Biology subjects

Karunarathne, A.

Publications and source records attributed to Karunarathne, A..

5 recordsLinked to original sources

bioRxiv2024

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

Beta-adrenergic receptors ({beta}ARs) 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, {beta}ARs also broadcast non-canonical signaling activities from the cell interior membranes (endomembranes). Dysregulation of these receptor pathways underlies severe pathological conditions. Excessive {beta}AR 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 {beta}AR, emerging evidence indicates potential pathological implications of deeper endomembrane {beta}ARs, 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 {beta}AR signaling with pathology. Informed by the {beta}1AR-catecholamine interactions, we engineered an efficiently photo-labile, protected hydroxy {beta}1AR pro-ligand (OptoIso) to trigger {beta}AR 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 {beta}ARs in unmodified cells, given its ability to control deep organelle {beta}AR signaling, OptoIso will be a valuable experimental tool.

biochemistry↗

bioRxiv2023

Engineered blue-shifted melanopsins for subcellular optogenetics

Melanopsin (MeOp) is a G protein-coupled Receptor (GPCR) family photopigment, expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs) that display remarkable functional diversity. In addition to non-image-forming visual functions, MeOp also controls signaling underlying the retina development, circadian clock, mood, and behavior. MeOp is bistable, recycles retinal, and can function under low retinaldehyde availability. It also activates multiple G protein heterotrimers. Though MeOp could be a versatile optogenetic tool, its potential, especially its utility for subcellular signaling control, is hampered by the broader spectral sensitivity spanning the entire visible range. Here, we use a recently reported in silico technology called Automatic Rhodopsin Modeling (ARM) to identify blue-shifting mutations of MeOp and, ultimately, allow for imaging biosensors with red light without activating the opsin. Accordingly, ARM was used to construct validated quantum mechanics/molecular mechanics (QM/MM) models for mouse MeOp (mMeOp) to search and optimize a set of mutants featuring a blue-shifted light absorption. We demonstrate that four mutants of such can be successfully expressed and display the required resistance to activation by red light; however, they are activated by yellow, green, and blue light. Localized subcellular optical activation of these mutants in macrophage cells showed localized PIP3 generation and cell migration. Further characterization showed that MeOp blue-shifted mutants are also bistable. Altogether, our data demonstrate the computer-aided engineering feasibility of opsins with desired spectral properties for subcellular optogenetic applications.

biochemistry↗

bioRxiv2023

Spatiotemporal optical control of Gαq-PLCβ interactions

Cells experience time-varying and spatially heterogeneous chemokine signals in vivo, activating cell surface proteins, including G protein-coupled receptors (GPCRs). The Gq pathway activation by GPCRs is a major signaling axis with a broad physiological and pathological significance. Compared to other G members, GqGTP activates many crucial effectors, including PLC{beta} (Phospholipase C{beta}) and Rho GEFs (Rho guanine nucleotide exchange factors). PLC{beta} regulates many key processes, such as hematopoiesis, synaptogenesis, and cell cycle, and is therefore implicated in terminal - debilitating diseases, including cancer, epilepsy, Huntingtons Disease, and Alzheimers Disease. However, due to a lack of genetic and pharmacological tools, examining how the dynamic regulation of PLC{beta} signaling controls cellular physiology has been difficult. Since activated PLC{beta} 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 GqGTP-PLC{beta} interactions reversibly in user-defined cellular-subcellular regions on optical command. Using this newly gained PLC{beta} signaling control, our data indicate that the molecular competition between RhoGEFs and PLC{beta} for GqGTP determines the potency of Gq-GPCR-governed directional cell migration.

molecular biology↗

bioRxiv2023

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 {gamma} (G{gamma}) in a subtype-dependent manner, we investigated the molecular reasoning for this differential. We examined the prenylation efficacy of carboxy terminus (Ct) mutated G{gamma} in cells exposed to Fluvastatin and prenyl transferase inhibitors and monitored the subcellular localization of fluorescently tagged G{gamma} 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 G{gamma} 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 proteins prenylation ability, especially under suboptimal conditions. Given the cell and tissue-specific expression of different G{gamma} 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.

cell biology↗

bioRxiv2022

Molecular regulation of GPCR-G-protein-governed PIP3 generation and its adaptation

Phosphatidylinositol (3,4,5) trisphosphate (PIP3) is a plasma membrane-bound signaling phospholipid involved in many cellular signaling pathways that control crucial cellular processes and behaviors, including cytoskeleton remodeling, metabolism, chemotaxis, and apoptosis. Therefore, defective PIP3 signaling is implicated in various disease driving processes, including cancer metastasis, diabetes, obesity, and cardiovascular diseases. Upon activation by G protein-coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs), phosphoinositide-3-kinases (PI3Ks) phosphorylate phosphatidylinositol (4,5) bisphosphate (PIP2), generating PIP3. Interestingly, though the mechanisms are unclear, PIP3 produced upon GPCR activation attenuates within minutes, indicating a tight temporal regulation. Our data show the subcellular redistributions of G proteins govern this PIP3 attenuation in the presence of sustained receptor stimulation, and thus meet the definition of signaling adaptation. Interestingly the observed adaptation of PIP3 was G{gamma} subtype-dependent. Considering distinct cell-tissue-specific G{gamma} expression profiles, our findings not only demonstrate how the GPCR-induced PIP3 response is adapted but also show how diversely this adaptation process is regulated by the dominant G{gamma}s of a cell.

cell biology↗