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| Novel Genes in Cellular Cholesterol Metabolism |
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Although feeding high-cholesterol diets to humans raises LDL cholesterol levels,
there is considerable inter-individual variation in responsiveness that is currently
unexplained. To provide insight into the genes that might be involved, we have been
studying the effect of dietary cholesterol on gene expression in the liver utilizing
gene expression microarrays. In initial experiments we used a cDNA microarray to compare
gene expression between livers from C57BL/6J mice fed 0.5 versus 0.02 percent cholesterol
diets.
We found a novel gene, which was named StarD4, consistently down-regulated ~3-fold by
cholesterol feeding. We've used transgenic mouse models and cell culture reporter
assays to show this gene is regulated by the sterol-regulatory element binding protein
(SREBP) transcription factor. StarD4 is an interesting candidate gene in cholesterol
metabolism, as it consists entirely of a StAR-related lipid transfer (START) domain.
A START domain is a 210 amino acid globular protein domain with a hydrophobic cavity
for lipid binding. In collaboration with Steven Burley's lab, we solved the X-ray crystal
structure of StarD4 (see image), showing an internal cavity large enough to bind a
cholesterol molecule. There are fifteen proteins with START domains encoded by the human
genome, three of which have known lipid ligands. StAR, the prototype of this family,
delivers cholesterol to mitochondria in certain tissues to begin steroid hormone synthesis.
MLN64 also binds cholesterol and likely plays a role in cholesterol transport from lysosomes
to the endoplasmic reticulum. Not all START domains bind sterols, as PCTP is a specific
cytosolic phosphatidylcholine transfer protein. Based on homology to StarD4, we described a
subfamily of three novel START domain proteins, StarD4, StarD5, and StarD6, which share ~30%
amino acid identity. These proteins can shuttle cholesterol among subcellular compartments
and current studies seek to determine the roles of these proteins in intracellular cholesterol
metabolism.
StarD4 and StarD5 are expressed in most tissues, with highest levels in
the liver, a tissue that plays a central role in cholesterol homeostasis. While StarD4
expression levels are regulated by cholesterol, StarD5 expression appears regulated by other
factors. When cells are subject to stress affecting the endoplasmic reticulum, StarD5
expression is induced. StarD4 and StarD5 may have different roles since they are subject
to different regulatory influences. Likewise, StarD6 likely functions in male fertility,
as its expression is limited to sperm and sperm precursor cells in the testis. In addition
to in vitro and cell culture experiments, mouse models with gene knockouts by homologous
recombination are being generated to shed more light on the physiological functions of StarD4,
StarD5, and StarD6.
In subsequent experiments aimed at identifying more novel genes involved in
cellular cholesterol metabolism, we used Affymetrix oligonucleotide
microarrays to identify genes in mouse liver that were regulated by a one
week, high cholesterol diet. Sixty-nine genes were consistently regulated
by dietary cholesterol in an analysis of three microarray experiments. Among
these genes were a number of known SREBP and LXR target genes. SREBP
regulates genes involved in cholesterol biosynthesis and uptake and LXR
regulates genes involved in cholesterol catabolism. A number of the novel
regulated genes were investigated in confirmatory RT-PCR experiments, a time
course of cholesterol feeding, in SREBP transgenic mice, and in mice treated
with an LXR agonist. In this analysis, we were able to identify three novel
putative SREBP target genes and three novel putative LXR target genes. Work
is now aimed at determining the function of two of these genes, Adam11 and a
newly cloned gene of the subtilisin protease family, Pcsk9.
Since Pcsk9 was cloned in our laboratory as a gene regulated by dietary
cholesterol, we hypothesized that Pcsk9 may have an important role in
cholesterol metabolism. This hypothesis was supported by work from other
laboratories that identified Pcsk9 mutations associated with a form of
familial hypercholesterolemia. In order to study the role of Pcsk9 in
cholesterol metabolism in mice, we used adenoviral mediated overexpression
of Pcsk9. These studies demonstrated that excess Pcsk9 leads to an increase
in plasma LDL cholesterol levels with normal HDL cholesterol levels. This
effect is due to a decrease in liver LDL receptor protein with normal LDL
receptor RNA levels. Using human hepatoma cells, we further showed that
Pcsk9 induces the degradation of the LDL receptor within the cell by a
non-proteasomal mechanism that likely involves the lysosome. Further
research is being conducted to increase our understanding of Pcsk9's role in
regulating LDL cholesterol levels.
Researcher:
Marc Waase, mwaase@rockefeller.edu
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