The process of cytokine hormone binding to a transmembrane receptor is a central theme to many cell signaling pathways. Cytokine hormones and their receptors have been utilized as therapeutic agents and/or targets for a wide variety of medical applications. Although the interaction of cytokine hormones and the extracellular domains of their receptors has been extensively characterized, how the hormone activates the full receptor molecule within the membrane and in the cytoplasm is still largely unknown. The absence of a model system to study such interactions has limited the field to cell-based activity assays, which have failed to provide a detailed picture of the biophysical interactions and dynamics within the membrane and inside the cell. In this study, semi-synthetic model systems of the full receptor molecule will be engineered by combining bacterially-expressed proteins and synthetic peptides providing a novel platform for biophysical characterization of the human growth hormone receptor. The components will be assembled by native chemical ligation to restore native peptide bonds. Site-directed spin labeling will be used to introduce paramagnetic molecules into various residues within the engineered receptor to probe its structural dynamics upon hormone binding using EPR spectroscopy. The modular nature of this novel approach will allow the incorporation of interchangeable parts within the system, a property that can be generalized to integral membrane systems that have otherwise been hard to isolate and characterize.
This project aims to study the interactions of hormones and their receptors at an unprecedented level of detail. The long term goal of this project is to develop the technology to engineer molecules that faithfully simulate hormone receptor molecules that span the cell membrane in an effort to elucidate the mechanisms involved in hormone function. The proposed model systems are not limited to hormone receptors and can be generalized to study many transmembrane proteins and the activation mechanisms of myriad cell signaling pathways. ? ? ?
| Rizk, Shahir S; Mukherjee, Somnath; Koide, Akiko et al. (2017) Targeted rescue of cancer-associated IDH1 mutant activity using an engineered synthetic antibody. Sci Rep 7:556 |
| Rizk, Shahir S; Kouadio, Jean-Louis K; Szymborska, Anna et al. (2015) Engineering synthetic antibody binders for allosteric inhibition of prolactin receptor signaling. Cell Commun Signal 13:1 |
| Rizk, Shahir S; Misiura, Agnieszka; Paduch, Marcin et al. (2012) Substance P derivatives as versatile tools for specific delivery of various types of biomolecular cargo. Bioconjug Chem 23:42-6 |
| Rizk, Shahir S; Paduch, Marcin; Heithaus, John H et al. (2011) Allosteric control of ligand-binding affinity using engineered conformation-specific effector proteins. Nat Struct Mol Biol 18:437-42 |
| Rizk, Shahir S; Luchniak, Anna; Uysal, Serdar et al. (2009) An engineered substance P variant for receptor-mediated delivery of synthetic antibodies into tumor cells. Proc Natl Acad Sci U S A 106:11011-5 |
