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Excessive Inorganic Phosphate Burden Perturbed Intracellular Signaling: Quantitative Proteomics and Phosphoproteomics Analyses.

ABSTRACT: Inorganic phosphate (Pi) is an essential nutrient for the human body which exerts adverse health effects in excess and deficit. High Pi-mediated cytotoxicity has been shown to induce systemic organ damage, though the underlying molecular mechanisms are poorly understood. In this study, we employed proteomics and phosphoproteomics to analyze Pi-mediated changes in protein abundance and phosphorylation. Bioinformatic analyses and literature review revealed that the altered proteins and phosphorylation were enriched in signaling pathways and diverse biological processes. Western blot analysis confirms the extensive change in protein level and phosphorylation in key effectors that modulate pre-mRNA alternative splicing. Global proteome and phospho-profiling provide a bird-eye view of excessive Pi-rewired cell signaling networks, which deepens our understanding of the molecular mechanisms of phosphate toxicity.

SUBMITTER: Hetz R

PROVIDER: S-EPMC8795896 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Excessive Inorganic Phosphate Burden Perturbed Intracellular Signaling: Quantitative Proteomics and Phosphoproteomics Analyses.

Hetz Rebecca R Beeler Erik E Janoczkin Alexis A Kiers Spencer S Li Ling L Willard Belinda B BB Razzaque Mohammed S MS He Ping P

Frontiers in nutrition 20220114

Inorganic phosphate (Pi) is an essential nutrient for the human body which exerts adverse health effects in excess and deficit. High Pi-mediated cytotoxicity has been shown to induce systemic organ damage, though the underlying molecular mechanisms are poorly understood. In this study, we employed proteomics and phosphoproteomics to analyze Pi-mediated changes in protein abundance and phosphorylation. Bioinformatic analyses and literature review revealed that the altered proteins and phosphoryla ...[more]

PMID: 35096927

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Project description:Inorganic phosphate (Pi) is a central nutrient and signal molecule for bacteria. Pi limitation was shown to increase the virulence of several phylogenetically diverse pathogenic bacteria with different lifestyles. Hypophosphatemia enhances the risk of death in patients due to general bacteremia and was observed after surgical injury in humans. Phosphate therapy, or the reduction of bacterial virulence by the administration of Pi or phosphate-containing compounds, is a promising anti-infective therapy approach that will not cause cytotoxicity or the emergence of antibiotic-resistant strains. The proof of concept of phosphate therapy has been obtained using primarily Pseudomonas aeruginosa (PA). However, a detailed understanding of Pi-induced changes at protein levels is missing. Using pyocyanin production as proxy, we show that the Pi-mediated induction of virulence is a highly cooperative process that occurs between 0.2 to 0.6 mM Pi. We present a proteomics study of PA grown in minimal medium supplemented with either 0.2 mM or 1 mM Pi and rich medium. About half of the predicted PA proteins could be quantified. Among the 1,471 dysregulated proteins comparing growth in 0.2 mM to 1 mM Pi, 1,100 were depleted under Pi-deficient conditions. Most of these proteins are involved in general and energy metabolism, different biosynthetic and catabolic routes, or transport. Pi depletion caused accumulation of proteins that belong to all major families of virulence factors, including pyocyanin synthesis, secretion systems, quorum sensing, chemosensory signaling, and the secretion of proteases, phospholipases, and phosphatases, which correlated with an increase in exoenzyme production and antibacterial activity. IMPORTANCE Antibiotics are our main weapons to fight pathogenic bacteria, but the increase in antibiotic-resistant strains and their consequences represents a major global health challenge, revealing the necessity to develop alternative antimicrobial strategies that do not involve the bacterial killing or growth inhibition. P. aeruginosa has been placed second on the global priority list to guide research on the development of new antibiotics. One of the most promising alternative strategies is the phosphate therapy for which the proof of concept has been obtained for P. aeruginosa. This article reports the detailed changes at the protein levels comparing P. aeruginosa grown under Pi-abundant and Pi-depleted conditions. These data describe in detail the molecular mechanisms underlying phosphate therapy. Apart from Pi, several other phosphate-containing compounds have been used for phosphate therapy and this study will serve as a reference for comparative studies aimed at evaluating the effect of alternative compounds.

Project description:In eukaryotes, chromosomal DNA is equally distributed to daughter cells during mitosis, whereas the number of chromosomes is halved during meiosis. Despite considerable progress in understanding the molecular mechanisms that regulate mitosis, there is currently a lack of complete understanding of the molecular mechanisms regulating meiosis. Here, we took advantage of the fission yeast Schizosaccharomyces pombe, for which highly synchronous meiosis can be induced, and performed quantitative proteomics and phosphoproteomics analyses to track changes in protein expression and phosphorylation during meiotic divisions. We compared the proteomes and phosphoproteomes of exponentially growing mitotic cells with cells harvested around meiosis I, or meiosis II in strains bearing either the temperature-sensitive pat1-114 allele or conditional ATP analog-sensitive pat1-as2 allele of the Pat1 kinase. Comparing pat1-114 with pat1-as2 also allowed us to investigate the impact of elevated temperature (25 °C versus 34 °C) on meiosis, an issue that sexually reproducing organisms face due to climate change. Using TMTpro 18plex labeling and phosphopeptide enrichment strategies, we performed quantification of a total of 4673 proteins and 7172 phosphosites in S. pombe. We found that the protein level of 2680 proteins and the rate of phosphorylation of 4005 phosphosites significantly changed during progression of S. pombe cells through meiosis. The proteins exhibiting changes in expression and phosphorylation during meiotic divisions were represented mainly by those involved in the meiotic cell cycle, meiotic recombination, meiotic nuclear division, meiosis I, centromere clustering, microtubule cytoskeleton organization, ascospore formation, organonitrogen compound biosynthetic process, carboxylic acid metabolic process, gene expression, and ncRNA processing, among others. In summary, our findings provide global overview of changes in the levels and phosphorylation of proteins during progression of S. pombe cells through meiosis at normal and elevated temperatures, laying the groundwork for further elucidation of the functions and importance of specific proteins and their phosphorylation in regulating meiotic divisions in this yeast.

Dataset Information

Excessive Inorganic Phosphate Burden Perturbed Intracellular Signaling: Quantitative Proteomics and Phosphoproteomics Analyses.

Publications

Excessive Inorganic Phosphate Burden Perturbed Intracellular Signaling: Quantitative Proteomics and Phosphoproteomics Analyses.

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