Biochemical and Biophysical Research Communications
Differentially expressed genes in iron-induced prion protein conversion
Introduction
Fatal neurodegenerative prion disorders result from protein misfolding [1]. Prion protein is the key component of prion diseases, also known as transmissible spongiform encephalopathies (TSEs), including scrapie in sheep, chronic wasting disease (CWD) in elk and deer, bovine spongiform encephalopathy (BSE) in cattle, and variant Creutzfeldt-Jakob disease (vCJD) in humans. During the progression of prior disease, a conformational change in normal prion protein (PrPc) to the disease-specific PrP isoform (PrPSc) causes resistance to protease digestion and insolubility in water, yielding a protease-resistant PrP (PrPres). PrPc has several functional roles, and studies have proposed a relationship between PrPc and metal homeostasis, particularly for iron and copper [2], [3]. Oxidative stress, including the redox process, has been observed in response to the imbalance in metal homeostasis in the diseased brain [4], [5]. Two oxidative states of iron are implicated in prion diseases [4], [6], [7], [8]. In our previous study, the conversion and intracellular accumulation of recombinant PrP (rPrP) were specifically derived from Fe(III) rather than Fe(II). Furthermore, Fe(III) does not induce PrPres formation by coming in direct contact with rPrP. Fe(III)-mediated rPrP conversion to PrPres requires a complex cellular environment [9]. Although the pathogenic mechanisms of neurodegeneration, particularly the generation of the infectious isoform of PrP (PrPsc), have been studied extensively [3], [5], [10], [11], factors for the acquisition of protease resistance are not completely defined. Microarray analysis has performed to obtain information regarding the factors involved in disease development and progression, and several studies have been conducted to identify gene expression changes related to prion disease [12], [13], [14]. In this study, we identified differentially expressed genes correlated with prion degeneration dependent on the oxidative states of iron using Affymetrix microarrays and total RNA samples extracted from cells treated with iron and rPrP. Gene ontology (GO) annotations were performed to determine the functional groupings of differentially regulated genes.
Section snippets
Generation of recombinant protein
Bovine rPrP was cloned in a pET23 vector and expressed in Escherichia coli BL21 (DE3). Induction was performed with 1 mM isopropyl β-d-1-thiogalactopyranosid (IPTG) for 16 h before cloning. Cells were resuspended and sonicated in cold phosphate-buffered saline (PBS) containing protease inhibitor cocktail and 5 mM EDTA. Samples were centrifuged at 50,000 × g for 30 min; solubilized with 20 mM sodium phosphate (pH 7.4), 0.5 M NaCl, 20 mM imidazole, and 6 M guanidine-HCl; and then sonicated
Confirmation of PK-resistant internalized rPrP
The presence of the disease-specific isoform of the prion protein in cells treated with two different oxidative states of iron (FeCl2 and FAC) and rPrP was confirmed by western blotting (Fig. 1A). Cells were exposed to 0.3, 0.4, or 0.5 mM of either FeCl2 or FAC. Iron concentration-dependent accumulation of internalized rPrP was observed in cells treated with FAC, whereas no signal was detected from the mock-exposed control. Additionally, an insignificant level of internalized rPrP was observed
Discussion
To identify genes encoding factors related to the accumulation and conversion of rPrP, we identified 97 genes that were differentially expressed in FAC-exposed cells using Affymetrix GeneChip Mouse 2.0 ST Arrays. Thirty genes were specifically selected as differentially regulated genes.
Neuronal degeneration and cell death are specific processes associated with the diseased brain [11], [18], [19]. Gzme, Gzmd, and Slpi genes, which are associated with cell death pathways, and Adm and Dpt genes,
Acknowledgements
This work was supported by a Grant (No. 2012E5200400) from the Korea Centers for Disease Control & Prevention, Ministry of Health and Welfare, South Korea and partially supported by the Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University.
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Cited by (1)
Omics of Prion Diseases
2017, Progress in Molecular Biology and Translational ScienceCitation Excerpt :A different experiment was performed in 2016, when Kim et al. administered either ferrous chloride (FeCl2) [Fe(II)] or ferric ammonium citrate (FAC) [Fe(III)] to hippocampal mouse HpL3-4 cells devoided of functional Prnp, subsequently exposed to bovine recombinant PrP. Following Fe(III) treatment, 97 genes were differentially expressed, while Fe(II) treatment produced moderate alterations; cell growth, cell maintenance, and intra- and extracellular transport were the most affected pathways.53 Since scrapie has been the first reported prion disorder back in mid-1700, and given that it is one of the few naturally occurring TSEs—besides CWD in cervids and BSE in cattle—prion-affected ovine models has been extensively studied in order to understand the mechanism underlying idiopathic prion diseases.