Cells must respond to internal and external stimuli for healthy function, growth, and differentiation. One primary mechanism by which cells do this is through modulation of the post-translational phosphorylation levels of many proteins. Disruption of, or aberrant protein phosphorylation levels results in numerous diseases including cancer, Type-II diabetes, obesity, and inflammation. One class of enzymes that modulate the phosphorylation state of proteins is the protein tyrosine phosphatases. Because of their important role, the rate at which these enzymes catalyze phosphate removal is crucial and is tightly regulated. This regulation can occur by allosteric mechanisms, including small molecule binding and post-translational modification. And, as recent data in the PI's lab suggests, regulation is also inherently controlled by the closing kinetics of the acid loop in these phosphatases. The long-term goal is to understand in more detail how the activity of these phosphatase enzymes is regulated. This goal will be achieved through three aims: 1) study of the allosteric mechanism of PTP1B inhibition, a human enzyme whose misregulation leads to Type-II diabetes, obesity, and certain breast cancers~ 2) characterize how the primary sequence of the acid loop in PTP1B and YopH (a phosphatase from the virulent Yersinia bacterium) controls the kinetics of loop motion~ and 3) characterize the activation mechanism of the human VHR phosphatase that results from binding to the pseudokinase, VRK-3. For these three aims the motions of the acid loop will be monitored by solution NMR relaxation dispersion techniques with focus on any alterations in loop motions due to small molecule binding, post-translational modification, mutation, or accessory protein binding. Any functional changes in these phosphatases as a result of these perturbations will be characterized by detailed kinetic studies including pH and isotope effects. The combination of these biophysical and biochemical techniques will give insight into the connections between motions and catalysis while informing on the regulatory mechanisms of medically important enzymes.
The covalent attachment and removal of phosphate groups on proteins is crucial for healthy growth, differentiation, and regulation of cells and disruption f these phosphate levels leads to a vast number of diseases. Proteins crucial to this regulation are the protein tyrosine phosphatases, which are the object of this proposal. Here, we aim to understand the mechanism by which these phosphatases are regulated, which will impact diseases such as cancer, type II diabetes, obesity, and bacterial infections.
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| Lipchock, James M; Hendrickson, Heidi P; Douglas, Bonnie B et al. (2017) Characterization of Protein Tyrosine Phosphatase 1B Inhibition by Chlorogenic Acid and Cichoric Acid. Biochemistry 56:96-106 |
| Lisi, George P; Loria, J Patrick (2016) Using NMR spectroscopy to elucidate the role of molecular motions in enzyme function. Prog Nucl Magn Reson Spectrosc 92-93:1-17 |
