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The advantage of channeling nucleotides for very processive functions.

ABSTRACT: Nucleoside triphosphate (NTP)s, like ATP (adenosine 5'-triphosphate) and GTP (guanosine 5'-triphosphate), have long been considered sufficiently concentrated and diffusible to fuel all cellular ATPases (adenosine triphosphatases) and GTPases (guanosine triphosphatases) in an energetically healthy cell without becoming limiting for function. However, increasing evidence for the importance of local ATP and GTP pools, synthesised in close proximity to ATP- or GTP-consuming reactions, has fundamentally challenged our view of energy metabolism. It has become evident that cellular energy metabolism occurs in many specialised 'microcompartments', where energy in the form of NTPs is transferred preferentially from NTP-generating modules directly to NTP-consuming modules. Such energy channeling occurs when diffusion through the cytosol is limited, where these modules are physically close and, in particular, if the NTP-consuming reaction has a very high turnover, i.e. is very processive. Here, we summarise the evidence for these conclusions and describe new insights into the physiological importance and molecular mechanisms of energy channeling gained from recent studies. In particular, we describe the role of glycolytic enzymes for axonal vesicle transport and nucleoside diphosphate kinases for the functions of dynamins and dynamin-related GTPases.

SUBMITTER: Zala D

PROVIDER: S-EPMC5473427 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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The advantage of channeling nucleotides for very processive functions.

Zala Diana D Schlattner Uwe U Desvignes Thomas T Bobe Julien J Roux Aurélien A Chavrier Philippe P Boissan Mathieu M

F1000Research 20170518

Nucleoside triphosphate (NTP)s, like ATP (adenosine 5'-triphosphate) and GTP (guanosine 5'-triphosphate), have long been considered sufficiently concentrated and diffusible to fuel all cellular ATPases (adenosine triphosphatases) and GTPases (guanosine triphosphatases) in an energetically healthy cell without becoming limiting for function. However, increasing evidence for the importance of local ATP and GTP pools, synthesised in close proximity to ATP- or GTP-consuming reactions, has fundamenta ...[more]

PMID: 28663786

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Project description:We have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP (cdiGMP), cdiAMP, and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis. Synthetic genetic array (SGA) and transcriptome analyses indicated that, while these compounds elicit some common reactions in yeast, there are also complex and distinctive responses to each of the three nucleotides. All three are capable of inhibiting eukaryotic cell growth, with the guanine nucleotides exhibiting stronger effects than cdiAMP. Mutations compromising mitochondrial function and chromatin remodeling show negative epistatic interactions with all three nucleotides. In contrast, certain mutations that cause defects in chromatin modification and ribosomal protein function show positive epistasis, alleviating growth inhibition by at least two of the three nucleotides. Uniquely, cdiGMP is lethal both to cells growing by respiration on acetate and to obligately fermentative petite mutants. cdiGMP is also synthetically lethal with the ribonucleotide reductase (RNR) inhibitor hydroxyurea. Heterologous expression of the human ppGpp hydrolase Mesh1p prevented the accumulation of ppGpp in the engineered yeast and restored cell growth. Extensive in vivo interactions between bacterial signaling molecules and eukaryotic gene function occur, resulting in outcomes ranging from growth inhibition to death. cdiGMP functions through a mechanism that must be compensated by unhindered RNR activity or by functionally competent mitochondria. Mesh1p may be required for abrogating the damaging effects of ppGpp in human cells subjected to bacterial infection.IMPORTANCE During infections, pathogenic bacteria can release nucleotides into the cells of their eukaryotic hosts. These nucleotides are recognized as signals that contribute to the initiation of defensive immune responses that help the infected cells recover. Despite the importance of this process, the broader impact of bacterial nucleotides on the functioning of eukaryotic cells remains poorly defined. To address this, we genetically modified cells of the eukaryote Saccharomyces cerevisiae (baker's yeast) to produce three of these molecules (cdiAMP, cdiGMP, and ppGpp) and used the engineered strains as model systems to characterize the effects of the molecules on the cells. In addition to demonstrating that the nucleotides are each capable of adversely affecting yeast cell function and growth, we also identified the cellular functions important for mitigating the damage caused, suggesting possible modes of action. This study expands our understanding of the molecular interactions that can take place between bacterial and eukaryotic cells.

Project description:INTRO: The ongoing challenge of accurately diagnosing infection in the ICU motivates a search for novel molecular diagnostics. In the current study, we tested the hypothesis that the informational content of circulating leukocytes differs, thereby allowing one to rank leukocyte populations on their potential to contribute to RNA diagnostics for pneumonia. METHODS: Sixteen patients (10 male, 6 female) at risk for VAP were entered into our IRB-approved study that collects blood and clinical data every 48 hours for up to 21 days. Four of the sixteen patients developed VAP as diagnosed and treated by the attending ICU physician. Previously reported blood protocols were used to isolate buffy coat, enriched neutrophil, and enriched monocyte populations by using negative selection. Cellular purity was assessed by FACS for one of the 4 VAP patients. Genome-wide expression analysis was performed on RMA-normalized signal from Affymetrix U133 2.0 Plus GeneChips. EDGE software (FDR=0.10) was used to determine changes in mRNA abundance over time for each cell population. RESULTS: During the 5-day window in which each of the four patients (all males) developed VAP, significant changes in gene expression were observed (Table 2), but the information content (number of genes altered) varied across leukocyte populations. These differences were not due to signal variance (coefficient of variation, CV) or differences in the number of samples available for analysis. Moreover, only 0.6% of the monocyte gene list overlaps with the neutrophil list, arguing that neutrophil contamination of monocyte populations is insufficient to explain the 40-fold difference in gene number. CONCLUSIONS: Enriched monocyte populations from circulating blood provide far more genes for study of the host response to VAP than the other 2 cell populations studied. This information should be considered when designing blood gene expression profiling experiments in patients at risk for sepsis. Keywords: time course Four patients over several timepoints measured three different cell types for VAP signal

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The advantage of channeling nucleotides for very processive functions.

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The advantage of channeling nucleotides for very processive functions.

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