Scientific Report 2006

Molecular Biology

Functional Characterization of Proteases via Combinatorial Libraries

J.L. Harris, J. Alves*

With the complete sequencing of genomes from multiple organisms, information on the repertoire of genes can be readily established. However, large gaps still remain in our knowledge of the biological role of most genes. These gaps are mainly due to the fact that most biological functions are regulated not at the gene or transcript level, but at the posttranslational level. In contrast to the situation in genomics, in which the changes in the content or amount of cellular DNA or RNA can be readily examined, monitoring translational and posttranslational dynamics of functional proteins on a genome-wide level is more difficult. Progress in our current understanding of biological processes is limited by the available tools that can be used to probe function at the posttranslational level. We are developing and applying technologies based on small-molecule protein modifiers to profile the active state of enzymes.

In collaboration with N. Winssinger, Université Louis Pasteur, Strasbourg, Germany, we have developed an encoding strategy that uses peptide nucleic acid (PNA) sequences. Encoding combinatorial libraries with PNA tags allows not only for the synthetic history of the library to be captured in the resulting molecule but also for spatial deconvolution of the molecules on DNA microarrays.

Using this technology, we have created encoded protease inhibitor and substrate libraries of thousands of molecules. These libraries have been applied to various biological systems and have resulted in the identification and characterization of proteases within those systems. For example, a cysteine protease from the house dust mite Dermatophagoides pteronyssinus was identified by using a 4000-member PNA-encoded inhibitor library. The identified protease plays a key role in allergic hypersensitivity through the selective degradation of CD25 from T cells.

Another example of functional characterization of protein activity is the profiling of the substrate specificity of proteases from Dengue virus, the etiologic agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Using 2 substrate libraries of approximately 160,000 members, we characterized the structure-activity relationship of the NS3 protease from the 4 Dengue virus serotypes and facilitated the development of inhibitors of the virus.

Publications

Harris, J.L. Protease substrate profiling. In: Enzyme assays: high-throughput screening, genetic selection and fingerprinting. Reymond, J.-L. (Ed.). Wiley-VCH, New York, 2006, p. 303.

Harris, J.L., Winssinger, N. PNA encoding (PNA = peptide nucleic acid): from solution-based libraries to organized microarrays. Chem. Eur. J. 11:6792, 2005.

Li, J., Lim, S.P., Beer, D., Patel, V., Wen, D., Tumanut, C., Tully, D.C., Williams, J.A., Jiricek, J., Priestle, J.P., Harris, J.L., Vasudevan, S.G. Functional profiling of recombinant NS3 proteases from all four serotypes of Dengue virus using tetrapeptide and octapeptide substrate libraries. J. Biol. Chem. 280:28766, 2005.

Petrassi, H.M., Williams, J.A., Li, J., Tumanut, C., Ek, J., Nakai, T., Masick, B., Backes, B.J., Harris, J.L. A strategy to profile prime and non-prime proteolytic substrate specificity. Bioorg. Med. Chem. Lett. 15:3162, 2005.

Winssinger, N., Harris, J.L. Microarray-based functional protein profiling using peptide nucleic acid-encoded libraries. Expert Rev. Proteomics 2:937, 2005.