Title of Team Project:
Bridging the In Vitro – In Vivo Gap: Probing mechanisms of the control of protein-lipid and protein-protein interactions in adipogenesis and intracellular lipid signaling.
In 24 words or less this project will:
This project will train students
to characterize lipid-protein and protein-protein interactions both in
isolation (in vitro) and in living cells (in vivo).
Primary Faculty Co-Advisors:
Vince J. LiCata
Ph.D. – Biophysical Chemist
Jacqueline M.
Stephens, Ph.D. – Cell Biologist
Off-campus Participant:
Not yet
identified. The proteins to be studied
in this project are currently a focus of a number of pharmaceutical companies
due to their involvement in the development of obesity, type II diabetes and
atherosclerosis. Drs. LiCata and
Stephens have numerous colleagues at Bristol Meyers Squibb, Pfizer, Monsanto,
Merck, 3D-Pharmaceuticals, and Glaxo-Wellcome.
A suitable participant will be identified within the first year of the
project.
Prospectus:
Intracellular
lipid signaling, lipid metabolism and its regulation, and lipid linked cellular
differentiation are all complex biological processes involving a wide array of
intracellular macromolecules. This IGERT
team will focus on several proteins which are central players in these
inter-related processes: adipocyte lipid
binding protein (ALBP), liver fatty acid binding protein (LFABP), and
peroxisome proliferator activated receptors gamma and alpha (PPARg and PPARa). On this team students would utilize
biophysical and macromolecular approaches to characterize the interactions of
these proteins with each other and between these proteins and their lipid
ligands, and then use this information to begin to understand and control the
specifics of these interactions within living cells.
ALBP
and LFABP are small (14.5kDa) b-barrel proteins that bind and transport
a wide variety of intracellular lipids.
ALBP is exclusively expressed in fat cells (adipocytes) and in
macrophages, while LFABP was first isolated from liver, but is also expressed
in a variety of other cell types (1).
The tertiary structures of the two proteins are nearly identical, but
their lipid binding characteristics, their interactions with synthetic
membranes, and their surface charge distributions are all completely different
(1-4).
Peroxisome
proliferator-activated receptors (PPARs) are nuclear transcriptional regulators
that are activated by a variety of fatty acids and fatty acid analogs (5). The PPARs are primary regulators of lipid
metabolism and lipid storage. Because of
their documented primary roles in adipogenesis (6), and in the development of
type II diabetes and atherosclerosis (7,8), the PPARs are currently a major
pharmaceutical target for agonist design.
There are 3 PPAR subtypes in mammals: a, d, and g. The a and g subtypes are
highly conserved across species, and are primary regulators of lipid metabolism
and storage. PPARg
is a key initiator of adipocyte differentiation (6), and is a high affinity
receptor for synthetic antidiabetic agents (7).
PPARs
a
and g in liver cells have recently been shown to
interact directly with LFABP in vivo, and this interaction is postulated to be
a central element of a lipid signaling pathway (9). Preliminary evidence from this IGERT team
suggests that direct interaction between ALBP and the PPARs does not occur
(experiments performed by A Joubert during the 2000 NSF-REU Summer Program,
prior to formation of this IGERT team). PPARg regulates
expression of ALBP in adipocytes, and it has been recently demonstrated that
ALBP is present in significant amounts in the adipocyte nucleus, and that its
presence modulates the activity of PPARg (10).
Thus, even if ALBP and PPARg do not
directly interact, their functions are linked via lipid transfer or via
competition for available lipid. A
long-range goal of this team is to address the mechanism of lipid transfer in
both the LFABP-PPAR interaction and the ALBP-PPAR functional linkage.
Some
of the specific aims of this IGERT project will be:
1)
To characterize the in vitro interaction thermodynamics of LFABP and PPARs a and g,
and to assay for the presence of direct interactions between ALBP and PPARs a and g,
using titration calorimetry and analytical ultracentrifugation. The involvement of electrostatic interactions
and water in the binding reactions will be investigated by quantitating the
binding as a function of ionic and osmotic conditions, respectively.
2)
To perform crystal structure-based docking calculations on the LFABP-PPAR
partners, and the ALBP-PPAR partners to predict potential docking orientations
and identify potential mutation targets for modulating the interactions of the
proteins. Calculations already performed with ALBP and LFABP alone suggest that
helix 2, which is near the ligand entry/exit portal on both proteins, is a
strong candidate for mutagenesis studies.
While most of the charge surfaces of the two proteins are similar, this
region is highly positively charged in ALBP but highly negatively charged in
LFABP (2). Specifically, glu26, asp27,
and asp34 in helix 2 of LFABP would be changed to alanines in anticipation that
this would weaken or abolish LFABP’s direct interaction with PPARs a and g.
3)
To investigate the mechanism of lipid transfer and/or competition for lipid
between the LBPs and the PPARs. Using
fluorescent lipids, we will follow the kinetics of transfer of lipids between
LFABP and PPARs a
and g and between ALBP and PPARs a and g. Experiments will be performed in both
potential transfer directions: mixing of lipid bound LBPs with unligated PPARs,
and mixing of lipid bound PPARs with unligated LBPs. Accomplishment of this specific aim will
require careful characterization of the direct binding of the labeled lipids to
the LBPs and PPARs in isolation.
4)
The conditions identified in vitro that result in the modulation of lipid
binding to the LBPs and/or PPARs will be tested in vivo by altering the ionic
and osmotic environments of cultured adipocytes, macrophages, and hepatocytes.
A defined set of functionally relevant assays will be performed to determine
how these changes have altered intracellular processes specifically linked to
LBPs and PPARs, including:
a) assay of relative expression and total intracellular
concentrations of LBPs and PPARs by Western blot analysis.
b) assay of nuclear versus cytosolic distribution of LBPs
(via cell fractionation
and Western blot analysis). The Stephens laboratory
routinely fractionates adipocytes to examine the subcellular distribution of a
variety of different proteins including the PPARs. These studies will allow us
to determine if the import and export of LBPs into the nucleus is regulated by
PPAR and LBP ligands.
c) assay of total intracellular free
fatty acid concentrations
d) examine the interactions of LBPs
and PPARs over a time course of adipocyte development. These experiments will
be performed in the 3T3-L1 cells and protein interactions will be examined over
a time course of differentiation to determine if there are different
interaction in differentiating cells and mature adipocytes.
5)
Site directed mutants of LFABP and ALBP which have been shown in vitro to alter
lipid binding and/or LBP-PPAR interactions in vitro will be introduced into
cultured cells via stable or transient transfections to determine if predicted
enhancement or inhibition of processes involving LBP-PPAR linkages are
confirmed in vivo.
Number of IGERT apprentices to be
recruited by this team and departments from which they will probably
originate:
Up
to two students will be recruited to serve on this research team. Allison Joubert, an incoming Biological
Sciences Ph.D. student, has already agreed to be part of this team.
Consistency with the Macromolecular
Education, Research and Training theme:
Proteins
are high-performance macromolecules.
Their study requires many of the same concepts (radius of gyration,
polyelectrolyte effects, solution thermodynamics and spectroscopy) needed for
polymer research. Design of
protein-based medical devices requires expertise in biochemical, polymer, and
cellular physiology research.
Understanding the forces that control protein function in the cellular
environment requires understanding their interaction with solvent and the
impact of all intensive and extensive system properties known to differ between
the in vitro and in vivo environments.
Testing the predictions of in vitro investigations requires the ability
to work directly in the living intracellular environment. A basic understanding of the mechanisms
underlying protein structure and function is required for the exploitation of
these systems for other applications.
How does the proposed project form a
vector cross-product of existing research themes by the participants?
Existing Research Directions: The
research effort in the LiCata laboratory is primarily focused on the
examination of the biophysical properties of soluble proteins. The primary
focus of the Stephens’ laboratory is centered on the molecular pathogenesis of
insulin resistance and type II diabetes in adipocytes.
New research direction: In this
research proposal, we will exploit the strengths of both laboratories in the
examination of proteins involved in lipid transport, lipid metabolism, and
lipid regulation of gene expression. The
implications and findings from in vitro studies in the LiCata laboratory will
be tested directly in living cells in the Stephens laboratory. We
predict that these studies may lead to insights into the molecular mechanisms
regulating energy homeostasis and may contribute to understanding the defects
underlying obesity and type II diabetes.
How do students benefit from the
team-oriented research, beyond what would be available to them from either
advisor separately:
These two major approaches to understanding a particular biological system: molecular biophysics and cellular biology, are rarely ever bridged, even via collaboration between two different laboratories. Training on this IGERT team will give students the training and understanding to span this divide. Students trained across these disciplines will be poised to make advances in the understanding and control of macromolecular interactions within living systems.
Briefly describe the level of support
available to individual faculty or off-campus participant (i.e., without IGERT)
Dr. LiCata is currently funded by the National Science Foundation and the Louisiana Board of Regents. Dr. Stephens is currently funded by the National Institutes of Health and the American Diabetes Association.
Interdisciplinary strengths of the team
project
The synergy of biophysics (LiCata) with cell biology (Stephens) will lead to a marked increase in the capabilities and breadth of research in both laboratories and will likely enhance our understanding of protein/protein interactions which contribute to the pathogenesis of obesity and type II diabetes. Moreover, these studies will allow the advanced training of students in both of these important areas of investigation.
Commitment of faculty and of-campus
participants to work side-by-side with apprentice-level students. The
principal investigators on this research team are fully committed to the
overall educational paradigm of this IGERT proposal. Dr. LiCata is an assistant professor in the
Departments of Biological Sciences and Chemistry and works on a daily basis
with his research group and personally mentors his students in carrying out
experimental protocols and also the “business” of an active experimentalist. Dr. Stephens, an associate professor in the
Department of Biological Sciences, also maintains an active personal research
program and interacts daily with her laboratory members. Both principal investigators are excited
about the possibility of personally working side-by-side on a project of 2-6
weeks with students in the “master-apprentice” model presented in this IGERT
proposal. At the very least, this
time commitment would be fulfilled around Christmas (1 month break between
semesters), during the summer semester, or taking by advantage of Spring Break.
References