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eSymposia | Proteomics in Cell Biology and Disease


Phosphoproteomics revealed excessive phosphate burden perturbed intracellular signaling pathways


Sep 21, 2020 12:00am ‐ Sep 21, 2020 12:00am

Description

Phosphoproteomics revealed excessive phosphate burden perturbed intracellular signaling pathways Phosphoproteomics revealed excessive phosphate burden perturbed intracellular signaling pathways Ping He1, Alexis Janoczkin1, Spencer Kiers1, Erik Beeler1, Belinda B. Willard2, Mohammed S. Razzaque 3 1 Department of Biochemistry, Lake Erie College of Osteopathic Medicine, Erie, PA, USA 2 Proteomics and Metabolomics Core, Cleveland Clinic Lerner Research Institute, Cleveland, Ohio, USA. 3 Department of Pathology, Lake Erie College of Osteopathic Medicine, Erie, PA, USA Phosphate (Pi) is an essential nutrient for the human body that maintains and modulates normal cellular functions. Insufficient intake of dietary Pi leads to skeletal deformities, whereas Pi overload causes cytotoxicity. Preclinical and clinical studies have shown systemic organ damages induced by Pi-mediated cytotoxicity. However, the underlying molecular mechanisms are poorly understood. In our earlier studies, we have shown abnormally high Pi-mediated profound changes in protein expression and phosphorylation in components regulating AKT and mitogen-activated protein kinase (MAPK) signaling cascades, endoplasmic reticulum (ER) stress, and epithelial-mesenchymal transition (EMT). To expand our understanding of Pi-rewired signaling networks, we employed proteomics and phosphoproteomics to systematically analyze Pi-mediated changes in protein abundance and protein phosphorylation. HEK293 cells were treated with 1mM, 10mM (pro-survival) and 40mM (pro-death) Pi for 24 hours in triplicate settings. After trypsinization, the digested peptides were desalted followed by TMT labeling. For global proteomics, the TMT labeled samples were separated using a high pH reversed-phase HPLC method, and the collections were combined into 4 fractions. For phosphoproteomics, the peptides were PO3-enriched followed by TMT labeling. The labeled phosphopeptide samples were combined without fractionation. The peptides were analyzed on a Thermo Scientific Fusion Lumos mass spectrometry system, and the data was analyzed by Sequest programs to search against the human UniProtKB database for peptide/protein identification and quantification. A total of 4704 proteins and 5041 phosphopeptides were identified of which 4249 peptides had quantitative values. We thereafter compared the protein abundance and protein phosphorylation changes between 1mM and 10mM, and 1mM and 40mM Pi treated groups. Compared to 1mM Pi treated groups, 10mM Pi treatment caused one protein (Calmodulin-3) down-regulated over 2 folds and 20 phosphopeptides from 12 proteins down-regulated over 1.5 folds. In contrast to 10mM Pi, 40mM Pi treatment elicited more pronounced changes in both global protein expression and protein phosphorylation. The treatment resulted in over 2-fold change in 18 downregulated proteins and 44 upregulated proteins. It led to over 1.5-fold change in 560 downregulated phosphopeptides from 187 proteins and 19 upregulated phosphopeptides from 16 proteins. Bioinformatic pathway analysis and literature searching revealed that the differentially expressed and phosphorylated proteins were enriched in signal transduction pathways (such as Calcium and AKT signaling) and diverse biological processes (such as Cell cycle/DNA repair, apoptosis, ER stress, and mRNA alternative splicing). Molecular cross-talks were also identified among the Pi-induced pathological pathways. Further biological validation studies are underway to verify the changes of the selected candidates triggered by excessive Pi and to explore their pathological functions in the context of high Pi. Phosphoproteomics-based investigation of Pi-mediated alterations of the global phosphorylation landscape will offer a panoramic view of Pi-associated signaling networks, which will deepen our understanding of molecular mechanisms of phosphate toxicity and likely to provide the potential therapeutic targets to reduce high Pi-related pathologies.

Speaker(s):

  • Ping He, MD, PhD, Lake Erie College of Osteopathic Medicine (LECOM)

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