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1.
J Chem Inf Model ; 64(6): 2045-2057, 2024 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-38447156

RESUMO

Free-energy profiles for the activation/deactivation of the ß2-adrenergic receptor (ADRB2) with neutral antagonist and inverse agonist ligands have been determined with well-tempered multiple-walker (MW) metadynamics simulations. The inverse agonists carazolol and ICI118551 clearly favor single inactive conformational minima in both the binary and ternary ligand-receptor-G-protein complexes, in accord with the inverse-agonist activity of the ligands. The behavior of neutral antagonists is more complex, as they seem also to affect the recruitment of the G-protein. The results are analyzed in terms of the conformational states of the well-known microswitches that have been proposed as indicators of receptor activity.


Assuntos
Agonismo Inverso de Drogas , Receptores Adrenérgicos beta 2 , Receptores Adrenérgicos beta 2/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Ligantes
2.
Cell ; 187(6): 1460-1475.e20, 2024 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-38428423

RESUMO

Apelin is a key hormone in cardiovascular homeostasis that activates the apelin receptor (APLNR), which is regarded as a promising therapeutic target for cardiovascular disease. However, adverse effects through the ß-arrestin pathway limit its pharmacological use. Here, we report cryoelectron microscopy (cryo-EM) structures of APLNR-Gi1 complexes bound to three agonists with divergent signaling profiles. Combined with functional assays, we have identified "twin hotspots" in APLNR as key determinants for signaling bias, guiding the rational design of two exclusive G-protein-biased agonists WN353 and WN561. Cryo-EM structures of WN353- and WN561-stimulated APLNR-G protein complexes further confirm that the designed ligands adopt the desired poses. Pathophysiological experiments have provided evidence that WN561 demonstrates superior therapeutic effects against cardiac hypertrophy and reduced adverse effects compared with the established APLNR agonists. In summary, our designed APLNR modulator may facilitate the development of next-generation cardiovascular medications.


Assuntos
Receptores de Apelina , Fármacos Cardiovasculares , Desenho de Fármacos , Receptores de Apelina/agonistas , Receptores de Apelina/química , Receptores de Apelina/ultraestrutura , Microscopia Crioeletrônica , Proteínas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Humanos , Fármacos Cardiovasculares/química
3.
Function (Oxf) ; 5(2): zqae003, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38486977

RESUMO

G protein regulation by regulators of G protein signaling (RGS) proteins play a key role in vascular tone maintenance. The loss of Gi/o and Gq/11 regulation by RGS2 and RGS5 in non-pregnant mice is implicated in augmented vascular tone and decreased uterine blood flow (UBF). RGS2 and 5 are closely related and co-expressed in uterine arteries (UA). However, whether and how RGS2 and 5 coordinate their regulatory activities to finetune G protein signaling and regulate vascular tone are unclear. Here, we determined how the integrated activity of RGS2 and 5 modulates vascular tone to promote UBF. Using ultrasonography and pressure myography, we examined uterine hemodynamics and myogenic tone (MT) of UA of wild type (WT), Rgs2-/-, Rgs5-/-, and Rgs2/5 dbKO mice. We found that MT was reduced in Rgs5-/- relative to WT or Rgs2-/- UA. Activating Gi/o with dopamine increased, whereas exogenous cAMP decreased MT in Rgs5-/- UA to levels in WT UA. Dual deletion of Rgs2 and 5 abolished the reduced MT due to the absence of Rgs5 and enhanced dopamine-induced Gi/o effects in Rgs2/5 dbKO UA. Conversely, and as in WT UA, Gi/o inhibition with pertussis toxin or exogenous cAMP decreased MT in Rgs2/5 dbKO to levels in Rgs5-/- UA. Inhibition of phosphodiesterases (PDE) concentration-dependently decreased and normalized MT in all genotypes, and blocked dopamine-induced MT augmentation in Rgs2-/-, Rgs5-/-, and Rgs2/5 dbKO UA. We conclude that Gi/o augments UA MT in the absence of RGS2 by a novel mechanism involving PDE-mediated inhibition of cAMP-dependent vasodilatation..


Assuntos
Dopamina , Transdução de Sinais , Camundongos , Animais , Constrição , Proteínas de Ligação ao GTP/metabolismo , Hemodinâmica
4.
Methods Mol Biol ; 2754: 33-54, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38512659

RESUMO

Alzheimer's disease, a progressive neurological disorder, is characterized by the accumulation of neurofibrillary tangles and senile plaques by Tau and amyloid-ß, respectively, in the brain microenvironment. The misfolded protein aggregates interact with several components of neuronal and glial cells such as membrane lipids, receptors, transporters, enzymes, cytoskeletal proteins, etc. Under pathological conditions, Tau interacts with several G-protein-coupled receptors (GPCRs), which undergoes either receptor signaling or desensitization followed by internalization of the protein complex. The purinergic GPCR, P2Y12 which is expressed in microglial cells, plays a key role in its activation and migration. Microglial cells sense and migrate to the site of injury aided by P2Y12 receptor that interacts with ADP released from damaged cells. P2Y12 receptor also interacts with misfolded Tau accumulated at the extracellular space and promotes receptor-mediated internalization. Immunocolocalization and co-immunoprecipitation studies demonstrated the interaction of Tau species with the P2Y12 receptor. Later, in-silico analyses were carried out with the repeat domain of Tau (TauRD), which has been identified as the interacting partner of P2Y12 receptor by in-vitro studies. Molecular docking and molecular dynamics simulation studies show the stability and the type of interaction in TauRD-receptor complex. Tau interaction with P2Y12 receptor plays a significant role in maintaining the active state of microglia which could lead to neuroinflammation and neuronal damage in AD brain. Hence, blocking P2Y12-Tau interaction and P2Y12-mediated Tau internalization in microglial cells could be possible therapeutic strategies in downregulating the severity of neuroinflammation in AD.


Assuntos
Doença de Alzheimer , Simulação de Dinâmica Molecular , Humanos , Simulação de Acoplamento Molecular , Receptores Purinérgicos P2Y12/metabolismo , Antagonistas do Receptor Purinérgico P2Y , Doenças Neuroinflamatórias , Doença de Alzheimer/metabolismo , Microglia/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas tau/metabolismo
5.
BMC Plant Biol ; 24(1): 167, 2024 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-38438916

RESUMO

BACKGROUND: Generating elite rice varieties with high yield and superior quality is the main goal of rice breeding programs. Key agronomic traits, including grain size and seed germination characteristics, affect the final yield and quality of rice. The RGA1 gene, which encodes the α-subunit of rice G-protein, plays an important role in regulating rice architecture, seed size and abiotic stress responses. However, whether RGA1 is involved in the regulation of rice quality and seed germination traits is still unclear. RESULTS: In this study, a rice mutant small and round grain 5 (srg5), was identified in an EMS-induced rice mutant library. Systematic analysis of its major agronomic traits revealed that the srg5 mutant exhibited a semi-dwarf plant height with small and round grain and reduced panicle length. Analysis of the physicochemical properties of rice showed that the difference in rice eating and cooking quality (ECQ) between the srg5 mutant and its wild-type control was small, but the appearance quality was significantly improved. Interestingly, a significant suppression of rice seed germination and shoot growth was observed in the srg5 mutant, which was mainly related to the regulation of ABA metabolism. RGA1 was identified as the candidate gene for the srg5 mutant by BSA analysis. A SNP at the splice site of the first intron disrupted the normal splicing of the RGA1 transcript precursor, resulting in a premature stop codon. Additional linkage analysis confirmed that the target gene causing the srg5 mutant phenotype was RGA1. Finally, the introduction of the RGA1 mutant allele into two indica rice varieties also resulted in small and round rice grains with less chalkiness. CONCLUSIONS: These results indicate that RGA1 is not only involved in the control of rice architecture and grain size, but also in the regulation of rice quality and seed germination. This study sheds new light on the biological functions of RGA1, thereby providing valuable information for future systematic analysis of the G-protein pathway and its potential application in rice breeding programs.


Assuntos
Oryza , Oryza/genética , Sementes/genética , Germinação/genética , Melhoramento Vegetal , Grão Comestível/genética , Proteínas de Ligação ao GTP
6.
J Agric Food Chem ; 72(11): 5645-5658, 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38462712

RESUMO

The present study evaluated the effects of sodium butyrate (SB) supplementation on exocrine and endocrine pancreatic development in dairy calves. Fourteen male Holstein calves were alimented with either milk or milk supplemented with SB for 70 days. Pancreases were collected for analysis including staining, immunofluorescence, electron microscopy, qRT-PCR, Western blotting, and proteomics. Results indicated increased development in the SB group with increases in organ size, protein levels, and cell growth. There were also exocrine enhancements manifested as higher enzyme activities and gene expressions along with larger zymogen granules. Endocrine benefits included elevated gene expression, more insulin secretion, and larger islets, indicating a rise in ß-cell proliferation. Proteomics and pathway analyses pinpointed the G protein subunit alpha-15 as a pivotal factor in pancreatic and insulin secretion pathways. Overall, SB supplementation enhances pancreatic development by promoting its exocrine and endocrine functions through G protein regulation in dairy calves.


Assuntos
Suplementos Nutricionais , Proteômica , Animais , Bovinos/genética , Masculino , Ácido Butírico/farmacologia , Suplementos Nutricionais/análise , Pâncreas , Proteínas de Ligação ao GTP
7.
Int J Mol Sci ; 25(5)2024 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-38473855

RESUMO

In order to determine the behavior of the right ventricle, we have reviewed the existing literature in the area of cardiac remodeling, signal transduction pathways, subcellular mechanisms, ß-adrenoreceptor-adenylyl cyclase system and myocardial catecholamine content during the development of left ventricular failure due to myocardial infarction. The right ventricle exhibited adaptive cardiac hypertrophy due to increases in different signal transduction pathways involving the activation of protein kinase C, phospholipase C and protein kinase A systems by elevated levels of vasoactive hormones such as catecholamines and angiotensin II in the circulation at early and moderate stages of heart failure. An increase in the sarcoplasmic reticulum Ca2+ transport without any changes in myofibrillar Ca2+-stimulated ATPase was observed in the right ventricle at early and moderate stages of heart failure. On the other hand, the right ventricle showed maladaptive cardiac hypertrophy at the severe stages of heart failure due to myocardial infarction. The upregulation and downregulation of ß-adrenoreceptor-mediated signal transduction pathways were observed in the right ventricle at moderate and late stages of heart failure, respectively. The catalytic activity of adenylate cyclase, as well as the regulation of this enzyme by Gs proteins, were seen to be augmented in the hypertrophied right ventricle at early, moderate and severe stages of heart failure. Furthermore, catecholamine stores and catecholamine uptake in the right ventricle were also affected as a consequence of changes in the sympathetic nervous system at different stages of heart failure. It is suggested that the hypertrophied right ventricle may serve as a compensatory mechanism to the left ventricle during the development of early and moderate stages of heart failure.


Assuntos
Insuficiência Cardíaca , Infarto do Miocárdio , Humanos , Ventrículos do Coração/metabolismo , Insuficiência Cardíaca/metabolismo , Infarto do Miocárdio/metabolismo , Cardiomegalia/metabolismo , Miocárdio/metabolismo , Receptores Adrenérgicos beta/metabolismo , Catecolaminas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Adenilil Ciclases/metabolismo
8.
Int J Mol Sci ; 25(5)2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38474044

RESUMO

Transglutaminase type 2 (TG2) is the most ubiquitously expressed and well characterized member of the transglutaminase family. It is a ubiquitous multifunctional enzyme implicated in the regulation of several cellular pathways that support the survival, death, and general homeostasis of eukaryotic cells. Due to its multiple localizations both inside and outside the cell, TG2 participates in the regulation of many crucial intracellular signaling cascades in a tissue- and cell-specific manner, making this enzyme an important player in disease development and progression. Moreover, TG2 is capable of modulating the tumor microenvironment, a process of dynamic tissue remodeling and biomechanical events, resulting in changes which influence tumor initiation, growth, and metastasis. Even if generally related to the Ca2+-dependent post-translational modification of proteins, a number of different biological functions have been ascribed to TG2, like those of a peptide isomerase, protein kinase, guanine nucleotide binder, and cytosolic-nuclear translocator. With respect to cancer, TG2's role is controversial and highly debated; it has been described both as an anti- and pro-apoptotic factor and is linked to all the processes of tumorigenesis. However, numerous pieces of evidence support a tissue-specific role of TG2 so that it can assume both oncogenic and tumor-suppressive roles.


Assuntos
Neoplasias , Proteína 2 Glutamina gama-Glutamiltransferase , Humanos , Proteínas de Ligação ao GTP/metabolismo , Transglutaminases/metabolismo , Transdução de Sinais , Microambiente Tumoral
9.
Int J Mol Sci ; 25(5)2024 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-38474167

RESUMO

Melatonin is a neuroendocrine hormone that regulates the circadian rhythm and many other physiological processes. Its functions are primarily exerted through two subtypes of human melatonin receptors, termed melatonin type-1 (MT1) and type-2 (MT2) receptors. Both MT1 and MT2 receptors are generally classified as Gi-coupled receptors owing to their well-recognized ability to inhibit cAMP accumulation in cells. However, it remains an enigma as to why melatonin stimulates cAMP production in a number of cell types that express the MT1 receptor. To address if MT1 can dually couple to Gs and Gi proteins, we employed a highly sensitive luminescent biosensor (GloSensorTM) to monitor the real-time changes in the intracellular cAMP level in intact live HEK293 cells that express MT1 and/or MT2. Our results demonstrate that the activation of MT1, but not MT2, leads to a robust enhancement on the forskolin-stimulated cAMP formation. In contrast, the activation of either MT1 or MT2 inhibited cAMP synthesis driven by the activation of the Gs-coupled ß2-adrenergic receptor, which is consistent with a typical Gi-mediated response. The co-expression of MT1 with Gs enabled melatonin itself to stimulate cAMP production, indicating a productive coupling between MT1 and Gs. The possible existence of a MT1-Gs complex was supported through molecular modeling as the predicted complex exhibited structural and thermodynamic characteristics that are comparable to that of MT1-Gi. Taken together, our data reveal that MT1, but not MT2, can dually couple to Gs and Gi proteins, thereby enabling the bi-directional regulation of adenylyl cyclase to differentially modulate cAMP levels in cells that express different complements of MT1, MT2, and G proteins.


Assuntos
Melatonina , Humanos , Receptores de Melatonina/metabolismo , Melatonina/farmacologia , Células HEK293 , Receptor MT1 de Melatonina/metabolismo , Receptor MT2 de Melatonina/metabolismo , Proteínas de Ligação ao GTP/metabolismo
10.
Int J Mol Sci ; 25(5)2024 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-38474231

RESUMO

Melanoma, a highly aggressive skin cancer, is characterized by rapid progression and high mortality. Recent advances in molecular pathogenesis have shed light on genetic and epigenetic changes that drive melanoma development. This review provides an overview of these developments, focusing on molecular mechanisms in melanoma genesis. It highlights how mutations, particularly in the BRAF, NRAS, c-KIT, and GNAQ/GNA11 genes, affect critical signaling pathways. The evolution of diagnostic techniques, such as genomics, transcriptomics, liquid biopsies, and molecular biomarkers for early detection and prognosis, is also discussed. The therapeutic landscape has transformed with targeted therapies and immunotherapies, improving patient outcomes. This paper examines the efficacy, challenges, and prospects of these treatments, including recent clinical trials and emerging strategies. The potential of novel treatment strategies, including neoantigen vaccines, adoptive cell transfer, microbiome interactions, and nanoparticle-based combination therapy, is explored. These advances emphasize the challenges of therapy resistance and the importance of personalized medicine. This review underlines the necessity for evidence-based therapy selection in managing the increasing global incidence of melanoma.


Assuntos
Melanoma , Neoplasias Cutâneas , Humanos , Melanoma/patologia , Neoplasias Cutâneas/patologia , Mutação , Transdução de Sinais , Proteínas de Ligação ao GTP/metabolismo
11.
Ann Clin Transl Neurol ; 11(3): 819-825, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38327089

RESUMO

INTRODUCTION: COXPD23 is a rare mitochondrial disease caused by biallelic pathogenic variants in GTPBP3. We report on two siblings with a mild phenotype. CASE REPORTS: The young boy presented with global developmental delay, ataxic gait and upper limbs tremor, and the older sister with absence seizures and hypertrophic cardiomyopathy. Respiratory chain impairment was confirmed in muscle. DISCUSSION: Reviewed cases point toward clustering around two prevalent phenotypes: an early-onset presentation with severe fatal encephalopathy and a late milder presentation with global developmental delay/ID and cardiopathy, with the latter as, is the main feature. Our patients showed an intermediate phenotype with intrafamilial variability.


Assuntos
Doenças Mitocondriais , Convulsões , Masculino , Humanos , Mitocôndrias , Fenótipo , Proteínas de Ligação ao GTP
12.
Biosci Rep ; 44(3)2024 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-38372298

RESUMO

Septin proteins are a subfamily of closely related GTP-binding proteins conserved in all species except for higher plants and perform essential biological processes. Septins self-assemble into heptameric or octameric complexes and form higher-order structures such as filaments, rings, or gauzes by end-to-end binding. Their close association with cell membrane components makes them central in regulating critical cellular processes. Due to their organisation and properties, septins function as diffusion barriers and are integral in providing scaffolding to support the membrane's curvature and stability of its components. Septins are also involved in vesicle transport and exocytosis through the plasma membrane by co-localising with exocyst protein complexes. Recently, there have been emerging reports of several human and animal diseases linked to septins and abnormalities in their functions. Most of our understanding of the significance of septins during microbial diseases mainly pertains to their roles in bacterial infections but not viruses. This present review focuses on the known roles of septins in host-viral interactions as detailed by various studies.


Assuntos
Septinas , Viroses , Animais , Humanos , Septinas/genética , Septinas/metabolismo , Proteínas de Ligação ao GTP , Citoesqueleto/metabolismo , Citoplasma/metabolismo , Viroses/genética
13.
ACS Chem Neurosci ; 15(5): 1026-1041, 2024 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-38387042

RESUMO

In consideration of the limited number of FDA-approved drugs for autism spectrum disorder (ASD), significant efforts have been devoted to identifying novel drug candidates. Among these, 5-HT7R modulators have garnered considerable attention due to their potential in alleviating autism-like behaviors in ASD animal models. In this study, we designed and synthesized biphenyl-3-ylmethylpyrrolidines 3 and biphenyl-3-yl-dihydroimidazoles 4 as 5-HT7R modulators. Through extensive biological tests of 3 and 4 in G protein and ß-arrestin signaling pathways of 5-HT7R, it was determined that 2-(2'-methoxy-[1,1'-biphenyl]-3-yl)-4,5-dihydro-1H-imidazole 4h acted as a 5-HT7R antagonist in both signaling pathways. In in vivo study with Shank3-/- transgenic (TG) mice, the self-grooming behavior test was performed with 4h, resulting in a significant reduction in the duration of self-grooming. In addition, an immunohistochemical experiment with 4h restored reduced neurogenesis in Shank3-/- TG mice, which is confirmed by the quantification of doublecortin (DCX) positive neurons, suggesting the promising therapeutic potential of 4h.


Assuntos
Transtorno do Espectro Autista , Compostos de Bifenilo , Animais , Camundongos , Serotonina , beta-Arrestinas , Transdução de Sinais , Camundongos Transgênicos , Proteínas de Ligação ao GTP , Modelos Animais de Doenças , Proteínas dos Microfilamentos , Proteínas do Tecido Nervoso
14.
Front Immunol ; 15: 1303089, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38348040

RESUMO

Guanylate binding proteins (GBPs) are an evolutionarily ancient family of proteins that are widely distributed among eukaryotes. They belong to the dynamin superfamily of GTPases, and their expression can be partially induced by interferons (IFNs). GBPs are involved in the cell-autonomous innate immune response against bacterial, parasitic and viral infections. Evolutionary studies have shown that GBPs exhibit a pattern of gene gain and loss events, indicative for the birth-and-death model of evolution. Most species harbor large GBP gene clusters that encode multiple paralogs. Previous functional and in-depth evolutionary studies have mainly focused on murine and human GBPs. Since rabbits are another important model system for studying human diseases, we focus here on lagomorphs to broaden our understanding of the multifunctional GBP protein family by conducting evolutionary analyses and performing a molecular and functional characterization of rabbit GBPs. We observed that lagomorphs lack GBP3, 6 and 7. Furthermore, Leporidae experienced a loss of GBP2, a unique duplication of GBP5 and a massive expansion of GBP4. Gene expression analysis by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and transcriptome data revealed that leporid GBP expression varied across tissues. Overexpressed rabbit GBPs localized either uniformly and/or discretely to the cytoplasm and/or to the nucleus. Oryctolagus cuniculus (oc)GBP5L1 and rarely ocGBP5L2 were an exception, colocalizing with the trans-Golgi network (TGN). In addition, four ocGBPs were IFN-inducible and only ocGBP5L2 inhibited furin activity. In conclusion, from an evolutionary perspective, lagomorph GBPs experienced multiple gain and loss events, and the molecular and functional characteristics of ocGBP suggest a role in innate immunity.


Assuntos
Lagomorpha , Animais , Coelhos , Humanos , Camundongos , Lagomorpha/metabolismo , Proteínas de Transporte , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Imunidade Inata/genética , Interferons/metabolismo
15.
Acta Neuropathol Commun ; 12(1): 24, 2024 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-38331815

RESUMO

Myelin sheath abnormality is the cause of various neurodegenerative diseases (NDDs). G-proteins and their coupled receptors (GPCRs) play the important roles in myelination. Gnao1, encoding the major Gα protein (Gαo) in mammalian nerve system, is required for normal motor function. Here, we show that Gnao1 restricted to Schwann cell (SCs) lineage, but not neurons, negatively regulate SC differentiation, myelination, as well as re-myelination in peripheral nervous system (PNS). Mice lacking Gnao1 expression in SCs exhibit faster re-myelination and motor function recovery after nerve injury. Conversely, mice with Gnao1 overexpression in SCs display the insufficient myelinating capacity and delayed re-myelination. In vitro, Gnao1 deletion in SCs promotes SC differentiation. We found that Gnao1 knockdown in SCs resulting in the elevation of cAMP content and the activation of PI3K/AKT pathway, both associated with SC differentiation. The analysis of RNA sequencing data further evidenced that Gnao1 deletion cause the increased expression of myelin-related molecules and activation of regulatory pathways. Taken together, our data indicate that Gnao1 negatively regulated SC differentiation by reducing cAMP level and inhibiting PI3K-AKT cascade activation, identifying a novel drug target for the treatment of demyelinating diseases.


Assuntos
Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Animais , Camundongos , Proteínas de Ligação ao GTP , Mamíferos/metabolismo , Bainha de Mielina/metabolismo , Sistema Nervoso Periférico/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células de Schwann
16.
Sci Signal ; 17(823): eabl5880, 2024 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-38349968

RESUMO

The neuropeptide relaxin-3 is composed of an A chain and a B chain held together by disulfide bonds, and it modulates functions such as anxiety and food intake by binding to and activating its cognate receptor RXFP3, mainly through the B chain. Biased ligands of RXFP3 would help to determine the molecular mechanisms underlying the activation of G proteins and ß-arrestins downstream of RXFP3 that lead to such diverse functions. We showed that the i, i+4 stapled relaxin-3 B chains, 14s18 and d(1-7)14s18, were Gαi/o-biased agonists of RXFP3. These peptides did not induce recruitment of ß-arrestin1/2 to RXFP3 by GPCR kinases (GRKs), in contrast to relaxin-3, which enabled the GRK2/3-mediated recruitment of ß-arrestin1/2 to RXFP3. Relaxin-3 and the previously reported peptide 4 (an i, i+4 stapled relaxin-3 B chain) did not exhibit biased signaling. The staple linker of peptide 4 and parts of both the A chain and B chain of relaxin-3 interacted with extracellular loop 3 (ECL3) of RXFP3, moving it away from the binding pocket, suggesting that unbiased ligands promote a more open conformation of RXFP3. These findings highlight roles for the A chain and the N-terminal residues of the B chain of relaxin-3 in inducing conformational changes in RXFP3, which will help in designing selective biased ligands with improved therapeutic efficacy.


Assuntos
Relaxina , Relaxina/farmacologia , Relaxina/química , Relaxina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Proteínas de Ligação ao GTP/metabolismo , Domínios Proteicos , beta-Arrestinas/metabolismo
17.
Cells ; 13(3)2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38334664

RESUMO

Small G proteins (e.g., Rac1) play critical regulatory roles in islet ß-cell function in health (physiological insulin secretion) and in metabolic stress (cell dysfunction and demise). Multiple regulatory factors for these G proteins, such as GDP dissociation inhibitors (GDIs), have been implicated in the functional regulation of these G proteins. The current set of investigations is aimed at understanding impact of chronic hyperglycemic stress on the expression and subcellular distribution of three known isoforms of RhoGDIs (RhoGDIα, RhoGDIß, and RhoGDIγ) in insulin-secreting ß-cells. The data accrued in these studies revealed that the expression of RhoGDIß, but not RhoGDIα or RhoGDIγ, is increased in INS-1 832/13 cells, rat islets, and human islets. Hyperglycemic stress also promoted the cleavage of RhoGDIß, leading to its translocation to the nuclear compartment. We also report that RhoGDIα, but not RhoGDIγ, is associated with the nuclear compartment. However, unlike RhoGDIß, hyperglycemic conditions exerted no effects on RhoGDIα's association with nuclear fraction. Based on these observations, and our earlier findings of the translocation of Rac1 to the nuclear compartment under the duress of metabolic stress, we conclude that the RhoGDIß-Rac1 signaling module promotes signals from the cytosolic to the nucleus, culminating in accelerated ß-cell dysfunction under metabolic stress.


Assuntos
Células Secretoras de Insulina , Inibidor beta de Dissociação do Nucleotídeo Guanina rho , Animais , Humanos , Ratos , Proteínas de Ligação ao GTP/metabolismo , Células Secretoras de Insulina/metabolismo , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/metabolismo , Inibidor gama de Dissociação do Nucleotídeo Guanina rho/metabolismo
18.
Nat Commun ; 15(1): 1334, 2024 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-38351103

RESUMO

G protein-coupled receptors (GPCRs) bind to different G protein α-subtypes with varying degrees of selectivity. The mechanism by which GPCRs achieve this selectivity is still unclear. Using 13C methyl methionine and 19F NMR, we investigate the agonist-bound active state of ß1AR and its ternary complexes with different G proteins in solution. We find the receptor in the ternary complexes adopts very similar conformations. In contrast, the full agonist-bound receptor active state assumes a conformation differing from previously characterised activation intermediates or from ß1AR in ternary complexes. Assessing the kinetics of binding for the agonist-bound receptor with different G proteins, we find the increased affinity of ß1AR for Gs results from its much faster association with the receptor. Consequently, we suggest a kinetic-driven selectivity gate between canonical and secondary coupling which arises from differential favourability of G protein binding to the agonist-bound receptor active state.


Assuntos
Proteínas de Ligação ao GTP , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Ligação Proteica
19.
Proc Natl Acad Sci U S A ; 121(8): e2317893121, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38346183

RESUMO

Physics-based simulation methods can grant atomistic insights into the molecular origin of the function of biomolecules. However, the potential of such approaches has been hindered by their low efficiency, including in the design of selective agonists where simulations of myriad protein-ligand combinations are necessary. Here, we describe an automated input-free path searching protocol that offers (within 14 d using Graphics Processing Unit servers) a minimum free energy path (MFEP) defined in high-dimension configurational space for activating sphingosine-1-phosphate receptors (S1PRs) by arbitrary ligands. The free energy distributions along the MFEP for four distinct ligands and three S1PRs reached a remarkable agreement with Bioluminescence Resonance Energy Transfer (BRET) measurements of G-protein dissociation. In particular, the revealed transition state structures pointed out toward two S1PR3 residues F263/I284, that dictate the preference of existing agonists CBP307 and BAF312 on S1PR1/5. Swapping these residues between S1PR1 and S1PR3 reversed their response to the two agonists in BRET assays. These results inspired us to design improved agonists with both strong polar head and bulky hydrophobic tail for higher selectivity on S1PR1. Through merely three in silico iterations, our tool predicted a unique compound scaffold. BRET assays confirmed that both chiral forms activate S1PR1 at nanomolar concentration, 1 to 2 orders of magnitude less than those for S1PR3/5. Collectively, these results signify the promise of our approach in fine agonist design for G-protein-coupled receptors.


Assuntos
Receptores Acoplados a Proteínas G , Receptores de Lisoesfingolipídeo , Receptores de Lisoesfingolipídeo/metabolismo , Receptores de Esfingosina-1-Fosfato , Proteínas de Ligação ao GTP , Medições Luminescentes
20.
Mol Med ; 30(1): 28, 2024 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-38383297

RESUMO

BACKGROUND: Lung cancer is the leading cause of cancer-related death worldwide. The sex differences in the occurrence and fatality rates of non-small cell lung cancer (NSCLC), along with its association with estrogen dependence, suggest that estrogen receptors (ERs) contribute to the development of NSCLC. However, the influence of G protein-coupled estrogen receptor (GPER1) on NSCLC remains to be determined. Escape from ferroptosis is one of the hallmarks of tumor discovered in recent years. In this context, the present study evaluated whether GPER1 promotes NSCLC progression by preventing ferroptosis, and the underlying mechanism through which GPER1 protects against ferroptosis was also explored. METHODS: The effects of GPER1 on the cytotoxicity of H2O2, the ferroptosis inducer RSL3, and Erastin were assessed using the CCK8 assay and plate cloning. Lipid peroxidation levels were measured based on the levels of MDA and BODIPY™581/591C11. GPER1 overexpression and knockdown were performed and G1 was used, and the expression of SCD1 and PI3K/AKT/mTOR signaling factors was measured. Immunofluorescence analysis and immunohistochemistry were performed on paired specimens to measure the correlation between the expression of GPER1 and SCD1 in NSCLC tissues. The effect of GPER1 on the cytotoxicity of cisplatin was measured in vitro using the CCK8 assay and in vivo using xenograft tumor models. RESULTS: GPER1 and G1 alleviated the cytotoxicity of H2O2, reduced sensitivity to RSL3, and impaired lipid peroxidation in NSCLC tissues. In addition, GPER1 and G1 promoted the protein and mRNA expression of SCD1 and the activation of PI3K/AKT/mTOR signaling. GPER1 and SCD1 expression were elevated and positively correlated in NSCLC tissues, and high GPER1 expression predicted a poor prognosis. GPER1 knockdown enhanced the antitumor activity of cisplatin in vitro and in vivo. CONCLUSION: GPER1 prevents ferroptosis in NSCLC by promoting the activation of PI3K/AKT/mTOR signaling, thereby inducing SCD1 expression. Therefore, treatments targeting GPER1 combined with cisplatin would exhibit better antitumor effects.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Ferroptose , Neoplasias Pulmonares , Humanos , Feminino , Masculino , Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Pulmonares/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Cisplatino/farmacologia , Lipogênese , Peróxido de Hidrogênio/farmacologia , Peróxido de Hidrogênio/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Estrogênios , Receptores de Estrogênio/metabolismo , Proteínas de Ligação ao GTP , Estearoil-CoA Dessaturase/metabolismo
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