Structural and Functional Studies on Human Type 2 Cystatins

Abstract: Proteolytic enzymes are enzymes that hydrolyze peptide bonds in peptides and proteins. This ability to carry out protein degradation is essential for many cellular and extracellular processes that occur in all living organisms. Cysteine peptidases constitute a class of proteolytic enzymes. Among these, papain-like (C1) peptidases are the mostly studied ones. Inhibitors regulate proteolytic activities in the body. During more that two decades, cystatins have been seen as the natural inhibitors of papain-like peptidases. It has been simple to divide them into three major groups and there have been no larger differences between them when it comes to peptidase binding ability. In this thesis, the finding and characterization of human cystatins E and F are described. These cystatins show signs of adaptation to a more specific function thanks to their restricted localization and structural peculiarities, which are also reflected in their inhibition patterns. They give rise to a new branch of cystatins. In addition to this, the confirmation of the novel cystatin function to act as mammalian legumain (belonging to the C13 family of peptidases) inhibitors, the localization of the binding site and the finding that this feature is only applicable to some cystatins, are also described. These factors revealed that cystatins make up a system more complicated than that believed from the beginning. We were urged to dig deeper into this issue and we found plenty of information in the crystal structure of human cystatin D, which allowed us to follow up the studies on the C1 peptidase binding site, but also to establish the preliminary structural requirements for legumain inhibition by cystatins. These requirements were then confirmed by producing cystatin D variants that do inhibit mammalian legumain. Thus, this thesis work has contributed to a better understanding of the inhibitory ability of type 2 cystatins. The pieces of the cystatin puzzle start to fall into place and give a clearer picture of how such small proteins can be responsible for regulating the activity of such important peptidases in the body.