G Protein Research Articles

C9orf72 controls hepatic lipid metabolism by regulating SREBP1 transport.

Sterol regulatory element binding transcription factors (SREBPs) play a crucial role in lipid homeostasis. They are processed and transported to the nucleus via COPII, where they induce the expression of lipogenic genes. COPII maintains the homeostasis of organelles and plays an essential role in the protein secretion pathways in eukaryotes. The formation of COPII begins at endoplasmic reticulum exit sites (ERES), and is regulated by SEC16A, which provides a platform for the assembly of COPII. However, there have been few studies on the changes in SEC16A protein levels. The repetitive expansion of the hexanucleotide sequence GGGGCC within the chromosome 9 open reading frame 72 (C9orf72) gene is a prevalent factor in the development of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we found that the absence of C9orf72 leads to a decrease in SEC16A protein levels, resulting in reduced localization of the guanine nucleotide exchange factor SEC12 at the ERES. Consequently, the small GTP binding protein SAR1 is unable to bind the endoplasmic reticulum normally, impairing the assembly of COPII. Ultimately, the disruption of SREBPs transport decreases de novo lipogenesis. These results suggest that C9orf72 acts as a novel role in regulating lipid homeostasis and may serve as a potential therapeutic target for obesity.

Cholesterol 25-Hydroxylase Protects Against Diabetic Kidney Disease by Regulating ADP Ribosylation Factor 4.

Cholesterol 25-hydroxylase (CH25H), an enzyme involved in cholesterol metabolism, regulates inflammatory responses and lipid metabolism. However, its role in kidney disease is not known. The author found that CH25H transcript is expressed mostly in glomerular and peritubular endothelial cells and that its expression increased in human and mouse diabetic kidneys. Global deletion of Ch25h in Leprdb/db mice aggravated diabetic kidney disease (DKD), which is associated with increased endothelial cell apoptosis. Treatment of 25-hydroxycholesterol (25-HC), the product of CH25H, alleviated kidney injury in Leprdb/db mice. Mechanistically, 25-HC binds to GTP-binding protein ADP-ribosylation factor 4 (ARF4), an essential protein required for maintaining protein transport in the Golgi apparatus. Interestingly, ARF4's GTPase-activating protein ASAP1 is also predominantly expressed in endothelial cells and its expression increased in DKD. Suppression of ARF4 activity by deleting ARF4 or overexpressing ASAP1 results in endothelial cell death. These results indicatethat 25-HC binds ARF4 to inhibit its interaction with ASAP1, and thereby resulting in enhanced ARF4 activity to confer renoprotection. Therefore, treatment of 25-HC improves kidney injury in DKD in part by restoring ARF4 activity to maintain endothelial cell survival. This study provides a novel mechanism and a potential new therapy for DKD.

DRG2 is required for surface localization of PD-L1 and the efficacy of anti-PD-1 therapy.

More than half of tumor patients with high PD-L1 expression do not respond to anti-PD-1/PD-L1 therapy, and the underlying mechanisms are yet to be clarified. Here we show that developmentally regulated GTP-binding protein 2 (DRG2) is required for response of PD-L1-expressing tumors to anti-PD-1 therapy. DRG2 depletion enhanced IFN-γ signaling and increased the PD-L1 level in melanoma cells. However, it inhibited recycling of endosomal PD-L1 and reduced surface PD-L1 levels, which led to defects in interaction with PD-1. Anti-PD-1 did not expand effector-like T cells within DRG2-depleted tumors and failed to improve the survival of DRG2-depleted tumor-bearing mice. Cohort analysis revealed that patients bearing melanoma with low DRG2 protein levels were resistant to anti-PD-1 therapy. These findings identify DRG2 as a key regulator of recycling of endosomal PD-L1 and response to anti-PD-1 therapy and provide insights into how to increase the correlation between PD-L1 expression and response to anti-PD-1 therapy.

The Arf-GAPs, AoAge1 and AoAge2, regulate diverse cellular processes, conidiation, trap formation, and secondary metabolism in Arthrobotrys oligospora

Guanine nucleotide-binding proteins of the ADP ribosylation factor (Arf) family and their activating proteins (Arf-GAPs) are essential for diverse biological processes. Here, two homologous Arf-GAPs, Age1 (AoAge1) and Age2 (AoAge2), were identified in the widespread nematode-trapping fungus Arthrobotrys oligospora. Our results demonstrated that AoAge1, especially AoAge2, played crucial roles in mycelial growth, sporulation, trap production, stress response, mitochondrial activity, DNA damage, endocytosis, reactive oxygen species production, and autophagy. Notably, transcriptome data revealed that approximately 62.7% of the genes were directly or indirectly regulated by AoAge2, and dysregulated genes in Aoage2 deletion were enriched in metabolism, ribosome biogenesis, secondary metabolite biosynthesis, and autophagy. Furthermore, Aoage2 inactivation caused a substantial reduction in several compounds compared to the wild-type strain. Based on these results, a regulatory network for AoAge1 and AoAge2 was proposed and verified using a yeast two-hybrid assay. Based on our findings, AoAge1 and AoAge2 are essential for vegetative growth and mycelial development. Specifically, AoAge2 is required for sporulation and trapping morphogenesis. Our results demonstrated the critical functions of AoAge1 and AoAge2 in mycelial growth, diverse cellular processes, and pathogenicity, offering deep insights into the functions and regulatory mechanisms of Arf-GAPs in nematode-trapping fungi.

G-Protein and G-Protein Coupled Receptors: Implications in Regulation of Immune Response

The guanine nucleotide binding proteins (G proteins) act as molecular switches of ‘on’ and ‘off’ when bound to GTP and GDP respectively while the guanine protein coupled receptors (GPCRs) are membrane bound receptors whereby extracellular substances (ligands)communicate signals from these substances to an intracellular molecule the G-proteins which in turn bind and activate or inhibit downstream effect or molecules causing cellular responses. This review is aimed at exploring the concept and mechanism of G-proteins and GPCR and their implication in immune response. The GPCR can be activated by various physiological or pathological processes cellular metabolism, hormones, neuro-transmitters, chemokines, autocrines, paracrines, endocrine and exocrine secretions which play an important role in relaying or routing signals to several intracellular pathways. The signal transduction by the extracellular activation or inhibition of the GPCR mediate metabolic enzymes, ion channels, transporters, cellular gene transcription, migration, survival, activation, differentiation and cytokine secretion of immune cells resulting in the synthesis and regulation of embryonic development, gonadal development, learning /memory organismal homeostasis, hematopoiesis and immune dynamics. Therefore, G proteins and GPCRs signaling systems are key determinants in innate and adaptive immunity. The signal transduction of G-Protein and GPCR by cytokine chemotaxis as Chronic inflammatory mediators is associated with tumorigenesis, metastasis with potential antagonism for appropriate targeted therapy.

The M3 Muscarinic Acetylcholine Receptor Can Signal through Multiple G Protein Families.

The M3 muscarinic acetylcholine receptor (M3R) is a G protein-coupled receptor (GPCR) that regulates important physiologic processes, including vascular tone, bronchoconstriction, and insulin secretion. It is expressed on a wide variety of cell types, including pancreatic beta, smooth muscle, neuronal, and immune cells. Agonist binding to the M3R is thought to initiate intracellular signaling events primarily through the heterotrimeric G protein Gq. However, reports differ on the ability of M3R to couple to other G proteins beyond Gq. Using members from the four primary G protein families (Gq, Gi, Gs, and G13) in radioligand binding, GTP turnover experiments, and cellular signaling assays, including live cell G protein dissociation and second messenger assessment of cAMP and inositol trisphosphate, we show that other G protein families, particularly Gi and Gs, can also interact with the human M3R. We further show that these interactions are productive as assessed by amplification of classic second messenger signaling events. Our findings demonstrate that the M3R is more promiscuous with respect to G protein interactions than previously appreciated. SIGNIFICANCE STATEMENT: The study reveals that the human M3 muscarinic acetylcholine receptor (M3R), known for its pivotal roles in diverse physiological processes, not only activates intracellular signaling via Gq as previously known but also functionally interacts with other G protein families such as Gi and Gs, expanding our understanding of its versatility in mediating cellular responses. These findings signify a broader and more complex regulatory network governed by M3R and have implications for therapeutic targeting.

The long noncoding RNA CARDINAL attenuates cardiac hypertrophy by modulating protein translation.

One of the features of pathological cardiac hypertrophy is enhanced translation and protein synthesis. Translational inhibition has been shown to be an effective means of treating cardiac hypertrophy, although system-wide side effects are common. Regulators of translation, such as cardiac-specific long noncoding RNAs (lncRNAs), could provide new, more targeted therapeutic approaches to inhibit cardiac hypertrophy. Therefore, we generated mice lacking a previously identified lncRNA named CARDINAL to examine its cardiac function. We demonstrate that CARDINAL is a cardiac-specific, ribosome-associated lncRNA and show that its expression was induced in the heart upon pathological cardiac hypertrophy and that its deletion in mice exacerbated stress-induced cardiac hypertrophy and augmented protein translation. In contrast, overexpression of CARDINAL attenuated cardiac hypertrophy in vivo and in vitro and suppressed hypertrophy-induced protein translation. Mechanistically, CARDINAL interacted with developmentally regulated GTP-binding protein 1 (DRG1) and blocked its interaction with DRG family regulatory protein 1 (DFRP1); as a result, DRG1 was downregulated, thereby modulating the rate of protein translation in the heart in response to stress. This study provides evidence for the therapeutic potential of targeting cardiac-specific lncRNAs to suppress disease-induced translational changes and to treat cardiac hypertrophy and heart failure.

Identification of a Unique Genomic Region in Sweet Chestnut (Castanea sativa Mill.) That Controls Resistance to Asian Chestnut Gall Wasp Dryocosmus kuriphilus Yasumatsu.

The Asian chestnut gall wasp (ACGW) (Hymenoptera Dryocosmus kuriphilus Yasumatsu) is a severe pest of sweet chestnut (Castanea sativa Mill.) with a strong impact on growth and nut production. A comparative field trial in Central Italy, including provenances from Spain, Italy, and Greece, was screened for ACGW infestation over consecutive years. The Greek provenance Hortiatis expressed a high proportion of immune plants and was used to perform a genome-wide association study based on DNA pool sequencing (Pool-GWAS) by comparing two DNA pools from 25 susceptible and 25 resistant plants. DNA pools were sequenced with 50X coverage depth. Sequence reads were aligned to a C. mollissima reference genome and the pools were compared to identify SNPs associated with resistance. Twenty-one significant SNPs were identified and highlighted a small genomic region on pseudochromosome 3 (Chr 3), containing 12 candidate genes of three gene families: Cytochrome P450, UDP-glycosyltransferase, and Rac-like GTP-binding protein. Functional analyses revealed a putative metabolic gene cluster related to saccharide biosynthesis in the genomic regions associated with resistance that could be involved in the production of a toxic metabolite against parasites. The comparison with previous genetic studies confirmed the involvement of Chr 3 in the control of resistance to ACGW.

Insights into Calpain Activation and Rho-ROCK Signaling in Parkinson's Disease and Aging.

Parkinson's disease (PD), a progressive neurodegenerative disease, has no cure, and current therapies are not effective at halting disease progression. The disease affects mid-brain dopaminergic neurons and, subsequently, the spinal cord, contributing to many debilitating symptoms associated with PD. The GTP-binding protein, Rho, plays a significant role in the cellular pathology of PD. The downstream effector of Rho, Rho-associated kinase (ROCK), plays multiple functions, including microglial activation and induction of inflammatory responses. Activated microglia have been implicated in the pathology of many neurodegenerative diseases, including PD, that initiate inflammatory responses, leading to neuron death. Calpain expression and activity is increased following glial activation, which triggers the Rho-ROCK pathway and induces inflammatory T cell activation and migration as well as mediates toxic α-synuclein (α-syn) aggregation and neuron death, indicating a pivotal role for calpain in the inflammatory and degenerative processes in PD. Increased calpain activity and Rho-ROCK activation may represent a new mechanism for increased oxidative damage in aging. This review will summarize calpain activation and the role of the Rho-ROCK pathway in oxidative stress and α-syn aggregation, their influence on the neurodegenerative process in PD and aging, and possible strategies and research directions for therapeutic intervention.

Bayesian network models identify cooperative GPCR:G protein interactions that contribute to G protein coupling

Cooperative interactions in protein-protein interfaces demonstrate the interdependency or the linked network- like behavior and their effect on the coupling of proteins. Cooperative interactions also could cause ripple or allosteric effects at a distance in protein-protein interfaces. Although they are critically important in protein-protein interfaces, it is challenging to determine which amino acid pair interactions are cooperative. In this work we have used Bayesian network modeling, an interpretable machine learning method, combined with molecular dynamics trajectories to identify the residue pairs that show high cooperativity and their allosteric effect in the interface of G protein coupled receptor (GPCR) complexes with Gα subunits. Our results reveal six GPCR:Gα contacts that are common to the different Gα subtypes and show strong cooperativity in the formation of interface. Both the C-terminus helix5 and the core of the G protein are codependent entities and play an important role in GPCR coupling. We show that a promiscuous GPCR coupling to different Gα subtypes, makes all the GPCR:Gα contacts that are specific to each Gα subtype (Gαs, Gαi and Gαq). This work underscores the potential of data-driven Bayesian network modeling in elucidating the intricate dependencies and selectivity determinants in GPCR:G protein complexes, offering valuable insights into the dynamic nature of these essential cellular signaling components.

Septin 7 interacts with Numb to preserve sarcomere structural organization and muscle contractile function.

Here, we investigated the mechanisms by which aging-related reductions of the levels of Numb in skeletal muscle fibers contribute to loss of muscle strength and power, two critical features of sarcopenia. Numb is an adaptor protein best known for its critical roles in development, including asymmetric cell division, cell-type specification, and termination of intracellular signaling. Numb expression is reduced in old humans and mice. We previously showed that, in mouse skeletal muscle fibers, Numb is localized to sarcomeres where it is concentrated near triads; conditional inactivation of Numb and a closely related protein Numb-like (Numbl) in mouse myofibers caused weakness, disorganization of sarcomeres, and smaller mitochondria with impaired function. Here, we found that a single knockout of Numb in myofibers causes reduction in tetanic force comparable to a double Numb, Numbl knockout. We found by proteomics analysis of protein complexes isolated from C2C12 myotubes by immunoprecipitation using antibodies against Numb that Septin 7 is a potential Numb-binding partner. Septin 7 is a member of the family of GTP-binding proteins that organize into filaments, sheets, and rings, and is considered part of the cytoskeleton. Immunofluorescence evaluation revealed a partial overlap of staining for Numb and Septin 7 in myofibers. Conditional, inducible knockouts of Numb led to disorganization of Septin 7 staining in myofibers. These findings indicate that Septin 7 is a Numb-binding partner and suggest that interactions between Numb and Septin 7 are critical for structural organization of the sarcomere and muscle contractile function.

Septin 7 interacts with Numb to preserve sarcomere structural organization and muscle contractile function

Here, we investigated the mechanisms by which aging-related reductions of the levels of Numb in skeletal muscle fibers contribute to loss of muscle strength and power, two critical features of sarcopenia. Numb is an adaptor protein best known for its critical roles in development, including asymmetric cell division, cell-type specification, and termination of intracellular signaling. Numb expression is reduced in old humans and mice. We previously showed that, in mouse skeletal muscle fibers, Numb is localized to sarcomeres where it is concentrated near triads; conditional inactivation of Numb and a closely related protein Numb-like (Numbl) in mouse myofibers caused weakness, disorganization of sarcomeres, and smaller mitochondria with impaired function. Here, we found that a single knockout of Numb in myofibers causes reduction in tetanic force comparable to a double Numb, Numbl knockout. We found by proteomics analysis of protein complexes isolated from C2C12 myotubes by immunoprecipitation using antibodies against Numb that Septin 7 is a potential Numb-binding partner. Septin 7 is a member of the family of GTP-binding proteins that organize into filaments, sheets, and rings, and is considered part of the cytoskeleton. Immunofluorescence evaluation revealed a partial overlap of staining for Numb and Septin 7 in myofibers. Conditional, inducible knockouts of Numb led to disorganization of Septin 7 staining in myofibers. These findings indicate that Septin 7 is a Numb-binding partner and suggest that interactions between Numb and Septin 7 are critical for structural organization of the sarcomere and muscle contractile function.

Pediatric Central Nervous System Vascular Malformation : Pathological Review with Diagram.

Pediatric central nervous system (CNS) vascular malformations are a group of abnormal blood vessel formations within the brain or spinal cord in children. The most crucial point of pediatric CNS vascular malformation is that no golden standard classifications exist. In addition, there is a big gap in knowledge and the viewpoint of clinicians, radiologists, and pathologists. In addition, many genes associated with pediatric CNS vascular malformation, such as Sturge-Weber-Dimitri syndrome with guanine nucleotide-binding protein G(q) subunit alpha (GNAQ) gene mutation, and cavernous malformations with cerebral cavernous malformations 1 (CCM1), CCM2, and CCM3 gene mutation, were recently revealed. For proper therapeutic approaches, we must understand the lesions' characterizations in anatomical, morphological, and functional views. In this review, the author would like to provide basic pediatric CNS vascular malformation concepts with understandable diagrams. Thus, the author hopes that it might be helpful for the proper diagnosis and treatment of CNS pediatric vascular malformations.

RNA-Seq Analysis of Blastocoel Fluid-Conditioned Media From Euploid Embryos Reveals Higher Expression of Specific Mitochondrial Genes in Media From Embryos Associated With Positive Implantation Outcomes [ID 2683525

INTRODUCTION: This retrospective study measured gene expression using RNA-seq of blastocoel fluid-conditioned media from euploid embryos with known implantation status to identify genes associated with implantation outcomes. METHODS: Blastocoel fluid-conditioned media was obtained after biopsy (ploidy status via NextGen sequencing) of intracytoplasmic sperm injection-generated day 5 blastocysts. Media used for RNA-seq were from 10 euploid blastocysts with known implantation outcomes. RNA was extracted, libraries were prepared, NextSeq500 sequencing was performed, and then sequences were aligned to the human genome. After quality control, the total gene count was 15,284. DESeq2 was used to test for differential gene expression. Raw read counts were normalized and variance stabilizing transformations were applied across samples. Log fold change shrinkage methods were used for gene visualization and rankings. Wald test statistic was used to compare implantation outcomes. Adjusted false-discovery rate P value of <.05 was considered statistically significant for differential expressed genes. RESULTS: Several genes were identified in this analysis revealing differential expression when comparing implantation outcomes. Of interest were two mitochondrial genes (DNAJC19 and MT-TK) and the NOA1 (required for mitochondrial function) with higher expression in media from embryos resulting in successful implantation. CONCLUSION: This study implicates mitochondrial function as a key player during preimplantation development that may affect implantation outcomes. DNAJC19 acts to bring proteins into the mitochondrial matrix, MT-TK encodes a mitochondrial tRNA, and NOA1 is a GTP binding protein residing in the mitochondria. The media samples in this study were collected from euploid blastocysts; therefore, gene expression differences detected represent potential molecular markers for predicting implantation success.

Impacts of in-vitro zebularine treatment on genome-wide DNA methylation and transcriptomic profiles in Salix purpurea L.

Renewable energy resources such as biomass are crucial for a sustainable global society. Trees are a major source of lignocellulosic biomass, which can vary in response to different environmental factors owing to epigenetic regulation, such as DNA C-methylation. To investigate the effects of DNA methylation on plant development and wood formation, and its impacts on gene expression, with a focus on secondary cell wall (SCW)-associated genes, Salix purpurea plantlets were cloned from buds derived from a single hybrid tree for both treatment and control conditions. For the treatment condition, buds were exposed to 50 μM zebularine in vitro and a combined strategy of whole-genome bisulfite sequencing (WGBS) and RNA-seq was employed to examine the methylome and transcriptome profiles of different tissues collected at various time points under both conditions. Transcriptomic and methylome data revealed that most of the promoter and gene body demethylation had no marked effects on the expression profiles of genes. Nevertheless, gene expression tended to decrease with the increased methylation levels of genes with highly methylated promoters. Results indicated that demethylation is less evident in centromeric regions and sex chromosomes. Promoters of secondary cell wall-associated genes, such as 4-coumarate-CoA ligase-like and Rac-like GTP-binding protein RHO, were differentially methylated in the secondary xylem samples collected from two-month potted treated plants compared to control samples. Our results provide novel insights into DNA methylation and gene expression landscapes and a basis for investigating the epigenetic regulation of wood formation in S. purpurea as a model plant for bioenergy species.

Transcriptional Changes and Preservation of Muscle Protein Content in Hibernating Black Bears

Physical inactivity decreases mechanical load on the skeleton, which, when prolonged, leads to loss of muscle mass and strength in most mammalian species. In contrast, hibernating bears demonstrate limited loss of muscle mass and protein content over the prolonged periods (5-6 months) of inactivity and fasting during winter. This suggests that bears have natural adaptive mechanisms preserving muscle mass and functionality. To identify transcriptional changes that underlie molecular mechanisms attenuating muscle loss, we conducted a large-scale gene expression profiling (14,194 genes) in quadriceps muscle of adult black bear males, comparing hibernating animals (n=5) sampled in March, about 1 month before expected emergence, and summer active animals (n=5) using next generation sequencing (RNA-seq) of the transcriptome. In total, 336 differentially expressed (FDR<0.05) genes were up-regulated and 677 genes were down-regulated in muscle of hibernating bears. Gene set enrichment analysis with Reactome database showed significantly elevated proportion of overexpressed genes involved in eukaryotic translation initiation (FDR<0.0001) and elongation (FDR<0.0001) as well as in the mTORC1 mediated signaling pathway (FDR=0.021). In contrast, down-regulated genes were overrepresented among genes involved in catabolism of branched chain amino acid (BCAA: leucine, isoleucine and valine, FDR<0.0001) that suggests preservation of BCAA. These findings imply induction of protein biosynthesis through the mTORC1 signaling positively activated by availability of leucine in muscle during hibernation. Support for this conclusion comes from significant overexpression of Ras-related GTP-binding protein D (RRAGD, FC=2.12, FDR<0.0001), crucial regulator of the mTORC1 response to leucine availability, and up-regulation of eukaryotic translation initiation factor 4B (EIF4B, FC=1.95, FDR=0.012), downstream target of the mTORC1 mediated signaling. In addition to regulation of protein biosynthesis, activation of the mTOR signaling suppresses autophagy-dependent protein degradation. Consistent with reduction of autophagy, microtubule associated protein 1 light chain (MAP1LC3A, FC=-2.46, FDR<0.0001) and unc-51 like autophagy activating kinase (ULK1, FC=-1.69, FDR=0.052) were down-regulated in hibernating muscle. No expression differences were detected for two muscle atrophy markers, key members of the ubiquitin proteasome degradation, FBXO32 (Atrogin-1, MAFBX, FDR=0.85) and TRIM63 (MURF-1, FDR=0.63). The induction of protein biosynthesis and decrease in protein catabolism through the mTORC1 mediated signaling as response to BCAA availability likely contribute to the maintenance of muscle protein content through prolonged periods of immobility and fasting during hibernation. The follow up studies need to include metabolomic quantification of BCAA, phospho-proteomic assessment of key genes (RPS6KB1, RPS6) involved in the mTORC1 signaling and identification of upstream negative regulators suppressing BCAA catabolism in hibernating muscle. The work was supported by NIH COBRE under grant number [P20GM130443]. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

GmAGB1 plays a positive regulatory role in soybean defense responses

Heterotrimeric GTP-binding protein (G-proteins) complex, which consists of Gα, Gβ and Gγ subunits, plays critical roles in defense signaling. Arabidopsis genome contains only a single Gβ-encoding gene, AGB1. Loss function of AGB1 in Arabidopsis results in enhanced susceptibility to a wide range of pathogens. However, the function of soybean AGB1 in immunity has not been previously interrogated. Bioinformatic analysis indicated that there are four GmAGB1 homologous genes in soybean genome, sharing homology of 86%-97%. To overcome the functional redundancy of these GmAGB1 homologs, virus-induced gene silencing (VIGS) mediated by the bean pod mottle virus (BPMV) was used to silence these four genes simultaneously. As expected, these four GmAGB1 homologous genes were indeed silenced by a single BPMV-VIGS vector carrying a conserved fragments among these four genes. A dwarfed phenotype was observed in GmAGB1s-silenced soybean plants, suggesting that GmAGB1s play a crucial role in growth and development. Disease resistance analysis indicated that silencing GmAGB1s significantly compromised the resistance of soybean plants against Xanthomonas campestris pv. glycinea (Xag). This reduced resistance was correlated with the decreased accumulation of pathogen-induced reactive oxygen species (ROS) and the reduced activation of GmMPK3 in response to flg22, a conserved N-terminal peptide of flagellin protein. These results indicate that GmAGB1 functions as a positive regulator in disease resistance and GmAGB1 is indispensable for the ROS production and GmMPK3 activation induced by pathogen infection. Yeast two hybrid assay showed that GmAGB1 interacted with GmAGG1, suggesting that an evolutionary conserved heterotrimeric G protein complex similarly functions in soybean.

Potassium humate supplementation improves photosynthesis and agronomic and yield traits of foxtail millet

Foxtail millet is a highly nutritious crop, which is widely cultivated in arid and semi-arid areas worldwide. Humic acid (HA), as a common plant growth regulator, is used as an organic fertilizer and feed additive in agricultural production. However, the impact of potassium humate KH on the photosynthetic rate and yield of foxtail millet has not yet been studied. We explored the effects of KH application on the morphology, photosynthetic ability, carbon and nitrogen metabolism, and yield of foxtail millet. A field experiment was performed using six concentrations of KH (0, 20, 40, 80, 160, and 320 kg ha−1) supplied foliarly at the booting stage in Zhangza 10 cultivar (a widely grown high-yield variety). The results showed that KH treatment increased growth, chlorophyll content (SPAD), photosynthetic rate (Pn), transpiration rate (Tr), and stomatal conductance (Gs). In addition, soluble protein content, sugar content, and nitrate reductase activity increased in KH-treated plants. With increased KH concentration, the effects became more evident and the peak values of each factor were achieved at 80 kg ha−1. Photosynthetic rate showed significant correlation with SPAD, Tr, Gs, and soluble protein content, but was negatively correlated with intercellular CO2 concentration. Compared to that of the control, the yield of foxtail millet under the T2, T3, T4, and T5 (40, 80, 160, and 320 kg ha−1 of KH) treatments significantly increased by 6.0%, 12.7%, 10.5%, and 8.6%, respectively. Yield exhibited a significant positive correlation with Tr, Pn, and Gs. Overall, KH enhances photosynthetic rate and yield of foxtail millet, therefore it may be conducive to stable millet production. These findings may provide a theoretical basis for the green and efficient production of millet fields.

Structural determinants for activation of the Tau kinase CDK5 by the serotonin receptor 5-HT7R

BackgroundMultiple neurodegenerative diseases are induced by the formation and deposition of protein aggregates. In particular, the microtubule-associated protein Tau leads to the development of so-called tauopathies characterized by the aggregation of hyperphosphorylated Tau within neurons. We recently showed that the constitutive activity of the serotonin receptor 7 (5-HT7R) is required for Tau hyperphosphorylation and aggregation through activation of the cyclin-dependent kinase 5 (CDK5). We also demonstrated physical interaction between 5-HT7R and CDK5 at the plasma membrane suggesting that the 5-HT7R/CDK5 complex is an integral part of the signaling network involved in Tau-mediated pathology.MethodsUsing biochemical, microscopic, molecular biological, computational and AI-based approaches, we investigated structural requirements for the formation of 5-HT7R/CDK5 complex.ResultsWe demonstrated that 5-HT7R domains responsible for coupling to Gs proteins are not involved in receptor interaction with CDK5. We also created a structural model of the 5-HT7R/CDK5 complex and refined the interaction interface. The model predicted two conserved phenylalanine residues, F278 and F281, within the third intracellular loop of 5-HT7R to be potentially important for complex formation. While site-directed mutagenesis of these residues did not influence Gs protein-mediated receptor signaling, replacement of both phenylalanines by alanine residues significantly reduced 5-HT7R/CDK5 interaction and receptor-mediated CDK5 activation, leading to reduced Tau hyperphosphorylation and aggregation. Molecular dynamics simulations of 5-HT7R/CDK5 complex for wild-type and receptor mutants confirmed binding interface stability of the initial model.ConclusionsOur results provide a structural basis for the development of novel drugs targeting the 5-HT7R/CDK5 interaction interface for the selective treatment of Tau-related disorders, including frontotemporal dementia and Alzheimer’s disease.

Promiscuous G-protein activation by the calcium-sensing receptor.

The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca2+ concentration and maintains Ca2+ homeostasis1,2. It also mediates diverse cellular processes not associated with Ca2+ balance3-5. The functional pleiotropy of CaSR arises in part from its ability to signal through several G-protein subtypes6. We determined structures of CaSR in complex with G proteins from three different subfamilies: Gq, Gi and Gs. We found that the homodimeric CaSR of each complex couples to a single G protein through a common mode. This involves the C-terminal helix of each Gα subunit binding to a shallow pocket that is formed in one CaSR subunit by all three intracellular loops (ICL1-ICL3), an extended transmembrane helix 3 and an ordered C-terminal region. G-protein binding expands the transmembrane dimer interface, which is further stabilized by phospholipid. The restraint imposed by the receptor dimer, in combination with ICL2, enables G-protein activation by facilitating conformational transition of Gα. We identified a single Gα residue that determines Gq and Gs versus Gi selectivity. The length and flexibility of ICL2 allows CaSR to bind all three Gα subtypes, thereby conferring capacity for promiscuous G-protein coupling.