Search

RGS (Regulator of G protein signaling) proteins have long captured the fascination of researchers due to their intricate involvement across a wide array of signaling pathways within cellular systems. Their diverse and nuanced functions have positioned them as continual subjects of scientific inquiry, especially given the implications of certain family members in various cancer types. Of particular note in this context is RGS20, whose clinical relevance and molecular significance in hepatocellular carcinoma we have recently investigated. These investigations have prompted questions into the prevalence of pathogenic mutations within the RGS20 gene and the intricate network of interacting proteins that could contribute to the complex landscape of cancer biology. In our study, we aim to unravel the mutations within the RGS20 gene and the multifaceted interplay between RGS20 and other proteins within the context of cancer. Expanding on this line of inquiry, our research is dedicated to uncovering the intricate mechanisms of RGS20 in various cancers. In particular, we have redirected our attention to examining the role of RGS20 within hematological malignancies, with a specific focus on multiple myeloma and follicular lymphoma. These hematological cancers hold significant promise for further investigation, as understanding the involvement of RGS20 in their pathogenesis could unveil novel therapeutic strategies and treatment avenues. Furthermore, our exploration has extended to encompass the latest discoveries concerning the potential involvement of RGS20 in diseases affecting the central nervous system, thereby broadening the scope of its implications beyond oncology to encompass neurobiology and related fields. [ABSTRACT FROM AUTHOR]

GPR56, an adhesion G-protein coupled receptor (aGPCRs) with constitutive and ligand-promoted activity, is involved in many physiological and pathological processes. Whether the receptor's constitutive or ligand-promoted activation occur through the same molecular mechanism, and whether different activation modes lead to functional selectivity between G proteins is unknown. Here we show that GPR56 constitutively activates both G12 and G13. Unlike constitutive activation and activation with 3-α-acetoxydihydrodeoxygedunin (3αDOG), stimulation with an antibody, 10C7, directed against GPR56's extracellular domain (ECD) led to an activation that favors G13 over G12. An autoproteolytically deficient mutant, GPR56-T383A, was also activated by 10C7 indicating that the tethered agonist (TA) exposed through autocatalytic cleavage, is not required for this activation modality. In contrast, this proteolysis-resistant mutant could not be activated by 3αDOG indicating different modes of activation by the two ligands. We show that an N-terminal truncated GPR56 construct (GPR56-Δ1-385) is devoid of constitutive activity but was activated by 3αDOG. Similarly to 3αDOG, 10C7 promoted the recruitment of β-arrestin-2 but GPR56 internalization was β-arrestin independent. Despite the slow activation mode of 10C7 that favors G13 over G12, it efficiently activated the downstream Rho pathway in BT-20 breast cancer cells. These data show that different GPR56 ligands have different modes of activation yielding differential G protein selectivity but converging on the activation of the Rho pathway both in heterologous expressions system and in cancer cells endogenously expressing the receptor. 10C7 is therefore an interesting tool to study both the processes underlying GPR56 activity and its role in cancer cells. [ABSTRACT FROM AUTHOR]

Two-thirds of signaling hormones and one-third of approved drugs exert their effects by binding and modulating the G protein-coupled receptors (GPCRs) activation. While the activation mechanism for monomeric GPCRs has been well-established, little is known about GPCRs in dimeric form. Here, by combining transition pathway generation, extensive atomistic simulation-based Markov state models, and experimental signaling assays, we reveal an asymmetric, stepwise millisecond allosteric activation mechanism for the metabotropic glutamate receptor subtype 5 receptor (mGlu5), an obligate dimeric class C GPCR. The dynamic picture is presented that agonist binding induces dimeric ectodomains compaction, amplified by the precise association of the cysteine-rich domains, ultimately loosely bringing the intracellular 7-transmembrane (7TM) domains into proximity and establishing an asymmetric TM6-TM6 interface. The active inter-domain interface enhances their intra-domain flexibility, triggering the activation of micro-switches crucial for downstream signal transduction. Furthermore, we show that the positive allosteric modulator stabilizes both the active inter-domain 7TM interface and an open, extended intra-domain ICL2 conformation. This stabilization leads to the formation of a pseudo-cavity composed of the ICL2, ICL3, TM3, and C-terminus, which facilitates G protein coordination. Our strategy may be generalizable for characterizing millisecond events in other allosteric systems. Activation mechanism of GPCRs in dimeric form remains enigmatic. Here, the authors present a dynamic characterization of mGlu5, an obligate dimeric class C GPCR, suggesting an asymmetric, stepwise allosteric activation mechanism. [ABSTRACT FROM AUTHOR]

Guanosine Triphosphate (GTP)ases of Immunity Associated Proteins (GIMAP) are small G proteins that in vertebrates, regulate immunity, apoptosis, and autophagy. While previously thought to be limited to vertebrates and plants, this family of proteins was recently characterized in a diversity of dinoflagellates. However, information regarding the function of GIMAPs in dinoflagellates is lacking. Therefore, the goal of this study (conducted from March-November of 2022) was to assess the expression of GIMAPs in the cnidarian symbiont Breviolum minutum (formerly Symbiodinium minutum, referred to as Bm_GIMAPs) in response to low light (25 vs 8 μmol m−2 s−1 for 9 d), thermal stress (22 vs 28 °C for 48 h), and symbiotic state (cultured vs in hospite). These factors were selected due to their importance in the lifestyle of a photosynthetic symbiont and relevance to the ecologically important issue of coral bleaching. The results indicate Bm_GIMAP expression increased under both low light and elevated temperature, suggesting a role in the response to bleaching related stressors. Cultured B. minutum had lower baseline Bm_GIMAP expression than those in association with a cnidarian host, but a stronger response to thermal stress, suggesting their function is influenced by symbiosis. Lastly, further support for the responsiveness of GIMAPs to thermal stress was provided by a comparison with previously published RNA-seq data from cultured Symbiodiniaceae. Collectively, the results presented here represent the first functional investigation of GIMAPs in an ecologically important group of protists and provide a greater understanding of the molecular response of dinoflagellates to stressors related to bleaching. [ABSTRACT FROM AUTHOR]

Aerobic glycolysis has been recognized as a hallmark of human cancer. G protein pathway suppressor 2 (GPS2) is a negative regulator of the G protein-MAPK pathway and a core subunit of the NCoR/SMRT transcriptional co-repressor complex. However, how its biological properties intersect with cellular metabolism in breast cancer (BC) development remains poorly elucidated. Here, we report that GPS2 is low expressed in BC tissues and negatively correlated with poor prognosis. Both in vitro and in vivo studies demonstrate that GPS2 suppresses malignant progression of BC. Moreover, GPS2 suppresses aerobic glycolysis in BC cells. Mechanistically, GPS2 destabilizes HIF-1α to reduce the transcription of its downstream glycolytic regulators (PGK1 , PGAM1 , ENO1 , PKM2 , LDHA , PDK1 , PDK2 , and PDK4), and then suppresses cellular aerobic glycolysis. Notably, receptor for activated C kinase 1 (RACK1) is identified as a key ubiquitin ligase for GPS2 to promote HIF-1α degradation. GPS2 stabilizes the binding of HIF-1α to RACK1 by directly binding to RACK1, resulting in polyubiquitination and instability of HIF-1α. Amino acid residues 70–92 aa of the GPS2 N-terminus bind RACK1. A 23-amino-acid-long GPS2-derived peptide was developed based on this N-terminal region, which promotes the interaction of RACK1 with HIF-1α, downregulates HIF-1α expression and significantly suppresses BC tumorigenesis in vitro and in vivo. In conclusion, our findings indicate that GPS2 decreases the stability of HIF-1α, which in turn suppresses aerobic glycolysis and tumorigenesis in BC, suggesting that targeting HIF-1α degradation and treating with peptides may be a promising approach to treat BC. [Display omitted] • GPS2 suppresses glycolytic metabolism in breast cancer. • GPS2 suppresses glycolytic metabolism by downregulating the protein expression of HIF-1α. • GPS2 reduces HIF-1α protein stability through RACK1-mediated ubiquitination. • GPS2-derived peptide suppresses breast cancer by promoting HIF-1α degradation. [ABSTRACT FROM AUTHOR]

The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-24. Nonstandard abbreviations are available in the Notes. The manufacturing method of string cheese is similar to mozzarella, but the hot curd is extruded through narrow tubes or pipes, which align the protein fibers that provide the characteristic ability for consumers to pull strings from this cheese. Firmness is another important performance attribute for consumers who just bite into the string cheese without peeling off strings. There have only been a few studies on string cheese, but it is known that stringiness and firmness decrease during prolonged storage, which is a particular challenge for exporting string cheese. We explored 2 treatments to try to retain the stringiness and firmness of string cheese for longer storage periods. The techniques used were high-pressure processing (HPP; 600 MPa for 3 min) and reduced storage temperature (0°C). In other cheese varieties, these techniques have helped extend the performance shelf life. We tested these techniques using the 2 main types of commercial string cheese: direct acid string cheese (DASC) and cultured string cheese (CSC), which were obtained from 2 different manufacturing facilities. The DASC had higher fat (∼2.2%) and higher pH values (∼0.2 units) compared with the CSC. The CSC had higher protein content (∼3.4%), higher insoluble calcium content (∼8 mg insoluble Ca/g protein) and higher texture profile analysis (TPA) hardness values (∼4 N) compared with the DASC. Due to the compositional differences, the 2 varieties were statistically analyzed separately for all other attributes. In both cheese types, HPP caused an immediate reduction in stringiness, some solubilization of insoluble calcium, and a slight increase in the cheese pH values. High-pressure processing also caused a slight increase in TPA hardness of the CSC samples until 14 d (possibly due to a slight increase in cheese pH). The use of the 0°C storage temperature reduced proteolysis and helped retain firmness during storage. Low-temperature storage could help extend the performance shelf life of string cheese by a couple of months, but HPP was not suitable, as the process caused an immediate reduction in stringiness due to the disruption of the matrix induced by the HPP treatment. [ABSTRACT FROM AUTHOR]

The human respiratory syncytial virus (RSV) is a significant health concern, particularly for infants, young children, and the elderly. This virus is known to evolve continuously due to environmental factors and herd immunity. In light of this, our study aimed to analyze the genetic variability of the G protein in RSV-A and RSV-B genotypes in Kuwait from 2020 to 2022. Between January 2020 and September 2022, we collected 490 respiratory samples from hospitalized patients with acute respiratory tract infections. These samples were tested and confirmed positive for RSV using multiplex Real-Time PCR. Subsequently, the samples underwent nucleic acid sequencing using the advanced Nanopore sequencing technology to analyze the full-length G gene. Sequence analysis showed that 64 isolates (76%) were RSV-A, and 20 isolates (24%) were RSV-B. The G genes of RSV-A belonged to genotype GA2.3.5, while all the RSV-B genotypes belonged to GB5.0.5a. New lineages and sub-lineages of RSV-A and RSV-B were detected, indicating the circulation of new strains in Kuwait. Many unique and new amino acid changes, including insertions, were found in the G proteins of Kuwaiti isolates, with the highest variability in the second hypervariable region. An increased number of N and O-linked glycosylation sites were also identified in the G protein, which could speculate to alter the antigenicity of RSV. The identified changes in the G protein of RSV-A and RSV-B genotypes might result from immune pressure and could affect the antigenic characteristics of circulating strains in Kuwait. This could potentially lead to new RSV variants that can evade the immune response. Our in-depth analysis of the G proteins of both RSV-A and RSV-B could aid in the development of more potent treatments and vaccines. [ABSTRACT FROM AUTHOR]

The fusion protein of an engineered zymogen of microbial transglutaminase (EzMTG) with a protein G variant, EzMTG-pG, enabled the proximity-based, tag-free labeling of Lys65 in the heavy chain of a native IgG antibody (trastuzumab) with a Gln-donor peptidyl substrate functionalized with a fluorescent molecule. [ABSTRACT FROM AUTHOR]

Background Experimental Approach Key Results Conclusion and Implications There is increasing interest in developing FPR2 agonists (compound 43, ACT‐389949 and BMS‐986235) as potential pro‐resolving therapeutics, with ACT‐389949 and BMS‐986235 having entered phase I clinical development. FPR2 activation leads to diverse downstream outputs. ACT‐389949 was observed to cause rapid tachyphylaxis, while BMS‐986235 and compound 43 induced cardioprotective effects in preclinical models. We aim to characterise the differences in ligand‐receptor engagement and downstream signalling and trafficking bias profile.Concentration‐response curves to G protein dissociation, β‐arrestin recruitment, receptor trafficking and second messenger signalling were generated using FPR2 ligands (BMS‐986235, ACT‐389949, compound 43 and WKYMVm), in HEK293A cells. Log(τ/KA) was obtained from the operational model for bias analysis using WKYMVm as a reference ligand. Docking of FPR2 ligands into the active FPR2 cryoEM structure (PDBID: 7T6S) was performed using ICM pro software.Bias analysis revealed that WKYMVm and ACT‐389949 shared a very similar bias profile. In comparison, BMS‐986235 and compound 43 displayed approximately 5‐ to 50‐fold bias away from β‐arrestin recruitment and trafficking pathways, while being 35‐ to 60‐fold biased towards cAMP inhibition and pERK1/2. Molecular docking predicted key amino acid interactions at the FPR2 shared between WKYMVm and ACT‐389949, but not with BMS‐986235 and compound 43.In vitro characterisation demonstrated that WKYMVm and ACT‐389949 differ from BMS‐986235 and compound 43 in their signalling and protein coupling profile. This observation may be explained by differences in the ligand‐receptor interactions. In vitro characterisation provided significant insights into identifying the desired bias profile for FPR2‐based pharmacotherapy. [ABSTRACT FROM AUTHOR]

Increased protein provision might ameliorate muscle wasting and improve long-term outcomes in critically ill patients. The aim of the PRECISe trial was to assess whether higher enteral protein provision (ie, 2·0 g/kg per day) would improve health-related quality of life and functional outcomes in critically ill patients who were mechanically ventilated compared with standard enteral protein provision (ie, 1·3 g/kg per day). The PRECISe trial was an investigator-initiated, double-blinded, multicentre, parallel-group, randomised controlled trial in five Dutch hospitals and five Belgian hospitals. Inclusion criteria were initiation of invasive mechanical ventilation within 24 h of intensive care unit (ICU) admission and an expected duration of invasive ventilation of 3 days or longer. Exclusion criteria were contraindications for enteral nutrition, moribund condition, BMI less than 18 kg/m2, kidney failure with a no dialysis code, or hepatic encephalopathy. Patients were randomly assigned to one of four randomisation labels, corresponding with two study groups (ie, standard or high protein; two labels per group) in a 1:1:1:1 ratio through an interactive web-response system. Randomisation was done via random permuted-block randomisation in varying block sizes of eight and 12, stratified by centre. Participants, care providers, investigators, outcome assessors, data analysts, and the independent data safety monitoring board were all blinded to group allocation. Patients received isocaloric enteral feeds that contained 1·3 kcal/mL and 0·06 g of protein/mL (ie, standard protein) or 1·3 kcal/mL and 0·10 g of protein/mL (ie, high protein). The study-nutrition intervention was limited to the time period during the patient's ICU stay in which they required enteral feeding, with a maximum of 90 days. The primary outcome was EuroQoL 5-Dimension 5-level (EQ-5D-5L) health utility score at 30 days, 90 days, and 180 days after randomisation, adjusted for baseline EQ-5D-5L health utility score. This trial was registered with ClinicalTrials.gov (NCT04633421) and is closed to new participants. Between Nov 19, 2020, and April 14, 2023, 935 patients were randomly assigned. 335 (35·8%) of 935 patients were female and 600 (64·2%) were male. 465 (49·7%) of 935 were assigned to the standard protein group and 470 (50·3%) were assigned to the high protein group. 430 (92·5%) of 465 patients in the standard protein group and 419 (89·1%) of 470 patients in the high protein group were assessed for the primary outcome. The primary outcome, EQ-5D-5L health utility score during 180 days after randomisation (assessed at 30 days, 90 days, and 180 days), was lower in patients allocated to the high protein group than in those allocated to the standard protein group, with a mean difference of –0·05 (95% CI –0·10 to –0·01; p=0·031). Regarding safety outcomes, the probability of mortality during the entire follow-up was 0·38 (SE 0·02) in the standard protein group and 0·42 (0·02) in the high protein group (hazard ratio 1·14, 95% CI 0·92 to 1·40; p=0·22). There was a higher incidence of symptoms of gastrointestinal intolerance in patients in the high protein group (odds ratio 1·76, 95% CI 1·06 to 2·92; p=0·030). Incidence of other adverse events did not differ between groups. High enteral protein provision compared with standard enteral protein provision resulted in worse health-related quality of life in critically ill patients and did not improve functional outcomes during 180 days after ICU admission. Netherlands Organisation for Healthcare Research and Development and Belgian Health Care Knowledge Centre. [ABSTRACT FROM AUTHOR]

The molecular movement in single particle tracking (SPT) experiments shows a crucial role of diffusion in many biological processes such as signaling, cellular organization, transport mechanisms, and more. The SPT analysis detects not only classical Brownian motion but diffusion with other features. These include directed diffusion and confined motion. The behavior remains a challenging problem for several reasons. Due to the action of many physical processes, random trajectories of cellular molecules are segmented in different diffusive modes. Often their study requires sophisticated algorithms for the analysis of statistical properties. In this paper we consider the segment analysis for trajectories of G proteins and coupled-receptors in living cells. Their movement is often transient and switches among free diffusion, confined diffusion, directed diffusion, and immobility. Moreover, the confined segments can have both Gaussian and non-Gaussian statistics. The types of alternation of diffusive modes along the trajectories of G proteins and coupled-receptors are analyzed. [ABSTRACT FROM AUTHOR]

The effect of the diet modification (soybean and lupine addition) on the content of protein and amino acids (AA) in eggs was studied. Both the sampling day and the diet influenced the total protein content. In albumen, the lowest protein content (10.6%) was noted after administering a diet containing 25% lupine; in the same egg the yolk contained the most proteins (16.7%). In the content of nonessential AA (NAA) in egg yolks, differences were noted only for cysteine, with its the highest content in the yolks of the control group. The stable content of essential yolk amino acids (EAA) was observed only for isoleucine, leucine, tryptophan and phenylalanine. The highest contents of EAA and NAA were recorded in the yolks of the control group (~47 and ~53 g/100 g of protein, respectively) and in the group with 25% additions of lupine (~42 and ~51 g/100 g of protein, respectively). AA with constant content in the tested albumens were methionine, tryptophan and alanine. The highest content of EAA (>~42 g/100 g of protein) and NAA (>~62 g/100 g of protein) were determined in albumen of eggs determined in the group with at least 20% additions of lupine. The highest content of EAA for humans delivered eggs from groups 4–6 (with the addition of soy into the diet ≤5%). The protein sources used in the hen diet significantly influenced the content of protein and individual AA in the produced eggs. [ABSTRACT FROM AUTHOR]

In the investigation of heterotrimeric G protein‐mediated signal transduction in planta, their roles in the transmittance of low K+ stimuli remain to be elucidated. Here, we found that the primary root growth of wild‐type Arabidopsis was gradually inhibited with the decrease of external K+ concentrations, while the primary root of the mutants for G protein β subunit AGB1 and γ subunits AGG1, AGG2 and AGG3 could still grow under low K+ conditions (LK). Exogenous NAA application attenuated primary root elongation in agb1 and agg1/2/3 but promoted the growth in wild‐type seedlings under LK stress. Using ProDR5:GFP, ProPIN1:PIN1‐GFP and ProPIN2:PIN2‐GFP reporter lines, a diminishment in auxin concentration at the radicle apex and a reduction in PIN1and PIN2 efflux carrier abundance were observed in wild‐type roots under LK, a phenomenon not recorded in the agb1 and agg1/2/3. Further proteolytic and transcriptional assessments revealed an enhanced degradation of PIN1 and a suppressed expression of PIN2 in the wild‐type background under LK, contrasting with the stability observed in the agb1 and agg1/2/3 mutants. Our results indicate that the G protein β and γ subunits play pivotal roles in suppressing of Arabidopsis root growth under LK by modulating auxin redistribution via alterations in PIN1 degradation and PIN2 biosynthesis. [ABSTRACT FROM AUTHOR]

Nipah virus infection, one of the top priority diseases recognized by the World Health Organization, underscores the urgent need to develop effective countermeasures against potential epidemics and pandemics. Here, we identify a fully human single-domain antibody that targets a highly conserved cryptic epitope situated at the dimeric interface of the Nipah virus G protein (receptor binding protein, RBP), as elucidated through structures by high-resolution cryo-electron microscopy (cryo-EM). This unique binding mode disrupts the tetramerization of the G protein, consequently obstructing the activation of the F protein and inhibiting viral membrane fusion. Furthermore, our investigations reveal that this compact antibody displays enhanced permeability across the blood-brain barrier (BBB) and demonstrates superior efficacy in eliminating pseudovirus within the brain in a murine model of Nipah virus infection, particularly compared to the well-characterized antibody m102.4 in an IgG1 format. Consequently, this single-domain antibody holds promise as a therapeutic candidate to prevent Nipah virus infections and has potential implications for vaccine development. The fully human single-domain antibody n425 aginst NiV targets a conserved cryptic epitope on G protein and inhibits membrane fusion by disrupting tetramerization. These insights advance neutralization mechanisms and effective countermeasure design. [ABSTRACT FROM AUTHOR]

Different ligands stabilize specific conformations of the angiotensin II type 1 receptor (AT1R) that direct distinct signaling cascades mediated by heterotrimeric G proteins or β-arrestin. These different active conformations are thought to engage distinct intracellular transducers because of differential phosphorylation patterns in the receptor C-terminal tail (the "barcode" hypothesis). Here, we identified the AT1R barcodes for the endogenous agonist AngII, which stimulates both G protein activation and β-arrestin recruitment, and for a synthetic biased agonist that only stimulates β-arrestin recruitment. The endogenous and β-arrestin–biased agonists induced two different ensembles of phosphorylation sites along the C-terminal tail. The phosphorylation of eight serine and threonine residues in the proximal and middle portions of the tail was required for full β-arrestin functionality, whereas phosphorylation of the serine and threonine residues in the distal portion of the tail had little influence on β-arrestin function. Similarly, molecular dynamics simulations showed that the proximal and middle clusters of phosphorylated residues were critical for stable β-arrestin–receptor interactions. These findings demonstrate that ligands that stabilize different receptor conformations induce different phosphorylation clusters in the C-terminal tail as barcodes to evoke distinct receptor-transducer engagement, receptor trafficking, and signaling. Editor's summary: The specific patterns of phosphorylation in the intracellular tail of a G protein–coupled receptor (GPCR) are thought to act as a barcode that determines whether G proteins are stimulated or β-arrestins are recruited. Gareri et al. investigated the role of phosphorylation barcodes in signaling by the angiotensin II type 1 receptor (AT1R). AT1R elicits both G protein activation and β-arrestin recruitment in response to the endogenous agonist AngII but stimulates only β-arrestin recruitment in response to a synthetic biased agonist. The authors identified patterns of serine and threonine phosphorylation that stabilized specific conformations of β-arrestin and were necessary for β-arrestin function. These findings demonstrate the importance of phosphorylation barcodes in determining the consequences of AT1R activation. —Annalisa M. VanHook [ABSTRACT FROM AUTHOR]

Many neurotransmitter receptors activate G proteins through exchange of GDP for GTP. The intermediate nucleotide-free state has eluded characterization, due largely to its inherent instability. Here we characterize a G protein variant associated with a rare neurological disorder in humans. GαoK46E has a charge reversal that clashes with the phosphate groups of GDP and GTP. As anticipated, the purified protein binds poorly to guanine nucleotides yet retains wild-type affinity for G protein βγ subunits. In cells with physiological concentrations of nucleotide, GαoK46E forms a stable complex with receptors and Gβγ, impeding effector activation. Further, we demonstrate that the mutant can be easily purified in complex with dopamine-bound D2 receptors, and use cryo-electron microscopy to determine the structure, including both domains of Gαo, without nucleotide or stabilizing nanobodies. These findings reveal the molecular basis for the first committed step of G protein activation, establish a mechanistic basis for a neurological disorder, provide a simplified strategy to determine receptor-G protein structures, and a method to detect high affinity agonist binding in cells. Many neurotransmitters act on receptors coupled to GTP-binding G proteins. Here authors report the structure and activity of a mutant that locks the nucleotide-free and receptor-bound state of the G protein, leading to a rare neurological disorder. [ABSTRACT FROM AUTHOR]

Aims The Gα subunit is a major component of heterotrimeric G proteins, which play a crucial role in the development and pathogenicity of several model fungi. However, its detailed function in the causal agent of pear black spot (Alternaria alternata) is unclear. Our aim was to understand the characteristics and functions of AaGA1 in A. alternata. Methods and results AaGA1 was cloned from A. alternata in this study, which encodes 353 amino acids and has a "G-alpha" domain. Mutant ΔAaGA1 resulted in reduced vegetative growth, conidiation, and spore germination. Especially, mutant ΔAaGA1 produced only fewer conidia on the V8A medium, and spore formation-related genes AbaA, BrlA , and WetA were significantly downregulated. More tolerance against cell wall-inhibiting agents was observed after the deletion of AaGA1. Moreover, AaGA1 deletion led to a significant reduction in melanin and toxin production. Interestingly, deletion of AaGA1 resulted in defective appressorium-like formations, complete loss of the ability to penetrate cellophane, and decreased infection on non-wound inoculated tobacco leaves. Cell wall-degrading enzyme-related genes PME, CL, Cut2 , and LC were significantly downregulated in mutant ΔAaGA1 mutant, significantly reducing virulence on wound-inoculated pear fruits. Conclusions The G protein alpha subunit AaGA1 is indispensable for fungal development, appressorium-like formations, and pathogenicity in A. alternata. [ABSTRACT FROM AUTHOR]

Activation of G proteins through nucleotide exchange initiates intracellular signaling cascades essential for life processes. Under normal conditions, nucleotide exchange is regulated by the formation of G protein–G protein–coupled receptor complexes. Single point mutations in the Gα subunit of G proteins bypass this interaction, leading to loss of function or constitutive gain of function, which is closely linked with the onset of multiple diseases. Despite the recognized significance of Gα mutations in disease pathology, structural information for most variants is lacking, potentially due to inherent protein dynamics that pose challenges for crystallography. To address this, we leveraged an integrative spectroscopic and computational approach to structurally characterize seven of the most frequently observed and clinically relevant mutations in the stimulatory Gα subunit, GαS. A previously proposed allosteric model of Gα activation linked structural changes in the nucleotide-binding pocket with functionally important changes in interactions between switch regions. We investigated this allosteric connection in GαS by integrating data from variable temperature CD spectroscopy, which measured changes in global protein structure and stability, and molecular dynamics simulations, which observed changes in interaction networks between GαS switch regions. Additionally, saturation-transfer difference NMR spectroscopy was applied to observe changes in nucleotide interactions with residues within the nucleotide binding site. These data have enabled testing of predictions regarding how mutations in GαS result in loss or gain of function and evaluation of proposed structural mechanisms. The integration of experimental and computational data allowed us to propose a more nuanced classification of mechanisms underlying GαS gain-of-function and loss-of-function mutations. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum (P. falciparum), which causes the most severe form of malaria, if left untreated, has 24 h window in which it can cause severe illness and even death. The aim of this study was to create the most comprehensive and informative secretory-proteome possible by combining high-accuracy and high-sensitivity protein identification technology. In this study, we used Plasmodium falciparum 3D7 (Pf3D7) as the model parasite to develop a label-free quantification proteomic strategy with the main goal of identifying Pf3D7 proteins that are supposed to be secreted outside the infected erythrocytes in the spent media culture during the in-vitro study. The spent culture media supernatant was subjected to differential and ultra-centrifugation steps followed by total protein extraction, estimation, and in-solution digestion using trypsin, digested peptides were analyzed using Nano-LC coupled with ESI for MS/MS. MS/MS spectra were processed using Maxquant software (v2.1.4.0.). Non-infected erythrocytes incubated spent cultured media supernatant were considered as control. Out of discovered 38 proteins, proteins belonging to P. falciparum spp. were EGF-like protein (C0H544), Endoplasmic reticulum chaperone GRP170 (C0H5H0), Small GTP-binding protein sar1 (Q8I1S0), Erythrocyte membrane protein 1, PfEMP1 (Q8I639), aldehyde reductase (Q8ID61), Conserved Plasmodium proteins (Q8IEH3, Q8ILD1), Antigen 332, DBL-like protein (Q8IHN4), Fe-S cluster assembly protein (Q8II78), identified and chosen for further in-depth investigation. This study highlights the value of secretory Plasmodium proteins play crucial roles in various aspects of the disease progression and host-pathogen interactions which can serve as diagnostic markers for malaria infection. [ABSTRACT FROM AUTHOR]

Fermented chopped chili pepper (FCCP) is the most important fermented and processed chili pepper product, and most pepper varieties can be processed with obvious flavor differences. Many studies have tried to explain these differences using the results of component analysis, processing technology, the microbes involved, etc., but few have examined the characteristics of these varieties along with the flavor of products. We conducted a physiochemical characteristic analysis of 35 kinds of fresh chili peppers with different genotypes and made fermented products from 17 varieties, using principal component analysis (PCA) and cluster analysis (CA) to correlate their components with FCCP products. The results were as follows: (1) Protein, total acids, a* (representation of the object's red and green color), protopectin, and fruit thickness are the five key indexes that affect the quality of fresh chili peppers the most. (2) Protein, total acids, a*, protopectin, and fruit thickness are also the key indexes that affect the quality of FCCP. (3) Suitable ranges of fresh chili pepper to manufacture FCCP are 1.3–2.0 g/100 g for protein, 2.5–4.0 g/kg for total acids, 10–15 g/100 g for protopectin, 30–39 for a*, and 1.2–2.0 mm for fruit thickness. (4) Sensory appraisal shows crispness is the most important mouthfeel sensation and can decrease during the process. The varieties with the top three scores were A12 (21G675), A13 (20Z663), and A14 (21Z698) with scores of 87.92, 74.08, and 74.15, respectively. The varieties in the top three scores are pod peppers. The PCA and CA showed that pod peppers were the most suitable materials for making FCCP. Our results will benefit others in selecting fresh chili peppers for making FCCP and provide data support for directing processable variety breeding. [ABSTRACT FROM AUTHOR]

The blood-brain barrier (BBB) acquires unique properties to regulate neuronal function during development. The formation of the BBB, which occurs in tandem with angiogenesis, is directed by the Wnt/β-catenin signaling pathway. Yet the exact molecular interplay remains elusive. Our study reveals the G protein--coupled receptor GPR126 as a critical target of canonical Wnt signaling, essential for the development of the BBB's distinctive vascular characteristics and its functional integrity. Endothelial cell--specific deletion of the Gpr126 gene in mice induced aberrant vascular morphogenesis, resulting in disrupted BBB organization. Simultaneously, heightened transcytosis in vitro compromised barrier integrity, resulting in enhanced vascular permeability. Mechanistically, GPR126 enhanced endothelial cell migration, pivotal for angiogenesis, acting through an interaction between LRP1 and β1 integrin, thereby balancing the levels of β1 integrin activation and recycling. Overall, we identified GPR126 as a specifier of an organotypic vascular structure, which sustained angiogenesis and guaranteed the acquisition of the BBB properties during development. [ABSTRACT FROM AUTHOR]

GNAO1 mutated in pediatric encephalopathies encodes the major neuronal G protein Gαo. Of the more than 80 pathogenic mutations, most are single amino acid substitutions spreading across the Gαo sequence. We performed extensive characterization of Gαo mutants, showing abnormal GTP uptake and hydrolysis and deficiencies in binding Gβγ and RGS19. Plasma membrane localization of Gαo was decreased for a subset of mutations that leads to epilepsy; dominant interactions with GPCRs also emerged for the more severe mutants. Pathogenic mutants massively gained interaction with Ric8A and, surprisingly, Ric8B proteins, relocalizing them from cytoplasm to Golgi. Of these 2 mandatory Gα-subunit chaperones, Ric8A is normally responsible for the Gαi/Gαo, Gαq, and Gα12/Gα13 subfamilies, and Ric8B solely responsible for Gαs/Gαolf. Ric8 mediates the disease dominance when engaging in neomorphic interactions with pathogenic Gαo through imbalance of the neuronal G protein signaling networks. As the strength of Gαo-Ric8B interactions correlates with disease severity, our study further identifies an efficient biomarker and predictor for clinical manifestations in GNAO1 encephalopathies. Our work uncovers the neomorphic molecular mechanism of mutations underlying pediatric encephalopathies and offers insights into other maladies caused by G protein malfunctioning and further genetic diseases. [ABSTRACT FROM AUTHOR]

The accumulation of chromium in renal tissues promotes the generation of reactive oxygen species (ROS), leading to oxidative stress, genomic and cellular harm, and ultimately necrotic and apoptotic cell death induced by free radicals. Hence, the utilization of antioxidant phytochemicals becomes crucial for cellular defense against oxidative damage. This study endeavors to explore the potential protective effects of an aqueous avocado seed extract (ASE) on rabbit kidneys exposed to chromium‐induced damage. Fifteen adult rabbits were distributed into three groups: Group 1 was kept as the control. The second and third groups received a daily dose of K2Cr2O7 (5 mg/kg) intraperitoneally for 2 weeks. While the third group was given an oral dose of ASE (400 mg/kg). In rabbits administered with Cr (VI), kidney homogenates showed a marked increase in Malondialdehyde (MDA) (69.3 ± 4.1 nmol/g) along with a decrease in glutathione (59 ± 5.8 nmol/mg) content and the activity superoxide dismutase (SOD) (0.5 ± 0.05 U/mg protein), glutathione peroxidase (GPx) (16.7 ± 1.1 μmol/mg protein), and catalase (CAT) (73.8 ± 3.9 U/g protein) compared to the levels in control group. Also, the gene expression data for the enzymes SOD, GPx, and CAT dropped dramatically in kidney tissue following Cr (VI) injection. Additionally, Bowman's capsule and glomerulus showed degenerative alterations in the kidney's histopathology and immunohistochemistry. ASE treatment when administered along with Cr (VI) enhanced the activity and gene expression of antioxidant enzymes and improved histopathological conditions. The findings of this study unequivocally show that avocado seed extract, which is rich in phenolic derivatives, is a potent nephroprotective agent that inhibits nephrotoxicity induced by Cr (VI) in rabbits. [ABSTRACT FROM AUTHOR]

Background and Purpose: Pancreatic islets are modulated by cross‐talk among different cell types and paracrine signalling plays important roles in maintaining glucose homeostasis. Urocortin 3 (UCN3) secreted by pancreatic β cells activates the CRF2 receptor (CRF2R) and downstream pathways mediated by different G protein or arrestin subtypes in δ cells to cause somatostatin (SST) secretion, and constitutes an important feedback circuit for glucose homeostasis. Experimental Approach: Here, we used Arrb1−/−, Arrb2−/−, Gsfl/fl and Gqfl/fl knockout mice, the G11‐shRNA‐GFPfl/fl lentivirus, as well as functional assays and pharmacological characterization to study how the coupling of Gs, G11 and β‐arrestin1 to CRF2R contributed to UCN3‐induced SST secretion in pancreatic δ cells. Key Results: Our study showed that CRF2R coupled to a panel of G protein and arrestin subtypes in response to UCN3 engagement. While RyR3 phosphorylation by PKA at the S156, S2706 and S4697 sites may underlie the Gs‐mediated UCN3‐ CRF2R axis for SST secretion, the interaction of SYT1 with β‐arrestin1 is also essential for efficient SST secretion downstream of CRF2R. The specific expression of the transcription factor Stat6 may contribute to G11 expression in pancreatic δ cells. Furthermore, we found that different UCN3 concentrations may have distinct effects on glucose homeostasis, and these effects may depend on different CRF2R downstream effectors. Conclusions and Implications: Collectively, our results provide a landscape view of signalling mediated by different G protein or arrestin subtypes downstream of paracrine UCN3‐ CRF2R signalling in pancreatic β‐δ‐cell circuits, which may facilitate the understanding of fine‐tuned glucose homeostasis networks. [ABSTRACT FROM AUTHOR]

Copyright of Mycosystema is the property of Mycosystema Editorial Board and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

To further promote the pretreatment technique, the immunoaffinity column (IAC), immunoaffinity magnetic (IAM) and multi-component immunoaffinity magnetic (multi-IAM) for mycotoxins were developed and evaluated systematically. Anti-mycotoxin-McAbs had immobilized on the CNBr-Sepharose 4B, carboxyl magnetic bead and protein G magnetic bead to prepare the IAC/IAM/multi-IAM, respectively. The capture efficiencies of mycotoxins (100 ng) reached 92.8% for IAC and exceeded 94.0% for IAM/multi-IAM. The multi-IAM showed more beneficial features in multiple targets, pretreatment time and recycling time than IAC/IAM, which could simultaneously pretreat AFB1/DON/ZEN in 15 min with three recycling times. The recoveries of IAC/IAM/multi-IAM coupled with ELISA ranged from 85.2 to 105.1%, with RSD between 4.0 and 13.2%. Moreover, the mycotoxin-positive authentic samples were detected from 1.1 to 749.6 ng/g by ELISA, and 1.2 to 762.7 ng/g by LC-MS/MS, with correlated R2 exceeding 0.9955. The proposed IAC/IAM/multi-IAM owned the desired performance, such as specificity, accuracy, simpleness, efficiency, cost-effectiveness, simultaneity and environmentally friendly. This study can provide the theoretical basis for three immunoaffinity pretreatment techniques systematically, which may guide their application under different requirements and scenarios. [ABSTRACT FROM AUTHOR]

Sodium fluoride (NaF) is a fluoride application recommended by the World Health Organization for its efficacy and safety in preventing dental caries. Gingival fibroblasts that constitute the majority of connective tissue cells play a major role in wound healing via the expression of growth factors, including fibroblast growth factor-2 (FGF-2) and transforming growth factor-beta (TGF-β). This study examined the effect of NaF mouthwash on FGF-2 and TGF-β expression in human gingival fibroblasts (HGnFs). Fibroblasts were exposed to a medium with 225 ppmF NaF for 1 min, then switched to either 15 ppmF NaF for continuous stimulation or no NaF for transient stimulation. Continuous NaF stimulation significantly increased the gene and protein expression of FGF-2 and TGF-β in HGnFs compared to controls, suggesting NaF's potential role in modulating periodontal tissue wound healing. Signaling pathway investigations showed the involvement of heterotrimeric GTP-binding proteins, calcium/calmodulin-dependent kinase II (CaMKII), and extracellular signal-regulated kinase (ERK) phosphorylation. Inhibiting CaMKII reduced NaF-induced FGF-2 and TGF-β expression, while ERK phosphorylation increased after NaF stimulation. These results highlight NaF mouthwash's potential in promoting wound healing in extraction sockets, particularly during the mixed dentition period. Understanding NaF's effects is clinically relevant due to the common use of fluoride products. [ABSTRACT FROM AUTHOR]

Development of optimal therapeutics for disease states that can be associated with increased membrane cholesterol requires better molecular understanding of lipid modulation of the drug target. Type 1 cholecystokinin receptor (CCK1R) agonist actions are affected by increased membrane cholesterol, enhancing ligand binding and reducing calcium signaling, while agonist actions of the closely related CCK2R are not. In this work, we identified a set of chimeric human CCK1R/CCK2R mutations that exchange the cholesterol sensitivity of these 2 receptors, providing powerful tools when expressed in CHO and HEK-293 model cell lines to explore mechanisms. Static, low energy, high-resolution structures of the mutant CCK1R constructs, stabilized in complex with G protein, were not substantially different, suggesting that alterations to receptor dynamics were key to altered function. We reveal that cholesterol-dependent dynamic changes in the conformation of the helical bundle of CCK receptors affects both ligand binding at the extracellular surface and G protein coupling at the cytosolic surface, as well as their interrelationships involved in stimulus-response coupling. This provides an ideal setting for potential allosteric modulators to correct the negative impact of membrane cholesterol on CCK1R. A previous study in PLOS Biology solved the cryo-EM structure of the cholecystokinin type 1 receptor (CCK1R) to gain insights into the mechanisms governing G protein selectivity. This Update Article provides a structural and biophysical characterization of the effects of cholesterol on ligand binding and G protein coupling at the receptor. [ABSTRACT FROM AUTHOR]

Background: The plant root system is critical for the absorption of water and nutrients, and have a direct influence on growth and yield. In cucumber, a globally consumed crop, the molecular mechanism of root development remains unclear, and this has implications for developing stress tolerant varieties. This study sought to determine the genetic patterns and related genes of cucumber root weight. A core cucumber germplasms population was used to do the GWAS analysis in three environments. Results: Here, we investigated four root-weight related traits including root fresh weight (RFW), root dry weight (RDW), ratio of root dry weight to root fresh weight (RDFW) and the comprehensive evaluation index, D-value of root weight (DRW) deduced based on the above three traits for the core germplasm of the cucumber global repository. According to the D-value, we identified 21 and 16 accessions with light and heavy-root, respectively. We also found that the East Asian ecotype accessions had significantly heavier root than other three ecotypes. The genome-wide association study (GWAS) for these four traits reveals that 4 of 10 significant loci (gDRW3.1, gDRW3.2, gDRW4.1 and gDRW5.1) were repeatedly detected for at least two traits. Further haplotype and expression analysis for protein-coding genes positioned within these 4 loci between light and heavy-root accessions predicted five candidate genes (i.e., Csa3G132020 and Csa3G132520 both encoding F-box protein PP2-B1 for gDRW3.1, Csa3G629240 encoding a B-cell receptor-associated protein for gDRW3.2, Csa4G499330 encodes a GTP binding protein for gDRW4.1, and Csa5G286040 encodes a proteinase inhibitor for gDRW5.1). Conclusions: We conducted a systematic analysis of the root genetic basis and characteristics of cucumber core germplasms population. We detected four novel loci, which regulate the root weight in cucumber. Our study provides valuable candidate genes and haplotypes for the improvement of root system in cucumber breeding. [ABSTRACT FROM AUTHOR]

Machine learning (ML) has transformed protein engineering by constructing models of the underlying sequence-function landscape to accelerate the discovery of new biomolecules. ML-guided protein design requires models, trained on local sequence-function information, to accurately predict distant fitness peaks. In this work, we evaluate neural networks' capacity to extrapolate beyond their training data. We perform model-guided design using a panel of neural network architectures trained on protein G (GB1)-Immunoglobulin G (IgG) binding data and experimentally test thousands of GB1 designs to systematically evaluate the models' extrapolation. We find each model architecture infers markedly different landscapes from the same data, which give rise to unique design preferences. We find simpler models excel in local extrapolation to design high fitness proteins, while more sophisticated convolutional models can venture deep into sequence space to design proteins that fold but are no longer functional. We also find that implementing a simple ensemble of convolutional neural networks enables robust design of high-performing variants in the local landscape. Our findings highlight how each architecture's inductive biases prime them to learn different aspects of the protein fitness landscape and how a simple ensembling approach makes protein engineering more robust. Machine learning accelerates protein engineering by predicting sequence-function relationships. Here, authors evaluate neural network architectures' ability to extrapolate beyond training data, finding simpler models excel in local design while convolutional models explore deeper sequence spaces. [ABSTRACT FROM AUTHOR]

Conjugation to a carrier protein is essential to give rise to the antigenicity of hapten. Three carrier proteins e.g. KLH (Keyhole Limpet hemocyanin), BSA (bovine serum albumin), and OVA (Ovalbumin) were used mostly. KLH is advantageous to the others, majorly owing to its strong immunogenicity and limited usage in other biological assays. However, the cost of obtaining Keyhole Limpet is high and the solubility of KLH is not as well as the other carriers, especially after hapten conjugation. Here, we extracted the shrimp hemocyanin (SHC) from Litopenaeus vannamei (L. vannamei), which is a commonly sea product worldwide. The high pure SHC could be acquired by two-step purification, with a production yield of > 1 g proteins (98% pure) per 1 kg shrimp. Compared to KLH, the peptide-SHC conjugates exhibit higher solubility after hapten conjugation. Meanwhile, compared with KLH, SHC induces comparable antibody production efficiency in mammals, with or without conjugation. Furthermore, rabbit polyclonal antibodies or mouse monoclonal antibodies were generated by immunizing SHC-peptide conjugates, and the subsequent antibodies were confirmed to be used in western blot, immunofluorescence and immunohistochemistry. Therefore, we demonstrated that SHC may be used as a substitute for KLH in future antibody and vaccine development. [ABSTRACT FROM AUTHOR]

Atypical Chemokine Receptor 3 (ACKR3) belongs to the G protein-coupled receptor family but it does not signal through G proteins. The structural properties that govern the functional selectivity and the conformational dynamics of ACKR3 activation are poorly understood. Here, we combined hydrogen/deuterium exchange mass spectrometry, site-directed mutagenesis, and molecular dynamics simulations to examine the binding mode and mechanism of action of ACKR3 ligands of different efficacies. Our results show that activation or inhibition of ACKR3 is governed by intracellular conformational changes of helix 6, intracellular loop 2, and helix 7, while the DRY motif becomes protected during both processes. Moreover, we identified the binding sites and the allosteric modulation of ACKR3 upon β-arrestin 1 binding. In summary, this study highlights the structure-function relationship of small ligands, the binding mode of β-arrestin 1, the activation dynamics, and the atypical dynamic features in ACKR3 that may contribute to its inability to activate G proteins. [ABSTRACT FROM AUTHOR]

Background and Purpose Experimental Approach Key Results Conclusions and Implications Allosterism is a regulatory mechanism for GPCRs that can be attained by ligand‐binding or protein–protein interactions with another GPCR. We have studied the influence of the dimer interface on the allosteric properties of the A2A receptor and CB2 receptor heteromer.We have evaluated cAMP production, phosphorylation of signal‐regulated kinases (pERK1/2), label‐free dynamic mass redistribution, β‐arrestin 2 recruitment and bimolecular fluorescence complementation assays in the absence and presence of synthetic peptides that disrupt the formation of the heteromer. Molecular dynamic simulations provided converging evidence that the heteromeric interface influences the allosteric properties of the A2AR–CB2R heteromer.Apo A2AR blocks agonist‐induced signalling of CB2R. The disruptive peptides, with the amino acid sequence of transmembrane (TM) 6 of A2AR or CB2R, facilitate CB2R activation, suggesting that A2AR allosterically prevents the outward movement of TM 6 of CB2R for G protein binding. Significantly, binding of the selective antagonist SCH 58261 to A2AR also facilitated agonist‐induced activation of CB2R.It is proposed that the A2AR–CB2R heteromer contains distinct dimerization interfaces that govern its functional properties. The molecular interface between protomers of the A2AR–CB2R heteromer interconverted from TM 6 for apo or agonist‐bound A2AR, blocking CB2R activation, to mainly the TM 1/7 interface for antagonist‐bound A2AR, facilitating the independent opening of intracellular cavities for G protein binding. These novel results shed light on a different type of allosteric mechanism and extend the repertoire of GPCR heteromer signalling. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) control intracellular signaling cascades via agonist-dependent coupling to intracellular transducers including heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. In addition to their critical interactions with the transmembrane core of active GPCRs, all three classes of transducers have also been reported to interact with receptor C-terminal domains (CTDs). An underexplored aspect of GPCR CTDs is their possible role as lipid sensors given their proximity to the membrane. CTD-membrane interactions have the potential to control the accessibility of key regulatory CTD residues to downstream effectors and transducers. Here, we report that the CTDs of two closely related family C GPCRs, metabotropic glutamate receptor 2 (mGluR2) and mGluR3, bind to membranes and that this interaction can regulate receptor function. We first characterize CTD structure with NMR spectroscopy, revealing lipid composition-dependent modes of membrane binding. Using molecular dynamics simulations and structure-guided mutagenesis, we then identify key conserved residues and cancer-associated mutations that modulate CTD-membrane binding. Finally, we provide evidence that mGluR3 transducer coupling is controlled by CTD-membrane interactions in live cells, which may be subject to regulation by CTD phosphorylation and changes in membrane composition. This work reveals an additional mechanism of GPCR modulation, suggesting that CTD-membrane binding may be a general regulatory mode throughout the broad GPCR superfamily. [ABSTRACT FROM AUTHOR]

Certain amino acid sites of 5‐HT2AR play crucial roles in interacting with various G proteins. Hallucinogens and non‐hallucinogens both act on 5‐HT2AR but mediate different pharmacological effects, possibly due to the coupling of different G proteins. Therefore, this study identified the binding sites of hallucinogens and non‐hallucinogens with 5‐HT2AR through molecular docking. We conducted site mutation to examine the impact of these sites on G proteins, in order to find out the sites that can distinguish the pharmacological effects of hallucinogens and non‐hallucinogens. Our results indicate that I4.60A and S3.39A did not affect the ability of hallucinogens to activate Gq signaling, but significantly reduced Gs signaling activation by hallucinogens. These results suggest that S3.39 and I4.60 are important for the activation of Gs signaling by hallucinogens. [ABSTRACT FROM AUTHOR]

Wool is generated by hair follicles (HFs), which are crucial in defining the length, diameter, and morphology of wool fibers. However, the regulatory mechanism of HF growth and development remains largely unknown. Dermal papilla cells (DPCs) are a specialized cell type within HFs that play a crucial role in governing the growth and development of HFs. This study aims to investigate the proliferation and induction ability of ovine DPCs to enhance our understanding of the potential regulatory mechanisms underlying ovine HF growth and development. Previous research has demonstrated that microRNA-181a (miR-181a) was differentially expressed in skin tissues with different wool phenotypes, which indicated that miR-181a might play a crucial role in wool morphogenesis. In this study, we revealed that miR-181a inhibited the proliferation and induction ability of ovine DPCs by quantitative Real-time PCR (qRT-PCR), cell counting Kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU), flow cytometry, and alkaline phosphatase staining. Then, we also confirmed G protein subunit alpha i2 (GNAI2) is a target gene of miR-181a by dual luciferase reporter assay, qRT-PCR, and Western blot, and that it could promote the proliferation and induction ability of ovine DPCs. In addition, GNAI2 could also activate the Wnt/β-Catenin signaling pathway in ovine DPCs. This study showed that miR-181a can inhibit the proliferation and induction ability of ovine DPCs by targeting GNAI2 through the Wnt/β-Catenin signaling pathway. [ABSTRACT FROM AUTHOR]

Autotaxin (ATX) is a member of the ectonucleotide pyrophosphate/phosphodiesterase (ENPP) family; it is encoded by the ENPP2 gene. ATX is a secreted glycoprotein and catalyzes the hydrolysis of lysophosphatidylcholine to lysophosphatidic acid (LPA). LPA is responsible for the transduction of various signal pathways through the interaction with at least six G protein-coupled receptors, LPA Receptors 1 to 6 (LPAR1–6). The ATX–LPA axis is involved in various physiological and pathological processes, such as angiogenesis, embryonic development, inflammation, fibrosis, and obesity. However, significant research also reported its connection to carcinogenesis, immune escape, metastasis, tumor microenvironment, cancer stem cells, and therapeutic resistance. Moreover, several studies suggested ATX and LPA as relevant biomarkers and/or therapeutic targets. In this review of the literature, we aimed to deepen knowledge about the role of the ATX–LPA axis as a promoter of cancer development, progression and invasion, and therapeutic resistance. Finally, we explored its potential application as a prognostic/predictive biomarker and therapeutic target for tumor treatment. [ABSTRACT FROM AUTHOR]

Background and Purpose: Receptor activity‐modifying proteins (RAMPs) and melanocortin receptor accessory proteins (MRAPs) modulate expression and signalling of calcitonin and melanocortin GPCRs. Interactions with other GPCRs have also been reported. The cannabinoid receptors, CB1 and CB2, and two putative cannabinoid receptors, GPR18 and GPR55, exhibit substantial intracellular expression and there are discrepancies in ligand responsiveness between studies. We investigated whether interactions with RAMPs or MRAPs could explain these phenomena. Experimental Approach: Receptors and accessory proteins were co‐expressed in HEK‐293 cells. Selected receptors were studied at basal expression levels and also with enhanced expression produced by incorporation of a preprolactin signal sequence/peptide (pplss). Cell surface and total expression of receptors and accessory proteins were quantified using immunocytochemistry. Signalling was measured using cAMP (CAMYEL) and G protein dissociation (TRUPATH Gα13) biosensors. Key Results: MRAP2 enhanced surface and total expression of GPR18. Pplss‐GPR18 increased detection of cell surface MRAP2. MRAP1α and MRAP2 reduced GPR55 surface and total expression, correlating with reduced constitutive, but not agonist‐induced, signalling. GPR55, pplss‐CB1 and CB2 reduced detection of MRAP1α at the cell surface. Pplss‐CB1 agonist potency was reduced by MRAP2 in Gα13 but not cAMP assays, consistent with MRAP2 reducing pplss‐CB1 expression. Some cannabinoid receptors increased RAMP2 or RAMP3 total expression without influencing surface expression. Conclusions and Implications: Mutual influences on expression and/or function for specific accessory protein‐receptor pairings raises the strong potential for physiological and disease‐relevant consequences. Sequestration and/or hetero‐oligomerisation of cannabinoid receptors with accessory proteins is a possible novel mechanism for receptor crosstalk. LINKED ARTICLES: This article is part of a themed issue Therapeutic Targeting of G Protein‐Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetoc [ABSTRACT FROM AUTHOR]

G protein‐coupled receptors (GPCRs) are the most prominent receptors on the surface of the cell and play a central role in the regulation of cardiac and metabolic functions. GPCRs transmit extracellular stimuli to the interior of the cells by activating one or more heterotrimeric G proteins. The duration and intensity of G protein‐mediated signalling are tightly controlled by a large array of intracellular mediators, including the regulator of G protein signalling (RGS) proteins. RGS proteins selectively promote the GTPase activity of a subset of Gα subunits, thus serving as negative regulators in a pathway‐dependent manner. In the current review, we summarise the involvement of RGS proteins in cardiometabolic function with a focus on their tissue distribution, mechanisms of action and dysregulation under various disease conditions. We also discuss the potential therapeutic applications for targeting RGS proteins in treating cardiometabolic conditions and current progress in developing RGS modulators. LINKED ARTICLES: This article is part of a themed issue Therapeutic Targeting of G Protein‐Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetoc [ABSTRACT FROM AUTHOR]

Background and Purpose: Affinity‐based, selective orthosteric ligands for the muscarinic acetylcholine receptors (mAChRs) are difficult to develop due to high sequence homology across the five subtypes. Selectivity can also be achieved via the selective activation of a particular subtype or signalling pathway. Promisingly, a prior study identified compounds 6A and 7A as functionally selective and Gi biased compounds at the M2 mAChR. Here, we have investigated the activation of individual G protein subfamilies and the downstream signalling profiles of 6A and 7A at the M2 mAChR. Experimental Approach: G protein activation was measured with the TRUPATH assay in M2 mAChR FlpIn CHO cells. Activity in downstream signalling pathways was determined using the cAMP CAMYEL BRET sensor and assay of ERK 1/2 phosphorylation. Key Results: M2 mAChRs coupled to Gɑi1, GɑoA and Gɑs, but not Gɑq, in response to canonical orthosteric agonists. Compounds 6A and 7A did not elicit any G protein activation, cAMP inhibition or stimulation, or ERK 1/2 phosphorylation. Instead, a Schild analysis indicates a competitive, antagonistic interaction of compounds 6A and 7A with ACh in the Gɑi1 activation assay. Overexpression of the M2 mAChR may suggest an expression‐dependent activation profile of compounds 6A and 7A. Conclusions and Implications: These data confirm that the M2 mAChR preferentially couples to Gɑi/o and to a lesser extent to Gɑs in response to canonical orthosteric ligands. However, this study was not able to detect Gɑi bias of compounds 6A and 7A, highlighting the importance of cellular background when classifying new ligands. LINKED ARTICLES: This article is part of a themed issue Therapeutic Targeting of G Protein‐Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetoc [ABSTRACT FROM AUTHOR]

Rho of Plants (ROPs) constitute a plant-specific subset of small guanine nucleotide-binding proteins within the Cdc42/Rho/Rac family. These versatile proteins regulate diverse cellular processes, including cell growth, cell division, cell morphogenesis, organ development, and stress responses. In recent years, the dynamic cellular and subcellular behaviours orchestrated by ROPs have unveiled a notable connection to hormone-mediated organ development and physiological responses, thereby expanding our knowledge of the functions and regulatory mechanisms of this signalling pathway. This review delineates advancements in understanding the interplay between plant hormones and the ROP signalling cascade, focusing primarily on the connections with auxin and abscisic acid pathways, alongside preliminary discoveries in cytokinin, brassinosteroid, and salicylic acid responses. It endeavours to shed light on the intricate, coordinated mechanisms bridging cell- and tissue-level signals that underlie plant cell behaviour, organ development, and physiological processes, and highlights future research prospects and challenges in this rapidly developing field. [ABSTRACT FROM AUTHOR]

Classically, G protein-coupled receptors (GPCRs) promote signaling at the plasma membrane through activation of heterotrimeric Gαβγ proteins, followed by the recruitment of GPCR kinases and βarrestin (βarr) to initiate receptor desensitization and internalization. However, studies demonstrated that some GPCRs continue to signal from internalized compartments, with distinct cellular responses. Both βarr and Gβγ contribute to such non-canonical endosomal G protein signaling, but their specific roles and contributions remain poorly understood. Here, we demonstrate that the vasopressin V2 receptor (V2R)–βarr complex scaffolds Gβγ at the plasma membrane through a direct interaction with βarr, enabling its transport to endosomes. Gβγ subsequently potentiates Gαs endosomal translocation, presumably to regenerate an endosomal pool of heterotrimeric Gs. This work shines light on the mechanism underlying G protein subunits translocation from the plasma membrane to the endosomes and provides a basis for understanding the role of βarr in mediating sustained G protein signaling. A V2R–βarrestin–Gβγ complex forms at the plasma membrane and translocates to endosomes. This sheds light on G protein subunit translocation and βarr's role in sustained signaling. [ABSTRACT FROM AUTHOR]

Melanogenesis‐stimulated B16‐F10 cells enter in a quiescent state, present inhibited mitochondrial respiration and increased reactive oxygen species levels. These alterations suggest that these cells may be under redox signaling, allowing tumor survival. The aim of this study was to evaluate redox‐modified proteins in B16‐F10 cells after melanogenesis stimulation and rose bengal‐photodynamic therapy (RB‐PDT). A redox proteomics label‐free approach based on the biotin switch assay technique with biotin‐HPDP and N‐ethylmaleimide was used to assess the thiol‐oxidized protein profile. Aconitase was oxidized at Cys‐448 and Cys‐451, citrate synthase was oxidized at Cys‐202 and aspartate aminotransferase (Got2) was oxidized at Cys‐272 and Cys‐274, exclusively after melanogenesis stimulation. After RB‐PDT, only guanine nucleotide‐binding protein subunit beta‐2‐like 1 (Gnb2l1) was oxidized (Cys‐168). In contrast, melanogenesis stimulation followed by RB‐PDT led to the oxidation of different cysteines in Gnb2l1 (Cys‐153 and Cys‐249). Besides that, glyceraldehyde‐3‐phosphate dehydrogenase (Gapdh) presented oxidation at Cys‐245, peptidyl‐prolyl cis‐trans isomerase A (Ppia) was oxidized at Cys‐161 and 5,6‐dihydroxyindole‐2‐carboxylic acid oxidase (Tyrp1) was oxidized at Cys‐65, Cys‐30, and Cys‐336 after melanogenesis stimulation followed by RB‐PDT. The redox alterations observed in murine melanoma cells and identification of possible target proteins are of great importance to further understand tumor resistance mechanisms. [ABSTRACT FROM AUTHOR]

Memory processes rely on a molecular signaling system that balances the interplay between positive and negative modulators. Recent research has focused on identifying memory-regulating genes and their mechanisms. Phospholipase C beta 1 (PLCβ1), highly expressed in the hippocampus, reportedly serves as a convergence point for signal transduction through G protein-coupled receptors. However, the detailed role of PLCβ1 in memory function has not been elucidated. Here, we demonstrate that PLCβ1 in the dentate gyrus functions as a memory suppressor. We reveal that mice lacking PLCβ1 in the dentate gyrus exhibit a heightened fear response and impaired memory extinction, and this excessive fear response is repressed by upregulation of PLCβ1 through its overexpression or activation using a newly developed optogenetic system. Last, our results demonstrate that PLCβ1 overexpression partially inhibits exaggerated fear response caused by traumatic experience. Together, PLCβ1 is crucial in regulating contextual fear memory formation and potentially enhancing the resilience to trauma-related conditions. [ABSTRACT FROM AUTHOR]

Mitochondrial gene expression relies on mitoribosomes to translate mitochondrial mRNAs. The biogenesis of mitoribosomes is an intricate process involving multiple assembly factors. Among these factors, GTP-binding proteins (GTPBPs) play important roles. In bacterial systems, numerous GTPBPs are required for ribosome subunit maturation, with EngB being a GTPBP involved in the ribosomal large subunit assembly. In this study, we focus on exploring the function of GTPBP8, the human homolog of EngB. We find that ablation of GTPBP8 leads to the inhibition of mitochondrial translation, resulting in significant impairment of oxidative phosphorylation. Structural analysis of mitoribosomes from GTPBP8 knock-out cells shows the accumulation of mitoribosomal large subunit assembly intermediates that are incapable of forming functional monosomes. Furthermore, fPAR-CLIP analysis reveals that GTPBP8 is an RNA-binding protein that interacts specifically with the mitochondrial ribosome large subunit 16 S rRNA. Our study highlights the role of GTPBP8 as a component of the mitochondrial gene expression machinery involved in mitochondrial large subunit maturation. Multiple GTP-binding proteins (GTPBPs) aid ribosome maturation. Here, authors pinpoint GTPBP8's involvement in human mitoribosome maturation, demonstrating its specific binding to mitoribosomal large subunit RNA, which is necessary for ribosome assembly and protein synthesis. [ABSTRACT FROM AUTHOR]

The hypothalamic paraventricular nucleus (PVN) regulates sympathetic activity and blood pressure. The regulator of G protein signaling 2 (RGS2) is a negative G protein regulator, which selectively regulates Gaq signaling, a potential cause of hypertension. This study aimed to examine angiotensin II (ANG II)-G protein- RGS2 signaling on the central mechanisms of blood pressure control, sympathetic activation, and kidney function. The Sprague Dawley rats were infused with ANG II (200 ng/kg/min) via osmotic mini pump to induce hypertension. Adenovirus (AV) vectors encoding RGS2 was transfected into the PVN in vivo. By radio telemetry measurements, we found AV-RGS2 transfection to the PVN significantly attenuated the increase of mean arterial pressure in ANG II infusion rats from days 2-7 of the 2-week experiment (Day 7: ANG II + AV-RGS2 141.3 ± 10.0 mmHg vs. ANG II 166.9 ± 9.3 mmHg, p < 0.05). AV-RGS2 transfection significantly reduced the serum norepinephrine level and acute volume reflex and increased daily urine volume and sodium excretion in ANG II-infused hypertensive rats. AV-RGS2 transfection significantly reduced Gaq and PKC protein expressions within the PVN in ANG II infusion rats. In cultured mouse hypothalamic cells, real-time PCR study showed ANG II treatment increased mRNA expression of Gaq, Gas, and RGS2, and AV-RGS2 treatment decreased ANG II-induced mRNA expression of Gaq and Gas. Using confocal imagery, we found that AV-RGS2 attenuated the increase of calcium influx in ANG II-treated cells. Our results suggest that central overexpression of RGS2 in the PVN attenuated the increase of blood pressure and sympathetic outflow, and improves kidney excretory function in hypertensive rats. This may be via the alteration of ANG II-G-protein-RGS2 signaling in the central nervous system. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) are mainly regulated by GPCR kinase (GRK) phosphorylation and subsequent β-arrestin recruitment. The ubiquitously expressed GRKs are classified into cytosolic GRK2/3 and membrane-tethered GRK5/6 subfamilies. GRK2/3 interact with activated G protein βγ-subunits to translocate to the membrane. Yet, this need was not linked as a factor for bias, influencing the effectiveness of β-arrestin-biased agonist creation. Using multiple approaches such as GRK2/3 mutants unable to interact with Gβγ, membrane-tethered GRKs and G protein inhibitors in GRK2/3/5/6 knockout cells, we show that G protein activation will precede GRK2/3-mediated β-arrestin2 recruitment to activated receptors. This was independent of the source of free Gβγ and observable for Gs-, Gi- and Gq-coupled GPCRs. Thus, β-arrestin interaction for GRK2/3-regulated receptors is inseparably connected with G protein activation. We outline a theoretical framework of how GRK dependence on free Gβγ can determine a GPCR's potential for biased agonism. Due to this inherent cellular mechanism for GRK2/3 recruitment and receptor phosphorylation, we anticipate generation of β-arrestin-biased ligands to be mechanistically challenging for the subgroup of GPCRs exclusively regulated by GRK2/3, but achievable for GRK5/6-regulated receptors, that do not demand liberated Gβγ. Accordingly, GRK specificity of any GPCR is foundational for developing arrestin-biased ligands. Using cytosolic or membrane-tethered GRK2/3 mutants and G protein inhibitors, authors show that G protein activation, releasing free Gβγ, and GRK2/3-mediated β-arrestin recruitment are inseparably intertwined. This adds to the concept of biased agonism. [ABSTRACT FROM AUTHOR]

Grain size is one of the critical factors determining rice yield. Previous studies have found the grain-size-regulating function of Gα in rice. However, the regulatory mechanism underlying the development of rice grain mediated by Gα is still unclear. To reveal the functional mechanism of Gα in grain size regulation, a mutant of Gα (Gα-Cas9) was firstly constructed through a CRISPR/Cas9 strategy and was then grown in a greenhouse and field. The results showed that the seed length, plant height, 1000-grain weight, and spike length were significantly decreased in Gα-Cas9 compared to wild-type (WT) Pi-4b. During the grain filling stage, the increase in the grain dry weight of Pi-4b occurred earlier than that of Gα-Cas9. The total starch content and amylose content of matured grains of Pi-4b were higher than those of Gα-Cas9. Secondly, transcriptome sequencing analysis of Gα-Cas9 and Pi-4b during grain filling was performed to elucidate the functional pathways regulated by Gα. In total, 2867 and 4534 differentially expressed genes (DEGs) were discovered at 5 DAF and 10 DAF, and the starch and sucrose metabolism pathway enriched by DEGs was involved in grain size regulation mediated by Gα. Gα regulated the expression of starch-synthesis-related genes during grain filling, and the Gα protein interacted with OsNYC4 to trigger the sugar signaling pathway to promote starch accumulation in grain. Additionally, the GW2-WG1-OsbZIP47 pathway was switched off by Gα to relieve the inhibition of rice grain development. In this study, the results should provide new insights into the G protein signal transduction pathway. [ABSTRACT FROM AUTHOR]

Purpose: The objective of this study was to examine the molecular epidemiology and clinical characteristics of HMPV infection among children with ARIs in Nanjing. Methods: The respiratory samples were collected from 2078 children (≤ 14 years) with acute respiratory infections and were tested for HMPV using real-time RT-PCR. Amplification and sequencing of the HMPV G gene were followed by phylogenetic analysis using MEGA 7.0. Result: The detection rate of HMPV among children was 4.7% (97/2078), with a concentration in those under 5 years of age. Notably, the peak season for HMPV prevalence was observed in winter. Among the 97 HMPV-positive samples, 51.5% (50/97) were available for characterization of the HMPV G protein gene. Phylogenetic analysis indicated that the sequenced HMPV strains were classified into three sublineages: A2c111nt − dup (84.0%), B1 (2.0%), and B2 (14.0%). Conclusion: There was an incidence of HMPV among hospitalized children during 2021–2022 in Nanjing with A2c111nt − dup being the dominant strain. This study demonstrated the molecular epidemiological characteristics of HMPV among children with respiratory infections in Nanjing, China. [ABSTRACT FROM AUTHOR]

Receptor‐G protein promiscuity is frequently observed in class A G protein‐coupled receptors (GPCRs). In particular, GPCRs can couple with G proteins from different families (Gαs, Gαq/11, Gαi/o, and Gα12/13) or the same family subtypes. The molecular basis underlying the selectivity/promiscuity is not fully revealed. We recently reported the structures of kappa opioid receptor (KOR) in complex with the Gi/o family subtypes [Gαi1, GαoA, Gαz, and Gustducin (Gαg)] determined by cryo‐electron microscopy (cryo‐EM). The structural analysis, in combination with pharmacological studies, provides insights into Gi/o subtype selectivity. Given the conserved sequence identity and activation mechanism between different G protein families, the findings within Gi/o subtypes could be likely extended to other families. Understanding the KOR‐Gi/o or GPCR‐G protein selectivity will facilitate the development of more precise therapeutics targeting a specific G protein subtype. [ABSTRACT FROM AUTHOR]