Biology subjects

Oishi, A.

Publications and source records attributed to Oishi, A..

7 recordsLinked to original sources

bioRxiv2025

Structural basis and physiological significance of non-canonical Gs coupling to the prototypical Gi-coupled melatonin MT1 receptor

G protein-coupled receptors (GPCRs) transduce extracellular stimuli into intracellular signals by coupling to various heterotrimeric G proteins. However, the rules governing G protein preference remain largely elusive. MT1 and MT2 are prototypical Gi/o-coupled GPCRs responding to melatonin, a hormone secreted in a circadian manner. We show here that MT1, but not MT2, couples also to Gs proteins in vitro and activates the Gs/cAMP pathway upon long-term melatonin exposure in vivo, mimicking physiological dawn conditions. We solved the cryo-electron microscopy structure of the melatonin-MT1-Gs complex at 3.0[A] resolution, which revealed a strikingly distinct binding mode compared to the MT1-Gi complex. The third intracellular loop of MT1 emerges as a key stabilizer for Gs coupling, a feature previously unrecognized. This is the first solved receptor-Gs complex of a primary Gi-coupled GPCRs, providing new structural and functional insights into G protein selectivity and circadian switch of G protein coupling.

molecular biology↗

bioRxiv2025

Nocturnal intraocular pressure rise is regulated by norepinephrine via RHOB

Intraocular pressure (IOP), a key factor in glaucoma development, is regulated by aqueous humor (AH) dynamics, with inflow from the ciliary body and outflow through the trabecular meshwork (TM). IOP has a circadian rhythm entrained by sympathetic norepinephrine (NE) from the superior cervical ganglion. Herein, we investigated its underlying regulatory mechanisms in the TM. Through comprehensive gene expression analysis of human TM cells and mouse eyes, we identified 18 genes upregulated by NE stimulation, including the small GTPase RAS homologous protein B (RHOB). Promoter assays revealed RHOB upregulation via the cyclic adenosine monophosphate (cAMP) response element on its promotor. NE stimulation for 6-9 h increased RHOB level and cellular adhesion, and suppressed liquid permeability in the TM cells, indicating a time-dependent effect. RHOB deficiency increased TM macrophage phagocytosis and eliminated NE-induced suppression of phagocytosis and permeability, whereas RHOB overexpression had the opposite effect. Instillations of RHO or RHO-kinase inhibitors to mice eye reduced nocturnal and NE-induced IOP elevation. Our findings suggest that NE can elevate IOP via RHOB-mediated inhibition of TM phagocytosis, positioning RHOB as a potential glaucoma treatment target and IOP rhythm regulator.

pathology↗

bioRxiv2024

Callose Deficiency Modulates Plasmodesmata frequency and the intercellular space In Rice Anthers

Fertilization relies on pollen mother cells able to transit from mitosis-to-meiosis to supply gametes. This process involves remarkable changes at the molecular, cellular and physiological levels including (but not limited to) remodelling of cell wall. During meiosis onset, cellulose content at the pollen mother cell walls gradually declines with the concurrent deposition of the polysaccharide callose in anther locules. We aim to understand the biological significance of cellulose-to-callose turnover in pollen mother cells walls using electron microscopic analyses of rice flowers. Our observations indicate that in wild type anthers, the mitosis-to-meiosis transition coincides with a gradual reduction in the number of cytoplasmic connections called plasmodesmata. A mutant in the Oryza sativa callose synthase GSL5, impaired in callose accumulation in premeiotic and meiotic anthers, displayed a reduction in plasmodesmata frequency among pollen mother cells and tapetal cells suggesting a role for callose in plasmodesmata maintenance. In addition, a significant increase in cell-cell distance between pollen mother cells and impaired premeiotic cell shaping was observed in the mutant. The results suggest that cellulose-to-callose turnover during mitosis-meiosis transition is necessary to maintain cell-to-cell connections and optimal intercellular spacing among anther locular cells explaining the regulatory influence of callose metabolism during meiosis in flowering plants. HighlightsO_LICellulose-to-callose switch during meiosis onset in pollen mother cell walls correlates with changes in cytoplasmic connections called plasmodesmata, among meiocytes and between meiocytes and surrounding somatic cells. C_LIO_LIA mutant in GSL5 (glucan synthase like 5) affects callose deposition but no other cell wall component (e.g., cellulose) in premeiotic and meiotic anthers, except at dyad stage. C_LIO_LIImpaired callose synthesis is proposed to alter plasmodesmata frequency, the extracellular spacing and the shaping of pollen mother cells during mitosis-to-meiosis transition. C_LIO_LIIncrease in plasmodesmata frequency negatively correlates with cell-cell distance between pollen mother cells in both wildtype and a callose mutant. C_LI

plant biology↗

bioRxiv2022

Novel repertoire of tau biosensors to monitor pathological tau transformation and seeding activity in living cells

Aggregates of the tau protein are a well-known hallmark of several neurodegenerative diseases, collectively referred as tauopathies, including frontal temporal dementia and Alzheimers disease (AD). Monitoring the transformation process of tau from physiological monomers into pathological oligomers or aggregates in a high-throughput, quantitative manner and in a cellular context is still a major challenge in the field. Identifying molecules able to interfere with those processes is of high therapeutic interest. Here, we developed a series of inter- and intramolecular tau biosensors based on the highly sensitive Nanoluciferase (Nluc) binary technology (NanoBiT) able to monitor the pathological conformational change and self-interaction of tau in living cells. Our repertoire of tau biosensors reliably report i. molecular proximity of physiological full-length tau at microtubules; ii. changes in tau conformation and self-interaction associated with tau phosphorylation, as well as iii. tau interaction induced by seeds of recombinant tau or from mouse brain lysates of a mouse model of tau pathology. By comparing biosensors comprising different tau forms (i.e. full-length or short fragments, wild-type or the disease-associated tau(P301L) variant) further insights in the tau transformation process are obtained. Proof-of-concept data for the high-throughput suitability and identification of molecules interfering with the pathological tau transformation processes are presented. This novel repertoire of tau biosensors is aimed to boost the disclosure of molecular mechanisms underlying pathological tau transformation in living cells and to discover new drug candidates for tau-related neurodegenerative diseases.

cell biology↗

bioRxiv2021

Acylation of the incretin peptide exendin-4 directly impacts GLP-1 receptor signalling and trafficking

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor and mainstay therapeutic target for the treatment of type 2 diabetes and obesity. Recent reports have highlighted how biased agonism at the GLP-1R affects sustained glucose-stimulated insulin secretion through avoidance of desensitisation and downregulation. A number of GLP-1R agonists (GLP-1RAs) feature a fatty acid moiety to promote albumin binding in order to prolong their pharmacokinetics, but the potential for these ligand changes to influence GLP-1R signalling has rarely been investigated beyond potency assessments for cyclic adenosine monophosphate (cAMP). In this work we directly compare the prototypical GLP-1RA exendin-4 with its C-terminally acylated analogue, exendin-4-C16, for their relative propensities to recruit and activate G proteins and {beta}-arrestins, endocytic and post-endocytic trafficking profiles, and interactions with model and cellular membranes. Both ligands had similar cAMP potency but the exendin-4-C16 showed [~]2.5-fold bias towards G protein recruitment and a [~]60% reduction in {beta}-arrestin-2 recruitment efficacy compared to exendin-4, as well as reduced GLP-1R endocytosis and preferential targeting towards recycling pathways. These effects were associated with a reduced ability to promote the movement of the GLP-1R extracellular domain, as determined using a conformational biosensor approach, and a [~]70% increase in insulin secretion. Interactions with plasma membrane lipids were enhanced by the acyl chain. Exendin-4-C16 showed extensive albumin binding and was highly effective for lowering of blood glucose in mice over at least 72 hours. Overall, our study highlights the importance of a broad approach to the evaluation of GLP-1RA pharmacology. Significance statementAcylation is a common strategy to enhance the pharmacokinetics of peptide-based drugs. Our work shows how acylation can also affect various other pharmacological parameters, including biased agonism, receptor trafficking and interactions with the plasma membrane, which may be therapeutically important.

pharmacology and toxicology↗

bioRxiv2021

A PX-BAR protein Mvp1/SNX8 and a dynamin-like GTPase Vps1 drive endosomal recycling

Membrane protein recycling systems are essential for maintenance of the endosome-lysosome system. In yeast, retromer and Snx4 coat complexes are recruited to the endosomal surface where they recognize cargos. They sort cargo and deform the membrane into recycling tubules that bud from the endosome and target to the Golgi. Here, we reveal that the SNX-BAR protein, Mvp1, mediates an endosomal recycling pathway which is mechanistically distinct from the retromer and Snx4 pathways. Mvp1 deforms the endosomal membrane and sorts cargos containing a specific sorting motif into a membrane tubule. Subsequently, Mvp1 recruits the dynamin-like GTPase Vps1 to catalyze membrane scission and release of the recycling tubule. Similarly, SNX8, the human homolog of Mvp1, which has been also implicated in Alzheimers disease, mediates formation of an endosomal recycling tubule. Thus, we present evidence for a novel endosomal retrieval pathway that is conserved from yeast to humans. In BriefPX-BAR Mvp1 and dynamin-like GTPase Vps1 drive retromer independent endosomal recycling. HighlightsO_LIRetromer- and Snx4-independent endosomal recycling pathway discovered C_LIO_LISNX-BAR Mvp1 and dynamin-like GTPase Vps1 mediate cargo sorting into recycling tubules/vesicles in the absence of retromer function C_LIO_LIMvp1 together with retromer and Snx4 complexes contribute to proper endosome function C_LIO_LIMvp1 mediated recycling is evolutionary conserved from yeast to humans C_LIO_LICharacters: 43,934/45,000 (including spaces and main figure legends but excluding STAR Methods text, supplemental item legends, and References section) C_LI

cell biology↗

bioRxiv2020

Enterohepatic transcription factor CREB3L3 protects atherosclerosis via SREBP competitive inhibition

CREB3L3 is a membrane-bound transcription factor to maintain lipid metabolism in the liver and small intestine. CREB3L3 ablation in Ldlr-/- mice exacerbated hyperlipidemia with remnant ApoB-containing lipoprotein accumulation, developing enhanced aortic atheroma formation, whose extent was additive between liver- and intestine-specific deletion. Conversely, hepatic nuclear CREB3L3 overexpression markedly suppressed atherosclerosis with amelioration of hyperlipidemia. CREB3L3 directly upregulates anti-atherogenic FGF21 and ApoA4, whereas antagonizes hepatic SREBP-mediated lipogenic and cholesterogenic genes and regulates LXR-regulated genes involved in intestinal transport of cholesterol. CREB3L3 deficiency accumulates nuclear SREBP proteins. Because both transcriptional factors share the cleavage system for nuclear transactivation, full-length CREB3L3 and SREBPs on endoplasmic reticulum (ER) functionally inhibit each other. CREB3L3 competitively antagonizes SREBPs for ER-Golgi transport, resulting in ER retention and proteolytic activation inhibition at Golgi, and vice versa. Collectively, due to this new mechanistic interaction between CREB3L3 and SREBPs under atherogenic conditions, CREB3L3 has multi-potent protective effects against atherosclerosis.

physiology↗