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Regulation of PTP1B Activity and Insulin Resistance by Cellular Cholesterol
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Melendez, Juan Andre, Fasulo, Michael, Jourd'heuil, David, Boivin, Benoit, Chair
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2022-08
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Abstract
Protein Tyrosine phosphatase 1B (PTP1B), is an endoplasmic resident protein and a well-known
negative regulator of the insulin receptor, dephosphorylating Tyr-1162, and Tyr-1163, two
residues located in the activation loop of the insulin receptor. Mice lacking the PTPN1 gene
encoding for PTP1B exhibit increased insulin sensitivity and improved glucose tolerance. Apart
from its role in insulin signaling, mice lacking PTP1B show resistance to weight gain on a highfat
diet, increased basal metabolic rate, and decreased cholesterol levels. In addition, PTP1B was
previously identified in a proteome-wide mapping of cholesterol-interacting proteins in
mammalian cells. However, the relationship between PTP1B and cholesterol is still unclear. To
better understand the role of cholesterol on PTP1B function and on insulin signaling, we first used
an in silico approach to predict cholesterol-binding sites in the 3D structure of the phosphatase and
confirmed the binding sites through fluorescence binding studies and mass fingerprinting. We
confirmed that the association between PTP1B and cholesterol occurred in both in vitro and in
mammalian cells. In an attempt to understand whether cholesterol affects the ability of PTP1B to
dephosphorylate substrates, we performed activity assays in various conditions. We observed that
cholesterol could reduce and reactivate the reversibly oxidized form of PTP1B in vitro. Treatment
of mammalian cells with cholesterol confirmed that excess cholesterol kept PTP1B reduced, and
decreased Insulin Receptor phosphorylation and downstream signaling. In vivo results obtained
by exposing mice to a high cholesterol diet support a role in the cholesterol-mediated reduction of
PTP1B and decreased insulin sensitivity in the liver. We have established an electron tunneling
path between the allosteric site and the catalytic cysteine residue and used a redox-sensitive
fluorophore to measure electron tunneling in vitro. Hence, our results demonstrate for the first time
that cholesterol binds to PTP1B at an allosteric site and reduces the phosphatase to regulate its
activity and insulin signaling. Based on these results we propose a novel role for cholesterol in
activating enzymes and in the context of insulin resistance.