ApoE4 Domain Interaction

ApoE4 binds preferentially to very low density lipoproteins (VLDL), while apoE3 binds preferentially to high density lipoproteins (HDL) (Figure 1). We determined that the two domains in apoE4 interact and that this interaction is restricted to apoE4. Using a combination of x-ray crystallography, site-directed mutagenesis, and lipoprotein binding assays, we determined that Arg-61 (amino-terminal domain) and Glu-255 (carboxyl-terminal domain) mediate this domain interaction and that this interaction is responsible for the VLDL binding preference of apoE4 (4) (Figure 2). Our working hypothesis is that apoE4 domain interaction is a distinguishing feature of apoE4 that contributes to its unique metabolic properties.

This hypothesis has suggested a potential therapeutic approach targeting the detrimental effects of apoE4 in heart disease and neurodegeneration. We are collaborating with Drs. Fred Cohen and Irwin Kuntz (University of California, San Francisco) and using their DOCK program to identify small molecules that will bind to apoE4 in the vicinity of Arg-61 but not to apoE3. The expectation is that such a molecule will interfere with apoE4 domain interaction and blunt the effects of apoE4, converting apoE4 into an ‘apoE3-like’ molecule (Figure 2).

Novel Mouse Model of ApoE4.
We have also extended our structural studies on domain interaction to an animal model. In several species, including the mouse, apoE contains arginine and glutamic acid at positions equivalent to positions 112 and 255, respectively, in human apoE. However, all other species lack the critical human Arg-61 required for domain interaction. Their apoE contains threonine and, like human apoE3, displays a preference for HDL. When arginine was introduced at position 61 in mouse apoE, the mutant apoE displayed a preference for VLDL like that of human apoE4. Based on these results, we used a ‘knock-in’ gene targeting approach to introduce an arginine codon into the mouse gene to ‘humanize’ mouse apoE at position 61 and introduce domain interaction.

Proof That Domain Interaction Was Introduced in Vivo.
In heterozygous targeted mice, as in human apoE4 heterozygotes, the plasma level of the Arg-61 form is 20–40% lower than the level of the wild-type form. This characteristic pattern reflects the more rapid clearance of apoE4 from plasma. The ‘Arg-61’ mouse apoE also displays a preference for VLDL. These results demonstrate that domain interaction was successfully introduced in vivo, suggesting that this mouse model will reproduce the human apoE4 phenotype. Potentially, this model may provide new insight into the basic mechanisms by which apoE4 increases the risk for cardiovascular disease, neurodegeneration, and cognitive impairment.

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