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1.
J Biol Chem ; 299(2): 102834, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36572187

RESUMO

Under environmental stress, such as glucose deprivation, cells form stress granules-the accumulation of cytoplasmic aggregates of repressed translational initiation complexes, proteins, and stalled mRNAs. Recent research implicates stress granules in various diseases, such as neurodegenerative diseases, but the exact regulators responsible for the assembly and disassembly of stress granules are unknown. An important aspect of stress granule formation is the presence of posttranslational modifications on core proteins. One of those modifications is lysine acetylation, which is regulated by either a lysine acetyltransferase or a lysine deacetylase enzyme. This work deciphers the impact of lysine acetylation on an essential protein found in Saccharomyces cerevisiae stress granules, poly(A)-binding protein (Pab1). We demonstrated that an acetylation mimic of the lysine residue in position 131 reduces stress granule formation upon glucose deprivation and other stressors such as ethanol, raffinose, and vanillin. We present genetic evidence that the enzyme Rpd3 is the primary candidate for the deacetylation of Pab1-K131. Further, our electromobility shift assay studies suggest that the acetylation of Pab1-K131 negatively impacts poly(A) RNA binding. Due to the conserved nature of stress granules, therapeutics targeting the activity of lysine acetyltransferases and lysine deacetylase enzymes may be a promising route to modulate stress granule dynamics in the disease state.


Assuntos
Proteínas de Ligação a Poli(A) , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Grânulos de Estresse , Acetilação , Glucose/metabolismo , Lisina/metabolismo , Lisina Acetiltransferases/metabolismo , Proteínas de Ligação a Poli(A)/genética , Proteínas de Ligação a Poli(A)/metabolismo , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
2.
PLoS Genet ; 16(11): e1009220, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33253187

RESUMO

Cellular metabolism is tightly regulated by many signaling pathways and processes, including lysine acetylation of proteins. While lysine acetylation of metabolic enzymes can directly influence enzyme activity, there is growing evidence that lysine acetylation can also impact protein localization. As the Saccharomyces cerevisiae lysine acetyltransferase complex NuA4 has been implicated in a variety of metabolic processes, we have explored whether NuA4 controls the localization and/or protein levels of metabolic proteins. We performed a high-throughput microscopy screen of over 360 GFP-tagged metabolic proteins and identified 23 proteins whose localization and/or abundance changed upon deletion of the NuA4 scaffolding subunit, EAF1. Within this, three proteins were required for glycogen synthesis and 14 proteins were associated with the mitochondria. We determined that in eaf1Δ cells the transcription of glycogen biosynthesis genes is upregulated resulting in increased proteins and glycogen production. Further, in the absence of EAF1, mitochondria are highly fused, increasing in volume approximately 3-fold, and are chaotically distributed but remain functional. Both the increased glycogen synthesis and mitochondrial elongation in eaf1Δ cells are dependent on Bcy1, the yeast regulatory subunit of PKA. Surprisingly, in the absence of EAF1, Bcy1 localization changes from being nuclear to cytoplasmic and PKA activity is altered. We found that NuA4-dependent localization of Bcy1 is dependent on a lysine residue at position 313 of Bcy1. However, the glycogen accumulation and mitochondrial elongation phenotypes of eaf1Δ, while dependent on Bcy1, were not fully dependent on Bcy1-K313 acetylation state and subcellular localization of Bcy1. As NuA4 is highly conserved with the human Tip60 complex, our work may inform human disease biology, revealing new avenues to investigate the role of Tip60 in metabolic diseases.


Assuntos
Histona Acetiltransferases/metabolismo , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Acetilação , Glicogênio/biossíntese , Histona Acetiltransferases/genética , Lisina/metabolismo , Dinâmica Mitocondrial/genética , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência
3.
PLoS Genet ; 13(2): e1006626, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28231279

RESUMO

Eukaryotic cells form stress granules under a variety of stresses, however the signaling pathways regulating their formation remain largely unknown. We have determined that the Saccharomyces cerevisiae lysine acetyltransferase complex NuA4 is required for stress granule formation upon glucose deprivation but not heat stress. Further, the Tip60 complex, the human homolog of the NuA4 complex, is required for stress granule formation in cancer cell lines. Surprisingly, the impact of NuA4 on glucose-deprived stress granule formation is partially mediated through regulation of acetyl-CoA levels, which are elevated in NuA4 mutants. While elevated acetyl-CoA levels suppress the formation of glucose-deprived stress granules, decreased acetyl-CoA levels enhance stress granule formation upon glucose deprivation. Further our work suggests that NuA4 regulates acetyl-CoA levels through the Acetyl-CoA carboxylase Acc1. Altogether this work establishes both NuA4 and the metabolite acetyl-CoA as critical signaling pathways regulating the formation of glucose-deprived stress granules.


Assuntos
Acetilcoenzima A/genética , Acetiltransferases/genética , Glucose/metabolismo , Histona Acetiltransferases/genética , Proteínas de Saccharomyces cerevisiae/genética , Histona Acetiltransferases/biossíntese , Histona Acetiltransferases/metabolismo , Humanos , Lisina Acetiltransferase 5 , Proteínas Mutantes/biossíntese , Proteínas Mutantes/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Estresse Fisiológico/genética
4.
Metab Eng ; 52: 98-109, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30471359

RESUMO

The conversion of plant material into biofuels and high value products is a two-step process of hydrolysing plant lignocellulose and next fermenting the sugars produced. However, lignocellulosic hydrolysis not only frees sugars for fermentation it simultaneously generates toxic chemicals, including phenolic compounds which severely inhibit yeast fermentation. To understand the molecular basis of phenolic compound toxicity, we performed genome-wide chemogenomic screens in Saccharomyces cerevisiae to identify deletion mutants that were either hypersensitive or resistant to three common phenolic compounds found in plant hydrolysates: coniferyl aldehyde, ferulic acid and 4-hydroxybenzoic acid. Despite being similar in structure, our screen revealed that yeast utilizes distinct pathways to tolerate phenolic compound exposure. Furthermore, although each phenolic compound induced reactive oxygen species (ROS), ferulic acid and 4-hydroxybenzoic acid-induced a general cytoplasmic ROS distribution while coniferyl aldehyde-induced ROS partially localized to the mitochondria and to a lesser extent, the endoplasmic reticulum. We found that the glucose-6-phosphate dehydrogenase enzyme Zwf1, which catalyzes the rate limiting step of pentose phosphate pathway, is required for reducing the accummulation of coniferyl aldehyde-induced ROS, potentially through the sequestering of Zwf1 to sites of ROS accumulation. Our novel insights into biological impact of three common phenolic inhibitors will inform the engineering of yeast strains with improved efficiency of biofuel and biochemical production in the presence hydrolysate-derived phenolic compounds.


Assuntos
Acroleína/análogos & derivados , Ácidos Cumáricos/farmacologia , Fermentação , Redes e Vias Metabólicas/genética , Parabenos/farmacologia , Fenóis/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Acroleína/farmacologia , Estudo de Associação Genômica Ampla , Lignina , Via de Pentose Fosfato/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Triptofano/metabolismo
5.
PLoS Genet ; 10(1): e1004010, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24465216

RESUMO

Unbiased lipidomic approaches have identified impairments in glycerophosphocholine second messenger metabolism in patients with Alzheimer's disease. Specifically, we have shown that amyloid-ß42 signals the intraneuronal accumulation of PC(O-16:0/2:0) which is associated with neurotoxicity. Similar to neuronal cells, intracellular accumulation of PC(O-16:0/2:0) is also toxic to Saccharomyces cerevisiae, making yeast an excellent model to decipher the pathological effects of this lipid. We previously reported that phospholipase D, a phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2)-binding protein, was relocalized in response to PC(O-16:0/2:0), suggesting that this neurotoxic lipid may remodel lipid signaling networks. Here we show that PC(O-16:0/2:0) regulates the distribution of the PtdIns(4)P 5-kinase Mss4 and its product PtdIns(4,5)P2 leading to the formation of invaginations at the plasma membrane (PM). We further demonstrate that the effects of PC(O-16:0/2:0) on the distribution of PM PtdIns(4,5)P2 pools are in part mediated by changes in the biosynthesis of long chain bases (LCBs) and ceramides. A combination of genetic, biochemical and cell imaging approaches revealed that PC(O-16:0/2:0) is also a potent inhibitor of signaling through the Target of rampamycin complex 2 (TORC2). Together, these data provide mechanistic insight into how specific disruptions in phosphocholine second messenger metabolism associated with Alzheimer's disease may trigger larger network-wide disruptions in ceramide and phosphoinositide second messenger biosynthesis and signaling which have been previously implicated in disease progression.


Assuntos
Doença de Alzheimer/metabolismo , Complexos Multiproteicos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosforilcolina/toxicidade , Serina-Treonina Quinases TOR/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Membrana Celular/efeitos dos fármacos , Ceramidas/biossíntese , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina , Complexos Multiproteicos/biossíntese , Neurônios/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/biossíntese , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/biossíntese
6.
Proc Natl Acad Sci U S A ; 110(17): E1641-50, 2013 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-23572591

RESUMO

Recent global proteomic and genomic studies have determined that lysine acetylation is a highly abundant posttranslational modification. The next challenge is connecting lysine acetyltransferases (KATs) to their cellular targets. We hypothesize that proteins that physically interact with KATs may not only predict the cellular function of the KATs but may be acetylation targets. We have developed a mass spectrometry-based method that generates a KAT protein interaction network from which we simultaneously identify both in vivo acetylation sites and in vitro acetylation sites. This modified chromatin-immunopurification coupled to an in vitro KAT assay with mass spectrometry (mChIP-KAT-MS) was applied to the Saccharomyces cerevisiae KAT nucleosome acetyltransferase of histone H4 (NuA4). Using mChIP-KAT-MS, we define the NuA4 interactome and in vitro-enriched acetylome, identifying over 70 previously undescribed physical interaction partners for the complex and over 150 acetyl lysine residues, of which 108 are NuA4-specific in vitro sites. Through this method we determine NuA4 acetylation of its own subunit Epl1 is a means of self-regulation and identify a unique link between NuA4 and the spindle pole body. Our work demonstrates that this methodology may serve as a valuable tool in connecting KATs with their cellular targets.


Assuntos
Imunoprecipitação da Cromatina/métodos , Histona Acetiltransferases/metabolismo , Lisina/metabolismo , Espectrometria de Massas/métodos , Mapeamento de Interação de Proteínas/métodos , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Acetilação , Saccharomyces cerevisiae , Especificidade por Substrato
7.
PLoS Genet ; 7(2): e1001299, 2011 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-21347278

RESUMO

During Alzheimer's Disease, sustained exposure to amyloid-ß42 oligomers perturbs metabolism of ether-linked glycerophospholipids defined by a saturated 16 carbon chain at the sn-1 position. The intraneuronal accumulation of 1-O-hexadecyl-2-acetyl-sn-glycerophosphocholine (C16:0 PAF), but not its immediate precursor 1-O-hexadecyl-sn-glycerophosphocholine (C16:0 lyso-PAF), participates in signaling tau hyperphosphorylation and compromises neuronal viability. As C16:0 PAF is a naturally occurring lipid involved in cellular signaling, it is likely that mechanisms exist to protect cells against its toxic effects. Here, we utilized a chemical genomic approach to identify key processes specific for regulating the sensitivity of Saccharomyces cerevisiae to alkyacylglycerophosphocholines elevated in Alzheimer's Disease. We identified ten deletion mutants that were hypersensitive to C16:0 PAF and five deletion mutants that were hypersensitive to C16:0 lyso-PAF. Deletion of YDL133w, a previously uncharacterized gene which we have renamed SRF1 (Spo14 Regulatory Factor 1), resulted in the greatest differential sensitivity to C16:0 PAF over C16:0 lyso-PAF. We demonstrate that Srf1 physically interacts with Spo14, yeast phospholipase D (PLD), and is essential for PLD catalytic activity in mitotic cells. Though C16:0 PAF treatment does not impact hydrolysis of phosphatidylcholine in yeast, C16:0 PAF does promote delocalization of GFP-Spo14 and phosphatidic acid from the cell periphery. Furthermore, we demonstrate that, similar to yeast cells, PLD activity is required to protect mammalian neural cells from C16:0 PAF. Together, these findings implicate PLD as a potential neuroprotective target capable of ameliorating disruptions in lipid metabolism in response to accumulating oligomeric amyloid-ß42.


Assuntos
Glicerofosfolipídeos/metabolismo , Fosfolipase D/metabolismo , Fator de Ativação de Plaquetas/análogos & derivados , Fator de Ativação de Plaquetas/toxicidade , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Doença de Alzheimer/metabolismo , Linhagem Celular , Glicerofosfolipídeos/genética , Humanos , Metabolismo dos Lipídeos/genética , Mutação/genética , Neurônios/metabolismo , Fosfolipase D/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
8.
Mol Cell Biol ; 44(7): 273-288, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38961766

RESUMO

Here, we report a novel role for the yeast lysine acetyltransferase NuA4 in regulating phospholipid availability for organelle morphology. Disruption of the NuA4 complex results in 70% of cells displaying nuclear deformations and nearly 50% of cells exhibiting vacuolar fragmentation. Cells deficient in NuA4 also show severe defects in the formation of nuclear-vacuole junctions (NJV), as well as a decrease in piecemeal microautophagy of the nucleus (PMN). To determine the cause of these defects we focused on Pah1, an enzyme that converts phosphatidic acid into diacylglycerol, favoring accumulation of lipid droplets over phospholipids that are used for membrane expansion. NuA4 subunit Eaf1 was required for Pah1 localization to the inner nuclear membrane and artificially tethering of Pah1 to the nuclear membrane rescued nuclear deformation and vacuole fragmentation defects, but not defects related to the formation of NVJs. Mutation of a NuA4-dependent acetylation site on Pah1 also resulted in aberrant Pah1 localization and defects in nuclear morphology and NVJ. Our work suggests a critical role for NuA4 in organelle morphology that is partially mediated through the regulation of Pah1 subcellular localization.


Assuntos
Núcleo Celular , Metabolismo dos Lipídeos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Vacúolos , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Vacúolos/metabolismo , Núcleo Celular/metabolismo , Histona Acetiltransferases/metabolismo , Histona Acetiltransferases/genética , Fosfatidato Fosfatase/metabolismo , Fosfatidato Fosfatase/genética , Acetilação , Membrana Nuclear/metabolismo , Fosfolipídeos/metabolismo , Mutação
9.
J Biol Chem ; 287(6): 4139-47, 2012 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-22157760

RESUMO

The Saccharomyces cerevisiae iron-responsive transcription factor, Aft1, has a well established role in regulating iron homeostasis through the transcriptional induction of iron-regulon genes. However, recent studies have implicated Aft1 in other cellular processes independent of iron regulation such as chromosome stability. In addition, chromosome spreads and two-hybrid data suggest that Aft1 interacts with and co-localizes with kinetochore proteins; however, the cellular implications of this have not been established. Here, we demonstrate that Aft1 associates with the kinetochore complex through Iml3. Furthermore, like Iml3, Aft1 is required for the increased association of cohesin with pericentric chromatin, which is required to resist microtubule tension, and aft1Δ cells display chromosome segregation defects in meiosis. Our work defines a new role for Aft1 in chromosome stability and transmission.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Fúngicos/metabolismo , Proteínas do Citoesqueleto/metabolismo , Cinetocoros/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Ciclo Celular/genética , Cromatina/genética , Cromatina/metabolismo , Instabilidade Cromossômica/fisiologia , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos/fisiologia , Cromossomos Fúngicos/genética , Proteínas do Citoesqueleto/genética , Meiose/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Coesinas
10.
Genetics ; 221(4)2022 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-35608294

RESUMO

Acetyl-CoA Carboxylase 1 catalyzes the conversion of acetyl-CoA to malonyl-CoA, the committed step of de novo fatty acid synthesis. As a master regulator of lipid synthesis, acetyl-CoA carboxylase 1 has been proposed to be a therapeutic target for numerous metabolic diseases. We have shown that acetyl-CoA carboxylase 1 activity is reduced in the absence of the lysine acetyltransferase NuA4 in Saccharomyces cerevisiae. This change in acetyl-CoA carboxylase 1 activity is correlated with a change in localization. In wild-type cells, acetyl-CoA carboxylase 1 is localized throughout the cytoplasm in small punctate and rod-like structures. However, in NuA4 mutants, acetyl-CoA carboxylase 1 localization becomes diffuse. To uncover mechanisms regulating acetyl-CoA carboxylase 1 localization, we performed a microscopy screen to identify other deletion mutants that impact acetyl-CoA carboxylase 1 localization and then measured acetyl-CoA carboxylase 1 activity in these mutants through chemical genetics and biochemical assays. Three phenotypes were identified. Mutants with hyper-active acetyl-CoA carboxylase 1 form 1 or 2 rod-like structures centrally within the cytoplasm, mutants with mid-low acetyl-CoA carboxylase 1 activity displayed diffuse acetyl-CoA carboxylase 1, while the mutants with the lowest acetyl-CoA carboxylase 1 activity (hypomorphs) formed thick rod-like acetyl-CoA carboxylase 1 structures at the periphery of the cell. All the acetyl-CoA carboxylase 1 hypomorphic mutants were implicated in sphingolipid metabolism or very long-chain fatty acid elongation and in common, their deletion causes an accumulation of palmitoyl-CoA. Through exogenous lipid treatments, enzyme inhibitors, and genetics, we determined that increasing palmitoyl-CoA levels inhibits acetyl-CoA carboxylase 1 activity and remodels acetyl-CoA carboxylase 1 localization. Together this study suggests yeast cells have developed a dynamic feed-back mechanism in which downstream products of acetyl-CoA carboxylase 1 can fine-tune the rate of fatty acid synthesis.


Assuntos
Acetil-CoA Carboxilase , Histona Acetiltransferases , Lisina Acetiltransferases , Proteínas de Saccharomyces cerevisiae , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Coenzima A/metabolismo , Ácidos Graxos/metabolismo , Genômica , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Lipídeos , Lisina Acetiltransferases/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Esfingolipídeos/metabolismo
11.
Biophys J ; 100(10): L56-8, 2011 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-21575565

RESUMO

Gene expression noise varies with genomic position and is a driving force in the evolution of chromosome organization. Nevertheless, position effects remain poorly characterized. Here, we present a systematic analysis of chromosomal position effects by characterizing single-cell gene expression from euchromatic positions spanning the length of a eukaryotic chromosome. We demonstrate that position affects gene expression by modulating the size of transcriptional bursts, rather than their frequency, and that the histone deacetylase Sir2 plays a role in this process across the chromosome.


Assuntos
Efeitos da Posição Cromossômica/genética , Cromossomos Fúngicos/genética , Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2/metabolismo , Transcrição Gênica , Modelos Genéticos , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Sirtuína 2/genética , Processos Estocásticos
12.
Mol Syst Biol ; 6: 448, 2010 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-21179020

RESUMO

We previously reported a novel affinity purification (AP) method termed modified chromatin immunopurification (mChIP), which permits selective enrichment of DNA-bound proteins along with their associated protein network. In this study, we report a large-scale study of the protein network of 102 chromatin-related proteins from budding yeast that were analyzed by mChIP coupled to mass spectrometry. This effort resulted in the detection of 2966 high confidence protein associations with 724 distinct preys. mChIP resulted in significantly improved interaction coverage as compared with classical AP methodology for ∼75% of the baits tested. Furthermore, mChIP successfully identified novel binding partners for many lower abundance transcription factors that previously failed using conventional AP methodologies. mChIP was also used to perform targeted studies, particularly of Asf1 and its associated proteins, to allow for a understanding of the physical interplay between Asf1 and two other histone chaperones, Rtt106 and the HIR complex, to be gained.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Mapeamento de Interação de Proteínas , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Western Blotting , Histonas/metabolismo , Espectrometria de Massas , Análise Serial de Proteínas , Ligação Proteica , Saccharomyces cerevisiae/genética , Fatores de Transcrição
13.
Mol Cell Proteomics ; 8(4): 870-82, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19106085

RESUMO

Protein-protein interaction mapping has progressed rapidly in recent years, enabling the completion of several high throughput studies. However, knowledge of physical interactions is limited for numerous classes of proteins, such as chromatin-bound proteins, because of their poor solubility when bound to DNA. To address this problem, we have developed a novel method, termed modified chromatin immunopurification (mChIP), that allows for the efficient purification of protein-DNA macromolecules, enabling subsequent protein identification by mass spectrometry. mChIP consists of a single affinity purification step whereby chromatin-bound protein networks are isolated from mildly sonicated and gently clarified cellular extracts using magnetic beads coated with antibodies. We applied the mChIP method in Saccharomyces cerevisiae cells expressing endogenously tandem affinity purification (TAP)-tagged histone H2A or the histone variant Htz1p and successfully co-purified numerous chromatin-bound protein networks as well as DNA. We further challenged the mChIP procedure by purifying three chromatin-bound bait proteins that have proven difficult to purify by traditional methods: Lge1p, Mcm5p, and Yta7p. The protein interaction networks of these three baits dramatically expanded our knowledge of their chromatin environments and illustrate that the innovative mChIP procedure enables an improved characterization of chromatin-associated proteins.


Assuntos
Cromatina/metabolismo , Proteínas Nucleares/análise , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/análise , Saccharomyces cerevisiae/metabolismo , Imunoprecipitação da Cromatina , DNA Fúngico/metabolismo , Histonas/metabolismo , Proteínas Nucleares/isolamento & purificação , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/isolamento & purificação
14.
G3 (Bethesda) ; 11(11)2021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34849793

RESUMO

Metagenomic profiling of the human gut microbiome has discovered DNA from dietary yeasts like Saccharomyces cerevisiae. However, it is unknown if the S. cerevisiae detected by common metagenomic methods are from dead dietary sources, or from live S. cerevisiae colonizing the gut similar to their close relative Candida albicans. While S. cerevisiae can adapt to minimal oxygen and acidic environments, it has not been explored whether this yeast can metabolize mucin, the large, gel-forming, highly glycosylated proteins representing a major source of carbon in the gut mucosa. We reveal that S. cerevisiae can utilize mucin as their main carbon source, as well as perform both a transcriptome analysis and a chemogenomic screen to identify biological pathways required for this yeast to grow optimally in mucin. In total, 739 genes demonstrate significant differential expression in mucin culture, and deletion of 21 genes impact growth in mucin. Both screens suggest that mitochondrial function is required for proper growth in mucin, and through secondary assays we determine that mucin exposure induces mitogenesis and cellular respiration. We further show that deletion of an uncharacterized ORF, YCR095W-A, led to dysfunction in mitochondrial morphology and oxygen consumption in mucin. Finally, we demonstrate that Yps7, an aspartyl protease and homolog to mucin-degrading proteins in C. albicans, is important for growth on mucin. Collectively, our work serves as the initial step toward establishing how this common dietary fungus can survive in the mucus environment of the human gut.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Candida albicans , Humanos , Mucinas/genética , Saccharomyces cerevisiae/genética
15.
iScience ; 24(4): 102327, 2021 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-33889823

RESUMO

Adipic acid production by yeast fermentation is gaining attention as a renewable source of platform chemicals for making nylon products. However, adipic acid toxicity inhibits yeast growth and fermentation. Here, we performed a chemogenomic screen in Saccharomyces cerevisiae to understand the cellular basis of adipic acid toxicity. Our screen revealed that KGD1 (a key gene in the tricarboxylic acid cycle) deletion improved tolerance to adipic acid and its toxic precursor, catechol. Conversely, disrupting ergosterol biosynthesis as well as protein trafficking and vacuolar transport resulted in adipic acid hypersensitivity. Notably, we show that adipic acid disrupts the Membrane Compartment of Can1 (MCC) on the plasma membrane and impacts endocytosis. This was evidenced by the rapid internalization of Can1 for vacuolar degradation. As ergosterol is an essential component of the MCC and protein trafficking mechanisms are required for endocytosis, we highlight the importance of these cellular processes in modulating adipic acid toxicity.

16.
Autophagy ; 17(11): 3671-3689, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-33590792

RESUMO

Macrophage autophagy is a highly anti-atherogenic process that promotes the catabolism of cytosolic lipid droplets (LDs) to maintain cellular lipid homeostasis. Selective autophagy relies on tags such as ubiquitin and a set of selectivity factors including selective autophagy receptors (SARs) to label specific cargo for degradation. Originally described in yeast cells, "lipophagy" refers to the degradation of LDs by autophagy. Yet, how LDs are targeted for autophagy is poorly defined. Here, we employed mass spectrometry to identify lipophagy factors within the macrophage foam cell LD proteome. In addition to structural proteins (e.g., PLIN2), metabolic enzymes (e.g., ACSL) and neutral lipases (e.g., PNPLA2), we found the association of proteins related to the ubiquitination machinery (e.g., AUP1) and autophagy (e.g., HMGB, YWHA/14-3-3 proteins). The functional role of candidate lipophagy factors (a total of 91) was tested using a custom siRNA array combined with high-content cholesterol efflux assays. We observed that knocking down several of these genes, including Hmgb1, Hmgb2, Hspa5, and Scarb2, significantly reduced cholesterol efflux, and SARs SQSTM1/p62, NBR1 and OPTN localized to LDs, suggesting a role for these in lipophagy. Using yeast lipophagy assays, we established a genetic requirement for several candidate lipophagy factors in lipophagy, including HSPA5, UBE2G2 and AUP1. Our study is the first to systematically identify several LD-associated proteins of the lipophagy machinery, a finding with important biological and therapeutic implications. Targeting these to selectively enhance lipophagy to promote cholesterol efflux in foam cells may represent a novel strategy to treat atherosclerosis.Abbreviations: ADGRL3: adhesion G protein-coupled receptor L3; agLDL: aggregated low density lipoprotein; AMPK: AMP-activated protein kinase; APOA1: apolipoprotein A1; ATG: autophagy related; AUP1: AUP1 lipid droplet regulating VLDL assembly factor; BMDM: bone-marrow derived macrophages; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; BSA: bovine serum albumin; CALCOCO2: calcium binding and coiled-coil domain 2; CIRBP: cold inducible RNA binding protein; COLGALT1: collagen beta(1-O)galactosyltransferase 1; CORO1A: coronin 1A; DMA: deletion mutant array; Faa4: long chain fatty acyl-CoA synthetase; FBS: fetal bovine serum; FUS: fused in sarcoma; HMGB1: high mobility group box 1; HMGB2: high mobility group box 2: HSP90AA1: heat shock protein 90: alpha (cytosolic): class A member 1; HSPA5: heat shock protein family A (Hsp70) member 5; HSPA8: heat shock protein 8; HSPB1: heat shock protein 1; HSPH1: heat shock 105kDa/110kDa protein 1; LDAH: lipid droplet associated hydrolase; LIPA: lysosomal acid lipase A; LIR: LC3-interacting region; MACROH2A1: macroH2A.1 histone; MAP1LC3: microtubule-associated protein 1 light chain 3; MCOLN1: mucolipin 1; NBR1: NBR1, autophagy cargo receptor; NPC2: NPC intracellular cholesterol transporter 2; OPTN: optineurin; P/S: penicillin-streptomycin; PLIN2: perilipin 2; PLIN3: perilipin 3; PNPLA2: patatin like phospholipase domain containing 2; RAB: RAB, member RAS oncogene family; RBBP7, retinoblastoma binding protein 7, chromatin remodeling factor; SAR: selective autophagy receptor; SCARB2: scavenger receptor class B, member 2; SGA: synthetic genetic array; SQSTM1: sequestosome 1; TAX1BP1: Tax1 (human T cell leukemia virus type I) binding protein 1; TFEB: transcription factor EB; TOLLIP: toll interacting protein; UBE2G2: ubiquitin conjugating enzyme E2 G2; UVRAG: UV radiation resistance associated gene; VDAC2: voltage dependent anion channel 2; VIM: vimentin.


Assuntos
Autofagia , Colesterol/metabolismo , Células Espumosas/metabolismo , Gotículas Lipídicas/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Gotículas Lipídicas/fisiologia , Proteoma/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitinação
17.
Nat Genet ; 38(6): 610-1, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16736013
18.
Front Bioeng Biotechnol ; 8: 539902, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33154962

RESUMO

Synthetic biology has played a major role in engineering microbial cell factories to convert plant biomass (lignocellulose) to fuels and bioproducts by fermentation. However, the final product yield is limited by inhibition of microbial growth and fermentation by toxic phenolic compounds generated during lignocellulosic pre-treatment and hydrolysis. Advances in the development of systems biology technologies (genomics, transcriptomics, proteomics, metabolomics) have rapidly resulted in large datasets which are necessary to obtain a holistic understanding of complex biological processes underlying phenolic compound toxicity. Here, we review and compare different systems biology tools that have been utilized to identify molecular mechanisms that modulate phenolic compound toxicity in Saccharomyces cerevisiae. By focusing on and comparing functional genomics and transcriptomics approaches we identify common mechanisms potentially underlying phenolic toxicity. Additionally, we discuss possible ways by which integration of data obtained across multiple unbiased approaches can result in new avenues to develop yeast strains with a significant improvement in tolerance to phenolic fermentation inhibitors.

19.
Mol Cell Biol ; 26(8): 3018-28, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16581777

RESUMO

The SAS3-dependent NuA3 histone acetyltransferase complex was originally identified on the basis of its ability to acetylate histone H3 in vitro. Whether NuA3 is capable of acetylating histones in vivo, or how the complex is targeted to the nucleosomes that it modifies, was unknown. To address this question, we asked whether NuA3 is associated with chromatin in vivo and how this association is regulated. With a chromatin pulldown assay, we found that NuA3 interacts with the histone H3 amino-terminal tail, and loss of the H3 tail recapitulates phenotypes associated with loss of SAS3. Moreover, mutation of histone H3 lysine 14, the preferred site of acetylation by NuA3 in vitro, phenocopies a unique sas3Delta phenotype, suggesting that modification of this residue is important for NuA3 function. The interaction of NuA3 with chromatin is dependent on the Set1p and Set2p histone methyltransferases, as well as their substrates, histone H3 lysines 4 and 36, respectively. These results confirm that NuA3 is functioning as a histone acetyltransferase in vivo and that histone H3 methylation provides a mark for the recruitment of NuA3 to nucleosomes.


Assuntos
Cromatina/metabolismo , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Western Blotting , Histona Acetiltransferases/genética , Histonas/química , Histonas/genética , Lisina/química , Metilação , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
20.
Mol Cell Biol ; 26(21): 7871-9, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16923967

RESUMO

The ING (inhibitor of growth) protein family includes a group of homologous nuclear proteins that share a highly conserved plant homeodomain (PHD) finger domain at their carboxyl termini. Members of this family are found in multiprotein complexes that posttranslationally modify histones, suggesting that these proteins serve a general role in permitting various enzymatic activities to interact with nucleosomes. There are three members of the ING family in Saccharomyces cerevisiae: Yng1p, Yng2p, and Pho23p. Yng1p is a component of the NuA3 histone acetyltransferase complex and is required for the interaction of NuA3 with chromatin. To gain insight into the function of the ING proteins, we made use of a genetic strategy to identify genes required for the binding of Yng1p to histones. Using the toxicity of YNG1 overexpression as a tool, we showed that Yng1p interacts with the amino-terminal tail of histone H3 and that this interaction can be disrupted by loss of lysine 4 methylation within this tail. Additionally, we mapped the region of Yng1p required for overexpression of toxicity to the PHD finger, showed that this region capable of binding lysine 4-methylated histone H3 in vitro, and demonstrated that mutations of the PHD finger that abolish binding in vitro are no longer toxic in vivo. These results identify a novel function for the Yng1p PHD finger in promoting stabilization of the NuA3 complex at chromatin through recognition of histone H3 lysine 4 methylation.


Assuntos
Histonas/metabolismo , Lisina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Animais , Cromatina/metabolismo , Histona Acetiltransferases , Histonas/genética , Metilação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
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