Sulindac induces differentiation of glioblastoma stem cells making them more sensitive to oxidative stress.

Glioblastoma tumors (GBM) are very heterogeneous, being comprised of several cell subtypes, including glioblastoma stem cells (GSC). These tumors have a high rate of recurrence after initial treatment and one of the most prevalent theories to explain this is the cancer stem cell theory, which proposes that glioblastomas arise from mutations that transform normal neural stem cells (NSC) into GSC, which are highly resistant to oxidative stress and anti-cancer therapies. Sulindac is a non-steroidal anti-inflammatory drug (NSAID) that has been shown to protect the normal cells against oxidative damage by initiating a preconditioning response, but selectively sensitizes several cancer cell lines to agents that affect mitochondrial respiration, resulting in enhanced killing of the cancer cells. These effects of sulindac are independent of its NSAID activity. There is little information on the effect of sulindac on normal and cancer stem cells. To study the effect of sulindac on both normal and cancer stem cells, we have isolated normal neural stem cells (NSC), from mice hippocampi and glioblastoma stem cells (GSC) from a glioma cell line, U87. As expected from previous studies sulindac can protect normal astrocytes against oxidative stress. Sulindac induces differentiation of both NSC and GSC cells and sulindac upregulates neurogenesis in NSC. The differentiated NSC are also protected from oxidative stress damage, whereas the differentiation of GSC by sulindac increases the sensitivity of these cells to agents that cause oxidative stress. The S epimer of sulindac is more effective than the R epimer in inducing neuronal differentiation in both NSC and GSC. These results indicate that the ability of sulindac to induce GSC differentiation may have therapeutic value in preventing tumour recurrence.

Protective effects of taurine against oxidative stress in the heart of MsrA knockout mice.

Taurine has been shown to have potent anti-oxidant properties under various pathophysiological conditions. We reported previously a cellular dysfunction and mitochondrial damage in cardiac myocytes of methionine sulfoxide reductase A (MsrA) gene knockout mice (MsrA(-/-)). In the present study, we have explored the protective effects of taurine against oxidative stress in the heart of MsrA(-/-) mice with or without taurine treatment. Cardiac cell contractility and Ca(2+) dynamics were measured using cell-based assays and in vivo cardiac function was monitored using high-resolution echocardiography in the tested animals. Our data have shown that MsrA(-/-) mice exhibited a progressive cardiac dysfunction with a significant decrease of ejection fraction (EF) and fraction shortening (FS) at age of 8 months compared to the wild type controls at the same age. However, the dysfunction was corrected in MsrA(-/-) mice treated with taurine supplement in the diet for 5 months. We further investigated the cellular mechanism underlying the protective effect of taurine in the heart. Our data indicated that cardiac myocytes from MsrA(-/-) mice treated with taurine exhibited an improved cell contraction and could tolerate oxidative stress better. Furthermore, taurine treatment reduced significantly the protein oxidation levels in mitochondria of MsrA(-/-) hearts, suggesting an anti-oxidant effect of taurine in cardiac mitochondria. Our study demonstrates that long-term treatment of taurine as a diet supplement is beneficial to a heart that is vulnerable to environmental oxidative stresses.

MsrA protects cardiac myocytes against hypoxia/reoxygenation induced cell death.

Reactive oxygen species (ROS) are critical in tissue responses to ischemia-reperfusion. The enzyme methionine sulfoxide reductase-A (MsrA) is capable of protecting cells against oxidative damage by reversing damage to proteins caused by methionine oxidation or by decreasing ROS through a scavenger mechanism. The current study employed adenovirus mediated over-expression of MsrA in primary neonatal rat cardiac myocytes to determine the effect of this enzyme in protecting against hypoxia/reoxygenation in this tissue. Cells were transduced with MsrA encoding adenovirus and subjected to hypoxia/reoxygenation. Apoptotic cell death was decreased by greater than 45% in cells over-expressing MsrA relative to cells transduced with a control virus. Likewise total cell death as determined by levels of LDH release was dramatically decreased by MsrA over-expression. These observations indicate that MsrA is protective against hypoxia/reoxygenation stress in cardiac myocytes and point to MsrA as an important therapeutic target for ischemic heart disease.

Ecdysone induction of MsrA protects against oxidative stress in Drosophila.

The methionine sulfoxide reductases MsrA and MsrB reduce Met(O) to Met in epimer-specific fashion. In Drosophila, the major ecdysone induced protein is MsrA, which is regulated by the EcR-USP complex. We tested Kc cells for induction of MsrA, MsrB, EcR, and CAT by ecdysone and found that MsrA and the EcR were induced by ecdysone, but MsrB and CAT were not. When we tested for resistance to 20mM H2O2 toxicity, viability of Kc cells was reduced 3-fold. Pretreatment with 0.2 microM ecdysone for 48 h prior to exposure to H2O2, increased viability to 77% of controls. The EcR-deficient L57-3-11 knockout line was not responsive to ecdysone, and H2O2 resistance of both control and ecdysone-treated L57-3-11 cells was similar to that of the ecdysone-untreated Kc cells. These results show that hormonal regulation of MsrA is implicated in conferring protection against oxidative stress in the Drosophila model.

Gene structure, localization and role in oxidative stress of methionine sulfoxide reductase A (MSRA) in the monkey retina.

MSRA (EC 1.8.4.6) is a member of the methionine sulfoxide reductase family that can reduce methionine sulfoxide (MetO) in proteins. This repair function has been shown to protect cells against oxidative damage. In this study we have assembled the complete gene structure of msrA and identified the presence of two distinct putative promoters that generate three different transcripts. These transcripts were cloned by 5'RACE and code for three MSRA isoforms with different N-termini. The different forms of MSRA target to distinct intracellular regions. The main MSRA transcript (msrA1) had been previously shown to target the mitochondria. MsrA2 and 3 originate from a second promoter and target the cytosol and nuclei. In the monkey retina msrA message was detected mainly in the macular RPE-choroid region while its activity was measured mainly in the soluble fractions of fractionated neural retina and RPE-choroid. The MSRA protein is found throughout the retina but is especially abundant at the photoreceptor synapses, ganglion and Müller cells. Interestingly, MSRA was not detected in the mitochondria of the photoreceptor inner segments. The RPE in the peripheral retina shows very low levels of expression but the RPE in the macular region is strongly labeled. Targeted silencing of msrA message rendered cultured RPE cells more sensitive to oxidative damage suggesting a role for MSRA in RPE protection against oxidative stress. Collectively these data suggest MSRA may play an important role in protecting macular RPE from oxidative damage.

A simplified reconstitution of mRNA-directed peptide synthesis: activity of the epsilon enhancer and an unnatural amino acid.

The study of the early events in translation would be greatly facilitated by reconstitution with easily purified components. Here, Escherichia coli oligopeptide synthesis has been reconstituted using five purified recombinant His-tagged E. coli initiation and elongation factors. Highly purified ribosomes are required to yield products with strong dependencies on the translation factors. Based on HPLC separation of radiolabeled translation products from an mRNA encoding a tetrapeptide, approximately 80% of peptide products are full length, and the remaining 20% are the dipeptide and tripeptide products resulting from pausing or premature termination. Oligopeptide synthesis is enhanced when a commonly used epsilon (enhancer of protein synthesis initiation) sequence is included in the mRNA. The system incorporates a selectable, large, unnatural amino acid and may ultimately form the basis of a pure translation display technology for the directed evolution of peptidomimetic ligands and drug candidates. The recombinant clones can be exploited to prepare initiation factors and initiation complexes for structural studies, to study initiation and elongation in ribosomal peptide synthesis, and to screen for eubacterial-specific drugs.

Peptide methionine sulfoxide reductase from Escherichia coli and Mycobacterium tuberculosis protects bacteria against oxidative damage from reactive nitrogen intermediates.

Inducible nitric oxide synthase (iNOS) plays an important role in host defense. Macrophages expressing iNOS release the reactive nitrogen intermediates (RNI) nitrite and S-nitrosoglutathione (GSNO), which are bactericidal in vitro at a pH characteristic of the phagosome of activated macrophages. We sought to characterize the active intrabacterial forms of these RNI and their molecular targets. Peptide methionine sulfoxide reductase (MsrA; EC ) catalyzes the reduction of methionine sulfoxide (Met-O) in proteins to methionine (Met). E. coli lacking MsrA were hypersensitive to killing not only by hydrogen peroxide, but also by nitrite and GSNO. The wild-type phenotype was restored by transformation with plasmids encoding msrA from E. coli or M. tuberculosis, but not by an enzymatically inactive mutant msrA, indicating that Met oxidation was involved in the death of these cells. It seemed paradoxical that nitrite and GSNO kill bacteria by oxidizing Met residues when these RNI cannot themselves oxidize Met. However, under anaerobic conditions, neither nitrite nor GSNO was bactericidal. Nitrite and GSNO can both give rise to NO, which may react with superoxide produced by bacteria during aerobic metabolism, forming peroxynitrite, a known oxidant of Met to Met-O. Thus, the findings are consistent with the hypotheses that nitrite and GSNO kill E. coli by intracellular conversion to peroxynitrite, that intracellular Met residues in proteins constitute a critical target for peroxynitrite, and that MsrA can be essential for the repair of peroxynitrite-mediated intracellular damage.

Oxidative regulation of large conductance calcium-activated potassium channels.

Reactive oxygen/nitrogen species are readily generated in vivo, playing roles in many physiological and pathological conditions, such as Alzheimer's disease and Parkinson's disease, by oxidatively modifying various proteins. Previous studies indicate that large conductance Ca(2+)-activated K(+) channels (BK(Ca) or Slo) are subject to redox regulation. However, conflicting results exist whether oxidation increases or decreases the channel activity. We used chloramine-T, which preferentially oxidizes methionine, to examine the functional consequences of methionine oxidation in the cloned human Slo (hSlo) channel expressed in mammalian cells. In the virtual absence of Ca(2+), the oxidant shifted the steady-state macroscopic conductance to a more negative direction and slowed deactivation. The results obtained suggest that oxidation enhances specific voltage-dependent opening transitions and slows the rate-limiting closing transition. Enhancement of the hSlo activity was partially reversed by the enzyme peptide methionine sulfoxide reductase, suggesting that the upregulation is mediated by methionine oxidation. In contrast, hydrogen peroxide and cysteine-specific reagents, DTNB, MTSEA, and PCMB, decreased the channel activity. Chloramine-T was much less effective when concurrently applied with the K(+) channel blocker TEA, which is consistent with the possibility that the target methionine lies within the channel pore. Regulation of the Slo channel by methionine oxidation may represent an important link between cellular electrical excitability and metabolism.

Structure and mechanism of peptide methionine sulfoxide reductase, an "anti-oxidation" enzyme.

Peptide methionine sulfoxide reductase (MsrA) reverses oxidative damage to both free methionine and methionine within proteins. As such, it helps protect the host organism against stochastic damage that can contribute to cell death. The structure of bovine MsrA has been determined in two different modifications, both of which provide different insights into the biology of the protein. There are three cysteine residues located in the vicinity of the active site. Conformational changes in a glycine-rich C-terminal tail appear to allow all three thiols to come together and to participate in catalysis. The structures support a unique, thiol-disulfide exchange mechanism that relies upon an essential cysteine as a nucleophile and additional conserved residues that interact with the oxygen atom of the sulfoxide moiety.

Thiol-disulfide exchange is involved in the catalytic mechanism of peptide methionine sulfoxide reductase.

Peptide methionine sulfoxide reductase (MsrA; EC ) reverses the inactivation of many proteins due to the oxidation of critical methionine residues by reducing methionine sulfoxide, Met(O), to methionine. MsrA activity is independent of bound metal and cofactors but does require reducing equivalents from either DTT or a thioredoxin-regenerating system. In an effort to understand these observations, the four cysteine residues of bovine MsrA were mutated to serine in a series of permutations. An analysis of the enzymatic activity of the variants and their free sulfhydryl states by mass spectrometry revealed that thiol-disulfide exchange occurs during catalysis. In particular, the strictly conserved Cys-72 was found to be essential for activity and could form disulfide bonds, only upon incubation with substrate, with either Cys-218 or Cys-227, located at the C terminus. The significantly decreased activity of the Cys-218 and Cys-227 variants in the presence of thioredoxin suggested that these residues shuttle reducing equivalents from thioredoxin to the active site. A reaction mechanism based on the known reactivities of thiols with sulfoxides and the available data for MsrA was formulated. In this scheme, Cys-72 acts as a nucleophile and attacks the sulfur atom of the sulfoxide moiety, leading to the formation of a covalent, tetracoordinate intermediate. Collapse of the intermediate is facilitated by proton transfer and the concomitant attack of Cys-218 on Cys-72, leading to the formation of a disulfide bond. The active site is returned to the reduced state for another round of catalysis by a series of thiol-disulfide exchange reactions via Cys-227, DTT, or thioredoxin.

Reactive oxygen species and nitric oxide mediate plasticity of neuronal calcium signaling.

Reactive oxygen species (ROS) and nitric oxide (NO) are important participants in signal transduction that could provide the cellular basis for activity-dependent regulation of neuronal excitability. In young rat cortical brain slices and undifferentiated PC12 cells, paired application of depolarization/agonist stimulation and oxidation induces long-lasting potentiation of subsequent Ca(2+) signaling that is reversed by hypoxia. This potentiation critically depends on NO production and involves cellular ROS utilization. The ability to develop the Ca(2+) signal potentiation is regulated by the developmental stage of nerve tissue, decreasing markedly in adult rat cortical neurons and differentiated PC12 cells.

Peptide methionine sulfoxide reductase: biochemistry and physiological role.

The oxidation of methionine to methionine sulfoxide both in vivo and in vitro can lead to the loss of biological activity in a variety of proteins. This loss of activity can be reversed by an enzyme called methionine sulfoxide reductase. The gene for this enzyme has been cloned and sequenced from a variety of prokaryotic and eukaryotic cells, and the deduced amino acid sequence is very highly conserved. The mechanism of action of the bovine enzyme has been shown to involve a critical cysteine residue located at position 72 of the protein. In addition to its role as a "repair" enzyme, other evidence suggests that the enzyme may be involved in bacterial adherence and regulation of protein activity.

Molecular cloning and functional expression of a human peptide methionine sulfoxide reductase (hMsrA).

Oxidation of methionine residues in proteins to methionine sulfoxide can be reversed by the enzyme peptide methionine sulfoxide reductase (MsrA, EC 1.8.4.6). We cloned the gene encoding a human homologue (hMsrA) of the enzyme, which has an 88% amino acid sequence identity to the bovine version (bMsrA). With dot blot analyses based on RNA from human tissues, expression of hMsrA was found in all tissues tested, with highest mRNA levels in adult kidney and cerebellum, followed by liver, heart ventricles, bone marrow and hippocampus. In fetal tissue, expression was highest in the liver. No expression of hmsrA was detected in leukemia and lymphoma cell lines. To test if hMsrA is functional in cells, we assayed its effect on the inactivation time course of the A-type potassium channel ShC/B since this channel property strongly depends on the oxidative state of a methionine residue in the N-terminal part of the polypeptide. Co-expression of ShC/B and hMsrA in Xenopus oocytes significantly accelerated inactivation, showing that the cloned enzyme is functional in an in vivo assay system. Furthermore, the activity of a purified glutathione-S-transferase-hMsrA fusion protein was demonstrated in vitro by measuring the reduction of [3H]N-acetyl methionine sulfoxide.

Regulation of voltage-dependent K+ channels by methionine oxidation: effect of nitric oxide and vitamin C.

Methionine oxidation is known to alter functional properties of a transient A-type potassium channel expressed in Xenopus oocytes. We show here that nitric oxide (NO) slows down the K+ channel inactivation time course by oxidizing a critical methionine residue in the inactivation ball domain of the channel protein. We also demonstrate that the channel protein is protected from methionine oxidation by the enzyme methionine sulfoxide reductase and the antioxidant vitamin C.

Modulation of potassium channel function by methionine oxidation and reduction.

Oxidation of amino acid residues in proteins can be caused by a variety of oxidizing agents normally produced by cells. The oxidation of methionine in proteins to methionine sulfoxide is implicated in aging as well as in pathological conditions, and it is a reversible reaction mediated by a ubiquitous enzyme, peptide methionine sulfoxide reductase. The reversibility of methionine oxidation suggests that it could act as a cellular regulatory mechanism although no such in vivo activity has been demonstrated. We show here that oxidation of a methionine residue in a voltage-dependent potassium channel modulates its inactivation. When this methionine residue is oxidized to methionine sulfoxide, the inactivation is disrupted, and it is reversed by coexpression with peptide methionine sulfoxide reductase. The results suggest that oxidation and reduction of methionine could play a dynamic role in the cellular signal transduction process in a variety of systems.

Peptide methionine sulfoxide reductase contributes to the maintenance of adhesins in three major pathogens.

Pathogenic bacteria rely on adhesins to bind to host tissues. Therefore, the maintenance of the functional properties of these extracellular macromolecules is essential for the pathogenicity of these microorganisms. We report that peptide methionine sulfoxide reductase (MsrA), a repair enzyme, contributes to the maintenance of adhesins in Streptococcus pneumoniae, Neisseria gonorrhoeae, and Escherichia coli. A screen of a library of pneumococcal mutants for loss of adherence uncovered a MsrA mutant with 75% reduced binding to GalNAcbeta1-4Gal containing eukaryotic cell receptors that are present on type II lung cells and vascular endothelial cells. Subsequently, it was shown that an E. coli msrA mutant displayed decreased type I fimbriae-mediated, mannose-dependent, agglutination of erythrocytes. Previous work [Taha, M. K., So, M., Seifert, H. S., Billyard, E. & Marchal, C. (1988) EMBO J. 7, 4367-4378] has shown that mutants with defects in the pilA-pilB locus from N. gonorrhoeae were altered in their production of type IV pili. We show that pneumococcal MsrA and gonococcal PilB expressed in E. coli have MsrA activity. Together these data suggest that MsrA is required for the proper expression or maintenance of functional adhesins on the surfaces of these three major pathogenic bacteria.

ATP hydrolysis is not required for the dissociation of a substance P.BiP complex.

BiP is a member of the hsp70 family of proteins that is present in the endoplasmic reticulum where it functions as a molecular chaperone. Rapid quantitative assays have been used to study the effect of mutating BiP residue 229, located in the ATP binding site, from threonine to glycine. Although binding of ATP to the mutant BiP was not affected, the mutant protein possessed 10-20% of the wild-type BiP ATPase activity. Binding to a model peptide substrate, substance P (Brot et al. (1994) Proc. Natl. Acad. Sci. USA 91, 12120-12124), was twofold higher with mutant BiP at 4 degrees C than with wild-type BiP, and was ATP dependent. Under these conditions the substance P that was bound to mutant BiP, but not the wild-type, could be released by higher levels of ATP (5-10 microM), and the ratio of substance P released to ATP hydrolyzed was greater than 10. These results suggest that stoichiometric ATP hydrolysis is not required for release of a chaperone from its substrate.

Chromosomal localization of the mammalian peptide-methionine sulfoxide reductase gene and its differential expression in various tissues.

Peptide methionine sulfoxide reductase (MsrA; EC 1.8.4.6) is a ubiquitous protein that can reduce methionine sulfoxide residues in proteins as well as in a large number of methyl sulfoxide compounds. The expression of MsrA in various rat tissues was determined by using immunocytochemical staining. Although the protein was found in all tissues examined, it was specifically localized to renal medulla and retinal pigmented epithelial cells, and it was prominent in neurons and throughout the nervous system. In addition, blood and alveolar macrophages showed high expression of the enzyme. The msrA gene was mapped to the central region of mouse chromosome 14, in a region of homology with human chromosomes 13 and 8p21.

Cloning the expression of a mammalian gene involved in the reduction of methionine sulfoxide residues in proteins.

An enzyme that reduces methionine sulfoxide [Met(O)] residues in proteins [peptide Met(O) reductase (MsrA), EC 1.8.4.6; originally identified in Escherichia coli] was purified from bovine liver, and the cDNA encoding this enzyme was cloned and sequenced. The mammalian homologue of E. coli msrA (also called pmsR) cDNA encodes a protein of 255 amino acids with a calculated molecular mass of 25,846 Da. This protein has 61% identity with the E. coli MsrA throughout a region encompassing a 199-amino acid overlap. The protein has been overexpressed in E. coli and purified to homogeneity. The mammalian recombinant MsrA can use as substrate, proteins containing Met(O) as well as other organic compounds that contain an alkyl sulfoxide group such as N-acetylMet(O), Met(O), and dimethyl sulfoxide. Northern analysis of rat tissue extracts showed that rat msrA mRNA is present in a variety of organs with the highest level found in kidney. This is consistent with the observation that kidney extracts also contained the highest level of enzyme activity.