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Journal of Respiratory Biology and... Jun 2024The molecular mechanisms that regulate progressive pulmonary fibrosis remain poorly understood. Type 2 alveolar epithelial cells (AEC2s) function as adult stem cells in...
The molecular mechanisms that regulate progressive pulmonary fibrosis remain poorly understood. Type 2 alveolar epithelial cells (AEC2s) function as adult stem cells in the lung. We previously showed that there is a loss of AEC2s and a failure of AEC2 renewal in the lungs of idiopathic pulmonary fibrosis (IPF) patients. We also reported that beta-arrestins are the key regulators of fibroblast invasion, and beta-arrestin 1 and 2 deficient mice exhibit decreased mortality, decreased matrix deposition, and increased lung function in bleomycin-induced lung fibrosis. However, the role of beta-arrestins in AEC2 regeneration is unclear. In this study, we investigated the role and mechanism of Arrestin beta 1 (ARRB1) in AEC2 renewal and in lung fibrosis. We used conventional deletion as well as cell type-specific deletion of in mice and found that deficiency in fibroblasts protects mice from lung fibrosis, and the knockout mice exhibit enhanced AEC2 regeneration in vivo, suggesting a role of fibroblast-derived ARRB1 in AEC2 renewal. We further found that -deficient fibroblasts promotes AEC2 renewal in 3D organoid assays. Mechanistically, we found that CCL7 is among the top downregulated cytokines in deficient fibroblasts and CCL7 inhibits AEC2 regeneration in 3D organoid experiments. Therefore, fibroblast ARRB1 mediates AEC2 renewal, possibly by releasing chemokine CCL7, leading to fibrosis in the lung.
PubMed: 38736470
DOI: 10.35534/jrbtm.2024.10006 -
International Journal of Molecular... May 2024Cardiovascular outcome in Marfan syndrome (MFS) patients most prominently depends on aortic aneurysm progression with subsequent aortic dissection. Angiotensin II...
Cardiovascular outcome in Marfan syndrome (MFS) patients most prominently depends on aortic aneurysm progression with subsequent aortic dissection. Angiotensin II receptor blockers (ARBs) prevent aneurysm formation in MFS mouse models. In patients, ARBs only slow down aortic dilation. Downstream signalling from the angiotensin II type 1 receptor (AT1R) is mediated by G proteins and β-arrestin recruitment. AT1R also interacts with the monocyte chemoattractant protein-1 (MCP-1) receptor, resulting in inflammation. In this study, we explore the targeting of β-arrestin signalling in MFS mice by administering TRV027. Furthermore, because high doses of the ARB losartan, which has been proven beneficial in MFS, cannot be achieved in humans, we investigate a potential additive effect by combining lower concentrations of losartan (25 mg/kg/day and 5 mg/kg/day) with barbadin, a β-arrestin blocker, and DMX20, a C-C chemokine receptor type 2 (CCR2) blocker. A high dose of losartan (50 mg/kg/day) slowed down aneurysm progression compared to untreated MFS mice (1.73 ± 0.12 vs. 1.96 ± 0.08 mm, = 0.0033). TRV027, the combination of barbadin with losartan (25 mg/kg/day), and DMX-200 (90 mg/kg/day) with a low dose of losartan (5 mg/kg/day) did not show a significant beneficial effect. Our results confirm that while losartan effectively halts aneurysm formation in MFS mice, neither TRV027 alone nor any of the other compounds combined with lower doses of losartan demonstrate a notable impact on aneurysm advancement. It appears that complete blockade of AT1R function, achieved by administrating a high dosage of losartan, may be necessary for inhibiting aneurysm progression in MFS.
Topics: Animals; Marfan Syndrome; Mice; Losartan; Receptor, Angiotensin, Type 1; Signal Transduction; Angiotensin II Type 1 Receptor Blockers; Disease Models, Animal; Aortic Aneurysm; Male; beta-Arrestins; Receptors, CCR2; Mice, Inbred C57BL
PubMed: 38732244
DOI: 10.3390/ijms25095025 -
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; Mice; Alleles; Carrier Proteins; Cell Survival; Disease Models, Animal; Gene Deletion; Mutation, Missense; Retinal Cone Photoreceptor Cells; Retinal Pigment Epithelium; Retinitis Pigmentosa; Thioredoxins
PubMed: 38727583
DOI: 10.7554/eLife.90749 -
Psychopharmacology Jul 2024Medications are urgently needed to treat symptoms of drug withdrawal and mitigate dysphoria and psychiatric comorbidities that drive opioid abuse and relapse. ITI-333 is...
RATIONALE
Medications are urgently needed to treat symptoms of drug withdrawal and mitigate dysphoria and psychiatric comorbidities that drive opioid abuse and relapse. ITI-333 is a novel molecule in development for treatment of substance use disorders, psychiatric comorbidities, and pain.
OBJECTIVE
Characterize the preclinical profile of ITI-333 using pharmacological, behavioral, and physiological assays.
METHODS
Cell-based assays were used to measure receptor binding and intrinsic efficacy of ITI-333; animal models were employed to assess effects on opioid reinstatement, precipitated oxycodone withdrawal, and drug abuse liability.
RESULTS
In vitro, ITI-333 is a potent 5-HT receptor antagonist (K = 8 nM) and a biased, partial agonist at μ-opioid (MOP) receptors (K = 11 nM; lacking β-arrestin agonism) with lesser antagonist activity at adrenergic α (K = 28 nM) and dopamine D (K = 50 nM) receptors. In vivo, ITI-333 blocks 5-HT receptor-mediated head twitch and MOP receptor-mediated effects on motor hyperactivity in mice. ITI-333 alone is a naloxone-sensitive analgesic (mice) which suppresses somatic signs of naloxone-precipitated oxycodone withdrawal (mice) and heroin cue-induced reinstatement responding without apparent tolerance or physical dependence after chronic dosing (rats). ITI-333 did not acutely impair gastrointestinal or pulmonary function (rats) and was not intravenously self-administered by heroin-maintained rats or rhesus monkeys.
CONCLUSIONS
ITI-333 acts as a potent 5-HT receptor antagonist, as well a biased MOP receptor partial agonist with low intrinsic efficacy. ITI-333 mitigates opioid withdrawal/reinstatement, supporting its potential utility as a treatment for OUD.
Topics: Animals; Mice; Male; Substance Withdrawal Syndrome; Rats; Humans; Rats, Sprague-Dawley; Receptors, Opioid, mu; Serotonin 5-HT2 Receptor Antagonists; Substance-Related Disorders; Opioid-Related Disorders; Dose-Response Relationship, Drug; Oxycodone; Analgesics, Opioid; Self Administration; Cricetulus; CHO Cells
PubMed: 38710856
DOI: 10.1007/s00213-024-06578-w -
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