Quantification of GSTpi and JNK protein-protein interactions using backscatter interferometry.

摘要

Protein-protein interactions play a critical role in sustaining life and diseases can often result from mis-regulation of these protein-protein interactions. The interaction between glutathione S-transferase pi (GSTpi) and c-Jun N-terminal kinases (JNKs) is such a system that has been linked to cancer. GSTpi normally functions in the body to detoxify cells by removing foreign compounds, and JNKs participate in the Mitogen Activated Protein (MAP) kinase cascade, whose effects range from cell proliferation to programmed cell death (i.e., apoptosis). GSTpi is known to inhibit the apoptotic behavior of JNK proteins, which in turn can lead to cancer. It is hypothesized that GSTpi does so by binding directly to JNKs, and that this interaction depends upon factors such as the haplotype of GSTpi, or whether the JNKs are phosphorylated. It is then believed that such variables affect the affinity of these proteins for one another. However, these interactions have only been identified qualitatively. Thus, understanding quantitatively the interactions of GSTpi with JNKs in regards to these factors provides crucial insight towards manipulating the pathway for chemotherapies.;This project is aimed at determining the binding affinity constants of GSTpi and JNK proteins with relations to the above variables. To achieve this goal, we will use Backscatter Interferometry (BSI), a very sensitive technique that utilizes very small amount of sample and does not require labeling.;We have successfully expressed and purified a number of the necessary proteins to complete the study. Namely, we have purified GSTpi and the inactive (unphosphorylated) forms of JNK1α2 and JNK2α2, which are two isoforms that have been shown to bind to GSTpi. We have also obtained the active/phosphorylated form of JNK1α2 and JNK2α2, referred to as pJNK1α2/pJNK2α2 as we plan to study the effect of phosphorylation levels of JNKs on binding to GSTpi. We are currently in the process of purifying pJNKs. We were also able to obtain preliminary BSI data with GSTpi and JNK1α2 alone in solution, demonstrating the efficacy of BSI for use with small quantities of proteins. More testing is underway with GSTpi and the phosphorylated version of JNK1α2 and JNK2α2.