RNAseq analysis of the effects of inorganic long-chain polyphosphates on murine macrophages
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE131561 | GEO | 2020/07/07
REPOSITORIES: GEO
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Project description:This paper describes the development of a method that uses capillary gel electrophoresis (CGE) to analyze mixtures of inorganic polyphosphate ((P(i))(n)). Resolution of (P(i))(n) on the basis of n, the number of residues of dehydrated phosphate, is accomplished by CGE using capillaries filled with solutions of poly(N,N-dimethylacrylamide) (PDMA) and indirect detection by the UV absorbance of a chromophore, terephthalate, added to the running buffer. The method is capable of resolving peaks representing (P(i))(n) with n up to approximately 70; preparation and use of authentic standards enables the identification of peaks for (P(i))(n) with n = 1-10. The main advantages of this method over previously reported methods for analyzing mixtures of (P(i))(n) (e.g., gel electrophoresis, CGE using polyacrylamide-filled capillaries) are its resolution, convenience, and reproducibility; gel-filled capillaries are easily regenerated by pumping in fresh, low-viscosity solutions of PDMA. The resolution is comparable to that of ion-exchange chromatography and detection of (P(i))(n) by suppressed conductivity. The method is useful for analyzing (P(i))(n) generated by the dehydration of P(i) at low temperature (125-140 degrees C) with urea, in a reaction that may have been important in prebiotic chemistry. The method should also be useful for characterizing mixtures of other anionic, oligomeric, or polymeric species without an intrinsic chromophore (e.g., sulfated polysaccharides, oligomeric phospho-diesters).
| S-EPMC2926810 | biostudies-literature
Project description:Polyphosphates (polyP) are ancient molecules comprised of inorganic phosphates joined by high energy phosphoanhydride bonds in chains of 3-1000 units in length. In this study, we describe a system to produce polyP in mammalian cells by ectopic expression of the E. coli ppk1+ gene. Production of the EcPpk1 protein results in polyP accumulation throughout the cell, including the nucleus. Using transcriptomic and proteomic analyses, we demonstrate a broad impact of polyP on diverse pathways, including activation of the ERK1/2-EGR1 signaling axis. PolyP accumulation also results in redistribution of several chromatin bound proteins and a translation initiation factor from the nucleus to the cytoplasm. Our work will serve as a novel resource to interrogate polyP biology in higher eukaryotes.
2020-04-22 | GSE149011 | 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:IntroductionUbiquitously found in all life forms, inorganic polyphosphates (polyP) are linear polymers of repeated orthophosphate units. Present in intervertebral disc tissue, polyP was previously shown to increase extracellular matrix production in nucleus pulposus (NP) cells. However, the effects of polyP on human annulus fibrosus (hAF) cell metabolism is not known.Methods and resultsHere, hAF cells cultured in the presence of 0.5 to 1 mM polyP, chain length 22 (polyP-22), showed an increase in glycosaminoglycan content, proteoglycan and collagen synthesis, and aggrecan and collagen type 1 gene expression. Gene expression level of matrix metalloproteinases 1 was reduced while matrix metalloproteinases 3 level was increased in hAF cells treated with 1 mM polyP. Adenosine triphosphate (ATP) synthesis was also significantly increased in hAF cell culture 72 hours after the exposure to 1 mM polyP-22.ConclusionsPolyP thus has both anabolic and bioenergetic effects in AF cells, similar to that observed in NP cells. Together, these results suggest polyP as a potential energy source and a metabolic regulator of disc cells.
| S-EPMC8313173 | biostudies-literature
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: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:Purpose: The goal of this study is to identify differential expression profiles between WT and Setd2-KO in BMDMs induced by LPS for a time-course stimulaiton. Methods: We profiled RNA-seq in bone marrow-derived macrophages stimulated with LPS for 0h, 12h, 24h. Results: We find significant up and down regulated genes in Setd2-KO BMDMs under LPS stimulation.
2022-11-13 | GSE141474 | GEO
Project description:Primary human nasal epithelial cells collected by nasal brushing of healthy donors (data deposited at: EVA- EMBL-EBI; project ID: PRJEB42411; analyses: ERZ1700617) were plated (3x105 cells) and infected with SARS-CoV-2 viral particles 20A (EPI_ISL_514432-S66; MOI, 0.03). After 24 hours, the infected cells were treated with 18.5 microMolar long chain inorganic polyphosphates (polyP120) or vehicle (saline solution) as the negative control for the treatment. After 36 hours, the cells were lysed, and their RNA were extracted for RNAseq.
2024-05-31 | E-MTAB-13363 | biostudies-arrayexpress
Project description:Inorganic polyphosphates (polyPs) are linear polymers composed of repeated phosphate (PO4 3-) units linked together by multiple high-energy phosphoanhydride bonds. In addition to being a source of energy, polyPs have cytoprotective and antiviral activities. Here, we investigated the antiviral activities of long-chain polyPs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In molecular docking analyses, polyPs interacted with several conserved amino acid residues in angiotensin-converting enzyme 2 (ACE2), the host receptor that facilitates virus entry, and in viral RNA-dependent RNA polymerase (RdRp). ELISA and limited proteolysis assays using nano- LC-MS/MS mapped polyP120 binding to ACE2, and site-directed mutagenesis confirmed interactions between ACE2 and SARS-CoV-2 RdRp and identified the specific amino acid residues involved. PolyP120 enhanced the proteasomal degradation of both ACE2 and RdRp, thus impairing replication of the British B.1.1.7 SARS-CoV-2 variant. We thus tested polyPs for functional interactions with the virus in SARS-CoV-2-infected Vero E6 and Caco2 cells and in primary human nasal epithelial cells. Delivery of a nebulized form of polyP120 reduced the amounts of viral positive-sense genomic and subgenomic RNAs, of RNA transcripts encoding proinflammatory cytokines, and of viral structural proteins, thereby presenting SARS-CoV-2 infection in cells in vitro.
| S-EPMC8432949 | biostudies-literature
Project description:RNAseq analysis of the effects of inorganic long-chain polyphosphates on murine macrophages
| PRJNA544063 | ENA