Project: Research project

Project Details


DESCRIPTION (Adapted from the Investigator's Abstract): Recently, it has
been shown that peroxisome proliferators, an important class of tumor
promoters, activate novel members of the steroid hormone receptor
superfamily, the peroxisome proliferator-activated receptors (PPAR).
Several subtypes of PPAR have been discovered (alpha, beta, gamma), although
the predominant liver subtype (PPAR alpha) is the focus of the present
investigation. The present proposal will examine the sequence of events
initiated by peroxisome proliferators and culminated in altered gene
expression. First, the appropriate molecular tools will be developed for
the study of PPARalpha function. Recently, specific antibodies to PPARalpha
and an epitope-labeled receptor (PPARalpha-Flag) have been developed which
will help characterize the alpha subtype among the other possible forms.
Photoaffinity ligand probes and stable cells lines which express
PPARalpha-Flag will be pursued. Second, the subcellular localization of
PPARalpha will be examined. Whether PPARalpha is primarily cytosolic in the
inactivated state is an important consideration in receptor function.
Confocal microscopy will be used to examine PPARalpha distribution in the
inactivated and peroxisome proliferator-activated states. Third, the
composition of the PPARalpha-protein complex will be determined. The
specific interactions of PPARalpha with heat shock proteins and other
factors often associated with steroid hormone receptors will be examined
using several complementary approaches. Comparison of the receptor complex
before and after peroxisome proliferator treatment will be important in the
overall understanding of the signal transduction pathway. Fourth, determine
if phosphorylation is an important regulator of PPARalpha function.
Chemical and enzymatic digestion followed by phosphoamino and phosphopeptide
analysis will be used to examine which residues are phosphorylated on
PPARalpha and which are differentially affected by peroxisome proliferator
treatment. The importance of each phosphorylated residue will be examined
by introducing specific mutations in PPARalpha. Last, the process of
transport of PPARalpha across the nuclear membrane will be examined. If
confocal microscopy verifies that PPARalpha is present in the cytosol, the
process that governs its transport into the nucleus will be examined.
Fluorescently-labeled PPARalpha will be constructed and in vitro nuclear
uptake studies implemented and identification of proteins acting as
chaperones will be pursued. Taken together the proposed studies will
greatly increase our knowledge of PPARalpha's mechanism of regulation and
hence our understanding of an important class of carcinogen.
Effective start/end date12/1/964/30/09