Molecular Characterization of Propolis-Induced Cell Death in Saccharomyces cerevisiae

ABSTRACTPropolis, a natural product of plant resins, is used by the bees to seal holes in their honeycombs and protect the hive entrance. However, propolis has also been used in folk medicine for centuries. Here, we apply the power ofSaccharomyces cerevisiaeas a model organism for studies of genetics, cell biology, and genomics to determine how propolis affects fungi at the cellular level. Propolis is able to induce an apoptosis cell death response. However, increased exposure to propolis provides a corresponding increase in the necrosis response. We showed that cytochromecbut not endonuclease G (Nuc1p) is involved in propolis-mediated cell death inS. cerevisiae. We also observed that the metacaspaseYCA1gene is important for propolis-mediated cell death. To elucidate the gene functions that may be required for propolis sensitivity in eukaryotes, the full collection of about 4,800 haploidS. cerevisiaedeletion strains was screened for propolis sensitivity. We were able to identify 138 deletion strains that have different degrees of propolis sensitivity compared to the corresponding wild-type strains. Systems biology revealed enrichment for genes involved in the mitochondrial electron transport chain, vacuolar acidification, negative regulation of transcription from RNA polymerase II promoter, regulation of macroautophagy associated with protein targeting to vacuoles, and cellular response to starvation. Validation studies indicated that propolis sensitivity is dependent on the mitochondrial function and that vacuolar acidification and autophagy are important for yeast cell death caused by propolis.

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AT7519, a Potent Multi-Targeted CDK Inhibitor, Is Active in CLL Patient Samples Independent of Stage

Blood ◽

10.1182/blood.v112.11.3161.3161 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3161-3161

Author(s):

Vicky Lock ◽

Laurence Cooke ◽

Murray Yule ◽

Neil T Thompson ◽

K. Della Croce ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

B Cell ◽

Rna Polymerase Ii ◽

Parp Cleavage ◽

Regulation Of Transcription ◽

Cytotoxic Effects ◽

Rna Pol Ii ◽

Pol Ii ◽

Cyclin Dependent Kinases

Abstract Cyclin Dependent Kinases (CDKs) play a central role in the eukaryotic cell cycle. The activation of these kinases is modulated by the expression and binding of their regulatory cyclin partners. Their key role in cell cycle progression, coupled to evidence that pathways leading to their activation are deregulated in a number of human cancers makes them attractive therapeutic targets. More recently the role of CDKs 7, 8 and 9 in the regulation of transcription has been explored. CDK9 has been shown to play a role in the regulation of transcription via phosphorylation of RNA polymerase II (RNA pol II). The outcome of transcriptional inhibition via CDK9 exhibits significant variation between cell lines. B-Cell lymphoproliferative disorders, including CLL, rely on the expression of transcripts with a short half-life such as Mcl-1, Bcl-2 and XIAP for survival. In vitro studies have demonstrated that compounds with transcriptional inhibitory effects are effective pro-apoptotic agents in models of this disease. AT7519 is a potent inhibitor of cyclin dependent kinases 1, 2 and 9 and is currently in early phase clinical development. These studies profile the mechanism of action of AT7519 on CLL cells isolated from patients. Primary cell samples were isolated from a total of 15 patients with CLL with various stages of disease (8 Stage 0, 0/I or II and 7 Stage IV) and who were either treatment naïve or had received a variety of prior therapies. Patient samples were characterised for cytogenetic abnormalities (11q, 17p and 13q deletion or trisomy 12) as well IgVH mutation and ZAP70 expression. AT7519 was shown to induce apoptosis (by MTS, morphology and PARP cleavage) in these samples at concentrations of 100–700nM. AT7519 appears equally effective at inhibiting the survival of CLL cells harbouring a variety of mutations including those representative of patients that fall within poorer prognosis treatment groups. The amount of AT7519 required to induce cell death in 50% of the CLL cell population increased as exposure time was decreased but significant cell death was obtained at doses approximating to 1uM following 4–6h of treatment. These doses are equivalent to exposures achieved in ongoing AT7519 clinical studies indicating that cytotoxic doses can be achieved in patients on well tolerated schedules. The mechanism of AT7519 cytotoxic effects was investigated by western blotting for a variety of cell cycle and apoptotic markers following incubation with compound. Short term treatments (4–6h) resulted in inhibition of phosphorylation of the transcriptional marker RNA pol II and the downregulation of the anti-apoptotic protein Mcl-1. Additional antiapoptotic proteins including XIAP and Bcl-2 remained unchanged. The reduction in Mcl-1 protein levels was associated with an increase in the apoptotic marker cleaved PARP. No inhibition of cell cycle markers such as phospho-retinoblastoma protein was observed in the same samples suggesting that the cytotoxic effects of AT7519 in CLL patient samples is due to its transcriptional activity alone. Together the data suggest AT7519 offers a promising treatment strategy for patients with advanced B-cell leukemia and lymphoma.

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Pho85 Kinase, a Cyclin-Dependent Kinase, Regulates Nuclear Accumulation of the Rim101 Transcription Factor in the Stress Response of Saccharomyces cerevisiae

Eukaryotic Cell ◽

10.1128/ec.00247-09 ◽

2010 ◽

Vol 9 (6) ◽

pp. 943-951 ◽

Cited By ~ 13

Author(s):

Masafumi Nishizawa ◽

Mirai Tanigawa ◽

Michio Hayashi ◽

Tatsuya Maeda ◽

Yoshiaki Yazaki ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcription Factor ◽

Cellular Response ◽

Proteolytic Processing ◽

Yeast Cells ◽

Nuclear Accumulation ◽

Alkaline Stress ◽

Content Type ◽

Its Gene ◽

Alkaline Conditions

ABSTRACT The budding yeast Saccharomyces cerevisiae alters its gene expression profile in response to changing environmental conditions. The Pho85 kinase, one of the yeast cyclin-dependent kinases (CDK), is known to play an important role in the cellular response to alterations in parameters such as nutrient levels and salinity. Several genes whose expression is regulated, either directly or indirectly, by the Rim101 transcription factor become constitutively activated when Pho85 function is absent,. Because Rim101 is responsible for adaptation to alkaline conditions, this observation suggests an interaction between Pho85 and Rim101 in the response to alkaline stress. We have found that Pho85 affects neither RIM101 transcription, the proteolytic processing that is required for Rim101 activation, nor Rim101 stability. Rather, Pho85 regulates the nuclear accumulation of active Rim101, possibly via phosphorylation. Additionally, we report that Pho85 and the transcription factor Pho4 are necessary for adaptation to alkaline conditions and that PTK2 activation by Pho4 is involved in this process. These findings illustrate novel roles for the regulators of the PHO system when yeast cells cope with various environmental stresses potentially threatening their survival.

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The Interplay of Key Phospholipid Biosynthetic Enzymes and the Yeast V-ATPase Pump and their Role in Programmed Cell Death

10.5772/intechopen.97886 ◽

2021 ◽

Author(s):

Goldie Libby Sherr ◽

Chang-Hui Shen

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Death ◽

Programmed Cell Death ◽

Adaptive Response ◽

Mammalian Cells ◽

Biosynthetic Genes ◽

Yeast Saccharomyces Cerevisiae ◽

Growth Physiology ◽

Vacuolar Acidification ◽

Main Regulator

Exposure of the yeast Saccharomyces cerevisiae to environmental stress can influence cell growth, physiology and differentiation, and thus result in a cell’s adaptive response. During the course of an adaptive response, the yeast vacuoles play an important role in protecting cells from stress. Vacuoles are dynamic organelles that are similar to lysosomes in mammalian cells. The defect of a lysosome’s function may cause various genetic and neurodegenerative diseases. The multi-subunit V-ATPase is the main regulator for vacuolar function and its activity plays a significant role in maintaining pH homeostasis. The V-ATPase is an ATP-driven proton pump which is required for vacuolar acidification. It has also been demonstrated that phospholipid biosynthetic genes might influence vacuolar morphology and function. However, the mechanistic link between phospholipid biosynthetic genes and vacuolar function has not been established. Recent studies have demonstrated that there is a regulatory role of Pah1p, a phospholipid biosynthetic gene, in V-ATPase disassembly and activity. Therefore, in this chapter we will use Saccharomyces cerevisiae as a model to discuss how Pah1p affects V-ATPase disassembly and activity and how Pah1p negatively affect vacuolar function. Furthermore, we propose a hypothesis to describe how Pah1p influences vacuolar function and programmed cell death through the regulation of V-ATPase.

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Single-Cell Time-Lapse Observation Reveals Cell Shrinkage upon Cell Death in Batch Culture of Saccharomyces cerevisiae

mBio ◽

10.1128/mbio.03094-21 ◽

2021 ◽

Vol 12 (6) ◽

Author(s):

Setsu Kato ◽

Kenta Suzuki ◽

Taiki Kenjo ◽

Junya Kato ◽

Yoshiteru Aoi ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Death ◽

Stationary Phase ◽

Cell Survival ◽

Single Cell ◽

Batch Culture ◽

Time Lapse ◽

Cell Shrinkage ◽

Clonal Population ◽

Content Type

Cells display various behaviors even though they originate from a clonal population. Such diversity is also observed in cell survival in the stationary phase of Saccharomyces cerevisiae .

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pH Alkalinization by Chloroquine Suppresses Pathogenic Burkholderia Type 6 Secretion System 1 and Multinucleated Giant Cells

Infection and Immunity ◽

10.1128/iai.00586-16 ◽

2016 ◽

Vol 85 (1) ◽

Cited By ~ 3

Author(s):

Jennifer Chua ◽

Jeffrey L. Senft ◽

Stephen J. Lockett ◽

Paul J. Brett ◽

Mary N. Burtnick ◽

...

Keyword(s):

Cell Death ◽

Cell Biology ◽

Antibiotic Use ◽

Giant Cells ◽

Secretion System ◽

Multinucleated Giant Cells ◽

Content Type ◽

Acid Ph ◽

Virulence Protein ◽

System 1

ABSTRACT Burkholderia mallei and B. pseudomallei cause glanders and melioidosis, respectively, in humans and animals. A hallmark of pathogenesis is the formation of granulomas containing multinucleated giant cells (MNGCs) and cell death. These processes depend on type 6 secretion system 1 (T6SS-1), which is required for virulence in animals. We examined the cell biology of MNGC formation and cell death. We found that chloroquine diphosphate (CLQ), an antimalarial drug, inhibits Burkholderia growth, phagosomal escape, and subsequent MNGC formation. This depends on CLQ's ability to neutralize the acid pH because other alkalinizing compounds similarly inhibit escape and MNGC formation. CLQ inhibits bacterial virulence protein expression because T6SS-1 and some effectors of type 3 secretion system 3 (T3SS-3), which is also required for virulence, are expressed at acid pH. We show that acid pH upregulates the expression of Hcp1 of T6SS-1 and TssM, a protein coregulated with T6SS-1. Finally, we demonstrate that CLQ treatment of Burkholderia-infected Madagascar hissing cockroaches (HCs) increases their survival. This study highlights the multiple mechanisms by which CLQ inhibits growth and virulence and suggests that CLQ be further tested and considered, in conjunction with antibiotic use, for the treatment of diseases caused by Burkholderia.

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Mutual Cross Talk between the Regulators Hac1 of the Unfolded Protein Response and Gcn4 of the General Amino Acid Control of Saccharomyces cerevisiae

Eukaryotic Cell ◽

10.1128/ec.00123-13 ◽

2013 ◽

Vol 12 (8) ◽

pp. 1142-1154 ◽

Cited By ~ 9

Author(s):

Britta Herzog ◽

Blagovesta Popova ◽

Antonia Jakobshagen ◽

Hedieh Shahpasandzadeh ◽

Gerhard H. Braus

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Cross Talk ◽

Cellular Response ◽

Promoter Regions ◽

Content Type ◽

General Amino Acid Control ◽

Acid Control ◽

The Unfolded Protein Response ◽

Diploid Cells

ABSTRACTHac1 is the activator of the cellular response to the accumulation of unfolded proteins in the endoplasmic reticulum. Hac1 function requires the activity of Gcn4, which mainly acts as a regulator of the general amino acid control network providingSaccharomyces cerevisiaecells with amino acids. Here, we demonstrate novel functions of Hac1 and describe a mutual connection between Hac1 and Gcn4. Hac1 is required for induction of Gcn4-responsive promoter elements in haploid as well as diploid cells and therefore participates in the cellular amino acid supply. Furthermore, Hac1 and Gcn4 mutually influence their mRNA expression levels. Hac1 is also involved inFLO11expression and adhesion upon amino acid starvation. Hac1 and Gcn4 act through the same promoter regions of theFLO11flocculin. The results indicate an indirect effect of both transcription factors onFLO11expression. Our data suggest a complex mutual cross talk between the Hac1- and Gcn4-controlled networks.

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Interactions of Sen1, Nrd1, and Nab3 with Multiple Phosphorylated Forms of the Rpb1 C-Terminal Domain in Saccharomyces cerevisiae

Eukaryotic Cell ◽

10.1128/ec.05320-11 ◽

2012 ◽

Vol 11 (4) ◽

pp. 417-429 ◽

Cited By ~ 39

Author(s):

Karen Chinchilla ◽

Juan B. Rodriguez-Molina ◽

Doris Ursic ◽

Jonathan S. Finkel ◽

Aseem Z. Ansari ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase Ii ◽

Protein Interactions ◽

Noncoding Rna ◽

Protein Protein Interactions ◽

Coding Region ◽

Protein Coding ◽

Content Type ◽

Protein Coding Genes ◽

Terminal Domain

ABSTRACT The Saccharomyces cerevisiae SEN1 gene codes for a nuclear, ATP-dependent helicase which is embedded in a complex network of protein-protein interactions. Pleiotropic phenotypes of mutations in SEN1 suggest that Sen1 functions in many nuclear processes, including transcription termination, DNA repair, and RNA processing. Sen1, along with termination factors Nrd1 and Nab3, is required for the termination of noncoding RNA transcripts, but Sen1 is associated during transcription with coding and noncoding genes. Sen1 and Nrd1 both interact directly with Nab3, as well as with the C-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II. It has been proposed that Sen1, Nab3, and Nrd1 form a complex that associates with Rpb1 through an interaction between Nrd1 and the Ser 5 -phosphorylated (Ser 5 -P) CTD. To further study the relationship between the termination factors and Rpb1, we used two-hybrid analysis and immunoprecipitation to characterize sen1-R302W , a mutation that impairs an interaction between Sen1 and the Ser 2 -phosphorylated CTD. Chromatin immunoprecipitation indicates that the impairment of the interaction between Sen1 and Ser 2 -P causes the reduced occupancy of mutant Sen1 across the entire length of noncoding genes. For protein-coding genes, mutant Sen1 occupancy is reduced early and late in transcription but is similar to that of the wild type across most of the coding region. The combined data suggest a handoff model in which proteins differentially transfer from the Ser 5 - to the Ser 2 -phosphorylated CTD to promote the termination of noncoding transcripts or other cotranscriptional events for protein-coding genes.

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Signaling of Chloroquine-Induced Stress in the Yeast Saccharomyces cerevisiae Requires the Hog1 and Slt2 Mitogen-Activated Protein Kinase Pathways

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02393-13 ◽

2014 ◽

Vol 58 (9) ◽

pp. 5552-5566 ◽

Cited By ~ 11

Author(s):

Shivani Baranwal ◽

Gajendra Kumar Azad ◽

Vikash Singh ◽

Raghuvir S. Tomar

Keyword(s):

Saccharomyces Cerevisiae ◽

Histone Deacetylases ◽

Model Organism ◽

Biological Pathways ◽

Human Cells ◽

Mitogen Activated Protein Kinase ◽

Yeast Saccharomyces Cerevisiae ◽

Content Type ◽

Mitogen Activated Protein ◽

Glycerol 3 Phosphate Dehydrogenase

ABSTRACTChloroquine (CQ) has been under clinical use for several decades, and yet little is known about CQ sensing and signaling mechanisms or about their impact on various biological pathways. We employed the budding yeastSaccharomyces cerevisiaeas a model organism to study the pathways targeted by CQ. Our screening with yeast mutants revealed that it targets histone proteins and histone deacetylases (HDACs). Here, we also describe the novel role of mitogen-activated protein kinases Hog1 and Slt2, which aid in survival in the presence of CQ. Cells deficient in Hog1 or Slt2 are found to be CQ hypersensitive, and both proteins were phosphorylated in response to CQ exposure. CQ-activated Hog1p is translocated to the nucleus and facilitates the expression of GPD1 (glycerol-3-phosphate dehydrogenase), which is required for the synthesis of glycerol (one of the major osmolytes). Moreover, cells treated with CQ exhibited an increase in intracellular reactive oxygen species (ROS) levels and the effects were rescued by addition of reduced glutathione to the medium. The deletion of SOD1, the superoxide dismutase in yeast, resulted in hypersensitivity to CQ. We have also observed P38 as well as P42/44 phosphorylation in HEK293T human cells upon exposure to CQ, indicating that the kinds of responses generated in yeast and human cells are similar. In summary, our findings define the multiple biological pathways targeted by CQ that might be useful for understanding the toxicity modulated by this pharmacologically important molecule.

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Establishment of a fluorescent reporter of RNA-polymerase II activity to identify dormant cells

Nature Communications ◽

10.1038/s41467-021-23580-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Rasmus Freter ◽

Paola Falletta ◽

Omid Omrani ◽

Mahdi Rasa ◽

Katharine Herbert ◽

...

Keyword(s):

Stem Cell ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Cell Biology ◽

Cellular Response ◽

Cell Activity ◽

Isolation And Characterization ◽

Dormant Cells

AbstractDormancy, a reversible quiescent cellular state characterized by greatly reduced metabolic activity, protects from genetic damage, prolongs survival and is crucial for tissue homeostasis and cellular response to injury or transplantation. Dormant cells have been characterized in many tissues, but their identification, isolation and characterization irrespective of tissue of origin remains elusive. Here, we develop a live cell ratiometric fluorescent Optical Stem Cell Activity Reporter (OSCAR) based on the observation that phosphorylation of RNA Polymerase II (RNApII), a hallmark of active mRNA transcription elongation, is largely absent in dormant stem cells from multiple lineages. Using the small intestinal crypt as a model, OSCAR reveals in real time the dynamics of dormancy induction and cellular differentiation in vitro, and allows the identification and isolation of several populations of transcriptionally diverse OSCARhigh and OSCARlow intestinal epithelial cell states in vivo. In particular, this reporter is able to identify a dormant OSCARhigh cell population in the small intestine. OSCAR therefore provides a tool for a better understanding of dormant stem cell biology.

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Involvement of Pca1 in ROS-mediated apoptotic cell death induced by alpha-thujone in the fission yeast (Schizosaccharomyces pombe)

FEMS Yeast Research ◽

10.1093/femsyr/foaa022 ◽

2020 ◽

Vol 20 (4) ◽

Author(s):

Hizlan Hincal Agus ◽

Gizem Kok ◽

Ezgi Derinoz ◽

Didem Oncel ◽

Sedanur Yilmaz

Keyword(s):

Cell Death ◽

Schizosaccharomyces Pombe ◽

Cell Biology ◽

Apoptotic Cell ◽

Survival Rates ◽

Model Organism ◽

Apoptotic Cell Death ◽

Dependent Manner ◽

Induced Apoptosis ◽

Apoptotic Factors

ABSTRACT Alpha-thujone, widely used in beverages (1–5 mg/kg), is known to have cytotoxic effects, but the mode of action and the role of potential apoptotic proteins in yeast cell death should be unraveled. In this study, we used Schizosaccharomyces pombe, which is a promising unicellular model organism in mechanistic toxicology and cell biology, to investigate the involvement of pro-apoptotic factors in alpha-thujone-induced cell death. We showed alpha-thujone-induced ROS accumulation-dependent cytotoxicity and apoptosis. In addition, we used superoxide dismutase-deficient cells (sod1 and sod2 mutants) to understand the effect of oxidative stress. Alpha-thujone caused significant cytotoxicity and apoptotic cell death, particularly in sod mutants. Moreover, two potential apoptotic factors, pca1 and pnu1 (pombe caspase-1 and pombe nuc1) were investigated to understand which factor mediates alpha-thujone-induced cell death. Pca1-deficient cells showed increased survival rates and reduced apoptosis in comparison to parental cells after chemical treatment while pnu1 mutation did not cause any significant change and the response was found identical as of parental cells. Yeast responded to alpha-thujone in caspase-dependent manner which was very similar to that for acetic acid. In conclusion, alfa-thujone-induced apoptosis and accounting mechanisms, which were mediated by ROS and driven by Pca1, were clarified in the unicellular model, S. pombe.

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