Search Results (73)

The life-threatening disease pertussis, also known as whooping cough, is caused by a complex interplay of several virulence factors produced by the bacterium Bordetella (B.) pertussis. These include the AB-type protein toxin pertussis toxin (PT), the main causative agent of pertussis. After infection with B. pertussis, PT is released and binds to its human target cells, which internalize PT. The enzyme subunit of PT is then taken up into the cytosol, where it catalyzes the ADP-ribosylation of the α-subunit of inhibitory GTP-binding proteins from the Gαi type. This ultimately leads to the development of the characteristic clinical symptoms associated with pertussis. Pertussis is a vaccine-preventable but highly infectious respiratory disease, and especially younger children are prone to develop severe pertussis. Despite the vaccination, over the past few years, increasing case numbers have been reported globally. Moreover, treatment options are strongly limited to antibiotics and symptomatic treatment. Therefore, novel therapies against toxin-mediated diseases are urgently required, while AB-type toxins such as PT are promising pharmacological targets to combat these associated diseases. To identify novel pharmacological inhibitors for AB-type toxins, huge potential lies within the human proteome/peptidome. Endogenous protein or peptide inhibitors for bacterial toxins might have evolved as part of the innate immunity and are awaited to be discovered. The scientific community is committed to identify potential candidates through targeted screening or explorative hypothesis-driven approaches. This review summarizes the recent efforts in the identification and characterization of the human body’s own proteins and peptides that inhibit PT. PT-inhibiting peptides were found by unbiased screening of peptide libraries from human hemofiltrate or hypothesis-driven evaluation, and PT-neutralizing mechanisms were discovered in cell-based approaches. The identification of endogenous peptides and proteins, e.g., defensins and α₁-antitrypsin, as potent inhibitors of PT paves the way towards the development of novel therapeutic options against pertussis. Full article

Integrins and other cell adhesion molecules play a critical role in the migration and homing of leukocytes. This study investigates whether metastatic tumor cells can exploit leukocyte-like rolling and arrest mechanisms during early vascular steps of metastatic dissemination. B16 melanoma cell adhesion to activated bEnd.3 endothelial monolayers or immobilized VCAM-1 were analyzed under defined shear flow using a parallel-plate chamber. Function-blocking antibodies, divalent cation modulation, pertussis toxin, and low-temperature conditions were used as classical controls. B16-BL6 melanoma cells exhibited robust VLA-4-dependent rolling and arrest on activated endothelial monolayers and on immobilized VCAM-1 under physiological shear stresses (0.7–2 dyn/cm²), independent of chemokine-related Gαi signaling. These findings identify a chemokine-independent mechanism of VLA-4-mediated vascular capture by melanoma cells under shear flow, providing a potential mechanistic basis for early steps in metastatic dissemination. Full article

Bitter taste receptors (TAS2Rs) are G protein-coupled receptors best known for detecting bitter compounds in the oral cavity. However, their expression patterns and physiological roles in the liver remain largely unexplored. Here, we employed molecular and immunohistochemical approaches to demonstrate that multiple TAS2Rs are expressed in human Hep3B cells and mouse primary hepatocytes (MPHs) and co-localized with β-adrenergic receptors (βARs) at the bile canaliculi. Bioluminescence resonance energy transfer (BRET), cAMP assays, and Western blot analyses revealed that certain TAS2Rs exhibit ligand-dependent coupling preferences for the G protein subunits Gαi1, Gαi2, and Gαi3. This coupling leads to inhibition of cAMP production and a reduction in protein kinase A (PKA) substrate phosphorylation. Biochemical assays further showed that TAS2R activation significantly attenuates βAR-mediated lipolysis, as well as the production of glycerol and free fatty acid in both Hep3B cells and MPHs. These effects were partially reversed by small interfering RNA (siRNA)-mediated knockdown of TAS2Rs. Moreover, studies using a steatotic mouse model demonstrated that bitter compounds inhibit lipid droplet degradation, resulting in hepatic triacylglycerol accumulation. Collectively, these findings reveal a role for TAS2Rs in modulating hepatic lipid metabolism and highlight their potential as therapeutic targets for the prevention and treatment of liver diseases. Full article

The cuticular plate, a dense F-actin meshwork anchoring stereocilia in auditory hair cells (HCs), undergoes dynamic remodeling during development, but its structural transitions remain poorly understood. Here, we identified two distinct structural domains associated with this maturation. First, a transient F-actin-free area emerges within the lateral periphery of the developing cuticular plate, presenting as a crescent-shaped region that disappears upon HC maturation. Second, the lateral margin of the mature cuticular plate itself remodels into a persistent step-like structure, exhibiting cell-type-specific geometries in inner versus outer HCs. The consistent coincidence between Gαi-GPSM2 complex disruption and aberrant development of both structures in mutant mice implies a role for this complex in their formation. Additionally, microtubules spatially complemented F-actin distribution, suggesting coordinated cytoskeletal regulation. These findings revealed a sophisticated developmental program for cuticular plate maturation. Full article

Pertussis toxin (PTx) is a major virulence factor of Bordetella pertussis and an AB5-type exotoxin that disrupts host signaling. Its enzymatic A subunit ADP-ribosylates the α-subunit of inhibitory G proteins (Gα_i), preventing them from mediating receptor-induced inhibition of adenylyl cyclase (AC). This leads to unrestrained cAMP accumulation in host cells, a canonical mechanism underlying many pertussis disease manifestations. PTx works in concert with the bacterium’s adenylate cyclase toxin (ACT) to subvert immune defenses and establish infection. Interestingly, PTx exerts both cAMP-dependent and cAMP-independent effects. In addition to the well-known cAMP-mediated pathway, PTx’s B oligomer can engage host cell surface receptors to trigger signaling cascades independent of the A subunit’s catalytic activity. Such B oligomer-mediated pathways modulate cellular responses in the absence of ADP-ribosylation. This review provides a comprehensive analysis of PTx’s dual functionality, distinguishing its G_i protein-dependent elevation of cAMP from the noncanonical activities of the B oligomer. It also highlights how disruption of constitutive G_i signaling and the interplay between PTx and ACT shape host–pathogen interaction in pertussis pathogenesis. Full article

The G protein-coupled galanin receptors include three different subtypes: galanin receptor 1, 2 and 3 (GalR1, GalR2, GalR3). The neuropeptide galanin is the principal natural agonist of the galanin receptors, the so-called galaninergic system. Galanin-like peptide and spexin have also been identified as natural ligands of the galanin receptors. Galanin receptors are widely expressed in the brain; however, they can be found in other tissues, such as the skeletal muscle, the heart, and the gastrointestinal tract. The galaninergic system regulates diverse biological processes, including feeding behavior, neuroprotection, learning, memory, cardiovascular and renal function, and nociception. Its dysregulation is associated with various diseases, such as Alzheimer’s disease, diabetes mellitus, epilepsy, depression, and cancer. The stimulation of GalR1 and GalR3 leads to the Gαi/o-type G protein-mediated inhibition of cyclic AMP/protein kinase A, whereas GalR2 stimulation initiates phospholipase C activation via Gαq/11-type G proteins. A galanin-activated β-arrestin-dependent pathway has also been described for GalR2. In this review, we summarize the recent advances concerning galanin receptor signaling, including both the G protein-dependent and -independent pathways. A better understanding of the complex interplay of the signaling molecules, receptors, and various signaling pathways is crucial for the future development of specific agonists with therapeutic potential. Full article

The vomeronasal system (VNS) is directly linked to the various behavior and ecology of all animal species, and understanding it might help to prevent deer damage. We therefore histochemically analyzed the accessory olfactory bulb (AOB) and the vomeronasal organ (VNO) that, respectively, function as a primary integrative center and a peripheral receptor organ, in Hokkaido sika deer (Cervus nippon ssp. yesoensis). The AOB consisted of the vomeronasal nerve, glomerular, plexiform, and granule cell layers. We found that G protein α subunit i2 (Gαi2) and o (Gαo) that are, respectively, coupled with vomeronasal receptor type 1 and 2 families were strongly and weakly expressed in the glomerular layer, respectively. These properties of the AOB of sika deer were similar to those of other artiodactyl species, including wapiti. We then explored the sika deer VNO using 21 lectins that bind to each glycan structure. Although various cell types in the VNO had unique lectin binding profiles, all 21 lectins bound to the free border of the sensory epithelium, suggesting that various glycoconjugates are involved in pheromone detection in sika deer via the VNO. Furthermore, the reactivity of some lectins in the sensory epithelium and vomeronasal gland differed from those of roe deer and wapiti. Our findings suggest that the composition of glycoconjugates in the VNO differs among deer species. Full article

Heterotrimeric G_i proteins are crucial modulators of G protein-coupled receptor signaling, with Gα_i2 ubiquitously expressed and implicated in diverse physiological processes. Previous reports described partial lethality in Gnai2-deficient mice, but the timing and mechanism of death remained unclear. Here, we demonstrate that impaired neonatal respiratory adaptation contributes to mortality in Gnai2-deficient neonates. Despite normal Mendelian distribution at birth and no overt malformations, at least 20% of the expected Gnai2-deficient neonates died within minutes after birth, displaying abnormal breathing, cyanosis, and features resembling neonatal respiratory distress syndrome (RDS). Histological and ultrastructural analyses revealed reduced alveolar surface area, thickened septa, increased mesenchymal tissue, and impaired surfactant ultrastructure, despite unaltered alveolar surfactant phospholipid levels. These findings suggest that Gα_i2 modulates the structural deployment and functional organization of surfactant within alveoli, although the incomplete phenotype and survival of some neonates indicate a regulatory rather than indispensable role of Gα_i2. Our data underscore the complexity of neonatal respiratory adaptation and highlight potential systemic and intercellular mechanisms underlying alveolar stabilization. Full article

Background/Objectives: G protein-coupled receptors (GPCRs) can promote ligand-biased signaling, yet the mechanisms that promote bias are not well understood. We have shown that C-C Chemokine Ligand 19 (CCL19) and CCL21 promote ligand-biased internalization and signaling of C-C Chemokine Receptor 7 (CCR7) in T cells. The roles of GPCR kinases (GRKs) in regulating biased CCR7 internalization and biased signaling in T cells are unclear. GRK2 is a serine/threonine kinase that phosphorylates GPCRs in response to ligand binding and is recruited to the plasma membrane via its C-terminal pleckstrin homology domain to phosphatidylinositol 4,5-bisphosphate (PIP₂). Methods: Human embryonic kidney cells (HEK293) transfected to express wild-type and mutant GRK2 and human CCR7, human T cell lines harboring heterozygous deletions of GRK2, and naïve primary T cells from GRK2 heterozygous (GRK2^+/−) or GRK2^f/f CD4-Cre mice were used to examine the effects of GRK2 on ligand-induced CCR7 signaling in T cells. We used flow cytometry to assay the effect of GRK2 on CCR7 internalization, Fluorescence Resonance Energy Transfer (FRET) to define the effect of GRK2 on CCR7 activation of Gα_i isoforms and transwell migration assays to examine the effect of GRK2 on chemotaxis. Since chemotaxis via CCR7 is mediated by phospholipase Cγ1 (PLCγ1), Western blot assays were used to measure the effect of GRK2 during downstream signaling via phosphorylation of PLCγ1. Results: We found that following CCL19 binding, GRK2 promoted kinase-dependent CCR7 recruitment of arrestin-3, rapid CCR7 internalization and Gα_i3 recruitment to CCR7. In contrast, following binding of CCL21 to CCR7, GRK2 slowed CCR7 internalization, induced recruitment of Gα_i2 to the activated receptor_, and promoted chemotaxis. Since we have shown that CCL21 promotes chemotaxis via PLCγ1, we examined the effect of GRK2 on PLCγ1 activation and found that GRK2 had no effect on CCL21-mediated PLCγ1 phosphorylation. Conclusions: GRK2 promotes differential signaling downstream of CCR7 activation by CCL19 and CCL21 and provides a model for biased signaling downstream of a GPCR driven by GRK2. Full article

Metabolic syndrome (MetS) is a worldwide problem affecting at least one-third of the population. MetS patients have increased cardiovascular risk associated with an abnormal β-adrenergic response; however, it is not clear how MetS affects the cardiac β-adrenergic system. We analyzed cardiac function and the β-adrenergic response in an experimental model of MetS in rats by recording pressure–volume (PV) loops via an open-chest approach and performed a biochemical characterization of the cardiac β-adrenergic system through ELISA, radioligand binding assays, and Western blotting. Microscopy was employed to evaluate cardiac hypertrophy, fibrosis, and ultrastructure. MetS rats exhibited cardiac dysfunction, evidenced by a reduced cardiac output and ejection fraction, not explained by heart hypertrophy or fibrosis. MetS rats also had an elevated susceptibility to lethal arrhythmia following intra-cardiac administration of the non-selective β-adrenergic agonist isoproterenol, suggesting alterations in the β-adrenergic system. The total serum adrenaline and noradrenaline levels were higher in the MetS animals than those in the control group. The radioligand binding assays indicated no change in the βAR density; however, the Western blot analyses revealed decreased levels of Gα_s proteins and β-arrestin 1, but increased β₂AR and Gα_i protein levels. This study contributes to our understanding of how MetS can alter cardiac function, raising the risk of lethal arrhythmia induced by the β-adrenergic (fight or flight) response and underscores the relevance of therapeutically targeting MetS before its pathological progression toward cardiomyopathy. Full article

Adolescent binge drinking has lasting behavioral consequences by disrupting the endocannabinoid system (ECS) and depleting brain omega-3. The natural accumulation of omega-3 fatty acids in cell membranes is crucial for maintaining the membrane structure, supporting interactions with the ECS, and restoring synaptic plasticity and cognition impaired by prenatal ethanol (EtOH) exposure. However, it remains unclear whether omega-3 supplementation can mitigate the long-term effects on the ECS, endocannabinoid-dependent synaptic plasticity, and cognition following adolescent binge drinking. Here, we demonstrated that omega-3 supplementation during EtOH withdrawal increases CB1 receptors in hippocampal presynaptic terminals of male mice, along with the recovery of receptor-stimulated [³⁵S]GTPγS binding to Gαi/o proteins. These changes are associated with long-term potentiation (LTP) at excitatory medial perforant path (MPP) synapses in the dentate gyrus (DG), which depends on anandamide (AEA), transient receptor potential vanilloid 1 (TRPV1), and N-methyl-D-aspartate (NMDA) receptors. Finally, omega-3 intake following binge drinking reduced the time and number of errors required to locate the escape box in the Barnes maze test. Collectively, these findings suggest that omega-3 supplementation restores Barnes maze performance to levels comparable to those of control mice after adolescent binge drinking. This recovery is likely mediated by modulation of the hippocampal ECS, enhancing endocannabinoid-dependent excitatory synaptic plasticity. Full article

Metastatic prostate cancer occurs when the tumor spreads from the prostate gland to other parts of the body. Previous studies have shown that Gα_i2, a subunit of the heterotrimeric G protein complex, plays a critical role in inducing cell migration and invasion in prostate cancer cells in response to diverse stimuli. Rac1 is a small rho-GTPase, which is activated by the phosphoinositide 3-kinase (PI3K)/AKT pathway and plays an essential role during cell migration. Previous studies have shown that the knockdown of Gα_i2 attenuates cell migration without causing any reduction in basal Rac1 activity in both PC3 and DU145 cells, and has only marginal effects on the epidermal growth facotor (EGF)-induced increase in Rac1 activity. Therefore, Gα_i2 may be involved in the regulation of cell motility and invasion independently or downstream of Rac1 activation. In this study, we investigated the possible mechanism of Gα_i2 at the level of the Rac1-dependent activation of Wiskott-Aldrich Syndrome Protein)-family verprolin homologous protein2 (Wave2) and actin related protein 2/3 (Arp 2/3) proteins, downstream effectors of activated Rac1. PC3 cells with a stable overexpression of constitutively active Rac1 were transfected with control siRNA or Gα_i2 siRNA to knockdown endogenous Gα_i2 expression. Western blot analysis showed that the Rac1-dependent activation of Wave2 was impaired in the absence of Gα_i2. The overexpression of constitutively active Gα_i2 (Gα_i2-Q205L) in PC3 cells significantly increased cell migration compared to cells transfected with control plasmids. In the parallel experiments, a specific Gα_i2 inhibitor blocked G_iα2-Q205L-induced cell migration in PC3 cells. Furthermore, the Rac1 inhibitor did not block increased cell migration in PC3 cells overexpressing constitutively active Gα_i2. We conclude that activated Gα_i2 plays a crucial role in cell migration in prostate cancer cells independent of Rac1 activation. Full article

Background: Dopamine receptors (DRs) are G-protein-coupled receptors (GPCRs) found in the central nervous system (CNS). DRs are essential for mediating various downstream signaling cascades and play a critical role in regulating the dopaminergic nigrostriatal pathway, which is involved in motor control. Recently, mutations in DRD2 (WT), p.Ile212Phe (I212F), and p.Met345Arg (M345R) have been associated with hyperkinetic movement disorders and shown to alter heterotrimeric G-protein complex signaling and β-arrestin recruitment. Methods: To conduct a detailed investigation of the I212F and M345R functional phenotypes, we used the TRansdUcer PATHway (TRUPATH) assay to study heterotrimeric G-protein recruitment and the Parallel Receptorome Expression and Screening via Transcriptional Output (PRESTO-Tango) assay to evaluate transcriptional activation following arrestin translocation for β-arrestin recruitment. Results: In our study, we could confirm the reported mutant’s loss-of-function phenotype in β-arrestin 2 recruitment (reduced agonist potency and decreased maximal signaling efficacy in comparison to the WT). However, a detailed analysis of basal/constitutive activity also revealed a gain-of-function phenotype for mutant M345R. For a more comprehensive investigation of heterotrimeric G-protein complex signaling, we investigated the impact of WT mutants in combination with (i) a specifically suggested assay, and (ii) the most abundantly expressed heterotrimeric G-protein complex combinations in WT receptor-enriched regions. We were able to confirm the reported gain-of-function phenotype by Rodriguez-Contreras et al. and extend it by the use of the most abundant heterotrimeric G-protein subunits, Gα_oA and Gα_i1, β₁ and β₂, and γ₃ and γ₇, in mouse and human basal ganglia. Conclusions: Although our results indicate that the interaction of the two variants with the most highly expressed heterotrimeric G-protein complex subunit combinations also results in a gain-of-function phenotype, they also clearly demonstrate that the phenotype can be significantly altered, dependent on heterotrimeric G-protein complex expression. Full article

Background/Objectives: Omega-3 long-chain polyunsaturated fatty acids (PUFAs) support brain cell membrane integrity and help mitigate synaptic plasticity deficits. The endocannabinoid system (ECS) is integral to synaptic plasticity and regulates various brain functions. While PUFAs influence the ECS, the effects of omega-3 on the ECS, cognition, and behavior in a healthy brain remain unclear. Methods and Results: Here, we demonstrate that hippocampal synaptosomes from male mice fed an omega-3-rich diet exhibit increased levels of cannabinoid CB1 receptors (~30%), phospholipase C β1 (PLCβ1, ~30%), monoacylglycerol lipase (MAGL, ~30%), and cannabinoid receptor-interacting protein 1a (Crip1a, ~60%). Conversely, these synaptosomes show decreased levels of diacylglycerol lipase α (DAGLα, ~40%), synaptosomal-associated protein 25kDa (SNAP-25, ~30%), and postsynaptic density protein 95 (PSD-95, ~40%). Omega-3 intake also reduces Gαo and Gαi3 levels, though receptor-stimulated [³⁵S]GTPγS binding remains unaffected. Stimulation of the medial perforant path (MPP) induced long-term potentiation (LTP) in omega-3-fed mice. This LTP was dependent on group I metabotropic glutamate receptors (mGluR), 2 arachidonoylglycerol (2-AG), CB1 receptors, N-type Ca²⁺ channels, and actin filaments. Behaviorally, omega-3-fed mice displayed reduced exploratory behavior and significantly improved object discrimination in the novel object recognition test (NORT). They also spent more time in open arms and exhibited reduced freezing time in the elevated plus maze (EPM), indicative of reduced anxiety-like behavior. Conclusions: Our findings suggest that omega-3 leverages the ECS to enhance brain function under normal conditions. Full article

Neuropeptide FF (NPFF) is an endogenous octapeptide that was originally isolated from the bovine brain. It belongs to the RFamide family of peptides that has a wide range of physiological functions and pathophysiological effects. NPFF and its receptors, NPFFR1 and NPFFR2, abundantly expressed in rodent and human brains, participate in cardiovascular regulation. However, the expressions of NPFF and its receptors are not restricted within the central nervous system but are also found in peripheral organs, including the kidneys. Both NPFFR1 and NPFFR2 mainly couple to Gαi/o, which inhibits cyclic adenosine monophosphate (cAMP) production. NPFF also weakly binds to other RFamide receptors and the Mas receptor. Relevant published articles were searched in PubMed, Google Scholar, Web of Science, and Scopus. Herein, we review evidence for the role of NPFF in the regulation of blood pressure, in the central nervous system, particularly within the hypothalamic paraventricular nucleus and the brainstem, and the kidneys. NPFF is a potential target in the treatment of hypertension. Full article