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1.
J Biol Chem ; 294(3): 805-815, 2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30478173

RESUMO

5-Aminoimidazole-4-carboxamide 1-ß-d-ribofuranoside (AICAR, or acadesine) is a precursor of the monophosphate derivative 5-amino-4-imidazole carboxamide ribonucleoside 5'-phosphate (ZMP), an intermediate in de novo purine biosynthesis. AICAR proved to have promising anti-proliferative properties, although the molecular basis of its toxicity is poorly understood. To exert cytotoxicity, AICAR needs to be metabolized, but the AICAR-derived toxic metabolite was not identified. Here, we show that ZMP is the major toxic derivative of AICAR in yeast and establish that its metabolization to succinyl-ZMP, ZDP, or ZTP (di- and triphosphate derivatives of AICAR) strongly reduced its toxicity. Affinity chromatography identified 74 ZMP-binding proteins, including 41 that were found neither as AMP nor as AICAR or succinyl-ZMP binders. Overexpression of karyopherin-ß Kap123, one of the ZMP-specific binders, partially rescued AICAR toxicity. Quantitative proteomic analyses revealed 57 proteins significantly less abundant on nuclei-enriched fractions from AICAR-fed cells, this effect being compensated by overexpression of KAP123 for 15 of them. These results reveal nuclear protein trafficking as a function affected by AICAR.


Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Proteômica , Ribonucleotídeos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Aminoimidazol Carboxamida/farmacocinética , Aminoimidazol Carboxamida/farmacologia , Núcleo Celular/química , Núcleo Celular/genética , Cromatografia de Afinidade , Ribonucleotídeos/farmacocinética , Ribonucleotídeos/farmacologia , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
2.
Curr Genet ; 66(6): 1163-1177, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32780163

RESUMO

Because metabolism is a complex balanced process involving multiple enzymes, understanding how organisms compensate for transient or permanent metabolic imbalance is a challenging task that can be more easily achieved in simpler unicellular organisms. The metabolic balance results not only from the combination of individual enzymatic properties, regulation of enzyme abundance, but also from the architecture of the metabolic network offering multiple interconversion alternatives. Although metabolic networks are generally highly resilient to perturbations, metabolic imbalance resulting from enzymatic defect and specific environmental conditions can be designed experimentally and studied. Starting with a double amd1 aah1 mutant that severely and conditionally affects yeast growth, we carefully characterized the metabolic shuffle associated with this defect. We established that the GTP decrease resulting in an adenylic/guanylic nucleotide imbalance was responsible for the growth defect. Identification of several gene dosage suppressors revealed that TAT1, encoding an amino acid transporter, is a robust suppressor of the amd1 aah1 growth defect. We show that TAT1 suppression occurs through replenishment of the GTP pool in a process requiring the histidine biosynthesis pathway. Importantly, we establish that a tat1 mutant exhibits synthetic sickness when combined with an amd1 mutant and that both components of this synthetic phenotype can be suppressed by specific gene dosage suppressors. Together our data point to a strong phenotypic connection between amino acid uptake and GTP synthesis, a connection that could open perspectives for future treatment of related human defects, previously reported as etiologically highly conserved.


Assuntos
AMP Desaminase/genética , Sistemas de Transporte de Aminoácidos/genética , Aminoidrolases/genética , Nucleosídeos de Purina/genética , Proteínas de Saccharomyces cerevisiae/genética , Guanosina Trifosfato/genética , Humanos , Nucleotídeos/genética , Fenótipo , Saccharomyces cerevisiae/genética
3.
Curr Genet ; 64(6): 1275-1286, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29721631

RESUMO

AICAR is the precursor of ZMP, a metabolite with antiproliferative properties in yeast and human. We aim at understanding how AICAR (and its active form ZMP) affects essential cellular processes. In this work, we found that ZMP accumulation is synthetic lethal with a hypomorphic allele of the ubiquitin-activating enzyme Uba1. A search for gene-dosage suppressors revealed that ubiquitin overexpression was sufficient to restore growth of the uba1 mutant upon AICAR treatment, suggesting that the ubiquitin pool is critical for cells to cope with AICAR. Accordingly, two mutants with constitutive low ubiquitin, ubp6 and doa1, were highly sensitive to AICAR, a phenotype that could be suppressed by ubiquitin overexpression. We established, by genetic means, that these new AICAR-sensitive mutants act in a different pathway from the rad6/bre1 mutants which were previously reported as sensitive to AICAR (Albrecht et al., Genetics 204:1447-1460, 2016). Two ubiquitin-conjugating enzymes (Ubc4 and Cdc34) and a ubiquitin ligase (Cdc4) were found to contribute to the ability of cells to cope with ZMP. This study illustrates the complexity of chemo-genetic interactions and shows how genetic analyses allow deciphering the implicated pathways, the individual gene effects, and their combined phenotypic contribution. Based on additivity and suppression patterns, we conclude that AICAR treatment shows synthetic interactions with distinct branches of the yeast ubiquitin pathway.


Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Ribonucleotídeos/farmacologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Ubiquitina , Ubiquitinação/efeitos dos fármacos , Aminoimidazol Carboxamida/farmacologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação/genética
4.
J Biol Chem ; 290(39): 23947-59, 2015 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-26283791

RESUMO

5-Aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside monophosphate (AICAR) is a natural metabolite with potent anti-proliferative and low energy mimetic properties. At high concentration, AICAR is toxic for yeast and mammalian cells, but the molecular basis of this toxicity is poorly understood. Here, we report the identification of yeast purine salvage pathway mutants that are synthetically lethal with AICAR accumulation. Genetic suppression revealed that this synthetic lethality is in part due to low expression of adenine phosphoribosyl transferase under high AICAR conditions. In addition, metabolite profiling points to the AICAR/NTP balance as crucial for optimal utilization of glucose as a carbon source. Indeed, we found that AICAR toxicity in yeast and human cells is alleviated when glucose is replaced by an alternative carbon source. Together, our metabolic analyses unveil the AICAR/NTP balance as a major factor of AICAR antiproliferative effects.


Assuntos
Adenina Fosforribosiltransferase/antagonistas & inibidores , Aminoimidazol Carboxamida/análogos & derivados , Proliferação de Células/efeitos dos fármacos , Nucleotídeos/metabolismo , Ribonucleotídeos/farmacologia , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Saccharomyces cerevisiae/metabolismo , Adenina Fosforribosiltransferase/genética , Adenina Fosforribosiltransferase/metabolismo , Aminoimidazol Carboxamida/farmacologia , Linhagem Celular , Proliferação de Células/genética , Humanos , Nucleotídeos/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
PLoS Pathog ; 10(6): e1004158, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24945274

RESUMO

The [Het-s] prion of the fungus Podospora anserina represents a good model system for studying the structure-function relationship in amyloid proteins because a high resolution solid-state NMR structure of the amyloid prion form of the HET-s prion forming domain (PFD) is available. The HET-s PFD adopts a specific ß-solenoid fold with two rungs of ß-strands delimiting a triangular hydrophobic core. A C-terminal loop folds back onto the rigid core region and forms a more dynamic semi-hydrophobic pocket extending the hydrophobic core. Herein, an alanine scanning mutagenesis of the HET-s PFD was conducted. Different structural elements identified in the prion fold such as the triangular hydrophobic core, the salt bridges, the asparagines ladders and the C-terminal loop were altered and the effect of these mutations on prion function, fibril structure and stability was assayed. Prion activity and structure were found to be very robust; only a few key mutations were able to corrupt structure and function. While some mutations strongly destabilize the fold, many substitutions in fact increase stability of the fold. This increase in structural stability did not influence prion formation propensity in vivo. However, if an Ala replacement did alter the structure of the core or did influence the shape of the denaturation curve, the corresponding variant showed a decreased prion efficacy. It is also the finding that in addition to the structural elements of the rigid core region, the aromatic residues in the C-terminal semi-hydrophobic pocket are critical for prion propagation. Mutations in the latter region either positively or negatively affected prion formation. We thus identify a region that modulates prion formation although it is not part of the rigid cross-ß core, an observation that might be relevant to other amyloid models.


Assuntos
Peptídeos beta-Amiloides/química , Amiloide/química , Proteínas Fúngicas/química , Modelos Moleculares , Príons/química , Alanina/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Sequência Conservada , Transferência de Energia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Príons/genética , Príons/metabolismo , Dobramento de Proteína , Estabilidade Proteica , Estrutura Secundária de Proteína , Desdobramento de Proteína , Alinhamento de Sequência
6.
J Biol Chem ; 289(24): 16844-54, 2014 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-24778186

RESUMO

5-Aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAr) is the precursor of the active monophosphate form (AICAR), a small molecule with potent anti-proliferative and low energy mimetic properties. The molecular bases for AICAR toxicity at the cellular level are poorly understood. Here, we report the isolation and characterization of several yeast AICAr-hypersensitive mutants. Identification of the cognate genes allowed us to establish that thiamine transporters Thi7 and Thi72 can efficiently take up AICAr under conditions where they are overexpressed. We establish that, under standard growth conditions, Nrt1, the nicotinamide riboside carrier, is the major AICAr transporter in yeast. A study of AICAR accumulation in human cells revealed substantial disparities among cell lines and confirmed that AICAr enters cells via purine nucleoside transporters. Together, our results point to significant differences between yeast and human cells for both AICAr uptake and AICAR accumulation.


Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Proteínas de Membrana Transportadoras/metabolismo , Ribonucleotídeos/farmacologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Aminoimidazol Carboxamida/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Proteínas de Membrana Transportadoras/genética , Camundongos , Mutação , Proteínas de Transporte de Nucleosídeos/genética , Proteínas de Transporte de Nucleosídeos/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Tiamina/metabolismo
7.
Mol Microbiol ; 82(6): 1392-405, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22050595

RESUMO

HET-s is a prion protein of the filamentous fungus Podospora anserina. An orthologue of this protein, called FgHET-s has been identified in Fusarium graminearum. The region of the FgHET-s protein corresponding to the prion forming domain of HET-s, forms amyloid fibrils in vitro. These fibrils seed HET-s(218-289) fibril formation in vitro and vice versa. The amyloid fold of HET-s(218-289) and FgHET-s(218-289) are remarkably similar although they share only 38% identity. The present work corresponds to the functional characterization of the FgHET-s(218-289) region as a prion forming domain in vivo. We show that FgHET-s(218-289) is capable of prion propagation in P. anserina and is able to substitute for the HET-s PFD in the full-length HET-s protein. In accordance with the in vitro cross-seeding experiments, we detect no species barrier between P. anserina and F. graminearum PFDs. We use the yeast Saccharomyces cerevisiae as a host to compare the prion performances of the two orthologous PFDs. We find that FgHET-s(218-289) leads to higher spontaneous prion formation rates and mitotic prion stability than HET-s(218-289). Then we analysed the outcome of HET-s(218-289)/FgHET-s(218-289) coexpression. In spite of the cross-seeding ability of HET-s(218-289) and FgHET-s(218-289), in vivo, homotypic polymerization is favoured over mixed fibril formation.


Assuntos
Fusarium/metabolismo , Expressão Gênica , Podospora/metabolismo , Príons/química , Príons/genética , Proteínas Fúngicas , Fusarium/química , Fusarium/genética , Podospora/química , Podospora/genética , Príons/metabolismo , Conformação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
8.
Elife ; 82019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30860478

RESUMO

Metabolism is a highly integrated process resulting in energy and biomass production. While individual metabolic routes are well characterized, the mechanisms ensuring crosstalk between pathways are poorly described, although they are crucial for homeostasis. Here, we establish a co-regulation of purine and pyridine metabolism in response to external adenine through two separable mechanisms. First, adenine depletion promotes transcriptional upregulation of the de novo NAD+ biosynthesis genes by a mechanism requiring the key-purine intermediates ZMP/SZMP and the Bas1/Pho2 transcription factors. Second, adenine supplementation favors the pyridine salvage route resulting in an ATP-dependent increase of intracellular NAD+. This control operates at the level of the nicotinic acid mononucleotide adenylyl-transferase Nma1 and can be bypassed by overexpressing this enzyme. Therefore, in yeast, pyridine metabolism is under the dual control of ZMP/SZMP and ATP, revealing a much wider regulatory role for these intermediate metabolites in an integrated biosynthesis network.


Assuntos
Proteínas Fúngicas/metabolismo , Regulação Neoplásica da Expressão Gênica , NAD/biossíntese , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Purinas/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenina/química , Trifosfato de Adenosina/química , Biomassa , Cromatografia Líquida , Genótipo , Proteínas de Homeodomínio/metabolismo , Homeostase , Niacina/química , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo
9.
Genetics ; 204(4): 1447-1460, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27707786

RESUMO

Identifying synthetic lethal interactions has emerged as a promising new therapeutic approach aimed at targeting cancer cells directly. Here, we used the yeast Saccharomyces cerevisiae as a simple eukaryotic model to screen for mutations resulting in a synthetic lethality with 5-amino-4-imidazole carboxamide ribonucleoside (AICAR) treatment. Indeed, AICAR has been reported to inhibit the proliferation of multiple cancer cell lines. Here, we found that loss of several histone-modifying enzymes, including Bre1 (histone H2B ubiquitination) and Set1 (histone H3 lysine 4 methylation), greatly enhanced AICAR inhibition on growth via the combined effects of both the drug and mutations on G1 cyclins. Our results point to AICAR impacting on Cln3 subcellular localization and at the Cln1 protein level, while the bre1 or set1 deletion affected CLN1 and CLN2 expression. As a consequence, AICAR and bre1/set1 deletions jointly affected all three G1 cyclins (Cln1, Cln2, and Cln3), leading to a condition known to result in synthetic lethality. Significantly, these chemo-genetic synthetic interactions were conserved in human HCT116 cells. Indeed, knock-down of RNF40, ASH2L, and KMT2D/MLL2 induced a highly significant increase in AICAR sensitivity. Given that KMT2D/MLL2 is mutated at high frequency in a variety of cancers, this synthetic lethal interaction has an interesting therapeutic potential.


Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Antineoplásicos/farmacologia , Evolução Molecular , Histonas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ribonucleotídeos/farmacologia , Saccharomyces cerevisiae/genética , Aminoimidazol Carboxamida/farmacologia , Ciclinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HCT116 , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Proteínas de Neoplasias/metabolismo , Ligação Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Tripeptidil-Peptidase 1
10.
J Cell Biol ; 203(4): 585-94, 2013 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-24247429

RESUMO

The microtubule cytoskeleton is a highly dynamic network. In dividing cells, its complex architecture not only influences cell shape and movement but is also crucial for chromosome segregation. Curiously, nothing is known about the behavior of this cellular machinery in quiescent cells. Here we show that, upon quiescence entry, the Saccharomyces cerevisiae microtubule cytoskeleton is drastically remodeled. Indeed, while cytoplasmic microtubules vanish, the spindle pole body (SPB) assembles a long and stable monopolar array of nuclear microtubules that spans the entire nucleus. Consequently, the nucleolus is displaced. Kinetochores remain attached to microtubule tips but lose SPB clustering and distribute along the microtubule array, leading to a large reorganization of the nucleus. When cells exit quiescence, the nuclear microtubule array slowly depolymerizes and, by pulling attached centromeres back to the SPB, allows the recovery of a typical Rabl-like configuration. Finally, mutants that do not assemble a nuclear array of microtubules are impaired for both quiescence survival and exit.


Assuntos
Ciclo Celular , Núcleo Celular/metabolismo , Microtúbulos/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Núcleo Celular/ultraestrutura , Complexo Dinactina , Dineínas/metabolismo , Viabilidade Microbiana , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Proteínas Mutantes/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo
11.
J Cell Biol ; 192(6): 949-57, 2011 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-21402786

RESUMO

Quiescence is defined as a temporary arrest of proliferation, yet it likely encompasses various cellular situations. Our knowledge about this widespread cellular state remains limited. In particular, little is known about the molecular determinants that orchestrate quiescence establishment and exit. Here we show that upon carbon source exhaustion, budding yeast can enter quiescence from all cell cycle phases. Moreover, using cellular structures that are candidate markers for quiescence, we found that the first steps of quiescence exit can be triggered independently of cell growth and proliferation by the sole addition of glucose in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. Importantly, glucose needs to be internalized and catabolized all the way down to glycolysis to mobilize quiescent cell specific structures, but, strikingly, ATP replenishment is apparently not the key signal. Altogether, these findings strongly suggest that quiescence entry and exit primarily rely on cellular metabolic status and can be uncoupled from the cell cycle.


Assuntos
Ciclo Celular/fisiologia , Metabolismo Energético , Trifosfato de Adenosina/metabolismo , Proliferação de Células , Glucose/metabolismo , Saccharomyces cerevisiae/fisiologia , Schizosaccharomyces/fisiologia , Transdução de Sinais/fisiologia
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