Enzymatic characterization of selenoprotein K and S and harnessing selenocysteine for protein engineering
Date
2016
Authors
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Publisher
University of Delaware
Abstract
Selenoproteins contain the 21st amino acid selenocsyteine (Sec). Twenty-five selenoproteins have been identified in the human selenoproteome, and they have been implicated in various diseases, such as cancer, cardiovascular diseases, and aging. However their biochemical function, in contrast to their biological study, is less characterized due to the challenge to make sufficient amounts of them. ☐ Selenoprotein K (SelK) and selenoprotein S (SelS) are two membrane selenoproteins that belong to a SelK/SelS family that contains intrinsically disordered regions. Both of them were shown to participate in antioxidant defense and to be involved in the endoplasmic reticulum associated protein degradation pathway. Nevertheless, their precise function is unknown. The work described in this thesis established methods to incorporate Sec into SelK and SelS, and to characterize their biochemical properties. SelS in isolation possesses an intra selenylsulfide bond that has disulfide reductase activity relying on the presence of Sec. SelS is also found to possess weak peroxidase activity toward H2O2. For the first time, we have shown evidence of trapping a selenenic acid intermediate, and the mutagenesis study also established that the contribution of selenylsulfide rendered SelS resistant to inactivation by reactive oxygen species (ROS). The other member of the SelK/SelS family, SelK, was identified to contain a diselenide bond, with an apparent redox potential of -254 mV. SelK was also characterized to have weak lipid peroxidase activity. In addition, SelK was found to have auto-proteolytic cleavage, which may play a regulatory role in vivo. ☐ In a separate research effort, we developed a new method to prepare selenoproteins, which we termed expressed selenoprotein ligation (ESL). The method incorporates Sec at any position in the protein, with no constraints on the fragment’s size and properties using expressed protein ligation. Sec’s high reactivity was exploited to facilitate challenging ligation reactions. Furthermore, the selenocysteine can be converted in a straightforward and selective fashion to an alanine or serine offering great flexibility in the selection of the ligation site. Alternatively, the selenocysteine can be used to introduce bioconjugates in a site-specific fashion. Thus, this method facilitates complicated ligations, increases overall yield, and introduces site-specific chemistry into the protein scaffold.