The association of GNB5 with Alzheimer disease revealed by genomic analysis restricted to variants impacting gene function.

Disease-associated variants identified from genome-wide association studies (GWASs) frequently map to non-coding areas of the genome such as introns and intergenic regions. An exclusive reliance on gene-agnostic methods of genomic investigation could limit the identification of relevant genes associated with polygenic diseases such as Alzheimer disease (AD). To overcome such potential restriction, we developed a gene-constrained analytical method that considers only moderate- and high-risk variants that affect gene coding sequences. We report here the application of this approach to publicly available datasets containing 181,388 individuals without and with AD and the resulting identification of 660 genes potentially linked to the higher AD prevalence among Africans/African Americans. By integration with transcriptome analysis of 23 brain regions from 2,728 AD case-control samples, we concentrated on nine genes that potentially enhance the risk of AD: AACS, GNB5, GNS, HIPK3, MED13, SHC2, SLC22A5, VPS35, and ZNF398. GNB5, the fifth member of the heterotrimeric G protein beta family encoding Gß5, is primarily expressed in neurons and is essential for normal neuronal development in mouse brain. Homozygous or compound heterozygous loss of function of GNB5 in humans has previously been associated with a syndrome of developmental delay, cognitive impairment, and cardiac arrhythmia. In validation experiments, we confirmed that Gnb5 heterozygosity enhanced the formation of both amyloid plaques and neurofibrillary tangles in the brains of AD model mice. These results suggest that gene-constrained analysis can complement the power of GWASs in the identification of AD-associated genes and may be more broadly applicable to other polygenic diseases.

Structure of the class D GPCR Ste2 dimer coupled to two G proteins.

G-protein-coupled receptors (GPCRs) are divided phylogenetically into six classes1,2, denoted A to F. More than 370 structures of vertebrate GPCRs (belonging to classes A, B, C and F) have been determined, leading to a substantial understanding of their function3. By contrast, there are no structures of class D GPCRs, which are found exclusively in fungi where they regulate survival and reproduction. Here we determine the structure of a class D GPCR, the Saccharomyces cerevisiae pheromone receptor Ste2, in an active state coupled to the heterotrimeric G protein Gpa1-Ste4-Ste18. Ste2 was purified as a homodimer coupled to two G proteins. The dimer interface of Ste2 is formed by the N terminus, the transmembrane helices H1, H2 and H7, and the first extracellular loop ECL1. We establish a class D1 generic residue numbering system (CD1) to enable comparisons with orthologues and with other GPCR classes. The structure of Ste2 bears similarities in overall topology to class A GPCRs, but the transmembrane helix H4 is shifted by more than 20 Å and the G-protein-binding site is a shallow groove rather than a cleft. The structure provides a template for the design of novel drugs to target fungal GPCRs, which could be used to treat numerous intractable fungal diseases4.

A self-inactivating invertebrate opsin optically drives biased signaling toward Gßγ-dependent ion channel modulation.

Animal opsins, light-sensitive G protein-coupled receptors, have been used for optogenetic tools to control G protein-dependent signaling pathways. Upon G protein activation, the Gα and Gßγ subunits drive different intracellular signaling pathways, leading to complex cellular responses. For some purposes, Gα- and Gßγ-dependent signaling needs to be separately modulated, but these responses are simultaneously evoked due to the 1:1 stoichiometry of Gα and Gßγ Nevertheless, we show temporal activation of G protein using a self-inactivating invertebrate opsin, Platynereis c-opsin1, drives biased signaling for Gßγ-dependent GIRK channel activation in a light-dependent manner by utilizing the kinetic difference between Gßγ-dependent and Gα-dependent responses. The opsin-induced transient Gi/o activation preferentially causes activation of the kinetically fast Gßγ-dependent GIRK channels rather than slower Gi/oα-dependent adenylyl cyclase inhibition. Although similar Gßγ-biased signaling properties were observed in a self-inactivating vertebrate visual pigment, Platynereis c-opsin1 requires fewer retinal molecules to evoke cellular responses. Furthermore, the Gßγ-biased signaling properties of Platynereis c-opsin1 are enhanced by genetically fusing with RGS8 protein, which accelerates G protein inactivation. The self-inactivating invertebrate opsin and its RGS8-fusion protein can function as optical control tools biased for Gßγ-dependent ion channel modulation.

Mechanisms of Gßγ Release upon GPCR Activation.

Gßγ release is a key event in the transduction of GPCR signals. However, the molecular mechanisms of this process have been unclear. A recent report by Knight et al. provides important clues into the sequence of events that lead to the liberation of Gßγ upon G protein activation by GPCRs.

Structural basis for KCTD-mediated rapid desensitization of GABAB signalling.

The GABAB (γ-aminobutyric acid type B) receptor is one of the principal inhibitory neurotransmitter receptors in the brain, and it signals through heterotrimeric G proteins to activate a variety of effectors, including G-protein-coupled inwardly rectifying potassium channels (GIRKs)1,2. GABAB-receptor signalling is tightly regulated by auxiliary subunits called KCTDs, which control the kinetics of GIRK activation and desensitization3-5. However, the mechanistic basis for KCTD modulation of GABAB signalling remains incompletely understood. Here, using a combination of X-ray crystallography, electron microscopy, and functional and biochemical experiments, we reveal the molecular details of KCTD binding to both GABAB receptors and G-protein ßγ subunits. KCTDs associate with the receptor by forming an asymmetric pentameric ring around a region of the receptor carboxy-terminal tail, while a second KCTD domain, H1, engages in a symmetric interaction with five copies of Gßγ in which the G-protein subunits also interact directly with one another. We further show that KCTD binding to Gßγ is highly cooperative, defining a model in which KCTD proteins cooperatively strip G proteins from GIRK channels to induce rapid desensitization following receptor activation. These results provide a framework for understanding the molecular basis for the precise temporal control of GABAB signalling by KCTD proteins.

Biasing Gßγ Downstream Signaling with Gallein Inhibits Development of Morphine Tolerance and Potentiates Morphine-Induced Nociception in a Tolerant State.

Opioid analgesics are widely used as a treatment option for pain management and relief. However, the misuse of opioid analgesics has contributed to the current opioid epidemic in the United States. Prescribed opioids such as morphine, codeine, oxycodone, and fentanyl are mu-opioid receptor (MOR) agonists primarily used in the clinic to treat pain or during medical procedures, but development of tolerance limits their utility for treatment of chronic pain. Here we explored the effects of biasing Gßγ signaling on tolerance development after chronic morphine treatment in vivo. We hypothesized that biasing Gßγ signaling with gallein could prevent activation of regulatory signaling pathways that result in tolerance to antinociceptive effects of MOR agonists. Gallein has been shown to bind to Gßγ and inhibit interactions of Gßγ with phospholipase-Cß3 (PLCß3) or G-protein-coupled receptor kinase 2 (GRK2) but not G-protein inwardly rectifying potassium (GIRK) channels. In mice, morphine-induced antinociception was evaluated in the 55°C warm water tail withdrawal assay. We used two paradigms for gallein treatment: administration during and after three times-daily morphine administration. Our results show that gallein cotreatment during repeated administration of morphine decreased opioid tolerance development and that gallein treatment in an opioid-tolerant state enhanced the potency of morphine. Mechanistically, our data suggest that PLCß3 is necessary for potentiating effects of gallein in an opioid-tolerant state but not in preventing the development of tolerance. These studies demonstrate that small molecules that target Gßγ signaling could reduce the need for large doses of opioid analgesics to treat pain by producing an opioid-sparing effect. SIGNIFICANCE STATEMENT: Biasing Gßγ signaling prevents tolerance to repeated morphine administration in vivo and potentiates the antinociceptive effects of morphine in an opioid-tolerant state. Mechanistically, phospholipase-Cß is necessary for potentiating effects of gallein in an opioid-tolerant state but not in preventing the development of tolerance. This study identifies a novel treatment strategy to decrease the development of tolerance to the analgesic effects of mu-opioid receptor agonists, which are necessary to improve pain treatment and decrease the incidence of opioid use disorder.

Multiple potassium channel tetramerization domain (KCTD) family members interact with Gßγ, with effects on cAMP signaling.

G protein-coupled receptors (GPCRs) initiate an array of intracellular signaling programs by activating heterotrimeric G proteins (Gα and Gßγ subunits). Therefore, G protein modifiers are well positioned to shape GPCR pharmacology. A few members of the potassium channel tetramerization domain (KCTD) protein family have been found to adjust G protein signaling through interaction with Gßγ. However, comprehensive details on the KCTD interaction with Gßγ remain unresolved. Here, we report that nearly all the 25 KCTD proteins interact with Gßγ. In this study, we screened Gßγ interaction capacity across the entire KCTD family using two parallel approaches. In a live cell bioluminescence resonance energy transfer-based assay, we find that roughly half of KCTD proteins interact with Gßγ in an agonist-induced fashion, whereas all KCTD proteins except two were found to interact through coimmunoprecipitation. We observed that the interaction was dependent on an amino acid hot spot in the C terminus of KCTD2, KCTD5, and KCTD17. While KCTD2 and KCTD5 require both the Bric-à-brac, Tramtrack, Broad complex domain and C-terminal regions for Gßγ interaction, we uncovered that the KCTD17 C terminus is sufficient for Gßγ interaction. Finally, we demonstrated the functional consequence of the KCTD-Gßγ interaction by examining sensitization of the adenylyl cyclase-cAMP pathway in live cells. We found that Gßγ-mediated sensitization of adenylyl cyclase 5 was blunted by KCTD. We conclude that the KCTD family broadly engages Gßγ to shape GPCR signal transmission.

N-terminal intrinsic disorder is an ancestral feature of Gγ subunits that influences the balance between different Gßγ signaling axes in yeast.

Activated G protein-coupled receptors promote the dissociation of heterotrimeric G proteins into Gα and Gßγ subunits that bind to effector proteins to drive intracellular signaling responses. In yeast, Gßγ subunits coordinate the simultaneous activation of multiple signaling axes in response to mating pheromones, including MAP kinase (MAPK)-dependent transcription, cell polarization, and cell cycle arrest responses. The Gγ subunit in this complex contains an N-terminal intrinsically disordered region that governs Gßγ-dependent signal transduction in yeast and mammals. Here, we demonstrate that N-terminal intrinsic disorder is likely an ancestral feature that has been conserved across different Gγ subtypes and organisms. To understand the functional contribution of structural disorder in this region, we introduced precise point mutations that produce a stepwise disorder-to-order transition in the N-terminal tail of the canonical yeast Gγ subunit, Ste18. Mutant tail structures were confirmed using circular dichroism and molecular dynamics and then substituted for the wildtype gene in yeast. We find that increasing the number of helix-stabilizing mutations, but not isometric mutation controls, has a negative and proteasome-independent effect on Ste18 protein levels as well as a differential effect on pheromone-induced levels of active MAPK/Fus3, but not MAPK/Kss1. When expressed at wildtype levels, we further show that mutants with an alpha-helical N terminus exhibit a counterintuitive shift in Gßγ signaling that reduces active MAPK/Fus3 levels whilst increasing cell polarization and cell cycle arrest. These data reveal a role for Gγ subunit intrinsically disordered regions in governing the balance between multiple Gßγ signaling axes.

Disruption of the interaction between mutationally activated Gαq and Gßγ attenuates aberrant signaling.

Heterotrimeric G protein stimulation via G protein-coupled receptors promotes downstream proliferative signaling. Mutations can occur in Gα proteins which prevent GTP hydrolysis; this allows the G proteins to signal independently of G protein-coupled receptors and can result in various cancers, such as uveal melanoma (UM). Most UM cases harbor Q209L, Q209P, or R183C mutations in Gαq/11 proteins, rendering the proteins constitutively active (CA). Although it is generally thought that active, GTP-bound Gα subunits are dissociated from and signal independently of Gßγ, accumulating evidence indicates that some CA Gα mutants, such as Gαq/11, retain binding to Gßγ, and this interaction is necessary for signaling. Here, we demonstrate that disrupting the interaction between Gßγ and Gαq is sufficient to inhibit aberrant signaling driven by CA Gαq. Introduction of the I25A point mutation in the N-terminal α helical domain of CA Gαq to inhibit Gßγ binding, overexpression of the G protein Gαo to sequester Gßγ, and siRNA depletion of Gß subunits inhibited or abolished CA Gαq signaling to the MAPK and YAP pathways. Moreover, in HEK 293 cells and in UM cell lines, we show that Gαq-Q209P and Gαq-R183C are more sensitive to the loss of Gßγ interaction than Gαq-Q209L. Our study challenges the idea that CA Gαq/11 signals independently of Gßγ and demonstrates differential sensitivity between the Gαq-Q209L, Gαq-Q209P, and Gαq-R183C mutants.

Gßγ subunits colocalize with RNA polymerase II and regulate transcription in cardiac fibroblasts.

Gßγ subunits mediate many different signaling processes in various compartments of the cell, including the nucleus. To gain insight into the functions of nuclear Gßγ signaling, we investigated the functional role of Gßγ signaling in the regulation of GPCR-mediated gene expression in primary rat neonatal cardiac fibroblasts. We identified a novel, negative, regulatory role for the Gß1γ dimer in the fibrotic response. Depletion of Gß1 led to derepression of the fibrotic response at the mRNA and protein levels under basal conditions and an enhanced fibrotic response after sustained stimulation of the angiotensin II type I receptor. Our genome-wide chromatin immunoprecipitation experiments revealed that Gß1 colocalized and interacted with RNA polymerase II on fibrotic genes in an angiotensin II-dependent manner. Additionally, blocking transcription with inhibitors of Cdk9 prevented association of Gßγ with transcription complexes. Together, our findings suggest that Gß1γ is a novel transcriptional regulator of the fibrotic response that may act to restrict fibrosis to conditions of sustained fibrotic signaling. Our work expands the role for Gßγ signaling in cardiac fibrosis and may have broad implications for the role of nuclear Gßγ signaling in other cell types.

Cantil: a previously unreported organ in wild-type Arabidopsis regulated by FT, ERECTA and heterotrimeric G proteins.

We describe a previously unreported macroscopic Arabidopsis organ, the cantil, named for its 'cantilever' function of holding the pedicel at a distance from the stem. Cantil development is strongest at the first nodes after the vegetative to reproductive inflorescence transition; cantil magnitude and frequency decrease acropetally. Cantils develop in wild-type Arabidopsis accessions (e.g. Col-0, Ws and Di-G) as a consequence of delayed flowering in short days; cantil formation is observed in long days when flowering is delayed by null mutation of the floral regulator FLOWERING LOCUS T. The receptor-like kinase ERECTA is a global positive regulator of cantil formation; therefore, cantils never form in the Arabidopsis strain Ler. ERECTA functions genetically upstream of heterotrimeric G proteins. Cantil expressivity is repressed by the specific heterotrimeric complex subunits GPA1, AGB1 and AGG3, which also play independent roles: GPA1 suppresses distal spurs at cantil termini, while AGB1 and AGG3 suppress ectopic epidermal rippling. These G protein mutant traits are recapitulated in long-day flowering gpa1-3 ft-10 plants, demonstrating that cantils, spurs and ectopic rippling occur as a function of delayed phase transition, rather than as a function of photoperiod per se.

Arabidopsis AGB1 participates in salinity response through bZIP17-mediated unfolded protein response.

BACKGROUND: Plant heterotrimeric G proteins respond to various environmental stresses, including high salinity. It is known that Gß subunit AGB1 functions in maintaining local and systemic Na+/K+ homeostasis to accommodate ionic toxicity under salt stress. However, whether AGB1 contributes to regulating gene expression for seedling's survival under high salinity remains unclear. RESULTS: We showed that AGB1-Venus localized to nuclei when facing excessive salt, and the induction of a set of bZIP17-dependent salt stress-responsive genes was reduced in the agb1 mutant. We confirmed both genetic and physical interactions of AGB1 and bZIP17 in plant salinity response by comparing salt responses in the single and double mutants of agb1 and bzip17 and by BiFC assay, respectively. In addition, we show that AGB1 depletion decreases nuclei-localization of transgenic mRFP-bZIP17 under salt stress, as shown in s1p s2p double mutant in the Agrobacteria-mediated transient mRFP-bZIP17 expression in young seedlings. CONCLUSIONS: Our results indicate that AGB1 functions in S1P and/or S2P-mediated proteolytic processing of bZIP17 under salt stress to regulate the induction of salinity-responsive gene expression.

Lipid transport protein ORP2A promotes glucose signaling by facilitating RGS1 degradation.

Heterotrimeric GTP-binding proteins (G proteins) are a group of regulators essential for signal transmission into cells. Regulator of G protein signaling 1 (AtRGS1) possesses intrinsic GTPase-accelerating protein (GAP) activity and could suppress G protein and glucose signal transduction in Arabidopsis (Arabidopsis thaliana). However, how AtRGS1 activity is regulated is poorly understood. Here, we identified a knockout mutant of oxysterol binding protein-related protein 2A, orp2a-1, which exhibits similar phenotypes to the arabidopsis g-protein beta 1-2 (agb1-2) mutant. Transgenic lines overexpressing ORP2A displayed short hypocotyls, a hypersensitive response to sugar, and lower intracellular AtRGS1 levels than the control. Consistently, ORP2A interacted with AtRGS1 in vitro and in vivo. Tissue-specific expression of 2 ORP2A alternative splicing isoforms implied functions in controlling organ size and shape. Bioinformatic data and phenotypes of orp2a-1, agb1-2, and the orp2a-1 agb1-2 double mutant revealed the genetic interactions between ORP2A and Gß in the regulation of G protein signaling and sugar response. Both alternative protein isoforms of ORP2A localized in the endoplasmic reticulum (ER), plasma membrane (PM), and ER-PM contact sites and interacted with vesicle-associated membrane protein-associated protein 27-1 (VAP27-1) in vivo and in vitro through their two phenylalanines in an acidic track-like motif. ORP2A also displayed differential phosphatidyl phosphoinositide binding activity mediated by the pleckstrin homology domain in vitro. Taken together, the Arabidopsis membrane protein ORP2A interacts with AtRGS1 and VAP27-1 to positively regulate G protein and sugar signaling by facilitating AtRGS1 degradation.

Arabidopsis G-protein ß subunit AGB1 represses abscisic acid signaling via attenuation of the MPK3-VIP1 phosphorylation cascade.

Heterotrimeric G proteins play key roles in cellular processes. Although phenotypic analyses of Arabidopsis Gß (AGB1) mutants have implicated G proteins in abscisic acid (ABA) signaling, the AGB1-mediated modules involved in ABA responses remain unclear. We found that a partial AGB1 protein was localized to the nucleus where it interacted with ABA-activated VirE2-interacting protein 1 (VIP1) and mitogen-activated protein kinase 3 (MPK3). AGB1 acts as an upstream negative regulator of VIP1 activity by initiating responses to ABA and drought stress, and VIP1 regulates the ABA signaling pathway in an MPK3-dependent manner in Arabidopsis. AGB1 outcompeted VIP1 for interaction with the C-terminus of MPK3, and prevented phosphorylation of VIP1 by MPK3. Importantly, ABA treatment reduced AGB1 expression in the wild type, but increased in vip1 and mpk3 mutants. VIP1 associates with ABA response elements present in the AGB1 promoter, forming a negative feedback regulatory loop. Thus, our study defines a new mechanism for fine-tuning ABA signaling through the interplay between AGB1 and MPK3-VIP1. Furthermore, it suggests a common G protein mechanism to receive and transduce signals from the external environment.

G protein gamma subunit, a hidden master regulator of GPCR signaling.

Heterotrimeric G proteins (αßγ subunits) that are activated by G protein-coupled receptors (GPCRs) mediate the biological responses of eukaryotic cells to extracellular signals. The α subunits and the tightly bound ßγ subunit complex of G proteins have been extensively studied and shown to control the activity of effector molecules. In contrast, the potential roles of the large family of γ subunits have been less studied. In this review, we focus on present knowledge about these proteins. Induced loss of individual γ subunit types in animal and plant models result in strikingly distinct phenotypes indicating that γ subtypes play important and specific roles. Consistent with these findings, downregulation or upregulation of particular γ subunit types result in various types of cancers. Clues about the mechanistic basis of γ subunit function have emerged from imaging the dynamic behavior of G protein subunits in living cells. This shows that in the basal state, G proteins are not constrained to the plasma membrane but shuttle between membranes and on receptor activation ßγ complexes translocate reversibly to internal membranes. The translocation kinetics of ßγ complexes varies widely and is determined by the membrane affinity of the associated γ subtype. On translocating, some ßγ complexes act on effectors in internal membranes. The variation in translocation kinetics determines differential sensitivity and adaptation of cells to external signals. Membrane affinity of γ subunits is thus a parsimonious and elegant mechanism that controls information flow to internal cell membranes while modulating signaling responses.

Gßγ mediates activation of Rho guanine nucleotide exchange factor ARHGEF17 that promotes metastatic lung cancer progression.

Metastatic lung cancer is a major cause of death worldwide. Dissemination of cancer cells can be facilitated by various agonists within the tumor microenvironment, including by lysophosphatidic acid (LPA). We postulate that Rho guanine nucleotide exchange factors (RhoGEFs), which integrate signaling cues driving cell migration, are critical effectors in metastatic cancer. Specifically, we addressed the hypothetical role of ARHGEF17, a RhoGEF, as a potential effector of Gßγ in metastatic lung cancer cells responding to LPA. Here, we show that ARHGEF17, originally identified as a tumor endothelial marker, is involved in tumor growth and metastatic dissemination of lung cancer cells in an immunocompetent murine model. Gene expression-based analysis of lung cancer datasets showed that increased levels of ARHGEF17 correlated with reduced survival of patients with advanced-stage tumors. Cellular assays also revealed that this RhoGEF participates in the invasive and migratory responses elicited by Gi protein-coupled LPA receptors via the Gßγ subunit complex. We demonstrate that this signaling heterodimer promoted ARHGEF17 recruitment to the cell periphery and actin fibers. Moreover, Gßγ allosterically activates ARHGEF17 by the removal of inhibitory intramolecular restrictions. Taken together, our results indicate that ARHGEF17 may be a valid potential target in the treatment of metastatic lung cancer.

GNB1 promotes hepatocellular carcinoma progression by targeting BAG2 to activate P38/MAPK signaling.

G-proteins are intracellular partners of G-protein-coupled receptors. As a member of the G-protein family, GNB1 has been shown to play a pro-cancer role in lung cancer and breast cancer. However, the biological function and detailed mechanisms of GNB1 in hepatocellular carcinoma progression are unclear. In this study, we investigated the effects of GNB1 and its possible mechanism of action in hepatocellular carcinoma (HCC). The clinical significance of GNB1 was evaluated in a large cohort of HCC patients, showing that GNB1 was overexpressed in HCC compared to adjacent normal liver tissues, and increased GNB1 expression was associated with poor prognosis. We also demonstrated that GNB1 enhances cell proliferation, colony formation, and cell migration and invasion in vitro and promotes the epithelial-to-mesenchymal transition process in HCC cells. Tumor xenograft model assay confirmed the oncogenic role of GNB1 in tumorigenicity in nude mice. Activation of P38 signaling was found in the GNB1 overexpressed HCC cells. Further intervention of P38 confirmed it as an important signaling pathway for the oncogenic role of GNB1 in HCC. Moreover, co-immunoprecipitation followed by liquid chromatograph-mass spectrometry identified that GNB1 exerted oncogenic functions via the interaction of BAG2 and activated P38 signaling pathway. Together, our results reveal that GNB1 plays a pivotal oncogenic role in HCC by promoting the P38 pathway via cooperating with BAG2. GNB1 may serve as a prognostic biomarker for patients with HCC.

Helicobacter pylori-induced aberrant demethylation and expression of GNB4 promotes gastric carcinogenesis via the Hippo-YAP1 pathway.

BACKGROUND: Helicobacter pylori (H. pylori) infection causes aberrant DNA methylation and contributes to the risk of gastric cancer (GC). Guanine nucleotide-binding protein subunit beta-4 (GNB4) is involved in various tumorigenic processes. We found an aberrant methylation level of GNB4 in H. pylori-induced GC in our previous bioinformatic analysis; however, its expression and underlying molecular mechanisms are poorly understood. METHODS: The expression, underlying signaling pathways, and clinical significance of GNB4 were analyzed in a local cohort of 107 patients with GC and several public databases. H. pylori infection was induced in in vitro and in vivo models. Methylation-specific PCR, pyrosequencing, and mass spectrometry analysis were used to detect changes in methylation levels. GNB4, TET1, and YAP1 were overexpressed or knocked down in GC cell lines. We performed gain- and loss-of-function experiments, including CCK-8, EdU, colony formation, transwell migration, and invasion assays. Nude mice were injected with genetically manipulated GC cells, and the growth of xenograft tumors and metastases was measured. Real-time quantitative PCR, western blotting, immunofluorescence, immunohistochemistry, chromatin immunoprecipitation, and co-immunoprecipitation experiments were performed to elucidate the underlying molecular mechanisms. RESULTS: GNB4 expression was significantly upregulated in GC and correlated with aggressive clinical characteristics and poor prognosis. Increased levels of GNB4 were associated with shorter survival times. Infection with H. pylori strains 26695 and SS1 induced GNB4 mRNA and protein expression in GC cell lines and mice. Additionally, silencing of GNB4 blocked the pro-proliferative, metastatic, and invasive ability of H. pylori in GC cells. H. pylori infection remarkably decreased the methylation level of the GNB4 promoter region, particularly at the CpG#5 site (chr3:179451746-179451745). H. pylori infection upregulated TET1 expression via activation of the NF-κB. TET binds to the GNB4 promoter region which undergoes demethylation modification. Functionally, we identified that GNB4 induced oncogenic behaviors of tumors via the Hippo-YAP1 pathway in both in vitro and in vivo models. CONCLUSIONS: Our findings demonstrate that H. pylori infection activates the NF-κB-TET1-GNB4 demethylation-YAP1 axis, which may be a potential therapeutic target for GC.

G Protein Subunit ß1 Facilitates Influenza A Virus Replication by Promoting the Nuclear Import of PB2.

G protein subunit ß1 (GNB1), the beta subunit of the G protein family, plays an important role in regulating transmembrane signal transduction. Although a recent study has demonstrated that GNB1 can bind the matrix protein 1 (M1) to facilitate M1 transport to budding sites and promote the release of progeny influenza A virus (IAV), whether the GNB1 protein has other functions in IAV replication requires further study. Here, we found that GNB1 promoted IAV replication, as virus yield decreased in GNB1 knockdown or knockout cells. GNB1 interacted with polymerase subunits PB2, PB1, and PA. Overexpressed GNB1 facilitated PB2 binding to importin α3, α5, and α7 promoting the nuclear import of PB2, enhancing viral RNA synthesis and polymerase activity. Altogether, our results demonstrated that GNB1 positively regulates virus replication by interacting with polymerase subunits and facilitating the nuclear import of PB2, which provide novel insights into the molecular mechanism of IAV. IMPORTANCE Until now, there has been only one article on the role of GNB1 in IAV budding. No study has investigated the role of GNB1 in IAV replication. In this study, our research demonstrated that GNB1 could increase the interaction between PB2 and the importin α isoform and mediate the nuclear import of PB2. Therefore, GNB1 could promote viral replication and transcription. Our results provide a better understanding of the molecular mechanisms of viral replication and provide potential antiviral drug targets.

EXTRA LARGE G-PROTEIN2 mediates cell death and hyperimmunity in the chitin elicitor receptor kinase 1-4 mutant.

Heterotrimeric G-proteins are signal transduction complexes that comprised three subunits, Gα, Gß, and Gγ, and are involved in many aspects of plant life. The noncanonical Gα subunit EXTRA LARGE G-PROTEIN2 (XLG2) mediates pathogen-associated molecular pattern (PAMP)-induced reactive oxygen species (ROS) generation and immunity downstream of pattern recognition receptors. A mutant of the chitin receptor component CHITIN ELICITOR RECEPTOR KINASE1 (CERK1), cerk1-4, maintains normal chitin signaling capacity but shows excessive cell death upon infection with powdery mildew fungi. We identified XLG2 mutants as suppressors of the cerk1-4 phenotype. Mutations in XLG2 complex partners ARABIDOPSIS Gß1 (AGB1) and Gγ1 (AGG1) have a partial cerk1-4 suppressor effect. Contrary to its role in PAMP-induced immunity, XLG2-mediated control of ROS production by RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD) is not critical for cerk1-4-associated cell death and hyperimmunity. The cerk1-4 phenotype is also independent of the co-receptor/adapter kinases BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) and SUPPRESSOR OF BIR1 1 (SOBIR1), but requires the E3 ubiquitin ligase PLANT U-BOX 2 (PUB2). XLG2 localizes to both the cell periphery and nucleus, and the cerk1-4 cell death phenotype is mediated by the cell periphery pool of XLG2. Integrity of the XLG2 N-terminal domain, but not its phosphorylation, is essential for correct XLG2 localization and formation of the cerk1-4 phenotype. Our results support a model in which XLG2 acts downstream of an unknown cell surface receptor that activates an NADPH oxidase-independent cell death pathway in Arabidopsis (Arabidopsis thaliana).