in VLDL maturation. Lower Panel) When present, apoA-V binding to membrane defects created by TG accumulation stabilizes the lumenal leaflet, promoting nascent lipid droplet budding toward the cytosol at the expense of lumenal lipid droplet formation (see arrows).

In the plasma compartment, despite its exceedingly low concentration, apoA-V exerts a profound effect on triglyceride homeostasis.  Results show that, as a component of triglyceride rich lipoproteins, apoA-V binds to glycosylphosphatidylinositol high density lipoprotein binding protein 1, thereby enhancing lipoprotein lipase mediated lipolysis of lipoprotein associated triacylglycerol.

In project 3 we are investigating the ability of apolipoproteins to solubilize phospholipid dispersions, generating a homogeneous population of water-soluble, nanometer scale lipid particles, termed nanodisks.  One goal of this research is to incorporate hydrophobic biomolecules and employ the resulting particles as water-soluble transport / delivery vehicles.  Recent success incorporating the polyene antibiotic amphotericin B, the isoprenoid all trans retinoic acid and the polyphenol, curcumin, illustrate the potential utility of these complexes.

Cell culture and in vivo studies in mice have revealed that nanodisk-associated biomolecules retain their biological activity and can be targeted to cell surface receptors via their intrinsically associated apolipoprotein scaffold component. An example of the potential utility of nanodisk technology relates to Barth Syndrome.

Barth Syndrome (BTHS) is an X-linked recessive disease manifest in young boys that results from mutations in the TAZ (tafazzin) gene locus. The TAZ gene product is a phospholipid transacylase that functions in remodeling cardiolipin molecular species. Cardiolipin is a specialized phospholipid that has unique structural properties and a subcellular location that is largely confined to the inner membrane of mitochondria. Cardiolipin serves a structural role in this membrane where is provides an optimal environment for proteins involved in the electron transport chain. These proteins function in ATP production and Barth Syndrome patients present with ultrastructural changes to mitochondria and metabolic abnormalities consistent with disrupted oxidative metabolism. We hypothesize that exogenous cardiolipin delivered to a TAZ knockdown HL60 cell culture model of BTHS will restore these cells to a normal phenotype. We predict that cardiolipin will be transported to mitochondria and utilized in lieu of de novo synthesized / remodeled cardiolipin. The ultimate goal of this project is to develop a treatment strategy based on administration of a water-soluble cardiolipin /protein formulation to BTHS patients as a means to bypass defective tafazzin enzyme activity. Overall, this concept may be considered lipid replacement therapy and proposed studies are designed to determine if cardiolipin delivery can ameliorate characteristic phenotypic features of this disease.

 

Revised: Friday, May 9, 2014 3:31 PM

 

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