BINDING KINETICS INVESTIGATION OF A PEPTIDE FRAGMENT OF THE PSYCHIATRIC RISK PROTEIN, DISC1, TO THE KINASE GSK3β

Abstract

GSK3β is a multipurpose serine-threonine kinase which plays a role in many signaling pathways. Abnormal Glycogen Synthase Kinase 3β (GSK3β) function has been implicated in many diverse disease states including mental illness. Its part in neurological disease stems from its abnormal activity within the WNT/β-catenin signaling pathway. One of the binding partners of GSK3β within the WNT pathway that is linked to psychiatric disease is Disrupted in Schizophrenia 1 (DISC1). DISC1 has been suspected to play a role in individuals presenting with mental illness. Research in this area is lacking in the amount of information known about how GSK3β and DISC1 interact together. In the presence of a WNT signal, DISC1 is thought to participate in the binding complex that inhibits GSK3β activity. This thesis aims to identify a potential binding site for a small fragment of the DISC1 protein. Studies were conducted using Surface Plasmon Resonance and kinetic enzyme assays. The ability to determine the location of DISC1 binding is essential to better understanding the mechanism behind DISC1s ability to inhibit GSK3β. Most of the work is centered around the production of a His-tagged GSK3β eukaryotic construct using a bacterial overexpression system and its subsequent isolation utilizing a series of affinity and ion exchange liquid chromatography columns. This process was optimized to obtain GSK3β with a purity level of 80% to use in subsequent studies. Binding interaction studies using surface plasmon resonance technology were conducted to determine the suspected binding site of a short 44 mer fragment of DISC1 on GSK3β. The binding interaction studies utilized competitive binding methods between the 44 mer fragment and a small peptide fragment of the GSK3β binding protein FRAT (FRATtide). FRATtide is a peptide with a known binding location on GSK3β. The work developed a likely model representing how the DISC1 fragment binds GSK3β in the presence of FRATtide.