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- Molecular and functional characterization of grapevine NIPs through heterologous expression in aqy-null Saccharomyces cerevisiaePublication . Farzana, Sabir; Gomes, Sara; Loureiro-Dias, Maria C.; Soveral, Graça; Prista, CatarinaPlant Nodulin 26-like Intrinsic Proteins (NIPs) are multifunctional membrane channels of the Major Intrinsic Protein (MIP) family. Unlike other homologs, they have low intrinsic water permeability. NIPs possess diverse substrate selectivity, ranging from water to glycerol and to other small solutes, depending on the group-specific amino acid composition at aromatic/Arg (ar/R) constriction. We cloned three NIPs (NIP1;1, NIP5;1, and NIP6;1) from grapevine (cv. Touriga Nacional). Their expression in the membrane of aqy-null Saccharomyces cerevisiae enabled their functional characterization for water and glycerol transport through stopped-flow spectroscopy. VvTnNIP1;1 demonstrated high water as well as glycerol permeability, whereas VvTnNIP6;1 was impermeable to water but presented high glycerol permeability. Their transport activities were declined by cytosolic acidification, implying that internal-pH can regulate NIPs gating. Furthermore, an extension of C-terminal in VvTnNIP6;1M homolog, led to improved channel activity, suggesting that NIPs gating is putatively regulated by C-terminal. Yeast growth assays in the presence of diverse substrates suggest that the transmembrane flux of metalloids (As, B, and Se) and the heavy metal (Cd) are facilitated through grapevine NIPs. This is the first molecular and functional characterization of grapevine NIPs, providing crucial insights into understanding their role for uptake and translocation of small solutes, and extrusion of toxic compounds in grapevine
- Exploring the three PIPs and three TIPs of grapevine for transport of water and atypical substrates through heterologous expression in aqy-null yeastPublication . Sabir, Farzana; Leandro, Maria José; Martins, Ana Paula; Loureiro-Dias, Maria; Moura, Teresa F.; Soveral, Graça; Prista, CatarinaAquaporins are membrane channels that facilitate the transport of water and other small molecules across the cellular membranes. We examined the role of six aquaporins of Vitis vinifera (cv. Touriga nacional) in the transport of water and atypical substrates (other than water) in an aqy-null strain of Saccharomyces cerevisiae. Their functional characterization for water transport was performed by stopped-flow fluorescence spectroscopy. The evaluation of permeability coefficients (Pf) and activation energies (Ea) revealed that three aquaporins (VvTnPIP2;1, VvTnTIP1;1 and VvTnTIP2;2) are functional for water transport, while the other three (VvTnPIP1;4, VvTnPIP2;3 and VvTnTIP4;1) are non-functional. TIPs (VvTnTIP1;1 and VvTnTIP2;2) exhibited higher water permeability than VvTnPIP2;1. All functional aquaporins were found to be sensitive to HgCl2, since their water conductivity was reduced (24–38%) by the addition of 0.5 mM HgCl2. Expression of Vitis aquaporins caused different sensitive phenotypes to yeast strains when grown under hyperosmotic stress generated by KCl or sorbitol. Our results also indicate that Vitis aquaporins are putative transporters of other small molecules of physiological importance. Their sequence analyses revealed the presence of signature sequences for transport of ammonia, boron, CO2, H2O2 and urea. The phenotypic growth variations of yeast cells showed that heterologous expression of Vitis aquaporins increased susceptibility to externally applied boron and H2O2, suggesting the contribution of Vitis aquaporins in the transport of these species
- Effect of ethanol on fluxes of water and protons across the plasma membrane of Saccharomyces cerevisiaePublication . Madeira, Ana; Leitão, Luís; Soveral, Graça; Dias, Patrícia; Prista, Catarina; Moura, Teresa; Loureiro-Dias, Maria C.Plasma membrane integrity, ability to transport substrates and maintenance of homeostasis represent obligatory requirements for efficient ethanol production by Saccharomyces cerevisiae. The effect of ethanol on water diffusion through the bilayer and on mediated water movements was evaluated by stopped flow spectro- scopy. Ethanol stimulated water diffusion and inhibited mediated water transport. In a strain overexpressing AQY1, the activation energy for water transport increased progressively (from 5.9 to 12.7 kcal mol1) for increasing ethanol concentrations (up to 12%v/v), indicating that mediated water transport lost importance as compared with water diffusion through the bilayer. The effect of ethanol on proton movements (inward by passive diffusion and outward through the PMA1 H1-ATPase) was evaluated by measuring the rate of extracellular alcalinization and acidification of unbuffered cell suspensions at different tem- peratures. Above 10% ethanol, H1 diffusion was strongly increased at 30 1C, but no effect was observed at 20 1C up to 12%, indicating the existence of a threshold above which ethanol has a marked effect. On H1 extrusion, ethanol had no effect at 20 1C, but induced a monotonous decrease at higher temperatures. Our results support the view that above a threshold of ethanol concentration, the membrane structure is disrupted, becoming very leaky to H1.
- Exploring the multifaceted potential of a peptide fraction derived from Saccharomyces cerevisiae metabolism: antimicrobial, antioxidant, antidiabetic, and anti-inflammatory propertiesPublication . Branco, Patrícia; Maurício, Elisabete Muchagato; Costa, Ana; Ventura, Diogo; Roma-Rodrigues, Catarina; Duarte, Maria Paula; Fernandes, Alexandra R.; Prista, CatarinaThe rising demand for minimally processed, natural, and healthier food products has led to the search for alternative and multifunctional bioactive food components. Therefore, the present study focuses on the functional proprieties of a peptide fraction derived from Saccharomyces cerevisiae metabolism. The antimicrobial activity of the peptide fraction is evaluated against various foodborne pathogens, including Candida albicans, Candida krusei, Escherichia coli, Listeria monocytogenes, and Salmonella sp. The peptide fraction antioxidant properties are assessed using FRAP and DPPH scavenging capacity assays. Furthermore, the peptide fraction’s cytotoxicity is evaluated in colorectal carcinoma and normal colon epithelial cells while its potential as an antidiabetic agent is investigated through -amylase and -glucosidase inhibitory assays. The results demonstrate that the 2–10 kDa peptide fraction exhibits antimicrobial effects against all tested microorganisms, except C. krusei. The minimal inhibitory concentration for E. coli, L. monocytogenes, and Salmonella sp. remains consistently low, at 0.25 mg/mL, while C. albicans requires a higher concentration of 1.0 mg/mL. Furthermore, the peptide fraction displays antioxidant activity, as evidenced by DPPH radical scavenging activity of 81.03%, and FRAP values of 1042.50 32.5 M TE/mL at 1.0 mg/mL. The peptide fraction exhibits no cytotoxicity in both tumor and non-tumoral human cells at a concentration up to 0.3 mg/mL. Moreover, the peptide fraction presents anti-inflammatory activity, significantly reducing the expression of the TNF gene by more than 29.7% in non-stimulated colon cells and by 50% in lipopolysaccharide-stimulated colon cells. It also inhibits the activity of the carbohydrate digestive enzymes -amylase (IC50 of 199.3 0.9 g/mL) and -glucosidase (IC20 of 270.6 6.0 g/mL). Overall, the findings showed that the peptide fraction exhibits antibacterial, antioxidant, anti-inflammatory, and antidiabetic activity. This study represents a step forward in the evaluation of the functional biological properties of S. cerevisiae bioactive peptides.