Supporting Information for: A highly efficient form of the selenocysteine insertion sequence element in protozoan parasites and its use in mammalian cells

Selenoproteins are an elite group of proteins containing a rare amino acid, selenocysteine (Sec), encoded by the codon, UGA. In eukaryotes, incorporation of Sec requires a Sec insertion sequence (SECIS) element, a stem–loop structure located in the 3\u27-untranslated regions of selenoprotein mRNAs. Here we report identification of a noncanonical form of SECIS element in Toxoplasma gondii and Neospora canine, single-celled apicomplexan parasites of humans and domestic animals. This SECIS has a GGGA sequence in the SBP2-binding site in place of AUGA previously considered invariant. Using a combination of computational and molecular techniques, we show that Toxoplasma and Neospora possess both canonical and noncanonical SECIS elements. The GGGA-type SECIS element supported Sec insertion in mammalian HEK 293 and NIH 3T3 cells and did so more efficiently than the natural mammalian SECIS elements tested. In addition, mammalian type I and type II SECIS elements mutated into the GGGA forms were functional but manifested decreased Sec insertion efficiency. We carried out computational searches for both AUGA and GGGA forms of SECIS elements in Toxoplasma and detected five selenoprotein genes, including one coding for a previously undescribed selenoprotein, designated SelQ, and two containing the GGGA form of the SECIS element. In contrast, the GGGA-type SECIS elements were not detected in mammals and nematodes. As a practical outcome of the study, we developed pSelExpress1, a vector for convenient expression of selenoproteins in mammalian cells. It contains an SBP2 gene and the most efficient tested SECIS element: an AUGA mutant of the GGGA-type Toxoplasma SelT structure

Naked mole-rats maintain healthy skeletal muscle and Complex IV mitochondrial enzyme function into old age

This project was funded by Newcastle University Centre for Brain Ageing and Vitality (supported by the Biotechnology and Biological Sciences Research Council, Engineering and Physical Sciences Research Council, Economic and Social Research Council and Medical Research Council [G0700718]), the UK NIHR Biomedical Research Centre in Age and Age Related Diseases award to the Newcastle upon Tyne Hospitals NHS Foundation Trust, MRC Centre for Neuromuscular Disease [G000608-1], The Wellcome Trust Centre for Mitochondrial Research [096919/Z/11/Z]

Roles of the 15-kDa Selenoprotein (Sep15) in Redox Homeostasis and Cataract Development Revealed by the Analysis of Sep 15 Knockout Mice

The 15-kDa selenoprotein (Sep15) is a thioredoxin-like, endoplasmic reticulum-resident protein involved in the quality control of glycoprotein folding through its interaction with UDP-glucose:glycoprotein glucosyltransferase. Expression of Sep15 is regulated by dietary selenium and the unfolded protein response, but its specific function is not known. In this study, we developed and characterized Sep15 KO mice by targeted removal of exon 2 of the Sep15 gene coding for the cysteinerich UDP-glucose:glycoprotein glucosyltransferase-binding domain. These KO mice synthesized a mutant mRNA, but the shortened protein product could be detected neither in tissues nor in Sep15 KO embryonic fibroblasts. Sep15 KO mice were viable and fertile, showed normal brain morphology, and did not activate endoplasmic reticulum stress pathways. However, parameters of oxidative stress were elevated in the livers of these mice. We found that Sep15 mRNA was enriched during lens development. Further phenotypic characterization of Sep15KO mice revealed a prominent nuclear cataract that developed at an early age. These cataracts did not appear to be associated with severe oxidative stress or glucose dysregulation.Wesuggest that the cataracts resulted from an improper folding status of lens proteins caused by Sep15 deficiency

Roles of the 15-kDa Selenoprotein (Sep15) in Redox Homeostasis and Cataract Development Revealed by the Analysis of Sep 15 Knockout Mice

The 15-kDa selenoprotein (Sep15) is a thioredoxin-like, endoplasmic reticulum-resident protein involved in the quality control of glycoprotein folding through its interaction with UDP-glucose:glycoprotein glucosyltransferase. Expression of Sep15 is regulated by dietary selenium and the unfolded protein response, but its specific function is not known. In this study, we developed and characterized Sep15 KO mice by targeted removal of exon 2 of the Sep15 gene coding for the cysteinerich UDP-glucose:glycoprotein glucosyltransferase-binding domain. These KO mice synthesized a mutant mRNA, but the shortened protein product could be detected neither in tissues nor in Sep15 KO embryonic fibroblasts. Sep15 KO mice were viable and fertile, showed normal brain morphology, and did not activate endoplasmic reticulum stress pathways. However, parameters of oxidative stress were elevated in the livers of these mice. We found that Sep15 mRNA was enriched during lens development. Further phenotypic characterization of Sep15KO mice revealed a prominent nuclear cataract that developed at an early age. These cataracts did not appear to be associated with severe oxidative stress or glucose dysregulation.Wesuggest that the cataracts resulted from an improper folding status of lens proteins caused by Sep15 deficiency

Study of Antioxidant Properties of Agents from the Perspective of Their Action Mechanisms

The creation and analysis of a large variety of existing methods for the evaluation of integrated antioxidant properties are quite relevant in connection with a range of biological mechanisms of the antioxidants (AO) action. In this work, the existing methods are correlated with mechanisms of antioxidant action. It is shown that the results obtained by various methods are mainly incomparable. This can be connected with the implementation of various mechanisms of antioxidant action in methods. The analysis of the literature data presented in this review indicates the difficulty of creating a universal method and the feasibility of using integrated approaches based on the use of several methods that implement and combine various mechanisms of the chemical conversion of antioxidants. This review describes methods for studying the chelating ability of antioxidants, except for methods based on electron and hydrogen atom transfer reactions, which are currently not widely covered in modern literature. With the description of each mechanism, special attention is paid to electrochemical methods, as the interaction of active oxygen metabolites of radical and non-radical nature with antioxidants has an electron/proton/donor-acceptor nature, which corresponds to the nature of electrochemical methods and suggests that they can be used to study the interaction. © 2020 by the authors.This work was financially supported by the Russian Science Foundation (Project No. 20-13-00142)

Влияние феофорбида а на темновое и фотоокисление олефинов

Peculiarities of pheophorbide’s a behavior in conditions of photosensitized and dark oxidation of natural olefin limonene have been analyzed. It is shown that phaeophorbide a is the photosensitizer of limonene oxidation, but it does not act upon its dark oxidation and it does not interreact with peroxides. Although pheophorbide is spent in reactions with radicals formed under H2O2 catalyzed decomposition, and it forms complexes with compounds including zinc and cobaltРассмотрены особенности поведения феофорбида а (Ф) в условиях фотосенсибилизированного и темнового окисления природного олефина лимонена. Показано, что феофорбид а является фотосенсибилизатором окисления лимонена, но не влияет на его темновое окисление и не взаимодействует с пероксидами. Однако Ф расходуется в реакциях с радикалами, образующимися при катализированном распаде Н2О2, и образует комплексы с соединениями цинка и кобальта

Redox Signaling by the RNA Polymerase III TFIIB-Related Factor Brf2.

TFIIB-related factor 2 (Brf2) is a member of the family of TFIIB-like core transcription factors. Brf2 recruits RNA polymerase (Pol) III to type III gene-external promoters, including the U6 spliceosomal RNA and selenocysteine tRNA genes. Found only in vertebrates, Brf2 has been linked to tumorigenesis but the underlying mechanisms remain elusive. We have solved crystal structures of a human Brf2-TBP complex bound to natural promoters, obtaining a detailed view of the molecular interactions occurring at Brf2-dependent Pol III promoters and highlighting the general structural and functional conservation of human Pol II and Pol III pre-initiation complexes. Surprisingly, our structural and functional studies unravel a Brf2 redox-sensing module capable of specifically regulating Pol III transcriptional output in living cells. Furthermore, we establish Brf2 as a central redox-sensing transcription factor involved in the oxidative stress pathway and provide a mechanistic model for Brf2 genetic activation in lung and breast cancer

Reduced Utilization of Selenium by Naked Mole Rats Due to a Specific Defect in GPx1 Expression

Naked mole rat (MR) Heterocephalus glaber is a rodent model of delayed aging because of its unusually long life span (\u3e28 years). It is also not known to develop cancer. In the current work, tissue imaging by x-ray fluorescence microscopy and direct analyses of trace elements revealed low levels of selenium in the MR liver and kidney, whereas MR and mouse brains had similar selenium levels. This effect was not explained by uniform selenium deficiency because methionine sulfoxide reductase activities were similar in mice and MR. However, glutathione peroxidase activity was an order of magnitude lower inMRliver and kidney than in mouse tissues. In addition, metabolic labeling of MR cells with 75Se revealed a loss of the abundant glutathione peroxidase 1 (GPx1) band, whereas other selenoproteins were preserved. To characterize theMRselenoproteome, we sequenced its liver transcriptome. Gene reconstruction revealed standard selenoprotein sequences except for GPx1, which had an early stop codon, and SelP, which had low selenocysteine content. When expressed inHEK293cells,MRGPx1waspresent in low levels,and its expression could be rescued neither by removing the early stop codon nor by replacing its SECIS element. In addition, GPx1 mRNAwas present in lower levels inMRliver than in mouse liver. To determine if GPx1 deficiency could account for the reduced selenium content, we analyzed GPx1 knock-out mice and found reduced selenium levels in their livers and kidneys. Thus, MR is characterized by the reduced utilization of selenium due to a specific defect in GPx1 expression

Blunted Neuronal Calcium Response to Hypoxia in Naked Mole-Rat Hippocampus

Naked mole-rats are highly social and strictly subterranean rodents that live in large communal colonies in sealed and chronically oxygen-depleted burrows. Brain slices from naked mole-rats show extreme tolerance to hypoxia compared to slices from other mammals, as indicated by maintenance of synaptic transmission under more hypoxic conditions and three fold longer latency to anoxic depolarization. A key factor in determining whether or not the cellular response to hypoxia is reversible or leads to cell death may be the elevation of intracellular calcium concentration. In the present study, we used fluorescent imaging techniques to measure relative intracellular calcium changes in CA1 pyramidal cells of hippocampal slices during hypoxia. We found that calcium accumulation during hypoxia was significantly and substantially attenuated in slices from naked mole-rats compared to slices from laboratory mice. This was the case for both neonatal (postnatal day 6) and older (postnatal day 20) age groups. Furthermore, while both species demonstrated more calcium accumulation at older ages, the older naked mole-rats showed a smaller calcium accumulation response than even the younger mice. A blunted intracellular calcium response to hypoxia may contribute to the extreme hypoxia tolerance of naked mole-rat neurons. The results are discussed in terms of a general hypothesis that a very prolonged or arrested developmental process may allow adult naked mole-rat brain to retain the hypoxia tolerance normally only seen in neonatal mammals