Purification of Cytosolic Sulfotransferases and Towards the Synthesis of an Affinity-based Protein Profiling Tool for 3’- Phosphoadenosine-5’-phosphate




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Sulfotransferases play a large role in phase II metabolism, which involves the modification of xenobiotics and endogenous compounds for subsequent elimination from the cell. This is done through the addition of a sulfuryl group, from the universal sulfate donor 3’-phosphoadenosine- 5’-phosphosulfate (PAPS), replacing the hydroxyl or amine group of a substrate, generating the sulfurylated product and 3’-phosphoadenosine-5’-phosphate (PAP). Subsequently, the sulfurylated product can then be exported out of the cell by the associated transmembrane multidrug resistance transporter (MRP). A mechanistic understanding of these enzymes mechanism of actions is of vital importance, as disease states, such as anemia, cancer, and autism, have been linked to dysregulation of sulfurylation. Currently, the state of the art for studying sulfotransferase activity includes radiometric, fluorometric, photometric, and mass spectrometric assay methods. The study of sulfotransferases through a chemical approach is comparatively understudied. This body of work lays the foundation for the study of the activity of these enzymes through the development of a chemical tool to study its protein interaction partners. In Chapter 1, a protocol for the expression and purification of six human cytosolic sulfotransferases is established. In later studies, these enzymes will be utilized to characterize fluorescent sulfotransferase sensors in vitro prior to applications in live cells. The interaction dependent fluorescence of the sulfurylated product will allow for the study of sulfotransferase activity. In Chapter 2, we demonstrate the initial synthetic steps of ((2R,3S,4R,5R)-5-(6-amino-2-(prop-2-yn- 1-ylamino)-9H-purin-9-yl)-4-hydroxy-3-(phosphonatooxy)tetrahydrofuran-2-yl)methyl phosphate (PAP-A). After treating cells with PAP-A, biotin azide will react with the tag on PAPA, allowing for isolation through Western blot analysis. Proteins in the isolated product can be identified through peptide mass fingerprinting. This tool will further our understanding of the role of PAP by identifying proteins that interact with PAP. Sulfurylation is a vital cellular process and by studying the enzymes involved, and the by-products of this process, we will improve our understanding of the roles these components play within living systems.



Nucleotides, Biocatalysis, Phosphates