bluuebunny/crossref_metadata_2025_split huggingface dataset:
Priming GPCR Signaling Through the Synergistic Effect of Two G Proteins
Authors: Gupte Tejas M. , Malik Rabia U. , Sommese Ruth F. , Ritt Michael , Sivaramakrishnan Sivaraj 
Published: November 2023

While individual GPCRs are known to activate one or more G proteins, the GPCR‐G protein interaction is viewed as a bi‐molecular event involving the formation of a ternary ligand‐GPCR‐G protein complex. We have designed a FRET‐based sensor to monitor and modulate GPCR – G protein interactions in live cells. Using this systematic protein affinity strength modulation (SPASM) approach we present evidence that individual GPCR‐G protein interactions can reinforce each other to enhance signaling through canonical downstream second messengers, a phenomenon we term GPCR priming. Specifically, we find that the presence of non‐cognate Gq protein enhances cAMP stimulated by two Gs coupled receptors, β2‐adrenergic receptor (β2‐AR) and D1 dopamine receptor (D1‐R). The C‐terminus of either the Gαs or Gαq subunit is sufficient to enhance Gα subunit activation and cAMP levels. Interaction of Gαs or Gαq C‐termini with the GPCR increases signaling potency suggesting an altered GPCR conformation as the underlying basis for GPCR priming. We propose three parallel mechanisms involving (1) sequential G protein interactions at the orthosteric site, (2) simultaneous allosteric and orthosteric G protein interactions, and (3) asymmetric GPCR dimers. GPCR priming suggests another layer of regulation in the classic GPCR ternary complex model, with broad implications for the multiplicity inherent in signaling networks.Support or Funding InformationResearch was funded by the American Heart Association Scientist Development Grant (13SDG14270009) & the NIH (1DP2 CA186752‐01 & 1‐R01‐GM‐105646‐01‐A1) to S.S. T.M.G is an NCBS‐inStem Career Development Fellow. R.F.S is a Life Sciences Research Foundation postdoctoral fellow.
Upregulation of RGS2: A New Mechanism for Pirfenidone Amelioration of Pulmonary Fibrosis
Authors: Xie Yan , Jiang Haihong , Zhang Qian , Mehrotra Suneet , Abel Peter W. , Toews Myron L. , Wolff Dennis W. , Rennard Stephen , Panettieri Reynold A. , Casale Thomas B. , Tu Yaping 
Published: November 2023

BackgroundPirfenidone was recently approved for treatment of idiopathic pulmonary fibrosis. However, the therapeutic dose of pirfenidone is very high, causing side effects that limit its doses and therapeutic effectiveness. Understanding the molecular mechanisms of action of pirfenidone could improve its safety and efficacy. Because activated fibroblasts are critical effector cells associated with the progression of fibrosis, this study investigated the genes that change expression rapidly in response to pirfenidone treatment of pulmonary fibroblasts and explored their contributions to the anti‐fibrotic effects of pirfenidone.MethodsWe used the GeneChip microarray to screen for genes that were rapidly up‐regulated upon exposure of human lung fibroblast cells to pirfenidone, with confirmation for specific genes by real‐time PCR and western blots. Biochemical and functional analyses were used to establish their anti‐fibrotic effects in cellular and animal models of pulmonary fibrosis.ResultsWe identified Regulator of G‐protein Signaling 2 (RGS2) as an early pirfenidone‐induced gene. Treatment with pirfenidone significantly increased RGS2 mRNA and protein expression in both a human fetal lung fibroblast cell line and primary pulmonary fibroblasts isolated from patients without or with idiopathic pulmonary fibrosis. Pirfenidone treatment or direct overexpression of recombinant RGS2 in human lung fibroblasts inhibited the profibrotic effects of thrombin, whereas loss of RGS2 exacerbated bleomycin‐induced pulmonary fibrosis and mortality in mice. Pirfenidone treatment reduced bleomycin‐induced pulmonary fibrosis in wild‐type but not RGS2 knockout mice.ConclusionsEndogenous RGS2 exhibits anti‐fibrotic functions. Upregulated RGS2 contributes significantly to the anti‐fibrotic effects of pirfenidone.Support or Funding InformationThis work was supported by NIH grant R01HL116849 to Y. Tu and T.B. Casale; R01HL097796, P01 HL114471 and P30 ES013508 to R.A. Panettieri; Nebraska State LB595 grant to Y. Tu and P.W. Abel; and the NIH National Center for Research Resources G20‐RR024001.
Functional Mechanisms of Novel G Protein Inhibitor Ligands As Vasodilators
Authors: Meleka Matthew M , Medcalf Matthew , Moeller Kevin D , Osei‐Owusu Patrick 
Published: November 2023

G‐protein coupled receptor (GPCR) signaling plays an essential role in physiologic homeostasis and cardiovascular health. Dysfunction of GPCR pathway is implicated in hypertension, a leading risk factor for cardiovascular diseases. Here, we examined the functional properties of novel Gq inhibitor ligands, YM‐254890, FR900359, and WU‐07047, and determined their effectiveness at blocking GPCR signaling in the resistance vasculature. Under ex‐vivo pressurized vessel preparation, we measured vascular reactivity of small mesenteric arteries isolated from 3–4 month old wild type mice. Vasoconstriction of mesenteric arteries was evoked by applying increasing concentrations of the α1‐adrenergic receptor agonist, phenylephrine (PE), in the presence or absence of Gq inhibitors. We found that WU‐07047 had the least effect on maximal contractile response to PE (FR[0.01 μM]: 0.8 ± 0.6% vs. YM[0.01 μM]: 12.8 ± 2.8% vs. WU[10 μM]: 34.8 ± 3.2% %), whereas FR was the most potent (FR[0.01uM]: LogIC50 −0.008 ± 0 vs. YM[0.01uM]: LogIC50 −0.49 ± 0 vs. WU: LogIC50 −64.95 ± 6.4). WU‐07047 and YM‐254890 inhibited vasoconstriction through G protein‐independent pathway by blocking L‐type calcium channels (LTCCs). Bioinformatics analysis revealed a high sequence similarity between binding sites for dihydropyridines in the pore‐forming subunit of LTCCs and the binding pocket for the novel Gq inhibitor ligands in Gqα subunit. We conclude that the novel Gq inhibitors decrease vascular tone by blocking G protein‐dependent and G protein‐independent signaling mechanisms mediating resistance artery vasoconstriction. These findings suggest that targeting signaling downstream of GPCRs may be a more effective therapeutic approach to managing hypertension and associated cardiovascular diseases.Support or Funding InformationPennsylvania Dept. of Health (CURE) Grant (PO)Margaret Q. Landenberger Research Grant (PO)AHA Scientist Development Grant (PO)National Science Foundation (CBET 1262176) (KDM)
Molecular Characteristics of FBXO44‐Mediated RGS2 Protein Degradation
Authors: Sjogren Benita 
Published: November 2023

Hypertension and heart failure are major health issues and there is a need to identify more effective treatments. Regulator of G protein Signaling 2 (RGS2) is highly expressed throughout the cardiovascular system and negatively regulates signaling via several G protein‐coupled receptors known to mediate vasoconstriction and hypertrophic responses. As a result, RGS2−/− mice are hypertensive, show enhanced responses to vasoconstrictors and lower tolerability to pressure overload. Conversely, over‐expression of RGS2 reduces cardiac hypertrophy and we have shown that pharmacologically enhanced RGS2 protein expression has functional effects on G protein signaling both in vitro and in vivo. Therefore, we hypothesize that enhancing RGS2 protein expression is a novel route in treating cardiovascular disease.RGS2 has a short protein half‐life due to rapid degradation through the ubiquitin‐proteasomal pathway. Thus, identifying the specific molecular mechanisms of this intricate system involved in RGS protein degradation will enable rational drug discovery efforts to develop small molecule stabilizers of RGS2 protein. In our previous work, we identified a multi‐component Cullin 4B/DNA Damage Binding protein 1/F‐box protein 44 (CUL4B/DDB1/FBXO44) E3 ligase responsible for mediating RGS protein degradation through high‐throughput siRNA screening. This work is now continuing by defining the molecular characteristics of this complex using biochemical approaches. FBXO44 belongs to a family of over 50 F‐box proteins that confer substrate recognition in Cullin‐RING E3 ligases. In our previous work, we demonstrated that FBXO44 associates with CUL4B and DDB1 through its conserved F‐box domain, in agreement with the canonical action of F‐box proteins. FBXO44 associates with RGS2 through its F‐box Associated (FBA) domain, which is much more variable.FBXO44 is closely related to FBXO2, however it does not bind the same substrates. Based on the homology between FBXO44 and FBXO2, as well as the known substrate recognition sites in FBXO2, we used site‐directed mutagenesis to identify the substrate recognition sites for RGS2 in FBXO44. Mutations in non‐conserved regions of the FBA domain revealed key interaction sites with RGS2. Ongoing work includes defining the corresponding residues in RGS2, responsible for FBXO44 binding, as well as investigating the role of posttranslational modifications and other mechanisms responsible for targeting RGS2 for protein degradation. These studies will aid in rational design of small molecule stabilizers of RGS2 protein that could serve as novel therapeutics in disease states associated with low RGS2 protein levels.Support or Funding InformationAmerican Heart Association (AHA 15SDG21630002)
Computational Prediction of Off‐Target Pharmacology for Discontinued Drugs
Authors: Rao Mohan , Liguori Michael , Mantena Srinivasa , Mittelstadt Scott , Blomme Eric , Van Vleet Terry 
Published: November 2023

Most small molecule drugs interact with unintended, often unknown targets that lead to a wide range of undesirable adverse effects in both pre‐clinical and clinical development and that result in safety‐related attrition. Undesired off‐target interactions are generally weak and quite often not detected using current drug discovery assays. Thus, improvement in the early identification of off‐target interactions is critical to reduce the drug attrition rates related to toxicities. In order to better identify potential off‐target interactions that could be linked to predictable safety issues, we have evaluated and designed a novel computational process to predict safety‐relevant low and high‐affinity interactions that are currently not covered in experimental polypharmacology screens. The result of these analyses termed Off Target Safety Assessment (OTSA) assesses more than 1500 mechanisms of action (target + actions). This approach exploits a highly curated training set of about 10 million compounds, with known in‐vitro activities, derived from patents, journals and internally installed external databases. To evaluate the tool, we used a panel of computational tools to predict both primary and secondary pharmacology for 1279 discontinued drug molecules. A total of 33,513 possible interactions were predicted for these drugs, of which 19,513 interactions (i.e. 15 interactions/compound) were associated with known safety warnings/issues at various levels. The top common safety associated off‐targets for these discontinued drugs are i) Adenosine receptor's ii) Opioid receptor's iii) PPAR's iv) PDE's v) Adrenoceptor's and vi) Potassium channel's. The computational OTSA's most often identified the right pharmacological targets, but also predicted a significant number of off‐targets that may be relevant to the observed in‐vivo effects.All authors are employees of AbbVie. The design, study conduct, and financial support for this research was provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.
THE ANTI‐APOPTOTIC EFFECT OF METHANOL LEAF EXTRACT OF AZADIRACHTA INDICA ON LEAD ACETATE‐INDUCED APOPTOSIS IN CARDIAC AND RENAL TISSUES OF MALE WISTAR RATS
Authors: Adeyeoluwa Temitayo Esther , Oyagbemi Ademola Adetokunbo , Adedapo Adeolu Alex , Omobowale Temidayo Olutayo 
Published: November 2023

Lead toxicity is an existing and ongoing challenge. The apoptotic effects of lead have been extensively studied and several herbal remedies have been proposed. In this study, the anti‐apoptotic effect of the methanol extract of Azadirachta indica (MEAI) was investigated.70 adult male Wistar rats were divided into 7 groups. Groups B, C and D were exposed orally to 1.0 mg/ml lead acetate and groups E, F and G exposed to 2.0 mg/ml for 6‐week duration, after which groups C and D were given 100 mg/kg and 200 mg/kg Azadirachta indica methanol leaf extract respectively, groups F and G given 100 mg/kg and 200 mg/kg Azadirachta indica methanol leaf extract respectively, and groups B and E withdrawn from lead treatment for another 6 weeks. Group A, the control group, was given clean drinking water for the duration of 12 weeks.Exposure to 1.0 mg/ml and 2 mg/ml lead (Pb) resulted in an increased expression of caspase 3 and cytochrome C, which were seen to increase with increased concentration of exposure while there was a reduction in the expression of Bcl‐2 in both the cardiac and renal tissues.At 100 mg/Kg treatment, MEAI induced a higher expression of Bcl‐2 in both the cardiac and renal tissues while at 200 mg/kg treatment, there was a reduction in the expressions of Bcl‐2 in the kidney tissues in the 1 mg/ml Lead (Pb) exposed groups. The treatment of rats that were exposed to 2.0 mg/ml Lead (Pb) with 100 mg/kg and 200 mg/kg MEAI, resulted in an increased expression of Bcl‐2 in the kidney tissues but a down regulation in the heart tissues. MEAI at both 100 mg/kg and 200 mg/kg treatment resulted in a reduced expression of cytochrome C in both tissuesThe results of this study show that at 100 mg/kg and 200 mg/kg concentrations of MEAI, there was an inhibition/reversal of the lead induced apoptosis observed in both the cardiac and renal tissues.
Efavirenz Treatment Activates XBP1 Splicing in Primary Hepatocytes: a Comparison Across Species and an Investigation into the Role of PXR
Authors: Heck Carley J.S. , Bumpus Namandje N. 
Published: November 2023

The non‐nucleoside reverse transcriptase inhibitor efavirenz (EFV) is used to treat human immunodeficiency virus (HIV) despite adverse effects, including hepatoxicity in some individuals. The mechanisms underlying this hepatotoxicity remain largely unknown, although the formation of the primary metabolite, 8OH‐EFV, has been linked to the activation of certain cell death pathways. In light of this, the present study tested whether EFV activates the endoplasmic reticulum (ER) stress response in primary hepatocytes. Splicing of X‐box‐binding protein 1 (XBP1) (a process activated by the ER stress response), was monitored using reverse transcriptase PCR following treatment of primary human and mouse hepatocytes with EFV (10 μM and 50 μM). Incubation of primary hepatocytes isolated from male C57Bl/6 mice with 50 μM EFV resulted in a ratio of spliced to unspliced XBP1 that was 8.8‐fold greater than the vehicle control. In human primary hepatocytes, an increase of 2.6‐fold was observed in response to EFV. In order to gain an understanding of the structural characteristics of EFV that render it a stimulator of XBP1 splicing, treatments were performed using a panel of EFV analogs as well as the 8OH‐EFV. Interestingly, an analog of EFV that differed from EFV in that it contains a trans‐alkene in place of the alkyne exhibited augmented XBP1 splicing response as compared to EFV, with a ratio of spliced to unspliced increased 29.4‐fold over vehicle control in hepatocytes isolated from mice. Several other analogs, including one in which the cyclopropyl ring is broken and the 8OH‐EFV metabolite, did not stimulate splicing of XBP1. Similar patterns of structural dependence were observed between human and mouse primary hepatocytes. Since EFV has been previously reported to activate the nuclear receptor pregnane X receptor (PXR), we tested whether PXR was required for the XBP1 splicing in response to EFV. Interestingly, we observed that XBP1 splicing following EFV treatment was increased in PXR null mice as compared to wild‐type C57Bl/6 mice (76.2‐fold in PXR null versus 8.8‐fold in wild‐type). Similarly, the above mentioned trans‐alkene analog also exhibited enhanced splicing of XBP1 in the PXR null mice as compared to that of wild‐type mice. Viability was measured in primary mouse hepatocytes during the above treatments. All analogs that increased XBP1 splicing also displayed a commensurate decrease in viability, except one in which the oxazinone carbonyl is replaced with a methyl group. In conclusion, EFV stimulates XBP1 splicing in mouse and human primary hepatocytes in a manner that does not appear to be dependent on metabolism to the primary 8OH‐EFV metabolite.Support or Funding InformationNIH R01GM103853 and NSF‐GRFP DGE‐1232825
Structural differences in gut microbiota lead to altered hepatic expression of Drug Disposition Genes (DDG) in mice
Authors: Guo Yukuang , Cho Sung‐joon , Chung Eunah , Park Joo‐Seop , Jeong Hyunyoung 
Published: November 2023

Hepatic drug metabolism is a major route of drug elimination, and it is mediated by multiple DDG. Varied expression and activity of DDG leads to inter‐individual variability in drug response. Results from previous studies comparing conventional and germ‐free mice showed that the presence of gut microbiota is associated with the differential hepatic DDG expression. The purpose of this study was to examine whether structural differences in gut microbiota lead to altered DDG expression in the liver. Male C57BL/6 mice from two different vendors [Jackson Laboratory (JAX) and Taconic Laboratory (TAC)] were cohoused with mice from the same or the other vendor for 4 weeks, creating the following 4 groups (n=6–7/group): JAX, TAC, coJAX (JAX mice cohoused with TAC), and coTAC (TAC mice cohoused with JAX). Mice were sacrificed and RNAs were extracted from the liver tissues. RNA sequencing was performed (n=3 mice/group), followed by bioinformatics analysis of 225 DDG. Principle component analysis of RNA‐sequencing data revealed that overall expression profiles of DDG were distinct in JAX mice while those for the other groups were similar to each other. A similar pattern was observed in results from previous gut microbiome analysis (i.e., 16S rRNA sequencing of fecal samples collected from the same group of mice), suggesting that the differential expression of DDG may be due to structural differences in gut microbiota. To identify DDG whose expression is influenced by gut microbiota, the RNA‐sequencing results of 225 DDG were further examined in livers from the 4 groups of mice. Preliminary statistical analysis revealed that 30 genes (14 phase I; 12 phase II; and 4 transporters) showed significantly different expression between JAX and TAC. The majority (i.e., 27) of these 30 genes showed no difference between coJAX and coTAC, suggesting that expression of these genes may be determined by differential gut microbiota. To further verify the RNA‐sequencing results, quantitative real‐time PCR was performed (n=6–7/group) for 9 genes that were selected based on biological significance and previously reported effects of gut microbiota on their expression. Among them, Cyp4a10 and Cyp4a14 showed 6.0‐ and 33.8‐fold higher expression in JAX, respectively, and Fmo5 showed 1.4‐fold lower expression in JAX. Our study suggests that structural differences in gut microbiota may underlie differential expression of DDG in the liver, potentially contributing to the inter‐individual variability in drug response.Support or Funding InformationThis study was supported by Chicago Biomedical Consortium.
Cytochrome P450 1A2 N‐terminal Site Directed Mutagenesis: Effects on Lipid Microdomain Localization
Authors: Lott Charles S. , Park Ji Won , Reed James R. , Backes Wayne L. 
Published: November 2023

The protein components of the cytochrome P450 monooxygenase system consist of cytochromes P450 (CYPs), NADPH‐dependent cytochrome P450 reductase (CPR), and cytochrome b5. They form functional complexes that catalyze the oxidation of exogenous and endogenous molecules, rendering them more water soluble. In eukaryotes, these membrane bound proteins are found in many tissues and are located primarily in the endoplasmic reticulum. The lipid milieu into which the P450 system proteins reside is heterogenous, consisting of ordered and disordered domains. Ordered domains have a higher content of saturated fatty acids, cholesterol, and sphingomyelin than disordered domains, whereas disordered domains have a higher content of phospholipids with more unsaturated fatty acid chains. When a bilayer membrane is treated with non‐ionic detergent such as Brij 98, the disordered domains readily solubilize whereas ordered regions remain intact. Our lab has shown that various P450 system proteins reside in different lipid microdomains. For example, despite their high level of amino acid sequence similarity, CYP1A2 and CYP1A1 reside in ordered and disordered regions, respectively. Furthermore, our lab has shown that the N‐terminal amino acid sequences of CYP1A2 and CYP1A1 contribute to their specific localization in the ER membrane. The goal of this study is to use site directed mutagenesis to identify which N‐terminal amino acids may be responsible in the lipid raft localization of CYP1A2 and how this effects CYP1A2 enzymatic activity. CYP1A2 N‐terminal amino acids were mutated to their CYP1A1 counterparts and expressed in HEK293‐T cells, and their microdomain localization determined. Although substitution of the NH2‐terminal amino acids of CYP1A2 with that of CYP1A1 leads to changes in their microdomain localization, the results show that Serine4Aspartic Acid and Proline5Phenylalanine mutations alone do not alter microdomain localization of the CYP1A proteins.Support or Funding InformationThis work was supported, in whole or in part, by National Institutes of Health, NIEHS, United StatesPublic Health Service Grants ES004344, and ES013648.