Scientific Report 2007
Mechanism of Activation of Nuclear Receptors
P.R. Griffin, S.A. Busby, M.J. Chalmers, S. Prasad, S.Y. Dai
use a wide range of technologies to study ligand activation of nuclear receptors.
Previously, we focused heavily on peroxisome proliferator-activated receptor γ
see following), but we are beginning to study several orphan nuclear receptors that
are implicated in a variety of disorders. Of particular interest are the orphan
receptors liver receptor homolog-1 and steroidogenic factor-1. We are building the
appropriate tools to begin comprehensive mechanistic studies of these 2 receptors.
Mechanistic Studies of Ligand Activation of PPARγ
is a multidomain ligand-dependent transcription factor. Activation of PPARγ
is regulated by binding of ligands to the receptor's ligand-binding domain,
which induces a change in the conformational dynamics of the domain-mediated dissociation
of corepressor molecules and forms suitable neoepitopes for binding coactivator
molecules. Ligands of PPARγ
are characterized as full or partial agonists on the basis of their ability to transactivate
target genes. Full agonists have maximal transcriptional activity, whereas partial
agonists have moderate activity. Although the structural and molecular determinants
of full-agonist regulation of PPARγ
have been studied in detail, the determinants for partial agonists have not been
completely characterized. We are using structural, biochemical, and cell-based techniques
to examine the mechanism of regulation of PPARγ
transcriptional activity by partial agonists.
exchange (HDX) coupled with mass spectrometry was used to characterize ligand-dependent
structural and dynamic changes in PPARγ.
Ligand-induced protection of amide exchange in a transcriptional complex composed
retinoic X receptor α,
and either steroid receptor coactivator-1 or steroid receptor coactivator-3 were
probed by using automated solution-phase HDX. We used fluorescence-based assays
to determine recruitment of coactivators and to measure the affinity of the receptor
heterodimer composed of PPARγ
and retinoic X receptor α.
2-hybrid genetic screen was used to probe the molecular determinants of ligand-dependent
We found that
the magnitude of PPARγ
agonism is regulated by coactivator recruitment selectivity of p160 coactivators.
The functional relevance of the genetic screen was further confirmed in a 3T3-L1
preadipocyte differentiation assay as an in vitro model of adipogenesis. We found
that conversion of the full-agonist phenotype to the partial-agonist phenotype and
vice versa was a function of the availability of specific p160 coactivators.
proteomic and genomic differential analysis, we have extended these studies to examine
the molecular components of PPARγ-mediated
insulin sensitization and adipogenesis after treatment of preadipocyte cells with
full and partial agonists of PPARγ.
Our goal is to determine unique components of the insulin sensitivity pathway and
dissociate them from components of the proadipogenic pathways that lead to the adverse
side effects common after therapy with PPARγ
full agonists. To complete this study, we are developing new strategies to improve
the detection of membrane- and lipid-associated proteins, and we are using new mass
spectrometry methods to measure the samples.
In other studies,
we are using coactivators as chemical tools to generate desired functional responses
and distinguish beneficial functions from adverse functions, a novel therapeutic
avenue for treating insulin resistance that has not yet been exploited. Our goals
are to determine the structure-activity relationships between PPARγ
ligands and their coactivator recruitment selectivity and to obtain PPARγ
ligands with preferences for specific coactivators. To this end, we have developed
a validated homogenous time-resolved fluorescence assay for ligand-dependent recruitment
of the coactivator to PPARγ
for a large-scale high-throughput screen to identify coactivator-selective agonists
of the receptor. We will do 3 different primary screenings of the National Institutes
of Health small-molecule chemical library. In each screen, we will target PPARγ
association with a different coactivator to obtain coactivator-specific agonists.
The results obtained from this research will provide molecular insight into how
recruitment of coactivators modulates activation of PPARγ
and will shed light on the role of specific coactivators in the pharmacologic behavior
Mechanistic Studies of Ligand Activation of the Estrogen Receptor
with scientists at Eli Lilly and Company, Indianapolis, Indiana, we used HDX to
characterize the estrogen receptor. As drug targets, estrogen receptors play important
roles in the treatment of multiple diseases, including breast cancer and osteoporosis.
Tamoxifen and raloxifene, modulators of estrogen receptors approved by the Food
and Drug Administration, have intriguing mixed agonism and antagonism effects depending
on the target tissue. Structural studies have revealed differences between complexes
consisting of the estrogen receptor and an agonist and complexes consisting of the
receptor and an antagonist and have provided insight into how these ligands interact
with the receptor. We have used HDX to examine an array of chemical compounds that
have different degrees of agonism/antagonism. We found an excellent correlation
between HDX profiles and pharmacologic properties, and we were able to classify
estrogen receptor ligands on the basis of HDX signatures. Discoveries derived from
this study will help in understanding tissue-specific activities of drugs targeted
to estrogen receptors and will facilitate future drug development programs.
S.A., Chalmers, M.J., Griffin, P.R.
Improving digestion efficiency under H/D exchange conditions with activated pepsinogen
columns. Int. J. Mass Spectrom. 259:130, 2007.
B.D., Chalmers, M.J., Busby, S.A., Mader, C.C., Southern, M.R., Tsinoremas, N.F.,
Griffin, P.R. The Deuterator:
software for the determination of backbone amide deuterium levels from H/D exchange
MS data. BMC Bioinformatics 8:156, 2007.
P., Ayala, I., Busby, S.A., Chalmers, M.J., Griffin, P.R., Rocca, J., Nick, H.S.,
Silverman, D.N. Structural
mobility in human manganese superoxide dismutase. Biochemistry 45:8209, 2006.