MsrB1 and MICALs regulate actin assembly and macrophage function via reversible stereoselective methionine oxidation.

Redox control of protein function involves oxidation and reduction of amino acid residues, but the mechanisms and regulators involved are insufficiently understood. Here, we report that in conjunction with Mical proteins, methionine-R-sulfoxide reductase B1 (MsrB1) regulates mammalian actin assembly via stereoselective methionine oxidation and reduction in a reversible, site-specific manner. Two methionine residues in actin are specifically converted to methionine-R-sulfoxide by Mical1 and Mical2 and reduced back to methionine by selenoprotein MsrB1, supporting actin disassembly and assembly, respectively. Macrophages utilize this redox control during cellular activation by stimulating MsrB1 expression and activity as a part of innate immunity. We identified the regulatory role of MsrB1 as a Mical antagonist in orchestrating actin dynamics and macrophage function. More generally, our study shows that proteins can be regulated by reversible site-specific methionine-R-sulfoxidation.

Thiol peroxidases mediate specific genome-wide regulation of gene expression in response to hydrogen peroxide.

Hydrogen peroxide is thought to regulate cellular processes by direct oxidation of numerous cellular proteins, whereas antioxidants, most notably thiol peroxidases, are thought to reduce peroxides and inhibit H(2)O(2) response. However, thiol peroxidases have also been implicated in activation of transcription factors and signaling. It remains unclear if these enzymes stimulate or inhibit redox regulation and whether this regulation is widespread or limited to a few cellular components. Herein, we found that Saccharomyces cerevisiae cells lacking all eight thiol peroxidases were viable and withstood redox stresses. They transcriptionally responded to various redox treatments, but were unable to activate and repress gene expression in response to H(2)O(2). Further studies involving redox transcription factors suggested that thiol peroxidases are major regulators of global gene expression in response to H(2)O(2). The data suggest that thiol peroxidases sense and transfer oxidative signals to the signaling proteins and regulate transcription, whereas a direct interaction between H(2)O(2) and other cellular proteins plays a secondary role.

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 (>28 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 in MR liver and kidney than in mouse tissues. In addition, metabolic labeling of MR cells with (75)Se revealed a loss of the abundant glutathione peroxidase 1 (GPx1) band, whereas other selenoproteins were preserved. To characterize the MR selenoproteome, 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 in HEK 293 cells, MR GPx1 was present 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 mRNA was present in lower levels in MR liver 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.

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 cysteine-rich 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 Sep15 KO 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. We suggest that the cataracts resulted from an improper folding status of lens proteins caused by Sep15 deficiency.

Characterization of surface-exposed reactive cysteine residues in Saccharomyces cerevisiae.

Numerous cellular processes are subject to redox regulation, and thiol-dependent redox control, acting through reactive cysteine (Cys) residues, is among the major mechanisms of redox regulation. However, information on the sets of proteins that provide thiol-based redox regulation or are affected by it is limited. Here, we describe proteomic approaches to characterize proteins that contain reactive thiols and methods to identify redox Cys in these proteins. Using Saccharomyces cerevisiae as a eukaryotic model organism, we identified 284 proteins with exposed reactive Cys and determined the identities of 185 of these residues. We then characterized subsets of these proteins as in vitro targets of major cellular thiol oxidoreductases, thioredoxin and glutaredoxin, and found that these enzymes can control the redox state of a significant number of thiols in target proteins. We further examined common features of exposed reactive Cys and compared them with an unbiased control set of Cys using computational approaches. This analysis (i) validated the efficacy of targeting exposed Cys in proteins in their native, folded state, (ii) quantified the proportion of targets that can be redox regulated via thiol oxidoreductase systems, and (iii) revealed the theoretical range of the experimental approach with regard to protein abundance and physicochemical properties of reactive Cys. From these analyses, we estimate that approximately one-fourth of exposed Cys in the yeast proteome can be regarded as functional sites, either subject to regulation by thiol oxidoreductases or involved in structural disulfides and metal binding.

Compartmentalization and regulation of mitochondrial function by methionine sulfoxide reductases in yeast.

Elevated levels of reactive oxygen species can damage proteins. Sulfur-containing amino acid residues, cysteine and methionine, are particularly susceptible to such damage. Various enzymes evolved to protect proteins or repair oxidized residues, including methionine sulfoxide reductases MsrA and MsrB, which reduce methionine (S)-sulfoxide (Met-SO) and methionine (R)-sulfoxide (Met-RO) residues, respectively, back to methionine. Here, we show that MsrA and MsrB are involved in the regulation of mitochondrial function. Saccharomyces cerevisiae mutant cells lacking MsrA, MsrB, or both proteins had normal levels of mitochondria but lower levels of cytochrome c and fewer respiration-competent mitochondria. The growth of single MsrA or MsrB mutants on respiratory carbon sources was inhibited, and that of the double mutant was severely compromised, indicating impairment of mitochondrial function. Although MsrA and MsrB are thought to have similar roles in oxidative protein repair each targeting a diastereomer of methionine sulfoxide, their deletion resulted in different phenotypes. GFP fusions of MsrA and MsrB showed different localization patterns and primarily localized to cytoplasm and mitochondria, respectively. This finding agreed with compartment-specific enrichment of MsrA and MsrB activities. These results show that oxidative stress contributes to mitochondrial dysfunction through oxidation of methionine residues in proteins located in different cellular compartments.

Ion mediated monolayer deposition of gold nanoparticles on microorganisms: discrimination by age.

A general strategy to target cells by nanoparticles for drug delivery, imaging, or diagnostics involves immunospecific binding between the probes and target molecules on the particles and on the cell surface, respectively. Usually, the macromolecular nature of the molecules requires a specific conformation to achieve the desired immunospecificity, and the extent of deposition of particles is limited by the number of receptor molecules present on the cell. In this report, we successfully obtain targeted binding by decorating the nanoparticle with simple ions, such as Ca(2+), without affecting the cell's vitality. The yeast cells for study, Saccharomyces cerevisiae, have no specific electrostatic affinity toward positive charge as confirmed by lysine-coated Au nanoparticles. The specificity of nanoparticle binding is found to be directly related to the metabolic vitality of the yeast cell (i.e., a significantly larger deposition occurs on a younger generation with higher metabolism than on older cells). The ion-mediated targeted deposition seems to be a general phenomenon for biologically important ions, as demonstrated by the contrast between Mg(2+) and (toxic) Cd(2+). The high density of (percolating) nanoparticle deposition as a monolayer on the cells, as a result of the large number of ion receptors on the cell surface, is shown to be a potential method for building bioelectronic devices. The use of ions as an interface to target cells can have possible applications in diagnosing diseases and making biosensors using live cells.

A functional link between housekeeping selenoproteins and phase II enzymes.

Sec (selenocysteine) is biosynthesized on its tRNA and incorporated into selenium-containing proteins (selenoproteins) as the 21st amino acid residue. Selenoprotein synthesis is dependent on Sec tRNA and the expression of this class of proteins can be modulated by altering Sec tRNA expression. The gene encoding Sec tRNA (Trsp) is a single-copy gene and its targeted removal in liver demonstrated that selenoproteins are essential for proper function wherein their absence leads to necrosis and hepatocellular degeneration. In the present study, we found that the complete loss of selenoproteins in liver was compensated for by an enhanced expression of several phase II response genes and their corresponding gene products. The replacement of selenoprotein synthesis in mice carrying mutant Trsp transgenes, wherein housekeeping, but not stress-related selenoproteins are expressed, led to normal expression of phase II response genes. Thus the present study provides evidence for a functional link between housekeeping selenoproteins and phase II enzymes.

Analysis of methionine/selenomethionine oxidation and methionine sulfoxide reductase function using methionine-rich proteins and antibodies against their oxidized forms.

Methionine (Met) residues are present in most proteins. However, this sulfur-containing amino acid is highly susceptible to oxidation. In cells, the resulting Met sulfoxides are reduced back to Met by stereospecific reductases MsrA and MsrB. Reversible Met oxidation occurs even in the absence of stress, is elevated during aging and disease, but is notoriously difficult to monitor. In this work, we computationally identified natural Met-rich proteins (MRPs) and characterized three such proteins containing 21-33% Met residues. Oxidation of multiple Met residues in MRPs with H(2)O(2) and reduction of Met sulfoxides with MsrA/MsrB dramatically influenced the mobility of these proteins on polyacrylamide gels and could be monitored by simple SDS-PAGE. We further prepared antibodies enriched for reduced and Met sulfoxide forms of these proteins and used them to monitor Met oxidation and reduction by immunoblot assays. We describe applications of these reagents for the analysis of MsrA and MsrB functions, as well as the development of the assay for high-throughput analysis of their activities. We also show that all Met sulfoxide residues in an MRP can be reduced by MsrA and MsrB. Furthermore, we prepared a selenomethionine form of an MRP and found that selenomethionine selenoxide residues can be efficiently reduced nonenzymatically by glutathione and other thiol compounds. Selenomethionine selenoxide residues were not recognized by antibodies specific for the Met sulfoxide form of an MRP. These findings, reagents, assays, and approaches should facilitate research and applications in the area of Met sulfoxide reduction, oxidative stress, and aging.

Functional diversity of cysteine residues in proteins and unique features of catalytic redox-active cysteines in thiol oxidoreductases.

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 promotes anti-inflammatory cytokine gene expression in macrophages and controls immune response in vivo.

Post-translational redox modification of methionine residues often triggers a change in protein function. Emerging evidence points to this reversible protein modification being an important regulatory mechanism under various physiological conditions. Reduction of oxidized methionine residues is catalyzed by methionine sulfoxide reductases (Msrs). Here, we show that one of these enzymes, a selenium-containing MsrB1, is highly expressed in immune-activated macrophages and contributes to shaping cellular and organismal immune responses. In particular, lipopolysaccharide (LPS) induces expression of MsrB1, but not other Msrs. Genetic ablation of MsrB1 did not preclude LPS-induced intracellular signaling in macrophages, but resulted in attenuated induction of anti-inflammatory cytokines, such as interleukin (IL)-10 and the IL-1 receptor antagonist. This anomaly was associated with excessive pro-inflammatory cytokine production as well as an increase in acute tissue inflammation in mice. Together, our findings suggest that MsrB1 controls immune responses by promoting anti-inflammatory cytokine expression in macrophages. MsrB1-dependent reduction of oxidized methionine in proteins may be a heretofore unrecognized regulatory event underlying immunity and inflammatory disease, and a novel target for clinical applications.

Selenoprotein deficiency and high levels of selenium compounds can effectively inhibit hepatocarcinogenesis in transgenic mice.

The micronutrient element selenium (Se) has been shown to be effective in reducing the incidence of cancer in animal models and human clinical trials. Selenoproteins and low molecular weight Se compounds were implicated in the chemopreventive effect, but specific mechanisms are not clear. We examined the role of Se and selenoproteins in liver tumor formation in TGFalpha/c-Myc transgenic mice, which are characterized by disrupted redox homeostasis and develop liver cancer by 6 months of age. In these mice, both Se deficiency and high levels of Se compounds suppressed hepatocarcinogenesis. In addition, both treatments induced expression of detoxification genes, increased apoptosis and inhibited cell proliferation. Within low-to-optimal levels of dietary Se, tumor formation correlated with expression of most selenoproteins. These data suggest that changes in selenoprotein expression may either suppress or promote tumorigenesis depending on cell type and genotype. Since dietary Se may have opposing effects on cancer, it is important to identify the subjects who will benefit from Se supplementation as well as those who will not.

CxxS: fold-independent redox motif revealed by genome-wide searches for thiol/disulfide oxidoreductase function.

Redox reactions involving thiol groups in proteins are major participants in cellular redox regulation and antioxidant defense. Although mechanistically similar, thiol-dependent redox processes are catalyzed by structurally distinct families of enzymes, which are difficult to identify by available protein function prediction programs. Herein, we identified a functional motif, CxxS (cysteine separated from serine by two other residues), that was often conserved in redox enzymes, but rarely in other proteins. Analyses of complete Escherichia coli, Campylobacter jejuni, Methanococcus jannaschii, and Saccharomyces cerevisiae genomes revealed a high proportion of proteins known to use the CxxS motif for redox function. This allowed us to make predictions in regard to redox function and identity of redox groups for several proteins whose function previously was not known. Many proteins containing the CxxS motif had a thioredoxin fold, but other structural folds were also present, and CxxS was often located in these proteins upstream of an alpha-helix. Thus, a conserved CxxS sequence followed by an alpha-helix is typically indicative of a redox function and corresponds to thiol-dependent redox sites in proteins. The data also indicate a general approach of genome-wide identification of redox proteins by searching for simple conserved motifs within secondary structure patterns.

Genomics perspective on disulfide bond formation.

Disulfide bond formation, reduction, and isomerization in substrate proteins are catalyzed by designated pathways composed of thiol-dependent enzymes. Disulfides are generated in oxidizing environments, such as bacterial periplasm and eukaryotic endoplasmic reticulum (ER), but could also be formed in the cytosol. Major contributors to the formation of intramolecular disulfides in proteins are thiol/disulfide oxidoreductases containing a conserved CxxC motif (two cysteines separated by two other residues), which in turn transfer reducing equivalents to adapter or membrane-bound oxidoreductases. Disulfide bond formation is accompanied by disulfide bond reduction and isomerization processes, allowing disulfide repair and quality control. Higher eukaryotes evolved a complex network of thiol/disulfide oxidoreductases that are involved in disulfide bond formation and isomerization and thiol-dependent protein retention. Emerging evidence suggests that these ER functions might be assisted by mammalian selenocysteine-containing oxidoreductases Sep15 and SelM.

Thiol peroxidase deficiency leads to increased mutational load and decreased fitness in Saccharomyces cerevisiae.

Thiol peroxidases are critical enzymes in the redox control of cellular processes that function by reducing low levels of hydroperoxides and regulating redox signaling. These proteins were also shown to regulate genome stability, but how their dysfunction affects the actual mutations in the genome is not known. Saccharomyces cerevisiae has eight thiol peroxidases of glutathione peroxidase and peroxiredoxin families, and the mutant lacking all these genes (∆8) is viable. In this study, we employed two independent ∆8 isolates to analyze the genome-wide mutation spectrum that results from deficiency in these enzymes. Deletion of these genes was accompanied by a dramatic increase in point mutations, many of which clustered in close proximity and scattered throughout the genome, suggesting strong mutational bias. We further subjected multiple lines of wild-type and ∆8 cells to long-term mutation accumulation, followed by genome sequencing and phenotypic characterization. ∆8 lines showed a significant increase in nonrecurrent point mutations and indels. The original ∆8 cells exhibited reduced growth rate and decreased life span, which were further reduced in all ∆8 mutation accumulation lines. Although the mutation spectrum of the two independent isolates was different, similar patterns of gene expression were observed, suggesting the direct contribution of thiol peroxidases to the observed phenotypes. Expression of a single thiol peroxidase could partially restore the growth phenotype of ∆8 cells. This study shows how deficiency in nonessential, yet critical and conserved oxidoreductase function, leads to increased mutational load and decreased fitness.

Diversity of plant methionine sulfoxide reductases B and evolution of a form specific for free methionine sulfoxide.

Methionine can be reversibly oxidized to methionine sulfoxide (MetO) under physiological conditions. Organisms evolved two distinct methionine sulfoxide reductase families (MSRA & MSRB) to repair oxidized methionine residues. We found that 5 MSRB genes exist in the soybean genome, including GmMSRB1 and two segmentally duplicated gene pairs (GmMSRB2 and GmMSRB5, GmMSRB3 and GmMSRB4). GmMSRB2 and GmMSRB4 proteins showed MSRB activity toward protein-based MetO with either DTT or thioredoxin (TRX) as reductants, whereas GmMSRB1 was active only with DTT. GmMSRB2 had a typical MSRB mechanism with Cys121 and Cys 68 as catalytic and resolving residues, respectively. Surprisingly, this enzyme also possessed the MSRB activity toward free Met-R-O with kinetic parameters similar to those reported for fRMSR from Escherichia coli, an enzyme specific for free Met-R-O. Overexpression of GmMSRB2 or GmMSRB4 in the yeast cytosol supported the growth of the triple MSRA/MSRB/fRMSR (Δ3MSRs) mutant on MetO and protected cells against H2O2-induced stress. Taken together, our data reveal an unexpected diversity of MSRBs in plants and indicate that, in contrast to mammals that cannot reduce free Met-R-O and microorganisms that use fRMSR for this purpose, plants evolved MSRBs for the reduction of both free and protein-based MetO.

Comparative genomics of thiol oxidoreductases reveals widespread and essential functions of thiol-based redox control of cellular processes.

AIMS: Redox regulation of cellular processes is an important mechanism that operates in organisms from bacteria to mammals. Much of the redox control is provided by thiol oxidoreductases: proteins that employ cysteine residues for redox catalysis. We wanted to identify thiol oxidoreductases on a genome-wide scale and use this information to obtain insights into the general principles of thiol-based redox control. RESULTS: Thiol oxidoreductases were identified by three independent methods that took advantage of the occurrence of selenocysteine homologs of these proteins and functional linkages among thiol oxidoreductases revealed by comparative genomics. Based on these searches, we describe thioredoxomes, which are sets of thiol oxidoreductases in organisms. Their analyses revealed that these proteins are present in all living organisms, generally account for 0.5%-1% of the proteome and that their use correlates with proteome size, distinguishing these proteins from those involved in core metabolic functions. We further describe thioredoxomes of Saccharomyces cerevisiae and humans, including proteins which have not been characterized previously. Thiol oxidoreductases occur in various cellular compartments and are enriched in the endoplasmic reticulum and cytosol. INNOVATION: We developed bioinformatics methods and used them to characterize thioredoxomes on a genome-wide scale, which in turn revealed properties of thioredoxomes. CONCLUSION: These data provide information about organization and properties of thiol-based redox control, whose use is increased with the increase in complexity of organisms. Our data also show an essential combined function of a set of thiol oxidoreductases, and of thiol-based redox regulation in general, in all living organisms.

Knockout of the 15 kDa selenoprotein protects against chemically-induced aberrant crypt formation in mice.

Evidence suggests that selenium has cancer preventive properties that are largely mediated through selenoproteins. Our previous observations demonstrated that targeted down-regulation of the 15 kDa selenoprotein (Sep15) in murine colon cancer cells resulted in the reversal of the cancer phenotype. The present study investigated the effect of Sep15 knockout in mice using a chemically-induced colon cancer model. Homozygous Sep15 knockout mice, and wild type littermate controls were given four weekly subcutaneous injections of azoxymethane (10 mg/kg). Sep15 knockout mice developed significantly (p<0.001) fewer aberrant crypt foci than controls demonstrating that loss of Sep15 protects against aberrant crypt foci formation. Dietary selenium above adequate levels did not significantly affect aberrant crypt foci formation in Sep15 knockout mice. To investigate molecular targets affected by loss of Sep15, gene expression patterns in colonic mucosal cells of knockout and wild type mice were examined using microarray analysis. Subsequent analyses verified that guanylate binding protein-1 (GBP-1) mRNA and protein expression were strongly upregulated in Sep15 knockout mice. GBP-1, which is expressed in response to interferon-γ, is considered to be an activation marker during inflammatory diseases, and up-regulation of GBP-1 in humans has been associated with a highly significant, increased five-year survival rate in colorectal cancer patients. In agreement with these studies, we observed a higher level of interferon-γ in plasma of Sep15 knockout mice. Overall, our results demonstrate for the first time, that Sep15 knockout mice are protected against chemically-induced aberrant crypt foci formation and that Sep15 appears to have oncogenic properties in colon carcinogenesis in vivo.

Selective reduction of methylsulfinyl-containing compounds by mammalian MsrA suggests a strategy for improved drug efficacy.

Identification of pathways of drug metabolism provides critical information regarding efficacy and safety of these compounds. Particularly challenging cases involve stereospecific processes. We found that broad classes of compounds containing methylsulfinyl groups are reduced to methylsulfides specifically by methionine sulfoxide reductase A, which acts on the S-stereomers of methionine sulfoxides, whereas the R-stereomers of these compounds could not be efficiently reduced by any methionine sulfoxide reductase in mammals. The findings of efficient reduction of S-methylsulfinyls and deficiency in the reduction of R-methylsulfinyls by methionine sulfoxide reductases suggest strategies for improved efficacy and decreased toxicity of drugs and natural compounds containing methylsulfinyls through targeted use of their enantiomers.

Boron stress activates the general amino acid control mechanism and inhibits protein synthesis.

Boron is an essential micronutrient for plants, and it is beneficial for animals. However, at high concentrations boron is toxic to cells although the mechanism of this toxicity is not known. Atr1 has recently been identified as a boron efflux pump whose expression is upregulated in response to boron treatment. Here, we found that the expression of ATR1 is associated with expression of genes involved in amino acid biosynthesis. These mechanisms are strictly controlled by the transcription factor Gcn4 in response to boron treatment. Further analyses have shown that boron impaired protein synthesis by promoting phosphorylation of eIF2α in a Gcn2 kinase dependent manner. The uncharged tRNA binding domain (HisRS) of Gcn2 is necessary for the phosphorylation of eIF2α in the presence of boron. We postulate that boron exerts its toxic effect through activation of the general amino acid control system and inhibition of protein synthesis. Since the general amino acid control pathway is conserved among eukaryotes, this mechanism of boron toxicity may be of general importance.