Optical Control of Cell-Surface and Endomembrane-Exclusive ß-Adrenergic Receptor Signaling.

Beta-adrenergic receptors (ßARs) are G protein-coupled receptors (GPCRs) that mediate catecholamine-induced stress responses, such as heart rate increase and bronchodilation. In addition to signals from the cell surface, ßARs also broadcast non-canonical signaling activities from the cell interior membranes (endomembranes). Dysregulation of these receptor pathways underlies severe pathological conditions. Excessive ßAR stimulation is linked to cardiac hypertrophy, leading to heart failure, while impaired stimulation causes compromised fight or flight stress responses and homeostasis. In addition to plasma membrane ßAR, emerging evidence indicates potential pathological implications of deeper endomembrane ßARs, such as inducing cardiomyocyte hypertrophy and apoptosis, underlying heart failure. However, the lack of approaches to control their signaling in subcellular compartments exclusively has impeded linking endomembrane ßAR signaling with pathology. Informed by the ß1AR-catecholamine interactions, we engineered an efficiently photo-labile, protected hydroxy ß1AR pro-ligand (OptoIso) to trigger ßAR signaling at the cell surface, as well as exclusive endomembrane regions upon blue light stimulation. Not only does OptoIso undergo blue light deprotection in seconds, but it also efficiently enters cells and allows examination of G protein heterotrimer activation exclusively at endomembranes. In addition to its application in the optical interrogation of ßARs in unmodified cells, given its ability to control deep organelle ßAR signaling, OptoIso will be a valuable experimental tool.

Spatiotemporal Optical Control of Gαq-PLCß Interactions.

Cells experience time-varying and spatially heterogeneous chemokine signals in vivo, activating cell surface proteins including G protein-coupled receptors (GPCRs). The Gαq pathway activation by GPCRs is a major signaling axis with broad physiological and pathological significance. Compared with other Gα members, GαqGTP activates many crucial effectors, including PLCß (Phospholipase Cß) and Rho GEFs (Rho guanine nucleotide exchange factors). PLCß regulates many key processes, such as hematopoiesis, synaptogenesis, and cell cycle, and is therefore implicated in terminal-debilitating diseases, including cancer, epilepsy, Huntington's Disease, and Alzheimer's Disease. However, due to a lack of genetic and pharmacological tools, examining how the dynamic regulation of PLCß signaling controls cellular physiology has been difficult. Since activated PLCß induces several abrupt cellular changes, including cell morphology, examining how the other pathways downstream of Gq-GPCRs contribute to the overall signaling has also been difficult. Here we show the engineering, validation, and application of a highly selective and efficient optogenetic inhibitor (Opto-dHTH) to completely disrupt GαqGTP-PLCß interactions reversibly in user-defined cellular-subcellular regions on optical command. Using this newly gained PLCß signaling control, our data indicate that the molecular competition between RhoGEFs and PLCß for GαqGTP determines the potency of Gq-GPCR-governed directional cell migration.

Selective C-H Activation of Unprotected Allylamines by Control of Catalyst Speciation.

An outstanding challenge in the Pd-catalyzed functionalization of allylamines is the control of stereochemistry. Terminal alkenes preferentially undergo Heck-type reactions, while internal alkenes may undergo a mixture of Heck and C-H activation reactions that give mixtures of stereochemical products. In the case of unprotected allylamines, the challenge in achieving C-H activation is that facile in situ formation of Pd nanoparticles leads to preferential formation of trans rather than cis-substituted products. In this study we have demonstrated the feasibility of using mono-protected amino acid (MPAA) ligands as metal protecting groups to prevent aggregation and reduction, allowing the selective synthesis of free cis-arylated allylamines. This method complements Heck-selective methods, allowing complete stereochemical control over the synthesis of cinnamylamines, an important class of amine that can serve as therapeutics directly or as advanced intermediates. To highlight the utility of the methodology, we have demonstrated rapid access to mu opioid receptor ligands.

CaaX-motif-adjacent residues influence G protein gamma (Gγ) prenylation under suboptimal conditions.

Prenylation is an irreversible post-translational modification that supports membrane interactions of proteins involved in various cellular processes, including migration, proliferation, and survival. Dysregulation of prenylation contributes to multiple disorders, including cancers and vascular and neurodegenerative diseases. Prenyltransferases tether isoprenoid lipids to proteins via a thioether linkage during prenylation. Pharmacological inhibition of the lipid synthesis pathway by statins is a therapeutic approach to control hyperlipidemia. Building on our previous finding that statins inhibit membrane association of G protein γ (Gγ) in a subtype-dependent manner, we investigated the molecular reasoning for this differential inhibition. We examined the prenylation of carboxy-terminus (Ct) mutated Gγ in cells exposed to Fluvastatin and prenyl transferase inhibitors and monitored the subcellular localization of fluorescently tagged Gγ subunits and their mutants using live-cell confocal imaging. Reversible optogenetic unmasking-masking of Ct residues was used to probe their contribution to prenylation and membrane interactions of the prenylated proteins. Our findings suggest that specific Ct residues regulate membrane interactions of the Gγ polypeptide, statin sensitivity, and extent of prenylation. Our results also show a few hydrophobic and charged residues at the Ct are crucial determinants of a protein's prenylation ability, especially under suboptimal conditions. Given the cell and tissue-specific expression of different Gγ subtypes, our findings indicate a plausible mechanism allowing for statins to differentially perturb heterotrimeric G protein signaling in cells depending on their Gγ-subtype composition. Our results may also provide molecular reasoning for repurposing statins as Ras oncogene inhibitors and the failure of using prenyltransferase inhibitors in cancer treatment.

Spatiotemporal optical control of Gαq-PLCß interactions.

Cells experience time-varying and spatially heterogeneous chemokine signals in vivo, activating cell surface proteins, including G protein-coupled receptors (GPCRs). The Gαq pathway activation by GPCRs is a major signaling axis with a broad physiological and pathological significance. Compared to other Gα members, GαqGTP activates many crucial effectors, including PLCß (Phospholipase Cß) and Rho GEFs (Rho guanine nucleotide exchange factors). PLCß regulates many key processes, such as hematopoiesis, synaptogenesis, and cell cycle, and is therefore implicated in terminal - debilitating diseases, including cancer, epilepsy, Huntington's Disease, and Alzheimer's Disease. However, due to a lack of genetic and pharmacological tools, examining how the dynamic regulation of PLCß signaling controls cellular physiology has been difficult. Since activated PLCß induces several abrupt cellular changes, including cell morphology, examining how the other pathways downstream of Gq-GPCRs contribute to the overall signaling has also been difficult. Here we show the engineering, validation, and application of a highly selective and efficient optogenetic inhibitor (Opto-dHTH) to completely disrupt GαqGTP-PLCß interactions reversibly in user-defined cellular-subcellular regions on optical command. Using this newly gained PLCß signaling control, our data indicate that the molecular competition between RhoGEFs and PLCß for GαqGTP determines the potency of Gq-GPCR-governed directional cell migration.

CaaX-motif adjacent residues control G protein prenylation under suboptimal conditions.

Prenylation is a universal and irreversible post-translational modification that supports membrane interactions of proteins involved in various cellular processes, including migration, proliferation, and survival. Thus, dysregulation of prenylation contributes to multiple disorders, including cancers, vascular diseases, and neurodegenerative diseases. During prenylation, prenyltransferase enzymes tether metabolically produced isoprenoid lipids to proteins via a thioether linkage. Pharmacological inhibition of the lipid synthesis pathway by statins has long been a therapeutic approach to control hyperlipidemia. Building on our previous finding that statins inhibit membrane association of G protein γ (Gγ) in a subtype-dependent manner, we investigated the molecular reasoning for this differential. We examined the prenylation efficacy of carboxy terminus (Ct) mutated Gγ in cells exposed to Fluvastatin and prenyl transferase inhibitors and monitored the subcellular localization of fluorescently tagged Gγ subunits and their mutants using live-cell confocal imaging. Reversible optogenetic unmasking-masking of Ct residues was used to probe their contribution to the prenylation process and membrane interactions of the prenylated proteins. Our findings suggest that specific Ct residues regulate membrane interactions of the Gγ polypeptide statin sensitivity, and prenylation efficacy. Our results also show that a few hydrophobic and charged residues at the Ct are crucial determinants of a protein's prenylation ability, especially under suboptimal conditions. Given the cell and tissue-specific expression of different Gγ subtypes, our findings explain how and why statins differentially perturb heterotrimeric G protein signaling in specific cells and tissues. Our results may provide molecular reasoning for repurposing statins as Ras oncogene inhibitors and the failure of using prenyltransferase inhibitors in cancer treatment.