Optimal trade-off between boosted tolerance and growth fitness during adaptive evolution of yeast to ethanol shocks.

BACKGROUND: The selection of Saccharomyces cerevisiae strains with higher alcohol tolerance can potentially increase the industrial production of ethanol fuel. However, the design of selection protocols to obtain bioethanol yeasts with higher alcohol tolerance poses the challenge of improving industrial strains that are already robust to high ethanol levels. Furthermore, yeasts subjected to mutagenesis and selection, or laboratory evolution, often present adaptation trade-offs wherein higher stress tolerance is attained at the expense of growth and fermentation performance. Although these undesirable side effects are often associated with acute selection regimes, the utility of using harsh ethanol treatments to obtain robust ethanologenic yeasts still has not been fully investigated. RESULTS: We conducted an adaptive laboratory evolution by challenging four populations (P1-P4) of the Brazilian bioethanol yeast, Saccharomyces cerevisiae PE-2_H4, through 68-82 cycles of 2-h ethanol shocks (19-30% v/v) and outgrowths. Colonies isolated from the final evolved populations (P1c-P4c) were subjected to whole-genome sequencing, revealing mutations in genes enriched for the cAMP/PKA and trehalose degradation pathways. Fitness analyses of the isolated clones P1c-P3c and reverse-engineered strains demonstrated that mutations were primarily selected for cell viability under ethanol stress, at the cost of decreased growth rates in cultures with or without ethanol. Under this selection regime for stress survival, the population P4 evolved a protective snowflake phenotype resulting from BUD3 disruption. Despite marked adaptation trade-offs, the combination of reverse-engineered mutations cyr1A1474T/usv1Δ conferred 5.46% higher fitness than the parental PE-2_H4 for propagation in 8% (v/v) ethanol, with only a 1.07% fitness cost in a culture medium without alcohol. The cyr1A1474T/usv1Δ strain and evolved P1c displayed robust fermentations of sugarcane molasses using cell recycling and sulfuric acid treatments, mimicking Brazilian bioethanol production. CONCLUSIONS: Our study combined genomic, mutational, and fitness analyses to understand the genetic underpinnings of yeast evolution to ethanol shocks. Although fitness analyses revealed that most evolved mutations impose a cost for cell propagation, combination of key mutations cyr1A1474T/usv1Δ endowed yeasts with higher tolerance for growth in the presence of ethanol. Moreover, alleles selected for acute stress survival comprising the P1c genotype conferred stress tolerance and optimal performance under conditions simulating the Brazilian industrial ethanol production.

The cAMP-PKA signalling crosstalks with CWI and HOG-MAPK pathways in yeast cell response to osmotic and thermal stress.

The yeast Saccharomyces cerevisiae is widely used in food and non-food industries. During industrial fermentation yeast strains are exposed to fluctuations in oxygen concentration, osmotic pressure, pH, ethanol concentration, nutrient availability and temperature. Fermentation performance depends on the ability of the yeast strains to adapt to these changes. Suboptimal conditions trigger responses to the external stimuli to allow homeostasis to be maintained. Stress-specific signalling pathways are activated to coordinate changes in transcription, translation, protein function, and metabolic fluxes while a transient arrest of growth and cell cycle progression occur. cAMP-PKA, HOG-MAPK and CWI signalling pathways are turned on during stress response. Comprehension of the mechanisms involved in the responses and in the adaptation to these stresses during fermentation is key to improving this industrial process. The scope of this review is to outline the advancement of knowledge about the cAMP-PKA signalling and the crosstalk of this pathway with the CWI and HOG-MAPK cascades in response to the environmental challenges heat and hyperosmotic stress.

p-synephrine induces transcriptional changes via the cAMP/PKA pathway but not cytotoxicity or mutagenicity in human gastrointestinal cells.

p-Synephrine (SN) is an alkaloid added to thermogenic formulations for weight loss that is predominantly absorbed in the human gastrointestinal tract (GI). As the adverse effects of SN on GI cells remain unclear, the aim of present study was to examine whether SN affected cell viability, cell cycle kinetics, genomic stability, redox status, and expression of cAMP/PKA pathway genes related to metabolism/energy homeostasis in stomach mucosa (MNP01) and colon adenocarcinoma (Caco-2) human cells. p-Synephrine at 25-5000 µM was not cytotoxic to both cell lines. At 2-200 µM, SN increased the formation of reactive oxygen species (ROS) but also enhanced levels of antioxidant defense molecules glutathione (GSH) and catalase (CAT) activity, which may account for the absence of cytotoxicity/mutagenicity in both cell lines. SN induced expression of the cAMP/PKA pathway genes ADCY3 and MAPK1 in MNP01 cells and MAPK1, GNAS, PRKACA, and PRKAR2A in Caco-2 cells, as well as modulated the transcription of genes related to cell proliferation (JUN; AKT1) and inflammation (RELA; TNF) in both cell lines. Therefore, the improved antioxidant state mitigated pro-oxidative effects attributed to SN. Evidence indicates that SN does not appear to exhibit adverse potential but modulated the cAMP/PKA pathway in human GI cell lines.

cAMP-PKA signal transduction specificity in Saccharomyces cerevisiae.

Living cells have developed a set of complex signaling responses, which allow them to withstand different environmental challenges. Signaling pathways enable the cell to monitor external and internal states and to articulate the appropriate physiological responses. Cellular signal transmission requires the dynamic formation of spatiotemporal controlled molecular interactions. One of the most important signaling circuits in Saccharomyces cerevisiae is the one controlled by cAMP-Protein Kinase A (PKA). In budding yeast, extracellular glucose and a plethora of signals related with growth and stress conditions regulate the intracellular cAMP levels that modulate PKA activity which in turn regulates a broad range of cellular processes. The cAMP-PKA signaling output requires a controlled specificity of the PKA responses. In this review we discuss the molecular mechanisms that are involved in the establishment of the specificity in the cAMP-PKA signaling pathway in S.cerevisiae.

Signaling pathways in cytoskeletal responses to plasma membrane depolarization in corneal endothelial cells.

In previous work, we reported that plasma membrane potential depolarization (PMPD) provokes cortical F-actin remodeling in bovine corneal endothelial (BCE) cells in culture, which eventually leads to the appearance of intercellular gaps. In kidney epithelial cells it has been shown that PMPD determines an extracellular-signal-regulated kinase (ERK)/Rho-dependent increase in diphosphorylated myosin light chain (ppMLC). The present study investigated the signaling pathways involved in the response of BCE cells to PMPD. Differently to renal epithelial cells, we observed that PMPD leads to a decrease in monophosphorylated MLC (pMLC) without affecting diphosphorylated MLC. Also, that the pMLC reduction is a consequence of cyclic adenosine 3',5'-monophosphate (cAMP)/protein kinase A (PKA) activation. In addition, we found evidence that the cAMP increase mostly depends on soluble adenylyl cyclase activity. Inhibition of this enzyme reduces the effect of PMPD on the cAMP rise, F-actin remodeling, and pMLC decrease. No changes in phosho-ERK were observed, although we could determine that RhoA undergoes activation. Our results suggested that active RhoA is not involved in the intercellular gap formation. Overall, the findings of this study support the view that, differently to renal epithelial cells, in BCE cells PMPD determines cytoskeletal reorganization via activation of the cAMP/PKA pathway.

G protein-coupled oestrogen receptor stimulation ameliorates iron- and ovariectomy-induced memory impairments through the cAMP/PKA/CREB signalling pathway.

Iron accumulation in the brain has been associated with neurodegenerative disorders, and imaging studies in humans indicate that iron content in brain regions correlates with poor performance in cognitive tasks. In rats, iron overload impairs memory retention in a variety of memory tasks. Although the effects of iron on cognition in rodents are extensively reported, no previous study has been conducted in female rats. The incidence of certain dementias, such as Alzheimer's disease, is higher in women after menopause compared to aged-matched men. The role of oestrogen depletion in memory deficits in menopausal women is still a matter of debate. The present study aimed to characterise the effects of iron overload on memory in female rats by investigating the effects of ovariectomy (OVX, an experimental model of oestrogen depletion) in rats submitted to iron overload, as well as examining the effects of G protein-coupled oestrogen receptor (GPER) agonism on memory impairments induced by iron and OVX. Female rats received iron (30 mg kg-1 , orally) or vehicle at postnatal days 12-14 and were submitted to OVX in adulthood. Results showed that either iron or OVX impaired memory for object placement and inhibitory avoidance. The selective GPER agonist G1, administered immediately after training, reversed both iron- and OVX-induced memory impairments. G1 effects were abolished by protein kinase A (PKA) inhibition, suggesting the involvement of the cAMP/PKA/CREB signalling pathway. The search for novel oestrogen agonists with positive effects on cognition may be promising for the development of treatments for memory disorders.

The activation of the chymotrypsin-like activity of the proteasome is regulated by soluble adenyl cyclase/cAMP/protein kinase A pathway and required for human sperm capacitation.

One of the first events of mammalian sperm capacitation is the activation of the soluble adenyl cyclase/cAMP/protein kinase A (SACY/cAMP/PKA) pathway. Here, we evaluated whether the increase in PKA activity at the onset of human sperm capacitation is responsible for the activation of the sperm proteasome and whether this activation is required for capacitation progress. Viable human sperm were incubated with inhibitors of the SACY/cAMP/PKA pathway. The chymotrypsin-like activity of the sperm proteasome was evaluated using a fluorogenic substrate. Sperm capacitation status was evaluated using the chlortetracycline assay and tyrosine phosphorylation. To determine whether proteasomal subunits were phosphorylated by PKA, the proteasome was immunoprecipitated and tested on a western blot using an antibody against phosphorylated PKA substrates. Immunofluorescence microscopy analysis and co-immunoprecipitation (IPP) were used to investigate an association between the catalytic subunit alpha of PKA (PKA-Cα) and the proteasome. The chymotrypsin-like activity of the sperm proteasome significantly increased after 5 min of capacitation (P < 0.001) and remained high for the remaining incubation time. Treatment with H89, KT5720 or KH7 significantly decreased the chymotrypsin-like activity of the proteasome (P < 0.001). IPP experiments indicated that PKA inhibition significantly modified phosphorylation of proteasome subunits. In addition, PKA-Cα colocalized with the proteasome in the equatorial segment and in the connecting piece, and co-immunoprecipitated with the proteasome. This is the first demonstration of sperm proteasome activity being directly regulated by SACY/PKA-Cα. This novel discovery extends our current knowledge of sperm physiology and may be used to manage sperm capacitation during assisted reproductive technology procedures.

Fibronectin modulates the endocannabinoid system through the cAMP/PKA pathway during human sperm capacitation.

Fibronectin (Fn) enhances human sperm capacitation via the cAMP/PKA pathway, and the endocannabinoid system participates in this process. Moreover, Fn has been linked to endocannabinoid system components in different cellular models, even though no evidence of such interactions in human sperm is available. Normal semen samples were evaluated over a 4-year period. Our findings suggest that (a) the capacitating effects of Fn were reversed by preincubating the sperm with a cannabinoid receptor 1 (CB1) or transient receptor potential cation channel subfamily V member 1 (TRPV1) antagonist ( p < 0.001 and p < 0.05, respectively); (b) cooperation between CB1 and TRPV1 may exist ( p < 0.01); (c) the activity of specific fatty acid amide hydroxylase (FAAH) decreased after 1 min ( p < 0.01) and increased after 60 min ( p < 0.01) of capacitation in the presence of Fn; (d) the effects of Fn on FAAH activity were prevented by preincubating spermatozoa with a protein kinase A (PKA) inhibitor ( p < 0.01); (e) Fn modulated both the cyclic adenosine monophosphate concentration and PKA activity ( p < 0.05) during early capacitation; and (f) FAAH was a PKA substrate modulated by phosphorylation. These findings indicate that Fn stimulates human sperm capacitation via the cAMP/PKA pathway through modulation of the endocannabinoid system. Understanding the functional competence of human spermatozoa is essential for facilitating clinical advances in infertility treatment and for developing novel contraceptive strategies.

Extracellular cAMP activates molecular signalling pathways associated with sperm capacitation in bovines.

STUDY QUESTION: Is extracellular cAMP involved in the regulation of signalling pathways in bovine sperm capacitation? SUMMARY ANSWER: Extracellular cAMP induces sperm capacitation through the activation of different signalling pathways that involve phospholipase C (PLC), PKC/ERK1-2 signalling and an increase in sperm Ca2+ levels, as well as soluble AC and cAMP/protein kinase A (PKA) signalling. WHAT IS KNOWN ALREADY: In order to fertilize the oocyte, ejaculated spermatozoa must undergo a series of changes in the female reproductive tract, known as capacitation. This correlates with a number of membrane and metabolic modifications that include an increased influx of bicarbonate and Ca2+, activation of a soluble adenylyl cyclase (sAC) to produce cAMP, PKA activation, protein tyrosine phosphorylation and the development of hyperactivated motility. We previously reported that cAMP efflux by Multidrug Resistance Protein 4 (MRP4) occurs during sperm capacitation and the pharmacological blockade of this inhibits the process. Moreover, the supplementation of incubation media with cAMP abolishes the inhibition and leads to sperm capacitation, suggesting that extracellular cAMP regulates crucial signalling cascades involved in this process. STUDY DESIGN, SIZE, DURATION: Bovine sperm were selected by the wool glass column method, and washed by centrifugation in BSA-Free Tyrode's Albumin Lactate Pyruvate (sp-TALP). Pellets were resuspended then diluted for each treatment. For in vitro capacitation, 10 to 15 × 106 SPZ/ml were incubated in 0.3% BSA sp-TALP at 38.5°C for 45 min under different experimental conditions. To evaluate the role of extracellular cAMP on different events associated with sperm capacitation, 10 nM cAMP was added to the incubation medium as well as different inhibitors of enzymes associated with signalling transduction pathways: U73122 (PLC inhibitor, 10 µM), Gö6983 (PKC inhibitor, 10 µM), PD98059 (ERK-1/2 inhibitor, 30 µM), H89 and KT (PKA inhibitors, 50 µM and 100 nM, respectively), KH7 (sAC inhibitor, 10 µM), BAPTA-AM (intracellular Ca2+ chelator, 50 µM), EGTA (10 µM) and Probenecid (MRPs general inhibitor, 500 µM). In addition, assays for binding to oviductal epithelial cells and IVF were carried out to test the effect of cAMP compared with other known capacitant agents such as heparin (60 µg/ml) and bicarbonate (40 mM). PARTICIPANTS/MATERIALS, SETTING, METHODS: Straws of frozen bovine semen (20-25 × 106 spermatozoa/ml) were kindly provided by Las Lilas, CIALE and CIAVT Artificial Insemination Centers. The methods used in this work include western blot, immunohistochemistry, flow cytometry, computer-assisted semen analysis, live imaging of Ca2+ and fluorescence scanning. At least three independent assays with bull samples of proven fertility were carried. MAIN RESULTS AND THE ROLE OF CHANCE: In the present study, we elucidate the molecular events induced by extracellular cAMP. Our results showed that external cAMP induces sperm capacitation, depending upon the action of PLC. Downstream, this enzyme increased ERK1-2 activation through PKC and elicited a rise in sperm Ca2+ levels (P < 0.01). Moreover, extracellular cAMP-induced capacitation also depended on the activity of sAC and PKA, and increased tyrosine phosphorylation, indicating that the nucleotide exerts a broad range of responses. In addition, extracellular cAMP-induced sperm hyperactivation and concomitantly increased the proportion of spermatozoa with high mitochondrial activity (P < 0.01). Finally, cAMP increased the in vitro fertilization rate compared to control conditions (P < 0.001). LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study performed with bovine cryopreserved spermatozoa. Studies in other species and with fresh samples are needed to extrapolate these data. WIDER IMPLICATIONS OF THE FINDINGS: These findings strongly suggest an important role of extracellular cAMP in the regulation of the signalling pathways involved in the acquisition of bull sperm fertilizing capability. The data presented here indicate that not only a rise, but also a regulation of cAMP levels is necessary to ensure sperm fertilizing ability. Thus, exclusion of the nucleotide to the extracellular space might be essential to guarantee the achievement of a cAMP tone, needed for all capacitation-associated events to take place. Moreover, the ability of cAMP to trigger such broad and complex signalling events allows us to hypothesize that cAMP is a self-produced autocrine/paracrine factor, and supports the emerging paradigm that spermatozoa do not compete but, in fact, communicate with each other. A precise understanding of the functional competence of mammalian spermatozoa is essential to generate clinical advances in the treatment of infertility and the development of novel contraceptive strategies. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas [PIP0 496 to S.P.-M.], Agencia Nacional de Promoción Científica y Tecológica [PICT 2012-1195 and PICT2014-2325 to S.P.-M., and PICT 2013-2050 to C.D.], Boehringer Ingelheim Funds, and the Swedish Farmers Foundation [SLF-H13300339 to J.M.]. The authors declare there are no conflicts of interests.

Role of calcium channels in bipolar disorder.

Bipolar disorder is characterized by a host of sleep-wake abnormalities that suggests that the reticular activating system (RAS) is involved in these symptoms. One of the signs of the disease is a decrease in high frequency gamma band activity, which accounts for a number of additional deficits. Bipolar disorder has also been found to overexpress neuronal calcium sensor protein 1 (NCS-1). Recent studies showed that elements in the RAS generate gamma band activity that is mediated by high threshold calcium (Ca2+) channels. This mini-review provides a description of recent findings on the role of Ca2+ and Ca2+ channels in bipolar disorder, emphasizing the involvement of arousal-related systems in the manifestation of many of the disease symptoms. This will hopefully bring attention to a much-needed area of research and provide novel avenues for therapeutic development.

Role of CREB on heme oxygenase-1 induction in adrenal cells: involvement of the PI3K pathway.

In addition to the well-known function of ACTH as the main regulator of adrenal steroidogenesis, we have previously demonstrated its effect on the transcriptional stimulation of HO-1 expression, a component of the cellular antioxidant defense system. In agreement, we hereby demonstrate that, in adrenocortical Y1 cells, HO-1 induction correlates with a significant prevention of the generation of reactive oxygen species induced by H2O2/Fe(2+) ACTH/cAMP-dependent activation of redox-imbalanced related factors such as NRF2 or NFκB and the participation of MAPKs in this mechanism was, however, discarded based on results with specific inhibitors and reporter plasmids. We suggest the involvement of CREB in HO-1 induction by ACTH/cAMP, as transfection of cells with a dominant-negative isoform of CREB (DN-CREB-M1) decreased, while overexpression of CREB increased HO-1 protein levels. Sequence screening of the murine HO-1 promoter revealed CRE-like sites located at -146 and -37 of the transcription start site and ChIP studies indicated that this region recruits phosphorylated CREB (pCREB) upon cAMP stimulation in Y1 cells. In agreement, H89 (PKA inhibitor) or cotransfection with DN-CREB-M1 prevented the 8Br-cAMP-dependent increase in luciferase activity in cells transfected with pHO-1[-295/+74].LUC. ACTH and cAMP treatment induced the activation of the PI3K/Akt signaling pathway in a PKA-independent mechanism. Inhibition of this pathway prevented the cAMP-dependent increase in HO-1 protein levels and luciferase activity in cells transfected with pHO-1[-295/+74].LUC. Finally, here we show a crosstalk between the cAMP/PKA and PI3K pathways that affects the binding of p-CREB to its cognate element in the murine promoter of the Hmox1 gene.

Calcitonin gene-related peptide inhibits autophagic-lysosomal proteolysis through cAMP/PKA signaling in rat skeletal muscles.

Calcitonin gene-related peptide (CGRP) is a neuropeptide released by motor neuron in skeletal muscle and modulates the neuromuscular transmission by induction of synthesis and insertion of acetylcholine receptor on postsynaptic muscle membrane; however, its role in skeletal muscle protein metabolism remains unclear. We examined the in vitro and in vivo effects of CGRP on protein breakdown and signaling pathways in control skeletal muscles and muscles following denervation (DEN) in rats. In isolated muscles, CGRP (10(-10) to 10(-6)M) reduced basal and DEN-induced activation of overall proteolysis in a concentration-dependent manner. The in vitro anti-proteolytic effect of CGRP was completely abolished by CGRP8-37, a CGRP receptor antagonist. CGRP down-regulated the lysosomal proteolysis, the mRNA levels of LC3b, Gabarapl1 and cathepsin L and the protein content of LC3-II in control and denervated muscles. In parallel, CGRP elevated cAMP levels, stimulated PKA/CREB signaling and increased Foxo1 phosphorylation in both conditions. In denervated muscles and starved C2C12 cells, Rp-8-Br-cAMPs or PKI, two PKA inhibitors, completely abolished the inhibitory effect of CGRP on Foxo1, 3 and 4 and LC3 lipidation. A single injection of CGRP (100 µg kg(-1)) in denervated rats increased the phosphorylation levels of CREB and Akt, inhibited Foxo transcriptional activity, the LC3 lipidation as well as the mRNA levels of LC3b and cathepsin L, two bona fide targets of Foxo. This study shows for the first time that CGRP exerts a direct inhibitory action on autophagic-lysosomal proteolysis in control and denervated skeletal muscle by recruiting cAMP/PKA signaling, effects that are related to inhibition of Foxo activity and LC3 lipidation.