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Prions are lipidated proteins that interact with endogenous lipids and metal ions. They also assemble into multimers and propagate into the infectious scrapie form known as PrP Sc The high-resolution structure of the infectious PrP Sc state remains unknown, and its analysis largely relies on detergent-based preparations devoid of endogenous ligands. Here we designed polymers that allow isolation of endogenous membrane:protein assemblies in native nanodiscs without exposure to conventional detergents that destabilize protein structures and induce fibrillization. A set of styrene-maleic acid (SMA) polymers including a methylamine derivative facilitated gentle release of the infectious complexes for resolution of multimers, and a thiol-containing version promoted crystallization. Polymer extraction from brain homogenates from Syrian hamsters infected with Hyper prions and WT mice infected with Rocky Mountain Laboratories prions yielded infectious prion nanoparticles including oligomers and microfilaments bound to lipid vesicles. Lipid analysis revealed the brain phospholipids that associate with prion protofilaments, as well as those that are specifically enriched in prion assemblies captured by the methylamine-modified copolymer. A comparison of the infectivity of PrP Sc attached to SMA lipid particles in mice and hamsters indicated that these amphipathic polymers offer a valuable tool for high-yield production of intact, detergent-free prions that retain in vivo activity. This native prion isolation method provides an avenue for producing relevant prion:lipid targets and potentially other proteins that form multimeric assemblies and fibrils on membranes., Competing Interests: Conflict of interest—M. O. and M. E. have filed a patent on related SMA copolymers and methods., (© 2020 Esmaili et al.)

In mammalian cultured cells, the cystine/glutamate exchange transport mediated by system x(c)- is important to maintain intracellular GSH levels. System x(c)- consists of two protein components, xCT and the heavy chain of 4F2 antigen. The activity of system x(c)- is induced by various stimuli, including electrophilic agents like diethyl maleate. In the present study, we have investigated the mechanism of the transcriptional regulation of xCT mRNA by diethyl maleate. The xCT gene consisted of twelve exons and sequence analysis identified four electrophile response element (EpRE)-like sequences between -230 and -1 in the 5'-flanking region, designated EpRE-1 to EpRE-4. To identify sequences mediating the constitutive and induced expression of xCT, a series of 5'-deletion mutants created from the 5'-flanking region were cloned into a luciferase reproter vector and transfected into BHK21 cells. The 5'-deletion analysis revealed that the sequence between -116 and -82 is essential for the basal expression and the sequence between -226 and -116 containing EpRE-1 is essential in response to diethyl maleate. Mutational analysis demonstrated that EpRE-1 is critically involved in the response to diethyl maleate. Other stress agents like arsenite, cadmium, and hydroquinone seemed to induce system x(c)- activity via the same sequence. Furthermore, the experiments using the mouse embryonic fibroblasts derived from the Nrf2-deficient mice revealed that the induction of xCT gene by electrophilic agents is mediated by Nrf2. EpRE occurs in a broad spectrum of genes for the proteins that are involved in the defense against xenobiotics and regulates their expression. The present results have demonstrated that xCT is a novel member of this protein family.

Footprinting of yeast DNA topoisomerase II and its NH2- and COOH-terminal truncation derivatives was carried out to map the locations of lysyl side chains that are involved in enzyme-DNA interaction, in the binding of ATP, or in interaction between domains of the same enzyme molecule. Several conclusions were drawn based on these measurements and the crystal structures of a 92-kDa fragment of the yeast enzyme and a 43-kDa fragment of Escherichia coli gyrase B-subunit. First, the footprinting results support the model previously inferred from the 92-kDa fragment crystal structure that the main site of DNA binding is comprised of a pair of semicircular grooves. Second, the binding of a nonhydrolyzable ATP analog to the yeast enzyme appears to affect citraconylation at a minimum of six lysines in the ATPase domain of each polypeptide. Two of these lysines are probably involved in contacting the nucleotide directly, and one probably becomes buried when the two ATPase domains of a dimeric enzyme come into contact upon ATP binding; for the others, changes in lysine reactivity appear to reflect allosteric changes following ATP binding. Third, from a comparison of the footprint of the intact enzyme and those of the truncated polypeptides comprised of either the NH2- or the COOH-terminal half of the intact polypeptide, it appears that there are few contacts between the NH2- and COOH-terminal half of yeast DNA topoisomerase II.

Inorganic cobalt was found to induce heme oxygenase activity in primary cultures of embryonic chick liver cells and to inhibit the induction of delta-aminolevulinate synthetase by the porphyrinogenic compounds allylisopropylacetamide, dicarbethoxy-1,4-dihydrocollidine, etiocholanolone, phenobarbital, Aroclor (R)1254, and secobarbital. Much smaller concentrations of Co2+ (5 muM) were required to inhibit delta-aminolevulinate synthetase than to induce heme oxygenase activity (50 muM). These effects of Co2+ on heme synthesis and heme degradation were potentiated by depletion of cellular glutathione content as a result of treatment with diethyl maleate. Cobalt inhibition of the induction of delta-aminolevulinate synthetase was of the same magnitude and probably involved the same mechanism as that produced by cobalt heme dimethyl ester and iron heme. The induction of heme oxygenase by cobalt could be blocked by cycloheximide. Plasma protein synthesis was not inhibited in the presence of concentrations of Co2+ which produced inhibition of delta-aminolevulinate synthetase or induction of heme oxygenase. Other metals such as Cd2+ and Cu2+ also inhibited the induction of delta-aminolevulinate synthetase by allylisopropylacetamide. These findings indicate that Co2+ can regulate heme metabolism directly in liver cells without intermediate actions on extrahepatic tissues. It is suggested that regulation of production of delta-aminolevulinate synthetase and heme oxygenase is mediated through the action of the metal ion rather than the metal in the form of a tetrapyrrole chelate.