An NMR‐Based Biosensor to Measure Stereospecific Methionine Sulfoxide Reductase Activities in Vitro and in Vivo**

ABSTRACT Two independent isolates of the gut commensal Lactobacillus johnsonii were sequenced. These isolates belonged to the same clonal lineage and differed mainly by a 40.8-kb prophage, LJ771, belonging to the Sfi11 phage lineage. LJ771 shares close DNA sequence identity with Lactobacillus gasseri prophages. LJ771 coexists as an integrated prophage and excised circular phage DNA, but phage DNA packaged into extracellular phage particles was not detected. Between the phage lysin gene and attR a likely mazE (“antitoxin”)/pemK (“toxin”) gene cassette was detected in LJ771 but not in the L. gasseri prophages. Expressed pemK could be cloned in Escherichia coli only together with the mazE gene. LJ771 was shown to be highly stable and could be cured only by coexpression of mazE from a plasmid. The prophage was integrated into the methionine sulfoxide reductase gene (msrA) and complemented the 5′ end of this gene, creating a protein with a slightly altered N-terminal sequence. The two L. johnsonii strains had identical in vitro growth and in vivo gut persistence phenotypes. Also, in an isogenic background, the presence of the prophage resulted in no growth disadvantage.

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Identification of Lactobacillus reuteri Genes Specifically Induced in the Mouse Gastrointestinal Tract

Applied and Environmental Microbiology ◽

10.1128/aem.69.4.2044-2051.2003 ◽

2003 ◽

Vol 69 (4) ◽

pp. 2044-2051 ◽

Cited By ~ 84

Author(s):

Jens Walter ◽

Nicholas C. K. Heng ◽

Walter P. Hammes ◽

Diane M. Loach ◽

Gerald W. Tannock ◽

...

Keyword(s):

Gastrointestinal Tract ◽

Reporter Gene ◽

Stress Responses ◽

Lactobacillus Reuteri ◽

Xylose Isomerase ◽

Methionine Sulfoxide ◽

Methionine Sulfoxide Reductase ◽

Chromosomal Dna

ABSTRACT Lactobacilli are common inhabitants of the gastrointestinal tracts of mammals and have received considerable attention due to their putative health-promoting properties. Little is known about the traits that enhance the ability of these bacteria to inhabit the gastrointestinal tract. In this paper we describe the development and application of a strategy based on in vivo expression technology (IVET) that enables detection of Lactobacillus reuteri genes specifically induced in the murine gut. A plasmid-based system was constructed containing ′ermGT (which confers lincomycin resistance) as the primary reporter gene for selection of promoters active in the gastrointestinal tract of mice treated with lincomycin. A second reporter gene, ′bglM (β-glucanase), allowed differentiation between constitutive and in vivo inducible promoters. The system was successfully tested in vitro and in vivo by using a constitutive promoter. Application of the IVET system with chromosomal DNA of L. reuteri 100-23 and reconstituted lactobacillus-free mice revealed three genes induced specifically during colonization. Two of the sequences showed homology to genes encoding xylose isomerase (xylA) and peptide methionine sulfoxide reductase (msrB), which are involved in nutrient acquisition and stress responses, respectively. The third locus showed homology to the gene encoding a protein whose function is not known. Our IVET system has the potential to identify genes of lactobacilli that have not previously been functionally characterized but which may be essential for growth of these bacteria in the gastrointestinal ecosystem.

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Methionine sulfoxide reductase 2 reversibly regulates Mge1, a cochaperone of mitochondrial Hsp70, during oxidative stress

Molecular Biology of the Cell ◽

10.1091/mbc.e14-09-1371 ◽

2015 ◽

Vol 26 (3) ◽

pp. 406-419 ◽

Cited By ~ 17

Author(s):

Praveen Kumar Allu ◽

Adinarayana Marada ◽

Yerranna Boggula ◽

Srinivasu Karri ◽

Thanuja Krishnamoorthy ◽

...

Keyword(s):

Oxidative Stress ◽

Methionine Sulfoxide ◽

Methionine Sulfoxide Reductase ◽

Yeast Cells ◽

Nucleotide Exchange ◽

New Paradigm ◽

Methionine Sulfoxide Reductases ◽

Mitochondrial Hsp70

Peptide methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in protein(s). Although these reductases have been implicated in several human diseases, there is a dearth of information on the identity of their physiological substrates. By using Saccharomyces cerevisiae as a model, we show that of the two methionine sulfoxide reductases (MXR1, MXR2), deletion of mitochondrial MXR2 renders yeast cells more sensitive to oxidative stress than the cytosolic MXR1. Our earlier studies showed that Mge1, an evolutionarily conserved nucleotide exchange factor of Hsp70, acts as an oxidative sensor to regulate mitochondrial Hsp70. In the present study, we show that Mxr2 regulates Mge1 by selectively reducing MetO at position 155 and restores the activity of Mge1 both in vitro and in vivo. Mge1 M155L mutant rescues the slow-growth phenotype and aggregation of proteins of mxr2Δ strain during oxidative stress. By identifying the first mitochondrial substrate for Mxrs, we add a new paradigm to the regulation of the oxidative stress response pathway.

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Expression of magA in Legionella pneumophila Philadelphia-1 Is Developmentally Regulated and a Marker of Formation of Mature Intracellular Forms

Journal of Bacteriology ◽

10.1128/jb.186.10.3038-3045.2004 ◽

2004 ◽

Vol 186 (10) ◽

pp. 3038-3045 ◽

Cited By ~ 16

Author(s):

Margot F. Hiltz ◽

Gary R. Sisson ◽

Ann Karen C. Brassinga ◽

Elizabeth Garduno ◽

Rafael A. Garduno ◽

...

Keyword(s):

Hela Cells ◽

Legionella Pneumophila ◽

Protein Profile ◽

Methionine Sulfoxide ◽

Growth Cycle ◽

Specific Protein ◽

Methionine Sulfoxide Reductase ◽

Developmental Cycle

ABSTRACT Legionella pneumophila displays a biphasic developmental cycle in which replicating forms (RFs) differentiate postexponentially into highly infectious, cyst-like mature intracellular forms (MIFs). Using comparative protein profile analyses (MIFs versus RFs), we identified a 20-kDa protein, previously annotated as “Mip-like” protein, that was enriched in MIFs. However, this 20-kDa protein shared no similarity with Mip, a well-characterized peptidyl-prolyl isomerase of L. pneumophila, and for clarity we renamed it MagA (for “MIF-associated gene”). We monitored MagA levels across the growth cycle (in vitro and in vivo) by immunoblotting and established that MagA levels increased postexponentially in vitro (∼3-fold) and nearly 10-fold during MIF morphogenesis in HeLa cells. DNA sequence analysis of the magA locus revealed an upstream divergently transcribed gene, msrA, encoding a peptide methionine sulfoxide reductase and a shared promoter region containing direct and indirect repeat sequences as well as −10 hexamers often associated with stationary-phase regulation. While MagA has no known function, it contains a conserved CXXC motif commonly found in members of the thioredoxin reductase family and in AhpD reductases that are associated with alkylhydroperoxide reductase (AhpC), suggesting a possible role in protection from oxidative stress. MIFs from L. pneumophila strain Lp02 containing a magA deletion exhibited differences in Giménez staining, as well as an apparent increase in cytopathology to HeLa cells, but otherwise were unaltered in virulence traits. As demonstrated by this study, MagA appears to be a MIF-specific protein expressed late in intracellular growth that may serve as a useful marker of development.

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In Vitro and In Vivo Oxidation of Methionine Residues in Small, Acid-Soluble Spore Proteins fromBacillus Species

Journal of Bacteriology ◽

10.1128/jb.180.10.2694-2700.1998 ◽

1998 ◽

Vol 180 (10) ◽

pp. 2694-2700 ◽

Cited By ~ 32

Author(s):

Christopher S. Hayes ◽

Berenice Illades-Aguiar ◽

Lilliam Casillas-Martinez ◽

Peter Setlow

Keyword(s):

Dna Binding ◽

Methionine Sulfoxide ◽

Small Degree ◽

Methionine Sulfoxide Reductase ◽

Mutant Form ◽

Wild Type ◽

Spore Proteins ◽

Methionine Residues

ABSTRACT Methionine residues in α/β-type small, acid-soluble spore proteins (SASP) of Bacillus species were readily oxidized to methionine sulfoxide in vitro by t-butyl hydroperoxide (tBHP) or hydrogen peroxide (H2O2). These oxidized α/β-type SASP no longer bound to DNA effectively, but DNA binding protected α/β-type SASP against methionine oxidation by peroxides in vitro. Incubation of an oxidized α/β-type SASP with peptidyl methionine sulfoxide reductase (MsrA), which can reduce methionine sulfoxide residues back to methionine, restored the α/β-type SASP’s ability to bind to DNA. Both tBHP and H2O2 caused some oxidation of the two methionine residues of an α/β-type SASP (SspC) in spores ofBacillus subtilis, although one methionine which is highly conserved in α/β-type SASP was only oxidized to a small degree. However, much more methionine sulfoxide was generated by peroxide treatment of spores carrying a mutant form of SspC which has a lower affinity for DNA. MsrA activity was present in wild-type B. subtilis spores. However, msrA mutant spores were no more sensitive to H2O2 than were wild-type spores. The major mechanism operating for dealing with oxidative damage to α/β-type SASP in spores is DNA binding, which protects the protein’s methionine residues from oxidation both in vitro and in vivo. This may be important in vivo since α/β-type SASP containing oxidized methionine residues no longer bind DNA well and α/β-type SASP-DNA binding is essential for long-term spore survival.

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Methionine Sulfoxide Reductase B Regulates the Activity of Ascorbate Peroxidase of Banana Fruit

Antioxidants ◽

10.3390/antiox10020310 ◽

2021 ◽

Vol 10 (2) ◽

pp. 310

Author(s):

Lu Xiao ◽

Guoxiang Jiang ◽

Huiling Yan ◽

Hongmei Lai ◽

Xinguo Su ◽

...

Keyword(s):

Ascorbate Peroxidase ◽

Posttranslational Modifications ◽

Stress Responses ◽

Redox State ◽

Methionine Sulfoxide ◽

Methionine Sulfoxide Reductase ◽

Banana Fruit ◽

Compound I

Ascorbate peroxidase (APX) is a key antioxidant enzyme that is involved in diverse developmental and physiological process and stress responses by scavenging H2O2 in plants. APX itself is also subjected to multiple posttranslational modifications (PTMs). However, redox-mediated PTM of APX in plants remains poorly understood. Here, we identified and confirmed that MaAPX1 interacts with methionine sulfoxide reductase B2 (MsrB2) in bananas. Ectopic overexpression of MaAPX1 delays the detached leaf senescence induced by darkness in Arabidopsis. Sulfoxidation of MaAPX1, i.e., methionine oxidation, leads to loss of the activity, which is repaired partially by MaMsrB2. Moreover, mimicking sulfoxidation by mutating Met36 to Gln also decreases its activity in vitro and in vivo, whereas substitution of Met36 with Val36 to mimic the blocking of sulfoxidation has little effect on APX activity. Spectral analysis showed that mimicking sulfoxidation of Met36 hinders the formation of compound I, the first intermediate between APX and H2O2. Our findings demonstrate that the redox state of methionine in MaAPX1 is critical to its activity, and MaMsrB2 can regulate the redox state and activity of MaAPX1. Our results revealed a novel post-translational redox modification of APX.

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An NMR-based biosensor to measure stereo-specific methionine sulfoxide reductase (MSR) activities in vitro and in vivo

10.1101/2020.05.14.092957 ◽

2020 ◽

Author(s):

Carolina Sánchez-López ◽

Natalia Labadie ◽

Verónica A. Lombardo ◽

Franco A. Biglione ◽

Bruno Manta ◽

...

Keyword(s):

Methionine Sulfoxide ◽

Methionine Sulfoxide Reductase ◽

Substrate Specificities ◽

Age Related ◽

Native Cell ◽

Highly Sensitive ◽

Sensitive Tool ◽

Single Reaction

AbstractOxidation of protein methionines to methionine-sulfoxides (MetOx) is associated with several age-related diseases. In healthy cells, MetOx is reduced to methionine by two families of conserved methionine sulfoxide reductase enzymes, MSRA and MSRB that specifically target the S- or R-diastereoisomers of methionine-sulfoxides, respectively. To directly interrogate MSRA and MSRB functions in cellular settings, we developed an NMR-based biosensor that we call CarMetOx to simultaneously measure both enzyme activities in single reaction setups. We demonstrate the suitability of our strategy to delineate MSR functions in complex biological environments that range from native cell lysates to zebrafish embryos. Thereby, we establish differences in substrate specificities between prokaryotic and eukaryotic MSRs and introduce CarMetOx as a highly sensitive tool for studying therapeutic targets of oxidative stress-related human diseases and redox regulated signaling pathways. Our approach further extends high-resolution in-cell NMR measurements of exogenously delivered biomolecules to an entire multicellular organism.

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METABOLIC CONSEQUENCES OF METHIONINE REDOX IN METHIONINE RESTRICTION

Innovation in Aging ◽

10.1093/geroni/igz038.398 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

pp. S106-S107

Author(s):

Kevin Thyne ◽

Yuhong Liu ◽

Adam B Salmon

Keyword(s):

Protein Function ◽

Methionine Sulfoxide ◽

Redox Status ◽

Significant Loss ◽

Methionine Sulfoxide Reductase ◽

Wild Type ◽

Entire Family ◽

Methionine Sulfoxide Reductase A ◽

Methionine Restriction

Abstract While caloric restriction (CR) provides highly robust improvements to longevity and health, dietary restriction of the essential amino acid methionine can provide similar benefits including improved metabolic function and increased longevity. Despite these similarities between CR and methionine restriction (MR), there is growing evidence to suggest they may be mediated by different mechanisms that require further elucidation. The sulfur side-chain of methionine is highly prone to oxidation, even in vivo, with redox changes of these residues potentially altering protein function and interfering with its use as a substrate. An entire family of enzymes, methionine sulfoxide reductases, have evolved in aerobic organisms to regulate the redox status of methionine. We tested the role of methionine sulfoxide reductase A (MsrA) in the physiological and metabolic benefits of MR. After three months of MR, mice lacking MsrA (MsrA KO) showed significant loss of weight, including both fat and lean mass, in comparison to wild-type mice under MR. Both MsrA KO and wild-type mice responded to MR with improvements to both glucose and insulin tolerance. However, MR MsrA KO mice showed lower HbA1c and reduced leptin compared to MR wild-type mice. Overall, our results show mice lacking MsrA have a stronger response to MR suggesting that methionine redox may play an important role in some of the mechanisms responsible for these metabolic outcomes. Further studies clarify whether MsrA could also be a potential regulator of the longevity benefits of MR.

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Oxidation of methionine residues activates the high-threshold heat-sensitive ion channel TRPV2

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1904332116 ◽

2019 ◽

Vol 116 (48) ◽

pp. 24359-24365 ◽

Cited By ~ 6

Author(s):

Tabea C. Fricke ◽

Frank Echtermeyer ◽

Johannes Zielke ◽

Jeanne de la Roche ◽

Milos R. Filipovic ◽

...

Keyword(s):

Transient Receptor Potential ◽

Receptor Potential ◽

Methionine Sulfoxide ◽

Ruthenium Red ◽

Methionine Sulfoxide Reductase ◽

High Threshold ◽

Temperature Dependent ◽

Excessive Heat ◽

Inward Currents

Thermosensitive transient receptor potential (TRP) ion channels detect changes in ambient temperature to regulate body temperature and temperature-dependent cellular activity. Rodent orthologs of TRP vanilloid 2 (TRPV2) are activated by nonphysiological heat exceeding 50 °C, and human TRPV2 is heat-insensitive. TRPV2 is required for phagocytic activity of macrophages which are rarely exposed to excessive heat, but what activates TRPV2 in vivo remains elusive. Here we describe the molecular mechanism of an oxidation-induced temperature-dependent gating of TRPV2. While high concentrations of H2O2 induce a modest sensitization of heat-induced inward currents, the oxidant chloramine-T (ChT), ultraviolet A light, and photosensitizing agents producing reactive oxygen species (ROS) activate and sensitize TRPV2. This oxidation-induced activation also occurs in excised inside-out membrane patches, indicating a direct effect on TRPV2. The reducing agent dithiothreitol (DTT) in combination with methionine sulfoxide reductase partially reverses ChT-induced sensitization, and the substitution of the methionine (M) residues M528 and M607 to isoleucine almost abolishes oxidation-induced gating of rat TRPV2. Mass spectrometry on purified rat TRPV2 protein confirms oxidation of these residues. Finally, macrophages generate TRPV2-like heat-induced inward currents upon oxidation and exhibit reduced phagocytosis when exposed to the TRP channel inhibitor ruthenium red (RR) or to DTT. In summary, our data reveal a methionine-dependent redox sensitivity of TRPV2 which may be an important endogenous mechanism for regulation of TRPV2 activity and account for its pivotal role for phagocytosis in macrophages.

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Methionine Sulfoxide Reductase B1 Regulates Hepatocellular Carcinoma Cell Proliferation and Invasion via the Mitogen-Activated Protein Kinase Pathway and Epithelial-Mesenchymal Transition

Oxidative Medicine and Cellular Longevity ◽

10.1155/2018/5287971 ◽

2018 ◽

Vol 2018 ◽

pp. 1-15 ◽

Cited By ~ 8

Author(s):

Qiang He ◽

Hui Li ◽

Fanzhi Meng ◽

Xiangjun Sun ◽

Xu Feng ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Proliferation ◽

Epithelial Mesenchymal Transition ◽

Mapk Pathway ◽

Methionine Sulfoxide ◽

Methionine Sulfoxide Reductase ◽

Mitogen Activated Protein Kinase ◽

Migration And Invasion ◽

Mesenchymal Transition

Methionine sulfoxide reductase B1 (MsrB1) is a member of the selenoprotein family, which contributes to the reduction of methionine sulfoxides produced from reactive oxygen species (ROS) by redox processes in energy pathways. However, few studies have examined the role of MsrB1 in human hepatocellular carcinoma (HCC). We observed that MsrB1 is highly expressed in HCC tissues and that its expression correlated with the prognoses of patients with HCC after hepatectomy. In vitro, knockdown of MsrB1 inhibits HCC cell growth by MTT and EdU proliferation assay, and MsrB1 interference enhances H2O2/trx-induced apoptosis. We observed that phosphorylation of the key proteins of the MAPK pathway, namely, ERK, MEK, and p53, was inhibited, but PARP and caspase 3 were increased, thus infecting mitochondrial integrity. In vivo, MsrB1 knockdown effectively inhibited tumor growth. Furthermore, MsrB1 knockdown reduced HCC cell migration and invasion in a transwell assay through inhibition of cytoskeletal rearrangement and spread. This change was linked to epithelial-mesenchymal transition (EMT) inhibition resulting from increases in E-cadherin expression and decreases in expression in TGF-β1, Slug, MMP-2/9, and so on. MsrB1 regulates HCC cell proliferation and migration by modulating the MAPK pathway and EMT. Thus, MsrB1 may be a novel therapeutic target with respect to the treatment of HCC.

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