A broad response to intracellular long-chain polyphosphate in human cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE149011 | GEO | 2020/04/22
REPOSITORIES: GEO
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Project description:Polyphosphates (PolyP) are highly anionic, linear polymers composed of long chains of inorganic phosphates linked together by phosphoanhydride bonds. PolyP chains are found in all kingdoms of life, and play roles in phosphate homeostasis, cell growth, infection, and blood coagulation. Unlike in bacteria and yeast, the mammalian enzymes responsible for the synthesis and degradation of polyP are unknown, which presents a challenge for the study of polyP in higher eukaryotes. To overcome this, we use a system to produce polyP inside of human cells through the ectopic expression of the E. coli Ppk1 synthetic enzyme. Here, we present the results from large-scale transcriptomic and proteomic analyses of polyP producing HEK293T cells. We uncovered >350 RNAs and 14 proteins that show changes following in vivo polyP accumulation. Follow up on specific hits revealed that the internal accumulation of polyP resulted in a significant decrease in the dehydrogenase/reductase DHRS2 without a corresponding increase in p53 phosphorylation, and activation of the ERK1/2-ERG1 signaling axis. Finally, following upregulation of internal polyP we observed the relocalization and redistribution of several nuclear proteins, including chromatin bound proteins DEK, TAF10, GTF2I and the translation initiation factor eIF5b. Altogether, our work serves as a novel resource to understand the impact of increased intracellular polyP in mammalian cells.
2020-09-24 | PXD018896 | Pride
Project description:In diverse cells from bacterial to mammalian species, inorganic phosphate is stored in long chains called polyphosphates (polyP). These near universal polymers, ranging from 3 to thousands of phosphate moieties in length, are associated with molecular functions including energy homeostasis, protein folding, and cell signaling. In many cell types, polyphosphate is concentrated in subcellular compartments or organelles. In the budding yeast S. cerevisiae, polyP synthesis by the membrane-bound VTC complex is coupled to its translocation into the lumen of the vacuole, a lysosome-related organelle, where it is stored at high concentrations. In contrast, ectopic expression of bacterial polyphosphate kinase, PPK, results in the toxic accumulation of polyP outside of the vacuole. In this study, we used label-free mass spectrometry to investigate the mechanisms underlying this toxicity. We find that PPK expression results in the activation of a stress response mediated in part by the Hog1 and Yak1 kinases, and Msn2/Msn4 transcription factors. This response is countered by the combined action of the Ddp1 and Ppx1 polyphosphatases that function together to counter polyP accumulation and downstream toxicity. In contrast, ectopic expression of previously proposed mammalian polyphosphatases did not impact PPK-mediated toxicity in the yeast model, suggesting either that these enzymes do not function directly as polyphosphatases in vivo or that they require co-factors unique to higher eukaryotes. Our work provides a mechanistic explanation for why polyP accumulation outside of lysosome-related organelles is toxic. Further, it serves as a resource for exploring how polyP may impact conserved biological processes at a molecular level.
2022-02-04 | PXD031326 | Pride
Project description:Polyphosphates (polyP) are chains of inorganic phosphates that can reach 1000s of residues in length. In Escherichia coli, polyP is produced by the polyP kinase (PPK) and is thought to play a protective role during the response to cellular stress. However, the pathways impacted by PPK activity and polyP accumulation remain poorly characterized. In this work we used label-free mass spectrometry to study the response of bacteria that cannot produce polyP (∆ppk mutants) during nutrient starvation and to identify novel pathways regulated by PPK. In response to nutrient starvation, we found 92 proteins significantly differentially expressed between wild-type and ∆ppkmutant cells. Wild-type cells were enriched for proteins related to amino acid biosynthesis and transport while Δppkmutants were enriched for proteins related to translation and ribosome biogenesis, suggesting that without PPK cells remain inappropriately primed for growth. From our data set, we were particularly interested in Arn and EptA proteins, which were downregulated in ∆ppk mutants compared to wild-type controls, because they play a role in lipid A modifications linked to polymyxin resistance. Using western blotting, we confirm differential expression of these and related proteins, and provide evidence that this mis-regulation in ∆ppk cells stems from a failure to induce the BasS/BasR two component system during starvation. We also show that ∆ppk mutants unable to upregulate Arn and EptA expression lack the respective L-Ara4N and pEtN modifications on lipid A. In line with this observation, loss of ppk restores polymyxin sensitivity in resistant strains carrying a constitutively active basR allele. Overall, we show a new role for PPK in lipid A modification and provide a rationale for targeting PPK to sensitize bacteria towards polymyxin treatment. We further anticipate that our proteomics work will provide an important resource for researchers interested in diverse pathways impacted by PPK.
2024-02-14 | PXD043566 | Pride
Project description:Inorganic polyphosphate (Poly P) is a polymer of various phosphate residues linked by phosphoanhydride bonds as in ATP. It is found in all cells in nature with roles in the origin and survival of species, particularly in bacteria. To study the role of the inorganic polyphosphate in bacteria, we obtained knockout mutants of polyP metabolism genes in Escherichia coli K12. We performed DNA microarray experiments of single mutants in polyphosphate kinase 1 (PPK1), exopolyphosphatase (PPX) and also with the double mutant (PPK1 and PPX). The mutant strains growth normally in LB medium but have different colony morphology phenotypes. All mutants have flagellation problems and a detail description of all gain and lost phenotypes o these strains will be published soon because we performed a complete phenotypic microarray study of all three mutant strains.
2011-06-15 | GSE29954 | GEO
Project description:Inorganic polyphosphate (polyP) is synthesized by bacteria in response to various stresses, but the mechanism of its regulation is unknown. Mutants of Escherichia coli lacking the RNA polymerase-binding transcription factor dksA are defective in polyP synthesis after a nutrient limitation stress, and this defect is reversed in a dksA greA mutant. In this work, we used RNA sequencing to compare transcription in wild-type, dksA, and dksA greA strains of E. coli before and after nutrient limitation, to identify genes whose expression pattern correlates with ability to synthesize polyP.
2020-03-11 | GSE144816 | GEO
Project description:The existing literature points towards the presence of robust mitochondrial mechanisms aimed at mitigating protein dyshomeostasis within the organelle. However, the precise molecular composition underlying these mechanisms remains unclear. Our data show that inorganic polyphosphate (polyP), a polymer well-conserved throughout evolution, is a component of these mechanisms. In mammals, mitochondria exhibit a significant abundance of polyP, and both our research and that of others have already highlighted its potent regulatory effect on bioenergetics. Given the intimate connection between energy metabolism and protein homeostasis, the involvement of polyP in proteostasis has also been demonstrated in several organisms. For example, polyP is a bacterial primordial chaperone, and its role in amyloidogenesis has already been established. Here, using HEK293 cells, our study reveals that the depletion of mitochondrial polyP leads to increased protein aggregation within the organelle, following stress exposure. Furthermore, mitochondrial polyP is able to bind to proteins, and these proteins differ under control and stress conditions. The depletion of mitochondrial polyP significantly affects the proteome under both control and stress conditions, while also exerting regulatory control over gene expression. Our findings suggest that mitochondrial polyP is a previously unrecognized, and potent component of mitochondrial proteostasis. The mass spectrometric raw files for this study are deposited here.
2024-06-28 | MSV000095178 | MassIVE
Project description:Polyphosphate (polyP) is a ubiquitous and abundant compound found in bacteria, fungi, algae, plant, and animals. Among roles of intracellular polyP in bacteria are resistance and survival in the stationary phase of the growth against stress and stringent condition. Therefore, intracellular polyP is considered as a virulence factor of bacteria. In contrast, exogenous polyP has an antimicrobial activity against a variety of microorganisms. To date, much has been numerous studies of antibacterial effect of polyP against gram positive bacteria and fungi while relatively little reports have been published concerning gram negative bacteria. Here we describe bactericidal effect of polyP against gram negative periodontopathic bacterium, Porphyromonas gingivalis, and the transcriptional change by polyP in this bacterium. The bacterial growth was inhibited by polyP (chain length of P3~P75) at concentration of 0.02~0.03%, but not by Pi and PPi at the concentrations. polyP75 was chosen for further experiments, which suppressed the further growth of P. gingivalis as low as 0.03%. polyP75 completely killed the bacterial cells at the concentration of 0.035%. Microarray analysis was employed to identify genes that showed a greater than 1.5-fold difference in the expression by polyP75 at the concentration of 0.03%. It was found that 155 genes were up-regulated and 173 were down-regulated. Down-regulated genes include groups of energy metabolism-related genes, cell envelope-related genes, and genes in relation to biosynthesis of cofactors, prosthetic groups and carriers. Among the down-regulated genes were several involved in DNA replication, cell division protein, and biosynthesis of purines, pyrimidines, nucleosides and nucleotides. In contrast, a large number of ribosomal proteins, transciptional regulators and transposases were up-regulated. Oxidative stress-related genes and iron storage proteins also appeared to be increased in the expression. Real-time PCR confirmed up- and down-regulation of some selected genes. The overall results suggest that polyP has a bactericidal activity against P. gingivalis, interfering with translation, energy metabolism, DNA replication, cell division, and biosynthesis of purines, pyrimidines, nucleoside and nucleotides. polyP may be used as an agent for prevention and treatment of periodontal infections. Polyp-treated vs. untreated intensity ratio data linked below as a supplementary file. Keywords: agent response
2008-05-30 | GSE11471 | GEO
Project description:Polyphosphate (polyP) is a ubiquitous and abundant compound found in bacteria, fungi, algae, plant, and animals. Among roles of intracellular polyP in bacteria are resistance and survival in the stationary phase of the growth against stress and stringent condition. Therefore, intracellular polyP is considered as a virulence factor of bacteria. In contrast, exogenous polyP has an antimicrobial activity against a variety of microorganisms. To date, much has been numerous studies of antibacterial effect of polyP against gram positive bacteria and fungi while relatively little reports have been published concerning gram negative bacteria. Here we describe bactericidal effect of polyP against gram negative periodontopathic bacterium, Porphyromonas gingivalis, and the transcriptional change by polyP in this bacterium. The bacterial growth was inhibited by polyP (chain length of P3~P75) at concentration of 0.02~0.03%, but not by Pi and PPi at the concentrations. polyP75 was chosen for further experiments, which suppressed the further growth of P. gingivalis as low as 0.03%. polyP75 completely killed the bacterial cells at the concentration of 0.035%. Microarray analysis was employed to identify genes that showed a greater than 1.5-fold difference in the expression by polyP75 at the concentration of 0.03%. It was found that 155 genes were up-regulated and 173 were down-regulated. Down-regulated genes include groups of energy metabolism-related genes, cell envelope-related genes, and genes in relation to biosynthesis of cofactors, prosthetic groups and carriers. Among the down-regulated genes were several involved in DNA replication, cell division protein, and biosynthesis of purines, pyrimidines, nucleosides and nucleotides. In contrast, a large number of ribosomal proteins, transciptional regulators and transposases were up-regulated. Oxidative stress-related genes and iron storage proteins also appeared to be increased in the expression. Real-time PCR confirmed up- and down-regulation of some selected genes. The overall results suggest that polyP has a bactericidal activity against P. gingivalis, interfering with translation, energy metabolism, DNA replication, cell division, and biosynthesis of purines, pyrimidines, nucleoside and nucleotides. polyP may be used as an agent for prevention and treatment of periodontal infections. Polyp-treated vs. untreated intensity ratio data linked below as a supplementary file. Keywords: agent response Twenty μg of each total RNA sample was used in separate hybridization experiments on identical arrays. The whole genome of 1,909 genes of P. gingivalis W83 (GenBank accession no. NC_002950) was submitted to NimbleGen System Inc. (Madison, WI) for microarray design and manufacture using maskless, digital micromirror technology. Five replicates of the genome were included per chip. An average of 19 different 60-base oligonucleotides (60-mer probes) represented each gene in the genome. Sixty-mer probes were selected such that each probe had at least three mismatches compared to all other 60-mers in the target genome. A quality control check (hybridization) was performed for each array, which contained on-chip control oligonucleotides. The arrays were analyzed using an Axon GenePix 4000B microarray scanner with associated software (Molecular Devices Corp., Sunnyvale, CA). Gene expression levels were calculated with NimbleScan Version 2.3 (NimbleGen). Relative signal intensities for each gene were generated using the Robust Multi-Array Average algorithm. The data were processed based on quantile normalization method using the R package. This normalization method aims to make the distribution of intensities for each array in a set of arrays the same. The background-adjusted, normalized, and log transformed intensity values were then analyzed using GeneSpring GX 7.3.1 (Silicon Genetics, Palo Alto, CA).
2008-05-30 | E-GEOD-11471 | biostudies-arrayexpress
Project description:It is very important to understand the mechanisms how bacteria become tolerant towards antibiotics during clinical therapy. In a previous study we showed that increased daptomycin (DAP) tolerance of Staphylococcus aureus was due to a point mutation in pitA (inorganic phosphate transporter) that led to intracellular accumulation of both inorganic phosphate (Pi) and polyphosphate (polyP). DAP tolerance in that pitA6 mutant differs from classical resistance mechanisms as there was no increase in minimal inhibitory concentration (MIC). In this study we demonstrate that DAP tolerance in the pitA6 mutant is not triggered by the accumulation of polyP. Transcriptome analysis revealed that about 234 genes were at least 2.0-fold differently expressed in the mutant. Particularly, genes involved in protein biosynthesis, carbohydrate and lipid metabolism as well as in replication and maintenance of DNA were downregulated. However, the most important change was the upregulation of the dlt-operon, which is induced by the accumulation of intracellular Pi. The GraXRS system, known as activator of both dlt and mprF, as well as surface charge, cell wall thickness or the content of wall teichoic acids (WTA) are not involved in DAP tolerance in the pitA6 mutant. In conclusion the DAP tolerance in the pitA6 mutant is due to an upregulation of the dlt-operon triggered directly or indirectly by the accumulation of Pi. WT strain HG003 and its pitA mutant were used to compare transcriptomic changes after 6 or 8 hours of growth in suspension in TSB medium
2016-02-10 | E-GEOD-77069 | biostudies-arrayexpress
Project description:Inorganic polyphosphate (polyP) is an ancestral, ubiquitous, and well-conserved polymer, which is present in all the studied organisms. It is formed by individual subunits of orthophosphate which are linked together by bonds structurally similar and isoenergetic to those found in ATP. While the metabolism and the physiological roles of polyP in some organisms, including bacteria and yeast, have already been described, the exact role of this polymer in mammalian physiology remains still poorly explored. In these organisms, polyP shows a co-localization with mitochondria. Accordingly, its role as a key regulator of bioenergetics has already been demonstrated by our group and others. Here, using Wild-type (Wt) and MitoPPX (cells enzymatically depleted of mitochondrial polyP) SH-SY5Y cells, we conducted a comprehensive study of the status of cellular physiology, using proteomics and metabolomics approaches. Our results suggested a clear dysregulation of mitochondrial physiology, especially of bioenergetics, in MitoPPX cells when compared with Wt. Interestingly, the effects induced by the enzymatically depletion of polyP are similar to those present in the mitochondrial dysfunction which is observed in neurodegenerative disorders and in neuronal aging. In fact, the role of polyP as a component of the cellular stress responses has already been proposed in diverse organisms, including mammals. Thereafter, our data could contribute to place the metabolism of mitochondrial polyP as a valid and innovative pharmacological target in these conditions.
2022-03-16 | PXD028185 | panorama