Structural and biochemical analysis of mammalian methionine sulfoxide reductase B2

Aachmann, Finn L.,Kwak, Geun‐,Hee,Del Conte, Rebecca,Kim, Hwa‐,Young,Gladyshev, Vadim N.,Dikiy, Alexander Wiley Subscription Services, Inc., A Wiley Company 2011 Proteins Vol.79 No.11

<P><B>Abstract</B></P><P>Methionine sulfoxide reductases are antioxidant enzymes that repair oxidatively damaged methionine residues in proteins. Mammals have three members of the methionine‐R‐sulfoxide reductase family, including cytosolic MsrB1, mitochondrial MsrB2, and endoplasmic reticulum MsrB3. Here, we report the solution structure of reduced Mus musculus MsrB2 using high resolution nuclear magnetic resonance (NMR) spectroscopy. MsrB2 is a β‐strand rich globular protein consisting of eight antiparallel β‐strands and three N‐terminal α‐helical segments. The latter secondary structure elements represent the main structural difference between mammalian MsrB2 and MsrB1. Structural comparison of mammalian and bacterial MsrB structures indicates that the general topology of this MsrB family is maintained and that MsrB2 more resembles bacterial MsrBs than MsrB1. Structural and biochemical analysis supports the catalytic mechanism of MsrB2 that, in contrast to MsrB1, does not involve a resolving cysteine (Cys). pH dependence of catalytically relevant residues in MsrB2 was accessed by NMR spectroscopy and the p<I>K</I><SUB>a</SUB> of the catalytic Cys162 was determined to be 8.3. In addition, the pH‐dependence of MsrB2 activity showed a maximum at pH 9.0, suggesting that deprotonation of the catalytic Cys is a critical step for the reaction. Further mobility analysis showed a well‐structured N‐terminal region, which contrasted with the high flexibility of this region in MsrB1. Our study highlights important structural and functional aspects of mammalian MsrB2 and provides a unifying picture for structure‐function relationships within the MsrB protein family. Proteins 2011; © 2011 Wiley‐Liss, Inc.</P>

Selenoprotein Gene Nomenclature

Gladyshev, Vadim N.,Arné,r, Elias S.,Berry, Marla J.,Brigelius-Flohé,é,, Regina,Bruford, Elspeth A.,Burk, Raymond F.,Carlson, Bradley A.,Castellano, Sergi,Chavatte, Laurent,Conrad, M American Society for Biochemistry and Molecular Bi 2016 The Journal of biological chemistry Vol.291 No.46

<P>The human genome contains 25 genes coding for selenocysteine-containing proteins (selenoproteins). These proteins are involved in a variety of functions, most notably redox homeostasis. Selenoprotein enzymes with known functions are designated according to these functions: TXNRD1, TXNRD2, and TXNRD3 (thioredoxin reductases), GPX1, GPX2, GPX3, GPX4, and GPX6 (glutathione peroxidases), DIO1, DIO2, and DIO3 (iodothyronine deiodinases), MSRB1 (methionine sulfoxide reductase B1), and SEPHS2 (selenophosphate synthetase 2). Selenoproteins without known functions have traditionally been denoted by SEL or SEP symbols. However, these symbols are sometimes ambiguous and conflict with the approved nomenclature for several other genes. Therefore, there is a need to implement a rational and coherent nomenclature system for selenoprotein-encoding genes. Our solution is to use the root symbol SELENO followed by a letter. This nomenclature applies to SELENOF (selenoprotein F, the 15-kDa selenoprotein, SEP15), SELENOH (selenoprotein H, SELH, C11orf31), SELENOI (selenoprotein I, SELI, EPT1), SELENOK (selenoprotein K, SELK), SELENOM (selenoprotein M, SELM), SELENON (selenoprotein N, SEPN1, SELN), SELENOO (selenoprotein O, SELO), SELENOP (selenoprotein P, SeP, SEPP1, SELP), SELENOS (selenoprotein S, SELS, SEPS1, VIMP), SELENOT (selenoprotein T, SELT), SELENOV (selenoprotein V, SELV), and SELENOW (selenoprotein W, SELW, SEPW1). This system, approved by the HUGO Gene Nomenclature Committee, also resolves conflicting, missing, and ambiguous designations for selenoprotein genes and is applicable to selenoproteins across vertebrates.</P>

Functional Diversity of Cysteine Residues in Proteins and Unique Features of Catalytic Redox-active Cysteines in Thiol Oxidoreductases

Dmitri E. Fomenko,Stefano M. Marino,Vadim N. Gladyshev 한국분자세포생물학회 2008 Molecules and cells Vol.26 No.3

Thiol-dependent redox systems are involved in regulation of diverse biological processes, such as response to stress, signal transduction, and protein folding. The thiol-based redox control is provided by mechanistically similar, but structurally distinct families of enzymes known as thiol oxidoreductases. Many such enzymes have been characterized, but identities and functions of the entire sets of thiol oxidoreductases in organisms are not known. Extreme sequence and structural divergence makes identification of these proteins difficult. Thiol oxidoreductases contain a redox-active cysteine residue, or its functional analog selenocysteine, in their active sites. Here, we describe computational methods for in silico prediction of thiol oxidoreductases in nucleotide and protein sequence databases and identification of their redox-active cysteines. We discuss different functional categories of cysteine residues, describe methods for discrimination between catalytic and noncatalytic and between redox and non-redox cysteine residues and highlight unique properties of the redox-active cysteines based on evolutionary conservation, secondary and three-dimensional structures, and sporadic replacement of cysteines with catalytically superior selenocysteine residues.

Selenoprotein MsrB1 deficiency exacerbates acetaminophen-induced hepatotoxicity <i>via</i> increased oxidative damage

Kim, Ki Young,Kwak, Geun-Hee,Singh, Mahendra Pratap,Gladyshev, Vadim N.,Kim, Hwa-Young Academic Press 2017 Archives of biochemistry and biophysics Vol.634 No.-

<P><B>Abstract</B></P> <P>Acetaminophen (APAP) overdose induces acute liver damage and failure <I>via</I> reactive oxygen species production and glutathione (GSH) depletion. Methionine sulfoxide reductase B1 (MsrB1) is an antioxidant selenoenzyme that specifically catalyzes the reduction of methionine <I>R</I>-sulfoxide residues. In this study, we used <I>MsrB1</I> gene-knockout mice and primary hepatocytes to investigate the effect of MsrB1 on APAP-induced hepatotoxicity. Analyses of histological alterations and serum indicators of liver damage showed that <I>MsrB1</I> <SUP>−/−</SUP> mice were more susceptible to APAP-induced acute liver injury than wild-type (<I>MsrB1</I> <SUP>+/+</SUP>) mice. Consistent with the <I>in vivo</I> results, primary <I>MsrB1</I> <SUP>−/−</SUP> hepatocytes displayed higher susceptibility to APAP-induced cytotoxicity than <I>MsrB1</I> <SUP>+/+</SUP> cells. MsrB1 deficiency increased hepatic oxidative stress after APAP challenge such as hydrogen peroxide production, lipid peroxidation, and protein oxidation levels. Additionally, basal and APAP-induced ratios of reduced-to-oxidized GSH (GSH/GSSG) were significantly lower in <I>MsrB1</I> <SUP>−/−</SUP> than in <I>MsrB1</I> <SUP>+/+</SUP> livers. Nrf2 nuclear accumulation and heme oxygenase-1 expression levels after APAP challenge were lower in <I>MsrB1</I> <SUP>−/−</SUP> than in <I>MsrB1</I> <SUP>+/+</SUP> livers, suggesting that MsrB1 deficiency attenuates the APAP-induced activation of Nrf2. Collectively, the results of this study suggest that selenoprotein MsrB1 plays a protective role against APAP-induced hepatotoxicity <I>via</I> its antioxidative function.</P> <P><B>Highlights</B></P> <P> <UL> <LI> MsrB1 deficiency increases APAP-induced hepatotoxicity. </LI> <LI> MsrB1 depletion enhances hepatic oxidative stress after APAP challenge. </LI> <LI> MsrB1 deficiency accelerates glutathione oxidation in response to APAP. </LI> <LI> MsrB1 deficiency attenuates APAP-induced Nrf2 activation. </LI> </UL> </P>

The selenoproteome of Clostridium sp. OhILAs: Characterization of anaerobic bacterial selenoprotein methionine sulfoxide reductase A

Kim, Hwa-Young,Zhang, Yan,Lee, Byung Cheon,Kim, Jae-Ryong,Gladyshev, Vadim N. Wiley Subscription Services, Inc., A Wiley Company 2009 Proteins Vol.74 No.4

<P>Selenocysteine (Sec) is incorporated into proteins in response to UGA codons. This residue is frequently found at the catalytic sites of oxidoreductases. In this study, we characterized the selenoproteome of an anaerobic bacterium, Clostridium sp. (also known as Alkaliphilus oremlandii) OhILA, and identified 13 selenoprotein genes, five of which have not been previously described. One of the detected selenoproteins was methionine sulfoxide reductase A (MsrA), an antioxidant enzyme that repairs oxidatively damaged methionines in a stereospecific manner. To date, little is known about MsrA from anaerobes. We characterized this selenoprotein MsrA which had a single Sec residue at the catalytic site but no cysteine (Cys) residues in the protein sequence. Its SECIS (Sec insertion sequence) element did not resemble those in Escherichia coli. Although with low translational efficiency, the expression of the Clostridium selenoprotein msrA gene in E. coli could be demonstrated by <SUP>75</SUP>Se metabolic labeling, immunoblot analyses, and enzyme assays, indicating that its SECIS element was recognized by the E. coli Sec insertion machinery. We found that the Sec-containing MsrA exhibited at least a 20-fold higher activity than its Cys mutant form, indicating a critical role of Sec in the catalytic activity of the enzyme. Furthermore, our data revealed that the Clostridium MsrA was inefficiently reducible by thioredoxin, which is a typical reducing agent for MsrA, suggesting the use of alternative electron donors in this anaerobic bacterium that directly act on the selenenic acid intermediate and do not require resolving Cys residues. Proteins 2009. © 2008 Wiley-Liss, Inc.</P>

Organization of the Mammalian Metabolome according to Organ Function, Lineage Specialization, and Longevity

Ma, S.,Yim, S.,Lee, S.G.,Kim, E.,Lee, S.R.,Chang, K.T.,Buffenstein, R.,Lewis, Kaitlyn N.,Park, Thomas J.,Miller, Richard A.,Clish, Clary B.,Gladyshev, Vadim N. Cell Press 2015 Cell metabolism Vol.22 No.2

Biological diversity among mammals is remarkable. Mammalian body weights range seven orders of magnitude and lifespans differ more than 100-fold among species. While genetic, dietary, and pharmacological interventions can be used to modulate these traits in model organisms, it is unknown how they are determined by natural selection. By profiling metabolites in brain, heart, kidney, and liver tissues of 26 mammalian species representing ten taxonomical orders, we report metabolite patterns characteristic of organs, lineages, and species longevity. Our data suggest different rates of metabolite divergence across organs and reveal patterns representing organ-specific functions and lineage-specific physiologies. We identified metabolites that correlated with species lifespan, some of which were previously implicated in longevity control. We also compared the results with metabolite changes in five long-lived mouse models and observed some similar patterns. Overall, this study describes adjustments of the mammalian metabolome according to lifespan, phylogeny, and organ and lineage specialization.

Regulation of HIF-1alpha Activity by Overexpression of Thioredoxin is Independent of Thioredoxin Reductase Status

Salvador Naranjo-Suarez,Dolph L. Hatfield,Bradley A. Carlson,Ryuta Tobe,유민혁,Petra A. Tsuji,Vadim N. Gladyshev 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.2

Under hypoxic conditions, cells activate a transcriptional response mainly driven by hypoxia-inducible factors (HIFs). HIF-1 stabilization and activity are known to be regulated by thioredoxin 1 (Txn1), but how the thioredoxin system regulates the hypoxic response is unknown. By examining the effects of Txn1 overexpression on HIF-1 function in HeLa, HT-29, MCF-7 and EMT6 cell lines, we found that this oxidoreductase did not stabilize HIF-1, yet could increase its activity. These effects were dependent on the redox function of Txn1. However, Txn1 deficiency did not affect HIF-1 hypoxic-stabilization and activity, and overexpression of thioredoxin reductase 1 (TR1), the natural Txn1 reductase, had no influence on HIF-1 activity. Moreover, overexpression of Txn1 in TR1 deficient HeLa and EMT6 cells was still able to increase HIF-1 hypoxic activity. These results indicate that Txn1 is not essential for HIF-1 hypoxic stabilization or activity, that its overex-pression can increase HIF-1 hypoxic activity, and that this effect is observed regardless of TR1 status. Thus, regulation of HIF-1 by the thioredoxin system depends on the specific levels of this system’s major components.

Poster Session : Model Organisms and Disease ; Regulation of methionine sulfoxide reduction by age, calorie restriction and dietary selenium in mice

( Sergey V. Nosvselov ),( Hwa Young Kim ),( Deame Hua ),( Byung Cheon Lee ),( Bertrand Friguet ),( Dolph L. Hatfield ),( Vadim N. Gladyshev ) 한국생화학분자생물학회 (구 한국생화학회) 2007 생화학분자생물학회 춘계학술발표논문집 Vol.2007 No.-

Cell Proliferation and Motility Are Inhibited by G1 Phase Arrest in 15-kDa Selenoprotein-Deficient Chang Liver Cells

Bang, Jeyoung,Huh, Jang Hoe,Na, Ji-Woon,Lu, Qiao,Carlson, Bradley A.,Tobe, Ryuta,Tsuji, Petra A.,Gladyshev, Vadim N.,Hatfield, Dolph L.,Lee, Byeong Jae Korean Society for Molecular and Cellular Biology 2015 Molecules and cells Vol.38 No.5

The 15-kDa selenoprotein (Sep15) is a selenoprotein residing in the lumen of the endoplasmic reticulum (ER) and implicated in quality control of protein folding. Herein, we established an inducible RNAi cell line that targets Sep15 mRNA in Chang liver cells. RNAi-induced Sep15 deficiency led to inhibition of cell proliferation, whereas cell growth was resumed after removal of the knockdown inducer. Sep15-deficient cells were arrested at the G1 phase by upregulating p21 and p27, and these cells were also characterized by ER stress. In addition, Sep15 deficiency led to the relocation of focal adhesions to the periphery of the cell basement and to the decrease of the migratory and invasive ability. All these changes were reversible depending on Sep15 status. Rescuing the knockdown state by expressing a silent mutant Sep15 mRNA that is resistant to siRNA also reversed the phenotypic changes. Our results suggest that SEP15 plays important roles in the regulation of the G1 phase during the cell cycle as well as in cell motility in Chang liver cells, and that this selenoprotein offers a novel functional link between the cell cycle and cell motility.

Selenophosphate synthetase 1 and its role in redox homeostasis, defense and proliferation

Na, Jiwoon,Jung, Jisu,Bang, Jeyoung,Lu, Qiao,Carlson, Bradley A.,Guo, Xiong,Gladyshev, Vadim N.,Kim, Jinhong,Hatfield, Dolph L.,Lee, Byeong Jae Elsevier 2018 FREE RADICAL BIOLOGY AND MEDICINE Vol.127 No.-

<P><B>Abstract</B></P> <P>Selenophosphate synthetase (SEPHS) synthesizes selenophosphate, the active selenium donor, using ATP and selenide as substrates. SEPHS was initially identified and isolated from bacteria and has been characterized in many eukaryotes and archaea. Two SEPHS paralogues, SEPHS1 and SEPHS2, occur in various eukaryotes, while prokaryotes and archaea have only one form of SEPHS. Between the two isoforms in eukaryotes, only SEPHS2 shows catalytic activity during selenophosphate synthesis. Although SEPHS1 does not contain any significant selenophosphate synthesis activity, it has been reported to play an essential role in regulating cellular physiology. Prokaryotic SEPHS contains a cysteine or selenocysteine (Sec) at the catalytic domain. However, in eukaryotes, SEPHS1 contains other amino acids such as Thr, Arg, Gly, or Leu at the catalytic domain, and SEPHS2 contains only a Sec. Sequence comparisons, crystal structure analyses, and ATP hydrolysis assays suggest that selenophosphate synthesis occurs in two steps. In the first step, ATP is hydrolyzed to produce ADP and gamma-phosphate. In the second step, ADP is further hydrolyzed and selenophosphate is produced using gamma-phosphate and selenide. Both SEPHS1 and SEPHS2 have ATP hydrolyzing activities, but Cys or Sec is required in the catalytic domain for the second step of reaction. The gene encoding SEPHS1 is divided by introns, and five different splice variants are produced by alternative splicing in humans. SEPHS1 mRNA is abundant in rapidly proliferating cells such as embryonic and cancer cells and its expression is induced by various stresses including oxidative stress and salinity stress. The disruption of the SEPHS1 gene in mice or <I>Drosophila</I> leads to the inhibition of cell proliferation, embryonic lethality, and morphological changes in the embryos. Targeted removal of SEPHS1 mRNA in insect, mouse, and human cells also leads to common phenotypic changes similar to those observed by in vivo gene knockout: the inhibition of cell growth/proliferation, the accumulation of hydrogen peroxide in mammals and an unidentified reactive oxygen species (ROS) in <I>Drosophila</I>, and the activation of a defense system. Hydrogen peroxide accumulation in SEPHS1-deficient cells is mainly caused by the down-regulation of genes involved in ROS scavenging, and leads to the inhibition of cell proliferation and survival. However, the mechanisms underlying SEPHS1 regulation of redox homeostasis are still not understood.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The main determinant for SEPHS activity is Cys or Sec in the catalytic domain. </LI> <LI> SEPHS1 makes homodimers or heterodimers with its splice variants. </LI> <LI> SEPHS1 interacts with many other proteins including SEPHS2. </LI> <LI> SEPHS1 plays a role in maintaining cellular redox homeostasis. </LI> <LI> Redox status by SEPHS1 is responsible for cell proliferation and defense. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>