• Dory, Ladislav (PI)

Project Details


DESCRIPTION (Adapted from the applicant's abstract): Apolipoprotein E is an
important component of circulating, cholesterol-rich lipoproteins. Defects
in its metabolism have pronounced effects on plasma cholesterol levels and
the development of atherosclerosis. A role for apo E has also been proposed
in the etiology of Alzheimer s disease and other neurodegenerative
disorders. The proposed work aims at the elucidation of two novel
posttranscriptional regulatory steps in apo E expression in macrophages,
discovered in this laboratory: a) translational down-regulation in response
to stimuli that act through the protein kinase C signaling pathway, and b)
rescue mechanism of newly-synthesized apo E, mediated by HDL and cAMP, which
inhibits intracellular degradation and leads to enhanced secretion. First,
we will test the hypothesis that protein kinase C activates a trans-acting
repressor protein(s), which binds to the 5' or 3' untranslated region of the
mature apo E transcript and inhibits translation. Studies are also proposed
to identify the corresponding cis-acting elements within the transcript.
The bulk of the proposed studies will utilize tools of molecular biology.
These will include the synthesis of modified apo E transcripts, in vitro
translation assays, coupled with gel mobility shift assays and UV
cross-linking studies. Second, the mechanism of HDL/cAMP-mediated rescue of
newly synthesized apo E from intracellular (lysosomal) degradation will be
examined in primary macrophages. These studies will utilize biochemical and
morphological approaches (immunoelectron microscopy) to test the hypothesis
that retroendocytosis of HDL coupled with cAMP production results in the
rescue of apo E in the endosomal compartment or the trans-Golgi network,
followed by their co-secretion. HDL processing will also be examined in the
J774 macrophage cell line (a cell line that appears defective in
mobilization of intracellular cholesterol into an HDL-accessible pool) and a
J774 cell line expressing human apo E3 (J774, E3), in order to examine HDL
retroendocytosis and the extent and mechanism of HDL-mediated increase in
apo E secretion, respectively. Macrophages or macrophage-like cells are
responsible for apo E synthesis in the arterial wall and brain. The results
of the proposed work will provide important information about the least-well
known aspect of apo E metabolism-regulation of expression and processing
within these cells.
Effective start/end date1/08/9130/06/04


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