Potential Use of Torulaspora delbrueckii As a New Source of Mannoproteins of Oenological Interest.

In this work, three MP extracts obtained from Torulaspora delbrueckii were added to red wine, and the changes in phenolic composition, color, and astringency were evaluated by HPLC-DAD-ESI-MS, tristimulus colorimetry, and sensory analysis, respectively. The MP extracts modified wine phenolic composition differently depending on the type of MP. Moreover, two MP extracts were able to reduce wine astringency. The fact that the MP-treated wines showed an increased flavanol content suggests the formation of MP-flavanol aggregates that remain in solution. Furthermore, the formation of these aggregates may hinder the interaction of flavanols with salivary proteins in the mouth. The effect of these MPs might be associated with their larger size, which could influence their ability to bind flavanols and salivary proteins. However, one of the astringent-modulating MPs also produced a loss of color, highlighting the importance of assessing the overall impact of MPs on the organoleptic properties of wine.

Possible Role of High-Molecular-Weight Salivary Proteins in Astringency Development.

Since the initial findings that food tannin/salivary protein interaction and subsequent precipitation is the main cause of the astringency development, numerous studies have concentrated on the supramolecular characterization of these bindings. Most of these works have focused on the low-molecular-weight salivary proteins, in particular proline-rich proteins, hardly considering the involvement of the high-molecular-weight salivary proteins (HMWSPs). Herein, different techniques such as fluorescence quenching, Isothermal Titration Calorimetry and HPLC-MS-DAD were employed to determine the occurrence of molecular interactions between three HMWSPs, namely, mucin, α-amylase and albumin, and a complex extract of tannins composed mainly of flavan-3-ols. The obtained results prove the capability of the three HMWSPs to effectively interact with the flavan-3-ol extract, involving different forces and action mechanisms. Flavan-3-ols are capable of interacting with mucins by a mechanism that includes the formation of stable ground-state complexes that led to approximately 90% flavan-3-ol precipitation, while for albumin and α-amylase, the interaction model of a "sphere of action" was established, which represented only 20% flavan-3-ol precipitation. These data highlight the relevance of including HMWSPs in astringency analyses, paying special heed to the role of mucins in the interaction and subsequent precipitation of dietary tannins.

Supramolecular Study of the Interactions between Malvidin-3-O-Glucoside and Wine Phenolic Compounds: Influence on Color.

Supramolecular study of the interactions between the major wine anthocyanin, malvidin-3-O-glucoside (Mv3G) and different wine phenolic compounds (quercetin 3-O-ß-glucopyranoside (QG), caffeic acid, (-)-epicatechin, (+)-catechin, and gallic acid) has been performed at two different molar ratios (1:1 and 1:2) in acidic medium where flavylium cation predominates (pH ≤ 2). Color variations have been evaluated by differential colorimetry using CIELAB color space. These studies have been complemented with isothermal titration calorimetry assays and molecular dynamics simulations. The color of Mv3G flavylium cation is modified by the interaction with QG toward more bluish and intense colors. Interaction constants between the anthocyanin and the different phenolic compounds were obtained, ranging from 9.72 × 108 M-1 for QG to 1.50 × 102 M-1 for catechin. Hydrophobic interactions and H-bonds are the main driving forces in the pigment/copigment aggregation, except for the interactions where caffeic acid is involved, in which hydrophobic interactions acquire greater preponderance.

Role of Anthocyanins in the Interaction between Salivary Mucins and Wine Astringent Compounds.

Wine astringency is a very complex sensation whose complete mechanism has not been entirely described. Not only salivary proline-rich proteins (PRPs) are involved in its development; salivary mucins can also play an important role. On the other hand, it has been described that anthocyanins can interact with PRPs, but there is no information about their potential role on the interactions with mucins. In this work, the molecular interactions between salivary mucins (M) and different wine phenolic compounds, such as catechin (C), epicatechin (E) and quercetin 3-ß-glucopyranoside (QG), as well as the effect of the anthocyanin malvidin 3-O-glucoside (Mv) on the interactions with mucins, were assessed by isothermal titration calorimetry (ITC). Results showed that the interaction between anthocyanin and mucins is stronger than that of both flavanols analyzed, since the affinity constant values were 10 times higher for anthocyanin than for catechin, the only flavanol showing interaction in binary assay. Moreover, at the concentration at which polyphenols are usually found in wine, flavonols seem not to be involved in the interactions with mucins. These results showed, for the first time, the importance of wine anthocyanins in the mechanisms of astringency involving high-molecular-weight salivary proteins like mucins.

First insights into the binding mechanism and colour effect of the interaction of grape seed 11S globulin with malvidin 3-O-glucoside by fluorescence spectroscopy, differential colorimetry and molecular modelling.

Recently, the search for alternative proteins endogenous to grapes to be used as wine colour protecting agents became an important research trend. In this study, the molecular interaction between the grape seed 11S globulin from winemaking by-product and malvidin-3-O-glucoside was investigated by fluorescence, differential colorimetry and molecular modelling. Fluorescence studies revealed the formation of grape seed protein- pigment complex whose KS was 8.5 × 104 M-1 and binding sites, n = 1.3. Malvidin-3-O-glucoside showed darker and more vivid bluish colour of in the presence of 11S globulin, suggesting the flavylium cation protection in a hydrophobic region of the protein. Docking analysis and molecular dynamics simulation indicated that malvidin-3-O-glucoside interacts mainly with the acidic subunit (40 kDa) of the 11S globulin monomer (60 kDa). An average of two hydrogen bonds and Van der Wall forces were the main interaction forces found for the protein-pigment complex, whose stability was confirmed by root-means-square deviation.

Effect of the addition of soluble polysaccharides from red and white grape skins on the polyphenolic composition and sensory properties of Tempranillo red wines.

Soluble polysaccharides from white (PSW) and red (PSR) grape skins were obtained to be evaluated as potential modulators of the unbalanced astringency of a Tempranillo red wine. The modulation of astringency was evaluated by a sensory panel and it seemed to be related to the changes in the polyphenolic profile. Isothermal Titration Calorimetry (ITC) studies, employed to characterize flavan-3-ol-polysaccharide interactions, showed that PSR decreased noticeably wine astringency causing a great flavan-3-ol loss (ca. 40 %), since they interacted more spontaneously with the flavan-3-ols (ca. ΔGtotal = -2.14 × 104 cal/mol) than PSW (ca. ΔGtotal = -1.32 × 104 cal/mol). The strength of these interactions seems to be related to the polysaccharide molecular size and to the presence of arabinogalactans in the structure. On the contrary, PSW showed no relevant effects on wine astringency. Furthermore, potential variations of color were also assessed and no deleterious effect was observed after the addition of any polysaccharide.

Effect of different yeast mannoproteins on the interaction between wine flavanols and salivary proteins.

Unbalanced wine astringency, caused by a gap between phenolic and technological grape maturities, is one of the consequences of the global climate change in the vitiviniculture. To resolve it, potential strategies are being currently used, like the addition of commercial yeast mannoproteins (MPs) to wines. In this work, the main interactions responsible for the wine astringent sensation, namely, interactions between human salivary proteins and wine flavanols have been studied by Dynamic Light Scattering (DLS) and liquid chromatography coupled to DAD and MS detectors (HPLC-DAD-MS), in presence or absence of two MPs with different saccharide/protein ratio. The results indicate that there are differences on the substrate specificity for each mannoprotein and that its action mechanism could change not only depending on the mannoprotein composition but also on the flavanol structure. MPs with elevated carbohydrate content could act thought the stabilization of soluble aggregates with human salivary proteins and flavanols, mainly non-galloylated flavanol oligomers, whereas MPs with higher protein percentage mostly could precipitate flavanols (mainly non-galloylated ones with low degree of polymerization) which partially prevents the formation of insoluble flavanol-salivary protein aggregates.

A novel checkpoint pathway controls actomyosin ring constriction trigger in fission yeast.

In fission yeast, the septation initiation network (SIN) ensures temporal coordination between actomyosin ring (CAR) constriction with membrane ingression and septum synthesis. However, questions remain about CAR regulation under stress conditions. We show that Rgf1p (Rho1p GEF), participates in a delay of cytokinesis under cell wall stress (blankophor, BP). BP did not interfere with CAR assembly or the rate of CAR constriction, but did delay the onset of constriction in the wild type cells but not in the rgf1Δ cells. This delay was also abolished in the absence of Pmk1p, the MAPK of the cell integrity pathway (CIP), leading to premature abscission and a multi-septated phenotype. Moreover, cytokinesis delay correlates with maintained SIN signaling and depends on the SIN to be achieved. Thus, we propose that the CIP participates in a checkpoint, capable of triggering a CAR constriction delay through the SIN pathway to ensure that cytokinesis terminates successfully.

Cell Wall Mannoproteins from Yeast Affect Salivary Protein-Flavanol Interactions through Different Molecular Mechanisms.

It is known that interactions between wine flavanols and salivary proline-rich proteins (PRPs) are one of the main factors responsible for wine astringency. The addition of commercial yeast mannoproteins (MPs) to wines has been pointed to as a possible tool to modulate the excessive astringency due to a lack of phenolic maturity at harvest time that might occur as a consequence of global climate change. The aim of this work was to study by isothermal titration calorimetry and molecular dynamics simulation the molecular mechanisms by which mannoproteins could modulate astringency elicited by tannins and if it can be influenced by mannoprotein composition. Results obtained indicate that the MPs assayed had an important impact on astringency through the formation of ternary aggregates with different solubilities or by preventing the flavanol-PRP interaction by a competitive mechanism, although in a different strength, depending on the size and the compositional characteristic of the mannoprotein.

Rgf1p (Rho1p GEF) is required for double-strand break repair in fission yeast.

Rho GTPases are conserved molecules that control cytoskeletal dynamics. These functions are expedited by Rho GEFs that stimulate the release of GDP to enable GTP binding, thereby allowing Rho proteins to initiate intracellular signaling. How Rho GEFs and Rho GTPases protect cells from DNA damage is unknown. Here, we explore the extreme sensitivity of a deletion mutation in the Rho1p exchange factor Rgf1p to the DNA break/inducing antibiotic phleomycin (Phl). The Rgf1p mutant cells are defective in reentry into the cell cycle following the induction of severe DNA damage. This phenotype correlates with the inability of rgf1Δ cells to efficiently repair fragmented chromosomes after Phl treatment. Consistent with this observation Rad11p (ssDNA binding protein, RPA), Rad52p, Rad54p and Rad51p, which facilitate strand invasion in the process of homology-directed repair (HDR), are permanently stacked in Phl-induced foci in rgf1Δ cells. These phenotypes are phenocopied by genetic inhibition of Rho1p. Our data provide evidence that Rgf1p/Rho1p activity positively controls a repair function that confers resistance against the anti-cancer drug Phl.

The putative exchange factor Gef3p interacts with Rho3p GTPase and the septin ring during cytokinesis in fission yeast.

The small GTP-binding proteins of the Rho family and its regulatory proteins play a central role in cytokinetic actomyosin ring assembly and cytokinesis. Here we show that the fission yeast guanine nucleotide exchange factor Gef3p interacts with Rho3p at the division site. Gef3p contains a putative DH homology domain and a BAR/IMD-like domain. The protein localized to the division site late in mitosis, where it formed a ring that did not constrict with actomyosin ring (cytokinetic actomyosin ring) invagination; instead, it split into a double ring that resembled the septin ring. Gef3p co-localized with septins and Mid2p and required septins and Mid2p for its localization. Gef3p interacts physically with the GTP-bound form of Rho3p. Although Gef3p is not essential for cell separation, the simultaneous disruption of gef3(+) and Rho3p-interacting proteins, such as Sec8p, an exocyst component, Apm1p, a subunit of the clathrin adaptor complex or For3p, an actin-polymerizing protein, yielded cells with strong defects in septation and polarity respectively. Our results suggest that interactions between septins and Rho-GEFs provide a new targeting mechanism for GTPases in cytokinesis, in this case probably contributing to Rho3p function in vesicle tethering and vesicle trafficking in the later steps of cell separation.

The checkpoint-dependent nuclear accumulation of Rho1p exchange factor Rgf1p is important for tolerance to chronic replication stress.

Guanine nucleotide exchange factors control many aspects of cell morphogenesis by turning on Rho-GTPases. The fission yeast exchange factor Rgf1p (Rho gef1) specifically regulates Rho1p during polarized growth and localizes to cortical sites. Here we report that Rgf1p is relocalized to the cell nucleus during the stalled replication caused by hydroxyurea (HU). Import to the nucleus is mediated by a nuclear localization sequence at the N-terminus of Rgf1p, whereas release into the cytoplasm requires two leucine-rich nuclear export sequences at the C-terminus. Moreover, Rgf1p nuclear accumulation during replication arrest depends on the 14-3-3 chaperone Rad24p and the DNA replication checkpoint kinase Cds1p. Both proteins control the nuclear accumulation of Rgf1p by inhibition of its nuclear export. A mutant, Rgf1p-9A, that substitutes nine serine potential phosphorylation Cds1p sites for alanine fails to accumulate in the nucleus in response to replication stress, and this correlates with a severe defect in survival in the presence of HU. In conclusion, we propose that the regulation of Rgf1p could be part of the mechanism by which Cds1p and Rad24p promote survival in the presence of chronic replication stress. It will be of general interest to understand whether the same is true for homologues of Rgf1p in budding yeast and higher eukaryotes.

The fission yeast cell wall stress sensor-like proteins Mtl2 and Wsc1 act by turning on the GTPase Rho1p but act independently of the cell wall integrity pathway.

Sensing stressful conditions that affect the cell wall reorganization is important for yeast survival. Here, we studied two proteins SpWsc1p and SpMtl2p with structural features indicative of plasma membrane-associated cell wall sensors. We found that Mtl2p and Wsc1p act by turning on the Rho1p GTPase. Each gene could be deleted individually without affecting viability, but the deletion of both was lethal and this phenotype was rescued by overexpression of the genes encoding either Rho1p or its GDP/GTP exchange factors (GEFs). In addition, wsc1Δ and mtl2Δ cells showed a low level of Rho1p-GTP under cell wall stress. Mtl2p-GFP (green fluorescent protein) localized to the cell periphery and was necessary for survival under different types of cell wall stress. Wsc1p-GFP was concentrated in patches at the cell tips, it interacted with the Rho-GEF Rgf2p, and its overexpression activated cell wall biosynthesis. Our results are consistent with the notion that cell wall assembly is regulated by two different networks involving Rho1p. One includes signaling from Mtl2p through Rho1p to Pck1p, while the second one implicates signaling from Wsc1p and Rgf2p through Rho1p to activate glucan synthase (GS). Finally, signaling through the mitogen-activated protein kinase (MAPK) Pmk1p remained active in mtl2Δ and wsc1Δ disruptants exposed to cell wall stress, suggesting that the cell wall stress-sensing spectrum of Schizosaccharomyces pombe sensor-like proteins differs from that of Saccharomyces cerevisiae.