Protease Signaling and Specificity using Single Molecule Detection:

 

       Protease specificity determination is a critical step in characterizing novel proteases and elucidating signaling pathways.  Given the large number of proteases (ca. 500-700) that are known to exist from human genome sequencing efforts [15], reasons that sensitive, reliable, and high-throughput methods to determine protease specificity must be developed.  Understanding the specificity of novel protease will increase our knowledge of biological signaling and allow for inhibitors to be designed for the treatment of diseases with aberrant proteolytic signaling cascades.

To advance our knowledge of protease specificity and signaling pathways, I have constructed a protein based fluorescence resonance energy transfer (FRET) library using the fluorescent proteins GFP and DsRed (and a second library using the cyan/yellow donor-acceptor FRET pair).  This library uses a randomized DNA linker region inserted between the donor-acceptor pair to produce substrates with varied amino acids located between the FRET proteins.  Kinetic assays have been performed and monitored using the increase in GFP fluorescence following hydrolysis to arrive at relative reaction velocities for a set of well characterized enzymes (TEV and thrombin).  These initial results demonstrated the ability of the enzymes tested to discriminate between different substrates and the resistance of GFP and DsRed to proteolysis.  Colony screening, using color development of individual E. coli colonies and restriction enzyme digests, were shown to eliminate those DNA samples in the library that contained stop codons and/or deletions.  Methods to screen this library for proteolytic activity using single-molecule detection are currently being developed.

Utilization of this library will be focused on; a) characterization of novel proteases (unknown specificity), b) developing a rapid drug screening platform using single-molecule detection studies, and c) monitoring protease activity in real time to elucidate single molecule binding and hydrolysis rates using fluorescently labeled proteases.  Human tissue kallikreins (hKs), which are encoded by the largest contiguous cluster of protease genes in the human genome [16] will be the focus of these advanced studies.  These enzymes are secreted serine proteases which are primarily known for their clinical applicability as cancer biomarkers.  HKs are involved in many cancer-related processes; cell-growth regulation, angiogenesis, invasion and metastasis, which makes them ideal targets for these studies.  In addition to the studies to determine reaction rates and inhibition constants, structural investigation (using x-ray crystallography) of these proteases will be initiated to determine the molecular basis of specificity and/or inhibition for these enzymes.

References.

 

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8.          Kravchuk, A.V., Zhao, L., Bruzik, K. S., Tsai, M. D., Engineering a catalytic metal binding site into a calcium-independent phosphatidylinositol-specific phospholipase C leads to enhanced stereoselectivity. Biochemistry., 2003. 42(8): p. 2422-30.

9.          Apiyo, D., et al., X-ray structure of the R69D phosphatidylinositol-specific phospholipase C enzyme: Insight into the role of calcium and surrounding amino acids in active site geometry and catalysis. Biochemistry, 2005. 44(30): p. 9980-9989.

10.        Iwasaki, Y., Tsubouchi, Y., Ichihashi, A., Nakano, H., Kobayashi, T., Ikezawa, H., Yamane, T., Two distinct phosphatidylinositol-specific phospholipase Cs from Streptomyces antibioticus. Biochimica et biophysica acta., 1998. 1391(1): p. 52-66.

11.        Zhao, L., Liu, Y., Bruzik, K. S., Tsai, M. D., A novel calcium-dependent bacterial phosphatidylinositol-specific phospholipase C displaying unprecedented magnitudes of thio effect, inverse thio effect, and stereoselectivity. Journal of the American Chemical Society., 2003. 125(1): p. 22-3.

12.        Chan, S.L. and V.C. Yu, Proteins of the Bcl-2 family in apoptosis signalling: From mechanistic insights to therapeutic opportunities. Clinical and Experimental Pharmacology and Physiology, 2004. 31(3): p. 119-128.

13.        Petros, A., E. Olejniczak, and S. Fesik, Structural biology of the Bcl-2 family of proteins. Biochim Biophys Acta, 2004. 1644(2-3): p. 83-94.

14.        Hinds, M.G., et al., Bim, Bad and Bmf: intrinsically unstructured BH3-only proteins that undergo a localized conformational change upon binding to prosurvival Bcl-2 targets. Cell Death and Differentiation, 2007. 14(1): p. 128-136.

15.        Southan, C., A genomic perspective on human proteases as drug targets. Drug Discovery Today, 2001. 6(13): p. 681-688.

16.        Borgono, C.A. and E.P. Diamandis, The emerging roles of human tissue kallikreins in cancer. Nature Reviews Cancer, 2004. 4(11): p. 876-890.