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Characterization of human Neuropsin (hOPN5) and development of new mutants for optogenetic stimulation of the Gq signaling cascade

dc.contributor.advisorBrügmann, Tobias Prof. Dr.
dc.contributor.authorSathya Narayanan, Udhaya Bhaskar
dc.rightsAttribution-NonCommercial-NoDerivatives 4.0 International*
dc.titleCharacterization of human Neuropsin (hOPN5) and development of new mutants for optogenetic stimulation of the Gq signaling cascadede
dc.contributor.refereeMoser, Tobias Prof. Dr.
dc.description.abstractengGq signaling plays a pivotal role in the physiological adaptation of cellular behavior based on the individual body's needs and it is also involved in the pathogenesis of many diseases including cancer, autoimmunity, pathological cardiac hypertrophy, and heart failure. However, the transition between physiological and pathological signaling and the underlying kinetics is not well understood since current approaches with pharmacological stimulation and genetic modulation lack spatiotemporal precision. In striking contrast, optogenetic G protein-coupled receptor (GPCR) stimulation offers an unprecedented advantage of high temporal and spatial precision with the chance of cell-specific expression. Therefore, an optogenetic GPCR would be an ideal tool to study the transitions between physiological and pathological Gq signaling and the underlying kinetics. In this thesis, I characterized wild-type (WT) Neuropsin (OPN5), an ultra-violet (UV) light-sensitive bistable GPCR, to prove its selective activation of the Gq pathway in heterologous expression systems and a transgenic mouse model. UV light-induced Inositol 1,4,5 trisphosphate (IP3) production as well as the activation of transient receptor potential canonical cation channel 6 (TRPC6) channels by Diacylglycerol (DAG) was demonstrated in transgenic HEK293 hOPN5 (WT) cells. Using Ca2+ imaging, with red-shifted Xanthene-based rhodamine like Ca2+ fluorescent indicator-1 (X-Rhod-1, 1.5 µM), the light sensitivity (3.26 ± 0.17 µW/mm2, n=17) and the most efficient wavelength for activation (388 ± 2.7 nm, n=14) were determined for hOPN5 (WT). I further compared light with pharmacological stimulation and also proved that light-stimulated Ca2+ transients were abolished in Gq/11 knock-out (KO) cells while the Ca2+ transients were preserved in Gi KO cells. G protein activated inward rectifying K+ (GIRK) channel assay excluded the promiscuous Gi coupling of hOPN5 (WT) in HEK293 cells. By combining structural modeling and sequence alignments, a few mutations were predicted around the 8 Å region of the retinal chromophore to alter the photo-excited state, thereby having a fine-tuned activation spectrum and altered kinetics. The expression rate and expression strength of the transgenic hOPN5 mutant cells along with chicken OPN5 (cOPN5) cells were analyzed. They were further characterized using Ca2+ imaging. The light sensitivities were ranging from (eLi50 in µW/mm2) 160 to 2. The most efficient wavelength for activation was shifted maximally from 388 ± 2.7 (hOPN5 (WT), n=10) to 406 ± 2.5 (cOPN5, n=10). Investigation of the retinal dependence among OPN5s hinted at the importance of threonine to alanine mutation. Taken together, hOPN5 is a selective optogenetic tool to control Gq signaling in cells, tissues, and organs. Further, the development of new hOPN5 mutants which are expected to have altered spectral properties would pave the way for a foundation to better understand the structural properties and important amino acid positions of OPN5 with the aid of Molecular dynamics (MD) simulations. Finally, the goal is to develop enhanced and highly efficient OPN5-based optogenetic tools in the future to control organ
dc.contributor.coRefereeKehlenbach, Ralph Prof. Dr.
dc.contributor.thirdRefereeSchlüter, Oliver Prof. Dr. Dr.
dc.subject.engGq signaling cascadede
dc.subject.engCalcium imagingde
dc.affiliation.instituteBiologische Fakultät für Biologie und Psychologiede
dc.subject.gokfullBiologie (PPN619462639)de
dc.notes.confirmationsentConfirmation sent 2024-02-20T15:15:01de

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