Using High-Resolution NMR to Examine the Components that Contribute to the Activity and Stability of Phosphatase of Regenerating Liver (PRL-1) and Their Dependence on the Redox State of Cysteine Residues

Abstract

Phosphatase of regenerating liver (PRL-1) has gained attention as an important therapeutic target because of its involvement in cancer and metastasis, where it promotes proliferation and cell motility. The protein is found at high concentration in numerous cancer tissues and when over-expressed induces cellular transformation. Elevated levels of PRL-1 have been shown to trigger metastasis and to promote cellular transformation and tumorigenesis. Because its enzyme activity contributes to both tumorigenesis and metastasis, this protein is an attractive target for therapeutic intervention. The problem with ubiquitously inhibiting PRL-1 is that this enzyme is also essential to cell survival and tissue regeneration under normal physiological conditions. Inhibition may result in nonspecific and unwanted cell death of healthy tissues. Targeting only the overactive form of an enzyme would provide a reasonable resolution to this problem, but it is currently unknown how PRL-1 prompts proliferation in some cells and differentiation in others. The long-term goal of this project is to identify the crucial structural and dynamic features that support the enzymatic activity of PRL-1 and characterize the different forms to identify ones appropriate to target for therapeutic intervention. In this work, we have shown that the full-length unmodified form of PRL-1 is primarily inactive based on its determined reduction potential (-364.3 ± 1.5 mV) and that in vivo, activation is likely conferred through a posttranslational modification at the C-terminus. Using an assortment of biophysical techniques, we have also shown that a mutant form of PRL-1 (PRL-1-C170S/C171S) is reduced and active and can serve as a model for posttranslationally modified PRL-1 at this site. A second goal of this dissertation project is to use PRL-1 as a model system for studying protein stability. An understanding of the forces that contribute to protein stability is crucial to the development of new biotechnology products with improved half-lives and lower immunogenicity. Because proteins are held together by numerous weak interactions, understanding the mechanisms by which stabilization is achieved is important to the design of new biotechnology products that better resist unfolding and aggregation. Mechanistic information describing how specific interactions influence stability is lacking, in part because the techniques typically used to study inherent stability do not provide sufficient detail. We used high-resolution solution NMR to examine the details of one aspect of stability in PRL-1. It is well-established that the oxidation state of cysteine residues in proteins are critical to overall physical stability. The presence of disulfide bonds most often imparts thermodynamic stability, and as such, engineered disulfide bonds have become a means for improving the viability of protein therapeutics. In some cases, however, disulfide bonds can diminish stability. PRL-1 has two discernable stable states, one of which contains a disulfide bond (oxidized, inactive) and is slightly less stable than the one that does not (reduced, active). To understand how the reduced form compensates for the loss of the disulfide bond, a series of mutants were analyzed that disrupted disulfide bond formation in distinct ways. The physical stability of each variant is unique with respect to the others, indicating that the stability of the whole protein depends on a combination of local, synergistic interactions with the Cys side chains. High-resolution solution NMR was used to provide site-specific and mechanistic information about how each interaction influences the protein's physical stability. This analysis reveals several key structural components that contribute to PRL-1's overall stability in the absence this disulfide bond. The results provide insight to explain how the reduced protein compensates for the loss of this structural feature, which has implications for its function in vivo, and how local instability may lead to aggregation.