Prion variants, species barriers, generation and propagation

Prion variants faithfully propagate across species barriers, but if the barrier is too high, new variants (mutants) are selected, as shown in a recent BMC Biology report. Protein sequence alteration can prevent accurate structural templating at filament ends producing prion variants

Chaperones that cure yeast artificial [PSI+] and their prion-specific effects

AbstractThe [PSI+] nonsense-suppressor determinant of Saccharomyces cerevisiae results from the ability of Sup35 (eRF3) translation termination factor to undergo prion-like aggregation [1]. Although this process is autocatalytic, in vivo it depends on the chaperone Hsp104, whose lack or overexpression can cure [PSI+] [2]. Overproduction of the chaperone protein Ssb1 increased the [PSI+] curing by excess Hsp104, although it had no effect on its own, and excess chaperone protein Ssa1 protected [PSI+] against Hsp104 [3,4]. We used an artificial [PSI+PS] based on the Sup35 prion-forming domain from yeast Pichia methanolica[5] to find other prion-curing factors. Both [PSI+PS] and [PSI+] have prion ‘strains’, differing in their suppressor efficiency and mitotic stability. We show that [PSI+PS] and a ‘weak’ strain of [PSI+] can be cured by overexpression of chaperones Ssa1, Ssb1 and Ydj1. The ability of different chaperones to cure [PSI+PS] showed significant prion strain specificity, which could be related to variation in Sup35 prion structure. Our results imply that homologs of these chaperones may be active against mammalian prion and amyloid diseases

Formation of Amyloid-Like Fibrils by Y-Box Binding Protein 1 (YB-1) Is Mediated by Its Cold Shock Domain and Modulated by Disordered Terminal Domains

YB-1, a multifunctional DNA- and RNA-binding nucleocytoplasmic protein, is involved in the majority of DNA- and mRNA-dependent events in the cell. It consists of three structurally different domains: its central cold shock domain has the structure of a β-barrel, while the flanking domains are predicted to be intrinsically disordered. Recently, we showed that YB-1 is capable of forming elongated fibrils under high ionic strength conditions. Here we report that it is the cold shock domain that is responsible for formation of YB-1 fibrils, while the terminal domains differentially modulate this process depending on salt conditions. We demonstrate that YB-1 fibrils have amyloid-like features, including affinity for specific dyes and a typical X-ray diffraction pattern, and that in contrast to most of amyloids, they disassemble under nearly physiological conditions

Therapeutic Targeting of the Mitochondria Initiates Excessive Superoxide Production and Mitochondrial Depolarization Causing Decreased mtDNA Integrity

<div><p>Mitochondrial dysregulation is closely associated with excessive reactive oxygen species (ROS) production. Altered redox homeostasis has been implicated in the onset of several diseases including cancer. Mitochondrial DNA (mtDNA) and proteins are particularly sensitive to ROS as they are in close proximity to the respiratory chain (RC). Mitoquinone (MitoQ), a mitochondria-targeted redox agent, selectively damages breast cancer cells possibly through damage induced via enhanced ROS production. However, the effects of MitoQ and other triphenylphosphonium (TPP⁺) conjugated agents on cancer mitochondrial homeostasis remain unknown. The primary objective of this study was to determine the impact of mitochondria-targeted agent [(MTAs) conjugated to TPP⁺: mitoTEMPOL, mitoquinone and mitochromanol-acetate] on mitochondrial physiology and mtDNA integrity in breast (MDA-MB-231) and lung (H23) cancer cells. The integrity of the mtDNA was assessed by quantifying the degree of mtDNA fragmentation and copy number, as well as by measuring mitochondrial proteins essential to mtDNA stability and maintenance (TFAM, SSBP1, TWINKLE, POLG and POLRMT). Mitochondrial status was evaluated by measuring superoxide production, mitochondrial membrane depolarization, oxygen consumption, extracellular acidification and mRNA or protein levels of the RC complexes along with TCA cycle activity. In this study, we demonstrated that all investigated MTAs impair mitochondrial health and decrease mtDNA integrity in MDA-MB-231 and H23 cells. However, differences in the degree of mitochondrial damage and mtDNA degradation suggest unique properties among each MTA that may be cell line, dose and time dependent. Collectively, our study indicates the potential for TPP⁺ conjugated molecules to impair breast and lung cancer cells by targeting mitochondrial homeostasis.</p></div

Acute total body ionizing gamma radiation induces long-term adverse effects and immediate changes in cardiac protein oxidative carbonylation in the rat.

Radiation-induced heart disease presents a significant challenge in the event of an accidental radiation exposure as well as to cancer patients who receive acute doses of irradiation as part of radiation therapy. We utilized the spontaneously hypertensive Wistar-Kyoto rat model, previously shown to demonstrate drug-induced cardiomyopathy, to evaluate the acute and long-term effects of sub-lethal total body gamma irradiation at two, four, and fifty-two weeks. We further examined irreversible oxidative protein carbonylation in the heart immediately following irradiation in the normotensive Wistar-Kyoto rat. Both males and females sustained weight loss and anemic conditions compared to untreated controls over a one-year period as reflected by reduced body weight and low red blood cell count. Increased inflammation was detected by elevated IL-6 serum levels selectively in males at four weeks. Serum cardiac troponin T and I analyses revealed signs of cardiomyopathy at earlier timepoints, but high variability was observed, especially at one year. Echocardiography at two weeks following 5.0Gy treatment revealed a significant decrease in cardiac output in females and a significant decrease in both diastolic and systolic volumes in males. Following 10.0Gy irradiation in the normotensive Wistar-Kyoto rat, the heart tissue showed an increase in total protein oxidative carbonylation accompanied by DNA damage indicated by an increase in γ-H2AX. Using proteomic analyses, we identified several novel proteins which showed a marked difference in carbonylation including those of mitochondrial origin and most notably, cardiac troponin T, one of the key proteins involved in cardiomyocyte contractility. Overall, we present findings of acute oxidative protein damage, DNA damage, cardiac troponin T carbonylation, and long-term cardiomyopathy in the irradiated animals

mtDNA gene expression and stability.

<p>Gene expression of <i>7S</i>, 12S (<i>RNR1</i>) and 16S (<i>RNR2</i>) transcripts (A-B) along with SSBP1 and TFAM protein levels (C-D) in MDA-MB-231 (A,C) and H23 (B,D) cell lines after 24 hours exposure to 0.02% DMSO or MitoT (dark gray bars), MitoQ (gray bars) or MitoCA (light gray bars) at 2μM. In A-B, bars represent the average log2 fold change normalized to <i>GAPDH</i> and the DMSO control. Statistical significance is expressed as asterisks at p<0.05 relative to the DMSO control. In C-D, bars signify the mean densitometry normalized to the corresponding DMSO treatment. For each endpoint, two independent experiments were performed (n = 3). Error bars signify +/-1 SEM.</p

Mitochondrial DNA integrity.

<p>mtDNA damage (A-B) and copy number (C-D) in MDA-MB-231 (A,C) and H23 (B,D) cells after exposure to DMSO [(0.02%) black bars], MitoT (dark gray bars), MitoQ (gray bars) or MitoCA (light gray bars) at 2μM for 24 hours. mtDNA fragmentation (A-B) was evaluated using PCR amplification of a long mitochondrial sequence relative to a short mitochondrial sequence. Band intensities of PCR products were quantitated using densitometry. In A-B, gels are representative images for PCR products. In C & D, mitochondrial copy number was assessed by amplification of short regions of house-keeping genes in both nDNA and mtDNA. Bars depict the mean ratio of long to short band intensities (A-B) or the mean ratio of mtDNA:nDNA (C-D) relative to the DMSO treatment +/-1 SEM. Asterisks show statistical significance at p<0.05 relative to the DMSO control at each time. For each assay, two independent experiments were performed (n = 3).</p