scholarly journals Proteomic analysis demonstrated transcription factor YRR1 gene deletion in Saccharomyces cerevisiae enhances its resistance to vanillin through the upregulation of transcriptional activator Haa1, coactivator Mbf1, and proteasome assembly chaperone Tma17 expression

2020 ◽  
Author(s):  
Wenyan Cao ◽  
Xinning Wang ◽  
Weiquan Zhao ◽  
Yu Shen ◽  
Wensheng Qin ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Ribosomal Proteins ◽  
Molecular Mechanisms ◽  
Transcriptional Activator ◽  
Wild Type ◽  
Translation Process ◽  
Enhanced Resistance ◽  
Yeast Strains ◽  
Transcriptomic Changes

Abstract Background: Vanillin is one of the major phenolic inhibitors in Saccharomyces cerevisiae for cellulosic ethanol production. Deleting transcription factor gene YRR1 improves vanillin resistance by promoting some translation-related processes that were confirmed at the transcription level in our previous studies. However, the known genes regulated by Yrr1 are not related to translation process. Therefore, in this work, we investigated the effects of proteomic changes on vanillin stress and YRR1 deletion to provide different perspectives from transcriptome analysis for comprehending the mechanisms of YRR1 deletion in yeast protective response to vanillin.Results: In wild-type cells, vanillin reduced the numbers of ribosomal proteins quantities and thereby inhibited cells’ translation. YRR1 deletion changed the quantities of 121 proteins which have no overlaps with transcriptomic changes. Of 112 proteins were up-regulated; 48 of 112 up-regulated proteins are involved in stress response, translational and basal transcriptional regulation. Fermentation data showed that the overexpression of HAA1, MBF1, and TMA17, which encode transcriptional activator, coactivator, and proteasome assembly chaperone, respectively, enhanced resistance to vanillin in S. cerevisiae. Conclusions: These results showed how YRR1 deletion increase vanillin resistance at protein level. This may advance our understanding of molecular mechanisms for YRR1 deletion to protect yeast from vanillin stress and offer novel targets of genetic engineering for designing inhibitor-resistant ethanologenic yeast strains.

scholarly journals Proteomic analysis revealed the roles of YRR1 deletion in enhancing the vanillin resistance of Saccharomyces cerevisiae

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Wenyan Cao ◽  
Weiquan Zhao ◽  
Bolun Yang ◽  
Xinning Wang ◽  
Yu Shen ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Level ◽  
Ribosomal Proteins ◽  
Pentose Phosphate ◽  
Wild Type ◽  
Transcription Level ◽  
Translation Process ◽  
Enhanced Resistance ◽  
Yeast Strains ◽  
In Cells

Abstract Background Vanillin is one of the important phenolic inhibitors in Saccharomyces cerevisiae for bioconversion of lignocellulosic materials and has been reported to inhibit the translation process in cells. In our previous studies, it was confirmed that the deletion of the transcription factor gene YRR1 enhanced vanillin resistance by promoting some translation-related processes at the transcription level. In this work, we investigated the effects of proteomic changes upon induction of vanillin stress and deletion of YRR1 to provide unique perspectives from a transcriptome analysis for comprehending the mechanisms of YRR1 deletion in the protective response of yeast to vanillin. Results In wild-type cells, vanillin reduced two dozens of ribosomal proteins contents while upregulated proteins involved in glycolysis, oxidative phosphorylation, and the pentose phosphate pathway in cells. The ratios of NADPH/NADP+ and NADH/NAD+ were increased when cells responded to vanillin stress. The differentially expressed proteins perturbed by YRR1 deletion were much more abundant than and showed no overlaps with transcriptome changes, indicating that Yrr1 affects the synthesis of certain proteins. Forty-eight of 112 upregulated proteins were involved in the stress response, translational and transcriptional regulation. YRR1 deletion increased the expression of HAA1-encoding transcriptional activator, TMA17-encoding proteasome assembly chaperone and MBF1-encoding coactivator at the protein level, as confirmed by ELISA. Cultivation data showed that the overexpression of HAA1 and TMA17 enhanced resistance to vanillin in S. cerevisiae. Conclusions Cells conserve energy by decreasing the content of ribosomal proteins, producing more energy and NAD(P)H for survival in response to vanillin stress. Yrr1 improved vanillin resistance by increasing the protein quantities of Haa1, Tma17 and Mbf1. These results showed the response of S. cerevisiae to vanillin and how YRR1 deletion increases vanillin resistance at the protein level. These findings may advance our knowledge of how YRR1 deletion protects yeast from vanillin stress and offer novel targets for genetic engineering of designing inhibitor-resistant ethanologenic yeast strains.

Mild temperature shock alters the transcription of a discrete class of Saccharomyces cerevisiae genes

1983 ◽  
Vol 3 (3) ◽  
pp. 457-465
Author(s):  
C H Kim ◽  
J R Warner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Temperature Shift ◽  
Restrictive Temperature ◽  
Ribosomal Protein Genes ◽  
Wild Type ◽  
Temperature Shock ◽  
Mild Temperature ◽  
Mutant Cells

In Saccharomyces cerevisiae the synthesis of ribosomal proteins declines temporarily after a culture has been subjected to a mild temperature shock, i.e., a shift from 23 to 36 degrees C, each of which support growth. Using cloned genes for several S. cerevisiae ribosomal proteins, we found that the changes in the synthesis of ribosomal proteins parallel the changes in the concentration of mRNA of each. The disappearance and reappearance of the mRNA is due to a brief but severe inhibition of the transcription of each of the ribosomal protein genes, although the total transcription of mRNA in the cells is relatively unaffected by the temperature shock. The precisely coordinated response of these genes, which are scattered throughout the genome, suggests that either they or the enzyme which transcribes them has unique properties. In certain S. cerevisiae mutants, the synthesis of ribosomal proteins never recovers from a temperature shift. Yet both the decline and the resumption of transcription of these genes during the 30 min after the temperature shift are indistinguishable from those in wild-type cells. The failure of the mutant cells to grow at the restrictive temperature appears to be due to their inability to process the RNA transcribed from genes which have introns (Rosbash et al., Cell 24:679-686, 1981), a large proportion of which appear to be ribosomal protein genes.

scholarly journals Ribosomal protein L30 is dispensable in the yeast Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (10) ◽  
pp. 5235-5243 ◽  
Author(s):  
D M Baronas-Lowell ◽  
J R Warner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Wild Type ◽  
Diploid Strain ◽  
Chain Reaction ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ribosomal Subunits ◽  
Strong Homology ◽  
Polymerase Chain

In the yeast Saccharomyces cerevisiae, L30 is one of many ribosomal proteins that is encoded by two functional genes. We have cloned and sequenced RPL30B, which shows strong homology to RPL30A. Use of mRNA as a template for a polymerase chain reaction demonstrated that RPL30B contains an intron in its 5' untranslated region. This intron has an unusual 5' splice site, C/GUAUGU. The genomic copies of RPL30A and RPL30B were disrupted by homologous recombination. Growth rates, primer extension, and two-dimensional ribosomal protein analyses of these disruption mutants suggested that RPL30A is responsible for the majority of L30 production. Surprisingly, meiosis of a diploid strain carrying one disrupted RPL30A and one disrupted RPL30B yielded four viable spores. Ribosomes from haploid cells carrying both disrupted genes had no detectable L30, yet such cells grew with a doubling time only 30% longer than that of wild-type cells. Furthermore, depletion of L30 did not alter the ratio of 60S to 40S ribosomal subunits, suggesting that there is no serious effect on the assembly of 60S subunits. Polysome profiles, however, suggest that the absence of L30 leads to the formation of stalled translation initiation complexes.

Ribosomal protein L30 is dispensable in the yeast Saccharomyces cerevisiae

1990 ◽  
Vol 10 (10) ◽  
pp. 5235-5243
Author(s):  
D M Baronas-Lowell ◽  
J R Warner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Wild Type ◽  
Diploid Strain ◽  
Chain Reaction ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ribosomal Subunits ◽  
Strong Homology ◽  
Polymerase Chain

In the yeast Saccharomyces cerevisiae, L30 is one of many ribosomal proteins that is encoded by two functional genes. We have cloned and sequenced RPL30B, which shows strong homology to RPL30A. Use of mRNA as a template for a polymerase chain reaction demonstrated that RPL30B contains an intron in its 5' untranslated region. This intron has an unusual 5' splice site, C/GUAUGU. The genomic copies of RPL30A and RPL30B were disrupted by homologous recombination. Growth rates, primer extension, and two-dimensional ribosomal protein analyses of these disruption mutants suggested that RPL30A is responsible for the majority of L30 production. Surprisingly, meiosis of a diploid strain carrying one disrupted RPL30A and one disrupted RPL30B yielded four viable spores. Ribosomes from haploid cells carrying both disrupted genes had no detectable L30, yet such cells grew with a doubling time only 30% longer than that of wild-type cells. Furthermore, depletion of L30 did not alter the ratio of 60S to 40S ribosomal subunits, suggesting that there is no serious effect on the assembly of 60S subunits. Polysome profiles, however, suggest that the absence of L30 leads to the formation of stalled translation initiation complexes.

Cadmium regulates copper homoeostasis by inhibiting the activity of Mac1, a transcriptional activator of the copper regulon, in Saccharomyces cerevisiae

2010 ◽  
Vol 431 (2) ◽  
pp. 257-265 ◽  
Author(s):  
Dong-Hyuk Heo ◽  
In-Joon Baek ◽  
Hyun-Jun Kang ◽  
Ji-Hyun Kim ◽  
Miwha Chang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Mechanisms ◽  
Cadmium Toxicity ◽  
Toxic Metal ◽  
Copper Metabolism ◽  
Transcriptional Activator ◽  
Northern Blotting ◽  
Model Systems ◽  
Transcriptional Level ◽  
Mechanisms Of Toxicity

Cadmium is a toxic metal and the mechanism of its toxicity has been studied in various model systems from bacteria to mammals. We employed Saccharomyces cerevisiae as a model system to study cadmium toxicity at the molecular level because it has been used to identify the molecular mechanisms of toxicity found in higher organisms. cDNA microarray and Northern blot analyses revealed that cadmium salts inhibited the expression of genes related to copper metabolism. Western blotting, Northern blotting and chromatin immunoprecipitation experiments indicated that CTR1 expression was inhibited at the transcriptional level through direct inhibition of the Mac1 transcriptional activator. The decreased expression of CTR1 results in cellular copper deficiency and inhibition of Fet3 activity, which eventually impairs iron uptake. In this way, cadmium exhibits a negative effect on both iron and copper homoeostasis.

scholarly journals A Nuclear Import Pathway for a Protein Involved in tRNA Maturation

1997 ◽  
Vol 139 (7) ◽  
pp. 1655-1661 ◽  
Author(s):  
Jonathan S. Rosenblum ◽  
Lucy F. Pemberton ◽  
Günter Blobel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Import ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Sequence Similarity ◽  
Wild Type ◽  
Major Pathway ◽  
Trna Processing ◽  
Transport Factors ◽  
Limited Sequence

A limited number of transport factors, or karyopherins, ferry particular substrates between the cytoplasm and nucleoplasm. We identified the Saccharomyces cerevisiae gene YDR395w/SXM1 as a potential karyopherin on the basis of limited sequence similarity to known karyopherins. From yeast cytosol, we isolated Sxm1p in complex with several potential import substrates. These substrates included Lhp1p, the yeast homologue of the human autoantigen La that has recently been shown to facilitate maturation of pre-tRNA, and three distinct ribosomal proteins, Rpl16p, Rpl25p, and Rpl34p. Further, we demonstrate that Lhp1p is specifically imported by Sxm1p. In the absence of Sxm1p, Lhp1p was mislocalized to the cytoplasm. Sxm1p and Lhp1p represent the karyopherin and a cognate substrate of a unique nuclear import pathway, one that operates upstream of a major pathway of pre-tRNA maturation, which itself is upstream of tRNA export in wild-type cells. In addition, through its association with ribosomal proteins, Sxm1p may have a role in coordinating ribosome biogenesis with tRNA processing.

Molecular Mechanism Regulating Foxo In Leukemia Initiating Cells of Chronic Myeloid Leukemia.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3391-3391
Author(s):  
Kazuhito Naka ◽  
Takayuki Hoshii ◽  
Yuko Tadokoro ◽  
Takako Ooshio ◽  
Yukio Kondo ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Chronic Myeloid Leukemia ◽  
Nuclear Localization ◽  
Myeloid Leukemia ◽  
Cellular Localization ◽  
Molecular Mechanisms ◽  
Imatinib Treatment ◽  
Cytoplasmic Localization ◽  
Wild Type ◽  
Imatinib Resistant

Abstract Abstract 3391 Chronic myeloid leukemia (CML) is caused by a defined genetic abnormality that generates BCR-ABL, a constitutively active tyrosine kinase. Although the development of imatinib, a small molecule inhibitor of ABL, represents a breakthrough in the treatment of CML, major part of patients treated in chronic phase CML are not off therapy due to resistance or intolerance. Recent studies have suggested that imatinib is a potent inhibitor against differentiated leukemia cells, but does not deplete leukemia-initiating cells (LICs) responsible for recurrence of CML. To date, therapeutics that can eradicate CML LICs, however, have remained under investigation. To overcome these clinical problems, here we studied the molecular mechanisms regulating maintenance of imatinib-resistant CML LICs by forkhead transcription factor Foxo3a. We first generated a mouse CML model by using retroviral induction of BCR-ABL-ires-GFP gene into mouse immature hematopoietic cells, and the cells were subsequently transplanted into irradiated recipient mice. These experiments showed that CML LICs were highly enriched in c-Kit+Lin−Sca-1+ (KLS+) population in BCR-ABL+ CML cells. Serial transplantation experiments for CML LICs originated from Foxo3a-deficient mice and littermate wild-type mice indicated that Foxo3a-deficiency reduced lethality of recipient mice at third transplantation. Although recipients that transplanted with wild-type LICs developed CML and acute lymphocytic leukemia (ALL) at third transplantation, we did not observe development of ALL or CML in recipients of Foxo3a deficient LICs after 45 days post-third transplantation, suggesting that the Foxo3a deficient LICs lose their potential to generate malignancies. In addition, a combination of Foxo3a deficiency and imatinib treatment led to efficient depletion of CML in vivo, indicating that Foxo3a plays an essential role for the maintenance of imatinib-resistant CML LICs (Naka et al., Nature 463, 676–680, 2010). Interestingly, when we examined sub-cellular localization of Foxo3a transcription factor in the CML LICs, we found two CML LIC populations; one population was the cells with nuclear localization of Foxo3a (Foxo3a transcription factor is active) and the other population was the cells with cytoplasmic localization of Foxo3a (Foxo3a is inactive). To understand the molecular mechanisms regulating Foxo3a in CML LICs, we next evaluated the activity of upstream BCR-ABL, PI3K, PDK1, and Akt signaling pathway by fluorescence immunohistochemistry. BCR-ABL activity that was determined by phosphorylation levels of CrkL, a down-stream target of BCR-ABL, was detected in almost all of the CML LICs. However, unexpectedly, phosphorylation levels of Akt in the CML LICs with nuclear localization of Foxo3a appeared to be lower than that in the CML LICs with cytoplasmic localization of Foxo3a, despite it is widely believed that BCR-ABL induces activation of Akt signal. Consistent with Akt phosphorylation status, we detected low levels phosphorylation of PDK1 and PI3K p85, upstream regulators for Akt, in the CML LICs with nuclear localization of Foxo3a. Interestingly, expression levels of the cell proliferation antigen Ki67 were lower in the CML LICs with nuclear Foxo3a than that in the CML LICs with cytoplasmic Foxo3a. These results suggest that Foxo3a responsible for maintenance of imatinib-resistant CML LICs may be regulated by molecular mechanisms that are involved in dormancy in CML LICs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The PAX3-FKHR fusion protein created by the t(2;13) translocation in alveolar rhabdomyosarcomas is a more potent transcriptional activator than PAX3.

1995 ◽  
Vol 15 (3) ◽  
pp. 1522-1535 ◽  
Author(s):  
W J Fredericks ◽  
N Galili ◽  
S Mukhopadhyay ◽  
G Rovera ◽  
J Bennicelli ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Fusion Protein ◽  
Target Genes ◽  
Transcriptional Activator ◽  
Wild Type ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Pediatric Solid Tumors ◽  
Single Polypeptide

Alveolar rhabdomyosarcomas are pediatric solid tumors with a hallmark cytogenetic abnormality: translocation of chromosomes 2 and 13 [t(2;13) (q35;q14)]. The genes on each chromosome involved in this translocation have been identified as the transcription factor-encoding genes PAX3 and FKHR. The NH2-terminal paired box and homeodomain DNA-binding domains of PAX3 are fused in frame to COOH-terminal regions of the chromosome 13-derived FKHR gene, a novel member of the forkhead DNA-binding domain family. To determine the role of the fusion protein in transcriptional regulation and oncogenesis, we identified the PAX3-FKHR fusion protein and characterized its function(s) as a transcription factor relative to wild-type PAX3. Antisera specific to PAX3 and FKHR were developed and used to examine PAX3 and PAX3-FKHR expression in tumor cell lines. Sequential immunoprecipitations with anti-PAX3 and anti-FKHR sera demonstrated expression of a 97-kDa PAX3-FKHR fusion protein in the t(2;13)-positive rhabdomyosarcoma Rh30 cell line and verified that a single polypeptide contains epitopes derived from each protein. The PAX3-FKHR protein was localized to the nucleus in Rh30 cells, as was wild-type PAX3, in t(2;13)-negative A673 cells. In gel shift assays using a canonical PAX binding site (e5 sequence), we found that DNA binding of PAX3-FKHR was significantly impaired relative to that of PAX3 despite the two proteins having identical PAX DNA-binding domains. However, the PAX3-FKHR fusion protein was a much more potent transcriptional activator than PAX3 as determined by transient cotransfection assays using e5-CAT reporter plasmids. The PAX3-FKHR protein may function as an oncogenic transcription factor by enhanced activation of normal PAX3 target genes.

scholarly journals Discovery of a Modified Transcription Factor Endowing Yeasts with Organic-Solvent Tolerance and Reconstruction of an Organic-Solvent-Tolerant Saccharomyces cerevisiae Strain

2008 ◽  
Vol 74 (13) ◽  
pp. 4222-4225 ◽  
Author(s):  
Ken Matsui ◽  
Shinya Teranishi ◽  
Shohei Kamon ◽  
Kouichi Kuroda ◽  
Mitsuyoshi Ueda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Organic Solvent ◽  
Carbonyl Compounds ◽  
Solvent Tolerance ◽  
Wild Type ◽  
Organic Solvent Tolerance ◽  
Organic Solvent Tolerant ◽  
Solvent Tolerant ◽  
Wild Type Yeast

ABSTRACT Organic-solvent tolerance in Saccharomyces cerevisiae strain KK-211, which was first isolated as an organic-solvent-tolerant strain, depends on point mutation (R821S) of the transcription factor Pdr1p. The integration of the PDR1 R821S mutation into wild-type yeast results in organic-solvent tolerance, and the PDR1 R821S mutant can reduce carbonyl compounds in organic solvents.

scholarly journals Flow-cytometric microglial sorting coupled with quantitative proteomics identifies moesin as a highly-abundant microglial protein with relevance to Alzheimer’s disease

2019 ◽  
Author(s):  
Sruti Rayaprolu ◽  
Tianwen Gao ◽  
Hailian Xiao ◽  
Supriya Ramesha ◽  
Laura D. Weinstock ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Cell Sorting ◽  
Ribosomal Proteins ◽  
Molecular Mechanisms ◽  
Synaptic Proteins ◽  
Wild Type ◽  
Proteomic Data ◽  
Core Set

AbstractBackgroundProteomic characterization of microglia provides the most proximate assessment of functionally relevant molecular mechanisms of neuroinflammation. However, microglial proteomics studies have been limited by low cellular yield and contamination by non-microglial proteins using existing enrichment strategies.MethodsWe coupled magnetic-activated cell sorting (MACS) and fluorescence activated cell sorting (FACS) of microglia with tandem mass tag-mass spectrometry (TMT-MS) to obtain a highly-pure microglial proteome and identified a core set of highly-abundant microglial proteins in adult mouse brain. We interrogated existing human proteomic data for Alzheimer’s disease (AD) relevance of highly-abundant microglial proteins and performed immuno-histochemical and in-vitro validation studies.ResultsQuantitative multiplexed proteomics by TMT-MS of CD11b+ MACS-enriched (N = 5 mice) and FACS-isolated (N = 5 mice), from adult wild-type mice, identified 1,791 proteins. A total of 203 proteins were highly abundant in both datasets, representing a core-set of highly abundant microglial proteins. In addition, we found 953 differentially enriched proteins comparing MACS and FACS-based approaches, indicating significant differences between both strategies. The FACS-isolated microglia proteome was enriched with cytosolic, endoplasmic reticulum, and ribosomal proteins involved in protein metabolism and immune system functions, as well as an abundance of canonical microglial proteins. Conversely, the MACS-enriched microglia proteome was enriched with mitochondrial and synaptic proteins and higher abundance of neuronal, oligodendrocytic and astrocytic proteins. From the 203 consensus microglial proteins with high abundance in both datasets, we confirmed microglial expression of moesin (Msn) in wild-type and 5xFAD mouse brains as well as in human AD brains. Msn expression is nearly exclusively found in microglia that surround Aβ plaques in 5xFAD brains. In in-vitro primary microglial studies, Msn silencing by siRNA decreased Aβ phagocytosis and increased lipopolysaccharide-induced production of the pro-inflammatory cytokine, tumor necrosis factor (TNF). In network analysis of human brain proteomic data, Msn was a hub protein of an inflammatory co-expression module positively associated with AD neuropathological features and cognitive dysfunction.ConclusionsUsing FACS coupled with TMT-MS as the method of choice for microglial proteomics, we define a core set of highly-abundant adult microglial proteins. Among these, we validate Msn as highly-abundant in plaque-associated microglia with relevance to human AD.

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