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Antibody-based tools for protein interactions
Antibodies, or immunoglobulins, are an important ingredient of our immune system that recognizes and neutralizes foreign material. We have developed recombinant technology that allows us to produce large numbers of antibody variants using phage display. Through iterative screening, antibodies can be identified that selectivity bind to a target with picomolar affinity. Antibodies have been widely used both in research labs - Western blotting, crystallization aids - and clinical settings - antibody-based drugs such as Herceptin. When applied to proteolytic enzymes, many of these antibodies have been found to interfere with substrate binding and thus act as potent inhibitors with potential diagnostic or therapeutic application.
 
Real-time enzymology in cells
Nanotechnology has great potential for aiding the characterization of biological processes in precise detail. We have developed "plasmon rulers" to observe conformational changes of single biomolecules. Our plasmon rulers are comprised of peptide-linked gold nanoparticle satellites around a core particle that enable monitoring of proteolytic events in real-time. Recently, we have applied our plasmon rulers within cells and the observed enzymatic reactions mediated by caspases. We have begun to quantify the individual contributions of different caspase members within the proteolytic cascade leading to apoptotic cell death.
 
Methods to determine protease selectivity
Determining the preferred substrate cleavage sequence of a protease is an important step toward understanding its role in health and disease. Knowledge of this sequence can aid in the design of new experimental tools for study as well as aid in the identification of endogenous protease substrates and signaling pathways. We have designed and successfully implemented a completely diverse fluorescence-based tetrapeptide positional scanning synthetic combinatorial library that allows for the rapid screening of proteases to determine their preferred residues at positions P1-P4 (Schecter and Berger nomenclature). We continue to apply this technology to many different types of proteases to gain insight into their biology.
 
Small molecule manipulation of protein-protein interactions
Many biological process are dependent on the interaction of two (or more) proteins in order to generate a signal. Many viral proteases, such as that found in human Kaposi's sarcoma-associated herpesvirus (KSHV), must dimerize in order to process the viral polyprotein. Hence, disruption of this dimerization event can be applied to halt the viral lifecycle and has therapeutical potential. Using small-molecule chemical libraries and other approaches, we have identified several lead targets and validated their interactions through nuclear magnetic resonance (NMR) spectroscopy.
 
Infectious disease
Many human pathogens, such as the causative agent of Chagas disease Trypanosoma cruzi, produce proteases that are attractive targets for therapeutics. We have studied the major cysteine protease of T. cruzi using a combination of approaches such as crystal structure determination, inhibitor design and optimization, and substrate selectivity analysis using the various libraries we have created. Through this insight we have developed potential antiparasitics based on protease inhibition using vinyl sulfones.
 
Imaging proteolysis in vivo
Both the selectivity and potency of antibodies enable their use in detecting and potentially treating disease. Our development of antibodies for type II transmembrane serine proteases (TTSPs) and MT-SP1 or matriptase, in particular, has allowed us to apply these tools to identify sites where active protease is present in whole organisms. Our recent efforts have shown that MT-SP1 is overexpressed in many cancer types and is a potential target for therapy. Moreover, our tools have also been applied to show that MT-SP1 and other TTSPs play important roles in biological processes in healthy conditions such as neural tube closure in developing mice.