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The inositol pyrophosphate pathway, a complex cell signaling network, plays a pivotal role in orchestrating vital cellular processes in the budding yeast, where it regulates cell cycle progression, growth, endocytosis, exocytosis, apoptosis, telomere elongation, ribosome biogenesis, and stress responses. This pathway has gained significant attention in pharmacology and medicine due to its role in generating inositol pyrophosphates, which serve as crucial signaling molecules not only in yeast, but also in higher eukaryotes. As targets for therapeutic development, genetic modifications within this pathway hold promise for disease treatment strategies, offering practical applications in biotechnology. The model organism Saccharomyces cerevisiae , renowned for its genetic tractability, has been instrumental in various studies related to the inositol pyrophosphate pathway. This review is focused on the Kcs1 and Vip1, the two enzymes involved in the biosynthesis of inositol pyrophosphate in S. cerevisiae , highlighting their roles in various cell processes, and providing an up-to-date overview of their relationship with phosphate homeostasis. Moreover, the review underscores the potential applications of these findings in the realms of medicine and biotechnology, highlighting the profound implications of comprehending this intricate signaling network.

Novel complexes of type [Cu(N-N)(dmtp) 2 (OH 2 )](ClO 4 ) 2 ·dmtp (( 1 ) N-N: 2,2'-bipyridine; ( 2 ) L: 1,10-phenantroline and dmtp: 5,7-dimethyl-1,2,4-triazolo[1,5- a ]pyrimidine) were designed in order to obtain biologically active compounds. Complexes were characterized as mononuclear species that crystallized in the space group P-1 of the triclinic system with a square pyramidal geometry around the copper (II). In addition to the antiproliferative effect on murine melanoma B16 cells, complex ( 1 ) exhibited low toxicity on normal BJ cells and did not affect membrane integrity. Complex ( 2 ) proved to be a more potent antimicrobial in comparison with ( 1 ), but both compounds were more active in comparison with dmtp-both against planktonic cells and biofilms. A stronger antimicrobial and antibiofilm effect was noticed against the Gram-positive strains, including methicillin-resistant Staphylococcus aureus (MRSA). Both electron paramagnetic resonance (EPR) and Saccharomyces cerevisiae studies indicated that the complexes were scavengers rather than reactive oxygen species promoters. Their DNA intercalating capacity was evidenced by modifications in both absorption and fluorescence spectra. Furthermore, both complexes exhibited nuclease-like activity, which increased in the presence of hydrogen peroxide.

Natural polyphenols are compounds with important biological implications which include antioxidant and metal-chelating characteristics relevant for their antimicrobial, antitumor, or antiaging potential. The mechanisms linking polyphenols and heavy metals in their concerted actions on cells are not completely elucidated. In this study, we used the model eukaryotic microorganism Saccharomyces cerevisiae to detect the action of widely prevalent natural polyphenols on yeast cells defective in the main components involved in essential heavy metal transport across the plasma membrane. We found that caffeic and gallic acids interfered with Zn accumulation, causing delays in cell growth that were alleviated by Zn supplementation. The flavones morin and quercetin interfered with both Mn and Zn accumulation, which resulted in growth improvement, but supplemental Mn and especially Zn turned the initially benefic action of morin and quercetin into potential toxicity. Our results imply that caution is needed when administering food supplements or nutraceuticals which contain both natural polyphenols and essential elements, especially zinc.

Oleandrin, the main component of Nerium oleander L. extracts, is a cardiotoxic glycoside with multiple pharmacological implications, having potential anti-tumoral and antiviral characteristics. Although it is accepted that the main mechanism of oleandrin action is the inhibition of Na + /K + -ATPases and subsequent increase in cell calcium, many aspects which determine oleandrin cytotoxicity remain elusive. In this study, we used the model Saccharomyces cerevisiae to unravel new elements accounting for oleandrin toxicity. Using cells expressing the Ca 2+ -sensitive photoprotein aequorin, we found that oleandrin exposure resulted in Ca 2+ influx into the cytosol and that failing to pump Ca 2+ from the cytosol to the vacuole increased oleandrin toxicity. We also found that oleandrin exposure induced Mn 2+ accumulation by yeast cells via the plasma membrane Smf1 and that mutants with defects in Mn 2+ homeostasis are oleandrin-hypersensitive. Our data suggest that combining oleandrin with agents which alter Ca 2+ or Mn 2+ uptake may be a way of controlling oleandrin toxicity.

Caffeine-a methylxanthine analogue of the purine bases adenine and guanine-is by far the most consumed neuro-stimulant, being the active principle of widely consumed beverages such as coffee, tea, hot chocolate, and cola. While the best-known action of caffeine is to prevent sleepiness by blocking the adenosine receptors, caffeine exerts a pleiotropic effect on cells, which lead to the activation or inhibition of various cell integrity pathways. The aim of this review is to present the main studies set to investigate the effects of caffeine on cells using the model eukaryotic microorganism Saccharomyces cerevisiae , highlighting the caffeine synergy with external cell stressors, such as irradiation or exposure to various chemical hazards, including cigarette smoke or chemical carcinogens. The review also focuses on the importance of caffeine-related yeast phenotypes used to resolve molecular mechanisms involved in cell signaling through conserved pathways, such as target of rapamycin (TOR) signaling, Pkc1-Mpk1 mitogen activated protein kinase (MAPK) cascade, or Ras/cAMP protein kinase A (PKA) pathway.

In this study we describe the synthesis and characterisation of a new hydrazone-based fluorescent compound that is able to selectively label the endoplasmic reticulum (ER) in yeast and mammalian living cells. The fluorescence properties of the compound depended on the DMSO/water ratio and on the pH. NMR experiments allowed determination of the conformation adopted in various environments. Apart from the convenient synthetic procedure, our compound displays low cell toxicity and blue emission compatible with filters routinely used in fluorescence microscopy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Transient potential receptor (TRP) channels are conserved cation channels found in most eukaryotes, known to sense a variety of chemical, thermal or mechanical stimuli. The Saccharomyces cerevisiae TRPY1 is a TRP channel with vacuolar localization involved in the cellular response to hyperosmotic shock and oxidative stress. In this study, we found that S. cerevisiae diploid cells with heterozygous deletion in TRPY1 gene are haploinsufficient when grown in synthetic media deficient in essential metal ions and that this growth defect is alleviated by non-toxic Mn 2+ surplus. Using cells expressing the Ca 2+ -sensitive photoprotein aequorin we found that Mn 2+ augmented the Ca 2+ flux into the cytosol under oxidative stress, but not under hyperosmotic shock, a trait that was absent in the diploid cells with homozygous deletion of TRPY1 gene. TRPY1 activation under oxidative stress was diminished in cells devoid of Smf1 (the Mn 2+ -high-affinity plasma membrane transporter) but it was clearly augmented in cells lacking Pmr1 (the endoplasmic reticulum (ER)/Golgi located ATPase responsible for Mn 2+ detoxification via excretory pathway). Taken together, these observations lead to the conclusion that increased levels of intracytosolic Mn 2+ activate TRPY1 in the response to oxidative stress.

Epigallocatechin-3-O-gallate (EGCG), the main green tea component, is intensively studied for its anti-oxidant, anti-inflammatory, anti-microbial and anti-cancer effects. In the present study, a screen on a Saccharomyces cerevisiae gene deletion library was performed to identify conditions under which EGCG had deleterious rather than beneficial effects. Two genes were identified whose deletion resulted in sensitivity to EGCG: FET3 and FTR1, encoding the components of the Fet3/Ftr1 high-affinity iron uptake system, also involved in Cu(I)/Cu(II) balance on the surface of yeast cells. The presence of EGCG in the growth medium induced the production of Cu(I), with deleterious effects on fet3Δ and ftr1Δ cells. Additionally, when combined, physiological surpluses of Cu(II) and EGCG acted in synergy not only against fet3Δ and ftr1Δ, but also against wild type cells, by generating surplus Cu(I) in the growth medium. The results imply that caution should be taken when combining EGCG-rich beverages/nutraceuticals with copper-rich foods., (Copyright © 2018. Published by Elsevier Ltd.)

Accumulation of heavy metals without developing toxicity symptoms is a phenotype restricted to a small group of plants called hyperaccumulators, whose metal-related characteristics suggested the high potential in biotechnologies such as bioremediation and bioextraction. In an attempt to extrapolate the heavy metal hyperaccumulating phenotype to yeast, we obtained Saccharomyces cerevisiae cells armed with non-natural metal-binding hexapeptides targeted to the inner face of the plasma membrane, expected to sequester the metal ions once they penetrated the cell. We describe the construction of S. cerevisiae strains overexpressing metal-binding hexapeptides (MeBHxP) fused to the carboxy-terminus of a myristoylated green fluorescent protein (myrGFP). Three non-toxic myrGFP-MeBHxP (myrGFP-H6, myrGFP-C6, and myrGFP-(DE)3) were investigated against an array of heavy metals in terms of their effect on S. cerevisiae growth, heavy metal (hyper) accumulation, and capacity to remove heavy metal from contaminated environments.

In this study we engineered yeast cells armed for heavy metal accumulation by targeting plant metallothioneins to the inner face of the yeast plasma membrane. Metallothioneins (MTs) are cysteine-rich proteins involved in the buffering of excess metal ions, especially Cu(I), Zn(II) or Cd(II). The cDNAs of seven Arabidopsis thaliana MTs (AtMT1a, AtMT1c, AtMT2a, AtMT2b, AtMT3, AtMT4a and AtMT4b) and four Noccaea caerulescens MTs (NcMT1, NcMT2a, NcMT2b and NcMT3) were each translationally fused to the C-terminus of a myristoylation green fluorescent protein variant (myrGFP) and expressed in Saccharomyces cerevisiae cells. The myrGFP cassette introduced a yeast myristoylation sequence which allowed directional targeting to the cytosolic face of the plasma membrane along with direct monitoring of the intracellular localization of the recombinant protein by fluorescence microscopy. The yeast strains expressing plant MTs were investigated against an array of heavy metals in order to identify strains which exhibit the (hyper)accumulation phenotype without developing toxicity symptoms. Among the transgenic strains which could accumulate Cu(II), Zn(II) or Cd(II), but also non-canonical metal ions, such as Co(II), Mn(II) or Ni(II), myrGFP-NcMT3 qualified as the best candidate for bioremediation applications, thanks to the robust growth accompanied by significant accumulative capacity.

To respond to metal surpluses, cells have developed intricate ways of defense against the excessive metallic ions. To understand the ways in which cells sense the presence of toxic concentration in the environment, the role of Ca 2+ in mediating the cell response to high Cu 2+ was investigated in Saccharomyces cerevisiae cells. It was found that the cell exposure to high Cu 2+ was accompanied by elevations in cytosolic Ca 2+ with patterns that were influenced not only by Cu 2+ concentration but also by the oxidative state of the cell. When Ca 2+ channel deletion mutants were used, it was revealed that the main contributor to the cytosolic Ca 2+ pool under Cu 2+ stress was the vacuolar Ca 2+ channel, Yvc1, also activated by the Cch1-mediated Ca 2+ influx. Using yeast mutants defective in the Cu 2+ transport across the plasma membrane, it was found that the Cu 2+ -dependent Ca 2+ elevation could correlate not only with the accumulated metal, but also with the overall oxidative status. Moreover, it was revealed that Cu 2+ and H 2 O 2 acted in synergy to induce Ca 2+ -mediated responses to external stress.

Lanthanides are a group of non-essential elements with important imaging and therapeutic applications. Although trivalent lanthanide ions (Ln³⁺) are used as potent blockers of Ca²⁺ channels, the systematic studies correlating Ln³⁺ accumulation and toxicity to Ca²⁺ channel blocking activity are scarce. In this study, we made use of the eukaryotic model Saccharomyces cerevisiae to investigate the correlation between Ln³⁺ accumulation, their toxicity and their capacity to block the exogenous stress-induced Ca²⁺ influx into the cytosol. It was found that the Ln³⁺ blocked the Ca²⁺ entry into the yeast cells only when present at concentration high enough to allow rapid binding to cell surface. At lower concentrations, Ln³⁺ were taken up by the cell, but Ca²⁺ blockage was no longer achieved. At 1 mM concentration, all ions from the Ln³⁺ series could block Ca²⁺ entry into cytosol with the exception of La³⁺, and to a lesser extent, Pr³⁺ and Nd³⁺. The plasma membrane Ca²⁺-channel Cch1/Mid1 contributed to La³⁺ and Gd³⁺ entry into the cells, with a significant preference for La³⁺. The results open the possibility to obtain cells loaded with controlled amounts and ratios of Ln³⁺.

To gain new insight into the antimicrobial potential of Ailanthus altissima Swingle, ethanol leaf extracts were evaluated for the antifungal effects against the model yeast Saccharomyces cerevisae. The extracts inhibited the yeast growth in a dose-dependent manner, and this effect could be augmented by heat shock, exposure to visible light or exposure to high concentrations of Ca(2+). Using transgenic yeast cells expressing the Ca(2+)-dependent photoprotein, aequorin, it was found that the leaf extracts induced cytosolic Ca(2+) elevation. Experiments on yeast mutants with defects in Ca(2+) transport demonstrated that the cytotoxicity of the A. altissima leaf extracts (AaLEs) was mediated by transient pulses of Ca(2+) ions which were released into the cytosol predominantly from the vacuole. The investigation of the antifungal synergies involving AaLEs may contribute to the development of optimal and safe combination therapies for the treatment of drug-resistant fungal infections.

The involvement of Ca(2+) in the response to high Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+), and Hg(2+) was investigated in Saccharomyces cerevisiae. The yeast cells responded through a sharp increase in cytosolic Ca(2+) when exposed to Cd(2+), and to a lesser extent to Cu(2+), but not to Mn(2+), Co(2+), Ni(2+), Zn(2+), or Hg(2+). The response to high Cd(2+) depended mainly on external Ca(2+) (transported through the Cch1p/Mid1p channel) but also on vacuolar Ca(2+) (released into the cytosol through the Yvc1p channel). The adaptation to high Cd(2+) was influenced by perturbations in Ca(2+) homeostasis. Thus, the tolerance to Cd(2+) often correlated with sharp Cd(2+)-induced cytosolic Ca(2+) pulses, while the Cd(2+) sensitivity was accompanied by the incapacity to rapidly restore the low cytosolic Ca(2+)., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Blueberries (Vaccinium corymbosum L.) are a rich source of antioxidants and their consumption is believed to contribute to food-related protection against oxidative stress. In the present study, the chemoprotective action of blueberry extracts against cadmium toxicity was investigated using a cadmium-hypersensitive strain of Saccharomyces cerevisiae. Four varieties of blueberries were used in the study, and it was found that the extracts with high content of total anthocyanidins exhibited significant protective effect against the toxicity of cadmium and H2O2. Both the blueberry extracts and pure cyanidin exhibited protective effects against cadmium in a dose-dependent manner, but without significantly interfering with the cadmium accumulation by the yeast cells. The results imply that the blueberry extracts might be a potentially valuable food supplement for individuals exposed to high cadmium., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Optical manipulation of Saccharomyces cerevisiae cells with high density green photons conferred protection against the deleterious effects of UV radiation. Combining chemical screening with UV irradiation of yeast cells, it was noted that the high density green photons relied on the presence of intact unfolded protein response (UPR) pathway to exert their protective effect and that the low Ca(2+) conditions boosted the effect. UPR chemical inducers tunicamycin, dithiotreitol and calcium chelators augmented the green light effect in a synergic action against UV-induced damage. Photo-manipulation of cells was a critical factor since the maximum protection was achieved only when cells were pre-exposed to green light., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Green tea extracts (GTEs) as well as their main component, the polyphenol epigallocatechin gallate (EGCG), are known for their versatile antioxidant, antimicrobial, antitumoral or anti-inflammatory effects. In spite of the huge beneficial action, there is increasing evidence that under certain conditions green tea and its components can be detrimental to living organisms. Using Saccharomyces cerevisiae strains with various defects in the response to oxidative stress, we found that GTEs or EGCG act in synergy with visible light, exhibiting either deleterious or protective effects depending on the solvent employed. Similar synergistic effects could be observed under singlet oxygen-generating conditions, such as light exposure in the presence of photosensitizers or UV-A irradiation, therefore solvent variance may represent a powerful tool to modulate the preparation of green tea extracts, depending on the intended target.

Superoxide dismutases (SODs) stand in the prime line of enzymatic antioxidant defense in nearly all eukaryotic cells exposed to oxygen, catalyzing the breakdown of the superoxide anionic radical to O(2) and H(2)O(2). Overproduction of superoxide correlates with numerous pathophysiological conditions, and although the native enzyme can be used as a therapeutic agent in superoxide-associated conditions, synthetic low molecular weight mimetics are preferred in terms of cost, administration mode, and bioavailability. In this study we make use of the model eukaryote Saccharomyces cerevisiae to investigate the SOD-mimetic action of a mononuclear mixed-ligand copper(II) complex, [CuCl(acac)(tmed)] (where acac is acetylacetonate anion and tmed is N,N,N',N'-tetramethylethylenediamine). Taking advantage of an easily reproducible phenotype of yeast cells which lack Cu-Zn SOD (Sod1p), we found that the compound could act either as a superoxide scavenger in the absence of native Sod1p or as a Sod1p modulator which behaved differently under various genetic backgrounds.

Pho84p, the protein responsible for the high-affinity uptake and transport of inorganic phosphate across the plasma membrane, is also involved in the low-affinity uptake of heavy metals in the Saccharomyces cerevisiae cells. In the present study, the effect of PHO84 overexpression upon the heavy metal accumulation by yeast cells was investigated. As PHO84 overexpression triggered the Ire1p-dependent unfolded protein response, abundant plasma membrane Pho84p could be achieved only in ire1Δ cells. Under environmental surplus, PHO84 overexpression augmented the metal accumulation by the wild type, accumulation that was exacerbated by the IRE1 deletion. The pmr1Δ cells, lacking the gene that encodes the P-type ATPase ion pump that transports Ca(2+) and Mn(2+) into the Golgi, hyperaccumulated Mn(2+) even from normal medium when overexpressing PHO84, a phenotype which is rather restricted to metal-hyperaccumulating plants.

One key step of the bioremediation processes designed to clean up heavy metal contaminated environments is growing resistant cells that accumulate the heavy metals to ensure better removal through a combination of biosorption and continuous metabolic uptake after physical adsorption. Saccharomyces cerevisiae cells can easily act as cation biosorbents, but isolation of mutants that are both hyperaccumulating and tolerant to heavy metals proved extremely difficult. Instead, mutants that are hypersensitive to heavy metals due to increased and continuous uptake from the environment were considered, aiming to use such mutants to reduce the heavy metal content of contaminated waters. In this study, the heavy metal hypersensitive yeast strain pmr1Delta was investigated for the ability to remove Mn2+, Cu2+, Co2+, or Cd2+ from synthetic effluents. Due to increased metal accumulation, the mutant strain was more efficient than the wild-type in removing Mn2+, Cu2+, or Co2+ from synthetic effluents containing 1-2 mM cations, with a selectivity and also in removing Mn2+ and Cd2+ from synthetic effluents containing 20-50 microM cations, with a selectivity Mn2+ > Cd2+.

Ni(2+) toxicity can be alleviated in yeast cells by exogenous L-histidine, but not by its enantiomer, D-histidine, nor by other natural L-amino acids tested. We studied the effect of L-histidine upon the accumulation and intracellular distribution of Ni(2+) and found that moderate L-histidine concentrations (less than or equal to those of Ni(2+)) increased cell tolerance without decreasing Ni(2+) accumulation. Although excess L-histidine appeared to lower Ni(2+) accumulation, the concomitant presence of Ni(2+) and L-histidine in the growth medium stimulated each other's uptake.

To identify new proteins involved in Mn2+ homeostasis, we isolated Mn(2+)-resistant mutants of Saccharomyces cerevisiae starting from a calcineurin-deficient, Mn2+ hypersensitive strain (delta cmp1 delta cmp2). The mutations were found to lie in the PMR1 gene, known to encode a "P-type" Ca(2+)-ATPase that transports Ca2+ and Mn2+ from the cytosol to the Golgi apparatus. A second gene, AHP1, was cloned as a suppressor of the Mn2+ tolerance of a delta cmp1 delta cmp2 pmr1 mutant. Ahp1p was recently described as a thioredoxin peroxidase type II, an antioxidant protein with alkyl hydroperoxide defense properties in yeast. AHP1 disruption in strain W303 decreased tolerance to Mn2+ and H2O2. We found that a GFP-Ahp1p fusion construct was in the cytosol when cells were grown in glucose, and in the mitochondria when cells were grown in oleate. Based on Mn2+ transport data, we concluded that Ahp1p is involved in cellular Mn2+ homeostasis in trafficking of Mn2+ from cytosol to mitochondria and from cytosol for export across the plasma membrane.

In a search for components involved in Mn2+ homeostasis in the budding yeast Saccharomyces cerevisiae, we isolated a mutant with modifications in Mn2+ transport. The mutation was found to be located in HIP1, a gene known to encode a high-affinity permease for histidine. The mutation, designated hip1-272, caused a frameshift that resulted in a stop codon at position 816 of the 1812-bp ORF. This mutation led to Mn2+ resistance, whereas the corresponding null mutation did not. Both hip1-272 cells and the null mutant exhibited low tolerance to divalent cations such as Co2+, Ni2+, Zn2+, and Cu2+. The Mn2+ phenotype was not influenced by supplementary histidine in either mutant, whereas the sensitivity to other divalent cations was alleviated by the addition of histidine. The cellular Mn2+ content of the hip1-272 mutant was lower than that of wild type or null mutant, due to increased rates of Mn2+ efflux. We propose that Hiplp is involved in Mn2+ transport, carrying out a function related to Mn2+ export.

The fate of Mn2+ inside Saccharomyces cerevisiae cells was monitored during import and export processes, using information from electron paramagnetic resonance (EPR) spectroscopy analysis. EPR spectra showed that when entering the cell in high number, manganese ions immediately precipitated. If recorded under conditions that favored manganese efflux, EPR spectra indicated that only osmotically free ions were exported and that bound manganese had to turn into the soluble form before being able to leave the cell.

The role of protein phosphatase 2B (PP2B/calcineurin) of Saccharomyces cerevisiae in the tolerance to divalent cations was investigated. PP2B-deficient mutants were found to be sensitive to MnCl2, but not to ZnCl2, CuCl2, NiCl2 and CoCl2. By measuring both manganese uptake and its efflux, it was found that the sensitivity of the mutant cells was due to an increase in manganese uptake and that the wild-type cells were able to prevent manganese entry into the cells, rather than export it in a more efficient manner. In the presence of the immunosuppressant FK506, the behavior of wild-type cells became similar to that of PP2B mutants. Out of various divalent cations tested, externally added magnesium ions were able to block manganese uptake in both wild-type and PP2B mutant strains.