-
ELife May 2024Retinitis pigmentosa (RP) is an inherited retinal disease in which there is a loss of cone-mediated daylight vision. As there are >100 disease genes, our goal is to...
Retinitis pigmentosa (RP) is an inherited retinal disease in which there is a loss of cone-mediated daylight vision. As there are >100 disease genes, our goal is to preserve cone vision in a disease gene-agnostic manner. Previously we showed that overexpressing TXNIP, an α-arrestin protein, prolonged cone vision in RP mouse models, using an AAV to express it only in cones. Here, we expressed different alleles of in the retinal pigmented epithelium (RPE), a support layer for cones. Our goal was to learn more of TXNIP's structure-function relationships for cone survival, as well as determine the optimal cell type expression pattern for cone survival. The C-terminal half of TXNIP was found to be sufficient to remove GLUT1 from the cell surface, and improved RP cone survival, when expressed in the RPE, but not in cones. Knock-down of HSP90AB1, a TXNIP-interactor which regulates metabolism, improved the survival of cones alone and was additive for cone survival when combined with TXNIP. From these and other results, it is likely that TXNIP interacts with several proteins in the RPE to indirectly support cone survival, with some of these interactions different from those that lead to cone survival when expressed only in cones.
Topics: Animals; Retinitis Pigmentosa; Retinal Cone Photoreceptor Cells; Disease Models, Animal; Mice; Carrier Proteins; Mutation, Missense; Cell Survival; Alleles; Gene Deletion; Thioredoxins; Retinal Pigment Epithelium
PubMed: 38727583
DOI: 10.7554/eLife.90749 -
Scientific Reports Apr 2024Chronic stress is associated with major depressive disorder (MDD). Increased glucocorticoid levels caused by uncontrolled release through the...
Chronic stress is associated with major depressive disorder (MDD). Increased glucocorticoid levels caused by uncontrolled release through the hypothalamic‒pituitary‒adrenal (HPA) axis can cause changes in the lipid content of the cellular plasma membrane. These changes are suspected to be involved in the development of depressive disorders. St. John's wort extract (SJW) Ze 117 has long been used as an alternative to synthetic antidepressants. Part of its effect may be due to an effect on the cellular lipid composition and thus on the properties of plasma membranes and receptor systems embedded therein. In this study, we investigated the effect of Ze 117 on that of dexamethasone and simvastatin. Dexamethasone increases the fluidity of C6 cell plasma membranes. This effect is counteracted by administration of Ze 117. Here we demonstrate that this is not due to a change in C16:1/16:0 and C18:1/18:0 ratios in C6 cell fatty acids. On the other hand, Ze 117 increased the cellular cholesterol content by 42.5%, whereas dexamethasone reduced cholesterol levels similarly to simvastatin. Lowering cholesterol levels by dexamethasone or simvastatin resulted in decreased β-arrestin 2 recruitment to the 5-HT receptor. This effect was counterbalanced by Ze 117, whereas the SJW extract had little effect on β-arrestin 2 recruitment in non-stressed cells. Taken together, in C6 cells, Ze 117 induces changes in membrane fluidity through its effect on cellular cholesterol metabolism rather than by affecting fatty acid saturation. This effect is reflected in an altered signal transduction of the 5-HT receptor under Ze 117 administration. The current in vitro results support the hypothesis that Ze 117 addresses relevant parts of the cellular lipid metabolism, possibly explaining some of the antidepressant actions of Ze 117.
Topics: Hypericum; Plant Extracts; Cholesterol; Membrane Fluidity; Dexamethasone; Cell Line, Tumor; Simvastatin; Glioma; Animals; Rats; Cell Membrane; Receptor, Serotonin, 5-HT1A; Fatty Acids
PubMed: 38684848
DOI: 10.1038/s41598-024-60562-0 -
F1000Research 2022Bovine congestive heart failure (BCHF) has become increasingly prevalent among feedlot cattle in the Western Great Plains of North America with up to 7% mortality in...
BACKGROUND
Bovine congestive heart failure (BCHF) has become increasingly prevalent among feedlot cattle in the Western Great Plains of North America with up to 7% mortality in affected herds. BCHF is an untreatable complex condition involving pulmonary hypertension that culminates in right ventricular failure and death. Genes associated with BCHF in feedlot cattle have not been previously identified. Our aim was to search for genomic regions associated with this disease.
METHODS
A retrospective, matched case-control design with 102 clinical BCHF cases and their unaffected pen mates was used in a genome-wide association study. Paired nominal data from approximately 560,000 filtered single nucleotide polymorphisms (SNPs) were analyzed with McNemar's test.
RESULTS
Two independent genomic regions were identified as having the most significant association with BCHF: the arrestin domain-containing protein 3 gene ( ), and the nuclear factor IA gene ( , mid- -values, 1x10 and 2x10 , respectively). Animals with two copies of risk alleles at either gene were approximately eight-fold more likely to have BCHF than their matched pen mates with either one or zero risk alleles at both genes (CI = 3-17). Further, animals with two copies of risk alleles at both genes were 28-fold more likely to have BCHF than all others ( -value = 1×10 , CI = 4-206). A missense variant in (C182Y) represents a potential functional variant since the C182 codon is conserved among all other jawed vertebrate species observed. A two-SNP test with markers in both genes showed 29% of 273 BCHF cases had homozygous risk genotypes in both genes, compared to 2.5% in 198 similar unaffected feedlot cattle. This and other DNA tests may be useful for identifying feedlot animals with the highest risk for BCHF in the environments described here.
CONCLUSIONS
Although pathogenic roles for variants in the and genes are unknown, their discovery facilitates classifying animals by genetic risk and allows cattle producers to make informed decisions for selective breeding and animal health management.
Topics: Animals; Cattle; Arrestins; Case-Control Studies; Cattle Diseases; Genetic Predisposition to Disease; Genome-Wide Association Study; Heart Failure; NFI Transcription Factors; Polymorphism, Single Nucleotide; Retrospective Studies
PubMed: 38680232
DOI: 10.12688/f1000research.109488.2 -
Biomolecules Mar 2024This study assessed the suitability of the complementarity-determining region 2 (CDR2) of the nanobody (Nb) as a template for the derivation of nanobody-derived peptides...
Computational Modeling and Characterization of Peptides Derived from Nanobody Complementary-Determining Region 2 (CDR2) Targeting Active-State Conformation of the β-Adrenergic Receptor (βAR).
This study assessed the suitability of the complementarity-determining region 2 (CDR2) of the nanobody (Nb) as a template for the derivation of nanobody-derived peptides (NDPs) targeting active-state β-adrenergic receptor (βAR) conformation. Sequences of conformationally selective Nbs favoring the agonist-occupied βAR were initially analyzed by the informational spectrum method (ISM). The derived NDPs in complex with βAR were subjected to protein-peptide docking, molecular dynamics (MD) simulations, and metadynamics-based free-energy binding calculations. Computational analyses identified a 25-amino-acid-long CDR2-NDP of Nb71, designated P4, which exhibited the following binding free-energy for the formation of the βAR:P4 complex (ΔG = -6.8 ± 0.8 kcal/mol or a Ki = 16.5 μM at 310 K) and mapped the βAR:P4 amino acid interaction network. In vitro characterization showed that P4 (i) can cross the plasma membrane, (ii) reduces the maximum isoproterenol-induced cAMP level by approximately 40% and the isoproterenol potency by up to 20-fold at micromolar concentration, (iii) has a very low affinity to interact with unstimulated βAR in the cAMP assay, and (iv) cannot reduce the efficacy and potency of the isoproterenol-mediated βAR/β-arrestin-2 interaction in the BRET-based recruitment assay. In summary, the CDR2-NDP, P4, binds preferentially to agonist-activated βAR and disrupts Gαs-mediated signaling.
Topics: Humans; Amino Acid Sequence; Complementarity Determining Regions; Cyclic AMP; Molecular Docking Simulation; Molecular Dynamics Simulation; Peptides; Protein Binding; Protein Conformation; Receptors, Adrenergic, beta-2; Single-Domain Antibodies
PubMed: 38672440
DOI: 10.3390/biom14040423 -
Frontiers in Molecular Biosciences 2024
PubMed: 38660374
DOI: 10.3389/fmolb.2024.1403161 -
BioRxiv : the Preprint Server For... Apr 2024The dopamine D1 receptor (D1R) has fundamental roles in voluntary movement and memory and is a validated drug target for neurodegenerative and neuropsychiatric...
The dopamine D1 receptor (D1R) has fundamental roles in voluntary movement and memory and is a validated drug target for neurodegenerative and neuropsychiatric disorders. However, previously developed D1R selective agonists possess a catechol moiety which displays poor pharmacokinetic properties. The first selective non-catechol D1R agonists were recently discovered and unexpectedly many of these ligands showed G protein biased signaling. Here, we investigate both catechol and non-catechol D1R agonists to validate potential biased signaling and examine if this impacts agonist-induced D1R endocytosis. We determined that most, but not all, non-catechol agonists display G protein biased signaling at the D1R and have reduced or absent Beta-arrestin recruitment. A notable exception was compound (Cmpd) 19, a non-catechol agonist with full efficacy at both D1R-G protein or D1R Beta-arrestin pathways. In addition, the catechol ligand A-77636 was a highly potent, super agonist for D1R Beta-arrestin activity. When examined for agonist-induced D1R endocytosis, balanced agonists SKF-81297 and Cmpd 19 induced robust D1R endocytosis while the G protein biased agonists did not. The Beta-arrestin super agonist, A-77636, showed significantly increased D1R endocytosis. Moreover, Beta-arrestin recruitment efficacy of tested agonists strongly correlated with total D1R endocytosis. Taken together, these results indicate the degree of D1R signaling functional selectivity profoundly impacts D1R endocytosis regardless of pharmacophore. The range of functional selectivity of these D1R agonists will provide valuable tools to further investigate D1R signaling, trafficking and therapeutic potential.
PubMed: 38659786
DOI: 10.1101/2024.04.15.589637 -
The Journal of Physical Chemistry. B May 2024The chemokine receptor CXCR4 is a critical target for the treatment of several cancer types and HIV-1 infections. While orthosteric and allosteric modulators have been...
The chemokine receptor CXCR4 is a critical target for the treatment of several cancer types and HIV-1 infections. While orthosteric and allosteric modulators have been developed targeting its extracellular or transmembrane regions, the intramembrane region of CXCR4 may also include allosteric binding sites suitable for the development of allosteric drugs. To investigate this, we apply the Gaussian Network Model (GNM) to the monomeric and dimeric forms of CXCR4 to identify residues essential for its local and global motions located in the hinge regions of the protein. Residue interaction network (RIN) analysis suggests hub residues that participate in allosteric communication throughout the receptor. Mutual residues from the network models reside in regions with a high capacity to alter receptor dynamics upon ligand binding. We then investigate the druggability of these potential allosteric regions using the site identification by ligand competitive saturation (SILCS) approach, revealing two putative allosteric sites on the monomer and three on the homodimer. Two screening campaigns with Glide and SILCS-Monte Carlo docking using FDA-approved drugs suggest 20 putative hit compounds including antifungal drugs, anticancer agents, HIV protease inhibitors, and antimalarial drugs. assays considering mAB 12G5 and CXCL12 demonstrate both positive and negative allosteric activities of these compounds, supporting our computational approach. However, functional assays based on the recruitment of β-arrestin to CXCR4 do not show significant agonism and antagonism at a single compound concentration. The present computational pipeline brings a new perspective to computer-aided drug design by combining conformational dynamics based on network analysis and cosolvent analysis based on the SILCS technology to identify putative allosteric binding sites using CXCR4 as a showcase.
Topics: Receptors, CXCR4; Allosteric Site; Ligands; Humans; Molecular Docking Simulation; Monte Carlo Method; Allosteric Regulation
PubMed: 38647430
DOI: 10.1021/acs.jpcb.4c00925 -
BioRxiv : the Preprint Server For... Apr 2024Alcohol use disorders (AUDs) impose an enormous societal and financial burden, and world-wide, alcohol misuse is the 7 leading cause of premature death. Despite this,...
Alcohol use disorders (AUDs) impose an enormous societal and financial burden, and world-wide, alcohol misuse is the 7 leading cause of premature death. Despite this, there are currently only 3 FDA approved pharmacological treatments for the treatment of AUDs in the United States. The neurotensin (Nts) system has long been implicated in modulating behaviors associated with alcohol misuse. Recently, a novel compound, SBI-553, that biases the action of Nts receptor 1 (NTSR1) activation, has shown promise in preclinical models of psychostimulant misuse. Here we investigate the efficacy of this compound to alter ethanol-mediated behaviors in a comprehensive battery of experiments assessing ethanol consumption, behavioral responses to ethanol, sensitivity to ethanol, and ethanol metabolism. Additionally, we investigated behavior in avoidance and cognitive assays to monitor potential side effects of SBI-553. We find that SBI-553 reduces binge-like ethanol consumption in mice without altering avoidance behavior or novel object recognition. We also observe sex-dependent differences in physiological responses to sequential ethanol injections in mice. In rats, we show that SBI-553 attenuates sensitivity to the interoceptive effects of ethanol (using a Pavlovian drug discrimination task). Our data suggest that targeting NTSR1 signaling may be promising to attenuate alcohol misuse, and adds to a body of literature that suggests NTSR1 may be a common downstream target involved in the psychoactive effects of multiple reinforcing substances.
PubMed: 38645173
DOI: 10.1101/2024.04.10.588903 -
Cellular Signalling Jul 2024G protein-coupled receptors (GPCRs) are a family of cell membrane receptors that couple and activate heterotrimeric G proteins and their associated intracellular...
G protein-coupled receptors (GPCRs) are a family of cell membrane receptors that couple and activate heterotrimeric G proteins and their associated intracellular signalling processes after ligand binding. Although the carboxyl terminal of the receptors is essential for this action, it can also serve as a docking site for regulatory proteins such as the β-arrestins. Prokineticin receptors (PKR1 and PKR2) are a new class of GPCRs that are able to activate different classes of G proteins and form complexes with β-arrestins after activation by the endogenous agonists PK2. The aim of this work was to define the molecular determinants within PKR2 that are required for β-arrestin-2 binding and to investigate the role of β-arrestin-2 in the signalling pathways induced by PKR2 activation. Our data show that PKR2 binds constitutively to β-arrestin-2 and that this process occurs through the core region of the receptor without being affected by the carboxy-terminal region. Indeed, a PKR2 mutant lacking the carboxy-terminal amino acids retains the ability to bind constitutively to β-arrestin-2, whereas a mutant lacking the third intracellular loop does not. Overall, our data suggest that the C-terminus of PKR2 is critical for the stability of the β-arrestin-2-receptor complex in the presence of PK2 ligand. This leads to the β-arrestin-2 conformational change required to initiate intracellular signalling that ultimately leads to ERK phosphorylation and activation.
Topics: beta-Arrestin 2; Humans; HEK293 Cells; Protein Binding; Receptors, G-Protein-Coupled; Animals; Receptors, Peptide; Signal Transduction; Binding Sites; Phosphorylation; Receptors, Gastrointestinal Hormone
PubMed: 38631405
DOI: 10.1016/j.cellsig.2024.111175 -
Hypertension (Dallas, Tex. : 1979) Jun 2024ANG (angiotensin II) elicits dipsogenic and pressor responses via activation of the canonical Gαq (G-protein component of the ATR [angiotensin type 1...
BACKGROUND
ANG (angiotensin II) elicits dipsogenic and pressor responses via activation of the canonical Gαq (G-protein component of the ATR [angiotensin type 1 receptor])-mediated ATR in the subfornical organ. Recently, we demonstrated that ARRB2 (β-arrestin 2) global knockout mice exhibit a higher preference for salt and exacerbated pressor response to deoxycorticosterone acetate salt. However, whether ARRB2 within selective neuroanatomical nuclei alters physiological responses to ANG is unknown. Therefore, we hypothesized that ARRB2, specifically in the subfornical organ, counterbalances maladaptive dipsogenic and pressor responses to the canonical ATR signaling.
METHODS
Male and female mice received intracerebroventricular injection of either adeno-associated virus (AAV)-Cre-GFP (green fluorescent protein) to induce brain-specific deletion of ARRB2 (). mice receiving ICV-AAV-GFP were used as control (). Infection with ICV-AAV-Cre primarily targeted the subfornical organ with few off targets. Fluid intake was evaluated using the 2-bottle choice paradigm with 1 bottle containing water and 1 containing 0.15 mol/L NaCl.
RESULTS
mice exhibited a greater pressor response to acute ICV-ANG infusion. At baseline conditions, mice exhibited a significant increase in saline intake compared with controls, resulting in a saline preference. Furthermore, when mice were subjected to water-deprived or sodium-depleted conditions, which would naturally increase endogenous ANG levels, mice exhibited elevated saline intake.
CONCLUSIONS
Overall, these data indicate that ARRB2 in selective cardiovascular nuclei in the brain, including the subfornical organ, counterbalances canonical ATR responses to both exogenous and endogenous ANG. Stimulation of the ATR/ARRB axis in the brain may represent a novel strategy to treat hypertension.
Topics: Animals; Subfornical Organ; Mice; Blood Pressure; Male; Homeostasis; beta-Arrestin 2; Female; Mice, Knockout; Angiotensin II; Brain; Receptor, Angiotensin, Type 1
PubMed: 38629290
DOI: 10.1161/HYPERTENSIONAHA.124.22874