210 - Role of Cholesterol and Oxysterols in Regulating PTP1B Activity

Abstract

Mass increase of adipose tissue can lead to obesity and be detrimental to health. Interestingly, Protein Tyrosine Phosphatase 1B (PTP1B) knockout mice are resistant to weight gain when subjected to a high fat diet and inhibition of PTP1B is linked to decreased cholesterol levels. PTP1B is a well-known regulator of the insulin and leptin signaling pathways: it can directly dephosphorylate the insulin receptor as well as IRS-1 to decrease and stop insulin signaling; and it can regulate the leptin pathway by dephosphorylating JAK2. Hence, PTP1B regulation of leptin and insulin signaling is thought to regulate food intake and sensitivity to glucose, respectively. However, the role of PTP1B in regulating cholesterol levels is unclear. A cholesterol binding domain has previously been identified in the C-tail of PTP1B. Hence we explored whether other sites could be identified in the primary sequence of PTP1B, and performed cholesterol binding studies using AutoDock to further characterize these sites. We identified new binding sites and confirmed their existence using NBD-cholesterol and by performing competition studies. Mutagenesis studies further confirmed the existence of cholesterol-binding sites in PTP1B. Since PTPs and PTP1B can be inactivated by reversible oxidation, we used oxidized lipids to understand whether interaction with oxysterols could also inactivate the enzyme and whether depleting cholesterol in cells affected the catalytic activity and substrate specificity of PTP1B. These studies are ongoing.