Metal-induced Protein Precipitation in E. coli
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Exploris 480
ORGANISM(S): Escherichia Coli
TISSUE(S): Cell Culture
SUBMITTER: 
grace bertles
LAB HEAD: Michael C. Fitzgerald
PROVIDER: PXD035711 | Pride | 2023-01-03
REPOSITORIES: Pride
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Project description:Described here is the application of thermodynamic stability measurements to study age-related differences in the protein folding and stability of proteins in a rodent model of ageing. Using the Stability of Proteins from Rates of Oxidation (SPROX) technique the thermodynamic stability profiles were generated for 807 proteins in brain cell lystaes from mice, aged 6- (n=7) and 18-months (n=9). The biological variability of the protein stability measurements was low and within the experimental error of SPROX. A total of 83 protein hits were detected with age-related stability differences in the brain samples. Remarkably, the large majority of the brain protein hits were destabilized in the old mice, and the hits were enriched in proteins that have slow turnover rates (p <0.07). Furthermore, 70% of the hits have been previously linked to ageing or age-related diseases. These results help validate the use of thermodynamic stability measurements to capture relevant age-related proteomic changes, and they establish a new biophysical link between these proteins and ageing.
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Project description:The proteins in an MCF-7 cell line were probed for tamoxifen (TAM) and n-desmethyl tamoxifen (NDT) induced stability changes using the Stability of Proteins from Rates of Oxidation (SPROX) technique in combination with two different quantitative proteomics strategies. Together the SILAC- and iTRAQ-SPROX experiments described here enabled over 1000 proteins to be assayed for TAM- and NDT- induced protein stability changes, and a total of 163 and 200 protein hits were identified in the TAM and NDT studies, respectively. A subset of high confidence hits were assessed for experimental links to the ER using a STRING analysis. TAM was shown to induce stabilization of Y-box binding protein 1 (YBX1), a protein recently shown to bind the estrogen receptor. TAM was shown to directly interact with purified recombinant YBX1 with Pulse Proteolysis (PP). These results support YBX1 as a direct protein target of TAM. Proteins with altered expression levels with TAM and NDT treatment were identified with SILAC quantitation. In total, 799 and 671 proteins were probed for TAM- and NDT- induced expression changes, respectively, and 49 and 30 proteins had altered expression. This work also involved the use of a novel data analysis strategy to identify protein targets of ligands using the SPROX technique.
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Project description:Manassantin A is a natural product that has been isolated from the perennial herb Saururus chinensis Baill and the aquatic plant Saururus cernuus. Manassantin A has been shown to possess potent hypoxia inducible factor 1 alpha (HIF-1α) inhibitory activity in a cell-based assay screen of thousands of natural products. Manassantin A holds promise as an anti-cancer drug since it has been shown to selectively target tumor cells over normal cells. Due to the complex biological pathways involved in cancer and hypoxia, it is difficult to determine the mode-of-action by which manassantin A inhibits HIF-1. While some of the biological activities of manassantin A have been discovered in various cell-based activity assays, the molecular basis of manassantin A’s biological activities is not well characterized. The proteins in a hypoxic MDA-MB-231 cell lysate were screened for interactions with manassantin A using large scale experiments to uncover novel manassantin A interactions that lead to the drug’s HIF-1 inhibition and anti-cancer activity. Two energetics-based approaches were utilized in this manassantin A mode-of-action study: iTRAQ-SPROX and SILAC-Pulse Proteolysis. In these energetics-based approaches, protein stability is measured using the chemical denaturant dependence of either a methionine oxidation reaction (iTRAQ-SPROX) or a thermolysin protease digest (SILAC-Pulse Proteolysis). Using boh of these techniques, the stability of proteins in the absence and presence of excess manassantin A was monitored to assess ligand-induced protein stability changes.
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