Mini G protein probes for active G protein– coupled receptors (GPCRs) in live cells

G protein–coupled receptors (GPCRs) are key signaling proteins that regulate nearly every aspect of cell function. Studies of GPCRs have benefited greatly from the development of molecular tools to monitor receptor activation and downstream signaling. Here, we show that mini G proteins are robust probes that can be used in a variety of assay formats to report GPCR activity in living cells. Mini G (mG) proteins are engineered GTPase domains of G subunits that were developed for struc- tural studies of active-state GPCRs. Confocal imaging revealed that mG proteins fused to fluorescent proteins were located diffusely in the cytoplasm and translocated to sites of receptor activation at the cell surface and at intracellular organ- elles. Bioluminescence resonance energy transfer (BRET) assays with mG proteins fused to either a fluorescent protein or luciferase reported agonist, superagonist, and inverse agonist activities. Variants of mG proteins (mGs, mGsi, mGsq, and mG12) corresponding to the four families of G subunits displayed appropriate coupling to their cognate GPCRs, allowing quantitative profiling of subtype-specific coupling to individual receptors. BRET between luciferase–mG fusion proteins and fluorescent markers indicated the presence of active GPCRs at the plasma membrane, Golgi apparatus, and endosomes. Complementation assays with fragments of NanoLuc luciferase fused to GPCRs and mG proteins reported constitutive receptor activity and agonist-induced activation with up to 20-fold increases in luminescence. We conclude that mG proteins are versatile tools for studying GPCR activation and coupling specificity in cells and should be useful for discovering and characterizing G protein sub- type–biased ligands

The Eating and Cooking Healthy (TEACH) Kitchen: A Research Protocol

Background: Diet-related chronic diseases, such as diabetes mellitus, hypertension, and hyperlipidemia have affected millions of individuals, resulting in disease-related complications and mortality. Strategies that may improve the outcome of chronic disease management include modification of lifestyle risk factors such as unhealthy diets. TEACH Kitchen is an experiential education program related to community nutrition, the goal of which is to teach patients management of chronic disease through dietary change. Methods: Adults (n=144) ≥18 years old and their children (n=144) 7-17 years old will complete four 2-hour sessions. Components of each session will include brief nutrition education (20 min), an interactive cooking session (1 hr), and after-dinner discussion (40 min). Pre- and post-session questionnaires will be administered to all participants for self-reported demographics, knowledge, attitude, and beliefs about healthy nutrition. Medical records will be used to collect information about adult participants’ demographics and clinical indicators (hemoglobin A1c, lipid profile, blood pressure, weight, height, and body mass index [BMI]). Descriptive analyses will be performed to determine socio-demographic characteristics using frequencies and proportions for all categorical data, and means for continuous variables. T-tests and multiple logistic regression analysis will be accomplished to compare the differences in means. Results: Differences in the pre- and post-session knowledge, attitude, and beliefs related to healthy eating will be evaluated for adults and children. The anticipated outcomes include enhanced education promoting healthy eating in the community, prevention of chronic disease complications related to poor diet, and prevention of obesity-related chronic diseases in children. Conclusions: Enhancement of chronic disease management among patients, and the prevention of obesity among children, can be accomplished through healthy cooking and diet

Mechanism of Copper(I)-Catalyzed 5‑Iodo-1,2,3-triazole Formation from Azide and Terminal Alkyne

5-Iodo-1,2,3-triazole (iodotriazole) can be prepared from a copper­(I)-catalyzed reaction between azide and terminal alkyne in the presence of an iodinating agent, with 5-protio-1,2,3-triazole (protiotriazole) as the side product. The increasing utilities of iodotriazoles in synthetic and supramolecular chemistry drive the efforts in improving their selective syntheses based on a sound mechanistic understanding. A routinely proposed mechanism takes the cue from the copper­(I)-catalyzed azide–alkyne cycloaddition, which includes copper­(I) acetylide and triazolide as the early and the late intermediates, respectively. Instead of being protonated to afford protiotriazole, an iodinating agent presumably intercepts the copper­(I) triazolide to give iodotriazole. The current work shows that copper­(I) triazolide can be iodinated to afford iodotriazoles. However, when the reaction starts from a terminal alkyne as under the practical circumstances, 1-iodoalkyne (iodoalkyne) is an intermediate while copper­(I) triazolide is bypassed on the reaction coordinate. The production of protiotriazole commences after almost all of the iodoalkyne is consumed. Using ¹H NMR to follow a homogeneous iodotriazole forming reaction, the rapid formation of an iodoalkyne is shown to dictate the selectivity of an iodotriazole over a protiotriazole. To ensure the exclusive production of iodotriazole, the complete conversion of an alkyne to an iodoalkyne has to, and can be, achieved at the early stage of the reaction

Loss-of-function of Endothelin receptor type A results in Oro-Oto-Cardiac syndrome

© 2020 Wiley Periodicals, Inc. Craniofacial morphogenesis is regulated in part by signaling from the Endothelin receptor type A (EDNRA). Pathogenic variants in EDNRA signaling pathway components EDNRA, GNAI3, PCLB4, and EDN1 cause Mandibulofacial Dysostosis with Alopecia (MFDA), Auriculocondylar syndrome (ARCND) 1, 2, and 3, respectively. However, cardiovascular development is normal in MFDA and ARCND individuals, unlike Ednra knockout mice. One explanation may be that partial EDNRA signaling remains in MFDA and ARCND, as mice with reduced, but not absent, EDNRA signaling also lack a cardiovascular phenotype. Here we report an individual with craniofacial and cardiovascular malformations mimicking the Ednra−/− mouse phenotype, including a distinctive micrognathia with microstomia and a hypoplastic aortic arch. Exome sequencing found a novel homozygous missense variant in EDNRA (c.1142A\u3eC; p.Q381P). Bioluminescence resonance energy transfer assays revealed that this amino acid substitution in helix 8 of EDNRA prevents recruitment of G proteins to the receptor, abrogating subsequent receptor activation by its ligand, Endothelin-1. This homozygous variant is thus the first reported loss-of-function EDNRA allele, resulting in a syndrome we have named Oro-Oto-Cardiac Syndrome. Further, our results illustrate that EDNRA signaling is required for both normal human craniofacial and cardiovascular development, and that limited EDNRA signaling is likely retained in ARCND and MFDA individuals. This work illustrates a straightforward approach to identifying the functional consequence of novel genetic variants in signaling molecules associated with malformation syndromes