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The Yale/NHLBI Proteomics Center (http://info.med.yale.edu/nhlbi-proteomics/) builds on the complementary expertise of 21 Yale faculty in 12 departments and of the HHMI Biopolymer/Keck Laboratory. One of the strengths of the Center is that it brings together faculty with highly regarded research programs in vascular biology, hematopoiesis, and hypertension with faculty who are leaders in designing the cell permeable synthetic biomolecule delivery systems that hold enormous promise for developing new strategies for disease treatment. The Center is further supported by faculty who are developing new approaches to the study of proteomics and who are experts in building the databases needed to effectively analyze, archive, and interpret the enormous amounts of protein expression data that will be produced by this research. Overall goals of the Center are to improve existing and to develop new:

• protein profiling and disease biomarker biotechnologies to identify proteins that play key roles in diseases related to vascular biology, hematopoiesis, and blood pressure regulation.
• synthetic peptide-based reagents to specifically modulate the activities of these key proteins in cells and tissues of interest.
• approaches to diagnose, more accurately classify, and understand diseases and conditions such as atherosclerosis, inflammation, blood diseases, hypertension, resistance to chemotherapy, and immunological rejection of transplanted tissues and organs.

Protein profiling biotechnologies and resources that will be implemented and improved are:

• MALDI-MS based protein disease biomarker analysis of serum and other biological fluids.
• Quantitative ICAT/LC-MS/MS protein profiling.
• Differential (fluorescence) 2D gel electrophoresis protein profiling.
• Phosphoproteome analysis based on MS analysis of phosphopeptide-enriched fractions from digests of cell extracts.
• Development of ribozyme and deoxyribozyme-based molecular biosensors for the large-scale analysis of selected proteins and their protein post-translational modifications.
• An Oracle based proteomics database is being developed to archive and to facilitate the retrieval, analysis, and cross- correlation of protein profiling data with mRNA expression, functional, protein interaction, and other relevant data available in other databases.

Several approaches are being taken to develop more effective synthetic peptide-based reagents to modulate the in vivo activities of selected proteins in cells and tissues of interest.

• To localize permeable peptides to different subcellular compartments we are:
  • Coupling antennapedia peptide and oligomeric D-arginine sequences with compartment-specific "signature sequences".
  • Optimizing the HIV-1 Tat protein transduction domain to deliver reagents into the cytosolic versus nuclear compartments of living cells.
  • Developing a method based on the TAT-transmembrane domain for delivering and selectively orienting reagents into the cell membrane.
• Peptide phage display is being used to identify peptide motifs capable of targeting proteins to specific cells (e.g., hematopoietic stem cells), vascular beds, and organs (e.g., heart).
• Protein grafting of essential recognition groups onto the avian pancreatic polypeptide protein scaffold is being used to produce miniature proteins that are pre-organized for binding target macromolecules with high affinity and that can be linked to the appropriate transducing peptide sequence to correctly target their in vivo site of action.
• Utilizing the above methodologies to understand the control of tumor necrosis factor signaling in the inflammatory response of vascular endothelial cells.

1. Development of protein and phosphoprotein profiling technology: Kathy Stone, Walter McMurray, William Konigsberg, Ken Williams (Mol. Biophys. & Biochem.) and Czabo Horvath (Chemical Engineering)
2. Engineering RNA molecular switches that respond to protein targets. Ron Breaker (Molecular, Cellular & Developmental Biology)
3. Biostatistics and Bioinformatics: Group Leader, Perry Miller (Medical Informatics)
   a. Quality control, quality assurance, and statistical analysis of protein expression data, Hongyu. Zhao (Epidemiology and Public Health)
   b. NHLBI/Yale Protein Expression Database, Kei Cheung (Medical Informatics)
   c. Functional and Interrelative Proteomics, Mark Gerstein (Mol. Biophysics & Biochem.)
4. Global Proteomic Approaches to Hematopoietic Differentiation: Group Leader, Sherman Weissman (Genetics)
   a. Molecular and functional analysis of myeloid differentiation, Sherman Weissman (Genetics)
   b. Molecular and functional correlates of myelodysplasia, Nancy Berliner (Medicine)
   c. Downstream targets of the homeodomain gene Pitx2 in hematopoietic cells, Bernie Forget (Medicine)
   d. Differential protein expression during early hematopoietic differentiation and mobilization, Diane Krause (Laboratory Medicine)
   e. Characterization of Evi-1-induced changes in protein expression during myelopoiesis, Arch Perkins (Pathology)
5. Hypertension
   a. Characterization of a regulated paracellular conductance involved in hypertension, Richard Lifton (Chair, Genetics)
6. Vascular Biology
   a. Protein expression profiling and phosphoproteome analysis of lipid rafts during angiogenesis, William Sessa (Pharmacology)
   b. Integrin engagement-mediated alterations in T cell HuR protein:protein interactions and posttranslational modifications, Jeffrey Bender (Internal Medicine)
   

1. Design of organelle-specific peptides for therapeutic disruption of protein-protein interactions, William Sessa (Pharmacology)
2. Intracellular delivery of peptides, proteins, and nucleic acids for studying cellular function, David Ward (Genetics)
3. Development of new technology to discover peptides that reduce inflammation, Sankar Ghosh (Immunobiology)
4. Protein-based vascular addressing and targeted cellular internalization, Frank Giordano (Internal Medicine)
5. Development of cell permeable miniature proteins as highly selective antagonists of protein-protein complexation in vivo, Alanna Schepartz (Chemistry)
6. Utility of cell permeable peptides to inhibit intracellular trafficking of signaling and adhesion molecules in vascular endothelial cells, Jordan Pober (Pathology)