VHH Activators and Inhibitors for Protein Kinase C Epsilon

Abstract

Protein kinase C epsilon (PKCε), which is one of the novel PKC isozymes, is widely expressed throughout the body and has important roles in the function of the nervous, cardiovascular and immune systems. In order to better understand PKCε regulated pathways, isozyme specific activity modulators are desperately needed. Such compounds could also be developed into drugs for diseases such as diabetes, cancer and Alzheimer’s disease, where PKCε dysregulation is implicated. This thesis describes the selection and characterization of PKCε specific llama single chain antibodies, known as VHHs. VHHs have several advantages compared to the conventional antibodies consisting of two heavy and two light chains. VHHs are small, easy to clone, convenient to genetically modify, remarkably stable, and tend to be highly specific for the target antigen. PKCε specific VHHs were selected in one round from an immune phage-VHH library using phage display. The VHHs that showed the strongest binding to human PKCε were produced in E. coli as monovalent proteins and tested for their ability to influence PKCε activity in in vitro kinase activity assays. Three VHHs (A10, C1 and D1) were found to increase the activity of PKCε, whereas three other VHHs (C7, E6 and G8) inhibited PKCε kinase activity. These effects were PKCε specific, since the VHHs had no effect on the activity of PKCδ, PKCθ or protein kinase A. A more detailed kinetic analysis revealed that the activators increased PKCε activity by increasing the maximum rate of the reaction and/or by speeding up the reaction. Furthermore, VHHs E6 and G8 were found to be non-competitive enzyme inhibitors. Interestingly, the binding site of all of the tested VHHs was in the catalytic domain of PKCε. Moreover, the binding of the VHHs was found to be conformation dependent and species specific. In order to study whether the VHHs could also influence PKCε activity in a cellular context, the VHHs were cloned into a mammalian expression vector with a C-terminal mCherry tag, and the VHH-mCherry proteins were transiently expressed in HeLa cells. Several cellular effects, such as changes in cell morphology, increases in cytotoxicity, and inhibition of PKCε downregulation, were observed for some of the VHH-mCherry constructs. The most direct indication that the VHHs can also influence PKCε activity in cells came from PKCε-EGFP translocation studies, where the activator A10-mCherry increased the rate of PKCε-EGFP translocation to the membranes in response to phorbol ester stimulation. In contrast, the inhibitors C7-mCherry and G8-mCherry decreased the rate of translocation. The results presented in this thesis demonstrate that highly specific VHHs against an intracellular antigen can be selected in one round from an immune phage-VHH library. Furthermore, it was shown that the selected VHHs can act as activators or inhibitors of PKCε activity in vitro and in cells. In addition to the potential therapeutic applications of the VHHs for diseases with aberrant PKCε signalling, these PKCε specific activity modulators will be valuable tools in research into PKCε regulated pathways