Crucial roles of Sp1 and epigenetic modifications in the regulation of the CLDN4 promoter in ovarian cancer cells

Claudins form a large family of tight junction proteins that have essential roles in the control of paracellular ion flux and the maintenance of cell polarity. Many studies have shown that several claudin family members are abnormally expressed in various cancers. In particular, CLDN4 (encoding claudin-4) is overexpressed in ovarian cancer. However, although CLDN4 overexpression is well established, the mechanisms responsible for this abnormal regulation remain unknown. In the present study, we delineate a small region of the CLDN4 promoter critical for its expression. This region contains two Sp1 sites, both of which are required for promoter activity. However, because of the ubiquitous expression of Sp1, these sites, although necessary, are not sufficient to explain the patterns of gene expression of CLDN4 in various ovarian tissues. We show that the CLDN4 promoter is further controlled by epigenetic modifications of the Sp1-containing critical promoter region. Cells that overexpress CLDN4 exhibit low DNA methylation and high histone H3 acetylation of the critical CLDN4 promoter region, and the reverse is observed in cells that do not express CLDN4. Moreover, the CLDN4-negative cells can be induced to express CLDN4 through treatment with demethylating and/or acetylating agents. Because CLDN4 is elevated in a large fraction of ovarian cancer, the mechanism leading to deregulation may represent a general pathway in ovarian tumorigenesis and may lead to novel strategies for therapy and an overall better understanding of the biology of this disease.     

MsrB1 (methionine-R-sulfoxide reductase 1) knock-out mice: roles of MsrB1 in redox regulation and identification of a novel selenoprotein form

Protein oxidation has been linked to accelerated aging and is a contributing factor to many diseases. Methionine residues are particularly susceptible to oxidation, but the resulting mixture of methionine R-sulfoxide (Met-RO) and methionine S-sulfoxide (Met-SO) can be repaired by thioredoxin-dependent enzymes MsrB and MsrA, respectively. Here, we describe a knock-out mouse deficient in selenoprotein MsrB1, the main mammalian MsrB located in the cytosol and nucleus. In these mice, in addition to the deletion of 14-kDa MsrB1, a 5-kDa selenoprotein form was specifically removed. Further studies revealed that the 5-kDa protein occurred in both mouse tissues and human HEK 293 cells; was down-regulated by MsrB1 small interfering RNA, selenium deficiency, and selenocysteine tRNA mutations; and was immunoprecipitated and recognized by MsrB1 antibodies. Specific labeling with (75)Se and mass spectrometry analyses revealed that the 5-kDa selenoprotein corresponded to the C-terminal sequence of MsrB1. The MsrB1 knock-out mice lacked both 5- and 14-kDa MsrB1 forms and showed reduced MsrB activity, with the strongest effect seen in liver and kidney. In addition, MsrA activity was decreased by MsrB1 deficiency. Liver and kidney of the MsrB1 knock-out mice also showed increased levels of malondialdehyde, protein carbonyls, protein methionine sulfoxide, and oxidized glutathione as well as reduced levels of free and protein thiols, whereas these parameters were little changed in other organs examined. Overall, this study established an important contribution of MsrB1 to the redox control in mouse liver and kidney and identified a novel form of this protein.     

A high-molecular-mass surface protein (Lsp) and methionine sulfoxide reductase B (MsrB) contribute to the ecological performance of Lactobacillus reuteri in the murine gut

Members of the genus Lactobacillus are common inhabitants of the gut, yet little is known about the traits that contribute to their ecological performance in gastrointestinal ecosystems. Lactobacillus reuteri 100-23 persists in the gut of the reconstituted Lactobacillus-free mouse after a single oral inoculation. Recently, three genes of this strain that were specifically induced (in vivo induced) in the murine gut were identified (38). We report here the detection of a gene of L. reuteri 100-23 that encodes a high-molecular-mass surface protein (Lsp) that shows homology to proteins involved in the adherence of other bacteria to epithelial cells and in biofilm formation. The three in vivo-induced genes and lsp of L. reuteri 100-23 were inactivated by insertional mutagenesis in order to study their biological importance in the murine gastrointestinal tract. Competition experiments showed that mutation of lsp and a gene encoding methionine sulfoxide reductase (MsrB) reduced ecological performance. Mutation of lsp impaired the adherence of the bacteria to the epithelium of the mouse forestomach and altered colonization dynamics. Homologues of lsp and msrB are present in the genomes of several strains of Lactobacillus and may play an important role in the maintenance of these bacteria in gut ecosystems.     

High-affinity and cooperative binding of oxidized calmodulin by methionine sulfoxide reductase

Methionines can play an important role in modulating protein-protein interactions associated with intracellular signaling, and their reversible oxidation to form methionine sulfoxides [Met(O)] in calmodulin (CaM) and other signaling proteins has been suggested to couple cellular redox changes to protein functional changes through the action of methionine sulfoxide reductases (Msr). Prior measurements indicate the full recovery of target protein activation upon the stereospecific reduction of oxidized CaM by MsrA, where the formation of the S-stereoisomer of Met(O) selectively inhibits the CaM-dependent activation of the Ca-ATPase. However, the physiological substrates of MsrA remain unclear, as neither the binding specificities nor affinities of protein targets have been measured. To assess the specificity of binding and its possible importance in the maintenance of CaM function, we have measured the kinetics of repair and the binding affinity between oxidized CaM and MsrA. Reduction of Met(O) in fully oxidized CaM by MsrA is sensitive to the protein fold, as repair of the intact protein is incomplete, with >6 Met(O) remaining in each CaM following MsrA reduction. In contrast, following proteolytic digestion, MsrA is able to fully reduce one-half of the oxidized methionines, indicating that surface-accessible Met(O) within folded proteins need not be substrates for MsrA repair. Mutation of the active site (i.e., C72S) in MsrA permitted equilibrium-binding measurements using both ensemble and single-molecule fluorescence correlation spectroscopy measurements. We observe cooperative binding of two MsrA to each CaMox with an apparent affinity (K = 70 +/- 10 nM) that is 3 orders of magnitude greater than the Michaelis constant (KM = 68 +/- 4 microM). The high-affinity and cooperative interaction between MsrA and CaMox suggests an important regulatory role of MsrA in the binding and reduction of Met(O) in functionally sensitive proteins, such that multiple MsrA proteins are recruited to simultaneously bind and reduce Met(O) in highly oxidized proteins. Given the suggested role of Met(O) in modulating reversible binding interactions between proteins associated with cellular signaling, these results indicate an ability of MsrA to selectively reduce Met(O) within highly surface-accessible sequences to maintain cellular function as part of an adaptive response to oxidative stress.     

Synergistic roles of Helicobacter pylori methionine sulfoxide reductase and GroEL in repairing oxidant-damaged catalase

Hypochlorous acid (HOCl) produced via the enzyme myeloperoxidase is a major antibacterial oxidant produced by neutrophils, and Met residues are considered primary amino acid targets of HOCl damage via conversion to Met sulfoxide. Met sulfoxide can be repaired back to Met by methionine sulfoxide reductase (Msr). Catalase is an important antioxidant enzyme; we show it constitutes 4-5% of the total Helicobacter pylori protein levels. msr and katA strains were about 14- and 4-fold, respectively, more susceptible than the parent to killing by the neutrophil cell line HL-60 cells. Catalase activity of an msr strain was much more reduced by HOCl exposure than for the parental strain. Treatment of pure catalase with HOCl caused oxidation of specific MS-identified Met residues, as well as structural changes and activity loss depending on the oxidant dose. Treatment of catalase with HOCl at a level to limit structural perturbation (at a catalase/HOCl molar ratio of 1:60) resulted in oxidation of six identified Met residues. Msr repaired these residues in an in vitro reconstituted system, but no enzyme activity could be recovered. However, addition of GroEL to the Msr repair mixture significantly enhanced catalase activity recovery. Neutrophils produce large amounts of HOCl at inflammation sites, and bacterial catalase may be a prime target of the host inflammatory response; at high concentrations of HOCl (1:100), we observed loss of catalase secondary structure, oligomerization, and carbonylation. The same HOCl-sensitive Met residue oxidation targets in catalase were detected using chloramine-T as a milder oxidant.     

Sp1 binding sites and an estrogen response element half-site are involved in regulation of the human progesterone receptor A promoter

Progesterone receptor gene expression is induced by estrogen in MCF-7 human breast cancer cells. Although it is generally thought that estrogen responsiveness is mediated through estrogen response elements (EREs), the progesterone receptor gene lacks an identifiable ERE. The progesterone receptor A promoter does, however, contain a half-ERE/Sp1 binding site comprised of an ERE half-site upstream of two Sp1 binding sites. We have used in vivo deoxyribonuclease I (DNase I) footprinting to demonstrate that the half-ERE/Sp1 binding site is more protected when MCF-7 cells are treated with estrogen than when cells are not exposed to hormone, suggesting that this region is involved in estrogen-regulated gene expression. The ability of the half-ERE/Sp1 binding site to confer estrogen responsiveness to a simple heterologous promoter was confirmed in transient cotransfection assays. In vitro DNase I footprinting and gel mobility shift assays demonstrated that Sp1 present in MCF-7 nuclear extracts and purified Sp1 protein bound to the two Sp1 sites and that the estrogen receptor enhanced Sp1 binding. In addition to its effects on Sp1 binding, the estrogen receptor also bound directly to the ERE half-site. Taken together, these findings suggest that the estrogen receptor and Sp1 play a role in activation of the human progesterone receptor A promoter.     

Effect of methionine sulfoxide reductase B1 (SelR) gene silencing on peroxynitrite-induced F-actin disruption in human lens epithelial cells

F-actin plays a crucial role in fundamental cellular processes, and is extremely susceptible to peroxynitrite attack due to the high abundance of tyrosine in the peptide. Methionine sulfoxide reductase (Msr) B1 is a selenium-dependent enzyme (selenoprotein R) that may act as a reactive oxygen species (ROS) scavenger. However, its function in coping with reactive nitrogen species (RNS)-mediated stress and the physiological significance remain unclear. Thus, the present study was conducted to elucidate the role and mechanism of MsrB1 in protecting human lens epithelial (hLE) cells against peroxynitrite-induced F-actin disruption. While exposure to high concentrations of peroxynitrite and gene silencing of MsrB1 by siRNA alone caused disassembly of F-actin via inactivation of extracellular signal-regulated kinase (ERK) in hLE cells, the latter substantially aggravated the disassembly of F-actin triggered by the former. This aggravation concurred with elevated nitration of F-actin and inactivation of ERK compared with that induced by the peroxynitrite treatment alone. In conclusion, MsrB1 protected hLE cells against the peroxynitrite-induced F-actin disruption, and the protection was mediated by inhibiting the resultant nitration of F-actin and inactivation of ERKs.     

Important role for methionine sulfoxide reductase in the oxidative stress response of Xanthomonas campestris pv. phaseoli

A methionine sulfoxide reductase gene (msrA) from Xanthomonas campestris pv. phaseoli has unique expression patterns and physiological function. msrA expression is growth dependent and is highly induced by exposure to oxidants and N-ethylmaleimide in an OxyR- and OhrR-independent manner. An msrA mutant showed increased sensitivity to oxidants but only during stationary phase.     

1H, 15N and 13C NMR assignments of mouse methionine sulfoxide reductase B2

A recombinant mouse methionine-r-sulfoxide reductase 2 (MsrB2ΔS) isotopically labeled with ¹⁵N and ¹⁵N/¹³C was generated. We report here the ¹H, ¹⁵N, and ¹³C NMR assignments of the reduced form of this protein. An erratum to this article can be found at     

Kinetic characterization of the catalytic mechanism of methionine sulfoxide reductase B from Neisseria meningitidis

Methionine sulfoxide reductases catalyze the thioredoxin-dependent reduction of methionine sulfoxide back to methionine. The methionine sulfoxide reductases family is composed of two structurally unrelated classes of enzymes named MsrA and MsrB, which display opposite stereoselectivities toward the sulfoxide function. Both enzymes are monomeric and share a similar three-step chemical mechanism. First, in the reductase step, a sulfenic acid intermediate is formed with a concomitant release of 1 mol of methionine per mol of enzyme. Then, an intradisulfide bond is formed. Finally, Msrs return back to reduced forms via reduction by thioredoxin. In the present study, it is shown for the Neisseria meningitidis MsrB that (1) the reductase step is rate-determining in the process leading to formation of the disulfide bond and (2) the thioredoxin-recycling process is rate-limiting. Moreover, the data suggest that within the thioredoxin-recycling process, the rate-limiting step takes place after the two-electron chemical exchange and thus is associated with the release of oxidized thioredoxin.     

Determination of the specific activities of methionine sulfoxide reductase A and B by capillary electrophoresis

A capillary electrophoresis (CE) method for the determination of methionine sulfoxide reductase A and methionine sulfoxide reductase B activities in mouse liver is described. The method is based on detection of the 4-(dimethylamino)azobenzene-4′-sulfonyl derivative of l-methionine (dabsyl Met), the product of the enzymatic reactions when either dabsyl l-methionine S-sulfoxide or dabsyl l-methionine R-sulfoxide is used as a substrate. The method provides baseline resolution of the substrates and, therefore, can be used to easily determine the purity of the substrates. The method is rapid (∼20 min sample to sample), requires no column regeneration, and uses very small amounts of buffers. Separation was performed by using a 75-μm internal diameter polyimide-coated fused silica capillary (no inside coating) with 60 cm total length (50 cm to the detector window). Samples were separated at 22.5 kV, and the separation buffer was 25 mM KH₂PO₄ (pH 8.0) containing 0.9 ml of N-lauroylsarcosine (sodium salt, 30% [w/v] solution) per 100 ml of buffer. Prior to use, the capillary was conditioned with the same buffer that also contained 25 mM sodium dodecyl sulfate. The CE method is compared with high-performance liquid chromatography (HPLC) as determined by comparing results from measurements of hepatic enzyme activities in mice fed either deficient or adequate selenium.