Cholesterol Metabolism-Role of Apo A-I and SR-B1

Project: Research project

Project Details

Description

DESCRIPTION (Provided by applicant): The two aims of this proposal are focused
on scavenger receptor B1 (SR-BI) and apolipoprotein Al (apoA-I) and their
function in the lipoprotein cholesteryl ester (CE) selective uptake pathway.
Previous studies with mice and rats and cultured human cells indicate that this
pathway plays a major role in the uptake of high density lipoprotein (HDL) CE
into the liver and steroidogenic cells. Studies in gene knockout mice show that
SR-BI is the receptor responsible for HDL CE selective uptake and that apoA-I
is the key HDL ligand for SR-BI. Recent studies indicate that SR-BI has
multiple effects on cellular cholesterol metabolism including changes in plasma
membrane properties. Aim 1 has four goals in which the mechanisms by which
SR-BI alters plasma membrane properties will be investigated. Goal 1 will test
the hypothesis that SR-BI is necessary for the formation of microvillar
channels and the cell surface localization of HDL particles on steroidogenic
cells. These experiments will compare wild type and SR-BI-deficient mice and
will use ultrastructural analysis at the electron microscope (EM) level and HDL
localization analyses at the light microscopic (LM) and EM levels. Goal 2 will
test the role of caveolin-1 in SR-BI-mediated HDL CE selective uptake by
comparisons of wild type and caveolin-1-deficient mice. These experiments
include morphological analyses and in vivo analysis of HDL CE selective uptake.
Goal 3 will test the hypothesis that SR-BI-induced alterations in plasma
membrane morphology reflect changes in membrane phospholipids. Tandem mass
spectrometry will be used to determine the phospholipid distribution and acyl
tail compositions in membranes prepared from two cell types in which SR-BI
alters membrane morphology, the mouse adrenal z. fasciculata cell and the
insect Sf9 cell. Goal 4 will test the hypothesis the SR-BI is assembled in the
plasma membrane of adrenal cells as a homo-oligomeric complex. Aim 2 is focused
on the key issue of how SR-BI recognizes its ligand during docking of the HDL
particle. These experiments use single cys mutants of apoA-I to provide
site-specific chemical cross-linking to SR-BI. Sites of cross-linking in SR-BI
will be determined by isolation of SR-BI peptides and identification by
matrix-assisted laser desorption ionization-time of flight mass spectrometry.
These experiments test the hypothesis that SR-BI recognizes apoA-I via its
amphipathic alpha-helical repeat units. Additionally, sites of cross-linking in
SR-BI will identify key residues and receptor domains involved in the
recognition of HDL particles. These studies will provide new and important
information relevant to the role of HDL in protecting against atherosclerotic
disease.
StatusFinished
Effective start/end date4/1/043/31/05

Funding

  • National Heart, Lung, and Blood Institute: $381,399.00

ASJC

  • Spectroscopy