UBC Theses and Dissertations
Structural requirements for the elastolytic and collagenolytic activities of cathepsins and the identification of exosite inhibitors Du, Xin
It has become increasingly transparent that substrate recognition and degradation of extracellular matrix proteins such as elastin and collagen by proteases require more than the binding to small areas around the scissile bond of the target protein. Important surface structures on proteases, known as exosites or the formation of protease complexes are likely required for the correct positioning and modification of the substrate. This thesis embarks on the identification of such exosites in lysosomal cysteine cathepsins and their involvement in protein unfolding that are required for their elastolytic and collagenolytic activities. In chapter 2, two exosites were identified in cathepsin V that are crucial for the degradation of insoluble elastin. Both exosites are distant from the active site of the protease. Replacement of both exosites completely abolished the elastolytic activity without affecting the general proteolytic efficacy of cathepsin V. Although the exact mechanism of contribution of these exosites to elastolysis is yet to be elucidated, the finding that the double exosite variant failed to bind to insoluble elastin implies that these exosites are involved in substrate recognition. In chapter 3, the involvement of exosites and the effect of protease oligomerization on the collagenase activity of cathepsin K was studied. Two mechanistic models including a cathepsin K/GAG tetramer and a dimer were proposed based on available crystal structures. Both models, although displaying different modes of GAG binding, share a number of important amino acid residues in their protein-protein interactions. Mutational, biochemical, and structural analysis revealed various mechanistic aspects of substrate specificity towards soluble and insoluble collagens, respectively. In chapter 4, a library of 1280 known drug derivatives was screened using a fluorometric polarization assay to identify potential exosite inhibitors that prevent the formation of active cathepsin K/GAG complexes. Two groups of compounds were identified: 1) polyanionic and 2) polyaromatic compounds, whose IC₅₀ values for the inhibition of soluble tropocollagen degradation were between 10 – 186 µM. Exosite inhibitors might have the advantage of overcoming the off-site effects of active site-directed inhibitors presently in development.
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