G-protein Complex Research Articles

Histotopography of α-Gustdusin- and T2R3-Expressing Lymphocytes in Mouse Spleen.

Histotopography of lymphocytes expressing bitter taste receptors of T2R family and α-gustducin (receptor-associated subunit of the G-protein complex) in mouse spleen in the norm and in 48 h after intraperitoneal administration of LPS was studied by the immunohistochemical method. Two populations of immunopositive lymphocytes expressing the above proteins were detected in the spleen; they were located in the marginal zones of lymphoid follicles and periarteriolar lymphoid sheaths. In most α-gustducin-positive lymphocytes, co-expression of α-gustducin and CD19 (B-cell marker) was found. Intraperitoneal administration of LPS significantly increased the number of gustducinergic lymphocytes in periarteriolar lymphoid sheaths. We hypothesize that spleen lymphocytes with gustducin signaling are involved in T-dependent immune response to the blood bacterial polysaccharides.

Abstract 364: The dopamine receptor D2 antagonist ONC201 has anti-tumorigenic activity in obesity-driven epithelial ovarian cancer

Abstract Objectives: ONC201 is an orally bioavailable dopamine receptor D2 antagonist that induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and TRAIL has been implicated in both the physiopathology of ovarian cancer (OC) and obesity. In addition, DRD2 is a G protein-coupled receptor that activates the trimeric G-protein complex Ras, modulating downstream metabolic pathways such as Akt-signaling which is highly dysregulated in both obesity and OC. ONC201 has demonstrated anti-tumorigenic activity in solid tumors, including uterine serous carcinoma, without significant toxicity in phase 1/2 trials. Thus, we sought to evaluate the anti-tumorigenic effects of ONC201 in human ovarian cancer (OC) cell lines and a high grade serous OC mouse model (K18-gT121+/-;p53fl/fl;Brca1fl/fl; KpB), using both obese and lean mice. Methods: The human epithelial OC cell lines (OVCAR3, SKOV3, IGROV, OVCAR433, and OVCAR5) were treated with ONC201 at doses of 0.01 to 250 μM. Cell proliferation and apoptosis were assessed by MTT and Annexin V assays, respectively. Adhesion and invasion were assessed by laminin and wound healing assays, respectively. For our animal study, we utilized the K18-gT121+/-; p53fl/fl;Brca1fl/fl (KpB) high grade serous OC mouse model. KpB mice were fed either a low fat diet (LFD, lean) with 10% calories from fat, or a high fat diet (HFD, obese) with 60% calories from fat in order to mimic diet-induced obesity. Following tumor onset, obese and lean mice were treated with vehicle or ONC201 (130 mg once a week, oral gavage) for 4 weeks (n = 8-10 mice/group). Results: ONC201 significantly decreased cell proliferation in a dose-dependent manner in all five cell lines, with a range of IC50s between 1 and 10μM. Moreover, ONC201 increased Annexin V expression in a dose-dependent manner in both the SKOV3 and OVCAR5 cell lines (p<0.05). Treatment with ONC201 reduced cell adhesion by 25-30% as well as invasion by 20-25% in both OC cell lines when compared to controls (p<0.05). HFD-fed mice (obese) had tumors that were significantly greater in size as compared to LFD-fed mice (lean) (p<0.05). ONC201 decreased tumor weight/size by 65% in the obese mice and 61% in the lean mice (p<0.01). Conclusions: ONC201 inhibited cell growth, induced apoptosis and decreased cellular adhesion and invasion in human OC cells. ONC201 was efficacious in reducing ovarian tumor growth in both obese and non-obese KpB mice. These results suggest that ONC201 holds promise as a novel targeted therapy in the treatment of OC. Citation Format: Allison Staley, Katherine Tucker, Ziwei Fang, Yali Fan, Wenchuan Sun, Yajie Yin, Yingao zhang, Varun Prabhu, Chunxiao Zhou, Victoria Bae-Jump. The dopamine receptor D2 antagonist ONC201 has anti-tumorigenic activity in obesity-driven epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 364.

ONC201 has anti-tumorigenic effects in obesity-driven endometrial cancer

Objective: ONC201 (Oncoceutics), a dopamine receptor D2 (DRD2) antagonist, is actively being explored in clinical trials for endometrial cancer (EC). DRD2 is a G protein-coupled receptor that activates the trimeric G-protein complex Ras, modulating downstream metabolic pathways such as Ras- and Akt-signaling, which are highly dysegulated in obesity-driven EC. Thus, we sought to investigate the antitumorigenic potential of ONC201 in a genetically engineered mouse model of endometrioid EC (LKB1fl/flp53fl/fl).

The Nucleotide-Dependent Interactome of Rice Heterotrimeric G-Protein α -Subunit.

The rice heterotrimeric G-protein complex, a guanine-nucleotide-dependent on-off switch, mediates vital cellular processes and responses to biotic and abiotic stress. Exchange of bound GDP (resting state) for GTP (active state) is spontaneous in plants including rice and thus there is no need for promoting guanine nucleotide exchange in vivo as a mechanism for regulating the active state of signaling as it is well known for animal G signaling. As such, a master regulator controlling the G-protein activation state is unknown in plants. Therefore, an ab initio approach is taken to discover candidate regulators. The rice Gα subunit (RGA1) is used as bait to screen for nucleotide-dependent protein partners. A total of 264 proteins are identified by tandem mass spectrometry of which 32 were specific to the GDP-bound inactive state and 22 specific to the transition state. Approximately, 10% are validated as previously identified G-protein interactors.

Spatial Cumulant Analysis to Study D2-Like Dopamine Receptor Dynamics on Plasma Membrane

Fluorescence fluctuation spectroscopy (FFS) measures physical properties of fluorescent samples by analyzing the statistical nature of signals measured by photodetectors. In biological contexts, the most frequently measured parameters include concentration, molecular brightness, and motion characteristics (i.e. diffusion coefficients). Molecular brightness holds salience because it can be used to determine the stoichiometry of homomeric and heteromeric interactions between fluorescent species. We use spatial cumulant analysis (SpCA), an adaptation of cumulant analysis of point measurements, to determine molecular brightness from images recorded using two-photon laser scanning microscopy. Spatial cumulant analysis combines the advantages of imaging and single point cumulant analysis. Imaging provides decreased photobleaching, the ability to measure fluctuations from a larger set of molecules, and spatial resolution of the measured parameters. Cumulants are favorable in FFS because they are additive for multiple molecular species and provide computational ease. Accurate determination of stoichiometry from molecular brightness can be difficult for several reasons including non-ideal photodetection, incomplete chromophore maturation, photobleaching, and resonance energy transfer. Our approach accounts for these complications. D2-like dopamine receptors (D2Rs) are cell surface receptors that couple to trimeric G-protein complexes. Activation of D2Rs catalyzes the dissociation of the G-protein complex into Gα and Gβγ subunits. Gα and Gβγ subunits mediate the action of D2Rs by interaction with downstream effectors including adenylyl cyclase, inwardly rectifying K+ channels, and voltage-gated Ca2+ channels. Physiologically, D2R activation inhibits cAMP production and Ca2+ influx while activating hyperpolarizing K+ currents. While it is known that D2Rs, G-proteins, and their effectors interact to produce these physiological effects, the details of these interactions are difficult to probe experimentally in live cells. We show how multicolor spatial cumulant analysis of labeled proteins of interest can accurately measure the stoichiometry of receptors, G-proteins, and downstream signaling targets.

Heterotrimeric G-Protein-Dependent Proteome and Phosphoproteome in Unstimulated Arabidopsis Roots.

The G-protein complex is a cytoplasmic on-off molecular switch that is set by plasma membrane receptors that activate upon binding of its cognate extracellular agonist. In animals, the default setting is the "off" resting state, while in plants, the default state is constitutively "on" but repressed by a plasma membrane receptor-like protein. De-repression appears to involve specific phosphorylation of key elements of the G-protein complex and possibly target proteins that are positioned downstream of this complex. To address this possibility, protein abundance and phosphorylation state are quantified in wild type and G-protein deficient Arabidopsis roots in the unstimulated resting state. A total of 3246 phosphorylated and 8141 non-modified protein groups are identified. It has been found that 428 phosphorylation sites decrease and 509 sites increase in abundance in the G-protein quadrupole mutant lacking an operable G-protein-complex. Kinases with known roles in G-protein signaling including MAP KINASE 6 and FERONIA are differentially phosphorylated along with many other proteins now implicated in the control of G-protein signaling. Taken together, these datasets will enable the discovery of novel proteins and biological processes dependent on G-protein signaling.

Clinical Characteristics of GNB1 and GNAS Mutations in an Unselected Cohort of 6,343 Patients with Hematologic Abnormalities

Genes encoding the alpha (GNAS) and beta (GNB1) subunits of the heterotrimeric G-protein complex are recurrently mutated in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Alterations in G-protein coupled receptors can affect signaling via the PI3K/AKT/mTOR and RAS/MAPK pathways, suggesting that GNB1/GNAS mutations may function similarly to mutations in RAS and tyrosine kinases in myeloid disease progression. However, unlike RAS mutations, GNB1/GNAS mutations are among the most commonly affected genes in clonal hematopoiesis of indeterminate potential (CHIP), suggestive of a distinct functional role in myeloid disease initiation.We evaluated 6343 unique patients who received gene panel sequencing at our institution as part of a diagnostic evaluation of known or suspected hematologic malignancy. We identified 68 patients that had at least 1 sample with a mutation in GNB1 (N =42), GNAS (N=24) or both (N=2) (Figure 1). Twenty-six patients had multiple samples (range 2-10) obtained at serial timepoints during the course of progression and treatment. GNB1 mutations affected codon 57 (K57E/M/N/T) in 38 out of 42 cases; the remaining were G53E, D76G, I81V, and K89R. GNAS mutations affected codon 201 (R201H/C) in 23 out of 24 cases.Forty patients had active myeloid neoplasms [AML (N=10), MDS (N=13), MPN (N=11), MDS/MPN (N=5), BPDCN (N=1)]. The distribution of myeloid diagnoses was similar among patients with GNB1 or GNAS mutations. Analysis of serial samples showed that GNB1/GNAS mutations were frequently acquired at the time of leukemic transformation and clinical progression. Among 5 patients with secondary AML, 2 patients acquired GNB1/GNAS mutations at the time of transformation from MDS to AML, and 3 patients were found to have GNB1/GNAS mutations in high-risk MDS prior to subsequent transformation to AML. After induction chemotherapy for AML, two patients had expanded or newly acquired GNB1 mutations at time of treatment failure.Notably, 15 out of the 40 (37.5%) patients with active myeloid disease had co-mutations in the MPN-associated genes JAK2, CALR, and MPL. Eleven of these patients had a clinical diagnosis of MPN, including myelofibrosis (n=5), essential thrombocytosis (n=3), polycythemia vera (n=2), and systemic mastocytosis (n=1). In the context of underlying oncogenic kinase alterations including JAK2 V617F, GNB1 mutations have been shown to promote resistance to kinase inhibitors in vitro. Fourteen out of fifteen patients had no history of ruxolitinib therapy, suggesting that these mutations arise during the course of disease progression in the absence of therapeutic selection pressures. The impact of GNB1/GNAS mutations on the clinical response to JAK inhibitors has not been evaluated.Twenty-eight patients in the cohort (41%) had no evidence of active myeloid disease, including 14 patients with a lymphoid malignancy [mature B-cell neoplasms (n=8), mature T cell neoplasms (n=3), plasma cell neoplasms (n=2), acute lymphoblastic leukemia (n=1)], 6 patients with AML in complete remission, and 8 patients without a clinical diagnosis of hematologic malignancy. Four of the AML patients had a GNAS or GNB1 mutation that was newly acquired or persistent at the time of complete remission following induction chemotherapy. All 4 patients underwent allogeneic stem cell transplant (HSCT) based on disease risk at diagnosis and are alive with median follow up of 2.5 years. In 1 patient, a new GNB1 mutation was identified immediately after HSCT in the context of 100% donor chimerism, suggesting donor-engrafted CHIP. All 8 patients without hematologic malignancy were evaluated for cytopenias or cytoses at the time of the identification of the GNB1/GNAS mutation.GNB1 and GNAS are recurrently mutated in diverse clinical and genetic contexts. In patients with active myeloid disease, MPN-associated mutations were common and GNB1/GNAS mutations were newly acquired at the time of clinical progression, suggesting a functional role similar to other gene mutations that activate mitogenic signaling pathways. However, many GNB1 and GNAS mutations are seen as sole mutations in the absence of active myeloid disease, suggesting that unlike other signaling mutations they can drive initiation of clonal hematopoiesis. The diverse clinical spectrum of patients with GNB1/GNAS mutations suggests that these mutations have distinct, context dependent effects in hematopoietic cells. [Display omitted] DisclosuresKim:Aushon Biosciences: Consultancy; LabCorp, Inc.: Consultancy; Papgene, Inc: Consultancy. Lane:N-of-one: Consultancy; Stemline Therapeutics: Research Funding.

Epigenetic control of pheromone MAPK signaling determines sexual fecundity in Candida albicans

Several pathogenic Candida species are capable of heritable and reversible switching between two epigenetic states, "white" and "opaque." In Candida albicans, white cells are essentially sterile, whereas opaque cells are mating-proficient. Here, we interrogate the mechanism by which the white-opaque switch regulates sexual fecundity and identify four genes in the pheromone MAPK pathway that are expressed at significantly higher levels in opaque cells than in white cells. These genes encode the β subunit of the G-protein complex (STE4), the pheromone MAPK scaffold (CST5), and the two terminal MAP kinases (CEK1/CEK2). To define the contribution of each factor to mating, C. albicans white cells were reverse-engineered to express elevated, opaque-like levels of these factors, either singly or in combination. We show that white cells co-overexpressing STE4, CST5, and CEK2 undergo mating four orders of magnitude more efficiently than control white cells and at a frequency approaching that of opaque cells. Moreover, engineered white cells recapitulate the transcriptional and morphological responses of opaque cells to pheromone. These results therefore reveal multiple bottlenecks in pheromone MAPK signaling in white cells and that alleviation of these bottlenecks enables efficient mating by these "sterile" cell types. Taken together, our findings establish that differential expression of several MAPK factors underlies the epigenetic control of mating in C. albicans We also discuss how fitness advantages could have driven the evolution of a toggle switch to regulate sexual reproduction in pathogenic Candida species.

Alteration of cell wall xylan acetylation triggers defense responses that counterbalance the immune deficiencies of plants impaired in the β-subunit of the heterotrimeric G-protein.

Arabidopsis heterotrimeric G-protein complex modulates pathogen-associated molecular pattern-triggered immunity (PTI) and disease resistance responses to different types of pathogens. It also plays a role in plant cell wall integrity as mutants impaired in the Gβ- (agb1-2) or Gγ-subunits have an altered wall composition compared with wild-type plants. Here we performed a mutant screen to identify suppressors of agb1-2 (sgb) that restore susceptibility to pathogens to wild-type levels. Out of the four sgb mutants (sgb10-sgb13) identified, sgb11 is a new mutant allele of ESKIMO1 (ESK1), which encodes a plant-specific polysaccharide O-acetyltransferase involved in xylan acetylation. Null alleles (sgb11/esk1-7) of ESK1 restore to wild-type levels the enhanced susceptibility of agb1-2 to the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM), but not to the bacterium Pseudomonas syringae pv. tomato DC3000 or to the oomycete Hyaloperonospora arabidopsidis. The enhanced resistance to PcBMM of the agb1-2 esk1-7 double mutant was not the result of the re-activation of deficient PTI responses in agb1-2. Alteration of cell wall xylan acetylation caused by ESK1 impairment was accompanied by an enhanced accumulation of abscisic acid, the constitutive expression of genes encoding antibiotic peptides and enzymes involved in the biosynthesis of tryptophan-derived metabolites, and the accumulation of disease resistance-related secondary metabolites and different osmolites. These esk1-mediated responses counterbalance the defective PTI and PcBMM susceptibility of agb1-2 plants, and explain the enhanced drought resistance of esk1 plants. These results suggest that a deficient PTI-mediated resistance is partially compensated by the activation of specific cell-wall-triggered immune responses.

Frozen in action: cryo-EM structure of a GPCR-G-protein complex.

Interaction with heterotrimeric G proteins is a hallmark of G-protein-coupled receptor (GPCR) family members, and it is the key step for a diverse range of cell-signaling cascades. A recent cryo-EM structure of the human calcitonin receptor (CTR) in complex with a G-protein heterotrimer reveals novel insights into receptor–G-protein coupling.

Phosphatidic acid binding inhibits RGS1 activity to affect specific signaling pathways in Arabidopsis.

Modulation of the active versus inactive forms of the Gα protein is critical for the signaling processes mediated by the heterotrimeric G-protein complex. We have recently established that in Arabidopsis, the regulator of G-protein signaling (RGS1) protein and a lipid-hydrolyzing enzyme, phospholipase Dα1 (PLDα1), both act as GTPase-activity accelerating proteins (GAPs) for the Gα protein to attenuate its activity. RGS1 and PLDα1 interact with each other, and RGS1 inhibits the activity of PLDα1 during regulation of a subset of responses. In this study, we present evidence that this regulation is bidirectional. Phosphatidic acid (PA), a second messenger typically derived from the lipid-hydrolyzing activity of PLDα1, is a molecular target of RGS1. PA binds and inhibits the GAP activity of RGS1. A conserved lysine residue in RGS1 (Lys259 ) is directly involved in RGS1-PA binding. Introduction of this RGS1 protein variant in the rgs1 mutant background makes plants hypersensitive to a subset of abscisic acid-mediated responses. Our data point to the existence of negative feedback loops between these two regulatory proteins that precisely modulate the level of active Gα, consequently generating a highly controlled signal-response output.

A Mutation in the G-Protein Gene GNB2 Causes Familial Sinus Node and Atrioventricular Conduction Dysfunction.

Familial sinus node and atrioventricular conduction dysfunction is a rare disorder that leads to paroxysmal dizziness, fatigue, and syncope because of a temporarily or permanently reduced heart rate. To date, only a few genes for familial sinus and atrioventricular conduction dysfunction are known, and the majority of cases remain pathogenically unresolved. We aim to identify the disease gene in a large 3-generation family (n=25) with autosomal dominant sinus node dysfunction (SND) and atrioventricular block (AVB) and to characterize the mutation-related pathomechanisms in familial SND+AVB. Genome-wide linkage analysis mapped the SND+AVB disease locus to chromosome 7q21.1-q31.1 (2-point logarithm of the odds score: 4.64; θ=0); in this region, targeted exome sequencing identified a novel heterozygous mutation (p.Arg52Leu) in the GNB2 gene that strictly cosegregated with the SND+AVB phenotype. GNB2 encodes the β2 subunit (Gβ2) of the heterotrimeric G-protein complex that is being released from G-protein-coupled receptors on vagal stimulation. In 2 heterologous expression systems (HEK-293T cells and Xenopus laevis oocytes), an enhanced activation of the G-protein-activated K+ channel (GIRK; Kir3.1/Kir3.4) was shown when mutant Gβ2 was coexpressed with Gγ2; this was in contrast to coexpression of mutant Gβ2-Gγ2 with other cardiac ion channels (HCN4, HCN2, and Cav1.2). Molecular dynamics simulations suggested a reduced binding property of mutant Gβ2 to cardiac GIRK channels when compared with native Gβ2. A GNB2 gene mutation is associated with familial SND+AVB and leads to a sustained activation of cardiac GIRK channels, which is likely to hyperpolarize the myocellular membrane potential and thus reduces their spontaneous activity. Our findings describe for the first time a role of a mutant G-protein in the nonsyndromic pacemaker disease because of GIRK channel activation.

Function of heterotrimeric G-protein γ subunit RGG1 in providing salinity stress tolerance in rice by elevating detoxification of ROS.

The present study provides evidence of a unique function of RGG1 in providing salinity stress tolerance in transgenic rice without affecting yield. It also provides a good example for signal transduction from the external environment to inside for enhanced agricultural production that withstands the extreme climatic conditions and ensures food security. The role of heterotrimeric G-proteins functioning as signalling molecules has not been studied as extensively in plants as in animals. Recently, their importance in plant stress signalling has been emerging. In this study, the function of rice G-protein γ subunit (RGG1) in the promotion of salinity tolerance in rice (Oryza sativa L. cv. IR64) was investigated. The overexpression of RGG1 driven by the CaMV35S promoter in transgenic rice conferred high salinity tolerance even in the presence of 200mM NaCl. Transcript levels of antioxidative genes, i.e., CAT, APX, and GR, and their enzyme activities increased in salinity-stressed transgenic rice plants suggesting a better antioxidant system to cope the oxidative-damages caused by salinity stress. The RGG1-induced signalling events that conferred tolerance to salinity was mediated by increased gene expression of the enzymes that scavenged reactive oxygen species. In salinity-stressed RGG1 transgenic lines, the transcript levels of RGG2, RGB, RGA, DEP1, and GS3 also increased in addition to RGG1. These observations suggest that most likely the stoichiometry of the G-protein complex was not disturbed under stress. Agronomic parameters, endogenous sugar content (glucose and fructose) and hormones (GA3, zeatin and IAA) were also higher in the transgenic plants compared with the wild-type plants. A BiFC assay confirmed the interaction of RGG1 with different stress-responsive proteins which play active roles in signalling and prevention of aggregation of proteins under stress-induced perturbation. The present study will help in understanding the G-protein-mediated stress tolerance in plants.

Integrated analysis of omics data using microRNA-target mRNA network and PPI network reveals regulation of Gnai1 function in the spinal cord of Ews/Ewsr1 KO mice.

BackgroundMultifunctional transcription factor (TF) gene EWS/EWSR1 is involved in various cellular processes such as transcription regulation, noncoding RNA regulation, splicing regulation, genotoxic stress response, and cancer generation. Role of a TF gene can be effectively studied by measuring genome-wide gene expression, i.e., transcriptome, in an animal model of Ews/Ewsr1 knockout (KO). However, when a TF gene has complex multi-functions, conventional approaches such as differentially expressed genes (DEGs) analysis are not successful to characterize the role of the EWS gene. In this regard, network-based analyses that consider associations among genes are the most promising approach.MethodsNetworks are constructed and used to show associations among biological entities at various levels, thus different networks represent association at different levels. Taken together, in this paper, we report contributions on both computational and biological sides.ResultsContribution on the computational side is to develop a novel computational framework that combines miRNA-gene network and protein-protein interaction network information to characterize the multifunctional role of EWS gene. On the biological side, we report that EWS regulates G-protein, Gnai1, in the spinal cord of Ews/Ewsr1 KO mice using the two biological network integrated analysis method. Neighbor proteins of Gnai1, G-protein complex subunits Gnb1, Gnb2 and Gnb4 were also down-regulated at their gene expression level. Interestingly, up-regulated genes, such as Rgs1 and Rgs19, are linked to the inhibition of Gnai1 activities. We further verified the altered expression of Gnai1 by qRT-PCR in Ews/Ewsr1 KO mice.ConclusionsOur integrated analysis of miRNA-transcriptome network and PPI network combined with qRT-PCR verifies that Gnai1 function is impaired in the spinal cord of Ews/Ewsr1 KO mice.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-016-0195-4) contains supplementary material, which is available to authorized users.

Manuscript title: antifungal proteins from moulds: analytical tools and potential application to dry-ripened foods.

Moulds growing on the surface of dry-ripened foods contribute to their sensory qualities, but some of them are able to produce mycotoxins that pose a hazard to consumers. Small cysteine-rich antifungal proteins (AFPs) from moulds are highly stable to pH and proteolysis and exhibit a broad inhibition spectrum against filamentous fungi, providing new chances to control hazardous moulds in fermented foods. The analytical tools for characterizing the cellular targets and affected pathways are reviewed. Strategies currently employed to study these mechanisms of action include 'omics' approaches that have come to the forefront in recent years, developing in tandem with genome sequencing of relevant organisms. These techniques contribute to a better understanding of the response of moulds against AFPs, allowing the design of complementary strategies to maximize or overcome the limitations of using AFPs on foods. AFPs alter chitin biosynthesis, and some fungi react inducing cell wall integrity (CWI) pathway. However, moulds able to increase chitin content at the cell wall by increasing proteins in either CWI or calmodulin-calcineurin signalling pathways will resist AFPs. Similarly, AFPs increase the intracellular levels of reactive oxygen species (ROS), and moulds increasing G-protein complex β subunit CpcB and/or enzymes to efficiently produce glutathione may evade apoptosis. Unknown aspects that need to be addressed include the interaction with mycotoxin production by less sensitive toxigenic moulds. However, significant steps have been taken to encourage the use of AFPs in intermediate-moisture foods, particularly for mould-ripened cheese and meat products.

Mahogunin-mediated regulation of Gαi localisation during mitosis and its effect on spindle positioning.

Mahogunin RING Finger 1 (MGRN1) is a ubiquitin E3 ligase known to affect spindle tilt in mitotic cells by regulating α-tubulin ubiquitination and polymerization. In cell culture systems we have found that expressing truncated mutants of MGRN1 leads to various other mitotic anomalies, such as lateral and angular spindle displacements. This seems to be independent of the MGRN1 ligase activity. Our experiments suggest that MGRN1 regulates the balance between the lower molecular weight monomeric Gαi and larger trimeric G-protein complex, along with its abundance in the ternary complex that regulates spindle positioning. The cytosolic isoforms of MGRN1 lead to the enrichment of monomeric Gαi in the cytosol and its subsequent recruitment at the plasma membrane. Excess Gαi at the cell cortex results in an imbalance in the assembly of the ternary complex regulating spindle positioning during mitosis. These observations seem independent of the ligase activity of MGRN1, although we cannot exclude the involvement of an intermediate player that acts as a substrate for MGRN1, and in turn, regulates Gαi.

The role of PLDα1 in providing specificity to signal-response coupling by heterotrimeric G-protein components in Arabidopsis.

Heterotrimeric G-proteins comprised of Gα, Gβ and Gγ subunits are important signal transducers in all eukaryotes. In plants, G-proteins affect multiple biotic and abiotic stress responses, as well as many developmental processes, even though their repertoire is significantly limited compared with that in metazoan systems. One canonical and three extra-large Gα, 1Gβ and 3Gγ proteins represent the heterotrimeric G-protein complex in Arabidopsis, and a single regulatory protein, RGS1, is one of the few known biochemical regulators of this signaling complex. This quantitative disparity between the number of signaling components and the range of processes they influence is rather intriguing. We now present evidence that the phospholipase Dα1 protein is a key component and modulator of the G-protein complex in affecting a subset of signaling pathways. We also show that the same G-protein subunits and their modulators exhibit distinct physiological and genetic interactions depending on specific signaling and developmental pathways. Such developmental plasticity and interaction specificity likely compensates for the lack of multiplicity of individual subunits, and helps to fine tune the plants' responses to constantly changing environments.

Heterotrimeric G-Protein Alpha (α) Subunit from a. Thaliana Forms Trimeric Structures in Solution

The heterotrimeric guanine nucleotide-binding proteins (G-proteins) mediate transmission of signals from G protein coupled receptors (GPCRs) to effector systems including ion channels, enzymes and intracellular second messengers in yeast, mammals, and plants. The complex is comprised of alpha (Gα), beta (Gβ) and gamma (Gγ) subunits; Gα has GTP binding and hydrolysis activity, and Gβ and Gγ interact with downstream effectors as a dimeric complex. Although the structure and activation mechanism for the mammalian complex are well known, these are still not fully understood in plants. In our group subunits of the heterotrimeric complexes from Arabidopsis thaliana and Oryza sativa (rice) are heterologously expressed in yeast and bacteria respectively. The recombinant proteins are then purified for biochemical characterization and structural investigations. Our overall aim is to gain insight into the activation and signaling mechanisms of the G-protein complex in plants through structural studies on the individual subunits as well as the in vitro reconstituted complex. The work in this thesis is undertaken with the primary aim of optimizing the purification protocol of the A. thaliana Gα subunit, GPA1, expressed in yeast to obtain sufficient quantities of homogeneous recombinant protein for biochemical, biophysical, and structural analyses. A further goal is the recombinant production of a truncated version of the wild-type GPA1, which lacks the N-terminal 36 amino acids (GPA1t) in E. coli. GPA1t is commonly used in structural studies due to its stability. Results show that the optimized purification procedure has improved the yield to 5.5 mg GPA1 from 0.5 liters P. pastoris culture which represents a fivefold increase compared to earlier results in the group. Nucleotide (GTP, GDP, GTPγS) binding to the recombinant GPA1 is confirmed by absorbance spectroscopy and circular dichroism spectropolorimetry (CD). Results indicate that the secondary structure elements of GPA1 are more stable when it binds GTPγS as compared to GDP-bound form. Dynamic light scattering (DLS) results and Native-PAGE analyses combined with small angle X-ray scattering (SAXS) measurements reveal that GPA1 has a tendency to form trimers in solution. SAXS data also shows that GPA1-GDP has a globular structure with flexible regions extending from the protein. We cloned GPA1t gene in BL21 cells using the pQE80-L vector and a purification procedure was developed for the isolation of the recombinant protein. Despite the low protein yield, preliminary biochemical and biophysical characterizations were carried out on this protein. DLS measurements confirmed the smaller/more compact size of the GPA1t compared to GPA1. According to the CD analyses its thermal stability is higher than that of GPA1. In future studies conditions for the co-existence of the two species (monomer and trimer) and its physiological significance need to be investigated. Moreover, shape models for both species need to be developed to understand AtGPA1 interactions with other components in the heterotrimer.

A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells.

G-protein coupled receptors (GPCRs) can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gαi1, Gαi2 and Gαi3 activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gαi subunit, and cp173Venus fused to the Gγ2 subunit as acceptor. The Gαi FRET biosensors constructs are expressed together with Gβ1 from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gαi FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gαi FRET sensor in single cells upon stimulation of several GPCRs, including the LPA2, M3 and BK2 receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gαi1, Gαi2 and Gαi3 activation will be valuable for live-cell measurements that probe Gαi activation.

Heterotrimeric G-proteins in Picea abies and their regulation in response to Heterobasidion annosum s.l. infection.

BackgroundHeterotrimeric G-proteins are important signalling switches, present in all eukaryotic kingdoms. In plants they regulate several developmental functions and play an important role in plant-microbe interactions. The current knowledge on plant G-proteins is mostly based on model angiosperms and little is known about the G-protein repertoire and function in other lineages. In this study we investigate the heterotrimeric G-protein subunit repertoire in Pinaceae, including phylogenetic relationships, radiation and sequence diversity levels in relation to other plant linages. We also investigate functional diversification of the G-protein complex in Picea abies by analysing transcriptional regulation of the G-protein subunits in different tissues and in response to pathogen infection.ResultsA full repertoire of G-protein subunits in several conifer species were identified in silico. The full-length P. abies coding regions of one Gα-, one Gβ- and four Gγ-subunits were cloned and sequenced. The phylogenetic analysis of the Gγ-subunits showed that PaGG1 clustered with A-type-like subunits, PaGG3 and PaGG4 clustered with C-type-like subunits, while PaGG2 and its orthologs represented a novel conifer-specific putative Gγ-subunit type. Gene expression analyses by quantitative PCR of P. abies G-protein subunits showed specific up-regulation of the Gα-subunit gene PaGPA1 and the Gγ-subunit gene PaGG1 in response to Heterobasidion annosum sensu lato infection.ConclusionsConifers possess a full repertoire of G-protein subunits. The differential regulation of PaGPA1 and PaGG1 indicates that the heterotrimeric G-protein complex represents a critical linchpin in Heterobasidion annosum s.l. perception and downstream signaling in P. abies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0676-1) contains supplementary material, which is available to authorized users.