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1.
FEBS Lett ; 598(13): 1655-1666, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38750637

RESUMO

Cymoxanil (CYM) is a widely used synthetic acetamide fungicide, but its biochemical mode of action remains elusive. Since CYM inhibits cell growth, biomass production, and respiration in Saccharomyces cerevisiae, we used this model to characterize the effect of CYM on mitochondria. We found it inhibits oxygen consumption in both whole cells and isolated mitochondria, specifically inhibiting cytochrome c oxidase (CcO) activity during oxidative phosphorylation. Based on molecular docking, we propose that CYM blocks the interaction of cytochrome c with CcO, hampering electron transfer and inhibiting CcO catalytic activity. Although other targets cannot be excluded, our data offer valuable insights into the mode of action of CYM that will be instrumental in driving informed management of the use of this fungicide.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Fungicidas Industriais , Mitocôndrias , Simulação de Acoplamento Molecular , Saccharomyces cerevisiae , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Fungicidas Industriais/farmacologia , Fungicidas Industriais/toxicidade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores
2.
Cell Death Differ ; 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38714881

RESUMO

The study of cell death mechanisms in fungi, particularly yeasts, has gained substantial interest in recent decades driven by the potential for biotechnological advancements and therapeutic interventions. Examples include the development of robust yeast strains for industrial fermentations and high-value compound production, novel food preservation strategies against spoilage yeasts, and the identification of targets for treating fungal infections in the clinic. In this review, we discuss a wide range of methods to characterize cellular alterations associated with yeast cell death, noting the advantages and limitations. We describe assays to monitor reversible events versus those that mark a commitment to cell death (point-of-no-return), as these distinctions are important to decipher the underlying regulatory mechanisms. Several well-known challenges remain, including the varied susceptibilities to death within a cell population and the delineation of detailed cell death mechanisms. The identification and characterization of morphologically distinct subsets of dying yeast cells within dynamic yeast populations provides opportunities to reveal novel vulnerabilities and survival mechanisms. Elucidating the intricacies of yeast regulated cell death (yRCD) will contribute to the advancement of scientific knowledge and foster breakthrough discoveries with broad-ranging implications.

3.
Biomolecules ; 14(4)2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38672489

RESUMO

Colorectal cancer (CRC) is a leading cause of death worldwide. Conventional therapies are available with varying effectiveness. Acetate, a short-chain fatty acid produced by human intestinal bacteria, triggers mitochondria-mediated apoptosis preferentially in CRC but not in normal colonocytes, which has spurred an interest in its use for CRC prevention/therapy. We previously uncovered that acetate-induced mitochondrial-mediated apoptosis in CRC cells is significantly enhanced by the inhibition of the lysosomal protease cathepsin D (CatD), which indicates both mitochondria and the lysosome are involved in the regulation of acetate-induced apoptosis. Herein, we sought to determine whether mitochondrial function affects CatD apoptotic function. We found that enhancement of acetate-induced apoptosis by CatD inhibition depends on oligomycin A-sensitive respiration. Mechanistically, the potentiating effect is associated with an increase in cellular and mitochondrial superoxide anion accumulation and mitochondrial mass. Our results provide novel clues into the regulation of CatD function and the effect of tumor heterogeneity in the outcome of combined treatment using acetate and CatD inhibitors.


Assuntos
Apoptose , Catepsina D , Neoplasias Colorretais , Mitocôndrias , Oligomicinas , Humanos , Acetatos/farmacologia , Apoptose/efeitos dos fármacos , Catepsina D/metabolismo , Catepsina D/antagonistas & inibidores , Linhagem Celular Tumoral , Respiração Celular/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Colorretais/tratamento farmacológico , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Oligomicinas/farmacologia
4.
Appl Microbiol Biotechnol ; 108(1): 10, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38170307

RESUMO

Due to increasing demand for high and stable crop production, human populations are highly dependent on pesticide use for growing and storing food. Environmental monitoring of these agrochemicals is therefore of utmost importance, because of their collateral effects on ecosystem and human health. Even though most current-use analytical methods achieve low detection limits, they require procedures that are too complex and costly for routine monitoring. As such, there has been an increased interest in biosensors as alternative or complementary tools to streamline detection and quantification of environmental contaminants. In this work, we developed a biosensor for environmental monitoring of tebuconazole (TEB), a common agrochemical fungicide. For that purpose, we engineered S. cerevisiae cells with a reporter gene downstream of specific promoters that are expressed after exposure to TEB and characterized the sensitivity and specificity of this model system. After optimization, we found that this easy-to-use biosensor consistently detects TEB at concentrations above 5 µg L-1 and does not respond to realistic environmental concentrations of other tested azoles, suggesting it is specific. We propose the use of this system as a complementary tool in environmental monitoring programs, namely, in high throughput scenarios requiring screening of numerous samples. KEY POINTS: • A yeast-based biosensor was developed for environmental monitoring of tebuconazole. •The biosensor offers a rapid and easy method for tebuconazole detection ≥ 5 µg L-1. •The biosensor is specific to tebuconazole at environmentally relevant concentrations.


Assuntos
Técnicas Biossensoriais , Fungicidas Industriais , Humanos , Saccharomyces cerevisiae/genética , Ecossistema , Monitoramento Ambiental
5.
Apoptosis ; 27(5-6): 368-381, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35362903

RESUMO

Proteins of the Bcl-2 protein family, including pro-apoptotic Bax and anti-apoptotic Bcl-xL, are critical for mitochondrial-mediated apoptosis regulation. Since yeast lacks obvious orthologs of Bcl-2 family members, heterologous expression of these proteins has been used to investigate their molecular and functional aspects. Active Bax is involved in the formation of mitochondrial outer membrane pores, through which cytochrome c (cyt c) is released, triggering a cascade of downstream apoptotic events. However, when in its inactive form, Bax is largely cytosolic or weakly bound to mitochondria. Given the central role of Bax in apoptosis, studies aiming to understand its regulation are of paramount importance towards its exploitation as a therapeutic target. So far, studies taking advantage of heterologous expression of human Bax in yeast to unveil regulation of Bax activation have relied on the use of artificial mutated or mitochondrial tagged Bax for its activation, rather than the wild type Bax (Bax α). Here, we found that cell death could be triggered in yeast cells heterologoulsy expressing Bax α with concentrations of acetic acid that are not lethal to wild type cells. This was associated with Bax mitochondrial translocation and cyt c release, closely resembling the natural Bax function in the cellular context. This regulated cell death process was reverted by co-expression with Bcl-xL, but not with Bcl-xLΔC, and in the absence of Rim11p, the yeast ortholog of mammalian GSK3ß. This novel system mimics human Bax α regulation by GSK3ß and can therefore be used as a platform to uncover novel Bax regulators and explore its therapeutic modulation.


Assuntos
Citocromos c , Saccharomyces cerevisiae , Ácido Acético , Animais , Apoptose/genética , Proteínas de Transporte , Citocromos c/genética , Citocromos c/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Mamíferos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismo
6.
Front Cell Dev Biol ; 9: 642375, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34249904

RESUMO

Acetic acid has long been considered a molecule of great interest in the yeast research field. It is mostly recognized as a by-product of alcoholic fermentation or as a product of the metabolism of acetic and lactic acid bacteria, as well as of lignocellulosic biomass pretreatment. High acetic acid levels are commonly associated with arrested fermentations or with utilization as vinegar in the food industry. Due to its obvious interest to industrial processes, research on the mechanisms underlying the impact of acetic acid in yeast cells has been increasing. In the past twenty years, a plethora of studies have addressed the intricate cascade of molecular events involved in cell death induced by acetic acid, which is now considered a model in the yeast regulated cell death field. As such, understanding how acetic acid modulates cellular functions brought about important knowledge on modulable targets not only in biotechnology but also in biomedicine. Here, we performed a comprehensive literature review to compile information from published studies performed with lethal concentrations of acetic acid, which shed light on regulated cell death mechanisms. We present an historical retrospective of research on this topic, first providing an overview of the cell death process induced by acetic acid, including functional and structural alterations, followed by an in-depth description of its pharmacological and genetic regulation. As the mechanistic understanding of regulated cell death is crucial both to design improved biomedical strategies and to develop more robust and resilient yeast strains for industrial applications, acetic acid-induced cell death remains a fruitful and open field of study.

7.
Int J Biol Macromol ; 171: 343-357, 2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33421469

RESUMO

Lactoferrin (Lf) is a bioactive milk-derived protein with remarkable wide-spectrum antifungal activity. To deepen our understanding of the molecular mechanisms underlying Lf cytotoxicity, the role of plasma membrane ergosterol- and sphingolipid-rich lipid rafts and their association with the proton pump Pma1p was explored. Pma1p was previously identified as a Lf-binding protein. Results showed that bovine Lf (bLf) perturbs ergosterol-rich lipid rafts organization by inducing intracellular accumulation of ergosterol. Using yeast mutant strains lacking lipid rafts-associated proteins or enzymes involved in the synthesis of ergosterol and sphingolipids, we found that perturbations in the composition of these membrane domains increase resistance to bLf-induced yeast cell death. Also, when Pma1p-lipid rafts association is compromised in the Pma1-10 mutant and in the absence of the Pma1p-binding protein Ast1p, the bLf killing activity is impaired. Altogether, results showed that the perturbation of lipid rafts and the inhibition of both Pma1p and V-ATPase activities mediate the antifungal activity of bLf. Since it is suggested that the combination of conventional antifungals with lipid rafts-disrupting compounds is a powerful antifungal approach, our data will help to pave the way for the use of bLf alone or in combination for the treatment/eradication of clinically and agronomically relevant yeast pathogens/fungi.


Assuntos
Antifúngicos/farmacologia , Lactoferrina/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , ATPases Translocadoras de Prótons/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Farmacorresistência Fúngica , Ergosterol/metabolismo , Filipina , Proteínas de Fluorescência Verde/análise , Microdomínios da Membrana/química , Mutação Puntual , ATPases Translocadoras de Prótons/biossíntese , ATPases Translocadoras de Prótons/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/biossíntese , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Vacúolos/efeitos dos fármacos , Vacúolos/enzimologia , beta-Ciclodextrinas/farmacologia
8.
Curr Med Chem ; 27(24): 4087-4108, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-29848266

RESUMO

Colorectal Cancer (CRC) is a major cause of cancer-related death worldwide. CRC increased risk has been associated with alterations in the intestinal microbiota, with decreased production of Short Chain Fatty Acids (SCFAs). SCFAs produced in the human colon are the major products of bacterial fermentation of undigested dietary fiber and starch. While colonocytes use the three major SCFAs, namely acetate, propionate and butyrate, as energy sources, transformed CRC cells primarily undergo aerobic glycolysis. Compared to normal colonocytes, CRC cells exhibit increased sensitivity to SCFAs, thus indicating they play an important role in cell homeostasis. Manipulation of SCFA levels in the intestine, through changes in microbiota, has therefore emerged as a potential preventive/therapeutic strategy for CRC. Interest in understanding SCFAs mechanism of action in CRC cells has increased in the last years. Several SCFA transporters like SMCT-1, MCT-1 and aquaporins have been identified as the main transmembrane transporters in intestinal cells. Recently, it was shown that acetate promotes plasma membrane re-localization of MCT-1 and triggers changes in the glucose metabolism. SCFAs induce apoptotic cell death in CRC cells, and further mechanisms have been discovered, including the involvement of lysosomal membrane permeabilization, associated with mitochondria dysfunction and degradation. In this review, we will discuss the current knowledge on the transport of SCFAs by CRC cells and their effects on CRC metabolism and survival. The impact of increasing SCFA production by manipulation of colon microbiota on the prevention/therapy of CRC will also be addressed.


Assuntos
Neoplasias Colorretais , Dieta , Fibras na Dieta , Ácidos Graxos Voláteis , Humanos
9.
DNA Repair (Amst) ; 73: 28-33, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30502926

RESUMO

Cisplatin is a highly effective chemotherapeutic drug acting as a DNA-damaging agent that induces apoptosis of rapidly proliferating cells. Unfortunately, cellular resistance still occurs. Mutations in p53 in a large fraction of tumor cells contribute to defects in apoptotic pathways and drug resistance. To uncover new strategies to eliminate tumors through a p53-independent pathway, we established a simplified model devoid of p53 to study cisplatin-induced regulated cell death, using the yeast Saccharomyces cerevisiae. We previously showed that cisplatin induces an active form of cell death accompanied by DNA condensation and fragmentation/degradation, but no significant mitochondrial dysfunction. We further demonstrated that proteasome inhibition, either with MG132 or genetically, increased resistance to cisplatin. In this study, we sought to determine how proteasome inhibition is important for cisplatin resistance by analyzing how it affects several phenotypes associated with the DNA damage response. We found MG132 does not seem to affect the activation of the DNA damage response or increase damage tolerance. Moreover, central modulators of the DNA damage response are not required for cisplatin resistance imparted by MG132. These results suggest the proteasome is involved in modulation of cisplatin toxicity downstream of DNA damage. Proteasome inhibitors can sensitize tumor cells to cisplatin, but protect others from cisplatin-induced cell death. Elucidation of this mechanism will therefore aid in the development of new strategies to increase the efficacy of chemotherapy.


Assuntos
Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Dano ao DNA , Inibidores de Proteassoma/farmacologia , Apoptose/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética
10.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1863(6): 576-583, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29496584

RESUMO

Changes in sphingolipid metabolism have been linked to modulation of cell fate in both yeast and mammalian cells. We previously assessed the role of sphingolipids in cell death regulation using a well characterized yeast model of acetic acid-induced regulated cell death, finding that Isc1p, inositol phosphosphingolipid phospholipase C, plays a pro-death role in this process. Indeed, isc1∆ mutants exhibited a higher resistance to acetic acid associated with reduced mitochondrial alterations. Here, we show that Isc1p is regulated by Sch9p under acetic acid stress, since both single and double mutants lacking Isc1p or/and Sch9p have the same resistant phenotype, and SCH9 deletion leads to a higher retention of Isc1p in the endoplasmic reticulum upon acetic acid exposure. We also found that the higher resistance of all mutants correlates with higher levels of endogenous mitochondrial phosphorylated long chain bases (LCBPs), suggesting that changing the sphingolipid balance in favour of LCBPs in mitochondria results in increased survival to acetic acid. In conclusion, our results suggest that Sch9p pathways modulate acetic acid-induced cell death, through the regulation of Isc1p cellular distribution, thus affecting the sphingolipid balance that regulates cell fate.


Assuntos
Ácido Acético/farmacologia , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fosfolipases Tipo C/metabolismo , Retículo Endoplasmático/genética , Mitocôndrias/genética , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fosfolipases Tipo C/genética
11.
Sci Rep ; 6: 39329, 2016 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-27982135

RESUMO

Silk-elastin-like proteins (SELPs) are a family of genetically engineered recombinant protein polymers exhibiting mechanical and biological properties suited for a wide range of applications in the biomedicine and materials fields. They are being explored as the next generation of biomaterials but low productivities and use of antibiotics during production undermine their economic viability and safety. We have developed an industrially relevant, scalable, fed-batch process for the high level production of a novel SELP in E. coli in which the commonly used antibiotic selection marker of the expression vector is exchanged for a post segregational suicide system, the separate-component-stabilisation system (SCS). SCS significantly augments SELP productivity but also enhances the product safety profile and reduces process costs by eliminating the use of antibiotics. Plasmid content increased following induction but no significant differences in plasmid levels were discerned when using SCS or the antibiotic selection markers under the controlled fed-batch conditions employed. It is suggested that the absence of competing plasmid-free cells improves host cell viability and enables increased productivity with SCS. With the process developed, 12.8 g L-1 purified SELP was obtained, this is the highest SELP productivity reported to date and clearly demonstrates the commercial viability of these promising polymers.


Assuntos
Biotecnologia/métodos , Elastina/metabolismo , Escherichia coli/metabolismo , Genética Microbiana/métodos , Proteínas Recombinantes/metabolismo , Seleção Genética , Elastina/genética , Escherichia coli/crescimento & desenvolvimento , Instabilidade Genômica , Viabilidade Microbiana , Plasmídeos , Proteínas Recombinantes/genética
12.
Microb Cell ; 3(10): 500-510, 2016 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-28357318

RESUMO

Mitochondrial outer membrane permeabilization is a key event in apoptosis processes leading to the release of lethal factors. We have previously shown that absence of the ADP/ATP carrier (AAC) proteins (yeast orthologues of mammalian ANT proteins) increased the resistance of yeast cells to acetic acid, preventing MOMP and the release of cytochrome c from mitochondria during acetic acid - induced apoptosis. On the other hand, deletion of POR1 (yeast voltage-dependent anion channel - VDAC) increased the sensitivity of yeast cells to acetic acid. In the present work, we aimed to further characterize the role of yeast VDAC in acetic acid - induced apoptosis and assess if it functionally interacts with AAC proteins. We found that the sensitivity to acetic acid resulting from POR1 deletion is completely abrogated by the absence of AAC proteins, and propose that Por1p acts as a negative regulator of acetic acid - induced cell death by a mechanism dependent of AAC proteins, by acting on AAC - dependent cytochrome c release. Moreover, we show that Por1p has a role in mitochondrial fusion that, contrary to its role in apoptosis, is not affected by the absence of AAC, and demonstrate that mitochondrial network fragmentation is not sufficient to induce release of cytochrome c or sensitivity to acetic acid - induced apoptosis. This work enhances our understanding on cytochrome c release during cell death, which may be relevant in pathological scenarios where MOMP is compromised.

13.
Mol Cell Endocrinol ; 415: 64-75, 2015 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-26265449

RESUMO

Thyroid cancer (TC) is frequently associated with BRAF or RAS oncogenic mutations and RET/PTC rearrangements, with aberrant RAF-MEK-ERK and/or PI3K pathway activation. BRAF underlies ERK activation in most TC cells, but not in TPC-1 cells with RET/PTC1 rearrangement. Here, we show that depletion of RAF-1, a RAF family member with a poorly defined role in TC, decreases proliferation and increases apoptosis in TPC-1 cells and, less significantly, in cells harboring a BRAF(V600E) or HRAS(G13R) mutations, but without affecting ERK activation. We further demonstrate that constitutive activation of ERKs in TPC-1 cells is not caused by mutations in 50 oncogenes and tumor suppressors prone to activate the ERK pathway, or affected by inhibition of BRAF, MEK1/2 or PI3K. Our data indicate that RAF-1 is important for the survival of TPC-1 cells independently of the classical MEK1/2-ERK activation, offering new perspectives on RET/PTC signaling and for the therapy of thyroid cancers.


Assuntos
Carcinoma/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Carcinoma Anaplásico da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , Apoptose , Carcinoma/metabolismo , Carcinoma Papilar , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas c-raf/genética , Câncer Papilífero da Tireoide , Carcinoma Anaplásico da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo
14.
Cell Signal ; 27(9): 1840-9, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26079297

RESUMO

Mitochondria function as the powerhouses of the cell for energy conversion through the oxidative phosphorylation process. Accumulation of dysfunctional mitochondria promotes a bioenergetic crisis and cell death by apoptosis. Yeast cells lacking Isc1p, an orthologue of mammalian neutral sphingomyelinase type 2, exhibit mitochondrial dysfunction and shortened lifespan associated with the accumulation of specific ceramide species and activation of the PP2A-like protein phosphatase Sit4p and of the Hog1p kinase. Here, we show that isc1Δ cells display hyperactivation of mitophagy that is suppressed by downregulating Sit4p, Hog1p or the TORC1-Sch9p pathway. Notably, isc1Δ cells also have high levels of Dnm1p associated with unbalanced mitochondrial fission, leading to mitochondrial fragmentation, and DNM1 deletion suppressed the oxidative stress sensitivity and shortened lifespan of isc1Δ cells. Moreover, Isc1p and Dnm1p physically interact, suggesting a possible regulatory role for Isc1p in mitochondrial dynamics. Overall, our work demonstrates that Isc1p-mediated ceramide signalling regulates mitophagy and mitochondrial dynamics in yeast with impact on mitochondrial function and lifespan. Since ceramides have been implicated in ageing and diseases associated with mitochondrial dysfunction, our findings suggest that therapeutic strategies targeting ceramide signalling may improve mitochondrial function and human healthspan.


Assuntos
Ceramidas/metabolismo , Dinâmica Mitocondrial/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mitofagia/fisiologia , Proteína Fosfatase 2/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/fisiologia , Fosfolipases Tipo C/deficiência , Humanos , Proteínas Quinases Ativadas por Mitógeno/genética , Proteína Fosfatase 2/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fosfolipases Tipo C/metabolismo
15.
FEMS Yeast Res ; 14(1): 160-78, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24103214

RESUMO

It has been established that sphingolipids are engaged in the regulation of apoptosis both as direct executors and as signalling molecules. However, the peculiarities of this class of bioactive lipids, namely the interconnectivity of their metabolic pathways, the specific subcellular localization where they are generated and the transport mechanisms involved, introduce a considerably high level of complexity in deciphering their role in the signalling and regulation of programmed cell death. Although yeast is undeniably a simple model, the conservation of the sphingolipid metabolism and of the core machinery engaged in regulated cell death has already provided valuable clues to the understanding of metabolic pathways involved in distinct cellular processes, including apoptosis. It can be anticipated that studies using this model system will further unravel mechanisms underlying the regulation of apoptosis by sphingolipids and contribute to novel therapeutic strategies against serious human diseases associated with dysfunction of sphingolipid-dependent cell death programmes.


Assuntos
Apoptose/efeitos dos fármacos , Saccharomyces cerevisiae/fisiologia , Controle Social Formal , Esfingolipídeos/metabolismo , Redes e Vias Metabólicas , Modelos Biológicos
16.
Microb Cell ; 1(9): 303-314, 2014 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-28357256

RESUMO

Acetic acid triggers apoptotic cell death in Saccharomyces cerevisiae, similar to mammalian apoptosis. To uncover novel regulators of this process, we analyzed whether impairing MAPK signaling affected acetic acid-induced apoptosis and found the mating-pheromone response and, especially, the cell wall integrity pathways were the major mediators, especially the latter, which we characterized further. Screening downstream effectors of this pathway, namely targets of the transcription factor Rlm1p, highlighted decreased cell wall remodeling as particularly important for acetic acid resistance. Modulation of cell surface dynamics therefore emerges as a powerful strategy to increase acetic acid resistance, with potential application in industrial fermentations using yeast, and in biomedicine to exploit the higher sensitivity of colorectal carcinoma cells to apoptosis induced by acetate produced by intestinal propionibacteria.

17.
BMC Genomics ; 14: 838, 2013 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-24286259

RESUMO

BACKGROUND: Acetic acid is mostly known as a toxic by-product of alcoholic fermentation carried out by Saccharomyces cerevisiae, which it frequently impairs. The more recent finding that acetic acid triggers apoptotic programmed cell death (PCD) in yeast sparked an interest to develop strategies to modulate this process, to improve several biotechnological applications, but also for biomedical research. Indeed, acetate can trigger apoptosis in cancer cells, suggesting its exploitation as an anticancer compound. Therefore, we aimed to identify genes involved in the positive and negative regulation of acetic acid-induced PCD by optimizing a functional analysis of a yeast Euroscarf knock-out mutant collection. RESULTS: The screen consisted of exposing the mutant strains to acetic acid in YPD medium, pH 3.0, in 96-well plates, and subsequently evaluating the presence of culturable cells at different time points. Several functional categories emerged as greatly relevant for modulation of acetic acid-induced PCD (e.g.: mitochondrial function, transcription of glucose-repressed genes, protein synthesis and modifications, and vesicular traffic for protection, or amino acid transport and biosynthesis, oxidative stress response, cell growth and differentiation, protein phosphorylation and histone deacetylation for its execution). Known pro-apoptotic and anti-apoptotic genes were found, validating the approach developed. Metabolism stood out as a main regulator of this process, since impairment of major carbohydrate metabolic pathways conferred resistance to acetic acid-induced PCD. Among these, lipid catabolism arose as one of the most significant new functions identified. The results also showed that many of the cellular and metabolic features that constitute hallmarks of tumour cells (such as higher glycolytic energetic dependence, lower mitochondrial functionality, increased cell division and metabolite synthesis) confer sensitivity to acetic acid-induced PCD, potentially explaining why tumour cells are more susceptible to acetate than untransformed cells and reinforcing the interest in exploiting this acid in cancer therapy. Furthermore, our results clearly establish a connection between cell proliferation and cell death regulation, evidencing a conserved developmental role of programmed cell death in unicellular eukaryotes. CONCLUSIONS: This work advanced the characterization of acetic acid-induced PCD, providing a wealth of new information on putative molecular targets for its control with impact both in biotechnology and biomedicine.


Assuntos
Ácido Acético/farmacologia , Apoptose/genética , Genes Fúngicos , Saccharomyces cerevisiae/genética , Metabolismo dos Carboidratos/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genoma Fúngico , Viabilidade Microbiana/efeitos dos fármacos , Mitocôndrias/genética , Processamento de Proteína Pós-Traducional/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Estresse Fisiológico
18.
PLoS One ; 8(9): e74240, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24040213

RESUMO

Specific ceramides are key regulators of cell fate, and extensive studies aimed to develop therapies based on ceramide-induced cell death. However, the mechanisms regulating ceramide cytotoxicity are not yet fully elucidated. Since ceramides also regulate growth and stress responses in yeast, we studied how different exogenous ceramides affect yeast cells. C2-phytoceramide, a soluble form of phytoceramides, the yeast counterparts of mammalian ceramides, greatly reduced clonogenic survival, particularly in the G2/M phase, but did not induce autophagy nor increase apoptotic markers. Rather, the loss of clonogenic survival was associated with PI positive staining, disorganization of lipid rafts and cell wall weakening. Sensitivity to C2-phytoceramide was exacerbated in mutants lacking Hog1p, the MAP kinase homolog of human p38 kinase. Decreasing sterol membrane content reduced sensitivity to C2-phytoceramide, suggesting sterols are the targets of this compound. This study identified a new function of C2-phytoceramide through disorganization of lipid rafts and induction of a necrotic cell death under hypo-osmotic conditions. Since lipid rafts are important in mammalian cell signaling and adhesion, our findings further support pursuing the exploitation of yeast to understand the basis of synthetic ceramides' cytotoxicity to provide novel strategies for therapeutic intervention in cancer and other diseases.


Assuntos
Parede Celular/efeitos dos fármacos , Ergosterol/metabolismo , Microdomínios da Membrana/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Esfingosina/análogos & derivados , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Parede Celular/química , Ergosterol/química , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Expressão Gênica , Humanos , Microdomínios da Membrana/química , Proteínas Quinases Ativadas por Mitógeno/deficiência , Proteínas Quinases Ativadas por Mitógeno/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Esfingosina/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
19.
DNA Repair (Amst) ; 12(6): 444-9, 2013 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-23602516

RESUMO

Cisplatin is a highly effective chemotherapeutic drug used in the treatment of several tumors. It is a DNA-damaging agent that induces apoptosis of rapidly proliferating cells, an important factor underlying its therapeutic efficacy. Unfortunately, cellular resistance occurs often. A large fraction of tumor cells harbor mutations in p53, contributing to defects in apoptotic pathways and drug resistance. However, cisplatin-induced apoptosis can also occur in p53 deficient cells; thus, elucidation of the molecular mechanism involved will potentially yield new strategies to eliminate tumors that have defects in the p53 pathway. Most of the studies in this field have been conducted in cultured mammalian cells, not amenable to systematic genetic manipulation. Therefore, we aimed to establish a simplified model devoid of a p53 ortholog to study cisplatin-induced programmed cell death (PCD), using the yeast Saccharomyces cerevisiae. Our results indicate cisplatin induces an active form of cell death in yeast, as this process was partially dependent on de novo protein synthesis and did not lead to loss of membrane integrity. Cisplatin also increased DNA condensation and fragmentation/degradation, but no significant mitochondrial dysfunction other than partial fragmentation. Co-incubation with the proteasome inhibitor MG132 increased resistance to cisplatin and, accordingly, yeast strains deficient in proteasome activity were more resistant to cisplatin than wild-type strains. Proteasome inhibitors can sensitize tumor cells to cisplatin, but protect others from cisplatin-induced cell death. Our results indicate inhibition of the proteasome protects budding yeast from cisplatin-induced cell death and validate yeast as a model to study the role of the proteasome in cisplatin-induced PCD. Elucidation of this mechanism will aid in the development of new strategies to increase the efficacy of chemotherapy.


Assuntos
Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Cisplatino/toxicidade , Inibidores de Proteassoma/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo
20.
FEBS Lett ; 587(2): 200-5, 2013 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-23220089

RESUMO

We have previously shown that the yeast Cathepsin D (CatD) Pep4p translocates from the vacuole to the cytosol during acetic acid-induced apoptosis and is required for efficient mitochondrial degradation, though its specific role in this process is still elusive. Here, we show that the protective role of Pep4p in acetic acid-induced apoptosis depends on its catalytic activity and is independent of the yeast voltage-dependent anion channel Por1p (which has no role on mitochondrial degradation) but dependent on AAC proteins, the yeast adenine nucleotide translocator. Our results demonstrate a differential interplay between yeast vacuolar CatD and mitochondrial proteins involved in apoptosis regulation.


Assuntos
Catepsina D/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Ácido Acético/farmacologia , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Transporte Biológico Ativo , Genes Fúngicos , Mitocôndrias/metabolismo , Mutação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Vacúolos/metabolismo
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