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Microbial production of cannabinoids promises to provide a consistent, cheaper, and more sustainable supply of these important therapeutic molecules. However, scaling production to compete with traditional plant-based sources is challenging. Our ability to make strain variants greatly exceeds our capacity to screen and identify high producers, creating a bottleneck in metabolic engineering efforts. Here, we present a yeast-based biosensor for detecting microbially produced Δ 9 -tetrahydrocannabinol (THC) to increase throughput and lower the cost of screening. We port five human cannabinoid G protein-coupled receptors (GPCRs) into yeast, showing the cannabinoid type 2 receptor, CB2R, can couple to the yeast pheromone response pathway and report on the concentration of a variety of cannabinoids over a wide dynamic and operational range. We demonstrate that our cannabinoid biosensor can detect THC from microbial cell culture and use this as a tool for measuring relative production yields from a library of Δ 9 -tetrahydrocannabinol acid synthase (THCAS) mutants. Microbial production of cannabinoids promises a cheaper and more sustainable route to these important therapeutic molecules, but strain improvement and screening is challenging. Here, the authors develop a yeast-based Δ9-tetrahydrocannabinol (THC) biosensor for screening microbial mutant libraries.

Strategies aimed at mimicking or enhancing the action of the incretin hormone glucagon-like peptide 1 (GLP-1) therapeutically improve glucose-stimulated insulin secretion (GSIS); however, it is not clear whether GLP-1 directly drives insulin secretion in pancreatic islets. Here, we examined the mechanisms by which GLP-1 stimulates insulin secretion in mouse and human islets. We found that GLP-1 enhances GSIS at a half-maximal effective concentration of 0.4 pM. Moreover, we determined that GLP-1 activates PLC, which increases submembrane diacylglycerol and thereby activates PKC, resulting in membrane depolarization and increased action potential firing and subsequent stimulation of insulin secretion. The depolarizing effect of GLP-1 on electrical activity was mimicked by the PKC activator PMA, occurred without activation of PKA, and persisted in the presence of PKA inhibitors, the KATP channel blocker tolbutamide, and the L-type Ca(2+) channel blocker isradipine; however, depolarization was abolished by lowering extracellular Na(+). The PKC-dependent effect of GLP-1 on membrane potential and electrical activity was mediated by activation of Na(+)-permeable TRPM4 and TRPM5 channels by mobilization of intracellular Ca(2+) from thapsigargin-sensitive Ca(2+) stores. Concordantly, GLP-1 effects were negligible in Trpm4 or Trpm5 KO islets. These data provide important insight into the therapeutic action of GLP-1 and suggest that circulating levels of this hormone directly stimulate insulin secretion by β cells.

G protein-coupled receptors (GPCRs) bind to different G protein α-subtypes with varying degrees of selectivity. The mechanism by which GPCRs achieve this selectivity is still unclear. Using 13 C methyl methionine and 19 F NMR, we investigate the agonist-bound active state of β 1 AR and its ternary complexes with different G proteins in solution. We find the receptor in the ternary complexes adopts very similar conformations. In contrast, the full agonist-bound receptor active state assumes a conformation differing from previously characterised activation intermediates or from β 1 AR in ternary complexes. Assessing the kinetics of binding for the agonist-bound receptor with different G proteins, we find the increased affinity of β 1 AR for G s results from its much faster association with the receptor. Consequently, we suggest a kinetic-driven selectivity gate between canonical and secondary coupling which arises from differential favourability of G protein binding to the agonist-bound receptor active state. The authors show G protein subtype selectivity at the β 1 -adrenergic receptor is driven by the binding kinetics of ternary complex formation. Bound to G protein, the receptor adopts conformations that differ from its agonist-bound solution states.

Receptor-activity–modifying proteins (RAMPs) are single transmembrane-spanning proteins which serve as molecular chaperones and allosteric modulators of G-protein–coupled receptors (GPCRs) and their signaling pathways. Although RAMPs have been previously studied in the context of their effects on Family B GPCRs, the coevolution of RAMPs with many GPCR families suggests an expanded repertoire of potential interactions. Using bioluminescence resonance energy transfer-based and cell-surface expression approaches, we comprehensively screen for RAMP interactions within the chemokine receptor family and identify robust interactions between RAMPs and nearly all chemokine receptors. Most notably, we identify robust RAMP interaction with atypical chemokine receptors (ACKRs), which function to establish chemotactic gradients for directed cell migration. Specifically, RAMP3 association with atypical chemokine receptor 3 (ACKR3) diminishes adrenomedullin (AM) ligand availability without changing G-protein coupling. Instead, RAMP3 is required for the rapid recycling of ACKR3 to the plasma membrane through Rab4-positive vesicles following either AM or SDF-1/CXCL12 binding, thereby enabling formation of dynamic spatiotemporal chemotactic gradients. Consequently, genetic deletion of either ACKR3 or RAMP3 in mice abolishes directed cell migration of retinal angiogenesis. Thus, RAMP association with chemokine receptor family members represents a molecular interaction to control receptor signaling and trafficking properties.

The development of therapeutic agonists for G protein-coupled receptors (GPCRs) is hampered by the propensity of GPCRs to couple to multiple intracellular signalling pathways. This promiscuous coupling leads to numerous downstream cellular effects, some of which are therapeutically undesirable. This is especially the case for adenosine A 1 receptors (A 1 Rs) whose clinical potential is undermined by the sedation and cardiorespiratory depression caused by conventional agonists. We have discovered that the A 1 R-selective agonist, benzyloxy-cyclopentyladenosine (BnOCPA), is a potent and powerful analgesic but does not cause sedation, bradycardia, hypotension or respiratory depression. This unprecedented discrimination between native A 1 Rs arises from BnOCPA’s unique and exquisitely selective activation of Gob among the six Gαi/o subtypes, and in the absence of β-arrestin recruitment. BnOCPA thus demonstrates a highly-specific Gα-selective activation of the native A 1 R, sheds new light on GPCR signalling, and reveals new possibilities for the development of novel therapeutics based on the far-reaching concept of selective Gα agonism. Wall et al. describe the selective activation of an adenosine A1 receptor-mediated intracellular pathway that provides potent analgesia in the absence of sedation or cardiorespiratory depression, paving the way for novel medicines based on the far-reaching concept of selective Gα agonism.

Patients diagnosed with lung squamous cell carcinoma (LUSC) have limited targeted therapies. We report here the identification and characterisation of BCL11A , as a LUSC oncogene. Analysis of cancer genomics datasets revealed BCL11A to be upregulated in LUSC but not in lung adenocarcinoma (LUAD). Experimentally we demonstrate that non-physiological levels of BCL11A in vitro and in vivo promote squamous-like phenotypes, while its knockdown abolishes xenograft tumour formation. At the molecular level we found that BCL11A is transcriptionally regulated by SOX2 and is required for its oncogenic functions. Furthermore, we show that BCL11A and SOX2 regulate the expression of several transcription factors, including SETD8 . We demonstrate that shRNA-mediated or pharmacological inhibition of SETD8 selectively inhibits LUSC growth. Collectively, our study indicates that BCL11A is integral to LUSC pathology and highlights the disruption of the BCL11A–SOX2 transcriptional programme as a novel candidate for drug development. Amongst the non-small cell lung cancers, to date, lung squamous cell carcinoma remains the most challenging to treat. Here the authors report BCL11A as an important factor which together with SOX2 can drive lung squamous cell carcinoma development and highlight a potential novel therapeutic candidate for this devastating disease.

IntroductionThe β2-adrenoceptor (β2AR) is a class A G protein-coupled receptor (GPCR). It is therapeutically relevant in asthma and chronic obstructive pulmonary disease (COPD), where β2AR agonists relieve bronchoconstriction. The β2AR is a prototypical GPCR for structural and biophysical studies. However, the molecular basis of agonist efficacy at the β2AR is not understood. We hypothesised that the kinetics of GPCR–G protein interactions could play a role in determining ligand efficacy. By studying a range of agonists with varying efficacy, we examined the relationship between ligand-induced mini-Gs binding to the β2AR and ligand efficacy, along with the ability of individual ligands to activate the G protein in cells.MethodsWe used NanoBRET technology to measure ligand-induced binding of purified Venus-mini-Gs to β2AR-nLuc in membrane preparations under both equilibrium and kinetic conditions. In addition, we examined the ability of these β2AR agonists to activate the heterotrimeric Gs protein, measured using the Gs-CASE protein biosensor in living cells. This assay detects a reduction in NanoBRET between the nano-luciferase (nLuc) donor on the Gα subunit and Venus acceptor on the Gγ upon Gs protein activation.ResultsThe 12 β2AR agonists under study revealed a broad range of ligand potency and efficacy values in the cellular Gs-CASE assays. Kinetic characterisation of mini-Gs binding to the agonist β2AR complex revealed a strong correlation between ligand efficacy values (Emax) and mini-Gs affinity ( K d) and its association rate ( k on). In contrast, there was no correlation between ligand efficacy and reported ligand dissociation rates (or residence times).ConclusionThe association rate ( k on) of the G protein to the agonist β2AR complex is directly correlated with ligand efficacy. These data support a model in which higher-efficacy agonists induce the β2AR to adopt a conformation that is more likely to recruit G protein. Conversely, these data did not support the role of agonist binding kinetics in determining the molecular basis of efficacy.

IP 3 receptor (IP 3 R)-mediated Ca 2+ transfer at the mitochondria-endoplasmic reticulum (ER) contact sites (MERCS) drives mitochondrial Ca 2+ uptake and oxidative metabolism and is linked to different pathologies, including Parkinson’s disease (PD). The dependence of Ca 2+ transfer efficiency on the ER-mitochondria distance remains unexplored. Employing molecular rulers that stabilize ER-mitochondrial distances at 5 nm resolution, and using genetically encoded Ca 2+ indicators targeting the ER lumen and the sub-mitochondrial compartments, we now show that a distance of ~20 nm is optimal for Ca 2+ transfer and mitochondrial oxidative metabolism due to enrichment of IP 3 R at MERCS. In human iPSC-derived astrocytes from PD patients, 20 nm MERCS were specifically reduced, which correlated with a reduction of mitochondrial Ca 2+ uptake. Stabilization of the ER-mitochondrial interaction at 20 nm, but not at 10 nm, fully rescued mitochondrial Ca 2+ uptake in PD astrocytes. Our work determines with precision the optimal distance for Ca 2+ flux between ER and mitochondria and suggests a new paradigm for fine control over mitochondrial function. The distance-dependence analysis of ER-mitochondria Ca 2+ transfer suggests that a distance of ∼20 nm between the organelles ensures maximal efficiency of mitochondrial Ca 2+ uptake and OXPHOS and rescues Ca 2+ uptake in Parkinson’s disease mitochondria.

Cryopreservation offers the potential to increase the availability of pancreatic islets for treatment of diabetic patients. However, current protocols, which use dimethyl sulfoxide (DMSO), lead to poor cryosurvival of islets. We demonstrate that equilibration of mouse islets with small molecules in aqueous solutions can be accelerated from > 24 to 6 h by increasing incubation temperature to 37 °C. We utilize this finding to demonstrate that current viability staining protocols are inaccurate and to develop a novel cryopreservation method combining DMSO with trehalose pre-incubation to achieve improved cryosurvival. This protocol resulted in improved ATP/ADP ratios and peptide secretion from β-cells, preserved cAMP response, and a gene expression profile consistent with improved cryoprotection. Our findings have potential to increase the availability of islets for transplantation and to inform the design of cryopreservation protocols for other multicellular aggregates, including organoids and bioengineered tissues.

The spatial and temporal control of polarity is fundamental to the survival of all organisms. Cells define their polarity using highly conserved mechanisms that frequently rely upon the action of small GTPases, such as Ras and Cdc42. Schizosaccharomyces pombe is an ideal system with which to study the control of cell polarity since it grows from defined tips using Cdc42-mediated actin remodeling. Here we have investigated the importance of Ras1-GTPase activity for the coordination of polarized cell growth during fission yeast mating. Following pheromone stimulation, Ras1 regulates both a MAPK cascade and the activity of Cdc42 to enable uni-directional cell growth towards a potential mating partner. Like all GTPases, when bound to GTP, Ras1 adopts an active conformation returning to an inactive state upon GTP-hydrolysis, a process accelerated through interaction with negative regulators such as GAPs. Here we show that, at low levels of pheromone stimulation, loss of negative regulation of Ras1 increases signal transduction via the MAPK cascade. However, at the higher concentrations observed during mating, hyperactive Ras1 mutations promote cell death. We demonstrate that these cells die due to their failure to coordinate active Cdc42 into a single growth zone resulting in disorganized actin deposition and unsustainable elongation from multiple tips. These results provide a striking demonstration that the deactivation stage of Ras signaling is fundamentally important in modulating cell polarity.