All ETDs from UAB

Advisory Committee Chair

Charles N Falany

Advisory Committee Members

Shannon M Bailey

Stephen Barnes

Matthew B Renfrow

Teresa W Wilborn

Document Type

Dissertation

Date of Award

2011

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The human cytosolic sulfotransferases are a family of phase II drug-metabolizing enzymes that conjugate a sulfonate moiety from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to a hydroxyl moeity on a substrate. SULT2B1b catalyzes the sulfation of hydroxysteroids as well as many xenobiotics; and is expressed extra-hepatically. SULT2B1b is a unique SULT because it has amino (N)- and carboxy (C)-terminal peptide extensions, undergoes post-translational modification, and is localized in both the cytosol and nucleus. Analysis of SULT2B1b revealed that the N- and C-terminal extensions of SULT2B1b are predicted to be intrinsically unstructured peptides. Size exclusion chromatography has revealed that SULT2B1b is enzymatically active as a homodimer, despite the proximity of the 45 AA C-terminal extension to the dimerization site of SULT2B1b. Nuclear translocation of SULT2B1b appears to be regulated by a PKA-dependent mechanism, because treatment of cells with db-cAMP showed a dose-dependent increase in nuclear translocation of SULT2B1b. Further, site-directed mutagenesis of SULT2B1b showed that Ser348 is associated with nuclear translocation of SULT2B1b. A Ser348 to Asp molecular mimic was generated to evaluate the structural and functional consequences of SULT2B1b phosphorylation. Molecular models of SULT2B1b-S348D showed a 27 Ǻ shift in peptides on the C-terminus of SULT2B1b. Further, recombinant SULT2B1b-S348D showed a 10-fold increase in sulfation activity and increased stability, compared to SULT2B1b. These substantial increases in activity and stability allowed for the determination of KD values for substrates and identification of serveral oxysterols as substrates for SULT2B1b. SULT2B1b mRNA and protein were detected in human brain, whereas neither SULT2A1, nor SULT1E1 were detected. Further, immunoreactive SULT2B1b protein was localized to neurons and oligodendrocytes. SULT2B1b expression and sulfation activity was identified in U-373MG glioblastom cells and SULT2B1b expression in U-373MG cells appears to be regulated by LXR activation. Evaluation of several types of human brain tumors by quantitative RT-PCR and immunoblot analysis demonstrated that SULT2B1b is over-expressed in gliboblastoma and late-state astrocytoma. Because SULT2B1b appears to conjugate neurosteroids, cholesterol, and oxysterols within the brain, understanding the regulatory role of SULT2B1b in the brain will help to elucidate the impact of SULT2B1b involvement in steroid and cholesterol regulation in normal brain and brain tumor physiology.

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