Protein-mediated Signaling Pathways Research Articles

Gasdermins: a dual role in pyroptosis and tumor immunity.

The gasdermin (GSDM) protein family plays a pivotal role in pyroptosis, a process critical to the body's immune response, particularly in combatting bacterial infections, impeding tumor invasion, and contributing to the pathogenesis of various inflammatory diseases. These proteins are adept at activating inflammasome signaling pathways, recruiting immune effector cells, creating an inflammatory immune microenvironment, and initiating pyroptosis. This article serves as an introduction to the GSDM protein-mediated pyroptosis signaling pathways, providing an overview of GSDMs' involvement in tumor immunity. Additionally, we explore the potential applications of GSDMs in both innovative and established antitumor strategies.

Bacterial Outer Membrane Vesicles Promote Lung Inflammatory Responses and Macrophage Activation via Multi-Signaling Pathways.

Emerging evidence suggests that Gram-negative bacteria release bacterial outer membrane vesicles (OMVs) and that these play an important role in the pathogenesis of bacterial infection-mediated inflammatory responses and organ damage. Despite the fact that scattered reports have shown that OMVs released from Gram-negative bacteria may function via the TLR2/4-signaling pathway or induce pyroptosis in macrophages, our study reveals a more complex role of OMVs in the development of inflammatory lung responses and macrophage pro-inflammatory activation. We first confirmed that various types of Gram-negative bacteria release similar OMVs which prompt pro-inflammatory activation in both bone marrow-derived macrophages and lung alveolar macrophages. We further demonstrated that mice treated with OMVs via intratracheal instillation developed significant inflammatory lung responses. Using mouse inflammation and autoimmune arrays, we identified multiple altered cytokine/chemokines in both bone marrow-derived macrophages and alveolar macrophages, suggesting that OMVs have a broader spectrum of function compared to LPS. Using TLR4 knock-out cells, we found that OMVs exert more robust effects on activating macrophages compared to LPS. We next examined multiple signaling pathways, including not only cell surface antigens, but also intracellular receptors. Our results confirmed that bacterial OMVs trigger both surface protein-mediated signaling and intracellular signaling pathways, such as the S100-A8 protein-mediated pathway. In summary, our studies confirm that bacterial OMVs strongly induced macrophage pro-inflammatory activation and inflammatory lung responses via multi-signaling pathways. Bacterial OMVs should be viewed as a repertoire of pathogen-associated molecular patterns (PAMPs), exerting more robust effects than Gram-negative bacteria-derived LPS.

Regulation of the tubulin polymerization-promoting protein by Ca2+/S100 proteins

To elucidate S100 protein-mediated signaling pathways, we attempted to identify novel binding partners for S100A2 by screening protein arrays carrying 19,676 recombinant glutathione S-transferase (GST)-fused human proteins with biotinylated S100A2. Among newly discovered putative S100A2 interactants, including TMLHE, TRH, RPL36, MRPS34, CDR2L, OIP5, and MED29, we identified and characterized the tubulin polymerization-promoting protein (TPPP) as a novel S100A2-binding protein. We confirmed the interaction of TPPP with Ca2+/S100A2 by multiple independent methods, including the protein array method, S100A2 overlay, and pulldown assay in vitro and in transfected COS-7 cells. Based on the results from the S100A2 overlay assay using various GST-TPPP mutants, the S100A2-binding region was identified in the C-terminal (residues 111–160) of the central core domain of a monomeric form of TPPP that is involved in TPPP dimerization. Chemical cross-linking experiments indicated that S100A2 suppresses dimer formation of His-tagged TPPP in a dose-dependent and a Ca2+-dependent manner. In addition to S100A2, TPPP dimerization is disrupted by other multiple S100 proteins, including S100A6 and S100B, in a Ca2+-dependent manner but not by S100A4. This is consistent with the fact that S100A6 and S100B, but not S100A4, are capable of interacting with GST-TPPP in the presence of Ca2+. Considering these results together, TPPP was identified as a novel target for S100A2, and it is a potential binding target for other multiple S100 proteins, including S100A6 and S100B. Direct binding of the S100 proteins with TPPP may cause disassembly of TPPP dimer formation in response to the increasing concentration of intracellular Ca2+, thus resulting in the regulation of the physiological function of TPPP, such as microtubule organization.

From cell surface to signalling and back: the life of the mammalian FSH receptor.

Follicle-stimulating hormone receptor (FSHR) is a class A G protein-coupled receptor that belongs to the subfamily of glycoprotein hormone receptors (GPHRs). The interaction of FSH with FSHR triggers downstream signaling pathways that play a central role in mammalian reproduction, such as folliculogenesis in females and the maintenance of spermatogenesis in males. This warrants a detailed investigation into FSHR, from its genesis, to the post-translational modifications that enable it to become functionally competent, followed by its trafficking to the cell membrane. Subsequently, FSH-stimulated Gs uncoupling and transduction of G protein-mediated signaling pathways takes place, after which the receptor undergoes β-arrestin-mediated internalization and may trigger other noncanonical signaling pathways. The majority of the FSH-FSHR complexes are recycled back to the cell surface and only a small proportion are routed to lysosomal degradation pathways, thus completing the lifecycle of the FSH receptor. Information about important epitopes and aspects of FSH receptor function has been gleaned from a number of sources, including structure-function studies on both naturally occurring and induced mutations, single nucleotide polymorphisms, peptides and antipeptide antibodies corresponding to predicted functional residues, X-ray crystallography analysis and high resolution imaging studies, in addition to the information available for the other GPHRs. In this review, we have traversed through the life cycle of the FSH receptor and discuss the reproductive pathophysiologies that could result from an impairment in receptor function, as may arise from defects during its journey from its birth to its degradation. Moreover, the unresolved questions and challenges that require exploration have been highlighted.

β-Arrestin 1 in Thyrotropin Receptor Signaling in Bone: Studies in Osteoblast-Like Cells.

A direct action of thyrotropin (TSH, thyroid-stimulating hormone) on bone precursors in humans is controversial. Studies in rodent models have provided conflicting findings. We used cells derived from a moderately differentiated osteosarcoma stably overexpressing human TSH receptors (TSHRs) as a model of osteoblast precursors (U2OS-TSHR cells) to investigate TSHR-mediated effects in bone differentiation in human cells. We review our findings that (1) TSHR couples to several different G proteins to induce upregulation of genes associated with osteoblast activity—interleukin 11 (IL-11), osteopontin (OPN), and alkaline phosphatase (ALPL) and that the kinetics of the induction and the G protein-mediated signaling pathways involved were different for these genes; (2) TSH can stimulate β-arrestin-mediated signal transduction and that β-arrestin 1 in part mediates TSH-induced pre-osteoblast differentiation; and (3) TSHR/insulin-like growth factor 1 (IGF1) receptor (IGF1R) synergistically increased OPN secretion by TSH and IGF1 and that this crosstalk was mediated by physical association of these receptors in a signaling complex that uses β-arrestin 1 as a scaffold. These findings were complemented using a novel β-arrestin 1-biased agonist of TSHR. We conclude that TSHR can signal via several transduction pathways leading to differentiation of this model system of human pre-osteoblast cells and, therefore, that TSH can directly regulate these bone cells.

Possible biased analgesic of hydromorphone through the G protein-over β-arrestin-mediated pathway: cAMP, CellKey™, and receptor internalization analyses

Morphine, fentanyl, and oxycodone are widely used as analgesics, and recently hydromorphone has been approved in Japan. Although all of these are selective for μ-opioid receptors (MORs) and have similar structures, their analgesic potencies and adverse effects (AEs) are diverse. Recent molecular analyses of MOR signaling revealed that the G protein-mediated signaling pathway causes analgesic effects and the β-arrestin-mediated signaling pathway is responsible for AEs. We used several cell-based analyses that selectively measure cellular responses activated by either G protein- or β-arrestin-mediated pathways. GloSensor™ cAMP, CellKey™, and receptor internalization assays were performed with four different types of cells stably expressing differentially labelled MOR. EC50 values measured by cAMP and CellKey™ assays had potencies in the order fentanyl ≤ hydromorphone < morphine ≤ oxycodone, all also exhibiting full agonist responses. However, in the internalization assay, only fentanyl elicited a full agonist response. Hydromorphone had the strongest potency next to fentanyl; however, contribution of the β-arrestin-mediated pathway was small, suggesting that its effect could be biased toward the G protein-mediated pathway. Based on these properties, hydromorphone could be chosen as an effective analgesic.

G Proteins and GPCRs in C. elegans Development: A Story of Mutual Infidelity.

Many vital processes during C. elegans development, especially the establishment and maintenance of cell polarity in embryogenesis, are controlled by complex signaling pathways. G protein-coupled receptors (GPCRs), such as the four Frizzled family Wnt receptors, are linchpins in regulating and orchestrating several of these mechanisms. However, despite being GPCRs, which usually couple to G proteins, these receptors do not seem to activate classical heterotrimeric G protein-mediated signaling cascades. The view on signaling during embryogenesis is further complicated by the fact that heterotrimeric G proteins do play essential roles in cell polarity during embryogenesis, but their activity is modulated in a predominantly GPCR-independent manner via G protein regulators such as GEFs GAPs and GDIs. Further, the triggered downstream effectors are not typical. Only very few GPCR-dependent and G protein-mediated signaling pathways have been unambiguously defined in this context. This unusual and highly intriguing concept of separating GPCR function and G-protein activity, which is not restricted to embryogenesis in C. elegans but can also be found in other organisms, allows for essential and multi-faceted ways of regulating cellular communication and response. Although its relevance cannot be debated, its impact is still poorly discussed, and C. elegans is an ideal model to understand the underlying principles.

Long non-coding RNAs in heart failure: an obvious lnc.

Heart failure is a life-threatening and costly ailment characterized by structural and functional impairment of the heart. Despite major advances in understanding protein-mediated transcriptional control and signaling pathways that underlie the cellular and interstitial alterations of heart failure, significant therapeutical breakthroughs for innovative treatments of this disease are still missing. The recent extensive profiling of the mammalian transcriptome has revealed a large number of long non-coding RNAs (lncRNAs) that play a diversity of important regulatory roles in gene expression. In here, we focus on a recent work by Ounzain and colleagues comprising genome-wide profiling of the cardiac transcriptome after myocardial infarction with an emphasis on the identification of novel heart-specific lncRNAs.

VGF, Which Is Induced Transcriptionally in Stroke Brain, Enhances Neurite Extension and Confers Protection Against Ischemia In Vitro.

Ischemic stroke is a devastating neural event as currently no therapies other than physical rehabilitation are available to enhance recovery after stroke. To identify endogenous mediators to repair stroke brain, we performed the expression profiling analysis of transcripts in the mouse photothrombotic stroke brain. Based on real-time PCR analysis, we found VGF, identified as a nerve growth factor (NGF)-regulated transcript, was induced transcriptionally in stroke brain at 1-7days after insult. The immunoreactivites of VGF were observed in the neurons around the ischemic core of stroke brain. Experiments with various inhibitors and plasmid transfections indicated that cAMP response element binding protein-mediated complex signaling pathways are possibly implicated in the NGF-mediated VGF expressions in vitro. Furthermore, the over-expression of VGF promoted neurite extensions and conferred protections from ischemic stress in vitro. These findings raise the possibility the application of VGF could be one of the promising therapeutic strategies to enhance recovery after stroke.

Synthesis of azabicycloalkanone amino acid and azapeptide mimics and their application as modulators of the prostaglandin F2α receptor for delaying preterm birth

Premature birth (&lt;37 weeks gestation) is the major cause of perinatal mortality and morbidity and has been steadily increasing worldwide. Towards the rational design of more effective therapeutic agents for inhibiting uterine contractions and prolonging gestation (a so-called tocolytic drug), our team has targeted the prostaglandin F2α receptor (FP) employing a peptidomimetic approach designed to provide modulators of this novel target. We identified first a lead peptide (PDC113) (1) based on the sequence of the second extracellular loop of FP on the basis that the loop itself might modulate receptor activation. Systematic study of the structure−activity relationships of 1 generated hypotheses concerning the conformation and side-chains responsible for activity that led to the synthesis of PDC113.31 (2), a potent all d-amino acid peptide, which has successfully completed Phase 1b clinical trials. Employing indolizidinone amino acids, peptide mimics were developed that served to probe the mechanism of FP modulation. For example, PDC113.824 (9) was shown to allosterically regulate FP activity contingent on the presence of prostaglandin F2α by a mechanism implicating biased signalling. Although attempts to understand the turn geometry responsible for the activity of 9 by replacement of its indolizidin-2-one moiety with other azabicycloalkanones failed to produce biologically active analogs, employment of aza-aminoacyl-proline analogs resulted in a series of FP modulators exhibiting distinct effects on different G protein-mediated signalling pathways. Our program has thus contributed novel probes for understanding the chemical biology of FP as well as new therapeutic agents with promise for inhibiting uterine contractions and preventing preterm birth.

Phosphoproteomics Combined with Quantitative 14-3-3-affinity Capture Identifies SIRT1 and RAI as Novel Regulators of Cytosolic Double-stranded RNA Recognition Pathway

Viral double-stranded RNA (dsRNA) is the most important viral structure recognized by cytosolic pattern-recognition receptors of the innate immune system, and its recognition results in the activation of signaling cascades that stimulate the production of antiviral cytokines and apoptosis of infected cells. 14-3-3 proteins are ubiquitously expressed regulatory molecules that participate in a variety of cellular processes, and 14-3-3 protein-mediated signaling pathways are activated by cytoplasmic dsRNA in human keratinocytes. However, the functional role of 14-3-3 protein-mediated interactions during viral dsRNA stimulation has remained uncharacterized. Here, we used functional proteomics to identify proteins whose phosphorylation and interaction with 14-3-3 is modulated by dsRNA and to characterize the signaling pathways activated during cytosolic dsRNA-induced innate immune response in human HaCaT keratinocytes. Phosphoproteome analysis showed that several MAPK- and immune-response-related signaling pathways were activated after dsRNA stimulation. Interactome analysis identified RelA-associated inhibitor, high-mobility group proteins, and several proteins associated with host responses to viral infection as novel 14-3-3 target proteins. Functional studies showed that RelA-associated inhibitor regulated dsRNA-induced apoptosis and TNF production. Integrated network analyses of proteomic data revealed that sirtuin1 was a central molecule regulated by 14-3-3s during dsRNA stimulation. Further experiments showed that sirtuin 1 negatively regulated dsRNA-induced NFκB transcriptional activity, suppressed expression of antiviral cytokines, and protected cells from apoptosis in dsRNA-stimulated and encephalomyocarditis-virus-infected keratinocytes. In conclusion, our data highlight the importance of 14-3-3 proteins in antiviral responses and identify RelA-associated inhibitor and sirtuin 1 as novel regulators of antiviral innate immune responses.

Candidate gene expression profiling in two contrasting tomato cultivars under chilling stress

Tomato (Solanum lycopersicum Mill.) is sensitive to chilling stress during all stages of plant development. Genetic variation for chilling tolerance exists between cultivated tomato and its related wild species, but intra-specific variation has not been thoroughly investigated so far. Seedlings of 63 tomato accessions were evaluated under low temperature and two contrasting cultivars were identified for the trait: Albenga and San Marzano, the former being more chillingtolerant. To clarify the molecular mechanisms of chilling tolerance in tomato, changes in candidate gene expressions in the two tomato genotypes were analysed, using quantitative RT-PCR. Candidate genes were chosen among those known to be induced by chilling and/or with putative roles in CBF/DREB and ROS-mediated pathways. Results show that besides a CBF regulon, whose function is conserved, ROS and C2H2-type zinc finger protein-mediated cold signalling pathways were also involved in chilling tolerance. Under the chilling stress, the up-regulation of respective transcripts was consistently higher in the chilling-tolerant genotype than in the chilling-sensitive ones.

Allosteric Modulation of a Cannabinoid G Protein-coupled Receptor: BINDING SITE ELUCIDATION AND RELATIONSHIP TO G PROTEIN SIGNALING

The cannabinoid 1 (CB1) allosteric modulator, 5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)-ethyl]-amide) (ORG27569), has the paradoxical effect of increasing the equilibrium binding of [(3)H](-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl]cyclohexan-1-ol (CP55,940, an orthosteric agonist) while at the same time decreasing its efficacy (in G protein-mediated signaling). ORG27569 also decreases basal signaling, acting as an inverse agonist for the G protein-mediated signaling pathway. In ligand displacement assays, ORG27569 can displace the CB1 antagonist/inverse agonist, N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide(SR141716A). The goal of this work was to identify the binding site of ORG27569 at CB1. To this end, we used computation, synthesis, mutation, and functional studies to identify the ORG27569-binding site in the CB1 TMH3-6-7 region. This site is consistent with the results of K3.28(192)A, F3.36(200)A, W5.43(279)A, W6.48(356)A, and F3.25(189)A mutation studies, which revealed the ORG27569-binding site overlaps with our previously determined binding site of SR141716A but extends extracellularly. Additionally, we identified a key electrostatic interaction between the ORG27569 piperidine ring nitrogen and K3.28(192) that is important for ORG27569 to act as an inverse agonist. At this allosteric site, ORG27569 promotes an intermediate conformation of the CB1 receptor, explaining ORG27569's ability to increase equilibrium binding of CP55,940. This site also explains ORG27569's ability to antagonize the efficacy of CP55,940 in three complementary ways. 1) ORG27569 sterically blocks movements of the second extracellular loop that have been linked to receptor activation. 2) ORG27569 sterically blocks a key electrostatic interaction between the third extracellular loop residue Lys-373 and D2.63(176). 3) ORG27569 packs against TMH6, sterically hindering movements of this helix that have been shown to be important for receptor activation.

Loss of cAMP-specific phosphodiesterase rescues spore development in G protein mutant in Dictyostelium

Cyclic AMP (cAMP) is an important intracellular signaling molecule for many G protein-mediated signaling pathways but the specificity of cAMP signaling in cells with multiple signaling pathways is not well-understood. In Dictyostelium, at least two different G protein signaling pathways, mediated by the Gα2 and Gα4 subunits, are involved with cAMP accumulation, spore production, and chemotaxis and the stimulation of these pathways results in the activation of ERK2, a mitogen-activated protein kinase that can down regulate the cAMP-specific phosphodiesterase RegA. The regA gene was disrupted in gα2− and gα4− cells to determine if the absence of this phosphodiesterase rescues the development of these G protein mutants as it does for erk2− mutants. The regA− mutation had no major effects on developmental morphology but enriched the distribution of the Gα mutant cells to the prespore/prestalk border in chimeric aggregates. The loss of RegA function had no effect on Gα4-mediated folate chemotaxis. However, the regA gene disruption in gα4− cells, but not in gα2− cells, resulted in a substantial rescue and acceleration of spore production. This rescue in sporulation required cell autonomous signaling because the precocious sporulation could not be induced through intercellular signaling in chimeric aggregates. However, intercellular signals from regA− strains increased the expression of the prestalk gene ecmB and accelerated the vacuolization of stalk cells. Intercellular signaling from the gα4−regA− strain did not induce ecmA gene expression indicating cell-type specificity in the promotion of prestalk cell development. regA gene disruption in a Gα4HC (Gα4 overexpression) strain did not result in precocious sporulation or stalk cell development indicating that elevated Gα4 subunit expression can mask regA− associated phenotypes even when provided with wild-type intercellular signaling. These findings indicate that the Gα2 and Gα4-mediated pathways provide different contributions to the development of spores and stalk cells and that the absence of RegA function can bypass some but not all defects in G protein regulated spore development.

Procontractile G protein–mediated signaling pathways antagonistically regulate smooth muscle differentiation in vascular remodeling

Procontractile G protein–mediated signaling pathways antagonistically regulate smooth muscle differentiation in vascular remodeling

Procontractile G protein–mediated signaling pathways antagonistically regulate smooth muscle differentiation in vascular remodeling

Vascular smooth muscle (Sm) cells (VSMCs) are highly plastic. Their differentiation state can be regulated by serum response factor (SRF), which activates genes involved in Sm differentiation and proliferation by recruiting cofactors, such as members of the myocardin family and ternary complex factors (TCFs), respectively. However, the extracellular cues and upstream signaling mechanisms regulating SRF-dependent VSMC differentiation under in vivo conditions are poorly understood. In this study, we show that the procontractile signaling pathways mediated by the G proteins G(12)/G(13) and G(q)/G(11) antagonistically regulate VSMC plasticity in different models of vascular remodeling. In mice lacking Gα(12)/Gα(13) or their effector, the RhoGEF protein LARG, RhoA-dependent SRF-regulation was blocked and down-regulation of VSMC differentiation marker genes was enhanced. This was accompanied by an excessive vascular remodeling and exacerbation of atherosclerosis. In contrast, Sm-specific Gα(q)/Gα(11) deficiency blocked activation of extracellular signal-regulated kinase 1/2 and the TCF Elk-1, resulting in a reduced VSMC dedifferentiation in response to flow cessation or vascular injury. These data show that the balanced activity of both G protein-mediated pathways in VSMCs is required for an appropriate vessel remodeling response in vascular diseases and suggest new approaches to modulate Sm differentiation in vascular pathologies.

Mechanisms of Platelet Activation and Integrin αIIβ3

Platelet aggregation is not only an essential part of hemostasis, but also initiates acute coronary syndrome or ischemic stroke. The precise understanding of the activation mechanism of platelet aggregation is fundamental for the development of more effective agents against platelet aggregation. Adenosine diphosphate, thrombin, and thromboxane A2 activate platelet integrin αIIbβ3 through G protein-coupled receptors. G protein-mediated signaling pathways, which are initiated by Gq, G12/G13 or Gi, include phospholipase C with calcium signaling, Rho signaling, protein kinase C and phosphatidylinositol 3-kinase. Rap1b, Ca2+ and diacylglycerol-regulated guanine nucleotide exchange factor I, Rap1-GTP-interacting adaptor molecule, and Akt are important proteins involved in G protein-mediated activation of integrin αIIbβ3. Binding of talin-1 and kindlin-3 to cytoplasmic domains of β3-integrin triggers a conformational change in the extracellular domains that increases its affinity for ligands, such as fibrinogen or von Willebrand factor. Fibrinogens act as bridges between adjacent platelets to generate a platelet aggregate.

Intranasal insulin affects adenyl cyclase system in rat tissues in neonatal diabetes

Abstract The changes in hormone-regulated adenylyl cyclase (AC) signaling system implicated in control of the nervous, cardiovascular and reproductive systems may contribute to complications of diabetes mellitus (DM). We investigated the functional state of AC system in the brain, myocardium, ovary and uterus of rats with neonatal DM and examined the influence of intranasally administered insulin on the sensitivity of this system to biogenic amines and polypeptide hormones. The regulatory effects of somatostatin and 5-HT1BR-agonist 5-nonyloxytryptamine acting via Gi protein-coupled receptors were significantly decreased in DM and partially restored in insulin-treated rats. The effects of hormones, activators of AC, are changed in tissue- and receptorspecific manner, and intranasal insulin restored the effects rather close to the level in control. In insulin-treated non-diabetic rats, AC stimulating effects of isoproterenol and relaxin in the myocardium and of human chorionic gonadotropin in the ovaries were decreased, while the effects of hormones, inhibitors of AC, were increased. These data indicate that with intranasal insulin, Gi protein-mediated signaling pathways continue to gain strength. The obtained data on the influence of hormones on AC system in the brain, myocardium, ovary and uterus allow looking anew into the mechanisms of therapeutic effects of intranasal insulin.

The binding protein BiP attenuates stress-induced cell death in soybean via modulation of the N-rich protein-mediated signaling pathway.

The molecular chaperone binding protein (BiP) participates in the constitutive function of the endoplasmic reticulum (ER) and protects the cell against stresses. In this study, we investigated the underlying mechanism by which BiP protects plant cells from stress-induced cell death. We found that enhanced expression of BiP in soybean (Glycine max) attenuated ER stress- and osmotic stress-mediated cell death. Ectopic expression of BiP in transgenic lines attenuated the leaf necrotic lesions that are caused by the ER stress inducer tunicamycin and also maintained shoot turgidity upon polyethylene glycol-induced dehydration. BiP-mediated attenuation of stress-induced cell death was confirmed by the decreased percentage of dead cell, the reduced induction of the senescence-associated marker gene GmCystP, and reduced DNA fragmentation in BiP-overexpressing lines. These phenotypes were accompanied by a delay in the induction of the cell death marker genes N-RICH PROTEIN-A (NRP-A), NRP-B, and GmNAC6, which are involved in transducing a cell death signal generated by ER stress and osmotic stress through the NRP-mediated signaling pathway. The prosurvival effect of BiP was associated with modulation of the ER stress- and osmotic stress-induced NRP-mediated cell death signaling, as determined in transgenic tobacco (Nicotiana tabacum) lines with enhanced (sense) and suppressed (antisense) BiP levels. Enhanced expression of BiP prevented NRP- and NAC6-mediated chlorosis and the appearance of senescence-associated markers, whereas silencing of endogenous BiP accelerated the onset of leaf senescence mediated by NRPs and GmNAC6. Collectively, these results implicate BiP as a negative regulator of the stress-induced NRP-mediated cell death response.

Extracellular proteinase formation in carbon starving Aspergillus nidulans cultures – physiological function and regulation

Extracellular proteinase formation in carbon depleted cultures of the model filamentous fungus Aspergillus nidulans was studied to elucidate its regulation and possible physiological function. As demonstrated by gene deletion, culture optimization, microbial physiological and enzymological experiments, the PrtA and PepJ proteinases of A. nidulans did not appear to play a decisive role in the autolytic decomposition of fungal cells under the conditions we tested. However, carbon starvation induced formation of the proteinases observable in autolytic cultures. Similar to other degradative enzymes, production of proteinase was regulated by FluG-BrlA asexual developmental signaling and modulated by PacC-dependent pH-responsive signaling. Under the same carbon starved culture conditions, alterations of CreA, MeaB or heterotrimeric G protein mediated signaling pathways caused less significant changes in the formation of extracellular proteinases. Taken together, these results indicate that while the accumulation of PrtA and PepJ is tightly coupled to the initiation of autolysis, they are not essential for autolytic cell wall degradation in A. nidulans. Thus, as Aspergillus genomes contain a large group of genes encoding proteinases with versatile physiological functions, selective control of proteinase production in fungal cells is needed for the improved industrial use of fungi.