Designing Switchable Opioid Peptides for Interrogating the Effects of Cell Type-Specific Opioid Receptor Activation
Geng, Lequn
2022
Abstract
Opioids are the most effective analgesics clinically available for treating severe pain, but they also cause adverse side effects such as addiction and respiratory suppression. Researchers and pharmaceutical companies have been trying to separate the desired outcomes of opioids from the unwanted ones, but this goal is yet to be achieved. This is in part due to the lack of understanding of how opioid receptors in different cell types and brain regions are linked to different opioid-induced effects. This thesis describes the engineering of a novel genetic tool termed M-PROBE. M-PROBE is aimed at activating opioid receptors in the animal brain with cell type specificity, and thus enabling the study of correlation between cell type and behavior. M-PROBE is a multi-module protein encoded by designer DNA sequences. It has three major components: an opioid peptide for activating opioid receptors, a protein switch for controlling the activity of the opioid peptide, and a transmembrane domain for displaying M-PROBE on the cell outer membrane. To allow versatile applications of M-PROBE, two types of protein switches were designed, one of which is controlled by light (photoswitchable), and the other by a small molecule (chemically-activated). All components of M-PROBE were first engineered through rational design. For the opioid peptide, met-enkephalin was identified as the optimal candidate based on a G-protein recruitment assay. For the protein switches, we selected the second light, oxygen, voltage sensing domain from oat as the starting point for the light switch, and an FK-506 binding protein previously engineered in-house as the starting point for the chemical switch. After re-engineering both proteins using circular permutation, they were confirmed to be capable of controlling a seven-amino-acid peptide, SsrA, in a binding assay. Lastly, an extracellular domain-truncated human CD4 protein was used as the transmembrane domain. After rational design, both the light switch and the chemical switch were improved using yeast surface-based directed evolution. I demonstrate that both directed revolution campaigns were highly effective in expanding the dynamic range (between the “closed” state and the “open” state) of the protein switches, and thus the theoretical overall performance of M-PROBE. Finally, the chemically-activated M-PROBE was verified using both G-protein recruitment assay and a downstream secondary messenger (cyclic AMP) assay. The engineering of M-PROBE has four main implications. First, M-PROBE is the first tool capable of activating any endogenous G protein-coupled receptors (GPCRs) with cell type specificity. This will expand our understanding of the endogenous GPCRs by providing a novel means of studying the correlation between cell type and behavior. The design principles of M-PROBE can be readily translated to other peptide GPCRs. Second, our re-engineered light and chemical switches have applications far beyond controlling opioid peptides. Since both switches can be applied to a range of short peptides, they open the door for studying many other cellular processes. Third, our directed evolution platform for engineering the light switch is the first one capable of labeling two states of a protein simultaneously. This design can be expanded to engineering other dual-state proteins. Lastly, M-PROBE itself may serve as a prototype of gene therapy that might solve the long-standing challenge of separating the analgesic effects of opioid from the adverse effects.Deep Blue DOI
Subjects
protein engineering optogenetics chemogenetics photoswitchable protein directed evolution circular permutation
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