Selective Targeting of Cell Signaling and Inflammatory Proteins Using Biologics PDF Download

Author: Jasdeep Kaur Mann
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Languages : en
Pages : 165

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Book Description
The main focus of this dissertation is to develop the technology that allows targeting of key cellular pathways such as cell signaling and inflammatory pathways, where dysregulations leads to a disease state. As part of the work, it provides an overview of protein therapeutics and highlights some of the progresses that have been made toward achieving the goal. Comparison is drawn among small molecules, antibodies and non-antibody protein scaffolds to offer a rationale for developing alternative strategies. The dissertation is divided into three sections: The first part describes the engineering of fibronectin type III (FN3) monobodies to target the ERK-2 signaling pathway. ERK-2, along with other mitogen activated protein kinases (MAPKs) forms evolutionarily conserved pathways in eukaryotes, and regulate changes in protein function and gene expression in response to stimuli at the cell surface. Several MAPK pathways exist in humans, which collectively control cell proliferation, apoptosis, and other adaptive behaviors of the cell. Correct activation of different MAPK pathways is crucial to generate appropriate biological responses, whereas overactive signaling results in a number of human diseases, including cancer and neurodegenerative diseases. MAPKs use modular binding interactions outside the catalytic site to achieve specificity of interaction. In particular, all MAPKs contain a common docking domain that selectively binds a linear peptide motif within their respective substrates and regulators. Using engineered monobody inhibitors, we disrupted the docking interaction and thus selectively inhibit the ERK-2 pathway. Monobodies are compact (MW = 11 kDa) and resemble an immunoglobulin domain yet fold without an intradomain disulfide bond. We show that the engineered monobodies inhibit the activity of ERK-2 in vitro by competitively inhibiting the binding of a D-peptide. They also inhibit ERK-2 dependent signaling in transfected mammalian cells and homologous signaling pathways in yeast and worms. The design strategy used in our study is general and a similar approach may be useful to engineer epitope-specific inhibitors against other molecules. The second part discusses the use of rationally designed peptides to target a signaling pathway. Peptides can provide effective and innovative solutions to treat diseases by delivering high biological activity with low toxicity. We demonstrated their relevance by engineering a helical peptide to potentiate NOD2 protein activity associated with Crohn's disease (CD). NOD2 is an intracellular sensor that recognizes the bacterial cell wall component, muramyl dipeptide (MDP), and is critical for the innate immune response in the GI tract. However, the molecular mechanisms by which mutations in NOD2 contribute to the disease are not well understood. Modeling CD associated mutations at a molecular level is important to identify new therapeutic targets and develop potential treatment options. We used computational and structural analysis to model the mechanism of NOD2 activation. The similarity of domain organization in NOD2 to other related proteins for which structural and biochemical data exist suggests an activation mechanism that includes ligand induced removal of auto-inhibition by the carboxy terminal ligand binding domain. Based on the analysis, we designed a peptide predicted to potentiate NOD2 response to MDP by inducing conformational change of NOD2. The designed peptide shows an increase in NOD2 activity for both wild type and CD risk mutants in the presence of MDP. In vitro biochemical assays show that the peptide binds directly to the LRR domain of NOD2 and has an allosteric effect on MDP binding. The peptide may contribute to the elucidation of the activation mechanism of NOD2 and help develop a novel therapeutic agent against CD. The last section describes the novel methods developed in our lab for use in high throughput assays. Protein complexes are common in nature but biochemical characterization of protein-protein interaction is laborious and expensive. We describe two methods that may help characterize protein-protein interaction. First, we show that a combination of yeast surface display and disulfide trapping can detect the formation of a broad spectrum of protein complexes on the yeast surface. This technique is referred to as stabilization of transient and unstable complexes by engineered disulfide (STUCKED). The technique uses co-expression of two potentially interacting proteins in the same yeast cell, one of which is designed to be anchored to the cell wall and the other is designed to be in a soluble form. A structure-based disulfide is then introduced between the subunits to covalently crosslink them so that the bound complex can be efficiently detected irrespective of the stability of the complex. We demonstrated that the technique can be applied to trap a diverse group of complexes, including a protein-peptide complex (MDM2-p53) and an oligomeric protein (streptavidin heterodimer). The method may be used to assist in the testing of rationally designed interactions as well as to develop a directed evolution study to identify novel protein-protein interactions. To study dynamic protein interactions in vivo, we developed a technique which involves the use of monomeric streptavidin (mSA) to induce selective biotinylation of protein molecules based on distance criteria. Selective biotinylation is achieved when mSA forms a noncovalent complex with a target protein and is used to recruit a photoactivatable biotin crosslinker to the target molecule. Photoactivation with UV leads to chemical crosslinking of nearby proteins and results in selective biotinylation, so that the modified proteins can be purified using immobilized streptavidin. By targeting a post translational modified enzyme, mSA induced biotinylation (MIB) can selectively biotinylate enzyme substrates for downstream analysis. The proposed technique is generic and can be easily modified to study different transient interactions.