Lipid electrophile adduction of Pin1 and effects on substrate binding & activity

Vanderbilt University, Nashville TN

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Abstract

DESCRIPTION (provided by applicant): Oxidative damage to cellular lipids is a contributing factor to aging, as well as many peripheral and central nervous system diseases. Reactive oxygen species-induced modification of polyunsaturated fatty acids results in the production of electrophilic metabolites capable of irreversibly modifying proteins, carbohydrates, and DNA. Examples of lipid electrophiles that have been extensively studied are 4-hydroxynonenal (4-HNE), 4- oxononenal (4-ONE), acrolein, and malondialdehyde. Adduction by lipid electrophiles distorts protein tertiary structure and typically has adverse effects on protein function. Despite an undeniable connection to aging as well as a multitude of diseases where oxidative stress occurs, the susceptibility of peptidyl-prolyl cis/trans isomerase A1 (Pin1) to adduction by different electrophiles has not been thoroughly investigated. In human colorectal carcinoma cells (RKO), I will map the adduction sites of Pin1 in vitro using proteomics. I will examine the potential downstream effects of 4-HNE and 4-ONE adduction to Pin1 in terms of substrate binding and prolyl isomerization activity. Furthermore, I propose that Pin1 modification by electrophiles results in an inability to catalyze the proline-directed isomerization of several Raf1 moieties necessary for dephosphorylation by protein phosphatase 2A, resulting in prolonged hyperphosphorylation/inactivation. This investigation represents the first study to mechanistically examine electrophile adduction of Pin1 and the subsequent regulatory fate of its substrates. The proposed research allows for significant insight into the cellular response to Pin1 protein modification as a consequence of lipid peroxidation.