G Protein Research Articles

Key Interaction Changes Determine the Activation Process of Human Parathyroid Hormone Type 1 Receptor.

The parathyroid hormone type 1 receptor (PTH1R) plays a crucial role in modulating various physiological functions and is considered an effective therapeutic target for osteoporosis. However, a lack of detailed molecular and energetic information about PTH1R limits our comprehensive understanding of its activation process. In this study, we performed computational simulations to explore key events in the activation process, such as conformational changes in PTH1R, Gs protein coupling, and the release of guanosine diphosphate (GDP). Our analysis identified kinetic information, including the rate-determining step, transition state, and energy barriers. Free-energy and structural analyses revealed that GDP could be released from the Gs protein when the binding cavity is partially open. Additionally, we predicted important residues, including potential pathogenic mutations, and verified their significance through site-directed mutations. These findings enhance our understanding of class B GPCR activation mechanisms. Furthermore, the methodology employed in this study can be applied to other biophysical systems.

Identifying disulfidptosis-related biomarkers in epilepsy based on integrated bioinformatics and experimental analyses.

One of the underlying mechanisms of epilepsy (EP), a brain disease characterized by recurrent seizures, is considered to be cell death. Disulfidptosis, a proposed novel cell death mechanism, is thought to play a part in the pathogenesis of epilepsy, but the exact role is unclear. The gene expression omnibus series (GSE) 33,000 and GSE63808 datasets were used to search for differentially expressed disulfidptosis-related molecules (DE-DRMs). A correlation between the DE-DRMs was discovered. Individuals with epilepsy were then used to investigate molecular clusters based on the expression of DE-DRMs. Following that, the best machine learning model which is validated by GSE143272 dataset and predictor molecules were identified. The correlation between predictive molecules and clinical traits was determined. Based on the in vitro and in vivo seizures models, experimental analyses were applied to verify the DE-DRMs expressions and the correlation between them. Nine molecules were identified as DE-DRMs: glycogen synthase 1 (GYS1), solute carrier family 3 member 2 (SLC3A2), solute carrier family 7 member 11 (SLC7A11), NADH:ubiquinone oxidoreductase core subunit S1 (NDUFS1), 3-oxoacyl-ACP synthase, mitochondrial (OXSM), leucine rich pentatricopeptide repeat containing (LRPPRC), NADH:ubiquinone oxidoreductase subunit A11 (NDUFA11), NUBP iron‑sulfur cluster assembly factor, mitochondrial (NUBPL), and NCK associated protein 1 (NCKAP1). NDUFS1 interacted with NDUFA11, NUBPL, and LRPPRC, while SLC3A2 interacted with SLC7A11. The optimal machine learning model was revealed to be the random forest (RF) model. G protein guanine nucleotide-binding protein alpha subunit q (GNAQ) was linked to sodium valproate resistance. The experimental analyses suggested an upregulated SLC7A11 expression, an increased number of formed SLC3A2 and SLC7A11 complexes, and a decreased number of formed NDUFS1 and NDUFA11 complexes. This study provides previously undocumented evidence of the relationship between disulfidptosis and EP. In addition to suggesting that SLC7A11 may be a specific DRM for EP, this research demonstrates the alterations in two disulfidptosis-related protein complexes: SLC7A11-SLC3A2 and NDUFS1-NDUFA11.

The Arabidopsis heterotrimeric G protein α subunit binds to and inhibits the inward rectifying potassium channel KAT1.

In animal cells, Gα subunit of the heterotrimeric G proteins can bind to both the N-terminal and C-terminal domains of G-protein-activated inwardly rectifying K+ channels (GIRKs) to inhibit their activities. In Arabidopsis guard cells, the Gα subunit GPA1 mediates multiple stimuli-regulated stomatal movements via inhibiting guard cell inward-rectifying K+ (K+in) current, but it remains unclear whether GPA1 directly interacts with and inhibits the activities of K+in channels. Here, we found that GPA1 interacted with the transmembrane domain rather than the intracellular domain of the Shaker family K+in channel KAT1. Two-Electrode Voltage-Clamp experiments in Xenopus oocytes demonstrated that GPA1 significantly inhibited KAT1 channel activity. However, GPA1 could not inhibit the assembly of KAT1 as well as KAT2 as homo- and hetero-tetramers and alter the subcellular localization and protein stability of these channels. In conclusion, these findings reveal a novel regulatory mechanism for Gα inhibition of the Shaker family K+in channel KAT1 via binding to its channel transmembrane domains but without affecting its subcellular localization, protein stability and the formation of functional homo- and hetero-tetramers. This suggests that in both animal and plant cells, Gα can regulate K+in channels through physical interaction, albeit with differing mechanisms of interaction and regulation.

Recurrent Small Variants in NESP55/NESPAS Associated with Broad GNAS Methylation Defects and Pseudohypoparathyroidism Type 1b.

Pseudohypoparathyroidism type 1B (PHP1B) is associated with epigenetic changes on the maternal allele of the imprinted GNAS gene that inhibit expression of the alpha subunit of Gs (Gsα), thereby leading to parathyroid hormone resistance in renal proximal tubule cells where expression of Gs from the paternal GNAS allele is normally silent. Although all patients with PHP1B show loss of methylation for the exon A/B differentially methylated region (DMR), some patients with autosomal dominant PHP1B (AD-PHP1B) and most patients with sporadic PHP1B have additional methylation defects that affect the DMRs corresponding to exons XL, AS1, and NESP. Because the genetic defect is unknown in most of these patients, we sought to identify the underlying genetic basis for AD-PHP1B in two multigenerational families with broad GNAS methylation defects and negative clinical exomes. Genome sequencing identified small GNAS variants in each family that were also present in unrelated PHP1B subjects in a replication cohort. Maternal transmission of one GNAS microdeletion showed reduced penetrance in some unaffected patients. Expression of AS transcripts was increased, and NESP was decreased, in cells from affected patients. These results suggest that the small deletion activate AS transcription leading to methylation of the NESP DMR with consequent inhibition of NESP transcription, and thereby provide a potential mechanism for PHP1B.

Taurine mechanism in preventing retinal cell damage from acute ocular hypertension through GTPBP3 regulation.

We aimed to explore the protective effects and underlying mechanisms of taurine on retinal cells during acute ocular hypertension (AOH)-induced damage. Retinal morphology, apoptosis, mitochondrial structure, electroretinography, expression of GTP binding protein 3 (GTPBP3), and molecules in the unfolded protein response (UPR) were examined in an AOH mouse model and wild-type (WT) mice with or without intravitreal injection of taurine. For in vitro experiments, the GTPBP3 expression and endoplasmic reticulum (ER) stress were examined in R28 cell line under hydrogen peroxide (H2O2)-induced damage or hypoxia/reoxygenation (H/R)-induced damage, with or without taurine pretreatment. Taurine pretreatment alleviated retinal damage caused by AOH modeling. The GTPBP3 expression level decreased after AOH injury, and taurine pretreatment reversed this reduction. Retinas with decreased GTPBP3 expression showed reduced retinal ganglion cell (RGC) function, which could be reversed by intravitreal taurine injection. In H2O2-, H/R-, and AOH-induced damage, UPR were activated and alleviated by taurine pretreatment. GTPBP3 knockdown in R28 cells also activated the UPR, which was alleviated by taurine. A UPR activator downregulated GTPBP3 levels in normal R28 cells, whereas a UPR inhibitor upregulated GTPBP3 levels in GTPBP3 knockdown R28 cells. In conclusion, this study provides important evidence that taurine prevents retinal cell damage in mice exposed to AOH and modulates GTPBP3 expression via the UPR pathway. Interventions targeting this mechanism can be used as potential therapeutic targets for AOH damage.

A2B adenosine receptor-triggered intracellular calcium mobilization: Cell type-dependent involvement of Gi, Gq, Gs proteins and protein kinase C.

Activation of PLCβ enzymes by G iβγ and G αq/11 proteins is a common mechanism to trigger cytosolic Ca 2+ increase. We and others reported that G αq/11 inhibitor FR900358 (FR) can inhibit both and G αq - and, surprisingly, G iβγ -mediated intracellular Ca 2+ mobilization. Thus, the G αi -G βγ -PLCβ-Ca 2+ signaling axis depends entirely on the presence of active G αq , which reasonably explained FR-inhibited G iβγ -induced Ca 2+ release. However, the conclusion that G iβγ signaling is controlled by G αq derives mostly from HEK293 cells. Here we show that indeed in HEK293 cells both G αq/11 siRNA and G αq/11 inhibitors diminished Ca 2+ increase triggered by native G q -coupled P2Y 1 receptors, or by transfected G i -coupled A 1 - or G s -coupled A 2B adenosine receptors (ARs). However, in T24 bladder cancer cells, G i inhibitor PTX, but not G αq/11 inhibitors, FR, YM254890 (YM) or G q/11 siRNA, inhibited Ca 2+ increase triggered by native A 2B AR activation. Simultaneous inactivation of G i and G s further suppressed A 2B AR-triggered Ca 2+ increase in T24 cells. The G αq/11 inhibitor YM fully and partially inhibited endogenous P2Y 1 - and β 2 -adrenergic receptor-induced Ca 2+ increase in T24 cells, respectively. PKC activator PMA partially diminished A 2B AR-triggered but completely diminished β 2 -adrenergic receptor-triggered Ca 2+ increase in T24 cells. Neither β-arrestin1 nor β-arrestin2 siRNA affected A 2B AR-mediated Ca 2+ increase. Unlike in T24 cells, YM inhibited native A 2B AR-triggered calcium mobilization in MDA-MB-231 breast cancer cells. Thus, G αq/11 is vital for Ca 2+ increase in some cell types, but G iβγ -mediated Ca 2+ signaling can be Gα q/11 -dependent or independent based on cell type and receptor activated. Besides G proteins, PKC also modulates cytosolic Ca 2+ increase depending on cell type and receptor.

A high copy suppressor screen identifies factors enhancing the allotopic production of subunit II of cytochrome c oxidase.

Allotopic expression refers to the artificial relocation of an organellar gene to the nucleus. Subunit 2 (Cox2) of cytochrome c oxidase, a subunit with two transmembrane domains (TMS1 and TMS2) residing in the inner mitochondrial membrane with a Nout-Cout topology, is typically encoded in the mitochondrial cox2 gene. In the yeast Saccharomyces cerevisiae, the cox2 gene can be allotopically expressed in the nucleus, yielding a functional protein that restores respiratory growth to a Δcox2 null mutant. In addition to a mitochondrial targeting sequence followed by its natural 15-residue leader peptide, the cytosol synthesized Cox2 precursor must carry one or several amino acid substitutions that decrease the mean hydrophobicity of TMS1 and facilitate its import into the matrix by the TIM23 translocase. Here, using a yeast strain that contains a COX2W56R gene construct inserted in a nuclear chromosome, we searched for genes whose overexpression could facilitate import into mitochondria of the Cox2W56R precursor and increase respiratory growth of the corresponding mutant strain. A COX2W56R expressing strain was transformed with a multicopy plasmid genomic library, and transformants exhibiting enhanced respiratory growth on non-fermentable carbon sources were selected. We identified three genes whose overexpression facilitates the internalization of the Cox2W56R subunit into mitochondria, namely: TYE7, RAS2 and COX12. TYE7 encodes a transcriptional factor, RAS2 a GTP-binding protein, and COX12 a non-core subunit of cytochrome c oxidase. We discuss potential mechanisms by which the TYE7, RAS2 and COX12 gene products could facilitate the import and assembly of the Cox2W56R subunit produced allotopically.

Sept10 and sept12 are expressed in specific proliferating cells in zebrafish brain.

Septins are a group of cytoskeletal GTP binding proteins which are involved in different cellular processes, like cell division, exocytosis and axon growth. Their function, especially in the nervous system, is not clear. In zebrafish 16 different septins are described and for some of them the expression in the brain is described. Interestingly, the expression pattern of several of them is highly specific. Here we describe the expression of sept10 and sept12 in the developing zebrafish brain and found that these show a very defined expression pattern. Interestingly, they show an overlap with a group, but not all proliferating PCNA positive cells in nervous tissue.

Activation of cellular responses by cyclic dinucleotides and Porphyromonas gingivalis lipopolysaccharide: a proteomic study on gingival fibroblasts

ABSTRACT Background Bacterial cyclic dinucleotides (CDNs), cyclic di-guanosine monophosphate (c-di-GMP), and cyclic di-adenosine monophosphate (c-di-AMP) upregulate interferon signaling proteins of human gingival fibroblasts (HGFs). However, the simultaneous effect of bacterial CDNs and lipopolysaccharides (LPS) on the HGF proteome is unknown. Aim The aim was to apply an unbiased proteomics approach to evaluate how simultaneous exposure to CDNs and Porphyromonas gingivalis (Pg) LPS affect the global proteome of HGFs. Methods The proteomic responses of HGFs were examined under three different treatment conditions (c-di-AMP+Pg LPS, c-di-GMP+Pg LPS, and Pg LPS alone) by label-free quantitative mass spectrometry analysis. Results Simultaneous exposure to CDNs and Pg LPS significantly upregulated innate immunity-related and interferon signaling-related proteins, such as ubiquitin-like protein ISG15 (ISG15), deoxynucleoside triphosphate triphosphohydrolase (SAMHD1), interferon regulatory factor 9 (IRF-9), interferon-induced GTP-binding protein Mx (MX)1, and MX2. Interferon signaling pathway was the most significantly regulated canonical pathway in both CDN treatment groups. Conclusion Simultaneous exposure to CDNs and Pg LPS stimulates the periodontal immune response by activating the anti-microbial cellular responses of HGFs with some notable differences from individual exposures.

Integrated NMR-crystallography-computational approach for molecular recognition studies of human Gαi3 protein by a small molecule inhibitor.

The small molecule IGGi-11 targets Gαi subunits of heterotrimeric guanine nucleotide-binding proteins. Gα subunits are activated by G-protein-coupled receptors in response to extracellular stimuli by accelerating the exchange of GDP for GTP, but they are also activated by intracellular proteins like GIV, of which elevated levels correlate with increased cell migration and cancer metastasis. IGGi-11 disrupts the interaction of Gαi proteins with GIV and inhibits pro-invasive traits of metastatic breast cancer cells without interfering with GPCR signaling. IGGi-11 is a competitive inhibitor but binds Gαi3 with a 10-fold lower affinity than GIV. To guide the design of higher affinity inhibitors, we aimed at obtaining high-resolution structural data on the complex. To facilitate its crystallization, we have removed the most flexible residues at the chain ends of Gαi3, identified by NMR. While Gαi3 crystals grown with excess IGGi-11 did not show the bound compound, computational docking and molecular dynamics simulations identified the interactions driving the molecular recognition. This approach revealed heterogeneous binding due to the symmetry of IGGi-11 chemical structure and to the elongated shape and flexibility of the binding site. Our results suggest that chemical modifications breaking IGGi-11 symmetry might yield inhibitors with higher affinity and potential as antimetastatic drugs.

The P2Y11–14 receptor is a double-edged regulator in stress-induced cellular senescence and aging: a narrative review

Cellular senescence is a process where cells reach the Hayflick number of divisions, leading to telomere dysfunction and genetic aberrations. Telomeres are bound by shelterin, preventing DNA repair proteins from accessing them, resulting in DNA damage and cellular senescence or apoptosis. Human telomerase deficiency is linked to various diseases, including aplastic anemia, dyskeratosis congenita, and early pulmonary fibrosis. Aging is characterized by a decline in cellular maintenance and repair processes, leading to the loss of hemostasis and functionality of tissues and organs over time. Purinergic receptors are essential for controlling healthy and diseased processes, activating adenosine 5′-triphosphate, and causing long-term and short-term processes. P2Y receptors, G protein coupled, have seven transmembrane-domain metabotropic receptors. Eight mammalian P2Y receptors have been cloned, with the earliest identified subtypes linked to Gq proteins, initiating the signaling pathway between phospholipase C and inositol 1,4,5-trisphosphate, and releasing Ca2+ from intracellular stores. Most interestingly, P2Y11 receptors activate and P2Y12 receptors, P2Y13 receptors, P2Y14 receptors inactivate adenylate cyclase via Gs and Gi proteins, respectively. In this review, we discuss the involvement of purinergic receptors, P2Y11, P2Y12, P2Y13 and P2Y14, in cellular senescence and aging.

Receptor-independent regulation of Gα13 by alpha-1-antitrypsin C-terminal peptides.

Alpha-1-antitrypsin (AAT), a circulating serine protease inhibitor, is an acute inflammatory response protein with anti-inflammatory functions. The C-terminal peptides of AAT are found in various tissues and have been proposed as putative bioactive peptides with multiple functions, but its mechanism of action remains unclear. We previously reported that a mouse AAT C-terminal peptide of 35 amino acids (mAAT-C1-35) penetrates plasma membrane and associates guanine nucleotide-binding protein subunit alpha 13 (Gα13). Here, we show that mAAT-C1-35 binds directly to the guanosine diphosphate (GDP)-bound form of Gα13 through the N-terminal region (mAAT-C1-17), thereby facilitating the interaction of Gα13・GDP with its effector proteins. The minimal sequence (mAAT-C3-16) and essential amino acid residue (Phe11) of mAAT-C1-17 were identified as being necessary for this effect. A molecular dynamics simulation for the Gα13・GDP-mAAT-C1-17 complex model showed that binding of mAAT-C1-17 to the region surrounded by switch regions of Gα13 stabilizes the flexible switch II and III regions, thereby maintaining their active conformation. In addition, mAAT-C1-35 activates the Gα13 signalling pathway in cells where Phe11 is required. Our study reveals the structure-based mechanism of action of AAT-C peptides in the regulation of Gα13, and demonstrates that AAT-C peptides represent a biological peptide capable of activating G protein signals in a manner that is independent of G-protein-coupled receptors.

The G protein inhibitor YM-254890 is an allosteric glue.

Given the prominence of G protein coupled receptors (GPCRs) as drug targets, targeting their immediate downstream effectors, G proteins, could be of immense therapeutic value. The discovery that the natural product YM-254890 (YM) can arrest uveal melanoma by specifically inhibiting constitutively active Gq/11without impacting other G protein families demonstrates the potential of this approach. However, efforts to find other G protein family-specific inhibitors have had limited success. Better understanding the mechanism of YM could facilitate efforts to develop other highly specific G protein inhibitors. We hypothesized that differences between the conformational distributions of various G proteins play an important role in determining he specificity of inhibitors like YM. To explore this hypothesis, we built Markov state models (MSMs) from molecular dynamics simulations of the Gα subunits of three different G proteins, as YM predominantly contacts Gα. We also modeled the heterotrimeric versions of these proteins where Gα is bound to the Gβγ heterodimer. We find that YM-sensitive Gα proteins have a higher probability of adopting YM-bound-like conformations than insensitive variants. There is also strong allosteric coupling between the YM- and Gβγ-binding interfaces of Gα. This allostery gives rise to positive cooperativity, wherein the presence of Gβγ enhances preorganization for YM binding. We predict that YM acts as an "allosteric" glue that allosterically stabilizes the complex between Gα and Gβγ despite the minimal contacts between YM and Gβγ.

Role of Rab10 in cocaine-induced behavioral effects is associated with GABAB receptor membrane expression in the nucleus accumbens.

Previous studies have demonstrated that Ras-related GTP-binding protein Rab10 (Rab10) plays a role in psychostimulant-induced behavioral effects. In this study, we showed that Rab10 in the nucleus accumbens (NAc) of male animals affects the development of cocaine-induced behavioral effects, which are associated with the plasma membrane expression of the GABAB heteroreceptor (GABABR). We performed flow cytometry, immunoendocytosis, pHluorin activity analysis, electrophysiology analysis, and open-field testing to explore the role of Rab10 in modulating the membrane expression and function of GABABR and its regulatory effect on cocaine-induced behavioral effects. Transcriptomics analysis showed that Rab10 was elevated following acute cocaine treatment. Membrane levels of Rab10 increased within day 1 of the cocaine treatment, subsequently decreasing at later time points. Rab10 deficiency in NAc regions significantly increased cocaine-inhibited membrane GABABR levels and inhibited cocaine-induced hyperlocomotion and behavioral sensitization. In addition, GAD 67 + -expressing neurons from NAc regions treated with cocaine revealed a significant decrease in Rab10 membrane expression. Furthermore, NAc neuron-specific Rab10 knockout resulted in a significant increase in the cocaine-inhibited membrane expression of GABABR, along with increased miniature inhibitory postsynaptic current (mIPSC) amplitude and attenuation of baclofen-amplified Ca2+ influx. These results uncover a new mechanism in which Rab10-GABABR signaling may serve as a potential pathway for regulating cocaine-induced behavioral effects.

Gnas Knockdown Induces Obesity and AHO Features in Early Zebrafish Larvae.

GNAS (Guanine Nucleotide-Binding Protein, Alpha Stimulating) is a complex gene that encodes the alpha subunit of the stimulatory G protein (Gsα), critical for signaling through various G protein-coupled receptors. Inactivating genetic and epigenetic changes in GNAS, resulting in Gsα deficiency, cause different variants of pseudohypoparathyroidism, which may manifest features of Albright hereditary osteodystrophy (AHO, a syndrome characterized by early-onset obesity and other developmental defects). Recent findings have linked Gsα deficiency with isolated, severe, early-onset obesity, suggesting it as a potential, underrecognized cause of monogenic, non-syndromic obesity. This study was prompted by identifying several GNAS variants of uncertain significance (VUSs) in pediatric patients presenting with unexplained, severe, early-onset obesity at Sidra Medicine in Qatar. To functionally characterize these variants, we developed the first zebrafish model of Gsα deficiency, offering numerous advantages over other model systems. This was achieved by knockdown of the ortholog through microinjection of translation-blocking Morpholino antisense oligonucleotides into the yolks of 1-8-cell-stage zebrafish embryos. The morphant larvae displayed an obese phenotype, marked by significantly enlarged yolk sacs, increased neutral lipid accumulation, and reduced metabolic rates, among other developmental abnormalities resembling those in AHO. This zebrafish model lays the foundation for efficient functional characterization of GNAS VUSs and paves the way for enhancing our understanding of Gsα deficiency-associated early-onset obesity.

CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

Mutations in LRRK2 are the most common genetic cause of Parkinson's disease (PD). LRRK2 protein contains two enzymatic domains: a GTPase (Roc-COR) and a kinase domain. Disease-causing mutations are found in both domains. Now, studies have focused largely on LRRK2 kinase activity, while attention to its GTPase function is limited. LRRK2 is a guanine nucleotide-binding protein, but the mechanism of direct regulation of its GTPase activity remains unclear and its physiological GEF is not known. Here, we identified CalDAG-GEFI (CDGI) as a physiological GEF for LRRK2. CDGI interacts with LRRK2 and increases its GDP to GTP exchange activity. CDGI modulates LRRK2 cellular functions and LRRK2-induced neurodegeneration in both LRRK2 Drosophila and mouse models. Together, this study identified the physiological GEF for LRRK2 and provides strong evidence that LRRK2 GTPase is regulated by GAPs and GEFs. The LRRK2 GTPase, GAP, or GEF activities have the potential to serve as therapeutic targets, which is distinct from the direct LRRK2 kinase inhibition.

Molecular Determinants for Guanine Binding in GTP-Binding Proteins: A Data Mining and Quantum Chemical Study.

GTP-binding proteins are essential molecular switches that regulate a wide range of cellular processes. Their function relies on the specific recognition and binding of guanine within their binding pockets. This study aims to elucidate the molecular determinants underlying this recognition. A large-scale data mining of the Protein Data Bank yielded 298 GTP-binding protein complexes, which provided a structural foundation for a systematic analysis of the intermolecular interactions that are responsible for the molecular recognition of guanine in proteins. It was found that multiple modes of non-bonded interactions including hydrogen bonding, cation-π interactions, and π-π stacking interactions are employed by GTP-binding proteins for binding. Subsequently, the strengths of non-bonded interaction energies between guanine and its surrounding protein residues were quantified by means of the double-hybrid DFT method B2PLYP-D3/cc-pVDZ. Hydrogen bonds, particularly those involving the N2 and O6 atoms of guanine, confer specificity to guanine recognition. Cation-π interactions between the guanine ring and basic residues (Lys and Arg) provide significant electrostatic stabilization. π-π stacking interactions with aromatic residues (Phe, Tyr, and Trp) further contribute to the overall binding affinity. This synergistic interplay of multiple interaction modes enables GTP-binding proteins to achieve high specificity and stability in guanine recognition, ultimately underpinning their crucial roles in cellular signaling and regulation. Notably, the NKXD motif, while historically considered crucial for guanine binding in GTP-binding proteins, is not universally required. Our study revealed significant variability in hydrogen bonding patterns, with many proteins lacking the NKXD motif but still effectively binding guanine through alternative arrangements of interacting residues.

Agonist activation to open the Gα subunit of the GPCR–G protein precoupled complex defines functional agonist activation of TAS2R5

G protein-coupled receptors (GPCRs) regulate multiple cellular responses and represent highly successful therapeutic targets. The mechanisms by which agonists activate the G protein are unclear for many GPCR families, including the bitter taste receptors (TAS2Rs). We ascertained TAS2R5 properties by live cell-based functional assays, direct binding affinity measurements using optical resonators, and atomistic molecular dynamics simulations. We focus on three agonists that exhibit a wide range of signal transduction in cells despite comparable ligand-receptor binding energies derived from direct experiment and computation. Metadynamics simulations revealed that the critical barrier to activation is ligand-induced opening of the G protein between the α-helical (AH) and Ras-like domains of Gα subunit from a precoupled TAS2R5-G protein state to the fully activated state. A moderate agonist opens the AH-Ras cleft from 22 Å to 31 Å with an energy gain of -4.8 kcal mol-1, making GDP water-exposed for signaling. A high-potency agonist had an energy gain of -11.1 kcal mol-1. The low-potency agonist is also exothermic for Gα opening, but with an energy gain of only -1.4 kcal mol-1. This demonstrates that TAS2R5 agonist-bound functional potencies are derived from energy gains in the transition from a precoupled complex at the level of Gα opening. Our experimental and computational study provides insights into the activation mechanism of signal transduction that provide a basis for rational design of new drugs.

Role and Interplay of Different Signaling Pathways Involved in Sciatic Nerve Regeneration.

Regeneration of the sciatic nerve is a sophisticated process that involves the interplay of several signaling pathways that orchestrate the cellular responses critical to regeneration. Among the key pathways are the mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/AKT, cyclic adenosine monophosphate (cAMP), and Janus kinase/signal transducers and transcription activators (JAK/STAT) pathways. In particular, the cAMP pathway modulates neuronal survival and axonal regrowth. It influences various cellular behaviors and gene expression that are essential for nerve regeneration. MAPK is indispensable for Schwann cell differentiation and myelination, whereas PI3K/AKT is integral to the transcription, translation, and cell survival processes that are vital for nerve regeneration. Furthermore, GTP-binding proteins, including those of the Ras homolog gene family (Rho), regulate neural cell adhesion, migration, and survival. Notch signaling also appears to be effective in the early stages of nerve regeneration and in preventing skeletal muscle fibrosis after injury. Understanding the intricate mechanisms and interactions of these pathways is vital for the development of effective therapeutic strategies for sciatic nerve injuries. This review underscores the need for further research to fill existing knowledge gaps and improve therapeutic outcomes.

β2-Chimaerin, a GTPase-Activating Protein for Rac1, Is a Novel Regulator of Hepatic Insulin Signaling and Glucose Metabolism.

Glucose homeostasis is a complex process regulated by multiple organs and hormones, with insulin playing a central role. Recent evidence underscores the role of small GTP-binding proteins, particularly Rac1, in regulating insulin secretion and glucose uptake. However, the role of Rac1-regulatory proteins in these processes remains largely unexplored. In this study, we investigated the role of β2-chimaerin, a Rac1-specific GTPase-activating protein (GAP), in glucose homeostasis using whole-body β2-chimaerin knockout mice. Our data revealed that β2-chimaerin deficiency results in improved glucose tolerance and enhanced insulin sensitivity in mice. These metabolic effects were associated with increased insulin-induced AKT phosphorylation in the liver and activation of downstream pathways that regulate gluconeogenesis and glycogen synthesis. We show that insulin activates Rac1 in the liver. However, β2-chimaerin deletion did not significantly alter Rac1 activation in this organ, suggesting that β2-chimaerin regulates insulin signaling via a Rac1-independent mechanism. These findings expand our understanding of Rac1 regulation in glucose metabolism, and identify β2-chimaerin as a novel modulator of hepatic insulin signaling, with potential implications for the development of insulin resistance and diabetes.