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NHLBI Proteomics Center
> Project Summaries
Yale/NHLBI Proteomics Center Project
Summaries Yale/NHLBI
Proteomics Center Project Summaries
A1. Development of Protein and Phosphoprotein Profiling Technology
K. Stone, W. McMurray, W. Konigsberg, K. Williams (Mol. Biophys. &
Biochem.) and C. Horvath (Chemical Engineering)
To bridge the gap between the enormous
potential of mass spectrometry to quantify protein expression and what is
available to NHLBI-supported investigators at Yale, we propose to substantially
improve and implement three protein profiling technologies:
- Isotope-coded affinity tag (ICAT) analysis
of protein expression - which is based on affinity isolation of cysteine-containing
tryptic peptides modified in vitro with a "light" or "heavy" stable isotope
version of the biotin-containing, ICAT reagent.
- Phosphoproteome profiling - which is based
on comparative analysis of affinity-isolated phosphopeptides obtained from
tryptic digests of control versus experimental samples.
- LC/MS analysis of tryptic digests of complex
protein extracts - which is based on comparative analysis of "3D" (scan number
versus m/z versus intensity) plots. This approach might be most effectively
used on the non ICAT-labeled tryptic peptide effluent from the avidin column
where it could be used to profile other protein post-translational
modifications as well as proteins which do not contain cysteine.
Two challenges that will be addressed are
improving the software available for analyzing MS-based protein profiling data
and extending the dynamic range of this technology so that lower abundance
proteins, which often play important regulatory roles, will be more easily
quantified.
A2. Engineering RNA Molecular
Switches That Respond To Protein Targets
R. Breaker (Molecular, Cellular & Developmental Biology)
The objective of this project is to engineer
RNA switches that serve as precision biosensor elements for the multiplex
detection of protein targets. The activities of ribozymes and deoxyribozymes can
be engineered to perform as allosteric enzymes that are modulated by specific
effectors. These RNAs undergo catalytic activation when bound to specific
effectors such as nucleotides, second messengers, enzyme cofactors,
pharmaceutical agents, proteins, and oligonucleotides. We propose to expand on
our initial successes in switch engineering to create ribozymes that are
modulated by binding to polypeptides of biological importance. Specifically, we
expect that designer molecular switches can be created that would enable the
construction of new RNA biochips or other biosensor systems for the large-scale
analysis of proteins. To this end we will pursue a two-part strategy for
creating protein-responsive RNA switches. In the first approach, we will employ
in vitro selection with a population of hammerhead self-cleaving ribozyme
constructs. These constructs (containing trillions of sequence variants) will be
subjected to allosteric selection to enrich for those rare molecules that
selectively recognize added protein targets and that subsequently catalyze RNA
cleavage. In the second approach, we will use a high-speed self-ligating
ribozyme as a catalytic platform for the allosteric selection of new
protein-dependent ribozymes. The RNA ligation activity of this ribozyme will
permit the high-throughput isolation of ribozymes for multiple protein targets
simultaneously using robotic selection protocols. Ultimately, these designer
RNAs will be immobilized to serve as “ribozyme pixels” that report the presence
or absence of specific protein targets.
A3. Biostatistics and
Bioinformatics
Group Leader, P. Miller (Medical Informatics)
A3a. Quality Control, Quality Assurance, And
Statistical Analysis Of Protein Expression Data, H. Zhao (Epidemiology
And Public Health)
The overall objective of this project is to
develop statistical methods for quality control and analysis of protein
expression analysis, and to collaborate with other investigators to apply these
methods to proteomics studies. More specifically, we will develop a statistical
model for the mass spectrometry data obtained from the ICAT technology through
controlled experiments with varying conditions and samples. These statistical
models will allow us to build further models that incorporate biological
variations and to develop a sound statistical procedure to assess the
statistical significance of the observed differences between two or more
conditions. In addition, we will implement various clustering algorithms and
develop statistical methods for identifying biomarkers to predict clinical
outcomes. The validity of our methods will be tested through our collaborations
with other investigators. We will also evaluate the Micromass MCAT software and
other available software and compare the results based on other methods and
those based on our developed models in an effort to improve our overall
approach. The computer programs developed from this project will be extensively
tested both with simulations and with data generated from this project, and
these programs will be integrated into the proposed NHLBI/YED database. These
programs will also be made available to the general scientific community at
Dr. Zhao’s group web site.
A3b.NHLBI/Yale Expression Database (YED): An
Interoperable Protein And mRNA Expression Database, K. Cheung (Medical
Informatics)
This
project will build an interoperable protein and mRNA expression database to
manage and analyze the large data sets that will be generated to quantify the
relative levels of expression of thousands of ICAT labeled peptides in hundreds
of samples annually. This database, the NHLBI/Yale Expression Database (YED),
will enable protein profiling data to be: (i) efficiently stored, queried, and
distributed; (ii) correlated with mRNA expression data in the
Yale Microarray Database (YMD),
which is currently being beta-tested by several laboratories at Yale; (iii)
linked with other related databases for functional annotation; and (iv) coupled
with a variety of analysis tools. The NHLBI/Yale Expression Database will be
made available to other NHLBI and non-NHLBI investigators both at Yale and
beyond. Capturing protein profiling data in a standardized database will greatly
facilitate the development of software tools for data retrieval, data sharing,
and analysis. The developers of YED data analysis tools can take advantage of
standardized structures to write programs to process the data efficiently. Data
returned by user’s queries can be directly input to such programs.
Alternatively, YED data can be exported as files in formats tailored to the
individual requirements of existing analysis programs. By linking YED to a
variety of analysis programs and creating a Web interface, biomedical
investigators who have little experience in biostatistics and computing can
readily carry out data analysis.
A3c. Functional And
Interrelative Proteomics
M. Gerstein (Mol. Biophysics & Biochem.)
We will develop methods and tools for analyzing
protein expression data. These methods will be principally based on integrating
protein expression values with other genomic information -- namely, mRNA
expression, protein-protein interactions, protein family and function,
occurrence of protein pseudogenes. We will start using available yeast data, we
will develop a formalism for scaling mRNA and protein data together, both for
individual genes and then across multi-gene categories of information. Then we
will expand our analysis formalism from yeast to human. We will initially focus
on interactions and networks relating to transcription factors in MPRO cell line
(in collaboration with S Weissman). Our protein analysis system will be
integrated with the proposed NHLBI/YED database (in collaboration with K Cheung
and K Williams). In particular, with K Williams we will try to identify and then
write algorithms needed to streamline ICAT data analysis and to make it as
suitable as possible for analyzing multiple samples. By the end of the grant,
one question that we hope to investigate in detail is the relationship between
protein expression, gene expression, and pseudogene occurrence. An mRNA species
can be reverse transcribed, post splicing, and inserted back into the genome to
create a functional template for a protein or a processed pseudogene. It would
be interesting to compare the expression of these processed mRNAs and their
homologs with the associated protein abundance, elucidating any possible control
splicing and mRNA processing has on protein abundance.
A4. Global Proteomic
Approaches To Hematopoietic Differentiation
Group Leader, S. Weissman (Genetics)
A4a. Molecular And Functional Analysis Of
Myeloid Differentiation
S. Weissman (Genetics)
Our overall goal is to obtain a genome wide
molecular description of lineage commitment and maturation for the hematopoietic
system, with emphasis on myeloid development. We have already accumulated large
amounts of data on RNA expression levels in normal neutrophils and stem cells,
as well as in the EML and MPRO cell lines at various stages of differentiation.
We have also obtained data on changes in protein levels, based on mass
spectrometric identification of spots from 2D gels. Our hope is to obtain a much
more complete determination of the amounts of most proteins in these cells and
to estimate stability of most proteins as a function of the site of
differentiation. We then plan to manipulate patterns of lineage determination
and development by use of RNAi inhibition of specific transcription factors and
kinases. mRNA and protein levels will be measured at various times after
induction of differentiation. As a complement to these experiments individual
protein levels will be transiently increased by exposure to proteins associated
with specific cell uptake mediators. To study lineage determination we will use
the EML cells as well as expanded normal hematopoietic precursor cells that
spontaneously differentiate into multiple lineages. We will test the hypothesis
that differentiation is associated with stabilization of specific chromatin
structures by selectively inhibiting DNA methylases, histone deacetylases or
acetylases and also by altering levels of polycomb or trithorax complex
proteins.
A4b. Molecular And Functional
Correlates Of Myelodysplasia
Arati Khanna-Gupta, and N. Berliner (Medicine)
MDS is a stem cell disorder characterized by
ineffective and disordered hematopoiesis and increased intramedullary apoptosis.
The challenge of MDS is two-fold, in that patients may die of infectious
complications of the quantitative and qualitative abnormalities of their mature
neutrophils, or they may die of leukemia. Analysis of MDS is therefore most
appropriately directed at understanding both the abnormalities of the
myelodysplastic neutrophil and the defect in the myelodysplastic stem cell.
We propose to examine global gene expression of
MDS cells in two ways. First, we will characterize the gene expression
correlates of abnormal function in neutrophils from MDS patients. We will assay
oxidative burst, granule secretion, and phagocytosis to define the abnormalities
of neutrophils before and after exposure to bacterial antigens. We will
correlate this with expression profiling using microarrays, differential
display, and the MS-based protein profiling described in this application.
Second, we will examine defective maturation of the primary stem cells from MDS
by analysis of patterns of gene expression during cytokine-induced
differentiation of primary marrow stem cells. These studies continue our
collaboration with Dr. Weissman on global gene expression of normal neutrophils
and cytokine-induced differentiation of myeloid cells.
We have identified over 40 patients being
treated for MDS at the West Haven VA Hospital. Peripheral blood and bone marrow
will be obtained for functional assays, RNA isolation, and protein extracts, and
will also be analyzed by cytokine-induced maturation.
A4c. Downstream Targets of the
Homeodomain Gene Pitx2 in Hematopoietic Cells, B. Forget (Medicine)
The homeodomain gene Pitx2 is
thought to play a role in hematopoiesis because it is preferentially expressed
in primitive hematopoietic stem/progenitor cells, as well as in stromal cell
lines that support the survival and proliferation of such cells in vitro. The
goal of this project is to identify proteins that are differentially expressed
in primary hematopoietic cells and stromal cell lines where the Pitx2 gene is
active compared to similar cells where the gene is inactive. In this way, we
hope to identify downstream target genes and gene products, the expression of
which is regulated by the transcription factor Pitx2. The model system to be
used is the Pitx2 knockout mouse model that is already present in our
laboratory. Heterozygous (Pitx2 +/ ) knockout mice will be mated to generate
both wild type (+/+) and homozygous null ( / ) embryos from which fetal livers
will be obtained as a source of cells for total cellular and nuclear proteins
for analysis. The fetal liver is a hematopoietic organ consisting primarily of
erythroid precursor cells at embryonic day 13.5. Homozygous embryos die beyond
that age, so there is no primary adult source of ( / ) tissue or cells. In
addition, embryonic stem (ES) cells homozygous for the Pitx2 gene disruption
that were developed in our laboratory will also serve as a source of proteins
for comparative analysis to proteins isolated from wild type ES cells. Finally,
we will also extract total cellular and nuclear proteins from the stromal cell
lines that we have shown to strongly express Pitx2 (line AFT024:supportive of
hematopoiesis)) or markedly underexpress it (line 2018: non supportive of
hematopoiesis). The extracted proteins from these different cell sources will
then be provided to the Protein Analysis Core for comparative protein profiling.
The results of profiling analysis will be compared to results of differential
expression of mRNA/cDNA from the same cell sources. These experiments will
hopefully identify novel proteins that are important for hematopoiesis.
A4d. Differential Protein
Expression During Early Hematopoietic Differentiation And Mobilization, D.
Krause (Laboratory Medicine)
In order to better understand the
mechanisms that control hematopoietic stem cell (HSC) self-renewal and
differentiation, we need to determine the changes in gene expression that occur
as hematopoietic stem cells initiate the differentiation process. Only then can
we dissect the molecular mechanisms responsible for this transition. We are
currently identifying the genes whose expression is tightly coordinated with the
earliest stages of HSC differentiation, by performing microarray analyses in
which we are comparing the gene expression profiles of highly purified human
CD34+CD38-, CD34+CD38+, and CD34- subpopulations. Human hematopoietic stem cells
express CD34, but do not express CD38. Fewer than 2% of CD34+ cells in the bone
marrow or mobilized peripheral blood are CD38-, but this population contains all
of the HSC's as determined using xenogeneic assays for hematopoietic
engraftment. Here, we propose to compare and contrast the gene and protein
expression patterns of these different purified subpopulations of human bone
marrow derived cells using microarrays as well as newly-emerging powerful
technologies in proteomics. Specifically, we will determine the extent to which
the protein profile varies between primary human CD34+CD38-, CD34+CD38+, and
CD34- hematopoietic stem and progenitor cells and we will assess the degree to
which expression levels for mRNA and protein compare for genes that are
differentially expressed in each of these human subpopulations. The proposed
studies will provide a new context within which to view a normal and
pathological hematopoiesis.
A4d. Characterization Of
Evi-1-Induced Changes In Protein Expression During Myelopoiesis, A. Perkins
(Pathology)
EVI1 is a zinc finger protein
involved in myeloid leukemia. We have shown that EVI1 interferes with normal
myelopoiesis as demonstrated by its ability to disrupt differentiation of a
cultured myeloid progenitor cell line (termed EML). Using mRNA expression
profiles, we have identified ~50 major differences that are due to Evi1
expression in myeloid cells. Based on these data, we hypothesize that EVI1
overexpression is causing the continued expression of key nuclear factors
involved in the maintenance of the pluripotent state. To assess this more
accurately, we propose to quantify nuclear proteins in EML cells that are
induced to differentiate either with or without expression of EVI1 and to then
identify those proteins that are differentially expressed using the ICAT/MS
methodology. We expect that differences in nuclear factors present in the two
populations will provide vital clues regarding the mechanism by which EVI1
interferes with normal blood cell maturation. We are also interested in
identifying the proteins with which EVI1 interacts. We will purify EVI1
complexes using the tandem affinity purification (TAP) system (see ref. 104 in
our application). The TAP system, in combination with LC/MS/MS will allow us to
determine the identities of these proteins. Further studies with mutant forms of
EVI1 will then be carried out using the ICAT technology to quantify the relative
amounts of proteins in each of the respective complexes - thus mapping the
portions of EVI1 protein required for the formation of these complexes. With
these projects, we hope to uncover key aspects of EVI1 molecular biology
important for leukemogenesis.
A5. Hypertension
A5a. Characterization Of A
Regulated Paracellular Conductance Involved In Hypertension, R. Lifton (Chair,
Genetics)
Hypertension is the most common disease in the United States. Its cause is
largely unknown. We have shown that mutations in WNK1 and WNK4, novel serine-threonine
kinases, result in hypertension (Science, 2001), implicating a previously
unknown signaling pathway in blood pressure homeostasis. The upstream regulators
and downstream targets of these kinases are presently unknown.
We are taking a proteomic approach
to this problem. We are engineering mice with either constitutively increased or
absent WNK signaling; we are also producing cell lines with inducible WNK
activity. In both systems, the effects of gain or loss of WNK activity will be
investigated using the ICAT/MS protocols for relative protein and phosphoprotein
levels in kidneys and cell lines with normal, increased, or absent WNK
signaling. We anticipate that increased WNK function will result in increased
phosphorylation of downstream targets of WNKs, potentially directly identifying
these elements of the pathway. We also anticipate that altered WNK function will
result in altered protein expression and/or phosphorylation of other proteins in
the cognate signaling pathway, potentially identifying both upstream and
downstream elements of the pathway. The role of such proteins will be further
evaluated by biochemical studies, for example examining the ability of WNKs to
directly phosphorylate putative downstream targets. This proteomic approach has
the capacity to rapidly identify elements of the WNK signaling cascade and to
provide important new insight into the molecular mechanisms underlying human
hypertension. Members of this pathway may represent new targets for development
of novel antihypertensive agents.
A6. Vascular Biology
A6a. Protein Expression
Profiling And Phosphoproteome Analysis Of Lipid Rafts During Angiogenesis, W.
Sessa (Pharmacology)
Lipid rafts or specialized rafts
domains, called caveolae, are sites of signal transduction in most cells. The
goals of this specific project are to:1) quantify the proteins enriched-in or
depleted from highly purified lipid rafts isolated from quiescent endothelial
cells, proliferating endothelial cells or cells differentiated into tube-like
structures in a three dimensional matrix, and 2) identify/quantify major
phosphoproteins in lipid rafts isolated from endothelial cells under the above
conditions. We have devised a method to purify lipid rafts domains from vascular
endothelial or smooth muscle cells using a combination of cell biological
approaches and will apply ICAT based derivatization for protein quantification
and identification.
A6b. Integrin
Engagement-Mediated Alterations In T Cell Hur Protein:Protein Interactions And
Posttranslational Modifications, J. Bender (Internal Medicine)
Description: Intercellular
adhesion is of paramount importance in a majority of immune responses, including
those that involve leukocyte-endothelial cell interactions. The leukocyte b2
integrin LFA-1 is a key T lymphocyte adhesion receptor which, when engaged,
leads to rapid nuclear-to-cytoplasmic translocation of an RNA binding protein,
HuR, stabilizing otherwise labile activation mRNA transcripts encoding immune
cytokines. The two major potential components of LFA-1-induced changes in HuR
are: (1) stimulus-dependent HuR protein-protein interactions, and (2) induced
posttranslational modifications, specifically phosphorylations.
Purified human T lymphocytes will
be activated through LFA-1 to identify those proteins with modulated cytoplasmic
levels upon LFA-1 engagement, including HuR as a positive control. We will use
this system as one of the first cellular protein extracts that will be subjected
to ICAT-based analysis of comparative protein expression. Parallel HuR
immunoprecipitates will be submitted for protein profiling to quantify the
activation-dependent increase in HuR and identify proteins bound to HuR This
will be one of the first "real" samples subjected to protein profiling. It will
be important to understand if there exists a threshold level of protein-protein
interaction for translocation.
In the second part of the work, we
will assess which cellular proteins, including HuR, are inducibly phosphorylated
in LFA-1-activated cells. The expectation is that HuR will be one of those
proteins. Those phosphopeptides which are elevated will be identified in a
data-dependent, LC-MS/MS experiment, which also should identify the sites of
phosphorylation. Protein-protein interactions and phosphorylation are likely
interdependent mechanisms in stimulus-initiated HuR function.
B. Development Of
Cell-Permeable, Synthetic Biotechnologies For Blocking Specific Protein: Protein
And Protein Post-Translational Modifications
Team Leader, W. Sessa (Pharmacology)
B1. Design Of
Organelle-Specific Peptides For Therapeutic Disruption Of Protein-Protein
Interactions, W. Sessa (Pharmacology)
Signal transduction largely occurs
through regulated protein-protein interactions and the extent of protein-protein
interactions can be regulated by the subcellular localization of the proteins in
question. The targeting of proteins to specific intracellular domains is
determined by organelle specific signal peptides that act as molecular zip
codes. Therefore, the goals of this project are: 1, to develop cell permeable
peptides based on AP and oligomeric D-arginine that will target to different
subcellular compartments in cells; and 2, to demonstrate that organelle specific
targeting influences protein:protein interactions or signal transduction in key
intracellular domains. We believe that such technology will vastly improve the
efficacy and specificity of cell permeable peptides and enhance their utility
for disrupting local protein:protein interactions in vivo.
B2. Intracellular Delivery Of
Peptides, Proteins, And Nucleic Acids For Studying Cellular Function, D. Ward
(Genetics)
The overall objective of this
project is to develop peptide delivery systems to efficiently introduce
biologically active molecules (proteins, fusion peptides, chimeric proteins or
nucleic acids) into living vascular and hematopoietic cells. Preliminary studies
have shown that the addition of C-terminal lysine residues (8-16) to the cell
transduction peptide of the HIV TAT protein results in a peptide that rapidly
translocates DNA and RNA species across the plasma membranes via electrostatic
interactions. When the same TAT peptide is coupled to a single-pass
transmembrane peptide domain (TMD) the resultant chimeric molecule is inserted
into the plasma membrane in an oriented fashion such that effector sequences can
be positioned on the exterior or cytoplasmic sides of the membrane. We propose
to apply these delivery vehicles to study protein-protein interactions in
cultured cells and in vivo. TAT-K16 peptides will be evaluated for their
efficiency in delivering RNAi molecules to disrupt the expression of specific
complex-forming proteins while TAT-TMD constructs carrying specific protein
interaction domains will be constructed and tested for their ability to disrupt
interactions with (or between) membrane- associated proteins. TAT-TMD peptides
will be also used to introduce receptor proteins, natural and chimeric, into
receptor free cells or tissues.
Transducing peptides with cell
type and/or tissue specificity developed by other investigators in the Program
will be subjected to similar modifications (K-16, TMD) and tested both for
retention of cell/tissue specificity and their intracellular distribution. Our
initial efforts will focus on interrupting interactions between
membrane-associated caveolin-1, nitrous oxide synthase, and signal transduction
proteins.
B3. Development Of New
Technology To Discover Peptides That Reduce Inflammation
S. Ghosh (Immunobiology & Molecular Biophysics and Biochemistry)
The major objectives of our
research proposal are to develop large scale screening approaches to help a)
identify vascular cell- and tissue-specific targeting sequences; and b) identify
more efficacious inhibitors of the NF-kB signaling pathway to influence vascular
inflammation. We will devise specific cell-based reporter systems for screening
mixtures of peptides of random sequences to help identify and progressively
narrow down the actual effective peptide in the mixture. The basic rationale
behind this project is to identify peptide-sequences that are effective in
inhibiting the desired target pathway (NF-kB) or demonstrate the ability to
discriminate between different cells (macrophages versus endothelial cells),
without constraining ourselves with pre-conceived notions about which sequences
should be chosen based exclusively on domain minimization strategies.
B4. Protein-Based Vascular
Addressing And Targeted Cellular Internalization
Y. Huang, R. Hickey, and F.Giordano (Internal Medicine)
This project is premised on the
general hypothesis that there are specific peptide motifs that target the
translocation of macromolecules from the intravascular space across the
endothelium, and that also target internalization into parenchymal cells. We
propose that these motifs are distinct from general membrane permeability
peptides such as the HIV TAT protein, and that they act by interaction with
specific cellular receptors or adhesion complexes. We have created an array of
diverse peptide phage display libraries and additional techniques, and have used
these to identify peptide sequences that are capable of a) translocation across
the endothelium, b) internalization into parenchymal cells (see Fig. 7., page 42
of proposal), and c) of carrying ‘cargo’ macromolecules into cells. In the
context of the general proposal, these peptide sequences will be made available
to individual investigators to facilitate studies of intracellular
protein-protein interactions. In the context of this specific project, we will
address the following aims: 1)Identify peptide motifs capable of targeting
proteins to specific vascular beds, and determine the receptors, adhesion
complexes or membrane proteins defining this specificity; 2) identify peptide
motifs capable of targeting proteins to specific organ parenchyma, and determine
the receptors, adhesion complexes or membrane proteins defining this
specificity; and 3) identify peptide motifs capable of cellular internalization
via receptor-mediated pathways, characterize the intracellular fate of these
peptides, and determine the receptors, adhesion complexes or membrane proteins
defining internalization.
B5. Development Of Cell
Permeable Miniature Proteins As Highly Selective Antagonists Of Protein-Protein
Complexation In Vivo, A. Schepartz (Chemistry)
Our laboratory has pioneered a
strategy called ‘protein grafting’ for the design of molecules – miniature
proteins - that recognize protein and DNA surfaces with high affinity and
unprecedented levels of selectivity. Our first miniature proteins targeted the
DNA half site recognized by the bZIP protein GCN4. The most successful of these
molecules, p007, binds specific DNA in the low nanomolar range under
physiological conditions and displays greater specificity than GCN4 itself. We
have also discovered miniature proteins that bind protein surfaces selectively
and with high affinity. One such molecule, PPBH3-1, folds cooperatively, binds
Bcl-2 and Bcl-XL 100-fold more tightly than the unstructured Bak BH3 domain, and
discriminates effectively against a range of non-specific proteins (see ref. 60
in application). Herein we propose to apply validated cell-permeation
technologies to deliver highly potent and selective miniature proteins to cells
and monitor the effects of these miniature proteins on a proteome-wide scale
using the mass spectrometric-based technology that is also proposed. In
addition, we will build on our recent discovery that the unique region of p007 –
a 6 residue type II polyproline helix – can transform other unstructured
peptides of appropriate sequence into folded miniature proteins with comparable
affinities and specificities. This discovery is very exciting in the context of
this proposal as it could be used, in combination with the transducing peptide
strategy described above, to quickly generate highly selective repressors of any
promoter recognized by a bZIP, bHLH, or even a homeodomain protein, or equally
potent inhibitors of any protein-protein interaction involving an
α-helix.
B6. Utility Of Cell Permeable
Peptides To Inhibit Intracellular Trafficking Of Signaling And Adhesion
Molecules In Vascular Endothelial Cells
Martin J. Kluger and J. Pober (Pathology)
TNF-mediated induction of
leukocyte adhesion molecules on vascular endothelial cells is a central event in
many types of tissue inflammation and inhibition of these processes is of proven
benefit in treatment of several inflammatory diseases. Both TNF signaling within
and sustained cell surface display of adhesion molecules on endothelium depend
upon regulated intracellular trafficking of specific proteins and these
trafficking processes are mediated via protein-protein interactions. The goal of
this project is to develop cell permeable peptides that can selectively disrupt
these interactions as well as the consequent trafficking events. Efficacy will
be tested in cultured human endothelial cells and in human skin grafts
transplanted on immunodeficient mice which retain human endothelial cell-lined
microvessels. A successful outcome of this contract may lead to the development
of new anti-inflammatory agents. |
