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Cady, Nathaniel, Melendez, Juan Andres, Fasullo, Michael, Jourd'heuil, David
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Spring 2024
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2024-04
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Abstract
It is well established that the catalytic activity of Protein tyrosine phosphatases (PTPs) is directly inhibited by cellular oxidants. In this process, reversible oxidation of their catalytic Cys residue can lead to the formation of a sulfenic acid, intra- or intermolecular disulfides, or to the formation of a cyclic sulfenamide form. Reversible oxidation of Cys215 in PTP1B (PTP1B-OX) leads to the formation of a cyclic sulfenamide and to other important changes in its structure, including the solvent exposure of the phosphotyrosine binding loop. We have shown that binding of 14-3-3ΞΆ to phosphoserine50 in the newly exposed phosphotyrosine binding loop was essential to stabilize PTP1B in its oxidized form, an important mechanistic insight into approaches to activate PTPs. In this process, we successfully developed an in vitro activity assay to measure the activity of PTP1B, enriched from cell lysate. We also showed that PTP1B was reversibly oxidized in cardiac hypertrophy and generated a PTP1B cardiomyocyte-specific knockout mouse model to study the inactivation of PTP1B in the heart. Importantly, the cardiac knockout of PTP1B caused a mild hypertrophic phenotype that was exacerbated by pressure overload. Inactivation of PTP1B lead to increased phosphorylation of Argonaute 2, which in turn prevented the association between Argonaute 2 and miR-208b and Argonaute 2-mediated silencing of Thrap1. In the process of these studies, we also updated a protocol for the modified cysteinyl labeling assay. Finally, we describe how cholesterol binds to PTP1B in cells, and how this interaction protects the PTP from reversible oxidation in cells and in mice treated with a high-cholesterol diet. Interestingly, our cell and animal work shows that cholesterol mediated PTP1B reduction prevents proper phosphorylation of the insulin receptor and cause insulin resistance. Overall, our studies bring new insight into the role of underlying mechanisms that regulate the reversible oxidation and reduction of PTP1B and shed light on the function of PTP1B in cardiac hypertrophy and insulin resistance.
Citation
Londhe, A. D. (2024). Characterization of regulators of PTP1B oxidation: A dissertation submitted to the University at Albany, State University of New York, in partial fulfillment of the requirements for the degree of Doctor of Philosophy in nanobioscience. SUNY Polytechnic Institute.